Proteomics of fish white muscle and Western blotting to detect putative allergens

A detailed workflow is provided for preparation from teleost fish white muscle of extracts for proteomics analysis. The protocol generates samples that can be analyzed by SWATH (Sequential Window data independent Acquisition of the Total High-resolution-Mass Spectra), a modern MS-based quantitative label free technology. The main steps for the extraction of three independent protein fractions, (1) soluble sarcoplasmic, (2) soluble myofibrillar, and (3) insoluble material, from fish white muscle are detailed. Coupled to the protein extraction protocol a Western blotting approach is outlined for detection of common fish allergens, in this case β-parvalbumin, in the white muscle sarcoplasmic protein fraction.PO MAR2020, Portugal2020, EU - Fundo Europeu dos Assuntos Marítimos e das Pescas (FEAMP), Project SEAFOODQual (MAR-01.03.01-FEAMP-0050) (Program: MAR2020-P01M03). Additional funds came from H2020-MSCA-RISE-2019, ICHTHYS (n° 872217). FCT provided CCMARs core funding (UIDB/04326/2020) and additional funds came from the operational programs CRESC Algarve 2020, COMPETE 2020, to EMBRC.PT ALG-01-0145-FEDER-022121.info:eu-repo/semantics/publishedVersio

Evolution and diversity of alpha-carbonic anhydrases in the mantle of the Mediterranean mussel (Mytilus galloprovincialis)

The α-carbonic anhydrases (α-CAs) are a large and ancient group of metazoan-specific enzymes. They generate bicarbonate from metabolic carbon dioxide and through calcium carbonate crystal formation play a key role in the regulation of mineralized structures. To better understand how α-CAs contribute to shell mineralization in the marine Mediterranean mussel (Mytilus galloprovincialis) we characterized them in the mantle. Phylogenetic analysis revealed that mollusc α-CA evolution was affected by lineage and species-specific events. Ten α-CAs were found in the Mediterranean mussel mantle and the most abundant form was named, MgNACR, as it grouped with oyster nacreins (NACR). Exposure of the Mediterranean mussel to reduced water salinity (18 vs 37 ppt), caused a significant reduction (p < 0.05) in mantle esterase activity and MgNACR transcript abundance (p < 0.05). Protonograms revealed multiple proteins in the mantle with α-CA hydratase activity and mapped to a protein with a similar size to that deduced for monomeric MgNACR. Our data indicate that MgNACR is a major α-CA enzyme in mantle and that by homology with oyster nacreins likely regulates mussel shell production. We propose that species-dependent α-CA evolution may contribute to explain the diversity of bivalve shell structures and their vulnerability to environmental changes.Agência financiadora European Union 605051 Portuguese national funds from FCT - Foundation for Science and Technology UID/Multi/04326/2019 operational programmes CRESC Algarve 2020 and COMPETE 2020 through project EMBRC FCT UID/Multi/04326/2019 SFRH/BPD/79105/2011 SFRH/BPD/89811/2012 EMBRIC (European Union's Horizon 2020 research and innovation programme) 654008 CAPES, Brazil 88887.116718/2016-00 PT ALG-01-0145-FEDER-022121info:eu-repo/semantics/publishedVersio

Chronic stress impairs the local immune response during cutaneous repair in gilthead sea bream (Sparus aurata, L.)

Scale removal in fish triggers a damage-repair program to re-establish the lost epidermis and scale and an associated local immune response. In mammals, chronic stress is known to delay wound healing and to modulate the cutaneous stress axis, but this is unstudied in teleost fish the most successful extant vertebrates. The present study was designed to test the hypothesis that chronic stress impairs cutaneous repair in teleost fish as a consequence of suppression of the immune response. The hypothesis was tested by removing the scales and damaging the skin on one side of the body of fish previously exposed for 4 weeks to a chronic crowding stress and then evaluating cutaneous repair for 1 week. Scale removal caused the loss of the epidermis although at 3 days it was re-established. At this stage the basement membrane was significantly thicker (p = 0.038) and the hypodermis was significantly thinner (p = 0.016) in the regenerating skin of stressed fish relative to the control fish. At 3 days, stressed fish also had a significantly lower plasma osmolality (p = 0.015) than control fish indicative of reduced barrier function. Chronic stress caused a significant down-regulation of the glucocorticoid receptor (gr) in skin before damage (time 0, p = 0.005) and of star at 3 and 7 days (p < 0.05) after regeneration relative to control fish. In regenerating skin key transcripts of cutaneous repair, pcna, colival and nunp9, and the inflammatory response, tgf beta 1, mpo and crtac2, were down-regulated (p < 0.05) by chronic stress. Irrespective of chronic stress and in contrast to intact skin many hyper pigmented masses, putative melanomacrophages, infiltrated the epidermis of regenerating skin. This study reveals that chronic stress suppresses the local immune response to scale removal and impairs the expression of key transcripts of wound healing. Elements of the stress axis were identified and modulated by chronic stress during cutaneous repair in gilthead seabream skin.European Regional Development Fund through COMPETEPortuguese Foundation for Science and Technology (FCT) [CCMAR/Multi/04326/2013, PTDC/MAR/ 122296/2010]FCT [UID/Multi/04326/ 2013, SFRH/BPD/79105/2011]Ministry of Science and Higher Education, Polan

Web evaluator for students: a case example

Getting the attention of students has always been a hard task for most teachers. In this context the use of new technologies can be taken into consideration for giving the teacher new means to incentive his students. The tool described in this article gives importance to the opinion of the students for their own evaluation. Thanks to it a group of students from the University of Minho was able to post questions to their colleagues and to evaluate their peer’s works on the discipline of Power Electronics Complement

Short- and long-term effects on growth and expression patterns in response to incubation temperatures in Senegalese sole

In this study, the short- and long-term effects of embryo incubation temperatures (16, 18 and 20 °C) on development and growth of the flatfish Senegalese sole (Solea senegalensis) was determined by investigating the expression patterns of the epigenetic regulators DNA methyltransferases (dnmt) and histone 3 (H3) and genes belonging to the retinoic acid (RA), insulin-like growth factors (IGFs) and hypothalamic-pituitary-thyroid (HPT) axes. Results indicated that egg incubation temperature affected embryo development, but not survival, and incubation at 16 °C significantly delayed development. Coincident with these effects, levels of muscle-specific dnmt3aa transcripts and histone H3 protein levels were significantly different between the 16 and 20 °C groups at hatch. The larvae from eggs incubated at 20 °C relative to the 16 °C group had significantly higher transcript levels of four genes belonging to the HPT axis (trhr1a, tshr, thrb and dio2), four genes of the RA axis (aldh1a2, cyp26a1, rara2, rarg), igfbp1 and the glycolytic enzyme gapdh2. Taken together the data suggest that higher egg incubation temperatures enhance energy production, which accelerates cell proliferation and larval development and that hatching is a key moment for the regulation of epigenetic mechanisms. Long-term effects of egg and larval incubation temperatures were revealed by a higher mRNA abundance of the thyroid-related genes tgb and tpo and the RA degrading enzyme cyp26a1 in pre- and metamorphic larvae when they were incubated at 20 °C as embryos and may be related to the earlier initiation of metamorphosis in the pelagic larval stages. Evaluation of growth in pelagic larvae and juveniles after weaning (one trial from 42 to 119 and another from 164 to 247 days post-hatch using a longitudinal approach) revealed that juveniles from embryos incubated at 20 °C had a higher growth rate. All these data demonstrate that the thermal regime during embryogenesis modulated mechanisms that regulate larval plasticity and caused imprinting evident in juvenile sole as persistent changes in key endocrinological pathways and growth performance.info:eu-repo/semantics/acceptedVersio

PTHrP-induced modifications of the sea bream (Sparus auratus) vertebral bone proteome

Endocrine factors play an essential role in the formation and turnover of the skeleton in vertebrates. In the present study sea bream vertebral bone transcripts for PTH1R and PTH3R were identified and the action of intermittent administration of parathyroid hormone related protein (PTHrP) on the proteome of vertebral bone was analysed. Treatment of immature sea bream (Sparus auratus, n = 6) for 5 days with homologous recombinant PTHrP(1–125; 150 ng/g body weight) modified bone metabolism and caused a significant (p < 0.05) reduction in both tartrate resistant acid phosphatase (TRACP) and alkaline phosphatase (ALP) in relation to control fish. However, the ratio of TRACP: ALP in PTHrP treated fish (1.3 to 2.2 cf. control) suggested it had an anabolic response. A sea bream vertebral bone proteome of 157 protein spots was generated and putative identity assigned to 118 (75.2%) proteins of which 72% had homology to proteins/transcripts from teleosts many of which have not previously been reported in teleost bone. Classification of bone proteins using gene ontology revealed those with protein or metal/ion (e.g., calcium, magnesium, zinc) binding (∼53%) activities were most abundant. The expression of eight proteins was significantly (p < 0.05) modified in the vertebra of PTHrP treated compared to control fish; three were up-regulated, betainehomocystein S-methyltransferase, glial fibrillary acidic protein, parvalbumin beta and five were down-regulated, annexin A5, apolipoprotein A1, myosin light chain 2, fast skeletal myosin light chain 3, troponin C. In conclusion, intermittent administration of PTHrP to sea bream is associated with an anabolic response in vertebral bone metabolism and modifies calcium binding proteins in the proteome

Dependent person in self-care: analysis of care needs

Background: The aim of this study was to assess the selfcare dependency levels of the dependent person at the time of home discharge and its relationship between (1) the degree of dependency of each self-care domain; (2) the previous dependency levels; and (3) the gender of the dependent person. It also aims to assess the relationship between the degree of dependency of each self-care domain, the length of admission, the length of dependency and the age of the dependent person at the time of discharge. Methods: A cross-sectional study was conducted. The sample comprised hospitalised adults and elderly in the medical services of a Portuguese hospital during the months of March, April and May of 2018. The data were collected by an interview conducted at the time of home discharge from the hospital medical ward. Results: The average age of dependent people of the sample is 80.7 years ( 10.1) with the majority being women (51.7%), with no statistical difference in the mean age according to gender (U = 2205.500; p > 0.05). They were hospitalised on average 11.4 days ( 33.2), most of them (44.0%) due to respiratory problems (85% of which were due to pneumonia). There were no statistically significant differences between the length of the hospital stay, the length of dependency and the participants’ gender (U = 2200.500, p > 0.05; U = 1688.000, p > 0.05). Medication intake was the highest dependency domain amongst participants (41.3%), followed by instrumental activities of daily living (40.6%) and bathing (39.9%). Conclusion: The amount of support required may vary according to the domain that the person is dependent. Thus, it is important to use a robust and reliable assessment tool that will be able to assess the degree of dependency on the various domains of self-care.info:eu-repo/semantics/publishedVersio

Molecular cloning and functional characterization of a monoterpene synthase isolated from the aromatic wild shrub Thymus albicans

The essential oil of Thymus albicans Hoffmanns. & Link, a native shrub from the Iberian Peninsula, is mainly composed of monoterpenes. In this study, a 1,8-cineole synthase was isolated from the 1,8-cineole chemotype. A partial sequence that lacked the complete plastid transit peptide but contained an extended C-terminal when compared to other related terpene synthases was generated by PCR and Rapid Amplification of cDNA Ends (RACE). The predicted mature polypeptide was 593 amino acids in length and shared 78% and 77% sequence similarity with the homologue 1,8-cineole synthase from Rosmarinus officinalis and Salvia officinalis, respectively. The putative protein possessed the characteristic conserved motifs of plant monoterpene synthases including the RRx(8)W and DDxxD motifs and phylogenetic analysis indicated that the amplified 1,8-cineole synthase bears greater sequence similarity with other 1,8-cineole synthases from Lamiaceae family relative to the terpene synthases from the genus Thymus. Functional expression of the recombinant protein in Escherichia coli revealed that in the presence of geranyl diphosphate (GPP) 1,8-cineole was the major product but that its production was too low for robust quantification. Other minor conversion products included a-pinene, beta-pinene, sabinene and beta-myrcene suggesting the isolated 1,8-cineole synthase may be a multi-product enzyme. To our knowledge, this is the first report of a functionally characterized monoterpene synthase from Thymus albicans.Ceratonia Project MONOTHYMUS, Universidade do Algarve, PortugalMinistry of Science, PortugalFCT under FEDER PT2020-Compete[FCT/UID/Multi/00631/2013/CEOT