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.

Cardiac MRI viability study and its role in revascularization- Excerpts from a tertiary care cardiac centre

Funding Acknowledgements Type of funding sources: None. Background Assessment of cardiac viability based revascularization has not convincingly demonstrated, to improve patient outcomes statistically even by large trials like STITCH and PPAR-2 using SPECT and PET analysis. Here we used cardiac viability by cardiac MRI to guide us for revascularization and also found out problems arising in the statistical analysis for the same Methods It is a retrospective observational longitudinal follow up study whereby patients who had ischemic cardiomyopathy (confirmed with coronary angiogram) and who were admitted with features of heart failure or with acute coronary syndrome and who subsequently underwent cardiac MRI viability testing during the period from 1/02/2017 to 31/01/2020 were included. Patients were excluded who had non ischemic cardiomyopathy. Using cardiac MRI- LVEF, RVEF, Wall motion severity Index and Total viability percentage were additionally computed and analyzed. Patients were deemed having viable myocardium on ≤50% LGE in cardiac MRI and final treatment of CABG, PCI or only medical management was analyzed for the Primary end points of CV mortality, non-fatal CVA and non-fatal AMI Results Based on the criteria total of 94 patients were selected for the study, 53 patients kept on only medical management, 19 patients underwent PCI and 22 patients had CABG. The baseline characteristics of the study population were an average age of 60years, male (76%) with Diabetes Mellitus(69%) and Hypertension (41.5%) in them. Coronary Angiogram showed that 10.6% patients had LM involvement, 92% had LAD disease, 72% patients had LCX lesion and 74% had RCA disease. While average Echo LVEF was 35.82%, Cardiac MRI based mean LVEF was 30.78%. It was found that patients who were kept only on medical management had higher Wall motion Severity Index (2.05) over patients who were treated with PCI (1.94) or CABG (1.80) (p = 0.006). Also it was found that the Total viability percentage was less in patients kept only on medical management (74%) vs patients who were treated with PCI (78%) or CABG (77.8%)(p = 0.08) .It was found by cardiac MRI that patients with significant LAD lesions with viable LAD territory, those who underwent CABG or PCI based therapy had lesser mortality(7.69%,10%) over patients kept only on medical management (23%) (p = 0.407). Among patients with significant LAD lesions with non-viable LAD territory, those who underwent CABG or only medical management had lesser mortality (11.5%) than patients who underwent PCI (50%) (p = 0.137). Conclusion(s) Cardiac MRI based viability testing may guide the physician for optimal treatment but it does not reach statistical significance. The reasons maybe different arterial segments having different viability and anatomical hazards acting as cofounding factors. Viability being a continuum process does not follow a strict cut off of 50% LGE and 100% acute occluded vessel may not allow LGE.

A standardised approach for determining heat tolerance in cotton using triphenyl tetrazolium chloride

Improving the heat tolerance of cotton is a major concern for breeding programs. To address this need, a fast and effect way of quantifying thermotolerant phenotypes is required. Triphenyl tetrazolium chloride (TTC) based enzyme viability testing following high-temperature stress can be used as a vegetative heat tolerance phenotype. This is because when live cells encounter a TTC solution, TTC undergoes a chemical reduction producing a visible, insoluble red product called triphenyl formazan, that can be quantified spectrophotometrically. However, existing TTC based cell viability assays cannot easily be deployed at the scale required in a crop improvement program. In this study, a heat stress assay (HSA) based on the use of TTC enzyme viability testing has been refined and improved for efficiency, reliability, and ease of use through four experiments. Sampling factors that may influence assay results, such as leaf age, plant water status, and short-term cold storage, were also investigated. Experiments conducted in this study have successfully downscaled the assay and identified an optimal sampling regime, enabling measurement of large segregating populations for application in breeding programs. The improved HSA methodology is important as it is proposed that long-term improvements in cotton thermotolerance can be achieved through the concurrent selection of superior phenotypes based on the HSA and yield performance in hot environments. Additionally, a new way of interpreting both heat tolerance and heat resistance was developed, differentiating genotypes that perform well at the time of a heat stress event and those that maintain a similar performance level to a non-stressed control.

METHOD FOR DRYING MAIZE POLLEN

Pollen managements consisted of suitable harvest timing, pollen drying to a certain misture content, pollen viability testing, and pollen storage. Maize pollen was sensitive to desiccation. This experiment was aimed to determine preservation technique in reducing pollen water content while maintaining its viability. Maize variety which used in this research was BIMA 3 as male line hybrid maize. Pollen viability testing used potassium iodide (KI) 1%. This experiment used randomized block design with 3 factors and 2 replicates, i.e. boron (H3BO3) fertilizers (0 and 3 kg ha-1 ), drying techniques (MgCl2, silica gel, zeolite, preservation in air-cond room (18±1 °C; RH43%), and drying periods (0, 2, 4, 6, 8 hours). The observation response of this experiment were viability and pollen weight changing. The result showed that there is no interaction between three factors to observation response, while drying technique and drying period combinations showed interactions to observation response. Pollen maize drying used silica gel for 4 hours was able to decline much pollen weight and maintain pollen viability better than other techniques.