Estimation of the Level of Abasic Sites in Plant mRNA Using Aldehyde Reactive Probe.

Oxidation of RNA is associated with the development of numerous disorders including Alzheimer's and Parkinson's diseases, amyotrophic lateral sclerosis (ALS), cancer, and diabetes. Additionally, a correlation has been found between increase in RNA oxidation and the process of aging. In plants, elevated level of oxidatively modified transcripts has been detected during alleviation of seeds dormancy and stress response. Increasing interest on the topic of RNA oxidative modifications requires elaboration of new laboratory techniques. So far, the most common method used for the assessment of RNA oxidation is quantification of 8-hydroxyguanine (8-OHG). However, reactive oxygen species (ROS) induce also numerous other changes in nucleic acids, including formation of abasic sites (AP-sites). Recently, the level of AP-sites in RNA has been measured with the use Aldehyde Reactive Probe (ARP). In the present chapter, we describe application of this technique for the evaluation of the level of AP-sites in plant transcripts.

Cadmium Stress Reprograms ROS/RNS Homeostasis in Phytophthora infestans (Mont.) de Bary.

Heavy metal pollution causes many soils to become a toxic environment not only for plants, but also microorganisms; however, little is known how heavy metal contaminated environment affects metabolism of phytopathogens and their capability of infecting host plants. In this study the oomycete Phytophthora infestans (Mont.) de Bary, the most harmful pathogen of potato, growing under moderate cadmium stress (Cd, 5 mg/L) showed nitro-oxidative imbalance associated with an enhanced antioxidant response. Cadmium notably elevated the level of nitric oxide, superoxide and peroxynitrite that stimulated nitrative modifications within the RNA and DNA pools in the phytopathogen structures. In contrast, the protein pool undergoing nitration was diminished confirming that protein tyrosine nitration is a flexible element of the oomycete adaptive strategy to heavy metal stress. Finally, to verify whether Cd is able to modify P. infestans pathogenicity, a disease index and molecular assessment of disease progress were analysed indicating that Cd stress enhanced aggressiveness of vr P. infestans towards various potato cultivars. Taken together, Cd not only affected hyphal growth rate and caused biochemical changes in P. infestans structures, but accelerated the pathogenicity as well. The nitro-oxidative homeostasis imbalance underlies the phytopathogen adaptive strategy and survival in the heavy metal contaminated environment.

Freezing Tolerance of Lolium multiflorum/Festuca arundinacea Introgression Forms is Associated with the High Activity of Antioxidant System and Adjustment of Photosynthetic Activity under Cold Acclimation.

Though winter-hardiness is a complex trait, freezing tolerance was proved to be its main component. Species from temperate regions acquire tolerance to freezing in a process of cold acclimation, which is associated with the exposure of plants to low but non-freezing temperatures. However, mechanisms of cold acclimation in Lolium-Festuca grasses, important for forage production in Europe, have not been fully recognized. Thus, two L. multiflorum/F. arundinacea introgression forms with distinct freezing tolerance were used herein as models in the comprehensive research to dissect these mechanisms in that group of plants. The work was focused on: (i) analysis of cellular membranes' integrity; (ii) analysis of plant photosynthetic capacity (chlorophyll fluorescence; gas exchange; gene expression, protein accumulation, and activity of selected enzymes of the Calvin cycle); (iii) analysis of plant antioxidant capacity (reactive oxygen species generation; gene expression, protein accumulation, and activity of selected enzymes); and (iv) analysis of Cor14b accumulation, under cold acclimation. The more freezing tolerant introgression form revealed a higher integrity of membranes, an ability to cold acclimate its photosynthetic apparatus and higher water use efficiency after three weeks of cold acclimation, as well as a higher capacity of the antioxidant system and a lower content of reactive oxygen species in low temperature.

Depressed Cardiac Mechanical Energetic Efficiency: A Contributor to Cardiovascular Risk in Common Metabolic Diseases-From Mechanisms to Clinical Applications.

Cardiac mechanical energetic efficiency is the ratio of external work (EW) to the total energy consumption. EW performed by the left ventricle (LV) during a single beat is represented by LV stroke work and may be calculated from the pressure-volume loop area (PVLA), while energy consumption corresponds to myocardial oxygen consumption (MVO2) expressed on a per-beat basis. Classical early human studies estimated total mechanical LV efficiency at 20-30%, whereas the remaining energy is dissipated as heat. Total mechanical efficiency is a joint effect of the efficiency of energy transfer at three sequential stages. The first step, from MVO2 to adenosine triphosphate (ATP), reflects the yield of oxidative phosphorylation (i.e., phosphate-to-oxygen ratio). The second step, from ATP split to pressure-volume area, represents the proportion of the energy liberated during ATP hydrolysis which is converted to total mechanical energy. Total mechanical energy generated per beat-represented by pressure-volume area-consists of EW (corresponding to PVLA) and potential energy, which is needed to develop tension during isovolumic contraction. The efficiency of the third step of energy transfer, i.e., from pressure-volume area to EW, decreases with depressed LV contractility, increased afterload, more concentric LV geometry with diastolic dysfunction and lower LV preload reserve. As practical assessment of LV efficiency poses methodological problems, De Simone et al. proposed a simple surrogate measure of myocardial efficiency, i.e., mechano-energetic efficiency index (MEEi) calculated from LV stroke volume, heart rate and LV mass. In two independent cohorts, including a large group of hypertensive subjects and a population-based cohort (both free of prevalent cardiovascular disease and with preserved ejection fraction), low MEEi independently predicted composite adverse cardiovascular events and incident heart failure. It was hypothesized that the prognostic ability of low MEEi can result from its association with both metabolic and hemodynamic alterations, i.e., metabolic syndrome components, the degree of insulin resistance, concentric LV geometry, LV diastolic and discrete systolic dysfunction. On the one part, an increased reliance of cardiomyocytes on the oxidation of free fatty acids, typical for insulin-resistant states, is associated with both a lower yield of ATP per oxygen molecule and lesser availability of ATP for contraction, which might decrease energetic efficiency of the first and second step of energy transfer from MVO2 to EW. On the other part, concentric LV remodeling and LV dysfunction despite preserved ejection fraction can impair the efficiency of the third energy transfer step. In conclusion, the association of low MEEi with adverse cardiovascular outcome might be related to a multi-step impairment of energy transfer from MVO2 to EW in various clinical settings, including metabolic syndrome, diabetes, hypertension and heart failure. Irrespective of theoretical considerations, MEEi appears an attractive simple tool which couldt improve risk stratification in hypertensive and diabetic patients for primary prevention purposes. Further clinical studies are warranted to estimate the predictive ability of MEEi and its post-treatment changes, especially in patients on novel antidiabetic drugs and subjects with common metabolic diseases and concomitant chronic coronary syndromes, in whom the potential relevance of MEE can be potentiated by myocardial ischemia.

Identification and epidemiological analysis of Perostrongylus falciformis infestation in Irish badgers.

BACKGROUND: The lungworm, Perostrongylus falciformis (fomerly known as Aelurostrongylus falciformis) has been identified in badgers (Meles meles) in Britain, the Russian Federation, Italy, Norway, Poland, Ukraine, Bosnia Herzegovina and Romania, while Aelurostrongylus pridhami has been reported from badgers in Spain. RESULTS: Pulmonary tissue from 1580 Irish badgers was examined and an estimated prevalence of 32.09% (95% CI: 29.79-34.45%) of this parasite was detected. Confirmation of its occurrence was made by PCR analysis on a subset of the population. CONCLUSION: Infestation was widely distributed throughout the Republic of Ireland, with a trend towards higher infestation risk in western versus eastern counties. In addition males were at a higher risk of infestation than females and juveniles were at a significantly higher risk than adult badgers.

Effect of cellular mass on chondrogenic differentiation during embryoid body formation.

One approach to cell differentiation is to use the natural capacity of pluripotent stem cells to form three germ layers via embryoid bodies (EB). However, unification of this process during in vitro culture remains challenging and many microenvironmental factors including the number of cells in the culture can influence differentiation patterns. The number of cells serves a crucial role as it determines access to nutrients, the distribution of oxygen concentration and cellular interactions, all of which influence the fate of the differentiated cells. The influence of EBs derived from human pluripotent cells on the chondrogenic potential of such cells is not well understood. For this reason, the present study sought to determine the effect of varying amounts of cells on the properties of EBs derived from human embryonic stem cells (BG01V cell line). In the present study, 500­2,000 cells per well were cultivated from 5 to 15 days in suspension cell culture. Expression of pluripotency genes and germ layer markers were evaluated in order to determine the EBs with the greatest and least mesodermal properties. Genes associated with pluripotency and chondrogenesis were also evaluated to assess the influence of suspension culture duration and EB size on chondrogenic differentiation. Immunofluorescence staining for pluripotent and chondrocyte­associated proteins confirmed successful differentiation into chondrocyte­like cells. Alcian blue staining confirmed deposition of proteoglycans. These results suggested that EBs formed in 500­cell wells possess the highest mesodermal and prochondrogenic properties. Differentiation of EBs into chondrocytes on day 5 in 500­cell wells was more efficient than in that observed in larger and older EBs.

Studies of Hematopoietic Cell Differentiation with a Ratiometric and Reversible Sensor of Mitochondrial Reactive Oxygen Species.

AIMS: Chronic elevations in cellular redox state are known to result in the onset of various pathological conditions, but transient increases in reactive oxygen species (ROS)/reactive nitrogen species (RNS) are necessary for signal transduction and various physiological functions. There is a distinct lack of reversible fluorescent tools that can aid in studying and unraveling the roles of ROS/RNS in physiology and pathology by monitoring the variations in cellular ROS levels over time. In this work, we report the development of ratiometric fluorescent sensors that reversibly respond to changes in mitochondrial redox state. RESULTS: Photophysical studies of the developed flavin-rhodamine redox sensors, flavin-rhodamine redox sensor 1 (FRR1) and flavin-rhodamine redox sensor 2 (FRR2), confirmed the reversible response of the probes upon reduction and re-oxidation over more than five cycles. The ratiometric output of FRR1 and FRR2 remained unaltered in the presence of other possible cellular interferants (metals and pH). Microscopy studies indicated clear mitochondrial localization of both probes, and FRR2 was shown to report the time-dependent increase of mitochondrial ROS levels after lipopolysaccharide stimulation in macrophages. Moreover, it was used to study the variations in mitochondrial redox state in mouse hematopoietic cells at different stages of embryonic development and maturation. INNOVATION: This study provides the first ratiometric and reversible probes for ROS, targeted to the mitochondria, which reveal variations in mitochondrial ROS levels at different stages of embryonic and adult blood cell production. CONCLUSIONS: Our results suggest that with their ratiometric and reversible outputs, FRR1 and FRR2 are valuable tools for the future study of oxidative stress and its implications in physiology and pathology. Antioxid. Redox Signal. 24, 667-679.

Thigh circumference as a useful predictor of body fat in adolescent girls with anorexia nervosa.

BACKGROUND/AIM: Girls with anorexia nervosa (AN) demonstrate severe depletion of body fat. The aim of this study was to determine an accurate anthropometric measurement for clinical assessment of fat depletion in girls with AN in connection with body composition measured by dual-energy X-ray absorptiometry (DXA). METHODS: In 64 female AN patients aged 12.8-23.1 years (mean 16.0 ± 1.8), body mass index (BMI), skinfold thickness (subscapular, abdominal and triceps), mid-upper arm and thigh circumference, fat mass (FM) and lean mass were determined and compared with the data of 71 controls. RESULTS: Girls with AN had lower anthropometric traits and were fat depleted compared to controls (14.9 ± 7.3 vs. 27.4 ± 6.4% of FM using DXA; all p < 0.001). BMI, thigh circumference and subscapular skinfold thickness demonstrated a very similar predictive value for DXA assessment of body fat. Based on the receiver-operating characteristic curve analysis and the determination of the positive predictive value, thigh circumference appeared the most specific and sensitive anthropometric predictor of fatness discriminating between AN and healthy girls, with the AUC value reaching 0.95 (95% CI = 0.92-0.97). Using a cutoff value of 49.6 cm, accuracy was 90.6%, sensitivity 93% and specificity 88.7%. CONCLUSIONS: Thigh circumference strongly correlates with DXA-FM and demonstrates a slight clinical advantage over BMI. This simple measurement might also serve as a useful predictor of body fatness in adolescent girls with AN and should therefore be further evaluated in independent cohort studies.

Is there relationship between the A1166C polymorphism of the angiotensin II receptor AT1 and plasma renin activity, insulin resistance and reduction of blood pressure after angiotensin-converting enzyme inhibitor therapy?

OBJECTIVES: The aim of this study was to assess the relationship between the A1166C polymorphism of the angiotensin AT1 receptor gene and reduction of blood pressure and pulse pressure in patients with mild and moderate arterial hypertension. Moreover, we sought to investigate the impact of insulin resistance and plasma renin activity on blood pressure reduction following treatment with perindopril depending on the A1166C polymorphism of the AT1 receptor gene. PATIENTS AND METHODS: The study included 64 patients with mild-to-moderate essential hypertension, with a mean age of 40.5 +/-16.4 years. Before and after treatment with angiotensin-converting enzyme inhibitors (ACEI) blood pressure measurement with a traditional method and ambulatory blood pressure monitoring were performed and blood samples were taken for laboratory investigation. RESULTS: The A1166C genotype distribution was: AA 53.1% in 34 patients, AC 43.8% in 28 patients, CC 3.1% in 2 patients. There were no statistically significant differences in the magnitude of blood pressure reduction and pulse pressure after treatment with perindopril between genotypes. Only in patients with genotype AA insulin resistance correlated with body mass index and only in these patients we observed a significant correlation between plasma renin activity and reduction of diastolic blood pressure. There was an inverse correlation between insulin resistance and reduction of systolic blood pressure only in patients with genotype AC. CONCLUSIONS: The A1166C polymorphism of the AT1 receptor gene is not associated with reduction of blood pressure after treatment with ACEI in patients with essential hypertension. There is a negative correlation between plasma renin activity and reduction of diastolic blood pressure only in patients with genotype AA. There is an inverse correlation between insulin resistance and systolic blood pressure only in patients with AC genotype.