Search Results (205)

Fennel (Foeniculum vulgare), a member of the Apiaceae family, is known for its culinary and medicinal uses, as well as its potential to cause allergic reactions. Thermal and nonthermal technologies are commonly applied during the production of fruit and vegetable-based products, which may contain allergenic proteins. Consequently, understanding how these processing treatments affect allergenicity is crucial for managing allergenic risks during manufacturing and for identifying technologies that can reduce the allergenic potential of the final products. Currently, there is limited information available on how thermal and ultrasonic processing methods affect the allergenic properties of fennel. The aim of this study was to investigate the effects of ultrasound and heat treatment on the in vitro immunoreactivity of protein extracts from fennel. After sonication and heat treatment, the protein extracts were analyzed by denaturing polyacrylamide gel electrophoresis, Western blot and enzyme-linked immunosorbent assay. All treatments altered the protein patterns of fennel and partially degraded proteins in the range of 15–80 kDa. In addition, all treatments reduced IgE binding, indicating lower allergenicity. Western blotting with IgE from fennel-allergic patients confirmed these effects. The ultrasound probe had the strongest effect, almost eliminating IgE reactivity for several allergens. Heat treatment reduced allergenicity by about 30%, while sonication showed a reduction of about 15% and lower. A larger sample size is needed to better understand the effects of these treatments and the differences in individual allergic responses. Full article

The immunofluorescence assay is widely used for cellular biology and diagnosis applications. Such an antigen–antibody detection system enables the assessment and visualization of the expression and localization of target proteins. In the classical indirect immunofluorescence assay, secondary antibodies are conjugated to fluorophores. However, conventional secondary antibodies have limited applications due to their large size (150 kDa). Moreover, as animal-derived products, secondary antibodies are associated with ethical concerns and batch-to-batch variability. In this study, we developed fluorescence-labeled recombinant nanobodies as secondary antibodies by utilizing previously established anti–mouse and anti–rabbit IgG secondary nanobodies in combination with the self-labeling SNAP-tag. Nanobodies, which are significantly smaller (15 kDa), are capable to detect primary antibodies produced in mice and rabbits. The SNAP-tag (20 kDa) enables site-specific binding of various O⁶-benzylguanine (BG)-modified fluorophores to the recombinant nanobodies. These recombinant nanobodies were produced using mammalian cell expression system, and their specific binding to mouse or rabbit antibodies was validated using flow cytometry and multi-color fluorescence microscopy. The low cost, easy of expression, purification and site-specific conjugation procedures for these anti–mouse and anti–rabbit IgG secondary nanobodies make them an attractive alternative to traditional secondary antibodies for indirect immunofluorescence assays. Full article

The objective of this study was to investigate the impact of thermal and homogenization pre-treatments on ultrafiltration (UF) efficiency and component retention in whole milk (WM). Four milk treatments were examined using a benchtop Optisep filtration system: skim milk (SM) processed in UF at 15 °C, homogenized WM processed in UF at 15 °C (T1), non-homogenized WM processed in UF at 15 °C (T2), and non-homogenized WM processed at 43 °C (T3). UF was performed using 10 kDa membranes to achieve 3× concentration, and the retention and flux rates were compared across treatments. Compositional changes were analyzed at each stage: initial, retentate (2X, 3X, Final), and permeate. The permeate flux varied across treatments; SM showed the highest initial permeate flux and achieved the target concentration in a shorter time. T1 maintained a steady permeate flux over time. T2 exhibited a steep decline in flux, reaching only a 2.5× concentration. In contrast, T3 initially displayed a higher permeate flux due to heat treatment and reduced viscosity. There were significant differences in compositional parameters. T1 retentate had significantly (p < 0.05) higher crude protein (CP), Ca, Mg, and Zn retention. Higher total solids (TS), non-protein nitrogen (NPN), and non-casein nitrogen (NCN), K, and P content were found in T3 permeate. The results from this study demonstrate that homogenization and heat treatment significantly impact the UF performance of WM, offering valuable approaches for achieving dairy product composition. Full article

Flax (Linum usitatissimum L.) seed is rich in α-linolenic acid, lignans, and fiber, which have potential health benefits. However, the potential toxicity of its cyanogenic glycosides limits its widespread use. The cytochrome P450 gene family is one of the largest gene families in plants and is involved in synthesizing phytohormones, secondary metabolites, and various defense compounds. Two P450 genes have been found to be important enzymes for the biosynthesis of cyanogenic glycosides in common sorghum (Sorghum bicolor (L.) Moench). However, the P450 gene family and its involvement in cyanogenic glycoside synthesis have been less studied in flax. In previous studies, we assembled a high-quality flax genome. In this study, a total of 412 P450 genes were identified in the flax genome, with molecular weights in the range of 7.42 kDa to 154.5 kDa and encoding amino acid lengths between 67 and 1378. These genes belonged to 48 families under eight clans and were distributed across 15 chromosomes. The number of introns varied from 0 to 14. Thirty-nine cis-acting elements were identified within 1500 bp upstream of the promoter, mainly related to the light response. There were 147 segmental duplications and 53 tandem duplication events among these P450 genes. Eleven genes potentially related to cyanogenic glycoside synthesis were identified by transcriptome analysis, and the RT-qPCR results verified the reliability of the transcriptome analysis. This study lays the foundation for the classification and functional study of the flax P450 gene family. The results will be useful for breeding new low-cyanogenic-glycoside flax varieties by genetic engineering. Full article

The blueberry fruit (Vaccinium corymbosum L.) exhibits a high content of bioactive compounds, including anthocyanins, that can be used as pigmenting agents, but they are mixed with sugars, which can hinder their utilization. The objective was to evaluate the use of aqueous two-phase extraction aided by centrifugation to separate bioactive compounds, particularly anthocyanins, from blueberry fruits, considering the reduction of sugars, for their use as pigmenting agents in a food product. A mixture of trisodium citrate (Na₃C₃H₅O(COO)₃; Na₃Cit) and polyethylene glycol ([HO-(CH₂CH₂O)n-CH₂OH]; poly (ethane-1,2-diol); PEG) with a molecular weight of 4 kDa was used. Based on the cloud point method, a binodal diagram was developed. After the evaluation of several systems with composition located on a tie line, conditions were identified to form biphasic systems with phases of equal volume. Passive sedimentation for 0, 15, and 30 min, followed by centrifugation and also passive sedimentation for 24 h without centrifugation, were evaluated. A system with 17.73% Na₃Cit, 21.33% PEG, 30 min of passive sedimentation, and 15 min of centrifugation at 2940× g produced an extract with a high concentration of soluble phenols (0.353 mg/mL) and anthocyanins (0.202 mg/mL) and, likewise, high antioxidant activity (910.0 mmol gallic acid equivalents per mL), with reduced sugar content, which demonstrated to have the potential to pigment food beverages with a reddish tone. Full article

C. perfringens type F isolates are a leading cause of food poisoning and antibiotic-associated diarrhea. Type F isolate virulence requires production of C. perfringens enterotoxin [CPE], which acts by forming large pore complexes in host cell plasma membranes. During GI disease, CPE is produced in the intestines when type F strains undergo sporulation. The toxin is then released into the intestinal lumen when the mother cell lyses at the completion of sporulation. Once present in the lumen, CPE encounters proteases. This study examined the in vitro, ex vivo, and in vivo processing of CPE by intestinal proteases and the effects of this processing on CPE activity. Results using purified trypsin or mouse intestinal contents detected the rapid cleavage of CPE to a major band of ~32 kDa and studies with Caco-2 cells showed that this processed CPE still forms large complexes and retains cytotoxic activity. When mouse small intestinal loops were challenged with CPE, the toxin caused intestinal histologic damage, despite rapid proteolytic processing of most CPE to 32 kDa within 15 min. Intestinal large CPE complexes became more stable with longer treatment times. These results indicate that CPE processing involving trypsin occurs in the intestines and the processed toxin retains enterotoxicity. Full article

The CPA gene family regulates ionic balance and pH homeostasis in cells, significantly contributing to plant stress tolerance. In this study, a total of 63 BrCPA gene family members were identified in the whole genome of Brassica rapa L. (B. rapa), and the three subfamily members were BrNHX (9), BrKEA (15), and BrCHX (39), respectively. The members of the BrCPA gene family encoded 303-1259 amino acids, with molecular weights in the range of 32,860.39~139,884.73 kDa, distributed on 10 chromosomes, and contained 17 conserved motifs, BrNHX and BraKEA, and the BrCPA gene family members had the same molecular weights on 10 chromosomes and contain 17 conserved motifs. The BrNHX and BraKEA subfamilies have more exons than the BrCHX subfamily. An analysis of promoter cis-acting elements in the BrCPA gene showed that members of this gene family contain TC-rich, LTR, MBS, and ARE stress response elements. In addition, transcriptome analysis revealed the expression of CPA genes in B. rapa under salt stress. The selected genes were verified by RT-qPCR. By detecting the Na⁺ and K⁺ flow rates in the root and chloroplast cells of salt-tolerant and salt-sensitive varieties after salt treatment, it was found that the rate of Na⁺ and K⁺ efflux from the root and chloroplast cells of salt-sensitive varieties was significantly higher than that of salt-tolerant varieties. This investigation marks the first systematic identification of the CPA gene family in B. rapa. This study further explores its expression patterns and the efflux rates of Na⁺ and K⁺ across salt-tolerant varieties, providing a theoretical basis for understanding the role of the CPA gene family in the salt stress response of B. rapa. Full article

This study investigated the effect of different culture agar media, derived from Bennett’s medium, on the antimicrobial activity of 15 Streptomyces sp. and 1 Lentzea sp. strains isolated from mining environments. The media were prepared from the standard Bennett’s medium by suppressing one, two, or three ingredients—yeast extract (YE), beef extract (BE), or casein (Cas)—while maintaining glucose (Gluc) or by substituting it with fructose (Fruc) or galactose (Gal) and keeping the same suppressions. The antimicrobial activity was investigated against Candida albicans ATCC 10231, Staphylococcus aureus ATCC 29213, Bacillus subtilis ATCC 6633, and Escherichia coli K12. The antimicrobial activity of actinomycete strains was positively influenced by media modifications, though the response was actinomycete strain and target pathogen-dependent. Unexpectedly, thirteen strains exhibited poor growth on a pure agar-agar medium, including six Streptomyces strains (AS34, AS3, BS59, BS68, BS69, and DAS104) that showed notable antimicrobial activity, with inhibition zone diameters ranging from 10.75 ± 1.06 to 18.00 ± 0.00 mm. Modifications of Bennett’s medium, including replacing glucose with fructose or galactose and maintaining yeast extract or both yeast extract and beef extract, induced and enhanced the antimicrobial activity of several actinomycete strains. Notably, the new media induced antimicrobial activity in strains that showed no activity in Bennett’s medium. They led, compared to Bennett’s medium, to the detection of eight additional active strains against S. aureus, eight against B. subtilis, six against E. coli, and four against C. albicans. This study is the first to explore the modification of Bennett’s medium, either by subtraction or substitution, in order to investigate the effect on antimicrobial activity of actinomycete strains. These results highlight the importance of the composition of culture media on inducing or boosting antimicrobial activity in Streptomyces and Lentzea. Full article

Acute kidney injury (AKI) is a syndrome characterized by a rise in creatinine or a decrease in urinary flow, according to the Kidney Disease Improving Global Outcomes (KDIGO) definition. It is diagnosed in 15% of inpatients and 50% of patients in the intensive care unit (ICU), and it is related to increased mortality. As part of a global effort aimed at the elimination of preventable deaths from AKI, there is a growing interest in identifying biomarkers that can be point-of-care and that are not influenced by the variability in patient characteristics in a relevant way. Neutrophil gelatinase-associated lipocalin (NGAL), particularly in its 25 kDa form, which is exclusively released by renal tubules, has emerged as a promising biomarker with potential use in the diagnosis of AKI in the critically ill, including its use in guiding the initiation and/or weaning of renal replacement therapy (RRT). The objective of this review is to summarize the current understanding of NGAL in acute settings, emphasizing biological and genomic insights. Full article

Serum amyloid A (SAA) is one of the most abundant acute-phase response proteins and has been extensively studied in vertebrates for its role in modulation of the inflammatory response and as a marker of disease diagnosis. By comparison, SAA is rarely identified in aquatic species and its physical functions are also not well studied. The present study identified the only one gene encoding SAA protein in oyster Crassostrea gigas. The open reading frame (ORF) of CgSAA was of 417 bp, encoding a putative polypeptide of 138 amino acid residues with a predicted molecular weight of 15.66 kDa. CgSAA was composed of a signal peptide (residues 1–22) and a conserved SAA domain (residues 36–138). The mRNA expression of CgSAA in normal individuals was detectable but at a low level, with the lowest expression level in the tissue of labial palp and a slightly higher expression level in hemocytes. The mRNA expression level of CgSAA was significantly up-regulated at 6 h (2.76-fold of that in control group, p < 0.01) post V. splendidus stimulation. It was also significantly induced under environmental stress at high temperature (34 °C) or low salinity (15‰ salinity). The recombinant protein rCgSAA was expressed in Escherichia coli and purified by affinity chromatography. After rCgSAA was injected into oysters or incubated with culture primary hemocytes, the mRNA expressions of the cytokines CgIL17-1, CgIL17-5, and CgTNF were all significantly up-regulated. The results collectively suggested that CgSAA, as a conserved acute-phase response protein in oyster, was quickly induced under environmental stress and promoted the expressions of cytokines, which provide fresh ideas for understanding the roles of SAA proteins in aquatic invertebrates. Full article

Background/Objectives: Broussonetia papyrifera is a tree-producing allergenic pollen that grows in varied climatic conditions worldwide and causes pollen allergies in susceptible humans. This study aimed to investigate B. papyrifera pollen morphology, pollen metabolome, pollen allergenicity, and climate change’s impact on the plant habitat suitability in the future. Methods: Tree pollen was collected in spring from different regions of Pakistan. Pollen samples were subjected to morphological analysis, Fourier transform infrared spectroscopy (FTIR), liquid chromatography–mass spectrometry (LC-MS/MS), and immunoblotting. Results: MaxEnt modeling predicted the tree’s future-growth invasion into new regions. Scanning electron microscopy (SEM) and FTIR displayed regional differences in pollen morphology and metabolome correlated to shifts in climatic variables. LC-MS/MS analysis detected four lipids that can potentially stimulate inflammatory responses. Pollen protein immunoblotting studies identified a putative 15 kDa novel allergen and verified previously known 40 kDa, 33 kDa, and 10 kDa allergens. B. papyrifera MaxEnt modeling through ACCESS1.0 and CCSM4 under 2-greenhouse gas emissions scenarios {representative concentration pathway (RCP) 4.5 and 8.5} projected the tree invasion by the years 2050 and 2070. Conclusions: The study findings demonstrate that differences in climatic variables affect B. papyrifera-pollen metabolome and predict the habitat suitability of the tree for invasion in the future. The study results provide a model system for studying other species’ pollen morphology, metabolome, future habitat suitability for plant invasion, and associated allergies in response to climate change. Full article

The discovery of effective cysteine protease inhibitors with crude protein kiwi extracts (CPKEs) has created novel challenges and prospects for pharmaceutical development. Despite extensive research on CPKEs, limited research has been conducted on treating atopic dermatitis (AD). Therefore, the objective of this work was to investigate the anti-inflammatory effects of CPKEs on TNF-α activation in a HaCaT cell model and in a DNCB (1-chloro-2, 4-dinitrochlorobenzene)-induced atopic dermatitis animal model. The molecular weight of the CPKE was determined using SDS-PAGE under non-reducing (17 kDa and 22 kDa) and reducing conditions (25 kDa, 22 kDa, and 15 kDa), whereas gelatin zymography was performed to examine the CPKE’s inhibitory impact on cysteine protease (actinidin and papain) activity. Moreover, the CPKE remains stable at 60 °C, with pH levels varying from 4 to 11, as determined by the azocasein assay. CPKE treatment decreased the phosphorylation of mitogen-activated protein kinase (MAPK) and Akt, along with the activation of nuclear factor-kappa B (NF-κB)-p65 in tumor necrosis factor-α (TNF-α)-stimulated HaCaT cells. Five-week-old BALB/c mice were treated with DNCB to act as an AD-like animal model. The topical application of CPKE to DNCB-treated mice for three weeks substantially decreased clinical dermatitis severity and epidermal thickness and reduced eosinophil infiltration and mast cells into ear and skin tissues. These findings imply that CPKE derived from kiwifruit might be a promising therapy option for inflammatory skin diseases such as AD. Full article

Objectives: The purpose of this study was to further characterize the ultrafiltration (UF) method for determining free saccharide levels in glycoconjugate vaccines and compare it with other methods used for the determination of free saccharide levels in meningococcal glycoconjugate vaccines. Methods: We performed experiments on both meningococcal glycoconjugates and capsular polysaccharides, and compared UF, deoxycholate (DOC) precipitation, and solid-phase extraction (SPE) methods. Meningococcal capsular polysaccharides from groups A (MenA), C (MenC), and W (MenW) were depolymerized and characterized using SEC-MALS (size-exclusion chromatography with multi-angle laser light scattering) to determine the molecular weight and hydrodynamic size and then subjected to UF. The free saccharide content was quantified using HPAEC-PAD (high-performance anion-exchange chromatography with pulsed amperometric detection). Results: The characterization of size-reduced group C polysaccharide revealed weight-average molecular mass (Mw) ranging from 22,200 g/mol to 287,300 g/mol and hydrodynamic radii of 3.7 to 19.5 nm. Pore size studies confirmed that polysaccharides with diameters up to 15 nm filtered through the 100 kDa cellulose membrane. The smallest PS fragment tested (22,200 g/mol, 7.4 nm diameter) was partially recovered from the 30 kDa membrane. For MenC-CRM₁₉₇, DOC yielded the lowest free saccharide content (<1%), UF gave moderate results (7–8%), and SPE showed the highest and most variable values (up to 15%). For MenA- and MenW-CRM₁₉₇, UF and DOC consistently provided low free saccharide levels (<2% and 3–11%, respectively). Conclusions: The upper limits on the size of free group C meningococcal polysaccharides that can be ultrafiltered were assessed. Differences in the relative amount of free saccharide were observed between various methods used to control meningococcal conjugate vaccines. Full article

Economic development increases and brings about issues such as the secure supply of food in a sustainable way. Phytases are enzymes catalyzing phytate hydrolysis to release phosphorus in an inorganic form. Animal feeds could be supplemented with bacterial phytases to increase their phosphorus and micronutrients bioavailability. To the best of our knowledge, this is the first report on the purification and characterization of an alkalophilic phytase from Cobetia marina. The purified newly isolated phytase from the halophilic Cobetia marina strain 439 appears to be appropriate for use as an additive in food and feed processing. Its molecular weight was determined to be 43 kDa by gel filtration and 40 kDa by SDS–polyacrylamide gel electrophoresis. The purified enzyme had maximum activity at pH 8.0 and 45 °C, while at 70 °C, it was 80% and about 50% at 80 °C for 40 min, showing its thermostability. Enzyme activity was retained at a broad pH range from 6.5 to 9.0. The half-life of the phytase of 15 min at pH 10 and 30 min at pH 4.0 was registered. The enzyme was proven to be with high substrate specificity. In addition, the purified phytase showed strong proteolytic tolerance against trypsin and pepsin. The pH profile, its thermostability, and proteolytic tolerance of the studied phytase as a halophilic bacterial product determine it as a unique candidate for application in agriculture, food, and feed industries. Full article

Whey proteins have anti-fatigue activity, but there are few studies that have reported the ameliorative effects of branched-chain amino acid (BCAA) oligopeptides from whey proteins on fatigue in mice. The purposes of this study were to establish a process for the preparation of BCAA oligopeptides from whey protein and to investigate the anti-fatigue activity of BCAA oligopeptides. Whey proteins were hydrolyzed by trypsin and flavourzyme and purified by ethanol precipitation and reversed-phase high performance liquid chromatography (RP-HPLC). Fraction D’ was found to contain the highest content of BCAAs and a high proportion of low-molecular-weight peptides (<1 kDa; content: 81.48%). Subsequently, mass spectrometry identified 15 BCAA oligopeptides in Fraction D’, including three dipeptides, six tripeptides, two tetrapeptides, and four pentapeptides. In addition, animal experiments showed that BCAA oligopeptides significantly prolonged the residence time on the rod and swimming time of mice. Further studies showed that BCAA oligopeptides remarkably reduced serotonin (5-hydroxytryptamine, 5-HT) synthesis in the brain by down-regulating the plasma-free tryptophan (F-Trp)/BCAA ratio, thereby alleviating fatigue. Therefore, BCAA oligopeptides can be used as an auxiliary functional dietary molecule in functional products to exert anti-fatigue activity by regulating 5-HT synthesis. Full article