Search

Amaranth is a promising staple food that produces seeds with excellent nutritional quality. Although cultivated species intended for grain production have interesting agronomic traits, relatively little is known about wild species, which can prosper in diverse environments and could be a rich genetic source for crop improvement. This work focuses on the proteomic comparison between the seeds of wild and cultivated amaranth species using polarity-based protein extraction and two-dimensional gel electrophoresis. Differentially accumulated proteins (DAPs) showed changes in granule-bound starch synthases and a wide range of 11S globulin isoforms. The electrophoretic profile of these proteins suggests that they may contain significant phosphorylation as post-translational modifications (PTMs), which were confirmed via immunodetection. These PTMs may impact the physicochemical functionality of storage proteins, with potential implications for seed agronomic traits and food system applications. Low-abundant DAPs with highly variable accumulation patterns are also discussed; these were involved in diverse molecular processes, such as genic regulation, lipid storage, and stress response. [ABSTRACT FROM AUTHOR]

Viral diseases of potatoes are among the main problems causing deterioration in the quality of tubers and loss of yield. The growth and development of potato plants largely depend on soil moisture. Prevention strategies require comprehensive protection against pathogens and abiotic stresses, including modeling the beneficial microbiome of agroecosystems combining microorganisms and immunostimulants. Chitosan and its derivatives have great potential for use in agricultural engineering due to their ability to induce plant immune responses. The effect of chitosan conjugate with caffeic acid (ChCA) in combination with Bacillus subtilis 47 on the transcriptional activity of PR protein genes and changes in the proteome of potato plants during potato virus Y (PVY) infection and drought was studied. The mechanisms of increasing the resistance of potato plants to PVY and lack of moisture are associated with the activation of transcription of genes encoding PR proteins: the main protective protein (PR-1), chitinase (PR-3), thaumatin-like protein (PR-5), protease inhibitor (PR-6), peroxidase (PR-9), and ribonuclease (PR-10), as well as qualitative and quantitative changes in the plant proteome. The revealed activation of the expression of marker genes of systemic acquired resistance and induced systemic resistance under the influence of combined treatment with B. subtilis and chitosan conjugate indicate that, in potato plants, the formation of resistance to viral infection in drought conditions proceeds synergistically. By two-dimensional electrophoresis of S. tuberosum leaf proteins followed by MALDI-TOF analysis, 10 proteins were identified, the content and composition of which differed depending on the experiment variant. In infected plants treated with ChCA, the synthesis of proteinaceous RNase P 1 and oxygen-evolving enhancer protein 2 was enhanced in conditions of normal humidity, and 20 kDa chaperonin and TMV resistance protein N-like was enhanced in conditions of lack of moisture. The virus coat proteins were detected, which intensively accumulated in the leaves of plants infected with potato Y-virus. ChCA treatment reduced the content of these proteins in the leaves, and in plants treated with ChCA in combination with Bacillus subtilis, viral proteins were not detected at all, both in conditions of normal humidity and lack of moisture, which suggests the promising use of chitosan derivatives in combination with B. subtilis bacteria in the regulation of plant resistance. [ABSTRACT FROM AUTHOR]

Results obtained from rat studies indicate that, even at low concentrations, mercurial species cause harmful effects on the kidneys, by inducing the nephrotic oxidative stress response. In the present work, Hg-associated proteins were identified as possible mercury-exposure biomarkers in rat kidneys exposed to low mercury chloride concentrations for 30 days (Hg-30) and 60 days (Hg-60), using metalloproteomic strategies. The renal proteomic profile was fractioned by two-dimensional electrophoresis and the mercury determinations in kidney samples, protein pellets and protein spots were performed using graphite furnace atomic absorption spectrometry. The characterization of Hg-associated protein spots and the analysis of differentially expressed proteins were performed by liquid chromatography, coupled with tandem mass spectrometry. Eleven Hg-associated protein spots with a concentration range of 79 ± 1 to 750 ± 9 mg kg−1 in the Hg-60 group were identified. The characterization and expression analyses allowed the identification of 53 proteins that were expressed only in the Hg-60 group, 13 "upregulated" proteins (p > 0.95) and 47 "downregulated" proteins (p < 0.05). Actin isoforms and hemoglobin subunits were identified in protein spots of the Hg-60 group, with mercury concentrations in the range of 138 to 750 mg kg−1, which qualifies these proteins as potential mercury-exposure biomarkers. [ABSTRACT FROM AUTHOR]

The resistance of silkworms to Bombyx mori nuclear polyhedrosis virus (BmNPV) is controlled by a major dominant gene and multiple modifying genes. Given the presence of modified genes, it is difficult to determine the main gene by positional cloning. In this study, the main anti-BmNPV gene of BmNPV-resistant silkworm variety N was introduced into the susceptible variety Su to breed the near-isogenic line SuN with BmNPV resistance. The infection process of BmNPV in the hemolymph of Su and SuN was analyzed using the cell analysis system TissueFAXS PLUS. According to the law of infection and proliferation, hemolymph was extracted every 6 h for two-dimensional electrophoresis (2-DE) analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Seven DEPs were found in comparisons between Su and SuN by 2-DE analysis. Among them, acid phosphatase, storage protein, and phenoloxidase can prevent pathogen invasion, which may play a role against BmNPV. Polyamine oxidase plays an important role in energy metabolism, which may be indirectly involved in the process of resisting BmNPV. Most of the transcriptional expression profiles of the seven DEPs were consistent with the 2-DE results. This study can provide a reference for the identification of anti-BmNPV genes and the breeding of BmNPV-resistant silkworm varieties. [ABSTRACT FROM AUTHOR]

Adipose-derived mesenchymal stem cells (ASCs) have the potential to differentiate into bone, cartilage, fat, and neural cells and promote tissue regeneration and healing. It is known that they can have variable responses to hypoxic conditions. In the present study, we aimed to explore diverse changes in the cells and secretome of ASCs under a hypoxic environment over time and to present the possibility of ASCs as therapeutic agents from a different perspective. The expression differences of proteins between normoxic and hypoxic conditions (6, 12, or 24 h) were specifically investigated in human ASCs using 2-DE combined with MALDI-TOF MS analysis, and secreted proteins in ASC-derived conditioned media (ASC-derived CM) were examined by an adipokine array. In addition, genetic and/or proteomic interactions were assessed using a DAVID and miRNet functional annotation bioinformatics analysis. We found that 64 and 5 proteins were differentially expressed in hypoxic ASCs and in hypoxic ASC-derived CM, respectively. Moreover, 7 proteins among the 64 markedly changed spots in hypoxic ASCs were associated with bone-related diseases. We found that two proteins, cathepsin D (CTSD) and cathepsin L (CTSL), identified through an adipokine array independently exhibited significant efficacy in promoting osteocyte differentiation in bone-marrow-derived mesenchymal stem cells (BM-MSCs). This finding introduces a promising avenue for utilizing hypoxia-preconditioned ASC-derived CM as a potential therapeutic approach for bone-related diseases. [ABSTRACT FROM AUTHOR]

Simple Summary: During the pupal stage, the midgut of Bombyx mori undergoes remarkable changes, condensing into the midgut lumen and, subsequently, undergoing decomposition. To comprehensively analyze this process of change, we utilized mass spectrometry to examine the midgut contents in the early and later stages of pupation. We detected a large number of enzymes involved in metabolism, proteases, inhibitors, chitin-binding proteins, chitinases, and storage proteins in the midgut contents during the pupal stage, indicating significant alterations within the pupal midgut lumen. Additionally, we have identified six proteases with potential gelatinolytic activity, which may be involved in the degradation of the midgut contents during the late stages of pupation. This research provides valuable insights into the mechanisms of tissue digestion and midgut remodeling in Bombyx mori pupae. The results serve as an important reference for comprehending the intricate processes taking place during this developmental stage in lepidopteran insects. The silkworm Bombyx mori, a lepidopteran insect, possesses an 8–10-day pupal stage, during which significant changes occur in the midgut, where it first condenses into the yellow body, and then undergoes decomposition. To gain insights into this transformation process, proteomics was performed on Bombyx mori midgut contents on day 2 and day 7 after pupation. The results revealed the identification of 771 proteins with more than one unique peptide. An analysis using AgriGO demonstrated that these proteins were predominantly associated with catalytic activity. Among the identified proteins, a considerable number were found to be involved in carbohydrate metabolism, amino acid metabolism, lipid metabolism, nucleic acid degradation, and energy support. Additionally, variations in the levels of certain proteases were observed between the midgut contents on day 2 and day 7 after pupation. An in-depth analysis of the two-dimensional electrophoresis of the midgut contents on day 7 after pupation led to the identification of twelve protein spots with potential gelatinolytic activity. Among these, six proteases were identified through mass spectrometry, including the p37k protease, vitellin-degrading protease, chymotrypsin-2, etc. These proteases may be responsible for the digestion of the yellow body during the later stages of pupal development. [ABSTRACT FROM AUTHOR]

Ammopiptanthus mongolicus, a traditional ethnic medicinal herb, is a rare broad-leaved evergreen shrub in the arid region of central Asia that can survive under extremely low temperatures during winter. In this study, we investigated the cold stress response of A. mongolicus leaves using physiological and proteomic approaches. Cold stress treatment increased the relative electrolyte leakage, proline, and soluble sugar levels and decreased the chlorophyll content in A. mongolicus leaves. Moreover, 93 differentially abundant proteins (DAPs) were identified using two-dimensional electrophoresis, of which 79 were further confirmed via tandem mass spectrometric analysis. The predicted functions of DAPs were mainly associated with photosynthesis in chloroplasts, reactive oxygen species scavenging, defense, and protein synthesis, folding, and degradation. A. mongolicus chitinase A (AmCHIA) is a cold-induced apoplast protein whose transcription is upregulated under cold, osmotic, high-salinity, and mechanical stresses. Recombinant AmCHIA expressed in Escherichia coli exhibits chitinase activity. Here, AmCHIA expression enhanced the cold tolerance of E. coli cells, suggesting that it may contribute to the cold adaptation of A. mongolicus after cold treatment. The present study not only provides important data for understanding the cold stress responses in plants but also serves as a basis for further exploration of the biological functions of cold-induced proteins in A. mongolicus using genetic approaches. [ABSTRACT FROM AUTHOR]

Protein adsorption is essential for determining material biocompatibility and promoting adherent cell growth. In this study, we focused on the a-plane structure of hydroxyapatite (HAp). This a-plane structure closely resembles the crystal plane where apatite is exposed in long bones. We conducted protein adsorption experiments using HAp ceramics with a preferred orientation to a-planes (aHAp), employing bovine serum albumin (BSA), lysozyme, and fetal bovine serum (FBS) as protein models to mimic the in vivo environment. Higher zeta potential and contact angle values were found in aHAp than in HAp ceramics fabricated from commercial HAp powder (iHAp). Bradford-quantified protein adsorption revealed BSA adsorption of 212 ng·mm−2 in aHAp and 28.4 ng mm−2 in iHAp. Furthermore, the Bradford-quantified protein adsorption values for FBS were 2.07 μg mm−2 in aHAp and 1.28 µg mm−2 in iHAp. Two-dimensional electrophoresis (2D-PAGE) showed a higher number of protein-derived major spots in aHAp (37 spots) than in iHAp (12 spots). Mass spectrometry analysis of the resulting 2D-PAGE gels revealed proteins adsorbed on aHAp, including secreted frizzled-related protein 3 and vitamin K epoxide reductase complex 1, which are involved in cellular bone differentiation. Overall, these proteins are expected to promote bone differentiation, representing a characteristic property of aHAp. [ABSTRACT FROM AUTHOR]

The number and identity of proteins and proteoforms presented in a single human cell (a cellular proteome) are fundamental biological questions. The answers can be found with sophisticated and sensitive proteomics methods, including advanced mass spectrometry (MS) coupled with separation by gel electrophoresis and chromatography. So far, bioinformatics and experimental approaches have been applied to quantitate the complexity of the human proteome. This review analyzed the quantitative information obtained from several large-scale panoramic experiments in which high-resolution mass spectrometry-based proteomics in combination with liquid chromatography or two-dimensional gel electrophoresis (2DE) were used to evaluate the cellular proteome. It is important that even though all these experiments were performed in different labs using different equipment and calculation algorithms, the main conclusion about the distribution of proteome components (proteins or proteoforms) was basically the same for all human tissues or cells. It follows Zipf's law and has a formula N = A/x, where N is the number of proteoforms, A is a coefficient, and x is the limit of proteoform detection in terms of abundance. [ABSTRACT FROM AUTHOR]

The fungus Ustilaginoidea virens, the causative agent of false smut in rice (Oryza sativa L.), is responsible for one of the severe grain diseases that lead to significant losses worldwide. In this research, microscopic and proteomic analyses were performed by comparing U. virens infected and non-infected grains of the susceptible and resistant rice varieties to provide insights into the molecular and ultrastructural factors involved in false smut formation. Prominent differentially expressed peptide bands and spots were detected due to false smut formation as revealed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis (2-DE) SDS-PAGE profiles and were identified using liquid chromatography-mass spectrometry (LC-MS/MS). The proteins identified from the resistant grains were involved in diverse biological processes such as cell redox homeostasis, energy, stress tolerance, enzymatic activities, and metabolic pathways. It was found that U. virens produces diverse degrading enzymes such as β-1, 3-endoglucanase, subtilisin-like protease, putative nuclease S1, transaldolase, putative palmitoyl-protein thioesterase, adenosine kinase, and DNase 1 that could discretely alter the host morphophysiology resulting in false smut. The fungus also produced superoxide dismutase, small secreted proteins, and peroxidases during the smut formation. This study revealed that the dimension of rice grain spikes, their elemental composition, moisture content, and the specific peptides produced by the grains and the fungi U. virens play a vital role in the formation of false smut. [ABSTRACT FROM AUTHOR]

Clubroot disease caused by Plasmodiophora brassicae Woronin (P. brassicae) has seriously influenced the production of Brassica rapa crops, but the interaction mechanism between P. brassicae and Brassica rapa is not clear. In our previous study, a differentially expressed protein, G15, was found between the roots of Chinese cabbage inoculated and un-inoculated with P. brassicae through two-dimensional electrophoresis (2-DE) and mass spectrometry, and G15 was matched with Bra011464. In this study, Bra011464 was found to have a 94% percent identity with Arabidopsis thaliana CAP, named BrCAP. The expression of BrCAP was the highest in the root compared with the stems and leaves of Chinese cabbage, and its expression in the roots of Chinese cabbage inoculated with P. brassicae was significantly higher than that in the control. The results were verified by real-time quantitative polymerase chain reaction (RT-qPCR) and in situ hybridization. Subcellular localization showed that BrCAP was localized on chloroplasts of leaf epidermal cells. To verify the function of BrCAP, it was found that the Arabidopsis thaliana mutant cap was more susceptible to infection with P. brassicae than the wild type (WT), which suggested that BrCAP has a potential role in the resistance progress of Chinese cabbage to P. brassicae. [ABSTRACT FROM AUTHOR]

Based on the known role of oxidative stress in the pathogenesis and progression of metabolic syndrome, we used two-dimensional gel electrophoresis with immunochemical detection of protein carbonyls (2D-Oxyblot) to characterize the carbonylated proteins induced by oxidative stress in spontaneously hypertensive rats/NDmcr-cp (CP), an animal model of metabolic syndrome. We also profiled the proteins that showed change of expression levels in their epididymal adipose tissue at the pre-symptomatic (6-week-old) and the symptomatic (25-week-old) stages of the metabolic syndrome. Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) combined with matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) was used to analyze proteins extracted from the epididymal adipose tissue. The up-regulated proteins identified at the pre-symptomatic stage were mainly associated with ATP production and redox reaction, while the down-regulated proteins found at the symptomatic stage were involved in antioxidant activity and the tricarboxylic acid (TCA) cycle. Further analysis using the 2D-Oxyblot showed significantly high carbonylation levels of gelsolin and glycerol-3-phosphate dehydrogenase [NAD+] at the symptomatic stage. These results suggest that reduced antioxidant capacity underlies the increased oxidative stress state in the metabolic syndrome. The identified carbonylated proteins, including gelsolin, are potential targets that may act as key regulators in the progression of the metabolic syndrome. [ABSTRACT FROM AUTHOR]

Mould development in foodstuffs is linked to both spoilage and the production of mycotoxins, provoking food quality and food safety concerns, respectively. The high-throughput technology proteomics applied to foodborne moulds is of great interest to address such issues. This review presents proteomics approaches useful for boosting strategies to minimise the mould spoilage and the hazard related to mycotoxins in food. Metaproteomics seems to be the most effective method for mould identification despite the current problems related to the bioinformatics tool. More interestingly, different high resolution mass spectrometry tools are suitable for evaluating the proteome of foodborne moulds able to unveil the mould's response under certain environmental conditions and the presence of biocontrol agents or antifungals, being sometimes combined with a method with limited ability to separate proteins, the two-dimensional gel electrophoresis. However, the matrix complexity, the high ranges of protein concentrations needed and the performing of multiple steps are some of the proteomics limitations for the application to foodborne moulds. To overcome some of these limitations, model systems have been developed and proteomics applied to other scientific fields, such as library-free data independent acquisition analyses, the implementation of ion mobility, and the evaluation of post-translational modifications, are expected to be gradually implemented in this field for avoiding undesirable moulds in foodstuffs. [ABSTRACT FROM AUTHOR]

Proteins play an essential regulatory role in the innate immune response of host plants following elicitation by either biotic or abiotic stresses. Isonitrosoacetophenone (INAP), an unusual oxime-containing stress metabolite, has been investigated as a chemical inducer of plant defence responses. Both transcriptomic and metabolomic studies of various INAP-treated plant systems have provided substantial insight into this compound's defence-inducing and priming capabilities. To complement previous 'omics' work in this regard, a proteomic approach of time-dependent responses to INAP was followed. As such, Nicotiana tabacum (N. tabacum) cell suspensions were induced with INAP and changes monitored over a 24-h period. Protein isolation and proteome analysis at 0, 8, 16 and 24 h post-treatment were performed using two-dimensional electrophoresis followed by the gel-free eight-plex isobaric tags for relative and absolute quantitation (iTRAQ) based on liquid chromatography and mass spectrometry. Of the identified differentially abundant proteins, 125 were determined to be significant and further investigated. INAP treatment elicited changes to the proteome that affected proteins from a wide range of functional categories: defence, biosynthesis, transport, DNA and transcription, metabolism and energy, translation and signalling and response regulation. The possible roles of the differentially synthesised proteins in these functional classes are discussed. Results indicate up-regulated defence-related activity within the investigated time period, further highlighting a role for proteomic changes in priming as induced by INAP treatment. [ABSTRACT FROM AUTHOR]

Simple Summary: Little is known about what happens in the canine gastrointestinal tract during episodes of acute diarrhea in dogs. In the present study, a proteomic approach was used to investigate at the time of inclusion, and at two subsequent time points (2 and 14 days after the onset of the clinical condition), changes in the digestive environment in a subset of dogs suffering from acute uncomplicated diarrhea. Four (groups of) proteins showed significant differences at two or more of the three time points investigated, mainly evidencing a reaction of the organism to the disease. Further studies are needed to confirm the present findings. Acute diarrhea is a very frequent condition affecting dogs; nevertheless, little is known about what happens in the GI tract during such conditions. Proteomics allows the study of proteins present in a specific biologic substrate, and fecal proteomic investigations have been recently implemented to study GI diseases in dogs. In the present study, the fecal protein profiles of eight dogs suffering from acute uncomplicated diarrhea at the time of inclusion was investigated for the first time, and then the same patients were followed, replicating two further evaluations at two subsequent time points (after 2 and 14 days from the first presentation), with the aim of gaining possible new insights regarding the pathologic changes in the gastrointestinal environment during such conditions. Two-dimensional gel electrophoresis (2-DE) was performed, followed by mass spectrometry. Nine spots, corresponding to four (groups of) proteins (i.e., albumin, alkaline phosphatase, chymotrypsin-C-like, and some immunoglobulins), showed significant differences at two or more of the three time points investigated, almost all behaving similarly and decreasing at T1 (2 days after the onset of the condition) and significantly increasing at T2 (14 days after the onset), mainly evidencing a reaction of the organism. Further studies including a greater number of patients and possibly different techniques are needed to confirm the present findings. [ABSTRACT FROM AUTHOR]

Copper-zinc superoxide dismutase (SOD1) is an antioxidant enzyme that catalyzes the disproportionation of superoxide anion to hydrogen peroxide and molecular oxygen (dioxygen). The yeast Saccharomyces cerevisiae lacking SOD1 (Δsod1) is hypersensitive to the superoxide anion and displays a number of oxidative stress-related alterations in its phenotype. We compared proteomes of the wild-type strain and the Δsod1 mutant employing two-dimensional gel electrophoresis and detected eighteen spots representing differentially expressed proteins, of which fourteen were downregulated and four upregulated. Mass spectrometry-based identification enabled the division of these proteins into functional classes related to carbon metabolism, amino acid and protein biosynthesis, nucleotide biosynthesis, and metabolism, as well as antioxidant processes. Detailed analysis of the proteomic data made it possible to account for several important morphological, biochemical, and physiological changes earlier observed for the SOD1 mutation. An example may be the proposed additional explanation for methionine auxotrophy. It is concluded that protein comparative profiling of the Δsod1 yeast may serve as an efficient tool in the elucidation of the mutation-based systemic alterations in the resultant S. cerevisiae phenotype. [ABSTRACT FROM AUTHOR]

For over four decades, mass spectrometry-based methods have provided a wealth of information relevant to various challenges in the field of cancers research. These challenges included identification and validation of novel biomarkers for various diseases, in particular for various forms of cancer. These biomarkers serve various objectives including monitoring patient response to the various forms of therapy, differentiating subgroups of the same type of cancer, and providing proteomic data to complement datasets generated by genomic, epigenetic, and transcriptomic methods. The same proteomic data can be used to provide prognostic information and could guide scientists and medics to new and innovative targeted therapies The past decade has seen a rapid emergence of epigenetics as a major contributor to carcinogenesis. This development has given a fresh momentum to MS-based proteomics, which demonstrated to be an unrivalled tool for the analyses of protein post-translational modifications associated with chromatin modifications. In particular, high-resolution mass spectrometry has been recently used for systematic quantification of chromatin modifications. Data generated by this approach are central in the search for new therapies for various forms of cancer and will help in attempts to decipher antitumor drug resistance. To appreciate the contribution of mass spectrometry-based proteomics to biomarkers discovery and to our understanding of mechanisms behind the initiation and progression of various forms of cancer, a number of recent investigations are discussed. These investigations also include results provided by two-dimensional gel electrophoresis combined with mass spectrometry. [ABSTRACT FROM AUTHOR]

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a crucial role in its life cycle. The Mpro-mediated limited proteolysis of the viral polyproteins is necessary for the replication of the virus, and cleavage of the host proteins of the infected cells may also contribute to viral pathogenesis, such as evading the immune responses or triggering cell toxicity. Therefore, the identification of host substrates of the viral protease is of special interest. To identify cleavage sites in cellular substrates of SARS-CoV-2 Mpro, we determined changes in the HEK293T cellular proteome upon expression of the Mpro using two-dimensional gel electrophoresis. The candidate cellular substrates of Mpro were identified by mass spectrometry, and then potential cleavage sites were predicted in silico using NetCorona 1.0 and 3CLP web servers. The existence of the predicted cleavage sites was investigated by in vitro cleavage reactions using recombinant protein substrates containing the candidate target sequences, followed by the determination of cleavage positions using mass spectrometry. Unknown and previously described SARS-CoV-2 Mpro cleavage sites and cellular substrates were also identified. Identification of target sequences is important to understand the specificity of the enzyme, as well as aiding the improvement and development of computational methods for cleavage site prediction. [ABSTRACT FROM AUTHOR]

Esophageal squamous cell carcinoma is a severe malignancy for its high mortality and poor prognosis. Mainstay chemotherapies cause serious side effects for their ways of inducing cell death. Oridonin is the main bioactive constituent from natural plants that has anticancer ability and weak side effects. The proteomics method is efficient to understand the anticancer mechanism. However, proteins identified by proteomics aimed at understanding oridonin's anticancer mechanism is seldom overlapped by different groups. This study used proteomics based on two-dimensional electrophoresis sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2-DE SDS-PAGE) integrated with mass spectrometry and Gene Set Enrichment Analysis (GSEA) to understand the anticancer mechanism of oridonin on esophageal squamous cell carcinoma (ESCC). The results showed that oridonin induced ESCC cell death via apoptosis by decreasing the protein expression of LASP1 and PDLIM1. [ABSTRACT FROM AUTHOR]

Simple Summary: Haberlea rhodopensis (Gesneriaceae) belongs to the group of so-called resurrection plants, which are able to lose more than 95% of the water in the cells and quickly restore their metabolism upon rehydration. The plant is characterized with a high ecological plasticity growing at altitude from 136 m to near 1700 m at different temperature, water, and light conditions. In its natural habitats, H. rhodopensis is often exposed to high temperatures during dry periods in the summer. In the present study, we investigated the accumulation of small heat shock proteins (sHSPs) and the extent of non-photochemical quenching during the downregulation of photosynthesis in H. rhodopensis under desiccation at optimum (23 °C) and high temperature (38 °C). Dehydration at high temperature has a detrimental effect on plant photosynthesis, thus leading to oxidative stress and damage of different important macromolecules in the cells. Plants accumulate different protective proteins when exposed to high temperatures, one of the most characteristic being sHSPs. They prevent irreversible aggregation of unfolded or aggregated proteins and facilitate their refolding. Plants possess more than 30 different of sHSPs, much more compared to mammals and microorganisms. Fourteen different isoforms of sHSPs accumulate during dehydration at 38 °C compared to 23 °C, where only two were detected. H. rhodopensis is an excellent model system to study the protective mechanisms under extreme dehydration at high temperature, thus helping plant breeders to create high temperature resistant crops. Haberlea rhodopensis belongs to the small group of angiosperms that can survive desiccation to air-dry state and quickly restore their metabolism upon rehydration. In the present study, we investigated the accumulation of sHSPs and the extent of non-photochemical quenching during the downregulation of photosynthesis in H. rhodopensis leaves under desiccation at optimum (23 °C) and high temperature (38 °C). Desiccation of plants at 38 °C caused a stronger reduction in photosynthetic activity and corresponding enhancement in thermal energy dissipation. The accumulation of sHSPs was investigated by Western blot. While no expression of sHPSs was detected in the unstressed control sample, exposure of well-hydrated plants to high temperature induced an accumulation of sHSPs. Only a faint signal was observed at 50% RWC when dehydration was applied at 23 °C. Several cross-reacting polypeptide bands in the range of 16.5–19 kDa were observed in plants desiccated at high temperature. Two-dimensional electrophoresis and immunoblotting revealed the presence of several sHSPs with close molecular masses and pIs in the range of 5–8.0 that differed for each stage of treatment. At the latest stages of desiccation, fourteen different sHSPs could be distinguished, indicating that sHSPs might play a crucial role in H. rhodopensis under dehydration at high temperatures. [ABSTRACT FROM AUTHOR]

Among grain pulses, lupins have recently gained considerable interest for a number of attractive nutritional attributes relating to their high protein and dietary fiber and negligible starch contents. The seeds of Lupinus albus (cv. Multitalia and Luxor, and the Modica ecotype); L. luteus (cv. Dukat, Mister, and Taper); and L. angustifolius (cv. Sonet) analyzed in this study were deposited within the germplasm collection of the Research Centre for Cereal and Industrial Crops of Acireale and were sowed in East Sicily in 2013/14. The collected seeds were analyzed for their multielemental micro- and macronutrient profiles, resulting in a wide variability between genotypes. Lupin seed flour samples were subjected to a defatting process using supercritical CO2, with oil yields dependent on the species and genotype. We determined the fatty acid profile and tocopherol content of the lupin oil samples, finding that the total saturated fatty acid quantities of different samples were very close, and the total tocopherol content was about 1500.00 µg/g FW. The proteomic analysis of the defatted lupin seed flours showed substantial equivalence between the cultivars of the same species of Lupinus albus and L. luteus. Moreover, the L. angustifolius proteome map showed the presence of additional spots in comparison to L. albus, corresponding to α-conglutins. Lupin, in addition to being a good source of mineral elements, also contributes vitamin E and, thanks to the very high content of gamma-tocopherols, demonstrates powerful antioxidant activity. [ABSTRACT FROM AUTHOR]

Watermelon is an important fruit that is widely distributed around the world. In particular, the production and consumption of watermelon in China ranks first in the world. Watermelon production is severely affected by a variety of biotic and abiotic stresses during cultivation, and polyploidization can promote stress resistance and yield. However, the morphological and physiological characteristics of tetraploid watermelon and the underlying molecular mechanisms are still poorly understood. In this study, we revealed that the leaves, fruits, and seeds of tetraploid watermelon were significantly larger than those of the diploid genotype. Some physiological characteristics, including photosynthetic rate (Pn) and stomatal conductance (Gs), were greater, whereas the intercellular CO2 concentration (Ci) and transpiration rate (Tr) were lower in tetraploid than in diploid watermelon. Two-dimensional gel electrophoresis combined with tandem mass spectrometry (MALDI-TOF/TOF) was performed to compare proteomic changes between tetraploid and diploid watermelon. A total of 21 differentially expressed proteins were identified; excluding the identical proteins, 8 proteins remained. Among them, four proteins were upregulated and four were downregulated in tetraploid versus diploid genotypes. qRT-PCR results showed inconsistencies in gene expression and protein accumulation, indicating a low correlation between gene expression and protein abundance. Generally, this study extends our understanding of the traits and molecular mechanisms of tetraploid watermelon and provides a theoretical basis for watermelon polyploid breeding. [ABSTRACT FROM AUTHOR]

The use of tumor markers aids in the early detection of cancer recurrence and prognosis. There is a hope that they might also be useful in screening tests for the early detection of cancer. Here, the question of finding ideal tumor markers, which should be sensitive, specific, and reliable, is an acute issue. Human plasma is one of the most popular samples as it is commonly collected in the clinic and provides noninvasive, rapid analysis for any type of disease including cancer. Many efforts have been applied in searching for "ideal" tumor markers, digging very deep into plasma proteomes. The situation in this area can be improved in two ways—by attempting to find an ideal single tumor marker or by generating panels of different markers. In both cases, proteomics certainly plays a major role. There is a line of evidence that the most abundant, so-called "classical plasma proteins", may be used to generate a tumor biomarker profile. To be comprehensive these profiles should have information not only about protein levels but also proteoform distribution for each protein. Initially, the profile of these proteins in norm should be generated. In our work, we collected bibliographic information about the connection of cancers with levels of "classical plasma proteins". Additionally, we presented the proteoform profiles (2DE patterns) of these proteins in norm generated by two-dimensional electrophoresis with mass spectrometry and immunodetection. As a next step, similar profiles representing protein perturbations in plasma produced in the case of different cancers will be generated. Additionally, based on this information, different test systems can be developed. [ABSTRACT FROM AUTHOR]

Tuberculosis, an infectious disease, is one of the leading causes of death worldwide. Drug-resistant tuberculosis exacerbates its threat. Despite long-term and costly treatment with second-line drugs, treatment failure rates and mortality remain high. Therefore, new strategies for developing new drugs and improving the efficiency of existing drug treatments are urgently needed. Our research team reported that PPs, a new class of potential anti-tuberculosis drug candidates, can inhibit the growth of drug-resistant Mycobacterium tuberculosis. Here, we report a synergistic effect of PPs with ethionamide (ETH), one of the second-line drugs, as a result of further research on PPs. While investigating gene expression changes based on microarray and 2DE (two-dimensional gel electrophoresis), it was found that PPs induced the greatest overexpression of Rv0560c in M. tuberculosis. Based on this result, a protein microarray using Rv0560c protein was performed, and it was confirmed that Rv0560c had the highest interaction with EthR, a repressor for EthA involved in activating ETH. Accordingly, a synergistic experiment was conducted under the hypothesis of increased susceptibility of ETH to M. tuberculosis by PPs. As a result, in the presence of 0.5× MIC PPs, ETH showed a growth inhibitory effect on drug-sensitive and -resistant M. tuberculosis even at a much lower concentration of about 10-fold than the original MIC of ETH. It is also suggested that the effect was due to the interaction between PPs and Rv2887, the repressor of Rv0560c. This effect was also confirmed in a mouse model of pulmonary tuberculosis, confirming the potential of PPs as a booster to enhance the susceptibility of M. tuberculosis to ETH in treating drug-resistant tuberculosis. However, more in-depth mechanistic studies and extensive animal and clinical trials are needed in the future. [ABSTRACT FROM AUTHOR]

Protein phosphorylation is a reversible post-translational modification (PTM) with major regulatory roles in many cellular processes. However, the analysis of phosphoproteins remains the most challenging barrier in the prevailing proteome research. Recent technological advances in two-dimensional electrophoresis (2-DE) coupled to mass spectrometry (MS) have enabled the identification, characterization, and quantification of protein phosphorylation on a global scale. Most research on phosphoproteins with 2-DE has been conducted using phosphostains. Nevertheless, low-abundant and low-phosphorylated phosphoproteins are not necessarily detected using phosphostains and/or MS. In this study, we report a comparative analysis of 2-DE phosphoproteome profiles using Pro-Q Diamond phosphoprotein stain (Pro-Q DPS) and chemical dephosphorylation of proteins with HF-P from longissimus thoracis (LT) muscle samples of the Rubia Gallega cattle breed. We found statistically significant differences in the number of identified phosphoproteins between methods. More specifically, we found a three-fold increase in phosphoprotein detection with the HF-P method. Unlike Pro-Q DPS, phosphoprotein spots with low volume and phosphorylation rate were identified by HF-P technique. This is the first approach to assess meat phosphoproteome maps using HF-P at a global scale. The results open a new window for 2-DE gel-based phosphoproteome analysis. [ABSTRACT FROM AUTHOR]

Antibiotics are used in plant in vitro tissue culture to eliminate microbial contamination or for selection in genetic transformation. Antibiotic timentin has a relatively low cytotoxic effect on plant tissue culture; however, it could induce an enduring growth-inhibiting effect in tobacco in vitro shoot culture that persists after tissue transfer to a medium without antibiotic. The effect is associated with an increase in oxidative stress injury in plant tissues. In this study, we assessed changes of reactive oxygen species accumulation, protein expression, and oxidative protein modification response associated with enduring timentin treatment-induced growth suppression in tobacco (Nicotiana tabacum L.) in vitro shoot culture. The study revealed a gradual 1.7 and 1.9-fold increase in superoxide (O2•−) content at the later phase of the propagation cycle for treatment control (TC) and post-antibiotic treatment (PA) shoots; however, the O2•− accumulation pattern was different. For PA shoots, the increase in O2•− concentration occurred several days earlier, resulting in 1.2 to 1.4-fold higher O2•− concentration compared to TC during the period following the first week of cultivation. Although no protein expression differences were detectable between the TC and PA shoots by two-dimensional electrophoresis, the increase in O2•− concentration in PA shoots was associated with a 1.5-fold increase in protein carbonyl modification content after one week of cultivation, and protein carbonylation analysis revealed differential modification of 26 proteoforms involved in the biological processes of photosynthesis and glycolysis. The results imply that the timentin treatment-induced oxidative stress might be implicated in nontranslational cellular redox balance regulation, accelerates the development of senescence of the shoot culture, and contributes to the shoot growth-suppressing effect of antibiotic treatment. [ABSTRACT FROM AUTHOR]

Two-dimensional electrophoresis gels (2DE, 2DEG) are the result of the procedure of separating, based on two molecular properties, a protein mixture on gel. Separated similar proteins concentrate in groups, and these groups appear as dark spots in the captured gel image. Gel images are analyzed to detect distinct spots and determine their peak intensity, background, integrated intensity, and other attributes of interest. One of the approaches to parameterizing the protein spots is spot modeling. Spot parameters of interest are obtained after the spot is approximated by a mathematical model. The development of the modeling algorithm requires a rich, diverse, representative dataset. The primary goal of this research is to develop a method for generating a synthetic protein spot dataset that can be used to develop 2DEG image analysis algorithms. The secondary objective is to evaluate the usefulness of the created dataset by developing a neural-network-based protein spot reconstruction algorithm that provides parameterization and denoising functionalities. In this research, a spot modeling algorithm based on autoencoders is developed using only the created synthetic dataset. The algorithm is evaluated on real and synthetic data. Evaluation results show that the created synthetic dataset is effective for the development of protein spot models. The developed algorithm outperformed all baseline algorithms in all experimental cases. [ABSTRACT FROM AUTHOR]

Iron (Fe) and manganese (Mn) are two essential elements for plants that compete for the same uptake transporters and show conflicting interactions at the regulatory level. In order to understand the differential response to both metal deficiencies in plants, two proteomic techniques (two-dimensional gel electrophoresis and label-free shotgun) were used to study the proteome profiles of roots from tomato plants grown under Fe or Mn deficiency. A total of 119 proteins changing in relative abundance were confidently quantified and identified, including 35 and 91 in the cases of Fe deficiency and Mn deficiency, respectively, with 7 of them changing in both deficiencies. The identified proteins were categorized according to function, and GO-enrichment analysis was performed. Data showed that both deficiencies provoked a common and intense cell wall remodelling. However, the response observed for Fe and Mn deficiencies differed greatly in relation to oxidative stress, coumarin production, protein, nitrogen, and energy metabolism. [ABSTRACT FROM AUTHOR]

An accuracy of ≥98% was achieved in sepsis diagnosis using serum samples from 30 sepsis patients and 68 healthy individuals and a high-performance two-dimensional polyacrylamide gel electrophoresis (HP-2D-PAGE) method developed here with deep learning and transfer learning algorithms. In this method, small-scale target domain data, which are collected to achieve our objective, are inputted directly into a model constructed with source domain data which are collected from a different domain from the target; target vectors are estimated with the outputted target domain data and applied to refine the model. Recognition performance of small-scale data is improved by reusing all layers, including the output layers of the neural network. Proteomics is generally considered the ultimate bio-diagnostic technique and provides extremely high information density in its two-dimensional electrophoresis images, but extracting the data has posed a basic problem. The present study is expected to solve that problem and will be an important breakthrough for practical utilization and future perspectives of proteomics in clinics after evaluation in clinical settings. [ABSTRACT FROM AUTHOR]

Proteomic analyses are rapid and powerful tools that are used to increase the understanding of cancer pathogenesis, discover cancer biomarkers and predictive markers, and select and monitor novel targets for cancer therapy. Feline injection-site sarcomas (FISS) are aggressive skin tumours with high recurrence rates, despite treatment with surgery, radiotherapy, and chemotherapy. Doxorubicin is a drug of choice for soft tissue sarcomas, including FISS. However, multidrug resistance is one of the major causes of chemotherapy failure. The main aim of the present study was to identify proteins that differentiate doxorubicin-resistant from doxorubicin-sensitive FISS using two-dimensional gel electrophoresis (2DE), followed by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Using the three-dimensional (3D) preclinical in ovo model, which resembles features of spontaneous fibrosarcomas, three significantly (p ≤ 0.05) differentially expressed proteins were identified in tumours grown from doxorubicin-resistant fibrosarcoma cell lines (FFS1 and FFS3) in comparison to the doxorubicin-sensitive one (FFS5): Annexin A5 (ANXA5), Annexin A3 (ANXA3), and meiosis-specific nuclear structural protein 1 (MNS1). Moreover, nine other proteins were significantly differentially expressed in tumours grown from the high doxorubicin-resistant cell line (FFS1) in comparison to sensitive one (FFS5). This study may be the first proteomic fingerprinting of FISS reported, identifying potential candidates for specific predictive biomarkers and research targets for doxorubicin-resistant FISS. [ABSTRACT FROM AUTHOR]

Dietary selenium putatively prevents oxidative damage, whereas excessive selenium may lead to animal disorder. In this study, we investigated the effects of low and excessive levels of dietary selenium on oxidative stress and mitochondrial proteins in mouse liver. Six to eight week old mice were fed a diet with low, excessive, or moderate (control) levels of selenium (sodium selenite). The selenium concentration and oxidative stress-related parameters in hepatic mitochondria were evaluated. Two-dimensional electrophoresis and mass spectrometry were applied to identify the differentially-expressed proteins associated with dietary selenium. The selenium content of the livers in mice with the low selenium diet was significantly lower than that of the control, while that of mice fed excessive levels was significantly higher. In both groups oxidative stress in hepatic mitochondria was found; accompanied by lower superoxide dismutase (SOD) and glutathione peroxidase (GPX) levels and higher malondialdehyde (MDA) content, compared with the control group. Furthermore, ten proteins in the hepatic mitochondria of the selenium-low or -excessive groups with more than two-fold differences in abundance compared with the control group were identified. The differentially-expressed proteins in hepatic mitochondria may be associated with dietary (low or excessive) selenium-induced oxidative stress. [ABSTRACT FROM AUTHOR]

Although radiotherapy is generally effective in the treatment of major nasopharyngeal carcinoma (NPC), this treatment still makes approximately 20% of patients radioresistant. Therefore, the identification of blood or biopsy biomarkers that can predict the treatment response to radioresistance and that can diagnosis early stages of NPC would be highly useful to improve this situation. Proteomics is widely used in NPC for searching biomarkers and comparing differentially expressed proteins. In this review, an overview of proteomics with different samples related to NPC and common proteomics methods was made. In conclusion, identical proteins are sorted as follows: Keratin is ranked the highest followed by such proteins as annexin, heat shock protein, 14-3-3σ, nm-23 protein, cathepsin, heterogeneous nuclear ribonucleoproteins, enolase, triosephosphate isomerase, stathmin, prohibitin, and vimentin. This ranking indicates that these proteins may be NPC-related proteins and have potential value for further studies. [ABSTRACT FROM AUTHOR]

The chitosan-induced coacervation of milk proteins was investigated using a proteomic approach. The addition of 0.8% chitosan to milk caused the milk proteins to coacervate after a 1 h incubation period. Approximately 86% of the milk proteins were present in the milk pellet fraction (MPF), and the protein concentration of the milk supernatant fraction (MSF) decreased from 29.4 ± 0.2 to 4.2 ± 0.6 mg/mL. SDS-PAGE analysis showed that the total intensities of serum albumin (BSA), αS-casein (αS-CN), β-casein (β-CN), κ-casein (κ-CN) and β-lactoglobulin (β-LG) in the MSF decreased to 8.5% ± 0.2%, 0.9% ± 0.3%, 0.7% ± 0.3%, 0.5% ± 0.2% and 15.0% ± 0.5%, respectively. Two-dimensional electrophoresis analysis indicated that αS1-, αS2-, β- and κ-CN and a fraction of the β-LG and BSA were found in the MSF following incubation with 0.8% chitosan. Isothermal titration calorimetry analysis indicated that binding of chitosan to milk proteins is an exothermic reaction based on binding titration curves of milk proteins dispersions with chitosan, and the enthalpy of binding (ΔH) and binding constant (Ka) were -7.85 × 104 cal/mol and 1.06 × 105/mol, respectively. These results suggested that the addition of 0.8% chitosan causes milk proteins to coacervate due to polysaccharide-protein interactions. [ABSTRACT FROM AUTHOR]

Rice (Oryza sativa L.) is the only widely cultivated gramineous crops that cannot be infected by rust fungi. To decipher the molecular basis of rice nonhost resistance (NHR) to Puccinia striiformis f. sp. tritici (Pst), the causal agent of wheat stripe rust, proteomic analysis was performed using the two-dimensional electrophoresis (2-DE) technique. The expressed proteins from rice leaves 24 and 48 h post inoculation with Pst and from mock-inoculated leaves were identified. Quantitative analysis revealed a total of 27 differentially expressed proteins in response to Pst inoculation. Most of these proteins fall into the category "response to stimulus" and are involved in basic resistance processes, such as glycerol-3-phosphate and hydrogen peroxide signaling. A homologue of wheat leaf rust resistance protein Lr10 was also identified, implicating multiple layers of plant defense are implicated in rice NHR to Pst. These results demonstrate an intrinsic relationship between host and nonhost resistance. Changes in abundance of these proteins, together with their putative functions reveal a comprehensive profile of rice NHR to Pst and provide new insights into plant immunity. [ABSTRACT FROM AUTHOR]

Soil contamination by chromium (Cr) has become an increasing problem worldwide as a result of extensive industrial activities. Chromium, especially hexavalent Cr, impairs the growth and productivity of plants. Although it has been proposed that plants could modify their metabolism to adapt to Cr stress by reprogramming the expression of genes, especially those related to the antioxidant system, damage response, and electron transport chain, evidence at the protein expression level is lacking. To better understand the precise mechanisms underlying Cr phytoxicity and the plant response to Cr exposure, the time-course of changes in the protein expression profile induced by short-term hexavalent Cr exposure (1, 6 and 24 h) were analyzed in maize leaves. Among the over 1200 protein spots detected reproducibly by two-dimensional electrophoresis (2-DE), 60 were found to be differentially accumulated during Cr stress treatment. Of the Cr-regulated proteins, 58 were identified using tandem mass spectrometry (MS/MS). The Cr-regulated proteins identified were mainly involved in ROS detoxification and defense responses (26%), photosynthesis and chloroplast organization (22%), post-transcriptional processing of mRNA and rRNA (12%), protein synthesis and folding (10%), the DNA damage response (5%), and the cytoskeleton (3%). The possible involvement of these Cr stress-responsive proteins in Cr phytoxicity and the plant response to Cr exposure in maize is discussed, taking into consideration the information available from other plant models. Our results provide preliminary evidence that will facilitate understanding the molecular mechanisms underlying Cr toxicity in maize. [ABSTRACT FROM AUTHOR]

The present study was undertaken to develop a rat model for monitoring the early development of breast cancer. Twelve female rats were divided into two groups of six rats that were either treated with N-methyl-N-nitrosourea to induce breast cancer or with bacterial lipopolysaccharide to induce inflammation. Serum samples taken from the rats prior to the treatment were used as controls. By the 14th week, presence of the tumor was detectable by contrast enhanced magnetic resonance imaging and confirmed by histopathology. When the serum proteins of the rats were examined by 2-dimensional electrophoresis (2-DE), no difference could be detected in the profiles of all proteins before and 18 weeks after administration of N-methyl-N-nitrosourea. However, higher expression of alpha-1B glycoprotein was detectable by 2-DE in serum samples of rats at the 18th week post-treatment with lipopolysaccharide. [ABSTRACT FROM AUTHOR]

A gel-based proteomics approach was used to screen for proteins of differential abundance between the saliva of smokers and those who had never smoked. Subjecting precipitated proteins from whole human saliva of healthy non-smokers to two-dimensional electrophoresis (2-DE) generated typical profiles comprising more than 50 proteins. While 35 of the proteins were previously established by other researchers, an additional 22 proteins were detected in the 2-DE saliva protein profiles generated in the present study. When the 2-DE profiles were compared to those obtained from subjects considered to be heavy cigarette smokers, three saliva proteins, including interleukin-1 receptor antagonist, thioredoxin and lipocalin-1, showed significant enhanced expression. The distribution patterns of lipocalin-1 isoforms were also different between cigarette smokers and non-smokers. The three saliva proteins have good potential to be used as biomarkers for the adverse effects of smoking and the risk for inflammatory and chronic diseases that are associated with it. [ABSTRACT FROM AUTHOR]

Staining of two-dimensional gels is a primary concern in proteomic studies using two-dimensional gel electrophoresis with respect to the number of proteins analyzed, the accuracy of spot quantification and reproducibility. In this review article, the efficiency of the most widely used dyes was investigated. Visible dyes (Coomassie blue and silver nitrate), fluorescent dyes (Sypro Ruby, Deep Purple) and cyanine labeled methods were compared. [ABSTRACT FROM AUTHOR]

Simple Summary: Canine diabetes is a serious disease, which can lead to numerous complications. There are limited data on urine proteomics in dogs, and none of the effect of diabetes mellitus on the urine proteome. In this study we aimed to analyze the protein composition of the urine collected from the healthy animals and compare it with two diabetic groups (normoalbuminuric and microalbuminuric). There are significant differences between these three groups, and we believe that the identified proteins hold promise as a potential diagnostic tool, which can be later on used in clinical practice, and for better understanding of the disease. In this study we aimed to analyze the protein composition of the urine collected from the healthy animals and compare it to the two diabetic groups (DM I normoalbuminuric diabetic dogs; DM II diabetic dogs with microalbuminuria). We tried to identify potential urinary proteins which could be up- or downregulated in diabetic patients even before the appearance of microalbuminuria. Methods: After obtaining urine, we performed two-dimensional electrophoresis, followed by Delta2D software analysis, which allowed for selection and identification with MALDI-TOF spectrometry, statistically significant differentially expressed proteins. Our study revealed 286 common protein spots on 2D gels from the diabetic and control group. From these proteins five were positively identified by MALDI-TOF MS. To further evaluate the five differentiating proteins, the Panther program was used to assign them to appropriate biological process. Conclusion: Significant number of identified proteins play a role in intracellular signaling—vesicle formation, bonding, transport through membranes. This may suggest that first signs of kidney diabetic cellular impairment may be seen in the urine composition before any clinical signs occur. [ABSTRACT FROM AUTHOR]

Changes in the physicochemical properties and structure of proteins derived from two malt varieties (Baudin and Guangmai) during wort boiling were investigated by differential scanning calorimetry, SDS-PAGE, two-dimensional electrophoresis, gel filtration chromatography and circular dichroism spectroscopy. The results showed that both protein content and amino acid composition changed only slightly during boiling, and that boiling might cause a gradual unfolding of protein structures, as indicated by the decrease in surface hydrophobicity and free sulfhydryl content and enthalpy value, as well as reduced a-helix contents and markedly increased random coil contents. It was also found that major component of both worts was a boiling-resistant protein with a molecular mass of 40 kDa, and that according to the two-dimensional electrophoresis and SE-HPLC analyses, a small amount of soluble aggregates might be formed via hydrophobic interactions. It was thus concluded that changes of protein structure caused by boiling that might influence beer quality are largely independent of malt variety. [ABSTRACT FROM AUTHOR]

Triticale is a wheat–rye hybrid with a higher abiotic stress tolerance than wheat and is better adapted for cultivation in light-type soils, where aluminum ions are present as Al-complexes that are harmful to plants. The roots are the first plant organs to contact these ions and the inhibition of root growth is one of the first plant reactions. The proteomes of the root apices in Al-tolerant and -sensitive plants were investigated to compare their regeneration effects following stress. The materials used in this study consisted of seedlings of three triticale lines differing in Al3+ tolerance, first subjected to aluminum ion stress and then recovered. Two-dimensional electrophoresis (2-DE) was used for seedling root protein separation followed by differential spot analysis using liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS/MS). The plants' tolerance to the stress was evaluated based on biometric screening of seedling root regrowth upon regeneration. Our results suggest that the Al-tolerant genotype can recover, without differentiation of proteome profiles, after stress relief, contrary to Al-sensitive genotypes that maintain the proteome modifications caused by unfavorable environments. [ABSTRACT FROM AUTHOR]

Alexandrium pacificum is a typical dinoflagellate that can cause harmful algal blooms, resulting in negative impacts on ecology and human health. The calcium (Ca2+) signal transduction pathway plays an important role in cell proliferation. Calmodulin (CaM) and CaM-related proteins are the main cellular Ca2+ sensors, and can act as an intermediate in the Ca2+ signal transduction pathway. In this study, the proteins that interacted with CaM of A. pacificum were screened by two-dimensional electrophoresis analysis and far western blots under different growth conditions including lag phase and high phosphorus and manganese induced log phase (HPM). The interactive proteins were then identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Four proteins were identified, including Ca2+/CaM-dependent protein kinase, serine/threonine kinase, annexin, and inositol-3-phosphate synthase, which all showed high expression levels under HPM. The gene expression levels encoding these four proteins were also up-regulated under HPM, as revealed by quantitative polymerase chain reaction, suggesting that the identified proteins participate in the Ca2+ transport channel and cell cycle regulation to promote cell division. A network of proteins interacting with CaM and their target proteins involved in the regulation of cell proliferation was raised, which provided new insights into the mechanisms behind the explosive growth of A. pacificum. [ABSTRACT FROM AUTHOR]

Simple Summary: During replication, DNA molecules undergo topological changes that affect supercoiling, catenation and knotting. To better understand this process and the role of topoisomerases, the enzymes that control DNA topology in in vivo, two-dimensional agarose gel electrophoresis were used to investigate the efficiency of three type II DNA topoisomerases, the prokaryotic DNA gyrase, topoisomerase IV and the human topoisomerase 2α, on partially replicated bacterial plasmids containing replication forks stalled at specific sites. The results obtained revealed that despite the fact these DNA topoisomerases may have evolved to accomplish specific tasks, they share abilities. To our knowledge, this is the first time two-dimensional agarose gel electrophoresis have been used to examine the ability of these topoisomerases to relax supercoiling in the un-replicated region and unlink pre-catenanes in the replicated one of partially replicated molecules in vitro. The methodology described here can be used to study the role of different topoisomerases in partially replicated molecules. DNA topoisomerases are the enzymes that regulate DNA topology in all living cells. Since the discovery and purification of ω (omega), when the first were topoisomerase identified, the function of many topoisomerases has been examined. However, their ability to relax supercoiling and unlink the pre-catenanes of partially replicated molecules has received little attention. Here, we used two-dimensional agarose gel electrophoresis to test the function of three type II DNA topoisomerases in vitro: the prokaryotic DNA gyrase, topoisomerase IV and the human topoisomerase 2α. We examined the proficiency of these topoisomerases on a partially replicated bacterial plasmid: pBR-TerE@AatII, with an unidirectional replicating fork, stalled when approximately half of the plasmid had been replicated in vivo. DNA was isolated from two strains of Escherichia coli: DH5αF' and parE10. These experiments allowed us to assess, for the first time, the efficiency of the topoisomerases examined to resolve supercoiling and pre-catenanes in partially replicated molecules and fully replicated catenanes formed in vivo. The results obtained revealed the preferential functions and also some redundancy in the abilities of these DNA topoisomerases in vitro. [ABSTRACT FROM AUTHOR]

Chocolate spot, which is caused by the necrotrophic fungus Botrytis fabae, is a major foliar disease occurring worldwide and dramatically reducing crop yields in faba bean (Vicia faba). Although chemical control of this disease is an option, it has serious economic and environmental drawbacks that make resistant cultivars a more sensible choice. The molecular mechanisms behind the defense against B. fabae are poorly understood. In this work, we studied the leave proteome in two faba bean genotypes that respond differently to B. fabae in order to expand the available knowledge on such mechanisms. For this purpose, we used two-dimensional gel electrophoresis (2DE) in combination with Matrix-Assisted Laser Desorption/Ionization (MALDI-TOF/TOF). Univariate statistical analysis of the gels revealed 194 differential protein spots, 102 of which were identified by mass spectrometry. Most of the spots belonged to proteins in the energy and primary metabolism, degradation, redox or response to stress functional groups. The MS results were validated with assays of protease activity in gels. Overall, they suggest that the two genotypes may respond to B. fabae with a different PSII protein repair cycle mechanism in the chloroplast. The differences in resistance to B. fabae may be the result of a metabolic imbalance in the susceptible genotype and of a more efficient chloroplast detoxification system in the resistant genotype at the early stages of infection. [ABSTRACT FROM AUTHOR]

Karenia mikimotoi is a well-known harmful algal bloom species. Blooms of this dinoflagellate have become a serious threat to marine life, including fish, shellfish, and zooplanktons and are usually associated with massive fish death. Despite the discovery of several toxins such as gymnocins and gymnodimines in K. mikimotoi, the mechanisms underlying the ichthyotoxicity of this species remain unclear, and molecular studies on this topic have never been reported. The present study investigates the fish-killing mechanisms of K. mikimotoi through comparative proteomic analysis. Marine medaka, a model fish organism, was exposed to K. mikimotoi for a three-part time period (LT25, LT50 and LT90). Proteins extracted from the whole fish were separated by using two-dimensional gel electrophoresis, and differentially expressed proteins were identified with reference to an untreated control. The change in fish proteomes over the time-course of exposure were analyzed. A total of 35 differential protein spots covering 19 different proteins were identified, of which most began to show significant change in expression levels at the earliest stage of intoxication. Among the 19 identified proteins, some are closely related to the oxidative stress responses, energy metabolism, and muscle contraction. We propose that oxidative stress-mediated muscle damage might explain the symptoms developed during the ichthyotoxicity test, such as gasping for breath, loss of balance, and body twitching. Our findings lay the foundations for more in-depth studies of the mechanisms of K. mikimotoi's ichthyotoxicity. [ABSTRACT FROM AUTHOR]

Jatropha curcas contains seeds with a high oil content, suitable for biodiesel production. After oil extraction, the remaining mass can be a rich source of enzymes. However, data from the literature describing physicochemical characteristics for a monomeric esterase from the J. curcas seed did not fit the electrostatic catapult model for esterases/lipases. We decided to reevaluate this J. curcas esterase and extend its characterization to check this apparent discrepancy and gain insights into the enzyme's potential as a biocatalyst. After anion exchange chromatography and two-dimensional gel electrophoresis, we identified the enzyme as belonging to the dienelactone hydrolase family, characterized by a cysteine as the nucleophile in the catalytic triad. The enzyme displayed a basic optimum hydrolysis pH of 9.0 and an acidic pI range, in contrast to literature data, making it well in line with the electrostatic catapult model. Furthermore, the enzyme showed low hydrolysis activity in an organic solvent-containing medium (isopropanol, acetonitrile, and ethanol), which reverted when recovering in an aqueous reaction mixture. This enzyme can be a valuable tool for hydrolysis reactions of short-chain esters, useful for pharmaceutical intermediates synthesis, due to both its high hydrolytic rate in basic pH and its stability in an organic solvent. [ABSTRACT FROM AUTHOR]

Advances in proteome research have opened the gateway to understanding numerous metabolic pathways and fundamental mechanisms involved in abiotic stress tolerance. In the present study, the antioxidant capacity of four tomato genotypes i.e., Kashi Amrit, Kashi Anupam, EC-317-6-1, and WIR-4360 was determined under drought stress to ascertain the scavenging potential for reactive oxygen species (ROS). A significant increase in the superoxide dismutase (SOD), Ascorbate peroxidase (APX), and catalase (CAT) activities in all the four genotypes under drought stress was observed, which seemed to be associated with a protective role against ROS (p < 0.001). Based on the antioxidant enzyme activities, a proteomic approach was applied to study differential protein expression in two selected genotypes from different species i.e., EC-317-6-1 (Solanum pimpinellifolium) and Kashi Amrit (Solanum lycopersicum) grown under irrigated, drought, and re-watering conditions. To reveal the protein network regulated under these conditions, two-dimensional gel electrophoresis was employed to identify and quantify the number of proteins in drought-sensitive (Kashi Amrit) and tolerant (EC-317-6-1) genotypes. Matrix-assisted laser desorption/ionization-time of flight analysis (MALDI-TOF) revealed a total of 453 spots after fine-tuning factors i.e., smoothness, saliency, and minimum area that responded to drought. Out of 453 total spots, 93 spots were identified in Kashi Amrit and 154 in EC-317-6-1 under irrigated conditions, whereas 4 spots were identified in Kashi Amrit and 77 spots in EC-317-6-1 under drought conditions. Furthermore, differentially expressed proteins were distinguished according to the fold change of their expression. Information provided in this report will be useful for the selection of proteins or genes in analyzing or improving drought tolerance in tomato cultivars. These findings may assist in the construction of a complete proteome database encompassing various divergent species which could be a valuable source for the improvement of crops under drought-stress conditions in the future. [ABSTRACT FROM AUTHOR]

Simple Summary: Liver plays an important role in lipid synthesis and muscle growth in poultry. The current study measured the growth traits and the proteome of Muscovy duck liver at 14, 28, 42, and 56 days, aiming at exploring the key regulatory proteins for intramuscular fat deposition and muscle growth. The results showed that Muscovy duck grew most rapidly at 28 vs. 42 days of age, subcutaneous and abdominal fat were deposited rapidly, but intramuscular fat content decreased. At the same time, the abundance of liver proteins regarding the tricarboxylic acid cycle and oxidoreduction increased significantly. This study provides a profile of the fat deposition and liver proteome for Muscovy duck. Liver is responsible for 90% of lipid synthesis in poultry; thus, it plays an important role in the growth of Muscovy ducks, which have a high fat deposition ability in a time-dependent manner. Therefore, male Muscovy ducks at 14, 28, 42, and 56 days were selected for body weight (BW), carcass weight (CW), subcutaneous fat thickness (SFT), abdominal fat weight (AFW), intramuscular fat content (IMF), and breast muscle fiber (BMF) diameter and density determination. Two-dimensional electrophoresis (2-DE) combining liquid chromatography linked to tandem mass spectrometry (LC-MS/MS) was used to analyze proteomic changes in liver at each stage. The BW, CW, AFW, SFT, and BMF diameter and density were significantly increased, while IMF content was significantly decreased at 28 to 42 days of age (p < 0.05). There were 57 differentially abundant protein (DEP) spots representing 40 proteins identified among the ages, in which 17, 41 and 4 spots were differentially abundant at 14 vs. 28, 28 vs. 42, and 42 vs. 56, respectively. Gene Ontology enrichment analysis found that DEPs were mostly enriched in the oxidation-reduction process, carboxylic acid metabolism, etc. Protein–protein interaction showed that catalase (CAT), triosephosphate isomerase (TPI), and protein disulfide-isomerase (PDI) were the key proteins responsible for the growth of Muscovy duck. In conclusion, 28 to 42 days of age is the crucial period for Muscovy ducks, and the ability of metabolism and antioxidants were significantly enhanced in liver. [ABSTRACT FROM AUTHOR]