Occurrence of Pseudomonas cichorii causing midrib rot of lettuce under hydroponic cultivation in Chile.

In Chile, lettuce is the vegetable that has increased in cultivated area the most in recent years, reaching 8,309 ha. The Coquimbo Region contributes the most to this growth in production with 3,284 ha in 2022 (ODEPA 2023). Most lettuce is grown under open field conditions, but there is significant production in greenhouses and an increase in hydroponic production systems (INIA 2017). During April to June 2021 and 2022 in the Coquimbo Region, butterhead-type lettuce seedlings (Lactuca sativa) cv. Neil, cultivated under a hydroponic system, showed severe brown to black lesions in the leaves and midrib (Figure S1). To determine the etiology of this problem, samples of diseased plants were taken. Pieces of symptomatic tissue were macerated, and the extract was spread on nutrient agar (NA) and on King's B medium (KB) and incubated at 23°C for 48 h. The bacterial colonies observed were predominantly circular, creamy-white in color with irregular margins and fluorescent in KB medium. Isolates were gram-negative strictly aerobic. LOPAT test (Lelliot et al. 1966) results of two selected isolates were: levan production (-), oxidase reaction (+), potato soft rot (-), arginine dihydrolase production (-), and tobacco hypersensitivity (+), which corresponds to the profile of Pseudomonas cichorii. Molecular identification was performed through amplification and sequencing of the 16S rRNA (GenBank Accessions No. OR540674 to OR540675), gyrB and rpoD genes (Hwang et al. 2005; Sarkar and Guttmann 2004) (GenBank Accessions No. OR558279 to OR558282). BLAST analysis of the 16S rRNA gene of the isolates resulted in a match with a 99.86% identity with P. cichorii type strain ATCC 10857 (NR_112070.1). BLAST analysis of gyrB and rpoD resulted in a match with a 100% (630/630 bp) and >99% (546/550 bp) identity respectively, with strains of P. cichorii. Five six-month-old lettuce plants cv. Desert Storm were pricked in the midrib with a toothpick smeared with a fresh colony grown on KB medium. Seven days after inoculation, the plants showed dark brown, watery lesions, characteristic of damage caused by P. cichorii (Figure S1). Bacteria were isolated again from the inoculated plants and were identified as P. cichorii using LOPAT and molecular identification techniques. Midrib rot caused by P. cichorii was reported as an emerging disease of greenhouse-grown lettuce by Cottyn et al. (2009). In Chile, P. cichorii was previously described affecting nectarine fruits (Pinto de Torres and Carreño Ibañez 1983) and reported as a pathogen of lettuce among others horticultural crops by Servicio Agrícola y Ganadero of the Government of Chile (Acuña 2008), but this is the first report of P. cichorii affecting hydroponic lettuce plants in Chile. These results will be the basis of future studies to evaluate the origin of the infection, the potential dissemination, and the implementation of disease management to avoid the damage caused by this bacterium in hydroponic systems in this crop of growing importance in Chile.

Development of a Prediction Model for Short-Term Remission of Patients with Crohn's Disease Treated with Anti-TNF Drugs.

Therapy with anti-tumor necrosis factor (TNF) has dramatically changed the natural history of Crohn's disease (CD). However, these drugs are not without adverse events, and up to 40% of patients could lose efficacy in the long term. We aimed to identify reliable markers of response to anti-TNF drugs in patients with CD. A consecutive cohort of 113 anti-TNF naive patients with CD was stratified according to clinical response as short-term remission (STR) or non-STR (NSTR) at 12 weeks of treatment. We compared the protein expression profiles of plasma samples in a subset of patients from both groups prior to anti-TNF therapy by SWATH proteomics. We identified 18 differentially expressed proteins (p ≤ 0.01, fold change ≥ 2.4) involved in the organization of the cytoskeleton and cell junction, hemostasis/platelet function, carbohydrate metabolism, and immune response as candidate biomarkers of STR. Among them, vinculin was one of the most deregulated proteins (p < 0.001), whose differential expression was confirmed by ELISA (p = 0.054). In the multivariate analysis, plasma vinculin levels along with basal CD Activity Index, corticosteroids induction, and bowel resection were factors predicting NSTR.

The thrombus proteome in stroke reveals a key role of the innate immune system and new insights associated with its etiology, severity, and prognosis.

BACKGROUND: Nowadays little is known about the molecular profile of the occluding thrombus of patients with ischemic stroke. OBJECTIVES: To analyze the proteomic profile of thrombi in patients who experienced an ischemic stroke in order to gain insights into disease pathogenesis. METHODS: Thrombi from an exploratory cohort of patients who experienced a stroke were obtained by thrombectomy and analyzed by sequential window acquisition of all theoretical spectra-mass spectrometry. Unsupervised k-means clustering analysis was performed to stratify patients who experienced a stroke. The proteomic profile was associated with both the neurological function (National Institute of Health Stroke Scale [NIHSS]) and the cerebral involvement (Alberta Stroke Program Early CT Score [ASPECTS]) prior to thrombectomy and the clinical status of patients at 3 months using the modified Rankin Scale. In an independent cohort of 210 patients who experienced a stroke, the potential role of neutrophils in stroke severity was interrogated. RESULTS: Proteomic analysis identified 580 proteins in thrombi, which were stratified into 4 groups: hemostasis, proteasome and neurological diseases, structural proteins, and innate immune system and neutrophils. The thrombus proteome identified 3 clusters of patients with distinctive severity, prognosis, and etiology of the stroke. A protein signature clearly distinguished atherothrombotic and cardioembolic strokes. Several proteins were significantly correlated with the severity of the stroke (NIHSS and ASPECTS). Functional proteomic analysis highlighted the prominent role of neutrophils in stroke severity. This was in line with the association of neutrophil activation markers and count with NIHSS, ASPECTS, and the modified Rankin Scale score 90 days after the event. CONCLUSION: The use of sequential window acquisition of all theoretical spectra-mass spectrometry in thrombi from patients who experienced an ischemic stroke has provided new insights into pathways and players involved in its etiology, severity, and prognosis. The prominent role of the innate immune system identified might pave the way for the development of new biomarkers and therapeutic approaches in this disease.

Pirfenidone Protects from UVB-Induced Photodamage in Hairless Mice.

BACKGROUND: Ultraviolet radiation (UV) is the main environmental factor that causes histological degenerative changes of the skin giving rise to a chronic process called photodamage. Non-melanoma skin cancer induced by UVB radiation is a result of a cascade of molecular events caused by DNA damage in epidermis cells, including persistent inflammation, oxidative stress, and suppression of T cell-mediated immunity. Retinoids such as tretinoin have been widely used in skin to treat photoaging and photodamage, though its secondary adverse effects have been recognized. Pirfenidone (PFD) has emerged as an antifibrogenic, anti-inflammatory and antioxidant agent, and in this work its efficacy was evaluated in a model of UVB-induced photodamage. METHODS: Epidermal, dermal, and inflammatory changes were measured by histomorphometric parameters. In addition, gene, and protein expression of key molecules in these processes were evaluated. RESULTS: Our results revealed an anti-photodamage effect of topical PFD with absence of inflammatory skin lesions determined by dermoscopy. In addition, PFD reduced elastosis, improved organization, arrangement, and deposition of dermal collagens, downregulated several pro-inflammatory markers such as NF-kB, IL-1, IL-6 and TNFα, and decreased keratinocyte damage. CONCLUSION: Topical pirfenidone represents a promising agent for the treatment of cell photodamage in humans. Clinical trials need to be carried out to explore this premise.

Data Processing and Analysis in Mass Spectrometry-Based Metabolomics.

Metabolomics is the latest of the omics sciences. It attempts to measure and characterize metabolites-small chemical compounds <1500 Da-on cells, tissue, or biofluids, which are usually products of biological reactions. As metabolic reactions are closer to the phenotype, metabolomics has emerged as an attractive science for various areas of research, including personalized medicine. However, due to the complexity of data obtained and the absence of curated databases for metabolite identification, data processing is the major bottleneck in this area since most technicians lack the required bioinformatics expertise to process datasets in a reliable and fast manner. The aim of this chapter is to describe the available tools for data processing that makes an inexperienced researcher capable of obtaining reliable results without having to undergo through huge parametrization steps.

The NtrYX Two-Component System of Paracoccus denitrificans Is Required for the Maintenance of Cellular Iron Homeostasis and for a Complete Denitrification under Iron-Limited Conditions.

Denitrification consists of the sequential reduction of nitrate to nitrite, nitric oxide, nitrous oxide, and dinitrogen. Nitrous oxide escapes to the atmosphere, depending on copper availability and other environmental factors. Iron is also a key element because many proteins involved in denitrification contain iron-sulfur or heme centers. The NtrYX two-component regulatory system mediates the responses in a variety of metabolic processes, including denitrification. A quantitative proteomic analysis of a Paracoccus denitrificans NtrY mutant grown under denitrifying conditions revealed the induction of different TonB-dependent siderophore transporters and proteins related to iron homeostasis. This mutant showed lower intracellular iron content than the wild-type strain, and a reduced growth under denitrifying conditions in iron-limited media. Under iron-rich conditions, it releases higher concentrations of siderophores and displayes lower nitrous oxide reductase (NosZ) activity than the wild-type, thus leading to nitrous oxide emission. Bioinformatic and qRT-PCR analyses revealed that NtrYX is a global transcriptional regulatory system that responds to iron starvation and, in turn, controls expression of the iron-responsive regulators fur, rirA, and iscR, the denitrification regulators fnrP and narR, the nitric oxide-responsive regulator nnrS, and a wide set of genes, including the cd1-nitrite reductase NirS, nitrate/nitrite transporters and energy electron transport proteins.

Discriminator for Cutaneous Leishmaniasis Using MALDI-MSI in a Murine Model.

Cutaneous leishmaniasis is a skin disease caused by flagellate protozoa of the genus Leishmania and transmitted by sandflies of the genus Lutzomyia. Around 1 million new cases occur in the world annually, with a total of 12 million people affected, mainly in rural areas with low access to health services and adequate treatments. In the area of the Americas, Colombia has one of the highest infection rates after Brazil. Topical treatments with pentamidine isethionate (PMD) present an attractive alternative due to their ease of application and low costs. However, cutaneous leishmaniasis lesions present nodules with seropurulent exudate that, when drying, form hyperkeratotic lesions, hindering the effective penetration of drugs for their treatment. The use of molecular histology techniques, such as MALDI-MSI, allow in situ evaluation of the penetration of the treatment to the sections of the dermis where the disease-causing parasite resides. However, the large volume of information generated makes it impossible to process it manually. Machine learning techniques allow the unsupervised processing of large amounts of information, generating prediction models for the classification of new information. This work proposes a low-cost method to generate cutaneous leishmaniasis detection and classification models using MALDI-MSI images taken from murine models. The proposed models allow a 95% efficiency when separating healthy samples from infected samples and an effectiveness of 67% when separating effectively treated samples from unsuccessfully treated samples.

First Report of bacterial speck caused by Pseudomonas syringae pv. tomato Race 1 affecting tomato in different Regions of Chile.

In Chile, tomato is one of the most widely cultivated vegetables, with around 5,000 ha for fresh market and 8,000 ha for processing industry. During recent years, symptoms of bacterial speck caused by Pseudomonas syringae pv. tomato, have been observed more frequently in tomato plants in different regions of Chile. This pathogen was first identified in Chile in 1987 (Latorre & Lolas, 1988) and the presence of an apparent new variant was reported in 2004 (Besoain et al. 2004). To characterize the pathogen that was affecting this crop, samples of diseased tomato plants were taken in three regions of Chile. The samples were collected in 2016 in Northern Chile in Lluta Valley from the Arica y Parinacota Region, and in Central Chile, in 2014 in Limache from Valparaíso Region and in 2015 in Pichidegua from O´Higgins Region. Affected tomato plants exhibited dark brown to black lesions surrounded by yellow halos in the leaves, and dark brown to black lesions in the stems, pedicels, and peduncles. Plants tissues were macerated, and the suspension was spread on King's B medium, resulting in fluorescent colonies visualized under 366 nm UV light. LOPAT tests results of three selected isolates from different Regions, were: levan production (+), oxidase reaction (-), potato soft rot (-), arginine dihydrolase production (-), and tobacco hypersensitivity (+) (Lelliot et al. 1966). Molecular identification was carried out by amplification and sequence analysis of housekeeping genes cts, encoding citrate synthase, gyrB, encoding DNA gyrase B, and rpoD, encoding sigma factor 70 (Hwang et al. 2005; Sarkar & Guttmann 2004) (GenBank Accessions No. OK001658-OK001666). BLAST analysis of cts and rpoD genes of the three isolates resulted in a match with a 100% identity (919 bp and 491 bp respectively) with Pseudomonas syringae pv. tomato strain B13-200 (GenBank: CP019871.1). BLAST analysis of gyrB gene of two isolates resulted in a match with a 100% identity (684 bp) and one isolate with 99.85% (683 bp) with Pseudomonas syringae pv. tomato strain B13-200. To identify the race 1, each strain was inoculated in five tomato plants cv. San Pedro, susceptible to both races of P. syringae pv. tomato, and cv. Rio Grande, resistant to race 0. The tomato plants were slightly wounded with a metal sponge and then sprayed with the bacterial suspension (108 CFU mL-1) of each isolate, including the reference strain DC3000 (race 0). Negative controls were sprayed with water. The plants inoculated with Chilean strains in both cv. San Pedro and cv. Rio Grande, showed symptoms of bacterial speck after 7 days. Plants inoculated with DC3000 strain showed symptoms only in cv. San Pedro, whereas control plants remained asymptomatic. Strains were re-isolated from symptomatic plants and identified by gene sequence analyses as Pseudomonas syryngae pv. tomato. This is the first report of Pseudomonas syryngae pv. tomato race 1 in Chile. Race 1 was previously reported in Canada (Lawton and MacNeill. 1986), in Italy (Buonaurio et al. 1996), in California (Arredondo and Davis 2000), in Portugal (Cruz et al. 2010), and in other states in the USA and countries in South America, Europe, Africa, and Australia, becoming the most commonly isolated race today (Cai et al 2011). These results will be the base for future studies of epidemiology, characterization, and virulence in order to explain the outbreak of this disease and the severity of symptoms observed.

Redox and global interconnected proteome changes in mice exposed to complex environmental hazards surrounding Doñana National Park.

Natural environments are receiving an increasing number of contaminants. Therefore, the evaluation and identification of early responses to pollution in these complex habitats is an urgent and challenging task. Doñana National Park (DNP, SW Spain) has been widely used as a model area for environmental studies because, despite its strictly protected core, it is surrounded by numerous threat sources from agricultural, mining and industrial activities. Since many pollutants often induce oxidative stress, redox proteomics was used to detect redox-based variations within the proteome of Mus spretus mice captured in DNP and the surrounding areas. Functional analysis showed that most differentially oxidized proteins are involved in the maintenance of homeostasis, by eliciting mechanisms to respond to toxic substances and oxidative stress, such as antioxidant and biotransformation processes, immune and inflammatory responses, and blood coagulation. Furthermore, changes in the overall protein abundance were also analysed by label-free quantitative proteomics. The upregulation of phase I and II biotransformation enzymes in mice from Lucio del Palacio may be an alert for organic pollution in the area located at the heart of DNP. Metabolic processes involved in protein turnover (proteolysis, amino acid catabolism, new protein biosynthesis and folding) were activated in response to oxidative damage to these biomolecules. Consequently, aerobic respiratory metabolism increased to address the greater ATP demands. Alterations of cholesterol metabolism that could cause hepatic steatosis were also detected. The proteomic detection of globally altered metabolic and physiological processes offers a complete view of the main biological changes caused by environmental pollution in complex habitats.

Diet switch and omega-3 hydroxy-fatty acids display differential hepatoprotective effects in an obesity/nonalcoholic fatty liver disease model in mice.

AIM: To study the effect of 18-hydroxy-eicosapentaenoic acid (18-HEPE) and 17-hydroxy-docosahexaenoic acid (17-HDHA) in a murine model of obesity/nonalcoholic fatty liver disease. METHODS: C57BL/6 mice were fed with standard chow diet (CD) or high-fat, fructose-enriched diet (HFD) for 16 wk. Then, three groups were treated for 14 d with either, diet switch (HFD for CD), 18-HEPE, or 17-HDHA. Weight and fasting glucose were recorded on a weekly basis. Insulin tolerance test was performed at the end of treatment. Histological analysis (HE and Masson's trichrome stain) and determination of serum insulin, glucagon, glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide, adiponectin and resistin were carried out as well as liver proteins by western blot. RESULTS: Mice treated with hydroxy-fatty acids 18-HEPE and 17-HDHA displayed no weight loss or improved insulin sensitivity. However, these mice groups showed a significant amelioration on serum GLP-1, adiponectin and resistin levels. Also, a significant reduction on inflammatory infiltrate was observed at both portal and lobular zones. Furthermore, up-regulation of PPARα/γ protein levels was observed in liver tissue and it was associated with decreased levels of NF-κB also determined by western blot analysis. On the other hand, diet switch regimen resulted in a marked improvement in most parameters including: weight loss, increased insulin sensitivity, decreased steatosis, restored levels of insulin, glucagon, leptin, adiponectin and resistin. However, no significant changes were observed regarding inflammatory infiltrate in this last group. CONCLUSION: 18-HEPE and 17-HDHA differentially exert hepatoprotective effects through up-regulation of nuclear receptors PPARα/γ and amelioration of serum adipokines profile.

A multicentric study to evaluate the use of relative retention times in targeted proteomics.

Despite the maturity reached by targeted proteomic strategies, reliable and standardized protocols are urgently needed to enhance reproducibility among different laboratories and analytical platforms, facilitating a more widespread use in biomedical research. To achieve this goal, the use of dimensionless relative retention times (iRT), defined on the basis of peptide standard retention times (RT), has lately emerged as a powerful tool. The robustness, reproducibility and utility of this strategy were examined for the first time in a multicentric setting, involving 28 laboratories that included 24 of the Spanish network of proteomics laboratories (ProteoRed-ISCIII). According to the results obtained in this study, dimensionless retention time values (iRTs) demonstrated to be a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups both intra- and inter-laboratories. iRT values also showed very low variability over long time periods. Furthermore, parallel quantitative analyses showed a high reproducibility despite the variety of experimental strategies used, either MRM (multiple reaction monitoring) or pseudoMRM, and the diversity of analytical platforms employed. BIOLOGICAL SIGNIFICANCE: From the very beginning of proteomics as an analytical science there has been a growing interest in developing standardized methods and experimental procedures in order to ensure the highest quality and reproducibility of the results. In this regard, the recent (2012) introduction of the dimensionless retention time concept has been a significant advance. In our multicentric (28 laboratories) study we explore the usefulness of this concept in the context of a targeted proteomics experiment, demonstrating that dimensionless retention time values is a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups.

Using environmental proteomics to assess pollutant response of Carcinus maenas along the Tunisian coast.

Biochemical responses to pollutants were studied at four Tunisia littoral sites using Carcinus maenas as a bioindicator. Proteomic analysis was used to assess the global impact of complex pollution mixtures, and to provide new biomarkers and basic insights into pollutant toxicity. Metal contents and metallothionein levels followed a gradient based on sampling sites: Bizerte ≫ Teboulba > Gargour~Mahres. Approximately 900 and 700 spots were resolved in digestive glands and gills, respectively. Gills from Bizerte animals had the maximum number of altered spots, mostly upregulated. In other locations, the number of altered spots in gills decreased in parallel to total metals in in the following order: Teboulba > Gargour > Mahres (mostly downregulated). Out of the 39 spots excised, ten proteins were identified in digestive glands and eight in gills. Digestive glands of Bizerte crabs had higher levels of ferritin, three vitellogenin forms and mannose-binding protein, while Gargour crabs had higher levels of four cryptocyanin forms. Gills of Bizerte crabs had higher levels of ferritin, three vitellogenins forms, lectin 4C, actin, and collagenolytic serine protease. Proteins with altered expression in crabs from Tunisia littoral are related to molting, oxidative stress and inflammation, innate immune response, and proteolysis.

Proteomic analysis through larval development of Solea senegalensis flatfish.

The post-embryonic development of the Senegalese sole, Solea senegalensis, a flatfish of growing interest in fisheries and aquaculture, is associated with drastic morpho-physiological changes during metamorphosis. Although in the last two decades knowledge on sole culture has notably increased, especially in Southern Europe, its progress was restricted due to lack of methods to control reproduction, improve larval quality and increase juvenile disease resistance. A limited knowledge of the physiological, molecular and genetic mechanisms involved is at the base of such limitation. A proteomic study was carried out to explore the molecular events that occur during S. senegalensis ontogenesis. Protein expression changes were monitored in larvae from 5 to 21 dph by combining 2DE and protein identification with de novo MS/MS sequencing. An average of 6177 ± 282 spots was resolved in 2DE gels. Hierarchical cluster analysis of the 705 selected spots grouped them in eight patterns. Thirty-four proteins were identified and assigned biological functions including structure, metabolism highlighting energy metabolism, transport, protein folding, stress response, chromatin organization and regulation of gene expression. These changes provide a sequential description of the molecular events associated with the biochemical and biological transformations that occur during sole larval development.

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.

Sensitivity to anti-Fas is independent of increased cathepsin D activity and adrenodoxin reductase expression occurring in NOS-3 overexpressing HepG2 cells.

Stable overexpression of endothelial nitric oxide synthase (NOS-3) in HepG2 cells (4TO-NOS) leads to increased nitro-oxidative stress and upregulation of the cell death mediators p53 and Fas. Thus, NOS-3 overexpression has been suggested as a useful antiproliferative mechanism in hepatocarcinoma cells. We aimed to identify the underlying mechanism of cell death induced by NOS-3 overexpression at basal conditions and with anti-Fas treatment. The intracellular localization of NOS-3, the nitro-oxidative stress and the mitochondrial activity were analysed. In addition, the protein expression profile in 4TO-NOS was screened for differentially expressed proteins potentially involved in the induction of apoptosis. NOS-3 localization in the mitochondrial outer membrane was not associated with changes in the respiratory cellular capacity, but was related to the mitochondrial biogenesis increase and with a higher protein expression of mitochondrial complex IV. Nitro-oxidative stress and cell death in NOS-3 overexpressing cells occurred with the expression increase of pro-apoptotic genes and a higher expression/activity of the enzymes adrenodoxin reductase mitochondrial (AR) and cathepsin D (CatD). CatD overexpression in 4TO-NOS was related to the apoptosis induction independently of its catalytic activity. In addition, CatD activity inhibition by pepstatin A was not effective in blocking apoptosis induced by anti-Fas. In summary, NOS-3 overexpression resulted in an increased sensitivity to anti-Fas induced cell death, independently of AR expression and CatD activity.

Proteomic analysis in caged Mediterranean crab (Carcinus maenas) and chemical contaminant exposure in Téboulba Harbour, Tunisia.

This study uses proteomics approach to assess the toxic effects of contaminants in the Mediterranean crab (Carcinus maenas) after transplantation into Téboulba fishing harbour. High levels of aliphatic and aromatic hydrocarbons were detected in sediments. Although their effects on vertebrates are well described, little is known about their early biological effects in marine invertebrates under realistic conditions. Protein expression profiles of crabs caged for 15, 30 and 60 days were compared to unexposed animals. Nineteen proteins with significant expression differences were identified by capLC-µESI-IT MS/MS and homology search on databases. Differentially expressed proteins were assigned to five different categories of biological function including: (1) chitin catabolism, (2) proteolysis, (3) exoskeleton biosynthesis, (4) protein folding and stress response, and (5) transport. The proteins showing major expression changes in C. maenas after different caging times may be considered as novel molecular biomarkers for effectively biomonitoring aquatic environment contamination.

[Intraductal papillary mucinous neoplasm and recurrent pancreatitis]. / Neoplasia intraductal papilar quística mucinosa del páncreas y pancreatitis recurrente.

Ohashi described for the first time the IPMN on 1982 as a pancreatic neoplasia with mucine cells forming papillae and producing dilatation of the main pancreatic duct or its branches. The IPMN represent the 1% of the pancreatic tumors and 5% of the cystic neoplasias. It is potentially malignant in a period of five years being more frequent in males between 60-70 and clinically these patients' presents as acute, recurrent or chronic pancreatitis, with an incidence of malignancy from 25% to 70%. CT scan and cholangio MRI allows the diagnosis, the variety, localization and possibility of determine malignancy. The treatment is the Whipple resection. We are reporting the case of an obese middle age male, being observed along the last 10 years because of recurrent pancreatitis with a cystic lesion of the head of the pancreas. The CT scan, endoscopic-ultrasound and the analysis of the liquid content suggested a mucinous lesion, reason why the patient underwent a pancreatic-duodenal resection. The histology study confirms the diagnosis of IPMN.

Neoplasia intraductal papilar quística mucinosa del páncreas y pancreatitis recurrente / Intraductal papillary mucinous neoplasm

La neoplasia Intraductal papilar quística mucinosa (NIPM) fue descrita por primera vez en 1982 por Ohashi, como una neoplasia del páncreas caracterizada por una proliferación de células neoplásicas mucinosas, que forman papilas y causan dilatación del ducto principal o sus ramas. Las NIPM representan el 1% de las neoplasias pancreáticas y el 25% de las neoplasias quísticas. Son potencialmente malignas, progresan desde una lesión benigna hasta carcinoma en un promedio de 5 años. Afecta con mayor frecuencia al sexo masculino entre 60 y 70 años, con síntomas de pancreatitis crónica o pancreatitis aguda recidivante y se localiza preferentemente en la cabeza y proceso uncinado. La clasificación depende de su localización, en variante ducto principal, variante rama lateral o mixto y la importancia radica en el pronóstico, con incidencia de cáncer que varía de 25% a 70%. Las imágenes (T.E.M y Colangio RM) nos permiten establecer el diagnóstico, la variedad, la localización, la extensión y determinar indicios de benignidad o malignidad. Además la eco endoscopía nos permite obtener una muestra y poder estudiar el líquido del quiste. El tratamiento de estas neoplasias es la duodeno pancreatectomía de Whipple. Reportamos un paciente obeso, con historia de pancreatitis recurrente, observado a lo largo de 10 años por una lesión quística del páncreas que creció en los últimos 24 meses. El estudio eco endoscópico y el análisis del líquido del quiste orientó al diagnóstico de NIPM por lo que se le sometió a una resección duodeno pancreática con evolución favorable. El estudio histológico confirmó el diagnóstico de NIPM.

Ohashi described for the first time the IPMN on 1982 as a pancreatic neoplasia with mucine cells forming papillae and producing dilatation of the main pancreatic duct or its branches. The IPMN represent the 1% of the pancreatic tumors and 5% of the cystic neoplasias. It is potentially malignant in a period of five years being more frequent in males between 60-70 and clinically these patients’ presents as acute, recurrent or chronic pancreatitis, with an incidence of malignancy from 25% to 70%. CT scan and cholangio MRI allows the diagnosis, the variety, localization and possibility of determine malignancy. The treatment is the Whipple resection. We are reporting the case of an obese middle age male, being observed along the last 10 years because of recurrent pancreatitis with a cystic lesion of the head of the pancreas. The CT scan, endoscopic-ultrasound and the analysis of the liquid content suggested a mucinous lesion, reason why the patient underwent a pancreatic-duodenal resection. The histology study confirms the diagnosis of IPMN.

Biochemical responses in seabream (Sparus aurata) caged in-field or exposed to benzo(a)pyrene and paraquat. Characterization of glutathione S-transferases.

Gilthead seabream (Sparus aurata) specimens were caged in-field at the Téboulba harbour or exposed to benzo(a)pyrene [B(a)P] or to paraquat [PQ] plus B(a)P, and several biochemical biomarker responses were investigated. Antioxidant enzymes, such as glutathione peroxidase, catalase and glutathione reductase, significantly increased in the in-field and B(a)P+PQ exposures, but were only moderately affected by B(a)P alone. Glucose-6-phosphate and 6-phosphogluconate dehydrogenases significantly diminished after in-field exposure. Different responses with biotransformation enzymes were observed: the P4501A-associated EROD activity was highly induced in response to B(a)P and B(a)P+PQ exposures, while total activity of the glutathione S-transferase (GST) was similar to control. However, after purification of the GST proteins by affinity chromatograpy and analysis by two-dimensional electrophoresis, nineteen highly reproducible isoforms were resolved. In addition, some of reproducible isoforms showed different and specific expression patterns in response to contaminants. Thus, proteomic analysis of the purified GST subunits is a reliable tool for ecotoxicological research, useful in polluted marine ecosystem as an effective biomarker of contamination.

Hepatic proteome changes in Solea senegalensis exposed to contaminated estuarine sediments: a laboratory and in situ survey.

Assessing toxicity of contaminated estuarine sediments poses a challenge to ecotoxicologists due to the complex geochemical nature of sediments and to the combination of multiple classes of toxicants. Juvenile Senegalese soles were exposed for 14 days in the laboratory and in situ (field) to sediments from three sites (a reference plus two contaminated) of a Portuguese estuary. Sediment characterization confirmed the combination of metals, polycyclic aromatic hydrocarbons and organochlorines in the two contaminated sediments. Changes in liver cytosolic protein regulation patterns were determined by a combination of two-dimensional electrophoresis with de novo sequencing by tandem mass spectrometry. From the forty-one cytosolic proteins found to be deregulated, nineteen were able to be identified, taking part in multiple cellular processes such as anti-oxidative defence, energy production, proteolysis and contaminant catabolism (especially oxidoreductase enzymes). Besides a clear distinction between animals exposed to the reference and contaminated sediments, differences were also observed between laboratory- and in situ-tested fish. Soles exposed in the laboratory to the contaminated sediments failed to induce, or even markedly down-regulated, many proteins, with the exception of a peroxiredoxin (an anti-oxidant enzyme) and a few others, when compared to reference fish. In situ exposure to the contaminated sediments revealed significant up-regulation of basal metabolism-related enzymes, comparatively to the reference condition. Down-regulation of basal metabolism enzymes, related to energy production and gene transcription, in fish exposed in the laboratory to the contaminated sediments, may be linked to sediment-bound contaminants and likely compromised the organisms' ability to deploy adequate responses against insult.