SDS-PAGE Research Articles

Bacterial ubiquitin ligases hijack the host deubiquitinase OTUB1 to inhibit MTORC1 signaling and promote autophagy

ABSTRACT Many bacterial pathogens have evolved effective strategies to interfere with the ubiquitination network to evade clearance by the innate immune system. Here, we report that OTUB1, one of the most abundant deubiquitinases (DUBs) in mammalian cells, is subjected to both canonical and noncanonical ubiquitination during Legionella pneumophila infection. The effectors SidC and SdcA catalyze OTUB1 ubiquitination at multiple lysine residues, resulting in its association with a Legionella-containing vacuole. Lysine ubiquitination by SidC and SdcA promotes interactions between OTUB1 and DEPTOR, an inhibitor of the MTORC1 pathway, thus suppressing MTORC1 signaling. The inhibition of MTORC1 leads to suppression of host protein synthesis and promotion of host macroautophagy/autophagy during L. pneumophila infection. In addition, members of the SidE family effectors (SidEs) induce phosphoribosyl (PR)-linked ubiquitination of OTUB1 at Ser16 and Ser18 and block its DUB activity. The levels of the lysine and serine ubiquitination of OTUB1 are further regulated by effectors that function to antagonize the activities of SidC, SdcA and SidEs, including Lem27, DupA, DupB, SidJ and SdjA. Our study reveals an effectors-mediated complicated mechanism in regulating the activity of a host DUB. Abbreviations: BafA1: bafilomycin A1; BMDMs: bone marrow-derived macrophages; DUB: deubiquitinase; Dot/Icm: defective for organelle trafficking/intracellular multiplication; DEPTOR: DEP domain containing MTOR interacting protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; L. pneumophila: Legionella pneumophila; LCV: L egionella-containing vacuole; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MTORC1: mechanistic target of rapamycin kinase complex 1; OTUB1: OTU deubiquitinase, ubiquitin aldehyde binding 1; PR-Ub: phosphoribosyl (PR)-linked ubiquitin; PTM: posttranslational modification; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SidEs: SidE family effectors; Ub: ubiquitin.

Gelatin and Collagen from Sheepskin.

Abattoirs dispose of sheepskins as solid waste due to low price and poor demand for sheepskin leather. In principle, as an alternative to being disposed of in landfill, sheepskins can serve as a source of the protein collagen or the hydrolysis product, gelatin. In this research, sheepskins collected from abattoirs were used as a source of collagen. Three extraction methods were compared: acid extraction, acid with enzymes, and alkali extraction. The extracted material was characterized using scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR), small angle X-ray scattering (SAXS), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The collagen and gelatin extraction yield ranged from 3.1% to 4.8% with the product purity determined by hydroxyproline, ranging from 7.8% for the alkali process to 59% and 68% for the acid and acid-enzyme processes. SDS PAGE showed that the acid process produced fragments with molecular weights in the range 100 to >250 kDa, while acid-enzyme resulted in smaller fragments, below 30 kDa. The FTIR region of the amide I band at 1800-1550 cm-1, which was used as an indicator of the collagen and gelatin content, showed that the gelatin dominated in the acid extracts, and the alkaline extract contained a large portion of keratin. SAXS was found to be a sensitive method for showing the presence of intact collagen fibrils in materials from all of the extraction methods, albeit at low concentrations. Herein, sheepskin is shown to be a useful source for collagen-gelatin material of varying molecular weights.

Thermo-Alkali-Tolerant Recombinant Laccase from Bacillus swezeyi and Its Degradation Potential against Zearalenone and Aflatoxin B1.

The enzymatic biodegradation of mycotoxins in food and feed has attracted the most interest in recent years. In this paper, the laccase gene from Bacillus swezeyi was cloned and expressed in Escherichia coli BL 21(D3). The sequence analysis indicated that the gene consisted of 1533 bp. The purified B. swezeyi laccase was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis -12% with an estimated molecular weight of 56.7 kDa. The enzyme is thermo-alkali-tolerant, displaying the optimal degradation of zearalenone (ZEN) and aflatoxin B1 (AFB1) at pH 8 and 9, with incubation temperatures of 55 and 50 °C, respectively, within 24 h. The degradation potentials of the 50 μg of the enzyme against ZEN (5.0 μg/mL) and AFB1 (2.5 μg/mL) were 99.60 and 96.73%, respectively, within 24 h. To the best of our knowledge, this is the first study revealing the recombinant production of laccase from B. swezeyi, its biochemical properties, and potential use in ZEN and AFB1 degradation in vitro and in vivo.

Synthesis and Characterization of Boronate Affinity Three-Dimensionally Ordered Macroporous Materials.

Sample pretreatment is a key step for qualitative and quantitative analysis of trace substances in complex samples. Cis-dihydroxyl (cis-diol) group-containing substances exist widely in biological samples and can be selectively bound by boronate affinity adsorbents. Based on this, in this article, we proposed a simple method for the preparation of novel spherical three-dimensionally ordered macropore (3DOM) materials based on a combination of the boronate affinity technique and colloidal crystal template method. The prepared 3DOM materials were characterized using Fourier transform-infrared spectroscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, and thermo-gravimetric analysis, and results showed that they possessed the characteristics of a high specific surface area, high porosity, and more boronic acid recognition sites. The adsorption performance evaluation results showed that the maximum adsorption capacity of the boron affinity 3DOMs on ovalbumin (OVA) could reach to 438.79 mg/g. Kinetic and isothermal adsorption experiments indicated that the boronate affinity 3DOM material exhibited a high affinity and selectivity towards OVA and adenosine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the proteins in egg whites was conducted and proved that the glycoprotein in the egg whites could be separated and enriched with a good performance. Therefore, a novel boronate affinity 3DOM material a with highly ordered and interconnected pore structure was prepared and could be applied in the separation and enrichment of molecules with cis-diol groups from complex samples with a good selectivity, efficiency, and high throughput.

Cysteine, sodium metabisulfite, and glutathione enhance crosslinking between proteins during high moisture meat analog extrusion processing and may improve the fibrousness of the products.

High moisture meat analog (HMMA) products processed using extrusion have become increasingly popular in the last few years. Because the formation of disulfide bonds is believed to play a critical role in the texturization mechanism, this study aimed to understand how chemical compounds capable of reducing disulfide bonds, specifically cysteine, sodium metabisulfite, and glutathione, affect the texture and the chemical interactions between the proteins. Wheat protein blended with cysteine, sodium metabisulfite, or glutathione at levels of 0, 0.5, 1.0, 2.5, 5.0, and 7.5 g kg-1 was extruded at three different temperatures (115, 140, and 165 °C) using a co-rotating twin-screw extruder. The feed rate (85 g min-1), the moisture content (600 g kg-1), and the screw speed (300 rpm) were kept constant. Unextruded and extruded material was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, polymeric protein fractionation, and sulfhydryl group/disulfide bond analysis. Extruded samples were further analyzed for their hardness and their anisotropic index. The inclusion of reductants significantly affected the structure of the obtained extrudates. Although reducing agents had a relatively small impact on the total amount of disulfide bonds, their action significantly enhanced crosslinking between the proteins. At select conditions, samples with high fibrousness were specifically obtained when cysteine or sodium metabisulfite was included at levels of 5.0 g kg-1. In the presence of reducing agents, it is believed that disulfide bonds are split earlier during the process without binding to them, giving the protein strands more time to unravel and align, leading to a better flow behavior and more fibrous products. © 2024 Society of Chemical Industry.

Gelatinization and fermentation synergy: investigating the protein digestibility, mineral bioaccessibility and microstructural transformations of black mash beans through Saccharomyces cerevisiae and Lactobacillus spp

ABSTRACT This study explored the nutritional qualities of black mash bean flour impacted by fermentation, gelatinization and their combination. Protease activity, in vitro protein digestibility (IVPD), and protein solubility were evaluated for both fermented and unfermented black mash bean flour samples. Interestingly, when compared to fermented samples and gelatinized mash bean flour (GMBF), raw mash bean flour (RMBF) showed the highest protein solubility. Due to enzymatic activity and protein hydrolysis, fermentation with Lactobacillus E14 and Saccharomyces cerevisiae MK-157 enhanced protein solubility. Prolonged fermentation timeframes were positively correlated with protease activity and IVPD, suggesting the degradation of complex proteins. The highest levels of IVPD (90.12%) and protease activity (5546.78 U/g) were detected in GMBF fermented by S. cerevisiae MK-157, indicating the impact of fermentation on protein breakdown. The Lactobacillus E14-fermented RMBF exhibited 5521.45 U/g of protease activity and 87.54% IVPD. The mineral content of the fermented samples was substantially greater than that of the unfermented GMBF. The increased calcium, magnesium, potassium, sodium, iron, zinc, and copper levels for the GMBF fermented by S. cerevisiae MK-157 indicated that fermentation facilitated the release of minerals from chelated compounds. Microscopic examination revealed higher protein quantities in the fermented flour as well as hydrolyzed and broken starch granules embedded in the protein matrix. Large mash bean proteins were shown to be proteolyzed during fermentation into smaller peptides by SDS PAGE. Storage proteins were extensively broken down during fermentation with S. cerevisiae MK-157, whereas Lactobacillus E14 displayed a more focused pattern of hydrolysis. The nutritional profile of black mash bean flour was improved by fermentation with Lactobacillus E14 and S. cerevisiae MK-157.

Methanol gel electrophoresis: Separation of human fast and slow myosin light chain 1 and other myofibrillar protein isoforms on a single gel format.

This report describes a novel sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) resolving gel format that consistently yields the electrophoretic separation of the fast and slow isoforms of human sarcomeric myosin light chain 1 (MLC1). The inclusion of methanol as a constituent of the resolving gel impacted the electrophoretic mobility of proteins across a broad range of molecular masses. There was greater separation of the fast and slow isoforms of human MLC1, as well as separation and high resolution of fast and slow isoforms of the three myosin heavy chain isoforms that are expressed in human skeletal muscle on the same gel format. Furthermore, the same resolving gel format substantially altered the electrophoretic mobility of at least one isoform of tropomyosin in human striated muscle. It is possible that the inclusion of methanol in SDS-PAGE resolving gels could improve the separation of other proteins that are expressed in muscle and in other tissues and cell types.

Characterization of tryptanthrin as an antibacterial reagent inhibiting Vibrio splendidus

Isolates of Vibrio splendidus are ubiquitously presented in various marine environments, and they can infect diverse marine culture animals, leading to high mortality and economic loss. Therefore, a control strategy of the infection caused by V. splendidus is urgently recommended. Tryptanthrin is a naturally extracted bioactive chemical with antimicrobial activity to other bacteria. In this study, the effects of tryptanthrin on the bacterial growth and virulence-related factors of one pathogenic strain V. splendidus AJ01 were determined. Tryptanthrin (10 μg/mL) could completely inhibit the growth of V. splendidus AJ01. The virulence-related factors of V. splendidus AJ01 were affected in the presence of tryptanthrin. Tryptanthrin resulted an increase in biofilm formation, but lead to reduction in the motility and hemolytic activity of V. splendidus cells. In the cells treated with tryptanthrin, two distinctly differentially expressed extracellular proteins, proteases and flagellum, were identified using SDS–PAGE combined with LC–MS. Real-time reverse transcriptase PCR confirmed that the genes involved in the flagellar formation and hemolysin decreased, whereas specific extracellular proteases and the genes involved in the biofilm formation were upregulated. Two previously annotated luxOVs genes were cloned, and their expression levels were analyzed at different cell densities. Molecular docking was performed to predict the interaction between LuxOVs and ATP/tryptanthrin. The two sigma-54-dependent transcriptional regulators showed similar ATP or tryptanthrin binding capacity but with different sites, and the direct competitive binding between ATP and tryptanthrin was present only in their binding to LuxO1. These results indicated that tryptanthrin can be used as a bactericide of V. splendidus by inhibiting the growth, bacterial flagella, and extracellular proteases, but increasing the biofilm. Sigma-54-dependent transcriptional regulator, especially the quorum sensing regulatory protein LuxO1, was determined to be the potential target of tryptanthrin.Key points• Tryptanthrin inhibited the growth of V. splendidus in a dose-dependent manner.• The effect of tryptanthrin on the virulence factors of V. splendidus was characterized.• LuxO was the potential target for tryptanthrin based on molecular docking.

Molecular Characterization of Methicillin-resistant Staphylococcus aureus (MRSA) Isolates from Ruminants

Background: Methicillin-resistant Staphylococcus aureus (MRSA) infection is one of the leading causes of infections in animals as well as human beings and is associated with significant morbidity, mortality, length of stay and cost burden. Methods: In this study, 725 samples of nasal swabs and milk were collected randomly from cattle, buffalo, sheep and goats and these samples were inoculated on Mannitol salt agar mixed with Oxacillin Resistance Selective Supplement for the molecular characterization of MRSA isolates through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and polymerase chain reaction (PCR) test. Result: Out of 725 samples, 171 were found positive for Oxacillin resistant S. aureus. The SDS-PAGE showed different bands of molecular weight 13, 17, 20, 24, 26, 28, 33, 36, 39, 43, 47, 59, 64, 72, 86, 97 and 121 kDa. An amplified mec A DNA fragment (137) of 533 base pairs (bp) and PCR product of fem A (133 isolates) of 510 bp were detected in isolates.

Cold plasma for enhancing covalent conjugation of ovalbumin-gallic acid and its functional properties

The utilization of cold plasma (CP) treatment to promote covalent conjugation of ovalbumin (OVA) and gallic acid (GA), as well as its functionality, were investigated. Results demonstrated that CP significantly enhanced the covalent grafting of OVA and GA. The maximum conjugation of GA, 24.33 ± 2.24 mg/g, was achieved following 45 s of CP treatment. Covalent conjugation between GA and OVA were confirmed through analyses of total sulfhydryl (-SH) group, Fourier transform infrared (FTIR) spectroscopy, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Unfolding of the OVA molecule occurred upon conjugation with GA, as evidenced by multiple spectroscopy analyses. Additionally, conjugation with GA resulted in significant improvements in the antioxidant activity and emulsifying properties of OVA. This study demonstrated that CP is a robust and sustainable technique for promoting the covalent conjugate of polyphenols and proteins, offering a novel approach to enhance the functional properties of proteins.

Investigation of cytogenetic, electrophoretic, histopathological and biochemical effects of walnut (juglans regia l.) Leaf extract in rats experimentally induced diabetes by streptozotocin

In this study, the effects of Juglans regia L. (JR) leaf extract on histological damage and cytogenetic, electrophoretic, and biochemical parameters in the liver and kidney tissues of diabetic rats were investigated. In the study, 60 male rats (Sprague-Dawley) were separated into six groups. According to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) data, it was determined that there were increases and decreases in different serum protein expressions because of the treatment of JR leaf extract in the diabetes group. In the kidney tissue of the diabetes group, an increase in volume in the glomeruli and narrowing of the Bowman's space as well as thickening of the walls of tubules were detected. Vacuolization and shedding were observed in the epithelial cells of tubules in the cortical regions in kidney. In diabetic + JR extract groups administered JR leaf extract at doses of 250 mg/kg and 500 mg/kg, serum AST and ALT levels were reduced compared to the diabetic group. Diabetic rats' livers showed spotted necrosis and fibrosis in the portal area, biliary tract proliferation, mild inflammation, increased vascularization, unicellular necrosis in hepatocytes, and sinusoidal dilatation. The JR leaf extract group did not exhibit these problems. JR leaf extract influences reducing hepatotoxic and oxidative damage due to diabetes and increasing the level of antioxidant enzymes. As a result, it was concluded that the application of JR leaf extract may have a protective effect against damage caused by diabetes.

High internal phase emulsions stabilized by physically modified mung bean protein isolates under different pHs

This research explored the impact of modified mung bean protein isolates (MBPIs) on the physicochemical, rheological, and structural properties of 70% oil-containing high internal phase emulsions (HIPEs). MBPIs (4% protein concentration) were subjected to heating (90 °C for 2 h) or sequential heating/ultrasonication (20 kHz, 120 W for 10 min) at different pHs (2, 7, and 10). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of modified MBPIs confirmed heat treatment at pH 2 hydrolyzed MBPI, unlike MBPIs heated at pH 7 and 10. Sequential heating/ultrasonication significantly reduced particle size, improved solubility and surface hydrophobicity of MBPIs, resulting in smaller droplet size and enhanced gel-like properties of HIPEs. Transmission electron microscopy revealed that sequentially heated/ultrasonicated MBPIs in the oil–water interface existed in a fibrillar or randomly aggregated structure at pH 2, a randomly aggregated structure at pH 7, and a particulate aggregated structure at pH 10. Overall, these structural modifications at varied pH conditions influenced MBPI adsorption at the HIPE droplet interface, impacting rheological features and promoting the potential use of protein-stabilized HIPEs across diverse applications.

Development of Drying Methodology for Intact Whole Buffalo Liver, Its Characterization, Shelf Life, and Evaluation of Palatability as Pet Treat

Globally, large quantity of animal byproducts is generated from the slaughter of food animals, but there is lack of research articles related to drying of these byproducts and its use as pet food. Therefore, this study was conducted with the aim of utilization of intact whole buffalo liver by drying for pet treat, evaluating its shelf life and palatability. The intact liver surface was superficially sliced, and the surface was pierced. Thereafter, the livers were pretreated in 3% sugar and 4% salt solution (1 : 3 w/v) for 3 h followed by microwaving for 4 min and hot air drying at 60°C for 40 h (designated as T2L). The livers which were dried the same as T2L except surface piercing were referred as T1L, whereas the livers dried only using hot air oven were referred as control (CL). The moisture and protein contents of the dried CL and T2L were found to be 28.46% and 14.29% and 43.85% and 52.76%, respectively. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) image of T2L revealed the presence of few low as well as high molecular weight protein bands which were absent in CL and T1L indicating a comparatively lower level of protein degradation in T2L. The shelf life of T1L and T2L samples based on microbiological and lipid oxidation analyses was found to be more than 60 days at 25 and 4°C. Palatability studies using dogs showed that all dried samples were highly palatable. Thus, it could be concluded that intact buffalo liver could be dried using surface slicing, with/without piercing followed by salt and sugar pretreatment, 4 min microwaving, and hot air drying at 60°C for 40 h. Future study should focus on the sensory properties such as aroma, texture, and flavor and sensory analysis of the dried liver by human.

Development of PCR-based markers for identification of wheat HMW glutenin Glu-1Bx and Glu-1By alleles

BackgroundIn common wheat (Triticum aestivum L.), allelic variations in the high-molecular-weight glutenin subunits Glu-B1 locus have important effects on grain end-use quality. The Glu-B1 locus consists of two tightly linked genes encoding x- and y-type subunits that exhibit highly variable frequencies. However, studies on the discriminating markers of the alleles that have been reported are limited. Here, we developed 11 agarose gel-based PCR markers for detecting Glu-1Bx and Glu-1By alleles.ResultsBy integrating the newly developed markers with previously published PCR markers, nine Glu-1Bx locus alleles (Glu-1Bx6, Glu-1Bx7, Glu-1Bx7*, Glu-1Bx7OE, Glu-1Bx13, Glu-1Bx14(−), Glu-1Bx14(+)/Bx20, and Glu-1Bx17) and seven Glu-1By locus alleles (Glu-1By8, Glu-1By8*, Glu-1By9, Glu-1By15/By20, Glu-1By16, and Glu-1By18) were distinguished in 25 wheat cultivars. Glu-1Bx6, Glu-1Bx13, Glu-1Bx14(+)/Bx20, Glu-1By16, and Glu-1By18 were distinguished using the newly developed PCR markers. Additionally, the Glu-1Bx13 and Glu-1Bx14(+)/Bx20 were distinguished by insertions and deletions in their promoter regions. The Glu-1Bx6, Glu-1Bx7, Glu-1By9, Glu-1Bx14(−), and Glu-1By15/By20 alleles were distinguished by using insertions and deletions in the gene-coding region. Glu-1By13, Glu-1By16, and Glu-1By18 were dominantly identified in the gene-coding region. We also developed a marker to distinguish between the two Glu-1Bx14 alleles. However, the Glu-1Bx14(+) + Glu-1By15 and Glu-1Bx20 + Glu-1By20 allele combinations could not be distinguished using PCR markers. The high-molecular-weight glutenin subunits of wheat varieties were analyzed by ultra-performance liquid chromatography and sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the findings were compared with the results of PCR analysis.ConclusionsSeven Glu-1Bx and four Glu-1By allele detection markers were developed to detect nine Glu-1Bx and seven Glu-1By locus alleles, respectively. Integrating previously reported markers and 11 newly developed PCR markers improves allelic identification of the Glu-B1 locus and facilitates more effective analysis of Glu-B1 alleles molecular variations, which may improve the end-use quality of wheat.

Effect of limited proteolysis and CaCl2 on the rheology, microstructure and in vitro digestibility of pea protein-carboxymethyl cellulose mixed gel

Limited proteolysis, CaCl2 and carboxymethyl cellulose (CMC) have individually demonstrated ability to increase the gel strength of laboratory-extracted plant proteins. However, the syneresis effects of their combination on the gelling capacity of commercial plant protein remains unclear. This was investigated by measuring the rheological property, microstructure and protein–protein interactions of gels formed from Alcalase hydrolyzed or intact pea proteins in the presence of 0.1 % CMC and 0–25 mM CaCl2. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed the molecular weight of pea protein in the mixture were < 15 kDa after hydrolysis. The hydrolysates showed higher intrinsic fluorescence intensity and lower surface hydrophobicity than the intact proteins. Rheology showed that the storage modulus (G') of hydrolyzed pea protein (PPH)-based gels sightly decreased compared to those of native proteins. 5–15 mM CaCl2 increased the G’ for both PP and PPH-based gels and decreased the strain in the creep-recovery test. Scanning electron microscopy (SEM) showed the presence of smaller protein aggregates in the PPH-based gels compared to PP gels and the gel network became denser, and more compact and heterogenous in the presence of 15 and 25 mM CaCl2. The gel dissociation assay revealed that hydrophobic interactions and hydrogen bonds were the dominant forces to maintain the gel structure. In vitro digestion showed that the soluble protein content in PPH-based gels was 10 ∼ 30 % higher compared to those of the PP counterpart. CaCl2 addition reduced protein digestibility with a concentration dependent behavior. The results obtained show contrasting effects of limited proteolysis and CaCl2 on the gelling capacity and digestibility of commercial pea proteins. These findings offer practical guidelines for developing pea protein-based food products with a balanced texture and protein nutrition through formulation and enzymatic pre-treatment.

Heat Treatment and Homogenization Influence the Gastric Digestion of Bovine Milk Protein in Growing Pigs as an Adult Human Model

BackgroundBovine milk processing influences the structure of the curd formed during gastric digestion, which may alter gastric protein hydrolysis and impact amino acid (AA) release into the small intestine. ObjectivesThis study aimed to determine the influence of heat treatment and homogenization on the gastric protein digestion and AA emptying of bovine milk. MethodsNine-wk-old pigs (n = 144) consumed either raw, pasteurized nonhomogenized (PNH), pasteurized homogenized (PH), or ultra-high-temperature homogenized (UHT) bovine milk for 10 d. On day 11, fasted pigs received the milk treatment (500 mL) before gastric contents were collected at 0, 20, 60, 120, 180, and 300 min postprandially. The apparent degree of gastric protein hydrolysis (based on the release of free amino groups), apparent gastric disappearance of individual proteins [based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel band intensity], and the gastric emptying of digested protein and AA were determined. ResultsDuring the first 60 min, the rate of apparent gastric protein hydrolysis was fastest in pigs fed UHT milk (0.29%/min compared with on average 0.07%/min in pigs fed raw, PNH, and PH milk). Differences in the apparent degree of gastric protein hydrolysis and emptying were reflected in the rate of digested protein entering the small intestine. The AA gastric emptying half-time was generally shorter in pigs fed PH and UHT milk than in pigs fed raw and PNH milk. For example, the gastric release of total essential AA was >2-fold faster (P < 0.01) in pigs fed PH or UHT milk than that in pigs fed raw or PNH milk (i.e., homogenized compared with nonhomogenized milk). ConclusionsHeat treatment and homogenization increased the apparent gastric degree of protein hydrolysis and the release of digested protein into the small intestine. However, the rate of AA entering the small intestine was mainly increased by homogenization.

Comparative study of a liposome and emulsion system with cinnamon essential oil on the quality and proteolysis of refrigerated minced pork.

Essential oils have been recognized for their strong antibacterial property, making them an innovative approach for preserving meat. However, their chemical instability and direct impact on meat proteins limit their application. To overcome these limitations, various loading systems have been explored. This study aimed to compare the effect of cinnamon essential oil (CEO) loaded in a liposome and emulsion system on the proteolysis of minced pork and to evaluate the advantages of each delivery system in preventing microorganism-induced quality deterioration of meat. Minced pork treated with CEO-liposomes exhibited lower pH, total volatile basic nitrogen (TVB-N), and total viable count (TVC) values than CEO-emulsions and provided better protection against microorganisms. SDS-polyacrylamide gel electrophoresis (PAGE) analysis confirmed that CEO-liposome was more effective in protecting proteins from degradation. Moreover, CEO-liposome produced lower amount of bitter amino acids and harmful biogenic amines. Antibacterial mechanisms indicated that CEO-liposome exhibited a stronger inhibitory effect against major spoilage bacteria in meat products by increasing cell membrane permeability. The membrane damage was further supported by an increase in conductivity and the leakage of nucleic acids. Compared to the CEO-emulsion system, CEO-liposome emerged as an effective preservative for minced pork. These results provided important theoretical support for using a bioactive compound delivery system to prevent microorganism-induced quality deterioration in meat.

Loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing K1-ST23 hypervirulent Klebsiella pneumoniae

ABSTRACT Objectives This study aimed at revealing the underlying mechanisms of the loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing hypervirulent Klebsiella pneumoniae (hvKp). Methods Here we longitudinally recovered 3 non-carbapenemase-producing K1-ST23 hvKp strains at a one-month interval (KP29105, KP29499 and KP30086) from an elderly male. Antimicrobial susceptibility testing, whole genome sequencing, transcriptomic sequencing, gene cloning, plasmid conjugation, quantitative real-time PCR (qRT-PCR), and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were conducted. Results Among the 3 hvKp strains, KP29105 was resistant to the third- and fourth-generation cephalosporins, KP29499 acquired resistance to both ceftazidime-avibactam and carbapenems, while KP30086 restored its susceptibility to ceftazidime-avibactam, imipenem and meropenem but retained low-level resistance to ertapenem. KP29105 and KP29499 carried plasmid-encoded genes bla CTX-M-15 and bla CTX-M-71, respectively, but KP30086 lost both. Cloning of gene bla CTX-M-71 and conjugation experiment of bla CTX-M-71-carrying plasmid showed that the transformant and transconjugant were susceptible to ceftazidime-avibactam but had a more than 8-fold increase in MICs. Supplementation with an outer membrane permeabilizer could reduce the MIC of ceftazidime-avibactam by 32 folds, indicating that porins play a key role in ceftazidime-avibactam resistance. The OmpK35 of the 3 isolates was not expressed, and the OmpK36 of KP29499 and KP30086 had a novel amino acid substitution (L359R). SDS-PAGE and qRT-PCR showed that the expression of porin OmpK36 of KP29499 and KP30086 was significantly down-regulated compared with KP29105. Conclusions In summary, we reported the rare ceftazidime-avibactam resistance in a non-carbapenemase-producing hvKp strain. Resistance plasmid carrying bla CTX-M-71 and mutated OmpK36 had a synergetic effect on the resistance.

Effect of Keratin Waste on Poly(ε-Caprolactone) Films: Structural Characterization, Thermal Properties, and Keratinocytes Viability and Proliferation Studies

Keratin extracted (KE) from chicken feathers was used for the production of composite films comprising poly(ε-caprolactone) (PCL) and keratin (PCL/KE films). The process involved the extraction of keratin from chicken feathers using a 0.1 M NaOH solution, followed by characterization via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The PCL was synthesized through the ring-opening polymerization (ROP) of ε-caprolactone (ԑ-CL) with Sn(Oct)2 as a catalyst. Films were prepared via solvent casting, including pure PCL films and those enriched with different weight percentages of KE (10%, 15%, 25%, and 30%). The films were characterized by differential scanning calorimetry (DSC), thermogravimetric analysis (TG), and scanning electron microscopy (SEM). SEM analysis revealed a more uniform incorporation of KE within the PCL matrix in the case of the 15% keratin-enriched film (PCL/KE15) as compared to other keratin percentages. The thermal analysis showed a positive influence of keratin on the thermal stability of the films. Keratinocytes viability and proliferation tests on the PCL/KE15 film demonstrated compatibility with cells. Collectively, these results hold relevance for potential biomedical applications of PCL/KE films.

Small ubiquitin-like modifier-tag and modified protein purification significantly increase the quality and quantity of recombinant African swine fever virus p30 protein.

African swine fever (ASF) is a highly virulent and contagious viral disease caused by the ASF virus (ASFV). It has a significant impact on swine production throughout the world, while existing vaccines and specific treatments remain ineffective. ASFV p30 is a potent antigenic protein that induces protective antibodies immediately after infection; however, most recombinant p30 is insoluble. This study aimed to improve the solubility, yield, and purity of recombinant p30 by tagging it with a small ubiquitin-like modifier (SUMO) and modifying the protein purification process. SUMO fused with ASFV p30 (SUMO-p30) and p30 alone were cloned and expressed in Escherichia coli. SUMO-p30 and p30 solubility and expression levels were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein purification was modified by combining ammonium sulfate precipitation method with affinity chromatography. In addition, large-scale production of all versions of p30 were compared using SDS-PAGE and western blotting, and the purified p30 was used to develop the indirect enzyme-linked immunosorbent assay (ELISA). The solubility and expression levels of SUMO-p30 were dramatically enhanced compared with that of p30. Modification of the purification process significantly increased purified and soluble SUMO-p30 and p30 yields by 6.59 and 1.02 μg/mL, respectively. Large-scale production confirmed that this procedure increased the quantity of recombinant p30 while maintaining protein purity and immunogenicity. The p30-based indirect ELISA was able to discriminate between positive and negative serum samples with statistically significant differences in mean optical density 450 values (p < 0.001). This study demonstrates the enhancement of solubility, purity, and yield of ASFV p30 expressed in E.coli by SUMO fusion tagging and combining ammonium sulfate precipitation with affinity chromatography for protein purification. These positive effects were sustained in large-scale production. Cleavage and removal of hexahistidine-SUMO tag from the fusion protein by protease may not be suitable when handling a large amount of the protein. However, the SUMO-fused p30 retained strong immunoreactivity to convalescent swine serum, indicating its application in immunization and diagnostic purposes. The expression and purification procedures in this study could be applied to increase solubility, quality, and quantity of other recombinant proteins as well.