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.     

Amphiphilic block copolymer/poly(dimethylsiloxane) (PDMS) blends and nanocomposites for improved fouling-release

Amphiphilic diblock copolymers, Sz6 and Sz12, consisting of a poly(dimethylsiloxane) block (average degree of polymerisation = 132) and a PEGylated-fluoroalkyl modified polystyrene block (Sz, average degree of polymerisation = 6, 12) were prepared by atom transfer radical polymerization (ATRP). Coatings were obtained from blends of either block copolymer (1–10 wt%) with a poly(dimethylsiloxane) (PDMS) matrix. The coating surface presented a simultaneous hydrophobic and lipophobic character, owing to the strong surface segregation of the lowest surface energy fluoroalkyl chains of the block copolymer. Surface chemical composition and wettability of the films were affected by exposure to water. Block copolymer Sz6 was also blended with PDMS and a 0.1 wt% amount of multiwall carbon nanotubes (CNT). The excellent fouling-release (FR) properties of these new coatings against the macroalga Ulva linza essentially resulted from the inclusion of the amphiphilic block copolymer, while the addition of CNT did not appear to improve the FR properties.     

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.     

Macrophage-tropic simian/human immunodeficiency virus chimeras use CXCR4, not CCR5, for infections of rhesus macaque peripheral blood mononuclear cells and alveolar macrophages

After the nearly complete and irreversible depletion of CD4(+) T lymphocytes induced by highly pathogenic simian/human immunodeficiency virus chimeric viruses (SHIVs) during infections of rhesus monkeys, tissue macrophages are able to sustain high levels (>10(6) viral RNA copies/ml) of plasma viremia for several months. We recently reported that the virus present in the plasma during the late macrophage phase of infection had acquired changes that specifically targeted the V2 region of gp120 (H. Imamichi et al., Proc. Natl. Acad. Sci. USA 99:13813-13818, 2002); some of these SHIV variants were macrophage-tropic (M-tropic). Those findings have been extended by examining the tropic properties, coreceptor usage, and gp120 structure of five independent SHIVs recovered directly from lymph nodes of late-stage animals. All of these tissue-derived SHIV isolates were able to infect alveolar macrophages. These M-tropic SHIVs used CXCR4, not CCR5, for infections of rhesus monkey PBMC and primary alveolar macrophages. Because the starting highly pathogenic T-tropic SHIV inoculum also utilized CXCR4, these results indicate that the acquisition of M-tropism in the SHIV-macaque system is not accompanied by a change in coreceptor usage. Compared to the initial T-tropic SHIV inoculum, tissue-derived M-tropic SHIVs from individual infected animals carry gp120s containing similar changes (specific amino acid deletions, substitutions, and loss of N-linked glycosylation sites), primarily within the V1 and/or V2 regions of gp120.     

Protein expression profiles distinguish between experimental invasive pulmonary aspergillosis and Pseudomonas pneumonia

We hypothesized that invasive pulmonary aspergillosis (IPA) may generate a distinctive proteomic signature in plasma and bronchoalveolar lavage (BAL). Proteins in plasma and BAL from two neutropenic rabbit models of IPA and Pseudomonas pneumonia were analyzed by SELDI-TOF MS. Hierarchical clustering analysis of plasma time course spectra demonstrated two clusters of peaks that were differentially regulated between IPA and Pseudomonas pneumonia (57 and 34 peaks, respectively, p<0.001). PCA of plasma proteins demonstrated a time-dependent separation of the two infections. A random forest analysis that ranked the top 30 spectral points distinguished between late Aspergillus and Pseudomonas pneumonias with 100% sensitivity and specificity. Based on spectral data analysis, three proteins were identified using SDS-PAGE and LC/MS and quantified using reverse phase arrays. Differences in the temporal sequence of plasma haptoglobin (p<0.001), apolipoprotein A1 (p<0.001) and transthyretin (p<0.038) were observed between IPA and Pseudomonas pneumonia, as was C-reactive protein (p<0.001). In summary, proteomic analysis of plasma and BAL proteins of experimental Aspergillus and Pseudomonas pneumonias demonstrates unique protein profiles with principal components and spectral regions that are shared in early infection and diverge at later stages of infection. Haptoglobin, apolipoprotein A1, transthyretin, and C-reactive protein are differentially expressed in these infections suggesting important contributions to host defense against IPA.     

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.     

A canine model of septic shock: balancing animal welfare and scientific relevance

A shock canine pneumonia model that permitted relief of discomfort with the use of objective criteria was developed and validated. After intrabronchial Staphylococcus aureus challenge, mechanical ventilation, antibiotics, fluids, vasopressors, sedatives, and analgesics were titrated based on algorithms for 96 h. Increasing S. aureus (1 to 8 x 10(9) colony-forming units/kg) produced decreasing survival rates (P = 0.04). From 4 to 96 h, changes in arterial-alveolar oxygen gradients, mean pulmonary artery pressure, IL-1, serum sodium levels, mechanical ventilation, and vasopressor support were ordered based on survival time [acute nonsurvivors (< or =24 h until death, n = 8) > or = subacute nonsurvivors (>24 to 96 h until death, n = 8) > or = survivors (> or =96 h until death, n = 22) (all P < 0.05)]. In the first 12 h, increases in lactate and renal abnormalities were greatest in acute nonsurvivors (all P < 0.05). Compared with survivors, subacute nonsurvivors had greater rises in cytokines and liver enzymes and greater falls in platelets, white cell counts, pH, and urine output from 24 to 96 h (all P < 0.05). Importantly, these changes were not attributable to dosages of sedation, which decreased in nonsurvivors [survivors vs. nonsurvivors: 5.0 +/- 1.0 vs. 3.8 +/- 0.7 ml x h(-1) x (fentanyl/midazolam/ medetomidine)(-1); P = 0.02]. In this model, the pain control regimen did not mask changes in metabolic function and lung injury or the need for more hemodynamic and pulmonary support related to increasing severity of sepsis. The integration into this model of both specific and supportive titrated therapies routinely used in septic patients may provide a more realistic setting to evaluate therapies for sepsis.     

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.