Gel Electrophoresis Of Proteins Research Articles

Characterization of Bila Tserkva winter common wheat cultivars with respect to marker loci

Aim. We studied special features of the sample of Bila Tserkva winter common wheat cultivars with respect to storage protein loci and some disease resistance genes. Methods. To identify alleles at gliadin and high-molecular-weight glutenin loci, acid polyacrylamide gel electrophoresis and SDS-electrophoresis of grain proteins were carried out. Alleles of markers for the disease resistance genes Lr34, Tsn1, and TDF_076_2D were identified using PCR. Results. The composition and frequencies of alleles at the Gli-1, Glu-1, and Gli-A3 loci were studied in samples of Bila Tserkva cultivars released before 2011 and after 2010. For the latter group of cultivars, alleles of the disease resistance gene markers were identified. We observed a significant increase in the frequency of the allele Gli-A1x(9) and a decrease in the frequency of Gli-A1c, as well as a tendency for an increase in the frequency of Gli-A3c. In the total sample of Bila Tserkva cultivars, nonrandom associations of pairs of certain storage protein alleles were revealed. Conclusions. The group of Bila Tserkva cultivars is similar to the previously studied groups of the Central Forest Steppe of Ukraine with respect to allele set and frequencies of the main prolamin loci, but differs in the frequencies of the disease resistance genes. A special feature of the group of Bila Tserkva cultivars is a high frequency of the allele c at the minor locus Gli-A3, as well as the allele association Gli-A1x Gli-B1l Gli-A3c.

Polysaccharide as a Separation Medium for Gel Electrophoresis

Gel electrophoresis and size exclusion chromatography (SEC) are vital techniques in biochemical research, employing gel matrix structures made of polysaccharides or synthetic polymers like polyacrylamide for the analysis and separation of macromolecules. Polysaccharides, such as agarose, offer safer alternatives to acrylamide. Polysaccharide gels, notably agarose, facilitate the analysis and purification of proteins and nucleic acids through a molecular sieving mechanism. Gel electrophoresis for proteins is mainly divided into denaturing and native methods. Denaturing electrophoresis with sodium dodecyl sulfate (SDS) simplifies protein migration but disrupts molecular interactions. Conversely, native gel electrophoresis, without SDS, allows proteins to migrate based on the running pH and the isoelectric point of the proteins, while nucleic acids consistently migrate toward the anode. The electrophoresis of proteins with variable charges presents complexes. This review focuses on the use of polysaccharides, particularly agarose, for native gel electrophoresis, highlighting their applications in separating macromolecules. It also discusses the applications and limitations of agarose gels when used as a matrix for electrophoresis. Such information should help in designing electrophoresis experiments using polysaccharides.

Different behavior of Ferguson plot between agarose and polyacrylamide gels

In this study, we conducted Ferguson plot analyses using both agarose and polyacrylamide gels in native electrophoresis and SDS-PAGE. The results revealed intriguing differences in the behavior of bovine serum albumin (BSA) and other model proteins. Specifically, BSA exhibited Ferguson plot slopes that were dependent on the oligomer size in agarose native gel electrophoresis, while such size-dependent behavior was not observed in native-PAGE or SDS-PAGE. These findings suggest that Ferguson plot analysis is a suitable approach when using agarose gel under the electrophoretic conditions employed in this study. Furthermore, our investigation extended to model proteins with acidic isoelectric points and larger molecular weights, namely Ferritin and caseinolytic peptidase B (ClpB). Notably, these proteins displayed distinct Ferguson plot slopes when subjected to agarose gel electrophoresis. Intriguingly, when polyacrylamide gel was employed, ClpB exhibited multiple bands, each with its unique Ferguson plot slope, deviating from the expected behavior based on molecular size. This divergence in Ferguson plot characteristics between agarose and polyacrylamide gels points to an interesting and complex interplay between protein properties and gel electrophoresis conditions.

Real-time and quantitative protein detection via polyacrylamide gel electrophoresis and online intrinsic fluorescence imaging

The detection of intrinsic protein fluorescence is a powerful tool for studying proteins in their native state. Thanks to its label-free and stain-free feature, intrinsic fluorescence detection has been introduced to polyacrylamide gel electrophoresis (PAGE), a fundamental and ubiquitous protein analysis technique, to avoid the tedious detection process. However, the reported methods of intrinsic fluorescence detection were incompatible with online PAGE detection or standard slab gel. Here, we fulfilled online intrinsic fluorescence imaging (IFI) of the standard slab gel to develop a PAGE-IFI method for real-time and quantitative protein detection. To do so, we comprehensively investigated the arrangement of the deep-UV light source to obtain a large imaging area compatible with the standard slab gel, and then designed a semi-open gel electrophoresis apparatus (GEA) to scaffold the gel for the online UV irradiation and IFI with low background noise. Thus, we achieved real-time monitoring of the protein migration, which enabled us to determine the optimal endpoint of PAGE run to improve the sensitivity of IFI. Moreover, online IFI circumvented the broadening of protein bands to enhance the separation resolution. Because of the low background noise and the optimized endpoint, we showcased the quantitative detection of bovine serum albumin (BSA) with a limit of detection (LOD) of 20 ng. The standard slab gel provided a high sample loading volume that allowed us to attain a wide linear range of 0.03–10 μg. These results indicate that the PAGE-IFI method can be a promising alternative to conventional PAGE and can be widely used in molecular biology labs.

Detecting PTP Protein-Protein Interactions by Fluorescent Immunoprecipitation Analysis (FIPA).

Identifying protein-protein interactions is crucial for revealing protein functions and characterizing cellular processes. Manipulating PPIs has become widespread in treating human diseases such as cancer, autoimmunity, and infections. It has been recently applied to the regulation of protein tyrosine phosphatases (PTPs) previously considered undruggable. A broad panel of methods is available for studying PPIs. To complement the existing toolkit, we developed a simple method called fluorescent immunoprecipitation analysis (FIPA). This method is based on coimmunoprecipitation followed by protein gel electrophoresis and fluorescent imaging to visualize components of a protein complex simultaneously on a gel. The FIPA allows the detection of proteins expressed under native conditions and is compatible with mass spectrometry identification of protein bands.

Inventory and quality control of biosample collection from pregnant women at different gestational ages to search for early biomarkers of pregnancy complications

Aim. To conduct an inventory and quality control of biosample collection from pregnant women at different gestational ages to search for early biomarkers of pregnancy complications.Material and methods. In this work, methods for assessing the sample preparation of biosamples were used, including the isolation of deoxyribonucleic acid (DNA)/ribonucleic acid from various biomaterials, polyacrylamide gel electrophoresis of protein, and database analysis.Results. Inventory and quality control of the collection (n=18390) was carried out, which confirmed the high safety of the biomaterial, regardless of storage period. The mean concentration of DNA was 69,96±6,56 ng/µl, extracellular DNA (ecDNA) — 0,20±0,02 ng/µl, ribonucleic acid — 38,16±5,69 ng/µl. DNA Integrity Number (DIN) >9, RNA integrity number (RIN) >7, A260/280 >1,8 were for all studied samples. Protein electrophoresis demonstrated no degradation of protein zones after longterm storage. The number of errors detected during the inventory was 84 (0,46% of all records in the database), while there were 64 donors with incomplete clinical information (15% of all donors in the collection).Conclusion. The necessity of mandatory implementation of standard operating procedures when creating and maintaining a collection, on the one hand, and periodic inventory with biosample quality assessment, on the other, has been demonstrated.

Protein Stability in Pasteurized Cow and Goat Milk using Protein Gel Electrophoresis

Milk is a nutritious beverage that contains whey, casein, and milk fat globule membrane (MFGM) proteins. Pasteurization kills pathogenic microorganisms in milk, making it safe to drink. A high heat load during processing can affect the protein quality of milk. In this study, protein gel electrophoresis (Native and SDS-PAGE) was used to view the protein profiles of retail cow and goat milk samples pasteurized at different temperatures (145, 165, or 280 °F) compared to commercially spray dried milk (356–482 °F). SDS-PAGE provided better resolution of the milk proteins compared to Native-PAGE. There were clear species differences in the MFGM proteins. This could be used to spot adulteration of goat milk with cow milk, however none of the samples showed signs of adulteration. There were no overt patterns of protein degradation with increasing pasteurization temperature. However drying reduced band intensity, especially MFGM and whey proteins in the goat milk sample. Whereas most samples came from different herds where genetics and environmental conditions varied, two of the goat milk samples were from the same herd, one fresh and one spray dried. It was evident from these two samples that spray drying can alter proteins in goat milk. Even so, if fresh milk is unavailable or unaffordable, dry milk remains a valuable protein source.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis of toxin proteins of Bacillus thuringiensis isolated from soil at the Liwa Botanical Garden

Bacillus thuringiensis is a spore-forming bacterium known to have the ability to synthesize proteins that are toxic against insect pests. Currently, the bacterial protein toxins are widely used as pesticides because they are environmentally friendly and safe for health. As a cosmopolitan microbe B. thuringiensis is widely distributed in nature with soil as its main habitat. Liwa Botanical Gardens is one among the conservation areas in Lampung Province of Indonesia where the soil type and conditions expected to good for the bacterial life of B. thuringiensis. This study aims to determine toxin proteins extracted from B. thuringiensis isolated from the soil of the Liwa Botanical Garden by their molecular weights. The proteins were quantified using Lowry method and the protein profiling performed using SDS PAGE electrophoresis. The results showed that there were two isolates, Bt 3 and Bt 5 which showed close protein concentrations, namely 0.679 mg/ml and Bt 5 with 1.313 mg/ml respectively. Both isolates also exhibit different molecular weights. Bt 3 isolate has a molecular weight of 110.59 kDa, while Bt 5 has a molecular weight of 76.97 kDa.

The Mobility of Eurasian Avian-like M2 Is Determined by Residue E79 Which Is Essential for Pathogenicity of 2009 Pandemic H1N1 Influenza Virus in Mice.

In 2009, a novel H1N1 influenza virus caused the first influenza pandemic of the 21st century. Studies have shown that the influenza M gene played important roles in the pathogenicity and transmissibility of the 2009 H1N1 pandemic ((H1N1)pdm09), whilst the underlying mechanism remains unclear. The influenza M gene encodes two proteins, matrix protein 1 and matrix protein 2, which play important roles in viral replication and assembly. In this study, it is found that the M2 protein of the (H1N1)pdm09 virus showed a lower mobility rate than the North America triple-reassortant influenza M2 protein in Polyacrylamide Gel Electrophoresis (PAGE). The site-directed mutations of the amino acids of (H1N1)pdm09 M2 revealed that E79 is responsible for the mobility rate change. Further animal studies showed that the (H1N1)pdm09 containing a single M2-E79K was significantly attenuated compared with the wild-type virus in mice and induced lower proinflammatory cytokines and IFNs in mouse lungs. Further in vitro studies indicated that this mutation also affected NLRP3 inflammasome activation. To reveal the reason why they have different mobility rates, a circular dichroism spectra assay was employed and showed that the two M2 proteins displayed different secondary structures. Overall, our findings suggest that M2 E79 is important for the virus replication and pathogenicity of (H1N1)pdm09 through NLRP3 inflammasome and proinflammatory response.

Toluidine blue O photosensitizer combined with caffeic acid improves antibacterial performance by increasing the permeability of cell membrane

Photodynamic therapy has always been an antibacterial tool for solving multi-drug resistant bacteria problem, but the side effects and the low efficiency due to the high aggregation and low solubility of photosensitizers limit its application. Due to the anti-inflammatory effect of caffeic acid, two novel photosensitizers (CA-1-TBO, CA-TBO) were synthesized by conjugating caffeic acid with toluidine blue O (TBO). The structures have been characterized by 1HNMR and high-resolution mass spectrometry. The UV-vis absorption, fluorescence spectra and the octanol-water partition coefficient of two photosensitizers were measured to evaluate their photophysical properties and hydrophilic/hydrophobic properties. Compared with parent TBO, the two modified photosensitizers have shown a higher quantum yield and kinetics constants of singlet oxygen, which has been supported by the simulation results of density functional theory. As drug-resistant representatives of gram-positive and gram-negative bacteria, respectively, S. aureus and P. aeruginosa have been used for in vitro antibacterial experiments. The sterilization efficiencies of the two modified photosensitizers far exceed that of parent TBO. The results of the octanol-water partition coefficient and fluorescence quantification showed that modified photosensitizers CA-1-TBO and CA-TBO could be more accumulated than parent TBO. Based on scanning electron microscopy images, protein gel electrophoresis, and the conductivity of the bacterial solution, the possible mechanism of improved antibacterial photodynamic efficiencies could be induced by membrane permeability due to the caffeic acid effect. The findings demonstrate the significant potential of natural phenolic compounds in the development of photosensitizer molecules with characteristics such as more efficient, biocompatible and less side effects.

Fate of pulse globulin proteins molecular Structure and composition on high moisture extrusion

Mungbean and lentil proteins are gaining attention for the production of high moisture extrusion (HME)-based meat- and dairy-analogues. However, understanding the mechanism of fibrous structure formation and molecular and higher-order changes in HME remains limited. Globulin protein isolates from mungbean (MBPI), green lentil (GLPI) and yellow pea (YPPI) along with commercial soy (CSPI) and yellow pea (CPPI) protein were investigated for their pasting properties using high-pressure rapid visco analyser (RVA), and the formation of anisotropic structures and molecular changes at constant HME conditions. Vicilin-rich MBPI showed higher viscosity on RVA, consumed higher specific mechanical energy (SME) during extrusion, and developed extrudates with higher textural strength and cutting force than proteins with both legumin and vicilin-like globulins, i.e. GLPI and YPPI. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and size-exclusion chromatography (SEC) of extrudates' soluble proteins showed dissociation of vicilin-like globulins into their smaller subunits. In contrast, legumin-like globulins were reaggregated into higher molecular weight soluble macromolecules. Further, SDS-PAGE of extrudates’ total protein revealed the involvement of legumin-like globulin in the macrostructure network of the extrudates. During HME, native unfolded structures were destroyed with conversion, predominately driving changes in the tertiary structure (thermal denaturation) and secondary structure with loss of helical structure to formation of random coil and beta-sheet structures. Protein extractability in different solvents was greatly reduced, with covalent and non-covalent interactions being majorly involved in the stabilisation of extrudates structure. Protein composition, and in particular vicilin-legumin ratio and protein conformation, determined the structural development and molecular changes in HME.

Production of branched-chain flavor compounds from hydrolysis of pork myofibrillar protein by optimizing the inoculation of Limosilactobacillus fermentum YZU-06 and Staphylococcus saprophyticus CGMCC 3475

This study aimed to develop a mixed starter of Limosilactobacillus fermentum YZU-06 and Staphylococcus saprophyticus CGMCC 3475 isolated from Jinhua ham for enhancing the production of branched-chain flavor compounds from pork myofibrillar protein. Different ratios of mixed strains (3:1, 2:1, 1:2, and 1:3) and single strains were employed to incubate with myofibrillar protein extract to detect viable counts, pH, proteolysis, and branched-chain volatiles at 24, 48 and 72 h of incubation. The result showed that the inoculated strains had acid-producing characteristics and bacterial suitability. The mixed starter of 2:1, 1:2, and 1:3 treatments exhibited a higher degree of protein degradation, as indicated by a 90% similarity through cluster analysis of myofibrillar protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Compared to the other treatment groups, 2:1 group showed a significantly higher peptide content of 123.58 μg/mL and a greater concentration of free amino acids (236.67 mg/L). Furthermore, 2:1 group obtained the highest levels of 3-methylbutanal (56.06 μmol/L), 2-methylpropanal (1.76 μmol/L), and 2-methylbutanal (0.48 μmol/L). Our findings emphasize the importance of appropriate proportions of L. fermentum YZU-06 and S. saprophyticus CGMCC 3475 for the starter to produce branched-chain flavor compounds of meat products.

Ultrasound and enzyme‐pretreated extraction for the valorization of pea pod proteins

AbstractPea pod (PP) is an agricultural by‐product with 10%–13% protein content which has a potential to be converted to value‐added ingredients. In this study, different protein extraction strategies including three different solvents (water, salt, and alkaline) and two different pretreatments (ultrasound [US] and enzyme such as pectinase and Viscozyme®) before conventional alkaline extraction were employed to maximize the extraction yield. The US‐pretreated alkaline extraction achieved the highest yield (21.1%) in protein extracts under optimum conditions (30% amplitude for 5 min). PP protein concentrates showed significant differences in solubility, sodium dodecyl sulfate–polyacrylamide gel electrophoresis protein patterns, scanning electron microscopy images, and color depending on the applied extraction method. All PP residues after extraction still remained protein (5.2–8.0 g/100 g) regardless of the extraction techniques, representing the challenges in protein extraction from agricultural by‐products with a lignocellulosic matrix. The combined use of US and enzyme treatments should be further investigated for higher extraction yields.Practical ApplicationsAgricultural wastes and by‐products could be potential protein sources; however, their lignocellulosic structure could be challenging for protein extraction. In the current study, different extraction strategies including ultrasound (US) and enzyme pretreatments were performed for the extraction of proteins from pea pods which is an agro‐industrial by‐product. Results suggest that US pretreatment may have promising applications for the extraction of valuable compounds from agricultural and industrial by‐products, contributing to sustainable food processing strategies.

Clonal diversity of Staphylococcus aureus isolates in clinical specimens from selected health facilities in Ethiopia

Staphylococcus aureus is among the top three causative agents of nosocomial infection in Ethiopia. The majority of studies in Ethiopia have focused on the epidemiology of S. aureus in hospital settings, with limited molecular genotyping results. Molecular characterization of S. aureus is essential for identification of strains, and contributes to the control and prevention of S. aureus infection. The aim of the current study was to determine the molecular epidemiology of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates recovered from clinical specimens in Ethiopia. A total of 161 MSSA and 9 MRSA isolates were characterized using pulsed-field gel electrophoresis (PFGE) and staphylococcal protein A (spa) typing. Based on the PFGE analysis, MSSA isolates were grouped into eight pulso-types groups (from A to I), while MRSA isolates clustered into three (A, B and C) pulso-types with more than 80% similarity. The spa typing analysis showed diversity of S. aureus with 56 distinct spa types. Spa type t355 was most prevalent (56/170, 32.9%), while eleven new spa types were detected including t20038, t20039, and t20042. The identified spa types were clustered into 15 spa-clonal complexes (spa-CCs) using BURP analysis; novel/unknown spa types were further subjected to MLST analysis. The majority of isolates belonged to spa-CC 152 (62/170, 36.4%), followed by spa-CC 121 (19/170, 11.2%), and spa-CC 005 (18 /170, 10.6%). Of the nine MRSA isolates, 2 (22.2%) were spa-CC 239 with staphylococcal cassette chromosome (SCC)mec III. These findings highlight the diversity of S. aureus strains in Ethiopia, as well as the presence of potentially epidemic strains circulating in the country necessitating further characterization of S. aureus for antimicrobial resistance detection and infection prevention purposes.

Harnessing the Topics of Cytochrome P450 Enzymology and Artemisinin to Teach a Semester-Long Biochemistry Laboratory Course

Eleven different laboratory experiments were designed and executed throughout a semester to provide a meaningful research experience for 23 undergraduate biochemistry majors at UTSA. The topic of the semester was based on the idea of exploring new aspects to enhance our understanding of how the human body uses cytochrome P450 enzymes to metabolize artemisinin, an endoperoxide-containing plant natural product used to treat malaria. Biochemical techniques introduced to the students included computational (dry lab) techniques: the use of bioinformatics tools to design plasmids for protein expression, software to analyze the 3-dimensional structure of proteins, metabolomics software to analyze enzyme-catalyzed reaction extracts by mass spectrometry, and docking software to dock ligands into the active site of proteins. In addition, students performed experimental (wet lab) techniques including: a transformation experiment to incorporate plasmid DNA into bacteria, an extraction of plasmid DNA from bacterial cells, performing enzymatic incubations and preparing samples for analysis by mass spectrometry, and running an SDS protein gel electrophoresis. The semester ended with the use of a web-based program to allow students to visualize the proteins they were studying throughout the semester with a virtual reality headset. This course was taught for the first time at the university, so this manuscript should inspire ideas for future biochemistry laboratory courses taught in the course based undergraduate research experiences (CUREs) format.

Qualitative and quantitative determination of proteins in extracts of some medicinal plants

Introduction. Global consumption of plant protein is increasing and high-fiber plants have health benefits through valuable phytochemicals. Plant proteins serve as an alternative to animal proteins to meet consumer demand on the one hand and reduce the risk of disease on the other. Material and Methods. We performed qualitative and quantitative determination of proteins in extracts by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and total content of proteins by Bradford assay. Results. Applied methods for the qualitative and quantitative determination of proteins in dry extracts of medicinal plants: Agrimonia eupatoria L., Cichorium intybus L., Galium verum L., and Solidago virgaurea L. confirm that proteins were detected in all dry extracts and were determined the total content of them. Conclusion. For all extracts obtained from aerial parts of: Agrimonia eupatoria, Cichorium intybus, Galium verum and Solidago virgaurea we find a low protein concentration, which implies minimizing allergic reactions and intolerance to the extracts studied.

Transcriptome responses of lactic acid bacteria isolated from kimchi under hydrogen peroxide exposure

In this study, five species of lactic acid bacteria (LAB) isolated from kimchi were analyzed in terms of their potential antioxidant activity. Latilactobacillus curvatus WiKim38, Companilactobacillus allii WiKim39, and Lactococcus lactis WiKim0124 exhibited higher radical scavenging activity, reducing power, and lipid peroxidation inhibition than the reference strain and tolerated hydrogen peroxide (H2O2) exposure up to a concentration of 2.5 mM. To investigate the antioxidant mechanism of LAB strains, transcriptomic and proteomic signatures were compared between the H2O2-exposed and untreated group using RNA sequencing and two-dimensional protein gel electrophoresis. Across all LAB strains, cell membrane responses and metabolic processes were the most prominent in the main categories of gene ontology classification, indicating that cellular components and interactions play an important role in oxidative stress responses. Thus, LAB strains isolated from kimchi could be considered for potential use in functional food production and in antioxidant starter cultures.

Quiescence of Escherichia coli Aerosols to Survive Mechanical Stress during High-Velocity Collection.

A low cutpoint wetted wall bioaerosol sampling cyclone (LCP-WWC), with an aerosol sampling flow rate of 300 L/min at 55″ H2O pressure drop and a continuous liquid outflow rate of about 0.2 mL/min, was developed by upgrading an existing system. The laboratory strain Escherichia coli MG1655 was aerosolized using a six-jet Collison Nebulizer and collected at high velocity using the LCP-WWC for 10 min with different collection liquids. Each sample was quantitated during a 15-day archiving period after aerosolization for culturable counts (CFUs) and gene copy numbers (GCNs) using microbial plating and whole-cell quantitative polymerase chain (qPCR) reaction. The samples were analyzed for protein composition and antimicrobial resistance using protein gel electrophoresis and disc diffusion susceptibility testing. Aerosolization and collection were followed by an initial period of quiescence or dormancy. After 2 days of archiving at 4 °C and RT, the bacteria exhibited increased culturability and antibiotic resistance (ABR), especially to cell wall inhibitors (ampicillin and cephalothin). The number of resistant bacteria on Day 2 increased nearly four-times compared to the number of cells at the initial time of collection. The mechanical stress of aerosolization and high-velocity sampling likely stunned the cells triggering a response of dormancy, though with continued synthesis of vital proteins for survival. This study shows that an increase in intensity in environmental conditions surrounding airborne bacteria affects their ability to grow and their potential to develop antimicrobial resistance.

Higher Protein Digestibility of Chicken Thigh than Breast Muscle in an In Vitro Elderly Digestion Model.

This study investigated the protein digestibility of chicken breast and thigh in an in vitro digestion model to determine the better protein sources for the elderly in terms of bioavailability. For this purpose, the biochemical traits of raw muscles and the structural properties of myofibrillar proteins were monitored. The thigh had higher pH, 10% trichloroacetic acid-soluble α-amino groups, and protein carbonyl content than the breast (p<0.05). In the proximate composition, the thigh had higher crude fat and lower crude protein content than the breast (p<0.05). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of myofibrillar proteins showed noticeable differences in the band intensities of tropomyosin α-chain and myosin light chain-3 between the thigh and breast. The intrinsic tryptophan fluorescence intensity of myosin was lower in the thigh than in the breast (p<0.05). Moreover, circular dichroism spectroscopy of myosin revealed that the thigh had higher α-helical and lower β-sheet structures than the breast (p<0.05). The cooked muscles were then chopped and digested in the elderly digestion model. The thigh had more α-amino groups than the breast after both gastric and gastrointestinal digestion (p<0.05). SDS-PAGE analysis of the gastric digesta showed that more bands remained in the digesta of the breast than that of the thigh. The content of proteins less than 3 kDa in the gastrointestinal digesta was also higher in the thigh than in the breast (p<0.05). These results reveal that chicken thigh with higher in vitro protein digestibility is a more appropriate protein source for the elderly than chicken breast.

Analysis of variable major protein antigenic variation in the relapsing fever spirochete, Borrelia miyamotoi, in response to polyclonal antibody selection pressure.

Borrelia miyamotoi is a tick-transmitted spirochete that is genetically grouped with relapsing fever Borrelia and possesses multiple archived pseudogenes that encode variable major proteins (Vmps). Vmps are divided into two groups based on molecular size; variable large proteins (Vlps) and variable small proteins (Vsps). Relapsing fever Borrelia undergo Vmp gene conversion at a single expression locus to generate new serotypes by antigenic switching which is the basis for immune evasion that causes relapsing fever in patients. This study focused on B. miyamotoi vmp expression when spirochetes were subjected to antibody killing selection pressure. We incubated a low passage parent strain with mouse anti-B. miyamotoi polyclonal antiserum which killed the majority population, however, antibody-resistant reisolates were recovered. PCR analysis of the gene expression locus in the reisolates showed vsp1 was replaced by Vlp-encoded genes. Gel electrophoresis protein profiles and immunoblots of the reisolates revealed additional Vlps indicating that new serotype populations were selected by antibody pressure. Sequencing of amplicons from the expression locus of the reisolates confirmed the presence of a predominant majority serotype population with minority variants. These findings confirm previous work demonstrating gene conversion in B. miyamotoi and that multiple serotype populations expressing different vmps arise when subjected to antibody selection. The findings also provide evidence for spontaneous serotype variation emerging from culture growth in the absence of antibody pressure. Validation and determination of the type, number, and frequency of serotype variants that arise during animal infections await further investigations.