Low-level lasers alter mRNA levels from traditional reference genes used in breast cancer cells

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Teixeira, A. F.; Canuto, K. S.; Rodrigues, J. A.; Fonseca, A. S.; Mencalha, A. L.

2017-07-01

Cancer is among the leading causes of mortality worldwide, increasing the importance of treatment development. Low-level lasers are used in several diseases, but some concerns remains on cancers. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a technique used to understand cellular behavior through quantification of mRNA levels. Output data from target genes are commonly relative to a reference that cannot vary according to treatment. This study evaluated reference genes levels from MDA-MB-231 cells exposed to red or infrared lasers at different fluences. Cultures were exposed to red and infrared lasers, incubated (4 h, 37 °C), total RNA was extracted and cDNA synthesis was performed to evaluate mRNA levels from ACTB, GUSB and TRFC genes by RT-qPCR. Specific amplification was verified by melting curves and agarose gel electrophoresis. RefFinder enabled data analysis by geNorm, NormFinder and BestKeeper. Specific amplifications were obtained and, although mRNA levels from ACTB, GUSB or TRFC genes presented no significant variation through traditional statistical analysis, Excel-based tools revealed that the use of these reference genes are dependent of laser characteristics. Our data showed that exposure to low-level red and infrared lasers at different fluences alter the mRNA levels from ACTB, GUSB and TRFC in MDA-MB-231 cells.

The relative contributions of scatter and attenuation corrections toward improved brain SPECT quantification

International Nuclear Information System (INIS)

Stodilka, Robert Z.; Msaki, Peter; Prato, Frank S.; Nicholson, Richard L.; Kemp, B.J.

1998-01-01

Mounting evidence indicates that scatter and attenuation are major confounds to objective diagnosis of brain disease by quantitative SPECT. There is considerable debate, however, as to the relative importance of scatter correction (SC) and attenuation correction (AC), and how they should be implemented. The efficacy of SC and AC for 99m Tc brain SPECT was evaluated using a two-compartment fully tissue-equivalent anthropomorphic head phantom. Four correction schemes were implemented: uniform broad-beam AC, non-uniform broad-beam AC, uniform SC+AC, and non-uniform SC+AC. SC was based on non-stationary deconvolution scatter subtraction, modified to incorporate a priori knowledge of either the head contour (uniform SC) or transmission map (non-uniform SC). The quantitative accuracy of the correction schemes was evaluated in terms of contrast recovery, relative quantification (cortical:cerebellar activity), uniformity ((coefficient of variation of 230 macro-voxels) x100%), and bias (relative to a calibration scan). Our results were: uniform broad-beam (μ=0.12cm -1 ) AC (the most popular correction): 71% contrast recovery, 112% relative quantification, 7.0% uniformity, +23% bias. Non-uniform broad-beam (soft tissue μ=0.12cm -1 ) AC: 73%, 114%, 6.0%, +21%, respectively. Uniform SC+AC: 90%, 99%, 4.9%, +12%, respectively. Non-uniform SC+AC: 93%, 101%, 4.0%, +10%, respectively. SC and AC achieved the best quantification; however, non-uniform corrections produce only small improvements over their uniform counterparts. SC+AC was found to be superior to AC; this advantage is distinct and consistent across all four quantification indices. (author)

Properties of the reverse transcription reaction in mRNA quantification

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Ståhlberg, Anders; Håkansson, Joakim; Xian, Xiaojie

2004-01-01

BACKGROUND: In most measurements of gene expression, mRNA is first reverse-transcribed into cDNA. We studied the reverse transcription reaction and its consequences for quantitative measurements of gene expression. METHODS: We used SYBR green I-based quantitative real-time PCR (QPCR) to measure...... the properties of reverse transcription reaction for the beta-tubulin, glyceraldehyde-3-phosphate dehydrogenase, Glut2, CaV1D, and insulin II genes, using random hexamers, oligo(dT), and gene-specific reverse transcription primers. RESULTS: Experimental variation in reverse transcription-QPCR (RT......-QPCR) was mainly attributable to the reverse transcription step. Reverse transcription efficiency depended on priming strategy, and the dependence was different for the five genes studied. Reverse transcription yields also depended on total RNA concentration. CONCLUSIONS: RT-QPCR gene expression measurements...

Urinary mRNA for the Diagnosis of Renal Allograft Rejection: The Issue of Normalization.

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Galichon, P; Amrouche, L; Hertig, A; Brocheriou, I; Rabant, M; Xu-Dubois, Y-C; Ouali, N; Dahan, K; Morin, L; Terzi, F; Rondeau, E; Anglicheau, D

2016-10-01

Urinary messenger RNA (mRNA) quantification is a promising method for noninvasive diagnosis of renal allograft rejection (AR), but the quantification of mRNAs in urine remains challenging due to degradation. RNA normalization may be warranted to overcome these issues, but the strategies of gene normalization have been poorly evaluated. Herein, we address this issue in a case-control study of 108 urine samples collected at time of allograft biopsy in kidney recipients with (n = 52) or without (n = 56) AR by comparing the diagnostic value of IP-10 and CD3ε mRNAs-two biomarkers of AR-after normalization by the total amount of RNA, normalization by one of the three widely used reference RNAs-18S, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Hypoxanthine-guanine phosphoribosyltransferase (HPRT)-or normalization using uroplakin 1A (UPK) mRNA as a possible urine-specific reference mRNA. Our results show that normalization based on the total quantity of RNA is not substantially improved by additional normalization and may even be worsened with some classical reference genes that are overexpressed during rejection. However, considering that normalization by a reference gene is necessary to ensure polymerase chain reaction (PCR) quality and reproducibility and to suppress the effect of RNA degradation, we suggest that GAPDH and UPK1A are preferable to 18S or HPRT RNA. © Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

DDAH2 mRNA expression is inversely associated with some cardiovascular risk-related features in healthy young adults.

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Puchau, Blanca; Hermsdorff, Helen Hermana M; Zulet, M Angeles; Martínez, J Alfredo

2009-01-01

The purpose of this study was to evaluate whether the mRNA expression profiles of three genes (PRMT1, DDAH2 and NOS3) are related to ADMA metabolism and signalling, and the potential relationships with anthropometrical, biochemical, lifestyle and inflammatory indicators in healthy young adults. An emphasis on the putative effect of different mRNA expression on cardiovascular risk-related features was paid. Anthropometrical measurements as well as lifestyle features were analyzed in 120 healthy young adults. Fasting blood samples were collected for the measurement of glucose and lipid profiles as well as the concentrations of selected inflammatory markers. Profiles of mRNA expression were assessed for PRMT1, DDAH2 and NOS3 genes from peripheral blood mononuclear cells. Regarding inflammatory biomarkers, DDAH2 was inversely associated with IL-6 and TNF-alpha. Moreover, subjects in the highest quintile of DDAH2 mRNA expression showed a reduced risk to have higher values of waist circumference, and to be more prone to show higher values of HDL-c. Interestingly, DDAH2 gene expression seemed to be related with some anthropometrical, biochemical, lifestyle and inflammatory indicators linked to cardiovascular risk in apparently healthy young adults, emerging as a potential disease marker.

DDAH2 mRNA Expression Is Inversely Associated with Some Cardiovascular Risk-Related Features in Healthy Young Adults

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Blanca Puchau

2009-01-01

Full Text Available The purpose of this study was to evaluate whether the mRNA expression profiles of three genes (PRMT1, DDAH2 and NOS3 are related to ADMA metabolism and signalling, and the potential relationships with anthropometrical, biochemical, lifestyle and inflammatory indicators in healthy young adults. An emphasis on the putative effect of different mRNA expression on cardiovascular risk-related features was paid. Anthropometrical measurements as well as lifestyle features were analyzed in 120 healthy young adults. Fasting blood samples were collected for the measurement of glucose and lipid profiles as well as the concentrations of selected inflammatory markers. Profiles of mRNA expression were assessed for PRMT1, DDAH2 and NOS3 genes from peripheral blood mononuclear cells. Regarding inflammatory biomarkers, DDAH2 was inversely associated with IL-6 and TNF-α. Moreover, subjects in the highest quintile of DDAH2 mRNA expression showed a reduced risk to have higher values of waist circumference, and to be more prone to show higher values of HDL-c. Interestingly, DDAH2 gene expression seemed to be related with some anthropometrical, biochemical, lifestyle and inflammatory indicators linked to cardiovascular risk in apparently healthy young adults, emerging as a potential disease marker.

The quantification of COMT mRNA in post mortem cerebellum tissue: diagnosis, genotype, methylation and expression

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Craig Ian W

2006-02-01

Full Text Available Abstract Background The COMT gene is located on chromosome 22q11, a region strongly implicated in the aetiology of several psychiatric disorders, in particular schizophrenia. Previous research has suggested that activity and expression of COMT is altered in schizophrenia, and is mediated by one or more polymorphisms within the gene, including the functional Val158Met polymorphism. Method In this study we examined the expression levels of COMT mRNA using quantitative RT-PCR in 60 post mortem cerebellum samples derived from individuals with schizophrenia, bipolar disorder, depression, and no history of psychopathology. Furthermore, we have examined the methylation status of two CpG sites in the promoter region of the gene. Results We found no evidence of altered COMT expression or methylation in any of the psychiatric diagnoses examined. We did, however, find evidence to suggest that genotype is related to COMT gene expression, replicating the findings of two previous studies. Specifically, val158met (rs165688; Val allele rs737865 (G allele and rs165599 (G allele all showed reduced expression (P COMT expression, with females exhibiting significantly greater levels of COMT mRNA. Conclusion The expression of COMT does not appear to be altered in the cerebellum of individuals suffering from schizophrenia, bipolar disorder or depression, but does appear to be influenced by single nucleotide polymorphisms within the gene.

Short interfering RNAs targeting a vampire-bat related rabies virus phosphoprotein mRNA.

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Ono, Ekaterina Alexandrovna Durymanova; Taniwaki, Sueli Akemi; Brandão, Paulo

The aim of this study was to assess the in vitro and in vivo effects of short-interfering RNAs (siRNAs) against rabies virus phosphoprotein (P) mRNA in a post-infection treatment for rabies as an extension of a previous report (Braz J Microbiol. 2013 Nov 15;44(3):879-82). To this end, rabies virus strain RABV-4005 (related to the Desmodus rotundus vampire bat) were used to inoculate BHK-21 cells and mice, and the transfection with each of the siRNAs was made with Lipofectamine-2000™. In vitro results showed that siRNA 360 was able to inhibit the replication of strain RABV-4005 with a 1log decrease in virus titter and 5.16-fold reduction in P mRNA, 24h post-inoculation when compared to non-treated cells. In vivo, siRNA 360 was able to induce partial protection, but with no significant difference when compared to non-treated mice. These results indicate that, despite the need for improvement for in vivo applications, P mRNA might be a target for an RNAi-based treatment for rabies. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

A pilot trial assessing urinary gene expression profiling with an mRNA array for diabetic nephropathy.

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Min Zheng

Full Text Available BACKGROUND: The initiation and progression of diabetic nephropathy (DN is complex. Quantification of mRNA expression in urinary sediment has emerged as a novel strategy for studying renal diseases. Considering the numerous molecules involved in DN development, a high-throughput platform with parallel detection of multiple mRNAs is needed. In this study, we constructed a self-assembling mRNA array to analyze urinary mRNAs in DN patients with aims to reveal its potential in searching novel biomarkers. METHODS: mRNA array containing 88 genes were fabricated and its performance was evaluated. A pilot study with 9 subjects including 6 DN patients and 3 normal controls were studied with the array. DN patients were assigned into two groups according to their estimate glomerular rate (eGFR: DNI group (eGFR>60 ml/min/1.73 m(2, n = 3 and DNII group (eGFR<60 ml/min/1.73 m(2, n = 3. Urinary cell pellet was collected from each study participant. Relative abundance of these target mRNAs from urinary pellet was quantified with the array. RESULTS: The array we fabricated displayed high sensitivity and specificity. Moreover, the Cts of Positive PCR Controls in our experiments were 24±0.5 which indicated high repeatability of the array. A total of 29 mRNAs were significantly increased in DN patients compared with controls (p<0.05. Among these genes, α-actinin4, CDH2, ACE, FAT1, synaptopodin, COL4α, twist, NOTCH3 mRNA expression were 15-fold higher than those in normal controls. In contrast, urinary TIMP-1 mRNA was significantly decreased in DN patients (p<0.05. It was shown that CTGF, MCP-1, PAI-1, ACE, CDH1, CDH2 mRNA varied significantly among the 3 study groups, and their mRNA levels increased with DN progression (p<0.05. CONCLUSION: Our pilot study demonstrated that mRNA array might serve as a high-throughput and sensitive tool for detecting mRNA expression in urinary sediment. Thus, this primary study indicated that mRNA array probably could be a

A selective splicing variant of hepcidin mRNA in hepatocellular carcinoma cell lines

International Nuclear Information System (INIS)

Toki, Yasumichi; Sasaki, Katsunori; Tanaka, Hiroki; Yamamoto, Masayo; Hatayama, Mayumi; Ito, Satoshi; Ikuta, Katsuya; Shindo, Motohiro; Hasebe, Takumu; Nakajima, Shunsuke; Sawada, Koji; Fujiya, Mikihiro; Torimoto, Yoshihiro; Ohtake, Takaaki; Kohgo, Yutaka

2016-01-01

Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncated peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells. - Highlights: • An aberrant splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene. • Absolute quantification of hepcidin mRNA by digital PCR amplification. • Hepatoma-derived cell lines have significant copies of variant-type hepcidin mRNA. • Truncated preprohepcidin is secreted from cells without posttranslational cleavage.

A selective splicing variant of hepcidin mRNA in hepatocellular carcinoma cell lines

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Toki, Yasumichi [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Sasaki, Katsunori, E-mail: k-sasaki@asahikawa-med.ac.jp [Department of Gastrointestinal Immunology and Regenerative Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Tanaka, Hiroki [Department of Legal Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Yamamoto, Masayo; Hatayama, Mayumi; Ito, Satoshi; Ikuta, Katsuya; Shindo, Motohiro; Hasebe, Takumu; Nakajima, Shunsuke; Sawada, Koji; Fujiya, Mikihiro [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Torimoto, Yoshihiro [Oncology Center, Asahikawa Medical University Hospital, Hokkaido 078-8510 (Japan); Ohtake, Takaaki; Kohgo, Yutaka [Department of Gastroenterology, International University of Health and Welfare Hospital, Tochigi 329-2763 (Japan)

2016-08-05

Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncated peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells. - Highlights: • An aberrant splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene. • Absolute quantification of hepcidin mRNA by digital PCR amplification. • Hepatoma-derived cell lines have significant copies of variant-type hepcidin mRNA. • Truncated preprohepcidin is secreted from cells without posttranslational cleavage.

High-level mRNA quantification of proliferation marker pKi-67 is correlated with favorable prognosis in colorectal carcinoma.

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Ihmann, Thomas; Liu, Jian; Schwabe, Wolfgang; Häusler, Peter; Behnke, Detlev; Bruch, Hans-Peter; Broll, Rainer; Windhövel, Ute; Duchrow, Michael

2004-12-01

The present study retrospectively examines the expression of pKi-67 mRNA and protein in colorectal carcinoma and their correlation to the outcome of patients. Immunohistochemistry and quantitative RT-PCR were used to analyze the expression of pKi-67 in 43 archival specimens of patients with curatively resected primary colorectal carcinoma, who were not treated with neo-adjuvant therapy. We determined a median pKi-67 (MIB-1) labeling index of 31.3% (range 10.3-66.4%), and a mean mRNA level of 0.1769 (DeltaC(T): range 0.01-0.69); indices and levels did not correlate. High pKi-67 mRNA DeltaC(T) values were associated with a significantly favorable prognosis, while pKi-67 labeling indices were not correlated to prognostic outcome. A multivariate analysis of clinical and biological factors indicated that tumor stage (UICC) and pKi-67 mRNA expression level were independent prognostic factors. Quantitatively determined pKi-67 mRNA can be a good and new prognostic indicator for primary resected colorectal carcinoma.

Influence of culture medium composition on relative mRNA abundances in domestic cat embryos.

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Hribal, R; Jewgenow, K; Braun, B C; Comizzoli, P

2013-04-01

Different culture conditions have been used to produce domestic cat embryos. As part of the in vitro procedures, the medium composition significantly affects the quality of the embryo development also. Quality assessments based on cleavage kinetics and blastomere symmetry are useful, but embryos also can differ in their relative gene expression patterns despite similar morphological characteristics. The aim of this study was to compare cat embryos produced with two different in vitro culture systems routinely used in two different laboratories [Smithsonian Conservation Biology Institute, Washington D.C., USA (SCBI) and Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany (IZW)]. Specifically, relative mRNA expression patterns of critical genes for pre-implantation embryo development were assessed in both conditions. Embryos were produced in parallel in both culture systems by IVF using frozen-thawed ejaculated semen in the United States and fresh epididymal sperm in Germany. Success of embryo development in vitro was recorded as well as relative mRNA abundances [DNA methyltransferases 1 and 3A (DNMT1, DNMT3A), gap junction protein alpha 1 (GJA1), octamer-binding transcription factor 4 [OCT4], insulin-like growth factors 1 and 2 receptors (IGF1R, IGF2R), beta-actin (ACTB)] in pools of days 4-5 morulae by semi-quantitative RT-PCR assay. Percentages of cleaved embryos were similar (p > 0.05) between both culture systems, regardless of the location. OCT4 mRNA abundance was higher (p culture system compared with those from the IZW system when epididymal sperm was used for IVF. No clear correlation between the expression pattern and the culture system could be found for all other genes. It is suggested that OCT4 expression might be affected by the media composition in some conditions and can be the indicator of a better embryo quality. © 2012 Blackwell Verlag GmbH.

Quantification of differential gene expression by multiplexed targeted resequencing of cDNA

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Arts, Peer; van der Raadt, Jori; van Gestel, Sebastianus H.C.; Steehouwer, Marloes; Shendure, Jay; Hoischen, Alexander; Albers, Cornelis A.

2017-01-01

Whole-transcriptome or RNA sequencing (RNA-Seq) is a powerful and versatile tool for functional analysis of different types of RNA molecules, but sample reagent and sequencing cost can be prohibitive for hypothesis-driven studies where the aim is to quantify differential expression of a limited number of genes. Here we present an approach for quantification of differential mRNA expression by targeted resequencing of complementary DNA using single-molecule molecular inversion probes (cDNA-smMIPs) that enable highly multiplexed resequencing of cDNA target regions of ∼100 nucleotides and counting of individual molecules. We show that accurate estimates of differential expression can be obtained from molecule counts for hundreds of smMIPs per reaction and that smMIPs are also suitable for quantification of relative gene expression and allele-specific expression. Compared with low-coverage RNA-Seq and a hybridization-based targeted RNA-Seq method, cDNA-smMIPs are a cost-effective high-throughput tool for hypothesis-driven expression analysis in large numbers of genes (10 to 500) and samples (hundreds to thousands). PMID:28474677

Expression of Panton-Valentine leukocidin mRNA among Staphylococcus aureus isolates associates with specific clinical presentations.

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Fangyou Yu

Full Text Available Panton-Valentine leukocidin (PVL; gene designation lukF/S-PV is likely an important virulence factor for Staphylococcus aureus (S. aureus, as qualitative expression of the protein correlates with severity for specific clinical presentations, including skin and soft tissue infections (SSTIs. Development of genetic approaches for risk-assessment of patients with S. aureus infections may prove clinically useful, and whether lukF/S-PV gene expression correlates with specific clinical presentations for S. aureus has been largely unexplored. In the present study, we quantified lukS-PV mRNA among 96 S. aureus isolates to determine whether expression levels correlated with specific clinical presentations in adults and children. Expression level of lukS-PV mRNA among isolates from skin and soft tissue infections (SSTIs was significantly greater than among isolates from blood stream infection (BSIs, and expression level of lukS-PV mRNA among BSI isolates from children was significantly greater than for BSI isolates among adults. Moreover, expression level of lukS-PV mRNA among community-acquired (CA isolates was significantly greater than for hospital-acquired (HA isolates. These data justify additional studies to determine the potential clinical utility for lukS-PV mRNA quantification as a predictive tool for severity of S. aureus infection.

Circadian transitions in radiation dose-dependent augmentation of mRNA levels for DNA damage-induced genes elicited by accurate real-time RT-PCR quantification

International Nuclear Information System (INIS)

Ishihara, Hiroshi; Tanaka, Izumi; Yakumaru, Haruko

2010-01-01

Molecular mechanisms of intracellular response after DNA-damage by exposure to ionizing radiation have been studied. In the case of cells isolated from living body of human and experimental animals, alteration of the responsiveness by physiological oscillation such as circadian rhythm must be considered. To examine the circadian variation in the response of p53-responsible genes p21, mdm2, bax, and puma, we established a method to quantitate their mRNA levels with high reproducibility and accuracy based on real-time reverse transcription polymerase chain reaction (RT-PCR) and compared the levels of responsiveness in mouse hemocytes after diurnal irradiation to that after nocturnal irradiation. Augmentations of p21 and mdm2 mRNA levels with growth-arrest and of puma mRNA before apoptosis were confirmed by time-course experiment in RAW264.7, and dose-dependent increases in the peak levels of all the RNA were shown. Similarly, the relative RNA levels of p21, mdm2, bax, and puma per glyceraldehyde-3-phosphate dehydrogenase (GAPDH) also increased dose-dependently in peripheral blood and bone marrow cells isolated from whole-body-irradiated mice. Induction levels of all messages reduced by half after nighttime irradiation as compared with daytime irradiation in blood cells. In marrow cells, nighttime irradiation enhanced the p21 and mdm2 mRNA levels than daytime irradiation. No significant difference in bax or puma mRNA levels was observed between nighttime and daytime irradiation in marrow cells. This suggests that early-stage cellular responsiveness in DNA damage-induced genes is modulated between diurnal and nocturnal irradiation. (author)

Analysis of mRNA expression of genes related to fatty acids synthesis in goose fatty liver

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Shuxia Xiang

2010-11-01

Full Text Available The aim of our study was to evaluate the effect of overfeeding on mRNA expression levels of genes involved in lipogenesis, in order to understand the mechanism of hepatic stea - tosis in the goose. Using Landes geese (Anser anser and Sichuan White geese (Anser cygnoides as experimental animals, we quantified the mRNA expression of lipogenic genes, acetyl-CoA carboxylase-α (ACCα and fatty acid synthase (FAS, and of two transcription factors, sterol regulatory element-binding proteins- 1 (SREBP-1 and carbohydrate responsive element-binding protein (ChREBP by real-time polymerase chain reaction (RTPCR, and measured the lipid and triglyceride (TG content in the liver and the plasma level of glucose, insulin and TG. Our results indicated that compared to the control group, the overfeeding induced an increase of the lipid and TG content in the liver and also of the plasma insulin and TG concentration in both breeds. However, the plasma glucose level decreased after overfeeding in the Sichuan White goose, and there was no evident change in the Landes goose. Lastly, the mRNA expression of ACCα, FAS, SREBP-1 and ChREBP in the overfed group was lower than in the control group in both breeds. We concluded that the lipogenesis pathway plays a role in overfeeding- induced hepatic steatosis and that the decreased mRNA level of related genes may be the indicator of hepatic steatosis.

Reliable PCR quantitation of estrogen, progesterone and ERBB2 receptor mRNA from formalin-fixed, paraffin-embedded tissue is independent of prior macro-dissection

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Tramm, Trine; Hennig, Guido; Kyndi, Marianne

2013-01-01

Gene expression analysis on messenger RNA (mRNA) purified from formalin-fixed, paraffin-embedded tissue is increasingly used for research purposes. Tissue heterogeneity may question specificity and interpretation of results from mRNA isolated from a whole slide section, and thresholds for minimal...... tumor content in the paraffin block or macrodissection are used to avoid contamination from non-neoplastic tissue. The aim was to test if mRNA from tissue surrounding breast cancer affected quantification of estrogen receptor α (ESR1), progesterone receptor (PGR) and human epidermal growth factor...... receptor 2 (ERBB2), by comparing gene expression from whole slide and tumor-enriched sections, and correlating gene expression from whole slide sections with corresponding immunohistochemistry. Gene expression, based on mRNA extracted from a training set (36 paraffin blocks) and two validation sets (133...

[Effects of lipopolysaccharides extracted from Porphyromonas endodontalis on the expression of IL-1beta mRNA and IL-6 mRNA in osteoblasts].

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Yang, Di; Li, Ren; Qiu, Li-Hong; Li, Chen

2009-04-01

To quantify the IL-1 beta mRNA and IL-6 mRNA expression induced by lipopolysaccharides (LPS)extracted from Porphyromonas endodontalis(P.e) in osteoblasts, and to relate P.e-LPS to bone absorption pathogenesis in lesions of chronical apical periodontitis. MG63 was treated with different concentrations of P.e-LPS(0-50 microg/mL) for different hours(0-24h). The expression of IL-1 beta mRNA and IL-6 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR).Statistical analysis was performed using one- way ANOVA and Dunnett t test with SPSS11.0 software package. The level of IL-1 beta mRNA and IL-6 mRNA increased significantly after treatment with P.e-LPS at more than 5 microg/mL (P<0.01)and for more than 1 hour (P<0.01), which indicated that P.e-LPS induced osteoblasts to express IL-1 beta mRNA and IL-6 mRNA in dose and time dependent manners. P.e-LPS may promote bone resorption in lesions of chronical apical periodontitis by inducing IL-1 beta mRNA and IL-6 mRNA expression in osteoblasts.

Epigenetic mechanisms involved in differential MDR1 mRNA expression between gastric and colon cancer cell lines and rationales for clinical chemotherapy

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Kim Kyung-Jong

2008-08-01

Full Text Available Abstract Background The membrane transporters such as P-glycoprotein (Pgp, the MDR1 gene product, are one of causes of treatment failure in cancer patients. In this study, the epigenetic mechanisms involved in differential MDR1 mRNA expression were compared between 10 gastric and 9 colon cancer cell lines. Methods The MDR1 mRNA levels were determined using PCR and real-time PCR assays after reverse transcription. Cytotoxicity was performed using the MTT assay. Methylation status was explored by quantification PCR-based methylation and bisulfite DNA sequencing analyses. Results The MDR1 mRNA levels obtained by 35 cycles of RT-PCR in gastric cancer cells were just comparable to those obtained by 22 cycles of RT-PCR in colon cancer cells. Real-time RT-PCR analysis revealed that MDR1 mRNA was not detected in the 10 gastric cancer cell lines but variable MDR1 mRNA levels in 7 of 9 colon cancer cell lines except the SNU-C5 and HT-29 cells. MTT assay showed that Pgp inhibitors such as cyclosporine A, verapamil and PSC833 sensitized Colo320HSR (colon, highest MDR1 expression but not SNU-668 (gastric, highest and SNU-C5 (gastric, no expression to paclitaxel. Quantification PCR-based methylation analysis revealed that 90% of gastric cancer cells, and 33% of colon cancer cells were methylated, which were completely matched with the results obtained by bisulfite DNA sequencing analysis. 5-aza-2'-deoxcytidine (5AC, a DNA methyltransferase inhibitor increased the MDR1 mRNA levels in 60% of gastric cells, and in 11% of colon cancer cells. Trichostatin A (TSA, histone deacetylase inhibitor increased the MDR1 mRNA levels in 70% of gastric cancer cells and 55% of colon cancer cells. The combined treatment of 5AC with TSA increased the MDR1 mRNA levels additively in 20% of gastric cancer cells, but synergistically in 40% of gastric and 11% of colon cancer cells. Conclusion These results indicate that the MDR1 mRNA levels in gastric cancer cells are significantly

The Prognostic Relevance of Sentinel Lymph Node Metastases Assessed by PHGR1 mRNA Quantification in Stage I to III Colon Cancer

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Satu Oltedal

2018-04-01

Full Text Available BACKGROUND: Regional lymph node (LN metastasis is a strong and well-established prognostic factor in colon cancer, and recent data suggest a prognostic value of detecting micrometastases and isolated tumor cells in regional LNs. The aim of the study was to investigate the clinical relevance of detecting sentinel lymph node (SLN metastases in colon cancer patients by measuring the novel metastasis marker PHGR1 mRNA. METHODS: Using quantitative reverse-transcription polymerase chain reaction, we measured PHGR1 mRNA levels in SLNs and primary tumors from 206 patients surgically treated for stage I to III colon cancer and 52 normal LNs from patients undergoing surgery for benign colon diseases. The prognostic impact of these findings was evaluated by Kaplan-Meier analysis and Cox proportional-hazards regression. RESULTS: Compared to normal LNs, elevated PHGR1 mRNA levels were detected in SLNs from 56 (89% of the 63 patients with pN+ disease. Furthermore, 68 (48% of the 143 node-negative (pN0 patients had elevated PHGR1 mRNA levels in SLNs, suggesting occult metastases. With a median follow-up of 7.2 years, a significantly shorter recurrence-free (P=.005 and disease-specific (P=.02 survival was observed in patients with elevated PHGR1 mRNA levels in SLNs. Multivariable modeling showed that the SLN PHGR1 mRNA level was an independent prognostic factor. However, when the survival analyses were restricted to pN0 patients, no significant prognostic information was found. CONCLUSION: Measuring PHGR1 mRNA in SLNs provided independent prognostic information on operable colon cancer patients but not in the pN0 subgroup.

Chitinase mRNA levels by quantitative PCR using the single standard DNA: acidic mammalian chitinase is a major transcript in the mouse stomach.

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Misa Ohno

Full Text Available Chitinases hydrolyze the β-1-4 glycosidic bonds of chitin, a major structural component of fungi, crustaceans and insects. Although mammals do not produce chitin or its synthase, they express two active chitinases, chitotriosidase (Chit1 and acidic mammalian chitinase (AMCase. These mammalian chitinases have attracted considerable attention due to their increased expression in individuals with a number of pathological conditions, including Gaucher disease, Alzheimer's disease and asthma. However, the contribution of these enzymes to the pathophysiology of these diseases remains to be determined. The quantification of the Chit1 and AMCase mRNA levels and the comparison of those levels with the levels of well-known reference genes can generate useful and biomedically relevant information. In the beginning, we established a quantitative real-time PCR system that uses standard DNA produced by ligating the cDNA fragments of the target genes. This system enabled us to quantify and compare the expression levels of the chitinases and the reference genes on the same scale. We found that AMCase mRNA is synthesized at extraordinarily high levels in the mouse stomach. The level of this mRNA in the mouse stomach was 7- to 10-fold higher than the levels of the housekeeping genes and was comparable to that the level of the mRNA for pepsinogen C (progastricsin, a major component of the gastric mucosa. Thus, AMCase mRNA is a major transcript in mouse stomach, suggesting that AMCase functions as a digestive enzyme that breaks down polymeric chitin and as part of the host defense against chitin-containing pathogens in the gastric contents. Our methodology is applicable to the quantification of mRNAs for multiple genes across multiple specimens using the same scale.

Quantitative evaluation of alternatively spliced mRNA isoforms by label-free real-time plasmonic sensing.

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Huertas, César S; Carrascosa, L G; Bonnal, S; Valcárcel, J; Lechuga, L M

2016-04-15

Alternative splicing of mRNA precursors enables cells to generate different protein outputs from the same gene depending on their developmental or homeostatic status. Its deregulation is strongly linked to disease onset and progression. Current methodologies for monitoring alternative splicing demand elaborate procedures and often present difficulties in discerning between closely related isoforms, e.g. due to cross-hybridization during their detection. Herein, we report a general methodology using a Surface Plasmon Resonance (SPR) biosensor for label-free monitoring of alternative splicing events in real-time, without any cDNA synthesis or PCR amplification requirements. We applied this methodology to RNA isolated from HeLa cells for the quantification of alternatively spliced isoforms of the Fas gene, involved in cancer progression through regulation of programmed cell death. We demonstrate that our methodology is isoform-specific, with virtually no cross-hybridization, achieving limits of detection (LODs) in the picoMolar (pM) range. Similar results were obtained for the detection of the BCL-X gene mRNA isoforms. The results were independently validated by RT-qPCR, with excellent concordance in the determination of isoform ratios. The simplicity and robustness of this biosensor technology can greatly facilitate the exploration of alternative splicing biomarkers in disease diagnosis and therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

Associations of hypoosmotic swelling test, relative sperm volume shift, aquaporin7 mRNA abundance and bull fertility estimates.

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Kasimanickam, R K; Kasimanickam, V R; Arangasamy, A; Kastelic, J P

2017-02-01

Mammalian sperm are exposed to a natural hypoosmotic environment during male-to-female reproductive tract transition; although this activates sperm motility in vivo, excessive swelling can harm sperm structure and function. Aquaporins (AQPs) is a family of membrane-channel proteins implicated in sperm osmoregulation. The objective was to determine associations among relative sperm volume shift, hypoosmotic swelling test (HOST), sperm aquaporin (AQP) 7 mRNA abundances, and sire conception rate (SCR; fertility estimate) in Holstein bulls at a commercial artificial insemination center. Three or four sires for each full point SCR score from -4 to +4 were included. Each SCR estimate for study bulls (N = 30) was based on > 500 services (mean ± SEM) of 725 ± 13 services/sire). Sperm from a single collection day (two ejaculates) from these commercial Holstein bulls were used. Relative mRNA expression of AQP7 in sperm was determined by polymerase chain reaction. Mean relative sperm volume shift and percentage of sperm reacted in a HOST (% HOST) were determined (400 sperm per bull) after incubating in isoosmotic (300 mOsm/kg) and hypoosmotic (100 mOsm/kg) solutions for 30 min. There was no correlation between %HOST and SCR (r = 0.28 P > 0.1). However, there was a positive correlation between relative sperm volume shift and SCR (r = 0.65, P 2) fertility sire groups. In conclusion, bulls with higher SCR had significantly greater AQP7 mRNA abundance in frozen-thawed sperm. This plausibly contributed to greater regulation of sperm volume shift, which apparently conferred protection from detrimental swelling and impaired functions. Copyright © 2016 Elsevier Inc. All rights reserved.

Molecular evolution of adiponectin in Carnivora and its mRNA expression in relation to hepatic lipidosis.

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Nieminen, Petteri; Rouvinen-Watt, Kirsti; Kapiainen, Suvi; Harris, Lora; Mustonen, Anne-Mari

2010-09-15

Adiponectin is a novel adipocyte-derived hormone with low circulating concentrations and/or mRNA expression in obesity and non-alcoholic fatty liver disease (NAFLD). The adiponectin mRNA of several Carnivora species was sequenced to enable further gene expression studies in this clade with potential experimental species to examine the connections of hypoadiponectinemia to hepatic lipidosis. In addition, adiponectin mRNA expression was studied in the retroperitoneal fat of the American mink (Neovison vison), as hepatic lipidosis with close similarities to NAFLD can be rapidly induced to the species by fasting. The mRNA expression was determined after overnight-7d of food deprivation and 28d of re-feeding and correlated to the liver fat %. The homologies between the determined carnivoran mRNA sequences and that of the domestic dog were 92.2-99.1%. As the mRNA expression was not affected by short-term fasting and did not correlate with the liver fat %, there seems to be no clear connection between adiponectin and the development of lipidosis in the American mink. In the future, the obtained sequences can be utilized in further studies of adiponectin expression in comparative endocrinology. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Expression of annexin-A1 and galectin-1 anti-inflammatory proteins and mRNA in chronic gastritis and gastric cancer.

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Jorge, Yvana Cristina; Mataruco, Mayra Mioto; Araújo, Leandro Pires; Rossi, Ana Flávia Teixeira; de Oliveira, Juliana Garcia; Valsechi, Marina Curado; Caetano, Alaor; Miyazaki, Kenji; Fazzio, Célia Sebastiana de Jesus; Thomé, Jorge Alberto; Rahal, Paula; Oliani, Sonia Maria; Silva, Ana Elizabete

2013-01-01

The anti-inflammatory proteins annexin-A1 and galectin-1 have been associated with tumor progression. This scenario prompted us to investigate the relationship between the gene and protein expression of annexin-A1 (ANXA1/AnxA1) and galectin-1 (LGALS1/Gal-1) in an inflammatory gastric lesion as chronic gastritis (CG) and gastric adenocarcinoma (GA) and its association with H. pylori infection. We analyzed 40 samples of CG, 20 of GA, and 10 of normal mucosa (C) by the quantitative real-time PCR (qPCR) technique and the immunohistochemistry assay. High ANXA1 mRNA expression levels were observed in 90% (36/40) of CG cases (mean relative quantification RQ = 4.26  ±  2.03) and in 80% (16/20) of GA cases (mean RQ = 4.38  ±  4.77). However, LGALS1 mRNA levels were high (mean RQ = 2.44  ±  3.26) in 60% (12/20) of the GA cases, while low expression was found in CG (mean RQ = 0.43 ± 3.13; P gastritis and gastric cancer, suggesting a strong association of these proteins with chronic gastric inflammation and carcinogenesis.

Automatic Drusen Quantification and Risk Assessment of Age-related Macular Degeneration on Color Fundus Images

NARCIS (Netherlands)

Grinsven, M.J.J.P. van; Lechanteur, Y.T.E.; Ven, J.P.H. van de; Ginneken, B. van; Hoyng, C.B.; Theelen, T.; Sanchez, C.I.

2013-01-01

PURPOSE: To evaluate a machine learning algorithm that allows for computer aided diagnosis (CAD) of non-advanced age-related macular degeneration (AMD) by providing an accurate detection and quantification of drusen location, area and size. METHODS: Color fundus photographs of 407 eyes without AMD

Verb aspect, alternations and quantification

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Svetla Koeva

2015-11-01

Full Text Available Verb aspect, alternations and quantification In this paper we are briefly discuss the nature of Bulgarian verb aspect and argue that the verb aspect pairs are different lexical units with different (although related meaning, different argument structure (reflecting categories, explicitness and referential status of arguments and different sets of semantic and syntactic alternations. The verb prefixes resulting in perfective verbs derivation in some cases can be interpreted as lexical quantifiers as well. Thus the Bulgarian verb aspect is related (in different way both with the potential for the generation of alternations and with the prefixal lexical quantification. It is shown that the scope of the lexical quantification by means of verbal prefixes is the quantified verb phrase and the scope remains constant in all derived alternations. The paper concerns the basic issues of these complex problems, while the detailed description of the conditions satisfying particular alternation or particular lexical quantification are subject of a more detailed study.

Interactions between the HIV-1 Unspliced mRNA and Host mRNA Decay Machineries

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Daniela Toro-Ascuy

2016-11-01

Full Text Available The human immunodeficiency virus type-1 (HIV-1 unspliced transcript is used both as mRNA for the synthesis of structural proteins and as the packaged genome. Given the presence of retained introns and instability AU-rich sequences, this viral transcript is normally retained and degraded in the nucleus of host cells unless the viral protein REV is present. As such, the stability of the HIV-1 unspliced mRNA must be particularly controlled in the nucleus and the cytoplasm in order to ensure proper levels of this viral mRNA for translation and viral particle formation. During its journey, the HIV-1 unspliced mRNA assembles into highly specific messenger ribonucleoproteins (mRNPs containing many different host proteins, amongst which are well-known regulators of cytoplasmic mRNA decay pathways such as up-frameshift suppressor 1 homolog (UPF1, Staufen double-stranded RNA binding protein 1/2 (STAU1/2, or components of miRNA-induced silencing complex (miRISC and processing bodies (PBs. More recently, the HIV-1 unspliced mRNA was shown to contain N6-methyladenosine (m6A, allowing the recruitment of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2, an m6A reader host protein involved in mRNA decay. Interestingly, these host proteins involved in mRNA decay were shown to play positive roles in viral gene expression and viral particle assembly, suggesting that HIV-1 interacts with mRNA decay components to successfully accomplish viral replication. This review summarizes the state of the art in terms of the interactions between HIV-1 unspliced mRNA and components of different host mRNA decay machineries.

Comparative study of label and label-free techniques using shotgun proteomics for relative protein quantification.

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Sjödin, Marcus O D; Wetterhall, Magnus; Kultima, Kim; Artemenko, Konstantin

2013-06-01

The analytical performance of three different strategies, iTRAQ (isobaric tag for relative and absolute quantification), dimethyl labeling (DML) and label free (LF) for relative protein quantification using shotgun proteomics have been evaluated. The methods have been explored using samples containing (i) Bovine proteins in known ratios and (ii) Bovine proteins in known ratios spiked into Escherichia coli. The latter case mimics the actual conditions in a typical biological sample with a few differentially expressed proteins and a bulk of proteins with unchanged ratios. Additionally, the evaluation was performed on both QStar and LTQ-FTICR mass spectrometers. LF LTQ-FTICR was found to have the highest proteome coverage while the highest accuracy based on the artificially regulated proteins was found for DML LTQ-FTICR (54%). A varying linearity (k: 0.55-1.16, r(2): 0.61-0.96) was shown for all methods within selected dynamic ranges. All methods were found to consistently underestimate Bovine protein ratios when matrix proteins were added. However, LF LTQ-FTICR was more tolerant toward a compression effect. A single peptide was demonstrated to be sufficient for a reliable quantification using iTRAQ. A ranking system utilizing several parameters important for quantitative proteomics demonstrated that the overall performance of the five different methods was; DML LTQ-FTICR>iTRAQ QStar>LF LTQ-FTICR>DML QStar>LF QStar. Copyright © 2013 Elsevier B.V. All rights reserved.

In vivo Identification and Specificity assessment of mRNA markers of hypoxia in human and mouse tumors

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Busk, Morten; Toustrup, Kasper; Sørensen, Brita S; Alsner, Jan; Horsman, Michael R; Jakobsen, Steen; Overgaard, Jens

2011-01-01

Tumor hypoxia is linked to poor prognosis, but identification and quantification of tissue hypoxia remains a challenge. The hypoxia-specificity of HIF-1α target genes in vivo has been questioned due to the confounding influence of other microenvironmental abnormalities known to affect gene expression (e.g., low pH). Here we describe a new technique that by exploiting intratumoral oxygenation heterogeneity allows us to identify and objectively rank the most robust mRNA hypoxia biomarkers. Mice carrying human (FaDu dd ) or murine (SCCVII) tumors were injected with the PET hypoxia tracer FAZA. Four hours post-injection tumors were removed, frozen, and crushed into milligram-sized fragments, which were transferred individually to pre-weighed tubes containing RNAlater and then weighed. For each fragment radioactivity per tissue mass and expression patterns of selected mRNA biomarkers were analyzed and compared. In both tumour models, fragmentation into pieces weighing 10 to 60 mg resulted in tissue fragments with highly variable relative content of hypoxic cells as evidenced by an up to 13-fold variation in FAZA radioactivity per mass of tissue. Linear regression analysis comparing FAZA retention with patterns of gene expression in individual tissue fragments revealed that CA9, GLUT1 and LOX mRNA levels were equally and strongly correlated to hypoxic extent in FaDu dd . The same link between hypoxia and gene expression profile was observed for CA9 and GLUT1, but not LOX, in SCCVII tumors. Apparent in vivo hypoxia-specificity for other putative molecular markers of tissue hypoxia was considerably weaker. The portrayed technique allows multiple pairwise measurements of mRNA transcript levels and extent of hypoxia in individual tumors at a smallest possible volumetric scale which (by limiting averaging effects inherent to whole-tumor analysis) strengthen the conclusiveness on true hypoxia-specificity of candidate genes while limiting the required number of tumors. Among

In vivo Identification and Specificity assessment of mRNA markers of hypoxia in human and mouse tumors

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Horsman Michael R

2011-02-01

Full Text Available Abstract Background Tumor hypoxia is linked to poor prognosis, but identification and quantification of tissue hypoxia remains a challenge. The hypoxia-specificity of HIF-1α target genes in vivo has been questioned due to the confounding influence of other microenvironmental abnormalities known to affect gene expression (e.g., low pH. Here we describe a new technique that by exploiting intratumoral oxygenation heterogeneity allows us to identify and objectively rank the most robust mRNA hypoxia biomarkers. Methods Mice carrying human (FaDudd or murine (SCCVII tumors were injected with the PET hypoxia tracer FAZA. Four hours post-injection tumors were removed, frozen, and crushed into milligram-sized fragments, which were transferred individually to pre-weighed tubes containing RNAlater and then weighed. For each fragment radioactivity per tissue mass and expression patterns of selected mRNA biomarkers were analyzed and compared. Results In both tumour models, fragmentation into pieces weighing 10 to 60 mg resulted in tissue fragments with highly variable relative content of hypoxic cells as evidenced by an up to 13-fold variation in FAZA radioactivity per mass of tissue. Linear regression analysis comparing FAZA retention with patterns of gene expression in individual tissue fragments revealed that CA9, GLUT1 and LOX mRNA levels were equally and strongly correlated to hypoxic extent in FaDudd. The same link between hypoxia and gene expression profile was observed for CA9 and GLUT1, but not LOX, in SCCVII tumors. Apparent in vivo hypoxia-specificity for other putative molecular markers of tissue hypoxia was considerably weaker. Conclusions The portrayed technique allows multiple pairwise measurements of mRNA transcript levels and extent of hypoxia in individual tumors at a smallest possible volumetric scale which (by limiting averaging effects inherent to whole-tumor analysis strengthen the conclusiveness on true hypoxia-specificity of candidate

Simultaneous detection of mRNA and protein stem cell markers in live cells

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Bao Gang

2009-04-01

Full Text Available Abstract Background Biological studies and medical application of stem cells often require the isolation of stem cells from a mixed cell population, including the detection of cancer stem cells in tumor tissue, and isolation of induced pluripotent stem cells after eliciting the expression of specific genes in adult cells. Here we report the detection of Oct-4 mRNA and SSEA-1 protein in live carcinoma stem cells using respectively molecular beacon and dye-labeled antibody, aiming to establish a new method for stem cells detection and isolation. Results Quantification of Oct-4 mRNA and protein in P19 mouse carcinoma stem cells using respectively RT-PCR and immunocytochemistry confirmed that their levels drastically decreased after differentiation. To visualize Oct-4 mRNA in live stem cells, molecular beacons were designed, synthesized and validated, and the detection specificity was confirmed using control studies. We found that the fluorescence signal from Oct-4-targeting molecular beacons provides a clear discrimination between undifferentiated and retinoic acid-induced differentiated cells. Using deconvolution fluorescence microscopy, Oct-4 mRNAs were found to reside on one side of the cytosol. We demonstrated that, using a combination of Oct-4 mRNA-targeting molecular beacon with SSEA-1 antibody in flow cytometric analysis, undifferentiated stem cells can be clearly distinguished from differentiated cells. We revealed that Oct-4 targeting molecular beacons do not seem to affect stem cell biology. Conclusion Molecular beacons have the potential to provide a powerful tool for highly specific detection and isolation of stem cells, including cancer stem cells and induced pluripotent stem (iPS cells without disturbing cell physiology. It is advantageous to perform simultaneous detection of intracellular (mRNA and cell-surface (protein stem cell markers in flow cytometric analysis, which may lead to high detection sensitivity and efficiency.

Whole-genome analysis of mRNA decay in Plasmodium falciparum reveals a global lengthening of mRNA half-life during the intra-erythrocytic development cycle.

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Shock, Jennifer L; Fischer, Kael F; DeRisi, Joseph L

2007-01-01

The rate of mRNA decay is an essential element of post-transcriptional regulation in all organisms. Previously, studies in several organisms found that the specific half-life of each mRNA is precisely related to its physiologic role, and plays an important role in determining levels of gene expression. We used a genome-wide approach to characterize mRNA decay in Plasmodium falciparum. We found that, globally, rates of mRNA decay increase dramatically during the asexual intra-erythrocytic developmental cycle. During the ring stage of the cycle, the average mRNA half-life was 9.5 min, but this was extended to an average of 65 min during the late schizont stage of development. Thus, a major determinant of mRNA decay rate appears to be linked to the stage of intra-erythrocytic development. Furthermore, we found specific variations in decay patterns superimposed upon the dominant trend of progressive half-life lengthening. These variations in decay pattern were frequently enriched for genes with specific cellular functions or processes. Elucidation of Plasmodium mRNA decay rates provides a key element for deciphering mechanisms of genetic control in this parasite, by complementing and extending previous mRNA abundance studies. Our results indicate that progressive stage-dependent decreases in mRNA decay rate function are a major determinant of mRNA accumulation during the schizont stage of intra-erythrocytic development. This type of genome-wide change in mRNA decay rate has not been observed in any other organism to date, and indicates that post-transcriptional regulation may be the dominant mechanism of gene regulation in P. falciparum.

A comparative study of digital PCR and real-time qPCR for the detection and quantification of HPV mRNA in sentinel lymph nodes of cervical cancer patients.

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Carow, Katrin; Read, Christina; Häfner, Norman; Runnebaum, Ingo B; Corner, Adam; Dürst, Matthias

2017-10-30

Qualitative analyses showed that the presence of HPV mRNA in sentinel lymph nodes of cervical cancer patients with pN0 status is associated with significantly decreased recurrence free survival. To further address the clinical potential of the strategy and to define prognostic threshold levels it is necessary to use a quantitative assay. Here, we compare two methods of quantification: digital PCR and standard quantitative PCR. Serial dilutions of 5 ng-5 pg RNA (≙ 500-0.5 cells) of the cervical cancer cell line SiHa were prepared in 5 µg RNA of the HPV-negative human keratinocyte cell line HaCaT. Clinical samples consisted of 10 sentinel lymph nodes with varying HPV transcript levels. Reverse transcription of total RNA (5 µg RNA each) was performed in 100 µl and cDNA aliquots were analyzed by qPCR and dPCR. Digital PCR was run in the RainDrop ® Digital PCR system (RainDance Technologies) using a probe-based detection of HPV E6/E7 cDNA PCR products with 11 µl template. qPCR was done using a Rotor Gene Q 5plex HRM (Qiagen) amplifying HPV E6/E7 cDNA in a SYBR Green format with 1 µl template. For the analysis of both, clinical samples and serial dilution samples, dPCR and qPCR showed comparable sensitivity. With regard to reproducibility, both methods differed considerably, especially for low template samples. Here, we found with qPCR a mean variation coefficient of 126% whereas dPCR enabled a significantly lower mean variation coefficient of 40% (p = 0.01). Generally, we saw with dPCR a substantial reduction of subsampling errors, which most likely reflects the large cDNA amounts available for analysis. Compared to real-time PCR, dPCR shows higher reliability. Thus, our HPV mRNA dPCR assay holds promise for the clinical evaluation of occult tumor cells in histologically tumor-free lymph nodes in future studies.

Single-cell mRNA transfection studies: delivery, kinetics and statistics by numbers.

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Leonhardt, Carolin; Schwake, Gerlinde; Stögbauer, Tobias R; Rappl, Susanne; Kuhr, Jan-Timm; Ligon, Thomas S; Rädler, Joachim O

2014-05-01

In artificial gene delivery, messenger RNA (mRNA) is an attractive alternative to plasmid DNA (pDNA) since it does not require transfer into the cell nucleus. Here we show that, unlike for pDNA transfection, the delivery statistics and dynamics of mRNA-mediated expression are generic and predictable in terms of mathematical modeling. We measured the single-cell expression time-courses and levels of enhanced green fluorescent protein (eGFP) using time-lapse microscopy and flow cytometry (FC). The single-cell analysis provides direct access to the distribution of onset times, life times and expression rates of mRNA and eGFP. We introduce a two-step stochastic delivery model that reproduces the number distribution of successfully delivered and translated mRNA molecules and thereby the dose-response relation. Our results establish a statistical framework for mRNA transfection and as such should advance the development of RNA carriers and small interfering/micro RNA-based drugs. This team of authors established a statistical framework for mRNA transfection by using a two-step stochastic delivery model that reproduces the number distribution of successfully delivered and translated mRNA molecules and thereby their dose-response relation. This study establishes a nice connection between theory and experimental planning and will aid the cellular delivery of mRNA molecules. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

TP53 and ATM mRNA expression in skin and skeletal muscle after low-level laser exposure.

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Guedes de Almeida, Luciana; Sergio, Luiz Philippe da Silva; de Paoli, Flavia; Mencalha, Andre Luiz; da Fonseca, Adenilson de Souza

2017-08-01

Low-level lasers are widespread in regenerative medicine, but the molecular mechanisms involved in their biological effects are not fully understood, particularly those on DNA stability. Therefore, this study aimed to investigate mRNA expression of genes related to DNA genomic stability in skin and skeletal muscle tissue from Wistar rats exposed to low-level red and infrared lasers. For this, TP53 (Tumor Protein 53) and ATM (Ataxia Telangiectasia Mutated gene) mRNA expressions were evaluated by real-time quantitative PCR (RT-qPCR) technique 24 hours after low-level red and infrared laser exposure. Our data showed that relative TP53 mRNA expression was not significantly altered in both tissues exposed to lasers. For ATM, relative mRNA expression in skin tissue was not significantly altered, but in muscle tissue, laser exposure increased relative ATM mRNA expression. Low-level red and infrared laser radiations alter ATM mRNA expression related to DNA stability in skeletal muscle tissue.

Proteomic Analysis of Sauvignon Blanc Grape Skin, Pulp and Seed and Relative Quantification of Pathogenesis-Related Proteins.

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Bin Tian

Full Text Available Thaumatin-like proteins (TLPs and chitinases are the main constituents of so-called protein hazes which can form in finished white wine and which is a great concern of winemakers. These soluble pathogenesis-related (PR proteins are extracted from grape berries. However, their distribution in different grape tissues is not well documented. In this study, proteins were first separately extracted from the skin, pulp and seed of Sauvignon Blanc grapes, followed by trypsin digestion and analysis by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS. Proteins identified included 75 proteins from Sauvignon Blanc grape skin, 63 from grape pulp and 35 from grape seed, mostly functionally classified as associated with metabolism and energy. Some were present exclusively in specific grape tissues; for example, proteins involved in photosynthesis were only detected in grape skin and proteins found in alcoholic fermentation were only detected in grape pulp. Moreover, proteins identified in grape seed were less diverse than those identified in grape skin and pulp. TLPs and chitinases were identified in both Sauvignon Blanc grape skin and pulp, but not in the seed. To relatively quantify the PR proteins, the protein extracts of grape tissues were seperated by HPLC first and then analysed by SDS-PAGE. The results showed that the protein fractions eluted at 9.3 min and 19.2 min under the chromatographic conditions of this study confirmed that these corresponded to TLPs and chitinases seperately. Thus, the relative quantification of TLPs and chitinases in protein extracts was carried out by comparing the area of corresponding peaks against the area of a thamautin standard. The results presented in this study clearly demonstrated the distribution of haze-forming PR proteins in grape berries, and the relative quantification of TLPs and chitinases could be applied in fast tracking of changes in PR proteins during grape growth and

Profiling and relative quantification of phosphatidylethanolamine based on acetone stable isotope derivatization

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Wang, Xiang [Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture (China); Hubei Key Laboratory of Lipid Chemistry and Nutrition (China); Wei, Fang, E-mail: willasa@163.com [Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture (China); Hubei Key Laboratory of Lipid Chemistry and Nutrition (China); Xu, Ji-qu; Lv, Xin; Dong, Xu-yan [Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture (China); Hubei Key Laboratory of Lipid Chemistry and Nutrition (China); Han, Xianlin [Center for Metabolic Origins of Disease, Sanford Burnham Prebys Medical Discovery Institute, Orlando, FL 32827 (United States); College of Basic Medical Sciences, Zhejiang Chinese Medical University, 548 Bingwen Road, Hangzhou, Zhejiang 310053 (China); Quek, Siew-young [School of Chemical Science, The University of Auckland, Auckland 1142 (New Zealand); Huang, Feng-hong [Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture (China); Hubei Key Laboratory of Lipid Chemistry and Nutrition (China); Chen, Hong, E-mail: chenhong@oilcrops.cn [Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture (China); Hubei Key Laboratory of Lipid Chemistry and Nutrition (China)

2016-01-01

Phosphatidylethanolamine (PE) is considered to be one of the pivotal lipids for normal cellular function as well as disease initiation and progression. In this study, a simple, efficient, reliable, and inexpensive method for the qualitative analysis and relative quantification of PE, based on acetone stable isotope derivatization combined with double neutral loss scan-shotgun electrospray ionization tandem-quadrupole mass spectrometry analysis (ASID-DNLS-Shotgun ESI-MS/MS), was developed. The ASID method led to alkylation of the primary amino groups of PE with an isopropyl moiety. The use of acetone (d{sub 0}-acetone) and deuterium-labeled acetone (d{sub 6}-acetone) introduced a 6 Da mass shift that was ideally suited for relative quantitative analysis, and enhanced sensitivity for mass analysis. The DNLS model was introduced to simultaneously analyze the differential derivatized PEs by shotgun ESI-MS/MS with high selectivity and accuracy. The reaction specificity, labeling efficiency, and linearity of the ASID method were thoroughly evaluated in this study. Its excellent applicability was validated by qualitative and relative quantitative analysis of PE species presented in liver samples from rats fed different diets. Using the ASID-DNLS-Shotgun ESI-MS/MS method, 45 PE species from rat livers have been identified and quantified in an efficient manner. The level of total PEs tended to decrease in the livers of rats on high fat diets compared with controls. The levels of PE 32:1, 34:3, 34:2, 36:3, 36:2, 42:10, plasmalogen PE 36:1 and lyso PE 22:6 were significantly reduced, while levels of PE 36:1 and lyso PE 16:0 increased. - Highlights: • A novel isotope reagent acetone was explored for the derivatization of PEs. • The labeling reaction was carried out under mild conditions with high specificity. • Enhanced detection sensitivity of PEs was achieved after derivatization. • The ASID-DNLS-Shotgun MS/MS method was used to relative quantification of PEs.

Network-Based Isoform Quantification with RNA-Seq Data for Cancer Transcriptome Analysis.

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Wei Zhang

2015-12-01

Full Text Available High-throughput mRNA sequencing (RNA-Seq is widely used for transcript quantification of gene isoforms. Since RNA-Seq data alone is often not sufficient to accurately identify the read origins from the isoforms for quantification, we propose to explore protein domain-domain interactions as prior knowledge for integrative analysis with RNA-Seq data. We introduce a Network-based method for RNA-Seq-based Transcript Quantification (Net-RSTQ to integrate protein domain-domain interaction network with short read alignments for transcript abundance estimation. Based on our observation that the abundances of the neighboring isoforms by domain-domain interactions in the network are positively correlated, Net-RSTQ models the expression of the neighboring transcripts as Dirichlet priors on the likelihood of the observed read alignments against the transcripts in one gene. The transcript abundances of all the genes are then jointly estimated with alternating optimization of multiple EM problems. In simulation Net-RSTQ effectively improved isoform transcript quantifications when isoform co-expressions correlate with their interactions. qRT-PCR results on 25 multi-isoform genes in a stem cell line, an ovarian cancer cell line, and a breast cancer cell line also showed that Net-RSTQ estimated more consistent isoform proportions with RNA-Seq data. In the experiments on the RNA-Seq data in The Cancer Genome Atlas (TCGA, the transcript abundances estimated by Net-RSTQ are more informative for patient sample classification of ovarian cancer, breast cancer and lung cancer. All experimental results collectively support that Net-RSTQ is a promising approach for isoform quantification. Net-RSTQ toolbox is available at http://compbio.cs.umn.edu/Net-RSTQ/.

Full-length mRNA sequencing uncovers a widespread coupling between transcription initiation and mRNA processing.

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Anvar, Seyed Yahya; Allard, Guy; Tseng, Elizabeth; Sheynkman, Gloria M; de Klerk, Eleonora; Vermaat, Martijn; Yin, Raymund H; Johansson, Hans E; Ariyurek, Yavuz; den Dunnen, Johan T; Turner, Stephen W; 't Hoen, Peter A C

2018-03-29

The multifaceted control of gene expression requires tight coordination of regulatory mechanisms at transcriptional and post-transcriptional level. Here, we studied the interdependence of transcription initiation, splicing and polyadenylation events on single mRNA molecules by full-length mRNA sequencing. In MCF-7 breast cancer cells, we find 2700 genes with interdependent alternative transcription initiation, splicing and polyadenylation events, both in proximal and distant parts of mRNA molecules, including examples of coupling between transcription start sites and polyadenylation sites. The analysis of three human primary tissues (brain, heart and liver) reveals similar patterns of interdependency between transcription initiation and mRNA processing events. We predict thousands of novel open reading frames from full-length mRNA sequences and obtained evidence for their translation by shotgun proteomics. The mapping database rescues 358 previously unassigned peptides and improves the assignment of others. By recognizing sample-specific amino-acid changes and novel splicing patterns, full-length mRNA sequencing improves proteogenomics analysis of MCF-7 cells. Our findings demonstrate that our understanding of transcriptome complexity is far from complete and provides a basis to reveal largely unresolved mechanisms that coordinate transcription initiation and mRNA processing.

mRNA Cancer Vaccines-Messages that Prevail.

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Grunwitz, Christian; Kranz, Lena M

2017-01-01

During the last decade, mRNA became increasingly recognized as a versatile tool for the development of new innovative therapeutics. Especially for vaccine development, mRNA is of outstanding interest and numerous clinical trials have been initiated. Strikingly, all of these studies have proven that large-scale GMP production of mRNA is feasible and concordantly report a favorable safety profile of mRNA vaccines. Induction of T-cell immunity is a multi-faceted process comprising antigen acquisition, antigen processing and presentation, as well as immune stimulation. The effectiveness of mRNA vaccines is critically dependent on making the antigen(s) of interest available to professional antigen-presenting cells, especially DCs. Efficient delivery of mRNA into DCs in vivo remains a major challenge in the mRNA vaccine field. This review summarizes the principles of mRNA vaccines and highlights the importance of in vivo mRNA delivery and recent advances in harnessing their therapeutic potential.

Principles of mRNA transport in yeast.

Science.gov (United States)

Heym, Roland Gerhard; Niessing, Dierk

2012-06-01

mRNA localization and localized translation is a common mechanism by which cellular asymmetry is achieved. In higher eukaryotes the mRNA transport machinery is required for such diverse processes as stem cell division and neuronal plasticity. Because mRNA localization in metazoans is highly complex, studies at the molecular level have proven to be cumbersome. However, active mRNA transport has also been reported in fungi including Saccharomyces cerevisiae, Ustilago maydis and Candida albicans, in which these events are less difficult to study. Amongst them, budding yeast S. cerevisiae has yielded mechanistic insights that exceed our understanding of other mRNA localization events to date. In contrast to most reviews, we refrain here from summarizing mRNA localization events from different organisms. Instead we give an in-depth account of ASH1 mRNA localization in budding yeast. This approach is particularly suited to providing a more holistic view of the interconnection between the individual steps of mRNA localization, from transcriptional events to cytoplasmic mRNA transport and localized translation. Because of our advanced mechanistic understanding of mRNA localization in yeast, the present review may also be informative for scientists working, for example, on mRNA localization in embryogenesis or in neurons.

Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis: interferon-beta treatment increases IL-10 mRNA expression while reducing IL-23 mRNA expression

DEFF Research Database (Denmark)

Krakauer, M.; Sorensen, P.; Khademi, M.

2008-01-01

volunteers served to confirm initial findings. mRNA was analyzed by real-time reverse transcriptase polymerase chain reaction (PCR). RESULTS: We found elevated expression of interleukin (IL)-23 and IL-10 in untreated MS patients. IFN-beta therapy increased IL-10 and decreased IL-23 expression independently...... of the regulatory cytokine IL-10. The elevated IL-23 mRNA levels in MS patients are noteworthy in view of the newly discovered IL-23-driven Th17 T-cell subset, which is crucial in animal models of MS. Since IFN-beta therapy resulted in decreased IL-23 mRNA levels, the Th17 axis could be another target of IFN...

Nucleolin Mediates MicroRNA-directed CSF-1 mRNA Deadenylation but Increases Translation of CSF-1 mRNA*

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Woo, Ho-Hyung; Baker, Terri; Laszlo, Csaba; Chambers, Setsuko K.

2013-01-01

CSF-1 mRNA 3′UTR contains multiple unique motifs, including a common microRNA (miRNA) target in close proximity to a noncanonical G-quadruplex and AU-rich elements (AREs). Using a luciferase reporter system fused to CSF-1 mRNA 3′UTR, disruption of the miRNA target region, G-quadruplex, and AREs together dramatically increased reporter RNA levels, suggesting important roles for these cis-acting regulatory elements in the down-regulation of CSF-1 mRNA. We find that nucleolin, which binds both G-quadruplex and AREs, enhances deadenylation of CSF-1 mRNA, promoting CSF-1 mRNA decay, while having the capacity to increase translation of CSF-1 mRNA. Through interaction with the CSF-1 3′UTR miRNA common target, we find that miR-130a and miR-301a inhibit CSF-1 expression by enhancing mRNA decay. Silencing of nucleolin prevents the miRNA-directed mRNA decay, indicating a requirement for nucleolin in miRNA activity on CSF-1 mRNA. Downstream effects followed by miR-130a and miR-301a inhibition of directed cellular motility of ovarian cancer cells were found to be dependent on nucleolin. The paradoxical effects of nucleolin on miRNA-directed CSF-1 mRNA deadenylation and on translational activation were explored further. The nucleolin protein contains four acidic stretches, four RNA recognition motifs (RRMs), and nine RGG repeats. All three domains in nucleolin regulate CSF-1 mRNA and protein levels. RRMs increase CSF-1 mRNA, whereas the acidic and RGG domains decrease CSF-1 protein levels. This suggests that nucleolin has the capacity to differentially regulate both CSF-1 RNA and protein levels. Our finding that nucleolin interacts with Ago2 indirectly via RNA and with poly(A)-binding protein C (PABPC) directly suggests a nucleolin-Ago2-PABPC complex formation on mRNA. This complex is in keeping with our suggestion that nucleolin may work with PABPC as a double-edged sword on both mRNA deadenylation and translational activation. Our findings underscore the complexity of

Increased FXYD1 and PGC-1α mRNA after blood flow-restricted running is related to fibre type-specific AMPK signalling and oxidative stress in human muscle

DEFF Research Database (Denmark)

Christiansen, Danny; Murphy, Robyn M; Bangsbo, Jens

2018-01-01

AIM: This study explored the effects of blood flow restriction (BFR) on mRNA responses of PGC-1α (total, 1α1, and 1α4) and Na+ ,K+ -ATPase isoforms (NKA; α1-3 , β1-3 , and FXYD1) to an interval running session, and determined if these effects were related to increased oxidative stress, hypoxia......). A muscle sample was collected before (Pre) and after exercise (+0h, +3h) to quantify mRNA, indicators of oxidative stress (HSP27 protein in type I and II fibres, and catalase and HSP70 mRNA), metabolites, and α-AMPK Thr172 /α-AMPK, ACC Ser221 /ACC, CaMKII Thr287 /CaMKII, and PLBSer16 /PLB ratios in type I...... of oxidative stress and type-I fibre ACC Ser221 /ACC ratio, but dissociated from muscle hypoxia, lactate, and CaMKII signalling. CONCLUSION: Blood flow restriction augmented exercise-induced increases in muscle FXYD1 and PGC-1α mRNA in men. This effect was related to increased oxidative stress and fibre type...

Nonparametric testing for DNA copy number induced differential mRNA gene expression

NARCIS (Netherlands)

van Wieringen, W.N.; van de Wiel, M.A.

2009-01-01

The central dogma of molecular biology relates DNA with mRNA. Array CGH measures DNA copy number and gene expression microarrays measure the amount of mRNA. Methods that integrate data from these two platforms may uncover meaningful biological relationships that further our understanding of cancer.

Breast Carcinoma Progression and Tumour Vascular Markers Related to Apoptotic Mechanisms

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Miroslava Bilecova-Rabajdova

2014-01-01

Full Text Available Background. In the last few years, the cancer research had tried to identify and characterize new biochemical and molecular pathways in which the inhibition induces prosurvival mechanisms. Our work describes the expression of two different members of apoptotic regulatory pathway and their relationship with a progression of breast carcinoma. Materials and Methods. We compared expression of genes related to apoptosis (DR6 and Gpm6B in the blood of patients suffering from stage I of breast cancer in different grades (I–IV, with healthy controls. After isolation of mRNA, transcription of mRNA into the cDNA was performed. The quantification of gene expression changes in DR6 and Gpm6B was detected by RT-PCR method. Analysis at the protein level was performed by the Western blot.Results. In statistical analysis of Dr6 mRNA level changes we detected significant increase starting in Grading 1 (G1 and reached maximal level in G3.This expression on mRNA levels was similar to protein levels, which copy rising tendency with maximal value in G3. The results of Gpm6B were significantly lower.Conclusion. This result showed that antiapoptotic signalling during neovascularization is increased significantly. It would be advisable in the future to study the influence of cytostatic treatment on the expression of genes related to apoptotic pathways and their relationship with progression of breast cancer tumours.

Effect of transportation stress on heat shock protein 70 concentration and mRNA expression in heart and kidney tissues and serum enzyme activities and hormone concentrations of pigs.

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Yu, Hong; Bao, En-Dong; Zhao, Ru-Qian; Lv, Qiong-Xia

2007-11-01

To determine the enzymatic and hormonal responses, heat shock protein 70 (Hsp70) production, and Hsp70 mRNA expression in heart and kidney tissues of transport-stressed pigs. 24 pigs (mean weight, 20 +/- 1 kg). Pigs were randomly placed into groups of 12 each. One group was transported for 2 hours. The other group was kept under normal conditions and used as control pigs. Sera were used to detect triiodothyronine, thyroxine, and cortisol concentrations and alanine aminotransferase, aspartate aminotransferase, and creatine kinase activities. The heart and kidneys of anesthetized pigs were harvested and frozen in liquid nitrogen for quantification of Hsp70 and Hsp70 mRNA. No significant differences were detected in serum alanine aminotransferase activity and triiodothyronine and cortisol concentrations between groups; however, the serum creatine kinase and aspartate aminotransferase activities and thyroxine concentrations were higher in transported pigs. Densitometric readings of western blots revealed that the amount of Hsp70 in heart and kidney tissues was significantly higher in transported pigs, compared with control pigs. Results of fluorescence quantitative real-time PCR assay revealed that the Hsp70 mRNA transcription in heart tissue, but not kidney tissue, was significantly higher in transported pigs, compared with control pigs. Transportation imposed a severe stress on pigs that was manifested as increased serum activities of aspartate aminotransferase and creatine kinase and increased amounts of Hsp70 and Hsp70 mRNA expression in heart and kidney tissues. Changes in serum enzyme activities were related to the tissue damage of transport-stressed pigs.

Multiplicity of expression of Na⁺,K⁺-ATPase alpha-subunit isoforms in the gill of Atlantic salmon: quantification and cellular localisation in response to salinity

DEFF Research Database (Denmark)

Madsen, Steffen; Kiilerich, Pia; Tipsmark, Christian Kølbæk

2009-01-01

but occasionally also on lamellae. Overall, the salinity-induced variation in labelling pattern and intensity matched the quantification data. In conclusion, the predominant switching of Na+,K+-ATPase -subunit isoform mRNA during salinity acclimation reflects a marked remodelling of mitochondrion-rich cells (MRCs...

Presence of albumin mRNA precursors in nuclei of analbuminemic rat liver lacking cytoplasmic albumin mRNA.

OpenAIRE

Esumi, H; Takahashi, Y; Sekiya, T; Sato, S; Nagase, S; Sugimura, T

1982-01-01

Analbuminemic rats, which lack serum albumin, were previously found to have no albumin mRNA in the cytoplasm of the liver. In the present study, the existence of nuclear albumin mRNA precursors in the liver of analbuminemic rats was examined by RNA X cDNA hybridization kinetics. Albumin mRNA precursors were present in the nuclei of analbuminemic rat liver at almost normal levels, despite the absence of albumin mRNA from the cytoplasm. Nuclear RNA of analbuminemic rat liver was subjected to el...

Tissue-specific mRNA expression profiling in grape berry tissues

Science.gov (United States)

Grimplet, Jerome; Deluc, Laurent G; Tillett, Richard L; Wheatley, Matthew D; Schlauch, Karen A; Cramer, Grant R; Cushman, John C

2007-01-01

Background Berries of grape (Vitis vinifera) contain three major tissue types (skin, pulp and seed) all of which contribute to the aroma, color, and flavor characters of wine. The pericarp, which is composed of the exocarp (skin) and mesocarp (pulp), not only functions to protect and feed the developing seed, but also to assist in the dispersal of the mature seed by avian and mammalian vectors. The skin provides volatile and nonvolatile aroma and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of organoleptic characteristics of wine. In order to understand the transcriptional network responsible for controlling tissue-specific mRNA expression patterns, mRNA expression profiling was conducted on each tissue of mature berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0. In order to monitor the influence of water-deficit stress on tissue-specific expression patterns, mRNA expression profiles were also compared from mature berries harvested from vines subjected to well-watered or water-deficit conditions. Results Overall, berry tissues were found to express approximately 76% of genes represented on the Vitis microarray. Approximately 60% of these genes exhibited significant differential expression in one or more of the three major tissue types with more than 28% of genes showing pronounced (2-fold or greater) differences in mRNA expression. The largest difference in tissue-specific expression was observed between the seed and pulp/skin. Exocarp tissue, which is involved in pathogen defense and pigment production, showed higher mRNA abundance relative to other berry tissues for genes involved with flavonoid biosynthesis, pathogen resistance, and cell wall modification. Mesocarp tissue, which is considered a nutritive tissue, exhibited a higher mRNA abundance of genes involved in cell wall function and

Tissue-specific mRNA expression profiling in grape berry tissues

Directory of Open Access Journals (Sweden)

Cramer Grant R

2007-06-01

Full Text Available Abstract Background Berries of grape (Vitis vinifera contain three major tissue types (skin, pulp and seed all of which contribute to the aroma, color, and flavor characters of wine. The pericarp, which is composed of the exocarp (skin and mesocarp (pulp, not only functions to protect and feed the developing seed, but also to assist in the dispersal of the mature seed by avian and mammalian vectors. The skin provides volatile and nonvolatile aroma and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of organoleptic characteristics of wine. In order to understand the transcriptional network responsible for controlling tissue-specific mRNA expression patterns, mRNA expression profiling was conducted on each tissue of mature berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0. In order to monitor the influence of water-deficit stress on tissue-specific expression patterns, mRNA expression profiles were also compared from mature berries harvested from vines subjected to well-watered or water-deficit conditions. Results Overall, berry tissues were found to express approximately 76% of genes represented on the Vitis microarray. Approximately 60% of these genes exhibited significant differential expression in one or more of the three major tissue types with more than 28% of genes showing pronounced (2-fold or greater differences in mRNA expression. The largest difference in tissue-specific expression was observed between the seed and pulp/skin. Exocarp tissue, which is involved in pathogen defense and pigment production, showed higher mRNA abundance relative to other berry tissues for genes involved with flavonoid biosynthesis, pathogen resistance, and cell wall modification. Mesocarp tissue, which is considered a nutritive tissue, exhibited a higher mRNA abundance of genes involved in cell

Electrochemical Branched-DNA Assay for Polymerase Chain Reaction-Free Detection and Quantification of Oncogenes in Messenger RNA

Energy Technology Data Exchange (ETDEWEB)

Lee, Ai Cheng; Dai, Ziyu; Chen, Baowei; Wu, Hong; Wang, Jun; Zhang, Aiguo; Zhang, Lurong; Lim, Tit-Meng; Lin, Yuehe

2008-12-01

We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcript in the population of messenger RNA (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify targets signal. The targets were captured on microplate well surfaces through cooperative sandwich hybridization prior to the labeling of bDNA. The activity of captured ALP was monitored by square-wave voltammetric (SWV) analysis of the electroactive enzymatic product in the presence of 1-napthyl-phosphate. The specificity and sensitivity of assay enabled direct detection of target transcript in as little as 4.6 ng mRNA without PCR amplification. In combination with the use of a well-quantified standard, the electrochemical bDNA assay was capable of direct use for a PCR-free quantitative analysis of target transcript in total mRNA population. The approach thus provides a simple, sensitive, accurate and quantitative tool alternate to the RQ-PCR for early disease diagnosis.

Protein functional features are reflected in the patterns of mRNA translation speed.

Science.gov (United States)

López, Daniel; Pazos, Florencio

2015-07-09

The degeneracy of the genetic code makes it possible for the same amino acid string to be coded by different messenger RNA (mRNA) sequences. These "synonymous mRNAs" may differ largely in a number of aspects related to their overall translational efficiency, such as secondary structure content and availability of the encoded transfer RNAs (tRNAs). Consequently, they may render different yields of the translated polypeptides. These mRNA features related to translation efficiency are also playing a role locally, resulting in a non-uniform translation speed along the mRNA, which has been previously related to some protein structural features and also used to explain some dramatic effects of "silent" single-nucleotide-polymorphisms (SNPs). In this work we perform the first large scale analysis of the relationship between three experimental proxies of mRNA local translation efficiency and the local features of the corresponding encoded proteins. We found that a number of protein functional and structural features are reflected in the patterns of ribosome occupancy, secondary structure and tRNA availability along the mRNA. One or more of these proxies of translation speed have distinctive patterns around the mRNA regions coding for certain protein local features. In some cases the three patterns follow a similar trend. We also show specific examples where these patterns of translation speed point to the protein's important structural and functional features. This support the idea that the genome not only codes the protein functional features as sequences of amino acids, but also as subtle patterns of mRNA properties which, probably through local effects on the translation speed, have some consequence on the final polypeptide. These results open the possibility of predicting a protein's functional regions based on a single genomic sequence, and have implications for heterologous protein expression and fine-tuning protein function.

Arc mRNA induction in striatal efferent neurons associated with response learning.

Science.gov (United States)

Daberkow, D P; Riedy, M D; Kesner, R P; Keefe, K A

2007-07-01

The dorsal striatum is involved in motor-response learning, but the extent to which distinct populations of striatal efferent neurons are differentially involved in such learning is unknown. Activity-regulated, cytoskeleton-associated (Arc) protein is an effector immediate-early gene implicated in synaptic plasticity. We examined arc mRNA expression in striatopallidal vs. striatonigral efferent neurons in dorsomedial and dorsolateral striatum of rats engaged in reversal learning on a T-maze motor-response task. Male Sprague-Dawley rats learned to turn right or left for 3 days. Half of the rats then underwent reversal training. The remaining rats were yoked to rats undergoing reversal training, such that they ran the same number of trials but ran them as continued-acquisition trials. Brains were removed and processed using double-label fluorescent in situ hybridization for arc and preproenkephalin (PPE) mRNA. In the reversal, but not the continued-acquisition, group there was a significant relation between the overall arc mRNA signal in dorsomedial striatum and the number of trials run, with rats reaching criterion in fewer trials having higher levels of arc mRNA expression. A similar relation was seen between the numbers of PPE(+) and PPE(-) neurons in dorsomedial striatum with cytoplasmic arc mRNA expression. Interestingly, in behaviourally activated animals significantly more PPE(-) neurons had cytoplasmic arc mRNA expression. These data suggest that Arc in both striatonigral and striatopallidal efferent neurons is involved in striatal synaptic plasticity mediating motor-response learning in the T-maze and that there is differential processing of arc mRNA in distinct subpopulations of striatal efferent neurons.

Relative quantification in seed GMO analysis: state of art and bottlenecks.

Science.gov (United States)

Chaouachi, Maher; Bérard, Aurélie; Saïd, Khaled

2013-06-01

Reliable quantitative methods are needed to comply with current EU regulations on the mandatory labeling of genetically modified organisms (GMOs) and GMO-derived food and feed products with a minimum GMO content of 0.9 %. The implementation of EU Commission Recommendation 2004/787/EC on technical guidance for sampling and detection which meant as a helpful tool for the practical implementation of EC Regulation 1830/2003, which states that "the results of quantitative analysis should be expressed as the number of target DNA sequences per target taxon specific sequences calculated in terms of haploid genomes". This has led to an intense debate on the type of calibrator best suitable for GMO quantification. The main question addressed in this review is whether reference materials and calibrators should be matrix based or whether pure DNA analytes should be used for relative quantification in GMO analysis. The state of the art, including the advantages and drawbacks, of using DNA plasmid (compared to genomic DNA reference materials) as calibrators, is widely described. In addition, the influence of the genetic structure of seeds on real-time PCR quantitative results obtained for seed lots is discussed. The specific composition of a seed kernel, the mode of inheritance, and the ploidy level ensure that there is discordance between a GMO % expressed as a haploid genome equivalent and a GMO % based on numbers of seeds. This means that a threshold fixed as a percentage of seeds cannot be used as such for RT-PCR. All critical points that affect the expression of the GMO content in seeds are discussed in this paper.

The yeast PNC1 longevity gene is up-regulated by mRNA mistranslation.

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Raquel M Silva

Full Text Available Translation fidelity is critical for protein synthesis and to ensure correct cell functioning. Mutations in the protein synthesis machinery or environmental factors that increase synthesis of mistranslated proteins result in cell death and degeneration and are associated with neurodegenerative diseases, cancer and with an increasing number of mitochondrial disorders. Remarkably, mRNA mistranslation plays critical roles in the evolution of the genetic code, can be beneficial under stress conditions in yeast and in Escherichia coli and is an important source of peptides for MHC class I complex in dendritic cells. Despite this, its biology has been overlooked over the years due to technical difficulties in its detection and quantification. In order to shed new light on the biological relevance of mistranslation we have generated codon misreading in Saccharomyces cerevisiae using drugs and tRNA engineering methodologies. Surprisingly, such mistranslation up-regulated the longevity gene PNC1. Similar results were also obtained in cells grown in the presence of amino acid analogues that promote protein misfolding. The overall data showed that PNC1 is a biomarker of mRNA mistranslation and protein misfolding and that PNC1-GFP fusions can be used to monitor these two important biological phenomena in vivo in an easy manner, thus opening new avenues to understand their biological relevance.

Direct qPCR quantification using the Quantifiler(®) Trio DNA quantification kit.

Science.gov (United States)

Liu, Jason Yingjie

2014-11-01

The effectiveness of a direct quantification assay is essential to the adoption of the combined direct quantification/direct STR workflow. In this paper, the feasibility of using the Quantifiler(®) Trio DNA quantification kit for the direct quantification of forensic casework samples was investigated. Both low-level touch DNA samples and blood samples were collected on PE swabs and quantified directly. The increased sensitivity of the Quantifiler(®) Trio kit enabled the detection of less than 10pg of DNA in unprocessed touch samples and also minimizes the stochastic effect experienced by different targets in the same sample. The DNA quantity information obtained from a direct quantification assay using the Quantifiler(®) Trio kit can also be used to accurately estimate the optimal input DNA quantity for a direct STR amplification reaction. The correlation between the direct quantification results (Quantifiler(®) Trio kit) and the direct STR results (GlobalFiler™ PCR amplification kit(*)) for low-level touch DNA samples indicates that direct quantification using the Quantifiler(®) Trio DNA quantification kit is more reliable than the Quantifiler(®) Duo DNA quantification kit for predicting the STR results of unprocessed touch DNA samples containing less than 10pg of DNA. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Short-term calorie restriction feminizes the mRNA profiles of drug metabolizing enzymes and transporters in livers of mice

International Nuclear Information System (INIS)

Fu, Zidong Donna; Klaassen, Curtis D.

2014-01-01

Calorie restriction (CR) is one of the most effective anti-aging interventions in mammals. A modern theory suggests that aging results from a decline in detoxification capabilities and thus accumulation of damaged macromolecules. The present study aimed to determine how short-term CR alters mRNA profiles of genes that encode metabolism and detoxification machinery in the liver. Male C57BL/6 mice were fed CR (0, 15, 30, or 40%) diets for one month, followed by mRNA quantification of 98 xenobiotic processing genes (XPGs) in the liver, including 7 uptake transporters, 39 phase-I enzymes, 37 phase-II enzymes, 10 efflux transporters, and 5 transcription factors. In general, 15% CR did not alter mRNAs of most XPGs, whereas 30 and 40% CR altered over half of the XPGs (32 increased and 29 decreased). CR up-regulated some phase-I enzymes (fold increase), such as Cyp4a14 (12), Por (2.3), Nqo1 (1.4), Fmo2 (5.4), and Fmo3 (346), and numerous number of phase-II enzymes, such as Sult1a1 (1.2), Sult1d1 (2.0), Sult1e1 (33), Sult3a1 (2.2), Gsta4 (1.3), Gstm2 (1.3), Gstm3 (1.7), and Mgst3 (2.2). CR feminized the mRNA profiles of 32 XPGs in livers of male mice. For instance, CR decreased the male-predominantly expressed Oatp1a1 (97%) and increased the female-predominantly expressed Oatp1a4 (11). In conclusion, short-term CR alters the mRNA levels of over half of the 98 XPGs quantified in livers of male mice, and over half of these alterations appear to be due to feminization of the liver. - Highlights: • Utilized a graded CR model in male mice • The mRNA profiles of xenobiotic processing genes (XPGs) in liver were investigated. • CR up-regulates many phase-II enzymes. • CR tends to feminize the mRNA profiles of XPGs

Short-term calorie restriction feminizes the mRNA profiles of drug metabolizing enzymes and transporters in livers of mice

Energy Technology Data Exchange (ETDEWEB)

Fu, Zidong Donna [Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Klaassen, Curtis D., E-mail: cklaasse@kumc.edu [Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS 66160 (United States)

2014-01-01

Calorie restriction (CR) is one of the most effective anti-aging interventions in mammals. A modern theory suggests that aging results from a decline in detoxification capabilities and thus accumulation of damaged macromolecules. The present study aimed to determine how short-term CR alters mRNA profiles of genes that encode metabolism and detoxification machinery in the liver. Male C57BL/6 mice were fed CR (0, 15, 30, or 40%) diets for one month, followed by mRNA quantification of 98 xenobiotic processing genes (XPGs) in the liver, including 7 uptake transporters, 39 phase-I enzymes, 37 phase-II enzymes, 10 efflux transporters, and 5 transcription factors. In general, 15% CR did not alter mRNAs of most XPGs, whereas 30 and 40% CR altered over half of the XPGs (32 increased and 29 decreased). CR up-regulated some phase-I enzymes (fold increase), such as Cyp4a14 (12), Por (2.3), Nqo1 (1.4), Fmo2 (5.4), and Fmo3 (346), and numerous number of phase-II enzymes, such as Sult1a1 (1.2), Sult1d1 (2.0), Sult1e1 (33), Sult3a1 (2.2), Gsta4 (1.3), Gstm2 (1.3), Gstm3 (1.7), and Mgst3 (2.2). CR feminized the mRNA profiles of 32 XPGs in livers of male mice. For instance, CR decreased the male-predominantly expressed Oatp1a1 (97%) and increased the female-predominantly expressed Oatp1a4 (11). In conclusion, short-term CR alters the mRNA levels of over half of the 98 XPGs quantified in livers of male mice, and over half of these alterations appear to be due to feminization of the liver. - Highlights: • Utilized a graded CR model in male mice • The mRNA profiles of xenobiotic processing genes (XPGs) in liver were investigated. • CR up-regulates many phase-II enzymes. • CR tends to feminize the mRNA profiles of XPGs.

Self-amplifying mRNA vaccines.

Science.gov (United States)

Brito, Luis A; Kommareddy, Sushma; Maione, Domenico; Uematsu, Yasushi; Giovani, Cinzia; Berlanda Scorza, Francesco; Otten, Gillis R; Yu, Dong; Mandl, Christian W; Mason, Peter W; Dormitzer, Philip R; Ulmer, Jeffrey B; Geall, Andrew J

2015-01-01

This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization. Copyright © 2015 Elsevier Inc. All rights reserved.

Identification and accurate quantification of structurally related peptide impurities in synthetic human C-peptide by liquid chromatography-high resolution mass spectrometry.

Science.gov (United States)

Li, Ming; Josephs, Ralf D; Daireaux, Adeline; Choteau, Tiphaine; Westwood, Steven; Wielgosz, Robert I; Li, Hongmei

2018-06-04

Peptides are an increasingly important group of biomarkers and pharmaceuticals. The accurate purity characterization of peptide calibrators is critical for the development of reference measurement systems for laboratory medicine and quality control of pharmaceuticals. The peptides used for these purposes are increasingly produced through peptide synthesis. Various approaches (for example mass balance, amino acid analysis, qNMR, and nitrogen determination) can be applied to accurately value assign the purity of peptide calibrators. However, all purity assessment approaches require a correction for structurally related peptide impurities in order to avoid biases. Liquid chromatography coupled to high resolution mass spectrometry (LC-hrMS) has become the key technique for the identification and accurate quantification of structurally related peptide impurities in intact peptide calibrator materials. In this study, LC-hrMS-based methods were developed and validated in-house for the identification and quantification of structurally related peptide impurities in a synthetic human C-peptide (hCP) material, which served as a study material for an international comparison looking at the competencies of laboratories to perform peptide purity mass fraction assignments. More than 65 impurities were identified, confirmed, and accurately quantified by using LC-hrMS. The total mass fraction of all structurally related peptide impurities in the hCP study material was estimated to be 83.3 mg/g with an associated expanded uncertainty of 3.0 mg/g (k = 2). The calibration hierarchy concept used for the quantification of individual impurities is described in detail. Graphical abstract ᅟ.

Noise stress changes mRNA expressions of corticotropin-releasing hormone, its receptors in amygdala, and anxiety-related behaviors

Directory of Open Access Journals (Sweden)

Evren Eraslan

2015-01-01

Full Text Available Noise is a psychological, environmental stressor that activates limbic sites in the brain. Limbic sites such as the amygdala and the amygdaloid corticotropin-releasing hormone (CRH system play an important role in integrating stress response. We investigated the association between noise exposures, CRH-related molecules in the amygdala, and behavioral alterations. In total 54 Sprague-Dawley rats were divided into the following three groups: Control (CON, acute noise exposure (ANE, and chronic noise exposure (CNE. The ANE group was exposed to 100 dB white noise only once in 4 h and the CNE group was exposed to the same for 4 h per day for 30 days. Expression profiles of CRH and its receptors CRH-R1 and CRH-R2 were analyzed by quantitative real-time polymerase chain reaction (qPCR. The same stress procedure was applied to the ANE and CNE groups for behavior testing. The anxiety responses of the animals after acute and chronic stress exposure were measured in the defensive withdrawal test. CNE upregulated CRH and CRH-R1 mRNA levels but downregulated CRH-R2 mRNA levels. ANE led to a decrease in both CRH-R1 and CRH-R2 expression. In the defensive withdrawal test, while the ANE increased, CNE reduced anxiety-like behaviors. The present study shows that the exposure of rats to white noise (100 dB leads to behavioral alterations and molecule-specific changes in the CRH system. Behavioral alterations can be related to these molecular changes in the amygdala.

Noise stress changes mRNA expressions of corticotropin-releasing hormone, its receptors in amygdala, and anxiety-related behaviors.

Science.gov (United States)

Eraslan, Evren; Akyazi, Ibrahim; Erg L-Ekiz, Elif; Matur, Erdal

2015-01-01

Noise is a psychological, environmental stressor that activates limbic sites in the brain. Limbic sites such as the amygdala and the amygdaloid corticotropin-releasing hormone (CRH) system play an important role in integrating stress response. We investigated the association between noise exposures, CRH-related molecules in the amygdala, and behavioral alterations. In total 54 Sprague-Dawley rats were divided into the following three groups: Control (CON), acute noise exposure (ANE), and chronic noise exposure (CNE). The ANE group was exposed to 100 dB white noise only once in 4 h and the CNE group was exposed to the same for 4 h per day for 30 days. Expression profiles of CRH and its receptors CRH-R1 and CRH-R2 were analyzed by quantitative real-time polymerase chain reaction (qPCR). The same stress procedure was applied to the ANE and CNE groups for behavior testing. The anxiety responses of the animals after acute and chronic stress exposure were measured in the defensive withdrawal test. CNE upregulated CRH and CRH-R1 mRNA levels but downregulated CRH-R2 mRNA levels. ANE led to a decrease in both CRH-R1 and CRH-R2 expression. In the defensive withdrawal test, while the ANE increased, CNE reduced anxiety-like behaviors. The present study shows that the exposure of rats to white noise (100 dB) leads to behavioral alterations and molecule-specific changes in the CRH system. Behavioral alterations can be related to these molecular changes in the amygdala.

Expression of mRNA for galanin, galanin-like peptide and galanin receptors 1-3 in the ovine hypothalamus and pituitary gland: effects of age and gender.

Science.gov (United States)

Whitelaw, Christine Margaret; Robinson, Jane Elizabeth; Chambers, George Ballantine; Hastie, Peter; Padmanabhan, Vasantha; Thompson, Robert Charles; Evans, Neil Price

2009-01-01

The neurotransmitters/neuromodulators galanin (GAL) and galanin-like peptide (GALP) are known to operate through three G protein-coupled receptors, GALR1, GALR2 and GALR3. The aim of this study was to investigate changes in expression of mRNA for galanin, GALP and GALR1-3 in the hypothalamus and pituitary gland, of male and female sheep, to determine how expression changed in association with growth and the attainment of reproductive competence. Tissue samples from the hypothalami and pituitary glands were analysed from late foetal and pre-pubertal lambs and adult sheep. Although mRNA for galanin and GALR1-3 was present in both tissues, at all ages and in both genders, quantification of GALP mRNA was not possible due to its low levels of expression. mRNA expression for both galanin and its receptors was seen to change significantly in both tissues as a function of age. Specifically, hypothalamic galanin mRNA expression increased with age in the male, but decreased with age in the female pituitary gland. mRNA expression for all receptors increased between foetal and pre-pubertal age groups and decreased significantly between pre-pubertal and adult animals. The results indicate that the expression of mRNA for galanin and its receptors changes dynamically with age and those significant differences exist with regard to tissue type and gender. These changes suggest that galaninergic neuroendocrine systems could be involved in the regulation of ovine growth and or the development of reproductive competence. The roles played by these systems in the sheep, however, may differ from other species, in particular the neuroendocrine link between nutrition and reproduction and GALR1's role in pituitary signalling.

CYP3A5 mRNA degradation by nonsense-mediated mRNA decay.

Science.gov (United States)

Busi, Florent; Cresteil, Thierry

2005-09-01

The total CYP3A5 mRNA level is significantly greater in carriers of the CYP3A5*1 allele than in CYP3A5*3 homozygotes. Most of the CYP3A5*3 mRNA includes an intronic sequence (exon 3B) containing premature termination codons (PTCs) between exons 3 and 4. Two models were used to investigate the degradation of CYP3A5 mRNA: a CYP3A5 minigene consisting of CYP3A5 exons and introns 3 to 6 transfected into MCF7 cells, and the endogenous CYP3A5 gene expressed in HepG2 cells. The 3'-untranslated region g.31611C>T mutation has no effect on CYP3A5 mRNA decay. Splice variants containing exon 3B were more unstable than wild-type (wt) CYP3A5 mRNA. Cycloheximide prevents the recognition of PTCs by ribosomes: in transfected MCF7 and HepG2 cells, cycloheximide slowed down the degradation of exon 3B-containing splice variants, suggesting the participation of nonsense-mediated decay (NMD). When PTCs were removed from pseudoexon 3B or when UPF1 small interfering RNA was used to impair the NMD mechanism, the decay of the splice variant was reduced, confirming the involvement of NMD in the degradation of CYP3A5 splice variants. Induction could represent a source of variability for CYP3A5 expression and could modify the proportion of splice variants. The extent of CYP3A5 induction was investigated after exposure to barbiturates or steroids: CYP3A4 was markedly induced in a pediatric population compared with untreated neonates. However, no effect could be detected in either the total CYP3A5 RNA, the proportion of splice variant RNA, or the protein level. Therefore, in these carriers, induction is unlikely to switch on the phenotypic CYP3A5 expression in carriers of CYP3A5*3/*3.

Three-Dimensional Mapping of mRNA Export through the Nuclear Pore Complex

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Steven J. Schnell

2014-11-01

Full Text Available The locations of transcription and translation of mRNA in eukaryotic cells are spatially separated by the nuclear envelope (NE. Plenty of nuclear pore complexes (NPCs embedded in the NE function as the major gateway for the export of transcribed mRNAs from the nucleus to the cytoplasm. Whereas the NPC, perhaps one of the largest protein complexes, provides a relatively large channel for macromolecules to selectively pass through it in inherently three-dimensional (3D movements, this channel is nonetheless below the diffraction limit of conventional light microscopy. A full understanding of the mRNA export mechanism urgently requires real-time mapping of the 3D dynamics of mRNA in the NPC of live cells with innovative imaging techniques breaking the diffraction limit of conventional light microscopy. Recently, super-resolution fluorescence microscopy and single-particle tracking (SPT techniques have been applied to the study of nuclear export of mRNA in live cells. In this review, we emphasize the necessity of 3D mapping techniques in the study of mRNA export, briefly summarize the feasibility of current 3D imaging approaches, and highlight the new features of mRNA nuclear export elucidated with a newly developed 3D imaging approach combining SPT-based super-resolution imaging and 2D-to-3D deconvolution algorithms.

Sodium arsenite-induced inhibition of cell proliferation is related to inhibition of IL-2 mRNA expression in mouse activated T cells

Energy Technology Data Exchange (ETDEWEB)

Conde, Patricia; Acosta-Saavedra, Leonor C.; Calderon-Aranda, Emma S. [Centro de Investigacion y de Estudios Avanzados, CINVESTAV, Seccion Toxicologia, P.O. Box 14-740, Mexico, D.F. (Mexico); Goytia-Acevedo, Raquel C. [Universidad Juarez del Estado de Durango, Facultad de Medicina, Gomez Palacio, Durango (Mexico)

2007-04-15

A proposed mechanism for the As-induced inhibition of cell proliferation is the inhibition of IL-2 secretion. However, the effects of arsenite on IL-2 mRNA expression or on the ERK pathway in activated-T cells have not yet been described. We examined the effect of arsenite on IL-2 mRNA expression, cell activation and proliferation in PHA-stimulated murine lymphocytes. Arsenite (1 and 10 {mu}M) decreased IL-2 mRNA expression, IL-2 secretion and cell proliferation. Arsenite (10 {mu}M) strongly inhibited ERK-phosphorylation. However, the partial inhibition (50%) of IL-2 mRNA produced by 1 {mu}M, consistent with the effects on IL-2 secretion and cell proliferation, could not be explained by the inhibition of ERK-phosphorylation, which was not affected at this concentration. The inhibition of IL-2 mRNA expression caused by 1 {mu}M could be associated to effects on pathways located downstream or parallel to ERK. Arsenite also decreased early activation (surface CD69{sup +} expression) in both CD4{sup +} and CD8{sup +}, and decreased total CD8{sup +} count without significantly affecting CD4{sup +}, supporting that the cellular immune response mediated by cytotoxic T cells is an arsenic target. Thus, our results suggest that arsenite decreases IL-2 mRNA levels and T-cell activation and proliferation. However, further studies on the effects of arsenite on IL-2 gene transcription and IL-2 mRNA stability are needed. (orig.)

Expression of Annexin-A1 and Galectin-1 Anti-Inflammatory Proteins and mRNA in Chronic Gastritis and Gastric Cancer

Directory of Open Access Journals (Sweden)

Yvana Cristina Jorge

2013-01-01

Full Text Available Objective. The anti-inflammatory proteins annexin-A1 and galectin-1 have been associated with tumor progression. This scenario prompted us to investigate the relationship between the gene and protein expression of annexin-A1 (ANXA1/AnxA1 and galectin-1 (LGALS1/Gal-1 in an inflammatory gastric lesion as chronic gastritis (CG and gastric adenocarcinoma (GA and its association with H. pylori infection. Methods. We analyzed 40 samples of CG, 20 of GA, and 10 of normal mucosa (C by the quantitative real-time PCR (qPCR technique and the immunohistochemistry assay. Results. High ANXA1 mRNA expression levels were observed in 90% (36/40 of CG cases (mean relative quantification RQ = 4.26 ± 2.03 and in 80% (16/20 of GA cases (mean RQ = 4.38 ± 4.77. However, LGALS1 mRNA levels were high (mean RQ = 2.44 ± 3.26 in 60% (12/20 of the GA cases, while low expression was found in CG (mean RQ = 0.43±3.13; P<0.01. Normal mucosa showed modest immunoreactivity in stroma but not in epithelium, while stroma and epithelium displayed an intense immunostaining in CG and GA for both proteins. Conclusion. These results have provided evidence that galectin-1 and mainly annexin-A1 are overexpressed in both gastritis and gastric cancer, suggesting a strong association of these proteins with chronic gastric inflammation and carcinogenesis.

Photobiomodulation effects on mRNA levels from genomic and chromosome stabilization genes in injured muscle.

Science.gov (United States)

da Silva Neto Trajano, Larissa Alexsandra; Trajano, Eduardo Tavares Lima; da Silva Sergio, Luiz Philippe; Teixeira, Adilson Fonseca; Mencalha, Andre Luiz; Stumbo, Ana Carolina; de Souza da Fonseca, Adenilson

2018-04-26

Muscle injuries are the most prevalent type of injury in sports. A great number of athletes have relapsed in muscle injuries not being treated properly. Photobiomodulation therapy is an inexpensive and safe technique with many benefits in muscle injury treatment. However, little has been explored about the infrared laser effects on DNA and telomeres in muscle injuries. Thus, the aim of this study was to evaluate photobiomodulation effects on mRNA relative levels from genes related to telomere and genomic stabilization in injured muscle. Wistar male rats were randomly divided into six groups: control, laser 25 mW, laser 75 mW, injury, injury laser 25 mW, and injury laser 75 mW. Photobiomodulation was performed with 904 nm, 3 J/cm 2 at 25 or 75 mW. Cryoinjury was induced by two applications of a metal probe cooled in liquid nitrogen directly on the tibialis anterior muscle. After euthanasia, skeletal muscle samples were withdrawn and total RNA extracted for evaluation of mRNA levels from genomic (ATM and p53) and chromosome stabilization (TRF1 and TRF2) genes by real-time quantitative polymerization chain reaction. Data show that photobiomodulation reduces the mRNA levels from ATM and p53, as well reduces mRNA levels from TRF1 and TRF2 at 25 and 75 mW in injured skeletal muscle. In conclusion, photobiomodulation alters mRNA relative levels from genes related to genomic and telomere stabilization in injured skeletal muscle.

Age-related changes in expression of neural cell adhesion molecule (NCAM) in heart

DEFF Research Database (Denmark)

Gaardsvoll, H; Krog, L; Zhernosekov, D

1993-01-01

, whereas it was clearly detectable in NCAM mRNA classes of 5.2 and 2.9 kb. Insertion of exons a and AAG between exons 12 and 13 was more pronounced in the 5.2 and 2.9 kb NCAM mRNAs than in the 6.7 kb mRNA at all ages. Insertions at the 12/13 junctions decreased in the 6.7 kb mRNA as compared to the 5.......2 and 2.9 kb mRNAs during postnatal development. Quantification of NCAM protein by enzyme-linked immunosorbent assay showed that NCAM protein amount decreased from a level of 0.93 microgram NCAM/mg total protein at birth to postnatal day 40 where a level of 0.21 microgram NCAM/mg total protein was found...

Colour thresholding and objective quantification in bioimaging

Science.gov (United States)

Fermin, C. D.; Gerber, M. A.; Torre-Bueno, J. R.

1992-01-01

Computer imaging is rapidly becoming an indispensable tool for the quantification of variables in research and medicine. Whilst its use in medicine has largely been limited to qualitative observations, imaging in applied basic sciences, medical research and biotechnology demands objective quantification of the variables in question. In black and white densitometry (0-256 levels of intensity) the separation of subtle differences between closely related hues from stains is sometimes very difficult. True-colour and real-time video microscopy analysis offer choices not previously available with monochrome systems. In this paper we demonstrate the usefulness of colour thresholding, which has so far proven indispensable for proper objective quantification of the products of histochemical reactions and/or subtle differences in tissue and cells. In addition, we provide interested, but untrained readers with basic information that may assist decisions regarding the most suitable set-up for a project under consideration. Data from projects in progress at Tulane are shown to illustrate the advantage of colour thresholding over monochrome densitometry and for objective quantification of subtle colour differences between experimental and control samples.

Cues, quantification, and agreement in language comprehension.

Science.gov (United States)

Tanner, Darren; Bulkes, Nyssa Z

2015-12-01

We investigated factors that affect the comprehension of subject-verb agreement in English, using quantification as a window into the relationship between morphosyntactic processes in language production and comprehension. Event-related brain potentials (ERPs) were recorded while participants read sentences with grammatical and ungrammatical verbs, in which the plurality of the subject noun phrase was either doubly marked (via overt plural quantification and morphological marking on the noun) or singly marked (via only plural morphology on the noun). Both acceptability judgments and the ERP data showed heightened sensitivity to agreement violations when quantification provided an additional cue to the grammatical number of the subject noun phrase, over and above plural morphology. This is consistent with models of grammatical comprehension that emphasize feature prediction in tandem with cue-based memory retrieval. Our results additionally contrast with those of prior studies that showed no effects of plural quantification on agreement in language production. These findings therefore highlight some nontrivial divergences in the cues and mechanisms supporting morphosyntactic processing in language production and comprehension.

mRNA related to insulin family in human placenta

International Nuclear Information System (INIS)

Younes, M.A.; D'Agostino, J.B.; Frazier, M.L.; Besch, P.K.

1986-01-01

The authors have previously reported that human term placenta contains mRNA displaying sequence homology to a rat preproinsulin I cDNA clone (p119). When placental poly(A + ) RNA was analyzed for homology to p119 by RNA/DNA blot hybridization, prominent hybridization was observed which was found by densitometric analysis to be three-fold higher than control. To further characterize this insulin-like message, a cDNA library was generated (approx.7000 transformants) using normal term cesarean-sectioned tissue to prepare placental poly(A + ) RNA templates. Five hundred transformants were initially screened by colony hybridization using a 32 P-labeled rat preproinsulin I cDNA as probe. Of the ten initial positives obtained, three were found to be true positives based on Southern hybridization analyses of the recombinant plasmids. Using Taq I digested pBr322 as a size marker, the cDNAs were found to be approximately 300 bp in length. Preliminary DNA sequencing using the Sanger dideoxy chain termination method has revealed that one of these clones displays significant homology to the 5' region of human insulin-like growth factors I and II

mRNA related to insulin family in human placenta

Energy Technology Data Exchange (ETDEWEB)

Younes, M.A.; D' Agostino, J.B.; Frazier, M.L.; Besch, P.K.

1986-03-01

The authors have previously reported that human term placenta contains mRNA displaying sequence homology to a rat preproinsulin I cDNA clone (p119). When placental poly(A/sup +/) RNA was analyzed for homology to p119 by RNA/DNA blot hybridization, prominent hybridization was observed which was found by densitometric analysis to be three-fold higher than control. To further characterize this insulin-like message, a cDNA library was generated (approx.7000 transformants) using normal term cesarean-sectioned tissue to prepare placental poly(A/sup +/) RNA templates. Five hundred transformants were initially screened by colony hybridization using a /sup 32/P-labeled rat preproinsulin I cDNA as probe. Of the ten initial positives obtained, three were found to be true positives based on Southern hybridization analyses of the recombinant plasmids. Using Taq I digested pBr322 as a size marker, the cDNAs were found to be approximately 300 bp in length. Preliminary DNA sequencing using the Sanger dideoxy chain termination method has revealed that one of these clones displays significant homology to the 5' region of human insulin-like growth factors I and II.

Short-term calorie restriction feminizes the mRNA profiles of drug metabolizing enzymes and transporters in livers of mice.

Science.gov (United States)

Fu, Zidong Donna; Klaassen, Curtis D

2014-01-01

Calorie restriction (CR) is one of the most effective anti-aging interventions in mammals. A modern theory suggests that aging results from a decline in detoxification capabilities and thus accumulation of damaged macromolecules. The present study aimed to determine how short-term CR alters mRNA profiles of genes that encode metabolism and detoxification machinery in the liver. Male C57BL/6 mice were fed CR (0, 15, 30, or 40%) diets for one month, followed by mRNA quantification of 98 xenobiotic processing genes (XPGs) in the liver, including 7 uptake transporters, 39 phase-I enzymes, 37 phase-II enzymes, 10 efflux transporters, and 5 transcription factors. In general, 15% CR did not alter mRNAs of most XPGs, whereas 30 and 40% CR altered over half of the XPGs (32 increased and 29 decreased). CR up-regulated some phase-I enzymes (fold increase), such as Cyp4a14 (12), Por (2.3), Nqo1 (1.4), Fmo2 (5.4), and Fmo3 (346), and numerous number of phase-II enzymes, such as Sult1a1 (1.2), Sult1d1 (2.0), Sult1e1 (33), Sult3a1 (2.2), Gsta4 (1.3), Gstm2 (1.3), Gstm3 (1.7), and Mgst3 (2.2). CR feminized the mRNA profiles of 32 XPGs in livers of male mice. For instance, CR decreased the male-predominantly expressed Oatp1a1 (97%) and increased the female-predominantly expressed Oatp1a4 (11). In conclusion, short-term CR alters the mRNA levels of over half of the 98 XPGs quantified in livers of male mice, and over half of these alterations appear to be due to feminization of the liver. Copyright © 2013 Elsevier Inc. All rights reserved.

Coordinated Regulations of mRNA Synthesis and Decay during Cold Acclimation in Arabidopsis Cells.

KAUST Repository

Arae, Toshihiro

2017-04-18

Plants possess a cold acclimation system to acquire freezing tolerance through pre-exposure to non-freezing low temperatures. The transcriptional cascade of C-repeat binding factors (CBFs)/dehydration response element-binding factors (DREBs) is considered a major transcriptional regulatory pathway during cold acclimation. However, little is known regarding the functional significance of mRNA stability regulation in the response of gene expression to cold stress. The actual level of individual mRNAs is determined by a balance between mRNA synthesis and degradation. Therefore, it is important to assess the regulatory steps to increase our understanding of gene regulation. Here, we analyzed temporal changes in mRNA amounts and half-lives in response to cold stress in Arabidopsis cell cultures based on genome-wide analysis. In this mRNA decay array method, mRNA half-life measurements and microarray analyses were combined. In addition, temporal changes in the integrated value of transcription rates were estimated from the above two parameters using a mathematical approach. Our results showed that several cold-responsive genes, including Cold-regulated 15a, were relatively destabilized, whereas the mRNA amounts were increased during cold treatment by accelerating the transcription rate to overcome the destabilization. Considering the kinetics of mRNA synthesis and degradation, this apparently contradictory result supports that mRNA destabilization is advantageous for the swift increase in CBF-responsive genes in response to cold stress.

The quantification of risk and tourism

Directory of Open Access Journals (Sweden)

Piet Croucamp

2014-01-01

Full Text Available Tourism in South Africa comprises 9.5% of Gross Domestic Product (GDP, but remains an underresearched industry, especially regarding the quantification of the risks prevailing in the social, political and economic environment in which the industry operates. Risk prediction, extrapolation forecasting is conducted largely in the context of a qualitative methodology. This article reflects on the quantification of social constructs as variables of risk in the tourism industry with reference to South Africa. The theory and methodology of quantification is briefly reviewed and the indicators of risk are conceptualized and operationalized. The identified indicators are scaled in indices for purposes of quantification. Risk assessments and the quantification of constructs rely heavily on the experience - often personal - of the researcher and this scholarly endeavour is, therefore, not inclusive of all possible identified indicators of risk. It is accepted that tourism in South Africa is an industry comprising of a large diversity of sectors, each with a different set of risk indicators and risk profiles. The emphasis of this article is thus on the methodology to be applied to a risk profile. A secondary endeavour is to provide for clarity about the conceptual and operational confines of risk in general, as well as how quantified risk relates to the tourism industry. The indices provided include both domesticand international risk indicators. The motivation for the article is to encourage a greater emphasis on quantitative research in our efforts to understand and manage a risk profile for the tourist industry.

Tumor protein D52 expression is post-transcriptionally regulated by T-cell intercellular antigen (TIA) 1 and TIA-related protein via mRNA stability.

Science.gov (United States)

Motohashi, Hiromi; Mukudai, Yoshiki; Ito, Chihiro; Kato, Kosuke; Shimane, Toshikazu; Kondo, Seiji; Shirota, Tatsuo

2017-05-04

Although tumor protein D52 (TPD52) family proteins were first identified nearly 20 years ago, their molecular regulatory mechanisms remain unclear. Therefore, we investigated the post-transcriptional regulation of TPD52 family genes. An RNA immunoprecipitation (RIP) assay showed the potential binding ability of TPD52 family mRNAs to several RNA-binding proteins, and an RNA degradation assay revealed that TPD52 is subject to more prominent post-transcriptional regulation than are TPD53 and TPD54. We subsequently focused on the 3'-untranslated region (3'-UTR) of TPD52 as a cis -acting element in post-transcriptional gene regulation. Several deletion mutants of the 3'-UTR of TPD52 mRNA were constructed and ligated to the 3'-end of a reporter green fluorescence protein gene. An RNA degradation assay revealed that a minimal cis -acting region, located in the 78-280 region of the 5'-proximal region of the 3'-UTR, stabilized the reporter mRNA. Biotin pull-down and RIP assays revealed specific binding of the region to T-cell intracellular antigen 1 (TIA-1) and TIA-1-related protein (TIAR). Knockdown of TIA-1/TIAR decreased not only the expression, but also the stability of TPD52 mRNA; it also decreased the expression and stability of the reporter gene ligated to the 3'-end of the 78-280 fragment. Stimulation of transforming growth factor-β and epidermal growth factor decreased the binding ability of these factors, resulting in decreased mRNA stability. These results indicate that the 78-280 fragment and TIA-1/TIAR concordantly contribute to mRNA stability as a cis -acting element and trans -acting factor(s), respectively. Thus, we here report the specific interactions between these elements in the post-transcriptional regulation of the TPD52 gene. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

mRNA localization mechanisms in Trypanosoma cruzi.

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Lysangela R Alves

Full Text Available Asymmetric mRNA localization is a sophisticated tool for regulating and optimizing protein synthesis and maintaining cell polarity. Molecular mechanisms involved in the regulated localization of transcripts are widespread in higher eukaryotes and fungi, but not in protozoa. Trypanosomes are ancient eukaryotes that branched off early in eukaryote evolution. We hypothesized that these organisms would have basic mechanisms of mRNA localization. FISH assays with probes against transcripts coding for proteins with restricted distributions showed a discrete localization of the mRNAs in the cytoplasm. Moreover, cruzipain mRNA was found inside reservosomes suggesting new unexpected functions for this vacuolar organelle. Individual mRNAs were also mobilized to RNA granules in response to nutritional stress. The cytoplasmic distribution of these transcripts changed with cell differentiation, suggesting that localization mechanisms might be involved in the regulation of stage-specific protein expression. Transfection assays with reporter genes showed that, as in higher eukaryotes, 3'UTRs were responsible for guiding mRNAs to their final location. Our results strongly suggest that Trypanosoma cruzi have a core, basic mechanism of mRNA localization. This kind of controlled mRNA transport is ancient, dating back to early eukaryote evolution.

Quantitation of TGF-beta1 mRNA in porcine mesangial cells by comparative kinetic RT/PCR: comparison with ribonuclease protection assay and in situ hybridization.

Science.gov (United States)

Ceol, M; Forino, M; Gambaro, G; Sauer, U; Schleicher, E D; D'Angelo, A; Anglani, F

2001-01-01

Gene expression can be examined with different techniques including ribonuclease protection assay (RPA), in situ hybridisation (ISH), and quantitative reverse transcription-polymerase chain reaction (RT/PCR). These methods differ considerably in their sensitivity and precision in detecting and quantifying low abundance mRNA. Although there is evidence that RT/PCR can be performed in a quantitative manner, the quantitative capacity of this method is generally underestimated. To demonstrate that the comparative kinetic RT/PCR strategy-which uses a housekeeping gene as internal standard-is a quantitative method to detect significant differences in mRNA levels between different samples, the inhibitory effect of heparin on phorbol 12-myristate 13-acetate (PMA)-induced-TGF-beta1 mRNA expression was evaluated by RT/PCR and RPA, the standard method of mRNA quantification, and the results were compared. The reproducibility of RT/PCR amplification was calculated by comparing the quantity of G3PDH and TGF-beta1 PCR products, generated during the exponential phases, estimated from two different RT/PCR (G3PDH, r = 0.968, P = 0.0000; TGF-beta1, r = 0.966, P = 0.0000). The quantitative capacity of comparative kinetic RT/PCR was demonstrated by comparing the results obtained from RPA and RT/PCR using linear regression analysis. Starting from the same RNA extraction, but using only 1% of the RNA for the RT/PCR compared to RPA, significant correlation was observed (r = 0.984, P = 0.0004). Moreover the morphometric analysis of ISH signal was applied for the semi-quantitative evaluation of the expression and localisation of TGF-beta1 mRNA in the entire cell population. Our results demonstrate the close similarity of the RT/PCR and RPA methods in giving quantitative information on mRNA expression and indicate the possibility to adopt the comparative kinetic RT/PCR as reliable quantitative method of mRNA analysis. Copyright 2001 Wiley-Liss, Inc.

Region specific regulation of glutamic acid decarboxylase mRNA expression by dopamine neurons in rat brain.

Science.gov (United States)

Lindefors, N; Brene, S; Herrera-Marschitz, M; Persson, H

1989-01-01

In situ hybridization histochemistry and RNA blots were used to study the expression of glutamic acid decarboxylase (GAD) mRNA in rats with or without a unilateral lesion of midbrain dopamine neurons. Two populations of GAD mRNA positive neurons were found in the intact caudate-putamen, substantia nigra and fronto-parietal cortex. In caudate-putamen, only one out of ten of the GAD mRNA positive neurons expressed high levels, while in substantia nigra every second of the positive neurons expressed high levels of GAD mRNA. Relatively few, but intensively labelled neurons were found in the intact fronto-parietal cerebral cortex. In addition, one out of six of the GAD mRNA positive neurons in the fronto-parietal cortex showed a low labeling. On the ipsilateral side, the forebrain dopamine deafferentation induced an increase in the number of neurons expressing high levels of GAD mRNA in caudate-putamen, and a decrease in fronto-parietal cortex. A smaller decrease was also seen in substantia nigra. However, the total number of GAD mRNA positive neurons were not significantly changed in any of these brain regions. The changes in the levels of GAD mRNA after the dopamine lesion were confirmed by RNA blot analysis. Hence, midbrain dopamine neurons appear to control neuronal expression of GAD mRNA by a tonic down-regulation in a fraction of GAD mRNA positive neurons in caudate-putamen, and a tonic up-regulation in a fraction of GAD mRNA positive neurons in fronto-parietal cortex and substantia nigra.

Evidence for a Complex Class of Nonadenylated mRNA in Drosophila

Science.gov (United States)

Zimmerman, J. Lynn; Fouts, David L.; Manning, Jerry E.

1980-01-01

The amount, by mass, of poly(A+) mRNA present in the polyribosomes of third-instar larvae of Drosophila melanogaster, and the relative contribution of the poly(A+) mRNA to the sequence complexity of total polysomal RNA, has been determined. Selective removal of poly(A+) mRNA from total polysomal RNA by use of either oligo-dT-cellulose, or poly(U)-sepharose affinity chromatography, revealed that only 0.15% of the mass of the polysomal RNA was present as poly(A+) mRNA. The present study shows that this RNA hybridized at saturation with 3.3% of the single-copy DNA in the Drosophila genome. After correction for asymmetric transcription and reactability of the DNA, 7.4% of the single-copy DNA in the Drosophila genome is represented in larval poly(A+) mRNA. This corresponds to 6.73 x 106 nucleotides of mRNA coding sequences, or approximately 5,384 diverse RNA sequences of average size 1,250 nucleotides. However, total polysomal RNA hybridizes at saturation to 10.9% of the single-copy DNA sequences. After correcting this value for asymmetric transcription and tracer DNA reactability, 24% of the single-copy DNA in Drosophila is represented in total polysomal RNA. This corresponds to 2.18 x 107 nucleotides of RNA coding sequences or 17,440 diverse RNA molecules of size 1,250 nucleotides. This value is 3.2 times greater than that observed for poly(A+) mRNA, and indicates that ≃69% of the polysomal RNA sequence complexity is contributed by nonadenylated RNA. Furthermore, if the number of different structural genes represented in total polysomal RNA is ≃1.7 x 104, then the number of genes expressed in third-instar larvae exceeds the number of chromomeres in Drosophila by about a factor of three. This numerology indicates that the number of chromomeres observed in polytene chromosomes does not reflect the number of structural gene sequences in the Drosophila genome. PMID:6777246

Lower FOXO3 mRNA expression in granulosa cells is involved in unexplained infertility.

Science.gov (United States)

Yamamoto, Hikaru; Yamashita, Yoshiki; Saito, Natsuho; Hayashi, Atsushi; Hayashi, Masami; Terai, Yoshito; Ohmichi, Masahide

2017-06-01

The aim of this study was to investigate whether FOXO1 and FOXO3 mRNA expression in granulosa cells is the cause of unexplained infertility. Thirty-one patients aged infertility and 18 with male partner infertility as a control group) whose serum anti-Müllerian hormone level was >0.5 ng/μL were enrolled in the study. All patients underwent oocyte retrieval under a short protocol from June 2012 to October 2013. Real-time PCR was carried out using mRNA extracted from granulosa cells retrieved from mature follicles. We compared FOXO1 and FOXO3 mRNA expression ratios in granulosa cells between the unexplained infertility group and the male infertility group. The relation between FOXO1 and FOXO3 mRNA expression ratios in granulosa cells and assisted reproduction technology clinical outcome was also examined. FOXO3 mRNA expression ratio was significantly lower in the unexplained infertility group than in the male infertility group. Moreover, FOXO3 mRNA expression ratio showed a positive correlation with both the number of retrieved oocytes and serum anti-Müllerian hormone level. A positive correlation was also identified between FOXO1 mRNA expression and total dose of hMG. As well, the number of retrieved oocytes in the unexplained infertility group was statistically lower than that in the male infertility group. A lower FOXO3 mRNA expression in granulosa cells leads to poor oocyte development in patients with unexplained infertility undergoing controlled ovarian stimulation for in vitro fertilization-embryo transfer. © 2017 Japan Society of Obstetrics and Gynecology.

Adipose tissue interleukin-18 mRNA and plasma interleukin-18: effect of obesity and exercise

DEFF Research Database (Denmark)

Leick, Lotte; Lindegaard, Birgitte; Stensvold, Dorthe

2007-01-01

resistance was tested. Furthermore, we speculated that acute exercise and exercise training would regulate AT IL-18 mRNA expression. RESEARCH METHODS AND PROCEDURES: Non-obese subjects with BMI women: n = 18; men; n = 11) and obese subjects with BMI >30 kg/m(2) (women: n = 6; men: n = 7...... of regular physical activity with improved insulin sensitivity.......OBJECTIVES: Obesity and a physically inactive lifestyle are associated with increased risk of developing insulin resistance. The hypothesis that obesity is associated with increased adipose tissue (AT) interleukin (IL)-18 mRNA expression and that AT IL-18 mRNA expression is related to insulin...

PAX5α and PAX5β mRNA expression in breast Cancer: Relation to ...

African Journals Online (AJOL)

Background: Many studies evaluated the role of paired box gene 5 (PAX5) in breast cancer. However, few investigated PAX5α and PAX5β isoforms individually. Objective: The aim of the present study is to evaluate mRNA expression of PAX5α and PAX5β in breast cancer and assessing their underlying pathological roles ...

Terahertz identification and quantification of penicillamine enantiomers

International Nuclear Information System (INIS)

Ji Te; Zhao Hongwei; Chen Min; Xiao Tiqiao; Han Pengyu

2013-01-01

Identification and characterization of L-, D- and DL- penicillamine were demonstrated by Terahertz time-domain spectroscopy (THz-TDS). To understand the physical origins of the low frequency resonant modes, the density functional theory (DFT) was adopted for theoretical calculation. It was found that the collective THz frequency motions were decided by the intramolecular and intermolecular hydrogen bond interactions. Moreover, the quantification of penicillamine enantiomers mixture was demonstrated by a THz spectra fitting method with a relative error of less than 3.5%. This technique can be a valuable tool for the discrimination and quantification of chiral drugs in pharmaceutical industry. (authors)

Impact of exogenous lipase supplementation on growth, intestinal function, mucosal immune and physical barrier, and related signaling molecules mRNA expression of young grass carp (Ctenopharyngodon idella).

Science.gov (United States)

Liu, Sen; Feng, Lin; Jiang, Wei-Dan; Liu, Yang; Jiang, Jun; Wu, Pei; Zeng, Yun-Yun; Xu, Shu-De; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu

2016-08-01

This study investigated the effects of exogenous lipase supplementation on the growth performance, intestinal growth and function, immune response and physical barrier function, and related signaling molecules mRNA expression of young grass carp (Ctenopharyngodon idella). A total of 450 grass carp (255.02 ± 0.34 g) were fed five diets for 60 days. There were 5 dietary treatments that included a normal protein and lipid diet containing 30% crude protein (CP) with 5% ether extract (EE), and the low-protein and high-lipid diets (28% CP, 6% EE) supplemented with graded levels of exogenous lipase supplementation activity at 0, 1193, 2560 and 3730 U/kg diet. The results indicated that compared with a normal protein and lipid diet (30% CP, 5% EE), a low-protein and high-lipid diet (28% CP, 6% EE) (un-supplemented lipase) improved lysozyme activities and complement component 3 contents in the distal intestine (DI), interleukin 10 mRNA expression in the proximal intestine (PI), and glutathione S-transferases activity and glutathione content in the intestine of young grass carp. In addition, in low-protein and high-lipid diets, optimal exogenous lipase supplementation significantly increased acid phosphatase (ACP) activities and complement component 3 (C3) contents (P exogenous lipase supplementation significantly decreased reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl (PC) contents (P exogenous lipase supplementation significantly elevated the mRNA levels of tight junction proteins (Occludin, zonula occludens 1, Claudin b, Claudin c and Claudin 3) (P exogenous lipase supplementation improved growth, intestinal growth and function, intestinal immunity, physical barrier, and regulated the mRNA expression of related signal molecules of fish. The optimal level of exogenous lipase supplementation in young grass carp (255-771 g) was estimated to be 1193 U kg(-1) diet. Copyright © 2016. Published by Elsevier Ltd.

T-lymphocyte cytokine mRNA expression in cystic echinococcosis.

Science.gov (United States)

Fauser, S; Kern, P

1997-04-01

In the present study we investigated cytokine mRNA expression by peripheral blood mononuclear cells (PBMC) from patients with cystic echinococcosis (CE) after stimulation with different antigens. By using reverse transcriptase polymerase chain reaction (RT-PCR) we could demonstrate that restimulation with crude Echinococcus granulosus antigen (Eg-Ag) induced or enhanced Th2 cytokine mRNA expression, especially IL-5 (by using antigen from sheep cyst fluid) in 23 out of 26 investigated CE patients and IL-10 (by using antigen from camel cyst fluid) in 10 out of 10 investigated CE patients. In contrast, IL-5 mRNA expression was absent in PBMC of healthy controls after Eg-Ag stimulation. To determine the specificity of this reaction we stimulated PBMC from 11 CE patients with crude Echinococcus multilocularis antigen (Em-Ag) and PBMC from 8 CE patients with Toxocara canis antigen (Tc-Ag). We found that the PBMC of patients showed a similar mRNA cytokine pattern on stimulation with Em-Ag when compared with Eg-Ag stimulation. The cytokine mRNA pattern on stimulation with Tc-Ag, however, resembled the cytokine mRNA pattern of unstimulated PBMC. Furthermore, the stimulation of PBMC with crude Mycobacterium tuberculosis antigen (H37Ra) and purified protein derivative (PPD) of M. tuberculosis revealed distinct IL-5 mRNA expression in all investigated CE patients, whereas in healthy controls IL-5 mRNA expression was very weak or totally absent. Thus, our results indicate an induction of Th2 cytokine mRNA expression in CE patients, which is frequently observed in parasite infections. Interestingly, this response persists after stimulation with tuberculosis antigens, which normally induce Th1 response.

Expression of calmodulin mRNA in rat olfactory neuroepithelium.

Science.gov (United States)

Biffo, S; Goren, T; Khew-Goodall, Y S; Miara, J; Margolis, F L

1991-04-01

A calmodulin (CaM) cDNA was isolated by differential hybridization screening of a lambda gt10 library prepared from rat olfactory mucosa. This cDNA fragment, containing most of the open reading frame of the rat CaMI gene, was subcloned and used to characterize steady-state expression of CaM mRNA in rat olfactory neuroepithelium and bulb. Within the bulb mitral cells are the primary neuronal population expressing CaM mRNA. The major CaM mRNA expressed in the olfactory mucosa is 1.7 kb with smaller contributions from mRNAs of 4.0 and 1.4 kb. CaM mRNA was primarily associated with the olfactory neurons and, despite the cellular complexity of the tissue and the known involvement of CaM in diverse cellular processes, was only minimally evident in sustentacular cells, gland cells or respiratory epithelium. Following bulbectomy CaM mRNA declines in the olfactory neuroepithelium as does olfactory marker protein (OMP) mRNA. In contrast to the latter, CaM mRNA makes a partial recovery by one month after surgery. These results, coupled with those from in situ hybridization, indicate that CaM mRNA is expressed in both mature and immature olfactory neurons. The program regulating CaM gene expression in olfactory neurons is distinct from those controlling expression of B50/GAP43 in immature, or OMP in mature, neurons respectively.

Complement mRNA in the mammalian brain: responses to Alzheimer's disease and experimental brain lesioning.

Science.gov (United States)

Johnson, S A; Lampert-Etchells, M; Pasinetti, G M; Rozovsky, I; Finch, C E

1992-01-01

This study describes evidence in the adult human and rat brain for mRNAs that encode two complement (C) proteins, C1qB and C4. C proteins are important effectors of humoral immunity and inflammation in peripheral tissues but have not been considered as normally present in brain. Previous immunocytochemical studies showed that C proteins are associated with plaques, tangles, and dystrophic neurites in Alzheimer's disease (AD), but their source is unknown. Combined immunocytochemistry and in situ hybridization techniques show C4 mRNA in pyramidal neurons and C1qB mRNA in microglia. Primary rat neuron cultures also show C1qB mRNA. In the cortex from AD brains, there were two- to threefold increases of C1qB mRNA and C4 mRNA, and increased C1qB mRNA prevalence was in part associated with microglia. As a model for AD, we examined entorhinal cortex perforant path transection in the rat brain, which caused rapid increases of C1qB mRNA in the ipsilateral, but not contralateral, hippocampus and entorhinal cortex. The role of brain-derived acute and chronic C induction during AD and experimental lesions can now be considered in relation to functions of C proteins that pertain to cell degeneration and/or cell preservation and synaptic plasticity.

Swift Quantification of Fenofibrate and Tiemonium methylsulfate Active Ingredients in Solid Drugs Using Particle Induced X-Ray Emission

International Nuclear Information System (INIS)

Bejjani, A.; Nsouli, B.; Zahraman, K.; Assi, S.; Younes, Gh.; Yazbi, F.

2011-01-01

The quantification of active ingredients (AI) in drugs is a crucial and important step in the drug quality control process. This is usually performed by using wet chemical techniques like LC-MS, UV spectrophotometry and other appropriate organic analytical methods. However, if the active ingredient contains specific heteroatoms (F, S, Cl), elemental IBA like PIXE and PIGE techniques, using small tandem accelerator of 1-2 MV, can be explored for molecular quantification. IBA techniques permit the analysis of the sample under solid form, without any laborious sample preparations. In this work, we demonstrate the ability of the Thick Target PIXE technique for rapid and accurate quantification of both low and high concentrations of active ingredients in different commercial drugs. Fenofibrate, a chlorinated active ingredient, is present in high amounts in two different commercial drugs, its quantification was done using the relative approach to an external standard. On the other hand, Tiemonium methylsulfate which exists in relatively low amount in commercial drugs, its quantification was done using GUPIX simulation code (absolute quantification). The experimental aspects related to the quantification validity (use of external standards, absolute quantification, matrix effect,...) are presented and discussed. (author)

Neurocognitive and neuroinflammatory correlates of PDYN and OPRK1 mRNA expression in the anterior cingulate in postmortem brain of HIV-infected subjects.

Science.gov (United States)

Yuferov, Vadim; Butelman, Eduardo R; Ho, Ann; Morgello, Susan; Kreek, Mary Jeanne

2014-01-09

Chronic inflammation may contribute to neuropsychological impairments in individuals with HIV, and modulation of this inflammatory response by opiate receptor ligands is important in light of the prevalence of drug use in HIV populations. Exogenous MOR and KOR agonists have differential effects on central nervous system (CNS) immunity and, while some data suggest KOR agonists are immunosuppressive, the KOR agonist dynorphin has been shown to stimulate human monocyte chemotaxis. In this study, we examined mRNA levels of endogenous opioid receptors OPRK1 and OPRM1, prodynorphin (PDYN), macrophage scavenger receptor CD163, and microglia/macrophage marker CD68 in the caudate and anterior cingulate of postmortem brains from HIV-positive and HIV-negative subjects. Brain tissues of HIV-infected (n = 24) and control subjects (n = 15) were obtained from the Manhattan HIV Brain Bank. Quantification of the gene mRNA was performed using SYBR Green RT-PCR. CD68 and CD163 were increased in HIV-positive (HIV+) compared to HIV-negative (HIV-) individuals in both brain regions. There were higher OPRK1 (P <0.005), and lower PDYN mRNA (P <0.005) levels in the anterior cingulate of HIV+ compared to HIV- subjects. This difference between the clinical groups was not found in the caudate. There was no difference in the levels of OPRM1 mRNA between HIV+ and HIV- subjects. Using linear regression analysis, we examined the relationship of OPRK1 and PDYN mRNA levels in the HIV+ subjects with seven cognitive domain T scores of a neuropsychological test battery. Within the HIV+ subjects, there was a positive correlation between anterior cingulate PDYN mRNA levels and better T-scores in the motor domain. Within the HIV+ subjects there were also positive correlations of both OPRK1 and PDYN mRNA levels with the anti-inflammatory marker CD163, but not with proinflammatory CD68 levels. In this setting, decreased PDYN mRNA may reflect a homeostatic mechanism to reduce monocyte

Cup regulates oskar mRNA stability during oogenesis.

Science.gov (United States)

Broyer, Risa M; Monfort, Elena; Wilhelm, James E

2017-01-01

The proper regulation of the localization, translation, and stability of maternally deposited transcripts is essential for embryonic development in many organisms. These different forms of regulation are mediated by the various protein subunits of the ribonucleoprotein (RNP) complexes that assemble on maternal mRNAs. However, while many of the subunits that regulate the localization and translation of maternal transcripts have been identified, relatively little is known about how maternal mRNAs are stockpiled and stored in a stable form to support early development. One of the best characterized regulators of maternal transcripts is Cup - a broadly conserved component of the maternal RNP complex that in Drosophila acts as a translational repressor of the localized message oskar. In this study, we have found that loss of cup disrupts the localization of both the oskar mRNA and its associated proteins to the posterior pole of the developing oocyte. This defect is not due to a failure to specify the oocyte or to disruption of RNP transport. Rather, the localization defects are due to a drop in oskar mRNA levels in cup mutant egg chambers. Thus, in addition to its role in regulating oskar mRNA translation, Cup also plays a critical role in controlling the stability of the oskar transcript. This suggests that Cup is ideally positioned to coordinate the translational control function of the maternal RNP complex with its role in storing maternal transcripts in a stable form. Published by Elsevier Inc.

The mRNA expression profile of metabolic genes relative to MHC isoform pattern in human skeletal muscles

DEFF Research Database (Denmark)

Plomgaard, Peter; Penkowa, Milena; Leick, Lotte

2006-01-01

The metabolic profile of rodent muscle is generally reflected in the myosin heavy chain (MHC) fiber-type composition. The present study was conducted to test the hypothesis that metabolic gene expression is not tightly coupled with MHC fiber-type composition for all genes in human skeletal muscle....... Triceps brachii, vastus lateralis quadriceps, and soleus muscle biopsies were obtained from normally physically active, healthy, young male volunteers, because these muscles are characterized by different fiber-type compositions. As expected, citrate synthase and 3-hydroxyacyl dehydrogenase activity...... of a broad range of metabolic genes. The triceps muscle had two- to fivefold higher MHC IIa, phosphofructokinase, and LDH A mRNA content and two- to fourfold lower MHC I, lipoprotein lipase, CD36, hormone-sensitive lipase, and LDH B and hexokinase II mRNA than vastus lateralis or soleus. Interestingly...

Identification of stress responsive genes by studying specific relationships between mRNA and protein abundance.

Science.gov (United States)

Morimoto, Shimpei; Yahara, Koji

2018-03-01

Protein expression is regulated by the production and degradation of mRNAs and proteins but the specifics of their relationship are controversial. Although technological advances have enabled genome-wide and time-series surveys of mRNA and protein abundance, recent studies have shown paradoxical results, with most statistical analyses being limited to linear correlation, or analysis of variance applied separately to mRNA and protein datasets. Here, using recently analyzed genome-wide time-series data, we have developed a statistical analysis framework for identifying which types of genes or biological gene groups have significant correlation between mRNA and protein abundance after accounting for potential time delays. Our framework stratifies all genes in terms of the extent of time delay, conducts gene clustering in each stratum, and performs a non-parametric statistical test of the correlation between mRNA and protein abundance in a gene cluster. Consequently, we revealed stronger correlations than previously reported between mRNA and protein abundance in two metabolic pathways. Moreover, we identified a pair of stress responsive genes ( ADC17 and KIN1 ) that showed a highly similar time series of mRNA and protein abundance. Furthermore, we confirmed robustness of the analysis framework by applying it to another genome-wide time-series data and identifying a cytoskeleton-related gene cluster (keratin 18, keratin 17, and mitotic spindle positioning) that shows similar correlation. The significant correlation and highly similar changes of mRNA and protein abundance suggests a concerted role of these genes in cellular stress response, which we consider provides an answer to the question of the specific relationships between mRNA and protein in a cell. In addition, our framework for studying the relationship between mRNAs and proteins in a cell will provide a basis for studying specific relationships between mRNA and protein abundance after accounting for potential

Tau mRNA 3'UTR-to-CDS ratio is increased in Alzheimer disease.

Science.gov (United States)

García-Escudero, Vega; Gargini, Ricardo; Martín-Maestro, Patricia; García, Esther; García-Escudero, Ramón; Avila, Jesús

2017-08-10

Neurons frequently show an imbalance in expression of the 3' untranslated region (3'UTR) relative to the coding DNA sequence (CDS) region of mature messenger RNAs (mRNA). The ratio varies among different cells or parts of the brain. The Map2 protein levels per cell depend on the 3'UTR-to-CDS ratio rather than the total mRNA amount, which suggests powerful regulation of protein expression by 3'UTR sequences. Here we found that MAPT (the microtubule-associated protein tau gene) 3'UTR levels are particularly high with respect to other genes; indeed, the 3'UTR-to-CDS ratio of MAPT is balanced in healthy brain in mouse and human. The tau protein accumulates in Alzheimer diseased brain. We nonetheless observed that the levels of RNA encoding MAPT/tau were diminished in these patients' brains. To explain this apparently contradictory result, we studied MAPT mRNA stoichiometry in coding and non-coding regions, and found that the 3'UTR-to-CDS ratio was higher in the hippocampus of Alzheimer disease patients, with higher tau protein but lower total mRNA levels. Our data indicate that changes in the 3'UTR-to-CDS ratio have a regulatory role in the disease. Future research should thus consider not only mRNA levels, but also the ratios between coding and non-coding regions. Copyright © 2017 Elsevier B.V. All rights reserved.

Collagen mRNA levels changes during colorectal cancer carcinogenesis

DEFF Research Database (Denmark)

Skovbjerg, Hanne; Anthonsen, Dorit; Lothe, Inger M B

2009-01-01

BACKGROUND: Invasive growth of epithelial cancers is a complex multi-step process which involves dissolution of the basement membrane. Type IV collagen is a major component in most basement membranes. Type VII collagen is related to anchoring fibrils and is found primarily in the basement membrane...... zone of stratified epithelia. Immunohistochemical studies have previously reported changes in steady-state levels of different alpha(IV) chains in several epithelial cancer types. In the present study we aimed to quantitatively determine the mRNA levels of type IV collagen (alpha1/alpha 4/alpha 6......) and type VII collagen (alpha1) during colorectal cancer carcinogenesis. METHODS: Using quantitative RT-PCR, we have determined the mRNA levels for alpha1(IV), alpha 4(IV), alpha 6(IV), and alpha1(VII) in colorectal cancer tissue (n = 33), adenomas (n = 29) and in normal tissue from the same individuals...

Quantification of rice bran oil in oil blends

Energy Technology Data Exchange (ETDEWEB)

Mishra, R.; Sharma, H. K.; Sengar, G.

2012-11-01

Blends consisting of physically refined rice bran oil (PRBO): sunflower oil (SnF) and PRBO: safflower oil (SAF) in different proportions were analyzed for various physicochemical parameters. The quantification of pure rice bran oil in the blended oils was carried out using different methods including gas chromatographic, HPLC, ultrasonic velocity and methods based on physico-chemical parameters. The physicochemical parameters such as ultrasonic velocity, relative association and acoustic impedance at 2 MHz, iodine value, palmitic acid content and oryzanol content reflected significant changes with increased proportions of PRBO in the blended oils. These parameters were selected as dependent parameters and % PRBO proportion was selected as independent parameters. The study revealed that regression equations based on the oryzanol content, palmitic acid composition, ultrasonic velocity, relative association, acoustic impedance, and iodine value can be used for the quantification of rice bran oil in blended oils. The rice bran oil can easily be quantified in the blended oils based on the oryzanol content by HPLC even at a 1% level. The palmitic acid content in blended oils can also be used as an indicator to quantify rice bran oil at or above the 20% level in blended oils whereas the method based on ultrasonic velocity, acoustic impedance and relative association showed initial promise in the quantification of rice bran oil. (Author) 23 refs.

DAG expression: high-throughput gene expression analysis of real-time PCR data using standard curves for relative quantification.

Directory of Open Access Journals (Sweden)

María Ballester

Full Text Available BACKGROUND: Real-time quantitative PCR (qPCR is still the gold-standard technique for gene-expression quantification. Recent technological advances of this method allow for the high-throughput gene-expression analysis, without the limitations of sample space and reagent used. However, non-commercial and user-friendly software for the management and analysis of these data is not available. RESULTS: The recently developed commercial microarrays allow for the drawing of standard curves of multiple assays using the same n-fold diluted samples. Data Analysis Gene (DAG Expression software has been developed to perform high-throughput gene-expression data analysis using standard curves for relative quantification and one or multiple reference genes for sample normalization. We discuss the application of DAG Expression in the analysis of data from an experiment performed with Fluidigm technology, in which 48 genes and 115 samples were measured. Furthermore, the quality of our analysis was tested and compared with other available methods. CONCLUSIONS: DAG Expression is a freely available software that permits the automated analysis and visualization of high-throughput qPCR. A detailed manual and a demo-experiment are provided within the DAG Expression software at http://www.dagexpression.com/dage.zip.

15 CFR 990.52 - Injury assessment-quantification.

Science.gov (United States)

2010-01-01

... (Continued) NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE OIL POLLUTION ACT..., trustees must quantify the degree, and spatial and temporal extent of such injuries relative to baseline. (b) Quantification approaches. Trustees may quantify injuries in terms of: (1) The degree, and...

FLT3-ITD and MLL-PTD influence the expression of MDR-1, MRP-1, and BCRP mRNA but not LRP mRNA assessed with RQ-PCR method in adult acute myeloid leukemia.

Science.gov (United States)

Nasilowska-Adamska, Barbara; Solarska, Iwona; Paluszewska, Monika; Malinowska, Iwona; Jedrzejczak, Wieslaw W; Warzocha, Krzysztof

2014-04-01

Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) and mixed-lineage leukemia gene-partial tandem duplication (MLL-PTD) are aberrations associated with leukemia which indicate unsatisfactory prognosis. Downstream regulatory targets of FLT3-ITD and MLL-PTD are not well defined. We have analyzed the expression of MDR-1, multidrug resistant protein-1 (MRP-1), breast cancer resistance protein (BCRP), and lung resistance protein (LRP) messenger RNA (mRNA) in relation to the mutational status of FLT3-ITD and MLL-PTD in 185 acute myeloid leukemia (AML) adult patients. The real-time quantitative polymerase chain reaction method was performed to assess the expression of the MDR-1, MRP-1, BCRP, and LRP mRNA, and the results were presented as coefficients calculated using an intermediate method according to Pfaffl's rule. Significantly higher expressions of MDR-1 mRNA were found in patients who did not harbor FLT3-ITD (0.20 vs. 0.05; p = 0.0001) and MRP-1 mRNA in patients with this mutation (0.96 vs. 0.70; p = 0.002) and of BCRP mRNA in patients with MLL-PTD (0.61 vs. 0.38; p = 0.03). In univariate analysis, the high expression of MDR-1 mRNA (≥0.1317) negatively influenced the outcome of induction therapy (p = 0.05), whereas the high expression of BCRP mRNA (≥1.1487) was associated with a high relapse rate (RR) (p = 0.013). We found that the high expression of MDR-1 (≥0.1317), MRP-1 (≥0.8409), and BCRP mRNA (≥1.1487) significantly influenced disease-free survival (DFS; p = 0.059, 0.032, and 0.009, respectively) and overall survival (0.048, 0.014, and 0.059, respectively). Moreover, a high expression of BCRP mRNA (≥1.1487) proved to be an independent prognostic factor for RR (p = 0.01) and DFS (p = 0.002) in multivariate analysis. The significant correlation between the expression of MDR-1, MRP-1, and BCRP mRNA and FLT3-ITD or MLL-PTD in AML patients requires further investigation.

Clinical Utility Of Blood E2F3 MRNA Assay In The Early Diagnosis Of Prostatic Cancer By Real-Time Polymerase Chain Reaction

International Nuclear Information System (INIS)

Zaki, M.M.; Elzayat, T.M.; Mahmoud, M.A.; El Hadidi, E.S.; Abdel Al Ahmed, H.; Mohamed, N.A.

2013-01-01

Background: Based on the fact that prostate cancer development and progression is the result of the interaction between different molecular mechanisms, many efforts have been devoted to the identification of new circulating genes that could serve as non invasive, reliable early diagnostic and prognostic markers and where their specific functions allow potential therapeutic targets. E2F3 is a member of E2F family of transcription factors involved in cell cycle regulatory functions. It was found that E2F3 is over-expressed in some tumors including bladder and prostate cancer. Aim: The aim of the study was to evaluate the clinical significance of peripheral blood E2F3 mRNA assay in the early diagnosis of patients with localized prostate cancer and to compare its expression in the blood of age-matched prostate cancer, benign prostatic hypertrophy and healthy males. Methods: This study was conducted on 25 patients with cancer prostate, 15 patients with benign prostatic hyperplasia (serving as a pathological control group) in addition to 10 healthy men (serving as a healthy control group). Blood samples were collected and tested for the detection of E2F3 mRNA gene by real time RT-PCR and prostate specific antigen (PSA) by electro chemiluminescence immunoassay. E2F3 mRNA results were reported in relative quantification, where the target and housekeeping gene (GAPDH) were amplified from the same sample in two separate reaction plates. Results were then compared between different samples relying on direct comparison of threshold cycle (CT) values. Finally, the normalized level of target gene expression was calculated by using the formula: 2 δδCT Results: Total PSA at the cutoff 4 ng/mL had a diagnostic sensitivity of 88%, specificity of 84%, positive predictive value of 88%, negative predictive value of 84% and diagnostic efficacy of 86%. E2F3 mRNA was statistically higher in cancer prostate group than in benign prostatic hyperplasia and healthy control groups. At the cut

[Impacts of the formula of Suoquanwan(SQW) on expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency].

Science.gov (United States)

Cao, Hong-Ying; Wu, Qing-He; Huang, Ping; He, Jin-Yang

2009-06-01

To observe the impacts of the formula of Suoquanwan (SQW) on the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency. The model rats were induced by adenine (250 mg/kg) for 4 weeks, then treated respectively with SQW or dDAVP. The expression of AQP-2 mRNA and AVPR-V2 mRNA in kidney of Yang-deficiency model by realtime fluorescence quantitative PCR method were investigated. In model rats, the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney decreased, dDAVP and SQW high dose could increased the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney. The others had no influence on the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney. SQW can increase the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency.

Staphylococcus aureus RNAIII binds to two distant regions of coa mRNA to arrest translation and promote mRNA degradation.

Directory of Open Access Journals (Sweden)

Clément Chevalier

2010-03-01

Full Text Available Staphylococcus aureus RNAIII is the intracellular effector of the quorum sensing system that temporally controls a large number of virulence factors including exoproteins and cell-wall-associated proteins. Staphylocoagulase is one major virulence factor, which promotes clotting of human plasma. Like the major cell surface protein A, the expression of staphylocoagulase is strongly repressed by the quorum sensing system at the post-exponential growth phase. Here we used a combination of approaches in vivo and in vitro to analyze the mechanism used by RNAIII to regulate the expression of staphylocoagulase. Our data show that RNAIII represses the synthesis of the protein through a direct binding with the mRNA. Structure mapping shows that two distant regions of RNAIII interact with coa mRNA and that the mRNA harbors a conserved signature as found in other RNAIII-target mRNAs. The resulting complex is composed of an imperfect duplex masking the Shine-Dalgarno sequence of coa mRNA and of a loop-loop interaction occurring downstream in the coding region. The imperfect duplex is sufficient to prevent the formation of the ribosomal initiation complex and to repress the expression of a reporter gene in vivo. In addition, the double-strand-specific endoribonuclease III cleaves the two regions of the mRNA bound to RNAIII that may contribute to the degradation of the repressed mRNA. This study validates another direct target of RNAIII that plays a role in virulence. It also illustrates the diversity of RNAIII-mRNA topologies and how these multiple RNAIII-mRNA interactions would mediate virulence regulation.

Mass spectrometry–based relative quantification of proteins in precatalytic and catalytically active spliceosomes by metabolic labeling (SILAC), chemical labeling (iTRAQ), and label-free spectral count

Science.gov (United States)

Schmidt, Carla; Grønborg, Mads; Deckert, Jochen; Bessonov, Sergey; Conrad, Thomas; Lührmann, Reinhard; Urlaub, Henning

2014-01-01

The spliceosome undergoes major changes in protein and RNA composition during pre-mRNA splicing. Knowing the proteins—and their respective quantities—at each spliceosomal assembly stage is critical for understanding the molecular mechanisms and regulation of splicing. Here, we applied three independent mass spectrometry (MS)–based approaches for quantification of these proteins: (1) metabolic labeling by SILAC, (2) chemical labeling by iTRAQ, and (3) label-free spectral count for quantification of the protein composition of the human spliceosomal precatalytic B and catalytic C complexes. In total we were able to quantify 157 proteins by at least two of the three approaches. Our quantification shows that only a very small subset of spliceosomal proteins (the U5 and U2 Sm proteins, a subset of U5 snRNP-specific proteins, and the U2 snRNP-specific proteins U2A′ and U2B′′) remains unaltered upon transition from the B to the C complex. The MS-based quantification approaches classify the majority of proteins as dynamically associated specifically with the B or the C complex. In terms of experimental procedure and the methodical aspect of this work, we show that metabolically labeled spliceosomes are functionally active in terms of their assembly and splicing kinetics and can be utilized for quantitative studies. Moreover, we obtain consistent quantification results from all three methods, including the relatively straightforward and inexpensive label-free spectral count technique. PMID:24448447

RNAontheBENCH: computational and empirical resources for benchmarking RNAseq quantification and differential expression methods

KAUST Repository

Germain, Pierre-Luc

2016-06-20

RNA sequencing (RNAseq) has become the method of choice for transcriptome analysis, yet no consensus exists as to the most appropriate pipeline for its analysis, with current benchmarks suffering important limitations. Here, we address these challenges through a rich benchmarking resource harnessing (i) two RNAseq datasets including ERCC ExFold spike-ins; (ii) Nanostring measurements of a panel of 150 genes on the same samples; (iii) a set of internal, genetically-determined controls; (iv) a reanalysis of the SEQC dataset; and (v) a focus on relative quantification (i.e. across-samples). We use this resource to compare different approaches to each step of RNAseq analysis, from alignment to differential expression testing. We show that methods providing the best absolute quantification do not necessarily provide good relative quantification across samples, that count-based methods are superior for gene-level relative quantification, and that the new generation of pseudo-alignment-based software performs as well as established methods, at a fraction of the computing time. We also assess the impact of library type and size on quantification and differential expression analysis. Finally, we have created a R package and a web platform to enable the simple and streamlined application of this resource to the benchmarking of future methods.

RNAontheBENCH: computational and empirical resources for benchmarking RNAseq quantification and differential expression methods

KAUST Repository

Germain, Pierre-Luc; Vitriolo, Alessandro; Adamo, Antonio; Laise, Pasquale; Das, Vivek; Testa, Giuseppe

2016-01-01

RNA sequencing (RNAseq) has become the method of choice for transcriptome analysis, yet no consensus exists as to the most appropriate pipeline for its analysis, with current benchmarks suffering important limitations. Here, we address these challenges through a rich benchmarking resource harnessing (i) two RNAseq datasets including ERCC ExFold spike-ins; (ii) Nanostring measurements of a panel of 150 genes on the same samples; (iii) a set of internal, genetically-determined controls; (iv) a reanalysis of the SEQC dataset; and (v) a focus on relative quantification (i.e. across-samples). We use this resource to compare different approaches to each step of RNAseq analysis, from alignment to differential expression testing. We show that methods providing the best absolute quantification do not necessarily provide good relative quantification across samples, that count-based methods are superior for gene-level relative quantification, and that the new generation of pseudo-alignment-based software performs as well as established methods, at a fraction of the computing time. We also assess the impact of library type and size on quantification and differential expression analysis. Finally, we have created a R package and a web platform to enable the simple and streamlined application of this resource to the benchmarking of future methods.

The effect of tissue decalcification on mRNA retention within bone for in-situ hybridization studies.

Science.gov (United States)

Walsh, L; Freemont, A J; Hoyland, J A

1993-06-01

Tissue decalcification is a routine part of the preparation of bone tissue for histological studies. Although in-situ hybridization has been employed to localize mRNA of collagenous and non-collagenous bone related proteins in skeletal tissue, little is known regarding the effects of decalcifying agents on mRNA retention within tissue. In this study in-situ hybridization using an oligonucleotide probe (i.e. a poly d(T) probe) to detect total messenger RNA has been employed to investigate the effects of the decalcifying agents nitric acid, formic acid and EDTA on mRNA retention compared to undeacalcified tissue. The results show that formalin fixation and EDTA decalcification preserve substantial amounts of mRNA within the tissue. In particular, this study illustrates that it is possible to perform in-situ hybridization on formalin fixed decalcified paraffin embedded tissue.

PAX5О± and PAX5ОІ mRNA expression in breast Cancer: Relation ...

African Journals Online (AJOL)

Manal Basyouni Ahmed

mRNA expression of PAX5a and PAX5b in breast cancer and assessing their underlying pathological roles through ... the molecular alterations that contribute to disease initiation and ... ring growth and survival of cancer cells [3]. PAX5 is ..... and CA15-3 are prognostic parameters for different molecular subtypes of · breast ...

Effects of branched-chain volatile fatty acids on lactation performance and mRNA expression of genes related to fatty acid synthesis in mammary gland of dairy cows.

Science.gov (United States)

Liu, Q; Wang, C; Guo, G; Huo, W J; Zhang, S L; Pei, C X; Zhang, Y L; Wang, H

2018-02-12

Branched-chain volatile fatty acids (BCVFA) supplements could promote lactation performance and milk quality by improving ruminal fermentation and milk fatty acid synthesis. This study was conducted to evaluate the effects of BCVFA supplementation on milk performance, ruminal fermentation, nutrient digestibility and mRNA expression of genes related to fatty acid synthesis in mammary gland of dairy cows. A total of 36 multiparous Chinese Holstein cows averaging 606±4.7 kg of BW, 65±5.2 day in milk (DIM) with daily milk production of 30.6±0.72 kg were assigned to one of four groups blocked by lactation number, milk yield and DIM. The treatments were control, low-BCVFA (LBCVFA), medium-BCVFA (MBCVFA) and high-BCVFA (HBCVFA) with 0, 30, 60 and 90 g BCVFA per cow per day, respectively. Experimental periods were 105 days with 15 days of adaptation and 90 days of data collection. Dry matter (DM) intake tended to increase, but BW changes were similar among treatments. Yields of actual milk, 4% fat corrected milk, milk fat and true protein linearly increased, but feed conversion ratio (FCR) linearly decreased with increasing BCVFA supplementation. Milk fat content linearly increased, but true protein content tended to increase. Contents of C4:0, C6:0, C8:0, C10:0, C12:0, C14:0 and C15:0 fatty acids in milk fat linearly increased, whereas other fatty acids were not affected with increasing BCVFA supplementation. Ruminal pH, ammonia N concentration and propionate molar proportion linearly decreased, but total VFA production and molar proportions of acetate and butyrate linearly increased with increasing BCVFA supplementation. Consequently, acetate to propionate ratios linearly increased. Digestibilities of DM, organic matter, CP, NDF and ADF also linearly increased. In addition, mRNA expressions of peroxisome proliferator-activated receptor γ, sterol regulatory element-binding factor 1 and fatty acid-binding protein 3 linearly increased, mRNA expressions of acetyl

Pressure overload stimulated cardiac hypertrophy leads to a rapid decrease in the mRNA for creatine kinase

International Nuclear Information System (INIS)

Boheler, K.; Popovich, B.; Dillmann, W.H.

1987-01-01

Cardiac hypertrophy (CH) leads to a decrease in creatine kinase (CK) enzymatic activity. To determine if the mRNA for CK also decreases with CH, they performed the following studies. Cardiac RNA was isolated from rats subjected to either abdominal aortic stenosis (AS) or sham surgery. Through Northern blot analysis, total cardiac RNA was quantitated with a CK specific 32 P-labelled cDNA clone. At 3 and 8 days post-constriction, the mRNA for CK decreases by 54.6 +/- 7% and 65.3 +/- 18% respectively, whereas the heart weight increases by 19% and 37% relative to controls. Further studies indicate that CK mRNA also decreases by 41.8% in hypothyroid rats (Tx) but decreases by a total of 68.1% in Tx rats subjected to 8 days of AS. Pressure overload stimulated CH leads to a rapid decrease in CK mRNA in normal and Tx rats. This CK mRNA decrease may account for the decreased efficiency of contraction seen in CH

Visfatin mRNA expression in human subcutaneous adipose tissue is regulated by exercise

DEFF Research Database (Denmark)

Frydelund-Larsen, Lone; Åkerström, Thorbjörn; Nielsen, Søren

2006-01-01

in abdominal subcutaneous adipose tissue and skeletal muscle biopsies obtained from healthy young men at time points 0, 3, 4.5, 6, 9, and 24 h in relation to either 3 h of ergometer cycle exercise at 60% of Vo(2 max) or rest. Adipose tissue visfatin mRNA expression increased threefold at the time points 3, 4......Visfatin [pre-beta-cell colony-enhancing factor (PBEF)] is a novel adipokine that is produced by adipose tissue, skeletal muscle, and liver and has insulin-mimetic actions. Regular exercise enhances insulin sensitivity. In the present study, we therefore examined visfatin mRNA expression.......5, and 6 h in response to exercise (n = 8) compared with preexercise samples and compared with the resting control group (n = 7, P = 0.001). Visfatin mRNA expression in skeletal muscle was not influenced by exercise. The exercise-induced increase in adipose tissue visfatin was, however, not accompanied...

Quantification in emission tomography

International Nuclear Information System (INIS)

Buvat, Irene

2011-11-01

The objective of this lecture is to understand the possibilities and limitations of the quantitative analysis of single photon emission computed tomography (SPECT) and positron emission tomography (PET) images. It is also to identify the conditions to be fulfilled to obtain reliable quantitative measurements from images. Content: 1 - Introduction: Quantification in emission tomography - definition and challenges; quantification biasing phenomena 2 - Main problems impacting quantification in PET and SPECT: problems, consequences, correction methods, results (Attenuation, scattering, partial volume effect, movement, un-stationary spatial resolution in SPECT, fortuitous coincidences in PET, standardisation in PET); 3 - Synthesis: accessible efficiency, know-how, Precautions, beyond the activity measurement

Accident sequence quantification with KIRAP

Energy Technology Data Exchange (ETDEWEB)

Kim, Tae Un; Han, Sang Hoon; Kim, Kil You; Yang, Jun Eon; Jeong, Won Dae; Chang, Seung Cheol; Sung, Tae Yong; Kang, Dae Il; Park, Jin Hee; Lee, Yoon Hwan; Hwang, Mi Jeong

1997-01-01

The tasks of probabilistic safety assessment(PSA) consists of the identification of initiating events, the construction of event tree for each initiating event, construction of fault trees for event tree logics, the analysis of reliability data and finally the accident sequence quantification. In the PSA, the accident sequence quantification is to calculate the core damage frequency, importance analysis and uncertainty analysis. Accident sequence quantification requires to understand the whole model of the PSA because it has to combine all event tree and fault tree models, and requires the excellent computer code because it takes long computation time. Advanced Research Group of Korea Atomic Energy Research Institute(KAERI) has developed PSA workstation KIRAP(Korea Integrated Reliability Analysis Code Package) for the PSA work. This report describes the procedures to perform accident sequence quantification, the method to use KIRAP`s cut set generator, and method to perform the accident sequence quantification with KIRAP. (author). 6 refs.

Accident sequence quantification with KIRAP

International Nuclear Information System (INIS)

Kim, Tae Un; Han, Sang Hoon; Kim, Kil You; Yang, Jun Eon; Jeong, Won Dae; Chang, Seung Cheol; Sung, Tae Yong; Kang, Dae Il; Park, Jin Hee; Lee, Yoon Hwan; Hwang, Mi Jeong.

1997-01-01

The tasks of probabilistic safety assessment(PSA) consists of the identification of initiating events, the construction of event tree for each initiating event, construction of fault trees for event tree logics, the analysis of reliability data and finally the accident sequence quantification. In the PSA, the accident sequence quantification is to calculate the core damage frequency, importance analysis and uncertainty analysis. Accident sequence quantification requires to understand the whole model of the PSA because it has to combine all event tree and fault tree models, and requires the excellent computer code because it takes long computation time. Advanced Research Group of Korea Atomic Energy Research Institute(KAERI) has developed PSA workstation KIRAP(Korea Integrated Reliability Analysis Code Package) for the PSA work. This report describes the procedures to perform accident sequence quantification, the method to use KIRAP's cut set generator, and method to perform the accident sequence quantification with KIRAP. (author). 6 refs

Exercise training and work task induced metabolic and stress-related mRNA and protein responses in myalgic muscles

DEFF Research Database (Denmark)

Sjøgaard, Gisela; Zebis, Mette Kreutzfeldt; Kiilerich, Kristian

2013-01-01

healthy controls. Those with myalgia performed similar to 7 hrs repetitive stressful work and were subsequently randomized to 10 weeks of specific strength training, general fitness training, or reference intervention. Muscles biopsies were taken from the trapezius muscle at baseline, after work and after...... 10 weeks intervention. The main findings are that the capacity of carbohydrate oxidation was reduced in myalgic compared with healthy muscle. Repetitive stressful work increased mRNA content for heat shock proteins and decreased levels of key regulators for growth and oxidative metabolism......The aim was to assess mRNA and/or protein levels of heat shock proteins, cytokines, growth regulating, and metabolic proteins in myalgic muscle at rest and in response to work tasks and prolonged exercise training. A randomized controlled trial included 28 females with trapezius myalgia and 16...

Analysis of alpha-synuclein in malignant melanoma - development of a SRM quantification assay.

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Charlotte Welinder

Full Text Available Globally, malignant melanoma shows a steady increase in the incidence among cancer diseases. Malignant melanoma represents a cancer type where currently no biomarker or diagnostics is available to identify disease stage, progression of disease or personalized medicine treatment. The aim of this study was to assess the tissue expression of alpha-synuclein, a protein implicated in several disease processes, in metastatic tissues from malignant melanoma patients. A targeted Selected Reaction Monitoring (SRM assay was developed and utilized together with stable isotope labeling for the relative quantification of two target peptides of alpha-synuclein. Analysis of alpha-synuclein protein was then performed in ten metastatic tissue samples from the Lund Melanoma Biobank. The calibration curve using peak area ratio (heavy/light versus concentration ratios showed linear regression over three orders of magnitude, for both of the selected target peptide sequences. In support of the measurements of specific protein expression levels, we also observed significant correlation between the protein and mRNA levels of alpha-synuclein in these tissues. Investigating levels of tissue alpha-synuclein may add novel aspect to biomarker development in melanoma, help to understand disease mechanisms and ultimately contribute to discriminate melanoma patients with different prognosis.

Genomic DNA-based absolute quantification of gene expression in Vitis.

Science.gov (United States)

Gambetta, Gregory A; McElrone, Andrew J; Matthews, Mark A

2013-07-01

Many studies in which gene expression is quantified by polymerase chain reaction represent the expression of a gene of interest (GOI) relative to that of a reference gene (RG). Relative expression is founded on the assumptions that RG expression is stable across samples, treatments, organs, etc., and that reaction efficiencies of the GOI and RG are equal; assumptions which are often faulty. The true variability in RG expression and actual reaction efficiencies are seldom determined experimentally. Here we present a rapid and robust method for absolute quantification of expression in Vitis where varying concentrations of genomic DNA were used to construct GOI standard curves. This methodology was utilized to absolutely quantify and determine the variability of the previously validated RG ubiquitin (VvUbi) across three test studies in three different tissues (roots, leaves and berries). In addition, in each study a GOI was absolutely quantified. Data sets resulting from relative and absolute methods of quantification were compared and the differences were striking. VvUbi expression was significantly different in magnitude between test studies and variable among individual samples. Absolute quantification consistently reduced the coefficients of variation of the GOIs by more than half, often resulting in differences in statistical significance and in some cases even changing the fundamental nature of the result. Utilizing genomic DNA-based absolute quantification is fast and efficient. Through eliminating error introduced by assuming RG stability and equal reaction efficiencies between the RG and GOI this methodology produces less variation, increased accuracy and greater statistical power. © 2012 Scandinavian Plant Physiology Society.

The RDE-10/RDE-11 complex triggers RNAi-induced mRNA degradation by association with target mRNA in C. elegans.

Science.gov (United States)

Yang, Huan; Zhang, Ying; Vallandingham, Jim; Li, Hua; Li, Hau; Florens, Laurence; Mak, Ho Yi

2012-04-15

The molecular mechanisms for target mRNA degradation in Caenorhabditis elegans undergoing RNAi are not fully understood. Using a combination of genetic, proteomic, and biochemical approaches, we report a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. Genetic analysis indicates that the RDE-10/RDE-11 complex acts in parallel to nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary siRNA. Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Deep sequencing reveals a fivefold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, while primary siRNA and microRNA biogenesis is normal. Therefore, the RDE-10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans.

Identification of stress responsive genes by studying specific relationships between mRNA and protein abundance

Directory of Open Access Journals (Sweden)

Shimpei Morimoto

2018-03-01

Full Text Available Protein expression is regulated by the production and degradation of mRNAs and proteins but the specifics of their relationship are controversial. Although technological advances have enabled genome-wide and time-series surveys of mRNA and protein abundance, recent studies have shown paradoxical results, with most statistical analyses being limited to linear correlation, or analysis of variance applied separately to mRNA and protein datasets. Here, using recently analyzed genome-wide time-series data, we have developed a statistical analysis framework for identifying which types of genes or biological gene groups have significant correlation between mRNA and protein abundance after accounting for potential time delays. Our framework stratifies all genes in terms of the extent of time delay, conducts gene clustering in each stratum, and performs a non-parametric statistical test of the correlation between mRNA and protein abundance in a gene cluster. Consequently, we revealed stronger correlations than previously reported between mRNA and protein abundance in two metabolic pathways. Moreover, we identified a pair of stress responsive genes (ADC17 and KIN1 that showed a highly similar time series of mRNA and protein abundance. Furthermore, we confirmed robustness of the analysis framework by applying it to another genome-wide time-series data and identifying a cytoskeleton-related gene cluster (keratin 18, keratin 17, and mitotic spindle positioning that shows similar correlation. The significant correlation and highly similar changes of mRNA and protein abundance suggests a concerted role of these genes in cellular stress response, which we consider provides an answer to the question of the specific relationships between mRNA and protein in a cell. In addition, our framework for studying the relationship between mRNAs and proteins in a cell will provide a basis for studying specific relationships between mRNA and protein abundance after

Elevation of D4 dopamine receptor mRNA in postmortem schizophrenic brain.

Science.gov (United States)

Stefanis, N C; Bresnick, J N; Kerwin, R W; Schofield, W N; McAllister, G

1998-01-01

The D4 dopamine (DA) receptor has been proposed to be a target for the development of a novel antipsychotic drug based on its pharmacological and distribution profile. There is much interest in whether D4 DA receptor levels are altered in schizophrenia, but the lack of an available receptor subtype-specific radioligand made this difficult to quantitate. In this study, we examined whether D4 mRNA levels are altered in different brain regions of schizophrenics compared to controls. Ribonuclease protection assays were carried out on total RNA samples isolated postmortem from frontal cortex and caudate brain regions of schizophrenics and matched controls. 32P-labelled RNA probes to the D4 DA receptor and to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), were hybridised with the RNA samples, digested with ribonucleases to remove unhybridised probe, and separated on 6% sequencing gels. Densitometer analysis on the subsequent autoradiogams was used to calculate the relative optical density of D4 mRNA compared to G3PDH mRNA. Statistical analysis of the data revealed a 3-fold higher level (P<0.011) of D4 mRNA in the frontal cortex of schizophrenics compared to controls. No increase was seen in caudate. D4 receptors could play a role in mediating dopaminergic activity in frontal cortex, an activity which may be malfunctioning in schizophrenia.

On the Confounding Effect of Temperature on Chemical Shift-Encoded Fat Quantification

Science.gov (United States)

Hernando, Diego; Sharma, Samir D.; Kramer, Harald; Reeder, Scott B.

2014-01-01

Purpose To characterize the confounding effect of temperature on chemical shift-encoded (CSE) fat quantification. Methods The proton resonance frequency of water, unlike triglycerides, depends on temperature. This leads to a temperature dependence of the spectral models of fat (relative to water) that are commonly used by CSE-MRI methods. Simulation analysis was performed for 1.5 Tesla CSE fat–water signals at various temperatures and echo time combinations. Oil–water phantoms were constructed and scanned at temperatures between 0 and 40°C using spectroscopy and CSE imaging at three echo time combinations. An explanted human liver, rejected for transplantation due to steatosis, was scanned using spectroscopy and CSE imaging. Fat–water reconstructions were performed using four different techniques: magnitude and complex fitting, with standard or temperature-corrected signal modeling. Results In all experiments, magnitude fitting with standard signal modeling resulted in large fat quantification errors. Errors were largest for echo time combinations near TEinit ≈ 1.3 ms, ΔTE ≈ 2.2 ms. Errors in fat quantification caused by temperature-related frequency shifts were smaller with complex fitting, and were avoided using a temperature-corrected signal model. Conclusion Temperature is a confounding factor for fat quantification. If not accounted for, it can result in large errors in fat quantifications in phantom and ex vivo acquisitions. PMID:24123362

Profiling and relative quantification of phosphatidylethanolamine based on acetone stable isotope derivatization.

Science.gov (United States)

Wang, Xiang; Wei, Fang; Xu, Ji-Qu; Lv, Xin; Dong, Xu-Yan; Han, Xianlin; Quek, Siew-Young; Huang, Feng-Hong; Chen, Hong

2016-01-01

Phosphatidylethanolamine (PE) is considered to be one of the pivotal lipids for normal cellular function as well as disease initiation and progression. In this study, a simple, efficient, reliable, and inexpensive method for the qualitative analysis and relative quantification of PE, based on acetone stable isotope derivatization combined with double neutral loss scan-shotgun electrospray ionization tandem-quadrupole mass spectrometry analysis (ASID-DNLS-Shotgun ESI-MS/MS), was developed. The ASID method led to alkylation of the primary amino groups of PE with an isopropyl moiety. The use of acetone (d0-acetone) and deuterium-labeled acetone (d6-acetone) introduced a 6 Da mass shift that was ideally suited for relative quantitative analysis, and enhanced sensitivity for mass analysis. The DNLS model was introduced to simultaneously analyze the differential derivatized PEs by shotgun ESI-MS/MS with high selectivity and accuracy. The reaction specificity, labeling efficiency, and linearity of the ASID method were thoroughly evaluated in this study. Its excellent applicability was validated by qualitative and relative quantitative analysis of PE species presented in liver samples from rats fed different diets. Using the ASID-DNLS-Shotgun ESI-MS/MS method, 45 PE species from rat livers have been identified and quantified in an efficient manner. The level of total PEs tended to decrease in the livers of rats on high fat diets compared with controls. The levels of PE 32:1, 34:3, 34:2, 36:3, 36:2, 42:10, plasmalogen PE 36:1 and lyso PE 22:6 were significantly reduced, while levels of PE 36:1 and lyso PE 16:0 increased. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification, characterization, and high-performance liquid chromatography quantification of process-related impurities in vonoprazan fumarate.

Science.gov (United States)

Liu, Lei; Cao, Na; Ma, Xingling; Xiong, Kaihe; Sun, Lili; Zou, Qiaogen

2016-04-01

High-performance liquid chromatography analysis of vonoprazan fumarate, a novel proton pump inhibitor drug revealed six impurities. These were identified by liquid chromatography with mass spectrometry. Further, the structures of the impurities were confirmed by synthesis followed by characterization by mass spectrometry, NMR spectroscopy, and infrared spectroscopy. On the basis of these data and knowledge of the synthetic scheme of vonoprazan fumarate, the previously unknown impurity was identified as 1-[5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrol-3-yl]-N-methyldimethylamine, which is a new compound. The possible mechanisms by which these impurities were formed were also discussed. A high-performance liquid chromatography method was optimized in order to separate, selectively detect, and quantify all process-related impurities of vonoprazan fumarate. The presented method has been validated in terms of linearity, limits of detection, and quantification, and response factors and, therefore, is highly suitable for routine analysis of vonoprazan fumarate related substances as well as stability studies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantification is Neither Necessary Nor Sufficient for Measurement

International Nuclear Information System (INIS)

Mari, Luca; Maul, Andrew; Torres Irribarra, David; Wilson, Mark

2013-01-01

Being an infrastructural, widespread activity, measurement is laden with stereotypes. Some of these concern the role of measurement in the relation between quality and quantity. In particular, it is sometimes argued or assumed that quantification is necessary for measurement; it is also sometimes argued or assumed that quantification is sufficient for or synonymous with measurement. To assess the validity of these positions the concepts of measurement and quantitative evaluation should be independently defined and their relationship analyzed. We contend that the defining characteristic of measurement should be the structure of the process, not a feature of its results. Under this perspective, quantitative evaluation is neither sufficient nor necessary for measurement

mRNA expression of 5-hydroxytryptamine 1B, 1D, and 1F receptors and their role in controlling the release of calcitonin gene-related peptide in the rat trigeminovascular system

DEFF Research Database (Denmark)

Amrutkar, Dipak V; Ploug, Kenneth B; Hay-Schmidt, Anders

2012-01-01

Triptans, a family of 5-hydroxytryptamine (5-HT) 1B, 1D, and 1F receptor agonists, are used in the acute treatment of migraine attacks. The site of action and subtypes of the 5-HT(1) receptor that mediate the antimigraine effect have still to be identified. This study investigated the mRNA expres......Triptans, a family of 5-hydroxytryptamine (5-HT) 1B, 1D, and 1F receptor agonists, are used in the acute treatment of migraine attacks. The site of action and subtypes of the 5-HT(1) receptor that mediate the antimigraine effect have still to be identified. This study investigated the m......RNA expression of these receptors and the role of 5-HT(1) receptor subtypes in controlling the release of calcitonin gene-related peptide (CGRP) in rat dura mater, trigeminal ganglion (TG), and trigeminal nucleus caudalis (TNC). The mRNA for each receptor subtype was quantified by quantitative real...

Expression of feeding-related peptide receptors mRNA in GT1-7 cell line and roles of leptin and orexins in control of GnRH secretion.

Science.gov (United States)

Yang, Ying; Zhou, Li-bin; Liu, Shang-quan; Tang, Jing-feng; Li, Feng-yin; Li, Rong-ying; Song, Huai-dong; Chen, Ming-dao

2005-08-01

To investigate the expression of feeding-related peptide receptors mRNA in GT1-7 cell line and roles of leptin and orexins in the control of GnRH secretion. Receptors of bombesin3, cholecystokinin (CCK)-A, CCK-B, glucagon-like peptide (GLP)1, melanin-concentrating hormone (MCH)1, orexin1, orexin2, neuromedin-B, neuropeptide Y (NPY)1 and NPY5, neurotensin (NT)1, NT2, NT3, and leptin receptor long form mRNA in GT1-7 cells were detected by reversed transcriptase-polymerase chain reaction. GT1-7 cells were treated with leptin, orexin A and orexin B at a cohort of concentrations for different lengths of time, and GnRH in medium was determined by radioimmunoassay (RIA). Receptors of bombesin 3, CCK-B, GLP1, MCH1, orexin1, neuromedin-B, NPY1, NPY5, NT1, NT3, and leptin receptor long form mRNA were expressed in GT1-7 cells, of which, receptors of GLP1, neuromedin-B, NPY1, and NT3 were highly expressed. No amplified fragments of orexin2, NT2, and CCK-A receptor cDNA were generated with GT1-7 RNA, indicating that the GT1-7 cells did not express mRNA of them. Leptin induced a significant stimulation of GnRH release, the results being most significant at 0.1 nmol/L for 15 min. In contrast to other studies in hypothalamic explants, neither orexin A nor orexin B affected basal GnRH secretion over a wide range of concentrations ranging from 1 nmol/L to 500 nmol/Lat 15, 30, and 60 min. Feeding and reproductive function are closely linked. Many orexigenic and anorexigenic signals may control feeding behavior as well as alter GnRH secretion through their receptors on GnRH neurons.

mRNA processing in yeast

International Nuclear Information System (INIS)

Stevens, A.

1982-01-01

Investigations in this laboratory center on basic enzymatic reactions of RNA. Still undefined are reactions involved in the conversion of precursors of mRA (pre-mRNA) to mRNA in eukaryotes. The pre-mRNA is called heterogeneous nuclear RNA and is 2 to 6 times larger than mRNA. The conversion, called splicing, involves a removal of internal sequences called introns by endoribonuclease action followed by a rejoining of the 3'- and 5'-end fragments, called exons, by ligating activity. It has not been possible yet to study the enzymes involved in vitro. Also undefined are reactions involved in the turnover or discarding of certain of the pre-mRNA molecules. Yeast is a simple eukaryote and may be expected to have the same, but perhaps simpler, processing reactions as the higher eukaryotes. Two enzymes involved in the processing of pre-mRNA and mRNA in yeast are under investigation. Both enzymes have been partially purified from ribonucleoprotein particles of yeast. The first is a unique decapping enzyme which cleaves [ 3 H]m 7 Gppp [ 14 C]RNA-poly (A) of yeast, yielding [ 3 H]m 7 GDP and is suggested by the finding that the diphosphate product, m 7 GpppA(G), and UDP-glucose are not hydrolyzed. The second enzyme is an endoribonuclease which converts both the [ 3 H] and [ 14 C] labels of [ 3 H]m 7 Gppp[ 14 C]RNA-poly(A) from an oligo(dT)-cellulose bound form to an unbound, acid-insoluble form. Results show that the stimulation involves an interaction of the labeled RNA with the small nuclear RNA. The inhibition of the enzyme by ethidium bromide and its stimulation by small nuclear RNA suggest that it may be a processing ribonuclease, requiring specific double-stranded features in its substrate. The characterization of the unique decapping enzyme and endoribonuclease may help to understand reactions involved in the processing of pre-mRNA and mRNA in eukaryotes

Endurance exercise induces mRNA expression of oxidative enzymes in human skeletal muscle late in recovery

DEFF Research Database (Denmark)

Leick, Lotte; Plomgaard, Peter S.; Grønløkke, L.

2010-01-01

exercise. To test the hypothesis that mRNA expression of many oxidative enzymes is up-regulated late in recovery (10-24 h) after exercise, male subjects (n=8) performed a 90-min cycling exercise (70% VO(2-max)), with muscle biopsies obtained before exercise (pre), and after 10, 18 and 24 h of recovery....... The mRNA expression of carnitine-palmitoyltransferase (CPT)I, CD36, 3-hydroxyacyl-CoA-dehydrogenase (HAD), cytochrome (Cyt)c, aminolevulinate-delta-synthase (ALAS)1 and GLUT4 was 100-200% higher at 10-24 h of recovery from exercise than in a control trial. Exercise induced a 100-300% increase...... in peroxisome proliferator-activated receptor gamma co-activator (PGC)-1alpha, citrate synthase (CS), CPTI, CD36, HAD and ALAS1 mRNA contents at 10-24 h of recovery relative to before exercise. No protein changes were detected in Cytc, ALAS1 or GLUT4. This shows that mRNA expression of several training...

Role of the mRNA export factor Sus1 in oxidative stress tolerance in Candida albicans.

Science.gov (United States)

Xiao, Chenpeng; Yu, Qilin; Zhang, Bing; Li, Jianrong; Zhang, Dan; Li, Mingchun

2018-02-05

In eukaryotes, the nuclear export of mRNAs is essential for gene expression. However, little is known about the role of mRNA nuclear export in the important fungal pathogen, Candida albicans. In this study, we identified C. albicans Sus1, a nucleus-localized protein that is required for mRNA export. Interestingly, the sus1Δ/Δ displayed hyper-sensitivity to extracellular oxidative stress, enhanced ROS accumulation and severe oxidative stress-related cell death. More strikingly, although the mutant exhibited normal activation of the expression of oxidative stress response (OSR) genes, it had attenuated activity of ROS scavenging system, which may be attributed to the defect in OSR mRNA export in this mutant. In addition, the virulence of the sus1Δ/Δ was seriously attenuated. Taken together, our findings provide evidence that the mRNA export factor Sus1 plays an important role in oxidative stress tolerance and pathogenesis. Copyright © 2018 Elsevier Inc. All rights reserved.

mRNA transfection of mouse and human neural stem cell cultures

OpenAIRE

McLenachan, Samuel; Zhang, D.; Palomo, A.B.; Edel, Michael John; Chen, F.K.

2013-01-01

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has ...

Course of c-myc mRNA expression in the regenerating mouse testis determined by competitive reverse transcriptase polymerase chain reaction.

Science.gov (United States)

Amendola, R

1994-11-01

The c-myc proto-oncogene is a reliable marker of the "G0-early G1" transition, and its down-regulation is believed to be necessary to obtain cellular differentiation. In murine spermatogenesis, the level of c-myc transcripts does not correlate with the rate of cellular division. Proliferation of supposed staminal spermatogonia to reproduce themselves is induced with a local 5 Gy X-ray dose in 90-day-old C57Bl/6 mice. c-myc quantification by a newly developed competitive reverse transcriptase polymerase chain reaction (RT-PCR) was carried out to follow the expression course of this proto-oncogene. Damage and restoration of spermatogenesis were analyzed at days 3, 6, 9, 10, 13, 30, and 60 after injury by relative testes/body weight determination and histological examination. Proliferative status was determined by histone H3 Northern blot analysis. c-myc mRNA level was 10 times higher after 3 days in the irradiated animals compared to the controls. An increasing number of copies were noted up to 10 days, but promptly decreased to the base level found for irradiated mice from 13 to 60 days. Interestingly, the expression of histone H3 detected S phase only in testes at 60 days from damage.

A novel link between Sus1 and the cytoplasmic mRNA decay machinery suggests a broad role in mRNA metabolism

Directory of Open Access Journals (Sweden)

Llopis Ana

2010-03-01

Full Text Available Abstract Background Gene expression is achieved by the coordinated action of multiple factors to ensure a perfect synchrony from chromatin epigenetic regulation through to mRNA export. Sus1 is a conserved mRNA export/transcription factor and is a key player in coupling transcription initiation, elongation and mRNA export. In the nucleus, Sus1 is associated to the transcriptional co-activator SAGA and to the NPC associated complex termed TREX2/THSC. Through these associations, Sus1 mediates the nuclear dynamics of different gene loci and facilitate the export of the new transcripts. Results In this study, we have investigated whether the yeast Sus1 protein is linked to factors involved in mRNA degradation pathways. We provide evidence for genetic interactions between SUS1 and genes coding for components of P-bodies such as PAT1, LSM1, LSM6 and DHH1. We demonstrate that SUS1 deletion is synthetic lethal with 5'→3' decay machinery components LSM1 and PAT1 and has a strong genetic interaction with LSM6 and DHH1. Interestingly, Sus1 overexpression led to an accumulation of Sus1 in cytoplasmic granules, which can co-localise with components of P-bodies and stress granules. In addition, we have identified novel physical interactions between Sus1 and factors associated to P-bodies/stress granules. Finally, absence of LSM1 and PAT1 slightly promotes the Sus1-TREX2 association. Conclusions In this study, we found genetic and biochemical association between Sus1 and components responsible for cytoplasmic mRNA metabolism. Moreover, Sus1 accumulates in discrete cytoplasmic granules, which partially co-localise with P-bodies and stress granules under specific conditions. These interactions suggest a role for Sus1 in gene expression during cytoplasmic mRNA metabolism in addition to its nuclear function.

Comparison of five DNA quantification methods

DEFF Research Database (Denmark)

Nielsen, Karsten; Mogensen, Helle Smidt; Hedman, Johannes

2008-01-01

Six commercial preparations of human genomic DNA were quantified using five quantification methods: UV spectrometry, SYBR-Green dye staining, slot blot hybridization with the probe D17Z1, Quantifiler Human DNA Quantification kit and RB1 rt-PCR. All methods measured higher DNA concentrations than...... Quantification kit in two experiments. The measured DNA concentrations with Quantifiler were 125 and 160% higher than expected based on the manufacturers' information. When the Quantifiler human DNA standard (Raji cell line) was replaced by the commercial human DNA preparation G147A (Promega) to generate the DNA...... standard curve in the Quantifiler Human DNA Quantification kit, the DNA quantification results of the human DNA preparations were 31% higher than expected based on the manufacturers' information. The results indicate a calibration problem with the Quantifiler human DNA standard for its use...

Consequences of metaphase II oocyte cryopreservation on mRNA content.

Science.gov (United States)

Chamayou, S; Bonaventura, G; Alecci, C; Tibullo, D; Di Raimondo, F; Guglielmino, A; Barcellona, M L

2011-04-01

We studied the consequences of freezing/thawing processes on mRNA contents in MII oocytes after slow-freezing/rapid thawing (SF/RT) and vitrification/warming (V/W) protocols, and compared the results to fresh MII oocytes. We quantified the nuclear transcript mRNA responsible for the translation of proteins belonging either to trans-regulatory protein family or to functional structural proteins such as proteins involved in DNA structural organization (NAP1L1, TOP1, H1F0H1), chromosomal structure maintenance (SMC, SCC3, RAD21, SMC1A, SMC1B, STAG3, REC8), mitochondrial energetic pathways (ATP5GJ, SDHC), cell cycle regulation and processes (CLTA, MAPK6, CKS2) and staminal cell potency-development competence stage (DPPA3, OCT4, FOXJ2). Surplus MII oocytes were donated from patients in IVF cycles and divided in three groups of 15 oocytes. Group 1 was comprised of non-cryopreserved oocytes and Groups 2 and 3 underwent SF/RT and V/W procedures, respectively. There was an overall decrease of mRNA extracted from cryopreserved oocytes compared to control group. Only 39.4% of mRNA content were preserved after SF/RT while 63.3% of mRNA content were maintained after V/W. Oocyte cryopreservation is associated with molecular injury associated with the decrease of stored mRNA. However the V/W protocol is more conservative than SF/RT resulting in a level of mRNA sufficient to maintain biologic functions in the subsequent fertilized oocyte. Copyright © 2011 Elsevier Inc. All rights reserved.

Characterization and relative quantification of phospholipids based on methylation and stable isotopic labeling[S

Science.gov (United States)

Cai, Tanxi; Shu, Qingbo; Liu, Peibin; Niu, Lili; Guo, Xiaojing; Ding, Xiang; Xue, Peng; Xie, Zhensheng; Wang, Jifeng; Zhu, Nali; Wu, Peng; Niu, Lili; Yang, Fuquan

2016-01-01

Phospholipids (PLs), one of the lipid categories, are not only the primary building blocks of cellular membranes, but also can be split to produce products that function as second messengers in signal transduction and play a pivotal role in numerous cellular processes, including cell growth, survival, and motility. Here, we present an integrated novel method that combines a fast and robust TMS-diazomethane-based phosphate derivatization and isotopic labeling strategy, which enables simultaneous profiling and relative quantification of PLs from biological samples. Our results showed that phosphate methylation allows fast and sensitive identification of the six major PL classes, including their lysophospholipid counterparts, under positive ionization mode. The isotopic labeling of endogenous PLs was achieved by deuterated diazomethane, which was generated through acid-catalyzed hydrogen/deuterium (H/D) exchange and methanolysis of TMS-diazomethane during the process of phosphate derivatization. The measured H/D ratios of unlabeled and labeled PLs, which were mixed in known proportions, indicated that the isotopic labeling strategy is capable of providing relative quantitation with adequate accuracy, reproducibility, and a coefficient of variation of 9.1%, on average. This novel method offers unique advantages over existing approaches and presents a powerful tool for research of PL metabolism and signaling. PMID:26733148

Anorexigenic and Orexigenic Hormone Modulation of Mammalian Target of Rapamycin Complex 1 Activity and the Regulation of Hypothalamic Agouti-Related Protein mRNA Expression

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Kenneth R. Watterson

2012-03-01

Full Text Available Activation of mammalian target of rapamycin 1 (mTORC1 by nutrients, insulin and leptin leads to appetite suppression (anorexia. Contrastingly, increased AMP-activated protein kinase (AMPK activity by ghrelin promotes appetite (orexia. However, the interplay between these mechanisms remains poorly defined. The relationship between the anorexigenic hormones, insulin and leptin, and the orexigenic hormone, ghrelin, on mTORC1 signalling was examined using S6 kinase phosphorylation as a marker for changes in mTORC1 activity in mouse hypothalamic GT1-7 cells. Additionally, the contribution of AMPK and mTORC1 signalling in relation to insulin-, leptin- and ghrelin-driven alterations to mouse hypothalamic agouti-related protein (AgRP mRNA levels was examined. Insulin and leptin increase mTORC1 activity in a phosphoinositide-3-kinase (PI3K- and protein kinase B (PKB-dependent manner, compared to vehicle controls, whereas increasing AMPK activity inhibits mTORC1 activity and blocks the actions of the anorexigenic hormones. Ghrelin mediates an AMPK-dependent decrease in mTORC1 activity and increases hypothalamic AgRP mRNA levels, the latter effect being prevented by insulin in an mTORC1-dependent manner. In conclusion, mTORC1 acts as an integration node in hypothalamic neurons for hormone-derived PI3K and AMPK signalling and mediates at least part of the assimilated output of anorexigenic and orexigenic hormone actions in the hypothalamus.

VDR mRNA overexpression is associated with worse prognostic factors in papillary thyroid carcinoma

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June Young Choi

2017-03-01

Full Text Available The purpose of this study was to assess the relationship between vitamin D receptor gene (VDR expression and prognostic factors in papillary thyroid cancer (PTC. mRNA sequencing and somatic mutation data from The Cancer Genome Atlas (TCGA were analyzed. VDR mRNA expression was compared to clinicopathologic variables by linear regression. Tree-based classification was applied to find cutoff and patients were split into low and high VDR group. Logistic regression, Kaplan–Meier analysis, differentially expressed gene (DEG test and pathway analysis were performed to assess the differences between two VDR groups. VDR mRNA expression was elevated in PTC than that in normal thyroid tissue. VDR expressions were high in classic and tall-cell variant PTC and lateral neck node metastasis was present. High VDR group was also associated with classic and tall cell subtype, AJCC stage IV and lower recurrence-free survival. DEG test reveals that 545 genes were upregulated in high VDR group. Thyroid cancer-related pathways were enriched in high VDR group in pathway analyses. VDR mRNA overexpression was correlated with worse prognostic factors such as subtypes of papillary thyroid carcinoma that are known to be worse prognosis, lateral neck node metastasis, advanced stage and recurrence-free survival.

Aberrant Expression of TNF-α and TGF-β1 mRNA in Spontaneous Abortion

Institute of Scientific and Technical Information of China (English)

Ji-fen HU; Hong-chu BAO; Feng-chuan ZHU; Cai-ling YOU

2004-01-01

Objective To investigate the aberrant expressions of TNF-α and TGF-β1 in peripheral blood mononuclear cells (PBMCs) and placental tissues in patients with early spontaneous abortionMethods Using the technique of semi-quantitative reverse transcript-polymerase chain reaction (RT-PCR), TNF-α mRNA and TGF-β1 mRNA in PBMCs were measured in spontaneous abortion group (30 cases), normal pregnancy group (25 cases) and nonpregnant group (25 cases). The expressive intension of TNF-α protein and TGF-β1 protein in placental tissues was also identified by immunohistochemistry.Results Both levels of TNF-α mRNA and TGF-β1 mRNA expressed in PBMCs were significantly different between the three groups respectively (P＜0. 05). Levels of TNF-α in syncytiotrophoblastic and cytotrophoblastic cells of the two aborted groups were substantially higher than those of the non-pregnant group (P＜0. 01), but the levels of TGF-β1 in syncytiotrophoblastic cells of the two aborted groups were markedly lower than those of the non-pregnant group (P＜0. 01).Conclusion There is potential relation between TGF-β1 at the fetomaternal interface and spontaneous abortion. TGF-β1 may contribute to the maintenance of pregnancy,and low-level expression of TGF-β1 may be associated with pregnancy failure.

Lung involvement quantification in chest radiographs

International Nuclear Information System (INIS)

Giacomini, Guilherme; Alvarez, Matheus; Oliveira, Marcela de; Miranda, Jose Ricardo A.; Pina, Diana R.; Pereira, Paulo C.M.; Ribeiro, Sergio M.

2014-01-01

Tuberculosis (TB) caused by Mycobacterium tuberculosis, is an infectious disease which remains a global health problem. The chest radiography is the commonly method employed to assess the TB's evolution. The methods for quantification of abnormalities of chest are usually performed on CT scans (CT). This quantification is important to assess the TB evolution and treatment and comparing different treatments. However, precise quantification is not feasible for the amount of CT scans required. The purpose of this work is to develop a methodology for quantification of lung damage caused by TB through chest radiographs. It was developed an algorithm for computational processing of exams in Matlab, which creates a lungs' 3D representation, with compromised dilated regions inside. The quantification of lung lesions was also made for the same patients through CT scans. The measurements from the two methods were compared and resulting in strong correlation. Applying statistical Bland and Altman, all samples were within the limits of agreement, with a confidence interval of 95%. The results showed an average variation of around 13% between the two quantification methods. The results suggest the effectiveness and applicability of the method developed, providing better risk-benefit to the patient and cost-benefit ratio for the institution. (author)

Fluorescent quantification of melanin.

Science.gov (United States)

Fernandes, Bruno; Matamá, Teresa; Guimarães, Diana; Gomes, Andreia; Cavaco-Paulo, Artur

2016-11-01

Melanin quantification is reportedly performed by absorption spectroscopy, commonly at 405 nm. Here, we propose the implementation of fluorescence spectroscopy for melanin assessment. In a typical in vitro assay to assess melanin production in response to an external stimulus, absorption spectroscopy clearly overvalues melanin content. This method is also incapable of distinguishing non-melanotic/amelanotic control cells from those that are actually capable of performing melanogenesis. Therefore, fluorescence spectroscopy is the best method for melanin quantification as it proved to be highly specific and accurate, detecting even small variations in the synthesis of melanin. This method can also be applied to the quantification of melanin in more complex biological matrices like zebrafish embryos and human hair. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

In human granulosa cells from small antral follicles, androgen receptor mRNA and androgen levels in follicular fluid correlate with FSH receptor mRNA

DEFF Research Database (Denmark)

Nielsen, M. E.; Rasmussen, I. A.; Kristensen, S. G.

2011-01-01

significantly with the expression of AMHRII, but did not correlate with any of the hormones in the follicular fluid. These data demonstrate an intimate association between AR expression in immature granulosa cells, and the expression of FSHR in normal small human antral follicles and between the follicular......Human small antral follicles (diameter 3-9 mm) were obtained from ovaries surgically removed for fertility preservation. From the individual aspirated follicles, granulosa cells and the corresponding follicular fluid were isolated in 64 follicles, of which 55 were available for mRNA analysis (24...... and to the follicular fluid concentrations of AMH, inhibin-B, progesterone and estradiol. AR mRNA expression in granulosa cells and the follicular fluid content of androgens both showed a highly significant positive association with the expression of FSHR mRNA in granulosa cells. AR mRNA expression also correlated...

An external standard method for quantification of human cytomegalovirus by PCR

International Nuclear Information System (INIS)

Rongsen, Shen; Liren, Ma; Fengqi, Zhou; Qingliang, Luo

1997-01-01

An external standard method for PCR quantification of HCMV was reported. [α- 32 P]dATP was used as a tracer. 32 P-labelled specific amplification product was separated by agarose gel electrophoresis. A gel piece containing the specific product band was excised and counted in a plastic scintillation counter. Distribution of [α- 32 P]dATP in the electrophoretic gel plate and effect of separation between the 32 P-labelled specific product and free [α- 32 P]dATP were observed. A standard curve for quantification of HCMV by PCR was established and detective results of quality control templets were presented. The external standard method and the electrophoresis separation effect were appraised. The results showed that the method could be used for relative quantification of HCMV. (author)

Green tea increases anti-inflammatory tristetraprolin and decreases pro-inflammatory tumor necrosis factor mRNA levels in rats

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Roussel Anne M

2007-01-01

Full Text Available Abstract Background Tristetraprolin (TTP/ZFP36 family proteins have anti-inflammatory activity by binding to and destabilizing pro-inflammatory mRNAs such as Tnf mRNA, and represent a potential therapeutic target for inflammation-related diseases. Tea has anti-inflammatory properties but the molecular mechanisms have not been completely elucidated. We hypothesized that TTP and/or its homologues might contribute to the beneficial effects of tea as an anti-inflammatory product. Methods Quantitative real-time PCR was used to investigate the effects of green tea (0, 1, and 2 g solid extract/kg diet on the expression of Ttp family genes (Ttp/Tis11/Zfp36, Zfp36l1/Tis11b, Zfp36l2/Tis11d, Zfp36l3, pro-inflammatory genes (Tnf, Csf2/Gm-csf, Ptgs2/Cox2, and Elavl1/Hua/Hur and Vegf genes in liver and muscle of rats fed a high-fructose diet known to induce insulin resistance, oxidative stress, inflammation, and TNF-alpha levels. Results Ttp and Zfp36l1 mRNAs were the major forms in both liver and skeletal muscle. Ttp, Zfp36l1, and Zfp36l2 mRNA levels were more abundant in the liver than those in the muscle. Csf2/Gm-csf and Zfp36l3 mRNAs were undetectable in both tissues. Tea (1 g solid extract/kg diet increased Ttp mRNA levels by 50–140% but Tnf mRNA levels decreased by 30% in both tissues, and Ptgs2/Cox2 mRNA levels decreased by 40% in the muscle. Tea (2 g solid extract/kg diet increased Elavl1/Hua/Hur mRNA levels by 40% in the liver but did not affect any of the other mRNA levels in liver or muscle. Conclusion These results show that tea can modulate Ttp mRNA levels in animals and suggest that a post-transcriptional mechanism through TTP could partially account for tea's anti-inflammatory properties. The results also suggest that drinking adequate amounts of green tea may play a role in the prevention of inflammation-related diseases.

Quantification of the impact of endometriosis symptoms on health-related quality of life and work productivity.

Science.gov (United States)

Fourquet, Jessica; Báez, Lorna; Figueroa, Michelle; Iriarte, R Iván; Flores, Idhaliz

2011-07-01

To quantify the impact of endometriosis-related symptoms on physical and mental health status, health-related quality of life, and work-related aspects (absenteeism, presenteeism, work productivity, and activity impairment). Cross-sectional quantitative study. Academic and research institution. Women (n = 193) with self-reported surgically diagnosed endometriosis from the Endometriosis Patient Registry at Ponce School of Medicine and Health Sciences (PSMHS). Anonymous questionnaire divided into three sections consisting of questions from the Patient Health Survey (SF-12), the Endometriosis Health Profile (EHP-5), and the Work Productivity and Activity Impairment Survey (WPAI). Quantification of impact of endometriosis symptoms on physical and mental health status, health-related quality of life, absenteeism, presenteeism, work productivity, and activity impairment. Patients had SF-12 scores denoting statistically significant disability in the physical and mental health components. They also reported an average of 7.41 hours (approximately one working day) of work time lost during the week when the symptoms are worse. In addition, the WPAI scores showed a high impact on work-related domains: 13% of average loss in work time (absenteeism), 65% of work impaired (presenteeism), 64% of loss in efficiency levels (work productivity loss), and 60% of daily activities perturbed (activity impairment). Endometriosis symptoms such as chronic, incapacitating pelvic pain and infertility negatively and substantially impact the physical and mental health status, health-related quality of life, and productivity at work of women. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae

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Susanne Huch

2016-10-01

Full Text Available The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs, or alternatively may imply a role for P bodies in mRNA stabilization.

Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

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Beatriz M. A. Fontoura

2013-07-01

Full Text Available Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses.

Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

Science.gov (United States)

Kuss, Sharon K.; Mata, Miguel A.; Zhang, Liang; Fontoura, Beatriz M. A.

2013-01-01

Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses. PMID:23872491

Low-level lasers on microRNA and uncoupling protein 2 mRNA levels in human breast cancer cells

Science.gov (United States)

Canuto, K. S.; Teixeira, A. F.; Rodrigues, J. A.; Paoli, F.; Nogueira, E. M.; Mencalha, A. L.; Fonseca, A. S.

2017-06-01

MicroRNA is short non-coding RNA and is a mediator of post-transcriptional regulation of gene expression. In addition, uncoupling proteins (UCPs) regulate thermogenesis, metabolic and energy balance, and decrease reactive oxygen species production. Both microRNA and UCP2 expression can be altered in cancer cells. At low power, laser wavelength, frequency, fluence and emission mode deternube photobiological responses, which are the basis of low-level laser therapy. There are few studies on miRNA and UCP mRNA levels after low-level laser exposure on cancer cells. In this work, we evaluate the micrRNA (mir-106b and mir-15a) and UCP2 mRNA levels in human breast cancer cells exposed to low-level lasers. MDA-MB-231 human breast cancer cells were exposed to low-level red and infrared lasers, total RNA was extracted for cDNA synthesis and mRNA levels by real time quantitative polymerase chain reaction were evaluated. Data show that mir-106b and mir-15a relative levels are not altered, but UCP2 mRNA relative levels are increased in MDA-MB-231 human breast cancer cells exposed to low-level red and infrared lasers at fluences used in therapeutic protocols.

Accumulation of catechins in tea in relation to accumulation of mRNA from genes involved in catechin biosynthesis.

Science.gov (United States)

Eungwanichayapant, P D; Popluechai, S

2009-02-01

Catechins are a group of polyphenols found in tea (Camellia sinensis var. sinensis) at high levels. They are beneficial for health. From the study on accumulation of catechins in shoots and mature leaves of a tea cultivar, Oolong No. 17, using high-performance liquid chromatography (HPLC), it was found that the amounts of most catechins in the shoots were higher than those in the mature leaves, with an exception of catechins gallate (CG) that was found in trace amounts in both the shoots and mature leaves. mRNA accumulation of genes involved in catechin synthesis was studied using reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that the mRNA accumulation of the genes were higher in the shoots than in the mature leaves. These genes included genes of phenylalanine ammonia-lyase 1 (PAL1; EC 4.3.1.5), chalcone synthase (CHS; EC 2.3.1.74), dihydroflavonol 4-reductase (DFR; EC 1.1.1.219), leucoanthocyanidin reductase (LCR; EC 1.17.1.3), and flavanone 3-hydroxylase (F3H; EC 1.14.11.9).

On the direct quantification of celecoxib in commercial solid drugs using the TT-PIXE and TT-PIGE techniques

International Nuclear Information System (INIS)

Nsouli, B.; Zahraman, K.; Bejjani, A.; Assi, S.; El-Yazbi, F.; Roumie, M.

2006-01-01

The quantification of the active ingredient (AI) in drugs is a crucial and important step in the drug quality control process. This is usually performed by using wet chemical techniques like LC-MS, UV spectrophotometry and other appropriate organic analytical methods. In the case of an active ingredient contains specific heteroatoms (F, S, Cl, etc.,), elemental IBA technique can be explored for molecular quantification. IBA techniques permit the analysis of the sample under solid form, without any laborious sample preparation. This is an advantage when the number of sample is relatively large. In this work, we demonstrate the ability of the thick target PIXE (TT-PIXE) and the TT-PIGE techniques for rapid and accurate quantification of celecoxib in commercial drugs. The experimental aspects related to the quantification validity are presented and discussed

Uncertainty quantification theory, implementation, and applications

CERN Document Server

Smith, Ralph C

2014-01-01

The field of uncertainty quantification is evolving rapidly because of increasing emphasis on models that require quantified uncertainties for large-scale applications, novel algorithm development, and new computational architectures that facilitate implementation of these algorithms. Uncertainty Quantification: Theory, Implementation, and Applications provides readers with the basic concepts, theory, and algorithms necessary to quantify input and response uncertainties for simulation models arising in a broad range of disciplines. The book begins with a detailed discussion of applications where uncertainty quantification is critical for both scientific understanding and policy. It then covers concepts from probability and statistics, parameter selection techniques, frequentist and Bayesian model calibration, propagation of uncertainties, quantification of model discrepancy, surrogate model construction, and local and global sensitivity analysis. The author maintains a complementary web page where readers ca...

Superposition Quantification

Science.gov (United States)

Chang, Li-Na; Luo, Shun-Long; Sun, Yuan

2017-11-01

The principle of superposition is universal and lies at the heart of quantum theory. Although ever since the inception of quantum mechanics a century ago, superposition has occupied a central and pivotal place, rigorous and systematic studies of the quantification issue have attracted significant interests only in recent years, and many related problems remain to be investigated. In this work we introduce a figure of merit which quantifies superposition from an intuitive and direct perspective, investigate its fundamental properties, connect it to some coherence measures, illustrate it through several examples, and apply it to analyze wave-particle duality. Supported by Science Challenge Project under Grant No. TZ2016002, Laboratory of Computational Physics, Institute of Applied Physics and Computational Mathematics, Beijing, Key Laboratory of Random Complex Structures and Data Science, Chinese Academy of Sciences, Grant under No. 2008DP173182

Profil Ekspresi mRNA Gen Murine Double Minute2, Kruppel-Like Factor4, dan c-Myc pada Fibrosarkoma

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- Humaryanto

2017-02-01

Full Text Available Abstrak Fibrosarkoma hanya terjadi 1–3% dari seluruh keganasan jaringan lunak. Hingga saat ini etiologi fibrosarkoma belum diketahui dengan pasti. Beberapa faktor dapat menjadi penyebab patogenesis fibrosarkoma antara lain radiasi, terpapar zat kimia tertentu, serta infeksi human herpes virus 8 (HHV8 dan Epstein-Barr virus (EBV. Penelitian terkini menunjukkan bahwa banyak sarkoma terkait dengan mutasi genetik. Penelitian ini bertujuan melihat profil ekspresi mRNA gen Krüppel-like Factor4, Murine Double Minute2, dan c-Myc pada fibrosarkoma menggunakan teknik real time PCR kuantitatif (quantitative real time PCR, qRT-PCR. Analisis data menggunakan metode kuantititatif relatif 2-ΔΔCt. Penelitian ini menggunakan 10 sampel kasus fibrosarkoma yang ditemukan di Kota Jambi dari tahun 2011–2015. Hasil ΔCt (+SD MDM2, KLF-4, dan c-Myc disusun dari nilai yang terkecil hingga tertinggi adalah 1,85±2,14; 2,06±3,86; 2,9±2,66 secara berurutan. Dibanding dengan level ekspresi dengan GAPDH sebagai housekeeping gene, gen MDM2 dan KLF-4 relatif menurun dua kali lipat, sedangkan gen c-Myc relatif menurun lebih dari tiga kali lipat. Simpulan, penelitian ini menunjukkan bahwa pada kasus fibrosarkoma, gen c-Myc disupresi lebih kuat dibanding dengan gen MDM2 dan KLF-4. Abstract Fibrosarcoma is a rare soft tissue sarcoma, reported only 1–3% of all soft tissue sarcomas. Like any other soft-tissue sarcomas the definitive caused has not yet understood. Recognized causes include exposure to ionizing radiation, various physical and chemical factors, infection with human herpes virus (HHV8 and Epstein-Barr virus (EBV. Current research indicates many sarcomas are associated with genetic mutations. In this study, we investigated profile of mRNA gene expression KLF4, MDM2, and c-Myc of RNA in fibrosarcoma cases. The genes expression was examined using quantitative real time PCR (qRT-PCR and we analyzed the relative gene expression using the 2-ΔΔCt method. Ten

Aging alters mRNA expression of amyloid transporter genes at the blood-brain barrier.

Science.gov (United States)

Osgood, Doreen; Miller, Miles C; Messier, Arthur A; Gonzalez, Liliana; Silverberg, Gerald D

2017-09-01

Decreased clearance of potentially toxic metabolites, due to aging changes, likely plays a significant role in the accumulation of amyloid-beta (Aβ) peptides and other macromolecules in the brain of the elderly and in the patients with Alzheimer's disease (AD). Aging is the single most important risk factor for AD development. Aβ transport receptor proteins expressed at the blood-brain barrier are significantly altered with age: the efflux transporters lipoprotein receptor-related protein 1 and P-glycoprotein are reduced, whereas the influx transporter receptor for advanced glycation end products is increased. These receptors play an important role in maintaining brain biochemical homeostasis. We now report that, in a rat model of aging, gene transcription is altered in aging, as measured by Aβ receptor gene messenger RNA (mRNA) at 3, 6, 9, 12, 15, 20, 30, and 36 months. Gene mRNA expression from isolated cerebral microvessels was measured by quantitative polymerase chain reaction. Lipoprotein receptor-related protein 1 and P-glycoprotein mRNA were significantly reduced in aging, and receptor for advanced glycation end products was increased, in parallel with the changes seen in receptor protein expression. Transcriptional changes appear to play a role in aging alterations in blood-brain barrier receptor expression and Aβ accumulation. Copyright © 2017 Elsevier Inc. All rights reserved.

Maternal separation induces hippocampal changes in cadherin-1 (CDH-1) mRNA and recognition memory impairment in adolescent mice.

Science.gov (United States)

de Azeredo, Lucas Araújo; Wearick-Silva, Luis Eduardo; Viola, Thiago Wendt; Tractenberg, Saulo Gantes; Centeno-Silva, Anderson; Orso, Rodrigo; Schröder, Nadja; Bredy, Timothy William; Grassi-Oliveira, Rodrigo

2017-05-01

In rodents, disruption of mother-infant attachment induced by maternal separation (MS) is associated with recognition memory impairment and long-term neurobiological consequences. Particularly stress-induced modifications have been associated to disruption of cadherin (CDH) adhesion function, which plays an important role in remodeling of neuronal connection and synaptic plasticity. This study investigated the sex-dependent effect of MS on recognition memory and mRNA levels of classical type I and type II CDH and the related β -catenin (β -Cat) in the hippocampus and prefrontal cortex of late adolescent mice. We provided evidence that the BALB/c mice exposed to MS present deficit in recognition memory, especially females. Postnatal MS induced higher hippocampal CDH-2 and CDH-8 mRNA levels, as well as an upregulation of CDH-1 in the prefrontal cortex in both males and females. MS-reared female mice presented lower CDH-1 mRNA levels in the hippocampus. In addition, hippocampal CDH-1 mRNA levels were positively correlated with recognition memory performance in females. MS-reared male mice exhibited higher β -Cat mRNA levels in the hippocampus. Considering sex-specific effects on CDH mRNA levels, it has been demonstrated mRNA changes in CDH-1, β -Cat, and CDH-6 in the hippocampus, as well as CDH-1, CDH-8 and CDH-11 in the prefrontal cortex. Overall, these findings suggest a complex interplay among MS, CDH mRNA expression, and sex differences in the PFC and hippocampus of adolescent mice. Copyright © 2017 Elsevier Inc. All rights reserved.

Multiple correlation analyses revealed complex relationship between DNA methylation and mRNA expression in human peripheral blood mononuclear cells.

Science.gov (United States)

Xie, Fang-Fei; Deng, Fei-Yan; Wu, Long-Fei; Mo, Xing-Bo; Zhu, Hong; Wu, Jian; Guo, Yu-Fan; Zeng, Ke-Qin; Wang, Ming-Jun; Zhu, Xiao-Wei; Xia, Wei; Wang, Lan; He, Pei; Bing, Peng-Fei; Lu, Xin; Zhang, Yong-Hong; Lei, Shu-Feng

2018-01-01

DNA methylation is an important regulator on the mRNA expression. However, a genome-wide correlation pattern between DNA methylation and mRNA expression in human peripheral blood mononuclear cells (PBMCs) is largely unknown. The comprehensive relationship between mRNA and DNA methylation was explored by using four types of correlation analyses and a genome-wide methylation-mRNA expression quantitative trait locus (eQTL) analysis in PBMCs in 46 unrelated female subjects. An enrichment analysis was performed to detect biological function for the detected genes. Single pair correlation coefficient (r T1 ) between methylation level and mRNA is moderate (-0.63-0.62) in intensity, and the negative and positive correlations are nearly equal in quantity. Correlation analysis on each gene (T4) found 60.1% genes showed correlations between mRNA and gene-based methylation at P correlation (R T4  > 0.8). Methylation sites have regulation effects on mRNA expression in eQTL analysis, with more often observations in region of transcription start site (TSS). The genes under significant methylation regulation both in correlation analysis and eQTL analysis tend to cluster to the categories (e.g., transcription, translation, regulation of transcription) that are essential for maintaining the basic life activities of cells. Our findings indicated that DNA methylation has predictive regulation effect on mRNA with a very complex pattern in PBMCs. The results increased our understanding on correlation of methylation and mRNA and also provided useful clues for future epigenetic studies in exploring biological and disease-related regulatory mechanisms in PBMC.

Viperin mRNA is a novel target for the human RNase MRP/RNase P endoribonuclease.

Science.gov (United States)

Mattijssen, Sandy; Hinson, Ella R; Onnekink, Carla; Hermanns, Pia; Zabel, Bernhard; Cresswell, Peter; Pruijn, Ger J M

2011-07-01

RNase MRP is a conserved endoribonuclease, in humans consisting of a 267-nucleotide RNA associated with 7-10 proteins. Mutations in its RNA component lead to several autosomal recessive skeletal dysplasias, including cartilage-hair hypoplasia (CHH). Because the known substrates of mammalian RNase MRP, pre-ribosomal RNA, and RNA involved in mitochondrial DNA replication are not likely involved in CHH, we analyzed the effects of RNase MRP (and the structurally related RNase P) depletion on mRNAs using DNA microarrays. We confirmed the upregulation of the interferon-inducible viperin mRNA by RNAi experiments and this appeared to be independent of the interferon response. We detected two cleavage sites for RNase MRP/RNase P in the coding sequence of viperin mRNA. This is the first study providing direct evidence for the cleavage of a mRNA by RNase MRP/RNase P in human cells. Implications for the involvement in the pathophysiology of CHH are discussed.

Temperature dependence of postmortem MR quantification for soft tissue discrimination

Energy Technology Data Exchange (ETDEWEB)

Zech, Wolf-Dieter; Schwendener, Nicole; Jackowski, Christian [University of Bern, From the Institute of Forensic Medicine, Bern (Switzerland); Persson, Anders; Warntjes, Marcel J. [University of Linkoeping, The Center for Medical Image Science and Visualization (CMIV), Linkoeping (Sweden)

2015-08-15

To investigate and correct the temperature dependence of postmortem MR quantification used for soft tissue characterization and differentiation in thoraco-abdominal organs. Thirty-five postmortem short axis cardiac 3-T MR examinations were quantified using a quantification sequence. Liver, spleen, left ventricular myocardium, pectoralis muscle and subcutaneous fat were analysed in cardiac short axis images to obtain mean T1, T2 and PD tissue values. The core body temperature was measured using a rectally inserted thermometer. The tissue-specific quantitative values were related to the body core temperature. Equations to correct for temperature differences were generated. In a 3D plot comprising the combined data of T1, T2 and PD, different organs/tissues could be well differentiated from each other. The quantitative values were influenced by the temperature. T1 in particular exhibited strong temperature dependence. The correction of quantitative values to a temperature of 37 C resulted in better tissue discrimination. Postmortem MR quantification is feasible for soft tissue discrimination and characterization of thoraco-abdominal organs. This provides a base for computer-aided diagnosis and detection of tissue lesions. The temperature dependence of the T1 values challenges postmortem MR quantification. Equations to correct for the temperature dependence are provided. (orig.)

Temperature dependence of postmortem MR quantification for soft tissue discrimination

International Nuclear Information System (INIS)

Zech, Wolf-Dieter; Schwendener, Nicole; Jackowski, Christian; Persson, Anders; Warntjes, Marcel J.

2015-01-01

To investigate and correct the temperature dependence of postmortem MR quantification used for soft tissue characterization and differentiation in thoraco-abdominal organs. Thirty-five postmortem short axis cardiac 3-T MR examinations were quantified using a quantification sequence. Liver, spleen, left ventricular myocardium, pectoralis muscle and subcutaneous fat were analysed in cardiac short axis images to obtain mean T1, T2 and PD tissue values. The core body temperature was measured using a rectally inserted thermometer. The tissue-specific quantitative values were related to the body core temperature. Equations to correct for temperature differences were generated. In a 3D plot comprising the combined data of T1, T2 and PD, different organs/tissues could be well differentiated from each other. The quantitative values were influenced by the temperature. T1 in particular exhibited strong temperature dependence. The correction of quantitative values to a temperature of 37 C resulted in better tissue discrimination. Postmortem MR quantification is feasible for soft tissue discrimination and characterization of thoraco-abdominal organs. This provides a base for computer-aided diagnosis and detection of tissue lesions. The temperature dependence of the T1 values challenges postmortem MR quantification. Equations to correct for the temperature dependence are provided. (orig.)

Effects of humic acid on DNA quantification with Quantifiler® Human DNA Quantification kit and short tandem repeat amplification efficiency.

Science.gov (United States)

Seo, Seung Bum; Lee, Hye Young; Zhang, Ai Hua; Kim, Hye Yeon; Shin, Dong Hoon; Lee, Soong Deok

2012-11-01

Correct DNA quantification is an essential part to obtain reliable STR typing results. Forensic DNA analysts often use commercial kits for DNA quantification; among them, real-time-based DNA quantification kits are most frequently used. Incorrect DNA quantification due to the presence of PCR inhibitors may affect experiment results. In this study, we examined the alteration degree of DNA quantification results estimated in DNA samples containing a PCR inhibitor by using a Quantifiler® Human DNA Quantification kit. For experiments, we prepared approximately 0.25 ng/μl DNA samples containing various concentrations of humic acid (HA). The quantification results were 0.194-0.303 ng/μl at 0-1.6 ng/μl HA (final concentration in the Quantifiler reaction) and 0.003-0.168 ng/μl at 2.4-4.0 ng/μl HA. Most DNA quantity was undetermined when HA concentration was higher than 4.8 ng/μl HA. The C (T) values of an internal PCR control (IPC) were 28.0-31.0, 36.5-37.1, and undetermined at 0-1.6, 2.4, and 3.2 ng/μl HA. These results indicate that underestimated DNA quantification results may be obtained in the DNA sample with high C (T) values of IPC. Thus, researchers should carefully interpret the DNA quantification results. We additionally examined the effects of HA on the STR amplification by using an Identifiler® kit and a MiniFiler™ kit. Based on the results of this study, it is thought that a better understanding of various effects of HA would help researchers recognize and manipulate samples containing HA.

A phase quantification method based on EBSD data for a continuously cooled microalloyed steel

Energy Technology Data Exchange (ETDEWEB)

Zhao, H.; Wynne, B.P.; Palmiere, E.J., E-mail: e.j.palmiere@sheffield.ac.uk

2017-01-15

Mechanical properties of steels depend on the phase constitutions of the final microstructures which can be related to the processing parameters. Therefore, accurate quantification of different phases is necessary to investigate the relationships between processing parameters, final microstructures and mechanical properties. Point counting on micrographs observed by optical or scanning electron microscopy is widely used as a phase quantification method, and different phases are discriminated according to their morphological characteristics. However, it is difficult to differentiate some of the phase constituents with similar morphology. Differently, for EBSD based phase quantification methods, besides morphological characteristics, other parameters derived from the orientation information can also be used for discrimination. In this research, a phase quantification method based on EBSD data in the unit of grains was proposed to identify and quantify the complex phase constitutions of a microalloyed steel subjected to accelerated coolings. Characteristics of polygonal ferrite/quasi-polygonal ferrite, acicular ferrite and bainitic ferrite on grain averaged misorientation angles, aspect ratios, high angle grain boundary fractions and grain sizes were analysed and used to develop the identification criteria for each phase. Comparing the results obtained by this EBSD based method and point counting, it was found that this EBSD based method can provide accurate and reliable phase quantification results for microstructures with relatively slow cooling rates. - Highlights: •A phase quantification method based on EBSD data in the unit of grains was proposed. •The critical grain area above which GAM angles are valid parameters was obtained. •Grain size and grain boundary misorientation were used to identify acicular ferrite. •High cooling rates deteriorate the accuracy of this EBSD based method.

Assessing mRNA nuclear export in mammalian cells by microinjection.

Science.gov (United States)

Lee, Eliza S; Palazzo, Alexander F

2017-08-15

The nuclear export of mRNAs is an important yet little understood part of eukaryotic gene expression. One of the easiest methods for monitoring mRNA export in mammalian tissue culture cells is through the microinjection of DNA plasmids into the nucleus and monitoring the distribution of the transcribed product over time. Here we describe how to setup a microscope equipped with a micromanipulator used in cell microinjections, and we explain how to perform a nuclear mRNA export assay and obtain the nuclear export rate for any given mRNA. Copyright © 2017 Elsevier Inc. All rights reserved.

Quantification of local mobilities

DEFF Research Database (Denmark)

Zhang, Y. B.

2018-01-01

A new method for quantification of mobilities of local recrystallization boundary segments is presented. The quantification is based on microstructures characterized using electron microscopy and on determination of migration velocities and driving forces for local boundary segments. Pure aluminium...... is investigated and the results show that even for a single recrystallization boundary, different boundary segments migrate differently, and the differences can be understood based on variations in mobilities and local deformed microstructures. The present work has important implications for understanding...

Absolute and direct microRNA quantification using DNA-gold nanoparticle probes.

Science.gov (United States)

Degliangeli, Federica; Kshirsagar, Prakash; Brunetti, Virgilio; Pompa, Pier Paolo; Fiammengo, Roberto

2014-02-12

DNA-gold nanoparticle probes are implemented in a simple strategy for direct microRNA (miRNA) quantification. Fluorescently labeled DNA-probe strands are immobilized on PEGylated gold nanoparticles (AuNPs). In the presence of target miRNA, DNA-RNA heteroduplexes are formed and become substrate for the endonuclease DSN (duplex-specific nuclease). Enzymatic hydrolysis of the DNA strands yields a fluorescence signal due to diffusion of the fluorophores away from the gold surface. We show that the molecular design of our DNA-AuNP probes, with the DNA strands immobilized on top of the PEG-based passivation layer, results in nearly unaltered enzymatic activity toward immobilized heteroduplexes compared to substrates free in solution. The assay, developed in a real-time format, allows absolute quantification of as little as 0.2 fmol of miR-203. We also show the application of the assay for direct quantification of cancer-related miR-203 and miR-21 in samples of extracted total RNA from cell cultures. The possibility of direct and absolute quantification may significantly advance the use of microRNAs as biomarkers in the clinical praxis.

Development of Quantification Method for Bioluminescence Imaging

International Nuclear Information System (INIS)

Kim, Hyeon Sik; Min, Jung Joon; Lee, Byeong Il; Choi, Eun Seo; Tak, Yoon O; Choi, Heung Kook; Lee, Ju Young

2009-01-01

Optical molecular luminescence imaging is widely used for detection and imaging of bio-photons emitted by luminescent luciferase activation. The measured photons in this method provide the degree of molecular alteration or cell numbers with the advantage of high signal-to-noise ratio. To extract useful information from the measured results, the analysis based on a proper quantification method is necessary. In this research, we propose a quantification method presenting linear response of measured light signal to measurement time. We detected the luminescence signal by using lab-made optical imaging equipment of animal light imaging system (ALIS) and different two kinds of light sources. One is three bacterial light-emitting sources containing different number of bacteria. The other is three different non-bacterial light sources emitting very weak light. By using the concept of the candela and the flux, we could derive simplified linear quantification formula. After experimentally measuring light intensity, the data was processed with the proposed quantification function. We could obtain linear response of photon counts to measurement time by applying the pre-determined quantification function. The ratio of the re-calculated photon counts and measurement time present a constant value although different light source was applied. The quantification function for linear response could be applicable to the standard quantification process. The proposed method could be used for the exact quantitative analysis in various light imaging equipment with presenting linear response behavior of constant light emitting sources to measurement time

Single step production of Cas9 mRNA for zygote injection.

Science.gov (United States)

Redel, Bethany K; Beaton, Benjamin P; Spate, Lee D; Benne, Joshua A; Murphy, Stephanie L; O'Gorman, Chad W; Spate, Anna M; Prather, Randall S; Wells, Kevin D

2018-03-01

Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary. A sequence from the mMalat1 gene was cloned downstream of the IRES/Cas9 cassette described above. An mRNA concentration curve was constructed with either commercially available Cas9 mRNA or the IRES/ Cas9/triplex, by injection into porcine zygotes. Blastocysts were genotyped to determine if differences existed in the percent of embryos modified. The concentration curve identified differences due to concentration and RNA type injected. Single step production of Cas9 mRNA provides an alternative source of Cas9 for use in zygote injections.

Chromatic and anisotropic cross-recurrence quantification analysis of interpersonal behavior

NARCIS (Netherlands)

Cox, R.F.A; van der Steen, Stephanie; Guevara Guerrero, Marlenny; Hoekstra, Lisette; van Dijk, Marijn; Webber, Charles; Ioana, Cornel; Marwan, Norbert

Cross-recurrence quantification analysis (CRQA) is a powerful nonlinear time-series method to study coordination and cooperation between people. This chapter concentrates on two methodological issues related to CRQA on categorical data streams, which are commonly encountered in the behavioral

Nonsense mutations in the human β-globin gene affect mRNA metabolism

International Nuclear Information System (INIS)

Baserga, S.J.; Benz, E.J. Jr.

1988-01-01

A number of premature translation termination mutations (nonsense mutations) have been described in the human α- and β-globin genes. Studies on mRNA isolated from patients with β 0 -thalassemia have shown that for both the β-17 and the β-39 mutations less than normal levels of β-globin mRNA accumulate in peripheral blood cells. (The codon at which the mutation occurs designates the name of the mutation; there are 146 codons in human β-globin mRNA). In vitro studies using the cloned β-39 gene have reproduced this effect in a heterologous transfection system and have suggested that the defect resides in intranuclear metabolism. The authors have asked if this phenomenon of decreased mRNA accumulation is a general property of nonsense mutations and if the effect depends on the location or the type of mutation. Toward this end, they have studied the effect of five nonsense mutations and two missense mutations on the expression of human β-globin mRNA in a heterologous transfection system. In all cases studied, the presence of a translation termination codon correlates with a decrease in the steady-state level of mRNA. The data suggest that the metabolism of a mammalian mRNA is affected by the presence of a mutation that affects translation

β-glucuronidase use as a single internal control gene may confound analysis in FMR1 mRNA toxicity studies.

Science.gov (United States)

Kraan, Claudine M; Cornish, Kim M; Bui, Quang M; Li, Xin; Slater, Howard R; Godler, David E

2018-01-01

Relationships between Fragile X Mental Retardation 1 (FMR1) mRNA levels in blood and intragenic FMR1 CGG triplet expansions support the pathogenic role of RNA gain of function toxicity in premutation (PM: 55-199 CGGs) related disorders. Real-time PCR (RT-PCR) studies reporting these findings normalised FMR1 mRNA level to a single internal control gene called β-glucuronidase (GUS). This study evaluated FMR1 mRNA-CGG correlations in 33 PM and 33 age- and IQ-matched control females using three normalisation strategies in peripheral blood mononuclear cells (PBMCs): (i) GUS as a single internal control; (ii) the mean of GUS, Eukaryotic Translation Initiation Factor 4A2 (EIF4A2) and succinate dehydrogenase complex flavoprotein subunit A (SDHA); and (iii) the mean of EIF4A2 and SDHA (with no contribution from GUS). GUS mRNA levels normalised to the mean of EIF4A2 and SDHA mRNA levels and EIF4A2/SDHA ratio were also evaluated. FMR1mRNA level normalised to the mean of EIF4A2 and SDHA mRNA levels, with no contribution from GUS, showed the most significant correlation with CGG size and the greatest difference between PM and control groups (p = 10-11). Only 15% of FMR1 mRNA PM results exceeded the maximum control value when normalised to GUS, compared with over 42% when normalised to the mean of EIF4A2 and SDHA mRNA levels. Neither GUS mRNA level normalised to the mean RNA levels of EIF4A2 and SDHA, nor to the EIF4A2/SDHA ratio were correlated with CGG size. However, greater variability in GUS mRNA levels were observed for both PM and control females across the full range of CGG repeat as compared to the EIF4A2/SDHA ratio. In conclusion, normalisation with multiple control genes, excluding GUS, can improve assessment of the biological significance of FMR1 mRNA-CGG size relationships.

Quantification in single photon emission computed tomography (SPECT)

International Nuclear Information System (INIS)

Buvat, Irene

2005-01-01

The objective of this lecture is to understand the possibilities and limitations of the quantitative analysis of single photon emission computed tomography (SPECT) images. It is also to identify the conditions to be fulfilled to obtain reliable quantitative measurements from images. Content: 1 - Introduction: Quantification in emission tomography - definition and challenges; quantification biasing phenomena; 2 - quantification in SPECT, problems and correction methods: Attenuation, scattering, un-stationary spatial resolution, partial volume effect, movement, tomographic reconstruction, calibration; 3 - Synthesis: actual quantification accuracy; 4 - Beyond the activity concentration measurement

Development of a VHH-Based Erythropoietin Quantification Assay

DEFF Research Database (Denmark)

Kol, Stefan; Beuchert Kallehauge, Thomas; Adema, Simon

2015-01-01

Erythropoietin (EPO) quantification during cell line selection and bioreactor cultivation has traditionally been performed with ELISA or HPLC. As these techniques suffer from several drawbacks, we developed a novel EPO quantification assay. A camelid single-domain antibody fragment directed against...... human EPO was evaluated as a capturing antibody in a label-free biolayer interferometry-based quantification assay. Human recombinant EPO can be specifically detected in Chinese hamster ovary cell supernatants in a sensitive and pH-dependent manner. This method enables rapid and robust quantification...

Quantification procedures in micro X-ray fluorescence analysis

International Nuclear Information System (INIS)

Kanngiesser, Birgit

2003-01-01

For the quantification in micro X-ray fluorescence analysis standardfree quantification procedures have become especially important. An introduction to the basic concepts of these quantification procedures is given, followed by a short survey of the procedures which are available now and what kind of experimental situations and analytical problems are addressed. The last point is extended by the description of an own development for the fundamental parameter method, which renders the inclusion of nonparallel beam geometries possible. Finally, open problems for the quantification procedures are discussed

Relative quantification of protein-protein interactions using a dual luciferase reporter pull-down assay system.

Directory of Open Access Journals (Sweden)

Shuaizheng Jia

Full Text Available The identification and quantitative analysis of protein-protein interactions are essential to the functional characterization of proteins in the post-proteomics era. The methods currently available are generally time-consuming, technically complicated, insensitive and/or semi-quantitative. The lack of simple, sensitive approaches to precisely quantify protein-protein interactions still prevents our understanding of the functions of many proteins. Here, we develop a novel dual luciferase reporter pull-down assay by combining a biotinylated Firefly luciferase pull-down assay with a dual luciferase reporter assay. The biotinylated Firefly luciferase-tagged protein enables rapid and efficient isolation of a putative Renilla luciferase-tagged binding protein from a relatively small amount of sample. Both of these proteins can be quantitatively detected using the dual luciferase reporter assay system. Protein-protein interactions, including Fos-Jun located in the nucleus; MAVS-TRAF3 in cytoplasm; inducible IRF3 dimerization; viral protein-regulated interactions, such as MAVS-MAVS and MAVS-TRAF3; IRF3 dimerization; and protein interaction domain mapping, are studied using this novel assay system. Herein, we demonstrate that this dual luciferase reporter pull-down assay enables the quantification of the relative amounts of interacting proteins that bind to streptavidin-coupled beads for protein purification. This study provides a simple, rapid, sensitive, and efficient approach to identify and quantify relative protein-protein interactions. Importantly, the dual luciferase reporter pull-down method will facilitate the functional determination of proteins.

Standardless quantification by parameter optimization in electron probe microanalysis

International Nuclear Information System (INIS)

Limandri, Silvina P.; Bonetto, Rita D.; Josa, Víctor Galván; Carreras, Alejo C.; Trincavelli, Jorge C.

2012-01-01

A method for standardless quantification by parameter optimization in electron probe microanalysis is presented. The method consists in minimizing the quadratic differences between an experimental spectrum and an analytical function proposed to describe it, by optimizing the parameters involved in the analytical prediction. This algorithm, implemented in the software POEMA (Parameter Optimization in Electron Probe Microanalysis), allows the determination of the elemental concentrations, along with their uncertainties. The method was tested in a set of 159 elemental constituents corresponding to 36 spectra of standards (mostly minerals) that include trace elements. The results were compared with those obtained with the commercial software GENESIS Spectrum® for standardless quantification. The quantifications performed with the method proposed here are better in the 74% of the cases studied. In addition, the performance of the method proposed is compared with the first principles standardless analysis procedure DTSA for a different data set, which excludes trace elements. The relative deviations with respect to the nominal concentrations are lower than 0.04, 0.08 and 0.35 for the 66% of the cases for POEMA, GENESIS and DTSA, respectively. - Highlights: ► A method for standardless quantification in EPMA is presented. ► It gives better results than the commercial software GENESIS Spectrum. ► It gives better results than the software DTSA. ► It allows the determination of the conductive coating thickness. ► It gives an estimation for the concentration uncertainties.

Clinical values of AFP, GPC3 mRNA in peripheral blood for prediction of hepatocellular carcinoma recurrence following OLT: AFP, GPC3 mRNA for prediction of HCC.

Science.gov (United States)

Wang, Yuliang; Shen, Zhongyang; Zhu, Zhijun; Han, Ruifa; Huai, Mingsheng

2011-03-01

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Annually, about 200,000 patients died of HCC in China. Liver transplantation (LT) holds great theoretical appeal in treating HCC. However, the high recurrence rate after transplantation is the most important limiting factor for long-term survival. To assess the value of alpha-fetoprotein (AFP) messenger RNA (mRNA), Glypican-3 (GPC3) mRNA-expressing cells in the peripheral blood (PB) for prediction of HCC recurrence following orthotopic liver transplantation (OLT). 29 patients with HCC who underwent OLT with a minimum clinical follow-up of 12 months were included in this retrospective study. We detected AFP mRNA, GPC3 mRNA-expressing cells in the PB by TaqMan real-time reverse transcriptase-polymerase chain reaction (RT-PCR), pre-, intra- and post-operatively. The early recurrence of patients was evaluated. 8 (28%), 15 (52%), and 9 (31%) patients had AFP mRNA detected pre-, intra-, and post-operatively, respectively. With 12 months of follow-up, HCC recurred in 7 (24%) patients. Univariate analysis revealed that positive pre- and post-operative AFP mRNA, TNM stage as well as vascular invasion were significant predictors for the HCC recurrence. Multivariate analysis revealed that being positive for AFP mRNA pre-operatively remained a significant risk factor for HCC recurrence after OLT. GPC3 mRNA was expressed in all PB samples. There was no significant difference in the expression levels of GPC3 mRNA between the HCC and control groups. There were no significant differences in GPC3 mRNA expression values between those patients with and without tumor recurrence. The pre-operative detection of circulating AFP mRNA-expressing cells could be a useful predictor for HCC recurrence following OLT. GPC3 mRNA-expressing cells in PB seem to have no diagnostic value.

Comparison of machine learning and semi-quantification algorithms for (I123)FP-CIT classification: the beginning of the end for semi-quantification?

Science.gov (United States)

Taylor, Jonathan Christopher; Fenner, John Wesley

2017-11-29

performance was lower for the local database than the research database for both semi-quantitative and machine learning algorithms. However, for both databases, the machine learning methods generated equal or higher mean accuracies (with lower variance) than any of the semi-quantification approaches. The gain in performance from using machine learning algorithms as compared to semi-quantification was relatively small and may be insufficient, when considered in isolation, to offer significant advantages in the clinical context.

Presence of High-Risk HPV mRNA in Relation to Future High-Grade Lesions among High-Risk HPV DNA Positive Women with Minor Cytological Abnormalities.

Directory of Open Access Journals (Sweden)

Hanna Johansson

Full Text Available Continuous expression of E6- and E7-oncogenes of high-risk human papillomavirus (HPV types is necessary for the development and maintenance of the dysplastic phenotype. The aim of the study was to determine the sensitivity and specificity of the APTIMA HPV mRNA assay (Hologic in predicting future development of high-grade cervical intraepithelial neoplasia (CIN among high-risk HPV-DNA-positive women with atypical squamous cells of undetermined significance (ASCUS or low-grade squamous epithelial lesion (LSIL cytology.Archived SurePath cervical samples of women ≥ 35 years of age with high-risk HPV DNA-positive ASCUS (n = 211 or LSIL, (n = 131 were tested for the presence of high-risk HPV E6/E7 mRNA using the APTIMA HPV assay, and the women were monitored for development of histopathologically verified CIN2+.Twenty-nine percent (61/211 of the women in the ASCUS group, and 34.3% (45/131 in the LSIL group developed CIN2+ within 4.5 years of follow-up. The prevalence of HPV mRNA was 90.0% (95% CI 85.9-94.0 among women with ASCUS and 95.4% (95% CI 91.8-99.0 among women with LSIL. The presence of HPV E6/E7 mRNA was associated with future development of CIN2+ among women with ASCUS and LSIL (p=0.02. The mRNA assay demonstrated high sensitivity in predicting future CIN2+ and CIN3 for index ASCUS (96.7%; 95% CI 87.6-99.4 and 100%; 95% CI 82.2-100, respectively and LSIL (97.8%, 95% CI 86.8-99.9 and 100%, 95% CI 79.9-100, respectively. The corresponding specificity was low, 12.7% (95% CI 7.9-19.3 and 5.8% (95% CI 2.2-13.6, for future CIN2+, respectively. The negative predictive value of the HPV mRNA assay for detecting future CIN3 was 100%, since no mRNA-negative woman developed CIN3 (0/27 as compared to 13.6% (43/315 of the mRNA-positive women (p = 0.03.The APTIMA mRNA assay demonstrated high sensitivity but low specificity in predicting future CIN2+ among women with minor cytological abnormalities. The assay had high negative predictive value for future

Presence of High-Risk HPV mRNA in Relation to Future High-Grade Lesions among High-Risk HPV DNA Positive Women with Minor Cytological Abnormalities

Science.gov (United States)

Johansson, Hanna; Bjelkenkrantz, Kaj; Darlin, Lotten; Dilllner, Joakim; Forslund, Ola

2015-01-01

Objective Continuous expression of E6- and E7-oncogenes of high-risk human papillomavirus (HPV) types is necessary for the development and maintenance of the dysplastic phenotype. The aim of the study was to determine the sensitivity and specificity of the APTIMA HPV mRNA assay (Hologic) in predicting future development of high-grade cervical intraepithelial neoplasia (CIN) among high-risk HPV-DNA-positive women with atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous epithelial lesion (LSIL) cytology. Methods Archived SurePath cervical samples of women ≥ 35 years of age with high-risk HPV DNA-positive ASCUS (n = 211) or LSIL, (n = 131) were tested for the presence of high-risk HPV E6/E7 mRNA using the APTIMA HPV assay, and the women were monitored for development of histopathologically verified CIN2+. Results Twenty-nine percent (61/211) of the women in the ASCUS group, and 34.3% (45/131) in the LSIL group developed CIN2+ within 4.5 years of follow-up. The prevalence of HPV mRNA was 90.0% (95% CI 85.9-94.0) among women with ASCUS and 95.4% (95% CI 91.8-99.0) among women with LSIL. The presence of HPV E6/E7 mRNA was associated with future development of CIN2+ among women with ASCUS and LSIL (p=0.02). The mRNA assay demonstrated high sensitivity in predicting future CIN2+ and CIN3 for index ASCUS (96.7%; 95% CI 87.6-99.4 and 100%; 95% CI 82.2-100, respectively) and LSIL (97.8%, 95% CI 86.8-99.9 and 100%, 95% CI 79.9-100, respectively). The corresponding specificity was low, 12.7% (95% CI 7.9-19.3) and 5.8% (95% CI 2.2-13.6), for future CIN2+, respectively. The negative predictive value of the HPV mRNA assay for detecting future CIN3 was 100%, since no mRNA-negative woman developed CIN3 (0/27) as compared to 13.6% (43/315) of the mRNA-positive women (p = 0.03). Conclusion The APTIMA mRNA assay demonstrated high sensitivity but low specificity in predicting future CIN2+ among women with minor cytological abnormalities. The assay had

Primary induction of vitellogenin mRNA in the rooster by 17beta-estradiol.

Science.gov (United States)

Burns, A T; Deeley, R G; Gordon, J I; Udell, D S; Mullinix, K P; Goldberger, R F

1978-01-01

We have studied the kinetics of vitellogenin mRNA accumulation in rooster liver after a primary injection of 17beta-estradiol. The levels of vitellogenin mRNA have been determined both by hybridization of total cellular RNA to vitellogenin cDNA and by translation of vitellogenin mRNA in a wheat germ cell-free system. The results obtained by both methods of analysis are in good agreement and indicate that vitellogenin mRNA is present in the liver of normal roosters at a level of 0-5 molecules per liver cell and increases in amount during the 3 days following injection of estrogen, reaching a level of almost 6000 molecules per cell at the peak of the response. The level of vitellogenin mRNA declined exponentially during the next 14 days with a half-life of 29 hr, reaching a level of less than 10 molecules per cell at 17 days after injection of the hormone. The levels of vitellogenin mRNA after stimulation with estrogen have been correlated with the in vivo rate of synthesis of the vitellogenin polypeptide. The results indicate that the rate of vitellogenin synthesis is closely correlated with the level of vitellogenin mRNA. On the basis of these findings, we conclude that vitellogenin mRNA does not exist in the liver in an untranslated form after withdrawal from estrogen. PMID:273910

Uncertainty Quantification in Alchemical Free Energy Methods.

Science.gov (United States)

Bhati, Agastya P; Wan, Shunzhou; Hu, Yuan; Sherborne, Brad; Coveney, Peter V

2018-05-02

Alchemical free energy methods have gained much importance recently from several reports of improved ligand-protein binding affinity predictions based on their implementation using molecular dynamics simulations. A large number of variants of such methods implementing different accelerated sampling techniques and free energy estimators are available, each claimed to be better than the others in its own way. However, the key features of reproducibility and quantification of associated uncertainties in such methods have barely been discussed. Here, we apply a systematic protocol for uncertainty quantification to a number of popular alchemical free energy methods, covering both absolute and relative free energy predictions. We show that a reliable measure of error estimation is provided by ensemble simulation-an ensemble of independent MD simulations-which applies irrespective of the free energy method. The need to use ensemble methods is fundamental and holds regardless of the duration of time of the molecular dynamics simulations performed.

In vivo MRS metabolite quantification using genetic optimization

Science.gov (United States)

Papakostas, G. A.; Karras, D. A.; Mertzios, B. G.; van Ormondt, D.; Graveron-Demilly, D.

2011-11-01

The in vivo quantification of metabolites' concentrations, revealed in magnetic resonance spectroscopy (MRS) spectra, constitutes the main subject under investigation in this work. Significant contributions based on artificial intelligence tools, such as neural networks (NNs), with good results have been presented lately but have shown several drawbacks, regarding their quantification accuracy under difficult conditions. A general framework that encounters the quantification procedure as an optimization problem, which is solved using a genetic algorithm (GA), is proposed in this paper. Two different lineshape models are examined, while two GA configurations are applied on artificial data. Moreover, the introduced quantification technique deals with metabolite peaks' overlapping, a considerably difficult situation occurring under real conditions. Appropriate experiments have proved the efficiency of the introduced methodology, in artificial MRS data, by establishing it as a generic metabolite quantification procedure.

In vivo MRS metabolite quantification using genetic optimization

International Nuclear Information System (INIS)

Papakostas, G A; Mertzios, B G; Karras, D A; Van Ormondt, D; Graveron-Demilly, D

2011-01-01

The in vivo quantification of metabolites' concentrations, revealed in magnetic resonance spectroscopy (MRS) spectra, constitutes the main subject under investigation in this work. Significant contributions based on artificial intelligence tools, such as neural networks (NNs), with good results have been presented lately but have shown several drawbacks, regarding their quantification accuracy under difficult conditions. A general framework that encounters the quantification procedure as an optimization problem, which is solved using a genetic algorithm (GA), is proposed in this paper. Two different lineshape models are examined, while two GA configurations are applied on artificial data. Moreover, the introduced quantification technique deals with metabolite peaks' overlapping, a considerably difficult situation occurring under real conditions. Appropriate experiments have proved the efficiency of the introduced methodology, in artificial MRS data, by establishing it as a generic metabolite quantification procedure

Lower glutamic acid decarboxylase 65kD mRNA and protein levels in the prefrontal cortex in schizoaffective disorder but not schizophrenia

Science.gov (United States)

Glausier, JR; Kimoto, S; Fish, KN; Lewis, DA

2014-01-01

Background Altered GABA signaling in the prefrontal cortex (PFC) has been associated with cognitive dysfunction in schizophrenia and schizoaffective disorder. PFC levels of the GABA-synthesizing enzyme glutamic acid decarboxylase 67kD (GAD67) has been consistently reported to be lower in these disorders, but the status of the second GABA-synthesizing enzyme, GAD65, remains unclear. Methods GAD65 mRNA levels were quantified in PFC area 9 by quantitative polymerase chain reaction from 62 subjects with schizophrenia or schizoaffective disorder and 62 matched healthy comparison subjects. GAD65 relative protein levels were quantified in a subset of subject pairs by confocal immunofluorescence microscopy. Results Mean GAD65 mRNA levels were 13.6% lower in schizoaffective disorder subjects, but did not differ in schizophrenia subjects, relative to their matched healthy comparison subjects. In the subjects with schizoaffective disorder, mean GAD65 protein levels were 19.4% lower and were correlated with GAD65 mRNA levels. Lower GAD65 mRNA and protein measures within schizoaffective disorder subjects was not attributable to factors commonly comorbid with the diagnosis. Conclusions In concert with previous studies, these findings suggest that schizoaffective disorder is associated with lower levels of both GAD65 and GAD67 mRNA and protein in the PFC, whereas subjects with schizophrenia have lower mean levels of only GAD67 mRNA and protein. Because cognitive function is generally better preserved in subjects with schizoaffective disorder relative to subjects with schizophrenia, these findings may support an interpretation that GAD65 down-regulation provides a homeostatic response complementary to GAD67 down-regulation expression that serves to reduce inhibition in the face of lower PFC network activity. PMID:24993056

Reprogramming the Dynamin 2 mRNA by Spliceosome-mediated RNA Trans-splicing

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Delphine Trochet

2016-01-01

Full Text Available Dynamin 2 (DNM2 is a large GTPase, ubiquitously expressed, involved in membrane trafficking and regulation of actin and microtubule cytoskeletons. DNM2 mutations cause autosomal dominant centronuclear myopathy which is a rare congenital myopathy characterized by skeletal muscle weakness and histopathological features including nuclear centralization in absence of regeneration. No curative treatment is currently available for the DNM2-related autosomal dominant centronuclear myopathy. In order to develop therapeutic strategy, we evaluated here the potential of Spliceosome-Mediated RNA Trans-splicing technology to reprogram the Dnm2-mRNA in vitro and in vivo in mice. We show that classical 3′-trans-splicing strategy cannot be considered as accurate therapeutic strategy regarding toxicity of the pre-trans-splicing molecules leading to low rate of trans-splicing in vivo. Thus, we tested alternative strategies devoted to prevent this toxicity and enhance frequency of trans-splicing events. We succeeded to overcome the toxicity through a 5′-trans-splicing strategy which also allows detection of trans-splicing events at mRNA and protein levels in vitro and in vivo. These results suggest that the Spliceosome-Mediated RNA Trans-splicing strategy may be used to reprogram mutated Dnm2-mRNA but highlight the potential toxicity linked to the molecular tools which have to be carefully investigated during preclinical development.

Changes in rRNA levels during stress invalidates results from mRNA blotting: Fluorescence in situ rRNA hybridization permits renormalization for estimation of cellular mRNA levels

DEFF Research Database (Denmark)

Hansen, M.C.; Nielsen, A.K.; Molin, Søren

2001-01-01

obtained by these techniques are compared between experiments in which differences in growth rates, strains, or stress treatments occur, the normalization procedure may have a significant impact on the results. In this report we present a solution to the normalization problem in RNA slot blotting...... the relative level of rRNA per cell, and slot blotting to rRNA probes, which estimates the level of rRNA per extracted total RNA, the amount of RNA per cell was calculated in a series of heat shock experiments with the gram-positive bacterium Lactococcus lactis. It was found that the level of rRNA per cell...... decreased to 30% in the course of the heat shock. This lowered ribosome level led to a decrease in the total RNA content, resulting in a gradually increasing overestimation of the mRNA levels throughout the experiment. Using renormalized cellular mRNA levels, the HrcA-mediated regulation of the genes...

The effects of dietary Myrtle (Myrtus communis) on skin mucus immune parameters and mRNA levels of growth, antioxidant and immune related genes in zebrafish (Danio rerio).

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Safari, Roghieh; Hoseinifar, Seyed Hossein; Van Doan, Hien; Dadar, Maryam

2017-07-01

Myrtle (Myrtus communis L., Myrtaceae) is a significant plant which naturally distributed around the globe. Although numerous studies have demonstrated the benefits of myrtle in different species, studies using the oral route are rare in the literature. In the present study, we evaluated the effect of myrtle intake on the antioxidant, immune, appetite and growth related genes as well as mucosal immune responses in zebrafish (Danio rerio) model. Zebrafish were fed control or myrtle (5, 10 and 20 g kg -1 myrtle) supplemented diets for sixty days. The results showed that, oral administration of Myrtle significantly improved mucosal immune responses (the activity of lysozyme, total Ig and protease). Furthermore, fish fed 20 g kg -1 showed remarkably higher antioxidant (sod and cat) enzymes gene expression compared other treatment. There were significant difference between myrtle fed fish and control group regarding tnf-alpha and lyz expression. Also, evaluation of growth (gh and igf1) related genes revealed remarkable upregulation in 20 g kg -1 myrtle treatment compared other myrtle treatments and control group. Similar results was observed regarding the mRNA levels of appetite related genes (ghrl) in zebrafish fed 20 g kg -1 myrtle. The present results indicated that dietary administration of myrtle improved mucosal immune parameters and altered mRNA levels of selected genes. These results on zebrafish model also highlights the potential use of Myrtle supplements as additive in human diets. Copyright © 2017 Elsevier Ltd. All rights reserved.

High ALK mRNA expression has a negative prognostic significance in rhabdomyosarcoma

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Bonvini, P; Zin, A; Alaggio, R; Pawel, B; Bisogno, G; Rosolen, A

2013-01-01

Background: Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in cancer, but its clinical and functional importance remain controversial. Mutation or amplification of ALK, as well as its expression levels assessed by conventional immunohistochemistry methods, has been linked to prognosis in cancer, although with potential bias because of the semi-quantitative approaches. Herein, we measured ALK mRNA expression in rhabdomyosarcoma (RMS) and determined its clinical impact on patients' stratification and outcome. Methods: Specimens were obtained from RMS patients and cell lines, and ALK expression was analysed by quantitative RT–PCR, western blotting, IHC, and copy number analysis. Results: High ALK mRNA expression was detected in the vast majority of PAX3/7-FOXO1-positive tumours, whereas PAX3/7-FOXO1-negative RMS displayed considerably lower amounts of both mRNA and protein. Notably, ALK mRNA distinguished unfavourable PAX3/7-FOXO1-positive tumours from PAX3/7-FOXO1-negative RMS (Ptumour size (PALK mRNA levels were of prognostic relevance by Cox univariate regression analysis and correlated with increased risk of relapse (P=0.001) and survival (P=0.01), whereas by multivariate analysis elevated ALK mRNA expression resulted a negative prognostic marker when clinical stage was not included. Conclusion: Quantitative assessment of ALK mRNA expression helps to improve risk stratification of RMS patients and identifies tumours with adverse biological characteristics and aggressive behaviour. PMID:24149177

Quantification of taurine in energy drinks using ¹H NMR.

Science.gov (United States)

Hohmann, Monika; Felbinger, Christine; Christoph, Norbert; Wachter, Helmut; Wiest, Johannes; Holzgrabe, Ulrike

2014-05-01

The consumption of so called energy drinks is increasing, especially among adolescents. These beverages commonly contain considerable amounts of the amino sulfonic acid taurine, which is related to a magnitude of various physiological effects. The customary method to control the legal limit of taurine in energy drinks is LC-UV/vis with postcolumn derivatization using ninhydrin. In this paper we describe the quantification of taurine in energy drinks by (1)H NMR as an alternative to existing methods of quantification. Variation of pH values revealed the separation of a distinct taurine signal in (1)H NMR spectra, which was applied for integration and quantification. Quantification was performed using external calibration (R(2)>0.9999; linearity verified by Mandel's fitting test with a 95% confidence level) and PULCON. Taurine concentrations in 20 different energy drinks were analyzed by both using (1)H NMR and LC-UV/vis. The deviation between (1)H NMR and LC-UV/vis results was always below the expanded measurement uncertainty of 12.2% for the LC-UV/vis method (95% confidence level) and at worst 10.4%. Due to the high accordance to LC-UV/vis data and adequate recovery rates (ranging between 97.1% and 108.2%), (1)H NMR measurement presents a suitable method to quantify taurine in energy drinks. Copyright © 2013 Elsevier B.V. All rights reserved.

mRNA transfection of mouse and human neural stem cell cultures.

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Samuel McLenachan

Full Text Available The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

mRNA Transfection of Mouse and Human Neural Stem Cell Cultures

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McLenachan, Samuel; Zhang, Dan; Palomo, Ana Belén Alvarez; Edel, Michael J.; Chen, Fred K.

2013-01-01

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages. PMID:24386231

mRNA transfection of mouse and human neural stem cell cultures.

Science.gov (United States)

McLenachan, Samuel; Zhang, Dan; Palomo, Ana Belén Alvarez; Edel, Michael J; Chen, Fred K

2013-01-01

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

Protein Structure and the Sequential Structure of mRNA

DEFF Research Database (Denmark)

Brunak, Søren; Engelbrecht, Jacob

1996-01-01

entries in the Brookhaven Protein Data Bank produced 719 protein chains with matching mRNA sequence, amino acid sequence, and secondary structure assignment, By neural network analysis, we found strong signals in mRNA sequence regions surrounding helices and sheets, These signals do not originate from......A direct comparison of experimentally determined protein structures and their corresponding protein coding mRNA sequences has been performed, We examine whether real world data support the hypothesis that clusters of rare codons correlate with the location of structural units in the resulting...... protein, The degeneracy of the genetic code allows for a biased selection of codons which may control the translational rate of the ribosome, and may thus in vivo have a catalyzing effect on the folding of the polypeptide chain, A complete search for GenBank nucleotide sequences coding for structural...

GABAergic Neurons in the Rat Medial Septal Complex Express Relaxin-3 Receptor (RXFP3 mRNA

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Hector Albert-Gascó

2018-01-01

Full Text Available The medial septum (MS complex modulates hippocampal function and related behaviors. Septohippocampal projections promote and control different forms of hippocampal synchronization. Specifically, GABAergic and cholinergic projections targeting the hippocampal formation from the MS provide bursting discharges to promote theta rhythm, or tonic activity to promote gamma oscillations. In turn, the MS is targeted by ascending projections from the hypothalamus and brainstem. One of these projections arises from the nucleus incertus in the pontine tegmentum, which contains GABA neurons that co-express the neuropeptide relaxin-3 (Rln3. Both stimulation of the nucleus incertus and septal infusion of Rln3 receptor agonist peptides promotes hippocampal theta rhythm. The Gi/o-protein-coupled receptor, relaxin-family peptide receptor 3 (RXFP3, is the cognate receptor for Rln3 and identification of the transmitter phenotype of neurons expressing RXFP3 in the septohippocampal system can provide further insights into the role of Rln3 transmission in the promotion of septohippocampal theta rhythm. Therefore, we used RNAscope multiplex in situ hybridization to characterize the septal neurons expressing Rxfp3 mRNA in the rat. Our results demonstrate that Rxfp3 mRNA is abundantly expressed in vesicular GABA transporter (vGAT mRNA- and parvalbumin (PV mRNA-positive GABA neurons in MS, whereas ChAT mRNA-positive acetylcholine neurons lack Rxfp3 mRNA. Approximately 75% of Rxfp3 mRNA-positive neurons expressed vGAT mRNA (and 22% were PV mRNA-positive, while the remaining 25% expressed Rxfp3 mRNA only, consistent with a potential glutamatergic phenotype. Similar proportions were observed in the posterior septum. The occurrence of RXFP3 in PV-positive GABAergic neurons gives support to a role for the Rln3-RXFP3 system in septohippocampal theta rhythm.

Kinetics of lipid-nanoparticle-mediated intracellular mRNA delivery and function

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Zhdanov, Vladimir P.

2017-10-01

mRNA delivery into cells forms the basis for one of the new and promising ways to treat various diseases. Among suitable carriers, lipid nanoparticles (LNPs) with a size of about 100 nm are now often employed. Despite high current interest in this area, the understanding of the basic details of LNP-mediated mRNA delivery and function is limited. To clarify the kinetics of mRNA release from LNPs, the author uses three generic models implying (i) exponential, (ii) diffusion-controlled, and (iii) detachment-controlled kinetic regimes, respectively. Despite the distinct differences in these kinetics, the associated transient kinetics of mRNA translation to the corresponding protein and its degradation are shown to be not too sensitive to the details of the mRNA delivery by LNPs (or other nanocarriers). In addition, the author illustrates how this protein may temporarily influence the expression of one gene or a few equivalent genes. The analysis includes positive or negative regulation of the gene transcription via the attachment of the protein without or with positive or negative feedback in the gene expression. Stable, bistable, and oscillatory schemes have been scrutinized in this context.

Regulation of mRNA Translation Is a Novel Mechanism for Phthalate Toxicity.

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Jun Ling

Full Text Available Phthalates are a group of plasticizers that are widely used in many consumer products and medical devices, thus generating a huge burden to human health. Phthalates have been known to cause a number of developmental and reproductive disorders functioning as endocrine modulators. They are also involved in carcinogenesis with mechanisms less understood. To further understand the molecular mechanisms of phthalate toxicity, in this study we reported a new effect of phthalates on mRNA translation/protein synthesis, a key regulatory step of gene expression. Butyl benzyl phthalate (BBP was found to directly inhibit mRNA translation in vitro but showed a complicated pattern of affecting mRNA translation in cells. In human kidney embryonic cell (HEK-293T, BBP increased cap-dependent mRNA translation at lower concentrations but showed inhibitory effect at higher concentrations. Cap-independent translation was not affected. On the other hand, mono (2-ethylhexyl phthalate (MEHP as a major metabolite of another important phthalate di (2-ethylhexyl phthalate (DEHP inhibited both can-dependent and -independent mRNA translation in vivo. In contrast, BBP and MEHP exhibited an overall promoting effect on mRNA translation in cancer cells. Mechanistic studies identified that the level and phosphorylation of eIF4E-BP (eIF4E binding protein and the amount of eIF4GI in eIF4F complex were altered in accordance with the effect of BBP on translation. BBP was also identified to directly bind to eIF4E, providing a further mechanism underlying the regulation of mRNA by phthalate. At the cellular level BBP inhibited normal cell growth but slightly promoted cancer cells (HT29 growth. Overall, this study provides the first evidence that phthalates can directly regulate mRNA translation as a novel mechanism to mediate their biological toxicities.

Polyomavirus BK replication in renal transplant recipients: combined monitoring of viremia and VP1 mRNA in urine

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Sara Astegiano

2010-06-01

Full Text Available Introduction. Human polyomavirus BK (BKV is worldwide distributed, with a seroprevalence rate of 70–90% in the adults. Following primary infection, BK remains latent in the renourinary tract as the epidemiologically most relevant latency site, and in B cell, brain, spleen and probably other tissues. Reactivation may occur in both immunocompetent subjects and immunocompromised patients. In renal transplantation, in the context of intense immunosuppression, viral replication may determine BKV-associated nephropathy (BKVAN with interstitial nephritis and/or ureteral stenosis in 1–10% of the patients and leading to graft failure and return to haemodialysis in 30 to 80% of the cases (5. Screening of BKV replication represents the basic strategy to predict early the onset of BKVAN and may allow for earlier intervention with reduced allograft loss (3, 4. Nowadays, replication of BKV is monitored by quantification of BKV-DNA in serum and urine (2. The aim of this study was to evaluated the role of BKV VP1 mRNA in urine as a marker of viral replication in renal transplant recipients.

Virus detection and quantification using electrical parameters

Science.gov (United States)

Ahmad, Mahmoud Al; Mustafa, Farah; Ali, Lizna M.; Rizvi, Tahir A.

2014-10-01

Here we identify and quantitate two similar viruses, human and feline immunodeficiency viruses (HIV and FIV), suspended in a liquid medium without labeling, using a semiconductor technique. The virus count was estimated by calculating the impurities inside a defined volume by observing the change in electrical parameters. Empirically, the virus count was similar to the absolute value of the ratio of the change of the virus suspension dopant concentration relative to the mock dopant over the change in virus suspension Debye volume relative to mock Debye volume. The virus type was identified by constructing a concentration-mobility relationship which is unique for each kind of virus, allowing for a fast (within minutes) and label-free virus quantification and identification. For validation, the HIV and FIV virus preparations were further quantified by a biochemical technique and the results obtained by both approaches corroborated well. We further demonstrate that the electrical technique could be applied to accurately measure and characterize silica nanoparticles that resemble the virus particles in size. Based on these results, we anticipate our present approach to be a starting point towards establishing the foundation for label-free electrical-based identification and quantification of an unlimited number of viruses and other nano-sized particles.

Impact of target mRNA structure on siRNA silencing efficiency: A large-scale study.

Science.gov (United States)

Gredell, Joseph A; Berger, Angela K; Walton, S Patrick

2008-07-01

The selection of active siRNAs is generally based on identifying siRNAs with certain sequence and structural properties. However, the efficiency of RNA interference has also been shown to depend on the structure of the target mRNA, primarily through studies using exogenous transcripts with well-defined secondary structures in the vicinity of the target sequence. While these studies provide a means for examining the impact of target sequence and structure independently, the predicted secondary structures for these transcripts are often not reflective of structures that form in full-length, native mRNAs where interactions can occur between relatively remote segments of the mRNAs. Here, using a combination of experimental results and analysis of a large dataset, we demonstrate that the accessibility of certain local target structures on the mRNA is an important determinant in the gene silencing ability of siRNAs. siRNAs targeting the enhanced green fluorescent protein were chosen using a minimal siRNA selection algorithm followed by classification based on the predicted minimum free energy structures of the target transcripts. Transfection into HeLa and HepG2 cells revealed that siRNAs targeting regions of the mRNA predicted to have unpaired 5'- and 3'-ends resulted in greater gene silencing than regions predicted to have other types of secondary structure. These results were confirmed by analysis of gene silencing data from previously published siRNAs, which showed that mRNA target regions unpaired at either the 5'-end or 3'-end were silenced, on average, approximately 10% more strongly than target regions unpaired in the center or primarily paired throughout. We found this effect to be independent of the structure of the siRNA guide strand. Taken together, these results suggest minimal requirements for nucleation of hybridization between the siRNA guide strand and mRNA and that both mRNA and guide strand structure should be considered when choosing candidate si

Simultaneous isolation of mRNA and native protein from minute samples of cells

DEFF Research Database (Denmark)

Petersen, Tonny Studsgaard; Andersen, Claus Yding

2014-01-01

Precious biological samples often lack a sufficient number of cells for multiple procedures, such as extraction of mRNA while maintaining protein in a non-denatured state suitable for subsequent characterization. Here we present a new method for the simultaneous purification of mRNA and native...... in their native state for traditional protein assays. We validated the procedure using neonatal rat ovaries and small numbers of human granulosa cells, demonstrating the extraction of mRNA suitable for gene expression analysis with simultaneous isolation of native proteins suitable for downstream characterization...... proteins from samples containing small numbers of cells. Our approach utilizes oligodeoxythymidylate [oligo(dT)25]-coated paramagnetic beads in an optimized reaction buffer to isolate mRNA comparable in quantity and quality to mRNA isolated with existing methods, while maintaining the proteins...

Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription

International Nuclear Information System (INIS)

Dreyfuss, G.; Adam, S.A.; Choi, Y.D.

1984-01-01

Exposure of intact cells to UV light brings about cross-linking of polyadenylated mRNA to a set of cytoplasmic proteins which are in direct contact with the mRNA in vivo. Substantial amounts of an additional protein of molecular weight 38,000 become cross-linked to the mRNA when cells are treated with inhibitors of mRNA synthesis (actinomycin D, camptothecin, and 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole) or after infection with vesicular stomatitis virus. Cordycepin, which inhibits polyadenylation but not mRNA synthesis, has no such effect. Inhibitors of protein synthesis and of rRNA synthesis are also without effect on 38K cross-linking to mRNA. The onset of the effect of inhibitors of mRNA synthesis on the UV cross-linkable interaction between mRNA and 38K is rapid and reaches a maximal level in less than 60 min, and it is completely and rapidly reversible. In cells treated with actinomycin D, the amount of 38K which becomes cross-linked to mRNA is proportional to the extent of inhibition of mRNA synthesis. The association of 38K with mRNA during transcriptional arrest does not require protein synthesis because simultaneous treatment with the protein synthesis inhibitor emetine does not interfere with it. The effectors which promote the interaction of 38K with mRNA do not affect the proteins which are in contact with polyadenylated heterogeneous nuclear RNA and do not markedly affect protein synthesis in the cell. The 38K protein can be isolated with the polyribosomal polyadenylated fraction from which it was purified, and monoclonal antibodies against it were prepared

Population density approach for discrete mRNA distributions in generalized switching models for stochastic gene expression.

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Stinchcombe, Adam R; Peskin, Charles S; Tranchina, Daniel

2012-06-01

We present a generalization of a population density approach for modeling and analysis of stochastic gene expression. In the model, the gene of interest fluctuates stochastically between an inactive state, in which transcription cannot occur, and an active state, in which discrete transcription events occur; and the individual mRNA molecules are degraded stochastically in an independent manner. This sort of model in simplest form with exponential dwell times has been used to explain experimental estimates of the discrete distribution of random mRNA copy number. In our generalization, the random dwell times in the inactive and active states, T_{0} and T_{1}, respectively, are independent random variables drawn from any specified distributions. Consequently, the probability per unit time of switching out of a state depends on the time since entering that state. Our method exploits a connection between the fully discrete random process and a related continuous process. We present numerical methods for computing steady-state mRNA distributions and an analytical derivation of the mRNA autocovariance function. We find that empirical estimates of the steady-state mRNA probability mass function from Monte Carlo simulations of laboratory data do not allow one to distinguish between underlying models with exponential and nonexponential dwell times in some relevant parameter regimes. However, in these parameter regimes and where the autocovariance function has negative lobes, the autocovariance function disambiguates the two types of models. Our results strongly suggest that temporal data beyond the autocovariance function is required in general to characterize gene switching.

Expressions of interferon-inducible genes IFIT1 and IFIT4 mRNA in PBMCs of patients with systemic lupus erythematosus

International Nuclear Information System (INIS)

Liu Chunyan; Chen Xingguo; Wang Zizheng

2009-01-01

To investigate the expression levels of interferon-inducible genes (IFIT1, IFIT4) in the peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE), and the relations between these genes expression levels and disease activity, the expression levels of IFIT1 and IFIT4 mRNA in the 95 patients with SLE and 48 normal controls were detected by Sybr green dye based real-time quantitative PCR method, and these genes expression levels were compared with anti-double strand DNA antibody. The associations between the expression levels of IFIT1, IFIT4 mRNA, anti-double strand DNA antibody and SLEDAI scores in patients with SLE were analyzed. The results showed that the expression levels of IFIT1, IFIT4 mRNA in the SLE patients were significantly higher than those of the normal controls (P<0.01). The expression levels of IFIT1, IFIT4 mRNA in the active SLE patients were higher than those of the inactive SLE patients (P<0.05). The real time expression levels of IFIT1 and IFIT4 mRNA showed positive correlations with each other (P<0.05) in patients with SLE. There was positively correlation between the expression levels of IFIT1, IFIT4 mRNA and the anti-double strand DNA antibody (P<0.05). The expression levels of IFIT1, IFIT4 mRNA in patients with SLE were significantly higher than those of the normal controls, and positively associated with SLEDAI scores, so they were helpful in evaluating SLE disease activity and severity. To inhibit the expressions of IFIT1, IFIT4 mRNA may provide a novel target for SLE treatment. (authors)

Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

International Nuclear Information System (INIS)

Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M.

1990-01-01

The authors have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen mRNA appears to be a basal property of noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because >99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing the authors to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures

Creatine kinase and alpha-actin mRNA levels decrease in diabetic rat hearts

International Nuclear Information System (INIS)

Popovich, B.; Barrieux, A.; Dillmann, W.H.

1987-01-01

Diabetic cardiomyopathy is associated with cardiac atrophy and isoenzyme redistribution. To determine if tissue specific changes occur in mRNAs coding for α-actin and creatine kinase (CK), they performed RNA blot analysis. Total ventricular RNA from control (C) and 4 wk old diabetic (D) rats were hybridized with 32 P cDNA probes for α-actin and CK. A tissue independent cDNA probe, CHOA was also used. Signal intensity was quantified by photodensitometry. D CK mRNA was 47 +/- 16% lower in D vs C. Insulin increases CK mRNA by 20% at 1.5 hs, and completely reverses the deficit after 4 wks. D α-actin mRNA is 66 +/- 18% lower in D vs C. Insulin normalized α-actin mRNA by 5 hs. CHOA mRNA is unchanged in D vs C, but D + insulin CHOA mRNA is 30 +/- 2% lower than C. In rats with diabetic cardiomyopathy, muscle specific CK and α-actin mRNAs are decreased. Insulin treatment reverses these changes

Standardless quantification by parameter optimization in electron probe microanalysis

Energy Technology Data Exchange (ETDEWEB)

Limandri, Silvina P. [Instituto de Fisica Enrique Gaviola (IFEG), CONICET (Argentina); Facultad de Matematica, Astronomia y Fisica, Universidad Nacional de Cordoba, Medina Allende s/n, (5016) Cordoba (Argentina); Bonetto, Rita D. [Centro de Investigacion y Desarrollo en Ciencias Aplicadas Dr. Jorge Ronco (CINDECA), CONICET, 47 Street 257, (1900) La Plata (Argentina); Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 1 and 47 Streets (1900) La Plata (Argentina); Josa, Victor Galvan; Carreras, Alejo C. [Instituto de Fisica Enrique Gaviola (IFEG), CONICET (Argentina); Facultad de Matematica, Astronomia y Fisica, Universidad Nacional de Cordoba, Medina Allende s/n, (5016) Cordoba (Argentina); Trincavelli, Jorge C., E-mail: trincavelli@famaf.unc.edu.ar [Instituto de Fisica Enrique Gaviola (IFEG), CONICET (Argentina); Facultad de Matematica, Astronomia y Fisica, Universidad Nacional de Cordoba, Medina Allende s/n, (5016) Cordoba (Argentina)

2012-11-15

A method for standardless quantification by parameter optimization in electron probe microanalysis is presented. The method consists in minimizing the quadratic differences between an experimental spectrum and an analytical function proposed to describe it, by optimizing the parameters involved in the analytical prediction. This algorithm, implemented in the software POEMA (Parameter Optimization in Electron Probe Microanalysis), allows the determination of the elemental concentrations, along with their uncertainties. The method was tested in a set of 159 elemental constituents corresponding to 36 spectra of standards (mostly minerals) that include trace elements. The results were compared with those obtained with the commercial software GENESIS Spectrum Registered-Sign for standardless quantification. The quantifications performed with the method proposed here are better in the 74% of the cases studied. In addition, the performance of the method proposed is compared with the first principles standardless analysis procedure DTSA for a different data set, which excludes trace elements. The relative deviations with respect to the nominal concentrations are lower than 0.04, 0.08 and 0.35 for the 66% of the cases for POEMA, GENESIS and DTSA, respectively. - Highlights: Black-Right-Pointing-Pointer A method for standardless quantification in EPMA is presented. Black-Right-Pointing-Pointer It gives better results than the commercial software GENESIS Spectrum. Black-Right-Pointing-Pointer It gives better results than the software DTSA. Black-Right-Pointing-Pointer It allows the determination of the conductive coating thickness. Black-Right-Pointing-Pointer It gives an estimation for the concentration uncertainties.

Automated Quantification of Pneumothorax in CT

Science.gov (United States)

Do, Synho; Salvaggio, Kristen; Gupta, Supriya; Kalra, Mannudeep; Ali, Nabeel U.; Pien, Homer

2012-01-01

An automated, computer-aided diagnosis (CAD) algorithm for the quantification of pneumothoraces from Multidetector Computed Tomography (MDCT) images has been developed. Algorithm performance was evaluated through comparison to manual segmentation by expert radiologists. A combination of two-dimensional and three-dimensional processing techniques was incorporated to reduce required processing time by two-thirds (as compared to similar techniques). Volumetric measurements on relative pneumothorax size were obtained and the overall performance of the automated method shows an average error of just below 1%. PMID:23082091

Uncertainty Quantification and Regional Sensitivity Analysis of Snow-related Parameters in the Canadian LAnd Surface Scheme (CLASS)

Science.gov (United States)

Badawy, B.; Fletcher, C. G.

2017-12-01

The parameterization of snow processes in land surface models is an important source of uncertainty in climate simulations. Quantifying the importance of snow-related parameters, and their uncertainties, may therefore lead to better understanding and quantification of uncertainty within integrated earth system models. However, quantifying the uncertainty arising from parameterized snow processes is challenging due to the high-dimensional parameter space, poor observational constraints, and parameter interaction. In this study, we investigate the sensitivity of the land simulation to uncertainty in snow microphysical parameters in the Canadian LAnd Surface Scheme (CLASS) using an uncertainty quantification (UQ) approach. A set of training cases (n=400) from CLASS is used to sample each parameter across its full range of empirical uncertainty, as determined from available observations and expert elicitation. A statistical learning model using support vector regression (SVR) is then constructed from the training data (CLASS output variables) to efficiently emulate the dynamical CLASS simulations over a much larger (n=220) set of cases. This approach is used to constrain the plausible range for each parameter using a skill score, and to identify the parameters with largest influence on the land simulation in CLASS at global and regional scales, using a random forest (RF) permutation importance algorithm. Preliminary sensitivity tests indicate that snow albedo refreshment threshold and the limiting snow depth, below which bare patches begin to appear, have the highest impact on snow output variables. The results also show a considerable reduction of the plausible ranges of the parameters values and hence reducing their uncertainty ranges, which can lead to a significant reduction of the model uncertainty. The implementation and results of this study will be presented and discussed in details.

Stereo-particle image velocimetry uncertainty quantification

International Nuclear Information System (INIS)

Bhattacharya, Sayantan; Vlachos, Pavlos P; Charonko, John J

2017-01-01

Particle image velocimetry (PIV) measurements are subject to multiple elemental error sources and thus estimating overall measurement uncertainty is challenging. Recent advances have led to a posteriori uncertainty estimation methods for planar two-component PIV. However, no complete methodology exists for uncertainty quantification in stereo PIV. In the current work, a comprehensive framework is presented to quantify the uncertainty stemming from stereo registration error and combine it with the underlying planar velocity uncertainties. The disparity in particle locations of the dewarped images is used to estimate the positional uncertainty of the world coordinate system, which is then propagated to the uncertainty in the calibration mapping function coefficients. Next, the calibration uncertainty is combined with the planar uncertainty fields of the individual cameras through an uncertainty propagation equation and uncertainty estimates are obtained for all three velocity components. The methodology was tested with synthetic stereo PIV data for different light sheet thicknesses, with and without registration error, and also validated with an experimental vortex ring case from 2014 PIV challenge. Thorough sensitivity analysis was performed to assess the relative impact of the various parameters to the overall uncertainty. The results suggest that in absence of any disparity, the stereo PIV uncertainty prediction method is more sensitive to the planar uncertainty estimates than to the angle uncertainty, although the latter is not negligible for non-zero disparity. Overall the presented uncertainty quantification framework showed excellent agreement between the error and uncertainty RMS values for both the synthetic and the experimental data and demonstrated reliable uncertainty prediction coverage. This stereo PIV uncertainty quantification framework provides the first comprehensive treatment on the subject and potentially lays foundations applicable to volumetric

Influenza A Virus NS1 Protein Promotes Efficient Nuclear Export of Unspliced Viral M1 mRNA.

Science.gov (United States)

Pereira, Carina F; Read, Eliot K C; Wise, Helen M; Amorim, Maria J; Digard, Paul

2017-08-01

Influenza A virus mRNAs are transcribed by the viral RNA-dependent RNA polymerase in the cell nucleus before being exported to the cytoplasm for translation. Segment 7 produces two major transcripts: an unspliced mRNA that encodes the M1 matrix protein and a spliced transcript that encodes the M2 ion channel. Export of both mRNAs is dependent on the cellular NXF1/TAP pathway, but it is unclear how they are recruited to the export machinery or how the intron-containing but unspliced M1 mRNA bypasses the normal quality-control checkpoints. Using fluorescent in situ hybridization to monitor segment 7 mRNA localization, we found that cytoplasmic accumulation of unspliced M1 mRNA was inefficient in the absence of NS1, both in the context of segment 7 RNPs reconstituted by plasmid transfection and in mutant virus-infected cells. This effect was independent of any major effect on steady-state levels of segment 7 mRNA or splicing but corresponded to a ∼5-fold reduction in the accumulation of M1. A similar defect in intronless hemagglutinin (HA) mRNA nuclear export was seen with an NS1 mutant virus. Efficient export of M1 mRNA required both an intact NS1 RNA-binding domain and effector domain. Furthermore, while wild-type NS1 interacted with cellular NXF1 and also increased the interaction of segment 7 mRNA with NXF1, mutant NS1 polypeptides unable to promote mRNA export did neither. Thus, we propose that NS1 facilitates late viral gene expression by acting as an adaptor between viral mRNAs and the cellular nuclear export machinery to promote their nuclear export. IMPORTANCE Influenza A virus is a major pathogen of a wide variety of mammalian and avian species that threatens public health and food security. A fuller understanding of the virus life cycle is important to aid control strategies. The virus has a small genome that encodes relatively few proteins that are often multifunctional. Here, we characterize a new function for the NS1 protein, showing that, as well as

Specific mixing facilitates the comparative quantification of phosphorylation sites with significant dysregulations

Energy Technology Data Exchange (ETDEWEB)

Liu, Jing [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Xu, Bo [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China); Liu, Zheyi; Dong, Mingming; Mao, Jiawei; Zhou, Ye; Chen, Jin [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Wang, Fangjun, E-mail: wangfj@dicp.ac.cn [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China); Zou, Hanfa [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China)

2017-01-15

Mass spectrometry (MS) based quantitative analyses of proteome and proteome post-translational modifications (PTMs) play more and more important roles in biological, pharmaceutical and clinical studies. However, it is still a big challenge to accurately quantify the proteins or proteins PTM sites with extreme relative abundances in comparative protein samples, such as the significantly dysregulated ones. Herein, a novel quantification strategy, Mixing at Specific Ratio (MaSR) before isotope labeling, had been developed to improve the quantification accuracy and coverage of extreme proteins and protein phosphorylation sites. Briefly, the comparative protein samples were firstly mixed together at specific ratios of 9:1 and 1:9 (w/w), followed with mass differentiate light and heavy isotope labeling, respectively. The extreme proteins and protein phosphorylation sites, even if the newly expressed or disappeared ones, could be accurately quantified due to all of the proteins' relative abundances had been adjusted to 2 orders of magnitude (1/9-9) by this strategy. The number of quantified phosphorylation sites with more than 20 folds changes was improved about 10 times in comparative quantification of pervanadate stimulated phosphoproteome of HeLa cells, and 134 newly generated and 21 disappeared phosphorylation sites were solely quantified by the MaSR strategy. The significantly up-regulated phosphorylation sites were mainly involved in the key phosphoproteins regulating the insulin-related pathways, such as PI3K-AKT and RAS-MAPK pathways. Therefore, the MaSR strategy exhibits as a promising way in elucidating the biological processes with significant dysregulations. - Highlights: • All the proteins' relative abundances were adjusted into 2 orders of magnitude (1/9-9). • The quantification accuracy and coverage of extreme proteins and protein phosphorylation sites had been improved. • The newly expressed or disappeared proteins and protein

Exploratory Bioinformatics Study of lncRNAs in Alzheimer’s Disease mRNA Sequences with Application to Drug Development

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T. Holden

2013-01-01

Full Text Available Long noncoding RNA (lncRNA within mRNA sequences of Alzheimer’s disease genes, namely, APP, APOE, PSEN1, and PSEN2, has been analyzed using fractal dimension (FD computation and correlation analysis. We examined lncRNA by comparing mRNA FD to corresponding coding DNA sequences (CDSs FD. APP, APOE, and PSEN1 CDSs select slightly higher FDs compared to the mRNA, while PSEN2 CDSs FDs are lower. The correlation coefficient for these sequences is 0.969. A comparative study of differentially expressed MAPK signaling pathway lncRNAs in pancreatic cancer cells shows a correlation of 0.771. Selection of higher FD CDSs could indicate interaction of Alzheimer’s gene products APP, APOE, and PSEN1. Including hypocretin sequences (where all CDSs have higher fractal dimensions than mRNA in the APP, APOE, and PSEN1 sequence analyses improves correlation, but the inclusion of erythropoietin (where all CDSs have higher FD than mRNA would suppress correlation, suggesting that HCRT, a hypothalamus neurotransmitter related to the wake/sleep cycle, might be better when compared to EPO, a glycoprotein hormone, for targeting Alzheimer’s disease drug development. Fractal dimension and entropy correlation have provided supporting evidence, consistent with evolutionary studies, for using a zebrafish model together with a mouse model, in HCRT drug development.

Whole farm quantification of GHG emissions within smallholder farms in developing countries

International Nuclear Information System (INIS)

Seebauer, Matthias

2014-01-01

The IPCC has compiled the best available scientific methods into published guidelines for estimating greenhouse gas emissions and emission removals from the land-use sector. In order to evaluate existing GHG quantification tools to comprehensively quantify GHG emissions and removals in smallholder conditions, farm scale quantification was tested with farm data from Western Kenya. After conducting a cluster analysis to identify different farm typologies GHG quantification was exercised using the VCS SALM methodology complemented with IPCC livestock emission factors and the cool farm tool. The emission profiles of four farm clusters representing the baseline conditions in the year 2009 are compared with 2011 where farmers adopted sustainable land management practices (SALM). The results demonstrate the variation in both the magnitude of the estimated GHG emissions per ha between different smallholder farm typologies and the emissions estimated by applying two different accounting tools. The farm scale quantification further shows that the adoption of SALM has a significant impact on emission reduction and removals and the mitigation benefits range between 4 and 6.5 tCO 2  ha −1  yr −1 with significantly different mitigation benefits depending on typologies of the crop–livestock systems, their different agricultural practices, as well as adoption rates of improved practices. However, the inherent uncertainty related to the emission factors applied by accounting tools has substantial implications for reported agricultural emissions. With regard to uncertainty related to activity data, the assessment confirms the high variability within different farm types as well as between different parameters surveyed to comprehensively quantify GHG emissions within smallholder farms. (paper)

Effects of corticosteroid on the expressions of neuropeptide and cytokine mRNA and on tenocyte viability in lateral epicondylitis

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Han Soo

2012-10-01

Full Text Available Abstract Background The purpose of this study was to determine the reaction mechanism of corticosteroid by analyzing the expression patterns of neuropeptides (substance P (SP, calcitonin gene related peptide (CGRP and of cytokines (interleukin (IL-1α, tumor growth factor (TGF-β after corticosteroid treatment in lateral epicondylitis. In addition, we also investigated whether corticosteroid influenced tenocyte viability. Methods The corticosteroid triamcinolone acetonide (TAA was applied to cultured tenocytes of lateral epicondylitis, and the changes in the mRNA expressions of neuropeptides and cytokines and tenocyte viabilities were analyzed at seven time points. Quantitative real-time polymerase chain reaction and an MTT assay were used. Results The expression of SP mRNA was maximally inhibited by TAA at 24 hours but recovered at 72 hours, and the expressions of CGRP mRNA and IL-1α mRNA were inhibited at 24 and 3 hours, respectively. The expression of TGF-β mRNA was not significant. Tenocyte viability was significantly reduced by TAA at 24 hours. Conclusions We postulate that the reaction mechanism predominantly responsible for symptomatic relief after a corticosteroid injection involves the inhibitions of neuropeptides and cytokines, such as, CGRP and IL-1α. However the tenocyte viability was compromised by a corticosteroid.

FoxP3 mRNA splice forms in synovial CD4+ T cells in rheumatoid arthritis and psoriatic arthritis

DEFF Research Database (Denmark)

Ryder, L Rebekka; Bartels, Else Marie; Woetmann, Anders

2012-01-01

Our aim was to elucidate the relative amount of the different splice forms of FoxP3 mRNA in CD4+ T cells in peripheral blood (PB) compared to synovial fluid (SF) in RA and PsA patients. FoxP3 mRNA was measured using a quantitative real-time PCR method. CD4+ T cells were isolated from 17 paired...... samples of PB and SF from RA and PsA patients, and PB from 10 controls. FoxP3fl and FoxP3Δ2 mRNA was significantly increased (6.7 and 2.1-fold, respectively) in PB CD4+ T cells from RA patients compared to controls. FoxP3fl and Δ2 mRNA in SF CD4+ T cells was increased compared to controls in sero......-negative RA and PsA, but not in sero-positive RA patients, who had a high FoxP3 expression in both PB and SF. The FoxP3Δ2Δ7 mRNA was barely detectable in patient samples, and not at all in healthy individuals. We provide evidence of an increased expression of FoxP3 splice forms in synovial CD4+ T cells from...

Detection of melatonin receptor mRNA in human muscle

International Nuclear Information System (INIS)

Li Lei

2004-01-01

To verify the expression of melatonin receptor mRNA in human, muscle, muscle beside vertebrae was collected to obtain total RNA and the mRNA of melatonin receptor was detected by RT-PCR method. The electrophoretic results of RT-PCR products by mt 1 and MT 2 primer were all positive and the sequence is corresponding with human melatonin receptor cDNA. It suggests that melatonin may act on the muscle beside vertebrae directly and regulate its growth and development. (authors)

The ApaH-like phosphatase TbALPH1 is the major mRNA decapping enzyme of trypanosomes.

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Susanne Kramer

2017-06-01

Full Text Available 5'-3' decay is the major mRNA decay pathway in many eukaryotes, including trypanosomes. After deadenylation, mRNAs are decapped by the nudix hydrolase DCP2 of the decapping complex and finally degraded by the 5'-3' exoribonuclease. Uniquely, trypanosomes lack homologues to all subunits of the decapping complex, while deadenylation and 5'-3' degradation are conserved. Here, I show that the parasites use an ApaH-like phosphatase (ALPH1 as their major mRNA decapping enzyme. The protein was recently identified as a novel trypanosome stress granule protein and as involved in mRNA binding. A fraction of ALPH1 co-localises exclusively with the trypanosome 5'-3' exoribonuclease XRNA to a special granule at the posterior pole of the cell, indicating a connection between the two enzymes. RNAi depletion of ALPH1 is lethal and causes a massive increase in total mRNAs that are deadenylated, but have not yet started 5'-3' decay. These data suggest that ALPH1 acts downstream of deadenylation and upstream of mRNA degradation, consistent with a function in mRNA decapping. In vitro experiments show that recombinant, N-terminally truncated ALHP1 protein, but not a catalytically inactive mutant, sensitises the capped trypanosome spliced leader RNA to yeast Xrn1, but only if an RNA 5' polyphosphatase is included. This indicates that the decapping mechanism of ALPH1 differs from the decapping mechanism of Dcp2 by leaving more than one phosphate group at the mRNA's 5' end. This is the first reported function of a eukaryotic ApaH-like phosphatase, a bacterial-derived class of enzymes present in all phylogenetic super-groups of the eukaryotic kingdom. The substrates of eukaryotic ApaH-like phosphatases are unknown. However, the substrate of the related bacterial enzyme ApaH, diadenosine tetraphosphate, is highly reminiscent of a eukaryotic mRNA cap.

Differential regulation of renal cyclooxygenase mRNA by dietary salt intake

DEFF Research Database (Denmark)

Jensen, B L; Kurtz, A

1997-01-01

RNA correlated directly with salt intake. We conclude that dietary salt intake influences renal cyclooxygenase mRNAs zone-specifically with opposite responses between cortex and medulla. Cortical COX II-mediated prostaglandin formation is probably important in low salt states whereas medullary COX I......Experiments were done to investigate the influence of dietary salt intake on renal cyclooxygenase (COX) I and II mRNA levels. To this end rats were fed either a low NaCl diet (LS; 0.02% NaCl wt/wt) or a high NaCl diet (HS diet; 4% NaCl wt/wt) for 5, 10 and 20 days. After 10 days Na excretion...... differed 760-fold, plasma renin activity and renin mRNA were increased eight- and threefold in LS compared to HS animals. Total renal COX I mRNA decreased 50% following the LS diet and did not change after the HS diet. Conversely, COX II mRNA declined after HS intake and transiently increased after salt...

Myeloperoxidase mRNA detection for lineage determination of leukemic blasts: retrospective analysis.

Science.gov (United States)

Crisan, D; Anstett, M J

1995-07-01

Myeloperoxidase (MPO) mRNA is an early myeloid marker; its detection in the morphologically and immunophenotypically primitive blasts of acute undifferentiated leukemia (AUL) establishes myeloid lineage and allows reclassification as acute myelogenous leukemia with minimal differentiation (AML-MO). We have previously reported a procedure for MPO mRNA detection by RT-PCR (reverse transcription-polymerase chain reaction) and an adaptation for use of routine hematology smears. This variant procedure allows retrospective analysis of mRNA and is used in the present study to evaluate the lineage of leukemic blasts in seven cases with morphology and cytochemistry consistent with AUL. All hematology smears used in this study were air-dried, unstained or Wright-stained and stored at room temperature for periods varying between 3 days and 2 years. MPO mRNA was detected in six cases, establishing the myeloid lineage of the blasts and the diagnosis of AML-MO. In the remaining case, the blasts were MPO mRNA negative, confirming the diagnosis of AUL. The RT-PCR procedure for retrospective mRNA analysis is useful in the clinical setting, due to its high specificity and sensitivity, speed (less than 24 h), safety (no radioactivity) and convenient use of routine hematology smears; it is particularly attractive in clinical situations when fresh or frozen specimens are no longer available at the time when the need for molecular diagnostics becomes apparent.

A glimpse at mRNA dynamics reveals cellular domains and rapid trafficking through granules

NARCIS (Netherlands)

Gemert, Alice Myriam Christi van

2011-01-01

mRNA transport and targeting are essential to gene expression regulation. Specific mRNA sequences can bind several proteins and together form RiboNucleoProtein particles (RNP). The various proteins within the RNP determine mRNA fate: translation, transport or decay. RNP composition varies with

Human apolipoprotein B (apoB) mRNA: Identification of two distinct apoB mRNAs, an mRNA with the apoB-100 sequence and an apoB mRNA containing a premature in-frame translational stop codon, in both liver and intestine

International Nuclear Information System (INIS)

Higuchi, K.; Hospattankar, A.V.; Law, S.W.; Meglin, N.; Cortright, J.; Brewer, H.B. Jr.

1988-01-01

Human apolipoprotein B (apoB) is present in plasma as two separate isoproteins, designated apoB-100 (512 kDa) and apoB-48 (250 kDa). ApoB is encoded by a single gene on chromosome 2, and a single nuclear mRNA is edited and processed into two separate apoB mRNAs. A 14.1-kilobase apoB mRNA codes for apoB-100, and the second mRNA, which codes for apoB-48, contains a premature stop codon generated by a single base substitution of cytosine to uracil at nucleotide 6,538, which converts the translated CAA codon coding for the amino acid glutamine at residue 2,153 in apoB-100 to a premature in-frame stop codon (UAA). Two 30-base synthetic oligonucleotides, designated apoB-Stop and apoB-Gln, were synthesized containing the complementary sequence to the stop codon (UAA) and glutamine codon (CAA), respectively. The combined results from these studies establish that both human intestine and liver contain the two distinct apoB mRNAs, an mRNA that codes for apoB-100 and an apoB mRNA that contains the premature stop codon, which codes for apoB-48. The premature in-frame stop codon is not tissue specific and is present in both human liver and intestine

Uncertainty quantification for hyperbolic and kinetic equations

CERN Document Server

Pareschi, Lorenzo

2017-01-01

This book explores recent advances in uncertainty quantification for hyperbolic, kinetic, and related problems. The contributions address a range of different aspects, including: polynomial chaos expansions, perturbation methods, multi-level Monte Carlo methods, importance sampling, and moment methods. The interest in these topics is rapidly growing, as their applications have now expanded to many areas in engineering, physics, biology and the social sciences. Accordingly, the book provides the scientific community with a topical overview of the latest research efforts.

Complexity on Acute Myeloid Leukemia mRNA Transcript Variant

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Carlo Cattani

2011-01-01

Full Text Available This paper deals with the sequence analysis of acute myeloid leukemia mRNA. Six transcript variants of mlf1 mRNA, with more than 2000 bps, are analyzed by focusing on the autocorrelation of each distribution. Through the correlation matrix, some patches and similarities are singled out and commented, with respect to similar distributions. The comparison of Kolmogorov fractal dimension will be also given in order to classify the six variants. The existence of a fractal shape, patterns, and symmetries are discussed as well.

Digital PCR for direct quantification of viruses without DNA extraction.

Science.gov (United States)

Pavšič, Jernej; Žel, Jana; Milavec, Mojca

2016-01-01

DNA extraction before amplification is considered an essential step for quantification of viral DNA using real-time PCR (qPCR). However, this can directly affect the final measurements due to variable DNA yields and removal of inhibitors, which leads to increased inter-laboratory variability of qPCR measurements and reduced agreement on viral loads. Digital PCR (dPCR) might be an advantageous methodology for the measurement of virus concentrations, as it does not depend on any calibration material and it has higher tolerance to inhibitors. DNA quantification without an extraction step (i.e. direct quantification) was performed here using dPCR and two different human cytomegalovirus whole-virus materials. Two dPCR platforms were used for this direct quantification of the viral DNA, and these were compared with quantification of the extracted viral DNA in terms of yield and variability. Direct quantification of both whole-virus materials present in simple matrices like cell lysate or Tris-HCl buffer provided repeatable measurements of virus concentrations that were probably in closer agreement with the actual viral load than when estimated through quantification of the extracted DNA. Direct dPCR quantification of other viruses, reference materials and clinically relevant matrices is now needed to show the full versatility of this very promising and cost-efficient development in virus quantification.

Search for antisense copies of beta-globin mRNA in anemic mouse spleen

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Taylor John M

2001-03-01

Full Text Available Abstract Background Previous studies by Volloch and coworkers have reported that during the expression of high levels of β-globin mRNA in the spleen of anemic mice, they could also detect small but significant levels of an antisense (AS globin RNA species, which they postulated might have somehow arisen by RNA-directed RNA synthesis. For two reasons we undertook to confirm and possibly extend these studies. First, previous studies in our lab have focussed on what is an unequivocal example of host RNA-directed RNA polymerase activity on the RNA genome of human hepatitis delta virus. Second, if AS globin species do exist they could in turn form double-stranded RNA species which might induce post-transcriptional gene silencing, a phenomenon somehow provoked in eukaryotic cells by AS RNA sequences. Results We reexamined critical aspects of the previous globin studies. We used intraperitoneal injections of phenylhydrazine to induce anemia in mice, as demonstrated by the appearance and ultimate disappearance of splenomegaly. While a 30-fold increase in globin mRNA was detected in the spleen, the relative amount of putative AS RNA could be no more than 0.004%. Conclusions Contrary to earlier reports, induction of a major increase in globin transcripts in the mouse spleen was not associated with a detectable level of antisense RNA to globin mRNA.

The hypoxic proteome is influenced by gene-specific changes in mRNA translation

International Nuclear Information System (INIS)

Koritzinsky, Marianne; Seigneuric, Renaud; Magagnin, Michael G.; Beucken, Twan van den; Lambin, Philippe; Wouters, Bradly G.

2005-01-01

Background and purpose: Hypoxia causes a rapid reduction in mRNA translation efficiency. This inhibition does not affect all mRNA species to the same extent and can therefore contribute significantly to hypoxia-induced differential protein expression. Our aim in this study was to characterize changes in gene expression during acute hypoxia and evaluate the contribution of regulation via mRNA translation on these changes. For each gene, the contribution of changes in mRNA abundance versus mRNA translation was determined. Materials and methods: DU145 prostate carcinoma cells were exposed to 4 h of hypoxia ( 2 ). Efficiently translated mRNAs were isolated by sedimentation through a sucrose gradient. Affymetrix microarray technology was used to evaluate both the transcriptional and translational contribution to gene expression. Results were validated by quantitative PCR. Results: One hundred and twenty genes were more than 4-fold upregulated by hypoxia in the efficiently translated fraction of mRNA, in comparison to only 76 genes at the level of transcription. Of the 50 genes demonstrating the largest changes in translation, 11 were found to be more than 2-fold over represented in the translated fraction in comparison to their overall transcriptional level. The gene with the highest translational contribution to its induction was CITED-2, which is a negative regulator of HIF-1 transcriptional activity. Conclusions: Gene-specific regulation of mRNA translation contributes significantly to differential gene expression during hypoxia

Impairment of FOS mRNA stabilization following translation arrest in granulocytes from myelodysplastic syndrome patients.

Science.gov (United States)

Feng, Xiaomin; Shikama, Yayoi; Shichishima, Tsutomu; Noji, Hideyoshi; Ikeda, Kazuhiko; Ogawa, Kazuei; Kimura, Hideo; Takeishi, Yasuchika; Kimura, Junko

2013-01-01

Although quantitative and qualitative granulocyte defects have been described in myelodysplastic syndromes (MDS), the underlying molecular basis of granulocyte dysfunction in MDS is largely unknown. We recently found that FOS mRNA elevation under translation-inhibiting stimuli was significantly smaller in granulocytes from MDS patients than in healthy individuals. The aim of this study is to clarify the cause of the impaired FOS induction in MDS. We first examined the mechanisms of FOS mRNA elevation using granulocytes from healthy donors cultured with the translation inhibitor emetine. Emetine increased both transcription and mRNA stability of FOS. p38 MAPK inhibition abolished the emetine-induced increase of FOS transcription but did not affect FOS mRNA stabilization. The binding of an AU-rich element (ARE)-binding protein HuR to FOS mRNA containing an ARE in 3'UTR was increased by emetine, and the knockdown of HuR reduced the FOS mRNA stabilizing effect of emetine. We next compared the emetine-induced transcription and mRNA stabilization of FOS between MDS patients and healthy controls. Increased rates of FOS transcription by emetine were similar in MDS and controls. In the absence of emetine, FOS mRNA decayed to nearly 17% of initial levels in 45 min in both groups. In the presence of emetine, however, 76.7±19.8% of FOS mRNA remained after 45 min in healthy controls, versus 37.9±25.5% in MDS (Pknowledge, this is the first report demonstrating attenuation of stress-induced FOS mRNA stabilization in MDS granulocytes.

RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome

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Dewey Colin N

2011-08-01

Full Text Available Abstract Background RNA-Seq is revolutionizing the way transcript abundances are measured. A key challenge in transcript quantification from RNA-Seq data is the handling of reads that map to multiple genes or isoforms. This issue is particularly important for quantification with de novo transcriptome assemblies in the absence of sequenced genomes, as it is difficult to determine which transcripts are isoforms of the same gene. A second significant issue is the design of RNA-Seq experiments, in terms of the number of reads, read length, and whether reads come from one or both ends of cDNA fragments. Results We present RSEM, an user-friendly software package for quantifying gene and isoform abundances from single-end or paired-end RNA-Seq data. RSEM outputs abundance estimates, 95% credibility intervals, and visualization files and can also simulate RNA-Seq data. In contrast to other existing tools, the software does not require a reference genome. Thus, in combination with a de novo transcriptome assembler, RSEM enables accurate transcript quantification for species without sequenced genomes. On simulated and real data sets, RSEM has superior or comparable performance to quantification methods that rely on a reference genome. Taking advantage of RSEM's ability to effectively use ambiguously-mapping reads, we show that accurate gene-level abundance estimates are best obtained with large numbers of short single-end reads. On the other hand, estimates of the relative frequencies of isoforms within single genes may be improved through the use of paired-end reads, depending on the number of possible splice forms for each gene. Conclusions RSEM is an accurate and user-friendly software tool for quantifying transcript abundances from RNA-Seq data. As it does not rely on the existence of a reference genome, it is particularly useful for quantification with de novo transcriptome assemblies. In addition, RSEM has enabled valuable guidance for cost

Advances in forensic DNA quantification: a review.

Science.gov (United States)

Lee, Steven B; McCord, Bruce; Buel, Eric

2014-11-01

This review focuses upon a critical step in forensic biology: detection and quantification of human DNA from biological samples. Determination of the quantity and quality of human DNA extracted from biological evidence is important for several reasons. Firstly, depending on the source and extraction method, the quality (purity and length), and quantity of the resultant DNA extract can vary greatly. This affects the downstream method as the quantity of input DNA and its relative length can determine which genotyping procedure to use-standard short-tandem repeat (STR) typing, mini-STR typing or mitochondrial DNA sequencing. Secondly, because it is important in forensic analysis to preserve as much of the evidence as possible for retesting, it is important to determine the total DNA amount available prior to utilizing any destructive analytical method. Lastly, results from initial quantitative and qualitative evaluations permit a more informed interpretation of downstream analytical results. Newer quantitative techniques involving real-time PCR can reveal the presence of degraded DNA and PCR inhibitors, that provide potential reasons for poor genotyping results and may indicate methods to use for downstream typing success. In general, the more information available, the easier it is to interpret and process the sample resulting in a higher likelihood of successful DNA typing. The history of the development of quantitative methods has involved two main goals-improving precision of the analysis and increasing the information content of the result. This review covers advances in forensic DNA quantification methods and recent developments in RNA quantification. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A fast and robust hepatocyte quantification algorithm including vein processing

Directory of Open Access Journals (Sweden)

Homeyer André

2010-03-01

Full Text Available Abstract Background Quantification of different types of cells is often needed for analysis of histological images. In our project, we compute the relative number of proliferating hepatocytes for the evaluation of the regeneration process after partial hepatectomy in normal rat livers. Results Our presented automatic approach for hepatocyte (HC quantification is suitable for the analysis of an entire digitized histological section given in form of a series of images. It is the main part of an automatic hepatocyte quantification tool that allows for the computation of the ratio between the number of proliferating HC-nuclei and the total number of all HC-nuclei for a series of images in one processing run. The processing pipeline allows us to obtain desired and valuable results for a wide range of images with different properties without additional parameter adjustment. Comparing the obtained segmentation results with a manually retrieved segmentation mask which is considered to be the ground truth, we achieve results with sensitivity above 90% and false positive fraction below 15%. Conclusions The proposed automatic procedure gives results with high sensitivity and low false positive fraction and can be applied to process entire stained sections.

Characterization of a major late herpes simplex virus type 1 mRNA.

Science.gov (United States)

Costa, R H; Devi, B G; Anderson, K P; Gaylord, B H; Wagner, E K

1981-05-01

A major, late 6-kilobase (6-kb) mRNa mapping in the large unique region of herpes simplex virus type 1 (HSV-1) was characterized by using two recombinant DNA clones, one containing EcoRI fragment G (0.190 to 0.30 map units) in lambda. WES.B (L. Enquist, M. Madden, P. Schiop-Stansly, and G. Vandl Woude, Science 203:541-544, 1979) and one containing HindIII fragment J (0.181 to 0.259 map units) in pBR322. This 6-kb mRNA had its 3' end to the left of 0.231 on the prototypical arrangement of the HSV-1 genome and was transcribed from right to left. It was bounded on both sides by regions containing a large number of distinct mRNA species, and its 3' end was partially colinear with a 1.5-kb mRNA which encoded a 35,000-dalton polypeptide. The 6-kb mRNA encoded a 155,000-dalton polypeptide which was shown to be the only one of this size detectable by hybrid-arrested translation encoded by late polyadenylated polyribosomal RNA. The S1 nuclease mapping experiments indicated that there were no introns in the coding sequence for this mRNA and that its 3' end mapped approximately 800 nucleotides to the left of the BglII site at 0.231, whereas its 5' end extended very close to the BamHI site at 0.266.

Relative Abundance of Integral Plasma Membrane Proteins in Arabidopsis Leaf and Root Tissue Determined by Metabolic Labeling and Mass Spectrometry

Science.gov (United States)

Bernfur, Katja; Larsson, Olaf; Larsson, Christer; Gustavsson, Niklas

2013-01-01

Metabolic labeling of proteins with a stable isotope (15N) in intact Arabidopsis plants was used for accurate determination by mass spectrometry of differences in protein abundance between plasma membranes isolated from leaves and roots. In total, 703 proteins were identified, of which 188 were predicted to be integral membrane proteins. Major classes were transporters, receptors, proteins involved in membrane trafficking and cell wall-related proteins. Forty-one of the integral proteins, including nine of the 13 isoforms of the PIP (plasma membrane intrinsic protein) aquaporin subfamily, could be identified by peptides unique to these proteins, which made it possible to determine their relative abundance in leaf and root tissue. In addition, peptides shared between isoforms gave information on the proportions of these isoforms. A comparison between our data for protein levels and corresponding data for mRNA levels in the widely used database Genevestigator showed an agreement for only about two thirds of the proteins. By contrast, localization data available in the literature for 21 of the 41 proteins show a much better agreement with our data, in particular data based on immunostaining of proteins and GUS-staining of promoter activity. Thus, although mRNA levels may provide a useful approximation for protein levels, detection and quantification of isoform-specific peptides by proteomics should generate the most reliable data for the proteome. PMID:23990937

2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation.

Science.gov (United States)

Choi, Junhong; Indrisiunaite, Gabriele; DeMirci, Hasan; Ieong, Ka-Weng; Wang, Jinfan; Petrov, Alexey; Prabhakar, Arjun; Rechavi, Gideon; Dominissini, Dan; He, Chuan; Ehrenberg, Måns; Puglisi, Joseph D

2018-03-01

Chemical modifications of mRNA may regulate many aspects of mRNA processing and protein synthesis. Recently, 2'-O-methylation of nucleotides was identified as a frequent modification in translated regions of human mRNA, showing enrichment in codons for certain amino acids. Here, using single-molecule, bulk kinetics and structural methods, we show that 2'-O-methylation within coding regions of mRNA disrupts key steps in codon reading during cognate tRNA selection. Our results suggest that 2'-O-methylation sterically perturbs interactions of ribosomal-monitoring bases (G530, A1492 and A1493) with cognate codon-anticodon helices, thereby inhibiting downstream GTP hydrolysis by elongation factor Tu (EF-Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate aminoacylated tRNAs in initial selection and proofreading. Our current and prior findings highlight how chemical modifications of mRNA tune the dynamics of protein synthesis at different steps of translation elongation.

Quantitative PCR--new diagnostic tool for quantifying specific mRNA and DNA molecules

DEFF Research Database (Denmark)

Schlemmer, B O; Sorensen, B S; Overgaard, J

2004-01-01

of a subset of ligands from the EGF system is increased in bladder cancer. Furthermore, measurement of the mRNA concentration gives important information such as the expression of these ligands correlated to the survival of the patients. In addition to the alterations at the mRNA level, changes also can occur...... at the DNA level in the EGF system. Thus, it has been demonstrated that the number of genes coding for the human epidermal growth factor receptor 2 (HER2) is increased in a number of breast tumors. It is now possible to treat breast cancer patients with a humanized antibody reacting with HER2...... of mRNA or DNA in biological samples. In this study quantitative PCR was used to investigate the role of the EGF (epidermal growth factor) system in cancer both for measurements of mRNA concentrations and for measurements of the number of copies of specific genes. It is shown that the mRNA expression...

Applying the breaks on gene expression - mRNA deadenylation by Pop2p

DEFF Research Database (Denmark)

Andersen, Kasper Røjkjær; Jonstrup, Anette Thyssen; Van, Lan Bich

When driving a car, control of the brakes is just as important as control of the accelerator pedal. Likewise, in gene expression, regulation of mRNA degradation is as important as regulation of its synthesis (Mühlemann, 2005). The rate-determining step of mRNA decay in eukaryotes seems to be the ......When driving a car, control of the brakes is just as important as control of the accelerator pedal. Likewise, in gene expression, regulation of mRNA degradation is as important as regulation of its synthesis (Mühlemann, 2005). The rate-determining step of mRNA decay in eukaryotes seems...... to be the shortening of the poly(A) tail (deadenylation), as this step is slower than the subsequent decapping and degradation of the mRNA body. The Mega-Dalton Ccr4-Not complex contains two exonucleases, Ccr4p and Pop2p, responsible for this process. It is not known at present why two conserved nucleases are needed...

A real-time PCR assay for the relative quantification of the tetrahydrocannabinolic acid (THCA) synthase gene in herbal Cannabis samples.

Science.gov (United States)

Cascini, Fidelia; Passerotti, Stella; Martello, Simona

2012-04-10

In this study, we wanted to investigate whether or not the tetrahydrocannabinolic acid (THCA) synthase gene, which codes for the enzyme involved in the biosynthesis of THCA, influences the production and storage of tetrahydrocannabinol (THC) in a dose-dependent manner. THCA is actually decarboxylated to produce THC, the main psychoactive component in the Cannabis plant. Assuming as the research hypothesis a correlation between the gene copy number and the production of THC, gene quantification could be useful in forensics in order to complement or replace chemical analysis for the identification and classification of seized Cannabis samples, thus distinguishing the drug-type from the fibre-type varieties. A real-time PCR assay for the relative quantification of the THCA synthase gene was then validated on Cannabis samples; some were seized from the illegal drug market and others were derived from experimental cultivation. In order to determine the gene copy number to compare high vs. low potency plants, we chose the ΔΔCt method for TaqMan reactions. The assay enabled single plants with zero, one, and two copies of the gene to be distinguished. As a result of this first part of the research on the THCA synthase gene (the second part will cover a study of gene expression), we found no correlation between THCA synthase gene copy number and the content of THC in the herbal Cannabis samples tested. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Isobaric Quantification of Cerebrospinal Fluid Amyloid-β Peptides in Alzheimer's Disease: C-Terminal Truncation Relates to Early Measures of Neurodegeneration.

Science.gov (United States)

Rogeberg, Magnus; Almdahl, Ina Selseth; Wettergreen, Marianne; Nilsson, Lars N G; Fladby, Tormod

2015-11-06

The amyloid beta (Aβ) peptide is the main constituent of the plaques characteristic of Alzheimer's disease (AD). Measurement of Aβ1-42 in cerebrospinal fluid (CSF) is a valuable marker in AD research, where low levels indicate AD. Although the use of immunoassays measuring Aβ1-38 and Aβ1-40 in addition to Aβ1-42 has increased, quantitative assays of other Aβ peptides remain rarely explored. We recently discovered novel Aβ peptides in CSF using antibodies recognizing the Aβ mid-domain region. Here we have developed a method using both Aβ N-terminal and mid-domain antibodies for immunoprecipitation in combination with isobaric labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for relative quantification of endogenous Aβ peptides in CSF. The developed method was used in a pilot study to produce Aβ peptide profiles from 38 CSF samples. Statistical comparison between CSF samples from 19 AD patients and 19 cognitively healthy controls revealed no significant differences at group level. A significant correlation was found between several larger C-terminally truncated Aβ peptides and protein biomarkers for neuronal damage, particularly prominent in the control group. Comparison of the isobaric quantification with immunoassays measuring Aβ1-38 or Aβ1-40 showed good correlation (r(2) = 0.84 and 0.85, respectively) between the two analysis methods. The developed method could be used to assess disease-modifying therapies directed at Aβ production or degradation.

HLA-G allelic variants are associated with differences in the HLA-G mRNA isoform profile and HLA-G mRNA levels

DEFF Research Database (Denmark)

Hviid, Thomas Vauvert F; Hylenius, Sine; Rørbye, Christina

2003-01-01

between mother and fetus in several ways. Finally, the expression of membrane-bound HLA-G and soluble HLA-G has been proposed to influence the outcome of pregnancy, and an aberrant HLA-G expression in pre-eclamptic placentas and spontaneous abortions has been reported. Here, an association between certain...... HLA-G polymorphisms and the mRNA levels of the different alternatively spliced HLA-G isoforms in first trimester trophoblast cell populations is reported. Several alternatively spliced HLA-G mRNA isoforms, including a 14-bp polymorphism in the 3'UTR end (exon 8) of the HLA-G gene, are expressed...

Effect of in vitro estrogenic pesticides on human oestrogen receptor α and β mRNA levels

DEFF Research Database (Denmark)

Theander Grünfeld, Heidi; Bonefeld-Jørgensen, Eva Cecilie

2004-01-01

of the ERα mRNA level, but only significantly for prochloraz, dieldrin, and tolchlofos-methyl. Alone no pesticides affected the ERβ mRNA level significantly, but chlorpyrifos increased the mRNA level weakly. Co-exposure with E2 elicited a significant increased ERβ mRNA level by prochloraz, fenarimol...

Regulation of mRNA translation influences hypoxia tolerance

International Nuclear Information System (INIS)

Koritzinsky, M.; Wouters, B.G.; Koumenis, C.

2003-01-01

Hypoxia is a heterogenous but common characteristic of human tumours and poor oxygenation is associated with poor prognosis. We believe that the presence of viable hypoxic tumor cells reflects in part an adaptation and tolerance of these cells to oxygen deficiency. Since oxidative phosphorylation is compromized during hypoxia, adaptation may involve both the upregulation of glycolysis as well as downregulation of energy consumption. mRNA translation is one of the most energy costly cellular processes, and we and others have shown that global mRNA translation is rapidly inhibited during hypoxia. However, some mRNAs, including those coding for HIF-1 α and VEGF, remain efficiently translated during hypoxia. Clearly, the mechanisms responsible for the overall inhibition of translation during hypoxia does not compromize the translation of certain hypoxia-induced mRNA species. We therefore hypothesize that the inhibition of mRNA translation serves to promote hypoxia tolerance in two ways: i) through conservation of energy and ii) through differential gene expression involved in hypoxia adaptation. We have recently identified two pathways that are responsible for the global inhibition of translation during hypoxia. The phosphorylation of the eukaryotic initiation factor eIF2 α by the ER resident kinase PERK results in down-regulation of protein synthesis shortly after the onset of hypoxia. In addition, the initiation complex eIF4F is disrupted during long lasting hypoxic conditions. The identification of the molecular pathways responsible for the inhibition of overall translation during hypoxia has rendered it possible to investigate their importance for hypoxia tolerance. We have found that mouse embryo fibroblasts that are knockout for PERK and therefore not able to inhibit protein synthesis efficiently during oxygen deficiency are significantly less tolerant to hypoxia than their wildtype counterparts. We are currently also investigating the functional significance

On the direct characterization and quantification of active ingredients in commercial solid drugs using PIXE, PIGE and ToF-SIMS techniques

Energy Technology Data Exchange (ETDEWEB)

Nsouli, B; Zahraman, K; Bejjani, A; Roumie, M; Noun, M [Ion Beam Analysis Laboratory, Lebanese Atomic Energy Commission - CNRS, P.O.Box: 11-8281 Beirut (Lebanon); Younes, G [Faculty of Sciences, Department of Chemistry - Beirut Arab University, Beirut (Lebanon); Assi, S; El-Yazbi, F; Mahmoud, R [Faculty of Pharmacy, Department of Pharmaceutical Analytical Chemistry - Beirut Arab University, Beirut (Lebanon); Thomas, J P [Institut de Physique Nucleaire de Lyon - Universite Claude Bernard Lyon 1 (France)

2010-01-15

The quantification of the active ingredient (AI) in drugs is a crucial and important step in the drug quality control process. This is usually performed by using wet chemical techniques like HPLC, LC-MS/MS, UV spectrophotometry and other appropriate organic analytical methods. In the case of an active ingredient contains specific heteroatoms (F, S, Cl, Br, ...), elemental IBA technique can be explored for molecular quantification. IBA techniques permit the analysis of the sample under solid form, without any laborious sample preparation. This is an advantage when the number of sample is relatively large. In this work, we demonstrate the ability of the Thick target PIXE (TT-PIXE) and the TT-PIGE techniques for rapid and accurate quantification of celecoxib and atorvastatin in commercial solid drugs. The experimental aspects related to the quantification validity are presented and discussed. (author)

On the direct characterization and quantification of active ingredients in commercial solid drugs using PIXE, PIGE and ToF-SIMS techniques

International Nuclear Information System (INIS)

Nsouli, B.; Zahraman, K.; Bejjani, A.; Roumie, M.; Noun, M.; Younes, G.; Assi, S.; El-Yazbi, F.; Mahmoud, R.; Thomas, J.P.

2010-01-01

The quantification of the active ingredient (AI) in drugs is a crucial and important step in the drug quality control process. This is usually performed by using wet chemical techniques like HPLC, LC-MS/MS, UV spectrophotometry and other appropriate organic analytical methods. In the case of an active ingredient contains specific heteroatoms (F, S, Cl, Br, ...), elemental IBA technique can be explored for molecular quantification. IBA techniques permit the analysis of the sample under solid form, without any laborious sample preparation. This is an advantage when the number of sample is relatively large. In this work, we demonstrate the ability of the Thick target PIXE (TT-PIXE) and the TT-PIGE techniques for rapid and accurate quantification of celecoxib and atorvastatin in commercial solid drugs. The experimental aspects related to the quantification validity are presented and discussed. (author)

An investigation of nutrient-dependent mRNA translation in Drosophila larvae

Directory of Open Access Journals (Sweden)

Sabarish Nagarajan

2014-10-01

Full Text Available The larval period of the Drosophila life cycle is characterized by immense growth. In nutrient rich conditions, larvae increase in mass approximately two hundred-fold in five days. However, upon nutrient deprivation, growth is arrested. The prevailing view is that dietary amino acids drive this larval growth by activating the conserved insulin/PI3 kinase and Target of rapamycin (TOR pathways and promoting anabolic metabolism. One key anabolic process is protein synthesis. However, few studies have attempted to measure mRNA translation during larval development or examine the signaling requirements for nutrient-dependent regulation. Our work addresses this issue. Using polysome analyses, we observed that starvation rapidly (within thirty minutes decreased larval mRNA translation, with a maximal decrease at 6–18 hours. By analyzing individual genes, we observed that nutrient-deprivation led to a general reduction in mRNA translation, regardless of any starvation-mediated changes (increase or decrease in total transcript levels. Although sugars and amino acids are key regulators of translation in animal cells and are the major macronutrients in the larval diet, we found that they alone were not sufficient to maintain mRNA translation in larvae. The insulin/PI3 kinase and TOR pathways are widely proposed as the main link between nutrients and mRNA translation in animal cells. However, we found that genetic activation of PI3K and TOR signaling, or regulation of two effectors – 4EBP and S6K – could not prevent the starvation-mediated translation inhibition. Similarly, we showed that the nutrient stress-activated eIF2α kinases, GCN2 and PERK, were not required for starvation-induced inhibition of translation in larvae. These findings indicate that nutrient control of mRNA translation in larvae is more complex than simply amino acid activation of insulin and TOR signaling.

Localization of insulin receptor mRNA in rat brain by in situ hybridization

International Nuclear Information System (INIS)

Marks, J.L.; Porte, D. Jr.; Stahl, W.L.; Baskin, D.G.

1990-01-01

Insulin receptor mRNA was demonstrated in rat brain slices by in situ hybridization with three 35 S-oligonucleotide probes and contact film autoradiography. Specificity was confirmed by showing that (a) excess unlabeled probe abolished the signal, (b) an oligonucleotide probe for rat neuropeptide Y mRNA showed a different distribution of hybridization signal, and (c) the distribution of insulin receptor binding was consistent with the distribution of insulin receptor mRNA. Insulin receptor mRNA was most abundant in the granule cell layers of the olfactory bulb, cerebellum and dentate gyrus, in the pyramidal cell body layers of the pyriform cortex and hippocampus, in the choroid plexus and in the arcuate nucleus of the hypothalamus

Interleukin-21 mRNA expression during virus infections

DEFF Research Database (Denmark)

Holm, Christian; Nyvold, Charlotte Guldborg; Paludan, Søren Riis

2006-01-01

and activational effects of IL-21 on different leukocytes come into play in vivo in an immune response has so far not been fully investigated. We show here for the first time in vivo, that IL-21 mRNA is produced in the spleen when mice are challenged with herpes simplex virus type 2 (HSV-2) or lymphocytic...... choriomeningitis virus (LCMV). We show in HSV-2 challenged mice that this production takes place in CD4+ T cell fractions and is absent in CD4+ T cell-depleted fractions. We also show that the peak of IL-21 mRNA production in both the HSV-2 and LCMV-challenged mice coincides with the onset of the adaptive immune...

Identification and Relative Quantification of Bioactive Peptides Sequentially Released during Simulated Gastrointestinal Digestion of Commercial Kefir.

Science.gov (United States)

Liu, Yufang; Pischetsrieder, Monika

2017-03-08

Health-promoting effects of kefir may be partially caused by bioactive peptides. To evaluate their formation or degradation during gastrointestinal digestion, we monitored changes of the peptide profile in a model of (1) oral, (2) gastric, and (3) small intestinal digestion of kefir. Matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analyses revealed clearly different profiles between digests 2/3 and kefir/digest 1. Subsequent ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry identified 92 peptides in total (25, 25, 43, and 30, partly overlapping in kefir and digests 1, 2, and 3, respectively), including 16 peptides with ascribed bioactivity. Relative quantification in scheduled multiple reaction monitoring mode showed that many bioactive peptides were released by simulated digestion. Most prominently, the concentration of angiotensin-converting enzyme inhibitor β-casein 203-209 increased approximately 10 000-fold after combined oral, gastric, and intestinal digestion. Thus, physiological digestive processes may promote bioactive peptide formation from proteins and oligopeptides in kefir. Furthermore, bioactive peptides present in certain compartments of the gastrointestinal tract may exert local physiological effects.

Quantification of three steroid hormone receptors of the leopard gecko (Eublepharis macularius), a lizard with temperature-dependent sex determination: their tissue distributions and the effect of environmental change on their expressions.

Science.gov (United States)

Endo, Daisuke; Park, Min Kyun

2003-12-01

Sex steroid hormones play a central role in the reproduction of all vertebrates. These hormones function through their specific receptors, so the expression levels of the receptors may reflect the responsibility of target organs. However, there was no effective method to quantify the expression levels of these receptors in reptilian species. In this study, we established the competitive-PCR assay systems for the quantification of the mRNA expression levels of three sex steroid hormone receptors in the leopard gecko. These assay systems were successfully able to detect the mRNA expression level of each receptor in various organs of male adult leopard geckoes. The expression levels of mRNA of these receptors were highly various depending on the organs assayed. This is the first report regarding the tissue distributions of sex steroid hormone receptor expressions in reptile. The effects of environmental conditions on these hormone receptor expressions were also examined. After the low temperature and short photoperiod treatment for 6 weeks, only the androgen receptor expression was significantly increased in the testes. The competitive-PCR assay systems established in this report should be applicable for various studies of the molecular mechanism underlying the reproductive activity of the leopard gecko.

Evaluation of mRNA markers for estimating blood deposition time: Towards alibi testing from human forensic stains with rhythmic biomarkers.

Science.gov (United States)

Lech, Karolina; Liu, Fan; Ackermann, Katrin; Revell, Victoria L; Lao, Oscar; Skene, Debra J; Kayser, Manfred

2016-03-01

Determining the time a biological trace was left at a scene of crime reflects a crucial aspect of forensic investigations as - if possible - it would permit testing the sample donor's alibi directly from the trace evidence, helping to link (or not) the DNA-identified sample donor with the crime event. However, reliable and robust methodology is lacking thus far. In this study, we assessed the suitability of mRNA for the purpose of estimating blood deposition time, and its added value relative to melatonin and cortisol, two circadian hormones we previously introduced for this purpose. By analysing 21 candidate mRNA markers in blood samples from 12 individuals collected around the clock at 2h intervals for 36h under real-life, controlled conditions, we identified 11 mRNAs with statistically significant expression rhythms. We then used these 11 significantly rhythmic mRNA markers, with and without melatonin and cortisol also analysed in these samples, to establish statistical models for predicting day/night time categories. We found that although in general mRNA-based estimation of time categories was less accurate than hormone-based estimation, the use of three mRNA markers HSPA1B, MKNK2 and PER3 together with melatonin and cortisol generally enhanced the time prediction accuracy relative to the use of the two hormones alone. Our data best support a model that by using these five molecular biomarkers estimates three time categories, i.e. night/early morning, morning/noon, and afternoon/evening with prediction accuracies expressed as AUC values of 0.88, 0.88, and 0.95, respectively. For the first time, we demonstrate the value of mRNA for blood deposition timing and introduce a statistical model for estimating day/night time categories based on molecular biomarkers, which shall be further validated with additional samples in the future. Moreover, our work provides new leads for molecular approaches on time of death estimation using the significantly rhythmic mRNA

Exogenous mRNA encoding tetanus or botulinum neurotoxins expressed in Aplysia neurons

NARCIS (Netherlands)

Mochida, Sumiko; Poulain, Bernard; Eisel, Ulrich; Binz, Thomas; Kurazono, Hisao; Niemann, Heiner; Tauc, Ladislav; Bullock, Theodore H.

1990-01-01

Injection of exogenous mRNA purified from various tissue preparations into cellular translation systems such as Xenopus oocytes has allowed expression of complex proteins (e.g., receptors for neurotransmitters). No evidence for expression of injected exogenous mRNA, however, has been reported in

SU-G-IeP1-07: Inaccuracy of Lesion Blood Flow Quantification Related to the Proton Density Reference Image in Arterial Spin Labeling MRI of Brain Tumors

International Nuclear Information System (INIS)

Jen, M; Johnson, J; Hou, P; Liu, H

2016-01-01

Purpose: Cerebral blood flow quantification in arterial spin labeling (ASL) MRI requires an estimate of the equilibrium magnetization of blood, which is often obtained by a set of proton density (PD) reference image. Normally, a constant blood-brain partition coefficient is assumed across the brain. However, this assumption may not be valid for brain lesions. This study aimed to evaluate the impact of lesion-related PD variations on ASL quantification in patients with brain tumors. Methods: MR images for posttreatment evaluation of 42 patients with brain tumors were retrospectively analyzed. These images were acquired on a 3T MRI scanner, including T2-weighted FLAIR, 3D pseudo-continuous ASL and post-contrast T1-weighted images. Anatomical images were coregistered with ASL images using the SPM software. Regions of interest (ROIs) of the enhancing and FLAIR lesions were manually drawn on the coregistered images. ROIs of the contralateral normal appearing tissues were also determined, with the consideration of approximating coil sensitivity patterns in lesion ROIs. Relative lesion blood flow (lesion/contralateral tissue) was calculated from both the CBF map (dependent on the PD) and the ΔM map for comparison. Results: The signal intensities in both enhancing and FLAIR lesions were significantly different than contralateral tissues on the PD reference image (p<0.001). The percent signal difference ranged from −15.9 to 19.2%, with a mean of 5.4% for the enhancing lesion, and from −2.8 to 22.9% with a mean of 10.1% for the FLAIR lesion. The high/low lesion-related PD signal resulted in inversely proportional under-/over-estimation of blood flow in both enhancing and FLAIR lesions. Conclusion: Significant signal differences were found between lesions and contralateral tissues in the PD reference image, which introduced errors in blood flow quantification in ASL. The error can be up to 20% in individual patients with an average of 5- 10% for the group of patients

SU-G-IeP1-07: Inaccuracy of Lesion Blood Flow Quantification Related to the Proton Density Reference Image in Arterial Spin Labeling MRI of Brain Tumors

Energy Technology Data Exchange (ETDEWEB)

Jen, M; Johnson, J; Hou, P; Liu, H [UT MD Anderson Cancer Center, Houston, TX (United States)

2016-06-15

Purpose: Cerebral blood flow quantification in arterial spin labeling (ASL) MRI requires an estimate of the equilibrium magnetization of blood, which is often obtained by a set of proton density (PD) reference image. Normally, a constant blood-brain partition coefficient is assumed across the brain. However, this assumption may not be valid for brain lesions. This study aimed to evaluate the impact of lesion-related PD variations on ASL quantification in patients with brain tumors. Methods: MR images for posttreatment evaluation of 42 patients with brain tumors were retrospectively analyzed. These images were acquired on a 3T MRI scanner, including T2-weighted FLAIR, 3D pseudo-continuous ASL and post-contrast T1-weighted images. Anatomical images were coregistered with ASL images using the SPM software. Regions of interest (ROIs) of the enhancing and FLAIR lesions were manually drawn on the coregistered images. ROIs of the contralateral normal appearing tissues were also determined, with the consideration of approximating coil sensitivity patterns in lesion ROIs. Relative lesion blood flow (lesion/contralateral tissue) was calculated from both the CBF map (dependent on the PD) and the ΔM map for comparison. Results: The signal intensities in both enhancing and FLAIR lesions were significantly different than contralateral tissues on the PD reference image (p<0.001). The percent signal difference ranged from −15.9 to 19.2%, with a mean of 5.4% for the enhancing lesion, and from −2.8 to 22.9% with a mean of 10.1% for the FLAIR lesion. The high/low lesion-related PD signal resulted in inversely proportional under-/over-estimation of blood flow in both enhancing and FLAIR lesions. Conclusion: Significant signal differences were found between lesions and contralateral tissues in the PD reference image, which introduced errors in blood flow quantification in ASL. The error can be up to 20% in individual patients with an average of 5- 10% for the group of patients

A novel nano-immunoassay method for quantification of proteins from CD138-purified myeloma cells: biological and clinical utility.

Science.gov (United States)

Misiewicz-Krzeminska, Irena; Corchete, Luis Antonio; Rojas, Elizabeta A; Martínez-López, Joaquín; García-Sanz, Ramón; Oriol, Albert; Bladé, Joan; Lahuerta, Juan-José; Miguel, Jesús San; Mateos, María-Victoria; Gutiérrez, Norma C

2018-05-01

Protein analysis in bone marrow samples from patients with multiple myeloma has been limited by the low concentration of proteins obtained after CD138 + cell selection. A novel approach based on capillary nano-immunoassay could make it possible to quantify dozens of proteins from each myeloma sample in an automated manner. Here we present a method for the accurate and robust quantification of the expression of multiple proteins extracted from CD138-purified multiple myeloma samples frozen in RLT Plus buffer, which is commonly used for nucleic acid preservation and isolation. Additionally, the biological and clinical value of this analysis for a panel of 12 proteins essential to the pathogenesis of multiple myeloma was evaluated in 63 patients with newly diagnosed multiple myeloma. The analysis of the prognostic impact of CRBN /Cereblon and IKZF1 /Ikaros mRNA/protein showed that only the protein levels were able to predict progression-free survival of patients; mRNA levels were not associated with prognosis. Interestingly, high levels of Cereblon and Ikaros proteins were associated with longer progression-free survival only in patients who received immunomodulatory drugs and not in those treated with other drugs. In conclusion, the capillary nano-immunoassay platform provides a novel opportunity for automated quantification of the expression of more than 20 proteins in CD138 + primary multiple myeloma samples. Copyright © 2018 Ferrata Storti Foundation.

Development of a qPCR Method for the Identification and Quantification of Two Closely Related Tuna Species, Bigeye Tuna (Thunnus obesus) and Yellowfin Tuna (Thunnus albacares), in Canned Tuna.

Science.gov (United States)

Bojolly, Daline; Doyen, Périne; Le Fur, Bruno; Christaki, Urania; Verrez-Bagnis, Véronique; Grard, Thierry

2017-02-01

Bigeye tuna (Thunnus obesus) and yellowfin tuna (Thunnus albacares) are among the most widely used tuna species for canning purposes. Not only substitution but also mixing of tuna species is prohibited by the European regulation for canned tuna products. However, as juveniles of bigeye and yellowfin tunas are very difficult to distinguish, unintentional substitutions may occur during the canning process. In this study, two mitochondrial markers from NADH dehydrogenase subunit 2 and cytochrome c oxidase subunit II genes were used to identify bigeye tuna and yellowfin tuna, respectively, utilizing TaqMan qPCR methodology. Two different qPCR-based methods were developed to quantify the percentage of flesh of each species used for can processing. The first one was based on absolute quantification using standard curves realized with these two markers; the second one was founded on relative quantification with the universal 12S rRNA gene as the endogenous gene. On the basis of our results, we conclude that our methodology could be applied to authenticate these two closely related tuna species when used in a binary mix in tuna cans.

Natural selection and algorithmic design of mRNA.

Science.gov (United States)

Cohen, Barry; Skiena, Steven

2003-01-01

Messenger RNA (mRNA) sequences serve as templates for proteins according to the triplet code, in which each of the 4(3) = 64 different codons (sequences of three consecutive nucleotide bases) in RNA either terminate transcription or map to one of the 20 different amino acids (or residues) which build up proteins. Because there are more codons than residues, there is inherent redundancy in the coding. Certain residues (e.g., tryptophan) have only a single corresponding codon, while other residues (e.g., arginine) have as many as six corresponding codons. This freedom implies that the number of possible RNA sequences coding for a given protein grows exponentially in the length of the protein. Thus nature has wide latitude to select among mRNA sequences which are informationally equivalent, but structurally and energetically divergent. In this paper, we explore how nature takes advantage of this freedom and how to algorithmically design structures more energetically favorable than have been built through natural selection. In particular: (1) Natural Selection--we perform the first large-scale computational experiment comparing the stability of mRNA sequences from a variety of organisms to random synonymous sequences which respect the codon preferences of the organism. This experiment was conducted on over 27,000 sequences from 34 microbial species with 36 genomic structures. We provide evidence that in all genomic structures highly stable sequences are disproportionately abundant, and in 19 of 36 cases highly unstable sequences are disproportionately abundant. This suggests that the stability of mRNA sequences is subject to natural selection. (2) Artificial Selection--motivated by these biological results, we examine the algorithmic problem of designing the most stable and unstable mRNA sequences which code for a target protein. We give a polynomial-time dynamic programming solution to the most stable sequence problem (MSSP), which is asymptotically no more complex

(1) H-MRS processing parameters affect metabolite quantification

DEFF Research Database (Denmark)

Bhogal, Alex A; Schür, Remmelt R; Houtepen, Lotte C

2017-01-01

investigated the influence of model parameters and spectral quantification software on fitted metabolite concentration values. Sixty spectra in 30 individuals (repeated measures) were acquired using a 7-T MRI scanner. Data were processed by four independent research groups with the freedom to choose their own...... + NAAG/Cr + PCr and Glu/Cr + PCr, respectively. Metabolite quantification using identical (1) H-MRS data was influenced by processing parameters, basis sets and software choice. Locally preferred processing choices affected metabolite quantification, even when using identical software. Our results......Proton magnetic resonance spectroscopy ((1) H-MRS) can be used to quantify in vivo metabolite levels, such as lactate, γ-aminobutyric acid (GABA) and glutamate (Glu). However, there are considerable analysis choices which can alter the accuracy or precision of (1) H-MRS metabolite quantification...

A refined methodology for modeling volume quantification performance in CT

Science.gov (United States)

Chen, Baiyu; Wilson, Joshua; Samei, Ehsan

2014-03-01

The utility of CT lung nodule volume quantification technique depends on the precision of the quantification. To enable the evaluation of quantification precision, we previously developed a mathematical model that related precision to image resolution and noise properties in uniform backgrounds in terms of an estimability index (e'). The e' was shown to predict empirical precision across 54 imaging and reconstruction protocols, but with different correlation qualities for FBP and iterative reconstruction (IR) due to the non-linearity of IR impacted by anatomical structure. To better account for the non-linearity of IR, this study aimed to refine the noise characterization of the model in the presence of textured backgrounds. Repeated scans of an anthropomorphic lung phantom were acquired. Subtracted images were used to measure the image quantum noise, which was then used to adjust the noise component of the e' calculation measured from a uniform region. In addition to the model refinement, the validation of the model was further extended to 2 nodule sizes (5 and 10 mm) and 2 segmentation algorithms. Results showed that the magnitude of IR's quantum noise was significantly higher in structured backgrounds than in uniform backgrounds (ASiR, 30-50%; MBIR, 100-200%). With the refined model, the correlation between e' values and empirical precision no longer depended on reconstruction algorithm. In conclusion, the model with refined noise characterization relfected the nonlinearity of iterative reconstruction in structured background, and further showed successful prediction of quantification precision across a variety of nodule sizes, dose levels, slice thickness, reconstruction algorithms, and segmentation software.

Influenza polymerase encoding mRNAs utilize atypical mRNA nuclear export.

Science.gov (United States)

Larsen, Sean; Bui, Steven; Perez, Veronica; Mohammad, Adeba; Medina-Ramirez, Hilario; Newcomb, Laura L

2014-08-28

Influenza is a segmented negative strand RNA virus. Each RNA segment is encapsulated by influenza nucleoprotein and bound by the viral RNA dependent RNA polymerase (RdRP) to form viral ribonucleoproteins responsible for RNA synthesis in the nucleus of the host cell. Influenza transcription results in spliced mRNAs (M2 and NS2), intron-containing mRNAs (M1 and NS1), and intron-less mRNAs (HA, NA, NP, PB1, PB2, and PA), all of which undergo nuclear export into the cytoplasm for translation. Most cellular mRNA nuclear export is Nxf1-mediated, while select mRNAs utilize Crm1. Here we inhibited Nxf1 and Crm1 nuclear export prior to infection with influenza A/Udorn/307/1972(H3N2) virus and analyzed influenza intron-less mRNAs using cellular fractionation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We examined direct interaction between Nxf1 and influenza intron-less mRNAs using immuno purification of Nxf1 and RT-PCR of associated RNA. Inhibition of Nxf1 resulted in less influenza intron-less mRNA export into the cytoplasm for HA and NA influenza mRNAs in both human embryonic kidney cell line (293 T) and human lung adenocarcinoma epithelial cell line (A549). However, in 293 T cells no change was observed for mRNAs encoding the components of the viral ribonucleoproteins; NP, PA, PB1, and PB2, while in A549 cells, only PA, PB1, and PB2 mRNAs, encoding the RdRP, remained unaffected; NP mRNA was reduced in the cytoplasm. In A549 cells NP, NA, HA, mRNAs were found associated with Nxf1 but PA, PB1, and PB2 mRNAs were not. Crm1 inhibition also resulted in no significant difference in PA, PB1, and PB2 mRNA nuclear export. These results further confirm Nxf1-mediated nuclear export is functional during the influenza life cycle and hijacked for select influenza mRNA nuclear export. We reveal a cell type difference for Nxf1-mediated nuclear export of influenza NP mRNA, a reminder that cell type can influence molecular mechanisms. Importantly, we

Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning

International Nuclear Information System (INIS)

Han, J.H.; Stratowa, C.; Rutter, W.J.

1987-01-01

The authors have cloned a full-length putative rat pancreatic lysophospholipase cDNA by an improved mRNA isolation method and cDNA cloning strategy using [ 32 P]-labelled nucleotides. These new methods allow the construction of a cDNA library from the adult rat pancreas in which the majority of recombinant clones contained complete sequences for the corresponding mRNAs. A previously recognized but unidentified long and relatively rare cDNA clone containing the entire sequence from the cap site at the 5' end to the poly(A) tail at the 3' end of the mRNA was isolated by single-step screening of the library. The size, amino acid composition, and the activity of the protein expressed in heterologous cells strongly suggest this mRNA codes for lysophospholipase

Real-time RT-PCR analysis of mRNA decay: half-life of Beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines

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Barredo Julio C

2002-03-01

Full Text Available Abstract Background We describe an alternative method to determine mRNA half-life (t1/2 based on the Real-Time RT-PCR procedure. This approach was evaluated by using the β-actin gene as a reference molecule for measuring of mRNA stability. Results Human leukemia Nalm-6 and CCRF-CEM cells were treated with various concentrations of Actinomycin D to block transcription and aliquots were removed periodically. Total RNA was isolated and quantified using the RiboGreen® fluorescent dye with the VersaFluor Fluorometer System. One μg of total RNA was reverse transcribed and used as template for the amplification of a region of the β-actin gene (231 bp. To generate the standard curve, serial ten-fold dilutions of the pBactin-231 vector containing the cDNA amplified fragment were employed, β-actin mRNAs were quantified by Real-Time RT-PCR using the SYBR® Green I fluorogenic dye and data analyzed using the iCycle iQ system software. Using this method, the β-actin mRNA exhibited a half-life of 6.6 h and 13.5 h in Nalm-6 and CCRF-CEM cells, respectively. The t1/2 value obtained for Nalm-6 is comparable to those estimated from Northern blot studies, using normal human leukocytes (5.5 h. Conclusions We have developed a rapid, sensitive, and reliable method based on Real-Time RT-PCR for measuring mRNA half-life. Our results confirm that β-actin mRNA half-life can be affected by the cellular growth rate.

Benchmarking common quantification strategies for large-scale phosphoproteomics

DEFF Research Database (Denmark)

Hogrebe, Alexander; von Stechow, Louise; Bekker-Jensen, Dorte B

2018-01-01

Comprehensive mass spectrometry (MS)-based proteomics is now feasible, but reproducible quantification remains challenging, especially for post-translational modifications such as phosphorylation. Here, we compare the most popular quantification techniques for global phosphoproteomics: label-free...

Hedgehog signaling pathway is active in GBM with GLI1 mRNA expression showing a single continuous distribution rather than discrete high/low clusters.

Science.gov (United States)

Chandra, Vikas; Das, Tapojyoti; Gulati, Puneet; Biswas, Nidhan K; Rote, Sarang; Chatterjee, Uttara; Ghosh, Samarendra N; Deb, Sumit; Saha, Suniti K; Chowdhury, Anup K; Ghosh, Subhashish; Rudin, Charles M; Mukherjee, Ankur; Basu, Analabha; Dhara, Surajit

2015-01-01

Hedgehog (Hh) signaling pathway is a valid therapeutic target in a wide range of malignancies. We focus here on glioblastoma multiforme (GBM), a lethal malignancy of the central nervous system (CNS). By analyzing RNA-sequencing based transcriptomics data on 149 clinical cases of TCGA-GBM database we show here a strong correlation (r = 0.7) between GLI1 and PTCH1 mRNA expression--as a hallmark of the canonical Hh-pathway activity in this malignancy. GLI1 mRNA expression varied in 3 orders of magnitude among the GBM patients of the same cohort showing a single continuous distribution-unlike the discrete high/low-GLI1 mRNA expressing clusters of medulloblastoma (MB). When compared with MB as a reference, the median GLI1 mRNA expression in GBM appeared 14.8 fold lower than that of the "high-Hh" cluster of MB but 5.6 fold higher than that of the "low-Hh" cluster of MB. Next, we demonstrated statistically significant up- and down-regulation of GLI1 mRNA expressions in GBM patient-derived low-passage neurospheres in vitro by sonic hedgehog ligand-enriched conditioned media (shh-CM) and by Hh-inhibitor drug vismodegib respectively. We also showed clinically achievable dose (50 μM) of vismodegib alone to be sufficient to induce apoptosis and cell cycle arrest in these low-passage GBM neurospheres in vitro. Vismodegib showed an effect on the neurospheres, both by down-regulating GLI1 mRNA expression and by inducing apoptosis/cell cycle arrest, irrespective of their relative endogenous levels of GLI1 mRNA expression. We conclude from our study that this single continuous distribution pattern of GLI1 mRNA expression technically puts almost all GBM patients in a single group rather than discrete high- or low-clusters in terms of Hh-pathway activity. That is suggestive of therapies with Hh-pathway inhibitor drugs in this malignancy without a need for further stratification of patients on the basis of relative levels of Hh-pathway activity among them.

CBFA1 and topoisomerase I mRNA levels decline during cellular aging of human trabecular osteoblasts

DEFF Research Database (Denmark)

Christiansen, Mette; Kveiborg, M.; Kassem, M.

2000-01-01

In order to understand the reasons for age-related impairment of the function of bone forming osteoblasts, we have examined the steady-state mRNA levels of the transcription factor CBFA1 and topoisomerase I during cellular aging of normal human trabecular osteoblasts, by the use of semiquantitati...

Occupational Toluene Exposure Induces Cytochrome P450 2E1 mRNA Expression in Peripheral Lymphocytes

Science.gov (United States)

Mendoza-Cantú, Ania; Castorena-Torres, Fabiola; de León, Mario Bermúdez; Cisneros, Bulmaro; López-Carrillo, Lizbeth; Rojas-García, Aurora E.; Aguilar-Salinas, Alberto; Manno, Maurizio; Albores, Arnulfo

2006-01-01

Print workers are exposed to organic solvents, of which the systemic toxicant toluene is a main component. Toluene induces expression of cytochrome P450 2E1 (CYP2E1), an enzyme involved in its own metabolism and that of other protoxicants, including some procarcinogens. Therefore, we investigated the association between toluene exposure and the CYP2E1 response, as assessed by mRNA content in peripheral lymphocytes or the 6-hydroxychlorzoxazone (6OH-CHZ)/chlorzoxazone (CHZ) quotient (known as CHZ metabolic ratio) in plasma, and the role of genotype (5′-flanking region RsaI/PstI polymorphic sites) in 97 male print workers. The geometric mean (GM) of toluene concentration in the air was 52.80 ppm (10–760 ppm); 54% of the study participants were exposed to toluene concentrations that exceeded the maximum permissible exposure level (MPEL). The GM of urinary hippuric acid at the end of a work shift (0.041 g/g creatinine) was elevated relative to that before the shift (0.027 g/g creatinine; p < 0.05). The GM of the CHZ metabolic ratio was 0.33 (0–9.3), with 40% of the subjects having ratios below the GM. However, the average CYP2E1 mRNA level in peripheral lymphocytes was 1.07 (0.30–3.08), and CYP2E1 mRNA levels within subjects correlated with the toluene exposure ratio (environmental toluene concentration:urinary hippuric acid concentration) (p = 0.014). Genotype did not alter the association between the toluene exposure ratio and mRNA content. In summary, with further validation, CYP2E1 mRNA content in peripheral lymphocytes could be a sensitive and noninvasive biomarker for the continuous monitoring of toluene effects in exposed persons. PMID:16581535

Selective translation of the measles virus nucleocapsid mRNA by La protein

Directory of Open Access Journals (Sweden)

Yoshihisa eInoue

2011-08-01

Full Text Available Measles, caused by measles virus (MeV infection, is the leading cause of death in children because of secondary infections attributable to MeV-induced immune suppression. Recently, we have shown that wild-type MeVs induce the suppression of protein synthesis in host cells (referred to as "shutoff" and that viral mRNAs are preferentially translated under shutoff conditions in infected cells. To determine the mechanism behind the preferential translation of viral mRNA, we focused on the 5 untranslated region (UTR of nucleocapsid (N mRNA. The La/SSB autoantigen (La was found to specifically bind to an N-5UTR probe. Recombinant La enhanced the translation of luciferase mRNA containing the N-5UTR (N-fLuc, and RNA interference of La suppressed N-fLuc translation. Furthermore, recombinant MeV lacking the La-binding motif in the N-5UTR displayed delayed viral protein synthesis and growth kinetics at an early phase of infection. These results suggest that La induced predominant translation of N mRNA via binding to its 5UTR under shutoff conditions. This is the first report on a cellular factor that specifically regulates paramyxovirus mRNA translation.

[Influence of FPS on the expression of LDL-R mRNA in the liver tissues of hyperlipidemic rats].

Science.gov (United States)

Wu, Qing-he; Xing, Yan-hong; Rong, Xiang-lu; Huang, Ping

2007-08-01

To explore the effect of FPS on low-density lipoprotein acceptor (LDL-R) mRNA in the liver tissues of hyperlipidemic rats. Sixty healthy male SD rats were randomly divided into six groups: normal control, model control, Gynostemma pentaphyllum, FPS low dosage, FPS moderate dosage, and FPS high dosage group. Excepting the rats in the normal control group, the ones in other groups were all made rats' hyperlipidemic model by irrigating hyperlipidemic emulsion into the stomach and observed the expression of LDL-R mRNA in the liver tissues of rats of each group. Relative content of LDL-RmRNA in low and moderate dosage groups was notably higher than that inmodel group. The contents's difference was not remarkable between FPS moderate dosage group and Gynostemma pentaphyllum group. FPS can appreciably increase the expression of LDL-R mRNA in the liver tissues of hyperlipidemic rats and promote the elimination ofLDL-C to reduce serum cholesterol notably.

Chemokine-Like Receptor 1 mRNA Weakly Correlates with Non-Alcoholic Steatohepatitis Score in Male but Not Female Individuals

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Maximilian Neumann

2016-08-01

Full Text Available The chemokine-like receptor 1 (CMKLR1 ligands resolvin E1 and chemerin are known to modulate inflammatory response. The progression of non-alcoholic fatty liver disease (NAFLD to non-alcoholic steatohepatitis (NASH is associated with inflammation. Here it was analyzed whether hepatic CMKLR1 expression is related to histological features of NASH. Therefore, CMKLR1 mRNA was quantified in liver tissue of 33 patients without NAFLD, 47 patients with borderline NASH and 38 patients with NASH. Hepatic CMKLR1 mRNA was not associated with gender and body mass index (BMI in the controls and the whole study group. CMKLR1 expression was similar in controls and in patients with borderline NASH and NASH. In male patients weak positive correlations with inflammation, fibrosis and NASH score were identified. In females CMKLR1 was not associated with features of NAFLD. Liver CMKLR1 mRNA tended to be higher in type 2 diabetes patients of both genders and in hypercholesterolemic women. In summary, this study shows that hepatic CMKLR1 mRNA is weakly associated with features of NASH in male patients only.

Quantification of Cannabinoid Content in Cannabis

Science.gov (United States)

Tian, Y.; Zhang, F.; Jia, K.; Wen, M.; Yuan, Ch.

2015-09-01

Cannabis is an economically important plant that is used in many fields, in addition to being the most commonly consumed illicit drug worldwide. Monitoring the spatial distribution of cannabis cultivation and judging whether it is drug- or fiber-type cannabis is critical for governments and international communities to understand the scale of the illegal drug trade. The aim of this study was to investigate whether the cannabinoids content in cannabis could be spectrally quantified using a spectrometer and to identify the optimal wavebands for quantifying the cannabinoid content. Spectral reflectance data of dried cannabis leaf samples and the cannabis canopy were measured in the laboratory and in the field, respectively. Correlation analysis and the stepwise multivariate regression method were used to select the optimal wavebands for cannabinoid content quantification based on the laboratory-measured spectral data. The results indicated that the delta-9-tetrahydrocannabinol (THC) content in cannabis leaves could be quantified using laboratory-measured spectral reflectance data and that the 695 nm band is the optimal band for THC content quantification. This study provides prerequisite information for designing spectral equipment to enable immediate quantification of THC content in cannabis and to discriminate drug- from fiber-type cannabis based on THC content quantification in the field.

Sexual phenotype differences in zic2 mRNA abundance in the preoptic area of a protogynous teleost, Thalassoma bifasciatum.

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Katherine McCaffrey

Full Text Available The highly conserved members of the zic family of zinc-finger transcription factors are primarily known for their roles in embryonic signaling pathways and regulation of cellular proliferation and differentiation. This study describes sexual phenotype differences in abundances of zic2 mRNA in the preoptic area of the hypothalamus, a region strongly implicated in sexual behavior and function, in an adult teleost, Thalassoma bifasciatum. The bluehead wrasse (Thalassoma bifasciatum is a valuable model for studying neuroendocrine processes because it displays two discrete male phenotypes, initial phase (IP males and territorial, terminal phase (TP males, and undergoes socially-controlled protogynous sex change. Previously generated microarray-based comparisons suggested that zic2 was upregulated in the brains of terminal phase males relative to initial phase males. To further explore this difference, we cloned a 727 bp sequence for neural zic2 from field-collected animals. Riboprobe-based in situ hybridization was employed to localize zic2 signal in adult bluehead brains and assess the relative abundance of brain zic2 mRNA across sexual phenotypes. We found zic2 mRNA expression was extremely abundant in the granular cells of the cerebellum and widespread in other brain regions including in the thalamus, hypothalamus, habenula, torus semicircularis, torus longitudinalis, medial longitudinal fascicle and telencephalic areas. Quantitative autoradiography and phosphorimaging showed zic2 mRNA hybridization signal in the preoptic area of the hypothalamus was significantly higher in terminal phase males relative to both initial phase males and females, and silver grain analysis confirmed this relationship between phenotypes. No significant difference in abundance was found in zic2 signal across phenotypes in the habenula, a brain region not implicated in the control of sexual behavior, or cerebellum.

The effect of leptin receptor deficiency and fasting on cannabinoid receptor 1 mRNA expression in the rat hypothalamus, brainstem and nodose ganglion.

Science.gov (United States)

Jelsing, Jacob; Larsen, Philip Just; Vrang, Niels

2009-10-02

Despite ample evidence for the involvement of the endocannabinoid system in the control of appetite, food intake and energy balance, relatively little is known about the regulation of cannabinoid receptor 1 (CB(1)R) expression in respect to leptin signalling and fasting. In the present study, we examined CB(1)R mRNA levels in lean (Fa/?) and obese (fa/fa) male Zucker rats under basal and food-restricted conditions. Using stereological sampling principles coupled with semi-quantitative radioactive in situ hybridization we provide semi-quantitative estimates of CB(1)R mRNA expression in key appetite regulatory hypothalamic and brainstem areas, as well as in the nodose ganglia. Whereas no effect of fasting were determined on CB(1)R mRNA levels in the paraventricular (PVN) and ventromedial hypothalamic (VMH) nucleus, in the brainstem dorsal vagal complex or nodose ganglion of lean Zucker rats, CB(1)R mRNA levels were consistently elevated in obese Zucker rats pointing to a direct influence of disrupted leptin signalling on CB(1)R mRNA regulation.

Effect of rat ovary irradiation or OVX on the expression of COLI and TGF-β1 mRNA in the rat bone

International Nuclear Information System (INIS)

Gao Yanhong; Gao Jianjun; Jin Weifang; Wang Hongfu

2003-01-01

To observe the effects of exposure of rat ovary to radiation or OVX on the expression of TGF-β 1 and COLI in the rat bone. The mRNA levels of TGF-β 1 and COLI in rat tibiae were measured with RT-PCR after the rat ovaries were irradiated by 50 Gy of 137 Cs γ-rays or OVX. For both the radiation group and the OVX group, the COLI mRNA level in the rat bone increased, whereas the TGF-β 1 decreased. Irradiation of ovary and OVX affect the expression of COLI and TGF-β 1 mRNA in bone probably in a similar way which is related to estrogen decrease

RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

KAUST Repository

Köster, Tino

2017-04-13

RNA–protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture – where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

KAUST Repository

Kö ster, Tino; Marondedze, Claudius; Meyer, Katja; Staiger, Dorothee

2017-01-01

RNA–protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture – where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

Postage for the messenger: Designating routes for Nuclear mRNA Export

Science.gov (United States)

Natalizio, Barbara J.; Wente, Susan R.

2013-01-01

Transcription of messenger(m) RNA occurs in the nucleus, making the translocation of mRNA across the nuclear envelope (NE) boundary a critical determinant of proper gene expression and cell survival. A major mRNA export route occurs via the NXF1-dependent pathway through the nuclear pore complexes (NPCs) embedded in the NE. However, recent findings have discovered new evidence supporting the existence of multiple mechanisms for crossing the NE, including both NPC-mediated and NE budding-mediated pathways. An analysis of the trans-acting factors and cis components that define these pathways reveals shared elements as well as mechanistic differences. We review here the current understanding of the mechanisms that characterize each pathway and highlight the determinants that influence mRNA transport fate. PMID:23583578

Nerve growth factor mRNA in brain: localization by in situ hybridization

International Nuclear Information System (INIS)

Rennert, P.D.; Heinrich, G.

1986-01-01

Nerve Growth Factor is a 118 amino acid polypeptide that plays an important role in the differentiation and survival of neurons. The recent discovery that a mRNA that encodes beta Nerve Growth Factor is present in brain suggests that the Nerve Growth Factor gene may not only regulate gene expression of peripheral but also of central neurons. To identify the site(s) of Nerve Growth Factor mRNA production in the brain and to determine which cells express the Nerve Growth Factor gene, the technique of in situ hybridization was employed. A 32P-labeled RNA probe complementary to Nerve Growth Factor mRNA hybridized to cells in the stratum granulosum of the dentate gyrus and the stratum pyramidale of the hippocampus. These observations identify for the first time cellular sites of Nerve Growth Factor gene expression in the central nervous system, and suggest that Nerve Growth Factor mRNA is produced by neurons

Uncertainty quantification and stochastic modeling with Matlab

CERN Document Server

Souza de Cursi, Eduardo

2015-01-01

Uncertainty Quantification (UQ) is a relatively new research area which describes the methods and approaches used to supply quantitative descriptions of the effects of uncertainty, variability and errors in simulation problems and models. It is rapidly becoming a field of increasing importance, with many real-world applications within statistics, mathematics, probability and engineering, but also within the natural sciences. Literature on the topic has up until now been largely based on polynomial chaos, which raises difficulties when considering different types of approximation and does no

Tristetraprolin regulation of interleukin 23 mRNA stability prevents a spontaneous inflammatory disease.

Science.gov (United States)

Molle, Céline; Zhang, Tong; Ysebrant de Lendonck, Laure; Gueydan, Cyril; Andrianne, Mathieu; Sherer, Félicie; Van Simaeys, Gaetan; Blackshear, Perry J; Leo, Oberdan; Goriely, Stanislas

2013-08-26

Interleukin (IL) 12 and IL23 are two related heterodimeric cytokines produced by antigen-presenting cells. The balance between these two cytokines plays a crucial role in the control of Th1/Th17 responses and autoimmune inflammation. Most studies focused on their transcriptional regulation. Herein, we explored the role of the adenine and uridine-rich element (ARE)-binding protein tristetraprolin (TTP) in influencing mRNA stability of IL12p35, IL12/23p40, and IL23p19 subunits. LPS-stimulated bone marrow-derived dendritic cells (BMDCs) from TTP(-/-) mice produced normal levels of IL12/23p40. Production of IL12p70 was modestly increased in these conditions. In contrast, we observed a strong impact of TTP on IL23 production and IL23p19 mRNA stability through several AREs in the 3' untranslated region. TTP(-/-) mice spontaneously develop an inflammatory syndrome characterized by cachexia, myeloid hyperplasia, dermatitis, and erosive arthritis. We observed IL23p19 expression within skin lesions associated with exacerbated IL17A and IL22 production by infiltrating γδ T cells and draining lymph node CD4 T cells. We demonstrate that the clinical and immunological parameters associated with TTP deficiency were completely dependent on the IL23-IL17A axis. We conclude that tight control of IL23 mRNA stability by TTP is critical to avoid severe inflammation.

Expression and significance of cyclooxygenase-2 mRNA in benign and malignant ascites

Science.gov (United States)

Lu, Jing; Li, Xiao-Feng; Kong, Li-Xia; Ma, Lin; Liao, Su-Huan; Jiang, Chang-You

2013-01-01

AIM: To investigate the mRNA expression of cyclooxygensae-2 (COX-2) in benign and malignant ascites, and to explore the difference in COX-2 mRNA expression among different diseases. METHODS: A total of 36 samples were collected from the Fifth Affiliated Hospital of Sun Yat-Sen University and divided into two experimental groups: benign ascites (n = 21) and malignant ascites (n = 15). Benign ascites included cirrhotic ascites (n = 10) and tuberculous ascites (n = 5). Malignant ascites included oophoroma (n = 7), cancer of colon (n = 5), cancer of the liver (n = 6), gastric cancer (n = 2), and bladder carcinoma (n = 1). The mRNA expression of COX-2 in ascites was examined with reverse transcriptase polymerase chain reaction (RT-PCR) technology, and the positive rate of COX-2 mRNA was compared between different diseases. RESULTS: The positive rate of COX-2 mRNA in malignant ascites was 42.9% (9/21), which was significantly higher than in benign ascites, 6.7% (1/15), difference being significant between these two groups (χ2 = 4.051, P = 0.044). The proportion of the positive rate in the malignant ascites was as follows: ovarian cancers 57.1% (4/7), colon cancer 40.0% (2/5), liver cancer 33.3% (2/6), gastric cancer 50.0% (1/2), and bladder cancer 0.00% (0/1). However, there was no significant difference in COX-2 mRNA expression among various tumors with malignant ascites (χ2 = 1.614, P = 0.806). Among the benign ascites, COX-2 mRNA levels were different between the tuberculous ascites (0/5) and cirrhotic ascites (1/10), but there was no significant difference (P = 1.000). CONCLUSION: COX-2 mRNA, detected by RT-PCR, is useful in the differential diagnosis of benign and malignant ascites, which also has potential value in the clinical diagnosis of tumors. PMID:24187465

Quantification analysis of CT for aphasic patients

International Nuclear Information System (INIS)

Watanabe, Shunzo; Ooyama, Hiroshi; Hojo, Kei; Tasaki, Hiroichi; Hanazono, Toshihide; Sato, Tokijiro; Metoki, Hirobumi; Totsuka, Motokichi; Oosumi, Noboru.

1987-01-01

Using a microcomputer, the locus and extent of the lesions, as demonstrated by computed tomography, for 44 aphasic patients with various types of aphasia were superimposed onto standardized matrices, composed of 10 slices with 3000 points (50 by 60). The relationships between the foci of the lesions and types of aphasia were investigated on the slices numbered 3, 4, 5, and 6 using a quantification theory, Type 3 (pattern analysis). Some types of regularities were observed on Slices 3, 4, 5, and 6. The group of patients with Broca's aphasia and the group with Wernicke's aphasia were generally separated on the 1st component and the 2nd component of the quantification theory, Type 3. On the other hand, the group with global aphasia existed between the group with Broca's aphasia and that with Wernicke's aphasia. The group of patients with amnestic aphasia had no specific findings, and the group with conduction aphasia existed near those with Wernicke's aphasia. The above results serve to establish the quantification theory, Type 2 (discrimination analysis) and the quantification theory, Type 1 (regression analysis). (author)

Performance of the Real-Q EBV Quantification Kit for Epstein-Barr Virus DNA Quantification in Whole Blood.

Science.gov (United States)

Huh, Hee Jae; Park, Jong Eun; Kim, Ji Youn; Yun, Sun Ae; Lee, Myoung Keun; Lee, Nam Yong; Kim, Jong Won; Ki, Chang Seok

2017-03-01

There has been increasing interest in standardized and quantitative Epstein-Barr virus (EBV) DNA testing for the management of EBV disease. We evaluated the performance of the Real-Q EBV Quantification Kit (BioSewoom, Korea) in whole blood (WB). Nucleic acid extraction and real-time PCR were performed by using the MagNA Pure 96 (Roche Diagnostics, Germany) and 7500 Fast real-time PCR system (Applied Biosystems, USA), respectively. Assay sensitivity, linearity, and conversion factor were determined by using the World Health Organization international standard diluted in EBV-negative WB. We used 81 WB clinical specimens to compare performance of the Real-Q EBV Quantification Kit and artus EBV RG PCR Kit (Qiagen, Germany). The limit of detection (LOD) and limit of quantification (LOQ) for the Real-Q kit were 453 and 750 IU/mL, respectively. The conversion factor from EBV genomic copies to IU was 0.62. The linear range of the assay was from 750 to 10⁶ IU/mL. Viral load values measured with the Real-Q assay were on average 0.54 log₁₀ copies/mL higher than those measured with the artus assay. The Real-Q assay offered good analytical performance for EBV DNA quantification in WB.

Quantifications and Modeling of Human Failure Events in a Fire PSA

International Nuclear Information System (INIS)

Kang, Dae Il; Kim, Kilyoo; Jang, Seung-Cheol

2014-01-01

USNRC and EPRI developed guidance, 'Fire Human Reliability Analysis Guidelines, NUREG-1921', for estimating human error probabilities (HEPs) for HFEs under fire conditions. NUREG-1921 classifies HFEs into four types associated with the following human actions: - Type 1: New and existing Main Control Room (MCR) actions - Type 2: New and existing ex-MCR actions - Type 3: Actions associated with using alternate shutdown means (ASD) - Type 4: Actions relating to the error of commissions (EOCs) or error of omissions (EOOs) as a result of incorrect indications (SPI) In this paper, approaches for the quantifications and modeling of HFEs related to Type 1, 2 and 3 human actions are introduced. This paper introduced the human reliability analysis process for a fire PSA of Hanul Unit 3. A multiplier of 10 was used to re-estimate the HEPs for the preexisting internal human actions. The HEPs for all ex- MCR actions were assumed to be one. New MCR human actions were quantified using the scoping analysis method of NUREG-1921. If the quantified human action were identified to be risk-significant, detailed approaches (modeling and quantification) were used for incorporating fire situations into them. Multiple HFEs for single human action were defined and they were separately and were separately quantified to incorporate the specific fire situations into them. From this study, we can confirm that the modeling as well as quantifications of human actions is very important to appropriately treat them in PSA logic structures

Quantifications and Modeling of Human Failure Events in a Fire PSA

Energy Technology Data Exchange (ETDEWEB)

Kang, Dae Il; Kim, Kilyoo; Jang, Seung-Cheol [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

2014-10-15

USNRC and EPRI developed guidance, 'Fire Human Reliability Analysis Guidelines, NUREG-1921', for estimating human error probabilities (HEPs) for HFEs under fire conditions. NUREG-1921 classifies HFEs into four types associated with the following human actions: - Type 1: New and existing Main Control Room (MCR) actions - Type 2: New and existing ex-MCR actions - Type 3: Actions associated with using alternate shutdown means (ASD) - Type 4: Actions relating to the error of commissions (EOCs) or error of omissions (EOOs) as a result of incorrect indications (SPI) In this paper, approaches for the quantifications and modeling of HFEs related to Type 1, 2 and 3 human actions are introduced. This paper introduced the human reliability analysis process for a fire PSA of Hanul Unit 3. A multiplier of 10 was used to re-estimate the HEPs for the preexisting internal human actions. The HEPs for all ex- MCR actions were assumed to be one. New MCR human actions were quantified using the scoping analysis method of NUREG-1921. If the quantified human action were identified to be risk-significant, detailed approaches (modeling and quantification) were used for incorporating fire situations into them. Multiple HFEs for single human action were defined and they were separately and were separately quantified to incorporate the specific fire situations into them. From this study, we can confirm that the modeling as well as quantifications of human actions is very important to appropriately treat them in PSA logic structures.

The potential role of IGF-I receptor mRNA in rats with diabetic retinopathy

Institute of Scientific and Technical Information of China (English)

匡洪宇; 邹伟; 刘丹; 史榕荇; 程丽华; 殷慧清; 刘晓民

2003-01-01

Objective To evaluate the potential role of insulin-like growth factor-1 receptor mRNA(IGF-IR mRNA) in the onset and development of retinopathy in diabetic rats.Methods A diabetic model was duplicated in Wistar rats. The early changes in the retina were examined using light and transmission electron microscopy. Expression of IGF-IR mRNA was analyzed using in situ hybridization.Results Weak expression of IGF-IR mRNA(5%) was found in retinas of normal rats, but was significantly increased (15% and 18%) in the retinas of diabetic rats after 3 and 6 months of diabetes (P＜0.01). In situ hybridization and morphological study demonstrated that there was a positive correlation between IGF-IR mRNA expression and retinal changes at various stages.Conclusion Increased IGF-IR mRNA might play an important role in the onset and development of diabetic retinopathy.

Associations of ACE Gene Insertion/Deletion Polymorphism, ACE Activity, and ACE mRNA Expression with Hypertension in a Chinese Population

Science.gov (United States)

He, Qingfang; Fan, Chunhong; Yu, Min; Wallar, Gina; Zhang, Zuo-Feng; Wang, Lixin; Zhang, Xinwei; Hu, Ruying

2013-01-01

Background The present study was designed to explore the association of angiotensin converting enzyme (ACE) gene insertion/deletion (I/D, rs4646994) polymorphism, plasma ACE activity, and circulating ACE mRNA expression with essential hypertension (EH) in a Chinese population. In addition, a new detection method for circulating ACE mRNA expression was explored. Methods The research was approved by the ethics committee of Zhejiang Provincial Center for Disease Prevention and Control. Written informed consent was obtained prior to the investigation. 221 hypertensives (cases) and 221 normotensives (controls) were interviewed, subjected to a physical examination, and provided blood for biochemical and genetic tests. The ACE mRNA expression was analyzed by real time fluorescent quantitative Reverse Transcription PCR (FQ-RT-PCR). We performed logistic regression to assess associations of ACE I/D genotypes, ACE activity, and ACE mRNA expression levels with hypertension. Results The results of the multivariate logistic regression analysis showed that the additive model (ID, DD versus II) of the ACE genotype revealed an association with hypertension with adjusted OR of 1.43(95% CI: 1.04-1.97), and ACE ID genotype with adjusted OR of 1.72(95% CI: 1.01-2.92), DD genotype with adjusted OR of 1.94(95% CI: 1.01-3.73), respectively. In addition, our data also indicate that plasma ACE activity (adjusted OR was 1.13(95% CI: 1.08-1.18)) was significantly related to hypertension. However, the plasma ACE mRNA expressions were not different between the cases and controls. Conclusion ACE I/D polymorphism and ACE activity revealed significant influence on hypertension, while circulating ACE mRNA expression was not important factors associated with hypertension in this Chinese population. The detection of circulating ACE mRNA expression by FQ-RT-PCR might be a useful method for early screening and monitoring of EH. PMID:24098401

Real-time PCR based on SYBR-Green I fluorescence: An alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions

Directory of Open Access Journals (Sweden)

Puisieux Alain

2003-10-01

Full Text Available Abstract Background Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive. Results We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC in peripheral blood mononuclear cells; the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy. Conclusion Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.

Naturally occurring BRCA2 alternative mRNA splicing events in clinically relevant samples

DEFF Research Database (Denmark)

Fackenthal, James D; Yoshimatsu, Toshio; Zhang, Bifeng

2016-01-01

patterns and thereby disrupt gene function. mRNA analyses are therefore among the tests used to interpret the clinical significance of some genetic variants. However, these could be confounded by the appearance of naturally occurring alternative transcripts unrelated to germline sequence variation...... to characterise the spectrum of naturally occurring BRCA2 mRNA alternate-splicing events. METHODS: mRNA was prepared from several blood and breast tissue-derived cells and cell lines by contributing ENIGMA laboratories. cDNA representing BRCA2 alternate splice sites was amplified and visualised using capillary...... or agarose gel electrophoresis, followed by sequencing. RESULTS: We demonstrate the existence of 24 different BRCA2 mRNA alternate-splicing events in lymphoblastoid cell lines and both breast cancer and non-cancerous breast cell lines. CONCLUSIONS: These naturally occurring alternate-splicing events...

Surface Enhanced Raman Spectroscopy (SERS) methods for endpoint and real-time quantification of miRNA assays

Science.gov (United States)

Restaino, Stephen M.; White, Ian M.

2017-03-01

Surface Enhanced Raman spectroscopy (SERS) provides significant improvements over conventional methods for single and multianalyte quantification. Specifically, the spectroscopic fingerprint provided by Raman scattering allows for a direct multiplexing potential far beyond that of fluorescence and colorimetry. Additionally, SERS generates a comparatively low financial and spatial footprint compared with common fluorescence based systems. Despite the advantages of SERS, it has remained largely an academic pursuit. In the field of biosensing, techniques to apply SERS to molecular diagnostics are constantly under development but, most often, assay protocols are redesigned around the use of SERS as a quantification method and ultimately complicate existing protocols. Our group has sought to rethink common SERS methodologies in order to produce translational technologies capable of allowing SERS to compete in the evolving, yet often inflexible biosensing field. This work will discuss the development of two techniques for quantification of microRNA, a promising biomarker for homeostatic and disease conditions ranging from cancer to HIV. First, an inkjet-printed paper SERS sensor has been developed to allow on-demand production of a customizable and multiplexable single-step lateral flow assay for miRNA quantification. Second, as miRNA concentrations commonly exist in relatively low concentrations, amplification methods (e.g. PCR) are therefore required to facilitate quantification. This work presents a novel miRNA assay alongside a novel technique for quantification of nuclease driven nucleic acid amplification strategies that will allow SERS to be used directly with common amplification strategies for quantification of miRNA and other nucleic acid biomarkers.

Identifying airway sensitizers: cytokine mRNA profiles induced by various anhydrides

International Nuclear Information System (INIS)

Plitnick, L.M.; Loveless, S.E.; Ladics, G.S.; Holsapple, M.P.; Smialowicz, R.J.; Woolhiser, M.R.; Anderson, P.K.; Smith, C.; Selgrade, M.J.K.

2003-01-01

Exposure to low molecular weight (LMW) chemicals in the workplace has been linked to a variety of respiratory effects. Within the LMW chemicals, one of the major classes involved in these effects are the acid anhydrides. The immunological basis of respiratory hypersensitivity involves CD4+ cells. By virtue of their induction of cytokines typical of CD4+ T-helper type 2 (Th2) cells--interleukin (IL)-4, 10, and 13--respiratory sensitizers may be identified and differentiated from contact sensitizers which induce Th1 cytokines (IL-2 and IFN-γ). Our previous work suggested that the ribonuclease protection assay (RPA) was useful in identifying the respiratory sensitizer, trimellitic anhydride (TMA), based on quantitative differences in Th2 cytokine mRNA as compared to the contact sensitizer dinitrochlorobenzene (DNCB). Therefore, the purpose of the studies described in this report was to expand the chemicals tested in the RPA. To this end, four acid anhydrides with known respiratory sensitization potential, TMA, maleic anhydride (MA), phthalic anhydride (PA) and hexahydrophthalic anhydride (HHPA), were tested. Although previously determined to induce immunologically equivalent responses in a local lymph node assay (LLNA), the initial dose chosen (2.5%) failed to induce Th2 cytokine mRNA expression. To determine if the lack of cytokine expression was related to dose, LLNAs were conducted at higher doses for each of the anhydrides. The highest doses evaluated (four- to six-fold higher than those used in the initial RPA) gave equivalent proliferative responses for the various anhydrides and were used for subsequent RPA testing. At these higher doses, significant increases in Th2 versus Th1 cytokine mRNA were observed for all anhydrides tested. These results suggest that the RPA has the potential to serve as a screen for the detection of LMW airway sensitizing chemicals. However, the basis for selecting immunologically equivalent doses may require some modification

IGF-1R mRNA expression is increased in obese children.

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Ricco, Rafaela Cristina; Ricco, Rubens Garcia; Queluz, Mariangela Carletti; de Paula, Mariana Teresa Sarti; Atique, Patricia Volpon; Custódio, Rodrigo José; Tourinho Filho, Hugo; Del Roio Liberatori, Raphael; Martinelli, Carlos Eduardo

2018-04-01

Obese children are often taller than age-matched subjects. Reports on GH and IGF-I levels in obese individuals are controversial, with normal and reduced GH-IGF-I levels having been reported in this group of patients. Thus, the aim of this study was to analyse insulin-like growth factor type 1 receptor (IGF-IR) mRNA expression in obese children. Forty-seven pre-pubertal children were included in this study: 29 were obese and taller than their target height, and 18 were normal eutrophic controls. Fasting blood samples were collected for IGF-IR mRNA expression in isolated lymphocytes and serum IGF-I, ALS, IGFBP-3, and IGFBP-1 concentration analysis. Relative IGF-IR gene expression (2 -ΔΔCT ) was significantly (P=0.025) higher in obese children (median 1.87) than in controls (1.15). Fourteen of the 29 obese subjects showed 2 -ΔΔCT values greater than or equal to 2, while only 2 individuals in the control group showed values above 2 (P=0.01). Obese children showed significantly (P=0.01) higher IGF-I concentrations than the control group (237ng/ml and 144ng/ml, respectively). Among obese patients, 65.5% had IGF-I values above the 75 percentile of the control group (P=0.02). ALS concentration was significantly (P=0.04) higher in the obese group, while IGFBP-3 levels were similar in obese and control children. IGFBP-1 concentration was lower in obese children, while insulin levels and HOMA-IR index were higher than in controls. The higher IGF-IR mRNA expression observed in obese children, associated with the higher IGF-I and ALS and the lower IGFBP-1 levels, suggest that the higher stature observed in these children may be due to increased IGF-I bioactivity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Construction and Quantification of the One Top model of the Fire Events PSA

International Nuclear Information System (INIS)

Kang, Dae Il; Lee, Yoon Hwan; Han, Sang Hoon

2008-01-01

KAERI constructed the one top model of the fire events PSA for Ulchin Unit 3 and 4 by using the 'mapping technique'. The mapping technique was developed for the construction and quantification of external events PSA models with a one top model for an internal events PSA. With 'AIMS', the mapping technique can be implemented by the construction of mapping tables. The mapping tables include fire rooms, fire ignition frequency, related initiating events, fire transfer events, and the internal PSA basic events affected by a fire. The constructed one top fire PSA model is based on previously conducted fire PSA results for Ulchin Unit 3 and 4. In this paper, we introduce the construction procedure and quantification results of the one top model of the fire events PSA by using the mapping technique. As the one top model of the fire events PSA developed in this study is based on the previous study, we also introduce the previous fire PSA approach focused on quantification

Rift Valley fever virus NSS gene expression correlates with a defect in nuclear mRNA export.

Science.gov (United States)

Copeland, Anna Maria; Van Deusen, Nicole M; Schmaljohn, Connie S

2015-12-01

We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NSS gene, but not the N, GN or NSM genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NSS, confirming that expression of NSS is likely responsible for this phenomenon. Published by Elsevier Inc.

BAY11 enhances OCT4 synthetic mRNA expression in adult human skin cells.

Science.gov (United States)

Awe, Jason P; Crespo, Agustin Vega; Li, You; Kiledjian, Megerditch; Byrne, James A

2013-02-06

The OCT4 transcription factor is involved in many cellular processes, including development, reprogramming, maintaining pluripotency and differentiation. Synthetic OCT4 mRNA was recently used (in conjunction with other reprogramming factors) to generate human induced pluripotent stem cells. Here, we discovered that BAY 11-7082 (BAY11), at least partially through an NF-κB-inhibition based mechanism, could significantly increase the expression of OCT4 following transfection of synthetic mRNA (synRNA) into adult human skin cells. We tested various chemical and molecular small molecules on their ability to suppress the innate immune response seen upon synthetic mRNA transfection. Three molecules - B18R, BX795, and BAY11 - were used in immunocytochemical and proliferation-based assays. We also utilized global transcriptional meta-analysis coupled with quantitative PCR to identify relative gene expression downstream of OCT4. We found that human skin cells cultured in the presence of BAY11 resulted in reproducible increased expression of OCT4 that did not inhibit normal cell proliferation. The increased levels of OCT4 resulted in significantly increased expression of genes downstream of OCT4, including the previously identified SPP1, DUSP4 and GADD45G, suggesting the expressed OCT4 was functional. We also discovered a novel OCT4 putative downstream target gene SLC16A9 which demonstrated significantly increased expression following elevation of OCT4 levels. For the first time we have shown that small molecule-based stabilization of synthetic mRNA expression can be achieved with use of BAY11. This small molecule-based inhibition of innate immune responses and subsequent robust expression of transfected synthetic mRNAs may have multiple applications for future cell-based research and therapeutics.

Crimean-Congo Hemorrhagic Fever Virus Nucleocapsid Protein Augments mRNA Translation.

Science.gov (United States)

Jeeva, Subbiah; Cheng, Erdong; Ganaie, Safder S; Mir, Mohammad A

2017-08-01

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne Nairovirus of the Bunyaviridae family, causing severe illness with high mortality rates in humans. Here, we demonstrate that CCHFV nucleocapsid protein (CCHFV-NP) augments mRNA translation. CCHFV-NP binds to the viral mRNA 5' untranslated region (UTR) with high affinity. It facilitates the translation of reporter mRNA both in vivo and in vitro with the assistance of the viral mRNA 5' UTR. CCHFV-NP equally favors the translation of both capped and uncapped mRNAs, demonstrating the independence of this translation strategy on the 5' cap. Unlike the canonical host translation machinery, inhibition of eIF4F complex, an amalgam of three initiation factors, eIF4A, eIF4G, and eIF4E, by the chemical inhibitor 4E1RCat did not impact the CCHFV-NP-mediated translation mechanism. However, the proteolytic degradation of eIF4G alone by the human rhinovirus 2A protease abrogated this translation strategy. Our results demonstrate that eIF4F complex formation is not required but eIF4G plays a critical role in this translation mechanism. Our results suggest that CCHFV has adopted a unique translation mechanism to facilitate the translation of viral mRNAs in the host cell cytoplasm where cellular transcripts are competing for the same translation apparatus. IMPORTANCE Crimean-Congo hemorrhagic fever, a highly contagious viral disease endemic to more than 30 countries, has limited treatment options. Our results demonstrate that NP favors the translation of a reporter mRNA harboring the viral mRNA 5' UTR. It is highly likely that CCHFV uses an NP-mediated translation strategy for the rapid synthesis of viral proteins during the course of infection. Shutdown of this translation mechanism might selectively impact viral protein synthesis, suggesting that an NP-mediated translation strategy is a target for therapeutic intervention against this viral disease. Copyright © 2017 American Society for Microbiology.

The mRNA expression of XRCC repair genes in mice after γ-ray radiation

International Nuclear Information System (INIS)

Wang Qin; Yue Jingyin; Li Jin; Mu Chuanjie; Fan Feiyue

2006-01-01

Objective: To investigate the role of XRCC repair genes in radioresistance of IRM-2 inbred mice. Methods: Northern hybridization was used to measure mRNA expression of XRCC1 and XRCC5 genes in IRM-2 inbred mice. ICR/JCL and 615 after exposure to different doses of γ-ray radiation at different postirradiation time. Results: The levels of XRCC1 and XRCC5 mRNA expression in control IRM-2 mice were higher significantly than those in their control parental mice (P<0.01 and P<0.05). The mRNA expression of XRCC genes in ICR/JCL and 615 mice all increased to some extent after exposure 1, 2 and 4 Gy radiation. But the levels were significantly higher at 2h postirradiation (P<0.05) . The levels of XRCC mRNA expression in IRM-2 mice did not increase significnatly compared with the control mice after exposure 1 and 2 Gy radiation. But the levels of XRCC1 and XRCC5 mRNA expression increased markedly at 4Gy 1h postirradiation (P<0.05 and P<0.01). Conclusion: The basal levels of XRCC1 and XRCC5 mRNA expression in IRM-2 mice were high. The high level of XRCC5 mRNA expression was involved in the repair of DNA double strand breaks induced by higher dose radiation, which perhaps was one of radioresistance causes of IRM-2 mice. (authors)

All-in-one detector of circulating mRNA based on a smartphone

Science.gov (United States)

Cmiel, Vratislav; Gumulec, Jaromir; Svoboda, Ondrej; Raudenska, Martina; Hudcova, Kristyna; Sekora, Jiri; Balogh, Jaroslav; Masarik, Michal; Provaznik, Ivo

2016-03-01

Metallothionein is significantly elevated in various tumors, notably in prostate cancer on both mRNA and protein level. We demonstrated a strong predictive potential of free circulating metallothionein 2A isoform mRNA for patients with this cancer. Circulating mRNA detection relies on expensive equipment and requires high level of expertise. In this work we developed compact "all-in-one" laboratory system which replace microvolume spectrophotometer, thermocycler and realtime PCR machines. We managed to design and construct a microprocessor controlled heating/cooling chamber that ensures required temperature gradient. The chamber includes implemented optical system to enable fluorescence excitation and fluorescence analysis using a smart-phone.

Selective Distance-Based K+ Quantification on Paper-Based Microfluidics.

Science.gov (United States)

Gerold, Chase T; Bakker, Eric; Henry, Charles S

2018-04-03

In this study, paper-based microfluidic devices (μPADs) capable of K + quantification in aqueous samples, as well as in human serum, using both colorimetric and distance-based methods are described. A lipophilic phase containing potassium ionophore I (valinomycin) was utilized to achieve highly selective quantification of K + in the presence of Na + , Li + , and Mg 2+ ions. Successful addition of a suspended lipophilic phase to a wax printed paper-based device is described and offers a solution to current approaches that rely on organic solvents, which damage wax barriers. The approach provides an avenue for future alkali/alkaline quantification utilizing μPADs. Colorimetric spot tests allowed for K + quantification from 0.1-5.0 mM using only 3.00 μL of sample solution. Selective distance-based quantification required small sample volumes (6.00 μL) and gave responses sensitive enough to distinguish between 1.0 and 2.5 mM of sample K + . μPADs using distance-based methods were also capable of differentiating between 4.3 and 6.9 mM K + in human serum samples. Distance-based methods required no digital analysis, electronic hardware, or pumps; any steps required for quantification could be carried out using the naked eye.

Bioinspired nanocomplex for spatiotemporal imaging of sequential mRNA expression in differentiating neural stem cells.

Science.gov (United States)

Wang, Zhe; Zhang, Ruili; Wang, Zhongliang; Wang, He-Fang; Wang, Yu; Zhao, Jun; Wang, Fu; Li, Weitao; Niu, Gang; Kiesewetter, Dale O; Chen, Xiaoyuan

2014-12-23

Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions.

Prognostic impact of clinical course-specific mRNA expression profiles in the serum of perioperative patients with esophageal cancer in the ICU: a case control study

Directory of Open Access Journals (Sweden)

Oshima Yoshiaki

2010-10-01

Full Text Available Abstract Background We previously reported that measuring circulating serum mRNAs using quantitative one-step real-time RT-PCR was clinically useful for detecting malignancies and determining prognosis. The aim of our study was to find crucial serum mRNA biomarkers in esophageal cancer that would provide prognostic information for post-esophagectomy patients in the critical care setting. Methods We measured serum mRNA levels of 11 inflammatory-related genes in 27 post-esophagectomy patients admitted to the intensive care unit (ICU. We tracked these levels chronologically, perioperatively and postoperatively, until the two-week mark, investigating their clinical and prognostic significance as compared with clinical parameters. Furthermore, we investigated whether gene expression can accurately predict clinical outcome and prognosis. Results Circulating mRNAs in postoperative esophagectomy patients had gene-specific expression profiles that varied with the clinical phase of their treatment. Multivariate regression analysis showed that upregulation of IL-6, VWF and TGF-β1 mRNA in the intraoperative phase (p = 0.016, 0.0021 and 0.009 and NAMPT and MUC1 mRNA on postoperative day 3 (p ®, Ono Pharmaceutical Co., Ltd. significantly correlated with MUC1 and NAMPT mRNA expression (p = 0.048 and 0.045. IL-6 mRNA correlated with hypercytokinemia and recovery from hypercytokinemia (sensitivity 80.9% and was a significant biomarker in predicting the onset of severe inflammatory diseases. Conclusion Chronological tracking of postoperative mRNA levels of inflammatory-related genes in esophageal cancer patients may facilitate early institution of pharamacologic therapy, prediction of treatment response, and prognostication during ICU management in the perioperative period.

Accumulation of defence-related transcripts and cloning of a chitinase mRNA from pea leaves (Pisum sativum L.) inoculated with Ascochyta pisi Lib

DEFF Research Database (Denmark)

Vad, Knud; de Neergaard, Eigil; Madriz-Ordeñana, Kenneth

1993-01-01

The race specific resistance of pea to Ascochyta pisi Lib. was shown to be exhibited as a hypersensitive response associated with the production of polyphenolic substances in epidermal and mesophyll cells. The levels of transcripts representing a pathogenesis-related (PR) protein (chitinase......) and an enzyme of phytoalexin biosynthesis (chalcone synthase) were shown to accumulate more rapidly during the hypersensitive response than during lesion development in the compatible interaction. A full-length (1143 bp) cDNA sequence of a pea chitinase (EC 3.2.1.14) (coding for an approx. 34 500 Da protein......) was deduced by combining the overlapping sequences of three clones obtained following PCR amplification of cDNA prepared from mRNA isolated 24 h after inoculation of pea leaves with Ascochyta pisi. The combined sequences were identified as a class I chitinase corresponding to the basic A1-chitinase enzyme...

Expression of galectin-9 mRNA in obese children with polymorphism of the lactase gene

Directory of Open Access Journals (Sweden)

A.E. Abaturov

2018-02-01

Full Text Available Background. The aim of the study is to investigate the association of expression of galectin-9 (Gal-9 mRNA and lactose malabsorption in obese children with polymorphism (SNP of the lactase gene (LCT and to study the efficacy of lactase deficiency therapy using exogenous lactase preparations. Materials and methods. Seventy obese children (BMI > 95th percentile and 16 children without obesity aged 6–18 years were examined. There was studied SNP LCT (material for investigation venous blood by real-time PCR, expression of Gal-9 mRNA (study material buccal epithelium by real-time PCR with reverse transcription, malabsorption of lactose by hydrogen breath test (HBT. Among obese children, 38 children with genotype C/C 13910 presented the first observation group, 32 children with phenotype identical genotypes C/T 13910 and T/T 13910, p > 0.05, presented the second group. Children from the first observation group also determined the level of expression of Gal-9 mRNA and lactose malabsorption after using exogenous lactase preparations. Results. The genotype C/C 13910 was determined in 38 (54.3 %, genotype C/T 13910 in 22 (31.4 % and genotype T/T in 10 (14.3 % patients. Malabsorption of lactose in children with genotype C/C 13910 averaged 32.7 ± 10.4 pmm, in children with genotypes C/T 13910 — 26.3 ± 4.9 pmm (p > 0.05 and with genotype T/T 13910 and was absent in children without obesity (p < 0.05. The average level of expression of Gal-9 mRNA in children with genotype C/C 13910 was 564.3 ± 32.8 RU DmRNA Gal-9/mRNA actin, in children with genotypes C/T and T/T 13910 — 61.04 ± 15.30 RU DmRNA Gal-9/mRNA actin, p < 0.01. It is of great importance that the children with genotype C/C 13910 and lactose malabsorption (n = 20 had the lowest average level of expression of Gal-9 mRNA (42.47 ± 13.30 RU DmRNA Gal-9/mRNA actin whereas the children with genotype C/C 13910 and without lactose malabsorption (n =18 had the largest level (1086

Separation and quantification of ropinirole and some impurities using capillary liquid chromatography

NARCIS (Netherlands)

Coufal, P.; Stulik, K.; Claessens, H.A.; Hardy, M.J.; Webb, M.

1999-01-01

Ropinirole, 4-[2-(dipropylamino)ethyl]-1,3-dihydro-2H-indol-2-one, is a potent anti-Parkinson’s disease drug developed by SmithKline Beecham Pharmaceuticals. Capillary liquid chromatography (CLC) was used for the separation and quantification of ropinirole and its five related impurities,

Screening women for cervical cancer carcinoma with a HPV mRNA test: first results from the Venice pilot program.

Science.gov (United States)

Maggino, Tiziano; Sciarrone, Rocco; Murer, Bruno; Dei Rossi, Maria Rosa; Fedato, Chiara; Maran, Michela; Lorio, Melania; Soldà, Marika; Zago, Fiorella; Giorgi Rossi, Paolo; Zorzi, Manuel

2016-08-23

HPV DNA-based screening is more effective than a Pap test in preventing cervical cancer, but the test is less specific. New HPV tests have been proposed for primary screening. The HPV mRNA test showed a similar or slightly lower sensitivity than the HPV DNA tests but with a higher specificity. We report the results of an organised HPV mRNA-based screening pilot program in Venice, Italy. From October 2011 to May 2014, women aged 25-64 years were invited to undergo a HPV mRNA test (Aptima). Those testing positive underwent cytological triage. Women with positive cytology were referred to colposcopy, whereas those with negative cytology were referred to repeat the HPV mRNA test 1 year later. The results of the HPV mRNA test program were compared with both the local historical cytology-based program and with four neighbouring DNA HPV-based pilot projects. Overall, 23 211 women underwent a HPV mRNA test. The age-standardised positivity rate was 7.0%, higher than in HPV DNA programs (6.8%; relative rate (RR) 1.11, 95% confidence interval (CI) 1.05-1.17). The total colposcopy referral was 5.1%, double than with cytology (2.6%; RR 2.02, 95% CI 1.82-2.25) but similar to the HPV DNA programs (4.8%; RR 1.02; 95% CI 0.96-1.08). The cervical intraepithelial neoplasia grade 2+ detection rate with HPV mRNA was greater than in the HPV DNA programs at baseline (RR 1.50; 95% CI 1.19-1.88) and not significantly lower at the 1-year repeat (RR 0.70; 95% CI 0.40-1.16). The overall RR was 1.29 (95% CI 1.05-1.59), which was much higher than with cytology (detection rate 5.5‰ vs 2.1‰; RR 2.50, 95% CI 1.76-3.62). A screening programme based on the HPV mRNA obtained results similar to those observed with the HPV DNA test. In routine screening programmes, even a limited increase in HPV prevalence may conceal the advantage represented by the higher specificity of HPV mRNA.

Investigation of mRNA expression for secreted frizzled-related protein 2 (sFRP2) in chick embryos.

Science.gov (United States)

Lin, Chung-Tien; Lin, Yu-Ting; Kuo, Tzong-Fu

2007-08-01

The roles of secreted frizzled-related protein 2 (sFRP2) in organ development of vertebrate animals are not well understood. We investigated expression of sFRP2 during embryogenesis of Arbor Acre broiler chicken eggs. Expression of sFRP2 was detected in the folds and lateral layer of developing brains. The sFRP2 signals in the developing eye were marked as a circle along the orbit. In younger embryos on days 3-6, the sFRP2 signals were consistent with growth of the sclerotome, suggesting that sFRP2 may be associated with somite development. Furthermore, with the exception of bones, sFRP2 mRNA was detectable in the interdigital tissue of embryos older than eight days as the limbs matured. This revealed that sFRP2 might play a role in myogenesis. In situ hybridization was also used to analyze the expression of sFRP2 in day 3-10 chick embryos. Signals were expressed in the gray matter of the developing brain coelom, including the optic lobe, metencephalon, myelencephalon, mesencephalon and diencephalon. The developing eyes contained an intercellular distribution of sFRP2 in the pigmented layer of the retina and photoreceptors. Furthermore, sFRP2 was expressed in the mantle layer of the neural tube and notochord. Based on these findings, it seems reasonable to suggest that sFRP2 may play an active role in embryogenesis, especially in development of the neural system, eyes, muscles and limbs.

Generation of human induced pluripotent stem cells using non-synthetic mRNA.

Science.gov (United States)

Rohani, L; Fabian, C; Holland, H; Naaldijk, Y; Dressel, R; Löffler-Wirth, H; Binder, H; Arnold, A; Stolzing, A

2016-05-01

Here we describe some of the crucial steps to generate induced pluripotent stem cells (iPSCs) using mRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribed mRNA. V. virus' 2'-O-Methyltransferase enzyme creates a cap1 structure found in higher eukaryotes and has higher translation efficiency compared to other methods. Use of the polymeric transfection reagent polyethylenimine proved superior to other transfection methods. The mRNA created via this method did not trigger an intracellular immune response via human IFN-gamma (hIFN-γ) or alpha (hIFN-α) release, thus circumventing the use of suppressors. Resulting mRNA and protein were expressed at high levels for over 48h, thus obviating daily transfections. Using this method, we demonstrated swift activation of pluripotency associated genes in human fibroblasts. Low oxygen conditions further facilitated colony formation. Differentiation into different germ layers was confirmed via teratoma assay. Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Photodynamic antisense regulation of mRNA having a point mutation with psoralen-conjugated oligonucleotide.

Science.gov (United States)

Higuchi, Maiko; Yamayoshi, Asako; Kobori, Akio; Murakami, Akira

2008-01-01

Nucleic acid-based drugs, such as antisense oligonucleotide, ribozyme, and small interfering RNA, are specific compounds that inhibit gene expression at the post-transcriptional level. To develop more effective nucleic acid-based drugs, we focused on photo-reactive antisense oligonucleotides. We have optimized the structure of psoralen-conjugated oligonucleotide to improve their sequence selectivity and photo-crosslinking efficiency. Previously, we reported that photo reactive oligonucleotides containing 2'-O-psoralenyl-methoxyethyl adenosine (2'-Ps-eom) showed drastic photo-reactivity with a strictly sequence specific manner in vitro. In this report, we evaluated the binding ability toward intracellular target mRNA. The 2'-Ps-eom selectively photo-cross-linked to the target mRNA extracted from cells. The 2'-Ps-eom also cross-linked to target mRNA in cells. Furthermore, 2'-Ps-eom did not cross-link to mRNA having a mismatch base. These results suggest that 2'-Ps-eom is a powerful antisense molecule to inhibit the expression of mRNA having a point mutation.

Synthesis and Review: Advancing agricultural greenhouse gas quantification

International Nuclear Information System (INIS)

Olander, Lydia P; Wollenberg, Eva; Tubiello, Francesco N; Herold, Martin

2014-01-01

Reducing emissions of agricultural greenhouse gases (GHGs), such as methane and nitrous oxide, and sequestering carbon in the soil or in living biomass can help reduce the impact of agriculture on climate change while improving productivity and reducing resource use. There is an increasing demand for improved, low cost quantification of GHGs in agriculture, whether for national reporting to the United Nations Framework Convention on Climate Change (UNFCCC), underpinning and stimulating improved practices, establishing crediting mechanisms, or supporting green products. This ERL focus issue highlights GHG quantification to call attention to our existing knowledge and opportunities for further progress. In this article we synthesize the findings of 21 papers on the current state of global capability for agricultural GHG quantification and visions for its improvement. We conclude that strategic investment in quantification can lead to significant global improvement in agricultural GHG estimation in the near term. (paper)

ZMS regulation of M2 muscarinic receptor mRNA stability requires protein factor

International Nuclear Information System (INIS)

Zhang Yongfang; Xia Zongqin; Hu Ya'er

2010-01-01

Aim The aim of this work is to study the elevation mechanism of ZMS on muscarinic M2 receptor mRNA expression. Methods Actinomycin D was added to cultured CHOm2 cells to stop the de novo synthesis of M2 receptor mRNA and samples were taken at various times to determine the time course of mRNA of M2 receptor with real-time quantitative RT-PCR. Half-life of M2 receptor mRNA and the effect of ZMS on the half-life was obtained from the slope of the exponential curves. Cycloheximide was added at 4 h prior to and 24 h after the addition of ZMS to examine the effect of de novo protein synthesis on the action of ZMS. Results The half-life of m2 mRNA was prolonged by ZMS treatment without cycloheximide (4.75±0.54 h and 2.13 h±0.23 h for ZMS and vehicle treated groups, respectively, P<0.05). When cycloheximide was added to the culture medium 4h prior to the addition of ZMS, the effect of ZMS in prolonging the half-life of m2 mRNA disappeared (3.06 h±0.23 h and 3.00 h±l.20 h for cells with and without ZMS, respectively). However, when the ZMS was added to the medium 24h prior to the addition of cycloheximide, the action of ZMS was not abolished by cycloheximide (half-life was 5.43 h±1.13 h and 2.46 h±0.09 h for cells with and without ZMS, respectively). Conclusion These data suggest that de novo protein synthesis was required for the increase in M2 mRNA stability induced by ZMS. (authors)

UCP2 mRNA expression is dependent on glucose metabolism in pancreatic islets

International Nuclear Information System (INIS)

Dalgaard, Louise T.

2012-01-01

Highlights: ► UCP2 mRNA levels are decreased in islets of Langerhans from glucokinase deficient mice. ► UCP2 mRNA up-regulation by glucose is dependent on glucokinase. ► Absence of UCP2 increases GSIS of glucokinase heterozygous pancreatic islets. ► This may protect glucokinase deficient mice from hyperglycemic damages. -- Abstract: Uncoupling Protein 2 (UCP2) is expressed in the pancreatic β-cell, where it partially uncouples the mitochondrial proton gradient, decreasing both ATP-production and glucose-stimulated insulin secretion (GSIS). Increased glucose levels up-regulate UCP2 mRNA and protein levels, but the mechanism for UCP2 up-regulation in response to increased glucose is unknown. The aim was to examine the effects of glucokinase (GK) deficiency on UCP2 mRNA levels and to characterize the interaction between UCP2 and GK with regard to glucose-stimulated insulin secretion in pancreatic islets. UCP2 mRNA expression was reduced in GK+/− islets and GK heterozygosity prevented glucose-induced up-regulation of islet UCP2 mRNA. In contrast to UCP2 protein function UCP2 mRNA regulation was not dependent on superoxide generation, but rather on products of glucose metabolism, because MnTBAP, a superoxide dismutase mimetic, did not prevent the glucose-induced up-regulation of UCP2. Glucose-stimulated insulin secretion was increased in UCP2−/− and GK+/− islets compared with GK+/− islets and UCP2 deficiency improved glucose tolerance of GK+/− mice. Accordingly, UCP2 deficiency increased ATP-levels of GK+/− mice. Thus, the compensatory down-regulation of UCP2 is involved in preserving the insulin secretory capacity of GK mutant mice and might also be implicated in limiting disease progression in MODY2 patients.

The ribosome structure controls and directs mRNA entry, translocation and exit dynamics

International Nuclear Information System (INIS)

Kurkcuoglu, Ozge; Doruker, Pemra; Jernigan, Robert L; Sen, Taner Z; Kloczkowski, Andrzej

2008-01-01

The protein-synthesizing ribosome undergoes large motions to effect the translocation of tRNAs and mRNA; here, the domain motions of this system are explored with a coarse-grained elastic network model using normal mode analysis. Crystal structures are used to construct various model systems of the 70S complex with/without tRNA, elongation factor Tu and the ribosomal proteins. Computed motions reveal the well-known ratchet-like rotational motion of the large subunits, as well as the head rotation of the small subunit and the high flexibility of the L1 and L7/L12 stalks, even in the absence of ribosomal proteins. This result indicates that these experimentally observed motions during translocation are inherently controlled by the ribosomal shape and only partially dependent upon GTP hydrolysis. Normal mode analysis further reveals the mobility of A- and P-tRNAs to increase in the absence of the E-tRNA. In addition, the dynamics of the E-tRNA is affected by the absence of the ribosomal protein L1. The mRNA in the entrance tunnel interacts directly with helicase proteins S3 and S4, which constrain the mRNA in a clamp-like fashion, as well as with protein S5, which likely orients the mRNA to ensure correct translation. The ribosomal proteins S7, S11 and S18 may also be involved in assuring translation fidelity by constraining the mRNA at the exit site of the channel. The mRNA also interacts with the 16S 3' end forming the Shine–Dalgarno complex at the initiation step; the 3' end may act as a 'hook' to reel in the mRNA to facilitate its exit

Regulation of the growth hormone (GH) receptor and GH-binding protein mRNA

Energy Technology Data Exchange (ETDEWEB)

Kaji, Hidesuke; Ohashi, Shin-Ichirou; Abe, Hiromi; Chihara, Kazuo [Kobe Univ. School of Medicine, Kobe (Japan)

1994-12-31

In fasting rats, a transient increase in growth hormone-binding protein (GHBP) mRNA levels was observed after 1 day, in muscle, heart, and liver, but not in fat tissues. The liver GH receptor (GHR) mRNA level was significantly increased after 1 day (but not after 5 days) of bovine GH (bGH) treatment in fed rats. Both the liver GHR mRNA level and the net increment of plasma IGF-I markedly decreased after 5 days of bGH administration in fasting rats. These findings suggest that GHR and GHBP mRNAs in the liver are expressed in a different way and that the expression of GHBP mRNA is regulated differently between tissues, at least in rats. The results also suggest that refractoriness to GH in a sustained fasting state might be beneficial in preventing anabolic effects of GH. In humans, GHR mRNA in lymphocytes, from subjects with either GH-deficiency or acromegaly, could be detected by the reverse transcription-polymerase chain reaction method. In one patient with partial GH insensitivity, a heterozygous missense mutation (P561T) was identified in the cytoplasmic domain of GHR. 15 refs., 4 figs.

Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

International Nuclear Information System (INIS)

Lazarus, Kyren A.; Zhao, Zhe; Knower, Kevin C.; To, Sarah Q.; Chand, Ashwini L.; Clyne, Colin D.

2013-01-01

Highlights: •LRH-1 is an orphan nuclear receptor that regulates tumor proliferation. •In breast cancer, high mRNA expression is associated with ER+ status. •In ER−ve cells, despite very low mRNA, we found abundant LRH-1 protein. •Our data show distinctly different LRH-1 protein isoforms in ER− and ER+ breast cancer cells. •This is due to differences in LRH-1 mRNA and protein stability rates. -- Abstract: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E 2 ), with LRH-1 mRNA transcript levels higher in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER− cells. However, the presence of LRH-1 protein in ER− cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER− breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER− compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E 2 , showed increased mRNA stability in ER− versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E 2 treatment, this effect mediated by ERα. Our data demonstrates that in ER− cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER− cells as well as ER− tumors suggests a possible role in the development of ER− tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER− and ER+ breast cancer

Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

Energy Technology Data Exchange (ETDEWEB)

Lazarus, Kyren A. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Environmental and Biotechnology Centre, Swinburne University, Hawthorn, Victoria 3122 (Australia); Zhao, Zhe; Knower, Kevin C. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); To, Sarah Q. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168 (Australia); Chand, Ashwini L. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Clyne, Colin D., E-mail: Colin.clyne@princehenrys.org [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168 (Australia)

2013-08-30

Highlights: •LRH-1 is an orphan nuclear receptor that regulates tumor proliferation. •In breast cancer, high mRNA expression is associated with ER+ status. •In ER−ve cells, despite very low mRNA, we found abundant LRH-1 protein. •Our data show distinctly different LRH-1 protein isoforms in ER− and ER+ breast cancer cells. •This is due to differences in LRH-1 mRNA and protein stability rates. -- Abstract: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E{sub 2}), with LRH-1 mRNA transcript levels higher in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER− cells. However, the presence of LRH-1 protein in ER− cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER− breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER− compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E{sub 2}, showed increased mRNA stability in ER− versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E{sub 2} treatment, this effect mediated by ERα. Our data demonstrates that in ER− cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER− cells as well as ER− tumors suggests a possible role in the development of ER− tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER− and ER+ breast cancer.

Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes.

Science.gov (United States)

Pardi, Norbert; Tuyishime, Steven; Muramatsu, Hiromi; Kariko, Katalin; Mui, Barbara L; Tam, Ying K; Madden, Thomas D; Hope, Michael J; Weissman, Drew

2015-11-10

In recent years, in vitro transcribed messenger RNA (mRNA) has emerged as a potential therapeutic platform. To fulfill its promise, effective delivery of mRNA to specific cell types and tissues needs to be achieved. Lipid nanoparticles (LNPs) are efficient carriers for short-interfering RNAs and have entered clinical trials. However, little is known about the potential of LNPs to deliver mRNA. Here, we generated mRNA-LNPs by incorporating HPLC purified, 1-methylpseudouridine-containing mRNA comprising codon-optimized firefly luciferase into stable LNPs. Mice were injected with 0.005-0.250mg/kg doses of mRNA-LNPs by 6 different routes and high levels of protein translation could be measured using in vivo imaging. Subcutaneous, intramuscular and intradermal injection of the LNP-encapsulated mRNA translated locally at the site of injection for up to 10days. For several days, high levels of protein production could be achieved in the lung from the intratracheal administration of mRNA. Intravenous and intraperitoneal and to a lesser extent intramuscular and intratracheal deliveries led to trafficking of mRNA-LNPs systemically resulting in active translation of the mRNA in the liver for 1-4 days. Our results demonstrate that LNPs are appropriate carriers for mRNA in vivo and have the potential to become valuable tools for delivering mRNA encoding therapeutic proteins. Copyright © 2015 Elsevier B.V. All rights reserved.

Dwell-Time Distribution, Long Pausing and Arrest of Single-Ribosome Translation through the mRNA Duplex.

Science.gov (United States)

Xie, Ping

2015-10-09

Proteins in the cell are synthesized by a ribosome translating the genetic information encoded on the single-stranded messenger RNA (mRNA). It has been shown that the ribosome can also translate through the duplex region of the mRNA by unwinding the duplex. Here, based on our proposed model of the ribosome translation through the mRNA duplex we study theoretically the distribution of dwell times of the ribosome translation through the mRNA duplex under the effect of a pulling force externally applied to the ends of the mRNA to unzip the duplex. We provide quantitative explanations of the available single molecule experimental data on the distribution of dwell times with both short and long durations, on rescuing of the long paused ribosomes by raising the pulling force to unzip the duplex, on translational arrests induced by the mRNA duplex and Shine-Dalgarno(SD)-like sequence in the mRNA. The functional consequences of the pauses or arrests caused by the mRNA duplex and the SD sequence are discussed and compared with those obtained from other types of pausing, such as those induced by "hungry" codons or interactions of specific sequences in the nascent chain with the ribosomal exit tunnel.

Sequence-engineered mRNA Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals

Science.gov (United States)

Thess, Andreas; Grund, Stefanie; Mui, Barbara L; Hope, Michael J; Baumhof, Patrick; Fotin-Mleczek, Mariola; Schlake, Thomas

2015-01-01

Being a transient carrier of genetic information, mRNA could be a versatile, flexible, and safe means for protein therapies. While recent findings highlight the enormous therapeutic potential of mRNA, evidence that mRNA-based protein therapies are feasible beyond small animals such as mice is still lacking. Previous studies imply that mRNA therapeutics require chemical nucleoside modifications to obtain sufficient protein expression and avoid activation of the innate immune system. Here we show that chemically unmodified mRNA can achieve those goals as well by applying sequence-engineered molecules. Using erythropoietin (EPO) driven production of red blood cells as the biological model, engineered Epo mRNA elicited meaningful physiological responses from mice to nonhuman primates. Even in pigs of about 20 kg in weight, a single adequate dose of engineered mRNA encapsulated in lipid nanoparticles (LNPs) induced high systemic Epo levels and strong physiological effects. Our results demonstrate that sequence-engineered mRNA has the potential to revolutionize human protein therapies. PMID:26050989

Critical points of DNA quantification by real-time PCR--effects of DNA extraction method and sample matrix on quantification of genetically modified organisms.

Science.gov (United States)

Cankar, Katarina; Stebih, Dejan; Dreo, Tanja; Zel, Jana; Gruden, Kristina

2006-08-14

Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary criterion by which to

La quantification en Kabiye: une approche linguistique | Pali ...

African Journals Online (AJOL)

... which is denoted by lexical quantifiers. Quantification with specific reference is provided by different types of linguistic units (nouns, numerals, adjectives, adverbs, ideophones and verbs) in arguments/noun phrases and in the predicative phrase in the sense of Chomsky. Keywords: quantification, class, number, reference, ...

Suppression of FAT/CD36 mRNA by human growth hormone in pancreatic β-cells

DEFF Research Database (Denmark)

Dalgaard, Louise Torp; Thams, Peter Grevsen; Gaarn, Louise Winkel

2011-01-01

of this study was to examine the effect of human growth hormone (hGH) on mRNAs of fatty acid transport and binding proteins expressed in pancreatic β-cells, and to examine this in relation to β-cell survival after exposure to fatty acids. hGH decreased mRNA levels of FAT/CD36, whereas mRNAs of GPR40, FASN, FABP...

Suppression of FAT/CD36 mRNA by human growth hormone in pancreatic ß-cells

DEFF Research Database (Denmark)

Dalgaard, Louise Torp; Thams, Peter Grevsen; Gaarn, Louise Winkel

2011-01-01

of this study was to examine the effect of human growth hormone (hGH) on mRNAs of fatty acid transport and binding proteins expressed in pancreatic ß-cells, and to examine this in relation to ß-cell survival after exposure to fatty acids. hGH decreased mRNA levels of FAT/CD36, whereas mRNAs of GPR40, FASN, FABP...

Inhibition of Xenograft tumor growth by gold nanoparticle-DNA oligonucleotide conjugates-assisted delivery of BAX mRNA.

Directory of Open Access Journals (Sweden)

Ji-Hyun Yeom

Full Text Available Use of non-biological agents for mRNA delivery into living systems in order to induce heterologous expression of functional proteins may provide more advantages than the use of DNA and/or biological vectors for delivery. However, the low efficiency of mRNA delivery into live animals, using non-biological systems, has hampered the use of mRNA as a therapeutic molecule. Here, we show that gold nanoparticle-DNA oligonucleotide (AuNP-DNA conjugates can serve as universal vehicles for more efficient delivery of mRNA into human cells, as well as into xenograft tumors generated in mice. Injections of BAX mRNA loaded on AuNP-DNA conjugates into xenograft tumors resulted in highly efficient mRNA delivery. The delivered mRNA directed the efficient production of biologically functional BAX protein, a pro-apoptotic factor, consequently inhibiting tumor growth. These results demonstrate that mRNA delivery by AuNP-DNA conjugates can serve as a new platform for the development of safe and efficient gene therapy.

Single-cell mRNA cytometry via sequence-specific nanoparticle clustering and trapping

Science.gov (United States)

Labib, Mahmoud; Mohamadi, Reza M.; Poudineh, Mahla; Ahmed, Sharif U.; Ivanov, Ivaylo; Huang, Ching-Lung; Moosavi, Maral; Sargent, Edward H.; Kelley, Shana O.

2018-05-01

Cell-to-cell variation in gene expression creates a need for techniques that can characterize expression at the level of individual cells. This is particularly true for rare circulating tumour cells, in which subtyping and drug resistance are of intense interest. Here we describe a method for cell analysis—single-cell mRNA cytometry—that enables the isolation of rare cells from whole blood as a function of target mRNA sequences. This approach uses two classes of magnetic particles that are labelled to selectively hybridize with different regions of the target mRNA. Hybridization leads to the formation of large magnetic clusters that remain localized within the cells of interest, thereby enabling the cells to be magnetically separated. Targeting specific intracellular mRNAs enablescirculating tumour cells to be distinguished from normal haematopoietic cells. No polymerase chain reaction amplification is required to determine RNA expression levels and genotype at the single-cell level, and minimal cell manipulation is required. To demonstrate this approach we use single-cell mRNA cytometry to detect clinically important sequences in prostate cancer specimens.

Convex geometry of quantum resource quantification

Science.gov (United States)

Regula, Bartosz

2018-01-01

We introduce a framework unifying the mathematical characterisation of different measures of general quantum resources and allowing for a systematic way to define a variety of faithful quantifiers for any given convex quantum resource theory. The approach allows us to describe many commonly used measures such as matrix norm-based quantifiers, robustness measures, convex roof-based measures, and witness-based quantifiers together in a common formalism based on the convex geometry of the underlying sets of resource-free states. We establish easily verifiable criteria for a measure to possess desirable properties such as faithfulness and strong monotonicity under relevant free operations, and show that many quantifiers obtained in this framework indeed satisfy them for any considered quantum resource. We derive various bounds and relations between the measures, generalising and providing significantly simplified proofs of results found in the resource theories of quantum entanglement and coherence. We also prove that the quantification of resources in this framework simplifies for pure states, allowing us to obtain more easily computable forms of the considered measures, and show that many of them are in fact equal on pure states. Further, we investigate the dual formulation of resource quantifiers, which provide a characterisation of the sets of resource witnesses. We present an explicit application of the results to the resource theories of multi-level coherence, entanglement of Schmidt number k, multipartite entanglement, as well as magic states, providing insight into the quantification of the four resources by establishing novel quantitative relations and introducing new quantifiers, such as a measure of entanglement of Schmidt number k which generalises the convex roof-extended negativity, a measure of k-coherence which generalises the \

Quantification analysis of CT for aphasic patients

Energy Technology Data Exchange (ETDEWEB)

Watanabe, S.; Ooyama, H.; Hojo, K.; Tasaki, H.; Hanazono, T.; Sato, T.; Metoki, H.; Totsuka, M.; Oosumi, N.

1987-02-01

Using a microcomputer, the locus and extent of the lesions, as demonstrated by computed tomography, for 44 aphasic patients with various types of aphasia were superimposed onto standardized matrices, composed of 10 slices with 3000 points (50 by 60). The relationships between the foci of the lesions and types of aphasia were investigated on the slices numbered 3, 4, 5, and 6 using a quantification theory, Type 3 (pattern analysis). Some types of regularities were observed on slices 3, 4, 5, and 6. The group of patients with Broca's aphasia and the group with Wernicke's aphasia were generally separated on the 1st component and the 2nd component of the quantification theory, Type 3. On the other hand, the group with global aphasia existed between the group with Broca's aphasia and that with Wernicke's aphasia. The group of patients with amnestic aphasia had no specific findings, and the group with conduction aphasia existed near those with Wernicke's aphasia. The above results serve to establish the quantification theory, Type 2 (discrimination analysis) and the quantification theory, Type 1 (regression analysis).

Rapid quantification and sex determination of forensic evidence materials.

Science.gov (United States)

Andréasson, Hanna; Allen, Marie

2003-11-01

DNA quantification of forensic evidence is very valuable for an optimal use of the available biological material. Moreover, sex determination is of great importance as additional information in criminal investigations as well as in identification of missing persons, no suspect cases, and ancient DNA studies. While routine forensic DNA analysis based on short tandem repeat markers includes a marker for sex determination, analysis of samples containing scarce amounts of DNA is often based on mitochondrial DNA, and sex determination is not performed. In order to allow quantification and simultaneous sex determination on minute amounts of DNA, an assay based on real-time PCR analysis of a marker within the human amelogenin gene has been developed. The sex determination is based on melting curve analysis, while an externally standardized kinetic analysis allows quantification of the nuclear DNA copy number in the sample. This real-time DNA quantification assay has proven to be highly sensitive, enabling quantification of single DNA copies. Although certain limitations were apparent, the system is a rapid, cost-effective, and flexible assay for analysis of forensic casework samples.

Quantification of mixed chimerism by real time PCR on whole blood-impregnated FTA cards.

Science.gov (United States)

Pezzoli, N; Silvy, M; Woronko, A; Le Treut, T; Lévy-Mozziconacci, A; Reviron, D; Gabert, J; Picard, C

2007-09-01

This study has investigated quantification of chimerism in sex-mismatched transplantations by quantitative real time PCR (RQ-PCR) using FTA paper for blood sampling. First, we demonstrate that the quantification of DNA from EDTA-blood which has been deposit on FTA card is accurate and reproducible. Secondly, we show that fraction of recipient cells detected by RQ-PCR was concordant between the FTA and salting-out method, reference DNA extraction method. Furthermore, the sensitivity of detection of recipient cells is relatively similar with the two methods. Our results show that this innovative method can be used for MC assessment by RQ-PCR.

The development of clinical activity in relapsing-remitting MS is associated with a decrease of FasL mRNA and an increase of Fas mRNA in peripheral blood

NARCIS (Netherlands)

Lopatinskaya, L.; Boxel van-Dezaire, A.H.H.; Barkhof, F.; Polman, C.H.; Lucas, C.J.; Nagelkerken, L.

2003-01-01

In this longitudinal study, we examined the expression of Fas, FasL, CCR3, CCR5 and CXCR3 mRNA in peripheral blood mononuclear cells (PBMCs) of secondary progressive (SP) and relapsing-remitting (RR) multiple sclerosis (MS) patients. In RR patients, FasL, CCR3 and CCR5 mRNA levels were increased

Expression of a serine protease (motopsin PRSS12) mRNA in the mouse brain: in situ hybridization histochemical study.

Science.gov (United States)

Iijima, N; Tanaka, M; Mitsui, S; Yamamura, Y; Yamaguchi, N; Ibata, Y

1999-03-20

Serine proteases are considered to play several important roles in the brain. In an attempt to find novel brain-specific serine proteases (BSSPs), motopsin (PRSS-12) was cloned from a mouse brain cDNA library by polymerase chain reaction (PCR). Northern blot analysis demonstrated that the postnatal 10-day mouse brain contained the most amount of motopsin mRNA. At this developmental stage, in situ hybridization histochemistry showed that motopsin mRNA was specifically expressed in the following regions: cerebral cortical layers II/III, V and VIb, endopiriform cortex and the limbic system, particularly in the CA1 region of the hippocampal formation. In addition, in the brainstem, the oculomotor nucleus, trochlear nucleus, mecencephalic and motor nuclei of trigeminal nerve (N), abducens nucleus, facial nucleus, nucleus of the raphe pontis, dorsoral motor nucleus of vagal N, hypoglossal nucleus and ambiguus nucleus showed motopsin mRNA expression. Expression was also found in the anterior horn of the spinal cord. The above findings strongly suggest that neurons in almost all motor nuclei, particularly in the brainstem and spinal cord, express motopsin mRNA, and that motopsin seems to have a close relation to the functional role of efferent neurons. Copyright 1999 Elsevier Science B.V.

Artesunate Reduces Serum Lipopolysaccharide in Cecal Ligation/Puncture Mice via Enhanced LPS Internalization by Macrophages through Increased mRNA Expression of Scavenger Receptors

Directory of Open Access Journals (Sweden)

Bin Li

2014-01-01

Full Text Available Innate immunity is the first line of defense in human beings against pathogen infection; monocytes/macrophages are the primary cells of the innate immune system. Recently, macrophages/monocytes have been discovered to participate in LPS clearance, and the clearance efficiency determines the magnitude of the inflammatory response and subsequent organ injury. Previously, we reported that artesunate (AS protected sepsis mice against heat-killed E. coli challenge. Herein, we further confirmed that AS protected cecal ligation/puncture (CLP sepsis mice. Its protection on sepsis mice was related to not only reduction of pro-inflammatory cytokines and serum LPS levels but also improvement of liver function. Based on the fact that AS did not directly bind and neutralize LPS, we hypothesized that the reduction of serum LPS level might be related to enhancement of LPS internalization and subsequent detoxification. Our results showed that AS increased FITC-LPS internalization by peritoneal macrophage and liver Kupffer cell, but enhancement of LPS internalization by AS was not related to the clathrin-dependent pathway. However, AS induced mRNA expression of important scavenger receptors (SRs; SR-A and MARCO mRNA expression was upregulated, suggesting that AS enhancement of LPS internalization and inhibition of pro-inflammatory cytokines was related to changes in mRNA expression of SRs.

NONOates regulate KCl cotransporter-1 and -3 mRNA expression in vascular smooth muscle cells.

Science.gov (United States)

Di Fulvio, Mauricio; Lauf, Peter K; Shah, Shalin; Adragna, Norma C

2003-05-01

Nitric oxide (NO) donors regulate KCl cotransport (KCC) activity and cotransporter-1 and -3 (KCC1 and KCC3) mRNA expression in sheep erythrocytes and in primary cultures of rat vascular smooth muscle cells (VSMCs), respectively. In this study, we used NONOates as rapid and slow NO releasers to provide direct evidence implicating NO as a regulator of KCC3 gene expression at the mRNA level. In addition, we used the expression of KCC3 mRNA to further investigate the mechanism of action of these NO donors at the cellular level. Treatment of VSMCs with rapid NO releasers, like NOC-5 and NOC-9, as well as with the direct NO-independent soluble guanylyl cyclase (sGC) stimulator YC-1, acutely increased KCC3 mRNA expression in a concentration- and time-dependent manner. The slow NO releaser NOC-18 had no effect on KCC3 gene expression. A specific NO scavenger completely prevented the NONOate-induced KCC3 mRNA expression. Inhibition of sGC with LY-83583 blocked the NONOate- and YC-1-induced KCC3 mRNA expression. This study shows that in primary cultures of rat VSMCs, the fast NO releasers NOC-9 and NOC-5, but not the slow NO releaser NOC-18, acutely upregulate KCC3 mRNA expression in a NO/sGC-dependent manner.

Anesthesia for euthanasia influences mRNA expression in healthy mice and after traumatic brain injury.

Science.gov (United States)

Staib-Lasarzik, Irina; Kriege, Oliver; Timaru-Kast, Ralph; Pieter, Dana; Werner, Christian; Engelhard, Kristin; Thal, Serge C

2014-10-01

Tissue sampling for gene expression analysis is usually performed under general anesthesia. Anesthetics are known to modulate hemodynamics, receptor-mediated signaling cascades, and outcome parameters. The present study determined the influence of anesthetic paradigms typically used for euthanization and tissue sampling on cerebral mRNA expression in mice. Naïve mice and animals with acute traumatic brain injury induced by controlled cortical impact (CCI) were randomized to the following euthanasia protocols (n=10-11/group): no anesthesia (NA), 1 min of 4 vol% isoflurane in room air (ISO), 3 min of a combination of 5 mg/kg midazolam, 0.05 mg/kg fentanyl, and 0.5 mg/kg medetomidine intraperitoneally (COMB), or 3 min of 360 mg/kg chloral hydrate intraperitoneally (CH). mRNA expression of actin-1-related gene (Act1), FBJ murine osteosarcoma viral oncogene homolog B (FosB), tumor necrosis factor alpha (TNFα), heat shock protein beta-1 (HspB1), interleukin (IL)-6, tight junction protein 1 (ZO-1), IL-1ß, cyclophilin A, micro RNA 497 (miR497), and small cajal body-specific RNA 17 were determined by real-time polymerase chain reaction (PCR) in hippocampus samples. In naïve animals, Act1 expression was downregulated in the CH group compared with NA. FosB expression was downregulated in COMB and CH groups compared with NA. CCI reduced Act1 and FosB expression, whereas HspB1 and TNFα expression increased. After CCI, HspB1 expression was significantly higher in ISO, COMB, and CH groups, and TNFα expression was elevated in ISO and COMB groups. MiR497, IL-6, and IL-1ß were upregulated after CCI but not affected by anesthetics. Effects were independent of absolute mRNA copy numbers. The data demonstrate that a few minutes of anesthesia before tissue sampling are sufficient to induce immediate mRNA changes, which seem to predominate in the early-regulated gene cluster. Anesthesia-related effects on gene expression might explain limited reproduciblity of real

Immediate-early gene response to repeated immobilization: Fos protein and arc mRNA levels appear to be less sensitive than c-fos mRNA to adaptation.

Science.gov (United States)

Ons, Sheila; Rotllant, David; Marín-Blasco, Ignacio J; Armario, Antonio

2010-06-01

Stress exposure resulted in brain induction of immediate-early genes (IEGs), considered as markers of neuronal activation. Upon repeated exposure to the same stressor, reduction of IEG response (adaptation) has been often observed, but there are important discrepancies in literature that may be in part related to the particular IEG and methodology used. We studied the differential pattern of adaptation of the IEGs c-fos and arc (activity-regulated cytoskeleton-associated protein) after repeated exposure to a severe stressor: immobilization on wooden boards (IMO). Rats repeatedly exposed to IMO showed reduced c-fos mRNA levels in response to acute IMO in most brain areas studied: the medial prefrontal cortex (mPFC), lateral septum (LS), medial amygdala (MeA), paraventricular nucleus of the hypothalamus (PVN) and locus coeruleus. In contrast, the number of neurons showing Fos-like immunoreactivity was only reduced in the MeA and the various subregions of the PVN. IMO-induced increases in arc gene expression were restricted to telencephalic regions and reduced by repeated IMO only in the mPFC. Double-labelling in the LS of IMO-exposed rats revealed that arc was expressed in only one-third of Fos+ neurons, suggesting two populations of Fos+ neurons. These data suggest that c-fos mRNA levels are more affected by repeated IMO than corresponding protein, and that arc gene expression does not reflect adaptation in most brain regions, which may be related to its constitutive expression. Therefore, the choice of a particular IEG and the method of measurement are important for proper interpretation of the impact of chronic repeated stress on brain activation.

Survey and Evaluate Uncertainty Quantification Methodologies

Energy Technology Data Exchange (ETDEWEB)

Lin, Guang; Engel, David W.; Eslinger, Paul W.

2012-02-01

The Carbon Capture Simulation Initiative (CCSI) is a partnership among national laboratories, industry and academic institutions that will develop and deploy state-of-the-art computational modeling and simulation tools to accelerate the commercialization of carbon capture technologies from discovery to development, demonstration, and ultimately the widespread deployment to hundreds of power plants. The CCSI Toolset will provide end users in industry with a comprehensive, integrated suite of scientifically validated models with uncertainty quantification, optimization, risk analysis and decision making capabilities. The CCSI Toolset will incorporate commercial and open-source software currently in use by industry and will also develop new software tools as necessary to fill technology gaps identified during execution of the project. The CCSI Toolset will (1) enable promising concepts to be more quickly identified through rapid computational screening of devices and processes; (2) reduce the time to design and troubleshoot new devices and processes; (3) quantify the technical risk in taking technology from laboratory-scale to commercial-scale; and (4) stabilize deployment costs more quickly by replacing some of the physical operational tests with virtual power plant simulations. The goal of CCSI is to deliver a toolset that can simulate the scale-up of a broad set of new carbon capture technologies from laboratory scale to full commercial scale. To provide a framework around which the toolset can be developed and demonstrated, we will focus on three Industrial Challenge Problems (ICPs) related to carbon capture technologies relevant to U.S. pulverized coal (PC) power plants. Post combustion capture by solid sorbents is the technology focus of the initial ICP (referred to as ICP A). The goal of the uncertainty quantification (UQ) task (Task 6) is to provide a set of capabilities to the user community for the quantification of uncertainties associated with the carbon

Survivin mRNA antagonists using locked nucleic acid, potential for molecular cancer therapy

DEFF Research Database (Denmark)

Fisker, Niels; Westergaard, Majken; Hansen, Henrik Frydenlund

2007-01-01

We have investigated the effects of different locked nucleic acid modified antisense mRNA antagonists against Survivin in a prostate cancer model. These mRNA antagonists were found to be potent inhibitors of Survivin expression at low nanomolar concentrations. Additionally there was a pronounced ...

Regulation of mouse hepatic CYP2D9 mRNA expression by growth and adrenal hormones.

Science.gov (United States)

Jarukamjorn, Kanokwan; Sakuma, Tsutomu; Jaruchotikamol, Atika; Oguro, Miki; Nemoto, Nobuo

2006-02-01

The constitutive expression of CYP2D9 is sexually dimorphic, namely, strong in males, but diminutive in females. Repetition of mimic growth hormone (GH) secretion pattern impressively returned the mRNA expression level to that in intact mice: the GH secretion pattern's regulation of CYP2D9 mRNA expression has been predominantly disrupted by exogenous GH-administration. The extensive decline of CYP2D9 mRNA expression becoming a sexually non-specific P450 in 9-week-old male mice exposed as neonates to monosodium L-glutamate (MSG) suggested that the male GH secretion pattern is a key to the regulation of male-specific CYP2D9 mRNA expression in adult mice. Dexamethasone (Dex) showed possibility to induce CYP2D9 mRNA expression in adult MSG-neonatally treated mice of either sex. However, the antagonism was observed by co-administration of Dex and GH in the males. Dex-administration in adrenalectomized mice significantly elevated CYP2D9 mRNA expression levels. These findings suggest that an adrenal hormone participates in the regulatory mechanism of CYP2D9 mRNA expression in association with GH.

Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes

OpenAIRE

Pardi, Norbert; Tuyishime, Steven; Muramatsu, Hiromi; Kariko, Katalin; Mui, Barbara L; Tam, Ying K; Madden, Thomas D; Hope, Michael J; Weissman, Drew

2015-01-01

In recent years, in vitro transcribed messenger RNA (mRNA) has emerged as a potential therapeutic platform. To fulfill its promise, effective delivery of mRNA to specific cell types and tissues needs to be achieved. Lipid nanoparticles (LNPs) are efficient carriers for short-interfering RNAs and have entered clinical trials. However, little is known about the potential of LNPs to deliver mRNA. Here, we generated mRNA-LNPs by incorporating HPLC purified, 1-methylpseudouridine-containing mRNA c...

Hepatic chemerin mRNA in morbidly obese patients with nonalcoholic fatty liver disease.

Science.gov (United States)

Kajor, Maciej; Kukla, Michał; Waluga, Marek; Liszka, Łukasz; Dyaczyński, Michał; Kowalski, Grzegorz; Żądło, Dominika; Berdowska, Agnieszka; Chapuła, Mateusz; Kostrząb-Zdebel, Anna; Bułdak, Rafał J; Sawczyn, Tomasz; Hartleb, Marek

The aim of this study was to investigate hepatic chemerin mRNA, serum chemerin concentration, and immunohistochemical staining for chemerin and and chemokine receptor-like 1 (CMKLR1) in hepatic tissue in 56 morbidly obese women with nonalcoholic fatty liver disease (NAFLD) and to search for a relationship with metabolic and histopathological features. Chemerin mRNA was assessed by quantitative real-time PCR, chemerin, and CMKLR1 immunohistochemical expression with specific antibodies, while serum chemerin concentration was assessed with commercially available enzyme-linked immunosorbent assays. Serum chemerin concentration reached 874.1 ±234.6 ng/ml. There was no difference in serum chemerin levels between patients with BMI steatosis, and definite nonalcoholic steatohepatitis (NASH). Liver chemerin mRNA was observed in all included patients and was markedly, but insignificantly, higher in those with BMI ≥ 40 kg/m2, hepatocyte ballooning, greater extent of steatosis, and definite NASH. Hepatic chemerin mRNA might be a predictor of hepatic steatosis, hepatocyte ballooning, and NAFLD activity score (NAS) but seemed not to be a primary driver regulating liver necroinflammatory activity and fibrosis. The lack of association between serum chemerin and hepatic chemerin mRNA may suggest that adipose tissue but not the liver is the main source of chemerin in morbidly obese women.

The actin binding cytoskeletal protein Moesin is involved in nuclear mRNA export.

Science.gov (United States)

Kristó, Ildikó; Bajusz, Csaba; Borsos, Barbara N; Pankotai, Tibor; Dopie, Joseph; Jankovics, Ferenc; Vartiainen, Maria K; Erdélyi, Miklós; Vilmos, Péter

2017-10-01

Current models imply that the evolutionarily conserved, actin-binding Ezrin-Radixin-Moesin (ERM) proteins perform their activities at the plasma membrane by anchoring membrane proteins to the cortical actin network. Here we show that beside its cytoplasmic functions, the single ERM protein of Drosophila, Moesin, has a novel role in the nucleus. The activation of transcription by heat shock or hormonal treatment increases the amount of nuclear Moesin, indicating biological function for the protein in the nucleus. The distribution of Moesin in the nucleus suggests a function in transcription and the depletion of mRNA export factors Nup98 or its interacting partner, Rae1, leads to the nuclear accumulation of Moesin, suggesting that the nuclear function of the protein is linked to mRNA export. Moesin localizes to mRNP particles through the interaction with the mRNA export factor PCID2 and knock down of Moesin leads to the accumulation of mRNA in the nucleus. Based on our results we propose that, beyond its well-known, manifold functions in the cytoplasm, the ERM protein of Drosophila is a new, functional component of the nucleus where it participates in mRNA export. Copyright © 2017 Elsevier B.V. All rights reserved.

Real-time PCR for the quantification of fungi in planta.

Science.gov (United States)

Klosterman, Steven J

2012-01-01

Methods enabling quantification of fungi in planta can be useful for a variety of applications. In combination with information on plant disease severity, indirect quantification of fungi in planta offers an additional tool in the screening of plants that are resistant to fungal diseases. In this chapter, a method is described for the quantification of DNA from a fungus in plant leaves using real-time PCR (qPCR). Although the method described entails quantification of the fungus Verticillium dahliae in lettuce leaves, the methodology described would be useful for other pathosystems as well. The method utilizes primers that are specific for amplification of a β-tubulin sequence from V. dahliae and a lettuce actin gene sequence as a reference for normalization. This approach enabled quantification of V. dahliae in the amount of 2.5 fg/ng of lettuce leaf DNA at 21 days following plant inoculation.

Rift Valley fever virus NS{sub S} gene expression correlates with a defect in nuclear mRNA export

Energy Technology Data Exchange (ETDEWEB)

Copeland, Anna Maria; Van Deusen, Nicole M.; Schmaljohn, Connie S., E-mail: Connie.s.schmaljohn.civ@mail.mil

2015-12-15

We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NS{sub S} gene, but not the N, G{sub N} or NS{sub M} genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NS{sub S}, confirming that expression of NS{sub S} is likely responsible for this phenomenon. - Highlights: • Rift Valley fever virus (RVFV) infection alters the localization of host mRNA. • mRNA accumulates in the nuclei of RVFV-infected but not mock-infected cells. • NS{sub S} is likely responsible for mRNA relocalization to the nucleus.

Cloning and tissue distribution of rat hear fatty acid binding protein mRNA: identical forms in heart and skeletal muscle

International Nuclear Information System (INIS)

Claffey, K.P.; Herrera, V.L.; Brecher, P.; Ruiz-Opazo, N.

1987-01-01

A fatty acid binding protein (FABP) as been identified and characterized in rat heart, but the function and regulation of this protein are unclear. In this study the cDNA for rat heart FABP was cloned from a λ gt11 library. Sequencing of the cDNA showed an open reading frame coding for a protein with 133 amino acids and a calculated size of 14,776 daltons. Several differences were found between the sequence determined from the cDNA and that reported previously by protein sequencing techniques. Northern blot analysis using rat heart FABP cDNA as a probe established the presence of an abundant mRNA in rat heart about 0.85 kilobases in length. This mRNA was detected, but was not abundant, in fetal heart tissue. Tissue distribution studies showed a similar mRNA species in red, but not white, skeletal muscle. In general, the mRNA tissue distribution was similar to that of the protein detected by Western immunoblot analysis, suggesting that heart FABP expression may be regulated at the transcriptional level. S1 nuclease mapping studies confirmed that the mRNA hybridized to rat heart FABP cDNA was identical in heart and red skeletal muscle throughout the entire open reading frame. The structural differences between heart FABP and other members of this multigene family may be related to the functional requirements of oxidative muscle for fatty acids as a fuel source

Cloning and tissue distribution of rat hear fatty acid binding protein mRNA: identical forms in heart and skeletal muscle

Energy Technology Data Exchange (ETDEWEB)

Claffey, K.P.; Herrera, V.L.; Brecher, P.; Ruiz-Opazo, N.

1987-12-01

A fatty acid binding protein (FABP) as been identified and characterized in rat heart, but the function and regulation of this protein are unclear. In this study the cDNA for rat heart FABP was cloned from a lambda gt11 library. Sequencing of the cDNA showed an open reading frame coding for a protein with 133 amino acids and a calculated size of 14,776 daltons. Several differences were found between the sequence determined from the cDNA and that reported previously by protein sequencing techniques. Northern blot analysis using rat heart FABP cDNA as a probe established the presence of an abundant mRNA in rat heart about 0.85 kilobases in length. This mRNA was detected, but was not abundant, in fetal heart tissue. Tissue distribution studies showed a similar mRNA species in red, but not white, skeletal muscle. In general, the mRNA tissue distribution was similar to that of the protein detected by Western immunoblot analysis, suggesting that heart FABP expression may be regulated at the transcriptional level. S1 nuclease mapping studies confirmed that the mRNA hybridized to rat heart FABP cDNA was identical in heart and red skeletal muscle throughout the entire open reading frame. The structural differences between heart FABP and other members of this multigene family may be related to the functional requirements of oxidative muscle for fatty acids as a fuel source.

Characterization of DNA polymerase. beta. mRNA: cell-cycle growth response in cultured human cells

Energy Technology Data Exchange (ETDEWEB)

Zmudzka, B Z; Fornace, A; Collins, J; Wilson, S H

1988-10-25

DNA polymerase ..beta.. (..beta..-polymerase) is a housekeeping enzyme involved in DNA repair in vertebrate cells. The authors used a cDNA probe to study abundance of ..beta..-polymerase mRNA in cultured human cells. The mRNA level in synchronized HeLa cells, representing different stages of the cell-cycle, varied only slightly. Contact inhibited fibroblasts AG-1522 contained the same level of mRNA as growing cells. The steady-state level of mRNA in fibroblasts is equivalent to 6 molecules per cell. The results indicate that the ..beta..-polymerase transcript is low abundance and is neither cell-cycles nor growth phase responsive.

Direct infusion-SIM as fast and robust method for absolute protein quantification in complex samples

Directory of Open Access Journals (Sweden)

Christina Looße

2015-06-01

Full Text Available Relative and absolute quantification of proteins in biological and clinical samples are common approaches in proteomics. Until now, targeted protein quantification is mainly performed using a combination of HPLC-based peptide separation and selected reaction monitoring on triple quadrupole mass spectrometers. Here, we show for the first time the potential of absolute quantification using a direct infusion strategy combined with single ion monitoring (SIM on a Q Exactive mass spectrometer. By using complex membrane fractions of Escherichia coli, we absolutely quantified the recombinant expressed heterologous human cytochrome P450 monooxygenase 3A4 (CYP3A4 comparing direct infusion-SIM with conventional HPLC-SIM. Direct-infusion SIM revealed only 14.7% (±4.1 (s.e.m. deviation on average, compared to HPLC-SIM and a decreased processing and analysis time of 4.5 min (that could be further decreased to 30 s for a single sample in contrast to 65 min by the LC–MS method. Summarized, our simplified workflow using direct infusion-SIM provides a fast and robust method for quantification of proteins in complex protein mixtures.

Group II intron inhibits conjugative relaxase expression in bacteria by mRNA targeting

Science.gov (United States)

Piazza, Carol Lyn; Smith, Dorie

2018-01-01

Group II introns are mobile ribozymes that are rare in bacterial genomes, often cohabiting with various mobile elements, and seldom interrupting housekeeping genes. What accounts for this distribution has not been well understood. Here, we demonstrate that Ll.LtrB, the group II intron residing in a relaxase gene on a conjugative plasmid from Lactococcus lactis, inhibits its host gene expression and restrains the naturally cohabiting mobile element from conjugative horizontal transfer. We show that reduction in gene expression is mainly at the mRNA level, and results from the interaction between exon-binding sequences (EBSs) in the intron and intron-binding sequences (IBSs) in the mRNA. The spliced intron targets the relaxase mRNA and reopens ligated exons, causing major mRNA loss. Taken together, this study provides an explanation for the distribution and paucity of group II introns in bacteria, and suggests a potential force for those introns to evolve into spliceosomal introns. PMID:29905149

Absolute quantitative proton NMR spectroscopy based on the amplitude of the local water suppression pulse. Quantification of brain water and metabolites

DEFF Research Database (Denmark)

Danielsen, E R; Henriksen, O

1994-01-01

Quantification in localized proton NMR spectroscopy has been achieved by various methods in recent years. A new method for absolute quantification is described in this paper. The method simultaneously rules out problems with B1 field inhomogeneity and coil loading, utilizing a relation between th......M and [NAA] = 9.15 +/- 0.74 nM. It is concluded that the quantification method is easily applied in vivo, and that the absolute concentrations obtained are similar to results in other studies except those relying on assumptions of the concentration of an internal reference. The advantage...

Negative regulation of neuromedin U mRNA expression in the rat pars tuberalis by melatonin.

Directory of Open Access Journals (Sweden)

Sayaka Aizawa

Full Text Available The pars tuberalis (PT is part of the anterior pituitary gland surrounding the median eminence as a thin cell layer. The characteristics of PT differ from those of the pars distalis (PD, such as cell composition and gene expression, suggesting that the PT has a unique physiological function compared to the PD. Because the PT highly expresses melatonin receptor type 1, it is considered a mediator of seasonal and/or circadian signals of melatonin. Expression of neuromedin U (NMU that is known to regulate energy balance has been previously reported in the rat PT; however, the regulatory mechanism of NMU mRNA expression and secretion in the PT are still obscure. In this study, we examined both the diurnal change of NMU mRNA expression in the rat PT and the effects of melatonin on NMU in vivo. In situ hybridization and quantitative PCR analysis of laser microdissected PT samples revealed that NMU mRNA expression in the PT has diurnal variation that is high during the light phase and low during the dark phase. Furthermore, melatonin administration significantly suppressed NMU mRNA expression in the PT in vivo. On the other hand, 48 h fasting did not have an effect on PT-NMU mRNA expression, and the diurnal change of NMU mRNA expression was maintained. We also found the highest expression of neuromedin U receptor type 2 (NMUR2 mRNA in the third ventricle ependymal cell layer, followed by the arcuate nucleus and the spinal cord. These results suggest that NMU mRNA expression in the PT is downregulated by melatonin during the dark phase and shows diurnal change. Considering that NMU mRNA in the PT showed the highest expression level in the brain, PT-NMU may act on NMUR2 in the brain, especially in the third ventricle ependymal cell layer, with a circadian rhythm.

An essential nuclear protein in trypanosomes is a component of mRNA transcription/export pathway.

Directory of Open Access Journals (Sweden)

Mariana Serpeloni

Full Text Available In eukaryotic cells, different RNA species are exported from the nucleus via specialized pathways. The mRNA export machinery is highly integrated with mRNA processing, and includes a different set of nuclear transport adaptors as well as other mRNA binding proteins, RNA helicases, and NPC-associated proteins. The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, a widespread and neglected human disease which is endemic to Latin America. Gene expression in Trypanosoma has unique characteristics, such as constitutive polycistronic transcription of protein-encoding genes and mRNA processing by trans-splicing. In general, post-transcriptional events are the major points for regulation of gene expression in these parasites. However, the export pathway of mRNA from the nucleus is poorly understood. The present study investigated the function of TcSub2, which is a highly conserved protein ortholog to Sub2/ UAP56, a component of the Transcription/Export (TREX multiprotein complex connecting transcription with mRNA export in yeast/human. Similar to its orthologs, TcSub2 is a nuclear protein, localized in dispersed foci all over the nuclei -except the fibrillar center of nucleolus- and at the interface between dense and non-dense chromatin areas, proposing the association of TcSub2 with transcription/processing sites. These findings were analyzed further by BrUTP incorporation assays and confirmed that TcSub2 is physically associated with active RNA polymerase II (RNA pol II, but not RNA polymerase I (RNA pol I or Spliced Leader (SL transcription, demonstrating participation particularly in nuclear mRNA metabolism in T. cruzi. The double knockout of the TcSub2 gene is lethal in T. cruzi, suggesting it has an essential function. Alternatively, RNA interference assays were performed in Trypanosoma brucei. It allowed demonstrating that besides being an essential protein, its knockdown causes mRNA accumulation in the nucleus and

Detection and quantification of Leveillula taurica growth in pepper leaves.

Science.gov (United States)

Zheng, Zheng; Nonomura, Teruo; Bóka, Károly; Matsuda, Yoshinori; Visser, Richard G F; Toyoda, Hideyoshi; Kiss, Levente; Bai, Yuling

2013-06-01

Leveillula taurica is an obligate fungal pathogen that causes powdery mildew disease on a broad range of plants, including important crops such as pepper, tomato, eggplant, onion, cotton, and so on. The early stage of this disease is difficult to diagnose and the disease can easily spread unobserved; for example, in pepper and tomato production fields and greenhouses. The objective of this study was to develop a detection and quantification method of L. taurica biomass in pepper leaves with special regard to the early stages of infection. We monitored the development of the disease to time the infection process on the leaf surface as well as inside the pepper leaves. The initial and final steps of the infection taking place on the leaf surface were consecutively observed using a dissecting microscope and a scanning electron microscope. The development of the intercellular mycelium in the mesophyll was followed by light and transmission electron microscopy. A pair of L. taurica-specific primers was designed based on the internal transcribed spacer sequence of L. taurica and used in real-time polymerase chain reaction (PCR) assay to quantify the fungal DNA during infection. The specificity of this assay was confirmed by testing the primer pair with DNA from host plants and also from another powdery mildew species, Oidium neolycopersici, infecting tomato. A standard curve was obtained for absolute quantification of L. taurica biomass. In addition, we tested a relative quantification method by using a plant gene as reference and the obtained results were compared with the visual disease index scoring. The real-time PCR assay for L. taurica provides a valuable tool for detection and quantification of this pathogen in breeding activities as well in plant-microbe interaction studies.

PPARg mRNA in the adult mouse hypothalamus: distribution and regulation in response to dietary challenges

Directory of Open Access Journals (Sweden)

Yang eLiu

2015-09-01

Full Text Available Peroxisome proliferator-activated receptor gamma (PPARg is a ligand-activated transcription factor that was originally identified as a regulator of peroxisome proliferation and adipocyte differentiation. Emerging evidence suggests that functional PPARg signaling also occurs within the hypothalamus. However, the exact distribution and identities of PPARg-expressing hypothalamic cells remains under debate. The present study systematically mapped PPARg mRNA expression in the adult mouse brain using in situ hybridization histochemistry. PPARg mRNA was found to be expressed at high levels outside the hypothalamus including the neocortex, the olfactory bulb, the organ of the vasculosum of the lamina terminalis, and the subfornical organ. Within the hypothalamus, PPARg was present at moderate levels in the suprachiasmatic nucleus and the ependymal of the 3rd ventricle. In all examined feeding-related hypothalamic nuclei, PPARg was expressed at very low levels that were close to the limit of detection. Using qPCR techniques, we demonstrated that PPARg mRNA expression was upregulated in the suprachiasmatic nucleus in response to fasting. Double in situ hybridization further demonstrated that PPARg was primarily expressed in neurons. Collectively, our observations provide a comprehensive map of PPARg distribution and regulation in the intact adult mouse hypothalamus.

The NO signaling pathway differentially regulates KCC3a and KCC3b mRNA expression.

Science.gov (United States)

Di Fulvio, Mauricio; Lauf, Peter K; Adragna, Norma C

2003-11-01

Nitric oxide (NO) donors and protein kinase G (PKG) acutely up-regulate K-Cl cotransporter-1 and -3 (KCC1 and KCC3) mRNA expression in vascular smooth muscle cells (VSMCs). Here, we report the presence, relative abundance, and regulation by sodium nitroprusside (SNP) of the novel KCC3a and KCC3b mRNAs, in primary cultures of rat VSMCs. KCC3a and KCC3b mRNAs were expressed in an approximate 3:1 ratio, as determined by semiquantitative RT-PCR analysis. SNP as well as YC-1 and 8-Br-cGMP, a NO-independent stimulator of soluble guanylyl cyclase (sGC) and PKG, respectively, increased KCC3a and KCC3b mRNA expression by 2.5-fold and 8.1-fold in a time-dependent manner, following a differential kinetics. Stimulation of the NO/sGC/PKG signaling pathway with either SNP, YC-1, or 8-Br-cGMP decreased the KCC3a/KCC3b ratio from 3.0+/-0.4 to 0.9+/-0.1. This is the first report on a differential regulation by the NO/sGC/PKG signaling pathway of a cotransporter and of KCC3a and KCC3b mRNA expression.

Myogenic, matrix and growth factor mRNA expression in human skeletal muscle: effect of contraction intensity and feeding

DEFF Research Database (Denmark)

Agergaard, Jakob; Reitelseder, Søren; Pedersen, T.G.

2013-01-01

. RESULTS: Relative muscle activity differed between HL and LL resistance exercise, whereas median power frequency was even, suggesting an equal muscle-fiber-type recruitment distribution. mRNA expression of Myf6, myogenin, and p21 was mostly increased, and myostatin was mostly depressed by HL resistance...

Influence of Temperature and Predation on Survival of Salmonella enterica Serovar Typhimurium and Expression of invA in Soil and Manure-Amended Soil▿

Science.gov (United States)

García, R.; Bælum, J.; Fredslund, L.; Santorum, P.; Jacobsen, C. S.

2010-01-01

The effects of three temperatures (5, 15, and 25°C) on the survival of Salmonella enterica serovar Typhimurium in topsoil were investigated in small microcosms by three different techniques: plate counting, invA gene quantification, and invA mRNA quantification. Differences in survival were related to the effect of protozoan predation. Tetracycline-resistant Salmonella serovar Typhimurium was inoculated into soil and manure-amended soil at 1.5 × 108 cells g soil−1. Population densities were determined by plate counting and by molecular methods and monitored for 42 days. Simultaneous extraction of RNA and DNA, followed by quantitative PCR, was used to investigate invA gene levels and expression. Analysis by these three techniques showed that Salmonella serovar Typhimurium survived better at 5°C. Comparing DNA and CFU levels, significantly higher values were determined by DNA-based techniques. invA mRNA levels showed a fast decrease in activity, with no detectable mRNA after an incubation period of less than 4 days in any of the soil scenarios. A negative correlation was found between Salmonella serovar Typhimurium CFU levels and protozoan most probable numbers, and we propose the role of the predator-prey interaction as a factor to explain the die-off of the introduced strain by both culture- and DNA quantification-based methods. The results indicate that temperature, manure, and protozoan predation are important factors influencing the survival of Salmonella serovar Typhimurium in soil. PMID:20562283

Quantification of collateral flow in humans: a comparison of angiographic, electrocardiographic and hemodynamic variables

NARCIS (Netherlands)

van Liebergen, R. A.; Piek, J. J.; Koch, K. T.; de Winter, R. J.; Schotborgh, C. E.; Lie, K. I.

1999-01-01

Evaluation of collateral vascular circulation according to hemodynamic variables and its relation to myocardial ischemia. There is limited information regarding the hemodynamic quantification of recruitable collateral vessels. Angiography of the donor coronary artery was performed before and during

A small RNA activates CFA synthase by isoform-specific mRNA stabilization.

Science.gov (United States)

Fröhlich, Kathrin Sophie; Papenfort, Kai; Fekete, Agnes; Vogel, Jörg

2013-11-13

Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5' end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5' untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability.

Kinetin improves IKBKAP mRNA splicing in patients with familial dysautonomia

Science.gov (United States)

Axelrod, Felicia B.; Liebes, Leonard; Gold-von Simson, Gabrielle; Mendoza, Sandra; Mull, James; Leyne, Maire; Norcliffe-Kaufmann, Lucy; Kaufmann, Horacio; Slaugenhaupt, Susan A.

2011-01-01

Familial dysautonomia (FD) is caused by an intronic splice mutation in the IKBKAP gene that leads to partial skipping of exon 20 and tissue-specific reduction in I-κ-B kinase complex associated protein/ elongation protein 1 (IKAP/ELP-1) expression. Kinetin (6-furfurylaminopurine) has been shown to improve splicing and increase wild-type IKBKAP mRNA and IKAP protein expression in FD cell lines and carriers. To determine if oral kinetin treatment could alter mRNA splicing in FD subjects and was tolerable, we administered kinetin to eight FD individuals homozygous for the splice mutation. Subjects received 23.5 mg/Kg/day for 28 days. An increase in wild-type IKBKAP mRNA expression in leukocytes was noted after eight days in six of eight individuals; after 28 days the mean increase as compared to baseline was significant (p=0.002). We have demonstrated that kinetin is tolerable in this medically fragile population. Not only did kinetin produce the desired effect on splicing in FD patients, but also that effect appears to improve with time despite lack of dose change. This is the first report of a drug that produces in vivo mRNA splicing changes in individuals with FD and supports future long-term trials to determine if kinetin will prove therapeutic in FD patients. PMID:21775922

Strategy study of quantification harmonization of SUV in PET/CT images

International Nuclear Information System (INIS)

Fischer, Andreia Caroline Fischer da Silveira

2014-01-01

In clinical practice, PET/CT images are often analyzed qualitatively by visual comparison of tumor lesions and normal tissues uptake; and semi-quantitatively by means of a parameter called SUV (Standardized Uptake Value). To ensure that longitudinal studies acquired on different scanners are interchangeable, and information of quantification is comparable, it is necessary to establish a strategy to harmonize the quantification of SUV. The aim of this study is to evaluate the strategy to harmonize the quantification of PET/CT images, performed with different scanner models and manufacturers. For this purpose, a survey of the technical characteristics of equipment and acquisition protocols of clinical images of different services of PET/CT in the state of Rio Grande do Sul was conducted. For each scanner, the accuracy of SUV quantification, and the Recovery Coefficient (RC) curves were determined, using the reconstruction parameters clinically relevant and available. From these data, harmonized performance specifications among the evaluated scanners were identified, as well as the algorithm that produces, for each one, the most accurate quantification. Finally, the most appropriate reconstruction parameters to harmonize the SUV quantification in each scanner, either regionally or internationally were identified. It was found that the RC values of the analyzed scanners proved to be overestimated by up to 38%, particularly for objects larger than 17mm. These results demonstrate the need for further optimization, through the reconstruction parameters modification, and even the change of the reconstruction algorithm used in each scanner. It was observed that there is a decoupling between the best image for PET/CT qualitative analysis and the best image for quantification studies. Thus, the choice of reconstruction method should be tied to the purpose of the PET/CT study in question, since the same reconstruction algorithm is not adequate, in one scanner, for qualitative

Application of Fuzzy Comprehensive Evaluation Method in Trust Quantification

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Shunan Ma

2011-10-01

Full Text Available Trust can play an important role for the sharing of resources and information in open network environments. Trust quantification is thus an important issue in dynamic trust management. By considering the fuzziness and uncertainty of trust, in this paper, we propose a fuzzy comprehensive evaluation method to quantify trust along with a trust quantification algorithm. Simulation results show that the trust quantification algorithm that we propose can effectively quantify trust and the quantified value of an entity's trust is consistent with the behavior of the entity.

HPLC Quantification of Cytotoxic Compounds from Aspergillus niger

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Paula Karina S. Uchoa

2017-01-01

Full Text Available A high-performance liquid chromatography method was developed and validated for the quantification of the cytotoxic compounds produced by a marine strain of Aspergillus niger. The fungus was grown in malt peptone dextrose (MPD, potato dextrose yeast (PDY, and mannitol peptone yeast (MnPY media during 7, 14, 21, and 28 days, and the natural products were identified by standard compounds. The validation parameters obtained were selectivity, linearity (coefficient of correlation > 0.99, precision (relative standard deviation below 5%, and accuracy (recovery > 96.

Micromethod for quantification of SH groups generated after reduction of monoclonal antibodies

International Nuclear Information System (INIS)

Escobar, Normando Iznaga; Morales, Alejo; Nunez, Gilda

1996-01-01

A simple, rapid, and reproducible micromethod for quantification of sulfhydryl (SH) groups generated after reduction of monoclonal antibody (MAb) disulfide bonds with 2-mercaptoethanol (2-ME) is described. The number of SH groups per molecule of antibody in the 2-ME and in the other reducing agents was calculated from the cysteine standard curve using Ellman's reagent to develop the yellow color. Results were plotted as absorbance at 405 nm vs. cysteine concentration (μg/mL). After subtraction of the background due to Ellman's reagent, a straight-line relationship passing through the origin was obtained. Absorption spectrum of the yellow products was controlled, and no significative differences were found between optical density at 412 nm and 405 nm. Using a small quantity of antibody in the order of 37 μg, the lowest detection limit for cysteine quantification was 0.03 μg. An excellent linear correlation was found between both cysteine concentration and absorbance (r = 0.999), and the mean value of the relative error in the quantification of cysteine from samples was 2.8%. A statistical Student t-test showed an excellent linearity and parallelism between cysteine standard and samples

Micromethod for quantification of SH groups generated after reduction of monoclonal antibodies

Energy Technology Data Exchange (ETDEWEB)

Escobar, Normando Iznaga; Morales, Alejo; Nunez, Gilda

1996-07-01

A simple, rapid, and reproducible micromethod for quantification of sulfhydryl (SH) groups generated after reduction of monoclonal antibody (MAb) disulfide bonds with 2-mercaptoethanol (2-ME) is described. The number of SH groups per molecule of antibody in the 2-ME and in the other reducing agents was calculated from the cysteine standard curve using Ellman's reagent to develop the yellow color. Results were plotted as absorbance at 405 nm vs. cysteine concentration ({mu}g/mL). After subtraction of the background due to Ellman's reagent, a straight-line relationship passing through the origin was obtained. Absorption spectrum of the yellow products was controlled, and no significative differences were found between optical density at 412 nm and 405 nm. Using a small quantity of antibody in the order of 37 {mu}g, the lowest detection limit for cysteine quantification was 0.03 {mu}g. An excellent linear correlation was found between both cysteine concentration and absorbance (r = 0.999), and the mean value of the relative error in the quantification of cysteine from samples was 2.8%. A statistical Student t-test showed an excellent linearity and parallelism between cysteine standard and samples.

The silence of MUC2 mRNA induced by promoter hypermethylation associated with HBV in Hepatocellular Carcinoma

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Ling Yang

2013-01-01

Full Text Available Abstract Background To evaluate the promoter methylation status of MUC2 gene and mRNA expression in patients with hepatocellular carcinoma. Methods We analyzed MUC2 methylation by MSP, and MUC2 mRNA by real-time PCR in 74 HCC. Results MUC2 mRNA were lower in HCC tissues (Mean -ΔCt = −4.70 than that in Non-HCC tissues (Mean -ΔCt = −2.98. Expression of MUC2 was elevated in only 23 (31.08% of the 74 HCC patients. MUC2 promoter was hypermethylated in 62.2% (46/74 of HCCs, and in only 18.9% (14/74 of non-tumor samples. MUC2 mRNA were lower in HCC patients with hypermethylation (Mean -ΔΔCt = −2.25 than those with demethylation (Mean -ΔΔCt = −0.22, and there is a decreased tendency for MUC2 mRNA in HCC patients with promoter hypermethylation (p = 0.011. There was a significantly correlation found between MUC2 mRNA and HBV and AFP in HCC. The loss of MUC2 mRNA and hypermethylation could be poor prognostic factors. After treated by 5-Aza-CdR and TSA, we found that MUC2 mRNA induced significantly in 7721, Huh7 and HepG2 cells. Conclusion The results suggested that MUC2 mRNA silenced by promoter hypermethylation is associated with high levels HBV in HCC.

Recruitment of Staufen2 Enhances Dendritic Localization of an Intron-Containing CaMKIIα mRNA

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Raúl Ortiz

2017-07-01

Full Text Available Regulation of mRNA localization is a conserved cellular process observed in many types of cells and organisms. Asymmetrical mRNA distribution plays a particularly important role in the nervous system, where local translation of localized mRNA represents a key mechanism in synaptic plasticity. CaMKIIα is a very abundant mRNA detected in neurites, consistent with its crucial role at glutamatergic synapses. Here, we report the presence of CaMKIIα mRNA isoforms that contain intron i16 in dendrites, RNA granules, and synaptoneurosomes from primary neurons and brain. This subpopulation of unspliced mRNA preferentially localizes to distal dendrites in a synaptic-activity-dependent manner. Staufen2, a well-established marker of RNA transport in dendrites, interacts with intron i16 sequences and enhances its distal dendritic localization, pointing to the existence of intron-mediated mechanisms in the molecular pathways that modulate dendritic transport and localization of synaptic mRNAs.

Time-course of 5-HT(6) receptor mRNA expression during memory consolidation and amnesia.

Science.gov (United States)

Huerta-Rivas, A; Pérez-García, G; González-Espinosa, C; Meneses, A

2010-01-01

Growing evidence indicates that antagonists of the 5-hydroxytryptamine (serotonin) receptor(6) (5-HT(6)) improve memory and reverse amnesia although the mechanisms involved are poorly understood. Hence, in this paper RT-PCR was used to evaluate changes in mRNA expression of 5-HT(6) receptor in trained and untrained rats treated with the 5-HT(6) receptor antagonist SB-399885 and amnesic drugs scopolamine or dizocilpine. Changes in mRNA expression of 5-HT(6) receptor were investigated at different times in prefrontal cortex, hippocampus and striatum. Data indicated that memory in the Pavlovian/instrumental autoshaping task was a progressive process associated to reduced mRNA expression of 5-HT(6) receptor in the three structures examined. SB-399885 improved long-term memory at 48h, while the muscarinic receptor antagonist scopolamine or the non-competitive NMDA receptor antagonist dizocilpine impaired it at 24h. Autoshaping training and treatment with SB-399885 increased 5-HT(6) receptor mRNA expression in (maximum increase) prefrontal cortex and striatum, 24 or 48h. The scopolamine-induced amnesia suppressed 5-HT(6) receptor mRNA expression while the dizocilpine-induced amnesia did not modify 5-HT(6) receptor mRNA expression. SB-399885 and scopolamine or dizocilpine were able to reestablish memory and 5-HT(6) receptor mRNA expression. These data confirmed previous memory evidence and of more interest is the observation that training, SB-399885 and amnesic drugs modulated 5-HT(6) receptor mRNA expression in prefrontal cortex, hippocampus and striatum. Further investigation in different memory tasks, times and amnesia models together with more complex control groups might provide further clues. Copyright 2009 Elsevier Inc. All rights reserved.

Early-life stress induces persistent alterationsin 5-HT1Areceptor and serotonin transporter mRNA expression in the adultrat brain.

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Javier A. Bravo

2014-04-01

Full Text Available Early-life experience plays a major role in the stress response throughout life. Neonatal maternal separation (MS is an animal model of depression with an altered serotonergic response. We hypothesize that this alteration may be caused by differences in 5-HT1A receptor and serotonin transporter (SERT mRNA expression in brain areas involved in the control of emotions, memory and fear as well as in regions controlling the central serotonergic tone.To test this, Sprague-Dawley rats were subjected to MS for 3h daily during post-natal days 2-12. As control, age matched rats were not separated (NS from their dams. When animals reached adulthood (11-13 weeks brain was extracted and mRNA expression of 5-HT1A receptor in amygdala, hippocampus and dorsal raphé nucleus (DRN and SERT in the DRN was analyzed through in-situ hybridisation.Densitometric analysis revealed that MS increased 5-HT1A receptor mRNA expression in the amygdala, and reduced its expression in the DRN, but no changes were observed in the hippocampus in comparison to NS controls. Also, MS reduced SERT mRNA expression in the DRN when compared to NS rats.These results suggest that early-life stress induces persistent changes in 5-HT1A receptor and SERT mRNA expression in key brain regions involved in the development of stress-related psychiatric disorders. The reduction in SERT mRNA indicates an alteration that is in line with clinical findings such as polymorphic variants in individuals with higher risk of depression. These data may help to understand how early-life stress contributes to the development of mood disorders in adulthood.

Responses of mRNA expression of PepT1 in small intestine to ...

African Journals Online (AJOL)

To study the effect of circulation small peptides concentration on mRNA expression in small intestine, graded amount of soybean small peptides (SSP) were infused into lactating goats through duodenal fistulas. Peptide-bound amino acid (PBAA) concentration in arterial plasma and the mRNA expression of PepT1 was ...

Iron overload in the liver diagnostic and quantification

Energy Technology Data Exchange (ETDEWEB)

Alustiza, Jose M. [Osatek SA, P Dr. Beguiristain 109, 20014, San Sebastian, Guipuzcoa (Spain)]. E-mail: jmalustiza@osatek.es; Castiella, Agustin [Osatek SA, P Dr. Beguiristain 109, 20014, San Sebastian, Guipuzcoa (Spain); Juan, Maria D. de [Osatek SA, P Dr. Beguiristain 109, 20014, San Sebastian, Guipuzcoa (Spain); Emparanza, Jose I. [Osatek SA, P Dr. Beguiristain 109, 20014, San Sebastian, Guipuzcoa (Spain); Artetxe, Jose [Osatek SA, P Dr. Beguiristain 109, 20014, San Sebastian, Guipuzcoa (Spain); Uranga, Maite [Osatek SA, P Dr. Beguiristain 109, 20014, San Sebastian, Guipuzcoa (Spain)

2007-03-15

Hereditary Hemochromatosis is the most frequent modality of iron overload. Since 1996 genetic tests have facilitated significantly the non-invasive diagnosis of the disease. There are however many cases of negative genetic tests that require confirmation by hepatic iron quantification which is traditionally performed by hepatic biopsy. There are many studies that have demonstrated the possibility of performing hepatic iron quantification with Magnetic Resonance. However, a consensus has not been reached yet regarding the technique or the possibility to reproduce the same method of calculus in different machines. This article reviews the state of the art of the question and delineates possible future lines to standardise this non-invasive method of hepatic iron quantification.

Iron overload in the liver diagnostic and quantification

International Nuclear Information System (INIS)

Alustiza, Jose M.; Castiella, Agustin; Juan, Maria D. de; Emparanza, Jose I.; Artetxe, Jose; Uranga, Maite

2007-01-01

Hereditary Hemochromatosis is the most frequent modality of iron overload. Since 1996 genetic tests have facilitated significantly the non-invasive diagnosis of the disease. There are however many cases of negative genetic tests that require confirmation by hepatic iron quantification which is traditionally performed by hepatic biopsy. There are many studies that have demonstrated the possibility of performing hepatic iron quantification with Magnetic Resonance. However, a consensus has not been reached yet regarding the technique or the possibility to reproduce the same method of calculus in different machines. This article reviews the state of the art of the question and delineates possible future lines to standardise this non-invasive method of hepatic iron quantification

Increased IL-17 and 22 mRNA expression in pediatric patients with otitis media with effusion.

Science.gov (United States)

Kwon, Oh Eun; Park, Sang Hyun; Kim, Sung Su; Shim, Haeng Seon; Kim, Min Gyeong; Kim, Young Il; Kim, Sang Hoon; Yeo, Seung Geun

2016-11-01

Middle ear effusion has been reported to be associated with immune responses in patients with otitis media with effusion (OME). Although various cytokines are involved in immunologic responses in patients with OME, no study to date has assessed the involvement of the pro-inflammatory cytokines interleukin (IL)-17 and IL-22. This study analyzed the levels of expression of IL-17 and IL-22 in the middle ear effusion of patients with OME. Patients aged Effusion fluid samples were obtained during surgery and levels of IL-17 and IL-22 mRNAs assessed by real-time PCR. IL-17 and IL-22 mRNA levels were compared in patients with effusion fluid positive and negative for bacteria; in patients with and without accompanying diseases, recurrent disease, and re-operation; and relative to fluid characteristics. The study cohort included 70 pediatric patients, 46 boys and 24 girls, of mean age 4.31 ± 2.11 years. The levels of IL-17 and IL-22 mRNA were higher in patients with than without sinusitis, but only IL-22 mRNA levels differed significantly (p < 0.05). The level of IL-17 mRNA was significantly higher in patients who did than did not undergo T&A (p < 0.05). The level of IL-22 expression was significantly higher in mucoid and purulent middle ear fluid samples than in serous fluid samples (p < 0.05). IL-17 and IL-22 mRNAs are involved in the pathophysiology of OME and are significantly higher in subjects with than without accompanying diseases. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Dis3- and exosome subunit-responsive 3′ mRNA instability elements

International Nuclear Information System (INIS)

Kiss, Daniel L.; Hou, Dezhi; Gross, Robert H.; Andrulis, Erik D.

2012-01-01

Highlights: ► Successful use of a novel RNA-specific bioinformatic tool, RNA SCOPE. ► Identified novel 3′ UTR cis-acting element that destabilizes a reporter mRNA. ► Show exosome subunits are required for cis-acting element-mediated mRNA instability. ► Define precise sequence requirements of novel cis-acting element. ► Show that microarray-defined exosome subunit-regulated mRNAs have novel element. -- Abstract: Eukaryotic RNA turnover is regulated in part by the exosome, a nuclear and cytoplasmic complex of ribonucleases (RNases) and RNA-binding proteins. The major RNase of the complex is thought to be Dis3, a multi-functional 3′–5′ exoribonuclease and endoribonuclease. Although it is known that Dis3 and core exosome subunits are recruited to transcriptionally active genes and to messenger RNA (mRNA) substrates, this recruitment is thought to occur indirectly. We sought to discover cis-acting elements that recruit Dis3 or other exosome subunits. Using a bioinformatic tool called RNA SCOPE to screen the 3′ untranslated regions of up-regulated transcripts from our published Dis3 depletion-derived transcriptomic data set, we identified several motifs as candidate instability elements. Secondary screening using a luciferase reporter system revealed that one cassette—harboring four elements—destabilized the reporter transcript. RNAi-based depletion of Dis3, Rrp6, Rrp4, Rrp40, or Rrp46 diminished the efficacy of cassette-mediated destabilization. Truncation analysis of the cassette showed that two exosome subunit-sensitive elements (ESSEs) destabilized the reporter. Point-directed mutagenesis of ESSE abrogated the destabilization effect. An examination of the transcriptomic data from exosome subunit depletion-based microarrays revealed that mRNAs with ESSEs are found in every up-regulated mRNA data set but are underrepresented or missing from the down-regulated data sets. Taken together, our findings imply a potentially novel mechanism of mRNA

Correlation of mRNA Expression and Signal Variability in Chronic Intracortical Electrodes.

Science.gov (United States)

Falcone, Jessica D; Carroll, Sheridan L; Saxena, Tarun; Mandavia, Dev; Clark, Alexus; Yarabarla, Varun; Bellamkonda, Ravi V

2018-01-01

The goal for this research was to identify molecular mechanisms that explain animal-to-animal variability in chronic intracortical recordings. Microwire electrodes were implanted into Sprague Dawley rats at an acute (1 week) and a chronic (14 weeks) time point. Weekly recordings were conducted, and action potentials were evoked in the barrel cortex by deflecting the rat's whiskers. At 1 and 14 weeks, tissue was collected, and mRNA was extracted. mRNA expression was compared between 1 and 14 weeks using a high throughput multiplexed qRT-PCR. Pearson correlation coefficients were calculated between mRNA expression and signal-to-noise ratios at 14 weeks. At 14 weeks, a positive correlation between signal-to-noise ratio (SNR) and NeuN and GFAP mRNA expression was observed, indicating a relationship between recording strength and neuronal population, as well as reactive astrocyte activity. The inflammatory state around the electrode interface was evaluated using M1-like and M2-like markers. Expression for both M1-like and M2-like mRNA markers remained steady from 1 to 14 weeks. Anti-inflammatory markers, CD206 and CD163, however, demonstrated a significant positive correlation with SNR quality at 14 weeks. VE-cadherin, a marker for adherens junctions, and PDGFR-β, a marker for pericytes, both partial representatives of blood-brain barrier health, had a positive correlation with SNR at 14 weeks. Endothelial adhesion markers revealed a significant increase in expression at 14 weeks, while CD45, a pan-leukocyte marker, significantly decreased at 14 weeks. No significant correlation was found for either the endothelial adhesion or pan-leukocyte markers. A positive correlation between anti-inflammatory and blood-brain barrier health mRNA markers with electrophysiological efficacy of implanted intracortical electrodes has been demonstrated. These data reveal potential mechanisms for further evaluation to determine potential target mechanisms to improve

Association of chemerin mRNA expression in human epicardial adipose tissue with coronary atherosclerosis

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Wang Linjie

2011-10-01

Full Text Available Abstract Background Growing evidence suggests that epicardial adipose tissue (EAT may play a key role in the pathogenesis and development of coronary artery disease (CAD by producing several inflammatory adipokines. Chemerin, a novel adipokine, has been reported to be involved in regulating immune responses and glucolipid metabolism. Given these properties, chemerin may provide an interesting link between obesity, inflammation and atherosclerosis. In this study, we sought to determine the relationship of chemerin expression in EAT and the severity of coronary atherosclerosis in Han Chinese patients. Methods Serums and adipose tissue biopsies (epicardial and thoracic subcutaneous were obtained from CAD (n = 37 and NCAD (n = 16 patients undergoing elective cardiac surgery. Gensini score was used to assess the severity of CAD. Serum levels of chemerin, adiponectin and insulin were measured by ELISA. Chemerin protein expression in adipose tissue was detected by immunohistochemistry. The mRNA levels of chemerin, chemR23, adiponectin and TNF-alpha in adipose tissue were detected by RT-PCR. Results We found that EAT of CAD group showed significantly higher levels of chemerin and TNF-alpha mRNA, and significantly lower level of adiponectin mRNA than that of NCAD patients. In CAD group, significantly higher levels of chemerin mRNA and protein were observed in EAT than in paired subcutaneous adipose tissue (SAT, whereas such significant difference was not found in NCAD group. Chemerin mRNA expression in EAT was positively correlated with Gensini score (r = 0.365, P P P P P P P > 0.05. Conclusions The expressions of chemerin mRNA and protein are significantly higher in EAT from patients with CAD in Han Chinese patients. Furthermore, the severity of coronary atherosclerosis is positive correlated with the level of chemerin mRNA in EAT rather than its circulating level.

Parallel mRNA, proteomics and miRNA expression analysis in cell line models of the intestine.

Science.gov (United States)

O'Sullivan, Finbarr; Keenan, Joanne; Aherne, Sinead; O'Neill, Fiona; Clarke, Colin; Henry, Michael; Meleady, Paula; Breen, Laura; Barron, Niall; Clynes, Martin; Horgan, Karina; Doolan, Padraig; Murphy, Richard

2017-11-07

To identify miRNA-regulated proteins differentially expressed between Caco2 and HT-29: two principal cell line models of the intestine. Exponentially growing Caco-2 and HT-29 cells were harvested and prepared for mRNA, miRNA and proteomic profiling. mRNA microarray profiling analysis was carried out using the Affymetrix GeneChip Human Gene 1.0 ST array. miRNA microarray profiling analysis was carried out using the Affymetrix Genechip miRNA 3.0 array. Quantitative Label-free LC-MS/MS proteomic analysis was performed using a Dionex Ultimate 3000 RSLCnano system coupled to a hybrid linear ion trap/Orbitrap mass spectrometer. Peptide identities were validated in Proteome Discoverer 2.1 and were subsequently imported into Progenesis QI software for further analysis. Hierarchical cluster analysis for all three parallel datasets (miRNA, proteomics, mRNA) was conducted in the R software environment using the Euclidean distance measure and Ward's clustering algorithm. The prediction of miRNA and oppositely correlated protein/mRNA interactions was performed using TargetScan 6.1. GO biological process, molecular function and cellular component enrichment analysis was carried out for the DE miRNA, protein and mRNA lists via the Pathway Studio 11.3 Web interface using their Mammalian database. Differential expression (DE) profiling comparing the intestinal cell lines HT-29 and Caco-2 identified 1795 Genes, 168 Proteins and 160 miRNAs as DE between the two cell lines. At the gene level, 1084 genes were upregulated and 711 were downregulated in the Caco-2 cell line relative to the HT-29 cell line. At the protein level, 57 proteins were found to be upregulated and 111 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Finally, at the miRNAs level, 104 were upregulated and 56 downregulated in the Caco-2 cell line relative to the HT-29 cell line. Gene ontology (GO) analysis of the DE mRNA identified cell adhesion, migration and ECM organization, cellular lipid

Disease quantification in dermatology

DEFF Research Database (Denmark)

Greve, Tanja Maria; Kamp, Søren; Jemec, Gregor B E

2013-01-01

Accurate documentation of disease severity is a prerequisite for clinical research and the practice of evidence-based medicine. The quantification of skin diseases such as psoriasis currently relies heavily on clinical scores. Although these clinical scoring methods are well established and very ...

Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis

DEFF Research Database (Denmark)

Krakauer, Martin; Sorensen, P; Khademi, M

2008-01-01

BACKGROUND: Interferon (IFN)-beta therapy in multiple sclerosis (MS) has been suggested to promote a deviation from T lymphocyte production of pathogenic Th1 cytokines to less detrimental Th2 cytokines, but this is still controversial. We studied patterns of in vivo blood mononuclear cell (MNC...... of any Th1 or Th2 cytokines. The largest changes in cytokine mRNA levels occurred early (~9-12 h) after an IFN-beta injection. CONCLUSION: We found no evidence of a Th1- or Th2-mRNA-promoting effect of IFN-beta therapy. The therapeutic effect of IFN-beta is more likely attributable to the induction...

Coordinated Regulations of mRNA Synthesis and Decay during Cold Acclimation in Arabidopsis Cells.

KAUST Repository

Arae, Toshihiro; Isai, Shiori; Sakai, Akira; Mineta, Katsuhiko; Hirai, Masami Yokota; Suzuki, Yuya; Kanaya, Shigehiko; Yamaguchi, Junji; Naito, Satoshi; Chiba, Yukako

2017-01-01

stress in Arabidopsis cell cultures based on genome-wide analysis. In this mRNA decay array method, mRNA half-life measurements and microarray analyses were combined. In addition, temporal changes in the integrated value of transcription rates were

[Expression of heat shock protein 70 and its mRNA in career exposure to manganese].

Science.gov (United States)

Chen, Wenwen; Shao, Hua; Chi, Mingfeng; Zhang, Zhihu; Shan, Yongle; Zou, Wei

2015-10-01

To analyze the expression levels of heat shock protein70 (HSPs70) and HSPs70 mRNA in different exposure to manganese, and research the neuroprotective effect on the career exposure to manganese. From 2008 to 2009, with cross-sectional study design, and in a locomotive and rolling stock works, by stratified random sampling method, the exposed sample consisted of 180 welders from different welding shops and 100 unexposed in the last three years, non-welder controls with age-matched workers of similar socioeconomic status from the same industry. The control workers had not been exposed to neurotoxic chemicals. The mRNA expressions of four different metabolic enzyme were detected by SYBR Green I quantitative real-time polymerase chain reaction. The expression levels of the two enzymes mRNA in different exposure to manganese were analyzed. The expressions of HSPs70 were detected by Western blot. The concentration of air manganese was determined by GFAAS. The average concentration of 8 h time (8h-TWA) was used to express the level of individual exposure to manganese, according to the air manganese workplace occupational exposure limit (8h-TWA=0.15 mg/m3), the exposed group is divided into high exposed group (>0.15 mg/m3) and low exposure group (<0.15 mg/m3). The individuals exposed to manganese dose of exposed group ((0.25±0.31) mg/m3) was higher than the control group ((0.06±0.02) mg/m3) (t=6.15, P=0.001); individuals exposed to manganese dose of high exposure group for (0.42±0.34) mg/m3, which was higher than low exposure group (0.09±0.07) mg/m3 (t=9.80, P=0.001). HSPs70 mRNA and protein of exposure group (5.65±0.21, 3.26±0.15) were higher than the reference group (0.41±0.03, 1.32±0.12) (t=18.91, t=8.68, P=0.001). HSP70 mRNA and protein of high exposure group (6.48±0.37, 3.67±0.26) were higher than the low exposure group (5.15±0.23, 3.02±0.19) (t=3.24, t=2.01, P=0.003, P=0.043). The expression of peripheral blood lymphocytes HSPs70 level and HSPs70 mRNA

In situ localization of chalcone synthase mRNA in pea root nodule development.

NARCIS (Netherlands)

Yang, W.C.; Canter Cremers, H.C.J.; Hogendijk, P.; Katinakis, P.; Wijffelman, C.A.; Franssen, H.J.; Kammen, van A.; Bisseling, T.

1992-01-01

In this paper studies on the role of flavonoids in pea root nodule development are reported. Flavonoid synthesis was followed by localizing chalcone synthase (CHS) mRNA in infected pea roots and in root nodules. In a nodule primordium, CHS mRNA is present in all cells of the primordium. Therefore it

Predictive biophysical modeling and understanding of the dynamics of mRNA translation and its evolution

Science.gov (United States)

Zur, Hadas; Tuller, Tamir

2016-01-01

mRNA translation is the fundamental process of decoding the information encoded in mRNA molecules by the ribosome for the synthesis of proteins. The centrality of this process in various biomedical disciplines such as cell biology, evolution and biotechnology, encouraged the development of dozens of mathematical and computational models of translation in recent years. These models aimed at capturing various biophysical aspects of the process. The objective of this review is to survey these models, focusing on those based and/or validated on real large-scale genomic data. We consider aspects such as the complexity of the models, the biophysical aspects they regard and the predictions they may provide. Furthermore, we survey the central systems biology discoveries reported on their basis. This review demonstrates the fundamental advantages of employing computational biophysical translation models in general, and discusses the relative advantages of the different approaches and the challenges in the field. PMID:27591251

Comparison of Suitability of the Most Common Ancient DNA Quantification Methods.

Science.gov (United States)

Brzobohatá, Kristýna; Drozdová, Eva; Smutný, Jiří; Zeman, Tomáš; Beňuš, Radoslav

2017-04-01

Ancient DNA (aDNA) extracted from historical bones is damaged and fragmented into short segments, present in low quantity, and usually copurified with microbial DNA. A wide range of DNA quantification methods are available. The aim of this study was to compare the five most common DNA quantification methods for aDNA. Quantification methods were tested on DNA extracted from skeletal material originating from an early medieval burial site. The tested methods included ultraviolet (UV) absorbance, real-time quantitative polymerase chain reaction (qPCR) based on SYBR ® green detection, real-time qPCR based on a forensic kit, quantification via fluorescent dyes bonded to DNA, and fragmentary analysis. Differences between groups were tested using a paired t-test. Methods that measure total DNA present in the sample (NanoDrop ™ UV spectrophotometer and Qubit ® fluorometer) showed the highest concentrations. Methods based on real-time qPCR underestimated the quantity of aDNA. The most accurate method of aDNA quantification was fragmentary analysis, which also allows DNA quantification of the desired length and is not affected by PCR inhibitors. Methods based on the quantification of the total amount of DNA in samples are unsuitable for ancient samples as they overestimate the amount of DNA presumably due to the presence of microbial DNA. Real-time qPCR methods give undervalued results due to DNA damage and the presence of PCR inhibitors. DNA quantification methods based on fragment analysis show not only the quantity of DNA but also fragment length.

Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

Directory of Open Access Journals (Sweden)

Žel Jana

2006-08-01

Full Text Available Abstract Background Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Results Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was

Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

Science.gov (United States)

Cankar, Katarina; Štebih, Dejan; Dreo, Tanja; Žel, Jana; Gruden, Kristina

2006-01-01

Background Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Results Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary

Lowering the quantification limit of the QubitTM RNA HS assay using RNA spike-in.

Science.gov (United States)

Li, Xin; Ben-Dov, Iddo Z; Mauro, Maurizio; Williams, Zev

2015-05-06

RNA quantification is often a prerequisite for most RNA analyses such as RNA sequencing. However, the relatively low sensitivity and large sample consumption of traditional RNA quantification methods such as UV spectrophotometry and even the much more sensitive fluorescence-based RNA quantification assays, such as the Qubit™ RNA HS Assay, are often inadequate for measuring minute levels of RNA isolated from limited cell and tissue samples and biofluids. Thus, there is a pressing need for a more sensitive method to reliably and robustly detect trace levels of RNA without interference from DNA. To improve the quantification limit of the Qubit™ RNA HS Assay, we spiked-in a known quantity of RNA to achieve the minimum reading required by the assay. Samples containing trace amounts of RNA were then added to the spike-in and measured as a reading increase over RNA spike-in baseline. We determined the accuracy and precision of reading increases between 1 and 20 pg/μL as well as RNA-specificity in this range, and compared to those of RiboGreen(®), another sensitive fluorescence-based RNA quantification assay. We then applied Qubit™ Assay with RNA spike-in to quantify plasma RNA samples. RNA spike-in improved the quantification limit of the Qubit™ RNA HS Assay 5-fold, from 25 pg/μL down to 5 pg/μL while maintaining high specificity to RNA. This enabled quantification of RNA with original concentration as low as 55.6 pg/μL compared to 250 pg/μL for the standard assay and decreased sample consumption from 5 to 1 ng. Plasma RNA samples that were not measurable by the Qubit™ RNA HS Assay were measurable by our modified method. The Qubit™ RNA HS Assay with RNA spike-in is able to quantify RNA with high specificity at 5-fold lower concentration and uses 5-fold less sample quantity than the standard Qubit™ Assay.

Cytochrome P450 1B1 mRNA levels in peripheral blood cells and exposure to polycyclic aromatic hydrocarbons in Chinese coke oven workers

Energy Technology Data Exchange (ETDEWEB)

Hanaoka, Tomoyuki; Tsugane, Shoichiro [Epidemiology and Biostatistics Division, National Cancer Center Research Institute East, 6-5-1 Kashiwanoha, Kashiwa-shi, 277-8577 Chiba (Japan); Yamano, Yuko; Kagawa, Jun [Tokyo Womens' Medical University, 8-1 Kawadacho, Shinjuku-ku, 162-8666 Tokyo (Japan); Pan, Guowei; Zhang, Shujuan [Liaoning Provincial Center for Disease Prevention and Control, 42-1 Jixian Street, 110005 Shenyang (China); Hara, Kunio [Institute for Science of Labour, 2-8-14 Miyamae-ku, 216-8501 Kawasaki (Japan); Ichiba, Masayoshi; Zhang, Jiusong [Saga Medical School, 5-1 Nabeshima, Saga-shi, 849-8501 Saga (Japan); Liu, Tiefu; Li, Landi [Angang Public Health and Anti-epidemic Station Lishan District, 23 Shengoushi Yutian Street, 114034 Anshan (China); Takahashi, Ken [University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, 807-8555 Kitakyushu (Japan)

2002-09-16

Cytochrome P450 1B1 (CYP1B1) is induced through the Ah receptor and is involved in the activation of polycyclic aromatic hydrocarbons (PAHs). To determine the validity of a quantitative analysis of CYP1B1 mRNA in peripheral human blood cells for the estimation of PAH exposure, a real-time quantitative polymerase chain reaction method was used to measure the relative levels of CYP1B1 mRNA in 37 Chinese coke oven workers and 13 control workers. A large inter-individual difference in the levels was observed. The average level of the CYP1B1 mRNA in workers at the top work site, where the PAH exposure level from the coke ovens was highest, was significantly higher than in workers at the middle site (P<0.01) or the controls (P=0.02). A non-significant positive correlation was found between the CYP1B1 mRNA levels and urinary 1-hydroxypyrene (R=0.22, P=0.13), and a significant correlation between these mRNA levels and urinary cotinine (R=0.33, P=0.02). It was interesting that a significant positive correlation between CYP1B1 mRNA and 1-hydroxypyrene was observed in subjects with the Leu/Leu type of CYP1B1 Leu432Val polymorphism (R=0.33, P=0.02, n=38) and a non-significant correlation in subjects with the Leu/Val and Val/Val types (R=-0.36, P=0.25, n=12), although the number of subjects in this strata analysis was small. Our preliminary study suggests that PAH exposure in coke ovens and smoking maybe associated with CYP1B1 mRNA levels in peripheral blood cells although mRNA is generally unstable and could be expressed following exposure to other agents.

The Expression of mRNA LMP1 Epstein-Barr Virus from FFPE Tumour Biopsy: a Potential Biomarker of Nasopharyngeal Carcinoma Diagnosis

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Daniel Joko Wahyono

2017-07-01

Full Text Available Nasopharyngeal carcinoma (NPC is a multifactorial disease that is endemic geographically in the world. Indonesian population has a highly incidence rate that is 6.2/100,000 people year. The pathogenesis of NPC is more directly reflected by carcinoma-specific viral transcriptional activity at the site of primary tumour. Epstein-Barr virus (EBV infection in NPC is reflected by the expression of EBV latent and lytic gene. In fact, mRNA Latent Membrane Protein 1 (LMP1 EBV expression was an important latent infection biomarker. The aim of this study was to determine a potential use of relative expression of mRNA LMP1 EBV from formalin-fixed paraffin embedded (FFPE tumour biopsy in NPC as a tumour biomarker. This reseach design was a cross sectional study. The samples were the archived specimens of FFPE tumour biopsy from NPC WHO-3 patient which were collected from untreated patients from 2014 in the Department of Pathology Anatomy, Prof. dr. Margono Soekarjo Hospital, Purwokerto. The expression of mRNA LMP1 EBV expression was determined by RT-PCR technique. The positivity of mRNA LMP1 EBV expression was 51.9%, indicating a moderate positivity. The result proved that the expression of mRNA LMP1 EBV from FFPE NPC WHO-3 tumour biopsy was a potential biomarker of NPC diagnosis. The molecular methods would improved the management of NPC, particularly in the histopathological diagnosis of NPC.

Relationship between PPARα mRNA expression and mitochondrial respiratory function and ultrastructure of the skeletal muscle of patients with COPD.

Science.gov (United States)

Zhang, Jian-Qing; Long, Xiang-Yu; Xie, Yu; Zhao, Zhi-Huan; Fang, Li-Zhou; Liu, Ling; Fu, Wei-Ping; Shu, Jing-Kui; Wu, Jiang-Hai; Dai, Lu-Ming

2017-11-02

Peripheral muscle dysfunction is an important complication in patients with chronic obstructive pulmonary disease (COPD). The objective of this study was to explore the relationship between the levels of peroxisome proliferator-activated receptor α (PPARα) mRNA expression and the respiratory function and ultrastructure of mitochondria in the vastus lateralis of patients with COPD. Vastus lateralis biopsies were performed on 14 patients with COPD and 6 control subjects with normal lung function. PPARα mRNA levels in the muscle tissue were detected by real-time PCR. A Clark oxygen electrode was used to assess mitochondrial respiratory function. Mitochondrial number, fractional area in skeletal muscle cross-sections, and Z-line width were observed via transmission electron microscopy. The PPARα mRNA expression was significantly lower in COPD patients with low body mass index (BMIL) than in both COPD patients with normal body mass index (BMIN) and controls. Mitochondrial respiratory function (assessed by respiratory control ratio) was impaired in COPD patients, particularly in BMIL. Compared with that in the control group, mitochondrial number and fractional area were lower in the BMIL group, but were maintained in the BMIN group. Further, the Z-line became narrow in the BMIL group. PPARα mRNA expression was positively related to mitochondrial respiratory function and volume density. In COPD patients with BMIN, mitochondria volume density was maintained, while respiratory function decreased, whereas both volume density and respiratory function decreased in COPD patients with BMIL. PPARα mRNA expression levels are associated with decreased mitochondrial respiratory function and volume density, which may contribute to muscle dysfunction in COPD patients.

Exploring Heterogeneous Multicore Architectures for Advanced Embedded Uncertainty Quantification.

Energy Technology Data Exchange (ETDEWEB)

Phipps, Eric T.; Edwards, Harold C.; Hu, Jonathan J.

2014-09-01

We explore rearrangements of classical uncertainty quantification methods with the aim of achieving higher aggregate performance for uncertainty quantification calculations on emerging multicore and manycore architectures. We show a rearrangement of the stochastic Galerkin method leads to improved performance and scalability on several computational architectures whereby un- certainty information is propagated at the lowest levels of the simulation code improving memory access patterns, exposing new dimensions of fine grained parallelism, and reducing communica- tion. We also develop a general framework for implementing such rearrangements for a diverse set of uncertainty quantification algorithms as well as computational simulation codes to which they are applied.

Effect of low-dose irradiation on expression of mRNA and protein. Pt.1. Induction of thioredoxin as radioprotective protein in human lymphocytes

International Nuclear Information System (INIS)

Hoshi, Yuko; Tanooka, Hiroshi; Wakasugi, Hiro; Miyasaki, Kunihisa

1997-01-01

To elucidate the mechanism of hormetic effect by low-dose ionizing radiation, we studied the expression of the thioredoxin (TRX) gene in human lymphocytes after irradiation. TRX is a radioprotector and a key protein regulating cellular functions through redox reaction. The major results obtained were as follows; (1) The peaks of TRX mRNA expression and protein synthesis in human lymphocytes appeared 6-8 hr after irradiation with 25cGy. (2) At 6 hr after irradiation, the optimum dose for induction of TRX mRNA and TRX protein in human lymphocytes appeared to be 25-50cGy. (3) Induction of expression TRX mRNA had individual variations about twice. (4) Lymphocytes prepared from fresh venous blood showed the lowest TRX mRNA level in other cells such a Jurkat cells, lymphocytes stimulated for now with IL-2 and CD3 and the immortalized cell line 1G8. (5) The optimal dose and time course of induction of TRX by low-dose radiation suggest that TRX is related to the radio-adaptive response. (author)

GMO quantification: valuable experience and insights for the future.

Science.gov (United States)

Milavec, Mojca; Dobnik, David; Yang, Litao; Zhang, Dabing; Gruden, Kristina; Zel, Jana

2014-10-01

Cultivation and marketing of genetically modified organisms (GMOs) have been unevenly adopted worldwide. To facilitate international trade and to provide information to consumers, labelling requirements have been set up in many countries. Quantitative real-time polymerase chain reaction (qPCR) is currently the method of choice for detection, identification and quantification of GMOs. This has been critically assessed and the requirements for the method performance have been set. Nevertheless, there are challenges that should still be highlighted, such as measuring the quantity and quality of DNA, and determining the qPCR efficiency, possible sequence mismatches, characteristics of taxon-specific genes and appropriate units of measurement, as these remain potential sources of measurement uncertainty. To overcome these problems and to cope with the continuous increase in the number and variety of GMOs, new approaches are needed. Statistical strategies of quantification have already been proposed and expanded with the development of digital PCR. The first attempts have been made to use new generation sequencing also for quantitative purposes, although accurate quantification of the contents of GMOs using this technology is still a challenge for the future, and especially for mixed samples. New approaches are needed also for the quantification of stacks, and for potential quantification of organisms produced by new plant breeding techniques.

Improved Method for PD-Quantification in Power Cables

DEFF Research Database (Denmark)

Holbøll, Joachim T.; Villefrance, Rasmus; Henriksen, Mogens

1999-01-01

n this paper, a method is described for improved quantification of partial discharges(PD) in power cables. The method is suitable for PD-detection and location systems in the MHz-range, where pulse attenuation and distortion along the cable cannot be neglected. The system transfer function...... was calculated and measured in order to form basis for magnitude calculation after each measurements. --- Limitations and capabilities of the method will be discussed and related to relevant field applications of high frequent PD-measurements. --- Methods for increased signal/noise ratio are easily implemented...

Characterization of the ptr5+ gene involved in nuclear mRNA export in fission yeast

International Nuclear Information System (INIS)

Watanabe, Nobuyoshi; Ikeda, Terumasa; Mizuki, Fumitaka; Tani, Tokio

2012-01-01

Highlights: ► We cloned the ptr5 + gene involved in nuclear mRNA export in fission yeast. ► The ptr5 + gene was found to encode nucleoporin 85 (Nup85). ► Seh1p and Mlo3p are multi-copy suppressors for the ptr5 mutation. ► Ptr5p/Nup85p functions in nuclear mRNA export through the mRNA export factor Rae1p. ► Ptr5p/Nup85p interacts genetically with pre-mRNA splicing factors. -- Abstract: To analyze the mechanisms of mRNA export from the nucleus to the cytoplasm, we have isolated eleven mutants, ptr [poly(A) + RNA transport] 1 to 11, which accumulate poly(A) + RNA in the nucleus at a nonpermissive temperature in Schizosaccharomyces pombe. Of those, the ptr5–1 mutant shows dots- or a ring-like accumulation of poly(A) + RNA at the nuclear periphery after shifting to the nonpermissive temperature. We cloned the ptr5 + gene and found that it encodes a component of the nuclear pore complex (NPC), nucleoporin 85 (Nup85). The ptr5–1 mutant shows no defects in protein transport, suggesting the specific involvement of Ptr5p/Nup85p in nuclear mRNA export in S. pombe. We identified Seh1p, a nucleoporin interacting with Nup85p, an mRNA-binding protein Mlo3p, and Sac3p, a component of the TREX-2 complex involved in coupling of nuclear mRNA export with transcription, as multi-copy suppressors for the ptr5–1 mutation. In addition, we found that the ptr5–1 mutation is synthetically lethal with a mutation of the mRNA export factor Rae1p, and that the double mutant exaggerates defective nuclear mRNA export, suggesting that Ptr5p/Nup85p is involved in nuclear mRNA export through Rae1p. Interestingly, the ptr5–1 mutation also showed synthetic effects with several prp pre-mRNA splicing mutations, suggesting a functional linkage between the NPCs and the splicing apparatus in the yeast nucleus.

The Quantification Process for the PRiME-U34i

International Nuclear Information System (INIS)

Hwang, Mee-Jeong; Han, Sang-Hoon; Yang, Joon-Eon

2006-01-01

In this paper, we introduce the quantification process for the PRIME-U34i, which is the merged model of ETs (Event Trees) and FTs (Fault Trees) for the level 1 internal PSA of UCN 3 and 4. PRiME-U34i has one top event. Therefore, the quantification process is changed to a simplified method when compared to the past one. In the past, we used the text file called a user file to control the quantification process. However, this user file is so complicated that it is difficult for a non-expert to understand it. Moreover, in the past PSA, ET and FT were separated but in PRiMEU34i, ET and FT were merged together. Thus, the quantification process is different. This paper is composed of five sections. In section 2, we introduce the construction of the one top model. Section 3 shows the quantification process used in the PRiME-U34i. Section 4 describes the post processing. Last section is the conclusions

MiR-520b suppresses proliferation of hepatoma cells through targeting ten-eleven translocation 1 (TET1) mRNA

Energy Technology Data Exchange (ETDEWEB)

Zhang, Weiying; Lu, Zhanping; Gao, Yuen [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China); Ye, Lihong [State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin (China); Song, Tianqiang, E-mail: tjchi@hotmai.com [Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China)

2015-05-08

Accumulating evidence indicates that microRNAs are able to act as oncogenes or tumor suppressor genes in human cancer. We previously reported that miR-520b was down-regulated in hepatocellular carcinoma (HCC) and its deregulation was involved in hepatocarcinogenesis. In the present study, we report that miR-520b suppresses cell proliferation in HCC through targeting the ten-eleven translocation 1 (TET1) mRNA. Notably, we identified that miR-520b was able to target 3′-untranslated region (3′UTR) of TET1 mRNA by luciferase reporter gene assays. Then, we revealed that miR-520b was able to reduce the expression of TET1 at the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blotting analysis. In terms of function, 5-ethynyl-2-deoxyuridine (EdU) incorporation and colony formation assays demonstrated that the forced miR-520b expression remarkably inhibited proliferation of hepatoma cells, but TET1 overexpression could rescue the inhibition of cell proliferation mediated by miR-520b. Furthermore, anti-miR-520b enhanced proliferation of hepatoma cells, whereas silencing of TET1 abolished anti-miR-520b-induced acceleration of cell proliferation. Then, we validated that the expression levels of miR-520b were negatively related to those of TET1 mRNA in clinical HCC tissues. Thus, we conclude that miR-520b depresses proliferation of liver cancer cells through targeting 3′UTR of TET1 mRNA. Our finding provides new insights into the mechanism of hepatocarcinogenesis. - Highlights: • TET1 is a novel target gene of miR-520b. • TET1 is upregulated in clinical HCC tissues. • MiR-520b is negatively correlated with TET1 in clinical HCC tissues. • MiR-520b depresses the proliferation of HCC cells through targeting TET1 mRNA.

Regulation and dysregulation of vitellogenin mRNA accumulation in daphnids (Daphnia magna)

Energy Technology Data Exchange (ETDEWEB)

Hannas, Bethany R.; Wang, Ying H.; Thomson, Susanne; Kwon, Gwijun; Hong, Li [Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695-7633 (United States); LeBlanc, Gerald A., E-mail: Gerald_LeBlanc@ncsu.edu [Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695-7633 (United States)

2011-01-25

The induction of vitellogenin in oviparous vertebrates has become the gold standard biomarker of exposure to estrogenic chemicals in the environment. This biomarker of estrogen exposure also has been used in arthropods, however, little is known of the factors that regulate the expression of vitellogenin in these organisms. We investigated changes in accumulation of mRNA products of the vitellogenin gene Vtg2 in daphnids (Daphnia magna) exposed to a diverse array of chemicals. We further evaluated the involvement of hormonal factors in the regulation of vitellogenin expression that may be targets of xenobiotic chemicals. Expression of the Vtg2 gene was highly responsive to exposure to various chemicals with an expression range spanning approximately four orders of magnitude. Chemicals causing the greatest induction were piperonyl butoxide, chlordane, 4-nonylphenol, cadmium, and chloroform. Among these, only 4-nonylphenol is recognized to be estrogenic. Exposure to several chemicals also suppressed Vtg2 mRNA levels, as much as 100-fold. Suppressive chemicals included cyproterone acetate, acetone, triclosan, and atrazine. Exposure to the estrogens diethylstilbestrol and bisphenol A had little effect on vitellogenin mRNA levels further substantiating that these genes are not induced by estrogen exposure. Exposure to the potent ecdysteroids 20-hydroxyecdysone and ponasterone A revealed that Vtg2 was subject to strong suppressive control by these hormones. Vtg2 mRNA levels were not significantly affected from exposure to several juvenoid hormones. Results indicate that ecdysteroids are suppressors of vitellogenin gene expression and that vitellogenin mRNA levels can be elevated or suppressed in daphnids by xenobiotics that elicit antiecdysteroidal or ecdysteroidal activity, respectively. Importantly, daphnid Vtg2 is not elevated in response to estrogenic activity.

Regulation and dysregulation of vitellogenin mRNA accumulation in daphnids (Daphnia magna)

International Nuclear Information System (INIS)

Hannas, Bethany R.; Wang, Ying H.; Thomson, Susanne; Kwon, Gwijun; Li Hong; LeBlanc, Gerald A.

2011-01-01

The induction of vitellogenin in oviparous vertebrates has become the gold standard biomarker of exposure to estrogenic chemicals in the environment. This biomarker of estrogen exposure also has been used in arthropods, however, little is known of the factors that regulate the expression of vitellogenin in these organisms. We investigated changes in accumulation of mRNA products of the vitellogenin gene Vtg2 in daphnids (Daphnia magna) exposed to a diverse array of chemicals. We further evaluated the involvement of hormonal factors in the regulation of vitellogenin expression that may be targets of xenobiotic chemicals. Expression of the Vtg2 gene was highly responsive to exposure to various chemicals with an expression range spanning approximately four orders of magnitude. Chemicals causing the greatest induction were piperonyl butoxide, chlordane, 4-nonylphenol, cadmium, and chloroform. Among these, only 4-nonylphenol is recognized to be estrogenic. Exposure to several chemicals also suppressed Vtg2 mRNA levels, as much as 100-fold. Suppressive chemicals included cyproterone acetate, acetone, triclosan, and atrazine. Exposure to the estrogens diethylstilbestrol and bisphenol A had little effect on vitellogenin mRNA levels further substantiating that these genes are not induced by estrogen exposure. Exposure to the potent ecdysteroids 20-hydroxyecdysone and ponasterone A revealed that Vtg2 was subject to strong suppressive control by these hormones. Vtg2 mRNA levels were not significantly affected from exposure to several juvenoid hormones. Results indicate that ecdysteroids are suppressors of vitellogenin gene expression and that vitellogenin mRNA levels can be elevated or suppressed in daphnids by xenobiotics that elicit antiecdysteroidal or ecdysteroidal activity, respectively. Importantly, daphnid Vtg2 is not elevated in response to estrogenic activity.

Cyclic-AMP mediated regulation of ABCB mRNA expression in mussel haemocytes.

Directory of Open Access Journals (Sweden)

Silvia Franzellitti

Full Text Available BACKGROUND: The multixenobiotic resistance system (MXR allows aquatic organisms to cope with their habitat despite high pollution levels by over-expressing membrane and intracellular transporters, including the P-glycoprotein (Pgp. In mammals transcription of the ABCB1 gene encoding Pgp is under cAMP/PKA-mediated regulation; whether this is true in mollusks is not fully clarified. METHODOLOGY/PRINCIPAL FINDINGS: cAMP/PKA regulation and ABCB mRNA expression were assessed in haemocytes from Mediterranean mussels (Mytilus galloprovincialis exposed in vivo for 1 week to 0.3 ng/L fluoxetine (FX alone or in combination with 0.3 ng/L propranolol (PROP. FX significantly decreased cAMP levels and PKA activity, and induced ABCB mRNA down-regulation. FX effects were abolished in the presence of PROP. In vitro experiments using haemocytes treated with physiological agonists (noradrenaline and serotonin and pharmacological modulators (PROP, forskolin, dbcAMP, and H89 of the cAMP/PKA system were performed to obtain clear evidence about the involvement of the signaling pathway in the transcriptional regulation of ABCB. Serotonin (5-HT decreased cAMP levels, PKA activity and ABCB mRNA expression but increased the mRNA levels for a putative 5-HT1 receptor. Interestingly, 5-HT1 was also over-expressed after in vivo exposures to FX. 5-HT effects were counteracted by PROP. Forskolin and dbcAMP increased PKA activity as well as ABCB mRNA expression; the latter effect was abolished in the presence of the PKA inhibitor H89. CONCLUSIONS: This study provides the first direct evidence for the cAMP/PKA-mediated regulation of ABCB transcription in mussels.

m-RNA mammaglobin expression in metastatic breast cancer patient at Medan city, Indonesia

Science.gov (United States)

Rimbun, S.; Siregar, Y.

2018-03-01

Breast cancer is the most common causes of women’s death in the world. Metastatic spread presents a major clinical problem in about 30% of the patients. The study aims to investigate the clinical reliability of mammaglobin mRNA as a marker of circulating cancer cells in breast cancer patients. The positivity of blood was analyzed in relation to clinical and pathological characteristics. This study was on 29 breast cancer patients (13 metastatic, 16 non- metastatic patients), where28 were invasive intraductal carcinoma type and 1 was invasive lobular carcinoma type. Breast cancer patients were according to the histologic grade into grade I (7 patients),grade II (6 patients) and grade III (15 patients). All individuals included in this study were subjected to detection of mammaglobin m-RNA of circulating tumor cells in peripheral blood using RT-PCR technique. Positivity for mammaglobin in blood samples was in 38% of patients with metastatic but not in the non-metastatic patients. The presence of mammaglobin correlated with metastatic tumor (P = 0.011). Mammaglobin overexpression in breast tissue was significantly positive in low-grade tumors (I and II).

Elemental quantification of airborne particulate matter in Bandung and Lembang area

International Nuclear Information System (INIS)

Sutisna; Achmad Hidayat; Dadang Supriatna

2004-01-01

ELEMENTAL QUANTIFICATION OF AIRBORNE PARTICULATE MATTER IN BANDUNG AND LEMBANG REGION: The contaminated airborne particulates by toxic gases and elements have a potential affect to the human health. Some toxic elements related to air pollution have carcinogenic affect. The quantification of those elements is important to monitor a level of pollutant contained in the airborne particulate. The aim of this work is to analyze the air particulate sample using instrumental neutron activation analysis and other related technique. Two sampling points of Bandung and Lembang that represent and urban and rural area respectively have been chosen to collect the air particulate sample. The samplings were carried out using Gent Stacked Filter Unit Sampler for 24 hours, and two cellulose filters of 8 μm and 0.45 μm pore size were used. Trace elements in the sample collected were determined using NAA based on a comparative method. Elemental distribution on PM 2.5 and PM 10 fraction of airborne particulate was analyzed, the enrichment factor was calculated using Al as reference elements, and the black carbons contents were determined using FEL Smoke Stain Reflectometer analyzed. The results are presented and discussed. (author)

Phase quantification in nanobainite via magnetic measurements and X-ray diffraction

Energy Technology Data Exchange (ETDEWEB)

Solano-Alvarez, W., E-mail: ws298@cam.ac.uk [Department of Materials Science and Metallurgy, University of Cambridge (United Kingdom); Abreu, H.F.G. [Departamento de Engenharia Metalúrgica e de Materiais, Universidade Federal do Ceará, Fortaleza (Brazil); Silva, M.R. da [Instituto de Física e Química, Universidade Federal de Itajubá, Itajubá, Minas Gerais (Brazil); Peet, M.J. [Department of Materials Science and Metallurgy, University of Cambridge (United Kingdom)

2015-03-15

Accurate phase quantification of nanostructured bainitic steel is of importance because of the nature of its percolating structure that controls many of its mechanical properties. X-ray diffraction is the technique of choice for such analysis, but magnetic methods can be more rapid and less sensitive to defect structures. In this study, the phase volume fractions measured using both of these techniques for the specific mixtures associated with nanostructured bainite have been compared and contrasted. An expression which relates the volume fraction and the saturation magnetization is obtained and its form is found to be consistent with previous work done on duplex stainless steels and TRIP steels. The fitting constants used in many of such analyses vary significantly so an attempt is made to rationalize the differences by considering the factors that determine the intrinsic saturation magnetization of ferrite. - Author-Highlights: • Magnetic phase quantification of nanobainite is presented for the first time. • Results are compared with x-ray diffraction. • Expression obtained that relates ferrite fraction and saturation magnetization. • Equation derived to calculate intrinsic saturation magnetization of ferrites. • These values agree with experimental data of the literature.

Enhanced Delivery and Potency of Self-Amplifying mRNA Vaccines by Electroporation in Situ

Directory of Open Access Journals (Sweden)

Kaustuv Banerjee

2013-08-01

Full Text Available Nucleic acid-based vaccines such as viral vectors, plasmid DNA (pDNA, and mRNA are being developed as a means to address limitations of both live-attenuated and subunit vaccines. DNA vaccines have been shown to be potent in a wide variety of animal species and several products are now licensed for commercial veterinary but not human use. Electroporation delivery technologies have been shown to improve the generation of T and B cell responses from synthetic DNA vaccines in many animal species and now in humans. However, parallel RNA approaches have lagged due to potential issues of potency and production. Many of the obstacles to mRNA vaccine development have recently been addressed, resulting in a revival in the use of non-amplifying and self-amplifying mRNA for vaccine and gene therapy applications. In this paper, we explore the utility of EP for the in vivo delivery of large, self-amplifying mRNA, as measured by reporter gene expression and immunogenicity of genes encoding HIV envelope protein. These studies demonstrated that EP delivery of self-amplifying mRNA elicited strong and broad immune responses in mice, which were comparable to those induced by EP delivery of pDNA.

Matrin 3 binds and stabilizes mRNA.

Directory of Open Access Journals (Sweden)

Maayan Salton

Full Text Available Matrin 3 (MATR3 is a highly conserved, inner nuclear matrix protein with two zinc finger domains and two RNA recognition motifs (RRM, whose function is largely unknown. Recently we found MATR3 to be phosphorylated by the protein kinase ATM, which activates the cellular response to double strand breaks in the DNA. Here, we show that MATR3 interacts in an RNA-dependent manner with several proteins with established roles in RNA processing, and maintains its interaction with RNA via its RRM2 domain. Deep sequencing of the bound RNA (RIP-seq identified several small noncoding RNA species. Using microarray analysis to explore MATR3's role in transcription, we identified 77 transcripts whose amounts depended on the presence of MATR3. We validated this finding with nine transcripts which were also bound to the MATR3 complex. Finally, we demonstrated the importance of MATR3 for maintaining the stability of several of these mRNA species and conclude that it has a role in mRNA stabilization. The data suggest that the cellular level of MATR3, known to be highly regulated, modulates the stability of a group of gene transcripts.

Self-sampling with HPV mRNA analyses from vagina and urine compared with cervical samples.

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Asciutto, Katrin Christine; Ernstson, Avalon; Forslund, Ola; Borgfeldt, Christer

2018-04-01

In order to increase coverage in the organized cervical screening program, self-sampling with HPV analyses has been suggested. The aim was to compare human papillomavirus (HPV) mRNA detection in vaginal and urine self-collected samples with clinician-taken cervical samples and the corresponding clinician-taken histological specimens. Self-collected vaginal, urine and clinician-taken cervical samples were analyzed from 209 women with the Aptima mRNA assay (Hologic Inc, MA, USA). Cervical cytology, colposcopy, biopsy and/or the loop electrosurgical excision procedure (LEEP) were performed in every examination. The sensitivity of the HPV mRNA test in detecting high-grade squamous intraepithelial lesions (HSIL)/adenocarcinoma in situ (AIS)/cancer cases was as follows: for the vaginal self-samples 85.5% (95% CI; 75.0-92.8), the urinary samples 44.8% (95% CI; 32.6-57.4), and for routine cytology 81.7% (95% CI; 70.7-89.9). For the clinician-taken cervical HPV samples the sensitivity of the HPV mRNA test in detecting HSIL/AIS/cancer was 100.0% (95% CI; 94.9-100.0). The specificity of the HPV mRNA was similar for the clinician-taken cervical HPV samples and the self-samples: 49.0% vs. 48.1%. The urinary HPV samples had a specificity of 61.9% and cytology had a specificity of 93.3%. The sensitivity of the Aptima HPV mRNA test in detecting HSIL/AIS/cancer from vaginal self-samples was similar to that of routine cytology. The Aptima HPV mRNA vaginal self-sampling analysis may serve as a complement in screening programs. Copyright © 2018 Elsevier B.V. All rights reserved.

Expression and clinicopathological significance of Mel-18 and Bmi-1 mRNA in gastric carcinoma.

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Lu, You-Wei; Li, Jin; Guo, Wei-Jian

2010-11-08

The Polycomb group (PcG) genes are a class of regulators responsible for maintaining homeotic gene expression throughout cell division. PcG expression is deregulated in some types of human cancer. Both Bmi-1 and Mel-18 are of the key PcG proteins. We investigate the expression and clinicopathological roles of Mel-18 and Bmi-1 mRNA in gastric cancer. The expression of Mel-18 and Bmi-1 in a series of 71 gastric cancer tissues and paired normal mucosal tissues distant from the tumorous lesion was assayed by quantitative real time RT-PCR. The correlation between Mel-18 and Bmi-1 mRNA expression, and between Mel-18 or Bmi-1 mRNA level and clinicopathological characteristics were analyzed. Expression of Mel-18 and Bmi-1 genes was variably detected, but overexpression of Bmi-1 mRNA and decreased expression of Mel-18 mRNA were the most frequent alteration. In addition, the expression of Bmi-1 and Mel-18 mRNA inversely correlates in gastric tumors. Moreover, a significant positive correlation between Bmi-1 overexpression and tumor size, depth of invasion, or lymph node metastasis, and a significant negative correlation between Mel-18 low-expression with lymph node metastasis or the clinical stage were observed. Our data suggest that Mel-18 and Bmi-1 may play crucial but opposite roles in gastric cancer. Decreased Mel-18 and increased Bmi-1 mRNA expression was associated with the carcinogenesis and progression of gastric cancer. It is possible to list Bmi-1 and Mel-18 as biomarkers for predicting the prognosis of gastric cancer.

La-related protein 1 (LARP1) represses terminal oligopyrimidine (TOP) mRNA translation downstream of mTOR complex 1 (mTORC1)

DEFF Research Database (Denmark)

Fonseca, Bruno; Zakaria, Chadi; Jia, J J

2015-01-01

is incompletely understood. Here, we report that LARP1 functions as a key repressor of TOP mRNA translation downstream of mTORC1. Our data show the following: (i) LARP1 associates with mTORC1 via RAPTOR; (ii) LARP1 interacts with TOP mRNAs in an mTORC1-dependent manner; (iii) LARP1 binds the 5′TOP motif...

Triage of Women with Low-Grade Cervical Lesions - HPV mRNA Testing versus Repeat Cytology

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Sørbye, Sveinung Wergeland; Arbyn, Marc; Fismen, Silje; Gutteberg, Tore Jarl; Mortensen, Elin Synnøve

2011-01-01

Background In Norway, women with low-grade squamous intraepithelial lesions (LSIL) are followed up after six months in order to decide whether they should undergo further follow-up or be referred back to the screening interval of three years. A high specificity and positive predictive value (PPV) of the triage test is important to avoid unnecessary diagnostic and therapeutic procedures. Materials and Methods At the University Hospital of North Norway, repeat cytology and the HPV mRNA test PreTect HPV-Proofer, detecting E6/E7 mRNA from HPV types 16, 18, 31, 33 and 45, are used in triage of women with ASC-US and LSIL. In this study, women with LSIL cytology in the period 2005–2008 were included (n = 522). Two triage methods were evaluated in two separate groups: repeat cytology only (n = 225) and HPV mRNA testing in addition to repeat cytology (n = 297). Histologically confirmed cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) was used as the study endpoint. Results Of 522 women with LSIL, 207 had biopsies and 125 of them had CIN2+. The sensitivity and specificity of repeat cytology (ASC-US or worse) were 85.7% (95% confidence interval (CI): 72.1, 92.2) and 54.4 % (95% CI: 46.9, 61.9), respectively. The sensitivity and specificity of the HPV mRNA test were 94.2% (95% CI: 88.7, 99.7) and 86.0% (95% CI: 81.5, 90.5), respectively. The PPV of repeat cytology was 38.4% (95% CI: 29.9, 46.9) compared to 67.0% (95% CI: 57.7, 76.4) of the HPV mRNA test. Conclusion HPV mRNA testing was more sensitive and specific than repeat cytology in triage of women with LSIL cytology. In addition, the HPV mRNA test showed higher PPV. These data indicate that the HPV mRNA test is a better triage test for women with LSIL than repeat cytology. PMID:21918682

Artifacts Quantification of Metal Implants in MRI

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Vrachnis, I. N.; Vlachopoulos, G. F.; Maris, T. G.; Costaridou, L. I.

2017-11-01

The presence of materials with different magnetic properties, such as metal implants, causes distortion of the magnetic field locally, resulting in signal voids and pile ups, i.e. susceptibility artifacts in MRI. Quantitative and unbiased measurement of the artifact is prerequisite for optimization of acquisition parameters. In this study an image gradient based segmentation method is proposed for susceptibility artifact quantification. The method captures abrupt signal alterations by calculation of the image gradient. Then the artifact is quantified in terms of its extent by an automated cross entropy thresholding method as image area percentage. The proposed method for artifact quantification was tested in phantoms containing two orthopedic implants with significantly different magnetic permeabilities. The method was compared against a method proposed in the literature, considered as a reference, demonstrating moderate to good correlation (Spearman’s rho = 0.62 and 0.802 in case of titanium and stainless steel implants). The automated character of the proposed quantification method seems promising towards MRI acquisition parameter optimization.

A Probabilistic Framework for Peptide and Protein Quantification from Data-Dependent and Data-Independent LC-MS Proteomics Experiments

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Richardson, Keith; Denny, Richard; Hughes, Chris; Skilling, John; Sikora, Jacek; Dadlez, Michał; Manteca, Angel; Jung, Hye Ryung; Jensen, Ole Nørregaard; Redeker, Virginie; Melki, Ronald; Langridge, James I.; Vissers, Johannes P.C.

2013-01-01

A probability-based quantification framework is presented for the calculation of relative peptide and protein abundance in label-free and label-dependent LC-MS proteomics data. The results are accompanied by credible intervals and regulation probabilities. The algorithm takes into account data uncertainties via Poisson statistics modified by a noise contribution that is determined automatically during an initial normalization stage. Protein quantification relies on assignments of component peptides to the acquired data. These assignments are generally of variable reliability and may not be present across all of the experiments comprising an analysis. It is also possible for a peptide to be identified to more than one protein in a given mixture. For these reasons the algorithm accepts a prior probability of peptide assignment for each intensity measurement. The model is constructed in such a way that outliers of any type can be automatically reweighted. Two discrete normalization methods can be employed. The first method is based on a user-defined subset of peptides, while the second method relies on the presence of a dominant background of endogenous peptides for which the concentration is assumed to be unaffected. Normalization is performed using the same computational and statistical procedures employed by the main quantification algorithm. The performance of the algorithm will be illustrated on example data sets, and its utility demonstrated for typical proteomics applications. The quantification algorithm supports relative protein quantification based on precursor and product ion intensities acquired by means of data-dependent methods, originating from all common isotopically-labeled approaches, as well as label-free ion intensity-based data-independent methods. PMID:22871168

The role of PET quantification in cardiovascular imaging.

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Slomka, Piotr; Berman, Daniel S; Alexanderson, Erick; Germano, Guido

2014-08-01

Positron Emission Tomography (PET) has several clinical and research applications in cardiovascular imaging. Myocardial perfusion imaging with PET allows accurate global and regional measurements of myocardial perfusion, myocardial blood flow and function at stress and rest in one exam. Simultaneous assessment of function and perfusion by PET with quantitative software is currently the routine practice. Combination of ejection fraction reserve with perfusion information may improve the identification of severe disease. The myocardial viability can be estimated by quantitative comparison of fluorodeoxyglucose ( 18 FDG) and rest perfusion imaging. The myocardial blood flow and coronary flow reserve measurements are becoming routinely included in the clinical assessment due to enhanced dynamic imaging capabilities of the latest PET/CT scanners. Absolute flow measurements allow evaluation of the coronary microvascular dysfunction and provide additional prognostic and diagnostic information for coronary disease. Standard quantitative approaches to compute myocardial blood flow from kinetic PET data in automated and rapid fashion have been developed for 13 N-ammonia, 15 O-water and 82 Rb radiotracers. The agreement between software methods available for such analysis is excellent. Relative quantification of 82 Rb PET myocardial perfusion, based on comparisons to normal databases, demonstrates high performance for the detection of obstructive coronary disease. New tracers, such as 18 F-flurpiridaz may allow further improvements in the disease detection. Computerized analysis of perfusion at stress and rest reduces the variability of the assessment as compared to visual analysis. PET quantification can be enhanced by precise coregistration with CT angiography. In emerging clinical applications, the potential to identify vulnerable plaques by quantification of atherosclerotic plaque uptake of 18 FDG and 18 F-sodium fluoride tracers in carotids, aorta and coronary arteries

60Co γ-irradiation enhances expression of GAP-43 mRNA in rat brain

International Nuclear Information System (INIS)

Su Bingyin; Cai Wenqin; Zhang Chenggang

2001-01-01

Objective: To study the relationship between the expression of GAP-43 mRNA and nerve regeneration in rat brain after 60 Co γ-irradiation. Methods: Wistar rats were subjected to whole-body irradiation with 8 Gy 60 Co γ-rays. The expression of GAP-43 was detected by in situ hybridization histochemistry using Dig-cRNA probe. Results: It was found that the expression of GAP-43 mRNA increased in the cerebral cortex, caudate, putamen, globus pallidum, thalamus and hypothalamus one week after 8 Gy 60 Co γ-irradiation. The peak of GAP-43 mRNA expression was observed in the fourth week and then began to decrease but still remained at a higher than normal level. However, it decreased to a low level after 7 weeks. Conclusion: Enhanced expression of GAP-43 mRNA after 60 Co γ-irradiation in rat brain is associated with nerve regeneration and reconstruction of synapse

Regulation of elastin synthesis in developing sheep nuchal ligament by elastin mRNA levels

International Nuclear Information System (INIS)

Davidson, J.M.; Smith, K.; Shibahara, S.; Tolstoshev, P.; Crystal, R.G.

1982-01-01

Levels of elastin production in explant culture of fetal sheep nuchal ligament and corresponding levels of translatable elastin mRNA were determined in parallel studies during a period of rapid growth of the embryo. The identity of the explant culture and cell-free proucts was confirmed by peptide mapping, immunoprecipitation, and the characteristic lack of histidine and methionine. Elastin production was quantitated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and radioimmune precipitation. The translation products could be labeled with methionine only when NH 2 -terminally donated as f-Met-tRNA/sup Met//sub f/. Explant cultures showed a large rise in elastin production from 70 days after conception to 150 days after conception. Cell free translation of RNA demonstrated a parallel in elastin mRNA levels and in elastin mRNA per cell. It appears, therefore, that the marked emphasis the differentiating muchal ligament places on elastin production is modulated, at least in part, by the quantities of available elastin in mRNA

Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells.

Science.gov (United States)

Van Tendeloo, V F; Ponsaerts, P; Lardon, F; Nijs, G; Lenjou, M; Van Broeckhoven, C; Van Bockstaele, D R; Berneman, Z N

2001-07-01

Designing effective strategies to load human dendritic cells (DCs) with tumor antigens is a challenging approach for DC-based tumor vaccines. Here, a cytoplasmic expression system based on mRNA electroporation to efficiently introduce tumor antigens into DCs is described. Preliminary experiments in K562 cells using an enhanced green fluorescent protein (EGFP) reporter gene revealed that mRNA electroporation as compared with plasmid DNA electroporation showed a markedly improved transfection efficiency (89% versus 40% EGFP(+) cells, respectively) and induced a strikingly lower cell toxicity (15% death rate with mRNA versus 51% with plasmid DNA). Next, mRNA electroporation was applied for nonviral transfection of different types of human DCs, including monocyte-derived DCs (Mo-DCs), CD34(+) progenitor-derived DCs (34-DCs) and Langerhans cells (34-LCs). High-level transgene expression by mRNA electroporation was obtained in more than 50% of all DC types. mRNA-electroporated DCs retained their phenotype and maturational potential. Importantly, DCs electroporated with mRNA-encoding Melan-A strongly activated a Melan-A-specific cytotoxic T lymphocyte (CTL) clone in an HLA-restricted manner and were superior to mRNA-lipofected or -pulsed DCs. Optimal stimulation of the CTL occurred when Mo-DCs underwent maturation following mRNA transfection. Strikingly, a nonspecific stimulation of CTL was observed when DCs were transfected with plasmid DNA. The data clearly demonstrate that Mo-DCs electroporated with mRNA efficiently present functional antigenic peptides to cytotoxic T cells. Therefore, electroporation of mRNA-encoding tumor antigens is a powerful technique to charge human dendritic cells with tumor antigens and could serve applications in future DC-based tumor vaccines.

Quantification of trace-level DNA by real-time whole genome amplification.

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Kang, Min-Jung; Yu, Hannah; Kim, Sook-Kyung; Park, Sang-Ryoul; Yang, Inchul

2011-01-01

Quantification of trace amounts of DNA is a challenge in analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. PCR-based methods including real-time PCR are highly sensitive and widely used for quantification of low-level DNA samples. However, ordinary PCR methods require at least one copy of a specific gene sequence for amplification and may not work for a sub-genomic amount of DNA. We suggest a real-time whole genome amplification method adopting the degenerate oligonucleotide primed PCR (DOP-PCR) for quantification of sub-genomic amounts of DNA. This approach enabled quantification of sub-picogram amounts of DNA independently of their sequences. When the method was applied to the human placental DNA of which amount was accurately determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES), an accurate and stable quantification capability for DNA samples ranging from 80 fg to 8 ng was obtained. In blind tests of laboratory-prepared DNA samples, measurement accuracies of 7.4%, -2.1%, and -13.9% with analytical precisions around 15% were achieved for 400-pg, 4-pg, and 400-fg DNA samples, respectively. A similar quantification capability was also observed for other DNA species from calf, E. coli, and lambda phage. Therefore, when provided with an appropriate standard DNA, the suggested real-time DOP-PCR method can be used as a universal method for quantification of trace amounts of DNA.

Collagen Quantification in Tissue Specimens.

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Coentro, João Quintas; Capella-Monsonís, Héctor; Graceffa, Valeria; Wu, Zhuning; Mullen, Anne Maria; Raghunath, Michael; Zeugolis, Dimitrios I

2017-01-01

Collagen is the major extracellular protein in mammals. Accurate quantification of collagen is essential in the biomaterials (e.g., reproducible collagen scaffold fabrication), drug discovery (e.g., assessment of collagen in pathophysiologies, such as fibrosis), and tissue engineering (e.g., quantification of cell-synthesized collagen) fields. Although measuring hydroxyproline content is the most widely used method to quantify collagen in biological specimens, the process is very laborious. To this end, the Sircol™ Collagen Assay is widely used due to its inherent simplicity and convenience. However, this method leads to overestimation of collagen content due to the interaction of Sirius red with basic amino acids of non-collagenous proteins. Herein, we describe the addition of an ultrafiltration purification step in the process to accurately determine collagen content in tissues.

Quantification of the 3D relative movement of external marker sets vs. bones based on magnetic resonance imaging.

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Sangeux, M; Marin, F; Charleux, F; Dürselen, L; Ho Ba Tho, M C

2006-11-01

Most in vivo knee kinematic analyses are based on external markers attached to the shank and the thigh. Literature data show that markers positioning and soft tissues artifacts affect the kinematic parameters of the bones true movement. Most of the techniques of quantification used were invasive. The aim of the present study was to develop and apply a non-invasive methodology to compute the relative movement between the bones and the markers. Magnetic resonance imaging acquisitions were performed on the right knee of eleven volunteers without knee injury. The subjects were equipped with external magnetic resonance imaging-compatible marker sets. A foot drive device allowed the subjects to perform an actively loaded knee extension. The whole volume of the subject's knee was processed for four sequentially held knee flexion positions during the knee movement. The bones and external marker sets geometry were reconstructed from magnetic resonance imaging images. Then a registration algorithm was applied to the bones and the relative movement of the thigh and shank marker sets with respect to their underlying bones was computed. The protocol resulted in a good geometrical accuracy and reproducibility. Marker sets movement differ from that of the bones with a maximum of 22 mm in translation and 15 degrees in rotation and it affects the knee kinematics. Marker sets relative movement modify the knee movement finite helical axes direction (range 10-35 degrees ) and localization (range 0-40 mm). The methodology developed can evaluate external marker set system to be used for kinematic analysis in a clinical environment.

Quantification of organic acids in beer by nuclear magnetic resonance (NMR)-based methods

International Nuclear Information System (INIS)

Rodrigues, J.E.A.; Erny, G.L.; Barros, A.S.; Esteves, V.I.; Brandao, T.; Ferreira, A.A.; Cabrita, E.; Gil, A.M.

2010-01-01

The organic acids present in beer provide important information on the product's quality and history, determining organoleptic properties and being useful indicators of fermentation performance. NMR spectroscopy may be used for rapid quantification of organic acids in beer and different NMR-based methodologies are hereby compared for the six main acids found in beer (acetic, citric, lactic, malic, pyruvic and succinic). The use of partial least squares (PLS) regression enables faster quantification, compared to traditional integration methods, and the performance of PLS models built using different reference methods (capillary electrophoresis (CE), both with direct and indirect UV detection, and enzymatic essays) was investigated. The best multivariate models were obtained using CE/indirect detection and enzymatic essays as reference and their response was compared with NMR integration, either using an internal reference or an electrical reference signal (Electronic REference To access In vivo Concentrations, ERETIC). NMR integration results generally agree with those obtained by PLS, with some overestimation for malic and pyruvic acids, probably due to peak overlap and subsequent integral errors, and an apparent relative underestimation for citric acid. Overall, these results make the PLS-NMR method an interesting choice for organic acid quantification in beer.

Quantification of organic acids in beer by nuclear magnetic resonance (NMR)-based methods

Energy Technology Data Exchange (ETDEWEB)

Rodrigues, J.E.A. [CICECO-Department of Chemistry, University of Aveiro, Campus de Santiago, 3810-193 Aveiro (Portugal); Erny, G.L. [CESAM - Department of Chemistry, University of Aveiro, Campus de Santiago, 3810-193 Aveiro (Portugal); Barros, A.S. [QOPNAA-Department of Chemistry, University of Aveiro, Campus de Santiago, 3810-193 Aveiro (Portugal); Esteves, V.I. [CESAM - Department of Chemistry, University of Aveiro, Campus de Santiago, 3810-193 Aveiro (Portugal); Brandao, T.; Ferreira, A.A. [UNICER, Bebidas de Portugal, Leca do Balio, 4466-955 S. Mamede de Infesta (Portugal); Cabrita, E. [Department of Chemistry, New University of Lisbon, 2825-114 Caparica (Portugal); Gil, A.M., E-mail: agil@ua.pt [CICECO-Department of Chemistry, University of Aveiro, Campus de Santiago, 3810-193 Aveiro (Portugal)

2010-08-03

The organic acids present in beer provide important information on the product's quality and history, determining organoleptic properties and being useful indicators of fermentation performance. NMR spectroscopy may be used for rapid quantification of organic acids in beer and different NMR-based methodologies are hereby compared for the six main acids found in beer (acetic, citric, lactic, malic, pyruvic and succinic). The use of partial least squares (PLS) regression enables faster quantification, compared to traditional integration methods, and the performance of PLS models built using different reference methods (capillary electrophoresis (CE), both with direct and indirect UV detection, and enzymatic essays) was investigated. The best multivariate models were obtained using CE/indirect detection and enzymatic essays as reference and their response was compared with NMR integration, either using an internal reference or an electrical reference signal (Electronic REference To access In vivo Concentrations, ERETIC). NMR integration results generally agree with those obtained by PLS, with some overestimation for malic and pyruvic acids, probably due to peak overlap and subsequent integral errors, and an apparent relative underestimation for citric acid. Overall, these results make the PLS-NMR method an interesting choice for organic acid quantification in beer.

Quantification of total phosphorothioate in bacterial DNA by a bromoimane-based fluorescent method.

Science.gov (United States)

Xiao, Lu; Xiang, Yu

2016-06-01

The discovery of phosphorothioate (PT) modifications in bacterial DNA has challenged our understanding of conserved phosphodiester backbone structure of cellular DNA. This exclusive DNA modification in bacteria is not found in animal cells yet, and its biological function in bacteria is still poorly understood. Quantitative information about the bacterial PT modifications is thus important for the investigation of their possible biological functions. In this study, we have developed a simple fluorescence method for selective quantification of total PTs in bacterial DNA, based on fluorescent labeling of PTs and subsequent release of the labeled fluorophores for absolute quantification. The method was highly selective to PTs and not interfered by the presence of reactive small molecules or proteins. The quantification of PTs in an E. coli DNA sample was successfully achieved using our method and gave a result of about 455 PTs per million DNA nucleotides, while almost no detectable PTs were found in a mammalian calf thymus DNA. With this new method, the content of phosphorothioate in bacterial DNA could be successfully quantified, serving as a simple method suitable for routine use in biological phosphorothioate related studies. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

ALS Associated Mutations in Matrin 3 Alter Protein-Protein Interactions and Impede mRNA Nuclear Export.

Science.gov (United States)

Boehringer, Ashley; Garcia-Mansfield, Krystine; Singh, Gurkaran; Bakkar, Nadine; Pirrotte, Patrick; Bowser, Robert

2017-11-06

Mutations in Matrin 3 have recently been linked to ALS, though the mechanism that induces disease in these patients is unknown. To define the protein interactome of wild-type and ALS-linked MATR3 mutations, we performed immunoprecipitation followed by mass spectrometry using NSC-34 cells expressing human wild-type or mutant Matrin 3. Gene ontology analysis identified a novel role for Matrin 3 in mRNA transport centered on proteins in the TRanscription and EXport (TREX) complex, known to function in mRNA biogenesis and nuclear export. ALS-linked mutations in Matrin 3 led to its re-distribution within the nucleus, decreased co-localization with endogenous Matrin 3 and increased co-localization with specific TREX components. Expression of disease-causing Matrin 3 mutations led to nuclear mRNA export defects of both global mRNA and more specifically the mRNA of TDP-43 and FUS. Our findings identify a potential pathogenic mechanism attributable to MATR3 mutations and further link cellular transport defects to ALS.

Aeroelastic Uncertainty Quantification Studies Using the S4T Wind Tunnel Model

Science.gov (United States)

Nikbay, Melike; Heeg, Jennifer

2017-01-01

This paper originates from the joint efforts of an aeroelastic study team in the Applied Vehicle Technology Panel from NATO Science and Technology Organization, with the Task Group number AVT-191, titled "Application of Sensitivity Analysis and Uncertainty Quantification to Military Vehicle Design." We present aeroelastic uncertainty quantification studies using the SemiSpan Supersonic Transport wind tunnel model at the NASA Langley Research Center. The aeroelastic study team decided treat both structural and aerodynamic input parameters as uncertain and represent them as samples drawn from statistical distributions, propagating them through aeroelastic analysis frameworks. Uncertainty quantification processes require many function evaluations to asses the impact of variations in numerous parameters on the vehicle characteristics, rapidly increasing the computational time requirement relative to that required to assess a system deterministically. The increased computational time is particularly prohibitive if high-fidelity analyses are employed. As a remedy, the Istanbul Technical University team employed an Euler solver in an aeroelastic analysis framework, and implemented reduced order modeling with Polynomial Chaos Expansion and Proper Orthogonal Decomposition to perform the uncertainty propagation. The NASA team chose to reduce the prohibitive computational time by employing linear solution processes. The NASA team also focused on determining input sample distributions.

Cutset Quantification Error Evaluation for Shin-Kori 1 and 2 PSA model

International Nuclear Information System (INIS)

Choi, Jong Soo

2009-01-01

Probabilistic safety assessments (PSA) for nuclear power plants (NPPs) are based on the minimal cut set (MCS) quantification method. In PSAs, the risk and importance measures are computed from a cutset equation mainly by using approximations. The conservatism of the approximations is also a source of quantification uncertainty. In this paper, exact MCS quantification methods which are based on the 'sum of disjoint products (SDP)' logic and Inclusion-exclusion formula are applied and the conservatism of the MCS quantification results in Shin-Kori 1 and 2 PSA is evaluated

mRNA Expression of Ovine Angiopoietin-like Protein 4 Gene in Adipose Tissues

Directory of Open Access Journals (Sweden)

Jing Zhang

2016-05-01

Full Text Available Angiopoietin-like protein 4 (ANGPTL4 is involved in a variety of functions, including lipoprotein metabolism and angiogenesis. To reveal the role of ANGPTL4 in fat metabolism of sheep, ovine ANGPTL4 mRNA expression was analyzed in seven adipose tissues from two breeds with distinct tail types. Forty-eight animals with the gender ratio of 1:1 for both Guangling Large Tailed (GLT and Small Tailed Han (STH sheep were slaughtered at 2, 4, 6, 8, 10, and 12 months of age, respectively. Adipose tissues were collected from greater and lesser omental, subcutaneous, retroperitoneal, perirenal, mesenteric, and tail fats. Ontogenetic mRNA expression of ANGPTL4 in these adipose tissues from GTL and STH was studied by quantitative real time polymerase chain reaction. The results showed that ANGPTL4 mRNA expressed in all adipose tissues studied with the highest in subcutaneous and the lowest in mesenteric fat depots. Months of age, tissue and breed are the main factors that significantly influence the mRNA expression. These results provide new insights into ovine ANGPTL4 gene expression and clues for its function mechanism.

mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development.

Science.gov (United States)

Kropp, Jenna; Khatib, Hasan

2015-01-01

In vitro production (IVP) systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage) embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerate conditioned media. Differential expression was confirmed by quantitative real-time PCR for fragments of mRNA POSTN and VSNL-1, in four additional biological replicates of media. To better understand the mechanisms of mRNA secretion into the media, the expression of a predicted RNA binding protein of POSTN, PUM2, was knocked down using an antisense oligonucleotide gapmer. Supplementation of a PUM2 gapmer significantly reduced blastocyst development and decreased secretion of POSTN mRNA into the media. Overall, differential mRNA expression in the media was repeatable and sets the framework for future study of mRNA biomarkers in in vitro culture media to improve predictability of reproductive performance.

SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export.

Science.gov (United States)

Müller-McNicoll, Michaela; Botti, Valentina; de Jesus Domingues, Antonio M; Brandl, Holger; Schwich, Oliver D; Steiner, Michaela C; Curk, Tomaz; Poser, Ina; Zarnack, Kathi; Neugebauer, Karla M

2016-03-01

Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1-7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1-7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3' untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3' ends. © 2016 Müller-McNicoll et al.; Published by Cold Spring Harbor Laboratory Press.

mRNA: From a chemical blueprint for protein production to an off-the-shelf therapeutic.

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Van Lint, Sandra; Heirman, Carlo; Thielemans, Kris; Breckpot, Karine

2013-02-01

Two decades ago, mRNA became the focus of research in molecular medicine and was proposed as an active pharmaceutical ingredient for the therapy of cancer. In this regard, mRNA has been mainly used for ex vivo modification of antigen-presenting cells (APCs), such as dendritic cells (DCs). This vaccination strategy has proven to be safe, well tolerated and capable of inducing tumor antigen-specific immune responses. Recently, the direct application of mRNA for in situ modification of APCs, hence immunization was shown to be feasible and at least as effective as DC-based immunization in pre-clinical models. It is believed that application of mRNA as an off-the-shelf vaccine represents an important step in the development of future cancer immunotherapeutic strategies. Here, we will discuss the use of ex vivo mRNA-modified DCs and "naked mRNA" for cancer immunotherapy focusing on parameters such as the employed DC subtype, DC activation stimulus and route of immunization. In addition, we will provide an overview on the clinical trials published so far, trying to link their outcome to the aforementioned parameters.

Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling

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Mo Min

2008-05-01

Full Text Available Abstract Background The polyadenylation of mRNA is one of the critical processing steps during expression of almost all eukaryotic genes. It is tightly integrated with transcription, particularly its termination, as well as other RNA processing events, i.e. capping and splicing. The poly(A tail protects the mRNA from unregulated degradation, and it is required for nuclear export and translation initiation. In recent years, it has been demonstrated that the polyadenylation process is also involved in the regulation of gene expression. The polyadenylation process requires two components, the cis-elements on the mRNA and a group of protein factors that recognize the cis-elements and produce the poly(A tail. Here we report a comprehensive pairwise protein-protein interaction mapping and gene expression profiling of the mRNA polyadenylation protein machinery in Arabidopsis. Results By protein sequence homology search using human and yeast polyadenylation factors, we identified 28 proteins that may be components of Arabidopsis polyadenylation machinery. To elucidate the protein network and their functions, we first tested their protein-protein interaction profiles. Out of 320 pair-wise protein-protein interaction assays done using the yeast two-hybrid system, 56 (~17% showed positive interactions. 15 of these interactions were further tested, and all were confirmed by co-immunoprecipitation and/or in vitro co-purification. These interactions organize into three distinct hubs involving the Arabidopsis polyadenylation factors. These hubs are centered around AtCPSF100, AtCLPS, and AtFIPS. The first two are similar to complexes seen in mammals, while the third one stands out as unique to plants. When comparing the gene expression profiles extracted from publicly available microarray datasets, some of the polyadenylation related genes showed tissue-specific expression, suggestive of potential different polyadenylation complex configurations. Conclusion An

SIRT1 and FOXO1 mRNA expression in PBMC correlates to physical activity in COPD patients

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Taka C

2017-11-01

Full Text Available Chihiro Taka, Ryuji Hayashi, Kazuki Shimokawa, Kotaro Tokui, Seisuke Okazawa, Kenta Kambara, Minehiko Inomata, Toru Yamada, Shoko Matsui, Kazuyuki Tobe First Department of Internal Medicine, Faculty of Medicine, University of Toyama, Sugitani, Toyama, Toyama, Japan Background: Physical activity (PA is considered as one of the most important prognostic predictors in chronic obstructive pulmonary disease (COPD patients. Longevity gene, SIRT1, is reported to be involved in the pathogenesis of COPD by regulating the signaling pathways of oxidative stress, inflammation, and aging. We hypothesize that SIRT1 and related genes are also associated with the benefits of PA in COPD patients.Methods: Eighteen COPD outpatients were enrolled in this study, and their PA level was assessed with an accelerometer. We assessed the SIRT1 and related genes mRNA expression levels in the peripheral blood mononuclear cells (PBMCs of the subjects. We carried out respiratory function testing, blood gas analysis, the 6-minute walk test, and measurement of the cross-sectional area of the erector spinae muscles (ESMCSA by chest computed tomography. We analyzed the association of PA with the results of each of the examinations.Results: The mean age was 72±9 years, and the mean forced expiratory volume in 1 second was 1.4±0.56 L (52%±19% predicted. Our findings revealed a correlation between the daily PA and ESMCSA. The SIRT1 and Forkhead box O (FOXO1 mRNA expression levels in PBMCs were positively correlated with moderate-PA time (r=0.60, p=0.008 for SIRT1 and r=0.59, p=0.01 for FOXO1. Keywords: COPD, accelerometer, mRNA, walking, sedentary, moderate

Effects of low dose radiation on expressions of ICAM-1 mRNA and protein in kidney of diabetic mice

International Nuclear Information System (INIS)

Zhang Chi; Li Xiaokun; Gong Shouliang; Liu Xiaoju; Zhao Xue; Liu Xiaoju; Zhao Xue; Shen Wenjie; Li Cai; Cai Lu

2010-01-01

Objective: To study the effects of low dose radiation (LDR) on the expressions of intercellular adhesion molecule-1 (ICAM-1) mRNA and protein in kidney of diabetes mellitus (DM) mice and illuminate that anti-inflammation of LDR is a main mechanism for diabetic therapy. Methods: The healthy and right age C57BL/6J mice were divided into 4 groups including control, DM, LDR and DM/LDR. The mice in DM and DM/LDR groups were injected intraperitoneally with streptozocin (STZ) to set up DM models. The mice in DM/LDR and LDR groups were irradiated with 25 mGy every other day for 4 weeks. The expressions of ICAM-1 mRNA and protein in kidney were detected with RT-PCR and Western blotting 2, 4, 8, 12 and 16 weeks after irradiation. Results: The expressions of ICAM-1 mRNA and protein in kidney had no significant difference among 4 groups before LDR (P>0.05). The expressions of ICAM-1 mRNA and protein 2 weeks after irradiation with LDR were higher than those in the other 3 groups (P<0.05). The expressions of ICAM-1 mRNA and protein in the DM/LDR group 4 weeks after irradiation were also significantly higher than those in non-DM groups (P<0.05), but still significantly lower than those in DM group (P<0.05), and the significant differences were kept to 16 weeks after irradiation. But the expressions of ICAM-1 mRNA and protein in LDR group were significantly higher than those in control group (P<0.05). IHC assay showed that the glomerular and tubular in DM and DM/LDR groups were abnormal and the quantities of the positive staining cells were significantly increased compared with non-DM groups. However the damage of glomerular and tubular in DM/LDR was significantly supressed compared with DM group and the positive staining cells were also decreased. Conclusion: Under the circumstance of DM, LDR can significantly decrease the expressions of ICAM-1 mRNA and protein in mouse kidney to relief the inflammation reaction in kidney; but in normal condition, LDR can improve the immunity and

Sequence, 'subtle' alternative splicing and expression of the CYYR1 (cysteine/tyrosine-rich 1) mRNA in human neuroendocrine tumors

International Nuclear Information System (INIS)

Vitale, Lorenza; Coppola, Domenico; Strippoli, Pierluigi; Frabetti, Flavia; Huntsman, Shane A; Canaider, Silvia; Casadei, Raffaella; Lenzi, Luca; Facchin, Federica; Carinci, Paolo; Zannotti, Maria

2007-01-01

CYYR1 is a recently identified gene located on human chromosome 21 whose product has no similarity to any known protein and is of unknown function. Analysis of expressed sequence tags (ESTs) have revealed high human CYYR1 expression in cells belonging to the diffuse neuroendocrine system (DNES). These cells may be the origin of neuroendocrine (NE) tumors. The aim of this study was to conduct an initial analysis of sequence, splicing and expression of the CYYR1 mRNA in human NE tumors. The CYYR1 mRNA coding sequence (CDS) was studied in 32 NE tumors by RT-PCR and sequence analysis. A subtle alternative splicing was identified generating two isoforms of CYYR1 mRNA differing in terms of the absence (CAG - isoform, the first described mRNA for CYYR1 locus) or the presence (CAG + isoform) of a CAG codon. When present, this specific codon determines the presence of an alanine residue, at the exon 3/exon 4 junction of the CYYR1 mRNA. The two mRNA isoform amounts were determined by quantitative relative RT-PCR in 29 NE tumors, 2 non-neuroendocrine tumors and 10 normal tissues. A bioinformatic analysis was performed to search for the existence of the two CYYR1 isoforms in other species. The CYYR1 CDS did not show differences compared to the reference sequence in any of the samples, with the exception of an NE tumor arising in the neck region. Sequence analysis of this tumor identified a change in the CDS 333 position (T instead of C), leading to the amino acid mutation P111S. NE tumor samples showed no significant difference in either CYYR1 CAG - or CAG + isoform expression compared to control tissues. CYYR1 CAG - isoform was significantly more expressed than CAG + isoform in NE tumors as well as in control samples investigated. Bioinformatic analysis revealed that only the genomic sequence of Pan troglodytes CYYR1 is consistent with the possible existence of the two described mRNA isoforms. A new 'subtle' splicing isoform (CAG + ) of CYYR1 mRNA, the sequence and

Engineering intranasal mRNA vaccines to enhance lymph node trafficking and immune responses.

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Li, Man; Li, You; Peng, Ke; Wang, Ying; Gong, Tao; Zhang, Zhirong; He, Qin; Sun, Xun

2017-12-01

Intranasal mRNA vaccination provides immediate immune protection against pandemic diseases. Recent studies have shown that diverse forms of polyethyleneimine (PEI) have potent mucosal adjuvant activity, which could significantly facilitate the delivery of intranasal mRNA vaccines. Nevertheless, optimizing the chemical structure of PEI to maximize its adjuvanticity and decrease its toxicity remains a challenge. Here we show that the chemical structure of PEI strongly influences how well nanocomplexes of PEI and mRNA migrate to the lymph nodes and elicit immune responses. Conjugating cyclodextrin (CD) with PEI600 or PEI2k yielded CP (CD-PEI) polymers with different CD/PEI ratios. We analyzed the delivery efficacy of CP600, CP2k, and PEI25k as intranasal mRNA vaccine carriers by evaluating the lymph nodes migration and immune responses. Among these polymers, CP2k/mRNA showed significantly higher in vitro transfection efficiency, stronger abilities to migrate to lymph nodes and stimulate dendritic cells maturation in vivo, which further led to potent humoral and cellular immune responses, and showed lower local and systemic toxicity than PEI25k/mRNA. These results demonstrate the potential of CD-PEI2k/mRNA nanocomplex as a self-adjuvanting vaccine delivery vehicle that traffics to lymph nodes with high efficiency. As we face outbreaks of pandemic diseases such as Zika virus, intranasal mRNA vaccination provides instant massive protection against highly variant viruses. Various polymer-based delivery systems have been successfully applied in intranasal vaccine delivery. However, the influence of molecular structure of the polymeric carriers on the lymph node trafficking and dendritic cell maturation is seldom studied for intranasal vaccination. Therefore, engineering polymer-based vaccine delivery system and elucidating the relationship between molecular structure and the intranasal delivery efficiency are essential for maximizing the immune responses. We hereby

Uncertainty Quantification of CFD Data Generated for a Model Scramjet Isolator Flowfield

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Baurle, R. A.; Axdahl, E. L.

2017-01-01

Computational fluid dynamics is now considered to be an indispensable tool for the design and development of scramjet engine components. Unfortunately, the quantification of uncertainties is rarely addressed with anything other than sensitivity studies, so the degree of confidence associated with the numerical results remains exclusively with the subject matter expert that generated them. This practice must be replaced with a formal uncertainty quantification process for computational fluid dynamics to play an expanded role in the system design, development, and flight certification process. Given the limitations of current hypersonic ground test facilities, this expanded role is believed to be a requirement by some in the hypersonics community if scramjet engines are to be given serious consideration as a viable propulsion system. The present effort describes a simple, relatively low cost, nonintrusive approach to uncertainty quantification that includes the basic ingredients required to handle both aleatoric (random) and epistemic (lack of knowledge) sources of uncertainty. The nonintrusive nature of the approach allows the computational fluid dynamicist to perform the uncertainty quantification with the flow solver treated as a "black box". Moreover, a large fraction of the process can be automated, allowing the uncertainty assessment to be readily adapted into the engineering design and development workflow. In the present work, the approach is applied to a model scramjet isolator problem where the desire is to validate turbulence closure models in the presence of uncertainty. In this context, the relevant uncertainty sources are determined and accounted for to allow the analyst to delineate turbulence model-form errors from other sources of uncertainty associated with the simulation of the facility flow.

Amitriptyline induces brain-derived neurotrophic factor (BDNF) mRNA expression through ERK-dependent modulation of multiple BDNF mRNA variants in primary cultured rat cortical astrocytes and microglia.

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Hisaoka-Nakashima, Kazue; Kajitani, Naoto; Kaneko, Masahiro; Shigetou, Takahiro; Kasai, Miho; Matsumoto, Chie; Yokoe, Toshiki; Azuma, Honami; Takebayashi, Minoru; Morioka, Norimitsu; Nakata, Yoshihiro

2016-03-01

A significant role of brain-derived neurotrophic factor (BDNF) has been previously implicated in the therapeutic effect of antidepressants. To ascertain the contribution of specific cell types in the brain that produce BDNF following antidepressant treatment, the effects of the tricyclic antidepressant amitriptyline on rat primary neuronal, astrocytic and microglial cortical cultures were examined. Amitriptyline increased the expression of BDNF mRNA in astrocytic and microglial cultures but not neuronal cultures. Antidepressants with distinct mechanisms of action, such as clomipramine, duloxetine and fluvoxamine, also increased BDNF mRNA expression in astrocytic and microglial cultures. There are multiple BDNF mRNA variants (exon I, IIA, IV and VI) expressed in astrocytes and microglia and the variant induced by antidepressants has yet to be elaborated. Treatment with antidepressants increased the expression of exon I, IV and VI in astrocyte and microglia. Clomipramine alone significantly upregulated expression of exon IIA. The amitriptyline-induced expression of both total and individual BDNF mRNA variants (exon I, IV and VI) were blocked by MEK inhibitor U0126, indicating MEK/ERK signaling is required in the expression of BDNF. These findings indicate that non-neural cells are a significant target of antidepressants and further support the contention that glial production of BDNF is crucial role in the therapeutic effect of antidepressants. The current data suggest that targeting of glial function could lead to the development of antidepressants with a truly novel mechanism of action. Copyright © 2016 Elsevier B.V. All rights reserved.

Dominance behaviour in a non-aggressive flatfish, Senegalese sole (Solea senegalensis and brain mRNA abundance of selected transcripts.

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Elvira Fatsini

Full Text Available Dominance is defined as the preferential access to limited resources. The present study aimed to characterise dominance in a non-aggressive flatfish species, the Senegalese sole (Solea senegalensis by 1 identifying dominance categories and associated behaviours and 2 linking dominance categories (dominant and subordinate with the abundance of selected mRNA transcripts in the brain. Early juveniles (n = 74, 37 pairs were subjected to a dyadic dominance test, related to feeding, and once behavioural phenotypes had been described the abundance of ten selected mRNAs related to dominance and aggressiveness was measured in the brain. Late juveniles were subjected to two dyadic dominance tests (n = 34, 17 pairs, related to feeding and territoriality and one group test (n = 24, 4 groups of 6 fish. Sole feeding first were categorized as dominant and sole feeding second or not feeding as subordinate. Three social behaviours (i. "Resting the head" on another fish, ii. "Approaching" another fish, iii. "Swimming above another" fish were associated with dominance of feeding. Two other variables (i. Total time occupying the preferred area during the last 2 hours of the 24 h test, ii. Organisms occupying the preferred area when the test ended were representative of dominance in the place preference test. In all tests, dominant fish compared to subordinate fish displayed a significantly higher number of the behaviours "Rest the head" and "Approaches". Moreover, dominant sole dominated the sand at the end of the test, and in the group test dominated the area close to the feed delivery point before feed was delivered. The mRNA abundance of the selected mRNAs related to neurogenesis (nrd2 and neuroplasticity (c-fos in dominant sole compared to subordinate were significantly different. This is the first study to characterise dominance categories with associated behaviours and mRNA abundance in Senegalese sole and provides tools to study dominance related problems in

Stereological quantification of mast cells in human synovium

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