SDS-PAGE Research Articles

Investigating the immunological activity of the Hsp70-P113 fusion protein for Mycoplasma ovipneumoniae detection: a groundbreaking study

BackgroundMycoplasmal pneumonia of sheep and goats (MPSG) is an important infectious disease that threatens sheep and goat production worldwide, and Mycoplasma ovipneumoniae (Movi) is one of the major aetiological agents causing MPSG. The aim of this study was to investigate the immunological activity of the Hsp70‒P113 fusion protein derived from Movi and to develop a serological assay for the detection of Movi.MethodsThis study involved codon optimization of the dominant antigenic regions of Movi heat shock protein 70 (Hsp70) and adhesin P113. Afterwards, the optimized sequences were inserted into the prokaryotic expression vector pET-30a( +) through tandem linking with the aid of a linker. Once a positive recombinant plasmid (pET-30a-rHsp70-P113) was successfully generated, the expression conditions were further refined. The resulting double gene fusion target protein (rHsp70‒P113) was subsequently purified using ProteinIso® Ni–NTA resin, and the reactivity of the protein was confirmed via SDS‒PAGE and Western blot analysis. An indirect enzyme-linked immunosorbent assay (i-ELISA) technique was developed to detect Movi utilizing the fusion protein as the coating antigen. The specificity, sensitivity, and reproducibility of all methods were assessed after each reaction parameter was optimized.ResultsThe resulting rHsp70-P113 protein had a molecular weight of approximately 51 kDa and was predominantly expressed in the supernatant. Western blot analysis demonstrated its favourable reactivity. The optimal parameters for the i-ELISA technique were as follows: the rHsp70-P113 protein was encapsulated at a concentration of 5 μg/mL; the serum was diluted at a ratio of 1:50; the HRP-labelled donkey anti-goat IgG was diluted at a ratio of 1:6,000. The results of the cross-reactivity assays revealed that the i-ELISA was not cross-reactive with other goat-positive sera against Mycoplasma mycodies subsp. capri (Mmc), Mycoplasma capricolum subsp. capripneumoniae (Mccp), Mycoplasma arginini (Marg), orf virus (ORFV) or enzootic nasal tumour virus of goats (ENTV-2). The sensitivity of this method is high, with a maximum dilution of up to 1:640. The results of the intra- and inter-batch replication tests revealed that the coefficients of variation were both less than 10%, indicating excellent reproducibility. The analysis of 108 clinical serum samples via i-ELISA and indirect haemagglutination techniques yielded significant findings. Among these samples, 43 displayed positive results, whereas 65 presented negative results, resulting in a positivity rate of 39.8% for the i-ELISA method. In contrast, the indirect haemagglutination technique identified 20 positive samples and 88 negative samples, resulting in a positivity rate of 18.5%. Moreover, a comparison between the two methods revealed a conformity rate of 78.7%.ConclusionThe results obtained in this study lay the groundwork for advancements in the use of an Movi antibody detection kit, epidemiological inquiry, and subunit vaccines.

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.

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.

Mechanistic insights into the interaction of Lycium barbarum polysaccharide with whey protein isolate: Functional and structural characterization

Protein-polysaccharide interactions are crucial for food system structure and stability. This study investigates the interaction of Lycium barbarum polysaccharide (LBP) at 0–2.00 % concentrations with whey protein isolate (WPI), focusing on functionality and structural changes. LBP covalently grafted onto WPI, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), forming WPI-LBP complexes with a maximum degree of grafting (DG) of 44.58 % at 2.00 % LBP. This grafting reduced WPI's surface hydrophobicity (H0) and improved solubility, emulsifying properties, and digestibility under certain conditions, with optimal antioxidant activity at 1.00 % LBP. Multispectral analysis and microscopy showed LBP grafting alters WPI's secondary, tertiary, crystalline, and micro/nanostructures. The comprehensive analysis indicates that the interaction between LBP and WPI involves covalent bonding, hydrogen bonding, hydrophobic interactions, and electrostatic forces, as supported by zeta potential and chemical forces results. These findings suggest LBP-protein complexes as promising food materials for enhancing functionality and stability in the food industry.

Multi-omics approach for understanding the response of Bacteroides fragilis to carbapenems

BackgroundThe prevalence of Bacteroides fragilis isolates resistant to first-line beta-lactam drugs is increasing, resulting in reduced treatment efficacy. Investigating the bacterial transcriptome and proteome can uncover links between bacterial genes and resistance mechanisms. In this study, we experimentally assessed in vitro the transcriptional and proteomic profiles of B. fragilis exposed to SICs of meropenem, an effective antimicrobial agent, collected from patients with intra-abdominal diseases at Astana City Hospital, Kazakhstan. MethodsB. fragilis was cultured in brain heart infusion broth and sub-cultured every 48 h for 8 days in media with and without meropenem. Total RNA was extracted from the liquid cultures using a commercial RNeasy mini kit, and strand-specific RNA sequencing (RNA-seq) was performed on the DNBSEQ platform. Raw RNA-seq data were retrieved from BioProject No. PRJNA531645 and uploaded to the NCBI Sequence Read Archive (accession no. SRX22081155). Proteins of B. fragilis were extracted and separated using sodium dodecyl sulphate–polyacrylamide gel electrophoresis, followed by analysis of the eluted peptides using liquid chromatography–tandem mass spectrometry. Cluster analysis utilised the Database for Annotation, Visualisation, and Integrated Discovery. ResultsThe subinhibitory concentration (SIC) of meropenem was determined to be 0.5 μg/L (minimum inhibitory concentration: 1). Mapping of reads to the reference genome identified 2477 expressed genes in all B. fragilis BFR KZ01 samples. Ten differentially expressed genes (DEGs) were common across comparison groups during and post-antibiotic exposure (wMEM vs. MEM2 and MEM2 vs. rMEM8); however, no substantially enriched Gene Ontology terms were identified. The cluster analysis highlighted a significant enrichment cluster (W-0560 oxidoreductase) of DEGs following antibiotic withdrawal. In total, 859 B. fragilis proteins were identified, with the expressions of three proteins, 3-oxoacyl-[acyl carrier protein] reductase, acetyl-CoA carboxylase biotin carboxylase subunit, and beta-ketoacyl-ACP synthase III, being upregulated in the enriched protein folding category. Notably, chaperone proteins such as FKBP-type peptidyl-prolyl cis-trans isomerases (involved in the cis-trans isomerisation of prolyl peptide bonds) and GroES (a co-chaperone functioning with GroEL) were also identified. ConclusionsUnder the influence of low doses of antibiotics defense mechanisms are activated which contribute to the emergence of resistance. These results provide insight into the response of B. fragilis to meropenem exposure, mainly at the SIC, contributing to the understanding bacterial survival strategies under stress conditions.

Maillard conjugates of whey protein and prebiotic dietary fibers: Emulsion properties, encapsulation efficiency and chemical stability after in vitro gastrointestinal digestion

The incorporation of dietary fibers into food formulations is a promising approach to developing new functional ingredients. In this study, we investigated the formation of Maillard conjugates using whey protein concentrate (WPC) combined with fructooligosaccharides (FOS) and xylooligosaccharides (XOS). These conjugates, innovatively utilized as emulsifiers for annatto seed oil, were subjected to in vitro gastrointestinal digestion to assess their chemical stability and effectiveness as encapsulating systems. Our study confirmed the occurrence of the Maillard reaction through different methods, including grafting degree (GD) assessment and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) to evaluate molecular weight variations in the proteins. The key innovation of our research lies in demonstrating the conjugation process through carbohydrate mass balance analysis using High-Performance Anion Exchange Chromatography coupled with Pulsed Amperometric Detection (HPAEC-PAD). The GD for the WPC-XOS and WPC-FOS was 46 ± 3 % and 56 ± 3 %, respectively. Our findings suggest that sugar quantification could serve as a novel approach to confirming the formation of Maillard conjugates. The emulsions stabilized by these conjugates exhibited larger oil droplet sizes compared to those stabilized by WPC alone yet maintained high encapsulation efficiency (≥ 90 %). Notably, the WPC-XOS emulsion demonstrated kinetic stability after 30 days of storage at 4°C, while the WPC-FOS emulsion showed signs of creaming after just 16 days. In vitro gastrointestinal digestion revealed that while the conjugates were susceptible to enzymatic degradation, the FOS and XOS sugars displayed gastrointestinal resistance of up to 82 % and 83 %, respectively. This study highlights the potential of WPC-XOS conjugates not only as effective emulsifiers but also as functional ingredients that enhance the nutritional and antioxidant properties of food products, presenting a novel approach to functional food development.

Antibacterial Activity and Cytotoxicity of the Novel Bacteriocin Pkmh.

Bacteriocins are a class of proteins produced by bacteria that are toxic to other bacteria. These bacteriocins play a role in bacterial competition by helping to inhibit potential competitors. In this study, we isolated and purified a novel bacteriocin Pkmh, different from the previously reported bacteriocin PA166, from Pseudomonas sp. strain 166 by ammonium sulfate precipitation, dialysis membrane method, ion exchange chromatography, and gel filtration chromatography. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) revealed that the molecular weight of Pkmh is approximately 35 kDa. Pkmh exhibited potent antimicrobial activity against bovine Mannheimia haemolytica (M. haemolytica) with low cytotoxicity, and lower hemolytic activity was observed. In addition, Pkmh retained antimicrobial activity at different pH ranges (2-10) and temperature conditions (40, 60, 80, 100 °C). Our analysis of its antimicrobial mechanism showed that Pkmh acts on bacterial cell membranes, increasing their permeability and leading to cell membrane rupture and death. In conclusion, Pkmh exhibited low hemolytic activity, low toxicity, and potent antibacterial effects, suggesting its potential as a promising candidate for clinical therapeutic drugs.

Preparation of micron-sized benzamidine-modified magnetic agarose beads for trypsin purification from fish viscera

The effective method for trypsin purification should be established because trypsin has important economic value. In this work, a novel and simple strategy was proposed for fabricating micron-sized magnetic Fe3O4@agarose-benzamidine beads (MABB) with benzamidine as a ligand, which can efficiently and selectively capture trypsin. The micro-sized MABB, with clear spherical core-shell structure and average particle size of 6.6 μm, showed excellent suspension ability and magnetic responsiveness in aqueous solution. The adsorption capacity and selectivity of MABB towards target trypsin were significantly better than those of non-target lysozyme. According to the Langmuir equation, the maximum adsorption capacity of MABB for trypsin was 1946 mg g−1 at 25 °C, and the adsorption should be a physical sorption process. Furthermore, the initial adsorption rate and half equilibrium time of MABB toward trypsin were 787.4 mg g−1 min−1 and 0.71 min, respectively. To prove the practicability, MABB-based magnetic solid-phase extraction (MSPE) was proposed, and the related parameters were optimized in detail to improve the purification efficiency. With Tris-HCl buffer (50 mM, 10 mM CaCl2, pH 8.0) as extraction buffer, Tris-HCl buffer (50 mM, 100 mM CaCl2, pH 8.0) as rinsing buffer, acidic eluent (0.01 M HCl, 0.5 M NaCl, pH 2.0) as eluent buffer and alkaline buffer (1 M Tris-HCl buffer, pH 10.0) as neutralization solution, the MABB-based MSPE was successfully used for trypsin purification from the viscera of grass carp (Ctenopharyngodon idella). The molecular weight of purified trypsin was determined as approximate 23 kDa through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified trypsin was highly active from 30 °C to 60 °C, with an optimum temperature of 50 °C, and was tolerant to pH variation, exhibiting 85 % of maximum enzyme activity from pH 7.0 to 10.0. The results demonstrated that the proposed MABB-based MSPE could effectively purify trypsin and ensure the biological activity of purified trypsin. Therefore, we believe that the novel MABB could be applicable for efficient purification of trypsin from complex biological systems.

Purification and characterization of α-amylase from Acanthoscelides obtectus (Say) (Coleoptera: Chrysomelidae)

Acanthoscelides obtectus is one of the most notorious pests of stored kidney beans (Phaseolus vulgaris) worldwide. Kidney beans are an important source of food for these insects. α-Amylase is the main carbohydrate-digesting enzyme in animals including insects. In the current study, the biochemical analysis revealed higher α-amylase activity (U/ml) in 3rd and 4th larval instars but decreased gradually in subsequent developmental stages. However, the specific activity (U/mg) interestingly was highest in 1st instar and decreased in further developmental stages. During qualitative analysis of α-amylase using starch-agar and native PAGE, the maximum zone of starch lysis and a prominent band on the gel was observed in 3rd and 4th larval stages. The molecular mass of the native enzyme was also estimated and found to be 30.34 kDa. The crude α-amylase was further purified by ammonium sulfate precipitation, gel filtration on a Sephadex G-75, and ion exchange chromatography on the DEAE cellulose column. The purified amylase was found to be a monomer with a molecular mass of 15 kDa. The specific activity of the purified enzyme increased from 1.74 U/mg in the crude sample to 166.35 U/mg in the final purification step resulting in 95-fold purification with a yield of 11.14%. Further characterization of purified α-amylase revealed a pH optimum of 7.0 and a temperature optimum of 35 °C. Lineweaver-Burk plot analysis revealed Km and Vmax to be 0.09% and 3.3 U/mL, respectively. Oxalic acid, tannic acid, and HgCl2 significantly inhibited the enzyme, while the Na+, Ca++, and Mg++ ions acted as activators. In conclusion, the study revealed, the highest α-amylase activity in 3rd and 4th larval stages of Acanthoscelides obtectus followed by native and SDS PAGE resulting in molecular mass of 30.34 and 15 kDa respectively.

Unveiling the transformative influence of sonochemistry on formation of whey protein isolate and green tea extract (WPI-GTE) conjugates

This study investigated the formation of conjugates between whey protein isolate (WPI) and green tea extract (GTE) using three methods: redox-pair (R), ultrasound-assisted redox-pair (RU), and ultrasonication (UL). Ultrasonication significantly reduced the reaction time for synthesizing WPI-GTE conjugates compared to the standard R method (p < 0.05). The UL methods had the highest conjugate yield determined by polyphenol binding (p < 0.05). Fourier-transform infrared spectroscopy (FTIR) and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the conjugate formation, indicating an increased molecular weight due to protein binding with polyphenols through covalent and non-covalent bonds. Conjugates produced via ultrasonication exhibited enhanced solubility, smaller particle size, better emulsifying capacity, and improved foaming ability compared to those formed using the traditional R method (p < 0.05). However, conjugates from the R method showed higher antioxidant activity, as evidenced by DPPH•and ABTS•+ scavenging activities (p < 0.05). In conclusion, WPI-GTE conjugates created through ultrasonic treatment demonstrate potential as dual-functional ingredients, serving as both antioxidant and emulsifier.

Different VH3-Binding Protein A Resins Show Comparable VH3-Binding Mediated By product Separation Capabilities Despite Having Varied Dynamic Binding Capacities Towards A VH3 Fab.

Protein A resins have been widely used for product capture during mAb, bispecific antibody (bsAb), and Fc-fusion protein purification. While Protein A ligands mainly bind the Fc region, many of them can also bind the VH3 domain. During mAb/bsAb purification, certain truncated byproducts may contain the same Fc region as the product but fewer numbers of the VH3 domain. In such a scenario, VH3-binding Protein A resins provide a potential means for byproduct separation based on the difference in VH3-binding valency. As the ligands of different VH3-binding Protein A resins are derived from distinct domains of the native Protein A, it would be interesting to know whether they possess comparable capabilities for separating species with the same Fc region but different numbers of VH3 domain. This study aims to explore the potential of different VH3-binding Protein A resins for separating antibody species with the same Fc region but different numbers of VH3 domain. The VH3 Fab was released from a VH3-containing mAb by papain digestion. Post digestion, the released VH3 Fab was purified sequentially using CaptureSelect CH1-XL and MabSelect SuRe affinity chromatography. The purified VH3 Fab was used as the load material to assess the dynamic binding capacity (DBC) of five VH3-binding Protein A resins (i.e., Amshpere A3, Jetted A50, MabCapture C, MabSelect and MabSelect PrismA). The potential of VH3-binding Protein A resins for separating species having the same Fc region but different numbers of VH3 domain was evaluated using an artificial mixture composed of the product and a truncated byproduct, which contained one and zero VH3 domain, respectively (both species contained the same Fc region). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to monitor Fab purification and separation of species containing the same Fc region but different numbers of VH3 domain. When loaded with an isolated VH3 Fab, different VH3-binding Protein A resins showed varied DBCs. Nevertheless, when these Protein A resins were used to separate a truncated byproduct, which contained the Fc region only without any VH3 domain, from the product, which included one VH3 domain in addition to the Fc region, they showed comparable capabilities for separating these two species. Although different VH3-binding Protein A resins showed varied DBCs towards a VH3 Fab, they exhibited comparable capabilities for separating species with the same Fc region but different numbers of VH3 domain.

Characterization and Evaluation of Native Bacillus thuringiensis Isolate T121 Toxic to Henosepilachna vigintioctopunctata (Coleoptera: Coccinellidae)1

Abstract The present study focused on the molecular characterization and toxicity analysis of the Bacillus thuringiensis Berliner (Bt) isolate T121 against Henosepilachna vigintioctopunctata (F.) (Coleoptera: Coccinellidae). Colonies of T121 growing on solid media were circular, with creamy white hues and raised centers. Scanning electron microscopy revealed the presence of cuboidal and spherical crystals in liquid media. Polymerase chain reaction screening confirmed the presence of binary toxin vip1 and vip2 in addition to cry3Aa gene. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis showed four prominent bands at 74, 65, 48, and 31 kDa. In toxicity assays, T121 exhibited a median lethal concentration of 4.675 µg/ml against H. vigintioctopunctata. These findings demonstrate that the Bt isolate T121 offers promising prospects for development as a component of a sustainable pest management strategy against H. vigintioctopunctata and perhaps other coleopteran pests.

Clickable Probes for Pathogen Proteasomes: Synthesis and Applications.

The 20S proteasome is a multimeric protease complex that is essential for proteostasis in the cell. Small molecule proteasome inhibitors are approved drugs for various cancers and are advancing clinically as antiparasitics. Although tools and technologies to study the 20S proteasome have advanced, only one probe is commercially available to image proteasome activity. This probe consists of a fluorescently labeled, peptidyl vinyl sulfone that binds to one or more of the catalytic proteasome subunits. Here, we synthesized two, active site-directed epoxyketone probes, LJL-1 and LJL-2, that were based on the peptidyl backbones of the anticancer drugs, carfilzomib and bortezomib, respectively. Each probe was conjugated, via click chemistry, to a bifunctional group comprising 5-carboxytetramethylrhodamine (TAMRA) and biotin to, respectively, visualize and enrich the 20S proteasome from protein extracts of two eukaryotic pathogens, Leishmania donovani and Trichomonas vaginalis. Depending on species, each probe generated a different subunit-binding profile by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and the biotin tag enabled the enrichment of the bound subunits which were then formally identified by proteomics. Species differences in the order of electrophoretic migration by the β subunits were also noted. Finally, both probes reacted specifically with the 20S subunits in contrast to the commercial vinyl sulfone probe that cross reacted with cysteine proteases. LJL-1 and LJL-2 should find general utility in the identification and characterization of pathogen proteasomes, and serve as reagents to evaluate the specificity and mechanism of binding of new antiparasitic proteasome inhibitors.

A Novel Application of Virus Like Particles in the Hemagglutination Inhibition Assay.

The hemagglutination inhibition (HI) assay is a traditional laboratory procedure for detection and quantitation of serum antibodies of hemagglutinating viruses containing the hemagglutinin (HA) gene. The current study aimed to investigate the novel use of virus like particles (VLP) as an antigen for the HI assay. VLPs were prepared from a strain of H5N1 using a baculovirus expression system. The VLPs were characterized using the hemagglutination test, Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and transmission electron microscopy. The comparative HI assay was performed using three different seed antigens: A/chicken/Mexico/232/94 (H5N2), A/chicken/Egypt/18-H/09(H5N1) and A/goose/Guangdong/1/1996(H5N1). The HI assay of serum antibody titrations using homologous antigens to these vaccinal seeds were compared to the VLP's antigens for the same serum. The HI titers were logically relevant to the similarity between VLP antigens and vaccinal seeds, indicating the VLPs behave similarly to the standard HI assay which uses inactivated whole virus as an antigen. VLPs could be considered as an alternative to the HI assay antigen as they show a relatedness between the similarity with vaccinal seed and serum antibodies. Compared to typical entire H5N1 viral antigen prepared in SPF eggs that require proper inactivation to avoid any public health risk, VLPs prepared in tissue culture, plants or insect cells are a safe, inexpensive and scalable alternative to inactivated whole virus antigen.

Structure of yuba films at the air/liquid interface as effected by the interfacial adsorption behavior of protein aggregates

The effects of adsorption behavior and assembly mechanism of proteins and lipids at the interface on the formation of yuba films were investigated. The thickness of yuba films increased rapidly from nano to micro scale within minutes according to the scanning electron microscopy (SEM) images. The confocal laser scanning microscope (CLSM), SEM images, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the formation of protein aggregates (40–100 nm) was an essential requirement for the development of yuba. Meanwhile, a relatively loose spatial structure was formed by protein aggregates under the influence of water vapor. This structure served as the foundation for incorporating lipids. Interfacial adsorption kinetics indicated that increasing the concentration (from 3 to 9 mg/mL) of protein aggregates enhanced the rearrangement rate. This finding demonstrated that the variations of interfacial protein aggregate concentration were a crucial factor leading to the non-linear growth of film thickness.

Proteomics analysis of carcinogenesis in a rat model of mammary cancer induced by DMBA (7,12-dimethylbenz[a]anthracene)

Background: Mammary cancer, called breast cancer in humans, results from the abnormal growth of cells in the mammary glands that attack the surrounding tissue. The process of carcinogenesis at the molecular level can be monitored through the production of proteins as biomarkers for carcinogenesis. 7,12-Dimethylbenz[a]anthracene (DMBA) is a known carcinogenic compound. This study aimed to analyze the proteomic profile as critical data regarding DMBA-induced carcinogenesis in Sprague‒Dawley rats. Methods: Experimental animals were divided into two groups: a treatment group given DMBA at a dose of 10 mg/kg (intramammary) at intervals of 48 hours for a total of 10 doses, and a negative control group that was not given any treatment. Measurement of the total protein concentration was carried out using a spectrophotometer, and the data were analyzed using a t-test, while the characterization of protein profiles was carried out based on molecular weight data using SDS‒PAGE. Mammary gland histopathology was evaluated by hematoxylin and eosin (H&amp;E) staining. Results: The results showed a significant (p&lt;0.05) increase of 27% in the total protein concentration in the rat mammary cancer model. The results of proteomic characterization showed a protein profile containing proteins of 187, 169, 68, 64, 53, 41, 24, 18, and 14 kDa, which were suspected to be HER-2, Nischarin, COX-2, Albumine, Vimentin, ACTB, TNF, p16, and fatty acid binding protein 3 (FABP3), respectively. Histopathology of the mammary glands showed an irregular and indistinct arrangement of the alveoli and extensive epithelial cell proliferation from the surface to the lumen of the mammary ducts, and the mammary stroma showed the formation of new epithelial cells, which were cancer cells that spread to surrounding tissue. Conclusions: The proteomic profile was strongly associated with morphological alterations in mammary carcinogenesis in a rat model of DMBA-induced mammary cancer.

Date nawah powder as a promising waste for β-mannanase production from a new isolate Aspergillus niger MSSFW, statistically improving production and enzymatic characterization

β-Mannanase producing fungus was isolated from coffee powder waste and identified as Aspergillus niger MSSFW (Gen Bank accession number OR668928). Dates nawah powder as industrial and agricultural waste was the most inducer of β-mannanase production. The Plackett–Burman and Central Composite designs were used to improve β-mannanase titer. Optimization studies enhanced the enzyme yield with approximate 13.50-times. β-Mannanase was purified by Sephadex G-150 gel filtration column and the molecular weight was estimated to be 60 kDa by SDS–PAGE. Crude and purified β-mannanase displayed maximum activity at temperature 60 °C and 50 °C, respectively. Crude β-mannanase showed an activation energy value 2.35-times higher than the purified enzyme. Activation energy for thermal denaturation of the purified β-mannanase was 1.08-times higher than that of the crude enzyme. Purified β-mannanase exhibited higher deactivation rate constant (Kd) and lower half-life (t0.5) and decimal reduction time (D-value) compared with the crude enzyme. Thermodynamic parameters of enthalpy, entropy, and free energy values for crude and purified β-mannanase were calculated. Substrate kinetic parameters suggested that the purified β-mannanase had a strong affinity toward locust bean gum by showing 3.44-times lower Km and 1.99-times higher Vmax compared to the crude enzyme.

Cloning and Optimization of Intracellular Expression of Human Interferon β-1a in Pichia pastoris GS115

Abstract Background: Interferon-beta (IFN-β) is a cytokine with a wide range of biological and pharmaceutical applications, including multiple sclerosis (MS), cancer, some autoimmune disorders, and viral infectious diseases. Thus, many studies have been performed to develop novel strategies for the high-yield production of functional IFN-β in a cost-effective approach. Here, we aimed to improve the intracellular expression of IFN-β-1a in Pichia pastoris. Materials and Methods: The gene of IFN-β-1a was successfully sub-cloned into the pPICZA vector. The recombinant vector was transfected to P. pastoris GS115 cells by electroporation. After screening positive P. pastoris transformants, the expression of IFN-β-1a was evaluated and the cultivation conditions, including temperature, time of incubation, and methanol concentration, were optimized. The protein expression levels were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Results: The double digestion with EcoRI and XhoI restriction enzymes and sequence analysis confirmed the correct sub-cloning of the IFN-β-1a gene into pPICZA. SDS-PAGE analysis showed that the highest level of IFN-β-1a (25 mg per 1 L of yeast culture) was produced with 2% methanol at 28°C after 72 h incubation. Conclusion: Optimization of cultivation conditions for intracellular expression of IFN-β-1a was successfully performed. This approach can be generally applied to improve the production yield and quality of other recombinant proteins in P. pastoris.

Phleum pratense-pollen adaptive variations and pollen microbiome investigation under different climatic regions and prospects of allergenicity.

Phleum pratense is an allergenic grass that pollinates in spring in Pakistan. Databases Allergenonline.org and Allergen.org record ten P. pratense allergens and their isoforms. Phl P 1, Phlp 5, and Phl p 11 are major P. pratense-pollen allergens with demonstrated basophil activity and skin test reactivity. Little is known about P. pratense pollen adaptive variations in different climatic regions and pollen-associated microbial diversity. In this study, we collected P. pratense-pollen and soils in the spring season 2022. Samples were collected from three climatic regions in Pakistan (R1, R2 and R3) with differences in mean monthly air temperature, mean monthly precipitation and elevation. The morphology of pollen was observed by light microscopy, scanning electron microscopy (SEM), biochemical fingerprint analysis, and composition of pollen were investigated by fourier-transform infrared spectroscopy (FTIR). The pollen-associated bacterial populations were identified through a Biolog GEN III microplate system. The pollen water-soluble proteins were isolated and stabilized in phosphate buffer saline (PBS) and tested for allergenicity responses through dot blots and western blots analysis. The morphology study found difference in pollen biochemical composition. Biolog identified Brevibacterium epidermidis and Pantoea agglomerans from P. pratense pollen. Protein extract quantification and sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) gel found decreased protein expression in R1 region pollen compared to R2 and R3 region pollen. Allergenicity studies found differential expression of beta-expansin and profilin allergens in pollen obtained from the three regions. Beta-expansin and profilin were suppressed in R1 pollen and expressed in compared to R2 and R3 pollen. This is the first study to identify B. epidermidis and P. agglomerans growth on P. pratense pollen. Variable allergen expression in P. pratense pollen has also been observed in different regions. Soil pH, an increase in mean monthly temperature and a decrease in mean monthly precipitation correlated with pollen biochemical composition, and reduced beta-expansin and profilin expression involved in pollen growth and development. The findings of this research are unique, which enhances basic knowledge and understanding of P. pratense-pollen associated microbiota and climate change impacts on the pollen allergen expression.

Separation methods for food protein purification and analysis

The extraction, separation, and purification of dietary proteins from a variety of food sources are crucial for their targeted use in food applications. To achieve this, proteins should be effectively separated from non-protein components such as cell wall structures, polysaccharides, and lipids. Traditional protein purification methods can be time-consuming, highlighting the need for automated, cost-effective, and sustainable alternatives. This comprehensive review critically assesses various protein purification instruments from an analytical perspective, weighing their advantages and disadvantages. The methods under evaluation include ultrafiltration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), fast protein liquid chromatography (FPLC), high-performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC), and microfluidic chips. Among these, FPLC stands out as an affordable and efficient technique that allows for high protein recovery. However, HPLC and UPLC provide faster results but may denature proteins, leading to lower recovery rates. Ultrafiltration is a cost-effective and straightforward method that doesn’t require complex equipment. Microchip-based approaches are emerging as innovative techniques for rapidly analyzing small samples. While SDS-PAGE is user-friendly, it denatures proteins, particularly those linked to other biomolecules. The choice of the most appropriate instrument depends on factors such as cost, energy efficiency, processing time, the characteristics of the target protein, desired outcomes, protein recovery, and resource availability. By critically examining these analytical instruments for protein purification, this review aims to assist researchers and practitioners in selecting the most suitable method for their specific needs, ultimately promoting efficient and successful protein purification endeavors in the field of food science and technology.