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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., (© 2024 Mural Oncology. Biotechnology Progress published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

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., (© 2020 John Wiley & Sons Ltd.)

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., (© 2020 Society of Cosmetic Scientists and the Société Française de Cosmétologie.)

The Bradford assay is one of the most commonly used methods for protein quantification. However, in proteomic research, the lysis buffer generally used for dissolving proteins can cause some interference to the assay. The dye reagent of classical Bradford assay contains 8.50% (w/v) phosphoric acid, which is an important factor relating to the color yield of the assay. In this study, the phosphoric acid content in dye reagent was increased to 9.35% (w/v), 10.20% (w/v), and 11.05% (w/v) to evaluate the changes of interference and the effects of lysis buffer on the interaction between proteins and dye reagent. Results show that lysis buffer not only causes background interference but also affects the protein-dye chromogenic process. Analysis of different components in the lysis buffer showed that carrier ampholyte is the main factor that introduces interference to the Bradford assay. Detergents are well-known interfering compounds in the Bradford assay, but CHAPS and octyl b-D-glucopyranoside only cause slight interference. When the amount of phosphoric acid was increased from 8.50%(w/v) to 10.20% (w/v), the sensitivity of the Bradford assay to proteins in lysis buffer was increased, and the interference delivered by lysis buffer was considerably reduced., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Isoelectric focusing (IEF) is a technique in protein research that has been used since 1975 to separate proteins based on their isoelectric point. When combined with sodium dodecyl sulfate polyacrylamide gel electrophoresis, this procedure allows for high-resolution separation of cellular proteins for analytical purposes. Laboratories perform IEF by (a) using carrier ampholytes that migrate through a gel to create the pH gradient or (b) using immobilized pH gradients (IPG) that contain ampholytes bound covalently to a gel. Here we describe an IEF system that uses immobilized pH gradient (IPG) strips that undergo the desired current and voltage setting to separate proteins based on its charge in the first dimension followed by SDS PAGE to generate a two-dimensional map of serum proteins.

Two-dimensional electrophoresis (2-DE) is a classical separation technique for complex protein mixtures based on a first-separation step by isoelectric point (pI) followed by SDS-PAGE. Due to its high resolving power (including protein isoforms) and the capability to display post-translational modifications, 2-DE has been the technique of choice in many studies focused in characterizing allergens. In this chapter, we describe a standard protocol which enables efficient protein separation and subsequent protein identification by tandem mass spectrometry (MALDI-MS/MS).

In the nowadays mainly mass spectrometry-driven world of Proteomics, one can frequently read and hear that 2D gel-based electrophoresis methods are no longer used for proteome analysis. This short review is a compilation of facts why the gel-based workflow is not dead.

Host cell proteins (HCP) are a problematic set of impurities in downstream processing (DSP) as they behave most similarly to the target protein during separation. Approaching DSP with the knowledge of HCP separation behavior would be beneficial for the production of high purity recombinant biologics. Therefore, this work was aimed at characterizing the separation behavior of complex mixtures of HCP during a commonly used method: anion-exchange chromatography (AEX). An additional goal was to evaluate the performance of a statistical methodology, based on the characterization data, as a tool for predicting protein separation behavior. Aqueous two-phase partitioning followed by two-dimensional electrophoresis provided data on the three physicochemical properties most commonly exploited during DSP for each HCP: pI (isoelectric point), molecular weight, and surface hydrophobicity. The protein separation behaviors of two alternative expression host extracts (corn germ and E. coli) were characterized. A multivariate random forest (MVRF) statistical methodology was then applied to the database of characterized proteins creating a tool for predicting the AEX behavior of a mixture of proteins. The accuracy of the MVRF method was determined by calculating a root mean squared error value for each database. This measure never exceeded a value of 0.045 (fraction of protein populating each of the multiple separation fractions) for AEX. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1453-1463, 2016., (© 2016 American Institute of Chemical Engineers.)

This unit describes basic protocols for efficient and reproducible protein solubilization from a variety of biological samples, including cultured animal cells and tissues, plant cells and tissues, bacteria, nuclei, other subcellular organelles, plasma, serum, and other biological fluids. The optimized extraction process is strongly sample dependent and cannot be described for every type of sample. Instead, typical protocols are provided as general guidelines and illustrate good starting points for optimization of sample preparation. These solubilization procedures take into account the constraints imposed by two-dimensional electrophoresis and are thus well suited for proteomic approaches., (Copyright © 2015 John Wiley & Sons, Inc.)

Separating proteins according to two different gel-electrophoretic methods not only increases the resolution power for highly complex samples when compared to one-dimensional separations, but is also a valuable tool for protein and protein complex characterization. There are a number of different electrophoresis methods which can be combined. The combination of isoelectric focusing under denaturing conditions and SDS polyacrylamide gel electrophoresis delivers the highest resolution of all bio-analytic techniques. This is a short review on the history and state of the art of two-dimensional electrophoresis methods, and contains some practical tips for high resolution 2D electrophoresis, which are based on several decades of experience with this method.

Objective: To identify the male molecular causes of failures of IVF (with a deficient binding of spermatozoa to the zona pellucida, without any obvious oocyte anomaly), which are undetected by classical sperm analysis., Design: Case-control prospective study., Setting: University hospital., Patient(s): Proteomic profiles of spermatozoa in patients with a complete failure of fertilization and no spermatozoa bound to the zona pellucida were compared with those of controls (men with normal fertilization and cleavage rates after classical IVF for tubal indication)., Intervention(s): All samples were analyzed by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) after being divided into three fractions according to their isoelectric point., Main Outcome Measure(s): Differentially expressed proteins between infertile men and controls were identified by mass spectrometry., Result(s): Seventeen proteins differentially expressed between cases and controls were found. Twelve of these proteins were identified by mass spectrometry, and two may influence gametes interaction: laminin receptor LR67 and L-xylulose reductase (P34H)., Conclusion(s): This study shows that 2D-DIGE might be useful in finding potential targets for diagnosis and prognosis of idiopathic infertility in IVF., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

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]

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]

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

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

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

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

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

This study aimed to identify proteins in seminal plasma that are related to sperm protection during the cryopreservation process. Semen samples from 14 boars were collected and two aliquots were separated and subjected to cryopreservation of semen and seminal plasma proteomic study. The ejaculates were subjected to a slow cooling curve. The thawed semen was resuspended and analyzed for vigor and motility. To study the seminal plasma proteins, they were obtained by centrifugation as whole sêmen and subjected to two-dimensional electrophoresis. The gels were stained, scanned, and analyzed using the PDQuest application. To search for markers freezability was made a division of the animals into two groups: good freezability (GFEs) and bad (PFEs), through the results of vigour and motility after thawing. The experimental design was completely randomized. Six animals were considered GFEs the group and had a mean sperm motility and vigor to 2.2 ± 0.8 and 41.8 ± 22.9, respectively, while the PFEs group consisted of eight animals had an average of 1.9 ± 0.6 to 26.8 ± 17.5 vigor and sperm motility. Twenty-eight spots were found that differed significantly between groups, corresponding to 16 proteins. Two of these were expressed with greated intensity in GFEs and corresponded to only one protein of seminal plasm, the Carbohydrate-binding protein. Soçoncluded that Carbohydrate-binding protein can be used as a freezer marker for the swine species. [ABSTRACT FROM AUTHOR]

Since Charles Darwin discovered growth movements of the shoot tip (coleoptile) toward light, the grass coleoptiles have long been used as a model system to study plant growth. Rapid growth of the coleoptile is vital for successful seed germination and early seedling establishment. However, the proteome changes underlying the rapid growth of the coleoptiles are not yet clear in the model plant maize (Zea mays). In the present study, we investigated proteome changes in vivo occurring in the rapidly growing coleoptiles of maize with two‐dimensional gel electrophoresis combined with mass spectrometry. A quantitative comparison of the proteomes at 1.5, 3, and 5 days after germination showed significant changes in protein profiles with coleoptile growth. As a result, 31 differential abundance proteins (DAPs) representing 44 protein spots were identified, of which 21 DAPs with increased abundances were implied in growth‐related processes, including translational initiation, transcription regulation, protein and other compound synthesis, H+‐transmembrane transport and cytoskeleton organization. The selected DAPs were confirmed by reverse transcription quantitative PCR and immunoblot analysis. We suggested that the rapid growth of the coleoptile is largely due to its ability to quickly enhance relevant cellular processes, especially the increased synthesis of the growth‐related DAPs. The content of indole‐3‐acetic acid, salicylic acid, and jasmonic acid decreased significantly, and the content of gibberellins first decreased and then increased during the elongation of coleoptile. This study provides new insight into the significance of proteome changes in coleoptile growth. [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]

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]

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]

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]

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]

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]

Background: Marine medaka is among the most popular models of fish species for ecotoxicology and environmental research and proteomic studies are useful tools for understanding the molecular responses of medaka upon exposure to different environmental stressors. The preparation of high-quality protein samples is the key to producing high-quality two-dimensional gel electrophoresis (2-DE) results for proteomic analysis. In recent years, Trizol-based protein extraction has been gaining popularity because of its promising performance in producing high-quality 2-DE as well as the convenience of the method. Methods: Three Trizol-based approaches (Trizol method, Aliquot Trizol method and Trizol method with a commercial clean-up kit) were used to extract proteins from a marine medaka sample and 2-DE profiles were produced. Quality of the 2-DE profiles and effectiveness of the extraction methods were evaluated. For comparison, two common protein extraction methods (lysis buffer method and trichloroacetic acid (TCA)/acetone precipitation extraction) were also applied in parallel to Trizol-based approaches. Results: Any of the three Trizol-based approaches produced a high-quality 2-DE profile of marine medaka compared with both lysis buffer method and TCA/acetone precipitation extraction. In addition, Trizol method with a commercial clean-up kit produced the best 2-DE profile in terms of background clarity, number of spots and resolution of proteins. Conclusions: Trizol-based approaches offered better choices than traditional protein extraction methods for 2-DE analysis of marine medaka. The modified version of Trizol method with a commercial clean-up kit was shown to produce the best 2-DE profile. [ABSTRACT FROM AUTHOR]

Objective: Liverworts possess historical adaptive strategies for abiotic stresses because they were the first plants that shifted from water to land. Proteomics is a state-of-the-art technique that can capture snapshots of events occurring at the protein level in many organisms. Herein, we highlight the comparison and optimization of an effective protein extraction and precipitation protocol for two-dimensional gel electrophoresis (2-DE) of liverworts. Results: We compared three different protein extraction methods, i.e.,1.5 M Tris–HCl (pH 8.8), 50 mM Tris–HCl (pH 7.5), and polyvinylpolypyrrolidone (PVPP) extraction, followed by three precipitation methods, i.e., 80% ethanol, 80% acetone, and 20% tricholoroacetic acid (TCA)–acetone, in a liverwort Dumortiera hirsuta. Among these methods, 50 mM Tris–HCl (pH 7.5) extraction, followed by 20% TCA–acetone precipitation, appeared to be more suitable for 2-DE. Furthermore, we performed modifications during protein washing, re-solubilization in rehydration buffer and isoelectric focusing (IEF). The modifications provided us better results in terms of protein yield, resolution, spot numbers, and intensities for 2-DE gels of D. hirsuta and other two liverworts, i.e., Marchantia paleacea and Plagiochasma appendiculatum. Furthermore, we randomly selected spots from the 2-DE gel of D. hirsuta and identified using mass spectrometry, which confirms the applicability of this protocol for liverworts proteomics. [ABSTRACT FROM AUTHOR]

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

Background: Paracoccidioidomycosis (PCM) is a systemic mycosis commonly found in Latin America that is caused by distinct species of Paracoccidioides genus: Paracoccidioides brasiliensis complex (S1, PS2, PS3 and PS4) and Paracoccidioides lutzii. Its pathobiology has been recently explored by different approaches to clarify the mechanisms of host-pathogen interactions underpinning PCM. The diversity of clinical forms of this disease has been attributed to both host- and fungus-related factors. Methodology/Principal findings: For better understanding of the molecular underpinnings of host-fungus interactions, we evaluated in vivo virulence of nine Paracoccidioides brasiliensis complex isolates and correlated it to protein expression profiles obtained by two-dimensional gel electrophoresis. Based on the recovery of viable fungi from mouse organs, the isolates were classified as those having low, moderate, or high virulence. Highly virulent isolates overexpressed proteins related to adhesion process and stress response, probably indicating important roles of those fungal proteins in regulating the colonization capacity, survival, and ability to escape host immune system reaction. Moreover, highly virulent isolates exhibited enhanced expression of glycolytic pathway enzymes concomitantly with repressed expression of succinyl-CoA ligase beta chain, a protein related to the tricarboxylic acid cycle. Conclusions/Significance: Our findings may point to the mechanisms used by highly virulent P. brasiliensis isolates to withstand host immune reactions and to adapt to transient iron availability as strategies to survive and overcome stress conditions inside the host. [ABSTRACT FROM AUTHOR]

Malassezia pachydermatis causes infections of the skin and mucous membranes, especially in individuals with metabolic, hormonal, and immunological disorders. The search for M. pachydermatis properties that differentiate isolates from healthy and infected animals may result in the identification of typically commensal and potentially pathogenic strains within the entire species. We aimed to determine and compare protein profiles of M. pachydermatis strains isolated from 30 dogs with clinical symptoms of otitis externa and 34 dogs without symptoms of any disease. Two-dimensional gel electrophoresis was applied, and proteins distinguishing the two groups of strains were identified by liquid chromatography coupled with tandem mass spectrometry. Significant differences were found between potentially pathogenic and commensal isolates. The most significant finding was the presence of nicotinamide adenine dinucleotide phosphate (NADP)-dependent mannitol dehydrogenase and ketol-acid reductoisomerase among M. pachydermatis strains obtained from dogs with otitis externa. Nevertheless, it is not clear whether they are associated directly with the pathogenicity or they play the role of fungal allergen. On the basis of these findings, we can conclude that there may be two distinct groups of M. pachydermatis strains—one typically commensal and the other with properties that enhance the infection process. These results may be used for more precise diagnosis and identification of potentially pathogenic strains in the future. [ABSTRACT FROM AUTHOR]

Background: The aim of this research was to compare excretory-secretory and somatic extract materials of Fasciola hepatica and F. gigantica to detect protein maps of two species. Methods: Twenty infected livers were collected from sheep in industrial slaughterhouse in Tehran, 2017–2019. Worms were detached from bile ducts, then recognized according to morphologic and morphometric criteria. After three times washing, worms were incubated in RPMI culture media and excretory-secretory products were collected. Worms were crushed and homogenized for preparation of somatic extract. Two Dimensional Electrophoresis gels were accomplished for both excretory-secretory material and somatic extracts. Gels were scanned with densitometer and analyzed with Same Spots software and protein spots were identified with Expasy database. Results: For both excretory-secretory products and somatic extract, protein spots were appeared with two-dimensional electrophoresis technique. Quantitative analysis showed 40 and 28 protein spots for excretory-secretory of F. hepatica and F. gigantica respectively. For somatic extract 19 and 12 protein spots were recognized for F. hepatica and F. gigantica in that order. Conclusion: The rate of expression of some proteins were more in F. hepatica while expression of other proteins was high in F. gigantica. The expression of protease enzyme was higher in F. gigantica than F. hepatica. These data could be considered for biochemical differentiation of Fasciola species and subsequently to design and prepare of antigens for diagnosis/vaccine development. [ABSTRACT FROM AUTHOR]

Obstructive sleep apnea (OSA) affects an estimated 20% of adults worldwide and has been associated with electrical and structural abnormalities of the atria, although the molecular mechanisms are not well understood. Here, we used two‐dimensional polyacrylamide gel electrophoresis (2D PAGE) coupled with nanoliquid chromatography‐tandem mass spectrometry (nanoLC‐MS/MS) to investigate the proteins that are dysregulated in the atria from severe and moderate apnea when compared to control. We found enzymes involved in the glycolysis, beta‐oxidation, electron transport chain and Krebs cycle to be down‐regulated. The data suggested that the dysregulated proteins may play a role in atrial pathology developing via chronic obstructive apnea and hypoxia. Our results are consistent with our previous 1D‐PAGE and nanoLC‐MS/MS study (Channaveerappa et al, J Cell Mol Med. 2017), where we found that some aerobic and anaerobic glycolytic and Krebs cycle enzymes were down‐regulated, suggesting that apnea may be a result of paucity of oxygen and production of ATP and reducing equivalents (NADH). The 2D‐PAGE study not only complements our current study, but also advances our understanding of the OSA. The complete mass spectrometry data are available via ProteomeXchange with identifier PXD011181. [ABSTRACT FROM AUTHOR]

The primary components of human hair shaft—keratin and keratin-associated proteins (KAPs), together with their cross-linked networks—are the underlying reason for its rigid structure. It is therefore requisite to overcome the obstacle of hair insolubility and establish a reliable protocol for the proteome analysis of this accessible specimen. The present study employed an alkaline-based method for the efficient isolation of hair proteins and subsequently examined them using gel-based proteomics. The introduction of two proteomic protocols, namely the conventional and modified protocol, have resulted in the detection of more than 400 protein spots on the two-dimensional gel electrophoresis (2DE). When compared, the modified protocol is deemed to improve overall reproducibility, whilst offering a quick overview of the total protein distribution of hair. The development of this high-performance protocol is hoped to provide a new approach for hair analysis, which could possibly lead to the discovery of biomarkers for hair in health and diseases in the future. [ABSTRACT FROM AUTHOR]

Abstract Objective We investigated effects of salazosulfapyridine (SASP) on the protein profile of cell surface (CS)-proteins of SW982, a human synovial sarcoma cell line, using biotinylation of CS-proteins and 2-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Methods SW982 cells were treated with SASP and its metabolites, sulfapyridine (SP) and 5-aminosalicylic acid (5ASA). Then the cells were treated with a membrane-impermeable biotinylating reagent. Biotinylated CS-proteins were isolated using NeutrAvidin-bound beads. CS-proteins affected by the drugs were detected by 2D-DIGE and subjected to mass spectrometry. Results By the 2D-DIGE analysis, in total 576 spots were detected, 29 out of which showed more than ±1.5-fold different intensity in the SASP-, SP-, and 5ASA-treated cells, compared to non-treated cells (p < 0.05). Interestingly, 7 out of the 29 spots changed their intensity only by SASP and 17 spots changed their intensity only by SP. We identified 9 protein from 15 out of the 29 spots, most of which were evidenced to exist on the cell surface by flow cytometry. Conclusion We found novel effects of SASP and its metabolites on SW982 cells by the combination of biotinylation of cell surface proteins and 2D-DIGE analysis. These data would help understanding of anti-rheumatic actions of SASP. Furthermore, the combination would be a useful method for the analysis of CS-proteins in various conditions. Highlights • Effects of salazosulfapyridine on cell surface proteins were analyzed by 2D-DIGE. • About 1/5 of the detected 576 spots were affected by the drug and its metabolites. • Most of the identified proteins were detected on the cell surface by flow cytometry. [ABSTRACT FROM AUTHOR]

To identify potential protein markers associated with tillering in rice, a d10 allelic mutant (JHCA) with a high-tillering dwarf phenotype and a wild-type cultivar (GLA4) with normal numbers of tillers were used to produce a hybrid (F1), and 2-DE (two-dimensional electrophoresis) analysis was conducted in seedling leaf sheaths and roots of the rice hybrid and its parents. A total of 61 protein spots representing qualitative or quantitative polymorphisms were detected. Among them, 38 spots were successfully identified as 23 different proteins by MALDI-TOF/MS. The identified proteins were further divided into P/As (presence/absence variations), PSs (position shift variations) and qVs (quantitative variations). Most of the identified polymorphic proteins exhibited tissue-specific expression. Two spot families, identified as a putative vitamin B12-independent methionine synthase and a putative phosphoglycerate mutase were found. The profiles of expression abundance of three iso-spots in each spot family showed a reciprocal relationship between the two parents. [ABSTRACT FROM AUTHOR]

[Display omitted] • The coffee protein profile differs before fermentation and after drying. • 2D-PAGE separation reveals significant differences in spot distribution to control. • Fermentation provides a valuable tool for modifying proteins and volatile compounds. This work aims to analyze the protein profile and volatile compounds of coffees fermented with the indigenous microbiota and with the co-inoculation of three yeasts (Saccharomyces cerevisiae , Torulaspora delbrueckii , and Candida parapsilosis). Two-dimensional gel electrophoresis (2D-PAGE), MALDI-ToF/ToF (MS/MS), and gas chromatography (GC–MS) were performed. A total of 72 "spots" were detected by 2D-PAGE. 16 spots were selected for identification by MALDI-ToF/ToF, and 12 were identified (11S protein, 13S globulin basic chain, 17.6 kDa class II heat shock protein (HSP17.6-CII), 18.0 kDa class I heat shock protein, Seed of Late Development Stage, Pru ar 1, and FAR-1 protein). 81 main volatile compounds were detected and classified into alcohols, acids, aldehydes, esters, hydrocarbons, pyrazines, furans, thiols, and pyridines/pyrrols. The difference between the identified volatile compounds and their concentrations was detected in the treatments with and without inoculation after drying. The compounds formed in green coffee during fermentation can participate in several reactions during roasting, presenting different sensory profiles and contributing to coffee quality. [ABSTRACT FROM AUTHOR]

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

Uterine leiomyomas are benign smooth muscle cell tumors that originate from the myometrium. In this study we focus on dysregulated chaperones associated with cell proliferation and apoptosis. Paired tissue samples of 15 leiomyomas and adjacent myometria were obtained and analyzed by two-dimensional gel electrophoresis (2-DE). Mass spectrometry was used for protein identification and western blotting for 2-DE data validation. The values of 6 chaperones were found to be significantly different in the leiomyoma when compared with the myometrium. A total of 4 proteins were upregulated in the leiomyoma and 2 proteins were downregulated. Calreticulin and 78 kDa glucose-regulated protein were further validated by western blotting because the first is considered a marker of cell proliferation, while the second protects against apoptotic cell death. In addition, we also validated the two downregulated proteins heat shock protein β-1 and heat shock 70 kDa protein 1A. Our study shows the existence of a dysregulation of chaperone proteins associated with leiomyoma development. Functional studies are needed to ascertain the role of these chaperones in the leiomyoma. This may be crucial for the further development of specific inhibitors against the activity of these proteins in order to block the growth of the leiomyoma. [ABSTRACT FROM AUTHOR]

Buckwheat (Fagopyrum esculentum Moench) blossom honey was analysed for enzymatic activity and protein content. Proteins were separated by one-dimensional denaturing electrophoresis (1DE) in sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) or resolved by two-dimensional denaturing gel electrophoresis (2DE). Using different mass spectrometry (MS) techniques, the main proteins of plant and honeybee Apis mellifera carnica, Pollmann origin were identified. Using direct MS analysis of honeybee proteome, a total of 87 proteins were identified in buckwheat honey. Proteins of honeybee origin included major royal jelly protein (MRJP), uncharacterized honeybee protein, high glutamic acid storage protein and enzymesαglucosidase,αamylase and glucosylceramidase. Separation of proteins by 1DE and 2DE revealed a smaller number of proteins, including 6 proteins of bee origin and 11 of yeast origin. In addition, catalase activity and glucose oxidase activity in buckwheat honey were estimated on the native PAGE. Our study demonstrates a useful approach to identification of honey proteins, which play an important role in human nutrition and immune defence system. [ABSTRACT FROM AUTHOR]

The aim of this study was the identification of proteins differentially represented in the serum proteome of seropositive dogs with (Group 1) and without (Group 2) clinical-pathologic signs consistent with ehrlichiosis compared to healthy control dogs. Serum samples were collected from 20 dogs of various breeds with naturally occurring ehrlichiosis (10 dogs belonged to Group 1 and 10 to Group 2) and 10 healthy dogs. Two-dimensional electrophoresis (2DE) of pooled serum for each of the group of dogs were run in triplicate. 2D image analysis showed 39 spots differently expressed between Group 1 and Group 2 compared with healthy ones. Mass spectrometry analysis allowed identification of 6 proteins: albumin, haptoglobin (Hp), alpha-1-antitrypsin (AAT), Retinol Binding Protein 4 (RBP-4), alpha-1-acid glycoprotein (AGP) and vitamin D-binding protein (VDBP). When a confirmatory study was performed for albumin, Hp, AAT and RBP-4 by using different assays, significant differences (P < 0.05) between diseased and healthy groups were observed. It can be concluded that there are significant changes in the serum proteome of dogs with ehrlichiosis with modifications in proteins related with the acute phase response such as Hp, albumin and AGP, with vitamin A transport such as RBP-4, with inhibitors of serine proteases and anti-inflammatory proteins such as AAT, and vitamin D metabolism and actin scavengers such as VDBP. [ABSTRACT FROM AUTHOR]

Key message : Osmoprimed seeds increased the abundance of pGlcT proteins. Osmopriming together with heat shock increased the abundance of RBR proteins. NAD-ME increased when osmoprimed and heat-shocked seeds were imbibed at low temperature. Abstract: The purpose of seed priming is to accelerate and synchronize germination and to increase stress tolerance through the activation of genes that function when seeds are exposed to unfavorable conditions. The objectives of this study were: (1) to evaluate the effects of osmopriming and heat-shock treatment on the germination of Eucalyptus urophylla seeds at different temperatures and (2) to analyze the seed proteome to elucidate the mechanisms of tolerance to thermal stress in primed and unprimed seeds. Untreated (control) seeds, osmoprimed (polyethylene glycol for 3 days) and redried seeds, and osmoprimed/heat-shocked (45 °C for 1 h) and redried seeds were germinated for 14 days under constant light (2 × 40 W fluorescent daylight tubes) at 9, 16, 22, 24, or 31 °C. Osmopriming, with or without heat-shock, led to a significant increase in the germination percentage at 9 °C and induced the highest germination speed index at 31 °C. According to proteomic analysis, osmoprimed seeds exhibited an increased abundance of several proteins, including sugar transport proteins, and this may have influenced the metabolic rate during germination. Osmopriming together with heat-shock treatment increased the abundance of proteins associated with regulation of the cell cycle suggesting that such proteins may be involved in protection against thermal stress. The Krebs cycle enzyme was increased when osmoprimed and heat-shocked seeds were imbibed at low temperature, possibly signifying increased synthesis of adenosine triphosphate. The results reported herein serve to explain some of the benefits of osmopriming/heat-shock treatment. [ABSTRACT FROM AUTHOR]

The growth rate and maximum biomass of Bacillus coagulans 2-6 were inhibited by lactate; inhibition by sodium lactate was stronger than by calcium lactate. The differences of protein expressions by B. coagulans 2-6 under the lactate stress were determined using two-dimensional electrophoresis coupled with mass spectrometric identification. Under the non-stress condition, calcium lactate stress and sodium lactate stress, the number of detected protein spots was 1,571 ± 117, 1,281 ± 231 and 904 ± 127, respectively. Four proteins with high expression under lactate stress were identified: lactate dehydrogenase, cysteine synthase A, aldo/keto reductase and ribosomal protein L7/L12. These proteins are thus potential targets for the reconstruction of B. coagulans to promote its resistance to lactate stress. [ABSTRACT FROM AUTHOR]

To survive at the body temperature of their hosts, pathogenic fungi have developed important molecular mechanisms, such as protein expression associated with growth at high temperatures. Thus, the aim of this study was to analyze the in vitro growth of Conidiobolus lamprauges at different temperatures and compare proteins expressed by two-dimensional electrophoresis (2D), for the pathogen cultivated at low (28°C) and high (37°C) temperatures. For the analysis of growth temperatures, five isolates of C. lamprauges from sick sheep were incubated at 20, 25, 30, 35 and 40°C and radial growth was measured every 24 hours. For the analysis of differential expression, protein extraction and twodimensional polyacrylamide gel electrophoresis were performed with C. lamprauges cultivated at 28°C and 37°C for 48 hours. The average radial growth was different at the temperatures tested, and 35°C was found to be the best growth temperature for all isolates. The optimum adjusted temperature ranged between 33.3°C and 34.8°C. The upper and lower limits of growth inhibition were 18°C and 42°C, respectively. Upon expression analysis, a total of 16 spots were differentially expressed, seven (7/16) proteins were downregulated and nine (9/16) were over-expressed at 37°C compared to 28°C. In addition, eight spots were present only at 28°C and six were present only at 37°C. It is suggested that C. lamprauges produces a profile of proteins that is related to thermoregulation triggered by the high temperature of the host. [ABSTRACT FROM AUTHOR]