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Adhesive-invasive E. coli has been suggested to be associated with the development of Crohn's disease (CD). It is assumed that they can provoke the onset of the inflammatory process as a result of the invasion of intestinal epithelial cells and then, due to survival inside macrophages and dendritic cells, stimulate chronic inflammation. In previous reports, we have shown that passage of the CD isolate ZvL2 on minimal medium M9 supplemented with sodium propionate (PA) as a carbon source stimulates and inhibits the adherent-invasive properties and the ability to survive in macrophages. This effect was reversible and not observed for the laboratory strain K12 MG1655. We were able to compare the isogenic strain AIEC in two phenotypes—virulent (ZvL2-PA) and non-virulent (ZvL2-GLU). Unlike ZvL2-GLU, ZvL2-PA activates the production of ROS and cytokines when interacting with neutrophils. The laboratory strain does not cause a similar effect. To activate neutrophils, bacterial opsonization is necessary. Differences in neutrophil NADH oxidase activation and ζ-potential for ZvL2-GLU and ZvL2-PA are associated with changes in membrane protein abundance, as demonstrated by differential 2D electrophoresis and LC-MS. The increase in ROS and cytokine production during the interaction of ZvL2-PA with neutrophils is associated with a rearrangement of the abundance of membrane proteins, which leads to the activation of Rcs and PhoP/Q signaling pathways and changes in the composition and/or modification of LPS. Certain isoforms of OmpA may play a role in the formation of the virulent phenotype of ZvL2-PA and participate in the activation of NADPH oxidase in neutrophils. [ABSTRACT FROM AUTHOR]

Background: Accelerated ageing is a method to assess seed storage performance by exposing the seeds to higher temperature and relative humidity to make seeds lose its vigour and viability quickly. The seed physiological parameters are then compared with the natural ageing to derive certain conclusions. But, the molecular pattern of seed deterioration under accelerated ageing varies compared to that of natural ageing. Methods: Chickpea, seed lots of variety JG-11 and Annigeri-1 were subjected to natural ageing in cloth bag with nine per cent initial seed moisture under ambient conditions. Simultaneously, representative sample of same seed lot were also aged at 41.C and 95±2% humidity up to 120 hours. Result: Both the ageing methods had significant negative effect on seed physiological and biochemical quality parameters. The germination potential of seeds tested after 48 hours of accelerated ageing (83.5%) was equal 10 months of natural ageing (85%) in JG-11, while also in Annigeri-1, 48 hours of accelerated ageing (78%) was nearly equal to 10 months of natural ageing (80%). The protein expression analyzed through 2D-PAGE at similar germination potential brought out by two independent ageing methods showed higher protein down regulation ratio (3.4) in accelerated ageing than in natural ageing (3.2) unveiling its rapidity in the seed deterioration process. Therefore, accelerated ageing can be used for predicting chickpea seed longevity. [ABSTRACT FROM AUTHOR]

Although one can trace the roots of proteomics well back into the 20th century, it is basically a discipline of the new millennium. At its outset, it was defined primarily by the technology available to analyze complex mixtures (basically 2D-gel electrophoresis, hybridizations/arrays and mass spectrometry) and what mainly set it aside from protein chemistry, that had flourished since the end of the second world war, was this use of unfractionated starting material as opposed to homogenous samples. Early on, two major new insights were quickly revealed: that the protein complement of cells was overwhelmingly involved in multiple protein–protein interactions and that it was nearly universally involved in a myriad of post-translational modifications. The revelations of the complex networks that result from these two phenomena have created a new understanding of cell biology that has affected our appreciation of such processes as transcription and translation, transmembrane signaling, differentiation, homeostasis and cell death. The development of these methods and strategies that principally characterize the field of proteomics depended heavily on the evolution of those that advanced protein chemistry, particularly during the last half of the twentieth century leading up to the elucidation of the human genome and will be briefly summarized in this article. In the last five decades of the 20th century, the original tools of protein chemistry combined and evolved to form the powerful largely mass spectrometry-based technology known as proteomics. This, together with the burgeoning science of genomics, has allowed the sensitive identification of proteins in complex samples, the principal targets of proteomics, including protein–protein interactions and post-translational modifications and their intricate roles in functional health and disease. [ABSTRACT FROM AUTHOR]

Simple Summary: Inulin-type fructans (ITFs), one of the best studied and most well-established prebiotic carbohydrates, have been shown to exert many health benefits to the host, including improvement in cardiovascular function. Various mechanisms are known to be involved in these processes, such as reduction of blood lipid levels by altering hepatic de novo lipogenesis, enhanced lipoprotein lipase activity in muscle, increased intestinal short-chain fatty acid production, elevated fecal excretion of bile salts and cholesterol and increased production of nitric oxide, known as endothelium-derived relaxing factor. Therefore, our overall aim was to assess the impact of complementary feeding with 4% of dried chicory root or 2% of native inulin on the aortic protein and gene changes in healthy nursery pigs. Our data clearly indicate that dietary inulin-type fructans have the potential to influence several structural and physiological alterations that are reflected both in the mRNA and protein levels in porcine aorta. In this study, two different ITFs sources were incorporated into a cereal-based diet to evaluate possible aortic protein and gene changes in nursery pigs. The animals were fed two different experimental diets from the 10th day of life, supplemented with either 4% of dried chicory root (CR) or with 2% of native inulin (IN). After a 40-day dietary intervention trial, pigs were sacrificed at day 50 and the aortas were harvested. Our data indicate that dietary ITFs have the potential to influence several structural and physiological changes that are reflected both in the mRNA and protein levels in porcine aorta. In contrast to our hypothesis, we could not show any beneficial effects of a CR diet on vascular functions. The direction of changes of several proteins and genes may indicate disrupted ECM turnover (COL6A1 and COL6A2, MMP2, TIMP3, EFEMP1), increased inflammation and lipid accumulation (FFAR2), as well as decreased activity of endothelial nitric oxide synthase (TXNDC5, ORM1). On the other hand, the IN diet may counteract a highly pro-oxidant environment through the endothelin–NO axis (CALR, TCP1, HSP8, PDIA3, RCN2), fibrinolytic activity (ANXA2), anti-atherogenic (CAVIN-1) and anti-calcification (LMNA) properties, thus contributing to the maintenance of vascular homeostasis. [ABSTRACT FROM AUTHOR]

Proteolysis is a post-translational modification (PTM) that affects the whole proteome. First regarded as only destructive, it is more precise than expected. It is finely regulated by other PTMs like phosphorylation. Aminopeptidase B (Ap-B), a M1 metallopeptidase, hydrolyses the peptide bond on the carbonyl side of basic residues at the NH 2 -terminus of peptides. 2D electrophoresis (2DE) was used to show that Ap-B is modified by phosphorylation. Detection of Ap-B by western blot after 2DE reveals several isoforms with different isoelectric points. Using alkaline phosphatase, Pro-Q Diamond phosphorylation-specific dye and kinase-specific inhibitors, we confirmed that Ap-B is phosphorylated. Phosphorylation can alter the structure of proteins leading to changes in their activity, localization, stability and association with other interacting molecules. We showed that Ap-B phosphorylation might delay its turnover. Our study illustrates the central role of the crosstalk between kinases and proteases in the regulation of many biological processes. • The metallopeptidase, hydrolyses the peptide bond on the carbonyl side of basic residues at the NH 2 -terminus of peptides. • Phosphorylation is responsible for the regulation of many proteases. • After 2D electrophoresis, Ap-B presents a "train of spots" signature which is specific of phosphorylation. • Specific dyes and kinase inhibitors were used to show that Ap-B is phosphorylated on serine, threonine and tyrosine residues. • Ap-B phosphorylation seems to delay its turnover. [ABSTRACT FROM AUTHOR]

The illicit use of dexamethasone and other glucocorticoids for cattle fattening in livestock production has been widely described; evidence for illegal treatments can be obtained by direct or indirect detection. In our previous study, we applied two-dimensional electrophoresis (2DE) to identify plasma protein markers of dexamethasone administration in veal calves. Comparison of 2DE maps obtained from blood samples before and after treatment showed the disappearance of two protein spots identified as serum paraoxonase/arylesterase 1 precursor (PON1). In the present study, we validated PON1 as a marker by analysing a larger number of samples treated with dexamethasone for illicit use. Analysis of samples from experimental treatment with other glucocorticoids, androgens and oestrogens confirmed that their influence on PON1 could be excluded. The specificity of the PON1 protein marker was verified on expected negative field samples to exclude interfering factors. However, there is poor statistical evidence to support a significant association between the outcome of PON1 and the considered variables. The results on field samples were compared with histological examination of the thymus as a biomarker of corticosteroid treatment monitored in the Italian histological plan for the control of growth promoters in animals. Two suspect cases were identified from two Piedmont farms where other animals had tested positive at histological examination. In conclusion, the absence of PON1 in the plasma of veal calves can indirectly reveal illicit dexamethasone treatment in individual animals and so identify suspect farms for further investigation. It is effective in a period ranging from 3 to about 10 days from illicit treatment, covering a time span that goes beyond the limits of official chemical controls and preceding histological controls on the thymus of slaughtered animals. PON1 detection in plasma can be coupled with other tests to identify illegal dexamethasone use on veal calf farms. [ABSTRACT FROM AUTHOR]

The aim of this study was to gain insight into pancreatic proteome changes induced by dexamethasone treatment. Sixweek-old male ICR mice were subcutaneously injected with or without dexamethasone (10 mg/kg body weight; n= 5-6 mice per group) for 4 weeks. Body weight, food intake, and fasting blood glucose were measured at baseline and at the end of 4 weeks. The mice were sacrificed and pancreases were removed, frozen, and stored at-75˚C until analysis. Proteins were extracted, twodimensional gel electrophoresis was performed and proteome profiles were examined with colloidal coomassie brilliant blue staining. Statistically significant protein spots (ANOVA, p<0.05) with 1.2-fold difference were excised and analyzed by LCMS/MS. Proteomic data were evaluated using Western blot analysis. High-dose dexamethasone induced hyperglycemia, reduced body weight, reduced food intake, and altered pancreatic proteins in mice. The affected proteins mainly belonged to cell redox homeostasis, metabolism, endoplasmic reticulum stress, translation, and signaling pathways. [ABSTRACT FROM AUTHOR]

Drought is a major abiotic stress that negatively influences crop yield. Breeding strategies for improved drought resistance require an improved knowledge of plant drought responses. We therefore applied drought to barley recombinant inbred lines and their parental genotypes shortly before tillering. A large-scale proteomic analysis of leaf and root tissue revealed proteins that respond to drought in a genotype-specific manner. Of these, Rubisco activase in chloroplast, luminal binding protein in endoplasmic reticulum, phosphoglycerate mutase, glutathione S-transferase, heat shock proteins and enzymes involved in phenylpropanoid biosynthesis showed strong genotype×environment interactions. These data were subjected to genetic linkage analysis and the identification of proteomic QTLs that have potential value in marker-assisted breeding programs. [ABSTRACT FROM AUTHOR]

Abstract Atherosclerosis and cancer are the leading causes of mortality around the world that share common pathogenic pathways. The aim of this study is the investigation of the protein profile of atherosclerotic plaque in order to find similar biomarker between cancer and atherosclerosis. The small pieces of human coronary artery containing advanced atherosclerotic plaque is obtained from patients during bypass surgery. Structural characterization of type V plaque, including fibrous connective tissue, necrotic lipid core, cholesterol clefts and calcium deposits are performed using high resolution transmission electron microscopy (HR-TEM). The protein profile of atherosclerosis plaque is also analyzed using 2-dimensional electrophoresis and matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF). TEM analysis shows that vascular smooth muscle cells (VSMCs) exhibit different and uncommon morphologies in atherosclerotic plaque which is correlated to the proliferative state of the cells. The proteomics analysis reveals proteins related to atherosclerosis formation including Mimecan, Ras Suppressor Protein-1 (RSUP-1) and Cathepsin D which identified as biomarker of cancerous tumors. The expression of Mimecan and RSUP-1 is down-regulated in atherosclerotic plaque while the expression of Cathepsin D is up-regulated. These data support that atherosclerotic plaque presents some degree of tumorgenesis with the significant activity of VSMCs as the key player in atherogenesis. Highlights • The protein profile of human atherosclerosis plaque is a analyzed using 2D electrophoresis and MALDI-TOF. • The proteomics analysis reveals proteins related to atherosclerosis formation including Mimecan, RSUP-1 and Cathepsin D. • The expression of Mimecan and RSUP-1 is down-regulated in atherosclerotic plaque. • The expression of Cathepsin D is up-regulated in atherosclerotic plaque. • The data support similar biomarker between cancer and atherosclerosis. [ABSTRACT FROM AUTHOR]

Purpose: Uveitis is a cause for concern in the developing countries like India. Its poor diagnosis and lack of proper therapeutics often cause blindness in children and young adults. Moreover, the exact mechanism of pathogenesis of different types of uveitis is still elusive. Modern proteomic techniques are found to be advantageous for an in‐depth understanding of the ocular physiology using proteomic diversity. Our aim was to identify unique proteins involved in the pathogenesis of autoimmune or noninfectious uveitis. Methods: Vitreous fluid samples (n = 90) were obtained from infectious (N = 34) and noninfectious (N = 56) uveitis patients, and their protein profiles were compared by analysing sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) and 2D electrophoresis. Unique proteins were identified through matrix‐assisted laser desorption ionization–time‐of‐flight mass spectrometry (MALDI‐TOF MS) and further studied for pathway analysis. Results: Protein spots having different molecular weights were observed in noninfectious vitreous fluid samples. Enzymatic digestion of these spots after MALDI‐TOF MS analysis revealed different proteins. We identified 25 different proteins through SDS‐PAGE and 22 through 2D electrophoresis. 50% of the proteins from SDS‐PAGE were associated with heterotrimeric G‐protein signalling pathway–rod outer segment phototransduction. 50% proteins from SDS‐PAGE and 20% from 2D electrophoresis revealed association with de novo purine biosynthesis. Carbonic anhydrase 1 and serpin B3 were found to be common in both analyses. Conclusion: High‐throughput proteomic and pathway analyses have exposed the potential association of these proteins with autoimmune pathogenesis in uveitis. The exact role of most of the proteins in autoimmune uveitis is yet to be unfurled. [ABSTRACT FROM AUTHOR]

Abstract We document the changes in sera and liver proteome during experimental hepatic fibrosis (HF) and its protection by resveratrol (Rsvtrl) in rats. HF was induced by N ′-Nitrosodimethylamine (NDMA) administration thrice a week for 21-days. Compared to the control group, significant (P < 0.05) elevations in ALT, AST, ALP, γGT and bilirubin occurred during fibrosis. H&E, IHC for α-SMA and reticulin stainings of liver demonstrated that collagen amassing and HF were concurrent. Rsvtrl-supplementation refurbished hepatic architecture and lessened collagen deposition. 2DE proteome analyses showed ~398 (pH, 3–10) and ~129 signatures (pH, 4–7) within 5–201 kDa range in liver and sera, respectively. Nearly, 45 spots in liver and 18 in sera were differentially expressed in fibrotic and Rsvtrl-supplemented animals. MALDI−TOF–MS/MS analysis revealed at least ten signatures as the potential biomarkers for HF, because of the significant changes (>2 fold) in their expression. Out of them, two signatures have not so far been characterized and we have accomplished it in this study. Our proteomics data provides new evidence on an overall involvement of carbohydrate, lipid, Ca2+ signaling and oxidative pathways during NDMA-induced HF. More importantly, the oxidative pathway appears to be the principal mediator in the NDMA-induced HF as well as the hepato-protection by Rsvtrl. Highlights • Proteomic study on fibrotic (HF) and resveratrol-supplemented rats is carried out. • 2D-MALDI-MS analysis revealed 10 major protein spots including 2 uncharacterized. • Carbohydrate, lipid and Ca2+ signaling pathway are inter-linked during HF. • Oxidative pathway appears principal to mediate liver damage. • Proteomic evidences suggest strong hepatoprotective potential of resveratrol. [ABSTRACT FROM AUTHOR]

In the present study, 155 Nellore cattle were genotyped for the CAPN4751 and UOGCAST polymorphisms and phenotyped for shear force (SF) at 1, 7 and 14 days aging. The effects of different genotypic combinations were evaluated on the Longissimus muscle proteomic profile using 2DE and mass spectrometry. A significant association was found between genotypes for UOGCAST and CAPN4751 and meat tenderness. The CC genotype for both markers was favorable for lesser shear force than TT. A total of 40 spots showed significant differential expression profiles (P < 0.05), of which eight had a main effect for the CAPN4751 marker, 11 for UOGCAST, two for both markers, and 19 had interactions between markers, including myosin (MYL1, MYL2, MYLPF and MYL6B), actin (ACTA1 and CAPZβ), troponin (TNNT1 and TNNT3) and heat shock proteins (HSPB6, HSPB1 and HSP70-2). The results demonstrated that UOGCAST and CAPN4751 genotypes led to variability on the expression of proteins that are involved in muscle metabolism, and consequently affect meat tenderness. [ABSTRACT FROM AUTHOR]

Abstract: The protein content and allergen composition was studied of cashews from 8 different origins (Benin, Brazil, Ghana, India, Ivory Coast, Mozambique, Tanzania, Vietnam), subjected to different in‐shell heat treatments (steamed, fried, drum‐roasted). On 2D electrophoresis, 9 isoforms of Ana o 1, 29 isoforms of Ana o 2 (11 of the acidic subunit, 18 of the basic subunit), and 8 isoforms of the large subunit of Ana o 3 were tentatively identified. Based on 1D and 2D electrophoresis, no difference in allergen content (Ana o 1, 2, 3) was detected between the cashews of different origins (P > 0.5), some small but significant differences were detected in allergen solubility between differently heated cashews. No major differences in N‐ and C‐terminal microheterogeneity of Ana o 3 were detected between cashews of different origins. Between the different heat treatments, no difference was detected in glycation, pepsin digestibility, or IgE binding of the cashew proteins. [ABSTRACT FROM AUTHOR]

Arthrospira platensis is a species of filamentous cyanobacteria whose high protein content renders it suitable as a nutritive supplement for human and animal diets. Furthermore it is easy to grow as it tolerates a broad range of environmental stresses such as extremes of pH, salinity and heavy metals. In this study, changes in the growth, pigments and proteome of A. platensis were analyzed under two major abiotic stresses limiting productivity, salinity and iron. The lower concentrations of stresses showed varied effects on fresh weight, growth rate, pigment constituents and soluble protein of A. platensis . A proteomic analysis was conducted using SDS-PAGE first to obtain an overview of gross changes, followed by 2-DE and mass spectrometry to identify specific proteins whose abundance was affected by these environmental stresses. Eighteen spots were differentially expressed under the stress conditions. Of these, six were found with increased abundance responding only to Fe 2+ stress, and five as a result of NaCl stress alone. Using LC–MS/MS, 37 proteins were identified within these spots. These proteins were classified into thirteen categories according to their function annotation. The present analysis could provide important data related to stress-tolerance by microalgae. [ABSTRACT FROM AUTHOR]

Contents: Parturition is one of the most important events in reproduction. Regardless of many studies, exact time for pregnancy termination and onset of parturition is impossible to determine. The aim of this study was to describe and to compare protein profile of plasma from healthy pregnant cows (n = 6) at following five time points: 2 weeks, 1 week before, at parturition, 1 week and 2 weeks after parturition to search for possible protein markers of parturition. Plasma samples were analysed by 1D and 2D electrophoresis, and selected spots were identified by mass spectrometry. Protein profile showed no uniform pattern. Seventy spots differed at least for one sampling point from the time point 2 weeks before parturition which served as reference. Thirty spots expressed higher intensity of staining 1 week as 2 weeks before parturition while 13 showed opposite relationship. Twenty two spots expressed higher intensity of staining at parturition as 2 weeks before delivery while 15 showed opposite relationship. Eighteen spots expressed higher intensity of staining 2 weeks before parturition as 1 week post‐partum while 2 showed opposite relationship. Fifteen spots expressed higher intensity of staining 2 weeks before parturition as 2 weeks after delivery while 14 showed opposite relationship. Thirty‐five proteins, belonging to different functional groups, were identified. Of them, 15 spots differed significantly between parturition and 2 weeks before delivery. Among them were metalloproteinase inhibitor and LDH which seem to be the most promising molecules considered as parturition markers due to their functions. [ABSTRACT FROM AUTHOR]

Background Bird fancier's lung (BFL) caused by repeated inhalation of avian proteins is the most common form of hypersensitivity pneumonitis. However, the exact identification of proteins involved is unknown, and serological test use for diagnosis need to be standardized. The objectives of this study were (i) to identify antigenic proteins from pigeon droppings (ii) to provide information about their location in avian matrices and (iii) to produce them in recombinant proteins to evaluate their diagnostic performances. Method Antigenic proteins of pigeon dropping extracts were investigated using 2-dimensional immunoblotting with sera from patients with BFL, asymptomatic exposed controls and healthy volunteers. We investigated the origin of these antigenic proteins by analyzing droppings, blooms and sera using a shotgun proteomic analysis. BFL-associated proteins were produced as recombinant antigens in E. coli and were assessed in ELISA with sera from patients (n = 25) and subject exposed controls (n = 30). These diagnostic performances were compared with those obtained by precipitin techniques (agar gel double diffusion, immunoelectrophoresis). Results We identified 14 antigenic proteins mainly located in droppings and blooms. These proteins were involved in either the digestive or immune systems of pigeons. Using the recombinant BFL-associated proteins: Immunoglobulin lambda-like polypeptide-1 (IGLL1: sensitivity: 76%; specificity: 100%; AUC: 0.93) and Proproteinase E (ProE: sensitivity: 84%; specificity: 80%; AUC: 0.85), the ELISA test showed better performance than precipitin assays with pigeon dropping extracts (sensitivity: 60%; specificity: 93.3%; AUC: 0.76). Conclusion IGLL1 and ProE were identified as the biomarkers of the disease. The use of these highly standardized antigens discriminates BFL cases from exposed subjects in serological assays. The results of this study offer new possibilities for the serological diagnosis of the disease. Clinical trial registration: ClinicalTrials.gov : Identifier NCT03056404 . [ABSTRACT FROM AUTHOR]

A procedure is developed for the determination of meat proteins by two-dimensional electrophoresis (2D). The procedure is intended for the quantitative determination of muscle proteins in samples of meat raw materials by a complex of proteomic technologies, including 2D-electrophoresis in a polyacrylamide gel and computer densitometry of the recorded electropherograms. [ABSTRACT FROM AUTHOR]

The Leishmania parasite is responsible for several diseases that affect humans and animals in more than 95 countries including Colombia. Despite the known toxicity shown by current drugs such as Glucantime or Amphotericin B, they are still used as treatments against the parasite. Natural antimicrobial peptides (AMPs) are considered promising agents for use against a broad spectrum of infectious diseases, they are considered promising and non-toxic therapeutic agents. AMPs are part of the immune response of organisms such as insects, from where they have been isolated and shown to have antibacterial and antifungal activity. Galleria mellonella moths express a broad repertoire of peptides in response to challenges with different organisms. This suggests an alternative for obtaining peptides with anti-parasitic activity. This study was based on the immunological challenge induced in Galleria mellonella larvae with Leishmania (viannia) panamensis promastigotes, with the goal of isolating a peptide fraction with antiparasitic activity. Non-challenged hemolymph and hemolymph obtained from the immunization with PBS and parasites were compared. Free hemocyte hemolymph from each treatment was analyzed by two-dimensional electrophoresis (2D gel). Furthermore, hemolymphs were fractionated by RP-HPLC. 2D gel analysis identified 4 spots in challenged hemolymph and 3 spots in PBS hemolymph. These spots were not present in the control hemolymph. On the other hand, fractions from RPHPLC of challenged hemolymph showed more antileishmania activity in comparison to the same fractions from the hemolymph control. This study may provide the basis for new antileishmanial peptides. [ABSTRACT FROM AUTHOR]

Issues regarding the structural diversity (heterogeneity) of an antibody molecule have been the subject of discussion along with the development of antibody drugs. Research on heterogeneity has been extensive in recent years, but no clear solution has been reached. Heterogeneity is also observed in catalytic antibody κ light chains (CLs). In this study, we investigated how the constant region domain of CLs concerns structural diversity because it is a simple and good example for elucidating heterogeneity. By means of cation-exchange chromatography, SDS-PAGE, and 2-dimensional electrophoresis for the CL, multimolecular forms consisting of different electrical charges and molecular sizes coexisted in the solution, resulting in the similar heterogeneity of the full length of CLs. The addition of copper ion could cause the multimolecular forms to change to monomolecular forms. Copper ion contributed greatly to the enrichment of the dimer form of CL and the homogenization of the differently charged CLs. Two molecules of the CL protein bound one copper ion. The binding affinity of the ion was 48.0 μM-1. Several divalent metal ions were examined, but only zinc showed a similar effect.--Hifumi, E., Taguchi, H., Kato, R., Uda, T. Role of the constant region domain in the structural diversity of human antibody light chains. [ABSTRACT FROM AUTHOR]

Ailanthus altissima pollen (AAP) is considered as an emerging cause of respiratory allergy in United States, Italy and Iran. However, the allergenic composition of AAP is still unknown and has yet to be characterized. The present study aimed to identify AAP allergens using a proteomics-based approach. For this purpose, optimized AAP protein extracts were analyzed using 1D- and 2D- gel electrophoresis and confronted to twenty sera from individuals with respiratory allergy during the AAP season. Candidate allergens were detected using the serum from an allergic patient with clinical history of AAP pollinosis. IgE-binding spots were identified using MALDI-TOF/TOF mass spectrometry and database searching. According to our results, AAP extracts were rich in proteins (up to 16.25 mg/ml) with a molecular-weight distribution ranging from 10 to 175 kDa. Two-D electrophoresis of AAP extracts revealed 125 protein spots from which 13 were IgE reactive. These IgE-binding proteins were identified as enolase, calreticulin, probable pectate lyase 6, conserved hypothetical protein and ras-related protein RHN1-like. By our knowledge, this study is the first report identifying AAP allergens. These findings will open up further avenues for the diagnosis and immunotherapy of the AAP allergy as well as for the cloning and molecular characterization of relevant allergens. Biological significance Ailanthus altissima colonizes new areas every year in Iran and is spreading aggressively worldwide. According to USDA, the tree of heaven is now present as an invasive plant in 30 states in US ( www.invasivespeciesinfo.gov/plants/treeheaven.shtml ) and come to dominate large areas in many regions. Up to now, several cases of allergy to A. altissima pollen have been reported in United States, Italy and Iran [1-4]. However, there is still no information on the sensitizing allergens and the molecular origin of these clinical symptoms, which constitutes a serious threat to patients suffering from respiratory allergies in these regions. To our knowledge, the current study describes, therefore, the first panel of proteins responsible for IgE-mediated A. altissima pollinosis by using a gel-based proteomic approach. This work represents the pioneer proteomic investigation on Simaroubaceae spp. and provides useful insights for further studies on the allergens of this widely distributed plant family. [ABSTRACT FROM AUTHOR]

Proteomics was used in the calanoid copepod Eurytemora affinis for screening of protein expression modifications induced by organic contaminants. The copepods were exposed in a continuous flow-through system for 86 h to environmentally relevant concentrations of contaminants representative of the pollution in the Seine Estuary (Haute-Normandie, France; diuron, 500 ng L−1; alkylphenol mixture, 1000 ng L−1). Proteome analysis of whole-body copepod extracts by 2-dimensional gel electrophoresis revealed that the contaminants induced modifications in protein expression, with the highest quantitative variations occurring after diuron exposure. Specifically, 88 and 41 proteins were differentially expressed after diuron and alkylphenol treatments, respectively. After mass spectrometry analysis, 51 (diuron exposure) and 15 (alkylphenol exposure) proteins were identified. The identified proteins were potentially related to energy metabolism, cell growth, nervous signal conductivity, excitotoxicity, oxidative stress response, and antioxidant defense. The data suggest a massive general disturbance of physiological functions of E. affinis after diuron exposure, whereas alkylphenols induced an alteration of a few targeted physiological functions. The protein expression signatures identified after contaminant exposure deserve further investigation in terms of the development of novel potential biomarkers for water quality assessment. Environ Toxicol Chem 2016;35:1860-1871. © 2015 SETAC [ABSTRACT FROM AUTHOR]

Neurocysticercosis (NCC) is one of the most prevalent parasitic infection of the brain and the most common cause of seizures in adults in tropical countries. Cysticercosis is caused by larvae of Taenia solium, a human tapeworm. Pig or humans are infected by ingestion of eggs in food contaminated by human feces. Diagnosis and treatment of pigs is a pillar of the control of the disease in a country. However current diagnostic tests are based on ELISA and/or Western blot using native antigens needing laboratory facilities not available in rural areas. Development of a pen side diagnostic test for swines, makes sense. Immunochromatographic test should be adapted for this purpose. To design it we started a bio-guided identification of new proteins in cysticercus fluid. Proteins were analyzed using ion exchange chromatography and 2D separation and were selected by Western blot analysis using sera from infected/non infected pigs. Spots from the Coomassie-stained gel corresponding to these proteins were then analyzed by mass spectroscopy and proteins were identified using a bank of Expressed Sequence Tags (EST) of T. solium . Eighteen new proteins of interest were identified and nine were selected for further development. [ABSTRACT FROM AUTHOR]

Along with the development of antibody drugs and catalytic antibodies, the structural diversity (heterogeneity) of antibodies has been given attention. For >20 yr, detailed studies on the subject have not been conducted, because the phenomenon presents many difficult and complex problems. Structural diversity provides some (or many) isoforms of an antibody distinguished by different charges, different molecular sizes, and modifications of amino acid residues. For practical use, the antibody and the subunits must have a defined structure. In recent work, we have found that the copper (Cu) ion plays a substantial role in solving the diversity problem. In the current study, we used several catalytic antibody light chains to examine the effect of the Cu ion. In all cases, the different electrical charges of the molecule converged to a single charge, giving 1 peak in cation‐exchange chromatography, as well as a single spot in 2‐dimensional gel electrophoresis. The Cu‐binding site was investigated by using mutagenesis, ultraviolet‐visible spectroscopy, atomic force microscope analysis, and molecular modeling, which suggested that histidine and cysteine residues close to the C‐terminus are involved with the binding site. The constant region domain of the antibody light chain played an important role in the heterogeneity of the light chain. Our findings may be a significant tool for preparing a single defined, not multiple, isoform structure.—Hifumi, E., Matsumoto, S., Nakashima, H., Itonaga, S., Arakawa, M., Katayama, Y., Kato, R., Uda T. A novel method of preparing the monoform structure of catalytic antibody light chain. FASEB J. 30, 895–908 (2016). www.fasebj.org [ABSTRACT FROM AUTHOR]

The aim of the study was the analysis of milk and milk fermented drink proteomes with the use of 2D electrophoresis. The criteria of proteins separation were the values of their isoelectric points (pI) and molecular weights (MW). Our results showed that milk and milk fermented drink proteomes consisted of 118 and 121 spots, respectively. The computer analysis revealed the identity of 95 spots in both proteomes. Non-identical spots indicated the changes resulting from the action of bacterial proteinases on milk proteins during the production of milk fermented drink. Proteolytic starter cultures applied to produce the milk fermented drink led to a partial hydrolysis of milk proteins to peptides and free amino acid residues. Results obtained led to confirm the suitability of 2D electrophoresis to observe the changes in the proteomes of milk products, as well as other food products after the application of technological processes. The development of the methods of proteomic analysis will let, in the future, eliminate the artefacts, which may limit the possibility of errors during proteins' identification. [ABSTRACT FROM AUTHOR]

It has been earlier hypothesized that lysogenic infection with Stx-encoding phages influences protein expression in the bacterial host, and therefore, some differentially expressed proteins could affect survival characteristics and pathogenicity. We compared the protein expression profiles of the host MG1655 and lysogens by 2D electrophoresis. Four different genes identified were all related to Fe/S subunit production, namely, nfuA, fdoH, sdhB and ftnA. To explore the role of nfuA in the biology of Stx prophage lysogeny, gene knockout experiments and phage lysogenic conversion were performed. The inactivation of nfuA caused the prophage to enter its lytic life cycle, especially under an iron-depleted condition. A similar activity was also detected in the Escherichia coli O157:H7 strain from which the Stx phage Min 27 was originally isolated. NfuA might be the positive regulator of genes controlling lysogenic cycle such as cI, cII and cIII since their transcriptional level was significantly reduced in nfuA deletion mutant as shown by qRT-PCR. We conclude that NfuA is essential for maintenance of Stx phage lysogeny in host's genetic reservoir under iron-deficient condition. [ABSTRACT FROM AUTHOR]

A simple and reproducible procedure for enrichment of a plasma protein subfraction suitable for two-dimensional polyacrylamide gel electrophoresis (2DE) was developed, using a Triton X-114-based cloud point extraction (CPE). Appropriate conditions for such a CPE procedure were found by SDS-PAGE to be a plasma protein concentration of about 10 mg/ml in 3% (w/v) Triton X-114. 2DE of proteins obtained by CPE of 400 μl of human plasma revealed about 200 spots constituting a spot pattern very different from the pattern of total plasma. The CPE procedure only had a limited contribution to the technical variation. Identification of about 60 spots, representing only 22 proteins, revealed that several proteins in the obtained subfraction were present in more isoforms or modifications. Among these were apolipoproteins (A-1, D, E, L1, and M), haptoglobin-related protein, phosphatidylcholine-sterol acyltransferase, serum amyloid A, and serum paraoxonase/arylesterase 1, which are proteins of a hydrophobic nature, as in plasma they relate to lipoprotein particles. Thus, Triton X-114-based CPE is a simple plasma prefractionation tool, attractive for detailed 2DE studies of hydrophobic plasma proteins and their isoforms or modifications. [ABSTRACT FROM AUTHOR]

Erwinia amylovora is a Gram-negative bacterium that causes the destructive disease fire blight affecting most members of the Rosaceae family, of which apple and pear are economically the most important hosts. E. amylovora has been considered as a homogeneous species in whole, although significant differences in virulence patterns have been observed. However, the underlying causes of the differences in virulence remain to be discovered. In a first-time comparative proteomic approach using E. amylovora , 2D differential in-gel electrophoresis (DIGE) was used to identify proteins that could explain the gradual difference in virulence between four different strains. Two important proteins were identified, FliC and CheY, both involved in flagella structure, motility and chemotaxis, which were more abundant in the least virulent strain. In the highly virulent strains the protein GalF, involved in amylovoran production, was more abundant, which was consistent with the higher expression of the gene and the higher amylovoran content in this strain in vitro. Together, these results confirm the involvement of amylovoran in virulence, but also imply an indirect role of flagellin in virulence as elicitor of plant defence. Biological significance This research provides new insights into our current understanding of the virulence of Erwinia amylovora . This plant-pathogen is considered a homogeneous species although different strains show differences in virulence. Despite the efforts made on the genomic level which resulted in the discovery of virulence factors, the reason for the different virulence patterns between strains has not yet been identified. In our lab we used a comparative proteomic approach, which has never been published before, to identify proteins involved in these differences between strains and hereby possibly involved in virulence. Our results provide interesting insights in virulence and present us with the opportunity to glance into the proteome of E. amylovora . [ABSTRACT FROM AUTHOR]

The present study was conducted to evaluate the effect of de-oiled castor cake on reproductive traits of crossbreed goats. Fourteen males were grouped into two lots ( n = 7/group), as described: group without de-oiled castor cake (WCC) and group fed with de-oiled castor cake (CC). Goats received two diets containing a mixture of Bermudagrass hay and concentrates with the same energy (73% total digestive nutrients) and protein content (15% crude protein) during 150 days, corresponding to ages from 40 (puberty) to 60 weeks. Blood plasma concentrations of urea, albumin, lactate dehydrogenase, creatinine, alanine aminotransferase and testosterone were determined. We also evaluated scrotal circumference, sperm parameters, quantitative aspects of spermatogenesis and daily sperm production (DSP), as well as the proteome of seminal plasma and sperm membrane. Seminal fluid and sperm proteins were analyzed by 2D SDS–PAGE and mass spectrometry. After 150 days of castor cake feeding, animals had no changes in the biochemical composition of blood plasma, suggesting the absence of intoxication by ingestion of ricin. There were no alterations in dry mater intake, weight gain, testis size, peripheral concentrations of testosterone, sperm concentration, motility and morphology. Sertoli and germ cell populations in the testis and DSP were not affected either. However, there were significant variations in the expression of five seminal plasma proteins and four sperm membrane proteins. In conclusion, the replacement of soybean meal by castor cake (with ricin concentrations of 50 mg/kg) did not interfere with the growth and core reproductive development of male goats. However, the diet with ricin altered the expression of certain seminal plasma and sperm membrane proteins, which play roles in sperm function and fertilization. Lower expression of these proteins may impair the ricin-fed animals to perform as high-fertility sires. [ABSTRACT FROM AUTHOR]

The Saanen is a highly productive breed, and for this reason, it has been raised in Brazil, but mostly under climate conditions completely different from where the breed originated. The objective of this study was to investigate variations in semen parameters and sperm membrane proteins from Saanen bucks ( n = 7) raised in Northeastern Brazil, during dry season (September, October, and November) and rainy season (March, April, and May). We showed that during the dry season, sperm motility, concentration, and the percentage of normal sperm decreased as compared to the rainy season. Rectal temperatures of bucks had no significant ( p > 0.05) variations during the dry and rainy seasons. However, temperatures of left and right skin testis were higher ( p < 0.05) during the dry as compared to the rainy season. Expression of three proteins (lysine-specific demethylase 5D, adenosine triphosphate (ATP) synthase subunit d, and radial spoke head protein 9 homolog) in sperm membrane were more intense in rainy season and only one protein (cytosol aminopeptidase) had greater expression in the dry season of the year. Our results show that mechanisms of testicular thermoregulation of Saanen bucks did not prevent a decrease in seminal parameters during the dry season. This deterioration may be related to reduced expression of proteins associated with important functions in sperm membrane. [ABSTRACT FROM AUTHOR]

• This research will be useful for meat processing industry to control the quality of frozen meat throughout the cold chain. • Large Ice crystals in the spaces between muscle fibers instigated ultrastructural changes in the chicken after freeze-thaw. • The truncated protein spots underwent a decline in intensities with increasing the number of freeze-thaw cycles. • The concentration of integral desmin of chicken meat was negatively associated with the number of freeze-thaw cycles. • MFT cycles caused a cycle-dependent reduction in the shear force of the chicken breast and the destruction of proteins. This study investigated the relationship of secondary protein structures with textural attributes of chicken breast subjected to 6 freeze (−20 °C) thaw (4 °C for 12 h) cycles. 2DE identified 78 distinct protein spots. Moreover, WB indicated a remarkable increase in the degree of desmin degradation during multiple freeze–thaw (MFT). In addition, the TEM micrographs revealed that MFT remarkably increased the spacing between the muscle fibers, especially from cycles 3 to 5, slightly decreased H-zone, but a remarkable shrinkage of Z-lines, as well as degradation of myofibril structures (Z-line, I-bands, and M-lines). DSC showed shifts in first and second endothermic transition peaks during the 3rd and 5th cycles. Viscoelastic graphs revealed a cycle-dependent decrease in gel-forming ability (G′) during MFT. Finally, the MFT chicken breast samples exhibited significantly decreased shear force in a cycle-dependent manner. This study highlighted the critical role of desmin in regulating myofibrillar degradation through various routes and mechanisms. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented about a study which is aimed at determining serum proteomics in fibromyalgia syndrome.

A simple synthetic α-methylene- δ-lactone, 1- isopropyl-2-methylene-1,2-dihydrobenzochromen-3-one, designated DL-3, was shown previously to induce apoptosis and significantly suppress cell metastatic potential in MDA- MB-231 breast cancer cells. The mechanisms through which DL-3 exerts its effects are poorly understood. The purpose of this study was to investigate the protein expression profiles in MDA- MB-231 cells exposed to the DL-3 treatment. Using 2D differential gel electrophoresis, a set of eight differentially expressed proteins (spot intensities which showed ≥1.25-fold change and statistical significance, p < 0.05, between the control and DL-3-treated group) were found and successfully identified by mass spectrometry ( MALDI- TOF/ MS). The proteomic results revealed that the presence of DL-3 in MDA- MB-231 cells led to the differential regulation of some proteins that are involved in the cell cycle progression, apoptosis, cytokinesis, modulation of transcription, cellular signaling, and vesicular trafficking. The function of other identified proteins is still unknown. Therefore, our data indicate new directions for the further studies of the pathways engaged in the anticancer action exerted by α-methylene- δ-lactones in cancer cells. [ABSTRACT FROM AUTHOR]

Abstract: Canine cardiopulmonary dirofilariosis caused by Dirofilaria immitis habitually develops as a chronic disease affecting pulmonary arteries, lung parenchyma and heart. Other organs like kidneys can also be involved. Renal pathology is a consequence of glomerulonephritis whose main sign is proteinuria. The aim of the present work is to identify proteins excreted in the urine of D. immitis infected dogs showing proteinuria, and the possible contribution of their loss to heartworm disease. Proteinuria is higher in microfilaremic (mf+) than in amicrofilaremic (mf−) dogs. Using bidimensional electrophoresis and mass spectrometry 9 different proteins from Canis lupus familiaris in the urine of both mf− and mf+ dogs were identified (serotransferrin isoform 6, serum albumin precursor, albumin, immunoglobulin gamma heavy chain D, apolipoprotein A-I, immunoglobulin lambda-like polypeptide 5-like, arginine esterase precursor, inmunoglobulin gamma heavy chain B and hemoglobin subunit alpha). Furthermore, 3 additional proteins were identified only in the urine of mf+ dogs, corresponding to dog fibrinogen alpha chain and immunoglobulin gamma heavy chain A and actin 2 homologous to a protein of Brugia malayi. The loss of these proteins and other in the urine of D. immitis infected dogs could affect the general condition of parasitized dogs through the interference in the cholesterol metabolism and O2 transport, among other mechanisms. [Copyright &y& Elsevier]

Corticosteroids have become the most widespread illegal growth promoters in veal calves and beef cattle. Testing for corticosteroids relies on either direct detection of compounds or their metabolites or indirect detection to identify changes in biological pathways. We used a comparative proteomic approach, based on two-dimensional electrophoresis (2DE), to identify plasma protein markers after short-term dexamethasone administration in veal calves. Twenty-three male Friesian veal calves were treated experimentally with dexamethasone sodium phosphate: 10 received low-dose administration of the drug (0.4 mg day–1per os) for 20 consecutive days (treatment group); 10 received the drug at therapeutic dosage (2–4 mg kg–1i.m.) for 3 consecutive days (comparison group). Three animals were not treated (control group). Plasma samples were collected from each animal at six time points (T1–T6; treatment and control group) and at four time points (T1–T4; comparison group) and stored at –80°C until analysis. Plasma proteins were quantified and analysed in triplicate by 2DE. The images were analysed with Bionumerics®software. Comparison of 2DE maps obtained from blood samples at T1 (before treatment) and at T6 (final sampling) showed a significant disappearance (p < 0.001) of two protein spots at T6 in the treatment group. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and immunoblotting identified these isoforms as serum paraoxonase/arylesterase 1 precursor (PON1). Synthesised in the liver and released into the blood, PON1 has an important role in lipid metabolism. The absence of variation of this protein in the comparison group suggests that the marker has good specificity for detecting illicit corticosteroid treatment. [ABSTRACT FROM AUTHOR]

Highlights: [•] HNE-induced proteomic changes in V79 cells were evaluated by 2D gel electrophoresis. [•] 23 proteins were upregulated and 19 proteins were downregulated by HNE. [•] Phosphoglycerate kinase 1 and Lamin C were downregulated by HNE. [•] RACK1 was upregulated by HNE and this was confirmed by Q-RT-PCR and Western blots. [•] The role of RACK1 in mediating HNE-induced apoptosis was confirmed. [ABSTRACT FROM AUTHOR]

Background Proteomic analysis was performed in post-nuclear supernatant (PNS) and Percoll-purified membranes (PM) prepared from fore brain cortex of rats exposed to increasing doses of morphine (10-50 mg/kg) for 10 days. Results In PNS, the 10 up (↑)- or down (↓)-regulated proteins exhibiting the largest morphineinduced change were selected, excised manually from the gel and identified by MALDI-TOF MS/MS: 1-(gi∣148747414, Guanine deaminase), ↑2.5x; 2-(gi∣17105370, Vacuolar-type proton ATP subunit B, brain isoform), ↑2.6x; 3-(gi∣1352384, Protein disulfide-isomerase A3), ↑3.4x; 4-(gi∣40254595, Dihydropyrimidinase-related protein 2), ↑3.6x; 5-(gi∣149054470, Nethylmaleimide sensitive fusion protein, isoform CRAa), ↑2.0x; 6-(gi∣42476181, Malate dehydrogenase, mitochondrial precursor), ↑1.4x; 7-(gi∣62653546, Glyceraldehyde-3- phosphate dehydrogenase), ↑1.6x; 8-(gi∣202837, Aldolase A), ↑1.3x; 9-(gi∣31542401, Creatine kinase B-type), ↓0.86x; 10-(gi∣40538860, Aconitate hydratase, mitochondrial precursor), ↑1.3x. The identified proteins were of cytoplasmic (1, 4, 5, 7, 9), cell membrane (2), endoplasmic reticulum (3) and mitochondrial (6, 8, 10) origin and 9 of them were significantly increased, 1.3-3.6x. The 4 out of 9 up-regulated proteins (4, 6, 7, 10) were described as functionally related to oxidative stress; the 2 proteins participate in genesis of apoptotic cell death. In PM, the 18 up (↑)- or down (↓)-regulated proteins were identified by LC-MS/MS and were of plasma membrane [Brain acid soluble protein, ↓2.1x; trimeric Gβ subunit, ↓2.0x], myelin membrane [MBP, ↓2.5x], cytoplasmic [Internexin, ↑5.2x; DPYL2, ↑4.9x; Ubiquitin hydrolase, ↓2.0x; 60S ribosomal protein, ↑2.7x; KCRB, ↓2.6x; Sirtuin-2, ↑2.5x; Peroxiredoxin-2, ↑2.2x; Septin-11, ↑2.2x; TERA, ↑2.1x; SYUA, ↑2.0x; Coronin-1A, ↓5.4x] and mitochondrial [Glutamate dehydrogenase 1, ↑2.7x; SCOT1, ↑2.2x; Prohibitin, ↑2.2x; Aspartate aminotransferase, ↓2.2x] origin. Surprisingly, the immunoblot analysis of the same PM resolved by 2D-ELFO indicated that the "active", morphine-induced pool of Gβ subunits represented just a minor fraction of the total signal of Gβ which was decreased 1.2x only. The dominant signal of Gβ was unchanged. Conclusion Brain cortex of rats exposed to increasing doses of morphine is far from being adapted. Significant up-regulation of proteins functionally related to oxidative stress and apoptosis suggests a major change of energy metabolism resulting in the state of severe brain cell "discomfort" or even death. [ABSTRACT FROM AUTHOR]

Fusarium oxysporum f. sp. cucumerinum (FOC) infects cucumber plants, causing significant yield losses. Chitosan is a natural biodegradable compound that has antifungal properties. To understand the inhibitory mechanism of chitosan against FOC, a comprehensive proteomic study was carried out for the identification of chitosan responsive proteins (CRPs) from the mycelia of chitosan-treated FOC. Two-dimensional gel electrophoresis (2-DE) coupled with LC-MS/MS analysis led to the identification of 62 differentially abundant CRPs. Functional classification of these CRPs revealed that most proteins were involved in metabolism and defense. Gene Ontology analysis revealed that the majority of the proteins were assigned in proteolysis and hydrolase activity. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that among the biologically active pathways in chitosan-treated FOC mycelia, 'carbohydrate metabolism' was enriched for most of the proteins. This study gives a snapshot of the molecular basis of fungal inhibition by chitosan resulting in disease resistance in cucumber plants after inoculation with chitosan-treated FOC by explaining how chitosan restricted disease severity (i.e., down-regulating the plant cell wall degrading enzymes, FOC self-attack, hindering FOC structural and functional protein biosynthesis and DNA biosynthesis and affecting FOC transporter proteins). This study contributes to putting more weight on using the bioactive natural compound chitosan as an antifungal material instead of applying chemical fungicides in agriculture. Chitosan has been used as one of the safe and effective alternatives to fungicides in controlling cucumber vascular wilt disease caused by Fusarium oxysporum f. sp. cucumerinum (FOC) that is responsible for severe production losses. Chitosan application showed a significant decrease in wilt disease severity compared to chitosan untreated FOC and showed an efficiency of 91.7% in reducing pathogenicity. A comprehensive proteomic investigation of chitosan-responsive proteins (CRPs) from the mycelia of chitosan-treated FOC was carried out in order to better understand the inhibitory mechanism of chitosan against FOC which led us to identify 62 differentially expressed CRPs. Our proteomic study revealed CRPs in FOC involved in a variety of functions, including disease inhibition in cucumber. This study depicts what happens inside the fungus following treatment with chitosan and how chitosan played the role of the maestro in influencing the synthesis of proteins responsible for the virulence of FOC and their respective pathways, rendering FOC unable to infect the cucumber plant and lose its pathogenic potential to cause wilt disease. The efficiency of chitosan in inhibiting certain proteins or specific pathways of FOC gives a golden opportunity in controlling vascular wilt, so we highly recommend applying chitosan in disease management under greenhouse conditions or in the open field [Display omitted] • Chitosan is a natural biodegradable compound has antifungal properties. • Mode of action of chitosan in controlling fungus virulence. • Fusarium metabolic pathways affected by chitosan • Chitosan enhances the Fusarium self-attack • Chitosan confers direct morphological and physiological changes to FOC [ABSTRACT FROM AUTHOR]

Channel catfish (Ictalurus punctatus) is the top species produced in US aquaculture and motile Aeromonas septicemia, caused by virulent Aeromonas hydrophila (vAh), is one of the most severe diseases that afflict catfish farms. Previously, vaccination of fish with extracellular proteins (ECP) of vAh was shown to produce a robust antibody-mediated immune response against vAh infection. In this study, we analyzed IgM transcripts that were differentially expressed in the head kidney and liver of ECP-immunized and mock-immunized (control) fish with emphasis on a variable domain of heavy chain. Quantitative PCR analysis indicated that immunized fish produced significantly more IgM transcripts than control fish. Full-length IgM heavy chain cDNA was cloned, which encoded typical IgM peptide, including signal peptide, variable domain (VH), constant domain (CH), and carboxyl terminal peptide. Great sequence diversity was revealed in a VH segment, with the third complementarity diversity region (CDR3) being most variable. Using germline VH gene grouping method, variants (clones) of VH characterized in this study belonged to nine VH families. The most unique variants (approximately 49%) were found in the VH2 family. Vaccinated fish apparently had more unique variants than in the control fish. There were 62% and 79% of unique variants in the head kidney and liver of vaccinated fish, respectively, while 44% and 27% unique variants in the head kidney and liver of control fish, respectively. Among the unique variants in VH2 family, approximately 87% of them were found in vaccinated fish. Two-dimensional gel electrophoresis of semi-purified IgM protein confirmed that matured IgM protein was as variable as IgM transcripts identified in this study, with isoelectric points crossing from 6 to 10. Results of this study provided insight into the molecular and genetic basis of antibody diversity and enriched our knowledge of the complex interplay between antigens and antibodies in Ictalurid catfish. [ABSTRACT FROM AUTHOR]

Highlights: [•] A glycaemia-reducing lupin seed protein is internalized by HepG2 cells. [•] The protein accumulates in the cytosol in an intact form. [•] The internalized protein is multiply phosphorylated. [ABSTRACT FROM AUTHOR]

Background: The extracellular space or apoplast forms a path through the whole plant and acts as an interface with the environment. The apoplast is composed of plant cell wall and space within which apoplastic fluid provides a means of delivering molecules and facilitates intercellular communications. However, the apoplastic fluid extraction from in planta systems remains challenging and this is particularly true for grapevine (Vitis vinifera L.), a worldwide-cultivated fruit plant. Large-scale proteomic analysis reveals the protein content of the grapevine leaf apoplastic fluid and the free interactive proteome map considerably facilitates the study of the grapevine proteome. Results: To obtain a snapshot of the grapevine apoplastic fluid proteome, a vacuum-infiltration-centrifugation method was optimized to collect the apoplastic fluid from non-challenged grapevine leaves. Soluble apoplastic protein patterns were then compared to whole leaf soluble protein profiles by 2D-PAGE analyses. Subsequent MALDI-TOF/TOF mass spectrometry of tryptically digested protein spots was used to identify proteins. This large-scale proteomic analysis established a well-defined proteomic map of whole leaf and leaf apoplastic soluble proteins, with 223 and 177 analyzed spots, respectively. All data arising from proteomic, MS and MS/MS analyses were deposited in the public database world-2DPAGE. Prediction tools revealed a high proportion of (i) classical secreted proteins but also of non-classical secreted proteins namely Leaderless Secreted Proteins (LSPs) in the apoplastic protein content and (ii) proteins potentially involved in stress reactions and/or in cell wall metabolism. Conclusions: This approach provides free online interactive reference maps annotating a large number of soluble proteins of the whole leaf and the apoplastic fluid of grapevine leaf. To our knowledge, this is the first detailed proteome study of grapevine apoplastic fluid providing a comprehensive overview of the most abundant proteins present in the apoplast of grapevine leaf that could be further characterized in order to elucidate their physiological function. [ABSTRACT FROM AUTHOR]

Abstract: Macrophages play a critical role at the crossroad between iron metabolism and immunity, being able to store and recycle iron derived from the phagocytosis of senescent erythrocytes. The way by which macrophages manage non-heme iron at physiological concentration is still not fully understood. We investigated protein changes in mouse bone marrow macrophages incubated with ferric ammonium citrate (FAC 10 μM iron). Differentially expressed spots were identified by nano RP-HPLC-ESI-MS/MS. Transcriptomic, metabolomics and western immunoblotting analyses complemented the proteomic approach. Pattern analysis was also used for identifying networks of proteins involved in iron homeostasis. FAC treatment resulted in higher abundance of several proteins including ferritins, cytoskeleton related proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at the membrane level, vimentin, arginase, galectin-3 and macrophage migration inhibitory factor (MIF). Interestingly, GAPDH has been recently proposed to act as an alternative transferrin receptor for iron acquisition through internalization of the GAPDH-transferrin complex into the early endosomes. FAC treatment also induced the up-regulation of oxidative stress-related proteins (PRDX), which was further confirmed at the metabolic level (increase in GSSG, 8-isoprostane and pentose phosphate pathway intermediates) through mass spectrometry-based targeted metabolomics approaches. This study represents an example of the potential usefulness of “integarated omics” in the field of iron biology, especially for the elucidation of the molecular mechanisms controlling iron homeostasis in normal and disease conditions. This article is part of a Special Issue entitled: Integrated omics. [Copyright &y& Elsevier]

Abstract: The soy-derived phytoestrogen genistein (GEN) has received attention for its potential benefits on the cardiovascular system by providing direct protection to cardiomyocytes against pathophysiological stresses. Here, we employed a proteomic approach to study the concentration-dependent effects of GEN treatments on cardiomyocytes. Cultured HL-1 cardiomyocytes were treated with low (1μM) and high (50μM) concentrations of GEN. Proteins were pre-fractionated by sequential hydrophilic/hydrophobic extraction and both protein fractions from each treatment group were separated by 2D gel electrophoresis (2DE). Overall, approximately 2,700 spots were visualized on the 2D gels. Thirty-nine and 99 spots changed in volume relative to controls (p<0.05) following the low- and high-concentration GEN treatments, respectively. From these spots, 25 and 62 protein species were identified by ESI-MS/MS and Mascot database searching, respectively. Identified proteins were further categorized according to their functions and possible links to cardioprotection were discussed. MetaCore gene ontology analysis suggested that 1μM GEN significantly impacted the anti-apoptosis process, and that both the low and high concentrations of GEN influenced the glucose catabolic process and regulation of ATPase activity. This proteomics study provides the first global insight into the molecular events triggered by GEN treatment in cardiomyocytes. [Copyright &y& Elsevier]

Abstract: This study used 2DE to investigate how Arabidopsis thaliana modulates protein levels in response to freezing stress after sub-lethal exposure at −10°C, both in cold-acclimated and in non-acclimated plants. A map was implemented in which 62 spots, corresponding to 44 proteins, were identified. Twenty-two spots were modulated upon treatments, and the corresponding proteins proved to be related to photosynthesis, energy metabolism, and stress response. Proteins demonstrated differences between control and acclimation conditions. Most of the acclimation-responsive proteins were either not further modulated or they were down-modulated by freezing treatment, indicating that the levels reached during acclimation were sufficient to deal with freezing. Anabolic metabolism appeared to be down-regulated in favor of catabolic metabolism. Acclimated plants and plants submitted to freezing after acclimation showed greater reciprocal similarity in protein profiles than either showed when compared both to control plants and to plants frozen without acclimation. The response of non-acclimated plants was aimed at re-modulating photosynthetic apparatus activity, and at increasing the levels of proteins with antioxidant-, molecular chaperone-, or post-transcriptional regulative functions. These changes, even less effective than the acclimation strategy, might allow the injured plastids to minimize the production of non-useful metabolites and might counteract photosynthetic apparatus injuries. [Copyright &y& Elsevier]

Abstract: Fruit ripening in Prunus persica involves a number of physiological changes, being one of the most significant the mesocarp softening in melting varieties. In order to get a better understanding of the molecular processes involved in this phenomenon, the protein accumulation patterns in firm and soft fruit of three peach and two nectarine melting flesh varieties were assessed using 2D gel analysis. A General Linear Model (GLM) two-way analysis of variance determined that 164 of the 621 protein spots analyzed displayed a differential accumulation associated with the softening process. Among them, only 14 proteins changed their accumulation in all the varieties assessed, including proteins mostly involved in carbohydrates and cell wall metabolism as well as fruit senescence. The analysis among varieties showed that 195 and 189 spots changed within the firm and soft fruit conditions, respectively. Despite the changes in relative abundance in the spot proteins, the proteome is conserved among varieties and during the transition from firm to soft fruit. Only two spots proteins exhibited a qualitative change in all the conditions assessed. These results are in agreement with the notion that Prunus persica commercial varieties have a narrow genetic background. [Copyright &y& Elsevier]

Background: Sesamia nonagrioides is a noctuid that feeds on maize, sugar cane and sorghum in North Africa and Southern Europe. Larvae reared under long day conditions pupate after 5 or 6 larval instars, whereas larvae reared under short day conditions enter diapause and undergo up to 12 molts before dying or pupating. To better understand the mechanism of larval development and diapause, we identified proteins with different expressions in the sixth instar of diapausing and non-diapausing larvae. Results: A total of 52 differentially regulated proteins were detected in the hemolymph of the diapausing or non-diapausing larvae at the beginning or end of the sixth instar. From these proteins, 11 were identified by mass spectrometry (MALDI-TOF MS or MALDI-TOF/TOF MS/MS): 5 were upregulated in the hemolymph of non-diapausing larvae and 6 in the hemolymph of the diapausing larvae. Interestingly, some proteins were expressed only in non-diapausing larvae but none was expressed only in the hemolymph of diapausing larvae. The possible functions of some of these proteins related to diapause maintenance or to larval-pupal metamorphosis are discussed. Conclusions: The 2-DE proteomic map of S. nonagrioides hemolymph shows differential protein expression in diapausing and non-diapausing larvae. Some proteins that showed higher expression in the diapausing larvae at the end of the sixth instar could be involved in JH level maintenance thus in the diapause status maintenance. On the contrary, other proteins that showed the highest expression or that were expressed only in the non-diapausing larvae could be involved in larval-pupal metamorphosis. [ABSTRACT FROM AUTHOR]

Abstract: Ripening of Cheddar cheese manufactured from raw milk (control) and milk HP-treated at 400MPa or 600MPa for 10min at 20°C was studied. Counts of non-starter lactic acid bacteria (NSLAB) were significantly reduced after HP-treatment of milk at 600MPa; however, NSLAB recovered after 60days of ripening. Overall, increased proteolysis and levels of free fatty acids were found in cheese manufactured from milk HP-treated at 600MPa. In addition, cheese manufactured from HP-treated milk was less hard, gummy and chewy compared to the control cheese; however, melting ability was significantly decreased in the former. Colour measurements indicated an increase in whiteness in cheese manufactured from HP-treated milk compared to the control cheese. Overall, HP treatment of milk at 600MPa prior to manufacture of Cheddar cheese decreased initial microbial counts and increased proteolysis during ripening. Industrial relevance: Raw milk cheese is in high demand by the consumer due to its unique traits; however, the cheese is prone to microbial contamination and flavour defects. The possibility of treating the milk with high-pressure prior to cheese manufacture helps to eliminate a number of risk factors, yet results in cheese with similar characteristics to cheese made from untreated raw milk. This could help to develop new innovative safe cheese, which could give a competitive advantage. [Copyright &y& Elsevier]

Abstract: Viral infections are known to have a detrimental effect on grapevine yield and performance, but there is still a lack of knowledge about their effect on the quality and safety of end products. Vines of Vitis vinifera cv. Nebbiolo clone 308, affected simultaneously by Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine virus A (GVA), and Rupestris stem pitting associated virus (RSPaV), were subjected to integrated analyses of agronomical performance, grape berry characteristics, instrumental texture profile, and proteome profiling. The comparison of performance and grape quality of healthy and infected vines cultivated in a commercial vineyard revealed similar shoot fertility, number of clusters, total yield, with significant differences in titratable acidity, and resveratrol content. Also some texture parameters such as cohesiveness and resilience were altered in berries of infected plants. The proteomic analysis of skin and pulp visualized about 400 spots. The ANOVA analysis on 2D gels revealed significant differences among healthy and virus-infected grape berries for 12 pulp spots and 7 skin spots. Virus infection mainly influenced proteins involved in the response to oxidative stress in the berry skin, and proteins involved in cell structure metabolism in the pulp. [Copyright &y& Elsevier]

Abstract: The protozoan parasite Leishmania donovani undergoes various developmental transitions during its infectious cycle that are triggered by environmental signals encountered inside insect and vertebrate hosts. Intracellular differentiation of the pathogenic amastigote stage is induced by pH and temperature shifts that affect protein kinase activities and downstream protein phosphorylation. Identification of parasite proteins with phosphotransferase activity during intracellular infection may reveal new targets for pharmacological intervention. Here we describe an improved protocol to trace this activity in L. donovani extracts at high resolution combining in-gel kinase assay and two-dimensional gel electrophoresis. This 2D procedure allowed us to identify proteins that are associated with amastigote ATP-binding, ATPase, and phosphotransferase activities. The 2D in-gel kinase assay, in combination with recombinant phospho-protein substrates previously identified by phospho-proteomics analyses, provides a novel tool to establish specific protein kinase–substrate relationships thus improving our understanding of Leishmania signal transduction with relevance for future drug development. [Copyright &y& Elsevier]

The protein pattern of murine macrophages cell line J774 has been investigated using 2D electrophoresis after combined action of bacterial endotoxins (LPS), heat shock treatment (HS), and administration of recombinant human Hsp70. The investigation demonstrated significant protective effect of HS and recombinant Hsp70 treatment applied before LPS introduction. This effect is apparently realized by means of several signal transduction systems. In the course of the investigation, we identified eight proteins that exhibited pronounced changes in their synthesis due to combined treatment. The data accumulated may shed light on molecular mechanisms underlying protective antiseptic action of HS and/or recombinant Hsp70 applied before LPS administration. [ABSTRACT FROM AUTHOR]

Abstract: An understanding of the diabetes-related changes in brain proteome composition in diabetes and treatment with Cynodon dactylon may provide insights to understand the brain function associated diabetes and metabolic protein mechanism responsible for this disease and in order to discover novel preventive and therapeutic drugs. We have performed a comprehensive proteomic analysis for comparison of rat brain proteome by using advance 2-dimensional electrophoresis (2DE) combined with mass spectrometry (MALDI-TOF-MS). We report here a comparison of alloxan-induced type 1 diabetic rats and C. dactylon-treated diabetic rats at 2weeks following diabetes onset. As a result, we identified two differentially expressed proteins from rat brain. The identified proteins were functionally classified into two groups: (i) metabolic signalling protein (PPP1R14D) and ii) vesicle transport signalling protein (RAB18). This study provides a preliminary reference map of normal rat brain that will form a basis for comparative studies on normal and pathological conditions of the brain connected with diabetes and may serve as a potential tool for clinical diagnosis, therapeutics and prognosis and may provide new insights into novel mechanisms and therapeutic targets for diabetes-associated neurological disorder. [ABSTRACT FROM AUTHOR]