Effect of recovery method on yield of bovine oocytes per ovary and their developmental competence after maturation, fertilization and culture in vitro

An important aim of an oocyte recovery method is to maximize the number of oocytes per ovary which can be employed for in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC). In this study, primary bovine oocytes were collected by 2 methods: aspiration of visible follicles (2 to 6mm in diameter) or surface dissection in which the ovary surface is finely dissected. The oocytes were classified on the basis of cumulus cover and cytoplasmic appearance. The total number of oocytes and the yield of good-quality oocytes recovered per ovary by surface dissection and aspiration were 44.2 and 13.9 and 13.5 and 4.6 (P<0.05), respectively. When a sample group of selected oocytes recovered by each method was measured, no significant difference was found in the mean diameter (144.11m vs 142.54m). A representative sample of good-quality oocytes recovered by each method was put through the IVM/IVF/IVC procedure: no significant difference in cleavage rate, cleavage index or blastocyst yield was found. However, when the blastocyst yield was compared on a per ovary basis, a significant difference was observed in favor of surface dissection (3.30+/-0.46 vs 0.96+/-0.16;P<0.05). When unselected oocytes recovered by surface dissection of the ovaries were put through the standard embryo production system, an average of 15.4 blastocysts per dam was obtained.     

Adaptations in coactivation after isometric resistance training.

Twenty sedentary male university students were randomly assigned to an experimental or a control group. The experimental group trained the knee extensors of one leg by producing 30 isometric extension maximal voluntary contractions (MVC) per day, three times per week for 8 wk. After 8 wk of training, extensor MVC in the trained leg increased 32.8% (P less than 0.05), but there was no change in vastus lateralis maximal integrated electromyographic activity (IEMGmax). The most important finding was that the degree of hamstring coactivation during extension MVC decreased by approximately 20% (P less than 0.05) after the 1st wk of training. Less pronounced adaptations occurred in the untrained leg: extension MVC force increased 16.2% (P less than 0.05), hamstring coactivity decreased 13% (P less than 0.05) after 2 wk of training, and vastus lateralis IEMGmax was unchanged. The same measures in legs of the control group were not changed during the study. There were no changes in flexion MVC, biceps femoris IEMGmax, or the degree of quadriceps coactivity during flexion MVC in either leg of the control or experimental group. A reduction in hamstring coactivity in the trained and untrained legs indicates that these muscles provide less opposing force to the contracting quadriceps. We conclude that this small but significant decrease in hamstring coactivation that occurs during the early stages of training is a nonhypertrophic adaptation of the neuromuscular system in response to static resistance training of this type.     

Effect of Lincocin Treatment on the Greening Process in Bean (Phaseolus vulgaris) Leaves

The effect of lincocin (a plastid protein synthesis inhibitor) treatment on the greening process of bean (Phaseolus vulgaris L.) leaves have been studied. In comparison with control leaves treated ones had a decreased rate of chloroplast development. They had a marked chlorophyll deficiency and a decreased chlorophyll a/b ratio. Some long and short wavelength forms of chlorophyll a were lacking as evidenced from the absorption spectra at 25°C and the fluorescence spectra at 77°K. The –¹⁴CO₂ fixation was inhibited by 80–90% in treated leaves. The fluorescence induced by the measuring light was greater in the treated leaves than in the control ones, and the kinetics of the decline of the relative fluorescence intensity were also different. Electron microscopic studies showed macrogranum-like structures and incomplete membrane vesicles in the treated plastids. After longer treatment a destruction of membranes was observed. The results indicate some structural and functional membrane deficiencies and instability of the membranes.     

Lecithotrophic development and metamorphosis in the Indo-West Pacific brittle star Ophiomastix venosa (Echinodermata: Ophiuroidea)

Summary

The larval development of the ophiocomid ophiuroid Ophiomastix venosais described using SEM. The gastrula transforms into a uniformly ciliated early larva which progressively changes into a lecithotrophic late premetamorphic larva with a continuous bilateral ciliated band. This stage is short-lived and equivalent to a highly reduced ophiopluteus. Comparisons between O. venosa and other ophiuroid species whose development has been investigated suggest that, whatever the developmental mode (lecithotrophic or planktotrophic), a pluteus stage always occurs in ophiuroids with planktonic development. Two metamorphic stages were identified, the late metamorphic larva differing from the early one by the closure of the larval mouth. The appearance of the permanent mouth marks the end of the metamorphosis. The postlarva still possesses remnants of larval features. The transformation of the reduced ophiopluteus into a barrel-shaped metamorphic larva with transverse ciliated bands, a vitellaria larva, is followed. The possible occurrence of a unique type of metamorphic larva in non-brooding ophiuroids is discussed. Verification of this, however, needs further SEM investigations on metamorphic larva from species having “regular” planktotrophic development.     

A Proteomic Investigation of Hepatic Resistance to Ascaris in a Murine Model

The helminth Ascaris causes ascariasis in both humans and pigs. Humans, especially children, experience significant morbidity including respiratory complications, growth deficits and intestinal obstruction. Given that 800 million people worldwide are infected by Ascaris, this represents a significant global public health concern. The severity of the symptoms and associated morbidity are related to the parasite burden and not all hosts are infected equally. While the pathology of the disease has been extensively examined, our understanding of the molecular mechanisms underlying resistance and susceptibility to this nematode infection is poor. In order to investigate host differences associated with heavy and light parasite burden, an experimental murine model was developed utilising Ascaris-susceptible and -resistant mice strains, C57BL/6J and CBA/Ca, respectively, which experience differential burdens of migratory Ascaris larvae in the host lungs. Previous studies identified the liver as the site where this difference in susceptibility occurs. Using a label free quantitative proteomic approach, we analysed the hepatic proteomes of day four post infection C57BL/6J and CBA/Ca mice with and without Ascaris infection to identify proteins changes potentially linked to both resistance and susceptibility amongst the two strains, respectively. Over 3000 proteins were identified in total and clear intrinsic differences were elucidated between the two strains. These included a higher abundance of mitochondrial proteins, particularly those associated with the oxidative phosphorylation pathway and reactive oxygen species (ROS) production in the relatively resistant CBA/Ca mice. We hypothesise that the increased ROS levels associated with higher levels of mitochondrial activity results in a highly oxidative cellular environment that has a dramatic effect on the nematode’s ability to successfully sustain a parasitic association with its resistant host. Under infection, both strains had increased abundances in proteins associated with the oxidative phosphorylation pathway, as well as the tricarboxylic acid cycle, with respect to their controls, indicating a general stress response to Ascaris infection. Despite the early stage of infection, some immune-associated proteins were identified to be differentially abundant, providing a novel insight into the host response to Ascaris. In general, the susceptible C57BL/6J mice displayed higher abundances in immune-associated proteins, most likely signifying a more active nematode cohort with respect to their CBA/Ca counterparts. The complement component C8a and S100 proteins, S100a8 and S100a9, were highly differentially abundant in both infected strains, signifying a potential innate immune response and the importance of the complement pathway in defence against macroparasite infection. In addition, the signatures of an early adaptive immune response were observed through the presence of proteins, such as plastin-2 and dipeptidyl peptidase 1. A marked decrease in proteins associated with translation was also observed in both C57BL/6J and CBA/Ca mice under infection, indicative of either a general response to Ascaris or a modulatory effect by the nematode itself. Our research provides novel insights into the in vivo host-Ascaris relationship on the molecular level and provides new research perspectives in the development of Ascaris control and treatment strategies.     

Investigation of protein induction in tumour vascular targeted strategies by MALDI MSI

Characterising the protein signatures in tumours following vascular-targeted therapy will help determine both treatment response and resistance mechanisms. Here, mass spectrometry imaging and MS/MS with and without ion mobility separation have been used for this purpose in a mouse fibrosarcoma model following treatment with the tubulin-binding tumour vascular disrupting agent, combretastatin A-4-phosphate (CA-4-P). Characterisation of peptides after in situ tissue tryptic digestion was carried out using Matrix-Assisted Laser Desorption/Ionisation-Mass Spectrometry (MALDI-MS) and Matrix-Assisted Laser Desorption/Ionisation-Ion Mobility Separation-Mass Spectrometry Imaging (MALDI IMS-MSI) to observe the spatial distribution of peptides. Matrix-Assisted Laser Desorption/Ionisation-Ion Mobility Separation-Tandem Mass Spectrometry (MALDI-IMS-MS/MS) of peaks was performed to elucidate any pharmacological responses and potential biomarkers. By taking tumour samples at a number of time points after treatment gross changes in the tissue were indicated by changes in the signal levels of certain peptides. These were identified as arising from haemoglobin and indicated the disruption of the tumour vasculature. It was hoped that the use of PCA-DA would reveal more subtle changes taking place in the tumour samples however these are masked by the dominance of the changes in the haemoglobin signals.     

Novel isosorbide di-ester compounds as inhibitors of acetylcholinesterase

We report herein that a variety of isosorbide di-esters, previously reported to be novel substrates for butyrylcholinesterase (BuChE, EC 3.1.1.8), are in fact inhibitors of the homologous enzyme acetylcholinesterase (AChE), with IC(50) values in the micromolar range. In vitro studies show that they are mixed inhibitors of the enzyme, and thus the ternary enzyme-inhibitor-substrate complex can form in acetylcholinesterase. This is rationalised by molecular modelling which shows that the compounds bind in the mid-gorge area. In this position, simultaneous substrate binding might be possible, but the hydrolysis of this substrate is prevented. The di-esters dock within the butyrylcholinesterase gorge in a very different manner, with the ester sidechain at the 5-position occupying the acyl pocket at residues Leu286 and Val288, and the 2-ester binding to Trp82. The carbonyl group of the 2-ester is susceptible to nucleophilic attack by Ser198 of the catalytic triad. The larger residues of the acyl pocket in acetylcholinesterase prevent binding in this manner. The results complement each other and explain the differing behaviours of the esters in the cholinesterase enzymes. These findings may prove very significant for future work.     

Topography and Land Cover of Watersheds Predicts the Distribution of the Environmental Pathogen Mycobacterium ulcerans in Aquatic Insects

Background

An understanding of the factors driving the distribution of pathogens is useful in preventing disease. Often we achieve this understanding at a local microhabitat scale; however the larger scale processes are often neglected. This can result in misleading inferences about the distribution of the pathogen, inhibiting our ability to manage the disease. One such disease is Buruli ulcer, an emerging neglected tropical disease afflicting many thousands in Africa, caused by the environmental pathogen Mycobacterium ulcerans. Herein, we aim to describe the larger scale landscape process describing the distribution of M. ulcerans.

Methodology

Following extensive sampling of the community of aquatic macroinvertebrates in Cameroon, we select the 5 dominant insect Orders, and conduct an ecological niche model to describe how the distribution of M. ulcerans positive insects changes according to land cover and topography. We then explore the generalizability of the results by testing them against an independent dataset collected in a second endemic region, French Guiana.

Principal Findings

We find that the distribution of the bacterium in Cameroon is accurately described by the land cover and topography of the watershed, that there are notable seasonal differences in distribution, and that the Cameroon model does not predict the distribution of M. ulcerans in French Guiana.

Conclusions/Significance

Future studies of M. ulcerans would benefit from consideration of local structure of the local stream network in future sampling, and further work is needed on the reasons for notable differences in the distribution of this species from one region to another. This work represents a first step in the identification of large-scale environmental drivers of this species, for the purposes of disease risk mapping.