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Background: The diagnostics of allergic occupational diseases is highly dependent on the quality of the allergen extracts and specific IgE tests available. To enhance the diagnostics of bovine-related occupational rhinitis, asthma and urticaria, we produced an in-house cow dander extract, assessed its allergen profile and performance in clinical tests, and compared it to commercial bovine dander extracts. Methods: One hundred patients with a suspected bovine-related occupational disease underwent skin prick tests (SPTs) with in-house and one to two commercial bovine dander extracts. Nasal allergen provocation tests were performed on 31 patients with suspected occupational rhinitis. We used Western blot to study the specific IgE-protein reactions from the serums of the patients with positive provocation tests, and identified allergens from immunoblot bands using tandem mass spectrometry. Results: Odorant-binding protein Bos d OBP, bovine serum albumin Bos d 6, and lipocalin Bos d 2 were identified as the major allergens. We found altogether 24 bovine dander allergens, of which several were formerly unknown. The in-house extract sensitivity and specificity in SPTs were 100% and 94%, in 87 patients respectively and SPTs appeared negative in 20 healthy controls. Nasal allergen provocation tests with inhouse extract detected occupational rhinitis with 100% sensitivity in 21 patients. Five healthy controls remained negative in the provocation tests. Conclusions: Three major and several minor allergens were found from bovine dander as a cause of occupational rhinitis. A sufficient concentration and variety of tested allergens were essential in the diagnostics of bovine-related occupational diseases.

Pregnancy loss predominantly occurs during the first 3–4 weeks due to fertilization failure or early embryonic losses in cattle. Insufficient biochemical communication between conceptus (embryo plus extraembryonic membranes) and endometrium has been suspected as the primary cause for early embryonic losses. If molecules regulating this communication were identified, molecular mechanisms associated with early pregnancy losses could be better understood. To identify candidate molecules as detection markers of non-pregnant or females undergoing embryonic loss, peripheral blood from embryo-transferred heifers on day 7 (day 0 = day of estrus) were collected on days 17 (pre-attachment), 20 (during attachment), and 22 (post-attachment), which were subjected to metabolome and global proteome iTRAQ analyses. The metabolome analysis partly divided serum components into pregnant or not. In the iTRAQ analysis, heatmap analysis with top 25 proteins was separated into pregnant or not on day 20 or 22. Furthermore, receiver operating characteristic curve (ROC) analysis identified five candidate proteins detecting non-pregnant heifers, of which SNX5 in day 22 serum had the highest area under the curve (AUC): 0.983. We also detected SNX5 in day 22 serum from non-pregnant heifers using western blotting. These results suggest that high SNX5 in day 22 serum could predict early pregnancy loss in heifers.

We aimed to understand the molecular-level changes occurring in the photosynthetic metabolic network in mutant chloro-plasts. We performed comparative liquid chromatography-mass spectrometry protein profiling of wild-type (WT) and chlorophyll-deficient melon (Cucumis melo L.) mutants. We identified 390 differentially expressed proteins and 81 shared proteins varied significantly in abundance, of which 76 were upregulated and 5 were downregulated. Differentially expressed proteins were involved in the following biological processes: binding, catalytic, structural, transporter, and antioxidant activities. The mutant had 6.08-fold higher expression of glutamate-1-semialdehyde 2,1-aminomutase (GSAM), an enzyme that synthesizes the chlorophyll precursor, 5-aminolevulinic acid, and a 5.02-fold higher expression of pyridoxal biosynthesis protein, a GSAM coenzyme. An RNA recognition motif-containing protein (RRM) decreased in expression by 5.22-fold. This suggests that GSAM and RRM are particularly relevant to chlorophyll deficiency.

Many studies have been conducted to elucidate the role of Type VI collagen in muscle and tendon, however, its role in oral tissues remains unclear. In this study, an α2(VI) deficient mouse (Col6α2-KO) model was used to examine the role of Type VI collagen in oral tissues. Tissue volume and mineral density were measured in oral tissues by µCT. Proteome analysis was performed using protein extracted from alveolar bone. In addition, alveolar bone was evaluated with a periodontitis induced model. µCT analysis showed the Col6α2-KO mice had less volume of alveolar bone, dentin and dental pulp, while the width of periodontal ligament (PDL) was greater than WT. The mineral density in alveolar bone and dentin were elevated in Col6α2-KO mice compared with WT. Our proteome analysis showed significant changes in proteins related to ECM organization and elevation of proteins associated with biomineralization in the Col6α2-KO mice. In induced periodontitis, Col6α2-KO mice had greater alveolar bone loss compared with WT. In conclusion, Type VI collagen has a role in controlling biomineralization in alveolar bone and that changes in the ECM of alveolar bone could be associated with greater bone loss due to periodontitis.

While wheat (Triticum aestivum L.) is a widely grown and enjoyed crop, the diverse and complex global situation and climate are exacerbating the instability of its supply. In particular, pre-harvest sprouting (PHS) is one of the major abiotic stresses that frequently occurs due to irregular climate conditions, causing serious damage to wheat and its quality. In this study, transcriptomic analysis with RNA-seq and proteomic analysis with LC-MS/MS were performed in PHS-treated spikes from two wheat cultivars presenting PHS sensitivity and tolerance, respectively. A total of 13,154 differentially expressed genes (DEGs) and 706 differentially expressed proteins (DEPs) were identified in four comparison groups between the susceptible/tolerant cultivars. Gene function and correlation analysis were performed to determine the co-profiled genes and proteins affected by PHS treatment. In the functional annotation of each comparative group, similar functions were confirmed in each cultivar under PHS treatment; however, in Keumgang PHS+7 (K7) vs. Woori PHS+7 (W7), functional annotations presented clear differences in the ”spliceosome” and ”proteasome” pathways. In addition, our results indicate that alternative splicing and ubiquitin–proteasome support the regulation of germination and seed dormancy. This study provides an advanced understanding of the functions involved in transcription and translation related to PHS mechanisms, thus enabling specific proposals for the further analysis of germination and seed dormancy mechanisms and pathways in wheat.

Drought is one of the most important constraints on the growth and productivity of many crops, including sorghum. However, as a primary sensing organ, the plant root response to drought has not been well documented at the proteomic level. In the present study, we compared physiological alteration and differential accumulation of proteins in the roots of sorghum (Sorghum bicolor) inbred line BT×623 response to Polyethylene Glycol (PEG)-induced drought stress at the seedling stage. Drought stress (up to 24 h after PEG treatment) resulted in increased accumulation of reactive oxygen species (ROS) and subsequent lipid peroxidation. The proline content was increased in drought-stressed plants. The physiological mechanism of sorghum root response to drought was attributed to the elimination of harmful free radicals and to the alleviation of oxidative stress via the synergistic action of antioxidant enzymes, such as superoxide dismutase, peroxidase, and polyphenol oxidase. The high-resolution proteome map demonstrated significant variations in about 65 protein spots detected on Coomassie Brilliant Blue-stained 2-DE gels. Of these, 52 protein spots were identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS) representing 49 unique proteins; the levels of 43 protein spots were increased, and 22 were decreased under drought condition. The proteins identified in this study are involved in a variety of cellular functions, including carbohydrate and energy metabolism, antioxidant and defense response, protein synthesis/processing/degradation, transcriptional regulation, amino acid biosynthesis, and nitrogen metabolism, which contribute jointly to the molecular mechanism of outstanding drought tolerance in sorghum plants. Analysis of protein expression patterns and physiological analysis revealed that proteins associated with changes in energy usage; osmotic adjustment; ROS scavenging; and protein synthesis, processing, and proteolysis play important roles in maintaining root growth under drought stress. This study provides new insight for better understanding of the molecular basis of drought stress responses, aiming to improve plant drought tolerance for enhanced yield.

Origanum L. (Lamiaceae) is an important genus of medicinal and aromatic plants used in traditional medicine since ancient times as culinary herbs and remedies. The aim of the present study was to evaluate the chemical composition, as well as the biochemical and cellular activities of freshly prepared Origanum majorana L. essential oil (OmEO) in an Alzheimer’s disease (AD) amyloid beta1-42 (Aβ1-42) rat model. OmEO (1% and 3%) was inhaled for 21 consecutive days, while Aβ1-42 was administered intracerebroventricularly to induce AD-like symptoms. Our data demonstrate that OmEO increased antioxidant activity and enhanced brain-derived neurotrophic factor (BDNF) expression, which in concert contributed to the improvement of cognitive function of animals. Moreover, OmEO presented beneficial effects on memory performance in Y-maze and radial arm-maze tests in the Aβ1-42 rat AD model.

Although some studies have been conducted on the effects of foreign protein expression on rice, the results vary with foreign gene types and protein expression. This study reveals the effects of fibroblast growth factor 21 (FGF21) expression on mature rice seeds in various aspects. Results revealed that the grain weight of the transgene rice was lower than that of non-transgenic wild-type. The sucrose content and ADP-glucose pyrophosphorylase (AGPase) activity in transgenic FGF21 rice were higher than that in non-transgenic wild-type rice, while changes in the starch content, starch branching enzyme (SBE), sucrose synthase (SuS), superoxide dismutase (SOD) and peroxidase (POD) activity were lower in transgenic FGF21 rice compared to non-transgenic wild-type. The scanning electron microscope results revealed that mature seeds of the transgenic FGF21 rice contained fewer vascular bundles with irregular arrangement compared to the wild-type. The mature seeds of CK and T1 rice lines were collected for proteome analysis, and 167 differentially expressed proteins (DEPs) were found. In addition, the most enriched pathways in both rice lines were determined to be amino sugar and nucleotide sugar metabolism and starch and sucrose metabolism, etc. This study laid the foundation for revealing the effects of exogenous protein expression on rice bioreactors.

The risk of rheumatoid arthritis (RA), an autoimmune disease, in the elderly population increases along with that of atherosclerosis, cardiovascular disease, type 2 diabetes, and Alzheimer's disease. Identifying specific biomarkers for RA can clarify the underlying molecular mechanisms and can aid diagnosis and patient care. To this end, the present study investigated the genes and proteins that are differentially expressed in RA using a mouse collagen-induced arthritis (CIA) model. We performed gene microarray and proteome array analyses using blood samples from the mice and found that 50 genes and 24 proteins were upregulated and 48 genes were downregulated by more than 2-fold in the CIA model relative to the control. The gene microarray and proteome array results were validated by evaluating the expression levels of select genes and proteins by real-time PCR and western blotting, respectively. We found that the level of integrin α2, which has not been previously reported as a biomarker of RA, was significantly increased in CIA mice as compared to controls. These findings provide a set of novel biomarkers that can be useful for diagnosing and evaluating the progression of RA.

In women, the flow of psoriasis is influenced by each phase of a woman’s life cycle. According to previous findings, significant changes in the levels of sex hormones affect the severity of the disease. Aim: The aim of this study was to identify the estrogen-responsive genes that could be responsible for the exacerbation of psoriasis in menopausal women. Methods: Skin samples of lesional skin donated by psoriasis patients (n = 5) were compared with skin samples of healthy volunteers (n = 5) using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The set of differentially expressed proteins was subjected to protein ontology analysis to identify differentially expressed estrogen-responsive proteins. The expression of discovered proteins was validated by qPCR and ELISA on four groups of female participants. The first group included ten psoriasis patients without menopause, the second included eleven postmenopausal patients, the third included five healthy volunteers without menopause, and the fourth included six postmenopausal volunteers. Moreover, the participants’ blood samples were used to assess the levels of estradiol, progesterone, and testosterone. Results: We found that the levels of estradiol and progesterone were significantly lower and the levels of testosterone were significantly higher in the blood of patients compared to the control. The protein ontology analysis of LC–MS/MS data identified six proteins, namely HMOX1, KRT19, LDHA, HSPD1, MAPK1, and CA2, differentially expressed in the lesional skin of female patients compared to male patients. ELISA and qPCR experiments confirmed differential expression of the named proteins and their mRNA. The genes encoding the named proteins were differentially expressed in patients compared to volunteers. However, KRT19 and LDHA were not differentially expressed when we compared patients with and without menopause. All genes, except MAPK1, were differentially expressed in patients with menopause compared to the volunteers with menopause. HMOX1, KRT19, HSPD1, and LDHA were differentially expressed in patients without menopause compared to the volunteers without menopause. However, no significant changes were found when we compared healthy volunteers with and without menopause. Conclusion: Our experiments discovered a differential expression of six estrogen-controlled genes in the skin of female patients. Identification of these genes and assessment of the changes in their expression provide insight into the biological effects of estrogen in lesional skin. The results of proteomic analysis are available via ProteomeXchange with identifier PXD021673.

SARS-CoV-2 is currently causing major havoc worldwide with its efficient transmission and propagation. To track the emergence as well as the persistence of mutations during the early stage of the pandemic, a comparative analysis of SARS-CoV-2 whole proteome sequences has been performed by considering manually curated 31,389 whole genome sequences from 84 countries. Among the 7 highly recurring (percentage frequency≥10%) mutations (Nsp2:T85I, Nsp6:L37F, Nsp12:P323L, Spike:D614G, ORF3a:Q57H, N protein:R203K and N protein:G204R), N protein:R203K and N protein: G204R are co-occurring (dependent) mutations. Nsp12:P323L and Spike:D614G often appear simultaneously. The highly recurring Spike:D614G, Nsp12:P323L and Nsp6:L37F as well as moderately recurring (percentage frequency between ≥1 and, Graphical abstract Unlabelled Image

The green-fruited Solanum habrochaites encodes a functional phytoene synthase 1 but fails to accumulate lycopene due to metabolic and structural blocks in chromoplast formation and carotenoid storage., Members of the tomato clade exhibit a wide diversity in fruit color, but the mechanisms governing inter-species diversity of coloration are largely unknown. The carotenoid profiles, carotenogenic gene expression and proteome profiles of green-fruited Solanum habrochaites (SH), orange-fruited S. galapagense, and red-fruited S. pimpinellifolium were compared with cultivated tomato [S. lycopersicum cv. Ailsa Craig (SL)] to decipher the molecular basis of coloration diversity. Green-fruited SH, though it showed normal expression of chromoplast-specific phytoene synthase1 and lycopene β-cyclase genes akin to orange/red-fruited species, failed to accumulate lycopene and β-carotene. The SH phytoene synthase1 cDNA encoded an enzymatically active protein, whereas the lycopene β-cyclase cDNA was barely active. Consistent with its green-fruited nature, SH’s fruits retained chloroplast structure and PSII activity, and had impaired chlorophyll degradation with high pheophorbide a levels. Comparison of the fruit proteomes with SL revealed retention of the proteome complement related to photosynthesis in SH. Targeted peptide monitoring revealed a low abundance of key carotenogenic and sequestration proteins in SH compared with tomato. The green-fruitedness of SH appears to stem from blocks at several critical steps regulating fruit-specific carotenogenesis namely the absence of chloroplast to chromoplast transformation, block in carotenoid biosynthesis, and a dearth of carotenoid sequestering proteins.

Key message: For the first time, a comprehensive proteome analysis was conducted on Brachypodium leaves under drought stress. Gradual changes in response to drought stress were monitored. Abstract: Drought is one of the major stress factors that dramatically affect the agricultural productivity worldwide. Improving the yield under drought is an urgent challenge in agriculture. Brachypodium distachyon is a model species for monocot plants such as wheat, barley and several potential biofuel grasses. In the current study, a comprehensive proteome analysis was conducted on Brachypodium leaves under different levels of drought application. To screen gradual changes upon drought, Brachypodium leaves subjected to drought for 4, 8 and 12 days were collected for each treatment day and relative water content of the leaves was measured for each time point. Cellular responses of Brachypodium were investigated through a proteomic approach involving two dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). Among 497 distinct spots in Brachypodium protein repertoire, a total of 13 differentially expressed proteins (DEPs) were identified as responsive to drought by mass spectrometry and classified according to their functions using bioinformatics tools. The biological functions of DEPs included roles in photosynthesis, protein folding, antioxidant mechanism and metabolic processes, which responded differentially at each time point of drought treatment. To examine further transcriptional expression of the genes that code identified protein, quantitative real time PCR (qRT-PCR) was performed. Identified proteins will contribute to the studies involving development of drought-resistant crop species and lead to the delineation of molecular mechanisms in drought response. © 2017, Springer Science+Business Media B.V. 115Z087 Kocaeli Üniversitesi We would like to thank Kocaeli University, Faculty of Medicine, Department of Medical Biology for technical support and MALDI-TOF/TOF analysis. This work was supported by TUBITAK Research Funds (Project No: 115Z087). Brachypodium seeds used in this study were kindly provided by Prof. Dr. Metin Tuna, Nam?k Kemal University, Department of Field Crops, Tekirda?, Turkey.

The metabolic status of individual cells in microbial cultures can differ, being relevant for biotechnology, environmental and medical microbiology. However, it is hardly understood in molecular detail due to limitations of current analytical tools. Here, we demonstrate that FACS in combination with proteomics can be used to sort and analyze cell populations based on their metabolic state. A previously established GFP reporter system was used to detect and sort single Corynebacterium glutamicum cells based on the concentration of branched chain amino acids (BCAA) using FACS. A proteomics workflow optimized for small cell numbers was used to quantitatively compare proteomes of a Delta aceE mutant, lacking functional pyruvate dehydrogenase (PD), and the wild type. About 800 proteins could be quantified from 1,000,000 cells. In the Delta aceE mutant BCAA production was coordinated with upregulation of the glyoxylate cycle and TCA cycle to counter the lack of acetyl CoA resulting from the deletion of aceE. Biological significance: Metabolic pathways in C glutamicum WT and Delta aceE, devoid of functional pyruvate dehydrogenase, were compared to understand proteome changes that contribute to the high production of branched chain amino acids (BCAA) in the Delta aceE strain. The data complements previous metabolome studies and corroborates the role of malate provided by the glyoxylate cycle and increased activity of glycolysis and pyruvate carboxylase reaction to replenish the TCA cycle. A slight increase in acetohydroxyacid synthase (ILV subunit B) substantiates the previously reported increased pyruvate pool in C. glutamicum Delta aceE, and the benefit of additional ilv gene cluster overexpression for BCAA production. (C) 2017 Elsevier B.V. All rights reserved.

Background Modularity is important for evolutionary innovation. The recombination of existing units to form larger complexes with new functionalities spares the need to create novel elements from scratch. In proteins, this principle can be observed at the level of protein domains, functional subunits which are regularly rearranged to acquire new functions. Results In this study we analyse the mechanisms leading to new domain arrangements in five major eukaryotic clades (vertebrates, insects, fungi, monocots and eudicots) at unprecedented depth and breadth. This allows, for the first time, to directly compare rates of rearrangements between different clades and identify both lineage specific and general patterns of evolution in the context of domain rearrangements. We analyse arrangement changes along phylogenetic trees by reconstructing ancestral domain content in combination with feasible single step events, such as fusion or fission. Using this approach we explain up to 70% of all rearrangements by tracing them back to their precursors. We find that rates in general and the ratio between these rates for a given clade in particular, are highly consistent across all clades. In agreement with previous studies, fusions are the most frequent event leading to new domain arrangements. A lineage specific pattern in fungi reveals exceptionally high loss rates compared to other clades, supporting recent studies highlighting the importance of loss for evolutionary innovation. Furthermore, our methodology allows us to link domain emergences at specific nodes in the phylogenetic tree to important functional developments, such as the origin of hair in mammals. Conclusions Our results demonstrate that domain rearrangements are based on a canonical set of mutational events with rates which lie within a relatively narrow and consistent range. In addition, gained knowledge about these rates provides a basis for advanced domain-based methodologies for phylogenetics and homology analysis which complement current sequence-based methods.

A variety of protein post-translational modifications has been identified that control many cellular functions. Phosphorylation studies in mycobacterial organisms have shown critical importance in diverse biological processes, such as intercellular communication and cell division. Recent technical advances in high-precision mass spectrometry have determined a large number of microbial phosphorylated proteins and phosphorylation sites throughout the proteome analysis. Identification of phosphorylated proteins with specific modified residues through experimentation is often laborintensive, costly and time-consuming. All these limitations could be overcome through the application of machine learning (ML) approaches. However, only a limited number of computational phosphorylation site prediction tools have been developed so far. This work aims to present a complete survey of the existing ML-predictors for microbial phosphorylation. We cover a variety of important aspects for developing a successful predictor, including operating ML algorithms, feature selection methods, window size, and software utility. Initially, we review the currently available phosphorylation site databases of the microbiome, the state-of-the-art ML approaches, working principles, and their performances. Lastly, we discuss the limitations and future directions of the computational ML methods for the prediction of phosphorylation.

Cutibacterium acnes, former Proprionibacterium acnes, is a heterogeneous species including acneic bacteria such as the RT4 strain, and commensal bacteria such as the RT6 strain. These strains have been characterized by metagenomic analysis but their physiology was not investigated until now. Bacteria were grown in different media, brain heart infusion medium (BHI), reinforced clostridial medium (RCM), and in sebum like medium (SLM) specifically designed to reproduce the lipid rich environment of the sebaceous gland. Whereas the RT4 acneic strain showed maximal growth in SLM and lower growth in RCM and BHI, the RT6 non acneic strain was growing preferentially in RCM and marginally in SLM. These differences were correlated with the lipophilic surface of the RT4 strain and to the more polar surface of the RT6 strain. Both strains also showed marked differences in biofilm formation activity which was maximal for the RT4 strain in BHI and for the RT6 strain in SLM. However, cytotoxicity of both strains on HaCaT keratinocytes remained identical and limited. The RT4 acneic strain showed higher inflammatory potential than the RT6 non acneic strain, but the growth medium was without significant influence. Both bacteria were also capable to stimulate β‐defensine 2 secretion by keratinocytes but no influence of the bacterial growth conditions was observed. Comparative proteomics analysis was performed by nano LC‐MS/MS and revealed that whereas the RT4 strain only expressed triacylglycerol lipase, the principal C. acnes virulence factor, when it was grown in SLM, the RT6 strain expressed another virulence factor, the CAMP factor, exclusively when it was grown in BHI and RCM. This study demonstrates the key influence of growth conditions on virulence expression by C. acnesand suggest that acneic and non acneic strains are related to different environmental niches.

Wheat represents one of the most important cereals for mankind. However, since wheat proteins are also the causative agent of several adverse reactions, during the last decades, consumers have shown an increasing interest in the old wheat genotypes, which are generally perceived as more “natural” and healthier than the modern ones. Comparison of nutritional value for modern and old wheat genotypes is still controversial, and to evaluate the real impact of these foods on human health comparative experiments involving old and modern genotypes are desirable. The nutritional quality of grain is correlated with its proteomic composition that depends on the interplay between the genetic characteristics of the plant and external factors related to the environment. We report here the label-free shotgun quantitative comparison of the metabolic protein fractions of two old Sicilian landraces (Russello and Timilia) and the modern variety Simeto, from the 2010–2011 and 2011–2012 growing seasons. The overall results show that Timilia presents the major differences with respect to the other two genotypes investigated. These differences may be related to different defense mechanisms and some other peculiar properties of these genotypes. On the other hand, our results confirm previous results leading to the conclusion that with respect to a nutritional value evaluation, there is a substantial equivalence between old and modern wheat genotypes. Data are available via ProteomeXchange with identifier &lt, PXD024204&gt

A conditioned medium of a cell culture is widely used for various biological applications and frequently analyzed to characterize the functional proteins responsible for observed biological functions. However, a large number of abundant proteins in fetal bovine serum (FBS), usually included in the conditioned medium of a mammalian cell culture medium, hampers in-depth proteomic analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS). For a deep proteomic analysis of a conditioned medium by LC-MS/MS, we developed a simple albumin depletion approach coupled with data-independent acquisition (DIA)-mode LC-MS/MS for the conditioned medium of mammalian cells in this study. The results showed that this approach enabled the detection of more than 3700 cell-derived proteins in the cell culture supernatant containing FBS. We further demonstrated the potency of this approach by analyzing proteins in the conditioned media of HeLa cells with and without tumor necrosis factor (TNF) stimulation: &gt, 40 differentially accumulated proteins, including four cytokines, upon TNF stimulation were identified in the culture media, which were hardly detected by conventional proteome approaches in the literature.

Seed germination is a critical phase in the plant life cycle, but the mechanism of seed germination is still poorly understood. In the present study, rice (Oryza sativa L. cv. Peiai 64S) seeds were sampled individually when they reached different germination stages, quiescent, germinated sensu stricto, germinated completely and seedling, and were used to study the changes in the embryo proteome. A total of 88 protein spots showed a significant change in abundance during germination in water, and the results showed an activation of metabolic processes. Cell division, cell wall synthesis, and secondary metabolism were activated at late seed germination and during preparation for subsequent seedling establishment. Cycloheximide (CHX) at 70 μM inhibited seedling establishment without an apparent negative effect on seed germination, while CHX at 500 μM completely blocked seed germination. We used this observation to identify the potentially important proteins involved in seed germination (coleoptile protrusion) and seedling establishment (coleoptile and radicle protrusion). Twenty-six protein spots, mainly associated with sugar/polysaccharide metabolism and energy production, showed a significant difference in abundance during seed germination. Forty-nine protein spots, mainly involved in cell wall biosynthesis, proteolysis as well as cell defense and rescue, were required for seedling establishment. The results help improve our understanding of the key events (proteins) involved in germination and seedling development.

Drought is one of the most important constraints on the growth and productivity of many crops, including sorghum. However, as a primary sensing organ, the plant root response to drought has not been well documented at the proteomic level. In the present study, we compared physiological alteration and differential accumulation of proteins in the roots of sorghum (Sorghum bicolor) inbred line BT&times, 623 response to Polyethylene Glycol (PEG)-induced drought stress at the seedling stage. Drought stress (up to 24 h after PEG treatment) resulted in increased accumulation of reactive oxygen species (ROS) and subsequent lipid peroxidation. The proline content was increased in drought-stressed plants. The physiological mechanism of sorghum root response to drought was attributed to the elimination of harmful free radicals and to the alleviation of oxidative stress via the synergistic action of antioxidant enzymes, such as superoxide dismutase, peroxidase, and polyphenol oxidase. The high-resolution proteome map demonstrated significant variations in about 65 protein spots detected on Coomassie Brilliant Blue-stained 2-DE gels. Of these, 52 protein spots were identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS) representing 49 unique proteins, the levels of 43 protein spots were increased, and 22 were decreased under drought condition. The proteins identified in this study are involved in a variety of cellular functions, including carbohydrate and energy metabolism, antioxidant and defense response, protein synthesis/processing/degradation, transcriptional regulation, amino acid biosynthesis, and nitrogen metabolism, which contribute jointly to the molecular mechanism of outstanding drought tolerance in sorghum plants. Analysis of protein expression patterns and physiological analysis revealed that proteins associated with changes in energy usage, osmotic adjustment, ROS scavenging, and protein synthesis, processing, and proteolysis play important roles in maintaining root growth under drought stress. This study provides new insight for better understanding of the molecular basis of drought stress responses, aiming to improve plant drought tolerance for enhanced yield.

BACKGROUND: Liver fibrosis is a consequence of chronic inflammation and is associated with protein changes within the hepatocytes structure. In this study, we aimed to investigate if this is reflected by the urinary proteome and can be explored to diagnose liver fibrosis in patients with chronic liver disease. METHODS: In a multicentre combined cross-sectional and prospective diagnostic test validation study, 129 patients with varying degrees of liver fibrosis and 223 controls without liver fibrosis were recruited. Additionally, 41 patients with no liver, but kidney fibrosis were included to evaluate interference with expressions of kidney fibrosis. Urinary low molecular weight proteome was analysed by capillary electrophoresis coupled to mass spectrometry (CE-MS) and a support vector machine marker model was established by integration of peptide markers for liver fibrosis. FINDINGS: CE-MS enabled identification of 50 urinary peptides associated with liver fibrosis. When combined into a classifier, LivFib-50, it separated patients with liver fibrosis (N = 31) from non-liver disease controls (N = 123) in cross-sectional diagnostic phase II evaluation with an area under the curve (AUC) of 0.94 (95% confidence intervals (CI): 0.89-0.97, p

Biliary atresia (BA) is a destructive inflammatory obliterative cholangiopathy of the neonate that affects various parts of the bile duct. If early diagnosis followed by Kasai portoenterostomy is not performed, progressive liver cirrhosis frequently leads to liver transplantation in the early stage of life. Therefore, prompt diagnosis is necessary for the rescue of BA patients. However, the prompt diagnosis of BA remains challenging because specific and reliable biomarkers for BA are currently unavailable. In this study, we discovered potential biomarkers for BA using deep proteome analysis by data-independent acquisition mass spectrometry (DIA–MS). Four patients with BA and three patients with neonatal cholestasis of other etiologies (non-BA) were recruited for stool proteome analysis. Among the 2110 host-derived proteins detected in their stools, 49 proteins were significantly higher in patients with BA and 54 proteins were significantly lower. These varying stool protein levels in infants with BA can provide potential biomarkers for BA. As demonstrated in this study, the deep proteome analysis of stools has great potential not only in detecting new stool biomarkers for BA but also in elucidating the pathophysiology of BA and other pediatric diseases, especially in the field of pediatric gastroenterology.

Sporadic cerebral amyloid angiopathy (CAA) is characterized by cerebrovascular amyloid-β (Aβ) deposition, which leads to lobar hemorrhage and dementia. Biological molecules affecting the development of CAA have not been fully characterized. In this study, we performed proteome analysis of biopsied leptomeningeal and cortical vessels obtained from 6 CAA patients and 5 non-CAA patients who underwent surgery for large lobar hemorrhages. We found that 6 proteins, including Aβ, apolipoprotein E (apoE), clusterin (CLU), albumin, complement C4 and vitronectin were significantly upregulated in the vessels of CAA patients as compared to non-CAA patients. ApoE and CLU were found in all CAA patients. We next examined the effects of apoE and CLU on the early phase of Aβ aggregation, using a simple yet powerful in vitro model of CAA, which recapitulates the intramural periarterial drainage pathway model. We found that physiological concentrations of apoE and CLU delayed the initiation time of amyloid growth kinetics in a concentration-dependent manner. These data indicate that apoE and CLU may act as extracellular chaperones to inhibit Aβ amyloid deposition in CAA.

Background Ulva prolifera belongs to green macroalgae and is the dominant species of green tide. It is distributed worldwide and is therefore subject to high-temperature stress during the growth process. However, the adaptation mechanisms of the response of U. prolifera to high temperatures have not been clearly investigated yet. Methods In this study, isobaric tags for relative and absolute quantitation (iTRAQ) labelling was applied in combination with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) to conduct comparative proteomic analysis of the response of U. prolifera to high-temperature stress and to elucidate the involvement of this response in adaptation mechanisms. Differentially expressed proteins (DEPs) of U. prolifera under high temperature (denote UpHT) compared with the control (UpC) were identified. Bioinformatic analyses including GO analysis, pathway analysis, and pathway enrichment analysis was performed to analyse the key metabolic pathways that underlie the thermal tolerance mechanism through protein networks. Quantitative real-time PCR and western blot were performed to validate selected proteins. Results In the present study, 1223 DEPs were identified under high temperature compared with the control, which included 790 up-regulated and 433 down-regulated proteins. The high-temperature stimulus mainly induced the expression of glutathione S-transferase, heat shock protein, ascorbate peroxidase, manganese superoxide dismutase, ubiquitin-related protein, lhcSR, rubisco activase, serine/threonine protein kinase 2, adenylate kinase, Ca2+-dependent protein kinase (CDPK), disease resistance protein EDS1, metacaspase type II, NDPK2a, 26S proteasome regulatory subunit, ubiquinone oxidoreductase, ATP synthase subunit, SnRK2s, and cytochrome P450. The down-regulated proteins were photosynthesis-related proteins, glutathione reductase, catalase-peroxidase, thioredoxin, thioredoxin peroxidase, PP2C, and carbon fixation-related proteins. Furthermore, biological index analysis indicated that protein content and SOD activity decreased; the value of Fv/Fm dropped to the lowest point after culture for 96 h. However, APX activity and MDA content increased under high temperature. Conclusion The present study implied an increase in proteins that were associated with the stress response, oxidative phosphorylation, the cytokinin signal transduction pathway, the abscisic acid signal transduction pathway, and the glutathione metabolism pathway. Proteins that were associated with photosynthesis, carbon fixation in photosynthesis organisms, and the photosynthesis antenna protein pathway were decreased. These pathways played a pivotal role in high temperature regulation. These novel proteins provide a good starting point for further research into their functions using genetic or other approaches. These findings significantly improve the understanding of the molecular mechanisms involved in the tolerance of algae to high-temperature stress.

Typically, nitrification is a two-stage microbial process and is key in wastewater treatment and nutrient recovery from waste streams. Changes in salinity represent a major stress factor that can trigger response mechanisms, impacting the activity and the physiology of bacteria. Despite its pivotal biotechnological role, little information is available on the specific response of nitrifying bacteria to varying levels of salinity. In this study, synthetic communities of ammonia-oxidizing bacteria (AOB Nitrosomonas europaea and/or Nitrosomonas ureae) and nitrite-oxidizing bacteria (NOB Nitrobacter winogradskyi and/or Nitrobacter vulgaris) were tested at 5, 10, and 30 mS cm-1 by adding sodium chloride to the mineral medium (0, 40, and 200 mM NaCl, respectively). Ammonia oxidation activity was less affected by salinity than nitrite oxidation. AOB, on their own or in combination with NOB, showed no significant difference in the ammonia oxidation rate among the three conditions. However, N. winogradskyi improved the absolute ammonia oxidation rate of both N. europaea and N. ureae. N. winogradskyi’s nitrite oxidation rate decreased to 42% residual activity upon exposure to 30 mS cm-1, also showing a similar behavior when tested with Nitrosomonas spp. The nitrite oxidation rate of N. vulgaris, as a single species, was not affected when adding sodium chloride up to 30 mS cm-1, however, its activity was completely inhibited when combined with Nitrosomonas spp. in the presence of ammonium/ammonia. The proteomic analysis of a co-culture of N. europaea and N. winogradskyi revealed the production of osmolytes, regulation of cell permeability and an oxidative stress response in N. europaea and an oxidative stress response in N. winogradskyi, as a result of increasing the salt concentration from 5 to 30 mS cm-1. A specific metabolic response observed in N. europaea suggests the role of carbon metabolism in the production of reducing power, possibly to meet the energy demands of the stress response mechanisms, induced by high salinity. For the first time, metabolic modifications and response mechanisms caused by the exposure to salinity were described, serving as a tool toward controllability and predictability of nitrifying systems exposed to salt fluctuations.

Background Ulva prolifera belongs to green macroalgae and is the dominant species of green tide. It is distributed worldwide and is therefore subject to high-temperature stress during the growth process. However, the adaptation mechanisms of the response of U. prolifera to high temperatures have not been clearly investigated yet. Methods In this study, isobaric tags for relative and absolute quantitation (iTRAQ) labelling was applied in combination with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) to conduct comparative proteomic analysis of the response of U. prolifera to high-temperature stress and to elucidate the involvement of this response in adaptation mechanisms. Differentially expressed proteins (DEPs) of U. prolifera under high temperature (denote UpHT) compared with the control (UpC) were identified. Bioinformatic analyses including GO analysis, pathway analysis, and pathway enrichment analysis was performed to analyse the key metabolic pathways that underlie the thermal tolerance mechanism through protein networks. Quantitative real-time PCR and western blot were performed to validate selected proteins. Results In the present study, 1223 DEPs were identified under high temperature compared with the control, which included 790 up-regulated and 433 down-regulated proteins. The high-temperature stimulus mainly induced the expression of glutathione S-transferase, heat shock protein, ascorbate peroxidase, manganese superoxide dismutase, ubiquitin-related protein, lhcSR, rubisco activase, serine/threonine protein kinase 2, adenylate kinase, Ca2+-dependent protein kinase (CDPK), disease resistance protein EDS1, metacaspase type II, NDPK2a, 26S proteasome regulatory subunit, ubiquinone oxidoreductase, ATP synthase subunit, SnRK2s, and cytochrome P450. The down-regulated proteins were photosynthesis-related proteins, glutathione reductase, catalase-peroxidase, thioredoxin, thioredoxin peroxidase, PP2C, and carbon fixation-related proteins. Furthermore, biological index analysis indicated that protein content and SOD activity decreased; the value of Fv/Fm dropped to the lowest point after culture for 96 h. However, APX activity and MDA content increased under high temperature. Conclusion The present study implied an increase in proteins that were associated with the stress response, oxidative phosphorylation, the cytokinin signal transduction pathway, the abscisic acid signal transduction pathway, and the glutathione metabolism pathway. Proteins that were associated with photosynthesis, carbon fixation in photosynthesis organisms, and the photosynthesis antenna protein pathway were decreased. These pathways played a pivotal role in high temperature regulation. These novel proteins provide a good starting point for further research into their functions using genetic or other approaches. These findings significantly improve the understanding of the molecular mechanisms involved in the tolerance of algae to high-temperature stress. Electronic supplementary material The online version of this article (10.1186/s12953-018-0145-5) contains supplementary material, which is available to authorized users.

Finding the keys to understanding the infectious process of Yersinia ruckeri was not a priority for many years due to the prompt development of an effective biotype 1 vaccine which was used mainly in Europe and USA. However, the gradual emergence of outbreaks in vaccinated fish, which have been reported since 2003, has awakened interest in the mechanism of virulence in this pathogen. Thus, during the last two decades, a large number of studies have considerably enriched our knowledge of many aspects of the pathogen and its interaction with the host. By means of both conventional and a variety of novel strategies, such as cell GFP tagging, bioluminescence imaging and optical projection tomography, it has been possible to determine three putative Y. ruckeri infection routes, the main point of entry for the bacterium being the gill lamellae. Moreover, a wide range of potential virulence factors have been highlighted by specific gene mutagenesis strategies or genome-wide transposon/plasmid insertion-based screening approaches, such us in vivo expression technology (IVET) and signature tagged mutagenesis (STM). Finally, recent proteomic and whole genomic analyses have allowed many of the genes and systems that are potentially implicated in the organism's pathogenicity and its adaptation to the host environmental conditions to be elucidated. Altogether, these studies contribute to a better understanding of the infectious process of Y. ruckeri in fish, which is crucial for the development of more effective strategies for preventing or treating enteric redmouth disease (ERM).

Background & Aims Chronic kidney disease (CKD) is a common complication after liver transplantation. Kidney biopsies cannot be easily performed before liver transplantation to predict patients at high risk for CKD. The aim of our study was to determine whether pre-, peri- and post-transplant factors, as well as peptides present in preliver transplant urine samples were associated with loss in kidney function at 6 months post-transplantation using proteome analysis. Methods Eighty patients who underwent a liver transplantation and that had pretransplant glomerular filtration rate (GFR) value of ≥60 mL/min/1.73 m² (MDRD) were included in the study. Results GFR decreased significantly after transplantation. At month 6 post-transplantation, 40 patients displayed a CKD, i.e. eGFR of

Thousands of genetic variants have been associated with complex traits through genome-wide association studies. However, the functional variants or mechanistic consequences remain elusive. Intermediate traits such as gene expression or protein levels are good proxies of the metabolic state of an organism. Proteome analysis especially can provide new insights into the molecular mechanisms of complex traits like obesity. The role of genetic variation in determining protein level variation has not been assessed in obesity. To address this, we design a large-scale protein quantitative trait locus (pQTL) analysis based on a set of 1129 proteins from 494 obese subjects before and after a weight loss intervention. This reveals 55 BMI-associated cis-pQTLs and trans-pQTLs at baseline and 3 trans-pQTLs after the intervention. We provide evidence for distinct genetic mechanisms regulating BMI-associated proteins before and after weight loss. Finally, by functional analysis, we identify and validate FAM46A as a trans regulator for leptin., Although many genetic variants are known for obesity, their function remains largely unknown. Here, in a weight-loss intervention cohort, the authors identify protein quantitative trait loci associated with BMI at baseline and after weight loss and find FAM46A to be a regulator of leptin in adipocytes.

Summary Conjugation of the small ubiquitin‐related modifier (SUMO) to protein substrates has an impact on stress responses and on development. We analyzed the proteome and phosphoproteome of mutants in this pathway. The mutants chosen had defects in SUMO ligase SIZ1, which catalyzes attachment of single SUMO moieties onto substrates, and in ligases PIAL1 and PIAL2, which are known to form SUMO chains. A total of 2657 proteins and 550 phosphopeptides were identified and quantified. Approximately 40% of the proteins and 20% of the phosphopeptides showed differences in abundance in at least one of the analyzed genotypes, demonstrating the influence of SUMO conjugation on protein abundance and phosphorylation. The data show that PIAL1 and PIAL2 are integral parts of the SUMO conjugation system with an impact on stress response, and confirm the involvement of SIZ1 in plant defense. We find a high abundance of predicted SUMO attachment sites in phosphoproteins (70% versus 40% in the total proteome), suggesting convergence of phosphorylation and sumoylation signals onto a set of common targets., Significance Statement The proteome of mutants in SUMO conjugation displays abundant changes in metabolic and regulatory pathway components, in particular those related to stress response. Phospho‐proteome analysis indicates that sumoylation and phosphorylation share a set of common targets, which may thereby integrate stimuli from different signaling cascades.

Leaf variegation is often the focus of plant breeding. Here, we studied a variegated mutant of Phalaenopsis aphrodite subsp. formosana, which is usually used as a parent of horticultural breeding, to understand its anatomic and genetic regulatory mechanisms in variegation. Chloroplasts with well-organized thylakoids and starch grains were found only in the mesophyll cells of green sectors but not of yellow sectors, confirming that the variegation belongs to the chlorophyll type. The two-dimensional electrophoresis and LC/MS/MS also reveal differential expressions of PsbP and PsbO between the green and yellow leaf sectors. Full-length cDNA sequencing revealed that mutant transcripts were caused by intron retention. When conditioning on the total RNA expression, we found that the functional transcript of PsbO and mutant transcript of PsbP are higher expressed in the yellow sector than in the green sector, suggesting that the post-transcriptional regulation of PsbO and PsbP differentiates the performance between green and yellow sectors. Because PsbP plays an important role in the stability of thylakoid folding, we suggest that the negative regulation of PsbP may inhibit thylakoid development in the yellow sectors. This causes chlorophyll deficiency in the yellow sectors and results in leaf variegation. We also provide evidence of the link of virus CymMV and the formation of variegation according to the differential expression of CymMV between green and yellow sectors.

The close relationship between diet and health is generally recognized and the growing wellness and consciousness, especially in developed countries, have led to increasing interest for old wheat genotypes, based on perceived health benefits. Although nutritional comparison between old and modern wheat varieties is still controversial, it is generally accepted that old wheat genotypes remained unchanged over the last hundred years. By contrast, modern wheat genotypes are derived by modification of old wheats during the so-called “Green-Revolution” in the second half of the 20th century focusing on obtaining properties in terms of higher grain yield. The present work reports the first comprehensive proteomic profiling and qualitative comparison at the molecular level of metabolic and Chloroform-Methanol (CM)-like protein fractions extracted from mature kernels of two old Sicilian durum wheat landraces, Russello and Timilia Reste Bianche, and Simeto, an improved durum wheat variety widespread in Italy and other Mediterranean countries and chosen as representative of the most widely commercial cultivars. The results obtained reveal that metabolic and CM-like protein fractions of old and modern genotypes present remarkably high similarity with only minor differences. This leads to the conclusion that from a food and nutritional perspective there is a substantial equivalence of the protein composition of the old and modern cultivars. Data are available via ProteomeXchange with identifier PXD014449 . Biological significance In recent years consumers have shown growing interest in the old wheat genotypes, which are generally perceived more “natural” and healthier than modern ones. However, comparison of nutritional value for modern and old wheat varieties is still controversial suggesting further studies. In particular proteome analysis of old and modern wheat genotypes is currently ongoing with particular focus on gluten proteins, whereas the metabolic protein fraction has not yet been investigated. In the present study, we conducted a comprehensive proteomic profile and qualitative comparison at the molecular level of metabolic and Chloroform-Methanol (CM)-like protein fractions of the old Sicilian landraces Russello and Timilia Reste Bianche and the modern cultivar Simeto by applying a shotgun approach. The results reveal that the metabolic and CM-like protein fractions of old and modern genotypes are remarkably similar with only minor differences, leading to the conclusion that from a food and nutritional perspective there is a substantial equivalence of these cultivars. These results may contribute to improved understanding of the relationship between protein profiles of old wheat genotypes and their potential benefits for human consumption.

Pseudomonas putida F1 can metabolize toluene, ethylbenzene, and benzene for growth. Previously, we identified proteins involved in the utilization of these compounds by P. putida F1 through culture in liquid media. However, it was unclear whether laboratory analysis of bacterial activity and catabolism accurately reflected the soil environment. We identified proteins involved in the degradation of toluene, ethylbenzene, and benzene growth in soil using two-dimensional gel electrophoresis (2-DE) or standard SDS-PAGE combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). According to 2-DE/LC-MS/MS analysis, 12 of 22 key enzymes involved in the degradation of toluene, ethylbenzene, and benzene were detected. In standard SDS-PAGE/LC-MS/MS analysis of soil with ethylbenzene, approximately 1,260 cellular proteins were identified in P. putida F1. All key enzymes and transporter and sensor proteins involved in ethylbenzene degradation were up-regulated similar to that noted in liquid cultures. In P. putida F1, aromatic hydrocarbon response in soil is the same as that observed in liquid media.

Sphingomonas elodea is a Gram-negative bacterium capable of producing ‘gellan gum’ exopolysaccharide that is the most extensively studied expolysaccharides of microbial origin. In this study, we investigated the phenotypic and proteomic alterations in S. elodea by homogeneously expressing both gelA and gelN involved in positive regulation and extracellular secretion of metabolites in gellan biosynthesis, respectively. Expression of six histidine-tagged GelA and GelN was determined by Western blot analysis. Successful expression of GelA and GelN resulted in both morphological changes of colonies and enhanced secretion of gellan into the growth medium (GelA, 21.2% more and GelN, 48.3% more) overexpressed compared to the wile-type. Comparative two-dimensional gel electrophoresis analysis revealed a differential proteome expression in S. elodea overexpressing GelA and GelN. Proteins up- or down-regulated by GelA and GelN overexpression were found to be mainly sugar transportation proteins, two-component regulatory proteins, and proteins involved in secretion pathways. The results suggest that the effect of GelA and GelN overexpression on gellan biosynthesis might be mainly caused by increased transportation of sugar units or enhanced exportation of gellan.

Background Dystrophic epidermolysis bullosa (DEB), a rare genodermatosis, is characterized by the formation of intra-epidermal blistering and the development of chronic nonhealing skin wounds. Recently, attempts have been made to develop cell-based therapies for this currently intractable disorder. The molecular mechanisms that govern directional migration of the adult stem cells, allowing their efficient and controlled homing to the skin affected with DEB, are poorly understood. The key mechanism that regulates recruitment of leukocytes and progenitor stem cells to distal anatomical tissues affected with disease is chemotaxis, which depends on the signaling molecules, chemokines, and acts primarily as part of the host defense and repair mechanism. Methods Comprehensive proteomic screening of chemokines in the blister fluids of DEB-affected mice was conducted to define the inflammatory and immune activities, thus providing potential to examine local biological mechanisms and define the protein signature within lesional skin as a potential marker of disease activity. Also, the therapeutic relevance of identified chemotactic pathways was investigated in vivo, providing a basis for future clinical investigations. Results Assessment of blister fluid-derived chemokines showed a persistent presence of several chemotactic molecules, including CXCL1 + 2 and CXCL5. The majority of blister-originated chemotactic signals were associated with preferential recruitment of CD45+CXCR2+ and CD11b+CXCR2+ leukocytes. Systemic transplantation of an enriched CXCR2 population of mouse adipose-derived stem cells (mADSC) into DEB-affected mice demonstrated effective recruitment of cells to the blistering skin under the influence of blister-derived ligands and deposition of therapeutic type VII collagen. Conclusions Collectively, these studies demonstrate that recruitment of mADSC into DEB skin is tightly controlled by disease-site chemotactic activities and suggest a potential mechanism for effective application of therapeutic stem cells for DEB.

Steroidogenesis requires coordination of the anabolic and catabolic pathways of lipid metabolism, but the profile of proteins associated with progesterone synthesis in cyclic and pregnant corpus luteum (CL) is not well-known in cattle. In Experiment 1, plasma progesterone level was monitored in cyclic cows (n = 5) and pregnant cows (n = 6; until d-90). A significant decline in the plasma progesterone level occurred at d-19 of cyclic cows. Progesterone level in abbatoir-derived luteal tissues was also determined at d 1 to 5, 6 to 13 and 14 to 20 of cyclic cows, and d-60 and -90 of pregnant cows (n = 5 each). Progesterone level in d-60 CL was not different from those in d 6 to 13 CL and d-90 CL, although the difference between d 6 to 13 and d-90 was significant. In Experiment 2, protein expression pattern in CL at d-90 (n = 4) was compared with that in CL of cyclic cows at d 6 to 13 (n = 5). Significant changes in the level of protein expression were detected in 32 protein spots by two-dimensional polyacrylamide gel electrophoresis (2-DE), and 23 of them were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Six proteins were found only in pregnant CL, while the other 17 proteins were found only in cyclic CL. Among the above 6 proteins, vimentin which is involved in the regulation of post-implantation development was included. Thus, the protein expression pattern in CL was disorientated from cyclic luteal phase to mid pregnancy, and alterations in specific CL protein expression may contribute to the maintenance of pregnancy in Korean native cows.

White rust, caused by Albugo candida, is a serious pathogen of Brassica juncea (Indian mustard) and poses a potential hazard to the presently developing canola-quality B. juncea industry worldwide. A comparative proteomic study was undertaken to explore the molecular mechanisms that underlie the defence responses of Brassica juncea to white rust disease caused by the biotrophic oomycete Albugo candida. Nineteen proteins showed reproducible differences in abundance between a susceptible (RH 819) and a resistant variety (CBJ 001) of B. juncea following inoculation with A. candida. The identities of all 19 proteins were successfully established through Q-TOF MS/MS. Five of these proteins were only detected in the resistant variety and showed significant differences in their abundance at various times following pathogen inoculation in comparison to mock-inoculated plants. Among these was a thaumatin-like protein (PR-5), a protein not previously associated with the resistance of B. juncea towards A. candida. One protein, peptidyl-prolyl cis/trans isomerase (PPIase) isoform CYP20-3, was only detected in the susceptible variety and increased in abundance in response to the pathogen. PPIases have recently been discovered to play an important role in pathogenesis by suppressing the host cell's immune response. For a subset of seven proteins examined in more detail, an increase in transcript abundance always preceded their induction at the proteome level. These findings are discussed within the context of the A. candida-Brassica juncea pathosystem, especially in relation to host resistance to this pathogen.

Chantier qualité GA; International audience; Within a population of Charolais young bulls, two extreme groups of longissimus thoracis muscle samples, classified according to Warner−Bratzler shear force (WBSF) of 55 °C grilled meat, were analyzed by 2D-electrophoresis. Muscle analyses were performed on 4 bulls of the “tender” group (WBSF = 27.7 ± 4.8 N) and 4 bulls of the “tough” group (WBSF = 41.2 ± 6.1 N), at 3 post-mortem times: D0, samples taken within 10 min post-mortem; D5 and D21, samples kept at 4 °C under vacuum during 5 and 21 days. Proteins of muscle samples were separated in two fractions based on protein solubility in Tris buffer: “soluble” and “insoluble”. Proteins of both fractions were separated by 2D-electrophoresis. Evolution of spots during the 3 post-mortem times was analyzed by hierarchical classification (HCA). Three clusters of proteins presenting similar evolution profiles provided accurate classification of post-mortem times and showed the translocation of some chaperone proteins and glycolytic enzymes from the soluble fraction to the insoluble fraction between D0 and D5. Cellular structure dismantlement and proteolysis was observed at D21. Effect of group (“tender” vs “tough”) on spot intensities was tested by ANOVA. At D0, higher quantity of proteins of the inner and outer membrane of mitochondria was found in the tender group suggesting a more extensive degradation of mitochondria that may be related to the apoptotic process.

Inflammatory response has recently been shown to induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), which either recovers proper ER function or activates apoptosis. Here we show that endotoxin (lipopolysaccharide = LPS) can lead to functional ER failure tentatively via a mitochondrion-dependent pathway in livers of rats. Histological examination did not reveal significant damage to liver in form of necroses. Electron microscopy displayed transparent rings appearing around morphologically unchanged mitochondria, which were identified as dilated ER. The spliced mRNA variant of X-box protein-1 (XBP1) and also the mRNA of 78 kDa glucose-regulated protein (GRP78) were up-regulated, both typical markers of ER stress. However, GRP78 was down-regulated at the protein level. A pro-apoptotic shift in the bax/bcl-XL mRNA ratio was not accompanied by translocation of apoptosis inducing factor (AIF) to the nucleus, suggesting that the cells entered a pre-apoptotic state, but apoptosis was not executed. Monooxygenase activity of p450, representing the detoxification system in ER, was decreased after administration of endotoxin. Biochemical analysis of proteins important for ER function revealed the impairment of protein folding, transport, and detoxification suggesting functional ER failure. We suggest that functional ER failure may be a reason for organ dysfunction upon excessive inflammatory response mediated by endotoxin.

Using histological analysis of the induction of secondary somatic embryogenesis (SSE) in cassava (Manihot esculenta Crantz) as a guide, we performed 2-DE for protein separation and matrix-assisted laser desorption ionization-time of flight-tandem mass spectrometry (MALDI-TOF-TOF-MS) for protein identification in cotyledons of cassava somatic embryos undergoing SSE. Reference map obtained by 2- DE within a pH range of 3–10 and a size range of 6–97 kDa revealed approximately 410 eletrophoretically resolved spots populated primarily by acidic (pI < 7) proteins with molecular masses between 30 and 75 kDa. Tryptic digestion of 163 of the most abundant spots led to the identification of 86 proteins with a protein identification success rate of 53%. In total, 43% of the identified proteins were involved in metabolism and energy and 11.6% in protein destination and storage. Others are, disease/defense (11.6%), transcription and protein synthesis (7%), signal transduction (5.8%), cell growth/division (3.5%), transporters (3.5%), cell structure (2.3%), secondary metabolism (1.2%) and other functional classes (10.5%). Our studies demonstrate that 2-DE-based proteomic approaches combined with histological studies can serve as tools for identifying protein markers for the developmental stages of cassava SE while providing clues on the underlying causes of the low rate of conversion of cassava somatic embryos into mature plants.

Two-dimensional electrophoresis was used to compare Longissimus sarcoplasmic protein abundance between two groups (tough meat and tender meat), defined on the basis of extreme Warner-Bratzler shear force values measured on cooked pork. Fourteen protein spots differed in quantity (P0.05) between the two groups and were identified. Adypocyte fatty acid binding protein and acyl-CoA binding protein involved in lipid traffic and in the control of gene expression regulating cell proliferation and differentiation, and Enoyl-CoA hydratase, aldose reductase and triosephosphate isomerase indirectly related to lipid metabolism were overrepresented in the tender group. The tender group was further characterized by increased levels of proteins involved in protein folding and polymerization (initiation factor elf-3beta, chaperonin subunit 2, profilin II). The results suggest that the lower post-cooking shear force could at least in part be related to muscle adipogenetic and/or myogenetic status of which the possible underlying mechanisms are discussed.

A glycosylphosphatidylinositol (GPI) anchor is a common but complex C-terminal post-translational modification of extracellular proteins in eukaryotes. Here we investigate the problem of correctly annotating GPI-anchored proteins for the growing number of sequences in public databases. We developed a computational system, called FragAnchor, based on the tandem use of a neural network (NN) and a hidden Markov model (HMM). Firstly, NN selects potential GPI-anchored proteins in a dataset, then HMM parses these potential GPI signals and refines the prediction by qualitative scoring. FragAnchor correctly predicted 91% of all the GPI-anchored proteins annotated in the Swiss-Prot database. In a large-scale analysis of 29 eukaryote proteomes, FragAnchor predicted that the percentage of highly probable GPI-anchored proteins is between 0.21% and 2.01%. The distinctive feature of FragAnchor, compared with other systems, is that it targets only the C-terminus of a protein, making it less sensitive to the background noise found in databases and possible incomplete protein sequences. Moreover, FragAnchor can be used to predict GPI-anchored proteins in all eukaryotes. Finally, by using qualitative scoring, the predictions combine both sensitivity and information content. The predictor is publicly available at http://navet.ics.hawaii.edu/~fraganchor/NNHMM/NNHMM.html.

Lotus (Nelumbo nucifera Gaertn.) seed proteome has been the focus of our studies, and we have recently established the first proteome dataset for its mature seed endosperm. The current study unravels the immature endosperm, as well as the embryo proteome, to provide a comprehensive dataset of the lotus seed proteins and a comparison between the mature and immature endosperm tissues across the seed’s development. One-dimensional gel electrophoresis (SDS-PAGE) linked with tandem mass spectrometry provided a protein inventory of the immature endosperm (122 non-redundant proteins) and embryo (141 non-redundant proteins) tissues. Comparing with the previous mature endosperm dataset (66 non-redundant proteins), a total of 206 non-redundant proteins were identified across all three tissues of the lotus seed. Results revealed some significant differences in proteome composition between the three lotus seed tissues, most notably between the mature endosperm and its immature developmental stage shifting the proteins from nutrient production to nutrient storage.

Rats and guinea pigs have frequently been used to study the development of the prostate and the mechanism of androgen action, but the mouse prostate has also become an attractive model for prostate research, because an enormous range of genetically altered mice is now available. However, the secretion of proteins in the mouse prostate has not yet been thoroughly investigated. In the present study, major secreted proteins from the ventral prostate (VP), dorso-lateral prostate (DLP), and anterior prostate (AP) of mice were identified by means of 2D-gel electrophoresis followed by MALDI-TOF mass spectrometric analysis. A quantitative reverse transcriptase-PCR method was further employed to examine the androgen-dependent transcriptional regulation of the identified proteins. Proteome analysis revealed that the VP secretes spermine-binding protein, serine protease inhibitor Kazal type-3, and a 91 kDa hypothetical scavenger receptor (AK035662). DLP and AP secrete a protein similar to immunoglobulin-binding protein, immunoglobulin-binding protein-like protein, and one of the experimental autoimmune prostatitis antigen proteins (EAPA2). Peroxiredoxin-6, glucose-regulated protein 78, zinc-α2-glycoprotein, and phospholipase Cα are also secreted. Castration of animals led to a decrease in the mRNAs of these secreted proteins, although the extents of changes varied greatlyamong different lobes. We present here an outlined view of mouse prostate secretion, which should contribute to an understanding of the biological functions of the prostate gland, as well as the androgen dependency of prostate secretion.

Two persistent myths, ingrained in the electrophoretic literature of the last thirty years, namely carbamylation and deamidation, have been recently dispelled (Herbert et al., J. Proteome Res. 2002, in press). We report here, for the first time, a noxious and unexpected artefact in proteome analysis: beta-elimination (or desulfuration), which results on the loss of an H(2)S group (34 Da) from cysteine (Cys) residues for protein focusing in the alkaline pH region. With such an elimination event, a dehydro alanine residue is generated at the Cys site. In turn, the presence of a double bond in this position elicits lysis of the peptide bond, generating a number of peptides of fairly large size from an intact protein. The first process seems to be favored by the electric field, probably due to the continuous harvesting of the SH(-) anion produced. The only remedy found to this noxious degradation pathway is the reduction and alkylation of all Cys residues prior to their exposure to the electric field. Alkylation appears to substantially reduce both beta-elimination and the subsequent amido bond lysis.

Extracellular vesicles (EVs) deliver biologically active cargos from donor cells to recipient cells for intercellular communication. Since the existence of RNA cargo was discovered, EVs have been considered to be useful drug-delivery systems. Specifically, EVs from bovine milk (mEV) are one of the most promising platforms, since bovine milk is a scalable source of EVs for mass production. However, it is still difficult to isolate pure EVs from bovine milk owing to the complexity of raw materials. Furthermore, the biocompatibility and immunotoxicity of mEVs are still unclear. In this study, we developed a new method for isolating bovine milk-derived EVs by employing acid treatment and ultracentrifugation. Isolated mEVs are spherical in shape, measure 120 nm in diameter and contain typical EV marker proteins, such as tetraspanins. Compared with the previously reported method, our method can isolate purer mEVs. When mEVs are contacted with the mouse macrophage cell line Raw264.7, mEVs are readily taken up by the cells without a cytotoxic effect, suggesting that mEVs can deliver the cargo molecules into cells. While systemic administration of mEVs into mice resulted in the absence of systemic toxicity, certain types of cytokines were slightly induced. No anaphylaxis effect was observed after serial administration of mEVs in mice. Thus, mEVs isolated using our method are well tolerated in vivo and may be useful for the drug-delivery application.

Das Nijmegen Breakage Syndrom (NBS) gehört als autosomal-rezessive Erkrankung zu den Chromosominstabilitätssyndromen und ist gekennzeichnet durch kraniofaziale Dysmorphie, Minderwuchs, Mikrozephalie, Immundefizienz und einem erhöhten Risiko für die Entwicklung maligner Tumoren. Da die Funktionen des Genprodukts Nibrin noch nicht vollständig geklärt sind, war es Ziel dieser Arbeit, Veränderungen und Modifikationen auf Proteinebene zu untersuchen. Für ein besseres Verständnis wurden hierfür homozygot nullmutante murine Fibroblasten aus einem Knockout-Modell bestrahlt und das Proteom mittels 2D- Gelelektrophorese dargestellt. Mit Hilfe der Maldi TOF Massenspektrometrie wurden die gegenüber heterozygoten Zellen differenziell exprimierten Proteine analysiert und durch die Datenbank identifiziert. Hierbei wurden 35 differenziell exprimierte Proteine mit einem signifikanten Mowse-Score gefunden, von denen 21 verstärkt und 14 vermindert exprimiert wurden. Nach funktionellen Gesichtspunkten gruppiert ergaben sich die Kategorien Energie- Metabolismus, Protein-Metabolismus, Chaperone, Zellzyklus/Proliferation, Signaltransduktion, Transkription/Translation, Zytoskelett und Kollagen. Besonders auffällig ist die hohe Anzahl detektierter Proteine, die den Gruppen der Heat Shock Proteine und der Proteasome angehören. Dies deutet auf deren Beteiligung an der Zellzykluskontrolle und Checkpointkontrolle hin, welche in Nibrindefizienten Zellen gestört sind und somit alternative Mechanismen gefragt sind. Ebenso kann deren verstärkte Aktivität mit der bei NBS-Patienten beobachteten Tumorgenese assoziiert sein. Möglich scheint auch eine Rolle Nibrins im Rahmen der ROS-Regulation und somit ein Fehlen Nibrins relevant für einige Merkmale des Nijmegen Breakage Syndroms. Bemerkenswert ist auch die Identifizierung von PCNA als Reparaturprotein. Auf Grund des Defizits von Nibrin scheint durch verstärkte Expression von PCNA versucht zu werden, die Checkpointregulation und Reparaturmechanismen aufrecht zu erhalten. In Zusammenschau könnte die klinische Variabilität des Krankheitsbildes in der Variation der verschiedenen Proteine und deren Interaktionen untereinander begründet liegen. Je nach Expressions- und Interaktionsmuster der Proteine ergeben sich unterschiedliche Symptome und deren verschieden starke Ausprägungen mit klinisch-praktischer Relevanz. So können Modifikationen bei der Behandlung von heterozygot betroffenen Erkrankten angewendet werden. Auch das individuelle Risiko, an Krebs zu erkranken, sowie ein möglicher Therapieerfolg im Sinne einer individualisierten Medizin können eingeschätzt werden. Darüber hinaus sind die hier detektierten, differenziell exprimierten Proteine interessante Kandidaten in Zusammenhang mit DNA-Reparatur, Checkpointkontrolle und posttranslationalen Modifikationen und für weitere, tiefer gehende Analysen der Funktionen Nibrins., In this thesis, differentially expressed proteins in the autosomal genetic disorder Nijmegen Breakage Syndrome were identified by proteome analysis. NBS patients show growth retardation und microcephaly, a characteristic facial appearance, immunodeficiency and an increased risk of cancer; in addition, NBS patient cells were found to be sensitive to ionizing irradiation und have higher levels of spontaneous chromosomal damage. Over 90 % of all patients are homozygous for a truncating mutation, producing two mutated forms of the NBS1 gene product nibrin. For a better understanding of the role of Nibrin, other proteins were identified whose abundance is altered in the absence of Nibrin. Homozygous null mutant fibroblasts of a murine model were exposed to ionising radiation, separated in a two-dimensional gel electrophoresis and then analyzed by MALDI-TOF mass spectrometry and by searching the Swiss Prot Database. In total, 35 proteins were identified which fall into different functional categories (metabolism, chaperones, cell cycle, signal transduction, transcription/translation, cytoskeleton, collagen). The results confirm Nibrin’s role in cell cycle and checkpoint control as well as tumorigenesis and indicate additional functions of Nibrin. The mechanisms operating in cells to combat DNA damage und mutation are further defined enabling a much more detailed insight into the processes disturbed in patient cells. A more thorough understanding of the chromosome instability syndrome NBS could contribute to further improvements in treatment and, with the introduction of individualised medicine, allow prediction of individual cancer risk und likely response to therapeutic regimes.

Clostridium cellulovorans is an anaerobic, cellulolytic bacterium, capable of effectively degrading various types of soft biomass. Its excellent capacity for degradation results from optimization of the composition of the protein complex (cellulosome) and production of non-cellulosomal proteins according to the type of substrates. In this study, we performed a quantitative proteome analysis to determine changes in the extracellular proteins produced by C. cellulovorans for degradation of several types of natural soft biomass. C. cellulovorans was cultured in media containing bagasse, corn germ, rice straw (natural soft biomass), or cellobiose (control). Using an isobaric tag method and a liquid chromatograph equipped with a long monolithic silica capillary column/mass spectrometer, we identified 372 proteins in the culture supernatant. Of these, we focused on 77 saccharification-related proteins of both cellulosomal and non-cellulosomal origins. Statistical analysis showed that 18 of the proteins were specifically produced during degradation of types of natural soft biomass. Interestingly, the protein Clocel_3197 was found and commonly involved in the degradation of every natural soft biomass studied. This protein may perform functions, in addition to its known metabolic functions, that contribute to effective degradation of natural soft biomass. Electronic supplementary material The online version of this article (doi:10.1186/s13568-014-0089-9) contains supplementary material, which is available to authorized users.

Background Finding the best extraction method of proteins from lysed cells is the key step for detection and identification in all proteomics applications. These are important to complement the knowledge about the mechanisms of interaction between plants and phytopathogens causing major economic losses. To develop an optimized extraction protocol, strains of Acidovorax citrulli, Pectobacterium carotovorum subsp. carotovorum and Ralstonia solanacearum were used as representative cells in the study of phytopathogenic bacteria. This study aims to compare four different protein extraction methods, including: Trizol, Phenol, Centrifugation and Lysis in order to determine which are more suitable for proteomic studies using as parameters the quantity and quality of extracted proteins observed in two-dimensional gels. Results The bacteria studied showed different results among the tested methods. The Lysis method was more efficient for P. carotovorum subsp. carotovorum and R. solanacearum phytobacteria, as well as simple and fast, while for A. citrulli, the Centrifugation method was the best. This evaluation is based on results obtained in polyacrylamide gels that presented a greater abundance of spots and clearer and more consistent strips as detected by two-dimensional gels. Conclusions These results attest to the adequacy of these proteins extraction methods for proteomic studies.