The ATP bioluminescence assay: a new application and optimization for viability testing in the parasitic nematode Haemonchus contortus

The parasitic gastrointestinal nematode Haemonchus contortus causes serious economic losses to agriculture due to infection and disease in small ruminant livestock. The development of new therapies requires appropriate viability testing, with methods nowadays relying on larval motility or development using procedures that involve microscopy. None of the existing biochemical methods, however, are performed in adults, the target stage of the anthelmintic compounds. Here we present a new test for the viability of H. contortus adults and exsheathed third-stage larvae which is based on a bioluminescent assay of ATP content normalized to total protein concentration measured using bicinchoninic acid. All the procedure steps were optimized to achieve maximal sensitivity and robustness. This novel method can be used as a complementary assay for the phenotypic screening of new compounds with potential antinematode activity in exsheathed third-stage larvae and in adult males. Additionally, it might be used for the detection of drug-resistant isolates.

Perioral Aerosol Sequestration Suction Device Effectively Reduces Biological Cross-Contamination in Dental Procedures

Objective The infection risk during dental procedures is a common concern for dental professionals which has increased due to coronavirus (severe acute respiratory syndrome coronavirus 2) pandemic. The development of devices to specifically mitigate cross-contamination by droplet/splatter is crucial to stop infection transmission. The objective of this study is to assess the effectiveness of a perioral suction device (Oral BioFilter, OBF) to reduce biological contamination spread during dental procedures. Materials and Methods Forty patients were randomized 1:1 to a standard professional dental hygiene treatment with OBF and without. Adenosine triphosphate (ATP) bioluminescence assay was used to evaluate the spread of potential contaminants. The total number of relative light units (RLU) from key dental operatory locations: operator’s face-shield, back of the surgical operator’s-gloves, patient’s safety-goggles, and instrumental table were measured. Percentage contamination reductions between control and OBF were compared. Statistical Analysis Primary outcome, total RLU, was analyzed by comparing the means of logged data, using a two-sided two-sample t-test. Secondary outcomes as RLU of logged data for the different locations were analyzed in the same way. Proportion of patients from whom different locations reported events (clean, acceptable, and failure) were analyzed by using Fisher’s exact test. Results For the whole dental environment, RLUs reduction (<150 units) achieved with OBF was 98.4% (97.4–99%). By dental operatory location the reduction in RLUs was from 99.6%, on the operator face-shield, to 83% on instrumental table. The control group reported a very high percentage of failures, (>300) being 100% on the surfaces closer to the patient’s mouth and decreasing to 70% on instrumental table. In contrast, the higher failure percentage in the OBF group was found on the patient’s goggles (40%), while the operator face-shield showed an absence of contamination. Conclusion OBF device has shown efficient reduction of biological aerosol cross-contamination during dental procedures as proved by ATP-bioluminescence assay. Nevertheless, for maximum safety, its use must be combined with standard protective gear such as goggles, face shield, and surgical gloves.

No-Touch Automated Disinfection System for Decontamination of Surfaces in Hospitals

Background: Hospital-acquired infections (HAIs) remain a common problem, which suggests that standard decontamination procedures are insufficient. Thus, new methods of decontamination are needed in hospitals. Methods: We assessed the effectiveness of a no-touch automated disinfection (NTD) system in the decontamination of 50 surfaces in 10 hospital rooms. Contamination of surfaces was assessed with a microbiological assay and an ATP bioluminescence assay. Unacceptable contamination was defined as > 100 colony forming units/100 cm2 in the microbiological assay, and as ≥ 250 relative light units in the ATP assay. Results: When measured with the microbiological assay, 11 of 50 surfaces had unacceptable contamination before NTD, and none of the surfaces had unacceptable contamination after NTD (p < 0.001). On the ATP bioluminescence assay, NTD decreased the number of surfaces with unacceptable contamination from 28 to 13, but this effect was non-significant (p = 0.176). On the microbiological assay taken before NTD, the greatest contamination exceeded the acceptable level by more than 11-fold (lamp holder, 1150 CFU/100 cm2). On the ATP bioluminescence assay taken before NTD, the greatest contamination exceeded the acceptable level by more than 43-fold (Ambu bag, 10,874 RLU). Conclusion: NTD effectively reduced microbiological contamination in all hospital rooms. However, when measured with the ATP bioluminescence assay, the reduction of contamination was not significant.

Current Perspective on Hospital Acquired Infection

Health care associated infections (HCAI) are a major complication faced by the healthcare sector leading to high morbidity and mortality. These infections are caused via the persistence of microbial pathogens in the hospital environment for extended periods (weeks to months) on contaminated surfaces. Foodborne illness is another significant source of infection in hospitals due to improper cleaning practices in the food operating sectors. Thus, frequent hygiene monitoring and efficient cleaning practices may reduce the rate of hospital-acquired infections. Contamination detection by traditional microbiological techniques is laborious, which has paved the way for the development of rapid biotechnological testing kits such as the ATP bioluminescence assay, which can be used as a rapid indicator of contamination.

Oleate prevents palmitate-induced mitochondrial dysfunction in chondrocytes

BACKGROUND : The clear association between obesity and osteoarthritis (OA) in joints not subjected to mechanical overload, together with the relationship between OA and metabolic syndrome (MS), suggests that there are systemic factors related to metabolic disorders that are involved in the metabolic phenotype of OA. The aim of this work is to study the effects of palmitate (PA) and oleate (OL), as the most abundant fatty acids (FA) present in the diet and serum, on cellular metabolism in an " in vitro " model of human chondrocytes. METHODS :.The Seahorse XF96 Analyzer was used tomeasure the mitochondrial, glycolytic function and the contribution of the mitochondrial oxidative phosphorilation system (OXPHOS) and glycolysis to the production of ATP, in the T/C-28a2 chondrocyte treated with to PA, OL and palmitate/oleate (PA/OL) ratio 1:2. Subsequently ATP bioluminescence assay kit was used for ATP quantification. To detect the presence of lipid droplets, two types of stains were performed and the amount of Triglycerides was quantified spectrophotometrically. RESULTS : PA, but not OL, produces mitochondrial dysfunction observed with a lower rate of OCR intended for the synthesis of ATP, coupling efficiency, maximal respiration and spare respiratory capacity. Glycolytic function showed lower rates for both glycolytic capacity and glycolytic reserve when cells were incubated withFA in relation to basal condition (BC). The production rate of ATP from OXPHOS showed lower values in chondrocytes incubated with any of the FAs. The evaluation of possible formation of Lipid droplets (LD) showed a significant increase of these structures in FA conditions, being significantly higher when the cells were incubated with OL. CONCLUSIONS : PA and OL show antagonistic effects in human chondrocytes; while increased levels of PA induce mitochondrial dysfunction and hinder the response of chondrocytes through the glycolytic pathway, OL shows a cytoprotective effect through which promotes the formation of triglycerides-rich LD, as well as the incorporation of PA.

Abstract 133: Cardiovascular Protective Effect of Thyroxine and Nano- Thyroxine Against Ischemic Insults

Introduction: Cardioprotection is a key purpose for therapeutic interventions in cardiology that aim to reduce infarct size, and thus prevent progression toward heart failure, after acute ischemic and cardiac arrest. Triiodothyronine/thyroxine (T3) therapy has been used to reverse myocardial stunning. Hyperthyroidism prevents the stunning with high dependence on the mitochondrial sodium-calcium exchanger and mitochondrial K+ channels. Hypothesis: This study aimed to investigate the positive protective role of T3 and PRO-AL 616 (Nano-T3) and PRO-AL 617 (Nano-T3 with Phosphocreatine, Pcr) in hypoxia-mediated cardiac cell insults, as well as its influences on vascularization and neuronal protection. Methods: The effects of T3 and Nano-T3 + Pcr on angiogenesis were studied in a CAM model. Their cardioprotective effect under hypoxia was studied using isolated neonatal cardiomyocytes treated with PBS (control), T3 (3 uM), Pcr (30 uM) and T3 + Pcr. Mitochondrial function and sarcomere integrity were studied using an ATP- bioluminescence assay, and cardiac Troponin T levels using flow cytometry, respectively. The effect of Nano-T3 on induced neuronal cells under hypoxia was also studied. Finally, Nano-T3-Cy7 were injected into mice tail veins to monitor their biodistribution in real time. Results: Nano-T3 activate the cell surface receptor αvβ3 and is distributed into the cytoplasm, but not the nucleus. Nano-T3 enhanced angiogenesis in the CAM model (~3 fold) versus T3. Under hypoxia, cardiac ATP improvement was achieved with T3+Pcr ( p <0.001) while maintaining normal Troponin T levels. ( Figure 1 ). Nano-T3 produced a significant upregulation of the neural protection markers, PAX6 and DLX2 by ~ 50%. In vivo, the Cy7 signal intensity was detected primarily in mice brains, and hearts, within minutes of administration. Conclusion: Nano-T3 + Pcr represents a potentially new therapeutic for the control of tissue damage in cardiac ischemia and resuscitation.