Susceptibility of Young Sheep to Oral Infection with Bovine Spongiform Encephalopathy Decreases Significantly after Weaning

Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy (TSE) (or prion disease) that is readily transmissible to sheep by experimental infection and has the shortest incubation period in animals with the ARQ/ARQ PRNP genotype (at codons 136, 154, and 171). Because it is possible that sheep in the United Kingdom could have been infected with BSE by being fed contaminated meat and bone meal supplements at the same time as cattle, there is considerable interest in the responses of sheep to BSE inoculation. Epidemiological evidence suggests that very young individuals are more susceptible to TSE infection; however, this has never been properly tested in sheep. In the present study, low doses of BSE were fed to lambs of a range of ages (∼24 h, 2 to 3 weeks, 3 months, and 6 months) and adult sheep. The incidence of clinical BSE disease after inoculation was high in unweaned lambs (∼24 h and 2 to 3 weeks old) but much lower in older weaned animals The incubation period was also found to be influenced by the genotype at codon 141 of the PRNP gene, as lambs that were LF heterozygotes had a longer mean incubation period than those that were homozygotes of either type. The results suggest that sheep in the United Kingdom would have been at high risk of BSE infection only if neonatal animals had inadvertently ingested contaminated supplementary foodstuffs.     

Position specificity in Chitonomyces (Ascomycota, Laboulbeniomycetes) on Laccophilus (Coleoptera, Dytiscidae): a molecular approach resolves a century-old debate

The occurrence of Laboulbeniomycete species consistently on a precise portion of beetle integument was investigated in 13 species of Chitonomyces ectoparasitic on the aquatic diving beetle Laccophilus maculosus (Coleoptera, Dytiscidae). The phenomenon was called "position specificity" by Roland Thaxter in 1896, yet the mechanism has remained unknown. By using molecular analysis of the nucSSU rRNA gene and the 5.8S and partial ITS1 rRNA regions, 13 species of Chitonomyces reported to exhibit position specificity on Laccophilus maculosus were placed neatly into pairs of morphotypes, resulting in synonomies and recognition of six phylogenetic species (one species is a triplet). Each phylogenetic species was located at corresponding positions on male and female beetles that make contact during mating. In addition, ecological data and video footage of the mating behaviors of Laccophilus confirmed that sexual transmission is the mechanism behind this enigmatic phenomenon.     

Mechanotransduction and focal adhesions

Cellular FAs (focal adhesions) respond to internal and external mechanical stresses which make them prime candidates for mechanotransduction. Recent observations showed that the FA proteins including vinculin, FAK (FA kinase) and p130Cas are crucial for the ability of cells to transmit forces and to generate cytoskeletal tension. When mechanically stimulated, cells respond by modulating the spreading area, remodel the actin cytoskeleton, activate actomyosin interactions, recruit integrins and reinforce FAs and cytoskeletal structures. These complex cellular responses are orchestrated such that mechanical stresses within the FA complex remained within a narrow range.     

Genetic Predictions of Prion Disease Susceptibility in Carnivore Species Based on Variability of the Prion Gene Coding Region

Mammalian species vary widely in their apparent susceptibility to prion diseases. For example, several felid species developed prion disease (feline spongiform encephalopathy or FSE) during the bovine spongiform encephalopathy (BSE) epidemic in the United Kingdom, whereas no canine BSE cases were detected. Whether either of these or other groups of carnivore species can contract other prion diseases (e.g. chronic wasting disease or CWD) remains an open question. Variation in the host-encoded prion protein (PrP^C) largely explains observed disease susceptibility patterns within ruminant species, and may explain interspecies differences in susceptibility as well. We sequenced and compared the open reading frame of the PRNP gene encoding PrP^C protein from 609 animal samples comprising 29 species from 22 genera of the Order Carnivora; amongst these samples were 15 FSE cases. Our analysis revealed that FSE cases did not encode an identifiable disease-associated PrP polymorphism. However, all canid PrPs contained aspartic acid or glutamic acid at codon 163 which we propose provides a genetic basis for observed susceptibility differences between canids and felids. Among other carnivores studied, wolverine (Gulo gulo) and pine marten (Martes martes) were the only non-canid species to also express PrP-Asp163, which may impact on their prion diseases susceptibility. Populations of black bear (Ursus americanus) and mountain lion (Puma concolor) from Colorado showed little genetic variation in the PrP protein and no variants likely to be highly resistant to prions in general, suggesting that strain differences between BSE and CWD prions also may contribute to the limited apparent host range of the latter.     

How to measure image quality in tissue-based diagnosis (diagnostic surgical pathology)

Background

Automated image analysis, measurements of virtual slides, and open access electronic measurement user systems require standardized image quality assessment in tissue-based diagnosis.

Aims

To describe the theoretical background and the practical experiences in automated image quality estimation of colour images acquired from histological slides.

Theory, material and measurements

Digital images acquired from histological slides should present with textures and objects that permit automated image information analysis. The quality of digitized images can be estimated by spatial independent and local filter operations that investigate in homogenous brightness, low peak to noise ratio (full range of available grey values), maximum gradients, equalized grey value distribution, and existence of grey value thresholds. Transformation of the red-green-blue (RGB) space into the hue-saturation-intensity (HSI) space permits the detection of colour and intensity maxima/minima. The feature distance of the original image to its standardized counterpart is an appropriate measure to quantify the actual image quality. These measures have been applied to a series of H&;E stained, fluorescent (DAPI, Texas Red, FITC), and immunohistochemically stained (PAP, DAB) slides. More than 5,000 slides have been measured and partly analyzed in a time series.

Results

Analysis of H&;E stained slides revealed low shading corrections (10%) and moderate grey value standardization (10 – 20%) in the majority of cases. Immunohistochemically stained slides displayed greater shading and grey value correction. Fluorescent stained slides are often revealed to high brightness. Images requiring only low standardization corrections possess at least 5 different statistically significant thresholds, which are useful for object segmentation. Fluorescent images of good quality only posses one singular intensity maximum in contrast to good images obtained from H&;E stained slides that present with 2 – 3 intensity maxima.

Conclusion

Evaluation of image quality and creation of formally standardized images should be performed prior to automatic analysis of digital images acquired from histological slides. Spatial dependent and local filter operations as well as analysis of the RGB and HSI spaces are appropriate methods to reproduce evaluated formal image quality.

     

Thermodynamic evidence of non-muscle myosin II-lipid-membrane interaction

A unique feature of protein networks in living cells is that they can generate their own force. Proteins such as non-muscle myosin II are an integral part of the cytoskeleton and have the capacity to convert the energy of ATP hydrolysis into directional movement. Non-muscle myosin II can move actin filaments against each other, and depending on the orientation of the filaments and the way in which they are linked together, it can produce contraction, bending, extension, and stiffening. Our measurements with differential scanning calorimetry showed that non-muscle myosin II inserts into negatively charged phospholipid membranes. Using lipid vesicles made of DMPG/DMPC at a molar ratio of 1:1 at 10 mg/ml in the presence of different non-muscle myosin II concentrations showed a variation of the main phase transition of the lipid vesicle at around 23 °C. With increasing concentrations of non-muscle myosin II the thermotropic properties of the lipid vesicle changed, which is indicative of protein-lipid interaction/insertion. We hypothesize that myosin tail binds to acidic phospholipids through an electrostatic interaction using the basic side groups of positive residues; the flexible, amphipathic helix then may partially penetrate into the bilayer to form an anchor. Using the stopped-flow method, we determined the binding affinity of non-muscle myosin II when anchored to lipid vesicles with actin, which was similar to a pure actin-non-muscle myosin II system. Insertion of myosin tail into the hydrophobic region of lipid membranes, a model known as the lever arm mechanism, might explain how its interaction with actin generates cellular movement.     

Implications of troponin testing in clinical medicine

During the past decade considerable research has been conducted into the use of cardiac troponins, their diagnostic capability and their potential to allow risk stratification in patients with acute chest pain. Determination of risk in patients with suspected myocardial ischaemia is known to be as important as retrospective confirmation of a diagnosis of myocardial infarction (MI). Therefore, creatine kinase (CK)-MB - the former 'gold standard' in detecting myocardial necrosis - has been supplanted by new, more accurate biomarkers.Measurement of cardiac troponin levels constitute a substantial determinant in assessment of ischaemic heart disease, the presentations of which range from silent ischaemia to acute MI. Under these conditions, troponin release is regarded as surrogate marker of thrombus formation and peripheral embolization, and therefore new therapeutic strategies are focusing on potent antithrombotic regimens to improve long-term outcomes. Although elevated troponin levels are highly sensitive and specific indicators of myocardial damage, they are not always reflective of acute ischaemic coronary artery disease; other processes have been identified that cause elevations in these biomarkers. However, because prognosis appears to be related to the presence of troponins regardless of the mechanism of myocardial damage, clinicians increasingly rely on troponin assays when formulating individual therapeutic plans.     

HOPE fixation of cytospin preparations of human cells for in situ hybridization and immunocytochemistry.

In primary or cultured cells, in situ hybridization (ISH) or immunocytochemistry (ICC) is often performed on tissue that has been fixed by paraformaldehyde or Carnoy's. Recently we reported an optimized HOPE (HEPES-glutamic acid buffer-mediated organic solvent protection effect) fixation protocol for ISH targeting mRNA in lung tissues. We have now examined whether HOPE fixation could also be used on in vitro cultured cells for targeting mRNA by ISH or proteins by ICC on cytospin preparations. Using the myeloid stem cell line KG-1a as a model system, we showed that HOPE fixation can be applied for ISH and ICC on cultured cells. HOPE can be used with cells and tissues and with a broad spectrum of immunohistocytochemical and molecular techniques.     

Environmental and Animal Characteristics as Factors Associated with American Cutaneous Leishmaniasis in Rural Locations with Presence of Dogs,Brazil

The aim of the study was to investigate the importance of dogs, other domesticated animals and environmental characteristics as risk factors in the epidemiology of American cutaneous leishmaniasis (ACL). A retrospective survey of cases of human ACL in the last ten years and visits to homes in rural locations were carried out in the municipality of Arapongas (southern Brazil) from 2008 to 2010. ACL in humans was significantly associated with a distance of up to 25 meters from the residence to a forest area (OR 5.08; 95% CI: 1.35–21.04), undergrowth area (OR 6.80; 95% CI: 1.69–45.33) and stream (OR 5.87; 95% CI: 1.15–24.59); banana plants near the residence (OR 5.98; 95% CI: 1.49–39.84), absence of ceiling below the roof in the residence (OR 7.30; 95% CI: 1.26–158.1), the dumping of trash in the forest area (OR 26.33; 95% CI: 7.32–93.46) and presence of ACL in dogs in the surrounding area (OR 4.39; 95% CI: 1.37–13.45). In dogs, ACL was associated with a distance of 25 to 50 meters and 51 to 100 meters, respectively, from the residence to a forest area (OR 2.59; 95% CI: 1.08–5.98; OR 3.29; 95% CI: 1.64–6.62), the presence of a stream up to 25 m from the residence (OR 6.23; 95% CI: 2.34–16.54) and banana plants near the residence (OR 0.45; 95% CI: 0.25–0.80). In the locations studied in the municipality of Arapongas (Brazil), the results reveal that canine infection increases the risk of human infection by ACL and the characteristics surrounding the residence increase the risk of infection in both humans and dogs. Thus, integrated environmental management could be a useful measure to avoid contact between humans and phlebotomines.     

Talin anchors and nucleates actin filaments at lipid membranes. A direct demonstration.

Platelet talin nucleates actin assembly as we show here directly by using rhodamine-phalloidin labelling of actin filaments. Nucleation by talin still occurs after reconstitution into liposomal bilayers. This is also demonstrated directly after protein-lipid double labelling and light microscopic imaging. Talin, thus, is the first actin binding protein for which anchoring and nucleation of actin filament growth at lipid interfaces have been visualized.