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.

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.

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.

Interferencia analítica en la determinación de troponina por anticuerpos heterófilos / Interference in the determination of Troponin due to heterophile antibodies

Resumen Por su elevada especificidad y sensibilidad, y a raíz de la tercera definición universal de infarto estas isoformas cardiacas han sido aceptadas en el ámbito internacional como los biomarcadaores de elección en la práctica clínica para el diagnóstico de síndrome coronario agudo, preferibles a la determinación de las enzimas creatina quinasa y su isoforma MB. Se presenta el caso de un varón de ochenta años, quien, de manera persistente, tuvo valores elevados de troponinas, pese a evolución clínica no compatible con síndrome coronario agudo ni otras causas de elevación de este biomarcador.

Abstract Due to its elevated specificity and sensitivity, and on being the third universal definition of myocardial infarction, these cardiac isoforms have been accepted internationally as the biomarkers of choice in clinical practice for the diagnosis of acute coronary syndrome, and are preferable to the determinations of creatine kinase and its MB isoform. A case is presented on an eighty year-old man, who persistently had elevated Troponin values, despite a clinical course that was incompatible with an acute coronary syndrome or any other causes of elevation of this biomarker.

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.

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.

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.

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.

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.

Redox proteomics as biomarker for assessing the biological effects of contaminants in crayfish from Doñana National Park.

Despite its environmental relevance and sensitivity, Doñana National Park (DNP) is under high ecological pressure. In crayfish (Procambarus clarkii), the utility of redox proteomics as a novel biomarker was evaluated in the aquatic ecosystems of DNP and its surroundings, where agricultural activity is a serious concern. After fluorescence labeling of reversibly oxidized Cys and 2-DE separation, the total density of proteins with reversibly oxidized thiols was found to be much higher in animals from the Matochal (MAT) and Rocina (ROC) streams, while no difference was found in crayfish from Partido (PAR) stream compared to those from the DNP core at Lucio del Palacio (the negative control). The 2-DE analysis revealed 35 spots with significant differences in thiol oxidation, among which 19 proteins were identified via MALDI-TOF/TOF. While 3 spots, identified as ferritin, showed higher oxidation levels in ROC, other identified proteins were more intense at MAT than at ROC (superoxide dismutase, protein disulfide isomerase and actin) or were overoxidized only in MAT (nucleoside diphosphate kinase, fructose-biphosphate aldolase, fatty acid-binding protein, phosphopyruvate hydratase). For most of the identified proteins, spots corresponding to different Cys oxidized forms were detected, and the native forms, without oxidized thiol groups were also found in some of them. Evidence of reversible oxidation was found for specific Cys residues, including Cys13 in ferritin as well as Cys76 and Cys108 in nucleoside diphosphate kinase. The identified thiol-oxidized proteins provide information about the metabolic pathways and/or physiological processes affected by pollutant-elicited oxidative stress.

Omics technologies and their applications to evaluate metal toxicity in mice M. spretus as a bioindicator.

Metals are important components of living organisms since many biological functions critically depend on their interaction with some metal in the cell. However, human activities have increased toxic metal levels in the terrestrial and aquatic ecosystems affecting living organisms. The impact of metals on cellular metabolism and global homeostasis has been traditionally assessed in free-living organisms by using conventional biomarkers; however, to obtain a global vision of metal toxicity mechanisms and the responses that metals elicit in the organisms, new analytical methodologies are needed. We review the use of omics approaches to assess the response of living organisms under metal stress illustrating the possibilities of different methodologies on the basis of our previous results. Most of this research has been based on free-living mice Mus spretus, a conventional bioindicator used to monitor metal pollution in Doñana National Park (DNP) (SW Spain), which is an important European biological reserve for migrating birds affected by agricultural, mining and industrial activities. The benefits of using omic techniques such as heterologous microarrays, proteomics methodologies (2-DE, iTRAQ®), metallomics, ionomics or metabolomics has been remarked; however, the complexity of these areas requires the integration of omics to achieve a comprehensive assessment of their environmental status. This article is part of a Special Issue entitled: Environmental and structural proteomics. BIOLOGICAL SIGNIFICANCE: This work presents new contributions in the study of environmental metal pollution in terrestrial ecosystems using Mus spretus mice as bioindicator in Doñana National Park (SW Spain) and surroundings. In addition, it has been demonstrated that the integration of omics multi-analytical approaches provides a very suitable approach for the study of the biological response and metal interactions in exposed and free-living mice (Mus musculus and Mus spretus, respectively) under metal pollution.

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.

Proteomics in HepG2 hepatocarcinoma cells with stably silenced expression of PRDX1.

Peroxiredoxin 1 (PRDX1) is a member of the peroxiredoxin family. Aberrant expression of PRDX1 has been described in various cancers. We investigated the significance of this up-regulation in non-challenged hepatocellularcarcinoma (HCC) cells by establishing a HepG2 cell line stably expressing a Prdx1 shRNA. Prdx1 silencing reversed, at least partially, the tumoural phenotype of HepG2 cells, resulting in morphological changes, delayed cell growth, down-regulation of transcripts for AFP, osteopontin and ß-catenin and decreased γ-glutamyl transpeptidase activity, and oppositely up-regulation of transcripts for E-cadherin and proapoptotic proteins (BAX, CASP3) and increased alkaline phosphatase and CASP3 activities. Proteomic profiling identified 16 spots differentially expressed in Prdx1-silenced cells. Most of the variations involved the down-regulation of proteins with pivotal roles in cell proliferation and differentiation, in agreement with the observed phenotypic changes. We also investigated the effect of Prdx1 silencing on thiol protein oxidation. Proteins prone to reversible cysteine oxidation play major physiological functions. Notably, the down-regulation and altered redox status of key enzymes of carbohydrate and amino acid metabolism suggested a disturbance of the Warburg effect and glutamine utilization, two major pathways in the proliferation of tumour cells. Overall, these observations suggest that PRDX1 acts as a pro-cancer protein in HCC HepG2 cells.

Identification of proteins containing redox-sensitive thiols after PRDX1, PRDX3 and GCLC silencing and/or glucose oxidase treatment in Hepa 1-6 cells.

The oxidation and reduction of cysteine thiols are thought to be a major mechanism for redox regulation. The aim of this study was to identify proteins with reactive thiols and determine their oxidation profiles under oxidative stress induced by simultaneous silencing of antioxidant defences (peroxiredoxin-1, peroxiredoxin-3, and the catalytic subunit of the glutamate-cysteine ligase), and/or treatment with glucose oxidase (GO). Using an approach that combined the labelling of reversibly oxidised cysteines, 2-DE protein separation and MS analysis, we identified 26 proteins with cysteines prone to reversible oxidation belonging to different functional classes. Among these proteins are those that have not been previously recognised as reversible oxidation targets, including cytoplasmic aspartate aminotransferase, proteasome subunit alpha type-6, heterogeneous nuclear ribonucleoproteins isoA2/B1 and A/B, and histidine triad nucleotide-binding protein 1. We provide the first evidence of reversible oxidation for specific cysteines, including Cys112 and Cys146 in glutamate dehydrogenase 1, Cys17 in actins, Cys5 in protein disulfide-isomerase A3, and Cys267 in the heat shock cognate 71 kDa protein. Silencing induced lower oxidative stress than GO treatment. Nevertheless, we detected some proteins particularly sensitive to oxidation by silencing. We hypothesised that these proteins may play a role in regulatory mechanisms by redox stress.

Relación entre la presión intrabdominal en diálisis peritoneal con las hernias y fugas / The link between intra-abdominal pressure in peritoneal dialysis and hernias and fugues

La presión intrabdominal normal es igual a la atmosférica (cero). En Diálisis Peritoneal con la introducción del líquido intraperitoneal, la presión intrabdominal aumenta. En varios estudios se aconseja que esta no supere los 16-20 cm. H2O. Además de las posibles molestias abdominales, una presión intrabdominal elevada puede tener relación con los problemas de la pared abdominal, como hernias y fugas y tener implicaciones en el transporte peritoneal y el déficit de ultrafiltración. Los objetivos del presente trabajo fueron: conocer los niveles de presión intrabdominal de los enfermos prevalentes en diálisis peritoneal, valorar los factores que influyen en llos valores de esta presión y estudiar retrospectivamente la relación entre presión intrabdominal y desarrollo de hernias y fugas. Realizamos un estudio transversal, observacional y retrospectivo de valoración de la presión intrabdominal en los enfermos prevalentes, estables, con más de tres meses en diálisis peritoneal. La presión intrabdominal se midió mediante el método descrito por Durand: paciente en decúbito supino, con el volumen peritoneal diurno. La presión intrabdominal final es la media entre las mediciones realizadas durante la inspiración y la espiración, se expresa en cm. de H2O, y se especifica el volumen drenado. También se realizó una medida en sedestación y en bipedestación. Se estudiaron 34 pacientes, 66% varones, edad media de 61.2±14 años, 3 con poliquistosis renal, un índice de comorbilidad de Charlson medio de 7,9, un índice de masa corporal medio de 27.4±4.2 y un tiempo medio en DP de 21±12 meses. El volumen medio diurno fue de 1796±385 mL y el nocturno de 2100±254 mL. Un 32% de los pacientes tenían antecedentes de cirugía abdominal y un 5% de hernias, reparadas antes del inicio de la diálisis peritoneal. La media de presión intrabdominal en decúbito fue de 17.5±4.1 cm. de H2O, y un volumen medio por superficie corporal de 1141±253 ml/m2. Un 23.5 % tenían una presión intrabdominal mayor a 20 cm. de H2O. En sedestación la media fue de 28±5.5 cm. de H2O y en bipedestación de 43.7±5.3 cm. de H2O. Los enfermos con presión intrabdominal > 20 cm. H2O tenían más porcentaje de hernias (50% vs 12 %) y fugas pericatéter (37 % vs. 12 %). Como principales conclusiones, podemos destacar que los niveles de presión intrabdominal de nuestros pacientes son algo más elevados que en otras series. A mayor edad, mayor comorbilidad y mayor índice de masa corporal, la presión intrabdominal es más elevada. Los enfermos con presión intrabdominal elevada presentaron más episodios de hernias y fugas (AU)

Normal intra-abdominal pressure is equal to atmospheric pressure (zero). In peritoneal dialysis the introduction of intra-peritoneal liquid increases intra-abdominal pressure. In various studies it is recommended that this does not exceed 16-20cm H2O. In addition to possible abdominal discomfort, high intra-abdominal pressure can be linked to problems with the abdominal wall, such as hernias and fugues, and have implications for peritoneal transport and ultrafiltration deficit. The aims of this study were the following: to find out the intra-abdominal pressure levels in the prevalent type of patients in peritoneal dialysis, to assess the factors influencing the values for this pressure and to study the relationship between intra-abdominal pressure and the development of hernias and fugues, retrospectively. A transversal, observational and retrospective study was conducted to measure intra-abdominal pressure in the prevalent, stable patients who had been on peritoneal dialysis for more than three months. Intra-abdominal pressure was measured using the method described by Durand: patient in a supine position, with diurnal peritoneal volume. The final intra-abdominal pressure is the average of the measurements taken during inspiration and expiration, is expressed in cm H2O and the volume drained is specified. Measurements were also taken in sitting and standing positions. 34 patients were studied, 66% of them male, with an average age of 61.2±14 years, 3 with polycystic kidney disease, an average Charlson comorbidity index of 7.9, an average body mass index of 27.4±4.2 and an average of 21±12 months on PD. Average diurnal volume was 1796±385 mL and nocturnal 2100±254 mL. 32% of the patients had a history of abdominal surgery and 5% of hernias, remedied before the start of peritoneal dialysis. The average intra-abdominal pressure lying down was 17.5±4.1cm H2O, with an average volume by body surface of 1141±253ml/m2. 23.5 % had an intra-abdominal pressure of over 20cm H2O. In a sitting position the average was 28±5.5cm H2O and standing up it was 43.7±5.3cm H2O. Patients with an intra-abdominal pressure of > 20cm H2O had a higher percentage of hernias (50% vs 12%) and pericatheter fugues (37% vs. 12%). As the principal conclusions, we would stress that the intra-abdominal pressure levels in our patients were rather higher than in other series. The greater the age, comorbidity and major body mass index, the higher the intra-abdominal pressure. Patients with high intra-abdominal pressure have more episodes of hernias and fugues (AU)

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.