Ultrastructural and cytochemical characterization of follicular cell types in bovine (Bos taurus) cumulus-oocyte complexes aspirated from small and medium antral follicles during the estrus cycle

In the Canadian Animal Genetic Resource Program, bull semen is donated in frozen or fresh (diluted) states. This study was designed to assess the cryopreservation of diluted bull semen shipped at 4°C overnight, and to determine the post-thaw quality of shipped semen using different straw volumes and freezing rates. Semen was collected from four breeding bulls (three ejaculates per bull). Semen was diluted in Tris-citric acid-egg yolk-glycerol (TEYG) extender, cooled to 4°C and frozen as per routine (control semen). After cooling to 4°C, a part of semen was removed and shipped overnight to the research laboratory via express courier (shipped semen). Semen was packaged in 0.25 or 0.5 ml straws and frozen in a programmable freezer using three freezing rates, i.e., -10, -25 or -40°C/min. Control semen was also shipped to the research laboratory. Post-thaw sperm motility characteristics were assessed using CASA, and post-thaw sperm plasma membrane, mitochondrial membrane potential and normal acrosomes were assessed using flow cytometry. Post-thaw sperm quality was greater in shipped semen as compared to control (P<0.001). The shipped semen packaged in 0.25 ml straws had better post-thaw sperm quality than in 0.5 ml straws (P<0.001). Freezing rate had no effect on post-thaw sperm quality. In conclusion, bull semen can be shipped overnight for subsequent cryopreservation and gene banking. Overnight shipping of semen was found advantageous for bull semen cryopreservation. Semen packaging in 0.25 ml straws yielded better post-thaw quality than 0.5 ml straws.     

Mechanism of stereoselective interaction between butyrylcholinesterase and ethopropazine enantiomers

Stereoselectivity of reversible inhibition of butyrylcholinesterase (BChE; EC 3.1.1.8) by optically pure ethopropazine [10-(2-diethylaminopropyl)phenothiazine hydrochloride] enantiomers and racemate was studied with acetylthiocholine (0.002–250 mM) as substrate. Molecular modelling resulted in the reaction between BChE and ethopropazine starting with the binding of ethopropazine to the enzyme peripheral anionic site. In the next step ethopropazine ‘slides down’ the enzyme gorge, resulting in interaction of the three rings of ethopropazine through π–π interactions with W82 in BChE. Inhibition mechanism was interpreted according to three kinetic models: A, B and C. The models differ in the type and number of enzyme–substrate, enzyme–inhibitor and enzyme–substrate–inhibitor complexes, i.e., presence of the Michaelis complex and/or acetylated BChE. Although, all three models reproduced well the BChE activity in absence of ethopropazine, model A was poor in describing inhibition with ethopropazine, while models B and C were better, especially for substrate concentrations above 0.2 mM. However model C was singled out because it approaches fulfilment of the one step-one event criteria, and confirms the inhibition mechanism derived from molecular modelling. Model C resulted in dissociation constants for the complex between BChE and ethopropazine: 61, 140 and 88 nM for R-enantiomer, S-enantiomer and racemate, respectively. The respective dissociation constants for the complexes between acetylated BChE and ethopropazine were 268, 730 and 365 nM. Butyrylcholinesterase had higher affinity for R-ethopropazine.     

Assessment of the Efficacy of Solutes from Extremophiles on Protein Aggregation in Cell Models of Huntington’s and Parkinson’s Diseases

Protein misfolding and deposition in the brain are implicated in the etiology of numerous neurodegenerative disorders. Here, organic solutes characteristic of microorganisms adapted to hot environments, were tested on experimental cell models of Huntington’s and Parkinson’s diseases. Diglycerol phosphate, di-myo-inositol phosphate, mannosylglycerate, and mannosylglyceramide were not toxic to the cells, at 10 mM concentration, but caused a decrease in cell density, which suggested an effect on proliferation. In contrast, mannosyl-lactate, an artificial analogue of mannosylglycerate, had a negative impact on cell viability. Concerning protein aggregation, inclusions of mutant huntingtin were reduced in the presence of diglycerol phosphate and di-myo-inositol phosphate, increased with mannosylglycerate, while mannosyl-lactate and mannosylglyceramide had no significant effect. α-Synuclein aggregation was not affected by the solutes tested, except for di-myo-inositol phosphate that led to a slight increased percentage of cells displaying visible aggregates. These solutes might be useful in the development of therapies for protein misfolding diseases.     

Release of ATP from avian Müller glia cells in culture

ATP can be released from neurons and act as a neuromodulator in the nervous system. Besides neurons, cortical astrocytes also are capable of releasing ATP from acidic vesicles in a Ca(2+)-dependent way. In the present work, we investigated the release of ATP from Müller glia cells of the chick embryo retina by examining quinacrine staining and by measuring the extracellular levels of ATP in purified Müller glia cultures. Our data revealed that glial cells could be labeled with quinacrine, a reaction that was prevented by incubation of the cells with 1μM bafilomycin A1 or 2μM Evans blue, potent inhibitors of vacuolar ATPases and of the vesicular nucleotide transporter, respectively. Either 50mM KCl or 1mM glutamate was able to decrease quinacrine staining of the cells, as well as to increase the levels of ATP in the extracellular medium by 77% and 89.5%, respectively, after a 5min incubation of the cells. Glutamate-induced rise in extracellular ATP could be mimicked by 100μM kainate (81.5%) but not by 100μM NMDA in medium without MgCl(2) but with 2mM glycine. However, both glutamate- and kainate-induced increase in extracellular ATP levels were blocked by 50μM of the glutamatergic antagonists DNQX and MK-801, suggesting the involvement of both NMDA and non-NMDA receptors. Extracellular ATP accumulation induced by glutamate was also blocked by incubation of the cells with 30μM BAPTA-AM or 1μM bafilomycin A1. These results suggest that glutamate, through activation of both NMDA and non-NMDA receptors, induces the release of ATP from retinal Müller cells through a calcium-dependent exocytotic mechanism.     

Molecular assessment of microbiota structure and dynamics along mixed olive oil and winery wastewaters biotreatment

The major parcel of the degradation occurring along wastewater biotreatments is performed either by the native microbiota or by added microbial inocula. The main aim of this study was to apply two fingerprinting methods, temperature gradient gel electrophoresis (TGGE) and length heterogeneity-PCR (LH-PCR) analysis of 16S rRNA gene fragments, in order to assess the microbiota structure and dynamics during mixed olive oil and winery wastewaters aerobic biotreatment performed in a jet-loop reactor (JLR). Sequence homology analysis showed the presence of bacterial genera Gluconacetobacter, Klebsiella, Lactobacillus, Novosphingobium, Pseudomonas, Prevotella, Ralstonia, Sphingobium and Sphingomonas affiliated with five main phylogenetic groups: alpha-, beta- and gamma-Proteobacteria, Firmicutes and Bacteroidetes. LH-PCR analysis distinguished eight predominant DNA fragments correlated with the samples showing highest performance (COD removal rates of 67 up to 75%). Cluster analysis of both TGGE and LH-PCR fingerprinting profiles established five main clusters, with similarity coefficients higher than 79% (TGGE) and 62% (LH-PCR), and related with hydraulic retention time, indicating that this was the main factor responsible for the shifts in the microbiota structure. Canonical correspondence analysis revealed that changes observed on temperature and O₂ level were also responsible for shifts in microbiota composition. Community level metabolic profile analysis was used to test metabolic activities in samples. Integrated data revealed that the microbiota structure corresponds to bacterial groups with high degradative potential and good suitability for this type of effluents biotreatments.     

Effect of repetitive potassium iodide on thyroid and cardiovascular functions in elderly rats

BackgroundTo date, paediatric thyroid cancer has been the most severe health consequence of the Chernobyl accident, caused by radioactive iodine (¹³¹I) aerosol's dispersion. WHO recommends a single dose of potassium iodide (KI) to reduce this risk. Following the Fukushima accident, it became obvious that repetitive doses of KI may be necessary due to multiple exposures to ¹³¹I. Knowledge about the effects of repeated ITB (Iodine Thyroid Blocking) is scarce and controversial. KI may affect the thyroid hormones synthesis; which is crucial for the cardiovascular function. Furthermore, myocardial and vascular endothelial tissues are sensitizes to subtle changes at the concentration of circulating pituitary and/or thyroid hormones.ObjectiveIn this preclinical study, we aimed to assess the effects of repeated ITB in elderly male rats.MethodsTwelve months old male Wistar rats were subjected to either KI or saline solution for eight days. Analyses were performed 24 h and 30 days after the treatment discontinuation.FindingsWe reported a significant increase (18%) in some urinary parameters related to renal function, a subtle decrease of plasma TSH level, a significant increase (379%) in renin and a significant decrease (50%) in aldosterone upon KI administration. At the molecular level, the expression of thyroid and cardiovascular genes was significantly affected by the treatment. However, in our experimental settlement, animal heart rate was not significantly affected thirty days after KI discontinuation. ECG patterns did not change after administration of KI, and arrhythmia was not observed in these conditions despite the PR-intervals decreased significantly. Cardiovascular physiology was preserved.ConclusionOur results indicate that repeated ITB in elderly rats is characterized by molecular modifications of cardiovascular key actors, particularly the Renin-angiotensin-aldosterone axis with a preserved physiological homeostasis. This new scientific evidence may be useful for the maturation of ITB guidelines especially for elderly sub-population.     

How to Improve the Early Diagnosis of Trypanosoma cruzi Infection: Relationship between Validated Conventional Diagnosis and Quantitative DNA Amplification in Congenitally Infected Children

Background

According to the Chagas congenital transmission guides, the diagnosis of infants, born to Trypanosoma cruzi infected mothers, relies on the detection of parasites by INP micromethod, and/or the persistence of T. cruzi specific antibody titers at 10–12 months of age.

Methodology and Principal Findings

Parasitemia levels were quantified by PCR in T. cruzi-infected children, grouped according to the results of one-year follow-up diagnosis: A) Neonates that were diagnosed in the first month after delivery by microscopic blood examination (INP micromethod) (n = 19) had a median parasitemia of 1,700 Pe/mL (equivalent amounts of parasite DNA per mL); B) Infants that required a second parasitological diagnosis at six months of age (n = 10) showed a median parasitemia of around 20 Pe/mL and 500 Pe/mL at 1 and 6 months old, respectively, and C) babies with undetectable parasitemia by three blood microscopic observations but diagnosed by specific anti - T. cruzi serology at around 1 year old, (n = 22), exhibited a parasitemia of around 5 Pe/mL, 800 Pe/mL and 20 Pe/mL 1, 6 and 12 month after delivery, respectively. T. cruzi parasites were isolated by hemoculture from 19 congenitally infected children, 18 of which were genotypified as DTU TcV, (former lineage TcIId) and only one as TcI.

Significance

This report is the first to quantify parasitemia levels in more than 50 children congenitally infected with T. cruzi, at three different diagnostic controls during one-year follow-up after delivery. Our results show that the parasite burden in some children (22 out of 51) is below the detection limit of the INP micromethod. As the current trypanocidal treatment proved to be very effective to cure T. cruzi - infected children, more sensitive parasitological methods should be developed to assure an early T. cruzi congenital diagnosis.