Search Results (695)

Background/Objectives: Cancer cells rely on metabolic reprogramming that is supported by altered mitochondrial redox status and an increased demand for NAD⁺. Over expression of Nampt, the rate-limiting enzyme of the NAD⁺ biosynthesis salvage pathway, is common in breast cancer cells, and more so in triple negative breast cancer (TNBC) cells. Targeting the salvage pathway has been pursued for cancer therapy. However, TNBC cells have heterogeneous responses to Nampt inhibition, which contributes to the diverse outcomes. There is a lack of imaging biomarkers to differentiate among TNBC cells under metabolic stress and identify which are responsive. We aimed to characterize and differentiate among a panel of TNBC cell lines under NAD-deficient stress and identify which subtypes are more dependent on the NAD salvage pathway. Methods: Optical redox imaging (ORI), a label-free live cell imaging microscopy technique was utilized to acquire intrinsic fluorescence intensities of NADH and FAD-containing flavoproteins (Fp) thus the mitochondrial redox ratio Fp/(NADH + Fp) in a panel of TNBC cell lines. Various fluorescence probes were then added to the cultures to image the mitochondrial ROS, mitochondrial membrane potential, mitochondrial mass, and cell number. Results: Various TNBC subtypes are sensitive to Nampt inhibition in a dose- and time-dependent manner, they have differential mitochondrial redox responses; furthermore, the mitochondrial redox indices linearly correlated with mitochondrial ROS induced by various doses of a Nampt inhibitor. Moreover, the changes in the redox indices correlated with growth inhibition. Additionally, the redox state was found fully recovered after removing the Nampt inhibitor. Conclusions: This study supports the utility of ORI in rapid metabolic phenotyping of TNBC cells under NAD-deficient stress to identify responsive cells and biomarkers of treatment response, facilitating combination therapy strategies. Full article

This study describes the applicability of the fluorescence polarization assay (FPA) based on the use of FITC-labeled oligosaccharide tracers of defined structure for the measurement of active lysozyme in hen egg white. Depending on the oligosaccharide chain length of the tracer, this method detects both the formation of the enzyme-to-tracer complex (because of lectin-like, i.e., carbohydrate-binding action of lysozyme) and tracer splitting (because of chitinase activity of lysozyme). Evaluation of the fluorescence polarization dynamics enables simultaneous measurement of the chitinase and lectin activities of lysozyme, which is crucial for its detection in complex biological systems. Hen egg white lysozyme (HEWL), unlike human lysozyme (HL), formed a stable complex with the chitotriose tracer that underwent no further transformations. This fact allows for easy measurement of the carbohydrate-binding activity of the HEWL. The results of the lysozyme activity measurement for hen egg samples obtained through the FPA correlated with the results obtained using the traditional turbidimetry method. The FPA does not have the drawbacks of turbidimetry, which are associated with the need to use bacterial cells that cannot be precisely standardized. Additionally, FPA offers advantages such as rapid analysis, the use of compact equipment, and standardized reagents. Therefore, the new express technique for measuring the lysozyme activity is applicable for evaluating the complex biomaterial, including for the purposes of food product quality control. Full article

Alfalfa is a widely grown forage with a high crude protein content. Clarifying the interactions between light quality and nitrogen level on yield and nitrogen metabolism can purposely improve alfalfa productivity in plant factories with artificial light (PFAL). In this study, the growth, quality, and nitrogen metabolism of alfalfa grown in PFAL were investigated using three nitrate-nitrogen concentrations (10, 15, and 20 mM, labeled as N₁₀, N₁₅, and N₂₀) and continuous light (CL) with two light qualities (red–blue and red–blue–green light, labeled as RB-C and RBG-C). The results showed that the adaptation performance of alfalfa to nitrogen concentrations differed under red–blue and red–blue–green CL. Plant height, stem diameter, leaf area, yield, Chl a + b, Chl a, Chl b, crude protein contents, and NiR activity under the RB-CN₁₅ treatment were significantly higher than RB-CN₁₀ and RB-CN₂₀ treatments. The RB-CN₂₀ treatment showed morphological damage, such as plant dwarfing and leaf chlorosis, and physiological damage, including the accumulation of proline, H₂O₂, and MDA. However, the difference was that under red–blue–green CL, the leaf area, yield, and Chl a + b, carotenoid, nitrate, and glutamate contents under RBG-CN₂₀ treatment were significantly higher than in the RBG-CN₁₀ and RBG-CN₁₅ treatments. Meanwhile, the contents of soluble sugar, starch, and cysteine were significantly lower. However, the crude protein content reached 21.15 mg·g⁻¹. The fresh yield, dry yield, stomatal conductance, leaf area, plant height, stem diameter, crude protein, GS, and free amino acids of alfalfa were positively correlated with increased green light. In addition, with the increase in nitrogen concentration, photosynthetic capacity, NiR, and GOGAT activities increased, promoting growth and improving feeding value. The growth, yield, photosynthetic pigments, carbon, nitrogen substances, and enzyme activities of alfalfa were significantly affected by the interaction between nitrogen concentration and light quality, whereas leaf/stem ratio and DPPH had no effect. In conclusion, RB-CN15 and RBG-CN₂₀ are suitable for the production of alfalfa in PFAL, and green light can increase the threshold for the nitrogen concentration adaptation of alfalfa. Full article

Irumamycin (Iru) is a complex polyketide with pronounced antifungal activity produced by a type I polyketide (PKS) synthase. Iru features a unique hemiketal ring and an epoxide group, making its biosynthesis and the structural diversity of related compounds particularly intriguing. In this study, we performed a detailed analysis of the iru biosynthetic gene cluster (BGC) to uncover the mechanisms underlying Iru formation. We examined the iru PKS, including the domain architecture of individual modules and the overall spatial structure of the PKS, and uncovered discrepancies in substrate specificity and iterative chain elongation. Two potential pathways for the formation of the hemiketal ring, involving either an olefin shift or electrocyclization, were proposed and assessed using ¹⁸O-labeling experiments and reaction activation energy calculations. Based on our findings, the hemiketal ring is likely formed by PKS-assisted double bond migration and TE domain-mediated cyclization. Furthermore, putative tailoring enzymes mediating epoxide formation specific to Iru were identified. The revealed Iru biosynthetic machinery provides insight into the complex enzymatic processes involved in Iru production, including macrocycle sculpting and decoration. These mechanistic details open new avenues for a targeted architecture of novel macrolide analogs through synthetic biology and biosynthetic engineering. Full article

Alterations in cellular energy metabolism are a hallmark of cancer and lactate dehydrogenase (LDH) enzymes are overexpressed in many cancers regardless of sufficient oxygen and functional mitochondria. Further, L-lactate plays signaling roles in multiple cell types. We evaluated the effect of singlet oxygen and hypochlorous acid (HOCl) on pig heart LDH-B, which shares 97% homology with human LDH-B. Singlet oxygen was generated photochemically using methylene blue or the chlorophyll metabolites, pheophorbide A and chlorin e6. Singlet oxygen induced protein crosslinks observed by SDS-PAGE under reducing conditions and inhibited LDH-B activity. Ascorbate, hydrocaffeic acid, glutathione and sodium azide were employed as singlet oxygen scavengers and shown to protect LDH-B. Using fluorescein-modified maleimide, no changes in cysteine availability as a result of singlet oxygen damage were observed. This was in contrast to HOCl, which induced the formation of disulfides between LDH-B subunits, thereby decreasing LDH-B labeling with fluorescein. HOCl oxidation inhibited LDH-B activity; however, disulfide reduction did not restore it. LDH-B cysteines were resistant to millimolar H₂O₂, chloramines and Angeli’s salt. In the absence of pyruvate, LDH-B enhanced NADH oxidation in a chain reaction initiated by singlet oxygen that resulted in H₂O₂ formation. Once damaged by either singlet oxygen or HOCl, NADH oxidation by LDH-B was impaired. Full article

Background: The aim of this study was to determine the effect of Sparstolonin B (SsnB) on cell proliferation and apoptosis in human breast cancer (MCF-7) and human ovarian epithelial cancer (OVCAR-3) cell lines in the presence and absence of estradiol hemihydrate (ES). Phosphoinositol-3 kinase (PI3K), phosphorylated protein kinase B alpha (p-AKT), phosphorylated mTOR (mechanistic target of rapamycin) signaling proteins, and sphingomyelin/ceramide metabolites were also measured within the scope of the study. Methods: The anti-proliferative effects of SsnB therapy were evaluated over a range of times and concentrations. Cell proliferation was determined by measuring the Proliferating Cell Nuclear Antigen (PCNA). PCNA was quantified by ELISA and cell distribution was assessed by immunofluorescence microscopy. MTT analysis was used to test the vitality of the cells, while LC-MS/MS was used to analyze the amounts of ceramides (CERs), sphingosine-1-phosphate (S1P), and sphingomyelins (SMs). TUNEL labeling was used to assess apoptosis, while immunofluorescence staining and enzyme-linked immunosorbent assay (ELISA) were used to measure the levels of PI3K, p-AKT, and p-mTOR proteins. Results: Sparstolonin B administration significantly decreased cell viability in MCF-7 and OVCAR-3 cells both in the presence and absence of ES, while it did not cause toxicity in healthy human fibroblasts. In comparison to controls, cancer cells treated with SsnB showed a significant drop in the levels of S1P, PI3K, p-AKT, and p-mTOR. In cancer cells cultured with SsnB, a significant increase in intracellular concentrations of C16-C24 CERs and apoptosis was observed. Conclusions: SsnB downregulated the levels of S1P, PI3K, p-AKT, and p-mTOR while reducing cell proliferation and promoting ceramide buildup and apoptosis. Full article

L-arginine: glycine amidinotransferase (AGAT) gained academic interest as the rate-limiting enzyme in creatine biosynthesis and its role in the regulation of creatine homeostasis. Of clinical relevance is the diagnosis of patients with AGAT deficiency but also the potential role of AGAT as therapeutic target for the treatment of another creatine deficiency syndrome, guanidinoacetate N-methyltransferase (GAMT) deficiency. Applying a stable isotope-labeled substrate method, we utilized ARG 15N₂ (ARG-δ2) and GLY 13C₂15N (GLY-δ3) to determine the rate of 1,2-13C₂,15N₃ guanidinoacetate (GAA-δ5) formation to assess AGAT activity in various mouse tissue samples and human-derived cells. Following modification and optimization of the assay, we analyzed AGAT activity in several mouse organs. The K_m and V_max of AGAT in mouse kidney for GLY-δ3 were 2.06 mM and 6.48 ± 0.26 pmol/min/mg kidney, and those for ARG-δ2, they were 2.67 mM and 2.17 ± 0.49 pmol/min/mg kidney, respectively. Our results showed that mouse kidneys had the highest levels of enzymatic activity, followed by brain and liver, with 4.6, 1.8, and 0.4 pmol/min/mg tissue, respectively. Both the heart and muscle had no detectable levels of AGAT activity. We noted that due to interference with arginase in the liver, performing the enzyme assay in liver homogenates required the addition of Nor-NOHA, an arginase inhibitor. In immortalized human cell lines, we found the highest levels of AGAT activity in RH30 cells, followed by HepaRG, HAP1, and HeLa cells. AGAT activity was readily detectable in lymphoblasts and leukocytes from healthy controls. In our assay, AGAT activity was not detectable in HEK293 cells, in human fibroblasts, and in the lymphoblasts of a patient with AGAT deficiency. Our results demonstrate that this enzyme assay is capable of accurately quantifying AGAT activity from both tissues and cells for diagnostic purposes and research. Full article

Fusarium oxysporum, the pathogen responsible for apple replant disease (ARD), is seriously threatening the apple industry globally. We investigated the antagonistic properties of Trichoderma strains against F. oxysporum HS2, aiming to find a biological control solution to minimize the dependence on chemical pesticides. Two of the thirty-one Trichoderma strains assessed through plate confrontation assays, L7 (Trichoderma atroviride) and M19 (T. longibrachiatum), markedly inhibited = F. oxysporum, with inhibition rates of 86.02% and 86.72%, respectively. Applying 1 × 10⁶ spores/mL suspensions of these strains notably increased the disease resistance in embryonic mung bean roots. Strains L7 and M19 substantially protected Malus robusta Rehd apple rootstock from ARD; the plant height, stem diameter, leaf number, chlorophyll content, and defense enzyme activity were higher in the treated plants than in the controls in both greenhouse and field trials. The results of fluorescent labeling confirmed the effective colonization of these strains of the root soil, with the number of spores stabilizing over time. At 56 days after inoculation, the M19 and L7 spore counts in various soils confirmed their persistence. These results underscore the biocontrol potential of L7 and M19 against HS2, offering valuable insights into developing sustainable ARD management practices. Full article

Oxysophocarpine (OSC), a quinolizidine alkaloid, shows neuroprotective potential, though its mechanisms are unclear. The aim of the present study was to investigate the neuroprotective effects of OSC through the nuclear factor erythroid 2−related factor 2 (Nrf2)/ heme oxygenase−1 (HO–1) signaling pathway using the HT–22 cell line. Assessments of cell viability were conducted utilizing the 3−(4,5−dimethylthiazol−2−yl)−2,5−diphenyltetrazolium bromide (MTT) assay. Assessments of oxidative stress (OS) were conducted through the quantification of reactive oxygen species (ROS). The integrity of the mitochondrial membrane potential (MMP) was scrutinized using fluorescent probe technology. Apoptosis levels were quantified using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The trafficking of Nrf2 within the cell nucleus was examined through immunofluorescence analysis. Furthermore, Western blotting (WB) was applied to evaluate the expression levels of proteins implicated in apoptosis and the Nrf2/HO–1 pathway. To further probe the influence of OSC on the overexpression of antioxidant enzymes, cells were subjected to transfection with HO–1 siRNA. The results showed that OSC inhibited glutamate-induced OS, as evidenced by reduced cell viability and ROS levels. Furthermore, the apoptotic condition induced by glutamate in HT–22 cells was significantly reduced following OSC treatment. More interestingly, the Nrf2/HO–1 signaling pathway was upregulated following OSC treatment. These results suggest that OSC can exert neuroprotective effects by regulating the Nrf2/HO–1 pathway to inhibit neuronal cell apoptosis, potentially aiding in the treatment of neurodegenerative diseases. Full article

Background: Acute and chronic pre-traumatic cigarette smoke exposure increases morbidity and mortality after trauma and hemorrhage. In mice with a genetic deletion of the H₂S-producing enzyme cystathione-γ-lyase (CSE^−/−), providing exogenous H₂S using sodium thiosulfate (Na₂S₂O₃) improved organ function after chest trauma and hemorrhagic shock. Therefore, we evaluated the effect of Na₂S₂O₃ during resuscitation from blunt chest trauma and hemorrhagic shock on CSE^−/− mice with pre-traumatic cigarette smoke (CS) exposure. Since H₂S is well established as being able to modify energy metabolism, a specific focus was placed on whole-body metabolic pathways and mitochondrial respiratory activity. Methods: Following CS exposure, the CSE^−/− mice underwent anesthesia, surgical instrumentation, blunt chest trauma, hemorrhagic shock for over 1 h (target mean arterial pressure (MAP) ≈ 35 ± 5 mmHg), and resuscitation for up to 8 h comprising lung-protective mechanical ventilation, the re-transfusion of shed blood, fluid resuscitation, and continuous i.v. noradrenaline (NoA) to maintain an MAP ≥ 55 mmHg. At the start of the resuscitation, the mice randomly received either i.v. Na₂S₂O₃ (0.45 mg/g_bodyweight; n = 14) or the vehicle (NaCl 0.9%; n = 11). In addition to the hemodynamics, lung mechanics, gas exchange, acid–base status, and organ function, we quantified the parameters of carbohydrate, lipid, and protein metabolism using a primed continuous infusion of stable, non-radioactive, isotope-labeled substrates (gas chromatography/mass spectrometry) and the post-mortem tissue mitochondrial respiratory activity (“high-resolution respirometry”). Results: While the hemodynamics and NoA infusion rates did not differ, Na₂S₂O₃ was associated with a trend towards lower static lung compliance (p = 0.071) and arterial PO₂ (p = 0.089) at the end of the experiment. The direct, aerobic glucose oxidation rate was higher (p = 0.041) in the Na₂S₂O₃-treated mice, which resulted in lower glycemia levels (p = 0.050) and a higher whole-body CO₂ production rate (p = 0.065). The mitochondrial respiration in the heart, kidney, and liver tissue did not differ. While the kidney function was comparable, the Na₂S₂O₃-treated mice showed a trend towards a shorter survival time (p = 0.068). Conclusions: During resuscitation from blunt chest trauma and hemorrhagic shock in CSE^−/− mice with pre-traumatic CS exposure, Na₂S₂O₃ was associated with increased direct, aerobic glucose oxidation, suggesting a switch in energy metabolism towards preferential carbohydrate utilization. Nevertheless, treatment with Na₂S₂O₃ coincided with a trend towards worsened lung mechanics and gas exchange, and, ultimately, shorter survival. Full article

The interaction between gut-residing microorganisms plays a critical role in the pathogenesis of Crohn’s disease (CD), where microbiome dysregulation can alter immune responses, leading to unresolved local inflammation. The aim of this study is to analyze the immunomodulatory properties of a recently developed probiotic + amylase blend in the SAMP1/YitFc (SAMP) mouse model of CD-like ileitis. Four groups of SAMP mice were gavaged for 56 days with the following treatments: 1) probiotic strains + amylase (0.25 mg/100 µL PBS); 2) only probiotics; 3) only amylase; PBS-treated controls. Ilea were collected for GeoMx Digital Spatial Profiler (DSP) analysis and histological evaluation. Histology assessment for inflammation indicated a significantly reduced level of ileitis in mice administered the probiotics + amylase blend. DSP analysis showed decreased abundance of neutrophils and increased abundance of dendritic cells, regulatory T cells, and macrophages, with a significant enrichment of five intracellular pathways related to apoptosis, in probiotics + amylase-treated mice. Increased apoptosis occurrence was confirmed by (TdT)- deoxyuridine triphosphate (dUTP)-biotin nick end labeling assay. Our data demonstrate a beneficial role of the probiotic and amylase blend, highlighting an increased apoptosis of innate immunity-associated cell subsets, thus promoting the resolution of inflammation. Hence, we suggest that the developed probiotic enzyme blend may be a therapeutic tool to manage CD and therefore is a candidate formulation to be tested in clinical trials. Full article

A large portion of the world’s population has lactose intolerance. Fundamentally, this condition occurs when the small intestine does not produce enough of the lactase enzyme, which digests the disaccharide lactose in milk. Lactose avoiders might unconsciously decide to limit or exclude milk and dairy products from their diets. This group includes people with lactose intolerance, people with an allergy to milk protein, vegans, and those expressing personal preferences. Lactose avoiders are often self-reported as being milk intolerant. In this review, specific amounts of lactose in different types of milk and milk products are presented. The amounts of micro- and macronutrients in them are compared with the daily requirements established by accepted sources. Foods are suggested that can play vital roles in permanently avoiding lactose-containing dairy products, for example, brussels sprouts, as a good source of vitamin B1; kale, as a source of vitamin K; and cereals at breakfast for vitamin B6. Attention is paid to mature cheeses as they are extremely beneficial for health due to their rich vitamin and elemental compositions, and they are also suitable for people with lactose intolerance due to their low lactose content. This information is rarely provided on packaging. In addition, the current state of labeling for the presence of lactose in food and pharmaceutical products is discussed. The term “hidden lactose” is introduced to include added lactose in unexpected foods, drinks, and even medicines. Full article

The target of traditional immunological detection methods for milk allergens is usually the whole β-lactoglobulin molecule. However, thermal processes and hydrolysis can destroy the epitope of β-lactoglobulin and interfere with its accurate detection and labeling in prepackaged foods, posing a health risk to milk-allergic patients. There currently remains a need to excavate and locate recognition sites for β-lactoglobulin in thermally processed and hydrolyzed products. Therefore, a stable epitope of β-lactoglobulin (CAQKKIIAEKTKIPAVFKIDA) was selected as the ideal recognition site, and an indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed using an antibody against this stable β-lactoglobulin epitope in order to improve the detection of β-lactoglobulin in thermally processed and hydrolyzed foods in this study. The stable epitope of β-lactoglobulin was selected using a molecular dynamics simulation, and the binding ability of anti-stable epitope antibodies was characterized using indirect ELISA and indirect competitive ELISA. The limit of detection (LOD) and limit of quantitation (LOQ) of the established ELISA were 0.25 and 1.07 mg·kg⁻¹, respectively. Furthermore, the developed ELISA only showed cross-reactivity to goat milk among 23 common foods, therefore exhibiting high specificity to bovine β-lactoglobulin. In addition, the developed ELISA was able to effectively detect β-lactoglobulin residue in processed commercial foods and hydrolyzed formula milk powder. Our findings provide a novel strategy for accurately detecting milk allergens based on stable epitope recognition in thermally processed and hydrolyzed foods. Full article

Labneh is a popular fermented dairy product, which contemporarily has diversified into a varied range of styles, formulated with the inclusion of multiple additives, and is sourced across the globe. This has driven labneh’s emergence as a complex product with varying textural and rheological characteristics. The lack of scientific literature about labneh products available in the United Arab Emirates (UAE) market and their characterization has prompted this study. A detailed UAE market analysis of labneh for label, formulation, nutrition, and price variability was conducted. Surveyed labneh products were categorized as unpackaged, multinational company (MNC), small and medium enterprise (SME), and specialty products. They differed in manufacturing, such as acid ± enzyme coagulation with/without post-fermentation heat treatment, and contained various stabilizers, emulsifiers, preservatives, and processing aids. Interestingly, almost equal proportions, 64.7% and 67%, of MNC and SME labneh contained additives, respectively. All MNC labneh were post-heat-treated, in contrast to only 7% of SME labneh. Organic labneh and non-bovine milk-based labneh are not yet widely available. The second part of the study involved the physicochemical characterization of a select number of packaged labneh that were categorized in accordance with fat content as high-fat (17–18%), full-fat (7.1–8%), and lite-fat (3.5–4.5%). High-fat labneh showed a significantly higher complex viscosity, complex modulus, hardness, adhesiveness, stringiness, and fracturability, followed by lite-fat labneh compared to full-fat labneh, especially when it contained pectin. Full-fat labneh with added gums (and starch) and high-fat labneh with gums showed a significantly lower complex modulus compared to their respective control labneh. This study highlights the variety of commercial labneh products available and differences in their formulation, manufacturing, and composition, and provides specific dependencies of materials with their physicochemical characteristics. Full article

Biological warfare agents are infectious microorganisms or toxins capable of harming or killing humans. Francisella tularensis is a potential bioterrorism agent that is highly infectious, even at very low doses. Biosensors for biological warfare agents are simple yet reliable point-of-care analytical tools. Developing highly sensitive, reliable, and cost-effective label-free DNA biosensors poses significant challenges, particularly when utilizing traditional techniques such as fluorescence, electrochemical methods, and others. These challenges arise primarily due to the need for labeling, enzymes, or complex modifications, which can complicate the design and implementation of biosensors. In this study, we fabricated Graphene Quantum dot (GQD)-functionalized biosensors for highly sensitive label-free DNA detection. GQDs were immobilized on the surface of screen-printed gold electrodes via mercaptoacetic acid with a thiol group. The single-stranded DNA (ssDNA) probe was also immobilized on GQDs through strong π−π interactions. The ssDNA probe can hybridize with the ssDNA target and form double-stranded DNA, leading to a decrease in the effect of GQD but a positive shift associated with the increase in DNA concentration. The specificity of the developed system was observed with different microorganism target DNAs and up to three-base mismatches in the target DNA, effectively distinguishing the target DNA. The response time for the target DNA molecule is approximately 1010 s (17 min). Experimental steps were monitored using UV/Vis spectroscopy, Atomic Force Microscopy (AFM), and electrochemical techniques to confirm the successful fabrication of the biosensor. The detection limit can reach 0.1 nM, which is two–five orders of magnitude lower than previously reported methods. The biosensor also exhibits a good linear range from 10⁵ to 0.01 nM and has good specificity. The biosensor’s detection limit (LOD) was evaluated as 0.1 nM from the standard calibration curve, with a correlation coefficient of R² = 0.9712, showing a good linear range and specificity. Here, we demonstrate a cost-effective, GQD-based SPGE/F. tularensis DNA test suitable for portable electrochemical devices. This application provides good perspectives for point-of-care portable electrochemical devices that integrate sample processing and detection into a single cartridge without requiring a PCR before detection. Based on these results, it can be concluded that this is the first enzyme-free electrochemical DNA biosensor developed for the rapid and sensitive detection of F. tularensis, leveraging the nanoenzyme and catalytic properties of GQDs. Full article