Search Results (4,027)

Anti-Porphyromonas gingivalis mechanisms of Berberis hemsleyana bark extract remain to be elucidated, and the anti-inflammatory activity of its n-butanol fraction (BNB) in RAW264.7 cells—mediated through suppression of the NF-κB pathway—require further validation. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the crude extract from B. hemsleyana were determined against Candida albicans, Escherichia coli, Porphyromonas gingivalis, Staphylococcus aureus and Streptococcus mutans. Scanning electron microscopy (SEM) and bacterial protein leakage assays were used to evaluate its antibacterial activity against P. gingivalis. High-performance liquid chromatography-mass spectrometry (LC-MS) was applied to analyze the ethanol extract of B. hemsleyana bark, leading to the screening of 47 compounds. The antibacterial mechanisms of the compounds were predicted through Network Pharmacology analysis and Molecular docking. Anti-inflammatory activity mediated via the NF-κB pathway was verified using an LPS-induced RAW264.7 cell inflammatory model. Specifically, the BNB showed a significant antibacterial effect on P. gingivalis. Meanwhile, it was confirmed that this fraction damaged the bacterial cell membrane structure, leading to the leakage of intracellular proteins in bacteria and thus impairing their infectivity. Network pharmacology analysis and molecular docking results indicated that B. hemsleyana bark’s biologically active compounds (Calenduloside E, Limonin, Acanthoside B, Dihydroberberine) antibacterial activity by regulating cytokines and cell apoptosis, thereby coordinating the body’s microbial homeostasis and inflammation. Additionally, BNB significantly reduced the secretion of the inflammatory cytokines IL-1β, TNF-α and IL-6 in vitro via the NF-κB pathway. The crude extract from the bark of B. hemsleyana has antibacterial and Anti-inflammatory activity. The n-butanol fraction showed a significant antibacterial effect on P. gingivalis. Full article

Reusable enzyme carriers are valuable for proteomic workflows, yet many supports are expensive or lack robustness. This study describes the covalent immobilization of recombinant trypsin on micrometer-sized corundum particles and assesses their performance in protein digestion and antibody analysis. The corundum surface was cleaned with potassium hydroxide, silanized with 3-aminopropyltriethoxysilane and activated with glutaraldehyde. Recombinant trypsin was then attached, and the resulting imines were reduced with sodium cyanoborohydride. Aromatic amino acid analysis (AAAA) estimated an enzyme loading of approximately 1 µg/mg. Non-specific adsorption of human plasma proteins was suppressed by blocking residual aldehydes with a Tris-glycine-lysine buffer. Compared with free trypsin, immobilization shifted the temperature optimum from 50 to 60 °C and greatly improved stability in 1 M guanidinium hydrochloride. Activity remained above 80% across several reuse cycles, and storage at 4 °C preserved functionality for weeks. When applied to digesting the NISTmAb, immobilized trypsin provided peptide yields and sequence coverage comparable to soluble enzyme and outperformed it at elevated temperatures. MALDI-TOF MS analysis of Herceptin digests yielded fingerprint spectra that correctly identified the antibody and achieved >60% sequence coverage. The combination of low cost, robustness and analytical performance makes corundum-immobilized trypsin an attractive option for research and routine proteomic workflows. Full article

Dipeptidyl peptidase-4 (DPP-4) is a protease that degrades incretin and inhibits the secretion of insulin. Consequently, DPP-4 inhibition promotes insulin secretion and prevents the onset of type 2 diabetes. Given the growing interest in food-derived DPP-4 inhibitory peptides as potential functional ingredients, buckwheat (Fagopyrum esculentum) represents a promising source; however, few studies have investigated the bioactivity of peptides derived from buckwheat flour hydrolysates. In this study, two DPP-4 inhibitory peptides, Ile-Pro-Trp and Ile-Pro-Leu, were identified through purification of buckwheat flour hydrolysate and liquid chromatography–tandem mass spectrometry analysis. In a rat oral glucose tolerance test (OGTT), a fraction of buckwheat flour hydrolysate, crudely purified by reverse-phase column chromatography, showed a non-significant trend toward reducing increases in blood glucose. To our knowledge, this study is the first to show that Ile-Pro-Trp isolated from food protein hydrolysates exhibits considerable DPP-4 inhibitory activity. Moreover, this is the first study identifying Ile-Pro-Ile as a DPP-4 inhibitor from a plant source. Full article

This study presents a comprehensive analysis of cysteine synthase A (CysK) from Limosilactobacillus reuteri LR1 (LreCysK), an enzyme involved in the biosynthesis of L-cysteine. This protein supports crucial cellular functions such as sulfur metabolism, antioxidant defense, detoxification, and protein synthesis. Previously, the gene encoding LreCysK was cloned, and the enzyme with His-tag on the N-terminus was obtained in active and soluble form. Here, kinetic parameters of the enzyme were determined by the previously developed high-pressure liquid chromatography (HPLC) and ninhydrin methods. It was found that LreCysK has similar K_M^OAS and k_cat as CysKs from Escherichia coli and from the model plant Arabidopsis thaliana. The thermal stability of LreCysK was studied using differential scanning calorimetry. It was revealed that the melting point of the enzyme increases to almost 90°C when Pyridoxal-5 phosphate (PLP) is added, indicating that the stability of the enzyme complex with PLP is relatively high. Structural studies revealed that LreCysK is a dimer, and its active site is similar to those of other enzymes, but exhibits some features characteristic of lactobacilli CysKs (GISA), as well as unique residues, such as Ile50. Also, the potential biotechnological applications of LreCysK are discussed. These findings enhance our understanding of LreCysK’s biochemical versatility and its potential applications in biotechnology and medicine. Full article

Gamma-aminobutyric acid (GABA) is a non-protein amino acid distributed in nature by different types of organisms and microorganisms. GABA has been widely studied for its different physiological functions and industrial applications. Its production is mainly carried out through fermentation processes using lactic acid bacteria (LAB), which are of particular interest because they are safe and possess high glutamate decarboxylase enzyme activity. However, GABA production can vary among different LAB species and is affected by culture conditions. Therefore, strain development and selection, as well as optimization of fermentation parameters, are essential to increase GABA yields and meet the needs of industrial demand. This review quantitatively analyzes recent advances in fermentative GABA production, showing a sustained increase in publications and a predominance of chromatography-based quantification methods (approximately 68%), mainly using pre-column derivatization. Optimized fermentation strategies, supported by statistical and artificial intelligence models, have achieved GABA concentrations of up to 90 mM. In parallel, in silico genomic and metabolic analyses revealed the widespread distribution of key GABA biosynthesis and transport genes among LAB, supporting their selection and engineering. Overall, the integration of advanced analytical methods, bioinformatics-guided strain selection, and computational process optimization emerges as a key strategy to enhance GABA productivity and support future industrial-scale applications. Full article

Cephalothrix cf. simula is a highly toxic ribbon worm of the class Palaeonemertea, known for its high concentrations of tetrodotoxin. Recent transcriptomic and proteomic studies across Nemertea have revealed that species from all classes possess a diverse array of protein and peptide toxins, which are associated with unicellular glands of the proboscis and the integument epithelium. Previous studies have identified a large number of putative toxins in the transcriptome of C. cf. simula; however, corresponding proteomic data have so far been lacking. This study presents the first comprehensive analysis of the mucus and proboscis proteome of C. cf. simula using high-performance liquid chromatography–tandem mass spectrometry. We identified three putative toxins in the proboscis and three in the mucus. Additionally, four cysteine-rich peptides with putative toxic activity were identified in the mucus and one in the proboscis. The expression of the corresponding genes in both tissues was quantified using quantitative real-time PCR. The toxin compositions of the proboscis and mucus showed clear signs of functional specialization, with no overlapping toxins and tissue-specific patterns of gene expression. Feeding experiments combined with transmission electron microscopy confirmed the involvement of specialized proboscis structures, pseudocnidae, in delivering toxins into the prey. Full article

Botulinum neurotoxin serotype A (BoNT/A) is the most potent known neurotoxin. While its light chain (LC) catalytic domain is a prime target for next-generation vaccines and therapeutics, the functional differences among BoNT/A subtype LCs (A1, A2, A3) remain to be definitively characterized, despite notable sequence variation. This work aimed to systematically compare the proteolytic activity and immunoprotective efficacy of recombinant BoNT/A1-LC, A2-LC, and A3-LC. Recombinant A1-LC-His, A2-LC-His, A3-LC-His, and A3-LC-Twin-Strep proteins were expressed in Escherichia coli (E. coli) and purified with affinity chromatography. Their proteolytic activity was assessed via in vitro SNAP-25 cleavage assays. The protective potency of these antigens was evaluated in a mouse model. In vitro cleavage assays revealed a substrate cleavage efficiency order of A2-LC > A1-LC > A3-LC. In vivo, both A1-LC and A2-LC immunization conferred robust, broad protection against high-dose challenges with all three toxin subtypes. In stark contrast, A3-LC provided only minimal protection against its homologous toxin and none against heterologous subtypes. Crucially, the functional deficit of A3-LC was confirmed to be an intrinsic property, as the A3-LC-TS variant, designed to exclude tag-specific interference, exhibited comparable low efficacy. According to structural research, A3-LC’s compromised function may be caused by a four-amino-acid loss. The inferior performance of A3-LC is inherent to its primary structure. This work identified A1-LC or A2-LC as the potential proteolytic activity molecule and vaccine antigen by demonstrating functional differences among BoNT/A subtype LCs. These findings provide crucial insights for developing subtype-specific countermeasures against botulism. Full article

Fine particulate matter (PM_2.5), which contains heavy metals such as Al, Fe, Mg, and Mn, among others, induces cognitive dysfunction through oxidative stress, neuroinflammation, and impaired mitochondria. This study evaluated the neuroprotective effects of a 40% ethanol extract of Polygonum multiflorum (EPM) on PM_2.5-induced cognitive dysfunction in a mouse model. Behavioral assessments demonstrated attenuated learning and memory impairment following EPM treatment. Redox homeostasis was restored through increased expression of superoxide dismutase (SOD) and glutathione (GSH) and decreased levels of malondialdehyde (MDA) and mitochondrial reactive oxygen species (mtROS) in the EPM group. Mitochondrial function was attenuated, as indicated by recovery of mitochondrial membrane potential and ATP levels. EPM inhibited neuroinflammation by downregulating the TLR4-MyD88-NF-κB pathway and maintaining blood–brain barrier integrity through the upregulation of tight junction proteins. It modulated neuronal apoptosis through the JNK pathway, reducing the accumulation of amyloid-beta and phosphorylated tau. Synaptic plasticity was preserved through upregulation of BDNF/TrkB signaling and cholinergic neurotransmission via regulation of acetylcholine (ACh), acetylcholinesterase (AChE), and choline acetyltransferase (ChAT). To standardize EPM, high-performance liquid chromatography (HPLC) confirmed the presence of the bioactive compound, tetrahydroxystilbene glucoside (TSG). These findings suggest that EPM may be a promising functional food candidate for mitigating PM_2.5-related cognitive impairments. Full article

Low-flow liquid chromatography has become the primary tool for advanced chromatographic analysis and is an indispensable technique for the sensitive detection of biomolecules. In this study, we developed a new 4-tritylphenyl methacrylate-based monolithic nano-column with an internal diameter of 20 µm for bioanalytical separations in nano-liquid chromatography (nano-LC). The composition of the monolith was optimized with regard to the monomer and porogenic solvent. The column was characterized using Fourier Transformed Infrared Spectroscopy (FT-IR) spectroscopy, scanning electron microscopy (SEM) and chromatographic analyses. Chromatographic characterization was performed using homologous alkylbenzenes (ABs) and polyaromatic hydrocarbons (PAHs), which facilitate hydrophobic and π–π interactions. Run-to-run and column-to-column reproducibility values were found to be <2.51% and 2.4–3.2%, respectively. The final monolith was then used to separate six standard proteins, including β-lactoglobulin A, carbonic anhydrase, ribonuclease A (RNase A), α-chymotrypsinogen (α-chym), lysozyme (Lys), cytochrome C (Cyt C) and myoglobin (Myo), as well as three dipeptides: Alanine-tyrosine (Ala-Tyr), Glycine-phenylalanine (Gly-Phe) and L-carnosine. The nano-column was then applied to profiling peptides and proteins in the MCF-7 cell line, enabling high-resolution peptide analysis. Full article

Few studies have examined the effects of puerarin (PE) on ruminant parameters and methane production. Therefore, we determined the degradation of PE in the rumen and evaluated the effect of PE on in vitro fermentation, methanogenesis, and microbial community structure. A completely randomized design was used for the in vitro fermentation, and 4 gradient dosages of PE (0 mg/kg, 50 mg/kg, 100 mg/kg, and 150 mg/kg of DM) were applied in this trial. The in vitro fermentation was carried out in three runs at 6 h and 48 h, with four replicates per treatment per time point. Each run included 40 samples: eight treatments × four replicates and eight blank samples. Based on the fermentation results, both the PE treatments and the control group (CON) at 48 h were chosen for further analysis to explore the effects of PE on the bacterial community structure. Meanwhile, we determined the degradation rate and degradation products of PE in vitro ruminal fluid using high-performance liquid chromatography (HPLC). In this trial, PE may be isomerized into daidzin by rumen microorganisms; the in vitro degradation results of PE indicated that 70% of PE was degraded within 6 h, with the degradation rate reaching nearly 85% by 12 h. The concentrations of NH₃-N and microbial crude protein (MCP) significantly increased linearly with the PE doses at 6 h (p = 0.01). The concentrations of MCP (p = 0.02) and propionate (p = 0.04) demonstrated a linear increase with increasing PE doses at 48 h. In contrast to microbial protein (MCP) and propionate, the acetate-to-propionate ratio decreased linearly with increasing PE doses at 48 h (p = 0.05). Additionally, the addition of PE linearly decreased methane production at 48 h (p = 0.01). Meanwhile, the relative abundances of g_UBA1217 (p = 0.03), g_UBA2810 (p = 0.04), and g_Succiniclasticum (p = 0.03) were significantly lower compared with the CON group. The results showed that PE can be degraded by rumen microflora. Furthermore, it can improve rumen fermentation parameters, increase the amount of protein synthesized by rumen microorganisms, and reduce methane production and the acetate-to-propionate ratio. PE could potentially be an effective strategy for methane mitigation; however, further research is needed to assess its in vivo effects in dairy cows over a longer period. Full article

This review highlights the growing relevance of ion-exchange nanofibrous membranes (IEX-NFMs) in membrane chromatography (MC) for protein purification, emphasising their structural advantages such as high porosity, tunable surface functionality, and low-pressure drops. While the adsorption of IEX-NFMs in MC is expanding due to their potential for high throughput and rapid mass transfer, a critical limitation remains: the precise binding capacity of these membranes is not well understood. Traditional experimental methods to evaluate protein–membrane interactions and optimise binding capacities are labour-intensive, time-consuming, and costly. Therefore, this review underscores the importance of computational modelling as a viable predictive approach to guide membrane design and performance prediction. Yet major obstacles persist, including the challenge of accurate representation of the complex and often irregular pore structures, as well as limited and/or oversimplified adsorption models. Along with molecular-scale simulations such as molecular dynamics (MD) simulations and quantum simulations, meso-scale simulations can provide insight into protein–fibre and protein–protein interactions under varying physicochemical conditions for larger time scales and lower computational burden. These tools can help identify key parameters such as binding accessibility, ionic strength effects, and surface charge density, which are essential for the rational design and performance prediction of IEX-NFMs. Moreover, integrating simulations with experimental validation can accelerate optimisation process while reducing cost. This technical review sets the foundation for a computationally driven design framework for multifunctional IEX-NFMs, supporting their use in next-generation chromatographic separations and broadening their applications in bioprocessing and analytical biotechnology. Full article

Olive leaf phenols are recognized for their antioxidant and anti-inflammatory properties. A hydroalcoholic extract of Olea europaea L. cv. Ogliarola leaves was recovered with an ultrasound-assisted extraction using green solvents. Phenol content was investigated by means of liquid chromatography coupled with photodiode array and mass spectrometer detectors. Extract cytotoxicity was determined in SH-SY5Y neuroblastoma cells by the MTT assay to establish non-cytotoxic concentrations. The effects of the extract under lipopolysaccharide-induced conditions were investigated by assessing oxidative stress and lipid peroxidation through malondialdehyde quantification using the thiobarbituric acid assay. Antiglycation capacity was examined with a BSA methylglyoxal model. In parallel, quantitative real-time PCR was employed to assess the modulation of inflammation- and oxidative stress-related genes (TLR4, NF-κB, IL-6, IL-8, Nrf2, and HO-1), providing molecular insights into the extract’s bioactivity. The extract did not exert cytotoxic effects at the selected concentrations and with modulated oxidative stress, lipid peroxidation, protein glycation, and gene expression profiles associated with inflammatory and redox pathways in neuronal cells. These data demonstrated that olive leaf extract, rich in phenols, influenced multiple biochemical and molecular endpoints relevant to neuronal physiology, supporting its potential application as a nutraceutical ingredient for the modulation of oxidative and glycation-related processes. Full article

Mespilus germanica L. is one of the two species of the genus Mespilus L., and is distributed in several regions, including Southeastern Europe, Anatolia, the Caucasus, and parts of the Middle East. The fruits of the plant are consumed as food, and the fruits, leaves, and stem bark are traditionally used for various systemic disorders, including gastrointestinal, respiratory, urinary tract, and skin conditions, as well as menstrual irregularities. In our study, the anti-inflammatory potential and antimicrobial activities of aqueous-methanolic extracts prepared from ripe (MGRF) and unripe fruits (MGUF), leaves (MGL), and stem bark (MGB) of M. germanica were evaluated in vitro. Quercetin, catechin, epicatechin, ellagic acid, chlorogenic acid, and caffeic acid were analyzed using high-performance liquid chromatography. MGL exhibited the strongest activity against Staphylococcus aureus (MIC = 8 µg/mL), while MGB was most active against Enterococcus faecalis (MIC = 4 µg/mL), and fruit extracts were effective against resistant Acinetobacter baumannii (MIC = 16–32 µg/mL). Membrane-protective effects were more pronounced in MGUF and MGB, whereas MGL demonstrated the highest protein stabilization activity. Leaves also contained the highest levels of chlorogenic acid and epicatechin. These findings support the traditional use of M. germanica, though further studies are required to explore its therapeutic potential. Full article

Cutaneous lupus erythematosus (CLE) can occur independently of lupus erythematosus. SLE, and its responsiveness to treatment, does not necessarily align with that of coexisting SLE. Extracellular vesicles (EVs) allow communication between cells and rapid delivery throughout the body. We hypothesized that EVs may support disease-specific inflammation in CLE and SLE patients. Plasma EVs from healthy controls (n = 5), CLE (n = 6), and dermatomyositis (n = 17) were purified by ultracentrifugation and size-exclusion chromatography, phenotyped by flow cytometry, and profiled by LC-MS/MS. Circulating EVs were mainly platelet-, endothelial-, and antigen-presenting cell-derived examples. CLE EVs harbored four proteins absent in the controls—mimecan, IFI27, fibulin-2, and snRNP B/B′ (anti-Sm an-tigens)—and their cumulative number increased with SLEDAI. Relative to the controls, 18 proteins were upregulated and 15 downregulated in CLE EVs. The number of upregulated proteins showed a trend toward a correlation with SLEDAI (r = 0.79, p = 0.06) but not with CLASI (r = 0.21). Among upregulated proteins, lysozyme C and hyaluronan-binding protein 2 tracked with cutaneous activity (CLASI r = 0.74 and r = 0.86) but not with systemic activity (SLEDAI r = 0.52 and r = 0.31). CLE plasma EVs were enriched in antigen-presenting cell markers and disease-related cargo, including anti-Sm antigens and proinflammatory proteins. Although overall protein diversity correlated primarily with systemic disease activity, a subset of proteins appeared to reflect cutaneous activity. Full article

Streptococcus suis is an important zoonotic pathogen responsible for severe infections in pigs and humans. Its capacity to survive within phagocytic cells is considered a key virulence mechanism that contributes to dissemination and persistence in host tissues. This study employed comparative proteomic profiling to investigate intracellular adaptation of S. suis serotypes 2 (SS2) and 14 (SS14) during infection of human U937 macrophages. Five isolates originating from humans and pigs were analyzed using gel electrophoresis with liquid chromatography–tandem mass spectrometry (GeLC–MS/MS), revealing 118 differentially expressed proteins grouped into 11 functional categories. Translation-related proteins represented the largest group (48%), including upregulated ribosomal subunits (30S: S2, S5, S7, S8, S12, S15; 50S: L1, L5, L18, L22, L24, L33, L35) and translation factors such as GidA/TrmFO and RimP. Enrichment of carbohydrate metabolism and DNA replication proteins, including phosphoenolpyruvate carboxylase (PEP), UDP-N-acetylglucosamine pyrophosphorylase (GlmU), and ATP-dependent DNA helicase RuvB, indicated metabolic reprogramming and stress adaptation under intracellular conditions. Stress-response proteins such as molecular chaperone DnaK were also induced, supporting their multifunctional, “moonlighting” roles in virulence and host interaction. Comparative analysis showed that SS2 expressed a broader range of adaptive proteins than SS14, consistent with its higher virulence potential. These findings reveal conserved intracellular responses centered on translation, energy metabolism, and stress tolerance, which enable S. suis to survive within human macrophages. Integration of these intracellular proteomic signatures with previous exoproteomic, peptidomic, and network-based studies highlights translational and metabolic proteins—particularly DnaK, enolase, elongation factor EF-Tu, and GlmU—as multifunctional candidates linking survival and immunogenicity. This work establishes a comparative proteomic foundation for understanding S. suis intracellular adaptation and highlights potential targets for future vaccine or therapeutic development against this zoonotic pathogen. Full article