A benign approach to the preparation of freshwater bryozoan statoblasts for scanning electron microscopy (SEM) imaging

Abstract

Several different species of freshwater Bryozoa, belonging to the genera Plumatella, Rumarcanella and Fredericella, were detected within the Northern Mallee Pipeline (NMP) system in Victoria, Australia, that required definitive identification. These organisms produce asexual buds called statoblasts, with valves composed of sclerotised chitin that bear minute micro-ornamentations of considerable taxonomical significance. Imaging and analysis of these distinctive micro-ornamentations using scanning electron microscopy (SEM) is often employed for species identification. Meticulous preparation of statoblast samples is therefore required that necessitates the removal of adhering debris, dehydration and drying—whilst mitigating specimen damage and distortion. This technical note describes an approach whereby each of these three steps have been individually designed to be as benign as possible, using mild detergent/sonication to remove debris, a gradual and gentle dehydration procedure using ethanol, and critical point drying. For the overall process, these methods are chosen to optimise control and to minimise the use of harsh and hazardous chemicals.     

The use of adjunctive GPIIb/IIIa inhibitors in patients with unstable angina/non-Q-wave MI undergoing percutaneous coronary intervention

Glycoprotein IIb/IIIa receptor inhibitors represent a relatively new therapeutic approach in the field of antiplatelet therapy. Following the development of abciximab a number of small molecule GPIIb/IIIa inhibitors have been introduced such as tirofiban and eptifibatide. In this fast-moving field the interventional cardiologist needs a framework to guide decision-making for the individual patient. This review covers the efficacy and safety data from the clinical trials of GPIIb/IIIa inhibitors in the context of patients undergoing percutaneous coronary intervention for unstable angina/non-Q-wave myocardial infarction. There is an increasing body of evidence to support the efficacy of GPIIb/IIIa inhibitors in reducing the risk of adverse ischemic events in high and low risk patients undergoing percutaneous coronary intervention. A number of unresolved efficacy and safety issues remain, including the duration of treatment before and after intervention; whether a reduction in the heparin dose would further decrease the risk of hemorrhage without affecting the periprocedural thrombotic rate in patients undergoing PTCA with adjunctive GPIIb/IIIa inhibitors; and the cost-effectiveness of this therapy. When a thorough analysis of cost-effectiveness has been made, it will be easier to advocate the widespread use of these agents in all patients undergoing coronary intervention.     

A high-throughput cloning system for reverse genetics in <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Background  

The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.     

Spatial and temporal population genetic survey of Bulinus globosus from Zanzibar: an intermediate host of Schistosoma haematobium

In Zanzibar, the freshwater hermaphroditic snail Bulinus globosus is the intermediate host of the human parasite Schistosoma haematobium . To shed light on the patterns of genetic variation within Bu. globosus , variation at six polymorphic microsatellite loci was assessed to quantify spatial genetic structure within eight populations and temporal changes in a further four populations of the snails. Limited genetic variation was observed within populations, possibly reflecting both partial-selfing and fluctuations in population size. Although a statistically significant heterozygote deficiency was observed, Bu. globosus appears to be a preferential out-crosser, which was consistent with the absence of genotypic linkage disequilibria. Genetic variation across populations was substantial, illustrating significant isolation-by-distance on a regional scale and that gene flow was restricted to nearby populations. The temporal survey showed that levels of genetic variation and inbreeding changed over a 1-year period, consistent with field observations of seasonal change. The results strongly support a snail demographic model of population expansion and contraction throughout the year with habitats re-colonized by aestivating or surviving snails from local refugia. This model might help explain co-evolution of snails and schistosomes in Zanzibar as local populations of Bu. globosus have varying degrees of susceptibility to S. haematobium .     

Urinary schistosomiasis on Zanzibar: application of two novel assays for the detection of excreted albumin and haemoglobin in urine

As part of a urinary schistosomiasis control programme on Zanzibar, an aged cross-sectional survey of 305 children from three schools on Unguja was conducted to investigate the relationships between levels of excreted albumin and haemoglobin in urine and Schistosoma haematobium infection status. Diagnosis was determined by standard parasitological methods, dipstick reagents for microhaematuria, visual inspection for macrohaematuria as well as collection of case-history questionnaire data for self-diagnosis. Prevalence of infection as determined by parasitology was 53.9% and approximately, one quarter of the children examined were anaemic (<11 g dl(-1)). A statistically significant negative association of blood haemoglobin levels of boys and S. haematobium infection intensity status was observed (rs=-0.23, P=0.005). Through sensitivity analysis of urine-albumin values it was determined that a concentration of above >40 mg l(-1), as measured with the HemoCue urine-albumin photometer, had sensitivity, specificity, positive and negative predictive values of 0.90, 0.83, 0.86 and 0.89 respectively against 'gold-standard' parasitology. There was a clear association of reported pain upon micturition for children with elevated urine-albumin levels, with an odds ratio of 20 to 1. Levels of excreted blood in urine were quantified with the HemoCue Plasma/Low Hb photometer. However, dipsticks remain the method of choice for urine-haemoglobin of 0.1 g l(-1) and below. Urine parameters over a 24-h period were assessed in a small sub-sample. Reductions in both albumin and haemoglobin excretion were observed in 11 children 54 days after praziquantel treatment. It was concluded that these rapid, high-through-put, portable HemoCue assays could play a role in better describing and monitoring the occurrence, severity and evolution of urinary schistosomiasis disease. The urine-albumin assay has particular promise as a biochemical marker of S. haematobium induced kidney- and upper urinary tract-morbidity.     

Immunoprophylaxis of respiratory syncytial virus: global experience

In sarcoidosis, host genetic factors are discussed as contributing to disease susceptibility and course. Since tumor necrosis factor (TNF)-α is a central mediator of granuloma formation and since elevated TNF-α levels are found during active phases of sarcoidosis, genetic polymorphisms correlating with influences on TNF-α levels are of special interest. The complete sequencing of the MHC region and the increase in the number of identified gene polymorphisms in this locus associated with TNF-α production offer the opportunity of detecting new genes associated with sarcoidosis and perhaps of defining disease-associated haplotypes that bear the potential of serving as predictive markers for this disease.     

Flexibility in crosstalk between H2B ubiquitination and H3 methylation in vivo

Histone H2B ubiquitination is a dynamic modification that promotes methylation of histone H3K79 and H3K4. This crosstalk is important for the DNA damage response and has been implicated in cancer. Here, we show that in engineered yeast strains, ubiquitins tethered to every nucleosome promote H3K79 and H3K4 methylation from a proximal as well as a more distal site, but only if in a correct orientation. This plasticity indicates that the exact location of the attachment site, the native ubiquitin-lysine linkage and ubiquitination cycles are not critical for trans-histone crosstalk in vivo. The flexibility in crosstalk also indicates that other ubiquitination events may promote H3 methylation.