SDS-PAGE Analysis Research Articles

Serodiagnosis of urogenital schistosomiasis and profiling of immunoreactive protein(s) in Schistosoma haematobium soluble egg and adult worm antigens.

To achieve schistosomiasis eradication plan by 2030, the development of efficient diagnosis is crucial. This study focuses on assessing the immunodiagnostic potential of S. haematobium (Sh) soluble egg antigen (SEA) and worm antigen (SWA) for urogenital schistosomiasis. Urine microscopy identified 50 S. haematobium-positive and 50 negative samples from a total of 500 examined. An additional 50 samples from a non-endemic area were included, bringing the total number of samples used for the assay to 150. Indirect ELISA immunoassays using SEA and SWA as the probing antigens evaluated 50 sera samples each from Sh positive, negative endemic (NE), and non-endemic (NNE) individuals. SDS-PAGE analysis of crude protein extracts was conducted, followed by Western blot analysis using primary antibodies from pooled Sh-infected sera samples. Diagnostic performance was evaluated using the area under the receiver operating characteristic (ROC) curve (AUC), sensitivity, and specificity. The AUC values for Sh SEA and SWA were 0.75 and 0.76 in NE samples, and 0.91 and 0.89 in NNE samples, respectively. Sensitivities 90 (95% CI: 78.64 - 95.65)/ 64.71 (95% CI: 52.17 - 75.92), and specificities 50 (95% CI: 36.64 - 63.36)/ 81.25 (95% CI: 63.56 - 92.79) were recorded for SEA and SWA, respectively in NE samples. In addition, sensitivities 90 (78.64 - 95.65)/ 92 (95% CI: 80.77 - 97.78), and specificities 72 (95% CI: 58.33 - 82.53)/ 72.00 (95% CI: 57.51 - 83.77) were recorded for SEA and SWA, respectively in NNE samples. The mean antibody titer against Sh SEA in infected samples was significantly higher than in non-infected samples (P <0.0001). Eight (8) immunoreactive protein bands; 4 each of SEA and SWA were identified, indicating potential for diagnostic tool development. Sh SEA and SWA demonstrate promise for diagnosing urogenital schistosomiasis in both endemic and non-endemic regions.

Development of Cashew and Pistachio Ladders through a Food-Processing Approach

Following successful oral immunotherapy (OIT) for peanut allergy using boiled peanuts (BOPI trial), this study investigated the potential of wet-thermal-processing-induced allergen modification, specifically soaking and boiling (1–4 h) to reduce the allergenicity of cashew and pistachio allergens. In addition, this study provides a foundation of understanding for developing safer forms of cashew/pistachio administration for application in OIT by gradual exposure to increasing doses of modified allergens with reduced potency as an “allergen ladder”. An SDS-PAGE analysis and an intrinsic-fluorescence spectroscopy revealed altered tertiary structures of the allergens, leading to their denaturation and even degradation. Notably, the reduction in both allergen-specific polyclonal IgG and human-specific IgE (sIgE) binding correlated with the treatment time, with the most significant decrease observed after 4 h of boiling. In contrast, shorter soaking treatments showed negligible effects on the IgE-binding capacity of these nuts, therefore indicating a further need for optimization. These findings indicate that extended boiling effectively reduced the amounts of the highly potent allergenic component Ana o 3 in cashew and Pis v 1 in pistachio, as confirmed by ELISA using polyclonal anti-Ana o 3 antibodies, and an immunoblot showed decreased IgE epitope binding in cashew and pistachio allergens, which further modified their allergenic profiles. This approach shows promise as a viable method for offering a safer therapeutic option for cashew/pistachio allergy.

Optimization, Purification and Characterization of Lipase from Streptomyces sp. A3301, with Application of Crude Lipase for Cooking Oily Wastewater Treatment

Streptomyces sp. A3301, which produces lipase isolated by Panyachanakul et al. [1]. This optimization was done for the subsequent purification and characterization of the biological lipase produced by the isolate. The results showed that the strain produced lipase with a maximum activity of 321 U/mL using the optimal medium and conditions (1.5 % (w/v) xylose and 2 % (w/v) yeast extract, pH 7.0 at 30 °C, 150 rpm for 3 days). The specific activity of the purified lipase was 27,000 U/mg, which was 544 times the pre-purification level, based on hydrophobic chromatography. After ion-exchange chromatography, the specific activity was 5,600 U/mg, which was 113 times the pre-purification level, with a single-peak purification profile. The purified lipase had a single band based on SDS-PAGE analysis and the molecular mass was 45 kDa. The optimum temperature and thermo-stability of A3301 lipase were 60 and 30 - 55 °C, respectively. The optimum pH of the purified enzyme was pH 9.0, and the enzyme was stable in the pH range of 8.0 - 9.0. The purified lipase was stable in acetone, chloroform and toluene, with a high relative activity of 63 - 76 %. The immobilized strain was then applied to oily-wastewater treatment. The strain can remove oil and grease in synthetic wastewater containing oil at 1 - 3 % (v/v), with removal rates of 100, 99.82 and 99.68 %, respectively, after incubation for 6 days. It was then applied to oily-wastewater treatment from a restaurant, achieving the highest degradation rates of 98.53 % after treatment for 6 days. In addition, it also affected the Biochemical Oxygen Demand (BOD) and Chemical Oxygen Demand (COD) values decreasing. HIGHLIGHTS Optimization of lipase production by Streptomyces A3301. Purification and characterization of the lipase produced by the Streptomyces A3301. Application of the immobilized Streptomyces A3301 strain in the treatment of oily cooking wastewater. The immobilized Streptomyces A3301 strain has the capability to remove oil and grease in both synthetic mediums and restaurant wastewater. GRAPHICAL ABSTRACT

Characterization of newly isolated Bacillus cereus D3 Co-producing α-amylase and protease and its application in food raw materials

Bacillus cereus, which effectively secretes various extracellular enzymes, has been widely used in the food, feed, and fermentation industries. One strain co-producing α-amylase and protease was newly isolated from Daqu and identified as B. cereus D3. It was shown that the production of α-amylase and protease co-produced by B. cereus D3 reached 13.89 ± 0.07 and 2468.45 ± 38.96 U/mL through optimization of fermentation conditions (nutrients (g/L): 20 of glucose, 20 of urea, 0.7 of MnCl2; incubation conditions: 24 h of incubation, initial medium pH of 7.0, temperature of 37 °C, 8% inoculum size, and 30% working volume). Moreover, the enzymatic genetic co-expression exhibited a similar pattern to the changes in enzymatic activities (growth-phase-dependent pattern). The two enzymes performed excellent thermal stability and were stable over wide pH ranges of 6.0–11.0 and 2.0–8.0, respectively. Finally, the hydrolysis degrees of raw materials including rice, sorghum, corn, and soybeans by α-amylase from B. cereus D3 reached 66.59 ± 4.21, 57.99 ± 4.39, 57.44 ± 4.67, and 38.01% ± 1.73%, respectively, producing glucose and maltose as the main products. The hydrolysis degrees of proteinous materials including mackerel heads and soybeans by protease from B. cereus D3 were 39.93 ± 1.99 and 30.44% ± 1.39%. SDS-PAGE analysis displayed extensive breakdown of macromolecular proteins, forming peptides of varied sizes. This is a systematic study on the co-production of α-amylase and protease by B. cereus, which is crucial to widen the understanding of this species co-producing multi-enzymes and explore its potential application in the food fermentation industry.

Sonication-Assisted Decellularization of Waste Tilapia (Oreochromis niloticus) Heads for Extracellular Matrix Extraction

Tilapia (Oreochromis niloticus), which is extensively farmed globally and ranks as the second most cultivated fish in the Philippines, generates significant amounts of waste that are often underutilized. One specific type of waste material consists of fish heads, which contain a valuable source of extracellular matrix (ECM). This study aims to evaluate the effects of sonication as a viable decellularization method for the extraction of ECM from tilapia fish heads. Particularly, two treatments were tested on the head samples: sonication-assisted decellularization (dWS) using a water bath sonicator, and decellularization without sonication (dNS), each with different contact times (5 min and 10 min). Histological analysis with H and E staining and DNA quantification revealed that sonication-assisted samples (dWS) showed a greater reduction in basophilic components and DNA content, achieving a 93.7% removal rate. These dWS samples also had the highest protein loss, retaining only 33.86% of the original protein. SDS–PAGE analysis indicated that both dWS and dNS samples maintained similar collagen structures, as evidenced by identical subunit bands. ATR–FTIR spectra confirmed the presence of collagen type I in all samples, detecting characteristic amides A, B, I, II, and III. The results revealed that varying treatments and contact times had significant effects on the physical and mechanical properties of the decellularized extracellular matrix (ECM). These findings highlight the effectiveness of sonication in the decellularization process, particularly for utilizing waste tilapia heads.

Caspase-3 interactins with calpain and cathepsin L: implications for protein stability and quality in fish fillets during postmortem storage

The degradation of structural proteins during fish postmortem storage is a critical factor affecting meat quality, leading to economic losses in the seafood industry. The complex interplay between caspase-3 and structural protein stability during fish postmortem storage remains largely unexplored. By treating grass carp fillets with a caspase-3 inhibitor, we establish a model for inhibiting apoptosis, delineating the role of caspase-3 in protein degradation. The effect of caspase-3 on grass carp proteins following postmortem storage and its cascade interaction with calpain and cathepsin L was further investigated. The result demonstrated that the destabilisation of the mitochondrial membrane during apoptosis led to modifications in the B-cell lymphoma (Bcl) family proteins, subsequently triggered the activation of caspase-9/caspase-3. Additionally, caspase-3 reduced the expression of the calpastatin and lysosomal membrane protein 1 (LAMP-1) gene, resulting in increased calpain and cathepsin L activity. SDS-PAGE and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis indicated that caspase-3 inhibitor treatment reduced actin fractures and preserved critical structural proteins. This investigation unveils the molecular mechanisms through which caspase-3 affects protein degradation in fish fillets during storage, providing valuable insights into the postmortem degradation processes.

Structural modification and functional improvement of lactoferrin through non-covalent and covalent binding to coffee polyphenol

Studies have suggested that milk may enhance or neutralize the bioavailability of coffee polyphenols, possibly due to reversible and irreversible interactions between coffee polyphenols and milk proteins. The effects of non-covalent and covalent binding of lactoferrin (BLF) to caffeic acid (CAA) on protein structure and function were investigated. SDS-PAGE analysis confirmed the covalent interaction between BLF and CAA. Multispectral experiments characterized the BLF-CAA complexes and conjugates, revealing alterations in the tertiary structure of the proteins in the BLF-CAA conjugates. Molecular docking and kinetics results demonstrated that hydrogen bonding, electrostatic interaction, and hydrophobic forces were the primary internal forces between CAA and BLF. When combined with CAA, the covalent conjugates exhibited superior functional properties including solubility, oxidation resistance, thermal stability, emulsification and foamability, bioaccessibility, and antimicrobial properties. This study offers a theoretical foundation and technical benchmark for the preparation of protein-based delivery vectors with synergistic effects.

Cold plasma-induced covalent binding with epigallo-catechin 3-gallate: A strategy for antigenicity reduction and antioxidant improvement of β-lactoglobulin

The application of cold plasma (CP) treatment was investigated for its effectiveness in promoting the covalent grafting of β-lactoglobulin (β-LG) and epigallo-catechin 3 -gallate (EGCG), as well as improving the antioxidant capacity of β-LG and eliminating its antigenicity. The results indicated that CP treatment significantly enhanced the covalent grafting of β-LG and EGCG. The maximum EGCG content in β-LG-EGCG complexes reached 47.08 ± 0.58 mg/g (with 45 s treatment), as confirmed by SDS-PAGE analysis and FTIR spectroscopy. Additionally, LC-MS/MS identifies two binding sites (T76 and K77) of EGCG in the β-LG-EGCG conjugates. After 60 s treatments, there was a significant reduction in the IgG binding capacity of β-LG-EGCG conjugates (29.97 ± 0.05%). Further analysis suggested that the change in the conformational epitopes of β-LG, due to the conjugating with EGCG, was likely the main factor responsible for the reduced binding Immunoglobulin G (IgG) capacity. The maximum antioxidant activities of β-LG-EGCG conjugates were found to be 974.14 ± 1.94 μmol TE g−1 for ABTS and 748.48 ± 9.66 μmol TE g−1 for DPPH). These values represent an eightfold increase in DPPH activity and a twofold increase in ABTS compared to the control sample. This study demonstrated that the CP treatment is highly effective technology for facilitating the covalent grafting of polyphenols to proteins, thereby enhancing their antioxidant properties and eliminate their antigenicity.

Designing mixed cationic/anionic supports to covalently immobilize/stabilize enzymes with high isoelectric point by enzyme adsorption and support-enzyme glutaraldehyde crosslinking

Ficin fully immobilized on Asp-agarose beads at pH 7 but not on an aminated support. This made enzyme adsorption plus glutaraldehyde modification non-viable for this enzyme. Modifying glyoxyl-agarose beads with mixtures of Asp and 1,6-hexamethylenediamine (HA) at different ratios, mixed anion/cation exchanger supports were built. Only if HA greatly exceed Asp in the support, immobilization did not work. While only using the Asp-agarose support immobilized enzyme molecules were only ionically adsorbed after glutaraldehyde treatment (visualized in SDS-PAGE analysis), the mixed supports gave covalent immobilization. The glutaraldehyde modification of these biocatalysts permitted to establish covalent bonds with the support, and this was more effective when using higher amounts of HA in the support. When around 60 % of the groups in the support were HA, the treatment with glutaraldehyde fully suppressed enzyme release from the support after boiling in SDS. The glutaraldehyde treated biocatalysts were more stable than just the adsorbed enzymes or the enzyme adsorbed only on Asp supports and then treated with glutaraldehyde (the optimal biocatalyst retained 90 % of the initial activity while the just adsorbed ficin retained 50 % of the initial activity). This strategy can be utilized to immobilize other proteins with high isoelectric points following this immobilization strategy.

Production of Tobacco Etch Virus Protease (TEV) Expressed in the Endotoxin-Free Bacillus subtilis and Its Application.

Tobacco Etch virus (TEV) protease is one of the most common tools for removing fusion tags, but no study has shown that TEV can be expressed at high levels in the GRAS host strain Bacillus subtilis and purified for further application. In this study, the fusion protein BsLysSN-TEV C/S-His-TEV consisting of a fusion tag, N-terminal domain of a lysyl-tRNA synthetase (BsLysSN) coded by B. subtilis lysS gene, placed at the N-terminus followed by an endoprotease TEV cleavage site and then the expression of this fusion protein in the cytoplasm of B. subtilis was investigated. The SDS-PAGE and Western-blot analysis demonstrated that His-TEV was overexpressed under the induction of IPTG. This result infers that His-TEV protease showed promising activity in the B. subtilis cytoplasm by the cleavage of the fusion protein. These cleavage products could be purified using the Ni-NTA column, which effectively cleaved the purified recombinant protein substrate, which can be applied in the protein purification process to remove the fusion tag. Significantly, since both His-TEV protease and the fusion recombinant protein substrate are expressed in the endotoxin-free host strain, the tag removal and purified product should be theoretically endotoxin-free, which could be a promising approach for producing therapeutic proteins and also for other relevant biomedical applications.

Impact of roasting time on the color, protein, water distribution and key volatile compounds of pork

The study aimed to determine the changes in physical and chemical indicators in pork during the roasting process and establish predictive models for industrial intelligent production in the future, the effects of roasting times (0–15 min) on the dynamic changes in the surface color, water, protein and the volatile compounds in pork were investigated. The results showed that during the roasting process, the L* value increased firstly (0–3 min) and then decreased (3–15 min), the a* value decreased firstly (0–12 min) and then increased (12–15 min), while the b* value increased (0–15 min). A total of 29 volatile compounds were identified by gas chromatography–mass spectrometry, 11 volatile differentiators were identified by partial least squares-discriminant analysis, and 14 key volatile compounds were selected by odor activity values analysis. The total water content significantly decreased and the water in meat migrated from high degrees of freedom to low degrees of freedom. Roasting for 6 min was a key turning point in the changes of the α-helix, β-sheet and random coil. In addition, SDS-PAGE analysis, and the changes in the contents of carbonyl, total and free sulfhydryl verified proteins oxidation in the pork during the roasting process.

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.

A novel diglycosidase for the transformation of naringin to naringenin and neohesperidose

A novel fungal diglycosidase that transforms naringin into naringenin and neohesperidose, a rare biotransformation, has been purified to homogeneity using a simple procedure involving precipitation of the enzyme from the culture filtrate of the fungal strain using 80 % saturation of ammonium sulphate, dissolving the precipitate in minimum volume of the buffer and dialysing that against the buffer. The purified enzyme gives single protein bands of molecular mass 64.6 kDa in SDS-PAGE analysis. The purity of the enzyme has been further confirmed by the appearance of single protein band in native page analysis. Using naringin as the substrate, the diglycosidase has Km and kcat values of 0.20 m mol L−1 and 0.66 s−1, respectively, at pH 4.0 and 313 K. The specific activity of the purified enzyme using naringin as the natural substrate is 1.018 katal/kg. The diglycosidase also transforms rutin into quercetin and rutinose but has no effect on hesperidin. The feasibilities of preparing neohesperidose from naringin and rutinose from rutin on milligram scales using the pure enzyme have been demonstrated. These results open the way for developing an enzymatic process for preparation of neohesperidose from naringin. The reported diglycosidase has immense future applications in food and pharmaceutical industries.

Impact of Lycium barbarum polysaccharides on wheat dough quality and hydration dynamics

This study investigates the effects of crude (LBP) and degraded (LBPD) polysaccharides from Lycium barbarum L. leaves on wheat dough hydration dynamics and quality. Enhancing wheat dough functional properties is crucial for improving baked goods. We assessed how these polysaccharides affect water-holding capacity, gluten structure, texture, and sensory attributes. LBP and LBPD had distinct yields (32.05% and 29.23%, respectively) and chemical compositions. LBP darkened the dough, while LBPD gave it a tan hue. Both decreased water-holding capacity, with LBPD showing a concentration-dependent effect. SDS-PAGE analysis revealed that LBP destabilized gliadin, whereas LBPD protected gluten proteins. Adding LBP and LBPD improved texture and extensographic properties, with LBPD having a more pronounced effect, especially in sensory acceptability. These findings suggest LBP and LBPD can significantly enhance wheat dough quality, providing valuable insights for the food industry.

Unraveling the abundance of vip3-type genes in Indian Bacillus thuringiensis across the agroclimatic landscape and impact of amino acid substitutions for safer agriculture

Vegetative insecticidal protein (vip) genes of Bacillus thuringiensis (Bt) are candidates for gene pyramiding in the resistance management of pests. The prevalence of vip genes in Bt isolates is relatively under-explored. Bt isolates recovered from 29 diverse sources in nine agro-climatic zones of India were screened for the presence of vip3-type genes by PCR with 4 sets of oligonucleotide primers. Out of 155 Bt isolates, 70.32 % (109) and 44.52 % (69) isolates were positive for amplification of partial vip3-type genes with primer sets 1 and 4, respectively. The primer set-2 was found to be more efficient for amplifying full-length genes (29.03 % /45 isolates) as compared with primer set-3 (3.23 %/ 5 isolates), also corroborated in the amplification of full-length vip3 genes in ten Bt BGSC strains used as reference. Frequency analysis revealed presence of vip3 genes in Bt isolates across all agro-climatic zones. Thus, Indian Bt isolates from diverse sources have a rich repertoire of vip3-type genes. Our study reports the highest number (45) of full-length vip3-type genes detected in a native Bt isolates collection, demonstrating enrichment of Indian Bt isolates for vip3 genes. Twelve of these genes have been cloned, sequenced, and out of these, six were found to be effective against Helicoverpa armigera in our laboratory previously. Comparison of substitutions in deduced amino acids sequence of these genes and expression of Vip3 proteins in SDS-PAGE analysis of selected native Bt isolates positive for full-length vip3-type genes indicated their biopesticidal potential.

Comparison of in vitro digestive characteristics of proteins from different sources in simulated elderly gastrointestinal conditions

This study aimed to evaluate the impact of in vitro digestive characteristics of five different protein sources (fish, crab, beef, chicken, and soy) in adults and elderly individuals. Simulated gastrointestinal tract degradation in the elderly significantly reduced the digestibility of all proteins. The Elderly model had difficulties digesting any protein that had a firm structure or was prone to aggregation. Of the five protein types, soy protein was the least easily digested. Ultraviolet spectroscopy and SDS-PAGE analysis revealed that beef and crab proteins contain myoglobin and hemocyanin, respectively, making their structure firm. Simultaneously, alkali-soluble proteins in meat were more likely to be digested by the elderly than salt-soluble proteins. The alkali-soluble protein percentage in fish protein was the highest, including the best in vitro digestion characteristics in the elderly population. This study provides novel and significant recommendations for the elderly's daily intake of various protein sources.

Abstract A085: Engineered novel bioactive Nectin-4 dimer protein significantly enhances the binding of TIGIT

Abstract The goals of this study are to: (a) create a novel soluble bioactive Nectin-4 dimer molecule mimicking its native dimer quaternary structure on the cell surface and (b) evaluate the recombinant Nectin-4 dimer binding potency to its receptor vs the monomer form, so that the dimer can be used as an immunogen and an antigen to increase the potential of cancer therapeutic antibody and vaccine discovery when targeting quaternary structural epitopes. Nectin-4 is a cell adhesion molecule belonging to the nectin family. It's involved in cell-cell junctions and plays a role in cancer progression by influencing cell adhesion, migration, and proliferation. Nectin-4 is overexpressed on various types of tumor cells including breast, lung, ovarian and pancreatic cancers. Nectin-4 has been recently identified as a novel ligand of T cell immunoglobulin and ITIM domain (TIGIT) and interaction between Nectin-4 and TIGIT inhibits NK cell cytotoxicity thus suppressing immune responses, allowing cancer cells to evade immune surveillance. Nectin-4 and Nectin-1 interact in a heterophilic manner, however in the context of cancer, altered expression of Nectin-4 and Nectin-1 can contribute to tumor progression. Nectin-4 is a type 1 single transmembrane protein, and can form homodimers on the cell surface. While therapeutics such as Enfortumab Vedotin, an antibody-drug conjugate, binds Nectin-4 on the surface of cancer cells, mimicking the Nectin-4 dimer quaternary structure can present many more critical opportunities for therapeutic antibody and vaccine discovery. To better mimic the native dimer conformation and quaternary structure, we designed a novel soluble Nectin-4 dimer with 3 extracellular domains fused to a dimer motif at the C-terminus. The Nectin-4 dimer protein was expressed and purified from HEK293 cells and confirmed by SDS-PAGE and Western blot analyses. We characterized the Nectin-4 dimer protein binding potency to TIGIT and Nectin-1 by ELISA. Very interestingly, the results demonstrated that the Nectin-4 dimer binding potency to TIGIT increased dramatically as measured by ELISA, compared to the Nectin-4 monomer protein. Conversely, the Nectin-4 dimer binding to Nectin-1 monomer was significantly decreased, compared to Nectin-4 monomer, as expected. We also studied Nectin-4 dimer immunogenicity using a Nectin-4 dimer DNA immunization to induce potent antibody responses in mice. In summary, these findings imply that the Nectin-4 dimer protein is bioactive and in the desirable conformation, resulting in an increased binding potency to its receptor TIGIT, reduced binding to Nectin-1 (as expected), and eliciting strong antibody responses in mice. This novel Nectin-4 dimer protein can be a very useful molecule for cancer research, therapeutic antibody and vaccine discovery. The Nectin-4 dimer protein can be used to: (i) elicit antibody responses against the dimer quaternary structure, (ii) screen antibodies targeting more conformational epitopes, and (iii) study the Nectin-4 and TIGIT ligand/receptor interactions. Citation Format: Christofer Walsh, Timothy Smith, Mikhail Rashkovskii, Richard Parent, Jean Qiu, Shixia Wang. Engineered novel bioactive Nectin-4 dimer protein significantly enhances the binding of TIGIT [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A085.

Optimization and evaluation of a chitosan-coated PLGA nanocarrier for mucosal delivery of Porphyromonas gingivalis antigens

Recent advances in understanding Alzheimer's disease (AD) suggest the possibility of an infectious etiology, with Porphyromonas gingivalis emerging as a prime suspect in contributing to AD. P. gingivalis may invade systemic circulation via weakened oral/intestinal barriers and then cross the blood-brain barrier (BBB), reaching the brain and precipitating AD pathology. Based on the proposed links between P. gingivalis and AD, a prospective approach is the development of an oral nanovaccine containing P. gingivalis antigens for mucosal delivery. Targeting the gut-associated lymphoid tissue (GALT), the nanovaccine may elicit both mucosal and systemic immunity, thereby hampering P. gingivalis ability to breach the oral/intestinal barriers and the BBB, respectively.The present study describes the optimization, characterization, and in vitro evaluation of a candidate chitosan-coated poly(lactic-co-glycolic acid) (PLGA-CS) nanovaccine containing a P. gingivalis antigen extract. The nanocarrier was prepared using the double emulsion solvent evaporation method and optimized for selected experimental factors, e.g. PLGA amount, surfactant concentration, w1/o phase ratio, applying a d-optimal statistical design to target the desired physicochemical criteria for its intended application. After nanocarrier optimization, the nanovaccine was characterized in terms of particle size, polydispersity index (PdI), ζ-potential, encapsulation efficiency (EE), drug loading (DL), morphology, and in vitro release profile, as well as for mucoadhesivity, stability under simulated gastrointestinal conditions, antigen integrity, in vitro cytotoxicity and uptake using THP-1 macrophages.The candidate PLGA-CS nanovaccine demonstrated appropriate physicochemical, mucoadhesive, and antigen release properties for oral delivery, along with acceptable levels of EE (55.3 ± 3.5 %) and DL (1.84 ± 0.12 %). The integrity of the encapsulated antigens remained uncompromised throughout NPs production and simulated gastrointestinal exposure, as confirmed by SDS-PAGE and Western blotting analyses. Furthermore, the nanovaccine showed effective in vitro uptake, while exhibiting low cytotoxicity. Taken together, these findings underscore the potential of PLGA-CS NPs as carriers for adequate antigen mucosal delivery, paving the way for further investigations into their applicability as vaccine candidates against P. gingivalis.

Expression and purification of SARS-CoV-2 receptor binding domain in Escherichia coli for diagnostic and therapeutic purposes

Background and purpose: SARS-CoV-2 causes a severe respiratory disease known as COVID-19 and is responsible for a global viral pandemic. The SARS-CoV-2 receptor binding domain (RBD) is located on the spike protein, which identifies and binds to the angiotensin-converting enzyme 2 (ACE2) receptor. The RBD is an important target for developing virus-neutralizing antibodies, vaccines, and inhibitors. Experimental approach: In this study, recombinant SARS-CoV-2 RBD was expressed in E. coli BL21 (DE3) and purified and its binding activity was determined. Purification was conducted using the Ni-NTA column. ELISA. flow cytometry assays were set to evaluate the binding ability of recombinant RBD to different anti-RBD antibodies and native ACE2 receptors on HEK293A cells, respectively. Findings/Results: The SDS-PAGE analysis revealed the corresponding band at 27 kDa in the culture after induction with 0.7 mM IPTG, while the corresponding band was not observed in the culture without IPTG induction. ELISA results showed that antibodies produced in the human sera could bind to the recombinant RBD protein and the commercial anti-RBD antibody. Also, flow cytometry analysis revealed that the recombinant RBD could bind to human ACE2 on the surface of HEK293A cells. Conclusion and implication: Our outcomes displayed that the recombinant RBD expressed in the E. coli strain has biological activity and can be used as an antigen for the development of diagnosis kits and vaccines as well as a tool for screening drugs against SASR-CoV-2.

Characterisation, antibacterial activity, and prebiotic potential of dried Hermetia illucens L. larvae and of their fractions

Abstract In this study, black soldier fly (BSF) dried larvae were fractionated into their defatted, lipid, protein, and chitin-rich fractions. The samples were then characterised in terms of proximate composition, analysis of amino acids, analysis of the molecular weight distribution of proteins via gel electrophoresis, and fatty acids analysis of the lipid fraction. The antibacterial activity of all fractions was determined against Escherichia coli F4 and Streptococcus suis, two common swine pathogens. Finally, the prebiotic activity of the whole larvae and of their protein isolate and chitin-rich pellet was determined for Limosilactobacillus reuteri, a naturally occurring species in the gastro-intestinal tract of the pig. The results on amino acid profile showed that the protein extraction method used was not fully effective in cleaving chitin-bound proteins, as expected, as the true protein content of the chitin-rich sample was 48.5%. This was in accordance with the SDS-PAGE analysis, where the molecular weight distribution of proteins in the protein isolate sample did not show band at higher molecular weight, which were instead present in the chitin-rich sample. The analysis of fatty acid (FA) profile showed that lauric acid was the most abundant FA, in accordance with the literature. The results on microbiological analyses highlighted how all nitrogen-containing samples showed antibacterial activity against E. coli, the highest being the defatted fraction at 1% with a decrease of −0.77 log CFU/mL, while only the lipid fraction against S. suis (−0.48 log CFU/mL at 1%). All tested samples showed prebiotic activity for L. reuteri, but it was noticed that the increase in chitin concentration resulted in the decrease of the prebiotic effect. By combining the results of both antibacterial and prebiotic tests, it could be inferred that chitin might be considered as an antibacterial compound.