Bioluminescence Method Research Articles

Effectiveness of Sanitization Assessment by ATP Bioluminescence Detection System as a Complementary Method in Vivarium: A five-year status report

Sanitization is an important activity performed in the laboratory animal facilities and periodical assessment of sanitization provides confidence in surface cleanliness as well as health status of animals in the vivarium. The primary objective was to evaluate routine disinfection and/or sanitization practices upon verifying the adenosine triphosphate (ATP) bioluminescence method, which further expressed as relative light units (RLU), a relatively easy and rapid method to interpret results within a minute after the swab sampling performed on any surface. A five-year data compilation showed that RLU values were within the in-house acceptable limits of animal rooms sampled from racks, isolators, doors, trolleys, cage changing stations, tables, walls and cage accessories. However, some of the materials such as racks and trolleys of high-traffic areas showed a significant increase in RLU values due to organic matters that might be present on the equipment surfaces but recorded values were well within the limits set by the facility. Additionally, contact plates were also used as confirmatory method to evaluate microbial monitoring in animal rooms including cage accessories and further historical values of RLU provided confidence to increase monthly contact plate sampling interval to a quarterly basis and followed as per the schedule. Moreover, representative samples from incoming animals were screened by microbial monitoring at regular intervals during the quarantine period and active sentinel samples also screened for serology or PCR as part of comprehensive health monitoring program. In conclusion, ATP method can be used to assess the real-time effectiveness of sanitization practices in vivarium as it provides immediate feedback to animal care personnel that enables corrective actions; hence the ATP bioluminescence is continued as one of the complementary methods at our laboratory animal facility.

High-sensitive detection of organophosphate and carbamate pesticide residues in milk based on bioluminescence method

Organophosphorus pesticides (OPs) and carbamate pesticides (CBs) are the most commonly used pesticides in agricultural cultivation. The pesticide residues in plant products can easily enter dairy cows through feed, resulting in the pesticide low-concentration residues in milk. Traditional acetylcholinesterase (AChE) inhibition-based colorimetric methods have low sensitivity and could not satisfy the detection of low-concentrations of OPs and CBs residues in dairy products. In this study, we combined firefly luciferase (FLuc)-mediated bioluminescence with the inhibition of AChE-catalyzed substrate activity by pesticides to develop a highly sensitive and rapid method for detecting OPs and CBs residues in milk. AChE breaks down D-luciferin acetate to produce D-luciferin, which is recognized by the FLuc and emits luminescence in the presence of ATP. However, the presence of OPs and CBs inhibits AChE, causing the reduction or disappearance of the luminescence signal. The luminescence signal can be detected using a hand-held luminescence photometer, eliminating the need for large instrumentation. The AChE-FLuc system accurately detected OPs and CBs in milk within 30 min, with detection limits of 7.89 ng/mL and 1.75 ng/mL, respectively. The sensitivity of this method is approximately ten times higher than that of traditional AChE inhibition methods, meeting the pesticide residue limits in milk set by China and the European Union.

Three birds with one stone: A nano zeolitic imidazolate framework-luciferase-antimicrobial peptide biocomposite-based biosensing platform for improving enzyme stability, detection sensitivity, and sterilization effect

Adenosine triphosphate-driven bioluminescence (ATP-BL) biosensors were widely employed for on-site screening of bacteria. However, unstable luciferase with strict storage conditions limited its field detection. Moreover, the traditional ATP-BL biosensors lacked the sterilization effect. In this study, a robust and multifunctional zeolitic imidazolate framework-90 decorated with luciferase and polyethylene glycol–antimicrobial peptides (ZIF-90@Luc-AMP) was prepared. It could not only enhance the activity and stability of luciferase at ambient temperatures (≥1 month) but also sensitively detect target bacteria via the bioluminescence (BL) method and kill them (three birds with one stone). During the detection process, the target pathogen Escherichia coli O157:H7 (E. coli O157:H7) was specifically captured on a phage-modified stir bar and formed a sandwich complex with ZIF-90@Luc-AMP. With the synergistic bacteriolysis of ZIF-90@Luc-AMP and the phage, the captured E. coli O157:H7 was decomposed and emitted adenosine triphosphate (ATP), achieving the sterilization effect. Meanwhile, the introduced luciferase catalyzed ATP to produce a BL signal, which was proportional to the concentration of E. coli O157:H7. Combining ZIF-90@Luc-AMP and a phage-labeled stir bar for target recognition and enrichment, the entire analytical process could be easily manipulated within 30 min with a low limit of detection (20 CFU/mL) through a portable ATP bioluminescent meter. The multifunctional ZIF-90@Luc-AMP biosensor with high stability, specificity, and sensitivity was also appropriate for rapid pathogen screening and antibacterial application in the wild.

Phage LysSA163-CBD mediated specific recognition coupled with ATP bioluminescence for the sensitive detection of viable Staphylococcus aureus in food matrices

BackgroundStaphylococcus aureus is a significant foodborne pathogen, commonly detected in milk and meat products. Conventional detection methods have limited sensitivity and specificity, which are time-consuming and susceptible to background interference from complex samples, and cannot effectively distinguish live and dead bacteria. ResultsHerein, we developed a novel adenosine triphosphate (ATP) bioluminescence method coupled with magnetic separation, which is based on phage-encoded endolysin LysSA163-CBD (CBD 163) for rapid and specific detection of viable Staphylococcus aureus. The expressed protein (CBD 163) was derived from the phage LSA2301 and was successfully expressed in Escherichia coli BL21 following an induction period of 4 h at 37 °C, with a molecular weight approximating 40 kDa. The optimal conditions for CBD-magnetic beads (cMBs) to capture S. aureus cells were determined to be 100 μL/mL cMBs at 25 °C for 30 min. The viable S. aureus cells were disrupted by the Cetyl trimethyl ammonium bromide (CTAB) to release intracellular ATP. Then, the ATP reacted with the firefly luciferase and D-Luciferin-based bioluminescence (BL) reagents solution to generate intensive BL signal. The CBD-magnetic separation-ATP bioluminescence (cMS-BL) assay was able to quickly detect viable S. aureus via ATP bioluminescence in 45 min, with a detection range from 5 × 103 to 5 × 107 CFU/mL and limit of detection (LOD) of 190 CFU/mL. Additionally, the cMS-BL method exhibited high specificity and anti-interference ability, which has been successfully applied to quantify S. aureus cells in crayfish-tail, chicken, and skim milk. Significance and noveltyThese results demonstrate the potential of CBD 163 as a versatile and robust biorecognition element for rapid and specific detection of viable S. aureus in food matrices. The proposed phage-derived bacteria-binding proteins-based protocol for BL detection shows various advantages, including high sensitivity, simple operation, and the capability to distinguish live bacteria, providing a strategy for designing high-quality biorecognition element toward foodborne pathogens.

Bioluminescence Inhibition Bioassay for Estimation of Snow Cover in Urbanised Areas within Boreal Forests of Krasnoyarsk City

It has been proposed that the level of air pollution in a city should be estimated based on the accumulation of pollutants in the snow cover of urban forests. This study presents a bioluminescence method for estimating the extent of snow cover pollution in the urbanised areas of boreal forests in Krasnoyarsk city. A bioluminescent assay involving NAD(P)H:FMN oxidoreductase (Red) and luciferase with luminous bacteria (BLuc) was employed to measure the concentrations of six heavy metals (As, Cd, Zn, Co, Hg, and Pb) in the snow cover. The tested snow samples demonstrated a correlation between the reduced activity of the enzyme system and variations in Cd concentration. Furthermore, the research indicated that the period of unfavourable meteorological conditions in Krasnoyarsk city resulted in a notable decline in the activity of the BLuc–Red enzyme system, which may be associated with elevated air pollution levels. This study underscores the potential of the bioluminescence method for monitoring environmental pollution in urban forested areas.

Online monitoring system for qualitative and quantitative analysis of bioaerosols by combined ATP bioluminescence assay with loop-mediated isothermal amplification

Rapid detection of airborne pathogens is crucial in preventing respiratory infections and allergies. However, technologies aiming to real-time analysis of microorganisms in air remain limited due to the sparse and complex nature of bioaerosols. Here, we introduced an online bioaerosol monitoring system (OBMS) comprised of integrated units including a rotatable stainless-steel sintered filter-based sampler, a lysis unit for extracting adenosine triphosphate (ATP), and a single photon detector-based fluorescence unit. Through optimization of the ATP bioluminescence method and establishment of standard curves between relative luminescence units (RLUs) and ATP as well as microbial concentration, we achieved simultaneous detection of bioaerosols' concentration and activity. Testing OBMS with four bacterial and two fungal aerosols at a sampling flow rate of 10 to 50 L/min revealed an outstanding collection efficiency of 95 % at 30 L/min. A single OBMS measurement takes only 8 min (sampling: 5 min; lysis and detection: 3 min) with detection limits of 3 Pcs/ms photons (2.9 × 103 and 292 CFU/m3 for Staphylococcus aureus and Candida albicans aerosol). In both laboratory and field tests, OBMS detected higher concentrations of bioaerosol compared to the traditional Andersen impactor and liquid biosampler. When combined OBMS with loop-mediated isothermal amplification (LAMP), the bioaerosol can be qualitative and quantitative analyzed within 40 min without the cumbersome procedures of sample pretreatment and DNA extraction. These results offer a high compressive and humidity resistance membrane filtration sampler and validate the potential of OBMS for online measurement of bioaerosol concentration and composition.

Clinical features and lymphocytes metabolic parameters of children 7-11 years with gallbladder dysfunction and with its association with giardiasis

Purpose. Investigation of the clinical features and metabolic status of blood lymphocytes in children aged 7-11 years with gallbladder dysfunction depending on the presence or absence of giardiasis.
 Materials and methods. Children aged 7–11 years with gallbladder dysfunction (GD) were examined in depending on the presence or absence of giardiasis. Enzyme activity in blood lymphocytes was determined by bioluminescent method.
 Results. There are diarrheal disorders are more common in clinic dysfunction of gallbladder and flatulence – for gallbladder dysfunction in association with giardiasis. Enlargement of the liver and cystic positive symptoms are also characterized by dysfunction of gallbladder on a background of giardiasis. Significant changes in the NAD(P)-dependent dehydrogenases lymphocytes activity found in children with gallbladder dysfunction: reduced levels of substrate flux at the terminal glycolysis reactions and the tricarboxylic acid cycle, which determines the inhibition, respectively, anaerobic and aerobic respiration of cells.
 Conclusion. Found that children with combined pathology had in lymphocytes, while maintaining the intensity of the terminal reactions of anaerobic glycolysis at the reference level reduced enzyme activity, determining the state of aerobic respiration. Independently from the presence or absence of giardiasis in children with gallbladder dysfunction reduced activity of the pentose phosphate cycle that characterizes the inhibition of the corresponding reactions of macromolecular synthesis.

On-chip bioluminescence biosensor for the detection of microbial surface contamination

Detection of microbial pathogens is important for food safety reasons, and for monitoring sanitation in laboratory environments and health care settings. Traditional detection methods such as culture-based and nucleic acid-based methods are time-consuming, laborious, and require expensive laboratory equipment. Recently, ATP-based bioluminescence methods were developed to assess surface contamination, with commercial products available. In this study, we introduce a biosensor based on a CMOS image sensor for ATP-mediated chemiluminescence detection. The original lens and IR filter were removed from the CMOS sensor revealing a 12 MP periodic microlens/pixel array on an area of 6.5 mm × 3.6 mm. UltraSnap swabs are used to collect samples from solid surfaces including personal electronic devices, and office and laboratory equipment. Samples mixed with chemiluminescence reagents were placed directly on the surface of the image sensor. Close proximity of the sample to the photodiode array leads to high photon collection efficiency. The population of microorganisms can be assessed and quantified by analyzing the intensity of measured chemiluminescence. We report a linear range and limit of detection for measuring ATP in UltraSnap buffer of 10–1000 nM and 225 fmol, respectively. The performance of the CMOS-based device was compared to a commercial luminometer, and a high correlation with a Pearson's correlation coefficient of 0.98589 was obtained. The Bland-Altman plot showed no significant bias between the results of the two methods. Finally, microbial contamination of different surfaces was analyzed with both methods, and the CMOS biosensor exhibited the same trend as the commercial luminometer.

Checking the Cleanliness of Surgical Instruments Using the Adenosine Triphosphate-Bioluminescence Assay

Purpose: Cleaning is crucial for reprocessing reusable surgical instruments (RSIs). In this study, we used the adenosine triphosphate (ATP) bioluminescence method to assess the cleanliness of RSIs according to the cleaning method used.Methods: From April 3 to June 2, 2023, surgical sets, including hemostatic forceps, needle holders, operating scissors, dressing forceps, and suction tubes, that arrived at the decontamination room in the central sterile supply department of one general hospital were selected through convenience sampling. For each of the three cleaning processes, 30 cleaned RSIs were sampled with test swabs for inspection, and the relative light unit value was measured using ATP test equipment.Results: Suction tubes, hemostatic forceps, and needle holders ranked low in cleanliness (<i>x</i><sup>2</sup>=11.69, <i>p</i>=.020), and the contamination rate after mechanical cleaning was significantly lower than that after manual cleaning (<i>x</i><sup>2</sup>=31.34, <i>p</i><.001).Conclusion: Surgical instruments treated by manual processing and those containing tubes were associated with the low cleanliness. Therefore, it is necessary to check whether surgical instruments, including tubes, are manually washed at an appropriate level, and regular monitoring will be required.

Synthesis and mechanism analysis of a non-toxic amine-based clay mineral surface hydration intercalation inhibitor

The surface hydration of clay minerals remains a serious challenge for drilling projects. Herein, we synthesized a non-toxic amine-based clay mineral surface hydration intercalation inhibitor (ODBB) whose structure was characterized using fourier transform infrared spectroscopy, mass spectrometry, and proton nuclear magnetic resonance spectroscopy. The inhibitory ability of ODBB was evaluated through isothermal adsorption, X-ray diffraction, thermogravimetric analysis, linear expansion, rolling recovery, and sedimentation stability. The biological toxicity of ODBB was tested with a bioluminescent bacteriological method. The inhibition mechanism was analysed using elemental analysis, atomic absorption spectroscopy, wettability measurement, zeta potential analysis, capacity of exchangeable cation, scanning electron microscopy, and molecular dynamics simulation. The addition of more than 0.5 wt% ODBB resulted in a reduction in the base spacing of Na-MMT/ODBB from 2.10 to 1.31 nm, while the derivative thermogravimetric curve showed a single peak at 50–200 °C. These results showed that the surface hydration of Na-MMT was completely inhibited. Meanwhile, an EC50 value of 93,000 indicated that ODBB was non-toxic. ODBB intercalated into the crystal layer space of Na-MMT, replaced interlayer cations (Na+), adsorbed on the surface (d001) of Na-MMT by electrostatic interactions, and formed hydrogen bonds with the oxygen atoms on the surface (d001). Furthermore, ODBB tightened the crystalline layer and reduced the base spacing through hydrogen bonding and electrostatic forces.

A bioluminescence method based on NLuc-TetR for the detection of nine Tetracyclines in milk

Trace antibiotic residues in animal foodstuff may lead to resistance and hazard human health, so simple and rapid detection methods have aroused multiple attention. Here, a novel bioluminescence pseudo-ELISA for the trace detection of multiple tetracyclines (TCs) in milk was designed and prepared based on a new fusion protein NLuc-TetR. The N-terminal of the bio-recognition motif TetR was linked to the C-terminal of the reporting motif nano-luciferase (NLuc) through SOE-PCR. Biolumescence signal showed that NLuc-TetR was specificity to nine TCs. And the acting pattern of substrate, NLuc-TetR, and TCs ternary complex was analyzed by molecular docking. With the optimal parameters, the half inhibition concentrations (IC50) of the developed bioluminescence pseudo-ELISA for nine TCs ranged from 0.39 to 1.44 ng mL−1, and the detection limits (LODs) ranged from 2 to 11 pg mL−1. The results demonstrated that the bioluminescence method based on NLuc-TetR is a promising method for routine screening measures for the residues of TCs in milk.

Can the adenosine triphosphate (ATP) bioluminescence assay be used as an indicator for hospital cleaning? - A pilot study.

In hospital cleaning, there is currently no standard for uniform monitoring of surface cleaning, either in Germany or internationally. One possibility for monitoring is the use of so-called objective methods for checking cleaning performance (e.g. fluorescence or adenosine triphosphate (ATP) method). The aim of the study was to monitor and evaluate the implementation of the adenosine triphosphate (ATP) bioluminescence assay as a cleaning indicator in everyday hospital cleaning, in order to verify its utility and effectiveness. In three phases, five frequently touched surfaces were examined with the ATP bioluminescence assay at different time points. 846 measurements were performed on the dermatology ward of a university hospital (phase 1), 1,350 measurements were performed on five different wards of the university hospital (phase 2), and 1,044 measurements were performed on five wards of another large hospital (phase 3). For this purpose, one structurally old and one structurally new ward as well as an intensive care unit (ICU), an outpatient clinic and a radiology department were selected for phases 2 and 3. With the ATP bioluminescence method, we were able to demonstrate a reduction in values after cleaning: before cleaning mean of ATP, 907 relative light units (RLU) (95% confidence interval [CI] 777; 1,038); after cleaning mean=286 RLU (CI=233; 495) (phase 1) and by intervention (five hours after daily cleaning mean=360 RLU (CI=303; 428); five hours after daily cleaning and two additional cleanings mean=128 RLU (CI=107; 152) (phase 3). The ATP values increased five hours after cleaning in phases 1 and 2, and eight hours after cleaning in phase 3. The structurally old wards had the highest ATP content, the ICU and the radiology department, among others, the lowest. In all phases, door handles showed both a reduction after cleaning or intervention and a subsequent increase in ATP values. Chair armrests, examination tables and door handles had high ATP values overall. The study shows ward differences both for cleaning effects and for the soiling characteristics of surfaces during the course of the day. In addition, it demonstrates the benefit of intermediate cleaning twice a day. It is noteworthy that structurally old stations and older inventory were more heavily soiled and, in some cases, more difficult to clean. The results show that the ATP bioluminescence method is suitable for detecting cleaning effects and can be used in everyday clinical practice for simple cleaning monitoring. Furthermore, it enables the detection of risk surfaces and easy-to-clean surfaces with significant re-soiling.

Metabolic and Morphological Aspects of Adaptation of Alkaliphilic Bacillus aequororis 5-DB and Alkali-Tolerant Bacillus subtilis ATCC 6633 to Changes in pH and Mineralization.

The goal of the study is to evaluate metabolic and morphological changes of the facultative alkaliphile Bacillus aequororis 5-DB and the weakly alkali-resistant B. subtilis ATCC 6633 in a wide pH range and at different NaCl concentrations. The alkaliphile B. aequororis 5-DB is shown to have a broader general resistance to adverse factors (wide pH range, 50 g/L NaCl) than a weakly alkali-tolerant strain of the same genus. This alkaliphile is also shown to have a significantly greater resistance not only to high pH but also to low pH in comparison with B. subtilis ATCC 6633. The resistance of B. aequororis 5-DB to low pH was expressed in higher metabolic activity, maintenance of ΔpH, and no significant cell damage. The selected set of methods (reduction of resazurin to resorufin by cell dehydrogenases, bioluminescent method for determining ATP, AFM, and measurement of intracellular pH) allows us to adequately assess the ability of microbial cells to withstand harsh environmental factors. Nonspecific resistance of B. aequororis 5-DB was proven using a complex of selected methods. Tolerance to a wide range of pH and high salt concentrations may be useful for biotechnological applications of the strain.

Features of the respiratory burst state of neutrophils and the activity of NAD(P)-dependent dehydrogenases in patients with widespread purulent peritonitis in the prognosis of the development of sepsis

Aim: Studying of the respiratory burst state and NAD(P)-dependent dehydrogenases activities in blood neutrophils features in the forecast of the development of abdominal sepsis in patients with widespread purulent peritonitis (WPP).
 Materials and methods. The study involved 50 patients with WPP in the preoperative period. Abdominal sepsis was developed by 35 patients (70.0%) from 5 to 10 days postoperative period, 15 patients (30.0%) hadn’t complications. The respiratory burst condition of blood neutrophils was examined using a chemiluminescent assay. Intracellular activity of the NAD- and NADP-dependent dehydrogenases was researched with using bioluminescent methods.
 Results. It was found that patients with WPP whose dynamics of the preoperative period will develop sepsis the chemiluminescent activity of blood neutrophils was characterized by a reduced level of spontaneous synthesis of the primary reactive oxygen species (ROS) and elevated levels of spontaneous synthesis of secondary ROS relative of the indicators identified in patients without subsequent complications. The feature of neutrophil metabolism in WPP patients without subsequent development of sepsis was high activity of the anaerobic lactate dehydrogenase reaction and decrease in activity of the NADP-dependent decarboxylating malate dehydrogenase. In patients with WPP and the subsequent development of sepsis was found high level of NAD-dependent substrates outflow citric acid cycle in the reaction of amino acid metabolism via glutamate dehydrogenase that may affect the activity of aerobic respiration in the neutrophils. Using correlation analysis was found that the intensity of the neutrophils respiratory burst in patients with no subsequent complications depends on the activity of anaerobic glycolysis.
 Conclusion. The established differences in the state of the respiratory burst and the activity of enzymes in neutrophils in patients with WPP, in depending on the subsequent development of the sepsis, determine the possibility of developing the method of forecasting complications and developing immunoactive therapy in the postoperative period of WPP.

Urinary ATP may be a biomarker of interstitial cystitis/bladder pain syndrome and its severity.

Urinary tract cells respond to bladder distension by releasing adenosine triphosphate (ATP). Patients with interstitial cystitis/bladder pain syndrome (IC/BPS) exhibit elevated urinary ATP levels compared to asymptomatic controls. This study aimed to evaluate the potential of urinary ATP as a non-invasive biomarker for IC/BPS and its correlation with symptom severity. We included 56 patients diagnosed with IC/BPS and 50 asymptomatic controls. Urine samples were collected from both groups. Urinary ATP levels were quantified using the luciferin-luciferase bioluminescence method. The severity of IC/BPS symptoms was assessed using the visual analogue score (VAS), Interstitial Cystitis Symptom Index (ICSI), and Interstitial Cystitis Problem Index (ICPI) from the O'Leary-Sant score. We specifically examined the correlation between symptom scores and urinary ATP levels in IC/BPS patients. Urinary ATP levels were significantly higher in IC/BPS patients compared to the control group (P < 0.0001). There was a significant positive correlation between urinary ATP concentrations and VAS, ICPI, and ICSI scores among IC/BPS patients (P < 0.0001). The threshold value for ATP concentration was set at 56.6 nM, with an area under the receiver operating characteristic (ROC) curve of 0.811 (95% CI 0.730 - 0.892). Our findings indicate that IC/BPS patients excrete elevated amounts of ATP in their urine. This suggests that urinary ATP might serve as a non-invasive biomarker for IC/BPS, with a predictive potential in terms of symptom severity.

Влияние внутриклеточной регуляции метаболизма на популяционный состав лимфоцитов периферической крови человека

Metabolic activity has a significant impact on the differentiation, proliferation and functioning of T cells. Different lymphocyte subpopulations use, to varying degrees, glycolysis and mitochondrial metabolism, whose main regulators are hypoxia-inducible factor 1-alpha (HIF-1α) and sirtuin 3 (SIRT3), respectively. The purpose of this paper was to study changes in the population composition of peripheral blood lymphocytes in humans depending on the level of the intracellular metabolic regulators SIRT3 and HIF-1α. Materials and methods. 227 residents of the city of Arkhangelsk and the Arkhangelsk Region were examined (mean age 42 ± 11 years). Absolute lymphocyte count was determined using the Sysmex XS 500i haematology analyser, while CD3+, CD4+, CD8+, CD10+, CD25+ and CD95+ phenotypes content, by indirect immunoperoxidase reaction. Intracellular adenosine triphosphate (ATP) concentration was measured using the luciferase bioluminescence method. HIF-1α and SIRT3 concentrations were measured in lymphocyte lysate using enzyme immunoassay. To divide the total sample into groups according to SIRT3 and HIF-1α content, k-means clustering was utilized. Results. Changes in SIRT3 and HIF-1α intracellular concentrations correlated with ATP concentration. It was found that in the group with high HIF-1α content, the proportion of CD4+, CD8+, CD10+ and CD25+ lymphocytes was greater than in the group with high SIRT3 concentration, which had a greater proportion of CD95+ lymphocytes. Thus, the content of intracellular metabolic regulators that regulate ATP production pathways in the cell, i.e. oxidative phosphorylation (SIRT3) and glycolysis (HIF-1α), affects the population composition of lymphocytes and is therefore important for assessing the immune response.

The Mechanism of Antimicrobial Activity of Conjugated Bile Acids against Lactic Acid Bacilli.

The mechanism underlying antimicrobial activity of conjugated bile acids against strains of lactic acid bacilli is not well understood. The purpose of this study was to investigate two typical conjugated bile acids (glycochenodeoxycholic acid and taurochenodeoxycholic acid) for their mechanisms of antimicrobial activity against four strains of different species of lactic acid bacilli at the physiological pH of the small intestine of humans. The bacterial cell membrane integrity, transmembrane potential, and transmembrane pH gradient were examined using the fluorescence probes SYTO 9 plus propidium iodide, 3,3'-dipropylthiadicarbocyanine iodide, and 5(6)-carboxyfluorescein diacetate N-succinimidyl ester, respectively. The intracellular ATP levels were measured by the firefly luciferase-based bioluminescence method. It was found that the antimicrobial activity of conjugated bile acids against the strains of lactic acid bacilli is strain-specific, and glycochenodeoxycholic acid showed significantly greater antimicrobial activity than taurochenodeoxycholic acid against the strains of lactic acid bacilli. The conjugated bile acids inhibited the growth of strains of lactic acid bacilli by disrupting membrane integrity, dissipating transmembrane potential, reducing the transmembrane pH gradient, and depleting intracellular ATP. In conclusion, the antimicrobial activity of conjugated bile acids against lactic acid bacilli is a multifactorial phenomenon. This study will provide valuable information for developing strategies to improve the ability of lactic acid bacilli to tolerate bile in vivo.

Effects of CatSper Stimulation and Inhibition by Progesterone and NNC on Human Sperm

Calcium ion enters the sperm through a specific calcium channel, CatSper. This voltage-sensitive channel is stimulated by intracellular alkalization and progesterone. This study is aimed at investigating the effects of CatSper inhibition or stimulation on sperm motility, viability, and sperm function regulators such as mitochondrial membrane potential (MMP), ATP, and reactive oxygen species (ROS) production. This study was performed on 30 semen samples of fertile volunteers, referred to Shiraz Fertility Center. The semen samples were diluted to 20 × 10 6 sperm/mL. The samples were divided randomly into control, solvent, progesterone (10 μM), NNC (2 μM), and NNC+progesterone groups. Sperm kinematics, viability, MMP, ATP content, and the amount of ROS production were assessed using VT-SPERM3.1, eosin staining, JC1 flow cytometry, bioluminescence, and chemiluminescence methods, respectively. Sperm viability and total and progressive motility were significantly decreased in the NNC and NNC+progesterone groups. The amplitude of lateral head displacement (ALH) and curvilinear velocity (VCL) was reduced in the NNC-containing groups. These parameters did not change in the progesterone group. ROS production by viable spermatozoa in the NNC and NNC+progesterone groups was significantly higher than the controls. MMP and ATP content did not show any significant difference with controls in none of the experimental groups. NNC inhibits the CatSper and reduces human sperm motility and viability. These harmful NNC effects were not due to their impact on MMP or ATP production but are likely because of intracellular calcium reduction and higher ROS production. Progesterone at 10 μM concentration had no significant effect and may not be a considerable stimulator for CatSper.

Functional and metabolic activity of blood neutrophilic granulocytes in children with erosive and ulcerative lesions of the stomach and duodenum with detected &lt;i&gt;Helicobacter pylori&lt;/i&gt; infection

The Helicobacter pylori bacterium is currently considered one of the leading etiopathogenetic factors in the formation of gastric and duodenal ulcer in children and adults. Despite many studies in this area, the mechanisms of phagocytic activity in response to exposure to Helicobacter pylori are not completely clear. The aim of the work was to obtain results on the functional and metabolic activity of blood neutrophilic granulocytes in children with Helicobacter pylori-associated erosive and ulcerative lesions of the stomach and duodenum. The object of the study are neutrophilic granulocytes isolated from the blood of patients and the control group. Samples were taken from 46 persons with Helicobacter pylori-associated erosive – ulcerative lesions of the stomach and duodenum aged 11 to 18 years and the control group, which consisted of 55 practically healthy persons who had this disease excluded in the same age range. A comparative analysis of the functional activity of cells using chemiluminescent analysis and metabolic activity by the bioluminescent method was carried out. Luminol was used as a chemiluminescence activator. The measurement of the functional activity of phagocytes was based on the determination of the base activity (spontaneous reaction) and the reserve capacity of the cells when they were exposed to the nonspecific inducer zymosan. There is a reduced activation index of neutrophils in patients relative to the control group, which may characterize reduced metabolic reserves of cells. In neutrophilic granulocytes, there is a decrease in G6PDG, an enzyme that triggers glycolysis along the pentose phosphate pathway and contributes to the reduction of nicotinamide adenine dinucleotide phosphate (NADP) to NADPH, which is necessary for the formation of reduced glutathione that binds oxidants. With its insufficiency, a decrease in the energy reserves of cells occurs. In neutrophilic blood granulocytes in children with erosive and ulcerative lesions of the stomach and duodenum with H. pylori infection, a decrease in metabolic reserves is observed, which is associated with inhibition of metabolic processes in cells.

New Uracil Analog with Exocyclic Methylidene Group Can Reverse Resistance to Taxol in MCF-7 Cancer Cells.

Taxol (Tx), a microtubule-stabilizing drug, has been widely used as a chemotherapeutic in several types of cancer. However, the development of resistance limited its application. One of the strategies used to prevent the emergence of drug resistance is combination treatment, involving at least two drugs. The aim of the current study was to assess if a new uracil analog, 3-p-bromophenyl-1-ethyl-5-methylidenedihydrouracil (U-359) can prevent the development of Tx resistance in breast cancer cells. The cytotoxicity of the new drug was tested in MCF-7 (hormone receptor (ER, PR) positive cell-line) and MCF-10A cell lines using MTT method. For the detection of apoptosis and necrosis, the Wright and Giemsa staining was used. Gene expression was measured by real-time PCR, and changes in the protein levels were evaluated by ELISA and bioluminescent method. We investigated the effect of Tx and U-359 on cancer MCF-7 and normal MCF-10A cells alone and in combination. Tx co-administered with U-359 inhibited proliferation of MCF-7 cells to 7% while the level of ATPase drastically decreased to 14%, compared with effects produced by Tx alone. The apoptosis process was induced through the mitochondrial pathway. These effects were not seen in MCF-10A cells, showing the wide safety margin. The obtained results have shown that U-359 produced a synergistic effect with Tx probably by reducing Tx resistance in MCF-7 cells. To elucidate the possible mechanism of resistance, expression of tubulin III (TUBIII), responsible for microtubule stabilization and tau and Nlp proteins, responsible for microtubule dynamics, was assessed. Combination of Tx with U-359 reduced overexpression of TUBIII and Nlp. Thus, U-359 may stand for a potential reversal agent for the treatment of MDR in cancer cells.