Differences In Relative Gene Expression Research Articles

Comparative gene expression and genomics reflect geographical divergence in the plant symbiotic and entomopathogenic fungal genus Metarhizium

Several species within the fungal genus Metarhizium can both infect insects and colonize plant roots. In Brazil, a specific subgroup within Metarhizium anisopliae s.str. named “subclade Mani 2” is frequently observed infecting above-ground insects, whereas sympatric M. robertsii and M. brunneum predominantly occur in the soil environment. Genotypic variability within the genus may be linked to adaptations to these different habitats. We present a comparative analysis of the complete genomes and the adhesin genes Mad1 and Mad2 of 14 Metarhizium isolates representing M. anisopliae Mani 2 (n = 6), M. robertsii (n = 5) and M. brunneum (n = 3). In addition, the relative gene expression of six selected target genes was compared in root exudate solution and insect cuticle suspension. We hypothesized that M. anisopliae Mani 2 is adapted to insect-pathogenicity in the above-ground environment, reflected by higher relative expression of pathogenicity-related genes. In contrast, M. robertsii and M. brunneum are adapted to the soil environment, hence hypothesized to have a higher expression of genes related to plant associations. Phylogenomic and adhesin phylogenetic trees revealed species differences but also intraspecific variability associated with the geographic origin of isolates. Differences in relative gene expression were observed, with one pathogenicity-related gene ( Pr1 ) being higher expressed in M. anisopliae . The insect adhesion Mad1 gene was more conserved than the plant adhesion Mad2 and similarly expressed in exudate solution, while Mad2 was highly expressed by all Brazilian isolates in both exudate and cuticle conditions. The variabilities observed correlated with different habitats and lifestyles, demonstrating the importance of selecting a diverse collection of isolates in genomic and gene expression studies. • Genotypic variation of Metarhizium correlates with geographic origin. • Intraspecific variation in Mad1 and Mad2 likely reflects adaptation to different habitats. • Variation in expression of host-associated genes reflects functional divergence within Metarhizium .

TMIC-55. IMMUNE DIGITAL-SPATIAL PROFILING AND CHARACTERIZATION OF GLIOMA MICROENVIRONMENT AFTER ADOPTIVE CELLULAR THERAPY

Abstract INTRODUCTION Our group has previously shown that Adoptive Cellular Therapy (ACT) is efficacious against CNS malignancies including medulloblastoma, brain stem glioma, and glioblastoma. In this study, we characterized the spatial distribution of immune cells within the tumor microenvironment after ACT and conducted regional genomic analysis in situ to obtain insights into the underlying cellular dynamics. This method enabled us to define specific differences in gene expression within the T cells in the treated versus untreated tumors including changes in gene expression of regulatory T cell (Treg) associated genes, such as Runx1 and TGF-β. We also found a significant increase in Batf3 expression associated with therapy. METHODS We performed in situ GeoMx Digital Spatial Profiling (DSP) and whole genome transcriptomics (RNA) of murine glioma KR-158B-luciferase tumors after adoptive cellular therapy. ACT was conducted in orthotopic KR158B tumor-bearing mice. Histological slides were sent to be stained for CD45, CD3, GFP (to mark hematopoietic stem cell-derived cells) and nuclei, and processed following GeoMx DSP workflow for RNA. Overall gene expression clustering profiles were conducted using a Principal Component Analysis (PCA), and relative gene expression differences were analyzed by unpaired t-tests. RESULTS/CONCLUSIONS We observed differential expression of a substantial number of genes in the ACT-treated group compared to control. Our findings also indicate that tumors treated with ACT cluster separately from control tumors in PCA analysis. Furthermore, we found downregulation of genes associated with the suppressive properties of regulatory T cells (Runx1 and TGF-β), and upregulation of dendritic cell characteristic genes (Batf3) and antigen presentation genes (H2-Ab1) in ACT-treated tumors vs control. These findings provide insights into the immune cell dynamics and specific genes that are differentially expressed within the tumor microenvironment. This could help improve current adoptive cellular therapies to treat brain tumors.

Abstract 98: Harnessing Novel Gene Expression Analyses To Identify Drivers Of Ear Regeneration In MRL Mice

Purpose: The “super-healing” MRL/MpJ mouse is uniquely able to regenerate ear punch wounds, while dorsal wounds heal in a typical scarring fashion. Identification of the genes responsible for ear regeneration in these mice could provide insights toward novel anti-scarring therapies. However, previous methods for identifying the genetic basis of species-/strain-specific traits are imprecise and extremely resource-intensive. Recent studies have revealed that, by outbreeding a strain with a trait of interest (here, regenerative ear healing), assessing the hybrid offspring for spatial/tissue-specific differences in relative gene expression from the two parent alleles (“differential allele-specific expression”) can reveal genes underlying that phenotype. We hypothesized that differential allele-specific expression could represent a novel approach to identify genes driving regenerative ear healing in MRL mice. Methods: MRL/MpJ (MRL) and CAST/EiJ (CAST) mouse genome sequences were obtained. Variant calling was performed to identify sites where the sequences differed. MRL and CAST mice were cross-bred, F1 offspring underwent dorsal excisional and ear punch wounding (standard protocols), and wounds were harvested on POD7. Following incubation in ammonium thiocyanate, epidermis and underlying cartilage (for ear wounds) were removed under a surgical microscope. Dorsal and ear wound dermis were digested, and inflammatory/immune cells (CD45+; chosen because prior studies have implicated altered wound inflammatory response in MRL regeneration) were isolated by FACS and subjected to bulk RNA-sequencing. Reads were mapped to the MRL or CAST genome based on strain-specific variants, and genes with significantly differing expression (Benjamini-Hochberg method, FDR=5%) from the MRL vs. CAST allele and from dorsal vs. ear wounds were identified. Results: Ear wounds in MRLxCAST hybrid mice exhibited grossly apparent regeneration over time, consistent with prior reports. Roughly equal proportions of RNA-seq reads mapped to MRL vs. CAST alleles, indicating minimal mapping bias. Principal component analysis demonstrated that reads clustered by parent allele (MRL vs. CAST) and wound site (ear vs. dorsum), with good reproducibility between biological replicates (n=4). Importantly, the number of genes with significantly different expression from MRL vs. CAST allele was substantially (>10-fold) greater in the ear than the dorsum (2531 vs. 159 unique genes. The majority of allele-specific expression in the dorsum was also observed in the ear, overlapping region; 878 shared genes vs. 159 unique to dorsum). Conclusions: In MRL hybrid mice, inflammatory cells exhibit markedly greater allele-specific gene expression in ear wounds (regenerative) than dorsal wounds (scarring). This finding would be consistent with MRL-specific gene cis-regulatory activity leading to the improved healing phenotype seen in the ear. Our results suggest that probing differential allele-specific gene expression may represent a powerful computational approach for identifying the genetic basis of complex traits in mammals. We will examine inflammatory and other cell types (e.g., fibroblasts) in order to identify specific genes with differential allele-specific expression in regenerative versus scarring wounds, with the ultimate goal of identifying novel targets for preventing scarring.

Influence of ochratoxin A on adaptation of Penicillium nordicum on a NaCl-rich dry-cured ham-based medium

Iberian dry-cured ham is an important meat product with high consumption worldwide. The special ecological conditions occurring throughout its ripening favour surface colonisation of filamentous fungi. Normally, moulds contribute to the development of the sensory qualities of the ham; however, some toxigenic species, such as Penicillium nordicum, are able to successfully adapt to the NaCl-rich environment found in dry-cured ham and produce ochratoxin A (OTA) in this product. Moreover, it was suggested that the biosynthesis of OTA by P. nordicum itself may support the adaptation to this food environment. However, this mechanism has not been completely elucidated yet. The objective of this work was to evaluate the influence of different concentrations of commercial OTA (cOTA, at 0, 0.2, 1 and 5 ppb) on growth rate, biosynthetic- and stress-related gene expression and OTA production by two P. nordicum strains (Pn15 and Pn69) on dry-cured ham based-media. Two NaCl conditions (0% and 10%) were evaluated for each cOTA level. In general, no intra-strain and inter-strain differences in growth rates were found among the conditions tested. The stress-related Hog1 gene expression of the strain Pn15 was affected by cOTA and NaCl concentration whilst the strain Pn69 was not affected by these variables. The expression of OTA-related otapks and otanps genes of the strain Pn15 was affected by several NaCl and cOTA combinations. However, the strain Pn69 showed no differences in relative gene expression. Regarding to OTA production, different behaviours were displayed by the two strains. The strain Pn15, which produced high OTA amounts by itself, produced OTA without the necessity of the presence of NaCl or cOTA as stressors. However, the presence of cOTA triggers OTA production by the weak OTA producing Pn69 in the absence of NaCl. In addition, although a moderate correlation was found between the expression of the OTA-related genes and mycotoxin produced by P. nordicum in the absence of NaCl, none was obtained between Hog1 gene expression and mycotoxin production. This study is a step forward for a better understanding of the ability of P. nordicum producers of OTA to colonise NaCl-rich habitats such as Iberian ham for proposing actions to minimise OTA contamination in this meat product.

Differential survivorship of congeneric ornamental fishes under forecasted climate changes are related to anaerobic potential

Two Amazonian closely related tetras – cardinal Paracheirodon axelrodi and green neon P. simulans – were artificially acclimatized to environmental chambers mimicking future climate change scenarios (mild, moderate and extreme), using a microcosm facility. P. simulans survived (100%) to all scenarios after 30 days exposure, while P. axelrodi presented decreasing survival percentages according to environmental severity. These differences may be the reflection of distinct natural acclimatization to microhabitats between the species, which differ in thermal conditions. Survival responses might be related to differences in relative gene expression of lactate dehydrogenase (Ldh), suggesting that P. axelrodi anaerobic potential is lower or non-existent compared to P. simulans, not tolerating long-term thermal challenges. Accordingly, increases in temperature and in CO2 levels caused increases in energy demand and resulted in activation of the anaerobic pathway, as demonstrated by the higher enzyme levels measured in head and tail portions of both species. Sustained anaerobic glycolysis is possible when fish live in challenging environments (low oxygen or high temperature). Our results clearly show that P. simulans has a larger scope for survival to higher energy demands due to its increased anaerobic potential compared to P. axelrodi.

BIF-1 Relative Gene Expression Study in Breast Cancer Tumor and Normal Adjacent Tissues using Real Time PCR

Introduction: Breast cancer is a complex disease due to several biological Levels of genetic and epigenetic changes that ultimately alter the cellular processes including proliferation, apoptosis, angiogenesis and the acquisition of an invasive phenotype results. BIF-1 (BAX interacting factor) is a tumor suppressor gene which is involved in the regulation of mitochondrial membrane bends and containing BAR domains that mediate interaction with BAX. The aim of this study was to investigate the relative gene expression differences of BIF-1 in human breast tumor compared with normal adjacent tissues using Real-Time PCR method. Materials & methods: This case-control study was implemented on 50 tumour and normal adjacent tissue samples of breast cancer patients. The patients had referred to Milad and Khatam hospitals in 2014. After collecting the samples, total RNA was extracted from tissue by TriPure RNA isolation kit. The cDNA was synthesized from extracted RNA by Revert AID First strand cDNA synthesis kit. Finally the BIF-1 gene expression was analyzed in tumor and normal adjacent tissues using SYBR green Real-Time PCR. Findings: Our results indicated that BIF-1 was down regulated in 34 breast cancer patients which was statically significant (P=0.001). This lower mean of BIF-1 gene expression in tumor tissues compared with normal adjacent ones confirmed its role as a tumor suppressor gene. Discussion & conclusions: Due to the significant difference in BIF-1 gene expression in breast tumor and normal tissue, this gene may candidate as a possible biomarker related to breast cancer.

Sex chromosome complement involvement in angiotensin receptor sexual dimorphism

This study aimed to define whether sex chromosome complement (SCC) may differentially modulate sex differences in relative gene expression of basal Agtr1a, Agtr2, and Mas1 receptors at fore/hindbrain nuclei and at medulla/cortical kidney.Samples were collected from gonadectomized male (XX and XY) and female (XX and XY) mice of the “four core genotypes” model. At brain level, a SCC effect at the area postrema was demonstrated. An increase in mRNA level of Agtr1a and Agtr1a/Agtr2 ratio in XY-SCC mice was associated with a decrease in Mas1 compared to XX-SCC mice. In the renal cortex, a SCC effect for Agtr2 and Mas1 was observed. Regardless of sex (male or female), XX-SCC mice expressed higher levels of mRNA Agtr2 and Mas1 than XY-SCC mice {F(1,12) = 6,126,p < 0.05; F(1,21) = 5,143,p < 0.05}. Furthermore, XX-female mice showed a significant increase in Mas1 expression compared to XY-female mice.These results reveal a SCC modulatory effect at central and kidney level on angiotensin receptor expression, with an enhancement of the vasodilatory arm in XX-mice and an increase in the vasoconstriction arm in XY-mice, which may underlie sex differences in the regulation of arterial pressure.

The gut microbiota modulates host amino acid and glutathione metabolism in mice.

The gut microbiota has been proposed as an environmental factor that promotes the progression of metabolic diseases. Here, we investigated how the gut microbiota modulates the global metabolic differences in duodenum, jejunum, ileum, colon, liver, and two white adipose tissue depots obtained from conventionally raised (CONV-R) and germ-free (GF) mice using gene expression data and tissue-specific genome-scale metabolic models (GEMs). We created a generic mouse metabolic reaction (MMR) GEM, reconstructed 28 tissue-specific GEMs based on proteomics data, and manually curated GEMs for small intestine, colon, liver, and adipose tissues. We used these functional models to determine the global metabolic differences between CONV-R and GF mice. Based on gene expression data, we found that the gut microbiota affects the host amino acid (AA) metabolism, which leads to modifications in glutathione metabolism. To validate our predictions, we measured the level of AAs and N-acetylated AAs in the hepatic portal vein of CONV-R and GF mice. Finally, we simulated the metabolic differences between the small intestine of the CONV-R and GF mice accounting for the content of the diet and relative gene expression differences. Our analyses revealed that the gut microbiota influences host amino acid and glutathione metabolism in mice.

Seasonal Differences in Relative Gene Expression of Putative Central Appetite Regulators in Arctic Charr (Salvelinus alpinus) Do Not Reflect Its Annual Feeding Cycle.

The highly seasonal anadromous Arctic charr (Salvelinus alpinus) was used to investigate the possible involvement of altered gene expression of brain neuropeptides in seasonal appetite regulation. Pro-opiomelanocortin (POMCA1, POMCA2), Cocaine and amphetamine regulated transcript (CART), Agouti related Peptide (AgRP), Neuropeptide Y (NPY) and Melanocortin Receptor 4 (MC4-R) genes were examined. The function of centrally expressed Leptin (Lep) in fish remains unclear, so Lep (LepA1, LepA2) and Leptin Receptor (LepR) genes were included in the investigation. In a ten months study gene expression was analysed in hypothalamus, mesencephalon and telencephalon of immature charr held under natural photoperiod (69°38’N) and ambient temperature and given excess feed. From April to the beginning of June the charr did not feed and lost weight, during July and August they were feeding and had a marked increase in weight and condition factor, and from November until the end of the study the charr lost appetite and decreased in weight and condition factor. Brain compartments were sampled from non-feeding charr (May), feeding charr (July), and non-feeding charr (January). Reverse transcription real-time quantitative PCR revealed temporal patterns of gene expression that differed across brain compartments. The non-feeding charr (May, January) had a lower expression of the anorexigenic LepA1, MC4-R and LepR in hypothalamus and a higher expression of the orexigenic NPY and AgRP in mesencephalon, than the feeding charr (July). In the telencephalon, LepR was more highly expressed in January and May than in July. These results do not indicate that changes in central gene expression of the neuropeptides investigated here directly induce seasonal changes in feeding in Arctic charr.

Assessment of CCL2 and CXCL8 chemokines in serum, bronchoalveolar lavage fluid and lung tissue samples from dogs affected with canine idiopathic pulmonary fibrosis

Canine idiopathic pulmonary fibrosis (CIPF) is a progressive disease of the lung parenchyma that is more prevalent in dogs of the West Highland white terrier (WHWT) breed. Since the chemokines (C-C motif) ligand 2 (CCL2) and (C-X-C motif) ligand 8 (CXCL8) have been implicated in pulmonary fibrosis in humans, the aim of the present study was to investigate whether these same chemokines are involved in the pathogenesis of CIPF. CCL2 and CXCL8 concentrations were measured by ELISA in serum and bronchoalveolar lavage fluid (BALF) from healthy dogs and WHWTs affected with CIPF. Expression of the genes encoding CCL2 and CXCL8 and their respective receptors, namely (C-C motif) receptor 2 (CCR2) and (C-X-C motif) receptor 2 (CXCR2), was compared in unaffected lung tissue and biopsies from dogs affected with CIPF by quantitative PCR and localisation of CCL2 and CXCL8 proteins were determined by immunohistochemistry.Significantly greater CCL2 and CXCL8 concentrations were found in the BALF from WHWTs affected with CIPF, compared with healthy dogs. Significantly greater serum concentrations of CCL2, but not CXCL8, were found in CIPF-affected dogs compared with healthy WHWTs. No differences in relative gene expression for CCL2, CXCL8, CCR2 or CXCR2 were observed when comparing lung biopsies from control dogs and those affected with CIPF. In affected lung tissues, immunolabelling for CCL2 and CXCL8 was observed in bronchial airway epithelial cells in dogs affected with CIPF. The study findings suggest that both CCL2 and CXCL8 are involved in the pathogenesis of CIPF. Further studies are required to determine whether these chemokines might have a clinical use as biomarkers of fibrosis or as targets for therapeutic intervention.

Single-cell analysis of lung adenocarcinoma cell lines reveals diverse expression patterns of individual cells invoked by a molecular target drug treatment.

BackgroundTo understand the heterogeneous behaviors of individual cancer cells, it is essential to investigate gene expression levels as well as their divergence between different individual cells. Recent advances in next-generation sequencing-related technologies have enabled us to conduct a single-cell RNA-Seq analysis of a series of lung adenocarcinoma cell lines.ResultsWe analyze a total of 336 single-cell RNA-Seq libraries from seven cell lines. The results are highly robust regarding both average expression levels and the relative gene expression differences between individual cells. Gene expression diversity is characteristic depending on genes and pathways. Analyses of individual cells treated with the multi-tyrosine kinase inhibitor vandetanib reveal that, while the ribosomal genes and many other so-called house-keeping genes reduce their relative expression diversity during the drug treatment, the genes that are directly targeted by vandetanib, the EGFR and RET genes, remain constant. Rigid transcriptional control of these genes may not allow plastic changes of their expression with the drug treatment or during the cellular acquisition of drug resistance. Additionally, we find that the gene expression patterns of cancer-related genes are sometimes more diverse than expected based on the founder cells. Furthermore, we find that this diversity is occasionally latent in a normal state and initially becomes apparent after the drug treatment.ConclusionsCharacteristic patterns in gene expression divergence, which would not be revealed by transcriptome analysis of bulk cells, may also play important roles when cells acquire drug resistance, perhaps by providing a cellular reservoir for gene expression programs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0636-y) contains supplementary material, which is available to authorized users.

Genes Involved in Maintaining the Bone Marrow Stroma Are Dysregulated in Patients with Myelofibrosis: Lenalidomide Treatment Upregulates SOCS3

Genes Involved in Maintaining the Bone Marrow Stroma Are Dysregulated in Patients with Myelofibrosis: Lenalidomide Treatment Upregulates SOCS3Background: Primary myelofibrosis (PMF) is the most aggressive of the myeloproliferative neoplasms (MPNs), a group of genetically and clinically heterogenous yet-related diseases with a functionally disturbed hematopoietic niche. Egress of these cells from the BM in patients with PMF, suggests a change in the crosstalk between hematopoietic stem cells (HSCs) and the BM microenvironment (particularly BM stromal cells). Lenalidomide, an immunomodulatory agent, which modulates inflammatory cytokine secretion, angiogenesis and the expression of adhesion molecules, likely has effects on the BM microenvironment.Methods: We studied the expression of a set of genes involved in the organization of the hematopoietic niche in peripheral blood and bone marrow (BM) mononuclear cell (MNC) samples from 32 patients with PMF who participated in a phase 2 trial of lenalidomide plus prednisone (Quintas-Cardama et al., J Clin Oncol 2009;27:4760-766). Sequential samples taken at 3, 6, 9, 12 and >14 months after starting lenalidomide therapy were also analyzed. Baseline BM samples were available for 9 patients and baseline PB samples available for 12 patients and used for our analysis. Quantitative reverse transcriptase-polymerase chain reaction was performed to measure the expression levels of SPARC, COX-2, CXCR4, Pax5C-terminus, SOCS3 and HIF-1a transcripts. β-actin was used as an internal control. In addition, we compared the gene expression levels at baseline with healthy control samples.Results: We found that at baseline (before treatment) COX-2 was upregulated, while CXCR4, Pax5 C-terminus, SOCS3 and HIF-1a were downregulated when compared with expression in healthy BM MNCs. Expression of SPARC, K-RAS,FOS and SOCS2 were not significantly different. In addition, there were no significant differences in relative gene expression between BM MNC and PB samples. We found no correlation between gene expression and JAK2 mutational status or cytogenetic abnormalities. Treatment with LP significantly increased the expression of SOCS3, suggesting that some of the clinical effects of lenalidomide (i.e., reduction in splenomegaly) may be due to a SOCS3-mediated reduction in JAK signaling.Conclusions: Upregulation of COX-2 and downregulation of CXCR4, Pax5C, SOCS3, and HIF-1a may reflect disruptions in the interactions between cells in the BM microenvironment in MF. Increases in SOCS3 expression upon treatment with lenalidomide may lead to reduced JAK-STAT signaling, which may account for some of its clinical effects in myelofibrosis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Brassica villosa, a system for studying non-glandular trichomes and genes in the Brassicas.

Brassica villosa is a wild Brassica C genome species with very dense trichome coverage and strong resistance to many insect pests of Brassica oilseeds and vegetables. Transcriptome analysis of hairy B. villosa leaves indicated higher expression of several important trichome initiation genes compared with glabrous B. napus leaves and consistent with the Arabidopsis model of trichome development. However, transcripts of the TRY inhibitory gene in hairy B. villosa were surprisingly high relative to B. napus and relative transcript levels of SAD2, EGL3, and several XIX genes were low, suggesting potential ancillary or less important trichome-related roles for these genes in Brassica species compared with Arabidopsis. Several antioxidant, calcium, non-calcium metal and secondary metabolite genes also showed differential expression between these two species. These coincided with accumulation of two alkaloid-like compounds, high levels of calcium, and other metals in B. villosa trichomes that are correlated with the known tolerance of B. villosa to high salt and the calcium-rich natural habitat of this wild species. This first time report on the isolation of large amounts of pure B. villosa trichomes, on trichome content, and on relative gene expression differences in an exceptionally hairy Brassica species compared with a glabrous species opens doors for the scientific community to understand trichome gene function in the Brassicas and highlights the potential of B. villosa as a trichome research platform.

TGF-beta1 Does Not Induce Senescence of Multipotent Mesenchymal Stromal Cells and Has Similar Effects in Early and Late Passages

Transforming growth factor-beta 1 (TGF-β1) stimulates a broad range of effects which are cell type dependent, and it has been suggested to induce cellular senescence. On the other hand, long-term culture of multipotent mesenchymal stromal cells (MSCs) has a major impact on their cellular physiology and therefore it is well conceivable that the molecular events triggered by TGF-β1 differ considerably in cells of early and late passages. In this study, we analyzed the effect of TGF-β1 on and during replicative senescence of MSCs. Stimulation with TGF-β1 enhanced proliferation, induced a network like growth pattern and impaired adipogenic and osteogenic differentiation. TGF-β1 did not induce premature senescence. However, due to increased proliferation rates the cells reached replicative senescence earlier than untreated controls. This was also evident, when we analyzed senescence-associated DNA-methylation changes. Gene expression profiles of MSCs differed considerably at relatively early (P 3 - 5) and later passages (P 10). Nonetheless, relative gene expression differences provoked by TGF-β1 at individual time points or in a time course dependent manner (stimulation for 0, 1, 4 and 12 h) were very similar in MSCs of early and late passage. These results support the notion that TGF-β1 has major impact on MSC function, but it does not induce senescence and has similar molecular effects during culture expansion.

Comparison of methods for relative quantification of gene ex- pression using real-time PCR

Quantitative real-time PCR (qPCR) has become a widely used tool for quantifying gene expression. Several methods for relative quantification have been developed, enabling rapid and reliable detection and quantification of specific nucleic acids. These methods, based on qPCR include: the standard curve method, the efficiency calibrated method and the 2−ΔΔCq method. Here we analyzed if these three methods generate comparable results. To evaluate their performance, we analyzed the expression of the nuclease gene MS53_0284 from Mycoplasma synoviae type strain WVU 1853 during in vitro infection of CEC-32 cells, using qPCR. As determined, all three methods generated comparable and reliable results when all necessary conditions were fulfiled. Also, the efficiency calibrated and the standard curve methods were more suitable for quantifying small differences in relative gene expression than the 2−ΔΔCq method.

Expression of the arginine deiminase pathway genes in Lactobacillus sakei is strain dependent and is affected by the environmental pH.

The adaptation of Lactobacillus sakei to a meat environment is reflected in its metabolic potential. For instance, the ability to utilize arginine through the arginine deiminase (ADI) pathway, resulting in additional ATP, represents a competitive benefit. In L. sakei CTC 494, the arc operon (arcABCTDR) shows the same gene order and organization as that in L. sakei 23K, the genome sequence of which is known. However, differences in relative gene expression were found, and these seemed to be optimal in different growth phases, namely, the highest relative gene expression level was in the end exponential growth phase in the case of L. sakei CTC 494 and in the mid-exponential growth phase of L. sakei 23K. Also, the environmental pH influenced the relative expression level of the arc operon, as shown for L. sakei CTC 494, with the highest relative expression level occurring at the optimal pH for growth (pH 6.0). Deviations from this optimal pH (pH 5.0 and pH 7.0) resulted in an overall decline of the relative expression level of all genes of the arc operon. Furthermore, a differential relative expression of the individual genes of the arc operon was found, with the highest relative gene expression occurring for the first two genes of the arc operon (arcA and arcB). Finally, it was shown that some L. sakei strains were able to convert agmatine into putrescine, suggesting an operational agmatine deiminase pathway in these strains, a metabolic trait that is undesirable in meat fermentations. This study shows that this metabolic trait is most probably encoded by a previously erroneously annotated second putative arc operon.

Comparison of methods for relative quantification of gene expression using real-time PCR

1 Univ. of Ljubljana, Biotechnical Fac., Dept. of Animal Science, Groblje 3, SI-1230 Domžale, Slovenia and rapid quantification results (Pfaffl, 2001; Yuan et al., 2006). Because of the lacking consensus on how to best perform qPCR, MIQE guidelines have been developed to uniform qPCR experiment setup, optimization and data analysis, making the protocols comparable between different research groups and organizations and ensuring the relevance, accuracy, correct interpretation and repeatability of the results (Bustin et al., 2009). When analyzing gene expression, qPCR data can be subjected to absolute or relative quantification (Livak and Schmittgen, 2001; Pfaffl, 2001; Yuan et al., 2006). Absolute quantification employs internal or external caliComparison of methods for relative quantification of gene expression using real-time PCR Quantitative real-time PCR (qPCR) has become a widely used tool for quantifying gene expression. Several methods for relative quantification have been developed, enabling rapid and reliable detection and quantification of specific nucleic acids. These methods, based on qPCR include: the standard curve method, the efficiency calibrated method and the 2−ΔΔCq method. Here we analyzed if these three methods generate comparable results. To evaluate their performance, we analyzed the expression of the nuclease gene MS53_0284 from Mycoplasma synoviae type strain WVU 1853 during in vitro infection of CEC-32 cells, using qPCR. As determined, all three methods generated comparable and reliable results when all necessary conditions were fulfiled. Also, the efficiency calibrated and the standard curve methods were more suitable for quantifying small differences in relative gene expression than the 2−ΔΔCq method.

Endothelin-1 Markedly Decreases the Blood Perfusion of Transplanted Pancreatic Islets in Rats

Background Transplantation of insulin-producing β-cells is the only available curative treatment for type 1 diabetes. However, graft function declines within the first years after transplantation, which may reflect inadequate vascular engraftment. Endothelin-1 (ET-1) is a potent vasoconstrictor whose production is regulated by both hypoxia and inflammation. Moreover, the plasma concentration of ET-1 is elevated in patients with type 1 diabetes. The aim of this study was to investigate the gene expression and effects of ET-1 and its 2 receptor antagonists, BQ123 and BQ788, on blood flow in syngeneic rat islet transplants. Methods Pancreatic islets from Wistar Furth rats were isolated and transplanted syngeneically under the kidney capsule. Transplant and kidney cortex blood flow was measured using laser Doppler flowmetry after administration of ET-1 via topical application, or after administration of BQ123 and BQ788 intravenously. The grafts and isolated islets were analyzed for mRNA expression of ET-1, ET A receptor, ET B receptor, and endothelin-converting enzyme 1 using by reverse-transcription polymerase chain reaction. Results ET-1 markedly decreased transplant blood flow (77.5 ± 4.4% 1 minute after administration; n = 6), whereas neither BQ123 nor BQ788 had vascular effects. No differences in relative gene expression between the grafts and freshly isolated control islets were seen for ET-1 (0.65 ± 0.14 [n = 8] vs 0.79 ± 0.24 [n = 5]), ET A receptor (0.37 ± 0.14 [n = 8] vs 0.25 ± 0.04 [n =5]), ET B receptor (4.78 ± 1.43 [n = 8] vs 1.94 ± 0.32 [n = 5]), or endothelin converting enzyme 1 (7.25 ± 1.88 [n = 8] vs 11.83 ± 0.95 [n = 5]) when expressed as 2 −ΔCt. Conclusion Exogenous ET-1 strongly affects the blood perfusion of transplanted islets, and endogenous levels can, if up-regulated, contribute to graft failure.

Dopage et androgènes

Canine idiopathic pulmonary fibrosis (CIPF) is a progressive disease of the lung parenchyma that is more prevalent in dogs of the West Highland white terrier (WHWT) breed. Since the chemokines (C-C motif) ligand 2 (CCL2) and (C-X-C motif) ligand 8 (CXCL8) have been implicated in pulmonary fibrosis in humans, the aim of the present study was to investigate whether these same chemokines are involved in the pathogenesis of CIPF. CCL2 and CXCL8 concentrations were measured by ELISA in serum and bronchoalveolar lavage fluid (BALF) from healthy dogs and WHWTs affected with CIPF. Expression of the genes encoding CCL2 and CXCL8 and their respective receptors, namely (C-C motif) receptor 2 (CCR2) and (C-X-C motif) receptor 2 (CXCR2), was compared in unaffected lung tissue and biopsies from dogs affected with CIPF by quantitative PCR and localisation of CCL2 and CXCL8 proteins were determined by immunohistochemistry.Significantly greater CCL2 and CXCL8 concentrations were found in the BALF from WHWTs affected with CIPF, compared with healthy dogs. Significantly greater serum concentrations of CCL2, but not CXCL8, were found in CIPF-affected dogs compared with healthy WHWTs. No differences in relative gene expression for CCL2, CXCL8, CCR2 or CXCR2 were observed when comparing lung biopsies from control dogs and those affected with CIPF. In affected lung tissues, immunolabelling for CCL2 and CXCL8 was observed in bronchial airway epithelial cells in dogs affected with CIPF. The study findings suggest that both CCL2 and CXCL8 are involved in the pathogenesis of CIPF. Further studies are required to determine whether these chemokines might have a clinical use as biomarkers of fibrosis or as targets for therapeutic intervention.

Using real time RT-PCR analysis to determine multiple gene expression patterns during XX and XY mouse fetal gonad development

New techniques are being applied to identify all the genes involved in mammalian gonad development and differentiation. As this list of genes increases, understanding the potential interactions between these genes will become increasingly difficult. We used a real time reverse transcription PCR (real time RTPCR) protocol to examine and compare the relative expression levels of 55 genes in individual mouse fetal gonads. Real time PCR analysis demonstrated that except for Sry, no differences in relative gene expression were detectable between XX and XY gonad/mesonephroi complexes at embryonic day (E)11.5. Following Sry peak expression at E11.5, a number of genes were expressed at significantly higher relative levels in E12–14 XY than XX gonads. Of six genes expressed at higher levels in E12.5–14 XX than XY gonads, three, Bmp2, Emx2, and Fgfr2, had not been reported previously. Our results caution that differential localization patterns observed with whole mount in situ hybridization techniques may not accurately reflect changes in transcript levels. We conclude that real time PCR is an efficient and powerful tool for studying multiple gene expression patterns during gonad development and differentiation, and can provide insight into gene interactions.