Search Results (267)

In order to innovatively develop high-activity ACE inhibitory peptides from edible fungi, the conditions for a double-enzymatic hydrolysis preparation of ACE inhibitory peptides from Flammulina velutipes were optimized by response surface methodology. After purification by macroporous resin, gel chromatography, and RP-HPLC, a crude peptide fraction was obtained; its ACE inhibition rate was 85.73 ± 0.95% (IC₅₀ = 0.83 ± 0.09 mg/mL). Based on LC-MS/MS sequencing, the four novel peptides, namely, FAGGP, FDGY, FHPGY, and WADP, were screened by computer analysis and molecular docking technology. The four peptides exhibited a binding energy between −9.4 and −10.3 kcal/mol, and formed hydrogen bonds with Tyr523, Ala354, and Glu384 in the S1 pocket, Tyr520 and His353 in the S2 pocket, and His383 in the HEXXH zinc-coordinating motif of ACE, indicating their good affinity with the ACE active site. The IC₅₀ values of the four ACE inhibitory peptides were 29.17, 91.55, 14.79, and 41.27 μM, respectively, suggesting that these peptides could potentially contribute to the development of new antihypertensive products. Full article

Mussels are nutrient-rich but perishable, resulting in substantial resource loss. A protease-producing strain (Bacillus velezensis Z-1, Mytilus edulis) isolated from marine sludge was used to hydrolyze mussels, producing Y-1, a hydrolysate with antioxidant activity. In this study, ultrafiltration, gel chromatography, and LC-MS/MS were employed to isolate and identify bioactive peptides from the hydrolysate. The results revealed that the hydrolysate exhibited antioxidant activity, pancreatic cholesterol esterase inhibitory activity, pancreatic lipase inhibitory activity, and α-glucosidase inhibitory activity. Molecular docking using AutoDock Tools 1.5.6 was performed to analyze the interactions of peptides with CD38 and Keap1, leading to the identification of five potentially bioactive peptides: VPPFY, IMLFP, LPFLF, FLPF, and FPRIM. These peptides formed hydrogen bonds and hydrophobic interactions with CD38 and Keap1, demonstrating strong DPPH radical scavenging and superoxide anion radical scavenging capacities. This study highlights the multifunctional bioactive potential of these peptides, offering insights into their therapeutic applications. The findings provide a novel approach for the effective utilization of mussel resources and highlight their potential application value in the development of functional foods. Full article

In this study, camel milk (CM) and Gir cow milk (GCM) were fermented through cofermentation via yeast–lactic cultures, i.e., Lacticaseibacillus rhamnosus (M9, MTCC 25516) and Saccharomyces cerevisiae (WBS2A, MG101828), and their antioxidant and antidiabetic effectiveness were studied. To optimize the growth conditions, the level of proteolysis was evaluated by exploring various inoculation levels (1.5, 2.0 and 2.5%) as well as incubation durations (0, 12, 24, 36 and 48 h). Peptides were extracted and purified through 2D gel electrophoresis as well as SDS–PAGE. Water-soluble extracts (WSEs) of ultrafiltered (UF) peptide fractions were evaluated via reversed-phase high-performance liquid chromatography (RP-HPLC) to identify the peptide segments. By applying the Peakview tool, peptide sequences obtained from liquid chromatography–mass spectrometry (LC/MS) were reviewed by comparison with those in the BIOPEP database. Furthermore, the elevated levels of TNF-α, IL-6, IL-1β and nitric oxide (NO) in RAW 267.4 cells treated with lipopolysaccharide (LPS) are considerably lower than those in cultured CM and GCM. Protein macromolecules in CMs and GCMs have been captured via confocal laser scanning microscopy (CLSM) and Fourier transform infrared (FTIR) spectroscopy both before and after fermentation. Full article

Background: Human proteins exist in numerous modifications—proteoforms—which are promising targets for biomarker studies. In this study, we aimed to generate comparative proteomics data, including proteoform patterns, from hepatocellular carcinoma (HCC) and nonmalignant liver tissues. Methods: To investigate protein profiles and proteoform patterns, we employed a panoramic, integrative top-down proteomics approach: two-dimensional gel electrophoresis (2DE) coupled with liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS). Results: We visualized over 2500 proteoform patterns per sample type, enabling the identification of distinct protein signatures and common patterns differentiating nonmalignant and malignant liver cells. Among these, 1270 protein patterns were uniformly observed across all samples. Additionally, 38 proteins—including pyruvate kinase PKM (KPYM), annexin A2 (ANXA2), and others—exhibited pronounced differences in proteoform patterns between nonmalignant and malignant tissues. Conclusions: Most proteoform patterns of the same protein were highly similar, with the dominant peak corresponding to theoretical (unmodified) protein parameters. However, certain proteins displayed altered proteoform patterns and additional proteoforms in cancer compared to controls. These proteins were prioritized for further characterization. Full article

Background: The present work is devoted to the biological effects of sol–gel-derived silica (Si)–copper (Cu) nanomaterials. Methods and Results: Tetraethyl orthosilane (TEOS) was used as a silica precursor; copper was introduced as a solution in ethanol with Cu(OH)₂. The obtained samples were denoted as Si/Cu (gel) and Si/Cu/500 (500 °C heat-treated). Their phase formation and morphology were studied by XRD and SEM. The antibacterial activity was tested by two Gram-positive bacteria, three Gram-negative bacteria, and two types of eukaryotic species. Most bacteria were more sensitive to Si/Cu/500 materials than to Si/Cu (gel). The yeasts were more sensitive to Si/Cu (gel). The new nanomaterials were tested for oxidant activity at pH 7.4 (physiological) and pH 8.5 (optimal) in three model systems by the chemiluminescent method. They significantly inhibited the generation of free radicals and ROS. This result underlines their potential as regulators of the free radical processes in living systems. The epithelial tumor cell lines appeared more sensitive than the non-transformed fibroblasts, likely due to their metabolic activity and proliferation rates, leading to greater accumulation of the substances. Using Daphnia magna, the ecotoxicity study showed that the LC₅₀ was reached at 1 mg/L of Si/Cu/500. Si/Cu (gel) was more toxic. Conclusions: Our results reveal the potential of these nanohybrids to be applied in living, eukaryotic systems. The cytotoxicity evaluation showed higher tolerance of normal, non-transformed cells, in concurrence with the oxidation tests. Full article

During radiotherapy, X-rays can deliver significant doses of ionising radiation to both cancerous and healthy tissue, often leading to undesirable side effects that compromise patient outcomes. While the cellular effects of such therapeutic X-ray exposures are well studied, the impact on extracellular matrix (ECM) proteins remains poorly understood. This study characterises the response of ECM proteins, including the major tissue components collagen I and fibronectin (FN), to X-ray doses similar to those used in clinical practice (50 Gy, as employed in breast radiotherapy, and 100 Gy), using a combination of gel electrophoresis, biochemical assays, and mass spectrometry-based peptide location fingerprinting (PLF) analysis. In purified protein solutions, 50 Gy X-ray exposure led to the fragmentation of constituent collagen I α chains. Irradiation of purified plasma FN (pFN) induced localised changes in peptide yields (detected by liquid chromatography and tandem mass spectrometry (LC-MS/MS) and PLF) and enhanced its binding to collagen I. In complex environments, such as newly synthesised fibroblast-derived ECM and mature ex vivo breast tissue, X-ray exposure induced peptide yield changes in not only collagen I and FN but also key basement membrane proteins, including collagen IV, laminin, and perlecan. Intracellular proteins associated with gene expression (RPS3, MeCP2), the cytoskeleton (moesin, plectin), and the endoplasmic reticulum (calnexin) were also found to be impacted. These X-ray-induced structural changes may impair the ECM integrity and alter cell–ECM interactions, with potential implications for tissue stiffening, fibrosis, and impaired wound healing in irradiated tissues. Full article

Chestnut blight, caused by Cryphonectria parasitica (Murrill) M.E. Bar, is a destructive fungal disease threatening chestnut cultivation and production. In response to the limitations of chemical control, biological control using antagonistic microbes has gained increasing attention. A rhizosphere-derived bacterium, strain D39, was isolated from healthy chestnut trees and identified as Bacillus amyloliquefaciens based on morphological characteristics and the phylogenetic analysis of 16S rRNA and gyrA genes. The antifungal activity of strain D39 against C. parasitica was evaluated using dual-culture and double-layer Oxford cup assays. The strain exhibited broad-spectrum and stable antagonistic effects and harbored five key genes associated with antimicrobial compound biosynthesis (srfAA, ituC, fenD, bmyB, and bacA), as confirmed by PCR. A 145 kDa extracellular protein with strong antifungal activity was extracted and purified by ammonium sulfate precipitation, DEAE ion-exchange chromatography, and Sephadex G-75 gel filtration. LC-MS analysis identified the protein as a serine peptidase belonging to the S8 family, and its structure was predicted using multiple bioinformatic tools. In pot experiments, treatment with the strain D39 significantly reduced disease severity, achieving control efficiencies of 66.07% and 70.89% at 10 and 20 days post-inoculation, respectively. These results demonstrate that B. amyloliquefaciens D39 has strong potential as a biocontrol agent against chestnut blight, offering an effective and environmentally friendly alternative for disease management. Full article

To validate the long-term performance of self-developed limestone calcined clay cement (LC3), this study evaluated the durability performance of LC3 produced using calcined excavated spoil. Results showed that LC3 exhibited a faster chloride adsorption rate than OPC, achieving peak binding capacity within 14 days, although its total chloride-binding capacity was slightly lower. The chloride diffusion coefficient of LC3 was approximately one order of magnitude lower than that of OPC, enhancing chloride resistance. However, LC3 demonstrated weaker carbonation resistance due to complete decomposition of portlandite (Ca(OH)₂) and ettringite (AFt), alongside partial degradation of calcium silicate hydrate (C-S-H) gels, resulting in pore structure coarsening. Compared to LC3 made with commercial metakaolin (K0), the self-developed LC3 using K1 and K2 clays from excavated spoil showed comparable chloride-binding capacity but slightly weaker chloride penetration resistance. Its carbonation resistance surpassed K0-based LC3. Overall, the self-developed LC3 matched commercial metakaolin-based LC3 in durability, validating the use of locally sourced clays. Producing LC3 from calcined excavated spoil addresses environmental challenges associated with spoil disposal while delivering satisfactory durability. Full article

In this study, a global response analysis was performed to explore the mechanism of action of Usnic acid and its synergy with Norfloxacin, a well-known quinolone antibiotic to which MRSA clinical isolates showed resistance (MIC, 500 µg/mL). A microdilution assay, a growth kinetics analysis, a microscopic analysis, and cell-based assays consistently showed that Usnic acid possesses strong anti-staphylococcal activity (MIC, 7.8 µg/mL), causes cell leakage, modulates efflux pump activity, and synergizes with Norfloxacin against the multi-drug-resistant clinical isolate MRSA 2071. Whole-cell proteome profiling using gel-free proteomics-based nano-LC-ESI-QTOF-MS/MS revealed several proteins whose expression was significantly modulated by Usnic acid and Norfloxacin alone or in combination. Usnic acid downregulated the abundance of RNA polymerase subunits (RpoB and RpoC), carbamoyl phosphate synthase large subunit (PyrAB), chaperone (GroEL), and adenylosuccinate synthetase (PurA). Interestingly, proteins found to be upregulated in the presence of Usnic acid and Norfloxacin included oxidative-stress-related proteins such as peroxidase (Tpx), alkyl hydroperoxide reductase (AphC), and general stress protein (UspA). This study clearly shows that Usnic acid affects numerous cellular targets and can potentiate the action of Norfloxacin. Furthermore, an in vivo study showed that UA at low concentrations prevents body weight gain, but changes in other tested toxicological parameters were found to be within normal limits. Thus, UA at low doses appears to be a promising candidate for repurposing old antibiotics through combination therapy against MRSA infections. Full article

Ralstonia solanacearum is an important pathogen causing bacterial wilt in pepper (Capsicum annuum L.). The concurrent infection of R. solanacearum and root-knot nematodes (Meloidogyne spp.) exacerbates the severity of bacterial wilt in pepper. Utilizing plant endophytic bacteria to control these mixed diseases is a viable strategy. Waltheria indica L. (Sterculiaceae) is a traditional medicine plant. A total of 209 endophytic bacteria were isolated from W. indica, and Pseudomonas aeruginosa W-126 showed an efficient antagonistic effect against R. solanacearum. Based on active compound tracking principles, a compound was isolated through silica gel column chromatography and preparative HPLC combined with TLC analysis. It was identified as phenazine-1-carboxamide (PCN) by spectral techniques (ESI-MS, ¹H-NMR, ¹³C-NMR). PCN displayed excellent inhibitory activity against R. solanacearum, with an EC₅₀ of 64.16 μg/mL in vitro. In addition, it showed certain nematocide activity, with an LC₅₀ value of 118.63 μg/mL at 72 h. PCN also showed certain inhibitory activity against five other phytopathogenic bacteria. The structure−activity relationship indicated that the phenazine skeleton and acylamide groups were the key pharmacophores for the activity of phenazine-related compounds against R. solanacearum. PCN controlled the complex diseases of R. solanacearum and M. incognita in a pot experiment, with respective 51.41 and 39.80% inhibitory rates. The exploration of secondary metabolites of biocontrol bacteria can provide reference for the development of novel and efficient pesticides. Full article

Ensuring the authenticity of Mozzarella di Bufala Campana (MdBC), a Protected Designation of Origin (PDO) cheese, is essential for regulatory enforcement and consumer protection. This study evaluates a multi-technology analytical platform developed to detect adulteration due to the addition of non-buffalo milk or non-PDO buffalo milk in PDO dairy buffalo products. Peripheral laboratories use gel electrophoresis combined with polyclonal antipeptide antibodies for initial screening, enabling the detection of foreign caseins, including those originating outside the PDO-designated regions. For more precise identification, Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) differentiates species by detecting proteotypic peptides. In cases requiring confirmation, nano-liquid chromatography coupled to electrospray tandem mass spectrometry (nano-LC-ESI-MS/MS) is used in central state laboratories for the highly sensitive detection of extraneous milk proteins in PDO buffalo MdBC cheese. On the other hand, analysis of the pH 4.6 soluble fraction from buffalo blue cheese identified 2828 buffalo-derived peptides and several bovine specific peptides, confirming milk adulteration. Despite a lower detection extent in the pH 4.6 insoluble fraction following tryptic hydrolysis, the presence of bovine peptides was still sufficient to verify fraud. This integrated proteomic approach, which combines electrophoresis and mass spectrometry technologies, significantly improves milk adulteration detection, providing a robust tool to face increasingly sophisticated fraudulent practices. Full article

Food-derived bioactive peptides have attracted considerable research interest and are increasingly utilized as functional ingredients in the food industry. In this study, the immunomodulatory peptides were isolated and purified from Thunnus albacares (T. albacares) enzymatic hydrolysates of muscles using gel chromatography and RP-HPLC, and their amino acid sequences were identified via LC-MS/MS. A total of six peptides were selected based on their affinity to toll-like receptors. Subsequently, these peptides were synthesized to confirm the immunomodulatory activities in vitro. Among all the tested peptides, two peptides, HDCDLLR and YGSVELDELGK, significantly enhanced cell proliferation and phagocytosis and increased the production of tumor necrosis factor-α (TNF-α), nitric oxide (NO), and interleukin-6 (IL-6). Molecular docking analysis indicated that these two peptides could stably bind to the receptors through hydrogen bonds and electrostatic and hydrophobic interactions. These findings suggested that peptides from enzymatic hydrolysates of T. albacares could be promising candidates for developing immunomodulatory agents in functional foods. Full article

Antimicrobial peptides (AMPs) are biocompatible and biodegradable, making them an attractive alternative to traditional antimicrobial agents and chemical preservatives. Here, a novel α-helix amphiphilic anionic AMP Lc149 was screened from a large yellow croaker (Larimichthys crocea) using a Bacillus subtilis expression system. Lc149 is a hypothesized protein fragment not annotated in the genome of a large yellow croaker. Both extracellular protein and recombinant Lc149 (rLc149) exhibited significant killing effects against Gram-negative Escherichia coli and Vibrio harveyi. Scanning and transmission electron microscopy revealed that rLc149 had the ability to disrupt bacterial cell membranes, causing irregular cell morphology, severe cell membrane damage, cytoplasm agglutination, and intracellular content leakage. Confocal laser scanning microscopy and flow cytometry further confirmed bacterial cell destruction and mortality rates of over 80%. Gel retardation assays and SDS-PAGE electrophoresis showed that rLc149 was unable to bind to bacterial DNA, but did reduce bacterial protein contents. Additionally, rLc149 maintained antibacterial activity against E. coli and V. harveyi upon exposure to temperatures of 25–100 °C, UV radiation time of 0–60 min, pH levels of 3–12, and different proteases. Biosafety assays revealed low hemolytic toxicity to erythrocytes of large yellow croaker, rabbit, and shrimp, and low cytotoxicity to large yellow croaker kidney cells and HEK 293T cells. More deeply, rLc149 also possessed significant killing activity against parasites. Therefore, rLc149 can be considered an antibacterial and antiparasitic drug in fisheries. Full article

Legumes, a dietary staple for centuries, have seen an influx of conventional and unconventional varieties to cater to human care conscious consumers. These legumes often undergo pretreatments like baking, soaking, or boiling to mitigate the presence of non-proteinogenic amino acids (NPAAs) and reduce associated health risks. The recent tara flour health scare, linked to the NPAA baikiain, emphasizes the need for robust analytical methods to ensure the safety and quality of both traditional and novel plant-based protein alternatives. While traditional techniques provide insights into protein and non-proteinogenic amino acid profiles, modern liquid chromatography-mass spectrometry (LC-MS) offers superior sensitivity and specificity for NPAA detection. This study employed an LC-QToF method with MS/MS analysis to comprehensively map the distribution of free NPAAs and proteinogenic amino acids (PAAs) in various legume samples. A total of 47 NPAAs and 20 PAAs were identified across the legume samples, with at least 7–14 NPAAs detected in each sample. Sulfur-containing NPAAs, such as S-methyl-L-cysteine, γ-glutamyl-S-methyl cysteine, and S-methyl homoglutathione, were predominantly found in Phaseolus and Vigna species. Cysteine and methionine were the sulfur-containing PAAs identified. Gel electrophoresis and soluble protein quantification were also conducted to understand legume protein composition holistically. This orthogonal approach provides a valuable tool for ensuring the overall quality of plant-based proteins and may aid in investigating food poisoning or outbreaks related to such products. Full article

Despite extensive research on Bacillus sp. as lignin degraders, the enzyme mechanisms involved, particularly in Bacillus cereus isolated from termite guts, remain unclear. In this study, the selected Bacillus cereus was fermented to extract the lignin-degrading enzymes to identify the enzymes responsible for lignin degradation using the sample substrate empty fruit bunch (EFB) as their sole carbon source. After 7 days of submerged fermentation (SmF), the crude enzyme was extracted, and SDS-PAGE gel was used to determine the weight of the proteins, and bands with sizes of 20 kDa–97 kDa were extracted for further analysis. The extracted proteins were partially characterized and sequenced using liquid chromatography–mass spectrometry (LC–MS/MS). The results identified 11 enzymes that are responsible for lignin degradation, such as 4-aminobutyrate aminotransferase (GABA), amidohydrolase, chemotaxis protein, serine hydrolase, GMC family protein, glycosyltransferase, phosphate binding protein PstS, ABC transporter ATP-binding protein, heme peroxidase, nitrate reductase, and nitrite reductase. The value of the mutual relationships between all the enzymes in Bacillus cereus indicates the synergistic mechanism under carbon scrutinization. Also, the peptides sequenced in this study identified various uncharacterized proteins and hypothetical proteins that might not be discovered for their protein functions. Further analysis is essential to uncover more lignin degradation enzymes that can work synergically for paper and pulp bioprocessing. Full article