A rapid method for assessing the distribution of gold labeling on thin sections.

Particulate gold labeling on ultrathin sections is in widespread use for antigen localization at the EM level. To extend the usefulness of gold labeling technology, we are evaluating different methods for sampling and estimating quantities of gold labeling. Here we present a simple, rapid, and unbiased method for assessing the relative pool sizes of immunogold labeling distributed over different cell compartments. The method uses a sampling approach developed for stereology in which a regular array of microscopic fields or linear scans is positioned randomly on labeled sections. From these readouts, gold particles are counted and assigned to identifiable cell structures to construct a gold labeling frequency distribution of those labeled compartments. Here we use ultrathin cryosections labeled for a range of different proteins and for a signaling lipid. We show by scanning labeled sections at the electron microscope that counting 100-200 particles on each of two grids is sufficient to obtain a reproducible and rapid assessment of the pattern of labeling proportions over 10-16 compartments. If more precise estimates of labeling proportions over individual compartments are required (e.g., to achieve coefficients of error of 10-20%), then 100-200 particles need to be counted over each compartment of interest.     

Apparatus for Conditioning Unlimited Quantities of Finished Waters for Enteric Virus Detection

An apparatus has been developed, constructed, and tested for conditioning unlimited quantities of finished waters for enteric virus detection.     

Microbial Community Comparisons as a Function of the Physical and Geochemical Conditions of Galápagos Island Fumaroles

Microbial communities at six fumarole fields on Sierra Negra and Alcedo volcanoes in the Galápagos Islands were examined to test how extreme geochemical conditions affect microbial biodiversity. The geologic substrates consist of basalt and rhyolite with varying amounts of alteration and sulfur precipitates. Collected samples of substrates varied in pH from 0–6, and substrate temperatures were within the mesophilic, thermophilic, and hyperthermophilic temperature ranges. Terminal restriction fragment length polymorphism (T-RFLP) analyses were done to assess the relationship of communities to each other as a function of geologic substrate, pH, and temperature. Comparative analyses of community diversity define two distinct clusters showing that the relationship between spatially separated microbial communities at the fumaroles is most influenced by the pH of the local environment.     

Quantitative Muscle MRI as an Assessment Tool for Monitoring Disease Progression in LGMD2I: A Multicentre Longitudinal Study

Background

Outcome measures for clinical trials in neuromuscular diseases are typically based on physical assessments which are dependent on patient effort, combine the effort of different muscle groups, and may not be sensitive to progression over short trial periods in slow-progressing diseases. We hypothesised that quantitative fat imaging by MRI (Dixon technique) could provide more discriminating quantitative, patient-independent measurements of the progress of muscle fat replacement within individual muscle groups.

Objective

To determine whether quantitative fat imaging could measure disease progression in a cohort of limb-girdle muscular dystrophy 2I (LGMD2I) patients over a 12 month period.

Methods

32 adult patients (17 male;15 female) from 4 European tertiary referral centres with the homozygous c.826C>A mutation in the fukutin-related protein gene (FKRP) completed baseline and follow up measurements 12 months later. Quantitative fat imaging was performed and muscle fat fraction change was compared with (i) muscle strength and function assessed using standardized physical tests and (ii) standard T1-weighted MRI graded on a 6 point scale.

Results

There was a significant increase in muscle fat fraction in 9 of the 14 muscles analyzed using the quantitative MRI technique from baseline to 12 months follow up. Changes were not seen in the conventional longitudinal physical assessments or in qualitative scoring of the T₁w images.

Conclusions

Quantitative muscle MRI, using the Dixon technique, could be used as an important longitudinal outcome measure to assess muscle pathology and monitor therapeutic efficacy in patients with LGMD2I.     

The management and restoration of damaged blanket bog in the north of Scotland

Summary

The blanket bogs of Caithness and Sutherland are the finest examples of their type in the world. Restricted to a few parts of the world where cool, oceanic climatic conditions prevail, Britain holds approximately 13% of the total global resource of blanket bog, of which the bogs of Caithness and Sutherland form the largest and most intact area. In recent times, extensive areas of the peatlands of Caithness and Sutherland have been damaged – principally through drainage and forestry. In 1994, the Royal Society for the Protection of Birds (RSPB) purchased Forsinard Estate in the heart of the peatlands as part of a EU LIFE funded project on blanket bog conservation. In partnership with Scottish Natural Heritage and Caithness and Sutherland Enterprise, this four year RSPB led project promoted a number of initiatives on awareness raising and ecotourism as well as a range of practical demonstrations on restoring damaged blanket bog. More recently, a follow up LIFE Peatlands Project was launched in 2001 where RSPB extended the partnership to include SNH, Forest Enterprise, Plantlife and the Forestry Commission. This paper gives an over-view of the partnership approach to the management and restoration of damaged blanket bog in Caithness and Sutherland.     

Interactions of La3+ with phosphatidylserine vesicles Binding,phase transition,leakage, 31P-NMR and fusion

The interaction of La³⁺ with phosphatidylserine vesicles is studied by differential scanning calorimetry, ¹⁴⁰La binding, ³¹P-NMR chemical shifts and relaxation rates, carboxyfluorescein and [¹⁴C]sucrose release, X-ray diffraction and freeze-fracture electron microscopy. In the presence of La³⁺ concentrations above 1 mM and an incubation temperature of 38°C, i.e., at the phase transition temperature of the complex La/phosphatidylserine, the binding ratio of La/lipid exceeds a $\text{1}\text{3}$ ratio, reaching saturation at a $\text{1}\text{2}$ ratio. Analysis, employing a modified Gouy-Chapman equation, indicates a significant increase in the intrinsic binding constant of La/phosphatidylserine when the La³⁺ concentration exceeds the threshold concentration for leakage. The analysis illustrates that at the molecular level the binding of La³⁺ can be comparable to or even weaker than that of Ca²⁺, but that even when present at smaller concentrations La³⁺ competes with and partially displaces Ca²⁺ from membranes or other negatively charged surfaces. The results suggest that the sequence La³⁺>Ca²⁺>Mg²⁺ reflects both the binding strength of these cations to phosphatidylserine as well as their ability to induce leakage, enhancement of ³¹P spin-lattice relaxation rates, fusion and other structural changes. The leakage, fusion, and other structural changes are more pronounced at the phase transition temperature of the La/lipid complex.     

Multiple-labelling immunoEM using different sizes of colloidal gold: alternative approaches to test for differential distribution and colocalization in subcellular structures

Various methods for quantifying cellular immunogold labelling on transmission electron microscope thin sections are currently available. All rely on sound random sampling principles and are applicable to single immunolabelling across compartments within a given cell type or between different experimental groups of cells. Although methods are also available to test for colocalization in double/triple immunogold labelling studies, so far, these have relied on making multiple measurements of gold particle densities in defined areas or of inter-particle nearest neighbour distances. Here, we present alternative two-step approaches to codistribution and colocalization assessment that merely require raw counts of gold particles in distinct cellular compartments. For assessing codistribution over aggregate compartments, initial statistical evaluation involves combining contingency table and chi-squared analyses to provide predicted gold particle distributions. The observed and predicted distributions allow testing of the appropriate null hypothesis, namely, that there is no difference in the distribution patterns of proteins labelled by different sizes of gold particle. In short, the null hypothesis is that of colocalization. The approach for assessing colabelling recognises that, on thin sections, a compartment is made up of a set of sectional images (profiles) of cognate structures. The approach involves identifying two groups of compartmental profiles that are unlabelled and labelled for one gold marker size. The proportions in each group that are also labelled for the second gold marker size are then compared. Statistical analysis now uses a 2 × 2 contingency table combined with the Fisher exact probability test. Having identified double labelling, the profiles can be analysed further in order to identify characteristic features that might account for the double labelling. In each case, the approach is illustrated using synthetic and/or experimental datasets and can be refined to correct observed labelling patterns to specific labelling patterns. These simple and efficient approaches should be of more immediate utility to those interested in codistribution and colocalization in multiple immunogold labelling investigations.