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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]

Context: Systemic events resulting from mother-embryo relation at the peri-implantation period may result in specific changes to the protein composition of serum and thus provide a source of biomarkers for early detection of pregnancy. Aims: We set out to use two different quantitative proteomic approaches to test this hypothesis by comparing heifer serum at the peri-implantation period to that from cycling heifers. Methods: Two-dimensional electrophoresis (2-DE) and isobaric tags were used for relative and absolute quantitation (iTRAQ) of proteins. Key results: These methods yielded complementary data indicating biomarker candidate proteins. Conclusions: Different proteomic methods provide different and complementary information that needs to be analysed in order to consider proteins as potential biomarkers. Implications: In order to characterise the proteome under specific conditions, the use of complementary techniques is advisable. Early pregnancy detection is essential to cattle reproductive management and production efficiency in farms, and proper methods to this aim require biomarkers which may be identified by proteomic methods. We describe the use of two proteomic techniques that give complementary information to perform a primary screening of proteins which may undergo systemic changes related to pregnancy establishment. The way to combine these methods may become of great use to detect changes related to a specific event such as early pregnancy. [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]

In this study, we aimed to characterize the liver protein profile of Chaohu ducks using two-dimensional electrophoresis and proteomics. The livers were quickly collected from 120 healthy, 84-day-old Chaohu ducks. The intramuscular fat (IMF) content of the left pectoralis muscle was determined using the Soxhlet extraction method. The total protein of liver tissues from the high and low IMF groups was extracted for proteomics. Functional enrichment analysis of the differentially expressed proteins (DEPs) was conducted using gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG). In total, 43 DEPs were identified. Functional enrichment analysis indicated that these DEPs were significantly related to four lipid metabolic processes: carboxylic acid metabolic process, ATP metabolic process, oxoacid metabolic process, and organic acid metabolic process. Three pathways correlated with lipid metabolism were identified using KEGG analysis: glycolysis/gluconeogenesis, pentose phosphate pathway, fructose, and mannose metabolism. Eight key proteins associated with lipid metabolism were identified: ALDOB, GAPDH, ENO1, RGN, TPI1, HSPA9, PRDX1, and GPX1. Protein–protein interaction analysis revealed that the glycolysis/gluconeogenesis pathway mediated the interaction relationship. Key proteins and metabolic pathways were closely related to lipid metabolism and showed a strong interaction in Chaohu ducks.

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]

In this experiment, the effect of salt stress on the root proteome pattern of Arg (tolerant to salinity) and Moghan3 (sensitive to salinity) cultivars of wheat was investigated by two-dimensional polyacrylamide gel electrophoresis. Salinity was applied at two levels of 0 and 150 mM in the third-leaf stage for three weeks under greenhouse conditions. The proteome analysis of the roots revealed 120 reproducible protein spots, among which 15 spots showed significant changes in expression. In Moghan3, four protein spots with a significant reduction in expression were identified. In the tolerant cultivar of Arg, 11 protein spots showed significant changes in expression, among which five protein spots had an increased expression and six protein spots had a decreased expression. These proteins were classified based on their function into several groups such as the proteins involved in metabolism and energy, reactive oxygen species scavenging and detoxification, the proteins associated with cell walls, and the proteins involved in folding and degradation. Probably, the tolerance to salt stress in the Arg cultivar was related to the increased expression of pyruvate dehydrogenase, phosphoglycerate mutase, catalase, and malate dehydrogenase proteins. While in the sensitive variety of Moghan3, the decrease in the expression of enolase, peroxidase, and glutathione peroxidase proteins under salinity stress conditions probably caused oxidative stress in the plants due to increased production of reactive oxygen species. These findings can be useful for improving the salt tolerance in the breeding programs of wheat. [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]

By using a combinational method of non-denaturing two-dimensional electrophoresis (2DE) and reverse staining, various water-soluble esterases in the mouse liver were separated and detected. When the effects of Zn2+ and imidazole were investigated, Zn2+ increased esterase activity while imidazole had no effect on esterase activity. After separation using non-denaturing 2DE and reverse staining, esterase activity on the 2DE gel was also increased by Zn2+, whereas there was no effect on esterase activity by the treatment of imidazole. In addition, the activity of various esterases was inhibited by 1 mM Fe2+ using esterase activity staining after the combination of non-denaturing 2DE and reverse staining. Furthermore, by 1 mM Fe2+, the activities of various esterases separated and detected using the combined method were found to be suppressed to 34-42% of that in the absence of Fe2+ after analyzing substrate changes using fluorometry. Based on the above, the combination of non-denaturing 2DE and reverse staining is applicable to search for substances that inhibit various esterase activities. [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]

Diabetic kidney disease (DKD) affects an estimated 20– 40% of type 2 diabetes patients and is among the most prevalent microvascular complications in this patient population, contributing to high morbidity and mortality rates. Currently, changes in albuminuria status are thought to be a primary indicator of the onset or progression of DKD, yet progressive nephropathy and renal impairment can occur in certain diabetic individuals who exhibit normal urinary albumin levels, emphasizing the lack of sensitivity and specificity associated with the use of albuminuria as a biomarker for detecting diabetic kidney disease and predicting DKD risk. According to the study, a non-invasive method for early detection or prediction of DKD may involve combining proteomic analytical techniques such second generation sequencing, mass spectrometry, two-dimensional gel electrophoresis, and other advanced system biology algorithms. Another category of proteins of relevance may now be provided by renal tissue biomarkers. The establishment of reliable proteomic biomarkers of DKD represents a novel approach to improving the diagnosis, prognostic evaluation, and treatment of affected patients. In the present review, a series of protein biomarkers that have been characterized to date are discussed, offering a theoretical foundation for future efforts to aid patients suffering from this debilitating microvascular complication. [ABSTRACT FROM AUTHOR]

In this study, a comprehensive investigation was undertaken to elucidate a simple triazole compound, 5-phenyl-1-(p-tolyl)-1 H-1,2,3-triazole (PPTT), its interactions with high-abundant proteins and identification of low-abundant proteins by serum proteomics. Employing a combination of spectroscopic techniques and computational chemistry, the interactions between PPTT and three high-abundance blood globular proteins, namely human serum albumin (HSA), human immunoglobulin G (HIgG), and hemoglobin (BHb), were explored, thereby ascertaining their binding constants and thermodynamic parameters at the molecular level. Subsequently, based on the differential proteomics, utilizing two-dimensional gel electrophoresis (2-DE) in conjunction with matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS), the research team isolated and identified differentially expressed low-abundance proteins in human blood serum samples following exposure to PPTT. The results showed that there were twenty highly expressed proteins identified from blood serum samples intervened by PPTT. Combining bioinformatics techniques, these proteins were classified, providing preliminary insights like preproprotein or precursors inhibiting the activity of elastase, defending and regulating the immune system, carrying lipid, and other functions into their biological functionalities. One of the differential proteins, apolipoprotein A-1 (ApoA-1) protein, was selected as a possible target to explore the mechanism of action of PPTT intervention on the related signaling pathways involved in human hepatocellular carcinomas(Hep G2) cells. These research findings offer scientifically sound guidance for further in-depth exploration, development, and application of the 1,2,3-triazole compound. [Display omitted] • PPTT has stronger interactions with three high-abundant proteins in blood. • Utilizing serum proteomics to exploit possible biomarkers related to PPTT. • ApoA-1 may be a target of PPTT on the cholesterol metabolism of Hep G2 cells. [ABSTRACT FROM AUTHOR]

Diabetes mellitus is associated with cognitive deficits in humans and animal models. These deficits are paralleled by neurophysiological and structural changes in the central nervous system, particularly in the hippocampus, which plays an important role in memory formation. We previously reported that the magnitude of long-term potentiation at hippocampal Schaffer collateral-CA1 synapses was significantly impaired in streptozotocin (STZ)-induced type 1 diabetic rats (STZ rats). The present study investigated the mechanisms underlying morphological changes in the hippocampus of STZ rats. We performed a proteomic analysis of the hippocampus of STZ rats using two-dimensional gel electrophoresis followed by mass spectrometry. The distribution of 14–3-3 proteins identified by the proteomic analysis was then examined using immunohistochemistry. The results obtained revealed that 14–3-3 η immunoreactivity in the dorsal hippocampus was weaker in STZ rats than in age-matched control rats. Moreover, the density of glial fibrillary acidic protein-immunoreactive astrocytes in the dorsal hippocampus of STZ rats was increased, whereas 14–3-3 η immunoreactivity in astrocytes and neurons in the dentate gyrus was significantly decreased. These results suggest that changes in 14–3-3 η expression are involved in hippocampal astrogliosis or/and neurogenesis in STZ rats. [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.

The production of high-quality pork is closely related to the growth and development of muscle tissue. The present article provides a comparative proteomic research of l. dorsi, b. femoris, m. brachiocephalicus during the pigs’ growth and development (at age of 60 days and 180 days). This work was supported by data of electrophoretic methods: one-dimensional electrophoresis according to Laemmli with densitometric assessment in the ImageJ software and two-dimensional electrophoresis according to O’Farrell method with its further processing on the software ImageMaster. The mass spectrometric identification was conducted with the help of the high-performance liquid chromatography (HPLC) system connected to a mass spectrometer; further the data were interpreted by search algorithm Andromeda. When comparing frequency diagrams of one-dimensional electrophoregrams of all three muscle tissues of weaned pigs, the greatest difference was observed for the muscle sample l. dorsi. Comparison of diagrams of muscle tissue samples taken for mature pigs showed a great similarity of all three studied muscles samples. Within the framework of the research, the Fold indicator was calculated. The exceeding its value by more than 2 units is generally considered to be a statistically significant difference. When analyzing two-dimensional electrophoretograms of weaned pigs’ muscles, 18 protein fractions were revealed with Fold > 2. When examining the muscle tissue of mature pigs, 15 of those proteins were found; the differences were mostly detected in the minor protein fractions. The mass spectrometric analysis of the cut bands with well-pronounced differences from the onedimensional electrophoretogram revealed 214 proteins involved to a greater extent in cellular and metabolic processes, physical activity and localization. Growth and development protein — semaphorin‑6B (96.78 kDa) — was revealed in muscle tissue of l. dorsi, a. Also in l. dorsi and b. femoris the growth and development proteins were found: cadherin‑13 (78.23 kDa), cadherin‑7 (87.01 kDa), the F‑actin-cap protein beta subunit (30.66 kDa), and two uncharacterized proteins at 65.60 kDa and 63.88 kDa.

Background: Tear proteomic analysis has become an important tool in medical and veterinary research. The tear collection method could influence the tear protein profile. This study aims to evaluate the protein profiles of dog tears collected using microcapillary tubes (MT), Schirmer tear strips (ST), and ophthalmic sponges (OS). Methods: The tear samples were collected using MT, ST, and OS. Tear protein profiles were analyzed using two-dimensional electrophoresis (2-DE) and the different protein spots' expression was compared. Fourteen protein spots were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: Tear protein concentrations ranged from 2.80 to 4.03 μg/μL, with no statistically significant differences among collection methods. Protein expression in each collection method differed in terms of both the number and intensity of the spots. There were 249, 327, and 330 protein spots found from tears collected with MT, ST, and OS, respectively. The proteins albumin, haptoglobin, and lactoferrin identified from OS were found to have higher spot intensities than other methods of collection. The use of MT demonstrated the downregulation of nine proteins. Conclusions: The recent study supported that tear protein analysis is affected by different tear collection methods. Although ST is commonly used for tear collection, it provides insufficient information to study particular tear proteins. [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]

The aim of this study was the comparison of label-free method with 2DE to other analytical method of bacterium Butyrivibrio fibrisolvens extracellular protein samples. Label-free quantification (LF) method performed in this work was compared with previously obtained results of proteomic analysis using two-dimensional electrophoresis (2DE) and nano-liquid chromatography coupled with mass spectroscopy (nLC/MS). B. fibrisolvens as an important plant fibre degrader was cultivated on four different carbon sources (including xylan, xylose, glucose, and a mixture of xylan with glucose). The impact of growth substrate on protein profile was assessed by six pair-wise comparisons evaluating the significantly differently abundant extracellular proteins. Gel-free and gel-based methods resulted in substantially dissimilar results. The LC-MS/MS approach detected substrate-dependent differences in transport and binding membrane proteins (TBP) and nucleotidase, while the 2DE approach detected substrate effect on proteins included in protein synthesis and butyrate synthesis. On the other hand, both methods observed differentially regulated proteins involved in the glycolytic pathway, however, the only shared enzyme (protein detected both by 2DE and LC-MS/MS approach) was fructose-bisphosphate aldolase. The LC-MS/MS approach detected a high abundance of separated peptides. However, it cannot be easily considered as supreme to 2DE analysis. Both methods differ in sample preparation, their advantages and limitations. The study findings indicate these methods can complement each other and together they elucidate better the metabolic functions of B. fibrisolvens. Comparison of LF and 2DE analysis showed limitations connected with a narrow optimised pH interval and number of affected proteins in 2DE in contrary to LF results. Butyrivibrio fibrisolvens culture showed the same proteomic changes in the presence of different saccharides as the whole rumen fluid in vivo. Selected proteins could be used for the monitoring of polysaccharides fermentation in the rumen and be used for optimisation hemicellulose degradation and overall feed utilisation. [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]

Diabetic retinopathy (DR) is the leading cause of blindness in human and animal patients. Early detection and treatment of the disease are important and can be facilitated by proteomic approaches providing biomarkers. Tear films were collected on Schirmer strips from 32 canine patients (12 diabetic dogs without changes in the retina, 8 diabetic dogs with signs of DR, and 12 control dogs). Two-dimensional electrophoresis was used to separate tear film proteins prior to their identification with matrix-assisted laser desorption/ionisation–tandem time-of-flight mass spectrometry and interrogation of protein function databases to find matches. Five significantly differentially expressed proteins were identified; of those, one was downregulated (2ʹ-5ʹ-oligoadenylate synthase 3) and four were upregulated in the tear film of two diabetic groups (Ras-related protein RAB-13; aldo-keto-reductase family 1 member C3; 28S ribosomal protein S31, mitochondrial; and 60S ribosomal protein L5). The differentially expressed proteins identified in the tear film were involved in signalling pathways associated with impaired protein clearance, persistent inflammation and oxidative stress. The results of our study confirm that the pathological process in the retina in the course of diabetes mellitus causes changes in the tear film proteome. [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]

Background: Chlorophyll and carotenoids, the most widely distributed lipophilic pigments in plants, contribute to fruit coloration during development and ripening. These pigments are assembled with pigment-protein complexes localized at plastid membrane. Pigment-protein complexes are essential for multiple cellular processes, however, their identity and composition in fruit have yet to be characterized. Results: By using BN-PAGE technique in combination with microscopy, we studied pigment-protein complexes and plastid transformation in the purified plastids from the exocarp of citrus fruit. The discontinuous sucrose gradient centrifugation was used to isolate total plastids from kumquat fruit, and the purity of isolated plastids was assessed by microscopy observation and western blot analysis. The isolated plastids at different coloring stages were subjected to pigment autofluorescence observation, western blot, two-dimensional electrophoresis analysis and BN-PAGE assessment. Our results demonstrated that (i) chloroplasts differentiate into chromoplasts during fruit coloring, and this differentiation is accompanied with a decrease in the chlorophyll/carotenoid ratio; (ii) BN-PAGE analysis reveals the profiles of macromolecular protein complexes among different types of plastids in citrus fruit; and (iii) the degradation rate of chlorophyll-protein complexes varies during the transition from chloroplasts to chromoplasts, with the stability generally following the order of LHCII > PS II core > LHC I > PS I core. Conclusions: Our optimized methods for both plastid separation and BN-PAGE assessment provide an opportunity for developing a better understanding of pigment-protein complexes and plastid transitions in plant fruit. These attempts also have the potential for expanding our knowledge on the sub-cellular level synchronism of protein changes and pigment metabolism during the transition from chloroplasts to chromoplasts. [ABSTRACT FROM AUTHOR]

The administration of high-dose clindamycin (CLI) along with penicillin is recommended for the treatment of streptococcal toxic-shock syndrome (STSS). However, we previously reported that a "subinhibitory dose" of CLI induced the expression of the NAD-glycohydrolase (NADase) exotoxin in an emm1-type Streptococcus pyogenes 1529 strain isolated from an STSS patient. In this study, we examine NADase induction by CLI treatment using an extracellular NADase activity assay instead of the previous two-dimensional gel electrophoresis assay. The examination revealed that CLI administration can induce NADase expression in a dose-dependent manner. We analyzed 23 CLI-susceptible strains (5 emm1 strains, 6 emm3 strains, 3 emm4 strains, 1 emm6 strain, 3 emm12 strains, 1 emm28 strain, and 4 emm89 strains), and 19 of the 23 strains showed similar NADase induction phenotypes to that shown in strain 1529. These results indicate that NADase induction by CLI treatment is not restricted to specific strains and it could be a standard phenotype among CLI-susceptible S. pyogenes strains. We also analyzed four CLI-resistant strains. All four strains showed increased extracellular NADase activities at high concentrations of CLI that did not inhibit bacterial growth. These results indicated that the subinhibitory dose of CLI was not the critical factor for NADase induction. [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]

Aim/Background: Cancer is still known to be a devastating disease condition. Even after getting advancements in multimodality therapies, the success rate with respect to patient survival is very limited. Hence there is an emerging need to find alternative medication strategies for cancer treatment. Recently seaweeds are getting their significance due to its tremendous medicinal properties. Among different category of seaweeds, Padina gymnospora which is a brown seaweed is known to possess many important bioactivities including antimicrobial activity, anti-oxidative property and anti-inflammatory. Role of this seaweed in treating oral cancer cells is not much explored in detail. Hence, this study aims to understand the significance of Padina gymnospora in oral cancer cell line -- SAS. Materials and Methods: The ethanol extract of Padina was treated to oral cancer cell line -- SAS and incubated for 24 hr. After incubation, MTT assay was performed to find the efficiency of the extract. Further the cells were taken for protein extraction and subjected to SDS PAGE, two-dimensional electrophoresis (2D) coupled with tandem mass spectrometry approach. The differentially expressed proteins were further identified and subjected to bioinformatic analysis. Results and Conclusion: It was observed that pro apoptotic proteins including BIK, BAK1, BID, BAD and CASP8 were highly upregulated in the Padina treated samples compared with untreated control samples. This data clearly demonstrates that Padina gymnospora induces the apoptosis in the oral cancer cells. This study paves us the way to explore more about the therapeutic potential of Padina gymnospora in treating the oral cancer cells. [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]

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]

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]

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.

Copyright of Journal of Chinese Institute of Food Science & Technology / Zhongguo Shipin Xuebao is the property of Journal of Chinese Institute of Food Science & Technology Periodical Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The widespread occurrence of drug-resistant bacteria has increased interest in alternatives to antibiotics for combatting bacterial infections, among which bacteriophages play an important role. The ability of phage proteins to induce an anti-phage immune response can significantly limit the effectiveness of treatment, which was the basis for the study described in this article. The aim of the study was to assess the effects of bacteriophages on the induction of an anti-phage humoral response in calves. The study was conducted using phage components of experimental preparations and sera from calves treated and not treated with phages. Levels of G, M and A immunoglobulins were analysed by ELISA. The assay plates were coated with whole Escherichia coli and Mannheimia haemolytica phages and selected phage proteins obtained in sodium dodecyl sulphate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis. Neutralisation of phages by immunoglobulins was assessed by determining phage titres using double-layer plates. The results confirmed an increased anti-phage response affecting all immunoglobulin classes in the calf sera. The highest significant (P ≤ 0.05) level of antibodies was observed for IgG in the sera of calves receiving phages. The phage neutralisation test showed a significant differences (P ≤ 0.05) in the reduction of phage titres in comparison to untreated calves. Despite the induction of an anti-phage response, no significant negative effect on the antibacterial activity of phages was observed in vitro. [ABSTRACT FROM AUTHOR]

This study aims to compare the grain protein profile of four Brazilian cowpea cultivars (BRS Aracê, BRS Itaim, BRS Pajeú, and BRS Xiquexique) by two-dimensional electrophoresis (2-DE) and principal component analysis (PCA). 2-DE efficiently separate cowpea protein profiles, showing high homogeneity among the four cultivars. In addition, the principal component analysis indicated that there is a difference in abundance of proteins among the cultivars. The cultivars BRS Aracê and BRS Xiquexique, both biofortified in iron and zinc, were separated from the cultivars BRS Itaim and BRS Pajeú. These results demonstrate that protein profiles can be used to discriminate cowpea varieties. [ABSTRACT FROM AUTHOR]

Common bean (Phaseolus vulgaris L.) seeds are an economical protein source rich in bioactive compounds with antioxidant activity. In this work, we evaluated processed seeds from two common bean cultivars regarding protein profile and antioxidant capacity before and after enzymatic digestion in vitro. We provided protein maps by two‐dimensional electrophoresis (2‐DE) of seeds germinated during 36 h and 72 h. The principal component analysis (PCA) showed that protein abundance had more variation on germination times than the cultivar ones. Seeds germinated for 36 h showed greater antioxidant capacity compared to germination for 72 h and cooking, before and after enzymatic digestion. In protein isolates, cooking associated with digestion provided better antioxidant capacity. Germination time influences protein expression as well as the antioxidant capacity of common beans. Furthermore, in vitro digestion increases the antioxidant capacity of cooked bean protein isolates. [ABSTRACT FROM AUTHOR]

The objective of this study is to evaluate the changes in the motility and kinetic patterns of canine spermatozoa, capacitated and decapacitated, after the addition of seminal plasma protein fractions with different molecular weight. It has been proposed that proteins in seminal plasma support the survival of the spermatozoa and exert a dual effect: capacitation and decapacitation. The seminal plasma from fresh ejaculates was subjected to chromatographic separation and four protein fractions were obtained. Computer-assisted sperm analysis was used to determine the sperm subpopulations with specific motion and kinetic characteristics after incubation with each of the four protein fractions. Two-dimensional electrophoresis of the fractions that exhibit a significant effect on the capacitation and decapacitation was performed. By sperm class analyser, capacitation changes were observed in the sperm subpopulation with a high curvilinear velocity and amplitude of lateral head displacement incubated with the seminal plasma protein fraction with a high molecular weight, which was also reflected in the decreased linearity, straightness, and progressive motility. The sperm subpopulation incubated with the seminal plasma protein fraction with a low molecular weight seemed to undergo a process of decapacitation (decreasing of the curvilinear velocity, increasing of the linearity, straightness and showing progressive motility). Despite their ample panorama of actions, the role of seminal plasma proteins regarding capacitation and decapacitation is still undetermined. [ABSTRACT FROM AUTHOR]

Natural variability of stress tolerance in halophytic plants is of significance both ecologically and in view of identifying molecular traits for salt tolerance in plants. Using ecophysiological and proteomic analyses, we address these phenomena in two Tunisian accessions of the oilseed halophyte, Cakile maritima Scop., thriving on arid and semi-arid Mediterranean bioclimatic stages (Djerba and Raoued, respectively), with a special emphasis on the leaves. Changes in biomass, photosynthetic gas exchange and pigment concentrations in C. maritima plants treated with three salinity levels (0, 100 and 300 mM NaCl) were monitored for 1 month. Comparative two-dimensional gel electrophoresis (2-DE) revealed 94 and 56 proteins of differential abundance in Raoued and Djerba accessions, respectively. These salinity-responsive proteins were mainly related to photosynthesis and oxidative phosphorylation (OXPHOS). Although Djerba accession showed a lower biomass productivity, it showed a slightly higher CO2 assimilation rate than Raoued accession when salt-treated. Photosynthesis impairment in both accessions under salinity was also suggested by the lower abundance of proteins involved in Calvin cycle and electron transfer. A significant increase of protein spots involved in the OXPHOS system was found in Djerba accession, suggesting an increase in mitochondrial respiration for increased ATP production under saline conditions, whereas a lesser pronounced trend was observed for Raoued accession. The latter showed in addition higher abundance of proteins involved in photorespiration. Salt-challenged plants of Djerba also likely developed mechanisms for scavenging ROS in leaves as shown by the increase in superoxide dismutase and thioredoxin, while an opposite trend was found in Raoued. [ABSTRACT FROM AUTHOR]

Aim. To isolate and purify protein complexes – ATP synthase and RuBisCO – from pea leaf chloroplasts and study the effect of a microbiological fertilizer “Extracon” and sulfonamide inhibitors acetazolamide and ethoxyzolamide on the enzymatic activity of these proteins. Materials and methods. Chloroplasts were isolated from the leaves of two-week-old pea sprouts, protein complexes of purified thylakoid membranes were solubilized with digitonin (10 mg of digitonin per 1 mg of protein), the protein concentration was determined according to Lowry. Native electrophoresis with displacement of the charge of the soluble protein fraction from the chloroplast stroma, as well as membrane proteins, was carried out in the modified system of Anderson et al., Kolisnichenko et al. A modified Lemmley system was applied to the protein electrophoresis in the polyacrylamide gel in the presence of sodium dodecyl sulfate. The methods of Alain and Hintsik, as well as Gomorrah were used to determine the ATPase activity in the polyacrylamide gel. Visualization of the carbonic anhydrase activity in the polyacrylamide gel was performed by the method of Edwards and Petton. Results and discussion. Using physicochemical methods of potentiometry, spectrophotometry the ATPase, carbonic anhydrase and esterase activities of the enzymes were studied. The results obtained indicate that specific carbonic anhydrase inhibitors (acetazolamide and ethoxyzolamide) also block the esterase and ATPase activity of the enzyme complexes. “Extracon” (a multifunctional microbiological preparation) almost 1.5 times increases the activity of the enzymes, showing a complex activating effect of the fertilizer on both light and dark reactions of photosynthesis. Conclusions. The method of identification and isolation of RuBisCO and ATP synthase on the basis of two-dimensional electrophoresis and electrophoretic elution has been proposed. It allows determining the presence of certain enzyme activity of complexes at first in SDS plates (express analysis) and further to study the effect of various factors of endogenous and exogenous origin on the enzymatic properties of electrophoretically pure enzymes. The use of two-dimensional electrophoresis as a tool for assessing the impact of various factors of endogenous and exogenous origin on the plant cell and the plant as a whole through constant monitoring of the work and activity of enzyme systems of the plant cell is promising.

Vibrio cholerae can cause pandemic cholera in humans. The waterborne bacterium is frequently isolated from aquatic products worldwide. However, current literature on the impact of aquatic product matrices on the survival and pathogenicity of cholerae is rare. In this study, the growth of eleven non-O1/0O139 V. cholerae isolates recovered from eight species of commonly consumed fish and shellfish was for the first time determined in the eight aquatic animal matrices, most of which highly increased the bacterial biomass when compared with routine trypsin soybean broth (TSB) medium. Secretomes of the V. cholerae isolates (draft genome size: 3,852,021–4,144,013 bp) were determined using two-dimensional gel electrophoresis (2DE-GE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques. Comparative secretomic analyses revealed 74 differential extracellular proteins, including several virulence- and resistance-associated proteins secreted by the V. cholerae isolates when grown in the eight matrices. Meanwhile, a total of 8,119 intracellular proteins were identified, including 83 virulence- and 8 resistance-associated proteins, of which 61 virulence-associated proteins were absent from proteomes of these isolates when grown in the TSB medium. Additionally, comparative genomic and proteomic analyses also revealed several strain-specific proteins with unknown functions in the V. cholerae isolates. Taken, the results in this study demonstrate that distinct secretomes and proteomes induced by the aquatic animal matrices facilitate V. cholerae resistance in the edible aquatic animals and enhance the pathogenicity of the leading waterborne pathogen worldwide. [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]

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

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

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]

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]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background: Our previous two-dimensional electrophoresis experiment showed that the expression of LASP1 in patients with endometriosis was significantly higher than that of control endometrium. However, the molecular mechanism by which LASP1 is regulated in endometriosis/adenomyosis is unknown. Methods: Herein, qPCR was performed to analyze the expression levels of LASP1 and miR-218-5p between endometriosis (Ems) cells and control cells. Fluorescence in situ hybridization was carried out to measure the expression level of miR-218-5p in ectopic endometrium versus normal endometrium. After miR-218-5p mimic or inhibitor were transfected, the transwell experiment was carried out to see the effect of miR-218-5p on the migration of endometrial stromal cells (ESCs). EdU was used to measure cell proliferation rate. Dual-luciferase reporter assay was used to verify the binding of hsa-miR-218-5p to the 3'UTR of LASP1. Western blot and immunofluorescence analysis were carried out to identify the protein expression pattern of LASP1 and EMT markers in endometrial tissue. Results: The miR-218-5p is mainly secreted from blood vessels and expressed in the muscle layer around the endometrium, which inhibits the expression level of LASP1 by binding the 3'UTR region of LASP1 in normal ESCs. Overexpression of miR-218-5p impedes the epithelial-to-mesenchymal transition (EMT) and prevents the migration of ESCs and the expression of Vimentin in Ems. Conclusions: Our findings revealed that miR-218-5p in endometrial microenvironment prevents the migration of ectopic endometrial stromal cells by inhibiting LASP1. [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]