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

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

In this study, the optimal hydrogen (H 2) production conditions of the high-efficiency H 2 -producing mutant strain Ethanoligenens harbinense YR-3 (carbon-nitrogen ratio 5.5, phosphate buffer 80 mM, initial pH 6.0, biotin 1.4 mg/L) are obtained by intermittent experiments. The maximum specific H 2 production rate of YR-3 (2.85 mol H 2 /mol glucose) was 1.4 times that of the wild strain ZGX4 (2.04 mol H 2 /mol glucose). The liquid-phase products are mainly ethanol and acetic acid, indicating that the metabolic pathway has not changed. Two-dimensional electrophoresis and mass spectrometry were used to compare and analyze the protein map differences between YR-3 and ZGX4. The results show that 1,6-fructose diphosphate aldolase and the flavoprotein in hydrogenase are highly expressed. This study will provide a theoretical basis for the genetic modification of high-efficiency H 2 -producing strains and the improvement of H 2 production capacity. [ABSTRACT FROM AUTHOR]

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

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.)

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

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]

Acacia mearnsii De Wild (black wattle) is one of the most important forestry crops in South Africa. The bark of this species contains high-quality tannins, while its wood is used for wood chips, charcoal and mining timber. One of the main problems faced by black wattle farmers is frost damage, thus there is an urgent need to develop frost-tolerant black wattle varieties. A quick method for the screening of frost tolerance variation would be beneficial for black wattle breeders and would result in the need for fewer families to undergo field trials. Frost tolerance in plants is directly related to their ability to undergo cold acclimation in response to changing weather conditions. During the cold acclimation process, many physiological changes occur, including modifications in protein expression. In this study, two-dimensional gel electrophoresis was used to investigate changes in protein expression among 40 black wattle families with varying levels of frost tolerance before and after a cold-stress treatment. The proteomic data generated from this was analysed using multivariate analysis. Six proteins were observed to be upregulated in frosttolerant black wattle families. The proteins were identified and have been previously shown to contribute to the protection of cellular membranes, maintenance of photosynthetic processes and the prevention of protein misfolding and aggregation. [ABSTRACT FROM AUTHOR]

Some health disorders, such as obesity and type 2 diabetes, are associated with a poor diet and low quality of the fat in it. The type and duration of the diet have an impact on the liver. This investigation uses the proteomic approach to identify changes in the mouse liver protein profile in adaptation to high-fat diets with different saturated fatty acid contents and linoleic acid (18:2n-6) to α-linolenic acid (18:3n-3) fatty acid ratios.

The recent advances in pigeon pea genomics, including high-quality whole genome and chloroplast genome sequence information helped develop improved varieties. However, a comprehensive Cajanus proteome, including the organelle proteome, is yet to be fully mapped. The spatial delineation of pigeon pea proteins at sub-cellular levels and inter-organelle communication could offer valuable insights into its defense mechanism against various stresses. However, the major bottleneck in the proteomic study is the lack of a suitable method of protein extraction and sample preparation compatible with two-dimensional gel electrophoresis (2D-PAGE), liquid chromatography-mass spectrometry (LCMS), or matrix-assisted laser desorption ionization-time of flight (MALDi-ToF). Our study introduces two efficient methods, one for isolating total proteins and another for organelle (chloroplast) proteins from various Cajanus spp. For total protein extraction, we have optimized a protocol using phenol in combination with a reducing agent (DTT) and protease inhibitor cocktail, also washing (6–7 times) with ice-cold acetone after overnight protein precipitation of total proteins. Our modified extraction method using phenol for total leaf protein yielded approximately 2-fold more proteins than the previously reported protocols from C. cajan (3.18 ± 0.11 mg/gm) and C. scarabaeoides (2.06 ± 0.08 mg/gm). We have also optimized a protocol for plastid protein extraction, which yielded 1.33 ± 0.25 mg/10 gm plastid proteins from C. cajan and 0.88 ± 0.19 mg/10 gm plastid proteins from C. scarabaeoides. The 2D-PAGE analysis revealed 678 ± 08 reproducible total protein spots from C. cajan and 597 ± 22 protein spots from C. scarabaeoides. Similarly, we found 566 ± 10 and 486 ± 14 reproducible chloroplast protein spots in C. cajan and C. scarabaeoides, respectively. We confirmed the plastid protein fractions through immunoblot analysis using antibodies against LHCb1/LHCⅡ type Ⅰ protein. We found both methods suitable for 2D-PAGE and mass spectrometry (MS). This is the first report on developing protocols for total and chloroplastic protein extraction of Cajanus spp. suitable for advanced proteomics research. [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]

Although helminth parasites have different life cycles, their hosts share similar immune responses involving Th2 cell-type. Here, we extracted proteins from the larvae of Anisakis simplex complex and Trichinella spiralis to identify common and specific antigens (or allergens) associated with the Th2 immune response. We performed two-dimensional electrophoresis analysis and Matrix-assisted laser desorption ionization–time of flight/time of flight (MALDI-TOF/TOF) experiments. We found 13 potentially immunogenic proteins, which included 5 spots specific to T. spiralis and 8 common to T. spiralis and A. simplex, by tandem mass spectrometry. These molecules were identified structurally as actin, tropomyosin, col cuticle N domain-containing protein, and heat shock proteins. We also identified molecules related to parasite-host immune modulation and interactions. Our results may contribute to reveal potential roles of immunological proteins in parasite-derived immune modulation. [ABSTRACT FROM AUTHOR]

Pregnancy has its own protein dynamics, reflecting the hormonal profile. Quantitative and qualitative changes in plasma protein profile may provide useful information about this condition. Any alterations may be a signal heralding clinical or subclinical pathology. The objective of our study was to compare the plasma protein profile between selected months of pregnancy in cows for a better understanding gestation course. For this purpose, we collected blood from healthy pregnant (n = 30; n = 6 for each pregnancy stage) and non‐pregnant (C; n = 6) Holstein‐Friesian cows during a routine veterinary examination. Collected samples were selected according to pregnancy month (first, second, third, sixth, and ninth), prepared, and separated by two‐dimensional electrophoresis. The Delta‐2D program compared and statistically evaluated scanned gel images from the appropriate months. The mean volume of the spots was considered. The MALDI TOF/TOF spectrometer was used to identify statistically significant proteins. There were 11 distinct proteins found, including peptidyl‐prolyl cis‐trans isomerase F, oligoribonuclease, and PRELI domain‐containing protein 3B (all of them have the lowest abundance in the C group), alpha‐1B‐glycoprotein, L‐gulonolactone oxidase, hemopexin (first month with higher abundance than control), alpha‐2‐HS‐glycoprotein (significantly higher abundance in the first month than in remaining groups), ermin (absent in the first month and lower abundance in the third and sixth months than in the remaining groups and control), endophilin‐A2 (significant differences between the control and the second, third, sixth, and ninth months), apolipoprotein A‐I (significant difference between control and the first and sixth months), alpha‐1‐antiproteinase (significant difference between control and the ninth month). The study demonstrated the distinctions between plasma protein composition and alterations during the pregnancy course which may potentially serve as diagnostic tools. [ABSTRACT FROM AUTHOR]

Two‐dimensional electrophoresis (2DE) is a gel‐based protein separation method based on size and charge which is commonly used for the characterization of host cell proteins (HCPs) during drug development in biotech and pharmaceutical companies. HCPs are a heterogenous mixture of proteins produced by host cells during a biologics drug manufacturing process. Different gel electrophoresis methods including traditional 2D SDS‐PAGE with silver and SYPRO Ruby fluorescent dye staining as well as two‐dimensional difference gel electrophoresis (2D‐DIGE) were compared for their relative abilities to characterize HCPs. SYPRO Ruby was shown to be more sensitive than silver stain in the traditional 2D gels both with and without product protein present. Silver stain also displayed a significant preference for staining acidic proteins over basic ones while SYPRO Ruby was more consistent in imaging proteins across different isoelectric points. The non‐traditional method of 2D‐DIGE provides high resolution and reproducibility when comparing samples with similar protein profiles but was limited in imaging HCP spots due to its narrow dynamic range. Overall, 2DE is a powerful tool to separate and characterize HCPs and is optimized by choosing the best stain or method for each specific application. Using a combination of two or more different 2DE staining methods, when possible, provides the most comprehensive coverage to support the characterization of a complex mixture like HCPs. However, in instances where only one staining method can be used, SYPRO Ruby is shown to be the more reliable, more sensitive, and easier to use traditional staining method for most HCP‐based applications. [ABSTRACT FROM AUTHOR]

Toxoplasmosis, while often asymptomatic and prevalent as a foodborne disease, poses a considerable mortality risk for immunocompromised individuals during pregnancy. Point-of-care serological tests that detect specific IgG and IgM in patient sera are critical for disease management under limited resources. Despite many efforts to replace the T. gondii total lysate antigens (TLAs) by recombinant antigens (rAgs) in commercial kits, while IgG detection provides significant specificity and sensitivity, IgM detection remains comparatively low in sensitivity. In this study, we attempted to identify novel antigens targeting IgM in early infection, thereby establishing an IgM on-site detection kit. Using two-dimensional gel electrophoresis (2DE) and mouse serum immunoblotting, three novel antigens, including EF1γ, PGKI, and GAP50, were indicated to target T. gondii IgM. However, rAg EF1γ was undetectable by IgM of mice sera in Western blotting verification experiments, and ELISA coated with PGKI did not eliminate cross-reactivity, in contrast to GAP50. Subsequently, the lateral flow reaction employing a strip coated with 0.3 mg/mL purified rAg GAP50 and exhibited remarkable sensitivity compared with the conventional ELISA based on tachyzoite TLA, which successfully identified IgM in mouse sera infected with tachyzoites, ranging from 10³ to 104 at 5 dpi and 104 at 7 dpi, respectively. Furthermore, by using standard T. gondii-infected human sera from WHO, the limit of detection (LOD) for the rapid fluorescence immunochromatographic test (FICT) using GAP50 was observed at 0.65 IU (international unit). These findings underline the particular immunoreactivity of GAP50, suggesting its potential as a specific biomarker for increasing the sensitivity of the FICT in IgM detection. [ABSTRACT FROM AUTHOR]

Aims Acinetobacter baumannii is a nosocomial pathogen known to be multidrug-resistant (MDR), especially to drugs of the carbapenem class. Several factors contribute to resistance, including efflux pumps, β -lactamases, alteration of target sites, and permeability defects. In addition, outer membrane proteins (OMPs), like porins are involved in the passage of antibiotics, and their alteration could lead to resistance development. This study aimed to explore the possible involvement of porins and OMPs in developing carbapenem resistance due to differential expression. Methods and results The antibiotic-susceptible and MDR isolates of A. baumannii were first studied for differences in their transcriptional levels of OMP expression and OMP profiles. The antibiotic-susceptible isolates were further treated with imipenem, and it was found that the omp genes were differentially expressed. Six of the nine genes studied were upregulated at 1 h of exposure to imipenem. Their expression gradually decreased with time, further confirmed by their OMP profile and two-dimensional gel electrophoresis. Conclusions This study could identify OMPs that were differentially expressed on exposure to imipenem. Hence, this study provides insights into the role of specific OMPs in antibiotic resistance in A. baumannii. [ABSTRACT FROM AUTHOR]

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

Copyright of Journal of China Medical University is the property of Journal of China Medical University Editorial 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.)

Two-dimensional electrophoresis (2-DE) is an important method for studying urinary proteomics, playing an important role in assessing changes in protein concentrations under different physiological conditions. This study aimed to evaluate the different proteins expressed in the urine of normal cycling, healthy cows. We attained the images from 2-DE of urinary proteins in cow urine. Then, we implemented and evaluated an image analysis protocol with 2D ImageMaster software. Using ImageMaster, different spots were identified. On two-dimensional electrophoresis gels, 40 protein spots from anionic fractions and 15 spots from the cationic fractions of urine were identified. These spots corresponded to a molecular weight (MW) of 10-113 kDa for anionic fractions, and a MW of 15-51 kDa in cationic fractions. There were 12 master spot features across both the gels. This dataset can serve as a foundation for further investigations into the functional roles of specific proteins and their implications in the biological system under study. [ABSTRACT FROM AUTHOR]

The aim of the present study was to determine the proteins in canine seminal plasma with direct effect on specific sperm parameters during fertilisation. The influence of seminal plasma proteins was fo-cused on three processes: capacitation, hyperactivation and decapacitation. Size exclusion chroma-tographic fractional separation of proteins from seminal plasma based on their molecular weights was performed. In addition, two-dimensional electrophoresis of two seminal protein fractions was done. The effect of protein molecules on the motion and kinetic parameters of the male gametes in in vitro conditions was monitored by computer-assisted sperm analysis. The activity of alkaline phosphatase, creatine kinase and alanine aminotransferase in whole seminal plasma and each chromatography fraction was measured spectrophotometrically using the BA-88A semi-automatic chemistry analyzer. The protein fractions with different molecular weights affected the in vitro motility and kinetic characteristics (curvilinear velocity, straightness, linearity and amplitude of lateral head displacement) of spermatozoa, leading to hyperactivation or initiating decapacitation kinetics changes. The detailed study of the seminal plasma proteome would add fundamental information about the processes associated with physiological changes occurring in spermatozoa before fertilisation. The study of the canine seminal plasma proteome could add relevant information about its effects on the fertilising ability of the male gametes and the changes occurring in them before fertilisation.

The aim of the present study was to determine the proteins in canine seminal plasma with direct effect on specific sperm parameters during fertilisation. The influence of seminal plasma proteins was focused on three processes: capacitation, hyperactivation and decapacitation. Size exclusion chromatographic fractional separation of proteins from seminal plasma based on their molecular weights was performed. In addition, two-dimensional electrophoresis of two seminal protein fractions was done. The effect of protein molecules on the motion and kinetic parameters of the male gametes in in vitro conditions was monitored by computer-assisted sperm analysis. The activity of alkaline phosphatase, creatine kinase and alanine aminotransferase in whole seminal plasma and each chromatography fraction was measured spectrophotometrically using the BA-88A semi-automatic chemistry analyzer. The protein fractions with different molecular weights affected the in vitro motility and kinetic characteristics (curvilinear velocity, straightness, linearity and amplitude of lateral head displacement) of spermatozoa, leading to hyperactivation or initiating decapacitation kinetics changes. The detailed study of the seminal plasma proteome would add fundamental information about the processes associated with physiological changes occurring in spermatozoa before fertilisation. The study of the canine seminal plasma proteome could add relevant information about its effects on the fertilising ability of the male gametes and the changes occurring in them before fertilisation. [ABSTRACT FROM AUTHOR]

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

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]

Boron, in the form of potassium tetraborate, has previously been found to be effective at inhibiting mango black spot disease, caused by Alternaria alternata. However, the mechanisms involved in this inhibition are largely unknown. In this study, A. alternata was treated in vitro with potassium tetraborate at a concentration of 5–10 mM for 48 or 72 h. The intracellular and mitochondrial proteins were extracted from mycelium and separated using two‐dimensional electrophoresis (2‐DE). Differentially expressed proteins (DEPs) were identified using bioinformatics tools and differences between protein spots were derived from mass spectrometry (MS). Using matrix‐assisted laser desorption ionization time‐of‐flight tandem mass spectrometry (MALDI‐ToF‐MS/MS), 96 intracellular and 56 mitochondrial DEPs were identified. The intracellular proteins identified were found to be involved in posttranslational modifications, protein turnover and chaperones, while the mitochondrial proteins were involved in electron transport chains. Our results demonstrate that various metabolic pathways are involved in the antifungal activity of boron. The differential expression of 20 genes was also verified at the mRNA level by reverse transcription‐quantitative PCR. Our study suggests that borate could be used as a potential substitute for synthetic fungicides to control this postharvest disease of mango fruits. [ABSTRACT FROM AUTHOR]

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

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]

Purpose: This study aimed to investigate the presence of autoantigens in the gastric juices of children. Methods: Gastric juice and serum samples were obtained from 53 children <15 years of age who underwent gastric endoscopy. Among these, 8, 22, and 23 participants were in the age groups 0-5, 6-10, and 11-15 years, respectively. These samples were analyzed using two-dimensional electrophoresis (2-DE), immunoblot analysis, and matrix-assisted laser desorption ionization-time of-flight mass spectrometry. Furthermore, we reviewed the histopathological findings and urease test results and compared them with the results of 2-DE and immunoblot analysis. Results: There were no statistically significant differences in urease test positivity, grades of chronic gastritis, active gastritis, or Helicobacter pylori infiltration of the antrum and body among the three age groups. Three distinct patterns of gastric juice were observed on 2-DE. Pattern I was the most common, and pattern III was not observed below the age of 5 years. Histopathological findings were significantly different among active gastritis (p=0.037) and H. pylori infiltration (p=0.060) in the gastric body. The immunoblots showed large spots at an approximate pH of 3-4 and molecular weights of 31-45 kDa. These distinct, large positive spots were identified as gastric lipase and pepsin A and C. Conclusion: Three enzymes, which are normally secreted under acidic conditions were identified as autoantigens. Further investigation of the pathophysiology and function of autoantigens in the stomach is required. [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]

Muscle tissue is one of the most dynamic and plastic tissues of the mammalian body and covers different roles, such as force generation and metabolic control. Muscular proteomics provides an important opportunity to reveal the molecular mechanisms behind muscle pathophysiology. To ensure successful proteomic analysis, it is necessary to have an efficient and reproducible protein extraction method. This study aimed to evaluate the efficacy of two different extraction protocols of muscle samples for two-dimensional gel electrophoresis. In particular, mouse muscle proteins were extracted by an SDS-based buffer (Method A) and by a UREA/CHAPS/DTE/TRIS solution (Method B). The efficacies of the methods were assessed by performing an image analysis of the 2DE gels and by statistical and multivariate analyses. The 2DE gels in both preparations showed good resolution and good spot overlapping. Methods A and B produced 2DE gels with different means of total spots, higher for B. Image analysis showed different patterns of protein abundance between the protocols. The results showed that the two methods extract and solubilize proteins with different chemical-physical characteristics and different cellular localizations. These results attest the efficacy and reproducibility of both protein extraction methods, which can be parallelly applied for comprehensive proteomic profiling of muscle tissue.

Post-translational modifications (PTMs), such as phosphorylation and O-N-acetyl-β-d-glucosaminylation (O-GlcNAcylation), are involved in the fine spatiotemporal regulation of protein functions, and their dynamic interplay is at the heart of protein language. The coexistence of phosphorylation and O-GlcNAcylation on a protein leads to the diversification of proteoforms. It is therefore essential to decipher the phosphorylation/O-GlcNAcylation interplay on protein species that orchestrates cellular processes in a specific physiological or pathophysiological context. However, simultaneous visualization of phosphorylation and O-GlcNAcylation patterns on a protein of interest remains a challenge. To map the proteoforms of a protein, we have developed an easy-to-use two-dimensional electrophoresis method with a single sample processing permitting simultaneous visualization of the phosphorylated and the O-GlcNAcylated forms of the protein of interest. This method, we termed 2D-WGA-Phos-tag-PAGE relies on proteoforms retardation by affinity gel electrophoresis. With this novel approach, we established the cartography of phospho- and glycoforms of αB-crystallin and desmin in the whole extract and the cytoskeleton protein subfraction in skeletal muscle cells. Interestingly, we have shown that the pattern of phosphorylation and O-GlcNAcylation depends of the subcellular subfraction. Moreover, we have also shown that proteotoxic stress condition increased the complexity of the pattern of PTMs on αB-crystallin., (© 2024 Wiley‐VCH GmbH.)

The prevalence of diet-related obesity is increasing dramatically worldwide, making it important to understand the associated metabolic alterations in the liver. It is well known that obesity is a multifactorial condition that is the result of complex integration between many gene expressions and dietary factors. Obesity alone or in conjunction with other chronic diseases such as diabetes and insulin resistance causes many health problems and is considered a major risk factor for developing non-alcoholic steatohepatitis (NASH) and cirrhosis. In this study, we aimed to understand the molecular mechanisms underlying early hepatic changes in the pathophysiology of high-fat diet (HFD)-induced abdominal obesity in rats. Hepatic protein profiles of normal diet and HFD-induced obesity for 24 weeks were analysed using two-dimensional differential gel electrophoresis (DIGE) and protein identification by MS. Fifty-two proteins were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), and computer-assisted DIGE image software analysis showed that eighteen major proteins were significantly differentially expressed between comparable groups, with 2·0–4·0-fold change/more ( P < 0·01). These proteins are regulated in response to a HFD, and differentially expressed proteins are involved in key metabolic pathways such as lipid metabolism, energy metabolism, detoxification, urea cycle and hepatic Ca homoeostasis. In addition, Western blot and immunohistochemistry of liver-specific arginase-1 (Arg-1) showed significant increased expression in the liver of high-fat-fed rats ( P < 0·01). Further, Arg-1 expression was correlated with NASH patients with obesity-related fibrosis (F0–F4). It is concluded that high-fat content may affect changes in liver pathways and may be a therapeutic target for obesity-related liver disease. Arg-1 expressions may be a potential pathological marker for assessing the progression of the disease.