Coexistence of Positive 99mTc-DPD Scintigraphy and Monoclonal Gammopathy: A Frequent Challenge.

Background: Cardiac involvement is common in amyloidosis, and the vast majority of cases of amyloid cardiomyopathy are attributed to primary amyloidosis or transthyretin amyloidosis (ATTR). Although the coexistence of scintigraphy suggestive of ATTR with monoclonal gammopathy of undetermined significance is well documented, the correct diagnosis is still challenging in non-referral centers. Methods: We performed a retrospective study reviewing all amyloid cardiomyopathy cases diagnosed at our center over the last 5 years, and described our experience and diagnostic approach. Results: During the last 5 years, 74 patients with positive scintigraphy were identified. Of these patients, 41 were included in this study as they had all necessary tests for a complete diagnosis. Two of these 41 patients had variant ATTR and 29 had wild-type ATTR. Ten patients had monoclonal gammopathy (24.4%), and it was consequently impossible to obtain a specific diagnosis. During follow-up, 14 patients (34.1%), five of them from the monoclonal gammopathy group, died, reflecting the severity of disease. Conclusions: In patients with ATTR-suggestive scintigraphy, monoclonal gammopathy frequently occurs concomitantly, thus not allowing to establish a specific diagnosis. A biopsy could only be replaced by genetic testing in selected cases.

The environmental quality of Doñana surrounding areas affects the immune transcriptional profile of inhabitant crayfish Procambarus clarkii.

This study aimed to identify differentially expressed genes in Procambarus clarkii crayfish collected from locations of different environmental qualities in the Doñana National Park surrounding areas. The pollution sustained by the crayfish was confirmed by their hepatopancreatic metal concentration. We generated forward and reverse libraries by suppression subtractive hybridization (SSH) to analyze the transcriptional profiles of crayfish from moderately and highly polluted zones in comparison with the control site within the Doñana Biological Reserve. Forty-three differentially expressed genes were detected, and most of them were identified as genes involved in a variety of biological functions, particularly in the innate immune response. To verify the SSH results and assess interindividual variability nine transcripts (ALP, AST, BTF3, CHIT, CTS, ferritin, HC, HC2, and SPINK4) were selected for absolute quantification by real-time qRT-PCR. The qRT-PCR data revealed substantial differences in the absolute amounts of the nine transcripts and confirmed their up- or down-regulation in the polluted sites. Additionally, a positive and significant linear correlation was found between the hepatopancreatic copper concentration and the levels of the transcripts encoding hemocyanins. Finally, the transcriptomic study was complemented with a detailed analysis of SNP profiles of the selected transcripts that revealed point mutations that might underlie adaptive response to environmental stress in P. clarkii. Overall, this work provides novel insights into the molecular pathways that could mediate the response to environmental pollutants in P. clarkii emphasizing the central role of the immune function and thus, should clearly benefit further immunotoxicological research in this organism.

Heterologous microarray analysis of transcriptome alterations in Mus spretus mice living in an industrial settlement.

This work demonstrates the successful application of a commercial oligonucleotide microarray containing Mus musculus whole-genome probes to assess the biological effects of an industrial settlement on inhabitant Mus spretus mice. The transcriptomes of animals in the industrial settlement contrasted with those of specimens collected from a nearby protected ecosystem. Proteins encoded by the differentially expressed genes were broadly categorized into six main functional classes. Immune-associated genes were mostly induced and related to innate and acquired immunity and inflammation. Genes sorted into the stress-response category were mainly related to oxidative-stress tolerance and biotransformation. Metabolism-associated genes were mostly repressed and related to lipid metabolic pathways; these included genes that encoded 11 of the 20 cholesterol biosynthetic pathway enzymes. Crosstalk between members of different functional categories was also revealed, including the repression of serine-protease genes and the induction of protease-inhibitor genes to control the inflammatory response. Absolute quantification of selected transcripts was performed via RT-PCR to verify the microarray results and assess interindividual variability. Microarray data were further validated by immunoblotting and by cholesterol and protein-thiol oxidation level determinations. Reported data provide a broad impression of the biological consequences of residing in an industrial area.

Targeted sequencing of selected functional genes in patients with wild-type transthyretin amyloidosis.

OBJECTIVE: Wild-type transthyretin (ATTRwt) amyloidosis is caused by the misfolding and deposition of the transthyretin protein (TTR) in the absence of mutations in the TTR gene. Studies regarding the variant form of ATTR amyloidosis (ATTRv) suggest that the presence of single-nucleotide polymorphisms (SNP) in genes other than the TTR, may influence the development of the disease. However, other genetic factors involved in the aetiopathogenesis of ATTRwt are currently unknown. This work investigates the presence of sequence variants in genes selected for their possible impact on ATTRwt amyloidosis. To do so, targeted sequencing of 84 protein-coding genes was performed in a cohort of 27 patients diagnosed with ATTRwt. RESULTS: After applying quality and frequency filtering criteria, 72 rare or novel genetic variants were found. Subsequent classification according to the ACMG-AMP criteria resulted in 17 variants classified as of uncertain significance in 14 different genes. To our knowledge, this is the first report associating novel gene variants with ATTRwt amyloidosis. In conclusion, this study provides potential insights into the aetiopathogenesis of ATTRwt amyloidosis by linking novel coding-gene variants with the occurrence of the disease.

Differential expression of the Gstp2 gene between the aboriginal species Mus spretus and the laboratory mouse Mus musculus.

Glutathione S-transferases (GSTs) are pivotal phase-II enzymes for detoxification of xenobiotics. Pi-class GSTs play key roles in determining cancer susceptibility. The laboratory mouse Mus musculus (Mm) has two GST-Pi-encoding genes; while MmGstp1 is the counterpart of the unique human and rat Pi-class GST gene, the function of MmGstp2 remains unclear because its expression is almost undetectable in liver and its product lacks activity against typical GST/GST-Pi substrates. Mus spretus (Ms) is an aboriginal mouse species of great interest as a bio-indicator in environmental pollution studies and a reservoir of novel allelic variants and phenotypes. Using absolute real-time RT-PCR, we demonstrate significant differences in the hepatic levels of GST-Pi-encoding mRNAs between both mouse species. Particularly, we found that the Gstp2 gene of M. spretus, unlike its M. musculus counterpart, attains relatively high steady-state level of expression (∼30molecules/pg of total liver RNA in mice dwelling in a non-polluted area). To test whether the interspecies difference in Gstp2 mRNA levels is due, at least in part, to evolutionary divergence in the promoter regions, we (i) sequenced the 5'-flanking regulatory regions of the two Gstp2 genes; (ii) used bioinformatics tools to identify differences in TF binding sites (TFBSs) and cis-regulatory modules; and (iii) extended the in silico results to a cell-based functional assay. We observed high sequence divergence (2.8%) and differences in TFBSs (32.6%) between the two Gstp2 promoters. We also show that constructs harbouring promoter fragments with species-specific cis-regulatory motifs displayed differential luciferase reporter activity, suggesting that these promoter sequence variations may determine, at least in part, the strong difference in Gstp2 mRNA levels between M. musculus and M. spretus. Additionally, the comparative analysis of the coding sequences predicts that the MsGstp2 product may be an active Pi-class GST because of a Pro(12) to Arg(12) substitution. Interestingly, free-living M. spretus mice dwelling at an industrial settlement displayed significantly higher amounts of transcripts for both GST-P1 and GST-P2 than those from a non-polluted area, suggesting that. M. spretus may optimise the response to pollution by co-evolving the expression levels of the two Pi-class GST genes. Overall, our data suggest that MsGstp2 may be one of the genes contributing to the natural resistance of M. spretus, facilitating its adaptation in a wild environment. Further insights into the functional roles of mouse Pi-class GSTs should be gained from the data reported in this work.

Omic approaches in environmental issues.

Biomonitoring requires the application of batteries of different biomarkers, as environmental contaminants induce multiple responses in organisms that are not necessarily correlated. Omic technologies were proposed as an alternative to conventional biomarkers since these techniques quantitatively monitor many biological molecules in a high-throughput manner and thus provide a general appraisal of biological responses altered by exposure to contaminants. As the studies using omic technologies increase, it is becoming clear that any single omic approach may not be sufficient to characterize the complexity of ecosystems. This work aims to provide a preliminary working scheme for the use of combined transcriptomic and proteomic methodologies in environmental biomonitoring. There are difficulties in working with nonmodel organisms as bioindicators when combining several omic approaches. As a whole, our results with heterologous microarrays in M. spretus and suppressive subtractive hybridization (SSH) in P. clarkii indicated that animals sustaining a heavy pollution burden exhibited an enhanced immune response and/or cell apoptosis. The proteomic studies, although preliminary, provide a holistic insight regarding the manner by which pollution shifts protein intensity in two-dimensional gel electrophoresis (2-DE), completing the transcriptomic approach. In our study, the sediment element concentration was in agreement with the intensity of protein expression changes in C. maenas crabs. In conclusion, omics are useful technologies in addressing environmental issues and the determination of contamination threats.

A Comparative Study between Swedish Interactive Thresholding Algorithm Faster and Swedish Interactive Thresholding Algorithm Standard in Glaucoma Patients.

PURPOSE: To compare the results of the new strategy Swedish Interactive Thresholding Algorithm (SITA) Faster to the results of SITA Standard in patients with glaucoma. METHODS: This was a cross-sectional study of 49 patients with glaucoma and previous experience with standard automated perimetry. Two consecutive tests were performed in random order, one with SITA Standard and another one with SITA Faster, in the studied eye of each patient. Comparisons were made for test time, mean deviation (MD), visual field index (VFI), and number of depressed points in pattern deviation map and total deviation map for every level of significance. RESULTS: The average test time was 56% shorter with SITA Faster (P < 0.001). The intraclass correlation coefficient (ICC) for MD and VFI showed excellent agreement between both strategies, ICC = 0.98 (95% confidence interval [CI]: 0.96, 0.99) and ICC = 0.97 (95% CI: 0.95, 0.99), respectively. For the number of depressed points in total deviation map and pattern deviation map, ICC demonstrated good agreement with values between 0.8 and 0.95. CONCLUSIONS: Our study shows that SITA Faster is a shorter test with strong agreement with SITA Standard parameters. These results suggest that SITA Faster could replace SITA Standard for glaucoma diagnosis.

Growth phase-dependent variations in transcript profiles for thioredoxin- and glutathione-dependent redox systems followed by budding and hyphal Candida albicans cultures.

We report the absolute transcription profiles of 24 genes coding for putative thioredoxin (Trx)- and glutathione (GSH)-dependent redox system components, accompanying the Candida albicans batch culture growth, under either yeast or hyphal conditions. All mRNAs investigated (plus the housekeeping ACT1) displayed significant alterations in their steady-state copy number. Collectively, these quantifications show that: (1) the most abundant mRNAs during active growth coded for putative thiol peroxidases (TSA1, PRX1, AHP11 and AHP12) and for donor thioredoxin Trx1p, i.e. those five mRNAs represented >74% of all transcript molecules quantified in a late exponential phase; (2) the transcripts under study differed between budding and hyphal cells not only in their abundance but also in their profiles throughout the growth stages; (3) mRNA amounts for four GSH-transferase putative genes (GTT12, GTT13, GTT14 and GST3) increased in the stationary phase in yeast but not under filamentous conditions. Hydrogen peroxide resistance, plus GSH, GSSG and reactive oxygen species contents, throughout yeast and hyphal growth, were also studied, and the differences observed were related to the transcript profiles.

Immune- and stress-related transcriptomic responses of Solea senegalensis stimulated with lipopolysaccharide and copper sulphate using heterologous cDNA microarrays.

The sole, Solea senegalensis, is a common flatfish of Atlantic and Mediterranean waters with a high potential for aquaculture. However, its cultivation is hampered by high sensitivity to different stresses and several infectious diseases. Improving protection from pathogens and stressors is thus a key step in reaching a standardized production. Fish were exposed to lipopolysaccharide (LPS), a mimetic of bacterial infections, and copper sulphate (CuSO(4)), used in aquaculture to control algae and outbreaks of infectious diseases. We employed a European flounder cDNA microarray to determine the transcriptomic responses of Senegalese sole to these exposures. Microarray analyses showed that many genes were altered in expression following both LPS and copper treatments in comparison to vehicle controls. Gene ontology analysis highlighted copper-specific induction of genes related to cellular adhesion and cell signalling, LPS-specific induction of genes related to the immune response, and a common induction of genes related to unfolded protein binding, intracellular transport/secretion and proteasome. Additionally transcripts for glutathione-S-transferases were down-regulated by LPS, and those for digestive enzymes were down-regulated by both treatments. We selected nine changing genes for absolute quantification of transcript copy numbers by real-time RT-PCR to validate microarray differential expression and to assess inter-individual variability in individual fishes. The quantitative RT-PCR data correlated highly with the microarray results. Overall, data reported provide novel insights into the molecular pathways that could mediate the immune and heavy metal stress responses in Senegalese sole and thus might have biotechnological applications in the culture of this important fish species.

Estimating the prevalence of allelic variants in the transthyretin gene by analysing large-scale sequencing data.

Transthyretin amyloidosis (ATTR amyloidosis) is a rare disease characterised by extracellular deposition of amyloid fibrils composed by transthyretin. ATTR amyloidosis can be sub-classified as wild-type ATTR (ATTR-wt) or as hereditary amyloidosis (ATTR-m); the prevalence of both types are likely underestimated. There are tools that can help us to study ATTR-m, as gnomAD database. Our primary aim was to estimate prevalence of variants, especially amyloidogenic variants, in the TTR gene using gnomAD database. We analysed TTR missense variants found in gnomAD. The variables studied were classified according to their clinical significance and according to the different populations. We found 71 missense variants in the TTR gene. Eleven variants were described as affects function variants (prevalence 1:230). The most frequently detected variant were c.424G>A (p.(Val142Ile)) (prevalence 1:332, MAF 0.00151) and c.148G>A (p.(Val50Met)) (prevalence 1:4924, MAF 0.000102), which represented 88% and 5%, respectively, of all affects function variants detected. Seventeen variants were classified as probably affects function, 29 as unknown variants, 4 as probably does not affect function and 10 as does not affect function variants. In terms of different populations, c.424G>A (p.(Val142Ile)) was especially prevalent in African population (MAF 0.01602; prevalence of 1:31) and c.148G>A (p.(Val50Met)) in European population (MAF 0.000179; prevalence of 1:2792). Prevalence of amyloidogenic variants in the general population was higher than prevalence heretofore described. This difference could be explained by incomplete penetrance of the disease, but other factors contributing to this fact, fundamentally the underdiagnosis of the disease.

Alternative splicing of c-fos pre-mRNA: contribution of the rates of synthesis and degradation to the copy number of each transcript isoform and detection of a truncated c-Fos immunoreactive species.

BACKGROUND: Alternative splicing is a widespread mechanism of gene expression regulation. Previous analyses based on conventional RT-PCR reported the presence of an unspliced c-fos transcript in several mammalian systems. Compared to the well-defined knowledge on the alternative splicing of fosB, the physiological relevance of the unspliced c-fos transcript in regulating c-fos expression remains largely unknown. This work aimed to investigate the functional significance of the alternative splicing c-fos pre-mRNA. RESULTS: A set of primers was designed to demonstrate that, whereas introns 1 and 2 are regularly spliced from primary c-fos transcript, intron 3 remains unspliced in part of total transcript molecules. Here, the two species are referred to as c-fos-2 (+ intron 3) and spliced c-fos (- intron 3) transcripts. Then, we used a quantitatively rigorous approach based on real-time PCR to provide, for the first time, the actual steady-state copy numbers of the two c-fos transcripts. We tested how the mouse-organ context and mouse-gestational age, the synthesis and turnover rates of the investigated transcripts, and the serum stimulation of quiescent cells modulate their absolute-expression profiles. Intron 3 generates an in-frame premature termination codon that predicts the synthesis of a truncated c-Fos protein. This prediction was evaluated by immunoaffinity chromatography purification of c-Fos proteins. CONCLUSION: We demonstrate that: (i) The c-fos-2 transcript is ubiquitously synthesized either in vivo or in vitro, in amounts that are higher or similar to those of mRNAs coding for other Fos family members, like FosB, DeltaFosB, Fra-1 or Fra-2. (ii) Intron 3 confers to c-fos-2 an outstanding destabilizing effect of about 6-fold. (iii) Major determinant of c-fos-2 steady-state levels in cultured cells is its remarkably high rate of synthesis. (iv) Rapid changes in the synthesis and/or degradation rates of both c-fos transcripts in serum-stimulated cells give rise to rapid and transient changes in their relative proportions. Taken as a whole, these findings suggest a co-ordinated fine-tune of the two c-fos transcript species, supporting the notion that the alternative processing of the precursor mRNA might be physiologically relevant. Moreover, we detected a c-Fos immunoreactive species corresponding in mobility to the predicted truncated variant.

Validation of commercial real-time PCR-arrays for environmental risk assessment: Application to the study of p,p´-DDE toxicity in Mus spretus mice liver.

Data on gene transcription profiles provide a comprehensive assessment of the toxic and defensive mechanisms that are triggered by pollutants. PCR-arrays have emerged as a reliable tool for analyzing the expression of a panel of relevant, pathway- or disease-focused genes under uniform cycling conditions. By using SYBR Green-optimized primer assays, it is possible to simultaneously amplify a sample with high specificity and amplification efficiencies. However, commercial PCR-arrays target a limited group of organisms, excluding most of those with environmental relevance, as is the case with Mus spretus mice. Our previous works with M. spretus showed a high sequence similarity between M. spretus and the model organism M. musculus allowing the use of commercial platforms with M. spretus. This work demonstrates the successful application of a commercial PCR-array designed for the model organism M. musculus to assess the biological effects caused by the organochlorine pesticide p,p´-DDE in a focused panel of stress-related genes in M. spretus mice. This cost-effective, easy-to-use platform detected quantitative gene profiling differences between M. spretus hepatic RNA samples and generated data highly concordant with those obtained by absolute qRT-PCR, the most sensitive method to quantify transcripts. This platform is also suitable for use in field studies with free-living M. spretus mice for routine environmental risk assessment. Our results provide a broad impression of the biological consequences of p,p´-DDE on the hepatic health of mice.

2D-DIGE as a proteomic biomarker discovery tool in environmental studies with Procambarus clarkii.

A 2D-DIGE/MS approach was used to assess protein abundance differences in the red swamp crayfish Procambarus clarkii from polluted aquatic ecosystems of Doñana National Park and surrounding areas with different pollution loads. Procambarus clarkii accumulated metals in the digestive glands and gills reflecting sediment concentrations. We first stated that, probably related to elements accumulation, pollution increased oxidative damage in P. clarkii tissues, as shown by the thiol oxidation status of proteins and MDA levels. In these animals, the altered redox status might be responsible for the deregulated abundance of proteins involved in cellular responses to oxidative stress including protein folding, mitochondrial imbalance and inflammatory processes. Interestingly, polluted P. clarkii crayfish also displayed a metabolic shift to enhanced aerobic glycolysis, most likely aimed at generating ATP and reduction equivalents in an oxidative stress situation that alters mitochondrial integrity. The deregulated proteins define the physiological processes affected by pollutants in DNP and its surrounding areas and may help us to unravel the molecular mechanisms underlying the toxicity of environmental pollutants. In addition, these proteins might be used as exposure biomarkers in environmental risk assessment. The results obtained might be extrapolated to many other locations all over the world and have the added value of providing information about the molecular responses of this environmentally and economically interesting animal. SIGNIFICANCE: Metal content in digestive gland and gills of P. clarkii crayfish reflects their contents in sediments at sites of Doñana National Park and its surroundings. Accumulation of essential and toxic transition metals is paralleled by clear signs of oxidative stress to lipids and proteins and by significant deregulation of many proteins involved in protein folding, mitochondrial respiratory imbalance and inflammatory response. These results indicate that P. clarkii is an excellent bioindicator to be used in aquatic ecosystems quality monitoring. Additionally, results evidence that the anthropogenic activities carried out around Doñana National Park represent an extremely serious threat to this unique Biosphere Reserve and pose a risk to the environment and their inhabitants health. The identified deregulated proteins provide information about the metabolic pathways and/or physiological processes affected by pollutant-elicited oxidative stress, may also be useful as biomarkers of environmental pollution and have the added value of providing information about the molecular responses of this environmentally and economically interesting animal.

Transcript copy number of genes for DNA repair and translesion synthesis in yeast: contribution of transcription rate and mRNA stability to the steady-state level of each mRNA along with growth in glucose-fermentative medium.

We quantitated the copy number of mRNAs (NTG1, NTG2, OGG1, APN1, APN2, MSH2, MSH6, REV3, RAD30) encoding different DNA repair enzymes and translesion-synthesis polymerases in yeast. Quantitations reported examine how the steady-state number of each transcript is modulated in association with the growth in glucose-fermentative medium, and evaluate the respective contribution of the rate of mRNA degradation and transcription initiation to the specific mRNA level profile of each gene. Each transcript displayed a unique growth-related profile, therefore altering the relative abundance of mRNAs coding for proteins with similar functions, as cells proceed from exponential to stationary phase. Nonetheless, as general trend, they exhibited maximal levels when cells proliferate rapidly and minimal values when cells cease proliferation. We found that previous calculations on the stability of the investigated mRNAs might be biased, in particular regarding those that respond to heat shock stress. Overall, the mRNAs experienced drastic increments in their stabilities in response to gradual depletion of essential nutrients in the culture. However, differences among the mRNA stability profiles suggest a dynamic modulation rather than a passive process. As general rule, the investigated genes were much more frequently transcribed during the fermentative growth than later during the diauxic arrest and the stationary phase, this finding conciliating low steady-state levels with increased mRNA stabilities. Interestingly, while the rate at which each gene is transcribed appeared as the only determinant of the number of mRNA copies at the exponential growth, later, when cell growth is arrested, the rate of mRNA degradation becomes also a key factor for gene expression. In short, our results raise the question of how important the respective contribution of transcription and mRNA stability mechanisms is for the steady-state profile of a given transcript, and how this contribution may change in response to nutrient-availability.

Global gene expression profiling using heterologous DNA microarrays to analyze alterations in the transcriptome of Mus spretus mice living in a heavily polluted environment.

Microarray platforms are a good approach for assessing biological responses to pollution as they enable the simultaneous analyses of changes in the expression of thousands of genes. As an omic and non-targeted methodology, this technique is open to unforeseen responses under particular environmental conditions. In this study, we successfully apply a commercial oligonucleotide microarray containing Mus musculus whole-genome probes to compare and assess the biological effects of living in a heavily polluted settlement, the Domingo Rubio stream (DRS), at the Huelva Estuary (SW Spain), on inhabitant free-living Mus spretus mice. Our microarray results show that mice living in DRS suffer dramatic changes in gene and protein expression compared with reference specimens. DRS mice showed alteration in the oxidative status of hepatocytes, with activation of both the innate and the acquired immune responses and the induction of chronic inflammation, accompanied by metabolic alterations that imply the accumulation of lipids in the liver (hepatic steatosis). The identified deregulated genes may be useful as biomarkers of environmental pollution.

Tissue, species, and environmental differences in absolute quantities of murine mRNAs coding for alpha, mu, omega, pi, and theta glutathione S-transferases.

This article reports the first absolute quantitative analysis of expression patterns of murine transcripts (Gsta1/2, Gsta3, Gsta4, Gstm1, Gstm2, Gstm3, Gsto1, Gstp1/2, Gstt1, Gstt2) coding for most glutathione S-transferases (GSTs) of alpha, mu, omega, pi, and theta classes. We examine how the steady-state numbers of transcripts are modulated in association with: three animal organs (liver, kidney, and lung) where extensive detoxification occurs; two species (Mus musculus and Mus spretus) representing common laboratory and aboriginal mice; and two genetic and animal living conditions (wild-derived inbred animals and free-living mice). Moreover, quantitations performed examine how the pulmonary steady-state Gst mRNA amounts are affected in M. musculus by paraquat (a superoxide generator), and in M. spretus by dwelling at a polluted area. The results point to complex tissue-, species-, and life condition-dependent expression of the investigated transcripts. Among others, they show: i) the ubiquity of most transcripts, except Gstm3 mRNA that was virtually absent or at very low amounts (< or = 0.001 molecules/pg) in kidney and lung of M. spretus; ii) unique expression profiles for each transcript and mouse organ examined; iii) outstanding species-specific differences in basal amounts of most Gst mRNAs, this effect being most apparent in the case of Gsta1/2, Gsta3, Gstm2, Gsto1, Gstt1, and Gstt2; iv) paraquat-induced upregulation of most Gst mRNAs, with the notable exception of those coding for theta class GSTs; v) a tendency for mice dwelling at a wildlife reserve of having lower and more homogeneous Gsta3 mRNA levels than those collected in an anthropogenic environment.

Absolute transcript levels of thioredoxin- and glutathione-dependent redox systems in Saccharomyces cerevisiae: response to stress and modulation with growth.

We report the co-ordinated fine-tune of mRNA molecules that takes place in yeast (Saccharomyces cerevisiae) in response to diverse environmental stimuli. We performed a systematic and refined quantification of the absolute expression patterns of 16 genes coding for thioredoxin- and glutathione-dependent redox system components. Quantifications were performed to examine the response to oxidants, to sudden temperature upshifts and in association with metabolic changes accompanying culture growth and to explore the contribution of mRNA decay rates to the differences observed in basal expression levels. Collectively, these quantifications show (i) vast differences in the steady-state amounts of the investigated transcripts, cTPxI being largely overexpressed compared with GPX1 during the exponential phase and GPX2 beyond this growth stage; (ii) drastic changes in the relative abundance of the transcripts in response to oxidants and heat shock; and (iii) a unique temporal expression profile for each transcript as cells proceed from exponential to stationary growth phase, yet with some general trends such as maximal or near-maximal basal amounts of most mRNA species at early growth stages when glucose concentration is high and cells are actively growing. Moreover, the results indicate that (i) the half-lives of the investigated transcripts are longer and distributed within a narrower range than previously reported global mRNA half-lives and (ii) transcriptional initiation may play an important role in modulating the significant alterations that most mRNAs exhibit in their steady-state levels along with culture growth.

iTRAQ analysis of hepatic proteins in free-living Mus spretus mice to assess the contamination status of areas surrounding Doñana National Park (SW Spain).

This work aims to develop and integrate new -omics tools that would be applicable to different ecosystem types for a technological updating of environmental evaluations. We used a 2nd-generation (iTRAQ-8plex) proteomic approach to identify/quantify proteins differentially expressed in the liver of free-living Mus spretus mice from Doñana National Park or its proximities. Mass spectrometry was performed in an LTQ Orbitrap system for iTRAQ reporter ion quantitation and protein identification using a Mus musculus database as reference. A prior IEF step improved the separation of the complex peptide mixture. Over 2000 identified proteins were altered, of which 118 changed by ≥2.5-fold in mice from at least two problem sites. Part of the results obtained with the iTRAQ analysis was confirmed by Western blot. Over 75% of the 118 proteins were upregulated in animals captured at polluted sites and only 16 proteins were downregulated. Upregulated proteins were involved in stress response; cell proliferation and apoptosis; signal transduction; metastasis or tumour suppression; xenobiotic export or vesicular trafficking; and metabolism. The downregulated proteins, all potentially harmful, were classified as oncoproteins and proteins favouring genome instability. The iTRAQ results presented here demonstrated that the survival of hepatic cells is compromised in animals living at polluted sites, which showed deep alterations in metabolism and the signalling pathways. The identified proteins may be useful as biomarkers of environmental pollution and provide insight about the metabolic pathways and/or physiological processes affected by pollutants in DNP and its surrounding areas.