SDS-PAGE Research Articles

Comparison of the Bioactive Properties of Honey Proteins from Floral Sources in Indonesia

The diversity of plant species in Indonesia has resulted in a wide variety of floral honey. The antioxidant activities of these Indonesian honeys have not been determined. Ten types of honey from indigenous Indonesian sources (9 monofloral; 1 heterofloral) were evaluated for their antioxidant activity using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging method. The Folin-Ciocalteu test was used to determine the total phenolic content, and an aluminum chloride colorimetric assay was used to determine the total flavonoid content. SDS–PAGE was used to determine the molecular weights of the honey proteins. The mean antioxidant activity of the honey samples, defined as the IC50 of DPPH, ranged from 5.46 to 34.42. The mean total phenol content ranged from 27.57 to 53.85 mg GAE/100 g, and the mean flavonoid content ranged from 3.58–15.67 mg CE/100 g. A protein of ~60 kDa was detected in all honey varieties. A positive correlation was observed between antioxidant activity, total phenols, and total flavonoids, indicating that their phenolic and flavonoid components confer the antioxidant activity of these honey varieties. To the best of our knowledge, indigenous Indonesian honey, especially monofloral Calliandra honey, has shown greater antioxidant activity than other kinds of honey worldwide.

Stability and integrity of self-assembled bovine parvovirus virus‑like particles (BPV‑VLPs) of VP2 and combination of VP1VP2 assisted by baculovirus-insect cell expression: a potential logistical platform for vaccine deployment

BackgroundBovine parvovirus (BPV) is an autonomous DNA virus with a smaller molecular size and subtle differences in its structural proteins, unlike other animal parvoviruses. More importantly, this virus has the potential to produce visible to silent economic catastrophes in the livestock business, despite receiving very little attention. Parvoviral virus-like particles (VLPs) as vaccines and as logistical platforms for vaccine deployment are well studied. However, no single experimental report on the role of VP1 in the assembly and stability of BPV-VLPs is available. Furthermore, the self-assembly, integrity and stability of the VLPs of recombinant BPV VP2 in comparison to VP1 VP2 Cap proteins using any expression method has not been studied previously. In this study, we experimentally evaluated the self-assembling ability with which BPV virus-like particles (VLPs) could be synthesized from a single structural protein (VP2) and by integrating both VP2 and VP1 amino acid sequences.MethodsIn silico and experimental cloning methods were carried out. His-tagged and without-His-tag VP2 and V1VP2-encoding amino acid sequences were cloned and inserted into pFastbacdual, and insect cell-generated recombinant protein was evaluated by SDS‒PAGE and western blot. Period of infectivity and expression level were determined by IFA. The integrity and stability of the BPV VLPs were evaluated by transmission electron microscopy. The secondary structure of the BPV VLPs from both VP2 and V1VP2 was analyzed by circular dichroism.ResultsOur findings show that VP2 alone was equally expressed and purified into detectable proteins, and the stability at different temperatures and pH values was not appreciably different between the two kinds of VLPs. Furthermore, BPV-VP2 VLPs were praised for their greater purity and integrity than BPV-VP1VP2 VLPs, as indicated by SDS‒PAGE. Therefore, our research demonstrates that the function of VP1 has no bearing on the stability or integrity of BPV-VLPs.ConclusionsIn summary, incredible physiochemically stable BPV VP2-derived VLPs have been found to be promising candidates for the development of multivalent vaccines and immunodiagnostic kits against enteric viruses and to carry heterogeneous epitopes for various economically important livestock diseases.

Surface Engineering of Escherichia coli to Display Its Phytase (AppA) and Functional Analysis of Enzyme Activities.

Escherichia coli phytase (AppA) is widely used as an exogenous enzyme in monogastric animal feed mainly because of its ability to degrade phytic acid or its salt (phytate), a natural source of phosphorus. Currently, successful recombinant production of soluble AppA has been achieved by gene overexpression using both bacterial and yeast systems. However, some methods for the biomembrane immobilization of phytases (including AppA), such as surface display on yeast cells and bacterial spores, have been investigated to avoid expensive enzyme purification processes. This study explored a homologous protein production approach for displaying AppA on the cell surface of E. coli by engineering its outer membrane (OM) for extracellular expression. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of total bacterial lysates and immunofluorescence microscopy of non-permeabilized cells revealed protein expression, whereas activity assays using whole cells or OM fractions indicated functional enzyme display, as evidenced by consistent hydrolytic rates on typical substrates (i.e., p-nitrophenyl phosphate and phytic acid). Furthermore, the in vitro results obtained using a simple method to simulate the gastrointestinal tract of poultry suggest that the whole-cell biocatalyst has potential as a feed additive. Overall, our findings support the notion that biomembrane-immobilized enzymes are reliable for the hydrolysis of poorly digestible substrates relevant to animal nutrition.

Unveiling the presence of insulin-like antidiabetic proteins in potato: A wholesome food

A study was taken up to explore the possibilities for the presence of Insulin like protein in potato tubers. The serological based Direct antigen coating-Enzyme linked immune sorbent assay (DAC-ELISA) clearly detected and quantified the presence of insulin like proteins in dormant and sprouted potato tubers. Presence of ILP was further confirmed in dormant or sprouted potato tuber by Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis where an expected size of approximately 10 kDa corresponding to that of Insulin standard was recorded. In the second phase, an in vivo antihyperglycemic activity was carried out by administering the lyophilized powder derived from the tubers of Indian potato varieties to streptozotocin-induced diabetic rats. Among the varieties, Kufri Surya and Kufri Pushkar reduced the blood sugar level to 29.7% and 18% respectively, whereas, the metformin drug reduced the blood sugar level to 23.6%. In addition, the antioxidant and antidiabetic compounds (other than insulin like proteins) of the same varieties were also studies where Kufri Surya was found to be rich in nutrients and antidiabetic compounds. This is the first report on presence of insulin like proteins in the tubers of Indian potato varieties. The study presents a lead to explore the genes expressing insulin like proteins and subsequent utilization in developing specialty insulin rich potato varieties either by biotechnological based gene transfer approaches or by traditional breeding approach.

Preventing thermal aggregation of ovalbumin through dielectric-barrier discharge plasma treatment and enhancing its emulsification properties

The impact of Dielectric-Barrier Discharge (DBD) plasma treatment on the prevention of heat-induced aggregation of Ovalbumin (OVA) and improvement in emulsification properties was investigated. Results highlighted the effective inhibition of thermal aggregation of OVA following exposure to plasma. Structural analysis revealed that the plasma-induced oxidation of sulfhydryl and intermolecular disulfide bonds played a pivotal role in inhibiting the thermal aggregation, considered by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), multiplies spectroscopy, and analysis of dynamic exchange of sulfhydryl-disulfide bonds. Meanwhile, the oxidation of exposed hydrophobic sites due to plasma treatment resulted in the transformation of the OVA molecule's surface from hydrophobic to hydrophilic, contributing significantly to the aggregation inhibition. Additionally, compared to an untreated sample of OVA, almost one-fold increase in emulsifying ability (EAI) and 1.5-fold in emulsifying stability (ESI) was observed after 4 min of plasma treatment. These findings demonstrated that plasma treatment not only enhanced the thermal stability of OVA, but also improved its emulsification properties.

Evaluation of genetic diversity of some Zucchini yellow mosaic virus-infected cucurbits based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, inter simple sequence repeat and start codon targeted molecular tools

Evaluation of genetic diversity of some Zucchini yellow mosaic virus-infected cucurbits based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, inter simple sequence repeat and start codon targeted molecular tools

Identification and enzymatic properties of arginine decarboxylase from Aspergillus oryzae.

Aspergillus oryzae spores, when sprinkled onto steamed rice and allowed to propagate, are referred to as rice "koji." Agmatine, a natural polyamine derived from arginine through the action of arginine decarboxylase (ADC), is abundantly produced by solid state-cultivated rice koji of A. oryzae RIB40 under low pH conditions, despite the apparent absence of ADC orthologs in its genome. Mass spectrometry imaging revealed that agmatine was accumulated inside rice koji at low pH conditions, where arginine was distributed. ADC activity was predominantly observed in substrate mycelia and minimally in aerial mycelia. Natural ADC was isolated from solid state-cultivated A. oryzae rice koji containing substrate mycelia, using ammonium sulfate fractionation, ion exchange, and gel-filtration chromatography. The purified protein was subjected to sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE), and the detected peptide band was digested for identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The gene AO090102000327 of strain RIB40 was identified, previously annotated as phosphatidylserine decarboxylase (PSD), and encoded a 483-amino acid peptide. Recombinant protein encoded by AO090102000327 was expressed in Escherichia coli cells cultivated at 20°C, resulting in the detection of 49 kDa and 5 kDa peptides. The protein exhibited pyruvoyl-dependent decarboxylase activity, favoring arginine over ornithine and showing no activity with phosphatidylserine. The gene was designated Ao-adc1. Ao-ADC1 expression in rice koji at pH 4-6 was confirmed through western blotting using the anti-Ao-ADC1 serum. These findings indicate that Ao-adc1 encodes arginine decarboxylase involved in agmatine production.IMPORTANCEGene AO090102000327 in A. oryzae RIB40, previously annotated as a PSD, falls into a distinct clade when examining the phylogenetic distribution of PSDs. Contrary to the initial PSD annotation, our analysis indicates that the protein encoded by AO090102000327 is expressed in the substrate mycelia area of solid state-cultivated A. oryzae rice koji and functions as an arginine decarboxylase (ADC). The clade to which Ao-ADC1 belongs includes three other Ao-ADC1 paralogs (AO090103000445, AO090701000800, and AO090701000802) that presumably encode ADC rather than PSDs. Regarding PSD, AO090012000733 and AO090005001124 were speculated to be nonmitochondrial and mitochondrial PSDs in A. oryzae RIB40, respectively.

Collagen derived from a giant African snail (Achatina achatina) for biomedical applications

Achatina achatina (AA) is a rich source of collagen due to its large size, but it is underutilized. Type I collagen was extracted from AA to serve as an alternative to existing collagen sources. The collagen was extracted at varying alkaline and temperature conditions to determine the optimal parameters that would give a high yield of acid-soluble collagen. The extracted collagen was characterised using X-ray diffraction, Fourier transform infrared (FTIR) spectrometry, thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC) to confirm the integrity and purity of the extracted collagen. The type of collagen was determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The α-1, α-2, and dimer electrophoresis bands confirmed that the collagen is type I, and the XRD data supported the findings. The highest collagen yield was obtained at 4 ℃ for 48 h, which decreased with increasing temperature due to the instability of the protein in acid at high temperatures. A cytotoxicity test was conducted using an Alamar blue assay. The AA collagen-treated normal prostate cell line (PNT2) showed no significant difference from the untreated control cells. The high-quality type I collagen extracted from AA has the potential for biomedical and other industrial applications.

Use of Pea Proteins in High-Moisture Meat Analogs: Physicochemical Properties of Raw Formulations and Their Texturization Using Extrusion.

A new form of plant-based meat, known as 'high-moisture meat analogs' (HMMAs), is captivating the market because of its ability to mimic fresh, animal muscle meat. Utilizing pea protein in the formulation of HMMAs provides unique labeling opportunities, as peas are both "non-GMO" and low allergen. However, many of the commercial pea protein isolate (PPI) types differ in functionality, causing variation in product quality. Additionally, PPI inclusion has a major impact on final product texture. To understand the collective impact of these variables, two studies were completed. The first study compared four PPI types while the second study assessed differences in PPI inclusion amount (30-60%). Both studies were performed on a Wenger TX-52 extruder, equipped with a long-barrel cooling die. Rapid-visco analysis (RVA) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated differences in protein solubility among the different PPI types. In general, lower protein solubility led to better product quality, based on visual evaluation. Cutting strength and texture profile analysis showed increasing PPI inclusion from 30-60% led to significantly higher product hardness (14,160-16,885 g) and toughness (36,690-46,195 g. s). PPI4 led to lower product toughness (26,110 and 33,725 g. s), compared to the other PPIs (44,620-60,965 g. s). Heat gelling capacity of PPI4 was also highest among PPI types, by way of least gelation concentration (LGC) and RVA. When compared against animal meat, using more PPI (50-60%) better mimicked the overall texture and firmness of beef steak and pork chops, while less PPI better represented a softer product like chicken breast. In summary, protein content and also functionality such as cold water solubility and heat gelation dictated texturization and final product quality. High cold water solubility and poor heat gelation properties led to excessive protein cross linking and thicker yet less laminated shell or surface layer. This led to lower cutting firmness and toughness, and less than desirable product texture as compared to animal meat benchmarks. On the other hand, pea proteins with less cold water solubility and higher propensity for heat gelation led to products with more laminated surface layer, and higher cutting test and texture profile analysis response. These relationships will be useful for plant-based meat manufacturers to better tailor their products and choice of ingredients.

Impact of Lipid Composition on Vesicle Protein Adsorption: A BSA Case Study.

Investigating the interaction between liposomes and proteins is of paramount importance in the development of liposomal formulations with real potential for bench-to-bedside transfer. Upon entering the body, proteins are immediately adsorbed on the liposomal surface, changing the nanovehicles' biological identity, which has a significant impact on their biodistribution and pharmacokinetics and ultimately on their therapeutic effect. Albumin is the most abundant plasma protein and thus usually adsorbs immediately on the liposomal surface. We herein report a comprehensive investigation on the adsorption of model protein bovine serum albumin (BSA) onto liposomal vesicles containing the zwitterionic lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), in combination with either cholesterol (CHOL) or the cationic lipid 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP). While many studies regarding protein adsorption on the surface of liposomes with different compositions have been performed, to the best of our knowledge, the differential responses of CHOL and DOTAP upon albumin adsorption on vesicles have not yet been investigated. UV-vis spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a strong influence of the phospholipid membrane composition on protein adsorption. Hence, it was found that DOTAP-containing vesicles adsorb proteins more robustly but also aggregate in the presence of BSA, as confirmed by DLS and TEM. Separation of liposome-protein complexes from unadsorbed proteins performed by means of centrifugation and size exclusion chromatography (SEC) was also investigated. Our results show that neither method can be regarded as a golden experimental setup to study the protein corona of liposomes. Yet, SEC proved to be more successful in the separation of unbound proteins, although the amount of lipid loss upon liposome elution was higher than expected. In addition, coarse-grained molecular dynamics simulations were employed to ascertain key membrane parameters, such as the membrane thickness and area per lipid. Overall, this study highlights the importance of surface charge and membrane fluidity in influencing the extent of protein adsorption. We hope that our investigation will be a valuable contribution to better understanding protein-vesicle interactions for improved nanocarrier design.

Use of Microbial L-asparaginase in Cancer Treatment -A Detail Review

Abstract The exceptional anticancer effects of l-asparaginases, which hydrolyze plasma l-asparagine and l-glutamine, have drew a lot of interest in recent years. This enzyme is effective against acute lymphoblastic leukemia (ALL) and lymphosarcoma, as well as ALL in children, neoplasia, and various other cancer .The l-asparaginase anticancer application technique has achieved amazing advances in modern oncology by depleting plasma l-asparagine to suppress cancer cell development, notably in youngsters. As bacterial alternative enzyme sources for anticancer therapy, high levels of l-asparaginase enzyme production by Escherichia coli, Erwinia species, Streptomyces, and Bacillus subtilis species are very desirable. Purification of L-asparaginase by Ammonium Sulphate precipitation and various chromatographic technique methods, and improvement of enzyme purity by dialysis confirmation of molecular weight with SDS page.

Preparation of pumpkin seed protein isolate nanoparticles by heat-assisted pH-shifting: Enhanced emulsification performance and dispersibility

A food-grade Pickering emulsifier was fabricated using heat-assisted pH-shifting (HP) method for enhancing the emulsion stability. The emulsification performance of the modified pumpkin seed protein isolate (PSPI) nanoparticles was initially tested. The results showed that the modified PSPI had substantially improved emulsifying capacity, with the internal oil phase volume fraction reaching up to 80%, and the emulsion remaining stable after centrifugation at 10,000 g for 60 min and 30 days of storage. The data from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), UV–visible, intrinsic fluorescence and fourier transform infrared spectroscopy (FTIR) indicated that the primary, secondary and tertiary structures of PSPI were disrupted, resulting in changes in interfacial tension, wettability, particle size, and dispersibility of the protein, which promoted the adsorption of modified PSPI nanoparticles to the surface of oil droplets and improved the emulsion stability. Overall, the HP modified PSPI is a promising Pickering emulsifier for various applications.

Innovative Reversed-Phase Chromatography Platform Approach for the Fast and Accurate Characterization of Membrane Vesicles' Protein Patterns.

Outer membrane vesicles (OMVs) have been widely explored to develop vaccine candidates for bacterial pathogens due to their ability to combine adjuvant properties with immunogenic activity. OMV expresses a variety of proteins and carbohydrate antigens on their surfaces. For this reason, there is an analytical need to thoroughly characterize the species expressed at their surface: we here present a simple and accurate reversed-phase ultrahigh-performance liquid chromatography (RP-UPLC) method developed according to quality by design principles. This work provides an analytical alternative to the classical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) characterization. The higher selectivity and sensitivity of the RP-UHPLC assay allow for the identification of additional protein species with respect to SDS-PAGE and facilitate its precise relative abundance quantification. According to validation results, the assay showed high accuracy, linearity, precision, repeatability, and a limit of quantification of 1% for less abundant proteins. This performance paves the way for improved production campaign consistency while also being analytically simple (no sample pretreatment required), making it suitable for routine quality control testing. In addition, the applicability of the assay to a wider range of vesicle classes (GMMA) was demonstrated.

Morinda Officinalis-derived extracellular vesicle-like particles: Anti-osteoporosis effect by regulating MAPK signaling pathway

BackgroundPostmenopausal osteoporosis (PMOP) is a systemic bone disease characterized by low bone mass and microstructural damage. Morinda Officinalis (MO) contains various components with anti-PMOP activities. Morinda Officinalis-derived extracellular vesicle-like particles (MOEVLPs) are new active components isolated from MO, and no relevant studies have investigated their anti-osteoporosis effect and mechanism. PurposeTo investigate the alleviating effect of MOEVLPs on PMOP and the underlying mechanism. MethodsDifferential centrifugation and ultracentrifugation were used to isolate MOEVLPs from MO. Transmission electron microscopy (TEM), flow nano analyzer, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), agarose gel electrophoresis, and thin-layer chromatography were employed to characterize MOEVLPs. PMOP mouse models were utilized to examine the anti-PMOP effect of MOEVLPs. H&E and immunohistochemical staining were used for drug safety and osteogenic effect assessment. Mouse embryo osteoblast precursor cells (MC3T3-E1) were used in vitro experiments. CCK-8 kit, alizarin red staining, proteomic, bioinformatic analyses, and western blot were used to explore the mechanism of MOEVLPs. ResultsIn this study, MOEVLPs from MO were successfully isolated and characterized. Animal experiments demonstrated that MOEVLPs exhibited specific femur targeting, were non-toxic to the heart, liver, spleen, lung, kidney, and aorta, and possessed anti-PMOP properties. The ability of MOEVLPs to strengthen bone formation was better than that of alendronate. In vitro experiments, results revealed that MOEVLPs did not significantly enhance osteogenic differentiation in MC3T3-E1 cells. Instead, MOEVLPs promoted the proliferation of MC3T3-E1 cells. Proteomic and bioinformatic analyses suggested that the proliferative effect of MOEVLPs was closely associated with the mitogen-activated protein kinase (MAPK) signaling pathway, particularly the altered expression of cAMP response element-binding protein (CREB) and ribosomal S6 kinase 1 (RSK1). Western blot results further confirmed these findings. ConclusionOur studies successfully isolated high-quality MOEVLPs and demonstrated that MOEVLPs can alleviate PMOP by promoting osteoblast proliferation through the MAPK pathway. MOEVLPs have the potential to become a novel and natural anti-PMOP drug.

Chitin Deacetylase from Bacillus aryabhattai TCI-16: Heterologous Expression, Characterization, and Deacetylation Performance.

Chitin deacetylase (CDA) removes the acetyl group from the chitin molecule to generate chitosan in a uniform, high-quality deacetylation pattern. Herein, BaCDA was a novel CDA discovered from our previously isolated Bacillus aryabhattai strain TCI-16, which was excavated from mangrove soil. The gene BaCDA was cloned and overexpressed in Escherichia coli BL21 (DE3) to facilitate its subsequent purification. The purified recombinant protein BaCDA was obtained at a concentration of about 1.2 mg/mL after Ni2+ affinity chromatography. The molecular weight of BaCDA was around 28 kDa according to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. In addition, BaCDA exhibited a significant deacetylation effect on colloidal chitin, and the deacetylation degree was measured from the initial 25.69 to 69.23% by Fourier transform infrared (FT-IR) spectroscopy. Scanning electron microscopy (SEM) observation showed that the surface of colloidal chitin after enzymatic digestion was rough, the crystal fibers disappeared, and the chitin structure was loose and porous with grooves. The results of electrospray ionization mass spectrometry (ESI-MS) showed that BaCDA had full-deacetylation activity against (GlcNAc)4. Molecular docking revealed that BaCDA had an open active pocket capable of binding to the GlcNAc unit. This study not only provides a novel enzymatic resource for the green and efficient application of chitin but also helps to deepen the understanding of the catalytic mechanism of CDA.

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.