Search Results (31)

The enzymes in honey can originate not only from bees and the plants from which the bees collect pollen and nectar but also from feed provided by beekeepers. Enzymes that hydrolyze sucrose—present in honey (α-glucosidase) or honey adulterated with invert syrup (β-fructofuranosidase)—can be distinguished using zymography, where enzymatic bands are detected with nitroblue tetrazolium (NBT) after sugar removal via ultrafiltration. This method enables the identification of honey produced in hives that have been improperly fed with invert syrup, leading to the mixture of natural honey and syrup, and offers a practical tool to detect indirect adulteration. The NBT assay, in combination with ultrafiltration, was used to determine the isoelectric point of honey bee α-glucosidases. The pI value of 6.63 for isoforms found in the head, midgut, and natural honey extracts during winter can be attributed to α-glucosidase III. Two additional isoforms with isoelectric points of 5.20 and 5.77 were observed in the midgut extract and may correspond to α-glucosidase I and II. The difference between α-glucosidase and β-fructofuranosidase was confirmed using a substrate specificity test, followed by thin-layer chromatography, where it was confirmed that α-glucosidase from natural honey, bee head, and bee midgut does not hydrolyze raffinose, in contrast to yeast β-fructofuranosidase. Full article

This paper presents the optimization of storage conditions for textile dosimeters for ultraviolet radiation measurements, which are based on cotton-woven fabric and nitroblue tetrazolium chloride (NBT) as a radiation-sensitive compound. The results of changes in light reflectance and color coordinates depending on the storage time of the samples over six months from their manufacturing under various storage conditions are presented. The results obtained for cotton—NBT dosimeters, unirradiated and irradiated with a UVC dose of 100 mJ/cm², stored under the following conditions were compared: (i) at room temperature (23–25 °C, humidity 40–60%), without access to light; (ii) in a fridge (3–5 °C, humidity 70–90%), without access to light; (iii) in a freezer (−17 to −20 °C, humidity 80–90%), without access to light; and (iv) at room temperature (23–25 °C, humidity 40–60%), with access to light. Additionally, it was presented that the cotton–NBT dosimeters were suitable for 2D measurement of UV radiation doses after a period of eight months. The obtained results complement previous studies on cotton–NBT textile dosimeters and are crucial for determining the conditions of use and the expiry date of such systems. Full article

Background/Objectives: Parietin (PTN), a blue-light absorbing pigment from Teloschistes spp. lichens, exhibit photosensitizing properties via Type I (superoxide anion, O₂^•−) and Type II (singlet oxygen, ¹O₂) mechanisms, inactivating bacteria in vitro after photoexcitation. We evaluate the in vitro antifungal activity of PTN against Candida tropicalis biofilms under actinic irradiation, its role in O₂^•− and ¹O₂ production, and the cellular stress response. Methods: Minimum inhibitory concentration (MIC) of PTN was determined in C. tropicalis NCPF 3111 under dark and actinic light conditions. Biofilm susceptibility was assessed at MIC/2, MIC, MICx2, MICx4, and MICx6 in the same conditions, and viability was measured by colony-forming units. Photodynamic mechanisms were examined using Tiron (O₂^•− scavenger) or sodium azide (¹O₂ quencher). O₂^•− production was measured by the nitro-blue tetrazolium (NBT) reduction and nitric oxide (NO) generation by Griess assay. Total antioxidant capacity was studied by FRAP (Ferrous Reduction Antioxidant Potency) assay and superoxide dismutase (SOD) activity by NBT assay. Results: Photoexcitation of PTN reduced C. tropicalis biofilm viability by four logs at MICx2. Sodium azide partially reversed the effect, whereas Tiron fully inhibited it, indicating the critical role of O₂^•−. PTN also increased O₂^•− and NO levels, enhancing SOD activity and FRAP. However, this antioxidant response was insufficient to prevent biofilm photoinactivation. Conclusions: Photoinactivation of C. tropicalis biofilms by PTN is primarily mediated by O₂^•−, with a minor contribution from ¹O₂ and an imbalance in NO levels. These findings suggest PTN is a promising photosensitizer for antifungal photodynamic therapy. Full article

This study aimed to evaluate differences in the production of reactive oxygen species (ROS) by peripheral blood neutrophils in healthy dogs, dogs with superficial pyoderma, and dogs with sterile neutrophilic dermatitis using the nitroblue tetrazolium (NBT) reduction test. Additionally, the study assessed the potential of the NBT reduction test as a diagnostic tool to differentiate between these clinical conditions. A total of 28 dogs were divided into three groups: healthy (n = 10), superficial pyoderma (n = 10), and sterile neutrophilic dermatitis (n = 8). The results showed significantly higher neutrophil activation in dogs with sterile neutrophilic dermatitis (19 ± 10%) compared with healthy dogs (7.3 ± 5%) (p < 0.01) and dogs with superficial pyoderma (10 ± 2.8%) (p < 0.05). Dogs with sterile neutrophilic dermatitis and a high positive result for Leishmania spp. antibodies (n = 3) exhibited even higher NBT reduction rates (30 ± 4.6%) compared with seronegative dogs with sterile neutrophilic dermatitis (n = 5) (13 ± 5.3%) (p < 0.05). These findings suggest that the NBT reduction test could be useful for detecting systemic neutrophil activation in sterile neutrophilic dermatitis, especially when associated with Leishmania infection. However, no significant differences were observed between healthy dogs and those with superficial pyoderma, indicating that the test may not be effective in detecting systemic neutrophil activation in superficial bacterial infections. Full article

Nowadays, synthetic polymer (plastic) particles are ubiquitous in the environment. It is known that for several decades microplastics (MPs) have been accumulating in the World Ocean, becoming available to a large variety of marine organisms. Particularly alarming is the accumulation of aging plastic particles, as the degradation processes of such particles increase their toxicity. The diverse display of negative properties of aging MPs and its effect on biota are still poorly understood. In this study, in vitro experiments modeling the interaction of pristine and UV-irradiated aging polypropylene (PP) fragments with hemocytes and mitochondria of bivalve mollusks Mytilus sp. were performed. The appearance of free radicals in the environment was recorded by spectral characteristics of indicator dyes—methylene blue (MB) and nitroblue tetrazolium (NBT). It was found that due to photooxidation, aging PP fragments sorbed more than threefold MB on their modified surface compared to pristine samples of this polymer. Using NBT, the formation of reactive oxygen species in seawater in the presence of pristine and photoactivated PP was recorded. It was also found that photodegraded PP fragments largely stimulated the development of lipid peroxidation processes in mitochondrial membranes and reduced the stability of hemocyte lysosome membranes compared to pristine PP fragments. In general, the results obtained concretize and supplement with experimental data the previously stated hypothesis of toxicity of aging MPs. Full article

This work presents a 2D radiochromic dosimeter for ultraviolet (UV) radiation measurements, based on cotton fabric volume-modified with nitroblue tetrazolium chloride (NBT) as a radiation-sensitive compound. The developed dosimeter is flexible, which allows it to adapt to various shapes and show a color change from yellowish to purple-brown during irradiation. The intensity of the color change depends on the type of UV radiation and is the highest for UVC (253.7 nm). It has been shown that the developed dosimeters (i) can be used for UVC radiation dose measurements in the range of up to 10 J/cm²; (ii) can be measured in 2D using a flatbed scanner; and (iii) can have the obtained images after scanning be filtered with a medium filter to improve their quality by reducing noise from the fabric structure. The developed cotton–NBT dosimeters can measure UVC-absorbed radiation doses on objects of various shapes, and when combined with a dedicated computer software package and a data processing method, they form a comprehensive system for measuring dose distributions for objects with complex shapes. The developed system can also serve as a comprehensive method for assessing the quality and control of UV radiation sources used in various industrial processes. Full article

Chronic granulomatous disease (CGD) is a group of rare primary inborn errors of immunity characterised by a defect in the phagocyte respiratory burst, which leads to severe and life-threatening infective and inflammatory complications. Despite recent advances in our understanding of the genetic and molecular pathophysiology of X-linked and autosomal recessive CGD, and growth in the availability of functional and genetic testing, there remain significant barriers to early and accurate diagnosis. In the current review, we provide an up-to-date summary of CGD pathophysiology, underpinning current methods of diagnostic testing for CGD and closely related disorders. We present an overview of the benefits of early diagnosis and when to suspect and test for CGD. We discuss current and historical methods for functional testing of NADPH oxidase activity, as well as assays for measuring protein expression of NADPH oxidase subunits. Lastly, we focus on genetic and genomic methods employed to diagnose CGD, including gene-targeted panels, comprehensive genomic testing and ancillary methods. Throughout, we highlight general limitations of testing, and caveats specific to interpretation of results in the context of CGD and related disorders, and provide an outlook for newborn screening and the future. Full article

The aim of the study was to investigate the relationship between the serum zonulin and the cellular immunity in patients with ulcerative colitis (UC) and Crohn’s disease (CD). The study included 97 patients, 13 (13.4%) patients with CD and 84 (86.6%) patients with UC. The concentration of zonulin in the serum was studied using the ELISA kits. The investigation of the circulated leukocyte subpopulation was carried out through flow cytometry. The functional activity of the circulating neutrophils was studied using the absorption capacity of the monodisperse polystyrene latex particles and indicators of oxygen-dependent metabolism in the nitroblue tetrazolium test. The serum zonulin concentration in CD patients was significantly higher compared with UC patients (p = 0.003). The zonulin concentration directly correlated with the functional activity of the circulating neutrophils. Patients with a zonulin concentration > 472.4 pg/mL had a significantly higher number of band neutrophils (p = 0.0104), CD3+CD8+ cells (p = 0.0212), NK cells (p = 0.0161), and lower–CD19+ cells (p = 0.0034). Among the IBD patients, zonulin was associated with IBD severity. An increase in the serum concentration of zonulin was associated with an increase in the functional activity of circulating neutrophils and an increase in the number of CD3+CD8+ cells, NK cells, and a decrease in the number of CD19+ cells. Full article

This study aimed to investigate the role of neutrophils in canine leishmaniosis by assessing neutrophil activation and its relationship with different states of L. infantum infection and antibody and IFN-γ production. Dogs were categorized into five groups: healthy-seronegative (n = 25), healthy-seropositive (n = 21), LeishVet-stage I (n = 25), Leishvet-stage II (n = 41), and LeishVet-stage III–IV (n = 16). Results of the nitroblue tetrazolium reduction test (NBT) showed significantly higher neutrophil activation in stage I (median:17.17, range: [7.33–31.50]%) compared to in healthy-seronegative (4.10 [1.20–18.00]%), healthy-seropositive (7.65 [3.98–21.74]%), stage II (6.50 [1.50–28.70]%), and stage III–IV (7.50 [3.00–16.75]%) groups (p < 0.0001). Healthy-seropositive dogs also displayed higher values than all groups except stage I. Stages II and III–IV did not show significant differences compared to healthy-seronegative. Regarding IFN-γ, stage I dogs had higher concentrations (median:127.90, range: [0–3998.00] pg/mL) than healthy-seronegative (0 [0–109.50] pg/mL) (p = 0.0002), stage II (9.00 [0–5086.00] pg/mL) (p = 0.045), and stage III–IV (3.50 [80.00–548.80] pg/mL) (p = 0.02) dogs. Stage II dogs showed increased IFN-γ compared to healthy-seronegative dogs (p = 0.015), while stage III–IV dogs had no significant differences compared to healthy-seronegative dogs (p = 0.12). Healthy-seropositive dogs had elevated IFN-γ concentrations compared to healthy-seronegative dogs (p = 0.001) and dogs in stage III–IV (p = 0.03). In conclusion, neutrophil activation was higher in dogs with mild disease and healthy-seropositive dogs, and a relationship between neutrophil activation and the production of IFN-γ was found. Full article

Various stresses can affect the quality and yield of crops, including vegetables. In this study, CRISPR/Cas9 technology was employed to examine the role of the ELONGATED HYPOCOTYL 5 (HY5) gene in influencing the growth of Chinese cabbage (Brassica rapa). Single guide RNAs (sgRNAs) were designed to target the HY5 gene, and deep-sequencing analysis confirmed the induction of mutations in the bZIP domain of the gene. To investigate the response of Chinese cabbage to endoplasmic reticulum (ER) stress, plants were treated with tunicamycin (TM). Both wild-type and hy5 mutant plants showed increased growth inhibition with increasing TM concentration. However, the hy5 mutant plants displayed less severe growth inhibition compared to the wild type. Using nitroblue tetrazolium (NBT) and 3,3′-diaminobenzidine (DAB) staining methods, we determined the amount of reactive oxygen species (ROS) produced under ER stress conditions, and found that the hy5 mutant plants generated lower levels of ROS compared to the wild type. Under ER stress conditions, the hy5 mutant plants exhibited lower expression levels of UPR- and cell death-related genes than the wild type. These results indicate that CRISPR/Cas9-mediated editing of the HY5 gene can mitigate growth inhibition in Chinese cabbage under stresses, improving the quality and yield of crops. Full article

The WRKY gene family in plants regulates the plant’s response to drought through regulatory networks and hormone signaling. AfWRKY20 (MT859405) was cloned from Amorpha fruticosa (A. fruticosa) seedlings using RT-PCR. The binding properties of the AfWRKY20 protein and the W-box (a DNA cis-acting element) were verified both in vivo and in vitro using EMSA and Dual-Luciferase activity assays. RT-qPCR detected that the total expression level of AfWRKY20 in leaves and roots was 22 times higher in the 30% PEG6000 simulated drought treatment compared to the untreated group. Under the simulated drought stress treatments of sorbitol and abscisic acid (ABA), the transgenic tobacco with the AfWRKY20 gene showed enhanced drought resistance at the germination stage, with significantly increased germination rate, green leaf rate, fresh weight, and root length compared to the wild-type (WT) tobacco. In addition, the superoxide dismutase (SOD) activity, chlorophyll content, and Fv/Fm ratio of AfWRKY20 transgenic tobacco were significantly higher than those of the WT tobacco under natural drought stress, while the malondialdehyde (MDA) content and 3,3′-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining levels were lower. The expression levels of oxidation kinase genes (NbSOD, NbPOD, and NbCAT) in transgenic tobacco under drought stress were significantly higher than those in WT tobacco. This enhancement in gene expression improved the ability of transgenic tobacco to detoxify reactive oxygen species (ROS). The survival rate of transgenic tobacco after natural drought rehydration was four times higher than that of WT tobacco. In summary, this study revealed the regulatory mechanism of AfWRKY20 in response to drought stress-induced ABA signaling, particularly in relation to ROS. This finding provides a theoretical basis for understanding the pathways of WRKY20 involved in drought stress, and offers genetic resources for molecular plant breeding aimed at enhancing drought resistance. Full article

Cellular mitochondrial function can be assessed using high-resolution respirometry that measures the O₂ consumption rate (OCR) across a number of cells. However, a direct measurement of cellular mitochondrial function provides valuable information and physiological insight. In the present study, we used a quantitative histochemical technique to measure the activity of succinate dehydrogenase (SDH), a key enzyme located in the inner mitochondrial membrane, which participates in both the tricarboxylic acid (TCA) cycle and electron transport chain (ETC) as Complex II. In this study, we determine the maximum velocity of the SDH reaction (SDH_max) in individual human airway smooth muscle (hASM) cells. To measure SDH_max, hASM cells were exposed to a solution containing 80 mM succinate and 1.5 mM nitroblue tetrazolium (NBT, reaction indicator). As the reaction proceeded, the change in optical density (OD) due to the reduction of NBT to its diformazan (peak absorbance wavelength of 570 nm) was measured using a confocal microscope with the pathlength for light absorbance tightly controlled. SDH_max was determined during the linear period of the SDH reaction and expressed as mmol fumarate/liter of cell/min. We determine that this technique is rigorous and reproducible, and reliable for the measurement of mitochondrial function in individual cells. Full article

A twelve-week feeding trial was conducted to investigate the effect of dietary supplementation of a functional immunostimulant mixture (FIM; nucleotides, β-glucan and vitamins C and E) on growth, feed utilization, innate immunity, digestive enzyme activity, hematological parameters, intestinal morphology and inflammatory gene expressions of olive flounder, Paralichthys olivaceus. A fish meal-based basal diet (control) was formulated, and three other diets were prepared by incorporating 0.5, 1.0 and 1.5% FIM into the basal diet (HB0.5, HB1.0 and HB1.5, respectively). Five replicate groups of fish (26.3 ± 0.1 g) were fed one of the experimental diets. Inclusion of 1.5% FIM in the diet significantly enhanced growth and feed utilization. Significantly higher plasma hemoglobin, hematocrit, total protein levels and lower plasma glucose level were observed in the HB1.5 group. Total immunoglobulin content, lysozyme, nitroblue tetrazolium, myeloperoxidase, superoxide dismutase, glutathione peroxidase and catalase activities and immune-related gene expressions of toll-like receptor and perforin were significantly increased at the highest inclusion level. FIM supplementation significantly increased villus height and goblet cell counts. Anti-inflammatory gene expressions were significantly upregulated at a 1.5% level. Dietary supplementation of 1.5% of FIM could improve growth and feed utilization, immune, hematological and intestinal histomorphological parameters of olive flounder. Full article

(1) Background: Ionic transport in Trypanosoma cruzi is the object of intense studies. T. cruzi expresses a Fe-reductase (TcFR) and a Fe transporter (TcIT). We investigated the effect of Fe depletion and Fe supplementation on different structures and functions of T. cruzi epimastigotes in culture. (2) Methods: We investigated growth and metacyclogenesis, variations of intracellular Fe, endocytosis of transferrin, hemoglobin, and albumin by cell cytometry, structural changes of organelles by transmission electron microscopy, O₂ consumption by oximetry, mitochondrial membrane potential measuring JC-1 fluorescence at different wavelengths, intracellular ATP by bioluminescence, succinate-cytochrome c oxidoreductase following reduction of ferricytochrome c, production of H₂O₂ following oxidation of the Amplex^® red probe, superoxide dismutase (SOD) activity following the reduction of nitroblue tetrazolium, expression of SOD, elements of the protein kinase A (PKA) signaling, TcFR and TcIT by quantitative PCR, PKA activity by luminescence, glyceraldehyde-3-phosphate dehydrogenase abundance and activity by Western blotting and NAD⁺ reduction, and glucokinase activity recording NADP⁺ reduction. (3) Results: Fe depletion increased oxidative stress, inhibited mitochondrial function and ATP formation, increased lipid accumulation in the reservosomes, and inhibited differentiation toward trypomastigotes, with the simultaneous metabolic shift from respiration to glycolysis. (4) Conclusion: The processes modulated for ionic Fe provide energy for the T. cruzi life cycle and the propagation of Chagas disease. Full article

Peach (Prunus persica (L.) Batsch) is a fruit tree of economic and nutritional importance, but it is very sensitive to drought stress, which affects its growth to a great extent. Lauric acid (LA) is a fatty acid produced in plants and associated with the response to abiotic stress, but the underlying mechanism remains unclear. In this study, physiological analysis showed that 50 ppm LA pretreatment under drought stress could alleviate the growth of peach seedlings. LA inhibits the degradation of photosynthetic pigments and the closing of pores under drought stress, increasing the photosynthetic rate. LA also reduces the content of O₂⁻, H₂O₂, and MDA under drought stress; our results were confirmed by Evans Blue, nitroblue tetrazolium (NBT), and DAB(3,3-diaminobenzidine) staining experiments. It may be that, by directly removing reactive oxygen species (ROS) and improving enzyme activity, i.e., catalase (CAT) activity, peroxidase (POD) activity, superoxide dismutase (SOD) activity, and ascorbate peroxidase (APX) activity, the damage caused by reactive oxygen species to peach seedlings is reduced. Peach seedlings treated with LA showed a significant increase in osmoregulatory substances compared with those subjected to drought stress, thereby regulating osmoregulatory balance and reducing damage. RNA-Seq analysis identified 1876 DEGs (differentially expressed genes) in untreated and LA-pretreated plants under drought stress. In-depth analysis of these DEGs showed that, under drought stress, LA regulates the expression of genes related to plant–pathogen interaction, phenylpropanoid biosynthesis, the MAPK signaling pathway, cyanoamino acid metabolism, and sesquiterpenoid and triterpenoid biosynthesis. In addition, LA may activate the Ca²⁺ signaling pathway by increasing the expressions of CNGC, CAM/CML, and CPDK family genes, thereby improving the drought resistance of peaches. In summary, via physiological and transcriptome analyses, the mechanism of action of LA in drought resistance has been revealed. Our research results provide new insights into the molecular regulatory mechanism of the LA-mediated drought resistance of peach trees. Full article