SDS-PAGE Research Articles

Characterisation of a casein-/whey protein concentrate–Antarctic krill oil emulsion system and improvement of its storage stability

Aims To develop Antarctic krill oil emulsions with casein and whey protein concentrate (WPC) and study their physicochemical properties and storage stability. Methods Emulsions were prepared by homogenisation and ultrasonication. The properties of the emulsions were investigated via ultraviolet ray spectroscopy, dynamic light scattering, confocal laser scanning microscope, sodium dodecyl sulphate–polyacrylamide gel electrophoresis, Fourier transform infra-red spectrometer, and fluorescence spectrum. Shelf life was predicted by the Arrhenius model. Results Casein- and WPC-krill oil emulsions were well formed; the mean particle diameters were less than 128.19 ± 0.64 nm and 158 ± 1.56 nm, the polymer dispersity indices were less than 0.26 ± 0.01 and 0.27 ± 0.01, and the zeta potential were around −46.88 ± 5.02 mV and −33.51 ± 2.68 mV, respectively. Shelf life was predicted to be 32.67 ± 1.55 days and 29.62 ± 0.65 days (40 °C), 27.69 ± 1.15 days and 23.58 ± 0.14 days (50 °C), 24.02 ± 0.15 days and 20.1 ± 0.08 days (60 °C). Conclusion The prepared krill oil emulsions have great potential to become a new krill oil supplement.

Exploring the impact of malondialdehyde and 4-hydroxy-2-nonena on myofibrillar protein structure: A proteomic analysis

In this investigation, the impacts of malondialdehyde (MDA), 4-hydroxy-2-nonena (HNE), and their combinations on the structural characteristics of myofibrillar proteins (MP) were examined using endogenous fluorescence, Fourier transform infrared spectrum (FTIR) and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). The potential binding mechanisms were also explored using LC-MS/MS and proteomics techniques. The findings indicated that various reactive carbonyl compounds (RCCs) displayed distinct oxidation performances in the oxidation of myofibrillar proteins. Samples containing HNE (HMP and MHMP groups) exhibited a more pronounced ability to oxidize MP than those containing MDA, as evidenced by their higher carbonyl content and lower sulfhydryl content. The MHMP group demonstrated the greatest surface hydrophobicity and Schiff base content but the lowest fluorescence intensity, suggesting that MDA and HNE synergistically altered MP conformation. Moreover, the alteration by RCCs notably modified the secondary structure of MP. Among these, HMP triggered the unfolding of MP molecules, resulting in the lowest α-helix and highest β-sheet ratio. The SDS‒PAGE results suggested that MDA oxidation promoted the crosslinking of MP molecules. At the same time, HNE caused the degradation of MP, as indicated by the decrease in the intensity of the high-molecular-weight bands. Proteomic analysis suggested that lysine residues distributed in the tail region of myosin were the main binding sites for MDA, whereas HNE preferentially modified methionine located at the myosin head. Additionally, HNE-induced protein degradation dominated RCC-induced oxidation reactions. These findings provide deeper insights into the mechanisms underlying quality changes induced by lipid oxidation products in MP-based food systems.

Impact of Flammulina velutipes polysaccharide on properties and structural changes of pork myofibrillar protein during the gel process in the absence or presence of oxidation

The present study aimed to investigate the impact of Flammulina velutipes polysaccharide (FVSP) on the rheological properties and structural alterations of myofibrillar protein (MP) and oxidized MP (OMP), utilizing techniques such as rhehometer, fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In the unoxidized system, the addition of 5.00% FVSP significantly improved (p < 0.05) the storage and loss moduli of the composite gel and promoted the α-helix to β-sheet transformation. These effects enhanced the protein's gel strength and water-holding capacity (WHC). In the oxidation system, 5.00% FVSP had significant effects (p < 0.05) on repair and improvement of the oxidized MP. These effects inhibited the cross-linking aggregation and degradation of the protein. In addition, the addition of FVSP significantly improved the gel properties of MPs after oxidation (p < 0.05), hindered fracture of the protein gel network structure. In summary, polysaccharides have a substantial effect on the functional characteristics of MP, and FVSP could potentially be applied in meat products.

Biofilm-producing Escherichia coli O104:H4 overcomes bile salts toxicity by expressing virulence and resistance proteins.

We investigated bile salts' ability to induce phenotypic changes in biofilm production and protein expression of pathogenic Escherichia coli strains. For this purpose, 82 pathogenic E. coli strains isolated from humans (n=70), and animals (n=12), were examined for their ability to form biofilms in the presence or absence of bile salts. We also identified bacterial proteins expressed in response to bile salts using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-electrophoresis) and liquid chromatography-mass spectrometry (LC-MS/MS). Lastly, we evaluated the ability of these strains to adhere to Caco-2 epithelial cells in the presence of bile salts. Regarding biofilm formation, two strains isolated from an outbreak in Republic of Georgia in 2009 were the only ones that showed a high and moderate capacity to form biofilm in the presence of bile salts. Further, we observed that those isolates, when in the presence of bile salts, expressed different proteins identified as outer membrane proteins (i.e. OmpC), and resistance to adverse growth conditions (i.e. F0F1, HN-S, and L7/L12). We also found that these isolates exhibited high adhesion to epithelial cells in the presence of bile salts. Together, these results contribute to the phenotypic characterization of E. coli O104: H4 strains.

A New Algorithm for Detecting GPN Protein Expression and Overexpression of IDC and ILC Her2+ Subtypes on Polyacrylamide Gels Associated with Breast Cancer

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) is used to identify protein presence, absence, or overexpression and usually, their interpretation is visual. Some published methods can localize the position of proteins using image analysis on images of SDS-PAGE gels. However, they cannot automatically determine a particular protein band’s concentration or molecular weight. In this article, a new methodology to identify the number of samples present in an SDS-PAGE gel and the molecular weight of the recombinant protein is developed. SDS-PAGE images of different concentrations of pure GPN protein were created to produce homogeneous gels. Then, these images were analyzed using the developed methodology called Image Profile Based on Binarized Image Segmentation (IPBBIS). It is based on detecting the maximum intensity values of the analyzed bands and produces the segmentation of images filtered by a binary mask. The IPBBIS was developed to identify the number of samples in an SDS-PAGE gel and the molecular weight of the recombinant protein of interest, with a margin of error of 3.35%. An accuracy of 0.9850521 was achieved for homogeneous gels and 0.91736 for heterogeneous gels of low quality.

Purification, and characterization of detergent-compatible serine protease from Bacillussafensis strain PRN1: A sustainable alternative to hazardous chemicals in detergent industry

Owing to vast therapeutic, commercial, and industrial applications of microbial proteases microorganisms from different sources are being explored. In this regard, the gut microbiota of Monopteruscuchia were isolated and examined for the production of protease. All the isolates were primarily and secondarily screened on skim milk and gelatin agar plates. The protease-positive isolates were characterized morphologically, biochemically, and molecularly. Out of the 20 isolated strains,6 belonging to five different genera viz.Bacillus,Priestia,Aeromonas,Staphylococcus, and Serratia demonstrated proteolytic activity. Bacillussafensis strain PRN1 demonstrated the highest protease production and, thus, the largest hydrolytic clear zones in both skim milk agar (15 ± 1 mm) and gelatin (16 ± 1 mm) plates. The optimized parameters (time, pH, temperature, carbon, nitrogen) for highest protease activity and microbial growth of B.safensis strain PRN1 includes 72 h (OD600 = 0.56,1303 U/mL), pH 8 (OD600 = 0.83, 403.29 U/mL), 40 °C (OD600 = 1.75, 1849.11 U/mL), fructose (OD600 = 1.22, 1502 U/mL), and gelatin (OD600 = 1.88, 1015.33 U/mL). The enzyme was purified to homogeneity using salt-precipitation and gel filtration chromatography. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the purified enzyme was a monomer of a molecular weight of ∼33 kDa. The protease demonstrated optimal activity at pH 8 and 60 °C. It was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), demonstrating that it belongs to the serine-proteases family. The compatibility of the enzyme with surfactants and commercial detergents demonstrates its potential use in the detergent industry. Furthermore, the purified enzyme showed antibacterial and blood-stain removal properties.

Production and characterization of immunoglobulin G anti-rLipL32 antibody as a biomarker for the diagnosis of leptospirosis.

Microscopic agglutination test (MAT) for the diagnosis of leptospirosis requires live cultures and is serovar-specific, while polymerase chain reaction (PCR) requires expensive equipment and sample preparation. The rLipL32 protein is conserved and can be used for the production of immunoglobulin G (IgG) anti-rLipL32 antibody, which can be used as a biomarker for leptospirosis diagnosis. This study aimed to produce and characterize an IgG anti-rLipL32 antibody as a biomarker for leptospirosis diagnosis. Escherichia coli rLipL32 was cultured and analyzed by PCR and sequencing. Cultures were used for rLipL32 protein expression and purification and the rLipL32 protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The rLipL32 protein was used to produce anti-rLipL32 serum and was analyzed by enzyme-linked immunosorbent assay (ELISA). Serum was purified to obtain IgG anti-rLipL32 antibody and characterized by SDS-PAGE and western blotting. PCR was able to amplify the LipL32 gene from E. coli rLipL32, and sequencing analysis showed 99.19% similarity with pathogenic Leptospira. SDS-PAGE analysis showed a 32-kDa band. ELISA results showed an increase in OD in anti-rLipL32 serum compared to preimmune serum. Western blotting results showed that the IgG anti-rLipL32 antibody was able to bind and cross-reacts with pathogenic Leptospira serovar but not with E. coli or Staphylococcus aureus. IgG anti-rLipL32 antibody has high specificity and sensitivity against Leptospira pathogens. These findings suggest that IgG anti-rLipL32 antibody is a promising biomarker for the diagnosis of leptospirosis.

Effects of gold and green kiwifruit juices on the physicochemical properties and tenderness of pork loin and antioxidant activity during incubation (24 h) in a pork model system.

Although pork loins is not a tough meat, they need to develop meat products with a soft texture for the elderly. This study focused on the physicochemical properties and tenderness characteristics of pork loin injected with green kiwifruit juice (GRJ) and gold kiwifruit juice (GOJ) during various incubation times. In addition, the antioxidant activities of hydrolysate derived from the hydrolysis of pork loin by kiwifruit juice protease were evaluated. The pork loin was injected with 10% and 20% GRJ and GOJ, under various incubation times (0, 4, 8, and 24 h). Then, the physicochemical properties and tenderness of pork loins were measured. 2,2- diphenyl-1-picrylhydrazyl radical scavenging activity and reducing power were conducted to determine hydrolysate's antioxidant activities derived from pork loin's hydrolysis by kiwifruit juice protease. GRJ had greater tenderizing ability than GOJ, even at the 10% addition. When kiwifruit juice was injected into pork loin, the tenderness increased with increasing incubation time. This was confirmed by the decrease in intensity of the myosin heavy chain (MHC) band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In particular, the MHC band decreased at 8 h for both 10% GRJ and 20% GOJ and at 4 h for 20% GRJ alone. The highest myofibril fragmentation index and peptide solubility were observed in pork loin treated with 20% GRJ compared to the other treatments during incubation. The 10% GRJ and 20% GOJ treatments showed similar levels of antioxidant activity of the protein hydrolysates in pork loin, and 20% GRJ showed the highest activity among the treatments. Kiwifruit juice had protease activity, and GRJ was more useful for tenderizing meat products than GOJ. Thus, GRJ at 10% could be a potential agent to tenderize and enrich the natural antioxidant activity through the proteolysis of pork loin.

Effect of high-hydrostatic pressure on the digestibility of egg yolk and granule.

The impact of high hydrostatic pressure (HHP) on protein digestibility of egg yolk and egg yolk granule was evaluated by static in vitro digestion using the standardized INFOGEST 2.0 method. The degree of hydrolysis (DH) and the phospholipid content were determined during digestion, and the protein and peptide profiles were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse phase-high pressure liquid chromatography (RP-HPLC). The results showed that HHP induced protein aggregation in egg yolk and granule, mainly by disulfide bridges, which were not disrupted in the oral phase. Proteolysis during the gastric phase improved egg yolk and granule protein solubility, regardless of whether HHP was applied. However, the extent of the samples' digestibility was not affected, with DH values ranging from 15% to 20%. During the intestinal phase, the DH of egg yolk protein (∼40%) was higher than that of the granule (∼25%), probably due to the denser structure of the granule reducing the accessibility of intestinal enzymes. The DH, peptide, and protein profiles of control and HHP-treated egg yolk showed similar protein digestion behaviors for both gastric and intestinal phases. Among the different proteins, only the digestibility of β-phosvitin in HHP-treated granule was enhanced. Consequently, applying HHP to granules represents an interesting process that improves the digestibility of phosvitin with the potential to generate bioactive phosvitin-derived phosphopeptides. PRACTICAL APPLICATION: High hydrostatic pressure, mainly used as a preservation process, did not impair the nutritional quality of the egg yolk and granule proteins but improved the susceptibility of phosvitin (protein contained in egg yolk) proteolysis to produce bioactive phosphopeptides. Consequently, applying HHP to granules represents an interesting process that improves the digestibility of phosvitin.

Relative Protein Profile of Philippine Cinnamomum (Schaeffer) Using Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Philippine Cinnamomum are trees with aromatic leaves and bark. It has several economic and medicinal uses, but concerns about protein profile are limited. Thus, this study was conducted to determine the relative protein component of the species by Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE). A total of 27 cinnamon species were run in Sodium Dodecyl Sulphate - Polyacrylamide Gel Electrophoresis (SDS-Page) with LSB (Laemli Sample Buffer) and Tris HCI (1:1) as sample buffer. Determining protein profiles is a tool for the systematic classification of the species. Results showed that Philippine Cinnamomum leaf samples have the same component proteins. The SDS-PAGE provided a similar protein banding pattern, although the same bands' intensity varies. Pronounced protein bands were observed corresponding to 50 to 75 kDa and 25 kDa, with additional minor bands indicating a higher protein content. The results in SDS-PAGE showed that the component proteins of Cinnamomum leaves were comparable regardless of geographical source and different stages of growth of the samples. However, the concentration of some component proteins may vary, as indicated by the difference in intensity of protein bands. Sodium dodecyl sulphate Polyacrylamide gel electrophoresis (SDS-PAGE) is a tool in the biochemical method of determining the relative protein profile of Cinnamomum species. It can be used to classify plants up to the species level. Based on the study results, Philippine Cinnamomum has a higher protein content that can be used in culinary, medicinal, and pharmaceuticals.

Investigation of genetic diversity in lentil genotypes obtain from different countries using SDS-PAGE methodology

Pulses are an important component of the agricultural production system, providing high-quality food to millions of people worldwide. Among pulses, lentil is an annual and self-pollinated crop known to be its high proteins, amino acids, dietary fibers and low fat in the world. In the current study, the characterization of 44 lentil genotypes was performed using SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) technique. The protein bands were scored according to a binary system as the present (1) or absent (0) that ranged 12-20 bands in lentil genotypes. The Jaccard’s coefficient of genetic dissimilarity (GD) was measured, and the mean GD was 0.216. Maximum genetic distance was found as 0.526 between Syria2 with Iraq3 and these genotypes might be recommended for future lentil breeding. AMOVA explored the presence of higher genetic variety within genotypes (83%) than among genotypes (17% variations). STRUCTURE algorithm separated lentil genotypes into two groups mainly on the basis of their geographic. The UPGMA clustering separated lentil genotypes into two main clusters A and B. The PCoA was also conducted to confirm the results of structure and UPGMA analyses. Findigs from the PCoA and structure analyses were in full agreement with those obtained by UPGMA. The results might be useful for researchers worldwide who are interested in lentil breeding.

Covalent conjugate of pea protein induced by cyanidin-3-O-glucoside quinone: The structural formation and functional properties

The unique structural of anthocyanins makes them vulnerable to degradation within food processing environments. Proteins commonly coexist with polyphenols in natural food matrices on a non-covalent and covalent basis, serving to stabilize protection for polyphenols. Here, three natural pea-extracted proteins, including albumin, vicilin, and legumin were bound to cyanidin-3-O-glucoside (C3G), and binding modes, conjugate structures and functions were characterized. Isothermal titration calorimetry showed a strongly exothermic interaction between C3G and proteins. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fourier transform infrared spectroscopy revealed that the C3G-quinone (C3GQ) formed by C3G oxidation established covalent cross-links with C–S and C–N in the amino acid residues of proteins, and could form more large molecular weight aggregates at an addition amount of 1 mM. The free sulfhydryl and amino contents of the three protein conjugates decreased with the addition of C3GQ. Due to the presence of 4–5 times more sulfhydryl groups in albumin as compared to globulin, albumin showed a 20% higher binding capacity for C3GQ than globulin. This consequently led to the highest antioxidant activity in the C3GQ-albumin conjugate. In contrast to native pea proteins, the introduction of covalent modifications induces changes in the secondary structure of the pea proteins. Specifically, the structure of albumin tends to exhibit increased orderliness, while the molecular flexibility of globular proteins was augmented. Thus, the conjugates have enhanced thermal stability. Our results affirmed that the stability of C3G can be more effectively improved through designing it to covalently interact with proteins relative to non-covalent interactions. This study aimed at offering novel theoretical underpinnings for the functional utilization of C3G and pea proteins in the food industry.

Abstract 6061: Development of target cell-penetrating scFv conjugate degrader for the selective inhibition of mutant, resistant KRAS cancer

Abstract The activation of mutations in the RAS gene, which is frequently observed in human cancers, induces downstream critical effectors, contributing to tumorigenesis. While attempts to directly target the GTP-bound, activated RAS have shown some success in certain cancer treatments, resistance remains a challenge. Our objective is to develop a cell-penetrating single-chain fragment variable (scFv) degrader that targets mutant KRAS for degradation. The scFv, binding specifically to antigens, can mitigate the side effects of non-specific binding seen with chemical drugs. However, due to the limited cell-penetrating capability of scFv, a delivery carrier is often necessary. In this study, a cancer targeting, and cell-penetrating synthetic peptide (CPP) was developed as a drug delivery carrier and added to the sequence of scFv. To this conjugate, a von Hippel-Lindau (VHL) expressing sequence was added to enable the scFv binding to mutant KRAS, enabling its proteasomal degradation. These conjugates were engineered as a plasmid DNA to be expressed as a protein conjugate in CHO cells. The purity of the scFv-VHL-CPP degrader was confirmed by SDS PAGE. In SPR results, the degrader not only exhibited high binding ability to mutant KRAS but also influenced the proliferation of KRAS mutant cells in cell viability tests. Mutant GTP-bound KRAS was reduced by the KRAS degrader in KRAS mutant cancer cells. The in vivo study further confirmed the degradation of the KRAS, as reflected by the significantly reduced tumor growth. In conclusion, a degrader protein binding to mutant KRAS that consists of cell penetrating peptide, scFv, and VHL is expected to become a new protein anticancer drug especially effective against mutant, resistant KRAS-mediated cancer cells. Especially, the design of the target cell penetrating peptide-scFv-VHL would be served as a platform for the target protein degrader development. Citation Format: Gookjin Yoon, Beom Soo Jo, Dong Woo Lee, Jinwook Yang, Min-Ho Park, Sanghui Seok, Jue Yeon Lee, Chong Pyoung Chung, Yoon Jeong Park. Development of target cell-penetrating scFv conjugate degrader for the selective inhibition of mutant, resistant KRAS cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6061.

Immunomodulatory responses of extracellular vesicles released by gram-positive fish pathogen Streptococcus parauberis

Bacterial extracellular vesicles (BEVs) are nanosized structures that play a role in intercellular communication and transport of bioactive molecules. Streptococcus parauberis is a Gram-positive pathogenic bacterium that causes “Streptococcosis” in fish. In this study, we isolated S. parauberis-derived extracellular vesicles (SpEVs), and then physicochemical and immunomodulatory properties were determined to elucidate their biological functions. Initially, the biogenesis of SpEVs was detected using field emission scanning electron microscopy, which revealed that secretory phase SpEVs attached to the outer surface of S. parauberis. SpEVs had an average particle diameter and zeta potential of 168.3 ± 6.5 nm and −17.96 ± 2.11 mV, respectively. Field emission transmission electron microscopy analysis confirmed the presence of round or oval-shaped SpEVs with clear membrane margins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results showed three sharp protein bands when SpEVs were stained with Coomassie blue. In vitro toxicity of SpEVs was assayed using the murine macrophage RAW 264.7 cells and we observed no significant (p < 0.05) viability reduction up to 50 μg/mL qRT-PCR results revealed that SpEVs-treated (5 and 10 μg/mL) RAW 264.7 cells significantly (p < 0.05) induced the mRNA of proinflammatory (Il1β, Il6, and Tnfα) and anti-inflammatory (Il10) cytokines in a concentration-dependent manner. In vivo immunomodulatory effects of SpEVs were investigated by injecting SpEVs (5 and 10 μg/fish) into adult zebrafish. Transcriptional analysis based on qRT-PCR indicates significant (p < 0.05) upregulation of proinflammatory (il1β, il6, and tnfα) and anti-inflammatory (il10) genes in a concentration-dependent manner in zebrafish kidney. Further, protein expression results in zebrafish spleen tissue confirmed the immunomodulatory activity of SpEVs. In conclusion, SpEVs display the characteristics of BEVs and immunomodulatory activities, suggesting their potential application as vaccine candidate.

Comprehensive Analysis of Bufadienolide and Protein Profiles of Gland Secretions from Medicinal Bufo Species.

Toad Venom (TV) is the dried product of toxic secretions from Bufo bufo gargarizans Cantor (BgC) or B. melanostictus Schneider (BmS). Given the increasing medical demand and the severe depletion of wild toads, a number of counterfeit TVs appeared on the market, posing challenges to its quality control. In order to develop an efficient, feasible, and comprehensive approach to evaluate TV quality, a thorough analysis and comparison of chemical compounds among legal species BgC and BmS, as well as the main confusion species B. andrewsi Schmidt (BaS) and B. raddei Strauch (BrS), were conducted by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS), high performance liquid chromatography (HPLC), sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Nano LC-MS/MS analyses. We identified 126 compounds, including free or conjugated bufadienolides, indole alkaloids and amino acids, among the four Bufo species. The content of main bufadienolides, such as gamabufotalin, bufotalin, bufalin, cinobufagin, and resibufogenin, and the total protein contents varied widely among 28 batches of TV due to their origin species. The sum of the five bufadienolides within the BgC, BmS, BaS, and BrS samples were 8.15-15.93%, 2.45-4.14%, 11.15-13.50%, and 13.21-14.68%, respectively. The total protein content of BgC (6.9-24.4%) and BaS (19.1-20.6%) samples were higher than that of BmS (4.8-20.4%) and BrS (10.1-13.7%) samples. Additionally, a total of 1357 proteins were identified. There were differences between the protein compositions among the samples of the four Bufo species. The results indicated that BgC TV is of the highest quality; BaS and BrS TV could serve as alternative resources, whereas BmS TV performed poorly overall. This research provides evidence for developing approaches to evaluate TV quality and selecting the proper Bufo species as the origin source of TV listed in the Chinese pharmacopoeia.

Comparative study of the commonly used protein quantitation assays on different Hymenoptera venoms: A fundamental aspect of Hymenoptera venom proteome analysis

Determination of protein concentration in Hymenoptera venoms requires an accurate and reproducible assay as the results will be used to support subsequent proteomic techniques employed in their analyses. However, all protein assay techniques have inherent strengths and weaknesses, demanding their assessment before selecting the most suitable platform for sample analysis. In this study, protein profiles of ant, honeybee, and wasp venoms, and bovine serum albumin (BSA) and hyaluronidase standards were qualitatively assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Their amino acid and protein concentration were quantitatively determined via Amino Acid Analysis (AAA). Amino acid concentration was determined via hydrolysis, derivatization, and chromatographic quantification. Protein concentration was estimated using four different protein concentration assays. The ratios of protein concentration in venom samples to protein standards were calculated, and the accuracy of the protein concentration assays was analysed relative to the concentration determined from AAA. SDS-PAGE analysis showed that BSA contained several protein bands, while hyaluronidase contained a mixture of peptide and protein bands. Ant and honeybee venoms contained a higher proportion of peptide bands, while wasp venom contained more protein bands. As determined by AAA, the ratio of protein concentration in Hymenoptera venoms varied between 1.01 and 1.11 to BSA, and between 0.96 and 1.06 to hyaluronidase. Overall, the Bradford assay was found to be the least accurate and the BCA assay was the most accurate in estimating protein concentration in Hymenoptera venoms. There was no significant advantage in using hyaluronidase as a standard or increasing incubation temperature of BCA assay when analysing Hymenoptera venoms. Diluent solutions containing phenol and human serum albumin interfered with Lowry-based assays.

Akkermansia muciniphila extracellular vesicles have a protective effect against hypertension.

Akkermansia muciniphila (Am) shows a beneficial role as a probiotic in the treatment of metabolic syndrome. However, the mechanism remains to be elucidated. We tested the hypothesis that Am extracellular vesicles (AmEVs) have a protective effect against hypertension. Extracellular vesicles purified from anaerobically cultured Am were characterized by nanoparticle tracking analysis, transmission electron microscopy, and silver stain after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). AmEVs (1.0 × 1010 log particles/L) or vehicles were added into organ baths to induce vasorelaxation. In addition, AmEVs (1.0 × 108 or 1.0 × 109 particles/kg) or vehicles were injected into the tail veins of Wistar-Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) weekly for 4 weeks. Peripheral blood mononuclear cells (PBMCs) and splenocytes isolated from both rat strains were analyzed by flow cytometry, RT-qPCR, and western blot. AmEVs affected neither vascular contraction nor endothelial relaxation in thoracic aortas. Moreover, AmEVs protected against the development of hypertension in SHRs without a serious adverse reaction. Additionally, AmEVs increased the population of T regulatory (Treg) cells and tended to reduce proinflammatory cytokines. These results indicate that AmEVs have a protective effect against hypertension without a serious adverse reaction. Therefore, it is foreseen that AmEVs may be utilized as a novel therapeutic for the treatment of hypertension.

Immunochemical Recognition of Bothrops rhombeatus Venom by Two Polyvalent Antivenoms.

The protein profile of Bothrops rhombeatus venom was compared to Bothrops asper and Bothrops atrox, and the effectiveness of antivenoms from the National Institute of Health of Colombia (INS) and Antivipmyn-Tri (AVP-T) of Mexico were analyzed. Protein profiles were studied with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and reverse-phase high-performance liquid chromatography (RP-HPLC). The neutralizing potency and the level of immunochemical recognition of the antivenoms to the venoms were determined using Western blot, affinity chromatography, and enzyme-linked immunosorbent assay (ELISA). Bands of phospholipase A2 (PLA2), metalloproteinases (svMPs) I, II, and III as well as serine proteinases (SPs) in the venom of B. rhombeatus were recognized by SDS-PAGE. With Western blot, both antivenoms showed immunochemical recognition towards PLA2 and svMP. INS showed 94% binding to B. rhombeatus venom and 92% to B. asper while AVP-T showed 90.4% binding to B. rhombeatus venom and 96.6% to B. asper. Both antivenoms showed binding to PLA2 and svMP, with greater specificity of AVP-T towards B. rhombeatus. Antivenom neutralizing capacity was calculated by species and mL of antivenom, finding the following for INS: B. asper 6.6 mgV/mL, B. atrox 5.5 mgV/mL, and B. rhombeatus 1.3 mgV/mL. Meanwhile, for AVP-T, the following neutralizing capacities were found: B. asper 2.7 mgV/mL, B. atrox 2.1 mgV/mL, and B. rhombeatus 1.4 mgV/mL. These results show that both antivenoms presented similarity between calculated neutralizing capacities in our trial, reported in a product summary for the public for the B. asper species; however, this does not apply to the other species tested in this trial.

Ultrasound-enhanced covalent reaction of gliadin: the inhibition of antigenicity and its potential mechanisms.

Wheat proteins can be divided into water/salt-soluble protein (albumin/globulin) and water/salt-insoluble protein (gliadins and glutenins (Glu)) according to solubility. Gliadins (Glia) are one of the major allergens in wheat. The inhibition of Glia antigenicity by conventional processing techniques was not satisfactory. In this study, free radical oxidation was used to induce covalent reactions. The effects of covalent reactions by high-intensity ultrasound (HIU) of different powers was compared. The enhancement of covalent grafting effectiveness between gliadin and (-)-epigallo-catechin 3-gallate (EGCG) was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry and Folin-Ciocalteu tests. HIU caused protein deconvolution and disrupted the intrastrand disulfide bonds that maintain the tertiary structure, causing a shift in the side chain structure, as proved by Fourier, fluorescence and Raman spectroscopic analysis. Comparatively, the antigenic response of the conjugates formed in the sonication environment was significantly weaker, while these conjugates were more readily hydrolyzed and less antigenic during simulated gastrointestinal fluid digestion. HIU-enhanced free radical oxidation caused further transformation of the spatial structure of Glia, which hid or destroyed the antigenic epitope, effectively inhibiting protein antigenicity. This study widened the application of polyphenol modification in the inhibition of wheat allergens. © 2024 Society of Chemical Industry.

Chemical Probes to Interrogate the Extreme Environment of Mosquito Larval Guts.

Mosquito control methods are vital to curtail the spread of life-threatening illnesses, such as dengue fever, malaria, and yellow fever. Vector control technologies must be selective to minimize deleterious effects on our ecosystem. Successful methods that control mosquito larva populations utilize the uniquely high alkaline nature of the midgut. Here, we present novel protected triazabutadienes (pTBD) that are deprotected under basic conditions of the larval midgut, releasing an aryl diazonium ion (ADI) that results in protein modification. The probes contain a bioorthogonal terminal alkyne handle, enabling a selective Cu-click reaction with an azidofluorophore for quantification by SDS PAGE and visualization using fluorescence microscopy. A control TBD, unable to release an ADI, did not label the midgut. We envision our chemical probes will aid in the development of new selective mosquito control methods, thus preventing the spread of mosquito-borne illnesses with minimal impact on other organisms in the ecosystem.