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LightCycler® 480 Real-Time PCR system: Innovative solutions for relative quantification

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... Twenty pairs of primers were designed to validate the RNA-seq data ( Table 6). Analysis of a particular gene was performed three times under identical conditions in a 25-μ l volume in the Roche Light Cycler 480 system (Reagent, Roche, Basel, Switzerland) 53 . The E (Efficiency)-method from Roche Applied Science was used to analyse the relative quantification of all target genes 53 . ...
... Analysis of a particular gene was performed three times under identical conditions in a 25-μ l volume in the Roche Light Cycler 480 system (Reagent, Roche, Basel, Switzerland) 53 . The E (Efficiency)-method from Roche Applied Science was used to analyse the relative quantification of all target genes 53 . The expression levels of the target genes were normalized by comparison with the β -actin reference gene 53 . ...
... The E (Efficiency)-method from Roche Applied Science was used to analyse the relative quantification of all target genes 53 . The expression levels of the target genes were normalized by comparison with the β -actin reference gene 53 . ...
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Adults of the lady beetle species Harmonia axyridis (Pallas) are bred artificially en masse for classic biological control, which requires egg-laying by the H. axyridis ovary. Development-related genes may impact the growth of the H. axyridis adult ovary but have not been reported. Here, we used integrative time-series RNA-seq analysis of the ovary in H. axyridis adults to detect development-related genes. A total of 28,558 unigenes were functionally annotated using seven types of databases to obtain an annotated unigene database for ovaries in H. axyridis adults. We also analysed differentially expressed genes (DEGs) between samples. Based on a combination of the results of this bioinformatics analysis with literature reports and gene expression level changes in four different stages, we focused on the development of oocyte reproductive stem cell and yolk formation process and identified 26 genes with high similarity to development-related genes. 20 DEGs were randomly chosen for quantitative real-time PCR (qRT-PCR) to validate the accuracy of the RNA-seq results. This study establishes a robust pipeline for the discovery of key genes using high-throughput sequencing and the identification of a class of development-related genes for characterization.
... Crossing point values for each gene were normalized to the respective crossing point values for the reference gene actin-DF. Data were presented as normalized ratios of genes along with error standard deviations estimated using the Roche Applied Science E-method (Tellmann & Geulen 2006). ...
Article
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Peanut genotypes from the US mini-core collection were analysed for changes in leaf proteins during reproductive stage growth under water-deficit stress. One- and two-dimensional gel electrophoresis (1- and 2-DGE) was performed on soluble protein extracts of selected tolerant and susceptible genotypes. A total of 102 protein bands/spots were analysed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) analysis. Forty-nine non-redundant proteins were identified, implicating a variety of stress response mechanisms in peanut. Lipoxygenase and 1l-myo-inositol-1-phosphate synthase, which aid in inter- and intracellular stress signalling, were more abundant in tolerant genotypes under water-deficit stress. Acetyl-CoA carboxylase, a key enzyme of lipid biosynthesis, increased in relative abundance along with a corresponding increase in epicuticular wax content in the tolerant genotype, suggesting an additional mechanism for water conservation and stress tolerance. Additionally, there was a marked decrease in the abundance of several photosynthetic proteins in the tolerant genotype, along with a concomitant decrease in net photosynthesis in response to water-deficit stress. Differential regulation of leaf proteins involved in a variety of cellular functions (e.g. cell wall strengthening, signal transduction, energy metabolism, cellular detoxification and gene regulation) indicates that these molecules could affect the molecular mechanism of water-deficit stress tolerance in peanut.
... Crossing point values for each gene were normalized to the respective crossing point values for the reference gene b-2 myoglobulin (housekeeping gene). Data are presented as normalized ratios of genes along with standard error using Roche Applied Science E-Method (Tellman and Olivier, 2006). ...
Article
Zearalenone (ZEN) is a mycotoxin from Fusarium species commonly found in many food commodities and are known to cause reproductive disorders, genotoxic and immunosuppressive effects. Although many studies have demonstrated the cytotoxic effects of ZEN, the mechanisms by which ZEN mediates its cytotoxic effects appear to differ according to cell type and route of exposure. Meantime, the available information on the neurotoxic effects of ZEN is very much limited. In the present study we evaluated the role of oxidative stress in ZEN mediated neurotoxicity in SH-SY5Y cells and investigated the possible underlying mechanism. ZEN induced ROS formation and elevated levels of MDA, loss of mitochondrial membrane potential (MMP) and increase in DNA damage in a dose dependent manner as assessed by COMET assay and agarose gel electrophoresis. However, there was no DNA damage by plasmid breakage assay at 6, 12 and 24 h time points. DAPI staining showed apoptotic nuclei at 12 and 24 h. Further, ZEN treated SH-SY5Y cells showed a marked suppressive effect on the neuronal gene expression. Use of an antioxidant N-acetylcysteine (NAC) reversed the toxin-induced generation of ROS and also attenuated loss of MMP. Collectively, these results suggest that ROS is the main upstream signal leading to increased ZEN mediated neurotoxicity in SH-SY5Y cells.
... Crossing point values for each gene were normalized to the respective crossing point values for the reference gene actin. Data are presented as normalized ratios of genes along with error standard deviations estimated using the Roche Applied Science E-method [34]. ...
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Transcriptome expression analysis in peanut to date has been limited to a relatively small set of genes and only recently has a significant number of ESTs been released into the public domain. Utilization of these ESTs for oligonucleotide microarrays provides a means to investigate large-scale transcript responses to a variety of developmental and environmental signals, ultimately improving our understanding of plant biology. We have developed a high-density oligonucleotide microarray for peanut using 49,205 publicly available ESTs and tested the utility of this array for expression profiling in a variety of peanut tissues. To identify putatively tissue-specific genes and demonstrate the utility of this array for expression profiling in a variety of peanut tissues, we compared transcript levels in pod, peg, leaf, stem, and root tissues. Results from this experiment showed 108 putatively pod-specific/abundant genes, as well as transcripts whose expression was low or undetected in pod compared to peg, leaf, stem, or root. The transcripts significantly over-represented in pod include genes responsible for seed storage proteins and desiccation (e.g., late-embryogenesis abundant proteins, aquaporins, legumin B), oil production, and cellular defense. Additionally, almost half of the pod-abundant genes represent unknown genes allowing for the possibility of associating putative function to these previously uncharacterized genes. The peanut oligonucleotide array represents the majority of publicly available peanut ESTs and can be used as a tool for expression profiling studies in diverse tissues.
... Crossing point values for each gene were normalized to the respective crossing point values for the reference gene RP-II. Data are presented as normalized ratios of genes along with standard error using Roche Applied Science E-Method (Tellmann and Olivier, 2006). ...
Article
T-2 toxin is a cytotoxic fungal secondary metabolite produced by different species of Fusarium such as F. sporotichioides, F. poae, F. equiseti, F. acuminatum etc. This class of mycotoxins causes a number of pathologies including nervous disorders, cardiovascular alterations, immunodepression and hemostatic derangements. In the present study, mechanism of T-2 toxin induced alteration of blood-brain barrier (BBB) permeability was assessed in terms of oxidative stress, gene expression of MMP-9, MMP-2 and their inhibitors TIMP-1 and TIMP-2, activation of inflammatory cytokines in both brain and peripheral tissue spleen. Gel zymography was used to show the activity of MMP-9 and MMP-2. The percutaneous exposure of 1 LD50 T2 toxin caused a reversible alteration in BBB permeability as observed by extravasation of Evans blue dye. Maximum dye level was observed on day 3 and reduced by day 7. A significant GSH depletion was observed on days 1 and 3. Brain ROS and lipid peroxidation levels increased significantly on 1 and 3 days and decreased by day 7. The SOD levels in brain showed significantly higher activity on 3 days (4-fold) and 7 days (5-fold) of toxin exposure compared to control. A similar trend was observed with catalase enzyme levels. The gene expression analysis of cNOS and iNOS showed varying levels of expression on different time points of post exposure. MMP-9 expression was significantly high on days 3 and 7 in brain with corresponding alteration in TIMP-1. MMP-2 and TIMP-2 showed no effect. Gene expression analysis of the inflammatory cytokines, IL-1α, IL-1β, IL-6 and TNF-α showed elevated levels on day 7 in brain. As spleen plays an important role in inflammatory response we analyzed MMP-9, MMP-2 and inflammatory cytokines in spleen. The MMP-9 was activated on day 7. MMP-2 activity was found to be elevated on 3 and 7 days and TIMP-2 mRNA level increased on 1 and 3 days in spleen. Inflammatory cytokines, IL-1 α, IL-1β, IL-6 and TNF-α showed elevated levels on days 1 and 3 in spleen indicating an early effect in spleen than in brain. In summary, the results of the study showed that the T-2 induced alteration in BBB permeability is mediated through oxidative stress, activation of MMP-9, and proinflammatory cytokines in brain as well as contribution from peripheral tissue spleen.
... 1.5: Roche Applied Science), according to the manufacturer's instructions (Technical Notes No. LC10, 13 and 16: Roche Applied Science). The Cp values of target and reference gene were respectively corrected with the PCR efficiencies of target and reference gene by the E-method instead of the ΔΔC Tmethod, according to the manufacturer's instructions [42]. The PCR efficiency of each target and reference gene was determined by a relative standard curve, which was generated for each target and reference gene using serial dilutions of first-strand cDNA solution obtained by the RT from the middle temporal cortex of a LPC patient, because PCR efficiency depends on primers and UPL probes (Technical Note No. LC 13: Roche Applied Science). ...
Article
Neuroinflammation is involved in the pathology of Alzheimer's disease (AD). Our major focus was to clarify whether neuroinflammation plays an important role in AD pathogenesis, particularly prior to the manifestation of overt dementia. We analyzed cytokine expression profiles of the brain, with focus on non-demented patients with increasing AD pathology, referred to as high pathology control (HPC) patients, who provide an intermediate subset between AD and normal control subjects, referred to as low pathology control (LPC) patients. With real-time PCR techniques, we found significant differences in interleukin (IL)-1β, 10, 13, 18, and 33, tumor necrosis factor-α (TNFα) converting enzyme (TACE), and transforming growth factor β1 (TGFβ1) mRNA expression ratios between HPC and AD patients, while no significant differences in the expression ratios of any cytokine tested here were observed between LPC and HPC patients. The cytokine mRNA expression ratios were determined as follows: first, cytokine mRNA levels were normalized to mRNA levels of a housekeeping gene, peptidyl-prolyl isomerase A (PPIA), which showed the most stable expression among ten housekeeping genes tested here; then, the normalized data of cytokine levels in the temporal cortex were divided by those in the cerebellum, which is resistant to AD pathology. Subsequently, the expression ratios of the temporal cortex to cerebellum were compared among LPC, HPC, and AD patient groups. Our results indicate that cytokines are more mobilized and implicated in the later AD stage when a significant cognitive decline occurs and develops than in the developmental course of AD pathology prior to the manifestation of overt dementia.
... Crossing point values for each gene was normalized to the respective crossing point values for the reference gene Gapdh. Data are presented as normalized ratios of genes along with standard error using the Roche Applied Science E-method [48]. ...
Article
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Japanese encephalitis (JE) is one of the leading causes of acute encephalopathy with the highest mortality rate of 30-50%. The purpose of this study was to understand complex biological processes of host response during the progression of the disease. Virus was subcutaneously administered in mice and brain was used for whole genome expression profiling by cDNA microarray. The comparison between viral replication efficiency and disease progression confirms the active role of host response in immunopathology and disease severity. The histopathological analysis confirms the severe damage in the brain in a time dependent manner. Interestingly, the transcription profile reveals significant and differential expression of various pattern recognition receptors, chemotactic genes and the activation of inflammasome. The increased leukocyte infiltration and aggravated CNS inflammation may be the cause of disease severity. This is the first report that provides a detailed picture of the host transcriptional response in a natural route of exposure and opens up new avenues for potential therapeutic and prophylactic strategies against Japanese encephalitis virus.
... Crossing point values for each gene were normalized to the respective crossing point values for the reference gene RP-II. Data are presented as normalized ratios of genes along with standard error using Roche Applied Science E-Method (Tellmann and Olivier, 2006). ...
Article
Nerve agents irreversibly inhibit acetylcholinesterase (AChE), leading to cholinergic crisis and death at acute exposure levels. The complexity, delayed onset, and persistent nature of nerve agent induced CNS effects need to be elucidated to block their multiple effects. In the present study gene expression and phosphoprotein profile of certain key neuronal proteins were studied after soman exposure. Quantitative real time PCR analysis of c-Fos, Bax, CREB and caspase 3 genes in the hippocampus, cortex and cerebellum showed that only c-Fos and Bax mRNA expression was increased significantly. Western blot analysis also confirmed the induction of c-Fos at early time points both at 0.5 and 1.0 LD(50) dose of soman exposure. Acute soman exposure caused perturbations in the phosphorylation status of ERK, JNK, p38 MAPK, CREB, c-Jun and NF-κB in all the three brain regions. The primary target for soman toxicity, AChE was inhibited in blood and brain up to 90%. Therapeutic treatment comprising of HI-6, atropine and diazepam has completely protected animals from death and reactivated soman inhibited AChE up to 40% in the plasma and RBC. This therapeutic regime also reduced soman induced Bax expression to near control levels, but could not reverse the soman induced changes in c-Fos expression and phosphorylation levels completely. Results suggest that exposure to soman caused persistent changes in these key brain proteins, which could lead to the development of complex neurotoxic effects and there is an urgent need for development of better drugs to stop multiple effects of nerve agents poisoning.
... The corresponding PCR efficiency was calculated from the slope of the standard curve with the formula of efficiency = 10 (−1/slope) . Relative quantification was done using the standard curve method with PCR efficiency correction (E-method) of the LightCycler 480 Software (Roche) (Tellmann, 2006). All samples were normalized to GAPDH expression on the same plate. ...
Article
Human embryonic stem cells (hESCs) are an important cellular model for studying ion channel function in the context of a human cardiac cell and will provide a wealth of information about both heritable arrhythmias and acquired electrophysiological disorders. However, detailed electrophysiological characterization of the important cardiac ion channels has been so far overlooked. Because mutations in the gene for the I(Ks) α subunit, KCNQ1, constitute the majority of long QT syndrome (LQT-1) cases, we have carried out a detailed biophysical analysis of this channel expressed in hESCs to establish baseline I(Ks) channel biophysical properties in cardiac myocytes derived from hESCs (hESC-CMs). I(Ks) channels are heteromultimeric proteins consisting of four identical α-subunits (KCNQ1) assembled with auxiliary β-subunits (KCNE1). We found that the half-maximal I(Ks) activation voltage in hESC-CMs and in myocytes derived from human induced pluripotent stems cells (hiPSC-CMs) falls between that of KCNQ1 channels expressed alone and with full complement of KCNE1, the major KCNE subunit expressed in hESC-CMs as shown by qPCR analysis. Overexpression of KCNE1 by transfection of hESC-CMs markedly shifted and slowed native I(Ks) activation implying assembly of additional KCNE1 subunits with endogenous channels. Our results in hESC-CMs, which indicate an I(Ks) subunit stoichiometry that can be altered by variable KCNE1 expression, suggest the possibility for variable I(Ks) function in the developing heart, in different tissues in the heart, and in disease. This establishes a new baseline for I(Ks) channel properties in myocytes derived from pluripotent stem cells and will guide future studies in patient-specific hiPSCs.
... The actin gene was used as a reference in all qRT- PCR experiments. Relative quantification analyses of all target genes were performed using the E (Efficiency)-method from Roche Applied Science (Tellmann and Geulen, 2006). The expression level of each target gene was normalized to the level of the reference gene actin. ...
Article
The failure of peg penetration into the soil leads to seed abortion in peanut. Knowledge of genes involved in these processes is comparatively deficient. Here, we used RNA-seq to gain insights into transcriptomes of aerial and subterranean pods. More than 2 million transcript reads with an average length of 396 bp were generated from one aerial (AP) and two subterranean (SP1 and SP2) pod libraries using pyrosequencing technology. After assembly, sets of 49 632, 49 952 and 50 494 from a total of 74 974 transcript assembly contigs (TACs) were identified in AP, SP1 and SP2, respectively. A clear linear relationship in the gene expression level was observed between these data sets. In brief, 2194 differentially expressed TACs with a 99.0% true-positive rate were identified, among which 859 and 1068 TACs were up-regulated in aerial and subterranean pods, respectively. Functional analysis showed that putative function based on similarity with proteins catalogued in UniProt and gene ontology term classification could be determined for 59 342 (79.2%) and 42 955 (57.3%) TACs, respectively. A total of 2968 TACs were mapped to 174 KEGG pathways, of which 168 were shared by aerial and subterranean transcriptomes. TACs involved in photosynthesis were significantly up-regulated and enriched in the aerial pod. In addition, two senescence-associated genes were identified as significantly up-regulated in the aerial pod, which potentially contribute to embryo abortion in aerial pods, and in turn, to cessation of swelling. The data set generated in this study provides evidence for some functional genes as robust candidates underlying aerial and subterranean pod development and contributes to an elucidation of the evolutionary implications resulting from fruit development under light and dark conditions.
... All samples were normalized to actin expression on the same plate. The relative mRNA level was calculated with the second derivative formula that corrects for PCR efficiency differences between target and reference gene [41]. ...
Article
Poly(ADP-ribose) polymerase-1 (PARP-1) and its cleavage products regulate cell viability and NF-kB activity when expressed in neurons. PARP-1 cleavage generates a 24kDa (PARP-124) and an 89kDa fragment (PARP-189). Compared to WT (PARP-1WT), the expression of an uncleavable PARP-1 (PARP-1UNCL) or of PARP-124 conferred protection from oxygen/glucose deprivation (OGD) or OGD/restoration of oxygen and glucose (ROG) damage in vitro, whereas expression of PARP-189 was cytotoxic. Viability experiments were performed in SH-SY5Y, a human neuroblastoma cell line, as well as in rat primary cortical neurons. Following OGD, the higher viability in the presence of PARP-1UNCL or PARP-124 was not accompanied with decreased formation of poly(ADP-riboses) or higher NAD levels. PARP-1 is a known cofactor for NF-kB, hence we investigated whether PARP-1 cleavage influences the inflammatory response. All PARP-1 constructs mimicked PARP-1WT in regards to induction of NF-kB translocation into the nucleus and its increased activation during ischemic challenge. However, expression of PARP-189 construct induced significantly higher NF-kB activity than PARP-1WT; and the same was true for NF-kB-dependent iNOS promoter binding activity. At a protein level, PARP-1UNCL and PARP-124 decreased iNOS (and lower levels of iNOS transcript) and COX-2, and increased Bcl-xL. The increased levels of NF-kB and iNOS transcriptional activities, seen with cytotoxic PARP-189, were accompanied by higher protein expression of COX-2 and iNOS (and higher levels of iNOS transcript) and lower protein expression of Bcl-xL. Taken together, these findings suggest that PARP-1 cleavage products may regulate cellular viability and inflammatory responses in opposing ways during in vitro models of "ischemia".
... Crossing point values for each gene were normalized to the respective crossing point values for the reference gene b-2 myoglobulin (housekeeping gene). Data are presented as normalized ratios of genes along with standard error using Roche Applied Science E-Method (Tellman and Olivier, 2006). ...
Article
Zearalenone (ZEN) is a mycotoxin from Fusarium species commonly found in many food commodities and are known to cause reproductive disorders, genotoxic and immunosuppressive effects. Although many studies have demonstrated the cytotoxic effects of ZEN, the mechanisms by which ZEN mediates its cytotoxic effects appear to differ according to cell type and route of exposure. Meantime, the available information on the neurotoxic effects of ZEN is very much limited. In the present study we evaluated the role of oxidative stress in ZEN mediated neurotoxicity in SH-SY5Y cells and investigated the possible underlying mechanism. ZEN induced ROS formation and elevated levels of MDA, loss of mitochondrial membrane potential and increase in DNA damage in a dose dependent manner as assessed by COMET assay and electrophoresis. However, there was no DNA damage by plasmid breakage assay at different time points. DAPI staining showed apoptotic nuclei at 12 and 24 h. Further, ZEN treated SH-SY5Y cells showed a marked suppressive effect on the neuronal gene expression. Use of an antioxidant N-acetylcysteine reversed the toxin-induced generation of ROS and also attenuated loss of MMP. Collectively, these results suggest that ROS is the main upstream signal leading to increased ZEN mediated neurotoxicity in SH-SY5Y cells.
... Crossing point values for each gene were normalized to the respective crossing point values for the reference gene b-2 myoglobulin (housekeeping gene). Data are presented as normalized ratios of genes along with standard error using Roche Applied Science E-Method (Tellman and Olivier, 2006). ...
Article
Zearalenone(ZEN) is a mycotoxin from Fusarium species commonly found in many food commodities and are known to cause reproductive disorders, genotoxic and immunosuppressive effects. Although many studies have demonstrated the cytotoxic effects of ZEN, the mechanisms by which ZEN mediates its cytotoxic effects appear to differ according to cell type and route of exposure. Meantime, the available information on the neurotoxic effects of ZEN is very much limited. In the present study we evaluated the role of oxidative stress in ZEN mediated neurotoxicity in SH-SY5Y cells and investigated the possible underlying mechanism. ZEN induced ROS formation and elevated levels of MDA, loss of mitochondrial membrane potential (MMP) and increase in DNA damage in a dose dependent manner as assessed by COMET assay and agarose gel electrophoresis. However, there was no DNA damage by plasmid breakage assay at 6, 12 and 24h time points. DAPI staining showed apoptotic nuclei at 12 and 24 h. Further, ZEN treated SH-SY5Y cells showed a marked suppressive effect on the neuronal gene expression. Use of an antioxidant N-acetylcysteine (NAC) reversed the toxin-induced generation of ROS and also attenuated loss of MMP. Collectively, these results suggest that ROS is the main upstream signal leading to increased ZEN mediated neurotoxicity in SH-SY5Y cells.
... Crossing point values for each target gene were normalized to the respective crossing point values for the reference gene RP-II. Data are presented as normalized ratios of genes along with standard error using Roche Applied Science E-Method [44]. ...
Article
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Recent alleged attacks with nerve agent sarin on civilians in Syria indicate their potential threat to both civilian and military population. Acute nerve agent exposure can cause rapid death or leads to multiple and long term neurological effects. The biochemical changes that occur following nerve agent exposure needs to be elucidated to understand the mechanisms behind their long term neurological effects and to design better therapeutic drugs to block their multiple neurotoxic effects. In the present study, we intend to study the efficacy of antidotes comprising of HI-6(1-[[[4-(aminocarbonyl)-pyridinio]-methoxy]-methyl]-2-[(hydroxyimino) methyl] pyridinium dichloride), atropine and midazolam on soman induced neurodegeneration and the expression of c-Fos, Calpain, and Bax levels in discrete rat brain areas. Therapeutic regime consisting of HI-6 (50 mg/kg, i.m), atropine (10 mg/kg, i.m) and midazolam (5 mg/kg, i.m) protected animals against soman (2xLD50, s.c) lethality completely at 2 h and 80% at 24 h. HI-6 treatment reactivated soman inhibited plasma and RBC cholinesterase up to 40%. Fluoro-Jade B (FJ-B) staining of neurodegenerative neurons showed that soman induced significant necrotic neuronal cell death, which was reduced by this antidotal treatment. Soman increased the expression of neuronal proteins including c-Fos, Bax and Calpain levels in the hippocampus, cerebral cortex and cerebellum regions of the brain. This therapeutic regime also reduced the soman induced Bax, Calpain expression levels to near control levels in the different brain regions studied, except a mild induction of c-Fos expression in the hippocampus. Rats that received antidotal treatment after soman exposure were protected from mortality and showed reduction in the soman induced expression of c-Fos, Bax and Calpain and necrosis. Results highlight the need for timely administration of better antidotes than standard therapy in order to prevent the molecular and biochemical changes and subsequent long term neurological effects induced by nerve agents.
... MDP0000336547 was selected as the reference gene [17]. Primer efficiencies and relative expression levels of targets were calculated using the Roche 480 Light Cycler software E-Method [18]. ...
Article
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Fruit abscission is a complex physiological process that is regulated by internal and environmental factors. During early development, apple fruit are exposed to extreme temperature fluctuations that are associated with premature fruit drop; however, their effect on fruit abscission is largely unknown. We hypothesized that fruit abscission is triggered by cold stress and investigated the molecular basis of premature fruit drop using RNA-Seq and metabolomics data from apple fruit undergoing abscission following cold stress in the field. Genes responsive to abscisic acid signaling and cell wall degradation were upregulated during abscission, consistent with the increased abscisic acid concentrations detected by liquid chromatography-mass spectrometry. We performed ex vivo cold shock experiments with excised tree subunits consisting of a branch, pedicel, and fruit. Abscission induction occurred in the cold-stressed subunits with concurrent upregulation of abscisic acid biosynthesis ( MdNCED1 ) and metabolism ( MdCYP707A ) genes, and ethylene biosynthesis ( MdACS1 ) and receptor ( MdETR2 ) genes in the pedicel. Another key finding was the activation of cytoplasmic streaming in abscission-zone cells detected by electron microscopy. Our results provide a novel insight into the molecular basis of fruit abscission physiology in response to cold stress in apple.
... Crossing point values for each gene were normalized to the respective crossing point values for the reference gene RP-II. Data are presented as normalized ratios of genes along with standard error using Roche Applied Science E-Method [17]. ...
Article
Abstract T-2 toxin is the most toxic trichothecene and a frequent contaminant in many agriculture products. Dietary ingestion represents the most common route of T-2 toxin exposure in humans. T-2 toxin exposure leads to many pathological conditions like nervous disorders, cardiovascular alterations, immune depression and dermal inflammation. However, the neuronal toxicity of T-2 toxin in vitro remains unclear. In the present study, we investigated the mechanism of T-2 toxin-induced apoptosis in human neuroblastoma cells (IMR-32). T-2 toxin was cytotoxic at a low concentration of 10 ng/ml. The 50 % inhibitory concentration (IC50) of T-2 toxin was found to be 40 ng/ml as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, crystal violet dye exclusion test and lactate dehydrogenase (LDH) leakage. T-2 toxin increased intracellular reactive oxygen species generation as early as 15 min and peaked at 60 min as analyzed by flow cytometry. Annexin V+propidium iodide staining showed time-dependent increase in percent apoptotic c e l l s . DNA gel electrophoresis showed oligonucleosomal DNA fragmentation typical of apoptotic cells. Additionally, casapse-3 activation and PARP cleavage indicated involvement of mitochondrial mediated caspase-dependent pathway of apoptosis. Cell cycle analysis revealed time-dependent increase in sub-G1 population of cells and significant up-regulation of CDK2, CDK6, cyclin A and p21 messenger RNA (mRNA) levels. Exposure to T-2 toxin induced the phosphorylation of extracellular signal-regulated kinase (ERK), p38-mitogenactivated protein kinase and c-jun N-terminal kinases (JNK). Analysis of human phospho-mitogen-activated protein kinase (MAPK) antibody array revealed timedependent increase in phosphorylation. Upstream of ERK pathway Grb2, Ras and Raf and downstream transcription factors c-fos and c-jun were significantly up-regulated. ZVAD- FMK and MAPK inhibitors (PD 98059, SB 203580 and ZM 336372) exposure prior to T-2 toxin treatment significantly decreased percent of apoptotic cells compared to only T-2 toxin-exposed cells. Results of the present study show that T-2 toxin at nanogram concentrations can induce apoptosis in human neuronal cells through multiple signal transduction pathways. The study provides possible leads for developing therapeutic approaches to prevent T-2 toxin-induced neurotoxicity.
... Immediately following the run, a melt curve analysis was conducted to check the specificity of the reaction by heat denaturing for 40 cycles at 0.2°C/s from 55 to 95°C. The amplification of the PCR reactions had an efficiency of 1.95, where 2 is the highest quality representing doubling each amplification cycle (Tellmann and Geulen, 2006) and an error value of 0.009 calculated as the mean squared error of the standard curve. No amplification was detected in the negative controls. ...
Article
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Changes in land management practices may have significant implications for soil microbial communities important in organic P turnover. Soil bacteria can increase plant P availability by excreting phosphatase enzymes which catalyze the hydrolysis of ester-phosphate bonds. Examining the diversity and abundance of alkaline phosphatase gene harboring bacteria may provide valuable insight into alkaline phosphatase production in soils. This study examined the effect of 20 years of no input organic (ORG), organic with composted manure (ORG + M), conventional (CONV) and restored prairie (PRA) management on soil P bioavailability, alkaline phosphatase activity (ALP), and abundance and diversity of ALP gene (phoD) harboring bacteria in soils from the northern Great Plains of Canada. Management system influenced bioavailable P (P < 0.001), but not total P, with the lowest concentrations in the ORG systems and the highest in PRA. Higher rates of ALP were observed in the ORG and ORG + M treatments with a significant negative correlation between bioavailable P and ALP in 2011 (r2 = 0.71; P = 0.03) and 2012 (r2 = 0.51; P = 0.02), suggesting that ALP activity increased under P limiting conditions. The phoD gene abundance was also highest in ORG and ORG + M resulting in a significant positive relationship between bacterial phoD abundance and ALP activity (r2 = 0.71; P = 0.009). Analysis of phoD bacterial community fingerprints showed a higher number of species in CONV compared to ORG and ORG + M, contrary to what was expected considering greater ALP activity under ORG management. In 2012, banding profiles of ORG + M showed fewer phoD bacterial species following the second manure application, although ALP activity is higher than in 2011. This indicates that a few species may be producing more ALP and that quantitative gene analysis was a better indicator of activity than the number of species present.
... The relative quantitative expression of each gene was obtained from a second derivative maximum analysis by determination of the crossing point value normalized to the crossing points values of reference gene GAPDH. Data were shown as normalized ratios of genes together with a standard error by means of Roche Applied Science E-Method (Tellmann and Geulen, 2006). ...
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The present study was aimed to evaluate the bio-control efficacy of Pediococcus pentosaceus isolated from traditional fermented dairy products originated from India, against the growth and zearalenone (ZEA) production of Fusarium graminearum. The cell-free supernatants of P. pentosaceus (PPCS) were prepared and chemical profiling was carried out by GC-MS and MALDI-TOF analysis. Chemical profiling of PPCS evidenced that, the presence of phenolic antioxidants, which are responsible for the antifungal activity. Another hand, MALDI-TOF analysis also indicated the presence of antimicrobial peptides. To know the antioxidant potential of PPCS, DPPH free radical scavenging assay was carried out and IC 50 value was determined as 32 ± 1.89 µL/mL. The antifungal activity of P. pentosaceus was determined by dual culture overlay technique and zone of inhibition was recorded as 47 ± 2.81%, and antifungal activity of PPCS on F. graminearum was determined by micro-well dilution and scanning electron microscopic techniques. The minimum inhibitory concentration (MIC) of PPCS was determined as 66 ± 2.18 µL/mL in the present study. Also a clear variation in the micromorphology of mycelia treated with MIC value of PPCS compared to untreated control was documented. Further, the mechanism of growth inhibition was revealed by ergosterol analysis and determination of reactive oxygen species (ROS) in PPCS treated samples. The effects of PPCS on mycelial biomass and ZEA production were observed in a dose-dependent manner. The mechanism behind the suppression of ZEA production was studied by reverse transcriptase qPCR analysis of ZEA metabolic pathway genes (PKS4 and PKS13), and results showed that there is a dose dependent down-regulation of target gene expression in PPCS treated samples. The results of the present study were collectively proved that, the antifungal and ZEA inhibitory activity of PPCS against F. graminearum and it may find a potential application in agriculture and food industry as a natural bio-controlling agent.
... To increase the template to a quantifiable concentration, a second set of cDNA was spiked with a known amount of DNA that was subtracted from the final concentration to calculate phoD transcript abundance. The amplification of the PCR reactions had an efficiency of 1.96, where 2 is the highest quality representing a doubling at each amplification cycle (Tellmann and Geulen, 2006) and an error value of 0.04 calculated as the mean squared error of the standard curve. No amplification was detected in the negative controls. ...
Article
Bacterial transformation of phosphorus (P) compounds in soil is largely dependent on soil microbial community function, and is therefore sensitive to anthropogenic disturbances such as fertilization or cropping systems. However, the effect of soil management on the transcription of bacterial genes that encode phosphatases, such as phoD, is largely unknown. This greenhouse study examined the effect of long-term management and P amendment on potential alkaline phosphatase (ALP) activity and phoD gene (DNA) and transcript (RNA) abundance. Soil samples (0–15 cm) were collected from the Glenlea Long-term Rotation near Winnipeg, Manitoba, to compare organic, conventional and prairie management systems. In the greenhouse, pots of soil from each management system were amended with P as either soluble mineral fertilizer or cattle manure and then planted with Italian ryegrass (Lolium multiforum). Soils from each pot were sampled for analysis immediately and after 30 and 106 days. Significant differences among the soil/P treatments were detected for inorganic P, but not the organic P in NaHCO3-extracts. At day 0, ALP activity was similar among the soil/P treatments, but was higher after 30 days for all P amendments in soil from organically managed plots. In contrast, ALP activity in soils under conventional and prairie management responded to increasing rates of manure only, with significant effects from medium and high manure application rates at 30 and 106 days. Differences in ALP activity at 30 days corresponded to the abundance of bacterial phoD genes, which were also significantly higher in soils under organic management. However, this correlation was not significant for transcript abundance. Next-generation sequencing allowed the identification of 199 unique phoD operational taxonomic units (OTUs) from the metagenome (soil DNA) and 35 unique OTUs from the metatranscriptome (soil RNA), indicating that a subset of phoD genes was being transcribed in all soils.
... Normalized relative ratio (NRR) = E t CT (target calibrator) -CT (target sample) / E r CT (reference calibrator) -CT (reference sample) [75]. All results were normalized to reference gene gluceraldehyde-3-phosphate dehydrogenase (GAPDH), selected after initial testing of three reference genes (GAPDH, β-actin and ribosomal RNA (18 S)). mRNA expression for each of the outcome parameters was compared among treatments at the different time points using a random intercept, random slope model in R [76]. ...
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Studies in humans have shown that haemostatic and inflammatory pathways both play important roles in the pathogenesis of joint disease. The aim of this study was to assess mRNA expression of haemostatic and inflammatory factors in cultured equine fibroblast-like synoviocytes exposed to lipopolysaccharide (LPS), fibrinogen and thrombin. Synovial membranes were collected from metacarpo-phalangeal joints of 6 skeletally mature horses euthanized for non-orthopaedic reasons. Passage 4 fibroblast-like synoviocytes were left non-treated or treated with either 0.1 μg/ml LPS, 5 mg/ml fibrinogen or 5 U/ml thrombin and harvested at time points 0, 6, 24 and 48 h. mRNA expression of serum amyloid A (SAA), interleukin-6 (IL-6), monocyte chemotactic protein 1 (MCP-1), tissue factor (TF), plasminogen activator inhibitor 1 (PAI-1), urokinase plasminogen activator (uPA), vascular endothelial growth factor (VEGF) and protease activator receptor 1 (PAR-1) was assessed using quantitative real time reverse transcriptase PCR. LPS caused a significant increase in mRNA expression of SAA, IL-6, MCP-1 and uPA, and a decrease in TF, PAI-1 and PAR-1 when compared to non-treated cells. Treatment with thrombin resulted in increased mRNA expression of SAA, IL-6, MCP-1 and PAI-1, and a decreased PAR-1 expression compared to non-treated cells. The fibrinogen-treated synoviocytes showed significantly increased mRNA expression of IL-6, MCP-1, TF and PAI-1, and decreased PAR-1 expression compared to non-treated cells. LPS, fibrinogen and thrombin induced an increased gene expression of inflammatory markers in isolated equine fibroblast-like synoviocytes. LPS caused changes in gene expression promoting increased fibrinolysis, while fibrinogen and thrombin changed the gene expression resulting potentially in reduced fibrinolysis. Overall, it appeared that both inflammatory and haemostatic stimuli affected expression of genes involved in inflammatory and haemostatic pathways, supporting their importance in equine joint diseases.
... All assays for each particular gene were performed in triplicate synchronously under identical conditions. Relative quantification analyses of all target genes were performed using the E (Efficiency)-method from Roche Applied Science [35]. The expression level of each target gene was normalized to the level of the reference gene. ...
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In order to obtain more valuable insights into the protein dynamics and accumulation of allergens in seeds during underground development, we performed a proteomic study on developing peanut seeds at seven different stages. A total of 264 proteins with altered abundance and contained at least one unique peptide was detected by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS). All identified proteins were classified into five functional categories as level 1 and 20 secondary functional categories as level 2. Among them, 88 identified proteins (IPs) were related to carbohydrate/ amino acid/ lipid transport and metabolism, indicating that carbohydrate/amino acid/ lipid metabolism played a key role in the underground development of peanut seeds. Hierarchical cluster analysis showed that all IPs could be classified into eight cluster groups according to the abundance profiles, suggesting that the modulatory patterns of these identified proteins were complicated during seed development. The largest group contained 41 IPs, the expression of which decreased at R 2 and reached a maximum at R3 but gradually decreased from R4. A total of 14 IPs were identified as allergen-like proteins by BLAST with A genome ( Arachis duranensis ) or B genome ( Arachis ipaensis ) translated allergen sequences. Abundance profile analysis of 14 identified allergens showed that the expression of all allergen proteins was low or undetectable by 2-DE at the early stages (R1 to R4), and began to accumulate from the R5 stage and gradually increased. Network analysis showed that most of the significant proteins were involved in active metabolic pathways in early development. Real time RT-PCR analysis revealed that transcriptional regulation was approximately consistent with expression at the protein level for 8 selected identified proteins. In addition, some amino acid sequences that may be associated with new allergens were also discussed.
... Crossing point values for each gene were normalized to the particular crossing point values for the reference gene GAPDH. Data are shown as normalized ratios of genes together with standard error by means of Roche Applied Science E-Method (Tellman and Olivier, 2006). ...
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The present study was aimed to establish the antagonistic effects of Ocimum sanctum L. essential oil (OSEO) on growth and zearalenone (ZEA) production of Fusarium graminearum. GC-MS chemical profiling of OSEO revealed the existence of 43 compounds and the major compound was found to be eugenol (34.7%). DPPH free radical scavenging activity (IC50) of OSEO was determined to be 8.5 μg/mL. Minimum inhibitory concentration and minimum fungicidal concentration of OSEO on F. graminearum were recorded as 1250 and 1800 μg/mL, respectively. Scanning electron microscope observations showed significant micro morphological damage in OSEO exposed mycelia and spores compared to untreated control culture. Quantitative UHPLC studies revealed that OSEO negatively effected the production of ZEA; the concentration of toxin production was observed to be insignificant at 1500 μg/mL concentration of OSEO. On other hand ZEA concentration was quantified as 3.23 μg/mL in OSEO untreated control culture. Reverse transcriptase qPCR analysis of ZEA metabolic pathway genes (PKS4 and PKS13) revealed that increase in OSEO concentration (250-1500 μg/mL) significantly downregulated the expression of PKS4 and PKS13. These results were in agreement with the artificially contaminated maize grains as well. In conlusion, the antifungal and antimycotoxic effects of OSEO on F. graminearum in the present study reiterated that, the essential oil of O. sanctum could be a promising herbal fungicide in food processing industries as well as grain storage centers.
... The crossing point of the genes was normalized to the respective crossing point values for the housekeeping gene (b-2 myoglobulin). Obtained gene expression results processed by Roche Applied Science E-Method and results were shown as normalized ratios with standard error (Tellmann and Geulen, 2006). ...
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Deoxynivalenol (DON) is Fusarium mycotoxin that is frequently found in many cereal-based foods, and its ingestion has a deleterious impact on human health. In this investigation, we studied the mechanism of DON-induced neurotoxicity and followed by cytoprotective efficacy of quercetin (QUE) in contradiction of DON-induced neurotoxicity through assessing the oxidative stress and apoptotic demise in the human neuronal model, i.e. SH-SY5Y cells. DON diminished the proliferation of cells in the manner of dose and time-dependent as revealed by cell viability investigations, i.e. MTT and lactate dehydrogenase assays. Additional studies, such as intracellular reactive oxygen species (ROS), lipid peroxidation (LPO), mito-chondrial membrane potential (MMP), DNA damage, cell cycle, and neuronal biomarkers (amino acid decarboxylase, tyrosine hydroxylase, and brain-derived neurotrophic factor) demonstrated that DON induces apoptotic demise in neuronal cells through oxidative stress intermediaries. On another hand, pre-treatment of neuronal cells with 1 mM of quercetin (QUE) showed decent viability upon exposure to 100 mM of DON. In detailed studies demonstrated that QUE (1 mM) pre-treated cells show strong attenuation efficiency against DON-induced ROS generation, LPO, MMP loss, DNA impairment, cell cycle arrest, and down-regulation of neuronal biomarkers. The consequences of the investigation concluded that QUE mitigates the DON-induced stress viz., decreased ROS production and LPO generation, upholding MMP and DNA integrity and regulation of neuronal biomarker gene expression in SH-SY5Y cells.
... Subtraction of the "calibrator" from each ∆CT-value leads to ∆∆CT. Fold induction was calculated based on the formula 2 -∆∆CT(Tellmann and Geulen, 2006). ...
Thesis
DLC1 is a tumor suppressor protein downregulated in gastric cancer. It is a negative regulator of RHOA, which is the major oncogenic driver mutation of human diffuse gastric cancer. Helicobacter infection leads to chronic gastric inflammation, which is a risk factor for the development of carcinoma. The Helicobacter toxin CagA activates numerous signaling pathways including RHOA. The role of DLC1 in Helicobacter-related gastric disease is unknown and was analyzed by this thesis. DLC1gt/+ mice showed increased gastric inflammatory infiltration. Involvement of DLC1 in the regulation of the immune response was confirmed by RT-qPCR analyses. Furthermore, DLC1 was shown to be localized to enterochromaffin-like cells. Quantitative gene expression analyses verified a crucial role of the tumor suppressor in homeostasis of gastric acid in vivo, thereby preventing the development of gastric cancer. This study further demonstrates an interaction between DLC1 and CagA. DLC1 was transcriptionally downregulated by CagA and the two proteins fulfilled antagonizing functions by complex formation. DLC1 counters the oncogenic signaling of CagA in vitro by promoting adhesion, suppressing proliferation and antagonizing CagA concerning the hypoxic stress response. DLC1 furthermore inhibited CagA-mediated G-protein-coupled RHOA activation. In vivo therapy of a preclinical model for human gastric cancer with an inhibitor of the RHO/ROCK-pathway efficiently reduced tumor growth. These findings propose inhibition of this pathway as a novel treatment strategy for human gastric cancer. In summary, this thesis postulates a protective role of DLC1 in initial steps of gastric disease by antagonizing CagA-mediated oncogenic signaling. Transcriptional downregulation of DLC1 by CagA promotes oncogenic effects and constitutes DLC1 as an early molecular marker for Helicobacter-related gastric disease. Due to the involvement of CagA and DLC1 in the regulation of RHOA, Helicobacter-related gastric disease can be assigned to diffuse genomically stable gastric cancer. This represents a new risk stratification for Helicobacter-infected gastric cancer patients. Suppression of tumor growth using an inhibitor of the RHOA downstream effector ROCK proposes DLC1 as a future druggable target in human gastric cancer.
... Crossing point values for each target gene were normalized to the respective crossing point values for the reference gene RP-II. Data are presented as normalized ratios of genes along with standard error using Roche Applied Science E-Method (Tellmann and Olivier 2006). RNA polymerase-II (RP-II) was used as an endogenous reference gene to normalize the target gene expression. ...
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Neuroprotection from nerve agent such as soman-induced neural damage is a major challenge for existing drugs. Nerve agent exposure can cause many neural effects in survivors arising mainly due to acetylcholinesterase (AChE) inhibition or death within minutes. Unraveling the mechanisms underlying the nerve agent-induced multiple neurological effects is useful to develop better and safe drugs. The present study aimed to understand the molecular response during soman exposure and to evaluate the neuroprotective efficacy of galantamine on nerve agent-induced neurotoxic changes. mRNA expression studies using quantitative real-time PCR revealed significant changes in S-100β, Gfap, c-fos, and Bdnf in the hippocampus and piriform cortex after soman (90 μg/kg, s.c) exposure. Immunoblot analysis showed acute soman exposure significantly increased the protein levels of neuroglial markers (S100-β and GFAP); c-Fos and protein oxidation in discrete rat brain areas indicate their role in nerve agent-induced neurotoxicity. Induction of BDNF levels during soman exposure may indicate the recovery mechanisms activation. AChE was inhibited in the blood and brain up to 82% after soman exposure. Antidotal treatment with galantamine alone (3 mg/kg) and galantamine plus atropine (10 mg/kg) has protected animals from nerve agent-induced intoxication, death, and soman-inhibited AChE up to 45% in the blood and brain. Animal received galantamine displayed increased levels of neuroprotective genes (nAChRα-7, Bcl-2, and Bdnf) in the brain suggest the neuroprotective value of galantamine. Neuroglial changes, c-Fos, and protein oxidation levels significantly reduced after galantamine and galantamine plus atropine treatment indicate their potential antidotal value in nerve agent treatment.
... The RT-PCR analysis consists of 10 min of cDNA synthesis at 50°C for one cycle, 5 min of polymerase activation at 95°C and followed by 35 cycles of PCR at 95°C for 10 s, and finally 60°C for 30 s for combined annealing and extension. The relative fold expression of test genes was measured with respect to the normalized reference gene (Tellman and Olivier, 2006). ...
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In the present study, for the first time, biomimetization of hydroxyapatite (HA) with Azadirachta indica (AI) was proposed and established its antioxidant, antibacterial, and anti-inflammatory potential on lipopolysaccharide (LPS). The ethanolic extract of AI was found rich with phenolics and flavonoids, and determined their concentration as 8.98 ± 1.41 mg gallic acid equivalents/g and 5.46 ± 0.84 mg catechin equivalents/g, respectively. The HA was prepared by sol-gel method from calcium nitrate tetrahydrate and orthophosphoric acid, and successfully biomimetization was performed with ethanolic extract of AI. The FTIR analysis settled that as-synthesized HA-AI composite was comprised of both HA and AI. The XRD pattern and Zeta potential revealed that the HA-AI composite was crystalline and negative in charge (−24.0 mV). The average-size distribution, shape, and size of the HA-AI composite was determined as 238.90 d.nm, spherical, and 117.90 nm from size distribution, SEM, and HR-TEM analysis, respectively. The SEM-EDX concluded that the HA-AI composite was comprised of elements of HA as well as AI. The HA-AI composite presented potential antioxidant activity and its EC50 values (dose required to inhibit about half of the radicals) for ABTS and DPPH assays were determined as 115.72 ± 2.33 and 128.51 ± 1.04 μg/ml, respectively. The HA-AI composite showed potent antibacterial activity, and minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) towards S. aureus (ATCC 700699) and E. coli (ATCC 10536) were correspondingly determined as 266.7 ± 28.87 and 600.0 ± 50.0 μg/ml, and 400.0 ± 86.6 and 816.7 ± 76.38 μg/ml. Most importantly, HA-AI composite presented the potential anti-inflammatory response toward lipopolysaccharide (LPS) in RAW 264.7 cells. The dose of 250 μg/ml of HA-AI composite has shown optimum protection against LPS-induced stress (1 μg/ml) by scavenging oxidants and regulating mitochondrial membrane potential (MMP), inflammatory and apoptotic factors. Thus, this study concluded that the impartation of potential biofunctional features to HA from plant sources through biomimetic approach is much beneficial and could find potential application in dentistry and orthopedic.
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The Wilms' tumour suppressor, WT1, is a zinc finger protein with key roles in normal development of the genitourinary system and tumourigenesis. Mutations or deletion of WT1 result in a spectrum of developmental disorders and susceptibility to Wilms' tumour in children. Ectopic expression of Wt1 associated with oncogenic functions has been observed in a large number of malignancies, including haematological and solid cancers. Although Wt1 is predominantly a nuclear protein in normal tissues, it is mostly cytoplasmic in the majority of Wt1-expressing tumours. Actin was identified in this study as a new WT1 interaction partner both in the nucleus and in the cytoplasm. We confirmed this interaction both in vitro and in vivo and started to explore its functional significance. Perturbation of the actin cytoskeleton moved Wt1 off the polysome fraction in the cytoplasm, cancelled its nucleo-cytoplasmic shuttling and altered Wt1 DNA- and RNA-binding abilities. These data have implications for Wt1 functions in relation to RNA metabolism and response to cytoskeletal alterations in cancer cells. Thus, our findings could shed more light on the functions of both these proteins and possibly pave way for the development of new cancer therapies.
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As an important natural enemy of aphids and other pests in agriculture, Harmonia axyridis has been widely used in classic biological control. The testis and male accessory gland (MAG) of H. axyridis are typically associated with the ability of egg-laying by multiple mating and influence mass artificial breeding for biological control. Development-related genes might impact the growth of adult testis and MAGs, but this has not yet been reported. Here, we use an integrative time-series analysis with RNA-seq in the testis and MAGs of H. axyridis adults to detect development-related genes. >10.3 Gb of clean data were obtained. All differentially expressed genes (DEG) between samples were analyzed with five databases for building a DEG annotation database. By combining previous reports, the DEG annotation database, and gene expression level changes in four different stages, the 26 DEGs related to testis and MAGs development were identified. To validate the expression profile, 15 random DEGs were chosen to perform RT-qPCR, and they were all in accordance with the RNA-Seq results. Taken together, this study established a robust pipeline for the discovery of key genes using RNA-seq and allowed for the class identification of development-related genes in the testis and MAGs for comprehensive characterization.
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Accumulation of advanced glycation end products (AGEs) is accompanied by increased free radical activity which contributes to ageing and the development or worsening of degenerative diseases. Apart from other physiological factors, AGEs are also an important biomarker for premature ageing. Here we report protein modifications (glycation) in a mouse model of lamin B receptor deficient icJ/icJ mice displaying skin defects similar to those of classical progeria. Therefore, we analysed AGE-modifications in protein extracts from various tissues of icJ/icJ mice. Our results demonstrated that pentosidine as well as argpyrimidine were increased in icJ/icJ mice indicating a modification specific increase in biomarkers of ageing, especially derived from glycolysis dependent methylglyoxal. Furthermore, the expression of AGE-preventing enzymes (Glo1, Fn3k) differed between icJ/icJ and control mice. The results indicate that not only lamin A but also the lamin B receptor may be involved in ageing processes.
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Abrin toxin is a plant glycoprotein, which is similar in structure and properties to ricin and is obtained from the seeds of Abrus precatorius (jequirity bean). Abrin is highly toxic, with an estimated human fatal dose of 0.1-1 μg/kg, and has caused death after accidental and intentional poisoning. Abrin is a potent biological toxin warfare agent. There are no chemical antidotes available against the toxin. Neurological symptoms like delirium, hallucinations, reduced consciousness and generalized seizures were reported in human poisoning cases. Death of a patient with symptoms of acute demyelinating encephalopathy with gastrointestinal bleeding due to ingestion of abrin seeds was reported in India. The aim of this study was to examine both dose and time-dependent transcriptional responses induced by abrin in the adult mouse brain. Mice (n=6) were exposed to 1 and 2 LD50 (2.83 and 5.66 μg/kg respectively) dose of abrin by intraperitoneal route and observed over 3 days. A subset of animals (n=3) were sacrificed at 1 and 2 day intervals for microarray and histopathology analysis. None of the 2 LD50 exposed animals survived till 3 days. The histopathological analysis showed the severe damage in brain and the infiltration of inflammatory cells in a dose and time dependent manner. The abrin exposure resulted in the induction of rapid immune and inflammatory response in brain. Clinical biochemistry parameters like lactate dehydrogenase, aspartate aminotransferase, urea and creatinine showed significant increase at 2-day 2 LD50 exposure. The whole genome microarray data revealed the significant regulation of various pathways like MAPK pathway, cytokine-cytokine receptor interaction, calcium signaling pathway, Jak-STAT signaling pathway and natural killer cell mediated toxicity. The comparison of differential gene expression at both the doses showed dose dependent effects of abrin toxicity. The real-time qRT-PCR analysis of selected genes supported the microarray data. This is the first report on host-gene response using whole genome microarray in an animal model after abrin exposure. The data generated provides leads for developing suitable medical counter measures against abrin poisoning.
Article
T-2 toxin is one of the most toxic among several trichothecenes involved in both human and animal poisoning cases. We investigated the biochemical and histological alterations behind inflammation and cutaneous injury caused by T-2 toxin. Swiss albino mice were exposed to T-2 toxin topically at doses of 0.5, 1 and 2 LD50 (2.97, 5.94 and 11.88mg/kg respectively) and observed till 3, 24 and 72h. Topical application of T-2 toxin resulted in skin oxidative stress in terms of increased reactive oxygen species generation, lipid peroxidation and neutrophil mediated myeloperoxidase activity. The histological alterations include degenerative changes like vacuolation, ballooning of basal keratinocytes and infiltration of inflammatory cells in dermis. The mRNA levels of skin pro-inflammatory cytokines TNF-α, IL-6, and IL-1β showed significant up regulation. Anti-inflammatory cytokines IL-10 showed significant up regulation at 24h whereas IL-4 showed down regulation for all the doses and time points. Gelatin zymography and immunoblot analysis of matrix metalloproteinases (MMP)-9 and 2 indicated MMP activation and their role in degenerative skin histological changes. Time dependent increase in inducible nitric oxide synthase levels was seen. Immunoblot analysis revealed significant increase in the levels of phosphorylated p38 mitogen activated protein kinase (MAPK). Flow cytometry analysis of propidium iodide stained epidermal cells showed increase in sub-G1 population at all the doses and time points indicating apoptosis. In summary, T-2 toxin induced skin inflammation and cutaneous injury is mediated through oxidative stress, activation of myeloperoxidase, MMP activity, increase in inflammatory cytokines, activation of p38 MAPK and apoptosis of epidermal cells leading to degenerative skin histological changes.
Article
The prevalence of osteoarthritis (OA) varies between joints. Cartilage in eight different joints was evaluated to elucidate the disparate susceptibilities between joints to post-traumatic OA (PTOA) and provide evidence for joint specific clinical treatments. The hypothesis was that cartilage in different joints would have varying cell death and anabolic gene expression profiles after injury. Adult equine cartilage explants were harvested from shoulder (SH), elbow (EL), carpal (CA), metacarpophalangeal (MC), patellofemoral (FP), tarsal (TA), metatarsophalangeal (MT), and proximal interphalangeal (PP) joints, and were injured by loading with 30 MPa within 1 second. Fractional dissipated energy, cell density, cell death, and gene expression were quantified. PP had the highest fractional dissipated energy (94%, 95% confidence interval [CI] 88-101%). Cell density was most dense in superficial zone in all samples, with MC and MT having the highest peak density. Injured samples had significantly higher cell death (13.5%, 95% CI 9.1-17.9%) than non-injured samples (6.8%, 95% CI 2.5-11.1%, p=0.016); however, cell death after injury was not significantly different between joints. Gene expression was significantly different between joints. CD-RAP expression in normal cartilage was lowest in FP (Cp=21, 95% CI -80-122). After injury, the change in CD-RAP expression increased and was highest in FP (147% relative increase after injury, 95% CI 64-213). Different joints have different baseline characteristics, including cell density and gene expression, and responses to injury, including energy dissipation and gene expression. These unique characteristics may explain differences in OA prevalence and suggest differences in susceptibility to PTOA. Understanding differences in the response to injury and potential susceptibility of OA can lead to the development of preventative or treatment strategies. Gene expression, cartilage injury, chondrocyte, multiphoton microscopy, cartilage biomechanical properties. The prevalence of OA is variable among joints; however, most laboratory studies are performed on a single joint - most commonly the knee and extrapolated to other joints such as the ankle or shoulder. A small number of studies have compared knee and ankle cartilage and reported differences in mechanical properties and gene expression. There are differences in baseline cell density and gene expression and differences in response to injury, including gene expression and cell death. This suggests that there are inherent differences leading to varying susceptibilities in OA prevalence among joints. Joint-specific treatments may improve OA therapies. Copyright © 2015. Published by Elsevier Ltd.
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Aerolysins are virulence factors belonging to the β pore-forming toxin (β-PFT) superfamily that are abundantly distributed in bacteria. More rarely, β-PFTs have been described in eukaryotic organisms. Recently, we identified a putative cytolytic protein in the snail, Biomphalaria glabrata, whose primary structural features suggest that it could belong to this β-PFT superfamily. In the present paper, we report the molecular cloning and functional characterization of this protein, which we call Biomphalysin, and demonstrate that it is indeed a new eukaryotic β-PFT. We show that, despite weak sequence similarities with aerolysins, Biomphalysin shares a common architecture with proteins belonging to this superfamily. A phylogenetic approach revealed that the gene encoding Biomphalysin could have resulted from horizontal transfer. Its expression is restricted to immune-competent cells and is not induced by parasite challenge. Recombinant Biomphalysin showed hemolytic activity that was greatly enhanced by the plasma compartment of B. glabrata. We further demonstrated that Biomphalysin with plasma is highly toxic toward Schistosoma mansoni sporocysts. Using in vitro binding assays in conjunction with Western blot and immunocytochemistry analyses, we also showed that Biomphalysin binds to parasite membranes. Finally, we showed that, in contrast to what has been reported for most other members of the family, lytic activity of Biomphalysin is not dependent on proteolytic processing. These results provide the first functional description of a mollusk immune effector protein involved in killing S. mansoni.
Article
OBJECTIVE To investigate the role of the major equine acute phase protein serum amyloid A (SAA) in inflammation of equine intraarticular tissues. SAMPLE Articular chondrocytes and fibroblast-like synoviocytes (FLSs) from 8 horses (4 horses/cell type). PROCEDURES Chondrocytes and FLSs were stimulated in vitro for various periods up to 48 hours with cytokines (recombinant interleukin [IL]-1β, IL-6, tumor necrosis factor-α, or a combination of all 3 [IIT]) or with recombinant SAA. Gene expression of SAA, IL-6, matrix metalloproteinases (MMP)-1 and -3, and cartilage-derived retinoic acid-sensitive protein were assessed by quantitative real-time PCR assay; SAA protein was evaluated by immunoturbidimetry and denaturing isoelectric focusing and western blotting. RESULTS All cytokine stimulation protocols increased expression of SAA mRNA and resulted in detectable SAA protein production in chondrocytes and FLSs. Isoforms of SAA in lysed chondrocytes and their culture medium corresponded to those previously detected in synovial fluid from horses with joint disease. When exposed to SAA, chondrocytes and FLSs had increased expression of IL-6, SAA, and MMP3, and chondrocytes had increased expression of MMP-1. Chondrocytes had decreased expression of cartilage-derived retinoic acid-sensitive protein. CONCLUSIONS AND CLINICAL RELEVANCE Upregulation of SAA in chondrocytes and FLSs stimulated with proinflammatory cytokines and the proinflammatory effects of SAA suggested that SAA may be involved in key aspects of pathogenesis of the joint inflammation in horses.
Article
A characteristic feature of peanut is the subterranean fructification, geocarpy, in which the gynophore ('peg'), a specialized organ that transitions from upward growth habit to downward outgrowth upon fertilization, drives the developing pod into the soil for subsequent development underground. As a step towards understanding this phenomenon, we explore the developmental dynamics of the peanut pod transcriptome at 11 successive stages. We identified 110 217 transcripts across developmental stages and quantified their abundance along a pod developmental gradient in pod wall. We found that the majority of transcripts were differentially expressed along the developmental gradient as well as identified temporal programs of gene expression, including hundreds of transcription factors. Thought to be an adaptation to particularly harsh subterranean environments, both up- and down-regulated gene sets in pod wall were enriched for response to a broad array of stimuli, like gravity, light and subterranean environmental factors. We also identified hundreds of transcripts associated with gravitropism and photomorphogenesis, which may be involved in the geocarpy. Collectively, this study forms a transcriptional baseline for geocarpy in peanut as well as provides a considerable body of evidence that transcriptional regulation in peanut aerial and subterranean fruits is complex.
Article
With 9 figures and 1 table AbstractOver the past five decades, cultivated peanut in China has been subjected to strong artificial selection in breeding programmes. To investigate the impact of artificial selection on expression diversity, we compared gene expression profiles in pod and leaf of five widespread cultivars in Southern China. In terms of tissues, hierarchical clustering analysis revealed that expression data of pod and leaf generated different dendrograms owing to artificial selection. K‐means analysis also showed that there were 16 gene expression patterns in leaf, while only eight in pod. In considering cultivars, a cultivar specificity index (τ) was employed to characterize expression patterns, which suggested that genes having 0.15 80% of all expression patterns. Additionally, the diversity of gene expression in pod among cultivars of the ‘YY7’ pedigree decreased from 23.8% to 3.9%. Taken together, nucleotide polymorphisms in regulatory elements owing to artificial selection led to low‐expression polymorphisms in both tissues and cultivars, contributed to the narrow genetic diversity and might be a driving force behind the breeding of cultivated peanut.
Article
T-2 toxin is the most toxic trichothecene and a frequent contaminant in many agriculture products. Dietary ingestion represents the most common route of T-2 toxin exposure in humans. T-2 toxin exposure leads to many pathological conditions like nervous disorders, cardiovascular alterations, immune depression and dermal inflammation. However, the neuronal toxicity of T-2 toxin in vitro remains unclear. In the present study, we investigated the mechanism of T-2 toxin-induced apoptosis in human neuroblastoma cells (IMR-32). T-2 toxin was cytotoxic at a low concentration of 10 ng/ml. The 50 % inhibitory concentration (IC50) of T-2 toxin was found to be 40 ng/ml as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, crystal violet dye exclusion test and lactate dehydrogenase (LDH) leakage. T-2 toxin increased intracellular reactive oxygen species generation as early as 15 min and peaked at 60 min as analyzed by flow cytometry. Annexin V + propidium iodide staining showed time-dependent increase in percent apoptotic cells. DNA gel electrophoresis showed oligonucleosomal DNA fragmentation typical of apoptotic cells. Additionally, casapse-3 activation and PARP cleavage indicated involvement of mitochondrial mediated caspase-dependent pathway of apoptosis. Cell cycle analysis revealed time-dependent increase in sub-G1 population of cells and significant up-regulation of CDK2, CDK6, cyclin A and p21 messenger RNA (mRNA) levels. Exposure to T-2 toxin induced the phosphorylation of extracellular signal-regulated kinase (ERK), p38-mitogen-activated protein kinase and c-jun N-terminal kinases (JNK). Analysis of human phospho-mitogen-activated protein kinase (MAPK) antibody array revealed time-dependent increase in phosphorylation. Upstream of ERK pathway Grb2, Ras and Raf and downstream transcription factors c-fos and c-jun were significantly up-regulated. Z-VAD-FMK and MAPK inhibitors (PD 98059, SB 203580 and ZM 336372) exposure prior to T-2 toxin treatment significantly decreased percent of apoptotic cells compared to only T-2 toxin-exposed cells. Results of the present study show that T-2 toxin at nanogram concentrations can induce apoptosis in human neuronal cells through multiple signal transduction pathways. The study provides possible leads for developing therapeutic approaches to prevent T-2 toxin-induced neurotoxicity.
Article
Largemouth bass ranavirus (LMBV) has caused mortality in largemouth bass and led to huge economic losses in the aquaculture industry. Here, we developed a novel, fast, and simple isothermal recombinase polymerase amplification assay (RPA) for LMBV detection with primers designed on the basis of a fragment of the major capsid protein gene. The reaction conditions were optimized and the RPA method was specific for LMBV, as the DNA of other four Iridoviridae viruses (Singapore grouper iridovirus, soft-shelled turtle iridovirus, tiger frog virus, and large yellow croaker iridovirus), white spot syndrome virus, grass carp reovirus, and healthy largemouth bass could not be amplified. The detection limit of LMBV-RPA was 89 copies/μL, which was comparable to the sensitivity determined by real-time PCR. Finally, the RPA method was validated to be a simple, convenient, rapid, and field diagnostic tool for LMBV detection using 10 clinical samples. In this paper, the RPA method is firstly applied to the diagnosis and monitoring of LMBV infection in the aquaculture industry of largemouth bass, which shows great prospects for on-site diagnostics of LMBV using nearly free instruments.
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