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 Superior Technique for Culturing and Karyotyping Solid Tumors

We have devised a new cell culture method where two types of culture plates made of normal tissue culture plastic (NTCP) and Primaria(TM), and two media, RPMI-1640 and a serum-free media LHC-9(TM), are used in combination with an in situ harvest. Multiple culture conditions in combination with in situ harvest has yielded many more clones for karyotyping than was possible with previous methods. Cells were cultured in 60 mm Petri plates instead of flasks and harvested in situ. Chromosome banding was performed using a highly purified trypsin "Enzar-T(TM)" and Leishman stain instead of ordinary trypsin and Giemsa stain. These modifications have increased the number of clones retrieved, rate of culture success, and quality of karyotypes in solid tumors.     

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.     

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.     

Potassium-resistant triple helix formation and improved intracellular gene targeting by oligodeoxyribonucleotides containing 7-deazaxanthine.

Triple helix formation by purine-rich oligonucleotides in the anti-parallel motif is inhibited by physiological concentrations of potassium. Substitution with 7-deazaxanthine (c7X) has been suggested as a strategy to overcome this effect. We have tested this by examining triple helix formation both in vitro and in vivo by a series of triple helix-forming oligonucleotides (TFOs) containing guanine plus either adenine, thymine, or c7X. The TFOs were conjugated to psoralen at the 5'end and were designed to bind to a portion of the supF mutation reporter gene. Using in vitro gel mobility shift assays, we found that triplex formation by the c7X-substituted TFOs was relatively resistant to the presence of 140 mM K+. The c7X-containing TFOs were also superior in gene targeting experiments in mammalian cells, yielding 4- to 5-fold higher mutation frequencies in a shuttle vector-based mutagenesis assay designed to detect mutations induced by third strand-directed psoralen adducts. When the phosphodiester backbone was replaced by a phosphorothioate one, the in vitro binding of the c7X-TFOs was not affected, but the efficiency of in vivo triple helix formation was reduced. These results indicate the utility of the c7X substitution for in vivo gene targeting experiments, and they show that the feasibility of the triplex anti-gene strategy can be significantly enhanced by advances in nucleotide chemistry.     

Electronic detectors for electron microscopy

Electron microscopy (EM) is an important tool for high-resolution structure determination in applications ranging from condensed matter to biology. Electronic detectors are now used in most applications in EM as they offer convenience and immediate feedback that is not possible with film or image plates. The earliest forms of electronic detector used routinely in transmission electron microscopy (TEM) were charge coupled devices (CCDs) and for many applications these remain perfectly adequate. There are however applications, such as the study of radiation-sensitive biological samples, where film is still used and improved detectors would be of great value. The emphasis in this review is therefore on detectors for use in such applications. Two of the most promising candidates for improved detection are: monolithic active pixel sensors (MAPS) and hybrid pixel detectors (of which Medipix2 was chosen for this study). From the studies described in this review, a back-thinned MAPS detector appears well suited to replace film in for the study of radiation-sensitive samples at 300 keV, while Medipix2 is suited to use at lower energies and especially in situations with very low count rates. The performance of a detector depends on the energy of electrons to be recorded, which in turn is dependent on the application it is being used for; results are described for a wide range of electron energies ranging from 40 to 300 keV. The basic properties of detectors are discussed in terms of their modulation transfer function (MTF) and detective quantum efficiency (DQE) as a function of spatial frequency.     

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.