Influence of <Emphasis Type="Italic">Moraxella</Emphasis> sp. colonization on the kidney proteome of farmed gilthead sea breams (<Emphasis Type="Italic">Sparus aurata</Emphasis>, L.)

Background  

Currently, presence of Moraxella sp. in internal organs of fish is not considered detrimental for fish farming. However, bacterial colonization of internal organs can affect fish wellness and decrease growth rate, stress resistance, and immune response. Recently, there have been reports by farmers concerning slow growth, poor feed conversion, and low average weight increase of fish farmed in offshore floating sea cages, often associated with internal organ colonization by Moraxella sp. Therefore, presence of these opportunistic bacteria deserves further investigations for elucidating incidence and impact on fish metabolism.     

Application of 2-D DIGE to formalin-fixed, paraffin-embedded tissues

The ability to investigate the proteome of formalin-fixed, paraffin-embedded (FFPE) tissues can be considered a major recent achievement in the field of clinical proteomics. However, gel-based approaches to the investigation of FFPE tissue proteomes have lagged behind, mainly because of insufficient quality of full-length protein extracts. Here, the 2-D DIGE technology was investigated for applicability to FFPE proteins, for internal reproducibility among replicate FFPE extracts, and for comparability between FFPE and fresh-frozen tissue profiles. The 2-D DIGE patterns obtained upon labeling and electrophoresis of replicate FFPE tissue extracts were highly reproducible, with satisfactory resolution and complexity. Moreover, the implementation of DIGE enabled to highlight and characterize the consistent differences found in the FFPE profiles compared with fresh-frozen profiles, represented by an acidic shift, directly correlated to the protein pI value, and by a reduction in spot signal intensity, directly correlated to molecular weight and percentage of lysine residues. Being constantly and reproducibly present in all FFPE tissue extract replicates at similar extents, these modifications do not appear to hinder the comparative analysis of FFPE tissue extracts by 2-D DIGE, opening the way to its application for the differential proteomic investigation of archival tissue repositories.     

The sheep milk fat globule membrane proteome

Milk fat globule membranes (MFGM) are three-layered structures that enclose fat droplets, and are composed by an internal monolayer of endoplasmic reticulum origin, surrounded by a bilayer derived from the apical membrane of the lactating cell. In this work, an optimized protein extraction method was applied to sheep MFGM, and extracts were subjected to SDS-PAGE separation followed by shotgun LC tandem mass spectrometry (GeLC-MS/MS) for identification and characterization. In total, 140 unique sheep MFGM proteins (MFGMPs) were identified. All protein identification data were subjected to Gene Ontology (GO) classification for localization and function. Moreover, the relative abundance of all identified MFGMPs was estimated by means of the normalized spectral abundance factor (NSAF) approach, and GO abundance classes were obtained. The data gathered in this work provide a detailed picture of the proteome expressed in healthy sheep MFGs, and lay the foundations for future studies on sheep lactation physiology and on its alterations in pathological conditions.     

Spillover of Drosophila suzukii between noncrop and crop areas: implications for pest management

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Comparison of attractants for monitoring Drosophila suzukii in sweet cherry orchards in Italy

The invasiveness of Drosophila suzukii Matsumura is causing sizable damage to soft fruits and cherry. In order to define a targeted strategy for D. suzukii management, it is important to have access to highly sensitive trapping tools for detection, monitoring and control that are also selective, practical in use, economic, and with low environmental impact. The aim of this study was to compare different combinations of traps and lures to define a best practice approach for D. suzukii monitoring in Italy, extended over a wide elevational gradient and landscape complexity. The high attractiveness was not always combined with high selectivity, as in some cases up to 95% of the Drosophila spp. captured were belonging to species different from D. suzukii. The commercial Droso‐Trap was very efficient providing high capture ability. Overall, the most attractive lure was Droskidrink while Suzukii Trap was the most selective one. Lure attractiveness and selectivity, however, changed during the season in relation to environmental temperatures and phenological developmental stages, suggesting the need for implementing different lures in different periods and for the different purposes (monitoring or mass trapping). In terms of trap management, Droskidrink showed the problem of bacterial‐gel formation, while Suzukii Trap and Pherocon SWD were easier to handle as they did not need to be checked at weekly interval. Finally, these results were gathered to convey the most efficient combination of trap and bait able to efficiently perform mass trapping and attract and kill technique.     

Effects of postmortem storage temperature on sea bass (Dicentrarchus labrax) muscle protein degradation: analysis by 2-D DIGE and MS

Storage conditions are known to be important for postmortem deterioration of fish muscle, and temperature is one of the factors with the strongest impact on this process. In order to shed light on the influence of temperature on the status of sea bass (Dicentrarchus labrax) muscle proteins during postmortem storage, a 2-D DIGE and mass spectrometry study was performed on fish kept at either 1 or 18°C for 5 days. As expected, the greatest alterations in sea bass filet protein composition were observed upon postmortem storage at 18°C, with distinct changes appearing in the 2-D protein profile after 5 days of storage at this temperature. In particular, degradation of the myofibrillar protein myosin heavy chain and of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, among the most abundant muscle proteins, could be clearly observed upon storage at higher temperatures. Although to a lesser extent, however, several proteins were observed to vary in abundance also upon storage for 5 days at 1°C. In particular, one of the most interesting observations was the rapid and significant decrease in the abundance of nucleoside diphosphate kinase B and phosphoglycerate mutase 2, which was observed also at low storage temperatures and appeared to be temperature-independent. The results of this study offer new knowledge on changes occurring in sea bass muscle proteins during postmortem storage at different temperatures and provide indications on protein degradation trends that might be useful for monitoring freshness of fish and quality of storage conditions.     

Proteomic analysis of formalin-fixed, paraffin-embedded lung neuroendocrine tumor samples from hospital archives

Hospital tissue repositories host an invaluable supply of diseased samples with matched retrospective clinical information. In this work, a recently optimized method for extracting full-length proteins from formalin-fixed, paraffin-embedded (FFPE) tissues was evaluated on lung neuroendocrine tumor (LNET) samples collected from hospital repositories. LNETs comprise a heterogeneous spectrum of diseases, for which subtype-specific diagnostic markers are lacking. Six archival samples diagnosed as typical carcinoid (TC) or small cell lung carcinoma (SCLC) were subjected to a full-length protein extraction followed by a GeLC-MS/MS analysis, enabling the identification of over 300 distinct proteins per tumor subtype. All identified proteins were categorized through DAVID software, revealing a differential distribution of functional classes, such as those involved in RNA processing, response to oxidative stress and ion homeostasis. Moreover, using spectral counting for protein abundance estimation and beta-binomial test as statistical filter, a list of 28 differentially expressed proteins was generated and submitted to pathway analysis by means of Ingenuity Pathway Analysis software. Differential expression of chromogranin-A (more expressed in TCs) and stathmin (more expressed in SCLCs) was consistently confirmed by immunohistochemistry. Therefore, FFPE hospital archival samples can be successfully subjected to proteomic investigations aimed to biomarker discovery following a GeLC-MS/MS label-free approach.     

Unusual radioresistance of nitrogen-fixing cultures of <Emphasis Type="Italic">Anabaena</Emphasis> strains

Nitrogen-fixing cultures of two species of the filamentous, heterocystous cyanobacterium Anabaena, namely Anabaena sp. strain L-31 and Anabaena torulosa were found to be highly tolerant to ⁶⁰Co gamma radiation. No adverse effect on diazotrophic growth and metabolism were observed up to a dose of 5 kGy. At higher doses, radiation tolerance showed a correspondence with the inherent osmotolerance, with Anabaena L-31 being the more radiation tolerant as well as osmotolerant strain. In Anabaena L-31, exposure to 6 kGy of gamma rays resulted in genome disintegration, but did not reduce viability. Irradiation delayed heterocyst differentiation and nitrogen fixation, and marginally affected diazotrophic growth. All the affected parameters recovered after a short lag, without any discernible postirradiation phenotype. The radiation tolerance of these Gram-negative photoautodiazotrophs is comparable with that of the adiazotrophic photoautotrophic cyanobacterium Chroococcidiopsis or adiazotrophic heterotroph Deinococcus radiodurans. This is the first report of extreme radioresistance in nitrogen-fixing Anabaena cultures.     

Dose to the skin in helical tomotherapy: Results of in vivo measurements with radiochromic films

PurposeThe aim of this study is to report results of measurements of dose to the skin in vivo with radiochromic EBT films in treatments with helical tomotherapy.Methods and materialsIn vivo measurements were performed by applying pieces of radiochromic films to the skin or to the inner side of thermoplastic mask before the treatment. The sites of treatment included scalp, brain, head and neck, cranio-spinal axis and lower limbs. Skin dosimetry was performed in a patient who experienced grade 3–4 acute side effects to the skin shortly after the first treatment sessions. For each patient we measured the setup errors using the daily MVCT acquired for image guidance of the treatment. EBT films were read with a flatbed Epson Expression scanner and images were processed with an in-house written routine.ResultsA total of 96 measurements of dose to the skin performed on 14 patients. The mean difference and standard error of the mean difference between measured and TPS-calculated dose was ?9.2% ± 2.6% for all treatments, ?6.6% ± 2.6% for head and neck treatments. These differences were statistically significant at the 0.05 significance level (t-Student test). Planned dose and dose range in the region of measurements were not correlated with dose discrepancy.ConclusionsRadiochromic EBT films are suitable detectors for surface dose measurements in tomotherapy treatments. Results show that TPS overestimates dose to the skin measured with EBT radiochromic films. In vivo skin measurements with EBT films are a useful tool for quality assurance of tomotherapy treatments, as the treatment planning system may not give accurate dose values at the surface.