Sodium Dodecyl Sulfate-polyacrylamide Gel Research Articles

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.

Extraction and characterization of collagen from the skin of Pterygoplichthys spp. (armored catfish) from Chiapas

Because of its biocompatibility and safety, collagen is a valuable biomaterial that is used in various industries. While traditionally sourced from terrestrial mammals, collagen extracted from fish waste is a promising alternative due to its chemical properties and low cost. In Mexico, Pterygoplichthys spp. is considered an invasive species, inedible, and of limited commercial importance due to the low muscle content and skin covered with rigid bony plates. This study aimed to extract and characterize collagen from the skin of Pterygoplichthys spp. obtained from two communities in Chiapas, Mexico. Our results show that the extracted collagen is type 1, with a yield of 43.0% (dry weight). Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed the presence of two alpha chains (α1, α2) and a beta and gamma component, consistent with type 1 collagen. Liquid chromatography coupled to mass spectrophotometry analysis identified peptide sequences homologous to those reported in other species. This study highlights the potential of Pterygoplichthys spp. skin collagen as a viable alternative to mammalian collagen. The efficiency of the extraction process and the identification of peptides resembling those of the other species underscore the feasibility of utilizing collagen from the skin of Pterygoplichthys spp. in industrial applications, offering a sustainable solution to environmental and economic challenges. Practical Application: Collagen extracted from the skin of Pterygoplichthys spp., an invasive fish species, can serve as a sustainable alternative to mammalian collagen, offering a cost-effective biomaterial with properties suitable for various industrial applications.

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.

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.

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.

Recovery of Proteases and Protease Inhibitors from Ganoderma spp. Cultivated in Amazonian Lignocellulose Wastes.

Ganoderma spp. are a great source of bioactive molecules. The production and recovery of bioactive molecules vary according to strain, growth substrate, and extraction solution. Variations in protease and their inhibitors in basidiomata from a commercial strain (G. lingzhi) and an Amazonian isolate (Ganoderma sp.) cultivated in Amazonian lignocellulosic wastes and extracted with different solutions are plausible and were investigated in our study. Basidiomata from cultivation in substrates based on açaí seed, guaruba-cedro sawdust and three lots of marupá sawdust were submitted to extraction in water, Tris-HCl, and sodium phosphate. Protein content, proteases, and protease inhibitors were estimated through different assays. The samples were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR). Tris-HCl provided higher protein extraction from Ganoderma sp. and higher caseinolytic, gelatinolytic, and fibrinolytic activity for G. lingzhi cultivated in açaí. Water extracts of Ganoderma sp., in general, exhibited higher trypsin and papain inhibitor activities compared to G. lingzhi. Extracts in Tris-HCl and sodium phosphate showed more intense protein bands in SDS-- PAGE, highlighting bands of molecular weights around 100, 50, and 30 kDa. FTIR spectra showed patterns for proteins in all extracts, with variation in transmittance according to substrate and extractor. Water extract from Amazonian Ganoderma sp. cultivated in marupá wastes are promising as a source of protease inhibitors, while the Tris-HCL extract of G. lingzhi from açaí cultivation stands out as a source of proteases with fibrinolytic, caseinolytic, and gelatinolytic activities.

Separation and purification of hyaluronic acid by [formula omitted] nano and micro particles coated with chitosan and silica

Hyaluronic acid (HA), a glycosaminoglycan, is comprised of alternating units of D-glucuronic acid and N-acetylglucosamine. This compound harbors numerous biomedical applications, including its use in pharmaceuticals, wound healing, osteoarthritis treatment, and drug delivery. Its unique composition and exceptional features, such as its high water-absorbing and retaining capacity, have also led to its use in the cosmetics industry. The employment of this biopolymer has given rise to an escalation in the request for its manufacture. The present investigation has explored the correlation between hyaluronic acid and chitosan and silica for the purpose of separation. Consequently, Iron oxide magnetic nano particles and micro particles were produced via co-precipitation method and were layered with chitosan and silica to purify the hyaluronic acid from the fermentation broth that was generated by Streptococcus Zooepidemicus. The size distribution and zeta potentials of the two kinds of particles were gauged with the aid of a dynamic laser light scattering apparatus and zeta potential meter (Malvern, Zeta master) respectively. The confirmation of the chemical structure of the Fe3O4 nanoparticles and Fe3O4 particles conjugated with chitosan and silica was accomplished through the utilization of Fourier Transform Infrared Spectroscopy (FT-IR). Protein contamination was thoroughly characterized by means of sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Nanodrop 2000/2000c spectrophotometers protein estimation method. The maximum HA adsorption capacity, under optimal pH conditions of 4, was determined to be 87 mg/g, 112 mg/g, 51 mg/g, and 44 mg/g for Fe3O4 −chitosan nanoparticle, Fe3O4 −chitosan micro particle, Fe3O4 −silica microparticle, and Fe3O4 −silica nanoparticle, respectively.

Ecotoxicological impact of heavy metals on wild mud crabs (Scylla olivacea) in Malaysia: An integrative approach of omics, molecular docking and human risk assessment

Mud crab, one of the aquatic organisms found in estuary areas, has become a significant economic source of seafood for communities due to its delectable taste. However, they face the threat of heavy metal contamination, which may adversely affect their biological traits. This study explored the comparison of the mud crabs collected from Setiu Wetland as a reference site, while Kuala Sepetang is an area that contains a higher concentration of heavy metals than Setiu Wetlands. Heavy metal levels were quantified using inductively coupled plasma mass spectrometry (ICP-MS), while proteomes were assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 1H nuclear magnetic resonance (NMR)-based metabolomics, respectively. Heavy metal contamination affects the proteome, metabolome, and putative molecular targets in mud crabs (Scylla olivacea), leading to oxidative stress. Mud crabs collected from the metal-polluted area of Kuala Sepetang in Perak had considerably elevated concentrations of nickel (Ni), copper (Cu), zinc (Zn), lead (Pb), chromium (Cr), and cadmium (Cd) in comparison to the reference site of Setiu Wetlands in Terengganu. The proteome analysis revealed an upregulation of the stress-response protein Hsp70, which triggered superoxide dismutase (SOD) and increased arginine kinase expression (5.47 fold) in the muscle tissue, results in the alteration of metabolite regulation in the mud crab from Kuala Sepetang. Additionally, in the muscle tissues of mud crabs obtained from Kuala Sepetang, uncharacterized myosin-tail 1 domain proteins and sarcoplasmic calcium-binding proteins were downregulated. The metabolomic investigation identified changes in metabolites associated with energy metabolism and osmoregulation. Exploration of docking analysis suggests potential connections between methylarsonic acid and essential proteins in mud crabs. These findings suggest that the presence of heavy metals disrupts physiological processes and highlights potential molecular targets that warrant further investigation.

Acoustofluidic separation of bacteria from platelets using tilted-angle standing surface acoustic wave

Separation of microscale objects, such as cells and bacteria, is an essential sample preparation step in various microfluidic applications in microbiology, chemistry, biology, and biomedicine. Microfluidic separation of bacteria from blood cells is highly in demand to prevent or investigate bacterial infectious diseases. Unlike red and white blood cells, platelets have a similar size distribution to that of bacteria, which makes bacteria separation from platelets technically challenging. Most previous techniques for microfluidic separation rely on size as a separation marker. These size-based separation methods are not applicable to separation of the same or similar-sized samples. Here, we propose an acoustofluidic method using tilted-angle standing surface acoustic wave (taSSAW) for label-free separation of the microscale objects of the same or similar size based on compressibility. From the experimental investigation with polymethylmethacrylate, polystyrene, and polycaprolactone microparticles of the same size, we found that less compressible objects migrated further when exposed to the taSSAW field formed within a microchannel. Based on the findings, we demonstrated that Escherichia coli (E. coli) can be separated from platelets because the taSSAW-induced acoustic radiation force acting on comparatively less compressible E. coli was greater than that applied to the platelets. Fluorescence microscopy, scanning electron microscope, hemocytometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and polymerase chain reaction were conducted to confirm the successful positive separation of bacteria from platelets with high purity of exceeding 96 % and recovery rate of exceeding 81 %.

Antibacterial Properties of Peptide and Protein Fractions from Cornu aspersum Mucus.

The discovery and investigation of new natural compounds with antimicrobial activity are new potential strategies to reduce the spread of antimicrobial resistance. The presented study reveals, for the first time, the promising antibacterial potential of two fractions from Cornu aspersum mucus with an MW < 20 kDa and an MW > 20 kDa against five bacterial pathogens-Bacillus cereus 1085, Propionibacterium acnes 1897, Salmonella enterica 8691, Enterococcus faecalis 3915, and Enterococcus faecium 8754. Using de novo sequencing, 16 novel peptides with potential antibacterial activity were identified in a fraction with an MW < 20 kDa. Some bioactive compounds in a mucus fraction with an MW > 20 kDa were determined via a proteomic analysis on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and bioinformatics. High homology with proteins and glycoproteins was found, with potential antibacterial activity in mucus proteins named aspernin, hemocyanins, H-lectins, and L-amino acid oxidase-like protein, as well as mucins (mucin-5AC, mucin-5B, mucin-2, and mucin-17). We hypothesize that the synergy between the bioactive components determined in the composition of the fraction > 20 kDa are responsible for the high antibacterial activity against the tested pathogens in concentrations between 32 and 128 µg/mL, which is comparable to vancomycin, but without cytotoxic effects on model eukaryotic cells of Saccharomyces cerevisiae. Additionally, a positive effect, by reducing the levels of intracellular oxidative damage and increasing antioxidant capacity, on S. cerevisiae cells was found for both mucus extract fractions of C. aspersum. These findings may serve as a basis for further studies to develop a new antibacterial agent preventing the development of antibiotic resistance.

Expression and basic biochemical characteristics of recombinant surfactant protein D of bottlenose dolphin (Tursiopstruncatus)

We previously identified surfactant protein D (SP-D) in the bottlenose dolphin Tursiops truncatus as a unique evolutionary factor of the cetacean pulmonary immune system. In this short report, recombinant SP-D of bottlenose dolphin (dSP-D) was synthesized in mammalian cells, and its properties were analyzed in vitro. The recombinant proteins were purified using Ni-carrier or Co-carrier. Sodium dodecyl sulfate poly-acrylamide gel electrophoresis and western blotting revealed a 50 kDa major band with minor secondary bands. Enzyme-linked immunosorbent assay-like methods revealed that recombinant dSP-D bonded to gram-positive and gram-negative bacterial walls. Our findings suggest the clinical usefulness of dSP-D for cetacean pneumonia.

Enzyme-glycosylated ovalbumin/chitosan oligosaccharide nanoparticles: Simulation of in vitro digestion for the targeted release of vitamin D3 and application for encapsulation in beverages

Vitamin D3 (VD3) was encapsulated in TGaseOVA–chitosan (COS) composite nanoparticles. Isothermal titration calorimetry was used to explain the hydrophobic interaction between TGaseOVA and VD3 at the molecular level. The 87.7 nm TGaseOVA–COS composite nanoparticles achieved an encapsulation efficiency of 96.34% and loading capacity of 4.68% for 9 μg/ml VD3. An in vitro digestion experiment revealed that TGaseOVA–COS–VD3 composite nanoparticles had good stability in simulated gastric fluid and only 32.67% of the composite nanoparticles were released in simulated intestinal fluid. Meanwhile, sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed that the composite nanoparticles had a release rate of 100% in simulated intestinal fluid. TGaseOVA–COS–VD3 composite nanoparticles had a VD3 concentration of 8.73 μg/ml after pasteurization, indicating that they provided substantial protection to VD3 during pasteurization. TGaseOVA–COS was subjected to a 1-month simulated shelf life test. High-performance liquid chromatography confirmed that it had a VD3 retention rate of 90%. The concentration-dependent toxicity of VD3 was detected by using Caco-2 cells. VD3 exhibited toxicity at the concentration of 11 μg/ml. Therefore, the embedding dose of 9 μg/ml used in this work was considered a safe dose. TGaseOVA–COS composite nanoparticles have excellent stability and good VD3 slow-release properties. They can be used in concentrated clear beverages and other food and beverage products. Therefore, they have potential for applications in the food and pharmaceutical industries.

Preparation of interleukin-1β-targeted nanobodies and their effects on apoptosis in hypoxic cardiomyocytes of mice

Objective: To prepare interleukin-1β-targeted nanoantibodies and observe their effects on apoptosis in hypoxic cardiomyocyte of mice. Methods: Using DNA recombination technology, the pET-16b and pHEN1 expression vectors were used to construct the prokaryotic expression plasmids of interleukin-1β-targeted nanobodies (pET-16b-4G6M-VHH, pET-16b-5BVP-VHH, pET-16b-5MVZ-VHH, pHEN1-4G6M-VHH, pHEN1-5BVP-VHH and pHEN1-5MVZ-VHH, where VHH is a variable domain of heavy chain antibody, 4G6M-VHH, 5BVP-VHH, 5MVZ-VHH were three interleukin-1β-targeted nanoantibodies respectively). The constructed plasmids were transferred into Escherichia coli Rosetta2 (DE3) for induction of expression and nickel column purification, respectively. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were employed to identify the expression product and purified product, and the enzyme-linked immune sorbent assay (ELISA) was performed to determine their affinity. The cardiomyocyte hypoxia model was used with the highest affinity IL-1β-targeted nanobody (pHEN1-5MVZ-VHH), and cell survival and apoptosis rates were detected (the experiment was divided into normal control group, hypoxia model group, blank plasmid group and 12.5, 25.0, 50.0 μg/ml pHEN1-5MVZ-VHH treatment groups). Results: SDS-PAGE and Western blotting results showed that the anti-interleukin-1β (IL-1β) nanobodies with a relative molecular mass of about 15 000 were successfully obtained. Likewise, ELISA results found that the nanobodies expressed in pHEN1 vector group had higher affinity for IL-1β antigen compared with pET-16b vector group (4G6M-VHH group: 3.20±0.03 vs 1.20±0.03, P<0.001; 5BVP-VHH group: 3.18±0.06 vs 1.21±0.02, P<0.001; 5MVZ-VHH group: 3.38±0.05 vs 1.62±0.04, P<0.001). Additionally, the results of cell survival assay and apoptosis assay detected that compared with the hypoxia model group, HL-1 cell activity was significantly increased in the 25.0 μg/ml and 50.0 μg/ml pHEN1-5MVZ-VHH treatment groups [(75.55±2.23)% vs (46.90±2.51)%, P<0.001; (74.36±1.96)% vs (46.90±2.51)%, P<0.001], and apoptosis rate was significantly reduced [(6.83±0.27)% vs (10.24±0.76)%, P<0.001; (6.68±0.38)% vs (10.24±0.76)%, P<0.001]. Conclusions: 4G6M-VHH, 5BVP-VHH, and 5MVZ-VHH are expressed by both pET-16b and pHEN1 expression vectors and the nanobodies produced by the pHEN1 vector display enhanced antigen affinity. Furthermore, in hypoxic cardiomyocytes, pHEN1-5MVZ-VHH treatment reduces cell apoptosis.

Cubosomes functionalized with antibodies as a potential strategy for the treatment of HER2-positive breast cancer

Breast cancers that overexpress human epidermal growth factor receptor 2 (HER2) have poor prognosis. Moreover, available chemotherapies cause numerous side effects due to poor selectivity. To advance more effective and safer therapies for HER2-positive breast cancer, we explored the fusion of drug delivery technology and immunotherapy. Our research led to the design of immunocubosomes loaded with panobinostat and functionalized with trastuzumab antibodies, enabling precise targeting of breast cancer cells that overexpress HER2. We characterised the nanostructure of cubosomes using small-angle X-ray scattering (SAXS), cryo-transmission electron microscopy (cryo-TEM), and dynamic light scattering (DLS). Moreover, we confirmed the integrity of the trastuzumab antibodies on the immunocubosomes by Fourier-transform infrared spectroscopy (FTIR) and sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, we found that panobinostat-loaded immunocubosomes were more cytotoxic, and in an uptake-dependant manner, towards a HER2-positive breast cancer cell line (SKBR3) compared to a cell line representing healthy cells (L929). These results support that the functionalization of cubosomes with antibodies enhances both the effectiveness of the loaded drug and its selectivity for targeting HER2-positive breast cancer cells.