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Many foods including edible oils contain 2-monochloropropane-1,3-diol (2-MCPD), a processing-induced chemical contaminant. Cardiotoxic effects have been shown to result from oral 2-MCPD exposure in rodents, but the underlying mechanisms of action remain poorly understood. We undertook a comprehensive multi-omics approach to assess changes at the transcriptomic, proteomic, and oxylipin levels in heart tissues from male F344 rats that were exposed to 0 or 40 mg/kg BW/day of 2-MCPD in the diet for 90 days, in a regulatory compliant rodent bioassay. Heart tissues were collected for RNA sequencing, quantitative PCR analysis, proteomic analysis via two-dimensional gel electrophoresis and mass spectrometry, and targeted lipidomic profiling by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS). Transcriptomic and proteomic data analyses revealed upregulation of immune/inflammatory response processes and downregulation of energy metabolism and cardiac structure and functions. Among differentially expressed gene–protein pairs, coronin-1A, a key leukocyte-regulating protein, emerged as markedly up-regulated. Oxylipin profiling highlighted a selective suppression of docosahexaenoic acid-derived metabolites, suggesting a disruption in cardioprotective lipid pathways. These findings suggest that 2-MCPD disrupts homeostasis through inflammatory activation and suppression of metabolic and cardiac function. This research provides insights into 2-MCPD's cardiotoxicity, emphasizing the need for further studies to support hazard characterization. [ABSTRACT FROM AUTHOR]

Inhalation of ambient particulate matter (PM) and ozone (O3) has been associated with increased cardiovascular morbidity and mortality. However, the interactive effects of PM and O3 on cardiac dysfunction and disease have not been thoroughly examined, especially at a proteomic level. The purpose of this study was to identify and compare proteome changes in spontaneously hypertensive (SH) rats co-exposed to concentrated ambient particulates (CAPs) and O3, with a focus on investigating inflammatory and metabolic pathways, which are the two major ones implicated in the pathophysiology of cardiac dysfunction. For this, we measured and compared changes in expression status of 9 critical pro- and anti-inflammatory cytokines using multiplexed ELISA and 450 metabolic proteins involved in ATP production, oxidative phosphorylation, cytoskeletal organization, and stress response using two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) in cardiac tissue of SH rats exposed to CAPs alone, O3 alone, and CAPs + O3. Proteomic expression profiling revealed that CAPs alone, O3 alone, and CAPs + O3 differentially altered protein expression patterns, and utilized divergent mechanisms to affect inflammatory and metabolic pathways and responses. Ingenuity Pathway Analysis (IPA) of the proteomic data demonstrated that the metabolic protein network centered by gap junction alpha-1 protein (GJA 1) was interconnected with the inflammatory cytokine network centered by nuclear factor kappa beta (NF-kB) potentially suggesting inflammation-induced alterations in metabolic pathways, or vice versa, collectively contributing to the development of cardiac dysfunction in response to CAPs and O3 exposure. These findings may enhance understanding of the pathophysiology of cardiac dysfunction induced by air pollution and provide testable hypotheses regarding mechanisms of action. [ABSTRACT FROM AUTHOR]

This study investigates the changes in hippocampal proteomic profiles during demyelination and remyelination using the cuprizone model. Employing two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry for protein profiling, we observed significant alterations in the expression of ketimine reductase mu-crystallin (CRYM) and protein disulfide isomerase A3 precursor (PDIA3) following exposure to and subsequent withdrawal from cuprizone. Immunohistochemical staining validated these protein expression patterns in the hippocampus, revealing that both PDIA3 and CRYM were downregulated in the hippocampal CA1 region during demyelination and upregulated during remyelination. Additionally, we explored the potential protective effects of CRYM and PDIA3 against cuprizone-induced demyelination by synthesizing cell-permeable Tat peptide-fusion proteins (Tat-CRYM and Tat-PDIA3) to facilitate their crossing through the blood–brain barrier. Our results indicated that administering Tat-CRYM and Tat-PDIA3 mitigated the reduction in proliferating cell and differentiated neuroblast counts compared to the group receiving cuprizone alone. Notably, Tat-PDIA3 demonstrated significant effects in enhancing myelin basic protein expression alongside phosphorylation of CREB in the hippocampus, suggesting its potential therapeutic role in the prevention or treatment of demyelination, and by extension, in conditions such as multiple sclerosis. [ABSTRACT FROM AUTHOR]

The polyketide antibiotic 2,4-diacetylphloroglucinol (DAPG) is a phenolic secondary metabolite produced by Pseudomonas spp. that serves as a biocontrol agent. DAPG is known to inhibit root growth and seed germination in plants. However, the effects of DAPG on leaf growth have not been elucidated yet. In this study, we found that applying DAPG at concentration above 30 µM suppresses the development of Arabidopsis leaves. Using an integrated proteomic analysis approach, we investigated the physio-molecular responses of Arabidopsis leaves under 10–100 µM DAPG treatment. Two-dimensional electrophoresis (2-DE)-based proteomic analysis identified 46 differentially expressed spots with at least a twofold change, corresponding to 37 proteins involved in stress and defense, metabolism, photosynthesis, and carbohydrate metabolism processes. Furthermore, we found that the expressions of stress and defense-related proteins were upregulated in response to DAPG, while those of photosynthesis-related proteins were downregulated. Additionally, RT-PCR result showed that the identified DAPG-responsive proteins in the proteomic analysis were similarly regulated at the transcriptional level. Collectively, our findings provide insights into the molecular mechanism of the response to DAPG in Arabidopsis leaves. [ABSTRACT FROM AUTHOR]

Plants initiate specific defense responses by recognizing conserved epitope peptides within the flagellin proteins derived from bacteria. Proteolytic cleavage of epitope peptides from flagellin by plant apoplastic proteases is thought to be crucial for the perception of the epitope by the plant receptor. However, the identity of the plant proteases involved in this process remains unknown. Here, we establish an efficient identification system for the target proteases in Arabidopsis apoplastic fluid; the method employs native two-dimensional electrophoresis followed by an in-gel proteolytic assay using a fluorescence-quenching peptide substrate. We designed a substrate to specifically detect proteolytic activity at the C-terminus of the flg22 epitope in flagellin and identified two plant subtilases, SBT5.2 and SBT1.7, as specific proteases responsible for the C-terminal cleavage of flg22. In the apoplastic fluid of Arabidopsis mutant plants deficient in these two proteases, we observe a decrease in the C-terminal cleavage of the flg22 domain from flagellin, leading to a decrease in the efficiency of flg22 epitope liberation. Consequently, defensive reactive oxygen species (ROS) production is delayed in sbt5.2 sbt1.7 double-mutant leaf disks compared to wild type following flagellin exposure. Plants initiate specific defense responses by recognizing flg22 peptide epitope derived from flagellin, the major structural protein of the bacterial flagellum. Here, the authors identified two plant subtilases, SBT5.2 and SBT1.7, as specific proteases responsible for cleaving the peptide epitope. [ABSTRACT FROM AUTHOR]

Purpose: Visceral leishmaniasis (VL) is a systemic and parasitic disease that is usually fatal if left untreated. VL is endemic in different parts of Iran and is caused mainly by Leishmania infantum. This study aimed to recognition immunoreactive proteins in amastigote-like and promastigote stages of L. infantum (Iranian strain) by antibodies present in the sera of VL patients. Methods: Total protein extract from amastigote-like and promastigote cells was separated by two-dimensional electrophoresis (2DE). To detect the immunoreactive proteins, 2DE immunoblotting method was performed using different pools of VL patients' sera. Results: Approximately 390 and 430 protein spots could be separated in 2DE profiles of L. infantum amastigote-like and promastigote stages, respectively. In immunoblotting method, approximately 295 and 135 immunoreactive proteins of amastigotes-like reacted with high antibody titer serum pool and low antibody titer serum pool, respectively. Approximately 120 and 85 immunoreactive proteins of promastigote extract were recognized using the high antibody titer sera pool and low antibody titer sera, respectively. Conclusion: The present study has recognized a number of antigenic diversity proteins based on the molecular weight and pH in amastigote-like and promastigote stages of L. infantum. These results provide us a new concept for further analysis development in the field of diagnosis biomarkers and vaccine targets. [ABSTRACT FROM AUTHOR]

Phaeocystis globosa is an important unicellular eukaryotic alga that can also form colonies. P. globosa can cause massive harmful algal blooms and plays an important role in the global carbon or sulfur cycling. Thus far, the ecophysiology of P. globosa has been investigated by numerous studies. However, the proteomic response of P. globosa to nitrogen depletion remains largely unknown. We compared four protein preparation methods of P. globosa for two-dimensional electrophoresis (2-DE) (Urea/Triton X-100 with trichloroacetic acid (TCA)/acetone precipitation; TCA/acetone precipitation; Radio Immuno Precipitation Assay (RIPA) with TCA/acetone precipitation; and Tris buffer). Results show that the combination of RIPA with TCA/acetone precipitation had a clear gel background and showed the best protein spot separation effect, based on which the proteomic response to nitrogen depletion was studied using 2-DE. In addition, we identified six differentially expressed proteins whose relative abundance increased or decreased more than 1.5-fold (P<0.05). Most proteins could not be identified, which might be attributed to the lack of genomic sequences of P. globosa. Under nitrogen limitation, replication protein-like, RNA ligase, and sn-glycerol-3-phosphate dehydrogenase were reduced, which may decrease the DNA replication level and ATP production in P. globosa cells. The increase of endonuclease III and transcriptional regulator enzyme may affect the metabolic and antioxidant function of P. globosa cells and induce cell apoptosis. These findings provide a basis for further proteomic study of P. globosa and the optimization of protein preparation methods of marine microalgae. [ABSTRACT FROM AUTHOR]

Epilepsy is one of the most common neurological diseases and affects 1–2% of the world's population. Epilepsy is associated with recurrent and unpredictable seizures. Since 20% of epileptic patients suffer from drug resistance, much research is being done to find new ways to treat epilepsy. One of the new therapies is low-frequency deep brain stimulation (L-DBS). Although this method has an anticonvulsant effect and is used as a new method for the treatment of drug-resistant epilepsy, the mechanism of its action is still unclear. Understanding the changes in the amount of expression of proteins and enzymes in the nervous system following the induction of L-DBS is helpful for understanding the mechanism of effects of L-DBS. In this study, rats were first kindled (Common laboratory model of seizures and epilepsy) by electrical kindling (kindled group) and then received the L-DBS (kindled + L-DBS group). Comparison and analysis of the proteome of kindled and L-DBS-treated rats were performed by comparative proteomics using two-dimensional gel electrophoresis. We observed that electrical kindling induced changes in a series of proteins, that normalized by L-DBS. We identified 23 of these proteins as a sample by MALDI TOF/TOF. The protein–protein interaction network was also drawn for these 23 proteins. The most important pathway attributed to these proteins is the cytoskeleton pathway. Our results showed that the cytoskeleton after destruction by electrical kindling was restored to the normal by L-DBS. [ABSTRACT FROM AUTHOR]

GM risk assessments are crucial for determination and prevention of potential adverse effects through early detection and proper evaluation of intended and potential unintended changes in molecular breeding. In this context, the concept of 'substantial equivalence' has been suggested for the safety tests of GM products for many years. This study evaluated differences between four types of Glycine max crops; transgenic soybean, its non-transgenic counterpart, gamma induced soybean mutant and its parental line, at proteomic level in context of substantial equivalence. The results revealed the ratio of differentially expressed protein spots to total protein spots in mutants compared to their parental line was 50.7%. This ratio was 41.2% in transgenic plants compared to their counterparts. Scatter plot analysis presented those mutant plants showed a wider spread than transgenic plants in terms of distribution of proteins. It was determined that up-regulated proteins in mutant plants have various biological roles in processes such as development, stress response, photorespiration and ribosomal subunit assembly. In transgenic plants, upregulated proteins were shown to have diverse biological roles in processes such as ribosomal subunit assembly, electron transport, amino acid and nucleic acid metabolism and cell wall biogenesis. Although debate on the potential unintended effects focuses on GM organisms, our results point out that the plants that gained new characteristics by various breeding methods should be characterized in all aspects case-by-case. [ABSTRACT FROM AUTHOR]

The objectives of the study are to identify bioactive peptides from goat colostrum and use in-silico analysis to describe their physicochemical properties. Total protein fractions in colostrum (1–3 days), transitional (4–20 days), and mature (after 25 days) goat milk samples were quantified in the biochemical studies. Colostrum (27.26 mg/ml), transitional (12.01 mg/ml), and mature (6.56 mg/ml) protein concentrations were measured. In comparison to the transitional and mature stages of goat milk, the results showed that protein fractions in the colostrum stage have the highest protein concentration. The fat, protein, lactose, total solids, and immunoglobulin percentages in goat colostrum are also high. In 2D electrophoresis, a total of 81 protein spots were observed between 6 and 72 kDa marker regions. Out of these 81 protein spots, about nine prominent quantitative protein spots numbered 12, 23, 25, 30, 34, 60, 75, 77 and 79 were analyzed through MALDI-TOF/MS and they were identified as K casein, Alpha Lactalbumin, Ferritin, Calmodulin dependent protein kinase, Prolactin, Lactoferrin, Lactoperoxidase, Lysozyme and Alpha-S2-casein proteins respectively. The molecular weights of K casein, Lactoferrin, Lactoperoxidase, and Alpha-S2-casein proteins were determined using in silico physicochemical characterisation to be 69564.89 kDa, 21455.34 kDa, 80365.85 kDa, and 26388.09 kDa, respectively. These proteins contain a higher concentration of alpha helices, extended strands, beta twists, and random coils, according to their secondary structure. The findings of this study will aid future research into the isolation of bioactive peptides and their biological roles. [ABSTRACT FROM AUTHOR]

In allergic individuals, ingestion of wheat can lead to wheat-dependent exercise-induced anaphylaxis (WDEIA). Many studies have been conducted to find WDEIA allergen–deficient wheat, including by generating omega-5 gliadin antibodies. However, the reported antibodies have not been specific enough to detect omega-5 gliadins encoded on the 1B chromosome. In this study, we generated monoclonal antibodies against the major allergens causing WDEIA, omega-5 gliadins. Using these antibodies (mono-O5B-1C10), we assessed accumulation of omega-5 gliadins in wild-type and nullisomic-tetrasomic (NT) lines of the wheat (Triticum aestivum) varieties Chinese Spring (CS) by one- and two-dimensional gel electrophoresis, followed by Coomassie blue staining or immunoblotting with mono-O5B-1C10. We also tested mono-O5B-1C10 for major omega-5 gliadins in various wheat germplasms. Our results thus demonstrate the specificity of mono-O5B-1C10 for major omega-5 gliadins and potentially useful for identifying of omega-5 gliadin–deficient wheat varieties that should not cause WDEIA. [ABSTRACT FROM AUTHOR]

Microgravity can cause abnormalities and even damage to the cardiovascular system. We investigated if differences exist in the expression level of proteins in myocardial tissues after exposure to simulated microgravity, this could allow to explore microgravity impact on the cardiovascular system. Tail-suspended rats were established as a microgravity model and myocardial tissues were isolated for further identification by 2-dimensional gel electrophoresis (2-DE) combined with mass spectrometry. 864 proteins in myocardial tissues were separated and 15 proteins were identified to be differentially expressed in simulated microgravity model group, including that nine proteins were significantly down-regulated, two were significantly up-regulated, and four barely expressed. The nine down-regulated proteins were further identified as superoxide dismutase (SOD), lactate dehydrogenase (LDH), methylbutenoyl-co-carboxylase, pyruvate kinase 3 or β-fibrinogen, enolase 3, calcineurin (CaN), and two unknown proteins. Particularly, expression of CaN was determined to be down-regulated through Western blotting analysis, which could potentially result in the cardiovascular disorders. Thus, down-regulation of CaN may lead to cardiovascular dysfunction under microgravity. Therefore, improving understanding of the underlying mechanisms of this could provide insights into cardiovascular disorders and potential targets for treatment of cardiovascular diseases under microgravity. [ABSTRACT FROM AUTHOR]

Metalloproteomics is an innovative methodology for identifying of protein-associated mercury. Thus, we analyzed the muscle proteome of Arapaima gigas (pirarucu), collected in the Madeira River of the Brazilian Amazon, to identify protein-associated mercury, with the aim of identifying possible mercury biomarkers in fish muscle tissue. After obtaining the protein pellet, we conducted two-dimensional electrophoresis (2D PAGE) to fractionate the muscle proteome. Total mercury in muscle tissue and protein pellets and mapping of mercury content in protein spots of the 2D PAGE gels was determined using graphite furnace atomic absorption spectrometry (GFAAS). The protein-associated mercury identification was performed using liquid chromatography coupled with sequence mass spectrometry (LC‒MS/MS). Total mercury determinations by GFAAS indicated concentrations on the order of 153 ± 1.90 mg kg−1 and 142 ± 1.50 mg kg−1 (total precipitation of protein fraction) and 139 ± 1.45 mg kg−1 (fractional precipitation of protein fraction) in muscle tissue and protein pellets, respectively. Mercury concentrations in the range of 48 ± 0.90 to 165 ± 3.00 mg kg−1 were found in twelve protein spots. Among the 2D PAGE protein spots, eleven Hg-binding proteins were identified using LC‒MS/MS, which showed characteristics of mercury exposure biomarkers for important metabolic functions, such as five parvalbumin isoforms, triosephosphate isomerase, cofilin 2 (muscle), and fructose-bisphosphate aldolases. [ABSTRACT FROM AUTHOR]

The present study was aimed to investigate the effect of docosanol on the protein expression profile of methicillin-resistant Staphylococcus aureus (MRSA). Thus, two-dimensional gel electrophoresis coupled with MALDI-TOF MS technique was utilized to identify the differentially regulated proteins in the presence of docosanol. A total of 947 protein spots were identified from the intracellular proteome of both control and docosanol treated samples among which 40 spots were differentially regulated with a fold change greater than 1.0. Prominently, the thiol-dependent antioxidant system and stress response proteins are downregulated in MRSA, which are critical for survival during oxidative stress. In particular, docosanol downregulated the expression of Tpx, AhpC, BshC, BrxA, and YceI with a fold change of 1.4 (p = 0.02), 1.4 (p = 0.01), 1.6 (p = 0.002), 4.9 (p = 0.02), and 1.4 (p = 0.02), respectively. In addition, docosanol reduced the expression of proteins involved in purine metabolic pathways, biofilm growth cycle, and virulence factor production. Altogether, these findings suggest that docosanol could efficiently target the antioxidant pathway by reducing the expression of bacillithiol and stress-associated proteins. [ABSTRACT FROM AUTHOR]

Reference and commercial samples of meat products (boiled and canned) were analyzed via classical, two-dimensional electrophoresis according to O'Farrell. Ten different protein products or their fragments were identified in the final stage of the procedure. They turned to be components of soy, milk, egg, pumpkin, and sunflower. They can be used as the targets of undeclared additives to develop faster and more sensitive quality control methods. [ABSTRACT FROM AUTHOR]

Hli proteins of the multicellular cyanobacterium Arthrospira platensis have been studied in this work. According to the NCBI database, three Hli genes found in the A. platensis genome encode proteins 47, 64, and 69 amino acid (aa). A. platensis cells were incubated under light stress (500 µmol photons/m2 s, 1 h). The association of Hli proteins with pigment–protein complexes of thylakoid membranes was then studied by two-dimensional electrophoresis and subsequent mass spectrometry. Only Hli 47 aa was detected in the composition of pigment–protein complexes by matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectrometry. The identified Hli47 protein was shown to be associated with photosystem II and a homolog of the HliC protein of Synechocystis sp. Bioinformatic analysis has shown that the amino acid sequence of the identified protein shows a higher homology with proteins of multicellular cyanobacteria and a lower of homology with the amino acid sequence of Hli proteins of unicellular cyanobacteria. [ABSTRACT FROM AUTHOR]

Takayasu arteritis (TAK) is an autoimmune systemic arteritis of unknown etiology. Although a number of investigators have attempted to determine biomarkers for diagnosing TAK, there exist no specific serological markers of this intractable disease. We undertook the exploration of novel serological markers which could be useful for an accurate diagnosis of TAK using an unbiased proteomics approach. The purified plasma samples from untreated patients with TAK and healthy individuals were separated by two-dimensional electrophoresis. The differentially expressed protein spots were detected by gel comparison and identified using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MS). Next, we validated plasma concentrations of identified proteins by enzyme-linked immunosorbent assay (ELISA). Two-dimensional electrophoresis and numerical analysis revealed 19 spots and 3 spot clusters whose sum of the sample averages was ≥ 0.01, and the average concentrations were ≥ 1.5 times in the patient group compared with the control group. Among them, 10 spots and spot clusters that met the condition of the average spot concentration being 2.5 times more than that in the control group were selected. After processing these spots using MS and conducting MS/MS ion search, we identified 10 proteins: apolipoprotein C-2 (ApoC-2), actin, apolipoprotein A-1, complement C3, kininogen-1, vitronectin, α2-macroglobulin, 14–3–3 protein ζ/δ, complement C4, and inter-α-trypsin inhibitor heavy chain H4 isoform 1 precursor. Finally, ELISA demonstrated that plasma ApoC-2 level was significantly elevated in patients with TAK compared with that in healthy individuals. Thus, ApoC-2 would be a promising candidate biomarker for TAK diagnosis. [ABSTRACT FROM AUTHOR]

Leishmania braziliensis is responsible for most cases of human tegumentary leishmaniasis (HTL) and has caused a wide range of clinical manifestations, including cutaneous (CL) and mucosal leishmaniasis (ML). The diagnosis is based on criteria that consider epidemiological data, clinical findings, and laboratory tests and is hard to establish. For laboratory tests, none of the assays available can be considered gold standards for disease detection. In addition, the Montenegro skin test, essential to supporting infectologists in the clinical management of the disease, is no longer available in Brazil. Thus, the aim of this study was to develop new targets to be used in diagnostic tests for HTL. In the first step, we carried out two-dimensional gel electrophoresis, followed by mass spectrometry, combined with heat map analysis and immunoproteomics approach, and disclosed eight proteins expressed in the amastigote stage specifically recognized by serum from CL and ML patients. A chimeric protein was designed based on the combination of thirteen linear B-cell epitopes, identified by immunoinformatics analysis, from L. braziliensis proteins. Our results showed that the strategy used in this work was successful in developing an antigen to be used in immunological assays (100.0% sensitivity and specificity) in the detection of HTL cases and in comparison with results obtained from an ELISA using soluble L. braziliensis antigen (SLb-Antigen) and immunofluorescence assay (Bio-Manguinhos/FIOCRUZ). The present technology opens the door for its use in field exams by means of an immunochromatographic test, which will be even more helpful in regions without laboratory structures. Key points • Rational strategy to develop antigens. • Integration between immunoproteomic and immunoinformatics analysis. • Chimeric protein shows high performance in HTL diagnosis. [ABSTRACT FROM AUTHOR]

All bacteria can survive and adapt to different stresses, such as fluctuations in temperature, pH oxidative, and osmotic pressure occurring in their surrounding environments. This study aims to evaluate the effects of a variety of stress conditions on the growth, and proteome of Raoultella planticola PTCC 1598. R. planticola cells were exposed to different values of temperatures, sodium chloride, pH, and hydrogen peroxide stresses. Among the stress conditions, oxidative stress, upon exposure to hydrogen peroxide (H2O2) at 4000 ppm concentration was selected for proteomics analysis in detail. Approximately, 1400 spots were identified in two-dimensional gel electrophoresis (2-DE). Among the identified spots, 85 spots were repeatable using 2D-Platinum software and eye confirmation and, nine protein spots were differentially expressed. Among nine proteins, six proteins identified successfully with an MASCOT score greater than 40 (p < 0.05) were 2,3-dihydroxybenzoate-2,3-dehydrogenase (oxidoreductase family), hypothetical protein G787-04832, periplasmic d-galactose-binding protein, uridine phosphorylase (glycosyltransferases), a single peptide match to cysteine-binding periplasmic protein, and NADP(H) nitroreductase. All identified proteins showed decreased level expression. Based on the obtained results, we concluded that hydrogen peroxide as an antiseptic compound could affect cell growth and proteomics of R. planticola. Therefore, we recommend using an antiseptic solution containing H2O2 to prevent the spread of R. planticola as a new emerging pathogen. [ABSTRACT FROM AUTHOR]

Arsenic (As) is an increasing threat across the globe, widely known as a non-threshold carcinogen, and it is reaching harmful values in several areas of the world. In this study, the effect of plant growth promoting bacteria (Microbacterium foliorum) on inorganic arsenic (Arsenate) phytoremediation by Melastoma malabathricum plants was investigated through histological analysis and proteome profiling of the M. malabathricum plants. Two-dimensional gel electrophoresis and transmission electron microscopy were used to conduct the proteome and histological analysis. When arsenic-treated cells were compared to untreated cells, substantial changes were found (1) severely altered the morphology of the cells, intensely disturbed; (2) the cell wall was thicker; (3) drastically changed the cytoplasm, the cells were polygonal in shape, different in size (scattered), and relatively dense. Compared to the control group, the ultra-structure of the root cells of the control group revealed intact cytoplasm, vacuole, and cell wall under exposure to As + bacteria that had a minor effect on the cell form. To further understand As + bacteria interaction, proteome profiling of the root cell was analyzed. The As-induced oxidative stress enrichment was confirmed by the up-regulation of tubulin, nucleoside diphosphate kinase, and major allergen during As + bacteria exposure It was observed that the profusion of proteins involved in defence, protein biogenesis, signaling, photosynthesis, nucleoside and energy metabolism was greater in As + bacteria as compared to the rooting out of As only. Overall, it can be obviously seen that the current study demonstrates the effectiveness of phytoremediation by M. foliorum on proteins involved and responsive pathways in dealing with As toxicity in M. malabathricum plant. [ABSTRACT FROM AUTHOR]

Agricultural productivity is threatened by increasing incidence of drought and the drought tolerant cyanobacteria offer a better solution in the restoration of soil fertility and productivity. The present study describes the comparative physiological response of the cyanobacterium Anabaena sp. acclimated and un-acclimated to desiccation stress induced by polyethylene glycol (10% PEG). While, the acclimated cyanobacterial cells grew luxuriantly with optimal chlorophyll content, photosynthetic activities and nitrogen fixation, the un-acclimated cells exhibited reduced growth rate, chlorophyll content, photosynthetic activities and nitrogen fixation. Distinct differences in the accumulation of lipid peroxidation products, proline and activity of superoxide dismutase were observed under identical growth conditions in the acclimated and un-acclimated cells. Desiccation-acclimated and un-acclimated cyanobacteria showed significant alterations in the abundance of important proteins in the proteome. Two-dimensional gel electrophoresis followed by MALDI-TOF–MS/MS analysis identified twelve proteins. The acclimated cells showed the up regulation of proteins such as Rubisco, fructose-bis-phosphate aldolase, fructose 1–6 bisphosphatase, phosphoglycerate dehydrogenase and elongation factors Tu and Ts as compared to un-acclimated cells. Therefore, the ability to maintain photosynthesis, antioxidants and increased accumulation of proteins related to energy metabolism helped the acclimated cyanobacterium Anabaena sp. to grow optimally under desiccation stress conditions. [ABSTRACT FROM AUTHOR]

Objectives: To reveal the potential mechanism and key determinants that contributed to the improved pectinase activity in Aspergillus niger mutant EIMU2, which was previously obtained by UV-mutagenesis from the wild-type A. niger EIM-6. Results: Proteomic analysis for Aspergillus niger EIMU2 by two-dimensional electrophoresis demonstrated that mutant EIMU2 harbored a multiple enzyme system for the degradation of pectin, mainly constituting by main-chain-cleaving enzymes polygalacturonase, pectate lyase, pectinesterase, and some accessory enzymes rhamnogalacturonan lyase and arabinofuranosidase. Further quantitatively differential proteomic analysis revealed that the quantities of four proteins, pectinesterase, rhamnogalacturonan lyase A, DNA-directed RNA polymerase A, and a hypothetical protein in strain EIMU2 were much higher than those in EIM-6. PCR amplification, sequencing and alignment analysis of genes for the two main members of pectin-degrading enzymes, pectate lyase and polygalacturonase showed that their sequences were completely consistent in A. niger EIM-6 and mutant EIMU2. Conclusions: The result demonstrated that the improved pectinase activity by UV-mutagenesis in A. niger EIMU2 was probably contributed to the up-regulated expression of rhamnogalacturonan lyase, or pectinesterase, which resulted in the optimization of synergy amongst different components of pectin-degrading enzymes. [ABSTRACT FROM AUTHOR]

Aims: Understanding the molecular responses of plant roots to the challenging environment contributes to engineering plants with improved stress tolerance. However, little has been done to understand rice (Oryza sativa L.) roots response to the toxic concentration of zinc (Zn) at the proteomic level. This study explored proteomic responses of young rice roots 5–6 days after sowing to 765 μM of Zn in a hydroponic set-up after 4-day exposure. Methods: Dye staining method and spectrophotometry were chosen for physiological response investigations. ICP-MS was used to determine metal content in rice seedlings. Two-dimensional gel electrophoresis was used for the proteomic study of root tissue. Results: Elevated Zn reduced Mg translocation to the shoots and Mn uptake, decreased plant growth, and increased cell death of roots. Zn stimulated the biosynthesis of glutathione but decreased ROS and malondialdehyde levels. In total, 42 Zn-responsive proteins were identified. Proteins involved in redox regulation, defense response, sulfur metabolism, and proteolysis were induced, while those of energy production and cell wall biogenesis were decreased. Conclusion: Roots of O. sativa seedlings could tolerate certain Zn excess (765 μM Zn for 4 days) by stimulating nutrient recycling and glycolysis, and production of defensive metabolites and proteins to maintain ion and redox homeostasis. Besides, adenosylhomocysteinase (AHCY) down-regulation may contribute to balanced transmethylations, and methionine salvage pathway appears important for the adaptation to Zn. Our results provide some new insight into a complex metabolic response of rice roots to Zn stress, which is related to both stress and defense mechanisms. [ABSTRACT FROM AUTHOR]

Background: Bile acids have been implicated in the mechanism by which Roux-en-Y gastric bypass (RYGB) can induce remission of type 2 diabetes (T2D). Our goal was to identify circulating proteins whose levels changed after RYGB when dysglycemic parameters normalized. Materials and Methods: This was a retrospective study of 26 participants who underwent RYGB. Blood proteins were identified using two-dimensional electrophoresis and mass spectroscopy. Complement proteins were measured using immunoassays and bile acids measured using ultra-high-performance liquid chromatography and mass spectroscopy. Results: A total of 7/452 blood proteins were found to change 2 days after RYGB. Complement component 3 (C3) was selected because of its regulation by bile acids and the glucoregulatory function of its proteolytically processed product C3adesArg or acylation-stimulating protein (ASP). The median (inter-quartile range/IQR) C3 level was 47.4 (34.5, 65.9) mg/dL before surgery decreasing to 40.9 (13.4, 64.1) mg/dL within 2 days after surgery (p = 0.0292). The median (IQR) ASP level increased from 2.8 (0.9, 7.3) nM before surgery to 8.0 (5.3, 14.1) nM within 2 days after surgery (p = 0.0016). ASP levels increased in 14/17 (82%) with T2D remission and in 6/6 with normoglycemia but decreased in 3/3 with persistent T2D. Of ten bile acids measured, the levels of ursodeoxycholic acid (UDCA) were significantly decreased after RYGB and the levels of taurodeoxycholic acid (TDCA) were significantly decreased with T2D remission. Conclusions: These data further support an association of C3 with glucose metabolism and implicate bile acids and ASP in the early remittive effects of RYGB on T2D. [ABSTRACT FROM AUTHOR]

Natural rubber or latex from the Hevea brasiliensis is an important commodity in various economic sectors in today's modern society. Proteins have been detected in latex since the early twentieth century, and they are known to regulate various biological pathways within the H. brasiliensis trees such as the natural rubber biosynthesis, defence against pathogens, wound healing, and stress tolerance. However, the exact mechanisms of the pathways are still not clear. Proteomic analyses on latex have found various proteins and revealed how they fit into the mechanisms of the biological pathways. In the past three decades, there has been rapid latex protein identification due to the improvement of latex protein extraction methods, as well as the emergence of two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). In this manuscript, we reviewed the methods of latex protein extraction that keeps on improving over the past three decades as well as the results of numerous latex protein identification and quantitation. [ABSTRACT FROM AUTHOR]

Key message: A comparative proteomic study between WT and SAR-compromised rsi1/fld mutant reveals a set of proteins having possible roles in the SAR development. A partly infected plant shows enhanced resistance during subsequent infection through the development of systemic acquired resistance (SAR). Mobile signals generated at the site of primary infection travel across the plant for the activation of SAR. These mobile signals are likely to cause changes in the expression of a set of proteins in the distal tissue, which contributes to the SAR development. However, SAR-specific proteome is not revealed for any plant. The reduced systemic immunity 1 (rsi1)/(allelic to flowering locus D; fld) mutant of Arabidopsis is compromised for SAR but shows normal local resistance. Here we report the SAR-specific proteome of Arabidopsis by comparing differentially abundant proteins (DAPs) between WT and fld mutant. Plants were either mock-treated or SAR-induced by primary pathogen inoculation. For proteomic analysis, samples were collected from the systemic tissues before and after the secondary inoculation. Protein identification was carried out by using two-dimensional gel electrophoresis (2-DE) followed by tandem mass spectrometry. Our work identified a total of 94 DAPs between mock and pathogen treatment in WT and fld mutant. The DAPs were categorized into different functional groups along with their subcellular localization. The majority of DAPs are involved in metabolic processes and stress response. Among the subcellular compartments, plastids contained the highest number of DAPs, suggesting the importance of plastidic proteins in SAR activation. The findings of this study would provide resources to engineer efficient SAR activation traits in Arabidopsis and other plants. [ABSTRACT FROM AUTHOR]

Kidney failure is one of the most important challenges in medicine. In this study, we used HEK-293 kidney cells to evaluate and compare changes in the expression of natural antibodies in patients with kidney failure and healthy controls. We analyzed the immunoreactivity of two groups of pooled sera from patient and control groups against antigens derived from HEK-293 cell lysates. The cell lysates were immunoblotted with the pooled sera from the groups. The results showed that the patients' sera contained IgM antibodies, which recognized enolase 1 proteins. [ABSTRACT FROM AUTHOR]

This study describes the optimization of a culture medium to improve protease production by Janthinobacterium sp. using response surface methodology (RSM) and comparative proteomic analysis. Using a one-variable-at-a-time approach, dextrose, Triton X-100, peptone, and pH were identified as significant factors affecting protease production. The optimized medium resulted in a 6.25-fold increase in protease activity when compared to that in the original medium. To investigate the metabolic changes in the optimized medium, extracellular and intracellular proteins were analyzed by two-dimensional gel electrophoresis. Some proteins essential to cell viability were highly expressed in the optimized medium and the outer membrane protein A was secreted at a higher level. These results can be useful for the industrial application of psychrophiles that produce cold-adapted enzymes. [ABSTRACT FROM AUTHOR]

Background: Sago palm (Metroxylon sagu) is a versatile crop and has been hailed as the next viable commodity in Sarawak, Malaysia. Sago palm can thrive in the harsh swampy peat, from low flooded areas to uplands and in acidic to neutral soils. Sago palm has been neglected and unfortunately, very little is known about the sago palm proteome. This study aimed to determine the best protein extraction method of Metroxylon sagu for the two-dimensional gel electrophoresis (2-DE) and its comparative analysis. Results: To perform good proteome research, the most critical step is to establish a method that gives the best quality of extracted total proteins. Five different protein extraction protocols: polyethylene glycol (PEG) fractionation method, phenol extraction method, TCA–acetone method, the combination of phenol and TCA–acetone extraction method and imidazole method were compared to develop an optimized protein extraction method for two-dimensional gel electrophoresis analysis of Metroxylon sagu. The PEG fractionation method was found to give the most reproducible gels with the highest number of spots and highest protein concentration followed by phenol extraction method. The lowest number of spots was observed in the imidazole method. The PEG fractionation method provides improved resolution and reproducibility of 2-DE and reduces the time required to analyze samples. Partitioning Rubisco by polyethylene glycol (PEG) fractionation provides clearer detection of low abundance protein. Hence, the results from this study propose PEG fractionation as the effective protein extraction method for 2-DE proteomic studies of Metroxylon sagu. Conclusion: In this study, the PEG fractionation method is considered as the best extraction method for 2-DE proteomic studies of Metroxylon sagu in terms of yield, gel quality, spot numbers, and quantities of proteins. [ABSTRACT FROM AUTHOR]

Clubroot disease, caused by Plasmodiophora brassicae, is one of the most devastating diseases affecting members of the Brassicaceae family. It is difficult to control by chemical or cultural means, and the molecular mechanisms underlying interactions with Brassica oleracea (cabbage) remain poorly understood. Herein, we used a proteomic approach to investigate B. oleracea–P. brassicae interactions during the early phases of infection in above-ground tissues. Proteins were isolated from the aerial parts of clubroot-susceptible (CT-18) and -resistant (YCR) cabbage cultivars at 5 days after inoculation with P. brassicae or buffer (mock) and resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. A total of 24 differentially modulated proteins were identified in at least two biological replicates, and exhibited altered expression between mock and P. brassicae treatments and/or in the different cabbage cultivars. Most of the identified proteins are involved in oxidative stress, abscisic acid (ABA) metabolism, glucose-mediated signalling and responses to stimuli. Resistant YCR plants harboured an increased abundance of ABA-responsive protein, fructose-bisphosphate aldolase and glucose sensor interaction protein compared with CT-18 plants in both mock and P. brassicae-treated samples, suggesting that they may mediate basal defences against P. brassicae infection in YCR. Specifically, we observed that susceptible (CT-18) plants expressed higher levels of cobalamin-independent methionine synthase than YCR, which may enhance susceptibility of the host. Further investigation of the identified proteins will likely facilitate the identification of key molecular determinants, potentially improving clubroot disease resistance in future cabbage crop species. [ABSTRACT FROM AUTHOR]

University of California at Berkeley I or UCB-1 pistachio rootstock is propagated from the cross between Pistacia integerrima male × Pistacia atlantica female. So far, no report has been presented on the proteomic profile of Pistacia genus. In this research, 7-month-old UCB-1 rootstocks that were produced by tissue culture method and grown in pots containing 1/3 perlite, 1/3 clay and 1/3 sand were exposed to the three different concentrations of NaCl including 0, 100 and 200 mM for 30 days in the controlled conditions in the greenhouse. In the first step, under these three different concentrations of NaCl, the content of malondialdehyde and activities of guaiacol peroxidase, superoxide dismutase, catalase and peroxidase were evaluated. Malondialdehyde content increased up to 100 mM NaCl and then decreased. Activities of guaiacol peroxidase, superoxide dismutase and catalase increased with increasing concentration of NaCl, while peroxidase activity reduced. In the second step, 0 and 100 mM NaCl were selected to evaluate changes in the proteomic profile of this rootstock using MALDI-TOF/TOF method. In this study, ribonucleoside-diphosphate reductase small chain, polcalcin Phl p 7-like and golgin subfamily A member 5 were identified for the first time in response to salinity stress and have not been previously reported to be involved in the response of plant under abiotic stresses. Moreover, in this study, five unknown proteins were identified in UCB-1 pistachio under salinity stress. [ABSTRACT FROM AUTHOR]

Background and aims: Avicennia marina is a pioneer mangrove species widely distributed along the southeast coast of China. It suffers, especially in the seedling stage, from the tidal flooding and anaerobic surroundings as it closes to the seaward side. The aim of this study is to assess the ability of A. marina seedlings to withstand different tidal inundations by physiological and proteomic approaches. Methods: To accomplish this, the artificial tidal inundations were mimicked as 0, 4, and 8 h per tidal cycle about 1 week. The physiological approaches and two-dimensional electrophoresis coupled with MALDI-TOF/TOF-MS technology were used to reveal the adaptive alterations of A. marina seedlings to tidal inundation. Results: There were profound changes in photosynthesis and chlorophyll fluorescence characteristics of A. marina seedling leaves under inundation treatment. Particularly, photosynthetic rate increased at 4 h inundation, while decreasing at 8 h inundation treatment compared with control (0 h). The results highlighted that A. marina seedlings could greatly upregulate the abundances of photosynthesis-related proteins, activate antioxidant and defense systems to efficiently suppress the reactive oxygen species (ROS) burst and cell damages, mobilize carbohydrate metabolism associated proteins, and then maintain cellular homeostasis, resulting in well adaptation to short-term tidal inundation. Conclusion: Overall, our results demonstrated that short-term inundation is beneficial, while long-term inundation is detrimental to A. marina. The results allow us to recognize the roles of specific proteins in molecular tolerance to tidal inundation in mangrove plants. [ABSTRACT FROM AUTHOR]

Systemic anaplastic large cell lymphoma (ALCL) is an aggressive T-cell lymphoma most commonly seen in children and young adults. The majority of pediatric ALCLs are associated with the t(2;5)(p23;q35) translocation which fuses the Anaplastic Lymphoma Kinase (ALK) gene with the Nucleophosmin (NPM) gene. The NPM-ALK fusion protein is a constitutively-active tyrosine kinase, and plays a major role in tumor pathogenesis. In an effort to advance novel diagnostic approaches and the understanding of the function of this fusion protein in cancer cells, we expressed in E. coli, purified and characterized human NPM-ALK fusion protein to be used as a standard for estimating expression levels in cultured human ALCL cells, a key tool in ALCL pathobiology research. We estimated that NPM-ALK fusion protein is expressed at substantial levels in both Karpas 299 and SU-DHL-1 cells (ca. 4–6 million molecules or 0.5–0.7 pg protein per cell; based on our in-house developed NPM-ALK ELISA; LOD of 40 pM) as compared to the ubiquitous β-actin protein (ca. 64 million molecules or 4.5 pg per lymphocyte). We also compared NPM-ALK/ β-actin ratios determined by ELISA to those independently determined by two-dimensional electrophoresis and showed that the two methods are in good agreement. [ABSTRACT FROM AUTHOR]

In the present study, the mechanisms of Aspergillus niger AM1 and AM2 resistance to white phosphorus were studied. It was shown that the presence of white phosphorus (P4) at a concentration of 0.25% in the medium had a marginal impact on the ratio of living to dead cells during fungal cultivation, which indicates a high resistance of the strains to P4. Observations made with electron microscopy showed an increase in the thickness of the fungal cell wall, which is a barrier to the penetration of white phosphorus. MALDI results revealed the biosynthesis of new protein enzymes that could potentially participate in the neutralization of white phosphorus. In addition, white phosphorus caused activation of the metabolism, accompanied by an increase in the number of mitochondria in the cells. [ABSTRACT FROM AUTHOR]

Metroxylon sagu Rottb. or locally known as sago palm is a tropical starch crop grown for starch production in commercial plantations in Malaysia, especially in Sarawak, East Malaysia. This plant species accumulate the highest amount of edible starch compared to other starch-producing crops. However, the non-trunking phenomenon has been observed to be one of the major issues restricting the yield of sago palm starch. In this study, proteomics approach was utilised to discover differences between trunking and non-trunking proteomes in sago palm leaf tissues. Total protein from 16 years old trunking and non-trunking sago palm leaves from deep peat area were extracted with PEG fractionation extraction method and subjected to two-dimensional gel electrophoresis (2D PAGE). Differential protein spots were subjected to MALDI-ToF/ToF MS/MS. Proteomic analysis has identified 34 differentially expressed proteins between trunking and non-trunking sago samples. From these protein spots, all 19 proteins representing different enzymes and proteins have significantly increased in abundance in non-trunking sago plant when subjected to mass spectrometry. The identified proteins mostly function in metabolic pathways including photosynthesis, tricarboxylic acid cycle, glycolysis, carbon utilization and oxidative stress. The current study indicated that the several proteins identified through differentially expressed proteome contributed to physical differences in trunking and non-trunking sago palm. [ABSTRACT FROM AUTHOR]

As one of the main indicators of nutritional values of foxtail millet, grain protein contents (GPCs) of seeds can be affected by soil water conditions. Studies in this report indicated that intensified drought in filling stage increased the GPCs. To gain better insight into the interaction mechanism between the GPCs and soil water deficits, the proteomic profiles of mature seeds under three drought stress levels (moderate drought-W2; combined drought-W1; severe drought-W0) were conducted on two varieties, Longgu6 (LG6) and Huangjinmiao (HJM) by two-dimensional electrophoresis, with acidic (pH 4–7) gels. There were 83 protein spots in total identified successfully in two comparison groups (W1 vs W2 and W0 vs W2), associated with four major biochemical and metabolic pathways, including storage protein, amino acid and protein metabolisms, energy metabolism, response to stress and defense, and chaperones. Further analysis revealed that the increase of GPCs in LG6 under both W0 and W1 may be owing to the up-regulated storage proteins and the up-regulated protein response to drought stress. The levels of DEPs related to protein and amino acid biosynthesis and starch metabolism increased greatly in LG6 under W1, and this may also result in the high GPC level of seeds in LG6 under W1. Protein biosynthesis was up-regulated and drought stress-related pathways were enhanced in HJM under W1, thereby resulting in the increase of GPC in HJM under W1. These results will contribute to the existing knowledge on the complexity of protein changes leading to the GPC changes in response to drought. [ABSTRACT FROM AUTHOR]

Background: Maize is one of the important cereal food crops in the world. High temperature stress causes adverse influence on plant growth. When plants are exposed to high temperatures, they produce heat shock proteins (HSPs), which may impart a generalized role in tolerance to heat stress. Proteome analysis was performed in plant to assess the changes in protein types and their expression levels under abiotic stress. The purpose of the study is to explore which proteins are involved in the response of the maize plant to heat shock treatment. Results: We investigated the responses of abundant proteins of maize leaves, in an Egyptian inbred line of maize "K1", upon heat stress through two-dimensional electrophoresis (2-DE) on samples of maize leaf proteome. 2-DE technique was used to recognize heat-responsive protein spots using Coomassie Brilliant Blue (CBB) and silver staining. In 2-D analysis of proteins from plants treated at 45 °C for 2 h, the results manifested 59 protein spots (4.3%) which were reproducibly detected as new spots where did not present in the control. In 2D for treated plants for 4 h, 104 protein spots (7.7%) were expressed only under heat stress. Quantification of spot intensities derived from heat treatment showed that twenty protein spots revealed clear differences between the control and the two heat treatments. Nine spots appeared with more intensity after heat treatments than the control, while four spots appeared only after heat treatments. Five spots were clearly induced after heat treatment either at 2 h or 4 h and were chosen for more analysis by LC-MSMS. They were identified as ATPase beta subunit, HSP26, HSP16.9, and unknown HSP/Chaperonin. Conclusion: The results revealed that the expressive level of the four heat shock proteins that were detected in this study plays important roles to avoid heat stress in maize plants. [ABSTRACT FROM AUTHOR]

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is the fundamental technique for protein separation by size. Applying this technology in capillary format, gaining high separation efficiency in a more automated way, is a key technology for size separation of proteins in the biopharmaceutical industry. However, unequivocal identification by online mass spectrometry (MS) is impossible so far, due to strong interference in the electrospray process by SDS and other components of the SDS-MW separation gel buffer. Here, a heart-cut two-dimensional electrophoretic separation system applying an electrically isolated valve with an internal loop of 20 nL is presented. The peak of interest in the CE (SDS) separation is transferred to the CZE-MS, where electrospray-interfering substances of the SDS-MW gel are separated prior to online electrospray ionization mass spectrometry. An online SDS removal strategy for decomplexing the protein-SDS complex is implemented in the second dimension, consisting of the co-injection of organic solvent and cationic surfactant. This online CE (SDS)-CZE-MS system allows MS characterization of proteoforms separated in generic CE (SDS), gaining additional separation in the CZE and detailed MS information. In general, the system can be applied to all kinds of proteins separated by CE (SDS). Here, we present results of the CE (SDS)-CZE-MS system on the analysis of several biopharmaceutically relevant antibody impurities and fragments. Additionally, the versatile application spectrum of the system is demonstrated by the analysis of extracted proteins from soybean flour. The online hyphenation of CE (SDS) resolving power and MS identification capabilities will be a powerful tool for protein and mAb characterization. [ABSTRACT FROM AUTHOR]

Main conclusion: The tobacco nectar proteome mainly consists of pathogenesis-related proteins with two glycoproteins. Expression of nectarins was non-synchronous, and not nectary specific. After secretion, tobacco nectar changed from sucrose rich to hexose rich. Floral nectar proteins (nectarins) play important roles in inhibiting microbial growth in nectar, and probably also tailoring nectar chemistry before or after secretion; however, very few plant species have had their nectar proteomes thoroughly investigated. Nectarins from Nicotiana tabacum (NT) were separated using two-dimensional gel electrophoresis and then analysed using mass spectrometry. Seven nectarins were identified: acidic endochitinase, β-xylosidase, α-galactosidase, α-amylase, G-type lectin S-receptor-like serine/threonine-protein kinase, pathogenesis-related protein 5, and early nodulin-like protein 2. An eighth nectarin, a glycoprotein with unknown function, was identified following isolation from NT nectar using a Qproteome total glycoprotein kit, separation by SDS-PAGE, and identification by mass spectrometry. Expression of all identified nectarins, plus four invertase genes, was analysed by qRT PCR; none of these genes had nectary-specific expression, and none had synchronous expression. The total content of sucrose, hexoses, proteins, phenolics, and hydrogen peroxide were determined at different time intervals in secreted nectar, both within the nectar tube (in vivo) and following extraction from it during incubation at 30 °C for up to 40 h in plastic tubes (in vitro). After secretion, the ratio of hexose to sucrose substantially increased for in vivo nectar, but no sugar composition changes were detected in vitro. This implies that sucrose hydrolysis in vivo might be done by fixed apoplastic invertase. Both protein and hydrogen peroxide levels declined in vitro but not in vivo, implying that some factors other than nectarins act to maintain their levels in the flower, after secretion. [ABSTRACT FROM AUTHOR]

Downy mildew is one of the most prevalent diseases that cause destructive damages to the production of cucumber worldwide. To understand the regulation mechanism underlying the responses of cucumber leaves to P. cubensis infection, a proteomic approach was used to profile protein variations in cucumber leaves during a incubation period of up to 72 h. Differentially accumulated proteins (DAPs) at different time points after inoculation were investigated. A total of 90 DAP spots were identified using an integrated approach involving two-dimensional gel electrophoresis and MALDI-TOF-TOF mass spectrometer. Functional classification showed that most DAPs were putatively involved in metabolic reaction, transportation, protein folding, proteolysis, cell proliferation, photosynthesis, glucose metabolism, translation elongation, damage response, and other closely related biological processes. Several antioxidants, such as Cu/Zn-superoxide dismutase and 2-cysteine peroxiredoxins, were significantly up-regulated under P. cubensis infection. These antioxidants may play roles in regulating potentially harmful reactive oxygen species under pathogen infections. Furthermore, changes in the photosynthetical system-related proteins and heat shock proteins were observed under P. cubensis infection. Our data provides fundamental resources for identifying candidate proteins and metabolic pathways involved in the responses of cucumber plants to P. cubensis infection. [ABSTRACT FROM AUTHOR]

Despite technological advances, two-dimensional electrophoresis (2DE) of biological fluids, such as vitreous, remains a major challenge. In this study, artificial neural network was applied to optimize the recovery of vitreous proteins and its detection by 2DE analysis through the combination of several solubilizing agents (CHAPS, Genapol, DTT, IPG buffer), temperature, and total voltage. The highest protein recovery (94.9% ± 4.5) was achieved using 4% (w/v) CHAPS, 0.1% (v/v) Genapol, 20 mM DTT, and 2% (v/v) IPG buffer. Two iterations were required to achieve an optimized response (580 spots) using 4% (w/v) CHAPS, 0.2% (v/v) Genapol, 60 mM DTT, and 0.5% (v/v) IPG buffer at 35 kVh and 25 °C, representing a 2.4-fold improvement over the standard initial conditions of the experimental design. The analysis of depleted vitreous using the optimized protocol resulted in an additional 1.3-fold increment in protein detection over the optimal output, with an average of 761 spots detected in vitreous from different vitreoretinopathies. Our results clearly indicate the importance of combining the appropriate amount of solubilizing agents with a suitable control of the temperature and voltage to obtain high-quality gels. The high-throughput of this model provides an effective starting point for the optimization of 2DE protocols. This experimental design can be adapted to other types of matrices. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) is a ubiquitous bimolecule that regulates various physiological processes in plants. Despite the widespread study of NO in plants, the involvement of NO in proteomic regulation has been poorly investigated compared to the physiological processes. To explore the NO-induced proteomic regulation in rice leaves, 2-week-old leaves were exposed to sodium nitroprusside for 12 h. Protein profiles were analyzed by two-dimensional gel electrophoresis. A total of 53 NO-regulated proteins were identified by the matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis. Out of these proteins, 41 were up-regulated, 7 were down-regulated, and 5 were newly induced. The identified proteins were involved in photosynthesis, carbohydrate and energy metabolism, growth and development, metabolite biosynthesis, signal transduction, lignin modification, and defense response. Importantly, several key proteins including glutathione reductase, monodehydroascorbate reductase, dehydroascorbate reductase, ascorbate peroxidase, superoxide dismutase, catalase, 2-cysteine peroxiredoxin, peroxidase (newly induced), and glutathione S-transferase were significantly up-regulated in rice leaves and these proteins were associated with the ascorbate–glutathione cycle and ROS homeostasis system in plants. Thus, the identification of several key and newly induced proteins has provided a new insight into NO-mediated overall molecular response in plants. [ABSTRACT FROM AUTHOR]

Widespread application of silver nanoparticles (AgNPs), due to their antibacterial and antifungal properties, increases their release into the environment and potential detrimental impact on living organisms. Plants may serve as a potential pathway for AgNPs bioaccumulation and a route into the food chain, hence investigation of AgNP phytotoxic effects are of particular importance. Since proteins are directly involved in stress response, studies of their abundance changes can help elucidate the mechanism of the AgNP-mediated phytotoxicity. In this study, we investigated proteomic changes in tobacco (Nicotiana tabacum) exposed to AgNPs and ionic silver (AgNO3). A high overlap of differently abundant proteins was found in root after exposure to both treatments, while in leaf, almost a half of the proteins exhibited different abundance level between treatments, indicating tissue-specific responses. Majority of the identified proteins were down-regulated in both tissues after exposure to either AgNPs or AgNO3; in roots, the most affected proteins were those involved in response to abiotic and biotic stimuli and oxidative stress, while in leaf, both treatments had the most prominent effect on photosynthesis-related proteins. However, since AgNPs induced higher suppression of protein abundance than AgNO3, we conclude that AgNP effects can, at least partially, be attributed to nanoparticle form. [ABSTRACT FROM AUTHOR]

Main conclusion: The present study is the first to integrate physiological and proteomic data providing information on Fe, Mn and Zn deficiency-responsive mechanisms of potato plants in vitro. Micronutrient deficiency is an important limiting factor for potato production that causes substantial tuber yield and quality losses. To under the underlying molecular mechanisms of potato in response to Fe, Mn and Zn deficiency, a comparative proteomic approach was applied. Leaf proteome change of in vitro-propagated potato plantlets subjected to a range of Fe-deficiency treatments (20, 10 and 0 μM Na-Fe-EDTA), Mn-deficiency treatments (1 and 0 μM MnCl2·4H2O) and Zn-deficiency treatment (0 μM ZnCl2) using two-dimensional gel electrophoresis was analyzed. Quantitative image analysis showed a total of 146, 55 and 42 protein spots under Fe, Mn and Zn deficiency with their abundance significantly altered (P < 0.05) more than twofold, respectively. By MALDI-TOF/TOF MS analyses, the differentially abundant proteins were found mainly involved in bioenergy and metabolism, photosynthesis, defence, redox homeostasis and protein biosynthesis/degradation under the metal deficiencies. Signaling, transport, cellular structure and transcription-related proteins were also identified. The hierarchical clustering results revealed that these proteins were involved in a dynamic network in response to Fe, Mn and Zn deficiency. All these metal deficiencies caused cellular metabolic remodeling to improve metal acquisition and distribution in potato plants. The reduced photosynthetic efficiency occurred under each metal deficiency, yet Fe-deficient plants showed a more severe damage of photosynthesis. More defence mechanisms were induced by Fe deficiency than Mn and Zn deficiency, and the antioxidant systems showed different responses to each metal deficiency. Reprogramming of protein biosynthesis/degradation and assembly was more strongly required for acclimation to Fe deficiency. The signaling cascades involving auxin and NDPKs might also play roles in micronutrient stress signaling and pinpoint interesting candidates for future studies. Our results first provide an insight into the complex functional and regulatory networks in potato plants under Fe, Mn and Zn deficiency. [ABSTRACT FROM AUTHOR]

Two-dimensional gel electrophoresis (2-DE) was combined with liquid chromatography-mass spectrometry (LC-MS/MS) to identify the differential proteomics of grass carp gills after hypoxic stress to better understand the roles of proteins in the hypoxic response and to explore the possible molecular mechanisms. Protein spots were obtained from a hypoxia-stressed group (372 ± 11 individuals) and a control group (406 ± 14 individuals) using the lmage Master 2D Platinum 7.0 analysis software. Fifteen protein spots were expressed differentially in the hypoxia-stressed group and varied significantly after exposure to the hypoxic conditions. In addition, these differential proteins were identified by mass spectrometry and then searched in a database. We found the expression and upregulation of the toll-like receptor 4, ephx1 protein, isocitrate dehydrogenase, L-lactate dehydrogenase, GTP-binding nuclear protein Ran, and glyceraldehyde-3-phosphate dehydrogenase; however, the expression of the keratin type II cytoskeletal 8, type I cytokeratin, ARP3 actin-related protein 3 homolog, thyroid hormone receptor alpha-A, ATP synthase subunit beta, citrate synthase, tropomyosin 2, and tropomyosin 3 were downregulated. Six proteins were found in the hypoxia-inducible factor-1 (HIF-1) signaling pathway. We concluded that the grass carp gill is involved in response processes, including energy generation, metabolic processes, cellular structure, antioxidation, immunity, and signal transduction, to hypoxic stress. To our knowledge, this is the first study to conduct a proteomics analysis of expressed proteins in the gills of grass carp, and this study will help increase the understanding of the molecular mechanisms involved in hypoxic stress responses in fish at the protein level. [ABSTRACT FROM AUTHOR]

Purpose: In this study, by using a two-dimensional (2D) electrophoresis-based experimental approach, we aimed at understanding the nature of alkali injuries and the underlying mechanisms. A secondary aim was to compare the effects of cross-linking (CXL) and amnion membrane transplantation (AMT) on corneal protein compositions at the end of the early repair phase after injured with alkali.Method: The right corneas of 24 rabbits were injured with a 1 N solution of NaOH. Groups were formed based on the adjuvant therapies as (1) healthy group, (2) control group, (3) CXL group, (4) AMT group. In addition to the therapies, a conventional medical treatment was applied to all groups. Left eyes were used as within-subject healthy corneas (1). The corneas were excised at day 21, and a comparative proteomic analysis was performed using 2D gel electrophoresis coupled with MALDI-TOF/TOF.Result: 2D gel electrophoresis revealed the presence seven protein spots whose abundance changed among the groups. Those proteins were SH3 domain-binding protein, plant homeodomain finger protein 23, S100 calcium binding protein A-11(S100 A11), keratin type 2 cytoskeletal 1 and 2, transketolase and glyceraldehyde 3-phosphate dehydrogenase. Ingenuity pathway analysis predicted that the observed changes may be linked to a central metabolic pathway, transforming growth factor beta 1. Canonical pathway analysis focused our attention to two different pathways, namely nicotinamide adenine dinucleotide repair pathway and non-oxidative branch of pentose phosphate pathway.Conclusion: Our results shed some light onto the molecular mechanisms affected by alkali injury and adjuvant treatments. Further research is needed to propose medically significant target molecules that may be used for novel drug developments for alkali injury. [ABSTRACT FROM AUTHOR]

Objective: To investigate the innate characters of 3 endometriosis (EMT) syndromes, blood stasis (BS), qi stagnation and blood stasis (QSBS) as well as Shen (Kidney) deficiency and blood stasis (KDBS) in terms of proteomics, lay a molecular biological basis for the differentiation of various blood stasis syndromes of EMT, establish a EMT microscopic syndrome differentiation and diagnosis system in terms of proteomics, discover the evolution principles and therapeutic targets of these EMT syndromes, and search their signifificant molecular markers and genetic intervention targets.Methods: Six specimens from the ectopic and entopic endometrium tissues of patients with EMT in each syndrome, BS, QSBS as well as KDBS, in the early proliferative phase of the menstrual cycle, and 6 specimens from normal endometrium tissues in the early proliferative phase of the menstrual cycle were obtained. Three groups were formed in each syndrome by mixing two random specimens in equal amount, and then their respective two-dimensional electrophoresis graphs were obtained after total protein extraction. Finally, the detected differences in protein expression were identifified through matrix-assisted laser desorption Ionization-time of flflight mass spectrometry (MALDI-TOF/MS) and protein database.Results: The results of differential proteins expressed in each syndrome were shown as follows: BS syndrome had 2 differential proteins in entopic endometrium and 1 differential protein in ectopic endometrium; KDBS syndrome had 3 in entopic endometrium and 3 in ectopic endometrium; and QSBS syndrome had 3 in entopic endometrium and 4 in ectopic endometrium. It was found out that annexin was highly expressed in both entopic and ectopic endometrium of KDBS syndrome; and myosin light chain 3 was highly expressed in both entopic and ectopic endometrium of QSBS syndrome.Conclusion: There are differential protein expressions among the 3 EMT syndromes, which might be the inner origin of syndrome characters, and these differential proteins might be the candidate biomarkers for the pathogenesis of various EMT syndromes. [ABSTRACT FROM AUTHOR]

Abstract: Using the sectional analysis of two-dimensional electrophoretic gels with liquid chromatography-mass spectrometry, proteoform profiles for individual genes expressed in cancer (glioblastoma) and normal (FLEH) cells were obtained. Profiles of more than 5000 genes were analyzed. It turned out that many genes encoding potential biomarkers of glioblastoma are characterized by sets of proteoforms that are different in normal and cancer cells. These proteoforms could be sources of highly specific markers and targets for therapy. Using a section analysis of two-dimensional electrophoretic gels with liquid chromatography by mass spectrometry, proteoform profiles were obtained for individual genes expressed in cancer (glioblastoma) and normal (FLEH) cells. Profiles of more than 5000 genes were analyzed. It turned out that many genes encoding potential biomarkers of glioblastoma are characterized by sets of proteoforms, which are different in normal and cancer cells. These proteoforms could be sources of highly specific markers and targets for therapy. [ABSTRACT FROM AUTHOR]

Ultraviolet A (UVA), the major component of solar UV irradiation, is an important environmental factor inducing damage to insects including cell death, photoreceptor damage, and oxidative stress. In order to improve understanding of the adaptation mechanisms of insect after UVA exposure, a comparative proteomic analysis was carried out to reveal differential protein expression in Ostrinia furnacalis. Three-day-old adults were treated with UVA for 1 h. Total proteins of control and UVA-treated insects were examined using two-dimensional electrophoresis (2-DE). 2-DE analysis demonstrated that 19 proteins were increased and 18 proteins were decreased significantly in O. furnacalis after UVA exposure, respectively. Thirty differentially expressed proteins were successfully identified by mass spectrometry. The identified proteins were involved in diverse biological processes, such as signal transduction, transport processing, cellular stress, metabolisms, and cytoskeleton organization. Our results reveal that the response patterns of O. furnacalis to UVA irradiation are complex and provide novel insights into the adaptation response to UVA irradiation stress. [ABSTRACT FROM AUTHOR]

The heart is characterized by a remarkable degree of heterogeneity. Since different cardiac pathologies affect different cardiac regions, it is important to understand molecular mechanisms by which these parts respond to pathological stimuli. In addition to already described left ventricular (LV)/right ventricular (RV) and transmural differences, possible baso-apical heterogeneity has to be taken into consideration. The aim of our study has been, therefore, to compare proteomes in the apical and basal parts of the rat RV and LV. Two-dimensional electrophoresis was used for the proteomic analysis. The major result of this study has revealed for the first time significant baso-apical differences in concentration of several proteins, both in the LV and RV. As far as the LV is concerned, five proteins had higher concentration in the apical compared to basal part of the ventricle. Three of them are mitochondrial and belong to the “metabolism and energy pathways” (myofibrillar creatine kinase M-type, L-lactate dehydrogenase, dihydrolipoamide dehydrogenase). Myosin light chain 3 is a contractile protein and HSP60 belongs to heat shock proteins. In the RV, higher concentration in the apical part was observed in two mitochondrial proteins (creatine kinase S-type and proton pumping NADH:ubiquinone oxidoreductase). The described changes were more pronounced in the LV, which is subjected to higher workload. However, in both chambers was the concentration of proteins markedly higher in the apical than that in basal part, which corresponds to the higher energetic demand and contractile activity of these segments of both ventricles. [ABSTRACT FROM AUTHOR]