Polyacrylamide Gel Research Articles

Effects of spray drying and freeze drying on the protein profile of whey protein concentrate.

Whey protein concentrate (WPC) is consumed for its high protein content. The structure and biological functionality of whey proteins in WPC powders may be affected by the drying technique applied. However, the specific impact of spray drying and freeze drying on the overall protein profile of whey protein derived from sweet whey streams at scale is unknown. Herein, we examine the effects of commercial-scale freeze drying and spray drying on WPC to determine which method better preserves bioactive whey proteins, with the goal of helping the dairy industry create high-value products that meet the growing consumer demand for functional dairy products. WPCs were produced from pasteurized liquid whey using either a commercial spray dryer or freeze dryer. A variety of analytical techniques, including enzyme-linked immunosorbent assay, polyacrylamide gel electrophoresis, and bottom-up proteomics using liquid chromatography-tandem mass spectroscopy were used to identify, quantify, and compare the retention of bioactive proteins in WPC before and after spray drying and freeze drying. In addition, the extent of denaturation was studied via solubility testing, differential scanning calorimetry, and hydrophobicity assessment. There was little to no difference in the retention or denaturation of key bioactive proteins between spray-dried and freeze-dried WPC powders. There was a higher percentage of select Maillard modifications in freeze-dried and spray-dried powders than in the control. The lack of significant differences between spray drying and freeze drying identified herein indicates that freeze drying does not meaningfully improve retention of bioactive proteins compared with spray drying when performed after multiple pasteurization steps. PRACTICAL APPLICATION: This study aimed to provide insight into the impacts of spray drying versus freeze drying on whey proteins. Overall, our results indicate that for commercial dairy processing that involves multiple rounds of pasteurization, freeze drying does not meaningfully improve the retention of bioactive proteins compared with spray drying. These findings may help the food and dairy industry make informed decisions regarding the processing of its whey protein products to optimize nutritional value.

Tetraspanin 5 orchestrates resilience to salt stress through the regulation of ion and reactive oxygen species homeostasis in rice.

Tetraspanins (TETs) are integral membrane proteins, characterized by four transmembrane domains and a unique signature motif in their large extracellular loop. They form dynamic supramolecular complexes called tetraspanin-enriched microdomains (TEMs), through interactions with partner proteins. In plants, TETs are involved in development, reproduction and immune responses, but their role in defining abiotic stress responses is largely underexplored. We focused on OsTET5, which is differentially expressed under various abiotic stresses and localizes to both plasma membrane and endoplasmic reticulum. Using overexpression and underexpression transgenic lines we demonstrate that OsTET5 contributes to salinity and drought stress tolerance in rice. OsTET5 can interact with itself in yeast, suggesting homomer formation. Immunoblotting of native PAGE of microsomal fraction enriched from OsTET5-Myc transgenic rice lines revealed multimeric complexes containing OsTET5, suggesting the potential formation of TEM complexes. Transcriptome analysis, coupled with quantitative PCR-based validation, of OsTET5-altered transgenic lines unveiled the differential expression patterns of several stress-responsive genes, as well as those coding for transporters under salt stress. Notably, OsTET5 plays a crucial role in maintaining the ionic equilibrium during salinity stress, particularly by preserving an elevated potassium-to-sodium (K+/Na+) ratio. OsTET5 also regulates reactive oxygen species homeostasis, primarily by modulating the gene expression and activities of antioxidant pathway enzymes and proline accumulation. Our comprehensive investigation underscores the multifaceted role of OsTET5 in rice, accentuating its significance in developmental processes and abiotic stress tolerance. These findings open new avenues for potential strategies aimed at enhancing stress resilience and making valuable contributions to global food security.

Antioxidant analysis of protein from fresh and dry leaf of Orthosiphon aristatus (Blume) Miq.

Orthosiphon aristatus (Blume) Miq. is a folklore plant consumed as brewed tea for various health benefits. The white variety of the plants leaf and stem are predominantly sold in the market in dried form, blooming as a natural herbal product. To date, no proteomics and antioxidant studies are available on the fresh and dried leaf protein extract of O. aristatus since most studies take an interest in the crude extracts of plants. Thereupon, this study focuses on the One-dimensional (1D) electrophoretic pattern of the fresh and dry leaf determined via sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, antioxidant proteins are currently being focused on for their link to controlling disease through their potential to destroy free radicals that are present in excess. Hence, an antioxidant assay was conducted for the fresh and dry leaf protein extract using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical assay and ferric reducing antioxidant power (FRAP) assay. The electrophoretic pattern obtained resulted in approximately 16 and 10 bands for the fresh and dry leaves respectively. As for the antioxidant activity, the dry leaf had higher radical scavenging activity and higher reducing power compared to the fresh leaf. The findings obtained provides insight in to the protein content and antioxidant activity found in the protein extract of fresh and dry leaves of O. aristatus. Hence, further detailed study of the protein extracts could further unveil their therapeutic potential.

Rhizosphere bacterial community influence on <i>Deschampsia antarctica</i> È Desv. adaptability in context of temperature near plants in local spatial scales of Galindez Island, Argentine Islands (the maritime Antarctic)

The effect of rhizosphere bacterial community index (Irbi) influence on the nine populations of Antarctic hair grass (Deschampsia antarctica) adaptability was studied in the Galindez Island (summer season 2017/18). Moreover, the corresponding influence indices Irbi (i=1÷9) and Irbpi (p=1÷5 for the most common bacteria) were evaluated as well. The objective was to compare the Irbi and Irbpi series with the united temperature influence index on plant populations (It1i(z)) series and the united quality latent index of adaptability Iq1i. The study used data on the rhizosphere metagenome composition based on 16S RNA analysis. Methods determining the plant number in populations, and measuring the morphometric indices of D. antarctica populations were used. Reserve and protective seed proteins spectra were studied by polyacrylamide gel electrophoresis. Method of extreme grouping the spatial variables of these indices was applied for nine populations to obtain a Iq1I and It1i and Irbi, Irbpi. Sets of united indices were compared by regression technique. A comparative statistical analysis of the It1i and Irbi, Irbpi sets in this season was carried out. This possible influence appeared to be individual for each D. antarctica studied population. In each population, part of the plants reacted positively to the bacteria influence, while the other part either did not react or reacted negatively. Dependence of the plant adaptation indices on rhizospheric bacterial communities z(x) is shown in our data. This means that the rhizosphere bacterial community and temperature-dependent rhizosphere bacteria (x) can play an active role in plant adaptation of D. antarctica populations (z) to individual temperature conditions in the microscale of Galindez Island from a biological point of view.

Selection of antibacterial aptamers against Pseudomonas plecoglossicida and analysis on their potential binding proteins

Pseudomonas plecoglossicida is one of the main pathogens causing visceral white spot disease of the large yellow croaker (Pseudosciaena crocea). Although antibiotics are used to control P. plecoglossicida infections, the long-term and large-scale use of antibiotics can lead to the development of drug-resistant bacteria as well as environmental pollution. Therefore, it is necessary to develop novel antibacterial agents to treat P. plecoglossicida infections. In this study, we first developed a two-step-centrifugation method to isolate live and dead P. plecoglossicida cells. Subsequently, we used the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) screening based on inhibition rate to directly isolate antibacterial aptamers from the random library without post-processing. We isolated five antibacterial aptamers, namely B1, B2, B4, B8, and B109, which showed good inhibitory effects against P. plecoglossicida. Among these, B4 showed the highest inhibitory effect (62.40 ± 4.17 %). Further analysis revealed no positive correlation between the inhibitory effect of aptamers and their affinities with the target bacterium. The B4- and B109-binding proteins were isolated from P. plecoglossicida by magnetic separation and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Mass spectrometry analysis revealed that the 26 kDa ribosomal protein L2 was the probable B4-binding protein, while the 26 kDa ribosomal protein S3 and 75 kDa ribosomal protein S1 or succinate dehydrogenase flavoprotein subunit were the probable B109-binding proteins. Structural and subcellular localization analyses of these potential B4- and B109-binding proteins were also conducted. Our findings suggest that the inhibitory activity of the antibacterial aptamers against P. plecoglossicida may be mediated via their interaction with the ribosomal proteins, which can interfere with the protein synthesis process in the bacterium, affecting its growth. These findings provide the scientific basis for the development of functional antibacterial aptamers and the elucidation of their mechanisms of action.

Effect of pH-shifting and ultrasound on soy/potato protein structure and gelation

The substitution of meat proteins with plant-based proteins from various sources is often motivated by nutritional considerations. However, the inherent limited solubility of plant proteins, which results in suboptimal techno-functional properties, remains a persistent challenge in food formulation. The purpose of this study was to investigate the impact of pH-shifting (P), high-intensity ultrasound (U), and ultrasound-assisted pH-shifting on the structure and solubility of soy protein isolate (SPI), potato protein isolate (PPI), and the soy/potato protein complex (SPI/PPI complex). Additionally, a detailed examination of the gel properties of SPI, PPI, and the SPI/PPI complex was conducted to assess the changes that occurred before and after modification. The results of particle size, zeta potential, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), free sulfhydryl (R-SH), surface hydrophobicity (H0), Fourier transform infrared spectrum (FTIR) and fluorescence intensity showed that P and U treatment promoted the unfolding of proteins structures and decreased particle size, and augmented protein-water interactions, leading to enhanced solubility. The pH-shift and ultrasound (PU) treatment further amplified these structural alterations in proteins. Consequently, the gelling characteristics of the modified proteins were substantially improved, with notable increases in hardness, elasticity, and water-holding capacity (WHC). The findings facilitate developing more targeted plant protein-based gel products that specific techno-functional attributes, concurrently allowing for modifications to their nutritional profiles.

Cereal barcoding by assessing intron length polymorphism of γ-tubulin genes

Aim. The aim of the study was to obtain typical DNA profiles for common cereal plants using a method of assessing γ-tubulin intron length polymorphism and to evaluate the possibility of using this gene for plant species barcoding. Methods. Cereals, in particular wheat, aegilops, barley, and rice, have been studied. The method of γ-tubulin intron length polymorphism assessment was used. Amplified DNA fragments were separated via polyacrylamide gel electrophoresis under non-denaturating conditions and were visualized via silver nitrate staining. Results. The analyzed cereal species-specific DNA profiles of amplicons of the γ-tubulin gene introns were obtained, which made it possible to differentiate the species. Fingerprinting data was used for cluster analysis and dendrogram construction. Conclusions. The feasibility of using the proposed cereal barcoding method has been established. The studied plant DNA profiles can be effectively used in assessing the quality of food products such as flour.

Identification of molecular markers linked to leaf rust resistance gene Lr13 in wheat

Aim. Identification of codominant molecular markers closely linked to the Lr13 leaf rust resistance gene. Methods. PCR with detection in a polyacrylamide gel. Hybridological analysis. Phytopathology evaluation. Statistical analysis. Results. PCR analysis of varieties carrying the Lr13 gene and near-isogenic lines of the Thatcher variety with genes for resistance to leaf rust Lr13 (TcLr13), Lr16 (TcLr16), Lr23 (TcLr23) and the F2 population TcLr13 x TcLr34 by the microsatellite loci Xbarc13, Xbarc55, Xgwm148, Xwmc261, Xgwm271, Xwmc272, Xwmc344, Xbarc361, Xgwm410, Xwmc474, Xwmc477 and Xgwm630 was performed. To test the two microsatellite loci Xgwm148 and Xwmc344 as candidate markers for the Lr13 gene, we used the F2 population, which was obtained from crossing two near-isogenic lines of the Thatcher variety with leaf rust resistance genes Lr13 (TcLr13) and Lr34 (TcLr34). Conclusions. When comparing the genotyping data of F2 hybrids and the phenotypic manifestation of resistance to leaf rust, it was determined that the microsatellite locus Xwmc344 is closely linked to the Lr13 gene.

Variability of superoxide dismutase isoenzymes in varieties and introgressive lines of wheat differing in drought resistance

Aim. To study the variability of the component composition of superoxide dismutase (SOD) isoenzymes under the influence of hyperthermia and water deficit in introgressive lines of wheat with different drought resistance. Methods. The activity of SOD isoforms was determined using native electrophoresis in an acidic medium in a polyacrylamide gel. Results. Under the impact of stress factors (elevated temperature, water deficit and their combined effects), a redistribution of the relative content of individual SOD isoforms was observed in wheat seedlings of drought-resistant and drought-susceptible genotypes compared to growing under favorable conditions. The most significant decrease occurred due to the joint action of water deficit and increased temperature. Under these conditions, enzyme isoforms of medium electrophoretic mobility practically disappeared. Conclusions. The presence of low-mobility isoforms of SOD in wheat seedlings under the impact of stress factors (elevated temperature and water deficit) can serve as an indicator of drought resistance of wheat plants.

Assessment of the genetic polymorphism of genus Pinus L. the oligotrophic peat bog "Boloto Mshana"

Aim. To evaluate the molecular genetic polymorphism of representatives of the genus Pinus L. on the territory of the "Boloto Mshana" nature reserve, as well as to test the hypothesis about their possible local hybridization Methods. DNA extraction using CTAB and DNeasy Plant Pro Kit, polymerase chain reaction (PCR) with primers for the chloroplast DNA and genes encoding β-tubulin (TBP markers), electrophoresis of DNA in a polyacrylamide gel with silver nitrate staining. Results. DNA profiles of P. mugo, P. sylvestris and probably P. uliginosa (21 samples in total) were analyzed using one chloroplast DNA marker and two DNA markers based on introns of β-tubulin genes (ТВР, сТВР). The intraspecific genetic polymorphism of the species was compared and the probability of the existence of hybrid forms was investigated. Conclusions. The marker based on the 2nd intron of the β-tubulin genes turned out to be the most effective and informative DNA marker for studying representatives of the Pinus L. genus. P. sylvestris showed less genetic polymorphism by the investigated genetic markers, compared to P. mugo and P. uliginosa. Possible hybrid forms (P. mugo ✕ P. sylvestris) or P. uliginosa were also discovered.

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.

Interaction between modified magnetic nanoparticles and human albumin: Kinetics and isotherm studies and application in protein depletion

Magnetic nanoparticles modified with tetraethyl orthosilicate (Fe3O4@TEOS) and bovine serum albumin (Fe3O4@TEOS@BSA) were evaluated as sorbent in albumin depletion from human serum samples by magnetic dispersive solid phase extraction. Characterization studies were carried out by X-ray diffraction, thermogravimetry, Fourier transform infrared spectroscopy, zeta potential, and scanning electron microscopy. Both nanoparticles also showed high thermal stability and pH-dependent surface charges. The human serum albumin adsorption protocol was optimized using a central composite rotatable design. Nanoparticle mass, pH, and albumin concentration were the most influential variables. Avrami's fractional order and Freundlich isotherm models best fitted the data for human albumin adsorption kinetic and isotherm studies for Fe3O4@TEOS and Fe3O4@TEOS@BSA, and the maximum adsorption capacities were 11.93 and 14.89 mg g−1, respectively. The protein desorption was influenced by the pH of samples and eluent volume. Electrophoresis in a polyacrylamide gel containing sodium dodecyl sulfate showed different patterns of serum protein bands when consecutive depletions were performed. The Fe3O4@TEOS showed greater affinity for HSA and efficiency in depletion. The process was versatile, and the depleted albumin proportion could be controlled by the nanoparticle masses. The proposed method is a powerful sample preparation technique for rapid, reliable, and specific depletion of albumin.

Simultaneous and sequential combination of techniques for the sustainable and extensive extraction of proteins and polyphenols from malt rootlets

The emergence of tecnhiques such as microwave-assisted extraction (MAE), enzyme-assisted extraction (EAE), ultrasounds-assisted extraction (UAE), and pressurized liquid extraction (PLE) has brough many benefits but their single use does not guarantee the hollistic extraction of target compounds from complex samples. Taking into account their different principles and that one technique may favour the extraction of certain compounds over others, this work proposes the combination of these techniques for improving extraction yields in the recovery of proteins, as example of large molecule, and phenolic compounds, as example of small molecule, from a recalcitrant sample. No single extraction technique recovered all proteins or phenolic compounds, even after a proper optimization. Among them, PLE was the most advantageous, enabling the extraction of 67 % proteins and 1.20 g gallic acid equivalent (GAE)/100 g sample. Analysis of samples by scanning electron microscopy (SEM) and separation of extracts by reversed-phase high performance liquid chromatography (RP-HPLC) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that PLE showed the most distinct efficiency among EAE, MAE, and UAE, whereas the latter three exhibited more similar results. Based in these results, ten different combinations (sequential and/or simultaneous) of these techniques were proposed. PLE in combination with another technique showed a great potential for the extraction of both large and small molecules. Particularly, the combination of EAE and PLE recovered 100 % of proteins and more than three times the phenolic compounds extracted by other methods reported in the literature, even revealing significant synergistic effects.

Physioxia rewires mitochondrial complex composition to protect stem cell viability

Human induced pluripotent stem cells (hiPSCs) are an invaluable tool to study molecular mechanisms on a human background. Culturing stem cells at an oxygen level different from their microenvironmental niche impacts their viability. To understand this mechanistically, dermal skin fibroblasts of 52 probands were reprogrammed into hiPSCs, followed by either hyperoxic (20 % O2) or physioxic (5 % O2) culture and proteomic profiling. Analysis of chromosomal stability by Giemsa-banding revealed that physioxic -cultured hiPSC clones exhibited less pathological karyotypes than hyperoxic (e.g. 6 % vs. 32 % mosaicism), higher pluripotency as evidenced by higher Stage-Specific Embryonic Antigen 3 positivity, higher glucose consumption and lactate production. Global proteomic analysis demonstrated lower abundance of several subunits of NADH:ubiquinone oxidoreductase (complex I) and an underrepresentation of pathways linked to oxidative phosphorylation and cellular senescence. Accordingly, release of the pro-senescent factor IGFBP3 and β-galactosidase staining were lower in physioxic hiPSCs. RNA- and ATAC-seq profiling revealed a distinct hypoxic transcription factor-binding footprint, amongst others higher expression of the HIF1α-regulated target NDUFA4L2 along with increased chromatin accessibility of the NDUFA4L2 gene locus. While mitochondrial DNA content did not differ between groups, physioxic hiPSCs revealed lower polarized mitochondrial membrane potential, altered mitochondrial network appearance and reduced basal respiration and electron transfer capacity. Blue-native polyacrylamide gel electrophoresis coupled to mass spectrometry of the mitochondrial complexes detected higher abundance of NDUFA4L2 and ATP5IF1 and loss of incorporation into complex IV or V, respectively. Taken together, physioxic culture of hiPSCs improved chromosomal stability, which was associated with downregulation of oxidative phosphorylation and senescence and extensive re-wiring of mitochondrial complex composition.

Escherichia coli alcohol dehydrogenase YahK is a protein that binds both iron and zinc.

Previous studies have highlighted the catalytic activity of Escherichia coli alcohol dehydrogenase YahK in the presence of coenzyme nicotinamide adenine dinucleotide (NAD) and metal zinc. Notably, competitive interaction between iron and zinc ligands has been shown to influence the catalytic efficiency of several key proteases. This study aims to unravel the intricate mechanisms underlying YahK's catalytic action, with a particular focus on the pivotal roles played by metal ions zinc and iron. The purified YahK protein from E. coli cells cultivated in LB medium was utilized to investigate its metal-binding properties through UV-visible absorption measurements and determination of metal content. Subsequently, the effects of excess zinc and iron on the metal-binding ability and alcohol dehydrogenase activity of the YahK protein were explored using M9 minimal medium. Furthermore, site-directed mutagenesis technology was employed to determine the iron-binding site location within the YahK protein. Polyacrylamide gel electrophoresis was conducted to examine the relationship between iron and zinc with respect to the YahK protein. The study confirmed the presence of iron and zinc in the YahK protein, with the zinc-bound form exhibiting enhanced catalytic activity in alcohol dehydrogenation reactions. Conversely, the presence of iron appears to play a pivotal role in maintaining overall stability of the YahK protein. Furthermore, experimental findings indicate that excessive zinc within M9 minimal medium can competitively bind to iron-binding sites on YahK, thereby augmenting its alcohol dehydrogenase activity. The dynamic binding of YahK to iron and zinc unveils its intricate regulatory mechanism as an alcohol dehydrogenase, thereby highlighting the possible physiological role of YahK in E. coli and its significance in governing cellular metabolic processes. This discovery provides a novel perspective for further investigating the specific impact of metal ion binding on YahK and E. coli cell metabolism.

Capillary-Assisted Assembly of Polymer Gel-Supported Lipid Bilayers.

The polymer-supported lipid bilayer represents an attractive supramolecular assembly in numerous biophysical and bioanalytical applications. The assembly of polymer-supported membranes with a polymer layer thickness of just a few nanometers is now well-established, but bilayer properties in such a membrane architecture are still influenced by the nearby solid substrate. Polymer-supported lipid bilayer systems with a several micrometers thick polymer layer will overcome this shortcoming. However, formation of a fluid lipid bilayer on a fully hydrated, micrometer thick polymer film using traditional methods (e.g., vesicle fusion and lipid monolayer deposition techniques) remains a challenging task due to the rather unfavorable interfacial conditions for bilayer formation in such a system. Here, we report for the first time on the facile capillary-assisted formation of a lipid bilayer on the surface of a fully hydrated, several micrometers thick polyacrylamide (PAA) gel, in which forced molecular crowding of lipids at the air-water interface of the capillary results in monolayer instability and collapse, thereby forming a lipid bilayer on the top of the polymer gel inside the capillary. Stable bilayer attachment on the surface of the polymer gel can be achieved via physisorption or specific chemical linkages (tethering) on both cross-linked and non-cross-linked PAA films. Unlike the traditional solid-supported lipid bilayer (SLB), the lipid lateral diffusion in the polymer gel-supported lipid bilayer is not anymore perturbed by a solid substrate. Instead, more like a plasma membrane, it is mainly influenced by the properties of the underlying polymer and the nature/distribution of polymer-bilayer attachments. Polymer gel-supported lipid bilayers built using the capillary-assisted assembly approach show attractive self-healing properties, resulting in superior long-term stability relative to the SLB. We hypothesize that the described capillary-assisted assembly method can be applied to a wide range of polymeric materials and lipid compositions, opening exciting opportunities as an advanced model membrane system.

Modifying physicochemical properties, rheology, and creaming stability of milk fat globule and membrane through ultrasound treatment

The healthy benefits of milk fat globules and membrane (MFGs/MFGM) ingredients are increasingly recognized in the dairy industry. In this research, we examined the effects of ultrasonic treatment on the physicochemical and rheological properties, as well as the emulsions stability of MFGs/MFGM derived from bovine raw milk. Fresh milk was subjected to sonication at frequencies of 20 kHz and 40 kHz, either individually or simultaneously, for durations of 5 min or 15 min, using work/rest cycles of 5 s on and 3 s off. Bovine milk, without any treatment, served as the control. Regardless of the intensity difference, ultrasonic treatment for 5 min resulted in more pronounced changes in the regions of Amide Ⅱ (1600–1500 cm−1), Amide Ⅲ (1500–1200 cm−1), and fingerprint region (1200–1900 cm−1) compared to both the 15 min treatments and control MFGs/MFGM. Principal component analysis (PCA) conducted on the entire spectra, as well as in the regions of Amide Ⅰ, Amide Ⅱ, and the fingerprint spectra, clustered the 5 min treatment distinctly from the control and MFGs/MFGM ultrasonically treated for 15 min. MFGs/MFGM samples following 20 kHz and 40 kHz synchronous treatment for 15 min exhibited lower absorbance bands at 1727–1726 cm−1, whereas a higher content at 1740 cm−1 was observed compared to control MFGs/MFGM. Additionally, a more significant reduction in the intramolecular β-sheet content in 20 + 40 kHz/ 15 min treatment was observed. According to the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) patterns, a diminished intensity of Periodic Acid Schiff 6/7 (PAS 6/7) bands was observed across all the MFGs/MFGM. Ultrasonic treatment retained more caseins while reducing the β –LG levels compared to the controls, enhancing the stability of MFGs/MFGM, except in MFGs/ MFGM subjected to 20 and 40 kHz simultaneously treated for 15 min. The irregular sphericity of fat globules was noted particularly in MFGs/MFGM treated at 20 kHz independently or in combination with 40 kHz for 15 min. According to the confocal laser scanning microscopy (CLSM), ultrasonic treatment facilitated the binding of caseins or whey proteins to the MFGs surface and induced flocculation of membrane proteins. Hierarchical cluster analysis (HCA) heat map further underscored the impact of ultrasonic treatments on the structural and compositional changes, as well as rheology and emulsions stability, of MFGs/MFGM.

Evaluation of a commercial Immunochromatographic Strip Assay (ICT) for rapid detection of bovine, porcine and human Rotavirus A

The most frequent diarrhoeal pathogen “Rotavirus” is commonly detected by Ribo Nucleic Acid- Polyacrylamide Gel Electrophoresis (RNA-PAGE), Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Immunoassay (Enzyme Linked Immunosorbent Assay) in humans. However, use of these tests is limited in animals therefore, penside test like Immunochromatographic test/Lateral Flow Assay (ICT/LFA) is necessary. The present study was carried out to compare the diagnostic efficacy of rapid commercial ICT strip assay, RNA-PAGE and RT-PCR for specific detection of Rotavirus A (RVA) from stool samples of calves, piglets and children. A total of 313 faecal samples were collected between November 2022 to April 2023 from children below 5years of age (n=100), calves (n=100) and piglets (n=113) ≤ 3 months of age and were screened by RNA-PAGE, RT-PCR and commercial ICT kit for rotavirus A.The overall positivity of RVA from human, calves and piglets using RNA-PAGE, RT-PCR and LFA was found to be 35% (35/100), 8% (8/100) and 14.16% (16/113), respectively. Higher positivity of rotavirus was recorded in male children (44.26%, 27/61) than in female children (20.51%, 8/39). The kappa agreement between LFA and RT-PCR was 0.79 (substantial agreement); between LFA and RNA-PAGE was 0.61 (substantially low agreement) and between RNA-PAGE and RT-PCR was found to be 0.75 (substantial agreement). Relative diagnostic sensitivity and specificity of LFA in comparison with RT-PCR was 74.54% and 98.44%, respectively and in comparison with RNA-PAGE was 72.97% and 93.47%, respectively. RNA-PAGE in comparison with RT-PCR is having diagnostic sensitivity and specificity of 65.45% and 99.6%, respectively. In conclusion, the ICT strip assay is a rapid, convenient and effective method with satisfactory efficacy for detection of RVA from children, calves and piglets. ICT fulfilled the WHO ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Delivered) criteria for point-of-care testing. It can be useful in determining rotavirus outbreaks in resource-limited settings.

Efficient purification of soluble receptor for advanced glycation end-products from Sus scrofa lung tissue and synthesis of its binding ligand, glycated bovine serum albumin

A receptor for advanced glycation end products (RAGE) has emerged as a crucial player in various pathological conditions due to its involvement in inflammation and cellular dysfunction. Its soluble isoform, sRAGE, has garnered significant attention for its competitive inhibitory effects and potential therapeutic applications. However, obtaining sRAGE with appropriate glycosylation patterns for binding to glycated proteins has been challenging, often requiring costly expression systems. Here, we present a novel approach for producing and purifying sRAGE from Sus scrofa lungs, bypassing the need for expensive expression systems. Previous protocols for sRAGE extraction faced reproducibility issues due to high viscosity and haemoglobin content of the solution. To address this, we developed a method for selective haemoglobin precipitation using a zinc-containing buffer, enabling purification via various chromatographic methods. Through a combination of chromatographic techniques, we obtained sRAGE in suitable purity, identified using HPLC-MS/MS. Additionally, producing glycated proteins for RAGE receptor activation often involved lengthy protocols or inadequate separation from reactants. Thus, we devised a rapid method for producing and purifying pure BSA glycated with ribose, addressing a critical gap in the field. Functional studies, conducted using Native PAGE, demonstrated the capability of purified proteins to bind to each other.

Comparative venom analysis between melanistic and normally coloured phenotypes of the common adder (Vipera berus).

Snake venom is an ecologically relevant functional trait directly linked with a snake's fitness and survival, facilitating predation and defence. Snake venom variation occurs at all taxonomic levels, but the study at the intraspecific level is still in its early stages. The common adder (Vipera berus) exhibits considerable variation in colour phenotypes across its distribution range. Melanistic (fully black) individuals are the subject of myths and fairytales, and in German folklore such 'hell adders' are considered more toxic than their normally coloured conspecifics despite any formal investigation. Here, we provide the first comparative analysis of venoms from melanistic and normally coloured common adders. Specifically, we compared the venom profiles by sodium dodecylsulfate polyacrylamide gel electrophoresis and reversed-phase high-performance liquid chromatography and tested the venoms' protease, phospholipase A2 and cytotoxic activities. Phospholipase A2 activity was similar in both phenotypes, whereas general protease activity was higher in the melanistic venom, which was also more cytotoxic at two concentrations (6.25 and 12.5 µg ml-1). These minor differences between the venoms of melanistic and normally coloured adders are unlikely to be of clinical relevance in the context of human envenomation. In light of our results, the claim that melanistic adders produce more toxic venom than their normally coloured conspecifics appears rooted entirely in folklore.