SDS-PAGE Research Articles

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&E) staining. Results: The results showed a significant (p<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.

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.

A comparison of the chemical compositions and nutrient values of the sanim and srithong varieties of the farmed golden apple snails (Gastropoda; Ampullariidae; Pomacea canaliculata)

The comparison of the Sanim (SN) and Srithong (ST) varieties of the farmed apple snail Pomacea canaliculata as an alternative protein source has shown that ST contained more hydroxyproline and possessed a greater firmness than SN. Nevertheless, both varieties yielded similar protein contents of 20.90–23.07 % and exhibited similar texture, color, cooking yields and cooking losses after cooking. Good palatability with high liking score was obtained from both snail meat samples, particularly color, texture, and overall attributes. Snail meats possessed good nutritive value with an amount of protein and low fat content, whereas SN contained higher protein and total energy(P < 0.05). Other nutritionally useful chemicals (such as calcium, sodium, iron, vitamins B1 and B2) are present. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results discovered that myosin heavy chain, paramyosin, and actin were the prevalent protein components. Differences in nitrogen content distribution were observed between SN and ST, in which alkaline-soluble protein was the major fraction. Amino acid composition and fatty acid content varied regarding snail varieties with a large amount of essential amino acids and good source of MUFA and PUFA. Therefore, both farmed SN and ST snails could be an effective alternative protein source with a significant amount of nutritive value.

Effects of different postharvest drying processes on flavonoid content and enzymatic activity of Styphnolobium japonicum (L.) Schott flowers for industrial and medicinal use

Traditionally, fresh S. japonicum flowers (SJF) and S. japonicum flowers buds (SJFB) are dried prior to further processing and use. Here, we investigated the ways in which drying techniques, including sun drying (SD), steam drying (STD), microwave drying (MD), hot air drying (HAD, 40 °C, 60 °C, 80 °C, 100 °C), and freeze drying (FD), alter the flavonoid composition of freshly-harvested SJF and SJFB. The flavonoid content of dried samples was determined by Ultra High Performance Liquid Chromatography-Diode Array Detector (UPLC-DAD). Overall, different drying techniques had significantly different effects on the RU content, ranging from 10.63 % (HAD-80 °C) to 34.13 % (HAD-100 °C) in SJF and from 18.91 % (HAD-100 °C) to 29.16 % (HAD-40 °C) and 30.53 % (SD) in SJFB. To clarify the mechanism by which drying affects the RU content of S. japonicum flowers, we studied the activity of a rutin-hydrolyzing enzyme (RHE) isolated from SJF and SJFB using multiple separation and assay methods. According to the Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) results, the apparent molecular weight of the purified RHE was approximately 38 kDa. According to UPLC-DAD, RHE catalyzes the production of quercetin (QU) from rutin (RU), but not from other flavonoid glycosides. Drying fresh SJF and SJFB at low and high temperatures can inhibit RHE activity and prevent RU hydrolysis. Therefore, subjecting freshly-harvest SJF to HAD-100 °C, and freshly-harvest SJFB to SD or HAD-40 °C, can greatly increase the RU content. In particular, HAD is viable for large-scale application due to its simplicity and industrial feasibility.

Blue Light Compromises Bacterial β-Lactamases Activity to Overcome β-Lactam Resistance.

In this study, we evaluated the effectiveness of antimicrobial blue light (aBL; 410 nm wavelength) against β-lactamase-carrying bacteria and the effect of aBL on the activity of β-lactamases. Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae strains carrying β-lactamases as well as a purified β-lactamase enzymes were studied. β-lactamase activity was assessed using a chromogenic cephalosporin hydrolysis assay. Additionally, we evaluated the role of porphyrins in the photoreaction, as well as protein degradation by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Finally, we investigated the bactericidal effect of combined aBL-ceftazidime exposure against a metallo-β-lactamase expressing P. aeruginosa strain. Our study demonstrated that aBL effectively killed β-lactamase-producing bacteria and reduced β-lactamase activity. After an aBL exposure of 1.52 J/cm2, a 50% reduction in enzymatic activity was observed in P. aeruginosa. Additionally, we found a 40% decrease in the photoreaction activity of porphyrins following an aBL exposure of 64.8 J/cm2. We also revealed that aBL reduced β-lactamase activity via protein degradation (after 136.4 J/cm2). Additionally, aBL markedly improved the bactericidal effect of ceftazidime (by >4-log10) in the metallo-β-lactamase P. aeruginosa strain. Our results provide evidence that aBL compromises bacterial β-lactamase activity, offering a potential approach to overcome β-lactam resistance in bacteria.

Impact of Co-60 γ-ray irradiation on the cross-linking and stability of fish collagen: Structural changes and digestibility

Irradiation can promote the cross-linking of collagen to enhance its biological properties, but the molecular changes in irradiated collagen have few been investigated. This study aimed to explore the impact of irradiation treatment on the molecular structure stabilization and cross-linking degree of skin collagen. The results showed that collagen in solution was more susceptible to Co-60 gamma irradiation than that in fish skin and in sponge forms. The solubility of collagen in solution decreased by more than 80% and the carbonyl content increased by more than 4 times compared with the control group after 12 kGy irradiation. Spectroscopic analysis and sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) showed that collagen in solution was cross-linked, especially at high irradiation doses. However, the triple helical structure of skin collagen and collagen sponge remained intact. Furthermore, irradiation also reduced the sensitivity of collagen to collagenase. Gastrointestinal digestion results showed that the antioxidant activity of collagen increased at 1–3 kGy doses and decreased at 9–12 kGy doses. Collagen irradiated at high doses (9–12 kGy) was more difficult to digest and had a larger average particle size. This study provides a potential new approach for the preparation of stable fishery-derived collagen with excellent antioxidant activity and resistance to digestion by irradiation treatment.

Production, purification and characterization of endo-polygalacturonase using novel strain of Bacillus pumilus through RSM

Polygalacturonases (PG) are the enzymes that cause depolymerization of pectin. In current study, bacterial strain was isolated from rotten sample and then purified. It was screened for endo-polygalacturonase activity using PSAM media. It had shown positive response for endo-PG activity. Bacterial DNA was isolated and 16 S rRNA gene amplification was done using universal primer pair of 8 F and 1492 R. Bacterial strain was also amplified by 16 S rRNA gene for sequencing. BLAST of gene sequence on NCBI database had shown that bacterial isolate was identified as Bacillus pumilus. Endo-polygalacturonase enzyme was produced by submerged fermentation to optimize culture conditions by RSM in JMP-12 software. The optimized parameters had shown maximum endo-polygalacturonase activity of 153.2 U/mL/min after using 3 g of orange peels as substrate, 3 mL of inoculum size, 6.5 pH buffer, 40°C temperature and 3 days of incubation. After optimizing fermentation conditions, endo-polygalacturonase was precipitated using 70 % concentration of ammonium sulfate. This partially precipitated sample was dialyzed with dialysis tube and used to find activity of endo-polygalacturonase (1620.6 U/mL/min). This sample was applied to gel filtration chromatography for further purification. Endo-polygalacturonase activity increased many times 2231.44 U/mL/min after gel filtration. The Molecular weight of endo-polygalacturonase was 46 kDa after SDS PAGE characterization. High Vmax value and alkaliphilic nature of endo-polygalacturonase will make it good candidate for food and feed industry near future.

What are the main proteins in the hemolymph of Haemaphysalis flava ticks?

Haemaphysalis flava is a notorious parasite for humans and animals worldwide. The organs of H. flava are bathed in hemolymph, which is a freely circulating fluid. Nutrients, immune factors, and waste can be transported to any part of the body via hemolymph. The main soluble components in hemolymph are proteins. However, knowledge of the H. flava proteome is limited. The hemolymph was collected from fully engorged H. flava ticks by leg amputation. Hemolymph proteins were examined by both blue native polyacrylamide gel electrophoresis (BN-PAGE) and sodium dodecyl sulfate PAGE (SDS-PAGE). Proteins extracted from the gels were further identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two bands (380 and 520 kDa) were separated from tick hemolymph by BN-PAGE and were further separated into four bands (105, 120, 130, and 360 kDa) by SDS-PAGE. LC-MS/MS revealed that seven tick proteins and 13 host proteins were present in the four bands. These tick proteins mainly belonged to the vitellogenin (Vg) family and the α-macroglobulin family members. In silico structural analysis showed that these Vg family members all had common conserved domains, including the N-terminus lipid binding domain (LPD-N), the C-terminus von Willebrand type D domain (vWD), and the domain of unknown function (DUF). Additionally, two of the Vg family proteins were determined to belong to the carrier protein (CP) by analyzing the unique N-terminal amino acid sequences and the cleaving sites. These findings suggest that the Vg family proteins and α-macroglobulin are the primary constituents of the hemolymph in the form of protein complexes. Our results provide a valuable resource for further functional investigations of H. flava hemolymph effectors and may be useful in tick management.

Biochemical characterisation and production kinetics of high molecular-weight (HMW) putative antibacterial proteins of insect pathogenic Brevibacillus laterosporus isolates

BackgroundBacterial genomes often encode structures similar to phage capsids (encapsulins) and phage tails which can be induced spontaneously or using genotoxic compounds such as mitomycin C. These high molecular-weight (HMW) putative antibacterial proteins (ABPs) are used against the competitive strains under natural environment. Previously, it was unknown whether these HMW putative ABPs originating from the insect pathogenic Gram-positive, spore-forming bacterium Brevibacillus laterosporus (Bl) isolates (1821L, 1951) are spontaneously induced during the growth and pose a detrimental effect on their own survival. Furthermore, no prior work has been undertaken to determine their biochemical characteristics.ResultsUsing a soft agar overlay method with polyethylene glycol precipitation, a narrow spectrum of bioactivity was found from the precipitated lysate of Bl 1951. Electron micrographs of mitomycin C- induced filtrates showed structures similar to phage capsids and contractile tails. Bioactivity assays of cell free supernatants (CFS) extracted during the growth of Bl 1821L and Bl 1951 suggested spontaneous induction of these HMW putative ABPs with an autocidal activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of spontaneously induced putative ABPs showed appearance of ~ 30 kDa and ~ 48 kDa bands of varying intensity across all the time intervals during the bacterial growth except in the initial hours. Statistically, spontaneously induced HMW putative ABPs of Bl 1951 exhibited a significant decrease in the number of viable cells of its producer strain after 18 h of growth in liquid. In addition, a significant change in pH and prominent bioactivity of the CFS of this particular time period was noted. Biochemically, the filtered supernatant derived from either Bl 1821L or Bl 1951 maintained bioactivity over a wide range of pH and temperature.ConclusionThis study reports the spontaneous induction of HMW putative ABPs (bacteriocins) of Bl 1821L and Bl 1951 isolates during the course of growth with potential autocidal activity which is critically important during production as a potential biopesticide. A narrow spectrum of putative antibacterial activity of Bl 1951 precipitate was found. The stability of HMW putative ABPs of Bl 1821L and Bl 1951 over a wide range of pH and temperature can be useful in expanding the potential of this useful bacterium beyond the insecticidal value.

Debridement efficacy of serine protease and formulated cream by In Vitro assessment against artificial wound eschar

Chronic wounds typically comprise of necrotic tissue and dried secretions, often culminating in the formation of a thick and tough layer of dead skin known as eschar. Removal of eschar is imperative to facilitate wound healing. Conventional approach for eschar removal involves surgical excision and grafting, which can be traumatic and frequently leads to viable tissue damage. There has been growing interest in the use of enzymatic agents for a gentler approach to debridement, utilizing proteolytic enzymes. In this study, a purified intracellular recombinant serine protease from Bacillus sp. (SPB) and its cream formulation were employed to evaluate their ability to degrade artificial wound eschar; composed of collagen, fibrin, and elastin. Degradation was assessed based on percentage weight reduction of eschar biomass, analysis via sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and scanning electron microscopy (SEM). Both SPB and its cream formulation were able to degrade up to 50 % artificial wound eschar, with the SPB cream maintaining its degradation efficiency for up to 24 hours. Additionally, the SPB-based cream demonstrated the ability to hydrolyze proteinaceous components of eschars individually (fibrin and collagen) as determined through qualitative assessment. These findings suggest that SPB holds promise for the debridement of wound eschar.

Isolation and Characterization of Cellulolytic Pseudomonas Aeruginosa from Teak Forest Soil for its Cellulase Production and Cellulolytic Activity

A cellulolytic bacterium was isolated from Tectona forest soil and provisionally identified as Pseudomonas aeruginosa. The bacterium showed the highest level of reducing sugar and extracellular protein production when incubated for 3 days at 40°C, pH 8.5 in Winstead’s broth having 1.5% carboxymethyl cellulose (CMC) and 0.2% Yeast Extract as carbon and nitrogen sources, respectively. The bacterium produced the maximum 462 µg/ml reducing sugar and 450 µg/ml extracellular proteins. Crude cellulase produced by the bacterium showed the highest carboxymethyl cellulase (CMCase) activity rather than filter paperase (FPase), Avicelase and β-Glucosidase activities. The crude cellulase showed highest CMCase activity when incubated for 90 minutes at pH 8.5, 25°C using CMC and Yeast Extract as carbon and nitrogen sources, respectively as well as in presence of Hg2+ ion and SDS (inhibitor and reductant). The highest enzyme activity was found to be 315 U/ml. The molecular weight of the crude cellulase was found as 33 kDa through SDS PAGE analysis. According to Worthington Enzyme Manual it may be Endoglucanase IV. The Chittagong Univ. J. B. Sci., Vol. 10(1 &amp;2):167-179, 2020

Co-crosslinking strategy for dual functionalization of small magnetic nanoparticles with redox probes and biological probes

Surface functionalization strategy is becoming a crucial bridge from magnetic nanoparticles (MNPs) to their broad bio-application. To realize the multiple functions of MNPs such as magnetic manipulation, target capture, and signal amplification in their use of electrochemical biosensing, co-crosslinking strategy was proposed here to construct dual-functionalized MNPs by combining ultra-sensitive redox moieties and specific biological probes. In this work, MNPs with a TEM size of 10 nm were synthesized by co-precipitation for amination and PEGylation to maintain colloid stability once dispersed in high-ionic-strength buffer (such as phosphate-buffered saline). Then, MNPs@IgG were prepared via the bis(sulfosuccinimidyl) suberate (BS3) cross-linker to conjugate these IgG onto the MNP surface, with a binding efficiency of 73%. To construct dual-functionalized MNPs, these redox probes of ferrocene-NHS (Fc) were co-crosslinked onto the MNP surface, together with IgG, by using BS3. The developed MNPs@Redox@IgG were characterized by SDS‒PAGE to identify IgG binding and by square wave voltammetry (SWV) to validate the redox signal. Additionally, the anti-CD63 antibodies were selected for the development of MNPs@anti-CD63 for use in the bio-testing of exosome sample capture. Therefore, co-crosslinking strategy paved a way to develop dual-functionalized MNPs that can be an aid of their potential utilization in diagnostic assay or electrochemical methods.Graphical

Dermatoprotective effect of Moringa oleifera leaf extract on sodium valproate-induced skin damage in rats

Valproic acid is an antiepileptic drug associated with skin-related issues like excessive hair growth, hair loss, and skin rashes. In contrast, Moringa oleifera, rich in nutrients and antioxidants, is gaining popularity worldwide for its medicinal properties. The protective properties of M. oleifera extract against skin-related side effects caused by valproic acid were investigated. Female rats were divided into control groups and experimental groups such as moringa, sodium valproate, and sodium valproate + moringa groups. A 70% ethanolic extract of moringa (0.3 g/kg/day) was given to moringa groups, and a single dose of sodium valproate (0.5 g/kg/day) was given to valproate groups for 15 days. In the skin samples, antioxidant parameters (such as glutathione, glutathione-S-transferase, superoxide dismutase, catalase, and total antioxidant capacity), as well as oxidant parameters representing oxidative stress (i.e. lipid peroxidation, sialic acid, nitric oxide, reactive oxygen species, and total oxidant capacity), were examined. Additionally, boron, hydroxyproline, sodium-potassium ATPase, and tissue factor values were determined. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was also carried out for protein analysis in the skin samples. The results showed that moringa could increase glutathione, total antioxidant capacity, sodium-potassium ATPase, and boron levels, while decreasing lipid peroxidation, sialic acid, nitric oxide, total oxidant capacity, reactive oxygen species, hydroxyproline, and tissue factor levels. These findings imply that moringa possesses the potential to mitigate dermatological side effects.

Leptin-mediated suppression of lipoprotein lipase cleavage enhances lipid uptake and facilitates lymph node metastasis in gastric cancer.

Lymph node metastasis (LNM) is the primary mode of metastasis in gastric cancer (GC). However, the precise mechanisms underlying this process remain elusive. Tumor cells necessitate lipid metabolic reprogramming to facilitate metastasis, yet the role of lipoprotein lipase (LPL), a pivotal enzyme involved in exogenous lipid uptake, remains uncertain in tumor metastasis. Therefore, the aim of this study was to investigate the presence of lipid metabolic reprogramming during LNM of GC as well as the role of LPL in this process. Intracellular lipid levels were quantified using oil red O staining, BODIPY 493/503 staining, and flow cytometry. Lipidomics analysis was employed to identify alterations in intracellular lipid composition following LPL knockdown. Protein expression levels were assessed through immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assays. The mouse popliteal LNM model was utilized to investigate differences in LNM. Immunoprecipitation and mass spectrometry were employed to examine protein associations. In vitro phosphorylation assays and Phos-tag sodium dodecyl-sulfate polyacrylamide gel electrophoresis assays were conducted to detect angiopoietin-like protein 4 (ANGPTL4) phosphorylation. We identified that an elevated intracellular lipid level represents a crucial characteristic of node-positive (N+) GC and further demonstrated that a high-fat diet can expedite LNM. LPL was found to be significantly overexpressed in N+ GC tissues and shown to facilitate LNM by mediating dietary lipid uptake within GC cells. Leptin, an obesity-related hormone, intercepted the effect exerted by ANGPTL4/Furin on LPL cleavage. Circulating leptin binding to the leptin receptor could induce the activation of inositol-requiring enzyme-1 (IRE1) kinase, leading to the phosphorylation of ANGPTL4 at the serine 30 residue and subsequently reducing its binding affinity with LPL. Moreover, our research revealed that LPL disrupted lipid homeostasis by elevating intracellular levels of arachidonic acid, which then triggered the cyclooxygenase-2/prostaglandin E2 (PGE2) pathway, thereby promoting tumor lymphangiogenesis. Leptin-induced phosphorylation of ANGPTL4 facilitates LPL-mediated lipid uptake and consequently stimulates the production of PGE2, ultimately facilitating LNM in GC.

Laccase/caffeic acid-catalyzed crosslinking coupled with galactomannan alters the conformational structure of ovalbumin and alleviates Th2-mediated allergic asthma

Ovalbumin (OVA) is the major allergenic protein that can induce T helper 2 (Th2)-allergic reactions, for which current treatment options are inadequate. In this study, we developed a polymerized hypoallergenic OVA product via laccase/caffeic acid (Lac/CA)-catalyzed crosslinking in conjunction with galactomannan (Man). The formation of high molecular weight crosslinked polymers and the IgG-binding were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The study indicated that Lac/CA-catalyzed crosslinking plus Man conjugation substantially altered secondary and tertiary structures of OVA along with the variation in surface hydrophobicity. Gastrointestinal digestion stability assay indicated that crosslinked OVA exhibited less resistance in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Mouse model study indicated that Lac-Man/OVA ameliorated eosinophilic airway inflammatory response and efficiently downregulated the expression of Th2-related cytokines (interleukin (IL)-4, IL-5, and IL-13), and upregulated IFN-γ and IL-10 expression. Stimulation of bone marrow-derived dendritic cells with Lac-Man/OVA suppressed the expression of phenotypic maturation markers (CD80 and CD86) and MHC class II molecules, and suppressed the expression levels of proinflammatory cytokines. The knowledge obtained in the present study offers an effective way to acquire a hypoallergenic OVA product that can have a therapeutic effect in alleviating OVA-induced allergic asthma.

Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening

Alpha-lactalbumin (α-LA) is a major whey protein found in breast milk and plays a crucial role in the growth and development of infants. In this study, Bacillus subtilis RIK1285 harboring AprE signal peptide (SP) was selected as the original strain for the production of α-LA. It was found that α-LA was identified in the pellet after ultrasonic disruption and centrifugation instead of in the fermentation supernatant. The original strain most likely only produced α-LA intracellular, but not extracellular. To improve the expression and secretion of α-LA in RIK1285, a library of 173 homologous SPs from the B. subtilis 168 genome was fused with target LALBA gene in the pBE-S vector and expressed extracellularly in RIK1285. SP YjcN was determined to be the best signal peptide. Bands in supernatant were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purified by nickel column to calculate the highest yield signal peptide. In addition, different promoters (PaprE, P43, and Pglv) were compared and applied. The results indicated that the strain RIK1285-pBE-Pglv-YjcN-LALBA had the highest α-LA yield, reaching 122.04 μg/mL. This study demonstrates successful expression and secretion of human α-LA in B. subtilis and establishes a foundation for simulating breast milk for infant formulas and developing bioengineered milk.

Experimental production and efficacy testing of mono-specific antibodies against the venom of carpet viper (Echis ocellatus) from savannah Nigeria

Echis ocellatus is one of the commonest snakes responsible for envenomation in Nigeria. Antivenom is the only effective treatment, but the country suffers from a limited supply of effective antivenom. This study therefore aimed to explore the feasibility of effective, mono-specific antibodies production through immunization in rabbits using the venom of Echis ocellatus from Nigeria. The World Health Organization guide on antivenom production was employed in the immunization and the resultant antibodies were purified using protein A agarose column chromatography. Antibody titer reached a high plateau by 2-month immunization, and SDS PAGE of the sera suggests the presence of intact immunoglobulins accompanied with the heavy (50 kDa) and light (25 kDa) chains. The venom has an intravenous LD50 of 0.35 mg/kg in mice, and the venom lethality at a challenge dose of 2 LD50 was effectively neutralized by the antibodies with a potency value of 0.83 mg venom per g antibodies. The antibodies also neutralized the procoagulant activity of the venom with an effective dose (ED) of 13 ± 0.66 μl, supporting its use for hemotoxic envenomation. The study establishes the feasibility of developing effective, mono-specific antibodies against the Nigerian Carpet viper.

Disulfide bond cleavage combined with critical pH induced unfolding and assembly of soy protein and its encapsulation effect on curcumin

The limited water solubility and bioactivity of hydrophobic phytochemicals can be improved and preserved by encapsulation technologies. In this research, a low-cost and low-energy encapsulation method was explored based on the self-assembly characteristics of soy protein (SP). The disulfide bond of SP was broken by Na2S2O5, and then the pH of SP was adjusted to the critical pH 10.5 with NaOH, which was the critical point of protein unfolding. The treated protein was added to curcumin (Cur), which was encapsulated in self-assembled protein nanoparticles during the subsequent neutralization process to obtain the soy protein-curcumin complex (SP-Cur). This process was known as the disulfide bond breaking combined with critical pH-driven technology. The cleavage of SP disulfide bonds was studied by the methods of 4, 4′-dithiopyridine (DPS) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). When the SP:Cur mass ratio was 1:1, the encapsulation efficiency (EE%) of Cur in SP-Cur obtained by disulfide bond breaking combined with the critical pH-driven method increased to 85.36 ± 1.03%. It was notably greater than the EE% of Cur in SP-Cur prepared by the pH-driven method (61.82 ± 1.54%) and the EE% of Cur in SP-Cur prepared by disulfide bond breaking treatment (66.38 ± 1.2%). The encapsulation of Cur in SP particles exhibited significant resistance to light and temperature, leading to enhanced stability. Soy protein treated with disulfide bond cleavage combined with critical pH-driven method can serve as a potential functional carrier to further incorporate hydrophobic compounds into food systems.