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

Vibrio parahaemolyticus uses bacterial secretion systems and integrative and conjugative elements (ICEs) to induce various diseases and to adapt to harsh environments, respectively. Information pertaining to the identity of secreted proteins and functional characterization of ICEs has been previously reported, but the relationship between these elements remains unclear. Herein we investigated secreted proteins of V. parahaemolyticus strains JHY20 and JHY20△ICE using two-dimensional gel electrophoresis and LC-MS/MS, which led to the identification of an ICE-associated secreted protein – dihydrolipoamide dehydrogenase (DLDH). Considering the data related to its physical and biochemical characterization, we predicted that DLDH is a novel immunogenic protein and associated with virulence in JHY20. Our findings indicate a potential relationship between ICE-associated transport and secreted proteins and shed light on the function of such transport mechanisms. We believe that our data should enhance our understanding of mobile genetic elements. [ABSTRACT FROM AUTHOR]

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

Male accessory gland (MAG) secretory proteins affect female reproductive physiology and behaviour when they are delivered to the female at the time of mating along with sperm. In our study, proteomic approaches were used to identify MAG proteins of Leucinodes orbonalis, a monophagous and destructive pest of brinjal. A set of 117 and 186 MAG spots were observed in virgin and mated males, respectively, using two‐dimensional gel electrophoresis (2D‐GE). Among the identified spots, 49 are unique to the virgin, whereas 118 are unique to mated MAGs, with 68 spots found to be common. The differentially expressed MAG proteins, 14 are up‐regulated, and 16 are down‐regulated in virgin after mating. We used matrix‐assisted laser desorption ionization (MALDI)–mass spectrometry (MS) to identify the 13 unique proteins in the MAG of virgin L. orbonalis and analysed the data of the Swiss‐Prot database using the Mascot search engine. The proteins were identified as proteolysis regulators, lipid transporters, olfactory proteins, metabolism‐related proteins, DNA‐binding proteins, and hexamerins. This is the first report on the gel‐based protein analysis of L. orbonalis MAGs. [ABSTRACT FROM AUTHOR]

Talazoparib (TAL) has been effectively used for the treatment of gBRCA1/2‐mutated HER2‐negative metastatic breast cancer. However, acquired resistance to TAL remains a major challenge that impedes the clinical success of TAL treatment. Therefore, elucidation of proteins and pathways that contribute to or are affected by the TAL resistance is urgently needed to improve the treatment response and provide novel treatment strategies for advanced metastatic breast cancers. Herein, we aimed to investigate the altered protein signatures in TAL‐resistant triple‐negative breast cancer (TNBC) cells by comparing with the TNBC parental cell line via proteomic analysis. After validation of TAL‐resistance by WST‐1 and Annexin V analysis, two‐dimensional gel electrophoresis (2DE)‐based proteomic analysis coupled to matrix‐assisted laser desorption/ionization (MALDI)–time of flight (TOF) mass spectrometry was performed to identify differentially regulated proteins. The findings revealed the identities of 10 differentially regulated proteins in TAL‐resistant TNBC cells whose bioinformatic analysis predicted changes in EGF/FGF signaling pathways as well as in the AMPK signaling pathway. In addition, phosphorylation/dephosphorylation dynamics were predicted to be altered in TAL‐resistant cells. The proteins identified in this study might be the targets to overcome TAL resistance for the treatment of TNBC. [ABSTRACT FROM AUTHOR]

Background: Both the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. H5N1 viruses can cause severe damage and are associated with a high mortality rate, but H9N2 viruses do not cause such outcomes. Our purpose was to use proteomics technology to study the differential expression of mitochondrial-related proteins related to H5N1 and H9N2 virus infections. Methods: According to the determined viral infection titer, A549 cells were infected with 1 multiplicity of infection virus, and the mitochondria were extracted after 24 h of incubation. The protein from lysed mitochondria was analyzed by the BCA method to determine the protein concentration, as well as SDS-PAGE (preliminary analysis), two-dimensional gel electrophoresis, and mass spectrometry. Differential protein spots were selected, and Western blotting was performed to verify the proteomics results. The identified proteins were subjected to GO analysis for subcellular localization, KEGG analysis for functional classification and signaling pathways assessment, and STRING analysis for functional protein association network construction. Results: In the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. Protein spots were further subjected to mass spectrometry identification and removal of redundancy, and 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The differential expression of 70-kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase was verified by Western blot, and the results were consistent with the proteomics findings. Functional analysis indicated that these differentially expressed proteins were primarily involved in apoptosis and metabolism. Conclusions: Compared with their expression in the H9N2 group, the differential expression of eight mitochondrial proteins in the H5N1 group led to host T cell activation, antigen presentation, stress response, ATP synthesis and cell apoptosis reduction, leading to higher pathogenicity of H5N1 than H9N2. [ABSTRACT FROM AUTHOR]

At present, there is not much research on proteomics in the field of postharvest research on fruits and vegetables. In some research groups, postharvest induction of resistance in fruit trees is a promising technique. In order to obtain a clear protein gel map, this paper establishes an optimization system for two-dimensional electrophoresis of pear leaf protein. Optimize the steps of protein extraction method, hydration operation mode of the strip, setting of isoelectric focusing (IEF) program, and protein loading. The results showed that the homogenate method achieved good results in pear leaves in different environments. There were fewer vertical and horizontal striations and more protein spots can be observed by naked eyes in the whole electrophoresis map, and the distribution of points is even and clear. The experimental method in this paper effectively improves the resolution and lays a foundation for the differential proteomics research of pear leaves in different environments. [ABSTRACT FROM AUTHOR]

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

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

Objective: Proper characterization of hydatid cyst fluid (HCF) is useful for diagnostic and follow up purposes of cystic echinococcosis/hydatidosis, which is an important zoonotic disease. In this regard, proteomics methods are very helpful. The present study was conducted to compare three protein extraction methods for HCF collected from sheep liver hydatid cysts including, trichloracetic acid (TCA)/Acetone precipitation, TCA/Acetone along with dialysis, and combination of 2-D Clean-up Kit and dialysis followed by two-dimensional electrophoresis (2-DE), to achieve better resolution in the proteomic characterization of HCF proteins. Results: The 2-DE of TCA/Acetone products showed a lot of smears in the background of gels; TCA/Acetone with dialysis showed greatly reduced smears while the 2-D Clean-up Kit together with dialysis showed sharp spots and least smears. Three-dimensional images of separated spots created by Progenesis SameSpots software showed the best result was achieved by 2-D Clean-up Kit and dialysis. [ABSTRACT FROM AUTHOR]

The proteostasis system of animals, including various types of protein modification during the growth stage, leads to an almost incomprehensible number of possible forms of protein, and each can regulate numerous functions. In the presented work, the composition of muscle tissue protein from different portions of piglets was studied to understand the main muscle protein formation. Comparative analysis of weaned piglets' main muscle protein from l. dorsi, biceps femoris, and brachiocephalicus were analyzed using two-dimensional electrophoresis. Changes in the staining intensity of protein fractions inherent in different muscles were revealed. As part of this work, candidate groups of pig muscle proteins have been selected. Eleven protein spots were revealed for the longest muscle of the back, and seven for the biceps; the muscles of the neck are characterized by indicators of low protein fraction volume. Among the proteins found, myosin light chains, phosphoglycerate mutase, troponins, and adenylate kinase is most likely present. The obtained results of protein identification in muscle tissues, obtained during the intensive growth period, will allow a more detailed understanding of protein regulation, function, and interactions in complex biological systems, which will subsequently be significantly important for biomonitoring health and predicting farm animals productivity. [ABSTRACT FROM AUTHOR]

Escherichia coli O157: H7 is a pathogenic bacterium which is recognized for the ability to resist multiple antibiotics; accordingly, complications occur in the treatment of infections caused by this bacterium. The Ib-M1 peptide and the IONP @ Ib-M1 bioconjugate have emerged as a new antimicrobial alternatives against E. coli O157: H7. The mechanism of action of Ib-M1 and IONP @ Ib-M1 against this bacterium is still unknown; therefore, the goal of this research was to identify the change in the proteins profile of E. coli O157: H7 after treatment with Ib-M1 and IONP @ Ib-M1 as a first step to determine its mechanism of action. For this, the proteins were obtained first, and then a two-dimensional electrophoresis was performed to finally determine the variability of the protein profiles. Once the protein profiles were obtained, an analysis of variance (ANOVA) was carried out. 72 differentially expressed proteins were identified, which can be connected to the effect on the bacterium's growth in the presence of Ib-M1 and IONP @ Ib-M. These proteins are involved in acyl groups transfer processes (Yhbs protein), lipoprotein translocation (LolA protein) and amino acid transport (GpmA protein), among others. [ABSTRACT FROM AUTHOR]

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

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Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen causing severe economic loss. However, the molecular mechanism of silkworm resistance to BmNPV and the interactions of this virus with the host during infection remain largely unclear. To explore the virus-binding proteins of silkworms, the midgut subcellular component proteins that may interact with BmNPV were analyzed in vitro based on one- and two-dimensional electrophoresis and far-western blotting combined with mass spectrometry (MS). A total of 24 proteins were determined to be specifically bound to budded viruses (BVs) in two subcellular fractions (mitochondria and microsomes). These proteins were involved in viral transportation, energy metabolism, apoptosis and viral propagation, and they responded to BmNPV infection with different expression profiles in different resistant strains. In particular, almost all the identified proteins were downregulated in the A35 strain following BmNPV infection. Interestingly, there were no virus-binding proteins identified in the cytosolic fraction of the silkworm midgut. Two candidate proteins, RACK1 and VDAC2, interacted with BVs, as determined with far-western blotting and reverse far-western blotting. We speculated that the proteins interacting with the virus could either enhance or inhibit the infection of the virus. The data provide comprehensive useful information for further research on the interaction of the host with BmNPV.

Eight protein substances that undergo changes during autolysis were found using the electrophoretic methods with the following mass spectrometric identification. The revealed protein substances have different origin: structural proteins (fragments of troponins T and myosin light chains), and metabolic proteins (creatine kinase, pyruvate kinase and alpha-enolase). The decomposition of the fractions of fast skeletal muscle troponin T in 28.0 kDa, 27 kDa and 26.5 kDa was most pronounced.Identification of constitutive proteins and detection of the products of post — mortem degradation of protein molecules make them suitable candidates for meat quality markers and the following study of these specific fragments will lead to better understanding of the proteolytic activities that take part in the post mortem muscle transformation into meat.

One of the ways to address the food quality issues facing the industry is the development of standardized and certified methods related to the conduct of in-depth studies of biochemical indicators of quality and safety of meat and meat products. The world laboratory practice in the field of food quality and safety shows a constant expansion of the list of controlled indicators of food raw materials and products. An important feature of the modern period in the development of biomedical and biotechnological research is the introduction of a whole complex of postgenomic technologies, which are based on a systematic approach to the study of the functioning of the mammalian proteome in various physiological and pathological conditions, including the formation and development of alimentary-dependent pathologies. In this regard, the problem of multilateral study of food products, in particular their identification, is the most relevant, because the modern technology of their production has undergone significant changes and requires the development of “gentle “ processing modes. They concern raw materials and auxiliary materials used at all stages of production. This and new technologies of production of protein products from plant raw materials, as well as the introduction of food raw materials and food additives of artificial origin and the excess introduction of additives of plant and animal origin can cause falsification of products, as well as affect the health of the consumer. Food quality assessment includes the control of components of finished products. It is most difficult to determine the proportion of muscle protein in multi-component meat products that have undergone heat treatment. Therefore, in practice, when assessing the quality of food products, there is a need to identify its real composition in accordance with the declared normative documents. Currently, a promising area of research in the field of determining the composition of finished food is the selection of biomarkers of various components. Therefore, it is important to develop a methodology for the identification of biochemical changes in food raw materials under the influence of technological factors using modern research methods. This paper provides an overview of the protein and peptide analysis methodology, including the latest technologies that are becoming increasingly important.

Abstract The aim of study was to isolate, characterize and analyse the protein profiles of Myoviridae-like bacteriophages obtained from M. haemolytica using MALDI TOF mass spectrometry. The material consisted of the M. haemolytica reference strain ATCC® BAA410, reference serotypes A1, A2, A5, A6, A7, A9, and A11, and wild-type isolates of serotype A1. Bacteriophage morphology was examined with a transmission electron microscope. The proteins were separated in SDS-PAGE and two-dimensional electrophoresis and characterized by MALDI-TOF. Among the phages obtained, seven were specific for strains A1, A2, A5, A6, A7 and 25, and PHL-1 was specific for the BAA410 strain. The protein profiles for the phages were very similar to one another, but differed from the reference phage in that they lacked protein fractions with molecular weights of 22.9, 56.3 and 73.1 kDa. 2D electrophoresis revealed significant differences in the size of proteins and their localization in the pH gradient. The most similar profiles were observed in phages specific for strains BAA-410 and A6. In all profiles two main spots were observed in the molecular weight range from 44 to 70 kDa at pH

The main objectives of the study were to: (1) deeply analyse the serum protein composition of Equus caballus, (2) assess the effectiveness of the high-abundant protein depletion and improve the concentration of medium- and low-abundant proteins. The analysis were performed on the blood plasma of three healthy part-Arabian mares. The implementation of two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation – time of flight mass spectrometry allowed us to establish a horse plasma proteome map. Serum proteins were resolved at pH 4 to 7, followed by 12% SDS-PAGE. As a result 136 spots were successfully identified, representing the products of 46 unique genes. Of these, 22 gene products have not been previously identified in horse serum/plasma samples using proteomic tools. Gene ontology analysis showed that almost 30% of all identified gene products belong to the coagulation and complement cascades. These results can undoubtedly serve as a useful and prospective prerequisite for the future analysis of horse plasma proteome changes in different physiological and pathophysiological conditions. The use of the medium- and low-abundant protein enrichment tool increased their abundance and allowed us to identify a higher number of protein gene products. The highest depletion efficiency was observed for the most abundant plasma proteins, that is albumin, IgG heavy chains and serotransferrin.

At present, different methods are used to accumulate functional peptides in meat raw materials, including the use of spontaneous microflora during autolysis, the use of the microbial enzymes (the application of starter cultures) and the use of the non-microbial enzymes (enzymes of animals and plant origin). Each method has its own specific characteristics of an impact on raw materials, which requires their detail study. This paper examines an effect of spontaneous microflora of fermented meat products from horsemeat on formation of biologically active peptides. Using the T-RFLP analysis, it was established that in air dried and uncooked smoked sausages produced with the use of the muscle tissue of horsemeat as a raw material, a significant proportion of microflora was presented by lactic acid microorganisms. The highest content of lactic acid microflora was observed in sample 1 (52.45 %), and the least in sample 3 (29.62 %). Sample 2 had the medium percent content of microflora compared to samples 1 and 3 — 38.82 %. It is necessary to note that about 25 % of microflora was unculturable; i.e., it had metabolic processes but did not grow on culture media. In the samples, the representatives of Actinobacteria and Pseudomonadales were found. Pathogenic and conditionally pathogenic microflora was not detected. Not only quantitative but also qualitative changes were observed in the studied samples. For example, in samples 1 and 2, the fractions of amilo-1,6-glucosidase, fast-type muscle myosin-binding-protein C; glucose-6-phosphate isomerase; fast skeletal muscle troponin I, phosphoglycerate kinase, pyruvate kinase and skeletal muscle actin were found, which were absent or reduced in sample 3. Therefore, in the studied product, good preservation of the main spectra of muscle proteins was observed, and the identified fractions, apparently, can be sources of new functional peptides. Not only quantitative but also qualitative changes were observed in the studied samples. For example, in samples 1 and 2, the C-terminal fragments of the myosin heavy chain were found, which were absent in sample 3. Also, the significant content of myoglobin was revealed in samples 2 and 3, and the myosin light chain was found in sample 1. Therefore, in the studied product, good preservation of muscle proteins myosin and myoglobin, which can be a source of new functional peptides, was observed. Based on the results of tandem mass-spectrometry, the proteins and natural short peptides present in the analyzed extracts were identified by the obtained masses. They belonged mainly to different peptides of equine myoglobin. Also, we identified several fragments, among which fast skeletal muscle troponin T and muscle creatine kinase were found. The obtained materials can be regarded as an experimental basis for the directed impact of starter cultures with a possibility to predict the protein and peptide composition of a finished product including with the aim of obtaining biologically active peptides.

The results of studying the effect of various temperatures on the protein composition of minced meat from porcine m. longissimus dorsi by two-dimensional electrophoresis are presented. The most complete distribution of protein fractions was observed in fresh raw minced meat, and when it was exposed to negative temperature, there was a sharp decrease in protein components (carbonic anhydrase 3, αβ-crystallin), as well as a decrease in the staining intensity of protein spots of the main constitutive fractions (tropomyosin alpha 1, myosin light chain 1). In the case of heat treatment, structural muscle proteins were retained with some changes in high molecular weight fractions, namely, protein molecules degraded to compounds with a simpler structure. It was noted that fractions of tropomyosin β-chain, triosephosphate isomerase 1, myosin light chains 2 were not detected after minced meat was frozen, while tropomyosin alpha 1 was retained in all samples.

Photosynthesis, an indispensable biological process of plants, produces organic substances for plant growth, during which photorespiration occurs to oxidize carbohydrates to achieve homeostasis. Although the molecular mechanism underlying photosynthesis and photorespiration has been widely explored, the crosstalk between the two processes remains largely unknown. In this study, we isolated and characterized a T-DNA insertion mutant of tomato (Solanum lycopersicum) named yellow leaf (yl) with yellowish leaves, retarded growth, and chloroplast collapse that hampered both photosynthesis and photorespiration. Genetic and expression analyses demonstrated that the phenotype of yl was caused by a loss-of-function mutation resulting from a single-copy T-DNA insertion in chaperonin 60α1 (SlCPN60α1). SlCPN60α1 showed high expression levels in leaves and was located in both chloroplasts and mitochondria. Silencing of SlCPN60α1 using virus-induced gene silencing and RNA interference mimicked the phenotype of yl. Results of two-dimensional electrophoresis and yeast two-hybrid assays suggest that SlCPN60α1 potentially interacts with proteins that are involved in chlorophyll synthesis, photosynthetic electron transport, and the Calvin cycle, and further affect photosynthesis. Moreover, SlCPN60α1 directly interacted with serine hydroxymethyltransferase (SlSHMT1) in mitochondria, thereby regulating photorespiration in tomato. This study outlines the importance of SlCPN60α1 for both photosynthesis and photorespiration, and provides molecular insights towards plant genetic improvement. [ABSTRACT FROM AUTHOR]

All‑trans retinoic acid (ATRA) and arsenic trioxide (As2O3) are currently first‑line treatments for acute promyelocytic leukemia (APL). However, a number of patients with APL are resistant to ATRA but still sensitive to As2O3, and the underlying mechanisms of this remain unclear. In the present study, two‑dimensional gel electrophoresis, mass spectrometry and other proteomic methods were applied to screen and identify the differentially expressed proteins between the retinoic acid‑sensitive cell lines and drug‑resistant cell lines. The results demonstrated that in retinoic acid‑resistant NB4‑R1 cells, the protein expression of cofilin‑1 was markedly increased compared with that in the drug‑sensitive NB4 cells. Subsequently, the effects of cofilin‑1 on As2O3‑induced apoptosis in NB4‑R1 cells were further investigated. The results revealed that cell viability was markedly suppressed and apoptosis was increased in the As2O3‑treated NB4‑R1 cells, with increased expression levels of cleaved‑poly (ADP‑ribose) polymerase and cleaved‑caspase 12. Cofilin‑1 expression was significantly decreased at both the mRNA and protein levels in the As2O3‑treated group compared with the control. Western blotting further revealed that As2O3 treatment decreased the cytoplasmic cofilin‑1 level but increased its expression in the mitochondrion. However, the opposite effects of As2O3 on the cytochrome C distribution were found in NB4‑R1 cells. This suggested that As2O3 can induce the transfer of cofilin‑1 from the cytoplasm to mitochondria and trigger the release of mitochondrial cytochrome C in NB4‑R1 cells. Moreover, cofilin‑1 knockdown by its specific short hairpin RNA significantly suppressed As2O3‑induced NB4‑R1 cell apoptosis and inhibited the release of mitochondrial cytochrome C. Whereas, overexpression of cofilin‑1 using a plasmid vector carrying cofilin‑1 increased the release of cytochrome C into the cytoplasm from the mitochondria in As2O3‑treated NB4‑R1 cells. In conclusion, cofilin‑1 played a role in As2O3‑induced NB4‑R1 cell apoptosis and it might be a novel target for APL treatment. [ABSTRACT FROM AUTHOR]

Canine mammary tumours (CMTs) are regarded as invasive with a high rate of recurrent and metastasis in intact female dogs. Tumour diagnosis, therefore, is an important step in predicting and monitoring tumour progression. This study was designed to identify protein expression on CMTs by employing a proteomic approach. The primary cell culture from benign mixed tumour, simple carcinoma, complex carcinoma and normal mammary gland were established, and two‐dimensional electrophoresis (2DE) was subsequently performed. The different spots on each sample type were collected for identification using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). The results indicated that cytokeratin 5 (CK5) and transketolase (TKT) were identified in benign mixed tumour cells and complex carcinoma cells. In contrast, cytokeratin 18 (CK18) and pyruvate kinase PKM were identified in simple carcinoma cells. Moreover, alpha‐2‐HS‐glycoprotein tumour antigen was identified specifically in complex carcinoma cells. In addition, ATP‐dependent 6‐phosphofructokinase platelet type and elongation factor 2 proteins were observed in benign cells. In conclusion, all expressed proteins in this study have been recognized for acting as their expression that differs from healthy mammary epithelial cells. Expectantly, this study identified the expressed proteins that might be useful in further diagnostic biomarker studies on CMTs. [ABSTRACT FROM AUTHOR]

The aim of this study was to analyze the effects of the cryopreservation process on the protein profile of ram sperm using two-dimensional electrophoresis (2-DE) coupled with mass spectroscopy. Semen was collected from five rams and cryopreserved in a Tris-based extender supplemented with glycerol and egg yolk as the main cryoprotectants. The fresh and post-thaw sperm total proteins were extracted and purified, followed by the 2-DE. The differential proteins in the stained gel were determined by mass spectrometry. The results indicated that there were 39 differential proteins between fresh sperm and frozen-thawed sperm. Among these proteins, the abundance of 28 proteins in fresh sperm was higher than those in post-thaw sperm (P < 0.05). However, 11 proteins in post-thaw sperm were up-regulated instead. The gene ontology (GO) analysis showed that most of differential proteins were implicated in cellular process, metabolism and regulation of the biological process. The networks of protein-protein interaction indicated a strong interaction among these differential proteins, which may be involved in sperm metabolism, acrosomal function, sperm motility, and reducing ROS level. In conclusion, the cryopreservation process modifies the proteome of ram sperm, which may be directly associated with ram sperm cryodamage, consequently influencing their fertility. Additionally, these differential proteins can be used as biomarkers for evaluation of frozen ram semen quality. • Cryopreservation alters the ram sperm proteome. • Comparison of the spermatozoa proteomes between fresh and frozen in rams. • ATP5B and PRDX5 may be used as biomarkers to evaluate the quality of ram semen [ABSTRACT FROM AUTHOR]

In this study, two bread wheat (Triticum aestivum L.) cultivars, Pishgam (drought-tolerant) and Shahryar (drought-sensitive), were grown in the greenhouse under control and moisture stress conditions. Based on phenological and morpho-physiological results, Pishgam was confirmed as a moisture stress tolerant cultivar. In the fallowing, at the start of heading time, its treated and untreated flag leaves were sampled for two-dimensional electrophoresis (2-DE) based on proteomics approach. Among approximately 263 protein spots appearing in two-dimensional gels, 23 and 10 protein spots were up- and down-regulated, respectively. Among these differentially expressed proteins, 11 proteins with more differences were identified by MALDI TOF/TOF MS which allocated to six functional protein groups involved in photosynthesis or respiration, carbohydrate metabolism, energy metabolism, chaperon, lipid metabolism and unknown function. We report this for the first time that chloroplastic acyl carrier protein synthase I and chloroplastic 20 kDa chaperonin proteins were significantly changed in wheat in response to moisture stress. [ABSTRACT FROM AUTHOR]

Introduction: 15‐deoxy‐Δ12,14‐prostaglandin J2 (15d‐PGJ2) causes neuronal apoptosis independently of its nuclear receptor, peroxysome‐proliferator activated receptor γ. Its membrane receptor, chemoattractant receptor‐homologous molecule expressed on Th2 cells (CRTH2), did not also mediate the neurotoxicity of 15d‐PGJ2. In the present study, we ascertained whether membrane targets beside CRTH2 were involved in the neurotoxicity of 15d‐PGJ2. Methods: Neuronal membrane targets for 15d‐PGJ2 were separated by two‐dimensional electrophoresis, identified by proteomic approach. Their localizations were detected by microscopic immunofluorescence study. Cell viability and apoptosis was evaluated by MTT‐reducing activity and caspase‐3 activity, respectively. Results: Voltage‐dependent anion channel 1 (VDAC1) was identified as one of membrane targets for 15d‐PGJ2. Modification of VDAC1 with 15d‐PGJ2 was detected by pull‐down assay. VDAC1 was detected in the plasma membrane and localized on the neuronal cell surface. VDAC1 was partially colocalized with membrane targets for 15d‐PGJ2. The anti‐VDAC antibody significantly attenuated the neurotoxicity of 15d‐PGJ2, accompanied by the suppression of the 15d‐PGJ2‐stimulated caspase‐3. Conclusion: These findings suggested that the plasmalemmal VDAC might be involved in the neurotoxicity of 15d‐PGJ2. [ABSTRACT FROM AUTHOR]

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

Vitrification is widely used in assisted reproductive technologies. However, the nervous system of vitrification offspring is of concern, and research on this is lacking. Vitrification-born mice (vitrification group), conventional in vitro fertilization-embryo transfer pregnancy-born mice (IVF group), and natural pregnancy-born mice (control group) were used to study the effects of vitrification of mouse embryos on protein levels in the brain of offspring. Proteins differentially expressed among the three groups were analyzed using proteomic methods, including two-dimensional electrophoresis, mass spectrometry, and bioinformatics analysis. Immunohistochemistry was used to verify the expression of differentially expressed proteins, such as Actb and Actg1, in each group. Twenty differentially expressed proteins in the brain tissue were identified using two-dimensional protein electrophoresis and mass spectrometry. Bioinformatics analysis revealed that these proteins were related to the development of anatomical structure, signal transduction, transport, cell differentiation, and stress response (biological processes) and the binding of molecules in vivo (molecular functions). The Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the differentially expressed proteins were involved in 54 pathways, including phagosome, metabolic pathway, apoptosis, and cysteine and methionine metabolism. Thus, embryo vitrification may cause some changes in the mouse brain at the protein level, necessitating further safety assessment. [ABSTRACT FROM AUTHOR]

Continuous and sustained production of good quality eggs and larvae is required to have economically viable and ecologically sustainable rearing of Seriola rivoliana. Nonetheless, the complete production cycle of these species has been challenging to achieve due to high mortality during embryonic and larval stages. The objective of the project was to study the expression of the proteins involved in the embryonic process of almaco jack. Proteomic characterization of fertilized eggs was performed using two-dimensional electrophoresis and mass spectrometry. Different vitellogenin proteins A, B, C and Ab, ß-actin, peroxiredoxin, superoxide dismutase 1, alpha subunit proteasome, and keratin II were identified to their functions related to embryonic development, energy metabolism, protein synthesis, cell structure, cytoskeleton, and antioxidant proteins with defense enzymatic activity. [ABSTRACT FROM AUTHOR]

Drought is the most important abiotic stress limiting wheat production worldwide. Triticum boeoticum , as wild wheat, is a rich gene pool for breeding for drought stress tolerance. In this study, to identify the most drought-tolerant and susceptible genotypes, ten T. boeoticum accessions were evaluated under non-stress and drought-stress conditions for two years. Among the studied traits, water-use efficiency (WUE) was suggested as the most important trait to identify drought-tolerant genotypes. According to the desirable and undesirable areas of the bi-plot, Tb5 and Tb6 genotypes were less and more affected by drought stress, respectively. Therefore, their flag-leaves proteins were used for two-dimensional gel electrophoresis. While, Tb5 contained a high amount of yield, yield components, and WUE, Tb6 had higher levels of water use, phenological related traits, and root related characters. Of the 235 spots found in the studied accessions, 14 spots (11 and 3 spots of Tb5 and Tb6, respectively) were selected for sequencing. Of these 14 spots, 9 and 5 spots were upregulated and downregulated, respectively. The identified proteins were grouped into six functional protein clusters, which were mainly involved in photosynthesis (36%), carbohydrate metabolism (29%), chaperone (7%), oxidation and reduction (7%), lipid metabolism and biological properties of the membrane (7%) and unknown function (14%). We report for the first time that MICP, in the group of lipid metabolism proteins, was significantly changed into wild wheat in response to drought stress. Maybe, the present-identified proteins could play an important role to understand the molecular pathways of wheat drought tolerance. We believe comparing and evaluating the similarity-identified proteins of T. boeoticum with the previously identified proteins of Aegilops tauschii, can provide a new direction to improve wheat tolerance to drought stress. • Of the 14 identified index spots, 9 and 5 spots were upregulated, and downregulated, under drought stress, respectively. • The identified proteins were mainly involved in photosynthesis process. • MICP protein, in the group of lipid metabolism proteins, was significantly changed in wild relatives of wheat (T. boeoticum) in response to drought stress. • MICP protein maybe play an important role to understand the molecular pathways of wheat drought tolerance genotypes. • While, the tolerant genotype contained a high amount of yield, yield components, and WUE; the susceptible genotype had higher levels of water use, phenological related traits, and belowground related characters. [ABSTRACT FROM AUTHOR]

Abiotrophia defectiva is a species of nutritionally variant streptococci that is found in human saliva and dental plaques and that has been associated with infective endocarditis. In our previous study, it was found that A. defectiva could bind specifically to saliva‐coated hydroxyapatite beads (SHA). This study identified a cell surface component of A. defectiva that promotes adherence to SHA beads. The binding of A. defectiva to SHA was reduced in the presence of antibodies against human proline‐rich protein (PRP); these results suggested that PRP may be a critical component mediating interactions between A. defectiva and the salivary pellicle. Two‐dimensional gel electrophoresis of whole A. defectiva cells followed by Far‐Western blotting was conducted by probing with synthetic peptides analogous to the binding region of PRP known as PRP‐C. The results indicate that an A. defectiva protein of 37 kDa interacts with PRP‐C. The results of amino‐terminal sequencing of the adhesive A. defectiva protein revealed significant similarity to glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). Recombinant GAPDH bound to immobilized PRP‐C in a dose‐dependent manner and binding of A. defectiva to SHA or to PRP was reduced in the presence of anti‐GAPDH antiserum. Western blotting or electron immunomicroscopic observations with anti‐GAPDH antiserum revealed that this protein was expressed in both cytosolic and cell wall fractions. These results suggest that A. defectiva could specifically bind to PRP via interactions with cell surface GAPDH; the findings suggest a mechanism underlying A. defectiva‐mediated adherence to saliva‐coated tooth surfaces. [ABSTRACT FROM AUTHOR]

Auricularia auricula (A. auricula) has been reported to have positive health effects. Therefore, this study was conducted to explore possible mechanisms of A. auricula‐induced anticancer activity in hepatocellular carcinoma (HCC) cells. First, using proliferative assay including MTT assay and real‐time cell electronic sensing technique, we founded that A. auricula has an antiproliferative effect on various cancer cell lines. Among five cancer cell lines, we focused on huh‐7 cell line, HCC cell line, due to that A. auricula has most dramatic antiproliferative effects on huh‐7 cell line. Following experiments, we founded that its antiproliferative effects was related with apoptosis‐inducing activities. For more investigation, a two‐dimensional electrophoresis based‐proteomic analysis (2DE‐GE) was employed for identification of possible target‐related proteins of A. auricula‐induced apoptosis. Among seven identified proteins, we focused on peroxiredoxin1 (PRDX1), which has been known as an anti‐oxidative enzyme. We confirmed downregulation of expression of PRDX1 following A. auricula treatment in mRNA and protein level. In order to obtain a more validation of the correlation of A. auricula‐induced cell death and anti‐oxidative enzyme. We investigated the level of anti‐oxidative enzymes, total glutathione (GSSG/GSH), and superoxide dismutase (SOD) levels in treated cells and PRDX1 gene‐silenced cells. GSH and SOD levels were decreased in the treated cells and PRDX1 gene‐silenced cells. Our findings suggest that A. auricula is a potent inducer of apoptosis in HCC cells via PRDX1‐inhibition pathways. Practical applications: Hepatocellular carcinoma (HCC) is among the leading causes of cancer‐related mortality. The principal treatment is surgical resection or liver transplantation. However, in most patients with HCC the diagnosis is often late, thereby excluding the patients from definitive surgical resection. Chemotherapy and radiotherapy are generally ineffective. Newer treatments are needed with several being in development. In this research article, we provide regulation mechanism of PRDX1 in HCC. PRDX1 has a proliferative effect and play a role in cancer development or progression. Overexpression of PRDX1 in cancer cells implies the role of PRDX1 in the cancer therapy. PRDX1 is currently being investigated as a new target for gene therapy in cancer. A. auricula is an apoptotic inducer of HCC cells through PRDX1 pathway. Regulation of PRDX1 in HCC may contribute to cancer treatment. Therefore, the potentials of targeting apoptosis would be a viable therapeutic strategy to improve the outcome of HCC patients. [ABSTRACT FROM AUTHOR]

Background and Objectives: Lactobacillus casei, an acid-resistant bacterium, has a protective role against the pathogens. So we aimed to determine the proteome of Lactobacillus casei ATCC39392 strain in response to different pHs of 5 and 7 using proteomic analysis. Materials and Methods: Supernatant and bacterial extraction of Lactobacillus casei ATCC39392 adapts at pHs 5 and 7 were isolated using sodium dodecyl sulfate--polyacrylamide gel and two-dimensional gel electrophoresis. The comparison of results showed that 7 protein spots were seen in pH 5 but not in pH 7. Afterward, they were excised and sent for MALDI TOF mass spectrometry to be identified. Results: Seven different proteins (four secretory and three structural) with different roles in human body health were identified. Prescribed proteins include putative cell wall associated Hydrolase, Glycoside Hydrolase, beta-N-Acetyl hexosaminidase, Histidine Kinase, Chaperonin, metal dependent Hydrolase and Lysozyme. Conclusion: Seven isolated proteins with anti-cancer and digestive impresses are proper subjects in therapy or drug delivery approaches especially oral drug usage for protection against stomach acidic area. [ABSTRACT FROM AUTHOR]

[Objective] In this study, we would search for the differentially expressed protein between the rapeseed addition line EE and rapeseed inoculated with the root-knot nematode by proteomic technique to screen the resistance-related protein against root-knot nematodes. [Method] Roots of the the resistant rapeseed addition line EE and susceptible rapeseed were used as materials. Differentially expressed proteins were screened by proteomic two-dimensional electrophoresis and Image Master 5. 0 software. Proteins were identified by MALDl-TOFTOF/ MS mass spectrometry. In addition, functional classification, metabolic pathway analysis, subcellular localization and proteins interaction analysis of identified proteins were carried using Gene Ontology, KEGG, WoLF PSORT and STRING. [Result] Madara and rapeseed addition line EE had 1184 and 1163 protein spots, respectively. 21 differentially expressed proteins were successfully identified by MALDlTOF/ TOF MS. Among these proteins, more than half of them were localized extracellularly (52. 38 %), followed by cytoplasm (28. 57 %), chloroplasts (4. 76 %), mitochondria (4. 76 %), and internal plasma reticulum (4. 76 %), vacuoles (4. 76 %). Analysis of protein interactions showed that endogenous a-amylase/ subtilisin inhibitors was connected wisth 3-isopropylmalate dehydratase large subunits, tubulin a-6 chain, epidermis-specific secreted glycoprotein EPl, BnaC04g03560D and BnaC06g34210D. [ Conclusion]The screened resistance-related proteins lay the foundation for cloning of resistance genes and breeding of new resistant varieties with molecular markers in future. [ABSTRACT FROM AUTHOR]

Copyright of Acta Scientiarum Polonorum seria Zootechnica is the property of West Pomeranian University of Technology 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.)

Fish intestine is an important constituent of the mucosal immune system. The gut and gut-associated lymphoid tissue construct a local immune environment. A Shewanella algae strain was previously reported to be a pathogen causing ascitic disease accompanied with intestinal inflammation in Cynoglossus semilaevis. This study aimed to investigate the intestine immune response in C. semilaevis to S. alga e infection at the protein level. Two-dimensional electrophoresis coupled with mass spectrometry proteomics was utilized to compare protein expression in the intestines from normal and S. algae -infected C. semilaevis. A total of 70 differentially expressed proteins (DEPs), consisting of 16 upregulated and 54 downregulated proteins, were identified in the intestine tissue of C. Semilaevis. These protein expression changes were further validated using western blot analysis and quantitative real-time PCR. Gene ontology enrichment analysis showed that these 70 DEPs could be assigned across three categories: "cellular components", "molecular function", and "biological process". Forty-one DEPs (six up-regulated and 35 down-regulated proteins) related to metabolic processes were identified. In addition, 20 DEPs (eight up-regulated and 12 down-regulated proteins) related to stress and immune responses were identified. A protein-protein interaction network generated by the STRING (Search Tool for the Retrieval of Interacting Genes/protein) revealed that 30 DEPs interacted with one another to form an integrated network. Among them, 29 DEPs were related to stress, immune, and metabolism processes. In the network, some of the immune related proteins (C9, FGB, KNG1, apolipoprotein A-IV-like, and PDIA3) were up-regulated and most DEPs involved in metabolism processes were down-regulated. These results indicate that the immune defense response of the intestine was activated and the intestinal function associated with metabolism processes was disturbed. This study provides valuable information for further research into the functions of these DEPs in fish. • The protein expression was compared in intestine of normal and S. algae -infected C. semilaevis. • Seventy differentially expressed proteins (DEPs) were identified. • Protein-protein interaction analysis indicated that 29 DEPs were interrelated. • The immune defense was activated and metabolic processes were disturbed in intestine. [ABSTRACT FROM AUTHOR]

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

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

Renal amyloidosis (RA) is a rare protein misfolding disorder that prompts progressive renal insufficiency. This study aimed to decipher proteomic changes in human sera to understand the pathophysiology and molecular mechanisms underlying the disease development, hence assisting in the diagnosis of RA. Serum proteomic analysis was performed using a gel-based approach followed by MALDI-TOF MS. RA patients with age and sex matched healthy volunteers were recruited from Max Super Speciality Hospital, New Delhi, India. Proteome profiles of serum revealed eight differentially expressed proteins namely, Zinc finger protein 624, Protein FAM183A, Calcium-binding mitochondrial carrier protein Scamc-3, V-type proton ATPase 116 kDa subunit A isoforms 2, Protein TXNRD3NB, ATP – dependent RNA helicase, Troponin C and Mitogen-activated protein kinase kinase kinase 7. These proteins were reported first time in RA. The increased levels of MAP3K7 and TROPONIN C were validated by bio-layer interferometry and their diagnostic accuracy was evaluated by ROC curve analysis. The differentially expressed proteins were predominantly associated with vesicular trafficking, transcriptional regulation, metabolic processes, apoptotic process and mitochondrial metabolism. The results indicate that these proteomic signatures may be considered as potential molecular targets for RA diagnostics and therapeutics subject to validation on large sample size. Abbreviations: AβP= Amyloid-beta protein, Aβ=Amyloid-beta, AL= Light chain amyloidosis, AA= Amyloid A, ALECT2= LECT2 amyloidosis, APS= Ammonium persulfate CKD= Chronic Kidney Diseases, EBRT= external beam radiation therapy, ESRD= End-Stage Kidney Disease, Glis2= Gli-similar 2, JNK= c-Jun NH 2-terminal kinase, MAPK= Mitogen-Activated Protein Kinase, MM=Multiple Myeloma, PHD= Prolyl hydroxylase, RA = Renal Amyloidosis, SAA= Serum Amyloid A, SD= Standard Deviation, Sepp= Selenoprotein, SCC= Squamous cell carcinoma, SDS= Sodium dodecyl sulfate, TEMED = tetramethyl ethylenediamine, TGF-Beta-1=Transforming growth factor- Beta-1, Trx = Thioredoxin, TrxR= Thioredoxin reductase [ABSTRACT FROM AUTHOR]

Glycoproteins are important biomarkers for cancers, while most glycoproteomics biomarkers suffering from low sensitivity and specificity due to their uncharacterized glycan structures. AZGP1 is a potential biomarker for salivary diagnostics of lung cancer, which is used as a model glycoprotein in this study for method development. We initially analyzed salivary N-glycoproteome by using lectin affinity chromatography and more than 300 N-glycoproteins were identified, including AZGP1. 7 gel spots of AZGP1 were resolved by two-dimensional gel electrophoresis and further confirmed by two-dimensional western blot as well as mass spectrometry. The isomeric glycan structures of AZGP1 in these spots were systematically characterized both at composition level and at structure level. Our results revealed 10 glycan compositions for salivary AZGP1, including core fucosylated glycans on Asn128 and sialylated glycans on Asn109 and Asn112. We further compared the glycan structures of salivary AZGP1 from lung cancer group and control group. Accordingly, 14 and 7 potential glycan structures were successfully revealed, respectively. In total, 15 glycan compositions and 22 potential glycan structures were identified and characterized for AZGP1, including some different structures with the same compositions. In particular, 5 potential glycan structures were identified as lung cancer unique signatures. Our developed strategy holds promise for thorough identification of glycan structures on a target glycoprotein biomarker. In-depth characterization of its glycan structures will ultimately enhance its sensitivity and specificity for cancer detection. Image 1 • Affinity chromatography and 2-DE were combined for the separation of glycoproteins. • Robust strategy was developed for glycan identification of target proteomic marker. • 15 glycan compositions and 22 glycan structures were characterized for saliva AZGP1. • Unique glycan structures of saliva AZGP1 were revealed for lung cancer. [ABSTRACT FROM AUTHOR]

Objectives: To evaluate improved protein extraction and two‐dimensional electrophoresis (2DE) separation methods with Japanese reference human hair (JRH); to determine whether fibre curvature is related to protein composition in curly and straight Japanese women's human hair (JHH) samples; and to identify proteins from JRH 2DE maps and expression differences between curly and straight JHH. Methods: Hair keratin and keratin‐associated proteins (KAPs) were extracted intact with dithiothreitol or tris(2‐carboxyethyl) phosphine from JRH or from curved or straight JHH. Extracted proteins were isoelectric‐focused on first‐dimensional pH gradient gel strips, then separated by molecular weight on laboratory‐made, second‐dimension, large format gels. The software compared protein abundance between duplicate 2DE gels of curved and straight JHH. Thirty‐eight proteins from a JRH 2DE gel were enzyme‐cleaved for MALDI‐TOF‐MS analysis to determine peptide composition, and where possible, de novo sequencing gave peptide sequence data. An in‐house human hair protein database incorporating ninety‐eight annotated protein sequences assisted MS analysis. Results: 2DE gels of tris(2‐carboxyethyl) phosphine‐extracted JRH improved keratin and KAP resolution and number compared to those of dithiothreitol‐extracted JRH and published commercially made second‐dimensional gels. Silver‐stained 2DE gels of the straight or curved JHH sets were remarkably similar. Over‐staining to reveal basic proteins caused poor resolution of the major acidic protein classes. Software comparisons of fifty‐nine resolved proteins revealed two were significantly different in abundance between curved and straight hairs but in insufficient amounts for MS analysis. MS identified twelve proteins from a JRH CBBG‐stained 2DE gel: six type II keratins, three type I keratins and three high sulphur proteins. A further eight were potential conformational isoforms and isoelectric variants of the identified proteins bringing the total to twenty identified or partially identified proteins. Conclusion: Root‐end human hair extraction with tris(2‐carboxyethyl) phosphine improves protein resolution and visualizes more proteins on large format 2DE gels. The two minor protein differences between duplicate straight or curved JHH 2DE gels were unlikely to change fibre structure from straight to curved hair. MS results confirmed that multiple isoforms exist of various hair proteins. Low sequence coverage prevented distinction between members in rows of homologous protein spots of similar molecular weight. [ABSTRACT FROM AUTHOR]

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

Objectives: The larval stage of Echinococcus granulosus in the internal organs of a human causes cystic hydatidosis. Thus, the identification of excretory/secretory (E/S) proteins from E. granulosus protoscoleces may help to discover new candidates for drug targets, along with immunodiagnostic and vaccine products. The purpose of this study was to assess the efficacy of albendazole and mebendazole on protein spots of E/S products of hydatid cyst protoscolices which can be helpful for detecting some target proteins for therapeutic purposes. Materials and Methods: In this experimental study, protoscoleces were divided into three groups in order to assess the effects of the drug. The first and the second groups were treated with albendazole/mebendazole, respectively, and the third group was considered as control. To determine the proteome spots, E/S proteins were precipitated with trichloroacetic acid/acetone and loaded on the isoelectric focusing gel, resulting in gel differentiation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the 20 mA constant current. In addition, the gels were stained with Coomassie Brilliant Blue. Finally, the protein spots resulted from 2-DE gels were analyzed using the Progenesis SameSpots software. Results: The comparison between the proteome gels of the treated groups and the control group showed that 35 protein spots are paired among which, 11 spots had significant differences in their expressions (P < 0.05). Conclusions: In general, the comparison between the expression levels of protein gel spots indicated the increasing expression of some protein spots while the suppression of the others. This suppression can be considered as a specific effect of the drugs on the E/S product of the protoscoleces of hydatid cyst. [ABSTRACT FROM AUTHOR]

Assays of protein contained in water-soluble fraction of encysting cells Colpoda cucullus Nag-1 by two-dimensional electrophoresis (2-D PAGE) and mass spectrometry (MS) revealed that the amount of β-tubulin abruptly increased in 2.5-10 h after encystment induction. Judging from the results that total α-tubulin content did not decrease much until 12 h after encystment induction, the result indicates that disassembly of microtubules may occur soon after encystment is induced. Therefore, we tried to visualize dynamics of microtubules. Immunofluorescence microscopy using anti-α-tubulin antibody indicated that disassembly of axonemal microtubules of cilia became within 1.5 h after encystment induction, and resorbed in 3 days. Although the cytoplasmic microtubules failed to be visualized clearly, encystmentdependent globulation of cells was promoted by taxol, an inhibitor of disassembly of microtubules. It is possible that a temporary formation of cytoplasmic microtubules may be involved in cell globulation. The phosphorylation level of actin (43 kDa) became slightly elevated just after encystment induction. Lepidosomes, the sticky small globes surrounding encysting cells, were vividly stained with Acti-stain 555 phalloidin, suggesting that 43-kDa actin or its homologues may be contained in lepidosomes. [ABSTRACT FROM AUTHOR]

One and two dimensional electrophoresis is a powerful technique for separation of proteins from complex biological sources. These are effective techniques of gene expression analysis at translational level especially for the species of which genome is not yet sequenced. High resolution electrophoresis separation of proteins from complex biological systems is crucial in protein science. Three protein extraction methods, such as Phenol, Multi detergent and TCA acetone were analyzed in the present investigation to find out appropriate protein extraction method amenable to one dimensional (1-D) and two dimensional (2-D) protein profiling of Haloxylon recurvum and H. salicornicum. Plant species selected for the present investigation are major components of saline and arid ecosystem and valuable source of food additives, nutritional ingredients, herbal medicines and fuel in the region. Phenolic components present in these plants are major hindrances for extraction of pure proteins. High concentrations of good quality proteins represented by clear, intense and more bands/spots in 1-D and 2-D gels were extracted with Phenol extraction method in comparison to TCA acetone and Multi detergent methods. This is possibly the first report on extraction and purification of proteins amenable to 1-D and 2-D proteome profiling of Haloxylon species of Indian arid zone, and forms a basis for further comparative proteome analysis of these two contrasting species with respect to salinity and drought tolerant ability for prospects. [ABSTRACT FROM AUTHOR]

Rice plants under field conditions experience various biotic and abiotic stresses and are adapted to survive using a molecular cross-talk of genes and their protein products based on the severity of a given stress. Seedlings of cultivated variety ASD16 (resistant to fungal disease, blast; tolerant to abiotic stress, salinity) were subjected to salt, drought, high temperature and low temperature stress as well as infection by Rhizoctonia solani and Xanthomonas oryzae pv. oryzae (causing reemerging diseases such as sheath blight and leaf blight), respectively, the sheath blight and bacterial leaf blight pathogens. Leaf proteome was analyzed using two-dimensional electrophoresis and differentially expressed proteins were identified using mass spectrometry. In addition to many other differentially expressed proteins, acidic endochitinase was found to be upregulated during fungal infection and drought treatment, and a germin-like protein upregulated during fungal infection and high temperature stress. These two proteins were further validated at the gene expression level using reverse transcription-PCR in dual stress experiments. Pot culture plants were subjected to fungal infection followed by drought and drought followed by fungal infection to validate chitinase gene expression. Similarly, plants subjected to fungal infections followed by high temperature stress and vice versa were used to validate the expression of germin-like protein-coding gene. The results of the present study indicate that chitinase and germin-like protein are potential targets for further exploration to develop rice plants resistant or tolerant to biotic and abiotic stresses. [ABSTRACT FROM AUTHOR]

Copyright of Revista Iberoamericana de Micologia is the property of Elsevier B.V. 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.)

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen causing severe economic loss. However, the molecular mechanism of silkworm resistance to BmNPV and the interactions of this virus with the host during infection remain largely unclear. To explore the virus-binding proteins of silkworms, the midgut subcellular component proteins that may interact with BmNPV were analyzed in vitro based on one- and two-dimensional electrophoresis and far-western blotting combined with mass spectrometry (MS). A total of 24 proteins were determined to be specifically bound to budded viruses (BVs) in two subcellular fractions (mitochondria and microsomes). These proteins were involved in viral transportation, energy metabolism, apoptosis and viral propagation, and they responded to BmNPV infection with different expression profiles in different resistant strains. In particular, almost all the identified proteins were downregulated in the A35 strain following BmNPV infection. Interestingly, there were no virus-binding proteins identified in the cytosolic fraction of the silkworm midgut. Two candidate proteins, RACK1 and VDAC2, interacted with BVs, as determined with far-western blotting and reverse far-western blotting. We speculated that the proteins interacting with the virus could either enhance or inhibit the infection of the virus. The data provide comprehensive useful information for further research on the interaction of the host with BmNPV. [ABSTRACT FROM AUTHOR]

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