Nitroblue Tetrazolium Research Articles

Low CO2 Levels Are Detrimental for In Vitro Plantlets through Disturbance of Photosynthetic Functionality and Accumulation of Reactive Oxygen Species

Photosynthesis of plantlets in tissue culture containers is not considered important, compared to photosynthesis of ex vitro plants, due to the exogenous source of carbohydrates present in tissue culture media. However, CO2 starvation can generate a burst of reactive oxygen species (ROS). We examined this phenomenon in tissue culture, since CO2 levels may become very low during the light period. The research was carried out with lily scales, regenerating adventitious bulblets, and with Arabidopsis seedlings. CO2 starvation was achieved by placing a small vial of concentrated KOH solution in the culture container. CO2 removal reduced the growth of regenerated lily bulblets by 33% or 23%, with or without sucrose in the medium, respectively. In Arabidopsis seedlings, CO2 removal decreased growth by 50% or 78% in the presence or absence of sucrose in the medium, respectively. Therefore, the addition of sucrose as a replacement for photosynthesis resulted in only partial recovery of growth. Staining with nitroblue tetrazolium (NBT) showed little to no ROS in ex vitro growing seedlings, while abundant ROS were detected in seedlings grown under in vitro CO2 starvation. Seedlings grown under normal tissue culture conditions (no CO2 withdrawal) showed low levels of ROS. In lily tissue culture, CO2 starvation decreased the maximum quantum efficiency of photosystem II (Fv/Fm) from 0.69 to 0.60, and in Arabidopsis from 0.76 to 0.62. Fv/Fm of ex vitro lily and Arabidopsis seedlings was 0.77 and 0.79, respectively. This is indicative of a disturbance in photosynthesis functionality and the occurrence of in vitro stress under reduced CO2 concentrations. We conclude that poor growth, in the absence of CO2, was partly due to strongly reduced photosynthesis, while the detrimental effects were most likely due to a burst of ROS.

NEUTROPHIL ACTIVITIES IN ADOLESCENTS WITH TYPE I DIABETES MELLITUS DEPENDING ON PERIODONTAL STATE.

The aim: To estimate the neutrophil activities in adolescents with type 1 diabetes mellitus (T1DM) depending on periodontal state. Materials and methods: A total of 93 individuals aged 12-16 years, including 62 T1DM patients and 31 healthy (H) controls, were included. Both groups were categorized into subgroups depending on their periodontal state. Phagocytic activity of neutrophils (PAN) the index of neutrophil activation (INA), and the percent of formazan-active neutrophils were evaluated using the spontaneous and the induced nitroblue tetrazolium (sNBT and iNBT) tests into oral rinses. Results: PAN was significantly higher in the healthy (H) controls with gingivitis compared with the individuals with gingival health (p < 0.0001). This parameter decreased significantly in the T1DM subjects, especially with periodontitis, compared with the H controls (p < 0.0001). The percent of formazan-active neutrophils and INA in the sNBT test increased in the T1DM patients with gingival health and continued to raise as periodontal state of adolescents with T1DM worsened (p<0.0001). The parameters of the iNBT test in the T1DM adolescents decreased with the periodontal disease development (p < 0.0001) that may demonstrate that superoxide production exhausts in diabetes, especially associated with periodontal disease. Conclusions: The sNBT test in studied adolescents showed that both periodontal disease and T1DM increase the rate of activated neutrophils (p<0,05).

Внеклеточные ловушки нейтрофилов: динамика образования, ассоциированная с переходом от ремиссии к активному ревматоидному артриту

Tne aim of the research. To evaluate the main parameters of neutrophil extracellular traps formation (NETosis) and their dynamics in rheumatoid arthritis (RA) patients associated with the transition from remission to active autoimmune infl ammation. Material and methods. A total of 37 patients (6 males and 31 females) with verifi ed RA according to the ACR/EULAR 2010 criteria were enrolled in the study and formed the main group. Th e comparison group was constituted by 30 healthy volunteers. Circulating neutrophils were isolated with one-step density gradient centrifugation using double layers of iohexol gradient in the original modifi cation. Th e quantitative composition of neutrophils was assessed through microscopy of smears stained according to May-Grunwald. Cell viability as well as non-specifi c activation were evaluated microscopically using trypan blue exclusion assay and nitroblue tetrazolium test, respectively. Neutrophil extracellular trap (NETs) formation was induced by 50 nM phorbol-12-myristate-13- acetate and assessed using fl uorescence microscopy. Results. Neutrophils isolated from RA patients in clinical remission have been revealed to have a higher ability for spontaneous and induced NETs formation than neutrophils from the comparison group. Th e transition from remission to active RA was characterised by a signifi cant increase in induced and, especially, spontaneous formation of NETs. Th e growth rate of spontaneous NET formation was 3.9-fold higher than the induced NETs formation. Spontaneous formation of NETs above 16.4 % may serve as a possible RA activation indicator. Neutrophils from ACPA-positive RA patients were found to have increased spontaneous and induced NETs formation compared to ACPA-negative RA patients. Conclusion. We have studied the main parameters of NETs formation and their dynamics associated with the transition from remission to active autoimmune infl ammation in RA patients. Th e activity of RA has a more pronounced eff ect on the intensity of NETs formation than the presence of ACPA. NETs may be considered a new potential diagnostic and prognostic biomarker

Effect of lidocaine on oxidative activity of peripheral blood phagocytes

BACKGROUND: Excessive production of reactive oxygen species (ROS) by leukocytes can cause damage to intrinsic tissues. The pathogenesis of sepsis is based on an excessive inflammatory response of the body. Several studies have reported the inhibitory effect of lidocaine on neutrophilic granulocytes.&#x0D; AIM: This study aimed to analyze the effect of lidocaine on the oxidative activity of phagocytes.&#x0D; MATERIALS AND METHODS: Blood from 16 healthy donors was used in this study. Leukocyte mass was extracted using spontaneous sedimentation. Half of the leukocyte samples were incubated in buffered physiological saline with lidocaine. The other half of the leukocyte samples were incubated in physiological saline without lidocaine. The generation of ROS was studied using two methods. Method 1 included a nitro blue tetrazolium (NBT) test), which is based on the ability of ROS to reduce NBT to insoluble diformazan. Method 2 was based on the chemiluminescence reaction. A culture of S. Aureus was used to induce the production of ROS.&#x0D; RESULTS: NBT test revealed a decrease in the oxidative activity of leukocytes in the presence of lidocaine by 18% (p 0.05). The study of luminol-dependent chemiluminescence of leukocyte suspension in the presence of lidocaine revealed a significant 2-fold decrease in both spontaneous and stimulated respiratory activity of cells.&#x0D; CONCLUSIONS: After incubation with lidocaine, phagocytes generated ROS to a significantly lower extent. However, their complete blockade was not recorded. This property of lidocaine may be used in clinical practice to treat an excessive inflammatory response in sepsis.

Nonspecific protective factors in patients with early latent syphilis (report 1)

The objective of this work is to study nonspecific protective factors in patients with latent early syphilis. The results of the study are based on the data of a comprehensive examination before treatment of 142 patients with early latent syphilis and 20 patients of the comparison group. Methods of investigation: serological methods for the diagnosis of syphilis complex of serological reactions, enzyme-linked immunosorbent assay (IgM, IgG), Indirect immunofluorescence reaction (RIF) RIF-200, RIF-abs; determination of cytokines IL-2, IL-6, IL-10, TNFα and INFγ in blood serum by enzyme-linked immunosorbent assay; determination of the phenotype of lymphocytes (CD-receptors), the concentration of circulating immune complexes, phagocytic number, phagocytic index, Nitroblue tetrazolium test. In the blood serum of patients the concentration of IL-2, IL-6, IL-10 and TNFα, INFγ was significantly increased. The concentration of IL-10 and IL-6 was also significantly increased in patients with more than 1 year of infection. The most significant disorders of the parameters of the metabolic activity of neutrophils were found in patients with an infection of more than 1 year; an imbalance in the circulating immune complexes concentration was also established. Under the increasing duration of the infection, the concentration of CD16+ in the blood serum progressively decreases. The content of pro-inflammatory and anti-inflammatory cytokines in the blood serum of patients with early latent syphilis was significantly increased and depended on the timing of infection. This relationship was most pronounced for IL-6 and IL-10. In patients with early latent syphilis there is also an imbalance between CD25+ and late activation factor HLA-DR against the background of a progressive decrease in the number of CD16+ lymphocytes. A direct relationship was established between the time of infection and the number of NK-cells. The revealed disorders may be the consequence of significant disorders on the part of neutrophils and may be one of the factors of the latent course of syphilitic infection.

Early Defense Mechanisms of Brassica oleracea in Response to Attack by Xanthomonas campestris pv. campestris.

Black rot disease, caused by Xanthomonas campestris pv. campestris (Xcc), results in significant yield losses in Brassica oleracea crops worldwide. To find black rot disease-resistant cabbage lines, we carried out pathogenicity assays using the scissor-clipping method in 94 different B. oleracea lines. By comparing the lesion areas, we selected a relatively resistant line, Black rot Resistance 155 (BR155), and a highly susceptible line, SC31. We compared the two cabbage lines for the Xcc-induced expression pattern of 13 defense-related genes. Among them, the Xcc-induced expression level of PR1 and antioxidant-related genes (SOD, POD, APX, Trx H, and CHI) were more than two times higher in BR155 than SC31. Nitroblue tetrazolium (NBT) and diaminobenzidine tetrahydrochloride (DAB) staining analysis showed that BR155 accumulated less Xcc-induced reactive oxygen species (ROS) than did SC31. In addition, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays showed that BR155 had higher antioxidant activity than SC31. This study, focused on the defense responses of cabbage during the early biotrophic stage of infection, indicated that Xcc-induced ROS might play a role in black rot disease development. We suggest that non-enzymatic antioxidants are important, particularly in the early defense mechanisms of cabbage against Xcc.

Dietary Guduchi (Tinospora cordifolia) enhanced the growth performance, antioxidative capacity, immune response and ameliorated stress-related markers induced by hypoxia stress in Nile tilapia (Oreochromis niloticus)

Nile tilapia can tolerate a wide range of farming conditions; however, fluctuations in the environmental conditions may impair their health status. The incorporation of medicinal herbs in aquafeed is suggested to overcome stressful conditions. In this study, dietary Guduchi (Tinospora cordifolia) was evaluated on the growth performance, antioxidative capacity, immune response, and resistance of Nile tilapia against hypoxia stress. Fish fed five diets incorporated with Guduchi at 0, 2, 4, 6, and 8 g/kg for 56 days then exposed with hypoxia stress for 72 h. The growth performance, feed intake, and feed efficiency ratio were significantly (P < 0.05) increased by including Guduchi in tilapia diets regardless of the inclusion level. Similarly, the lipase and protease activities were markedly (P < 0.05) increased in tilapia fed dietary Guduchi. The activities of lysozyme and bactericidal activities in serum and mucus, nitro-blue tetrazolium (NBT), and alternative complement activity (ACH50) were markedly (P < 0.05) enhanced in tilapia treated with Guduchi supplements regardless of the dose. Additionally, the activities of liver and intestinal superoxide dismutase, catalase, and glutathione peroxidase were markedly enhanced (P < 0.05) by including Guduchi in tilapia diets compared with the control. Before and after hypoxia stress, tilapia-fed dietary Guduchi had lower glucose and cortisol levels than fish-fed Guduchi-free diets (P < 0.05). In all groups, glucose and cortisol levels were markedly higher after hypoxia compared before hypoxia stress (P < 0.05). In conclusion, dietary Guduchi can be included at 5.17–5.49 g/kg to enhance the growth performance, digestive enzyme activity, immune and antioxidative responses, and the resistance of Nile tilapia against hypoxia stress.

Harnessing efficient in-situ H2O2 production via a KPF6/BiOBr photocatalyst for the degradation of polyethylene

• KPF 6 doped BiOBr were successfully synthesized using a simple one-pot hydrothermal method. • The photocatalytic H 2 O 2 production of 20 wt% KPF 6 /BiOBr is 1.96 times higher than pure BiOBr. • Plastic bags were decomposed by KPF 6 /BiOBr in conjunction with the in-situ generation of H 2 O 2 . • Hydroxyl radicals play a key role in the polyethylene degradation. • A potential mechanism for H 2 O 2 production and polyethylene degradation was proposed. The development of cost-effective dual-function photocatalytic materials to advance clean energy generation and storage combined with superior pollutant degradation performance has thus far remained challenging. For this study, potassium hexafluorophosphate (KPF 6 ) was introduced to modify BiOBr via a simple one-pot hydrothermal method, where uniformly dispersed KPF 6 on BiOBr nanosheets improved H 2 O 2 yields and the pollutant degradation performance. Such materials have proven to be critical for low bandgap and enhanced surface charge separation for efficient H 2 O 2 production. The optimized sample of 20 wt% KPF 6 /BiOBr showed a H 2 O 2 production rate of 53.36 mg·L –1 , which was much higher than that of pure BiOBr (27.19 mg·L –1 ). Nitroblue tetrazolium chloride (NBT) superoxide radical detection results suggested that two-step single-electron oxygen reduction was the primary pathway. More importantly, discarded polyethylene bags from supermarkets have been decomposed though the in-situ generation of H 2 O 2 using KPF 6 /BiOBr materials, and the mass loss of plastics was 2.33 times higher than that of the pure BiOBr. This research offers new perspectives toward the development of new functionalized materials for H 2 O 2 production, while improving capacities for the degradation of plastics.

Hematological and Hematopoietic Effects of Bactericidal Doses of Trans-Cinnamaldehyde and Thyme Oil on Cyprinus carpio Juveniles.

The effects of two potential antibacterial agents of plant origin: trans-cinnamaldehyde (TC) and thyme oil (TO) on the peripheral blood parameters and cellular composition of hematopoietic tissue of Cyprinus carpio were studied. Both phytochemicals were used in the doses based on the bactericidal concentrations against Aeromonas spp. developed earlier in in vitro study. The fish were fed for 2 weeks on a commercial feed supplemented with 10 μl/kg of TC or 20 μl/kg of TO. Groups TC1 and TO1 were fed diets containing phytochemicals daily, while groups TC2 and TO2 every 2 days. Control group and groups TC2 and TO2 on the remaining days were fed plain feed. Peripheral blood and head kidney hematopoietic tissue were sampled from all the fish at the end of the experiment. In all the groups, hematological values were within the reference ranges for the healthy common carp juveniles. However, blood hemoglobin (Hb) concentration and mean corpuscular hemoglobin concentration (MCHC) were significantly lower in all the groups exposed to TC and TO, while MCH in TC1, TO1, and TO2 compared to the control. TC and TO did not affect leukocyte count [white blood cell (WBC)], differential leukocyte count, the oxidative activity of phagocytes [nitroblue tetrazolium (NBT)], or thrombocyte count (Thro). No significant alterations were observed in the hematopoietic tissue. The results showed that TC and TO exhibited no considerable hematotoxic effects and trials of their use in the treatment of fish infected with Aeromonas spp. may be undertaken.

Study of in-vitro anti-inflammatory and immunomodulatory effect of Ayurvedic plants – Murva

Background and Aims: Toxic chemicals, pathogens and damaged cells trigger the inflammatory process and cause disease in the kidney, heart, brain, liver, lung and reproductive system. The present study evaluates the anti-inflammatory and immunomodulatory effect of Ayurvedic plants under the Murva category. Methods: Marsdenia tenacissima is an acceptable source of Murva. Sansevieria roxburghiana is considered as Murva in west Bengal. Methanol extracts of both plants were used to evaluate anti-inflammatory and immunomodulatory effects by certain in vitro methods. The in vitro anti-inflammatory effects of methanol extract Marsdenia tenacissima (MEMT) and methanol extract of Sansevieria roxburghiana (MESR) were assessed by heat and hypotonic induced haemolysis on the red blood cells of rats. Nitro blue tetrazolium (NBT) assay and inhibition of TNF-α release in DAL cell lines were conducted to assess the immunomodulatory potential of extracts. Results: MEMT, MESR and a combination of MEMT&amp;MESR showed significant (p&lt;0.001) inhibition of haemolysis on heat and hypotonic induced methods compared with the standard Diclofenac Sodium. In NBT reduction test, MEMT showed a more significant (p&lt;0.001) result than MESR. Similarly, the inhibition of TNF-α release was also significantly enhanced by 400 µg/ mL of MEMT. Conclusion: The present research results revealed that Murva is a safe Ayurveda drug for the treatment of cancer and cardiac disease with the protection of systemic immunity and set free from an inflammatory condition.

Transcription factor OsMADS25 improves rice tolerance to cold stress.

Cold stress is the limiting factor of rice growth and production, and it is important to clone cold stress tolerant genes and cultivate cold tolerance rice varieties. The MADS transcription factors play an important role in abiotic stress signaling in rice. This study showed that OsMADS25 was up-regulated by low temperature and abscisic acid (ABA), suggesting that OsMADS25 may be involved in ABA-dependent signaling. The OsMADS25 overexpression vector, pCambia1300-221-OsMADS25-Flag, was constructed and introduced into the rice variety Zhonghua 11 (ZH11) through Agrobacterium tumefacian-mediated genetic transformation. Two homozygous lines with high expression levels were selected for phenotypic identification. OsMADS25 overexpression lines show significantly improved cold stress tolerance and the sensitivity to ABA at the seedling stage of rice. Reactive oxygen species (ROS) was detected by diaminobenzidine (DAB) staining and nitroblue tetrazolium (NBT) staining. After treatment with cold stress, little ROS accumulation was observed in OsMADS25 overexpression lines compared to wild-type ZH11. In conclusion, OsMADS25 plays a role in scavenging reactive oxygen species (ROS) and could improve rice tolerance to cold stress involved in ABA-dependent pathway.

Formation of extracellular traps by circulating neutrophils and monocytes in rheumatoid arthritis patients: a study of new citrullinated autoantigen

Detection of subcellular structures containing typical citrullinated rheumatoid autoantigens in a single compartment presents a special interest, due to importance of anticitrulline autoantibodies for the autoimmune response in RA. Neutrophil and monocyte extracellular traps (NETs and ETs, respectively) may be considered such candidate structures. Our objective was to assess ability of blood neutrophils and monocytes from RA patients to generate NETs and ETs spontaneously and after in vitro induction.32 patients with verified RA and 30 healthy volunteers as controls were included into the study. Circulating neutrophils and monocytes were isolated with one-step density gradient centrifugation using three layers of ficoll-amidotrizoate gradient. Composition of isolated cellular fractions, their viability, and non-specific activation were evaluated microscopically using Trypan Blue exclusion test, as well as Nitro-Blue Tetrazolium test. The NETs were induced by phorbol-12-myristate-13-acetate, and ETs by bacterial LPS. Spontaneous and induced formation of extracellular traps was assessed using fluorescence microscopy. Neutrophil and monocyte fractions contained minute percentages of impurities and low extents of activated and dead cells. Spontaneous NET and ET formation in RA patients was significantly increased comparing to healthy controls. Neutrophils from ACPA-positive RA patients were found to have higher frequency of NET formation, compared to ACPA-negative RA patients. The monocytes did not demonstrate such differences between these subgroups. There were no substantial morphological differences in NETs and ETs patterns between the individuals from both groups. Induced extracellular trap production in RA was significantly higher compared to healthy controls. The level of myeloperoxidase-specific fluorescence in ETs was considerably lower than in NETs. NETs could probably be considered as a source of citrulline autoantigen participating in autoantibody production and stimulation of inflammatory autoimmune responses in RA, whereas ETs may play less important role in this process.

Growth Performance, Growth-Related Genes, Digestibility, Digestive Enzyme Activity, Immune and Stress Responses of de novo Camelina Meal in Diets of Red Seabream (Pagrus major).

Simple SummaryFish meal (FM) is the major protein source in aquafeed to achieve sustainable aquaculture production. However, the supply of FM is low due to high cost and low availability. There is ongoing conceited research to identify alternative viable protein sources to replace the finite FM. Novel camelina meal (CM) is a plant protein with high amino acids levels and has been tested as an alternative protein source in livestock feeding. However, there is limited information on supplementation of the CM in aquaculture diets. In this study, four diets were formulated to contain 0% plant protein, 205 g/kg soybean meal, and two levels of CM (205 g/kg and 330 g/kg) in diets fed to red seabream. Results indicated that adding CM 205 g/kg in diets-maintained growth performance, digestive enzyme activities, and nutrient digestibility regulated the immunity and stress resistance and modulated the growth-related genes in red seabream. These findings provide the first step in using novel CM and are essential for future practical formulations of feed for red seabream and other marine fish species.A 60-day experiment was designed to assess the effect of different ratios of fish meal (FM): camelina meal plant protein (CM) on growth response and relative gene expression of growth-promoting factors, feed utilization potency, digestive enzymes activities, apparent digestibility (ADC), stress response, non-specific immunity of Pagrus major. Four isonitrogenous (490.7 g/kg of crude protein) and isolipidic (91.5 g/kg total lipid) experimental diets were formulated and designated as camelina meal (CM0), soyabean meal (SBM20.5), CM20.5, and CM33 based on protein contents. At the end of the feed trial, significantly higher (p < 0.05) weight gain, specific growth rate, and feed intake but lower feed conversion ratio were recorded in fish fed CM0, SBM20.5, and CM20.5 than fish fed CM33. The lowest growth, feed utilization, enzyme activity, and digestibility were recorded in fish fed CM33. Significantly higher pepsin, amylase, and protease activities were observed in fish fed CM0, SBM20.5, and CM20.5 diets than fish fed CM33. The highest ADC of protein was recorded in fish fed CM0, SBM20.5, and CM20.5 diets. Hematocrit levels were depressed CM33 while total serum protein, total cholesterol, triglyceride, blood urea nitrogen, total bilirubin, aspartate aminotransferase, and alanine aminotransferase were not significantly changed by the inclusion of CM. Non-specific immune variables (lysozyme activity, peroxidase activity in serum and nitro blue tetrazolium) in fish fed CM0, SBM20.5, and CM20.5 were significantly higher than in fish fed CM33 diet. The superoxide dismutase of fish fed CM20.5 was not significantly different from CM0 and SBM20.5 (p > 0.05). Catalase and low salinity stress test show that CM0, SBM20.5, and CM20.5 were not significantly (p > 0.05) different, while CM33 was significantly lower than the rest of the diets. TBARs show that CM20.5 and CM33 diets were significantly different (p < 0.05), but CM20.5 was not significantly different from SBM20.5. Significantly higher hepatic IGF-1 and IGF-2 mRNA expression was found in fish-fed diet groups CM0, SBM20.5, and CM20.5 than fish fed CM33. The present study indicated that the addition of CM up 205 kg/kg to diet maintains growth, digestive enzymes, nutrient digestibility, immunity, stress resistance, and feed utilization efficiency of red sea bream.

Postharvest application of single, multi-walled carbon nanotubes and nanographene oxide improves rose keeping quality

ABSTRACT Keeping quality is frequently terminated by adverse water relations or oxidative stress. This study was designed to evaluate whether postharvest application of single, multi-walled carbon nanotubes and nanographene oxide (SWCNTs, MWCNTs, and NGO respectively) can improve keeping quality in rose, along with the processes involved. Dose-response curves were realised by applying five concentrations of SWCNTs, MWCNTs, and NGO (0–80, 0–80, and 0–2 mg L−1 respectively) either once as a foliar spray or continuously in the holding solution of different rose cultivars. At optimal concentration, CNTs and NGO through either application method extended keeping quality. CNTs improved water balance by promoting water uptake more than water loss, whereas NGO did not. Each nanomaterial at optimal concentration was also employed to assess various parameters underlying keeping quality. CNTs generally enhanced leaf stomatal conductance and transpiration. CNTs and NGO attenuated chlorophyll degradation, electrolyte leakage and lipid peroxidation due to the amplified triggering of both enzymatic and non-enzymatic antioxidants. In conclusion, the extension of keeping quality by postharvest application of CNTs and NGO was characterised by different modes of action. Both upgraded the antioxidant status, while CNTs additionally promoted water relations.Abbreviations: APX, ascorbate peroxidase; CAT, catalase; CNTs, carbon nanotubes; DW, dry weight; FW, fresh weight; GAE, gallic acid equivalent; NGO, nanographene oxide; MDA, malondialdehyde; MWCNTs, multi-walled CNTs; NBT, nitro-blue tetrazolium chloride; POD, peroxidase; RH, relative air humidity; ROS, reactive oxygen species; RUE, rutin equivalent; RWC, relative water content; SOD, superoxide dismutase; SWCNTs, single-walled CNTs.

Comparative Study of Antioxidant and Pro-Oxidant Properties of Homoleptic and Heteroleptic Copper Complexes with Amino Acids, Dipeptides and 1,10-Phenanthroline: The Quest for Antitumor Compounds.

In a search for new antitumoral agents, a series of homoleptic copper(II) complexes with amino acids and dipeptides, as well as heteroleptic complexes containing both dipeptides and 1,10-phenanthroline, were studied. Furthermore, a single-crystal structure containing alanyl-leucinato ([Cu3(AlaLeu)3(H2O)3(CO3)]·PF6·H2O), which is the first homotrinuclear carbonato-bridged copper(II) complex with a dipeptide moiety, is presented. To assess possible antitumor action mechanisms, we focused on the comparative analysis of pro- and antioxidant behaviors. Pro-oxidant activity, in which the reactive oxygen species (ROS) formed by the reaction of the complexes with H2O2 produce oxidative damage to 2-deoxy-d-ribose, was evaluated using the TBARS method. Additionally, the antioxidant action was quantified through the superoxide dismutase (SOD)-like activity, using a protocol based on the inhibitory effect of SOD on the reduction of nitrobluetetrazolium (NBT) by the superoxide anion generated by the xanthine/xanthine oxidase system. Our findings show that Cu–amino acid complexes are strong ROS producers and moderate SOD mimics. Conversely, Cu–dipeptide–phen complexes are good SOD mimics but poor ROS producers. The activity of Cu–dipeptide complexes was strongly dependent on the dipeptide. A DFT computational analysis revealed that complexes with high SOD-like activity tend to display a large dipole moment and condensed-to-copper charge, softness and LUMO contribution. Moreover, good ROS producers have higher global hardness and copper electrophilicity, lower copper softness and flexible and freely accessible coordination polyhedra.

Utility of Immunohistochemistry and Immunofluorescence in Determining the Pathogenic Variants of Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder of phagocytes due to defects in any of the five subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. An initial diagnosis of CGD is made by flow cytometry-based dihydrorhodamine assay or nitro blue tetrazolium test, which is further confirmed by molecular assays. Expression of five subunits of NADPH oxidase components by either flow cytometric or western blot analysis provides clues toward the potential gene targets which are subsequently confirmed by various genetic assays. Immunohistochemistry (IHC) and immunofluorescence (IF) have never been earlier used to determine the expression of different subunits of NADPH oxidase system. We evaluated the utility of IHC and IF in determining the underlying pathogenic variants of CGD. Twelve genetically confirmed cases of CGD, comprising of biopsy specimens (n = 6), tissue blocks from autopsy cases (n = 3), and cellblocks of cell pellet prepared from peripheral blood (n = 4) were included. IHC for p67phox and p47phox subunits and IF for cytochrome b558 were performed. All 4 cases with pathogenic variation of NCF2 gene showed loss of expression for p67phox subunit. Two cases with pathogenic variation of NCF1 gene showed loss of expression for p47phox subunit. Five cases, except a single case with CYBB gene pathogenic variation, showed loss of expression for cytochrome b558 on IF. Thus, loss of expression consistently matched with the underlying genetic defects assessed by sequencing. Our results confirm our hypothesis that IHC and IF are two rapid, economical, pathologist-friendly techniques providing pertinent information regarding the underlying pathogenic variants and such immuno-analysis can be easily performed on the tissue.

Clinical and immunological features of opisthorchiasis

Aim. To assess the clinical and immunological features in patients with chronic opisthorchiasis, depending on the duration of the infection.&#x0D; Methods. The first group consisted of 19 patients with the duration of the infection up to 1 year, the second group consisted of 21 patients with the duration of the infection between 1 and 5 years, the third group was formed of 23 patients with the duration of the disease more than 5 years, the control group 20 healthy individuals. Immunological research was carried out at the Clinical Diagnostic Center. Statistical processing was performed using Microsoft Excel 2010 and Statistica 6.0 software. The statistical significance of differences was determined by using the MannWhitney test (U-test) at the level of significance of p 0.05. The correlations were assessed by calculating Spearman's rank correlation coefficients.Results Clinical features of chronic opisthorchiasis were revealed in the disease duration groups of up to 1 year, from 1 year to 5 years, more than 5 years: the subclinical course was most common in the group of up to 1 year; cholangiohepatitis prevailed in the group of between 1 to 5 years, allergic skin syndrome, cholangiocholecystitis and pancreatitis dominated in the group of more than 5 years. The immune response in chronic opisthorchiasis was characterized by: up to 1 year lymphocytosis, increased levels of immunoglobulins M (IgM) and circulating immune complexes (CIC), a decrease in the number of T-lymphocytes (CD3+), as well as an increase in bactericidal activity of leukocytes (BAL); between 1 and 5 years monocytosis, increased levels of immunoglobulins M, immunoglobulins G and circulating immune complexes, a decrease in T-cytotoxic lymphocytes (CD8+) and nitro blue tetrazolium (NBT test), as well as an increase in NK cells and phagocytic activity of monocytes, more than 5 years eosinophilia.&#x0D; Conclusion. Common features of rearrangement of the immune system in opisthorchiasis: inflammatory changes in the hemogram, activation of humoral immunity with parallel suppression of the cellular component of the immune system, and increased phagocytosis.

Identification and characterization of AnICE1 and AnCBFs involved in cold tolerance from Ammopiptanthus nanus

The ICE–CBF–COR pathway plays a vital role in improving the cold tolerance of plants. As an evergreen small shrub, Ammopiptanthus nanus has a high tolerance to cold stress because of its special growth conditions. Regrettably, no cold-responsive genes in the ICE–CBF–COR pathway have been reported in A. nanus. In the current study, we isolated AnICE1, AnCBF1, and AnCBF2 in A. nanus and analyzed their sequence structure. Evolutionary analysis indicated that these genes are most closely related to those from Ammopiptanthus mongolicus, Glycine max, Spatholobus suberectus, and Cajanus cajan, all belonging to the Fabaceae. Expression analysis showed that the expression levels of these genes were induced under cold stress and treatment with several plant hormones. As a critical upstream regulator in the ICE–CBF–COR pathway, the function of AnICE1 was further identified. The subcellular localization indicated that AnICE1 is predominantly localized in the plasma membrane and less in the nucleus. Overexpression of AnICE1 in Arabidopsis thaliana improved seed germination and growth of transgenic seedlings during cold stress. Moreover, some physiological indices such as relative electrical conductivity, contents of proline and malondialdehyde, catalase activity, and Nitro Blue tetrazolium and 3.3′-diaminobenzidine staining were investigated by transient expression in A. nanus seedlings and stable overexpression in A. thaliana. These results indicated that AnICE1 enhanced cold tolerance in A. nanus and transgenic A. thaliana. This study is significant for understanding the cold-resistant mechanism of ICE and CBF genes in A. nanus.

Mitigating multiple stresses in Pangasianodon hypophthalmus with a novel dietary mixture of selenium nanoparticles and Omega-3-fatty acid

Effects of a novel dietary mixture of selenium nanoparticles (Se-NPs) and omega-3-fatty acids i.e., Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on mitigating arsenic pollution, high-temperature stress and bacterial infection were investigated in Pangasianodon hypophthalmus. To aim this, four isocaloric and iso-nitrogenous diets were prepared: control feed (no supplementation), Se-NPs at 0.2 mg kg−1 diet with EPA + DHA at 0.2, 0.4 and 0.6% as supplemented diets. Fish were reared under normal condition or concurrent exposure to arsenic (2.65 mg L−1), and temperature (34 °C) (As + T) stress for 105 days. The experiment was conducted with eight treatments in triplicates. Response to various stresses i.e., primary (cortisol), secondary (oxidative stress, immunity, and stress biomarkers) and tertiary stress response (growth performance, bioaccumulation and mortality due to bacterial infection) were determined. Supplementation of dietary Se-NPs at 0.2 mg kg−1 diet and EPA + DHA at 0.2 and 0.4% reduced the primary stress level. Exposure to arsenic and temperature (As + T) and fed with control diet and EPA + DHA at 0.6% aggravated the cortisol level. Anti-oxidative enzymes (Catalase, superoxide dismutase, glutathione peroxidase and glutathione-s-transferase) and immunity (Nitroblue tetrazolium, total protein, albumin, globulin, A:G ratio, total immunoglobulin and myeloperoxidase) of the fish were augmented by supplementation of Se-NPs and EPA + DHA at 0.2 and 0.4%. Neurotransmitter enzyme, HSP 70, Vitamin C were significantly enhanced (p < 0.01) with supplementation of Se-NPs at 0.2 mg kg−1 and EPA + DHA at 0.2 and 0.4%. Whereas total lipid, cholesterol, phospholipid, triglyceride and very low-density lipoprotein (VLDL) were reduced (p < 0.01) with the supplementation of Se-NPs at 0.2 mg kg−1 diet and EPA + DHA at 0.2 and 0.4%. Tertiary stress response viz. growth performance was also significantly enhanced with supplementation of Se-NPs at 0.2 mg kg−1 and EPA + DHA at 0.2 and 0.4% reared under As + T. Whereas arsenic bioaccumulation in fish tissues was significantly reduced with dietary supplementation of Se-NPs and EPA + DHA. Cumulative mortality and relative percentage survival were reduced with Se-NPs at 0.2 mg kg−1 and EPA + DHA at 0.2 and 0.4%. The investigation revealed that a novel combination of Se-NPs at 0.2 mg kg−1 and EPA + DHA at 0.4% followed by 0.2% has the potential to alleviate temperature stress, bacterial infection and arsenic pollution. Whereas diet containing Se-NPs at 0.2 mg kg−1 diet and EPA + DHA at 0.6% was noticeably enhanced the stress in P. hypophthalmus.

Non-specific factors of children protection with streptococcal tonsillitis and infected with herpes virus type 6

The aim of the work was to study the characteristics of the response of non-specific protective factors in tonsillitis of streptococcal etiology in children infected and uninfected with HHV-6 infection at different periods of the disease. Materials and research methods. In 78 children aged 3–15 years, inflammation markers and phagocytic activity of peripheral blood neutrophils (PAN) were determined using the nitroblue tetrazolium reduction test. The studies were carried out in the acute period and in the period of convalescence. Mathematical and statistical processing of the data obtained was carried out using Microsoft Excel 2003 and Statistica 6.0 programs. The significance of differences between the mean values was determined using the Student's test (t). Results. It was found that infection of children with HHV-6 leads to a decrease in the functional activity of neutrophils in the onset of streptococcal tonsillitis compared with uninfected patients (p&lt;0.05). Also, the acute period of streptococcal tonsillitis in patients with co-infection is accompanied by a violation of the functional reserve of the oxygen-dependent mechanism of bactericidal neutrophils: minimal opportunities were found in tonsillitis on the background of HHV-6 infection (p&lt;0.05). By the period of convalescence, a decrease in the indicators of the functional activity of neutrophils, increased in the acute period, was recorded, but in children infected with HHV-6, a complete recovery of indicators to the physiological norm did not occur. One of the features of streptococcal tonsillitis in children with HHV-6 infection is an increase in serum gamma globulin level (p˂0.05), lack of significant dynamics of decrease in the ASL-O indicator. Conclusions. The presence of VHL-6t infection in a child with streptococcal tonsillitis adversely affects the state of nonspecific protective factors, in particular PAN. The obtained data must be used to optimize the management of patients at different periods of the disease, including at the stages of dispensary observation