Bioluminescence Method Research Articles

Resistance to Acetylsalicylic Acid in Patients with Coronary Heart Disease Is the Result of Metabolic Activity of Platelets.

Sensitivity to acetylsalicylic acid (ASA) is important in the treatment of patients with coronary heart disease (CHD) after coronary artery bypass grafting (CABG). Patients were divided into ASA sensitive (sASA) and ASA resistant (rASA) by the activity of platelet aggregation induced arachidonic acid (ARA) together with ASA. Induced platelet aggregation activity was studied in sASA and rASA patients with CHD before and after CABG. The level of synthesis of primary and secondary reactive oxygen species (ROS) by platelets was determined using chemiluminescent analysis. The activity of NAD- and NADP-dependent dehydrogenases in platelets was determined by the bioluminescent method. It was found that the aggregation activity of platelets depended on the sensitivity of CHD patients to ASA and decreased during postoperative ASA therapy. The most pronounced differences in metabolic parameters of platelets in sASA and rASA patients were detected by Nox2 activity. The synthesis of secondary ROS by platelets of CHD patients did not depend on the sensitivity of patients to ASA but increased during postoperative treatment with ASA. The activity of NAD(P)-dependent dehydrogenases in platelets did not differ in sASA and rASA patients with CHD.

Novel Technology for Studying Insulin Secretion: Imaging and Quantitative Analysis by a Bioluminescence Method

We developed a method of video-rate bioluminescence imaging to visualize proteins secreted from living cells. A protein of interest was fused to Gaussia luciferase (GLase), and the luminescence signals of secreted GLase with coelenterazine (luciferin) were visualized at a video-rate of 30-500 ms/frame by using a water-cooled EM-CCD camera. We established a subclonal rat INS-1E cell line, named iGL cells, stably expressing the fusion protein of insulin and GLase (Insulin-GLase). By stimulation with high glucose, 3D-cultured iGL cells showed synchronized oscillatory secretion of insulin for over 1 h, as similarly observed in an isolated rat pancreatic islet. In 2D-cultured iGL cells, the luminescence images indicated that synchronized insulin secretion was localized in intercellular spaces between cells. Further, the relative amount of insulin secretion from iGL cells was easily determined with a luminometer, and we demonstrated that cell-cell interaction of beta cells is fundamental to increase glucose-stimulated insulin secretion by synchronization. Thus, iGL cells would be valuable for studying oscillatory insulin secretion and evaluating anti-diabetic drugs. Our bioluminescence imaging method with GLase could be generally used for investigating protein secretion in 2D and 3D cell culture systems.

Peculiarities of Platelet Metabolism in Patients with Acute Coronary Syndrome with Anxiety-Depressive Disorders and Informativity of Enzymes in the Forecast of Development of Cardiovascular Complications.

Anxiety–depressive disorders (ADD) are a risk factor of cardiovascular mortality in patients with coronary artery disease (CAD). Acute coronary syndrome (ACS) is the main clinical manifestation of a progressing CAD. Metabolic processes disorder in platelets can be one of the causes of cardiovascular complications in patients with ACS and concomitant ADD. We studied platelets metabolism and prognostic informativity of NAD(P)-dependent dehydrogenases of platelets in ACS patients with ADD in terms of forecasting cardiovascular complications development over a year of observation. The levels of NAD- and NADP-dependent dehydrogenases of platelets were determined by means of a bioluminescent method during the first 24 h after admission to hospital and in dynamics in 10 days. Among 315 examined patients, ADD was found in 161 (51.1%). ACS patients with concomitant ADD had both cytoplasmic and mitochondrial processes impairment in platelets that consisted in a decrease of energy metabolism intensity, inhibition of anaerobic glycolysis reactions and lipid catabolism. After 12 months of follow-up, 41 (25.5%) cardiovascular complications were detected in the group of ACS patients with ADD and 20 (13.0%) in the group of ACS patients without ADD. According to the results of the analysis of the neural network based on NAD(P)-dependent dehydrogenases of platelets activity in ACS patients with ADD, indicators were obtained that are informative for predicting the development of recurrent cardiovascular complications.

A modified method for preparation of fluorescent MantGDP bound CDC42

Small GTPase cycled between the GDP-bound inactive state and GTP-bound active state, catalyzed by guanine nucleotide exchange factors (GEFs). Guanine nucleotide exchange assay was a direct way to investigate the specificity, activity, and kinetics of GEFs. The N-methylanthraniloyl derivative of GDP (mantGDP), which was bound to small GTPase, served as a substitution for labeled small GTPase involved in bioluminescent, colorimetric, or radioactive methods due to its safety and sensitivity. In this study, we present an economical and efficient approach to prepare qualified mantGDP-bound CDC42, a member of the Rho GTPase family. In our protocol, with a Kd value of 0.048 μM, alkaline phosphatase hydrolysis of CDC42 increased mantGDP binding affinity to CDC42, allowing mant-nucleotide associating onto CDC42 more easily. Only 1.5-fold molar excess of mantGDP was required to prepare mantGDP-bound CDC42 without nonhydrolyzable GTP analog and high performance liquid chromatography. The mantGDP-bound CDC42 was verified to be efficient for measuring the guanine nucleotide exchange activity of VAV2.

DETECTION OF BACTERIAL ETIOLOGY USING BIOLUMINESCENCE METHOD ON CUP LIDS DUE TO THE CHANGE IN THEIR FREQUENCY OF USE – SHORT COMMUNICATION

Nowadays, cup with a drink is an attribute of a modern consumer. It is used for hot drinks (e.g. coffee, tea, hot chocolate) and cold drinks (e.g. fruit juices). Currently, there is a noticeable tendency to replace disposable cups with one reusable cup. The barrier to their reuse (especially the mouthpiece on the lid), is the lack of knowledge about its microbiological state. The aim of the presented research was to estimate the risk of bacterial etiology occurrence on disposable and reused lids. The overall metabolic activity of microorganisms was assessed by measuring ATP using the bioluminescence method. Detection of microorganisms was carried out on the abiotic surface (PP / PS), both on the lids of disposable cups used once and repeatedly. Contact with the personnel's hands was also considered. The results of the experiment indicate that the contact of the lids with the personnel's hands is not a reason for the increasing amount of ATP. The highest amounts of ATP were observed on lids of cups used repeatedly.

Rapid and highly sensitive detection of Escherichia coli O157:H7 in food with loop-mediated isothermal amplification coupled to a new bioluminescent assay.

Testing for bioluminescent pyrophosphate is a convenient method of DNA detection without complex equipments, but it is insufficiently sensitive and offers no particular time advantage over other rapid detection methods. The shortcomings of the traditional bioluminescent pyrophosphate method have been addressed by using 2-deoxyadenosine-5-(α-thio)-triphosphate (dATPαS) instead of dATP for LAMP, thus reducing the high background signal and generating a constant background value. In this study, LAMP coupled to a novel bioluminescent pyrophosphate assay was developed to detect E. coli O157:H7. The new method has a limit of detection of <10 copies/μL or 5CFU/mL; its sensitivity is higher than that of the conventional LAMP assay. Moreover, a food-borne pathogen can be detected when a single DNA template is included in the LAMP assay, making it 100 times more sensitive than the traditional LAMP method. Three hundred food samples were tested with this assay and the accuracy of detection was verified with a culture method and MALDI Biotyper. The assay only took 90-120 min and detected <10 copies of the pathogen. This method had the advantages of rapidity, sensitivity, and simplicity, so it is very competitive for the rapid and highly sensitive detection of food-borne pathogens.

Kinetics of Interaction of Digitonin and Its Analogues with HEK293 Cells Studied by the Bioluminescence Method

Kinetics of Interaction of Digitonin and Its Analogues with HEK293 Cells Studied by the Bioluminescence Method

Optimization potential of anaerobic biocatalytic processes using intracellular ATP concentration as the main criterion for decision making

The effective use of such an analytical indicator as the concentration of intracellular adenosine triphosphate (ATP) in the process of anaerobic transformation of sulfones was shown. The bioluminescent method for determining ATP evaluates to quickly evaluate the state of biocatalysts in such processes, to select the conditions for effective conducted of biotechnological processes and the functioning of cells, as well as media compositions used in the conversion of sulfur-containing compounds.

Bioluminescent method for the rapid screening of toxic heayy metals in environmental samples using Photobacterium leiognathi strain AK-MIE.

Assessment of eco-toxicant using bioluminescent bacterial assay is a widely used and globally accepted method. In this work, a new luminescent bacterium was isolated from squid (Loligo duvauceli) and identified as Photobacterium leiognathi strain AK-MIE using 16S rRNA, phylogeny analysis. The predicted optimum conditions by RSM were 2.76% (w/v) NaCl, 2.28% (w/v) peptone, 0.34% (w/v) yeast extract, and pH 6.83 with 541,211.80 RLU of luminescent production whereas the predicted optimum conditions by ANN were 2.21% (w/v) NaCl, 2.27% (w/v) peptone, 0.39% (w/v) yeast extract, and pH 6.94 which produced 541,986.20 RLU. The validation analysis of both RSM and ANN show 0.60% and 0.69% deviation from the predicted results indicating that both models provided good quality predictions with ANN showing a superior data fitting capability for non-linear regression analysis. Toxicity tests show strain AK-MIE was sensitive to mercury (concentration causing 50% inhibition or IC50 of 0.00978mgL-1), followed by cadmium (IC50 of 0.5288mgL-1), copper IC50 of (0.8117mgL-1), silver (IC50 of 1.109mgL-1), and lead (IC50 of 10.71mgL-1) which are more sensitive than previously isolated luminescent bacteria, suggesting that strain AK-MIE has the potential to be used in toxicity assessment of heavy metals in the environment. Based on the field trial results, several sediment samples from industrial areas in Bangi, Selangor managed to inhibit the bioluminescence of strain AK-MIE. Validation method carried out using ICP-MS proved the presence of several toxic heavy metal elements.

Metabolic changes in peripheral blood lymphocytes from children with recurrent respiratory infections

Objective: to examine activity and correlative relations for peripheral blood lymphocyte NAD (P)-dependent dehydrogenases in young children with recurrent respiratory infections with hypertrophy of the pharyngeal tonsils and bronchial obstructive syndrome. Methods. 89 children, aged 1–3 years, with recurrent respiratory infections were examined, including 35 children with hypertrophy of pharyngeal tonsils (HPT) and 54 children — with bronchial obstructive syndrome (BOS). Control group contained 20 age-matched healthy children. Activity and relations for peripheral blood lymphocyte for NAD(P)-dependent dehydrogenases were assessed by using bioluminescent method proposed by А.А. Savchenko and L.N. Suntsova (1989). Results. It was found that children with recurrent respiratory infections displayed altered enzyme status in peripheral blood lymphocytes. In particular, activity ribose-5-phosphate- and NAD(P)-dependent metabolic events as well as substrate flux via the tricarboxylic acid cycle were elevated that was paralleled with decreased lactate dehydrogenase anaerobic reaction, thereby implicating a role for malate-aspartate shunt in the energy turnover, substrate efflux from the tricarboxylic acid cycle into amino acid metabolic pathways as well as activity of glutathione reductase. Moreover, features of altered enzymatic profile in peripheral blood lymphocytes were uncovered, which depended on type of complication related to respiratory infection. In addition, children with hypertrophy of pharyngeal tonsils were featured with increased influx of lipid catabolism products into glycolysis, elevated level of malic enzyme activity and decreased pyruvate production. However, children with bronchial obstructive syndrome were found to have decreased glycerol-3-phosphate dehydrogenase activity resulting in lowered shunting activity of slow reactions in Krebs cycle and increased influx of amino acid metabolism intermediates into the tricarboxylic acid cycle. Reshaping of enzymatic profile in peripheral blood lymphocytes depended on type of complications coupled to respiratory infections (ENT-pathology or BOS syndrome). A correlation analysis revealed features of relationship between parameters of NAD(P)-dependent dehydrogenase activity in peripheral blood lymphocytes found in children with hypertrophy of pharyngeal tonsils and bronchial obstructive syndrome marked by quantity, modality and power of correlative links. Conclusion. Children with the recurrent respiratory infections require metabolic therapy aimed at restoring intracellular pathology-driven metabolic processes in immune cells.

An Adenosine Triphosphate Bioluminescence Method for Evaluating the Microbial Contamination of the Salad-Preparing Tables and Salad-Serving Dishes in Restaurants of Mashhad City, Iran

Introduction: Consumption of vegetable products is increasing commonly in the world because they are recognized as an important source of nutrients, vitamins, and fiber for humans. Salads are among the most widely used foods that are also known as the most contaminated foods in restaurants. This study was conducted to determine the microbial contamination of salad-preparing tables and salad-serving dishes. Materials and Methods: This study was conducted at 39 restaurants in Mashhad City in Iran during 2017. In this regard, 78 samples were determined from the salad preparing tables and serving dishes. Samples were tested for cleanliness status using Adenosine Triphosphate (ATP)-bioluminescence method as a rapid test. Results: According to the ATP bioluminescence device, 43.6% of the assessed dishes were clean, 38.5 % were dirty, and 17.9 % were not adequately clean. According to the results of ATP bioluminescence device for tables, 23.1 % of the tested tables passed the test, 15.4 % were classified in the caution status, and 61.5% failed. Conclusion: The ATP method has been widely used, especially in the food industry, as a rapid method for measuring general levels of cleanliness. However, this method is not a reliable replacement for measuring the microbial contamination. The ATP bioluminescence should not be used as a substitute for quantitation of microbial load.

Isolation and Characterization of AbTJ, an Acinetobacter baumannii Phage, and Functional Identification of Its Receptor-Binding Modules.

A. baumannii is an opportunistic pathogen and a major cause of various community-acquired infections. Strains of this species can be resistant to multiple antimicrobial agents, leaving limited therapeutic options, also lacking in methods for accurate and prompt diagnosis. In this context, AbTJ, a novel phage that infects A. baumannii MDR-TJ, was isolated and characterized, together with its two tail fiber proteins. Morphological analysis revealed that it belongs to Podoviridae family. Its host range, growth characteristics, stability under various conditions, and genomic sequence, were systematically investigated. Bioinformatic analysis showed that AbTJ consists of a circular, double-stranded 42670-bp DNA molecule which contains 62 putative open reading frames (ORFs). Genome comparison revealed that the phage AbTJ is related to the Acinetobacter phage Ab105-1phi (No. KT588074). Tail fiber protein (TFPs) gp52 and gp53 were then identified and confirmed as species-specific proteins. By using a combination of bioluminescent methods and magnetic beads, these TFPs exhibit excellent specificity to detect A. baumannii. The findings of this study can be used to help control opportunistic infections and to provide pathogen-binding modules for further construction of engineered bacteria of diagnosis and treatment.

Quantification of live Gram-positive bacteria via employing artificial antibacterial peptide-coated magnetic spheres as isolation carriers

A novel artificial antibacterial peptide was prepared by minor modification of the amino acids sequence of a natural peptide protonectin. Distinct from protonectin with natural broad-spectrum antibacterial activity, the designed peptide named as Phe11-protonectin displayed obviously improved selectivity towards Gram-positive (G+) bacteria. Adopting Phe11-protonectin as the recognition reagent, a bioluminescent method was established to quantify live G+ bacteria. Phe11-protonectin-coated magnetic spheres were employed as carriers for isolating G+ bacteria from sample matrixes. The isolated bacteria were lysed to release intracellular adenosine triphosphate, followed by bioluminescent signal collection for quantifying the amount of live G+ bacteria. Staphylococcus aureus, Streptococcus mutans and Bacillus cereus were adopted as model bacteria to explore the feasibility of this method. The linear range for G+ bacteria was 2.3 × 103 - 1.2 × 107 CFU mL−1, and the limit of detection was 2.2 × 102 CFU mL−1 (3σ). The quantification process composed of bacteria isolation, bacteria lysis and signal detection was completed within 33 min. Both Gram-negative and dead G+ bacteria displayed very minor interference to the method for quantifying live G+ bacteria. It has been successfully employed for four types of sample matrixes including peach juice, glucose injection, human urine and lake water, revealing its application prospect in food and drug safety control, clinical diagnosis and hygiene monitoring.

In vivo antitumour activity of Britanin against gastric cancer through nuclear factor-κB-mediated immune response.

Britanin was explored for the antitumour effect on gastric cancer, which is a sesquiterpene lactone (SL) extracted from Inula japonica. In the present study, cell viability assays were performed to evaluate the antiproliferation effect of Britanin on gastric cancer cells. Tumour development in BGC-823 cell-bearing nude mice was monitored in real-time after Britanin treatment via a bioluminescent imaging method. Western blotting analysis and enzyme-linked immunosorbent assays detected proteins associated with the nuclear factor (NF)-κB signalling pathway. Britanin can suppress the proliferation of gastric cancer cells in vitro and the growth of tumours in vivo. In the treatment group, decreased levels of p65 and phosphorylated (p)-p65 were observed. This indicated that NF-κB plays an important role in the antitumour effect of Britanin. Furthermore, considering the additional role of NF-κB in the immune system, the levels of the downstream molecules interleukin (IL)-2 and the cytokine IL-10 were subsequently determined in vivo. An increase in the IL-2 level and a decrease in the IL-10 level indicated that Britanin elicited an enhanced immune response. Britanin may be a promising candidate for gastric cancer chemotherapy, and its anticancer effect likely depends on an NF-κB-mediated immune response.

ATP MONITORING AS AN EXPRESS METHOD TO DETERMINE OBJECT CONTAMINATION

The article presents the results of indication of water and surface contamination. The researches were aimed at estimation of water safety and sanitary-hygienic condition of surfaces. To study the contamination of water samples and common surfaces, we used the traditional method and the bioluminescence-based express method. The bioluminescence method is based on determining the total amount of ATP (bacterial, somatic and extracellular) on contact surfaces and in water. The level of ATP (adenosine triphosphate) was determined using the Lumitester PD-30 (Kikkoman, Japan) luminometer device according to the manufacturer's instructions, using special test systems. The ATP bioluminescence method is commercially more available for simple and quick monitoring of the sanitary-hygienic control effectiveness according to the HACCP principles or international requirements. The traditional method of determining water (and other material) contamination was performed by inoculating water or surface wiping on a common nutrient medium and further cultivation under appropriate conditions. The strongest glowing reaction was observed in sea water, which can be explained by the presence of organic substances in it, while the bioluminescence values in potable (bottled) and filtered water tests were most close to control test. The results of testing the sanitary-hygienic condition of surfaces showed that the amount of adenosine triphosphate exceeded the limits in almost all tested objects. However, a slight exceeding of adenosine triphosphate level was observed on the internal surface of new (plastic) food containers. The investigations performed show that the bioluminescence-based express method can be used as a primary control that gives immediate information about the contamination of both surfaces and liquids. Using the bioluminescence method can shorten the time of the study and therefore reduce the cost of the test. However, when determination of qualitative and quantitative composition of the tested object’s microbiota is necessary, then the classical microbiological control must be performed.

Evaluation of ATP bioluminescence for monitoring surface hygiene in a hospital pharmacy cleanroom

The usefulness of the ATP bioluminescence method for monitoring surface hygiene was evaluated in a hospital pharmacy cleanroom. The sensitivity of the method was found to be appropriate for assessing the efficiency of cleaning and disinfection. ATP bioluminescence was superior to the traditional microbiological culture-based method for detecting unclean surfaces (p < .05).

Assessment of percent positive agreement between fluorescent marker and ATPase for environmental cleaning monitoring during sequential application in an intensive care unit

Terminal room cleaning is critical in preventing pathogen transmission; however, the optimal cleaning effectiveness assessment modality is still being investigated. We sequentially compared cleanliness assessment agreement between a fluorescent marker and an adenosine triphosphate bioluminescence method, finding no significant differences between modalities.

Evaluation of different detector types in measurement of ATP bioluminescence compared to colony counting method for measuring bacterial burden of hospital surfaces

The ATP bioluminescence method has been increasingly employed as a rapid, on-site detection method in nosocomial infections control. In this study, we used a paired design of monitoring methods, the colony counting method (C) and the ATP bioluminescence method, to evaluate environmental surfaces after disinfection. The ATP bioluminescence method included three detector types (B, P, and N). Every surface after disinfection was performed by combining two types of monitoring methods or detectors. There was no statistically significant difference in theATP content per surface siteamong samples from intensive care units (ICUs)and internal medicine wards using B (p = 0.435) and P (p = 0.260). According to the Spearman’s rank correlation coefficients, with the exception of the correlation between the ATP content values detected by B and P, the correlation between the values generated by the remaining methods/detectors was weak or lacking, whereasthe differences between the detectors were statistically significant. Therefore, there are differences between the ATP bioluminescence method and the colony counting method, also between different detectors.

Evaluation of the in vitro effects of commercial herbal preparations significant in African traditional medicine on platelets

BackgroundCommercial herbal medicines (CHMs) marketed as immune boosters are gaining wide popularity in South Africa, in the absence of control and regulatory guidelines. These commercially packaged and labelled herbal preparations, acquired in various retail outlets, are used without consulting either a conventional health provider or a traditional health practitioner. Although they are indicated for immune-boosting purposes, they might exert many other beneficial and unwanted effects on physiological systems. Platelets are crucial in haemostasis and important for the immunological system. The aim was to investigate the effect of the CHMs used to strengthen the immune system on the activity of human platelets.MethodsSix CHMs commonly used as African traditional medicines in Pretoria, South Africa, were tested for their effects on healthy, isolated human platelets, using a bioluminescence method. The tested herbal medicines were Intlamba Zifo™, Maphilisa™ Herbal medicine, Matla™ African medicine for all diseases, Ngoma™ Herbal Tonic Immune Booster, Stametta™ Body Healing Liquid, and Vuka Uphile™ Immune Booster and serial-diluted standards of each from 10 to 10,000 times. The luminol-enhanced luminescence activity of the platelets was measured after incubation with the herbal medicines and activation with phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP).ResultsFive herbal medicines, namely Intlamba Zifo™, Maphilisa™ Herbal medicine, Matla™ African medicine for all diseases, Stametta™ Body Healing Liquid, and Vuka Uphile™ Immune Booster exerted comparable weak inhibitory effects on both PMA and fMLP-induced platelets, which were concentration dependent at high doses, and inversely related to concentration at low doses. Intlamba Zifo™, Matla™ African medicine for all diseases, Stametta™ Body Healing Liquid, and Vuka Uphile™ exhibited weak, but non-systematic stimulatory effects at low doses, which were not statistically significant. Ngoma™ Herbal Tonic Immune Booster had weak, inhibitory effects at high doses and weak stimulatory effects that were inversely related to concentration at low doses.ConclusionThe findings suggest a potential beneficial role of the CHMs in the suppression of platelets’ reactivity and in enhancing the immune system. Caution, however, should be exercised as platelet inhibition and stimulation predispose to the risk of bleeding and thrombosis, respectively.

Metabolic support of the respiratory burst in blood and sputum neutrophils of patients with community-acquired pneumonia

The aim of the study was to investigate relationships between the respiratory burst in blood and sputum neutrophils and intracellular enzyme activity in patients with pneumonia. Methods. The study involved 82 patients with moderate to severe community-acquired pneumonia (CAP). The chemiluminescent assay was used to investigate synthesis of primary and secondary reactive oxygen species (ROS). The bioluminescent method was used to examine NAD(P)-dependent dehydrogenase activity in neutrophils. Results. Low-intensity respiratory burst in blood neutrophils and its predominant relations to the state of mitochondrial metabolism and malic enzyme activity were observed in healthy subjects. Blood neutrophils in patients with CAP were activated; the anaerobic respiration and mitochondrial metabolism increased in the cells. Additionally, intensity of the respiratory burst was related to terminal glycolysis reactions and the key reaction of the pentose phosphate cycle. Sputum neutrophils in patients with CAP were also activated. Conclusion. Abnormalities of metabolic support of the respiratory burst, such as decreased production of primary and secondary ROS, could appear under unfavorable conditions. Peroxidation increased in the cells, the intensity of terminal glycolysis reactions decreased, and substrates transferred from the citric acid cycle to amino acid exchange reactions. Under these conditions, malic enzyme remained the only substrate that stimulated the respiratory burst in sputum neutrophils, while substrate transfer from the tricarboxylic acid cycle inhibited synthesis of the secondary ROS.