Correlated three-dimensional light and electron microscopy reveals transformation of mitochondria during apoptosis

In addition to their role in cellular bioenergetics, mitochondria also initiate common forms of programmed cell death (apoptosis) through the release of proteins such as cytochrome c from the intermembrane and intracristal spaces. The release of these proteins is studied in populations of cells by western blotting mitochondrial and cytoplasmic fractions of cellular extracts, and in single cells by fluorescence microscopy using fluorescent indicators and fusion proteins. However, studying the changes in ultrastructure associated with release of proteins requires the higher resolution provided by transmission electron microscopy. Here, we have used fluorescence microscopy to characterize the state of apoptosis in HeLa cells treated with etoposide followed by electron microscopy and three-dimensional electron microscope tomography of the identical cells to study the sequence of structural changes. We have identified a remodelling of the inner mitochondrial membrane into many separate vesicular matrix compartments that accompanies release of proteins; however, this remodelling is not required for efficient release of cytochrome c. Swelling occurs only late in apoptosis after release of cytochrome c and loss of the mitochondrial membrane potential.     

Biotin- and Glycoprotein-Coated Microspheres as Surrogates for Studying Filtration Removal of Cryptosporidium parvum in a Granular Limestone Aquifer Medium

Members of the genus Cryptosporidium are waterborne protozoa of great health concern. Many studies have attempted to find appropriate surrogates for assessing Cryptosporidium filtration removal in porous media. In this study, we evaluated the filtration of Cryptosporidium parvum in granular limestone medium by the use of biotin- and glycoprotein-coated carboxylated polystyrene microspheres (CPMs) as surrogates. Column experiments were carried out with core material taken from a managed aquifer recharge site in Adelaide, Australia. For the experiments with injection of a single type of particle, we observed the total removal of the oocysts and glycoprotein-coated CPMs, a 4.6- to 6.3-log₁₀ reduction of biotin-coated CPMs, and a 2.6-log₁₀ reduction of unmodified CPMs. When two different types of particles were simultaneously injected, glycoprotein-coated CPMs showed a 5.3-log₁₀ reduction, while the uncoated CPMs displayed a 3.7-log₁₀ reduction, probably due to particle-particle interactions. Our results confirm that glycoprotein-coated CPMs are the most accurate surrogates for C. parvum; biotin-coated CPMs are slightly more conservative, while unmodified CPMs are markedly overly conservative for predicting C. parvum removal in granular limestone medium. The total removal of C. parvum observed in our study suggests that granular limestone medium is very effective for the filtration removal of C. parvum and could potentially be used for the pretreatment of drinking water and aquifer storage recovery of recycled water.     

Non-impactor-Based Methods for Sizing of Aerosols Emitted from Orally Inhaled and Nasal Drug Products (OINDPs)

The purpose of this article is to review non-impactor-based methods for measuring particle size distributions of orally inhaled and nasal pharmaceutical aerosols. The assessment of the size distributions of sprays and aerosols from orally inhaled and nasal drug products by methods not involving multi-stage cascade impaction may offer significant potential advantages in terms of labor savings and reducing the risk for operator-related errors associated with complex-to-undertake impactor-based methods. Indeed, in the case of nasal spray products, cascade impaction is inappropriate and alternative, and preferably non-invasive methods must be sought that minimize size-related bias associated with the measurement process for these relatively large droplets. This review highlights the options that are available to those involved with product quality assessments, providing guidance on relative strengths and weaknesses, as well as highlighting precautions that should be observed to minimize bias. The advent of Raman chemical imaging, which enables an estimate to be made of the proportion of each particle comprising active pharmaceutical ingredient(s) (APIs), necessitates a re-think about the value of classical microscopy image analysis as now being capable of providing API-relevant information from collected aerosols and sprays.     

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest¹, recent developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association ^{2, 3}. One small tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH (green fluorescent), with high specificity even in live cells ². The TC/biarsenical dye system offers far less steric constraints to the host protein than fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions ^4-7. We recently developed a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington disease ⁷. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).Download video file.^{(77M, mov)}     

Avian influenza (H5N1) viruses isolated from humans in Asia in 2004 exhibit increased virulence in mammals

The spread of highly pathogenic avian influenza H5N1 viruses across Asia in 2003 and 2004 devastated domestic poultry populations and resulted in the largest and most lethal H5N1 virus outbreak in humans to date. To better understand the potential of H5N1 viruses isolated during this epizootic event to cause disease in mammals, we used the mouse and ferret models to evaluate the relative virulence of selected 2003 and 2004 H5N1 viruses representing multiple genetic and geographical groups and compared them to earlier H5N1 strains isolated from humans. Four of five human isolates tested were highly lethal for both mice and ferrets and exhibited a substantially greater level of virulence in ferrets than other H5N1 viruses isolated from humans since 1997. One human isolate and all four avian isolates tested were found to be of low virulence in either animal. The highly virulent viruses replicated to high titers in the mouse and ferret respiratory tracts and spread to multiple organs, including the brain. Rapid disease progression and high lethality rates in ferrets distinguished the highly virulent 2004 H5N1 viruses from the 1997 H5N1 viruses. A pair of viruses isolated from the same patient differed by eight amino acids, including a Lys/Glu disparity at 627 of PB2, previously identified as an H5N1 virulence factor in mice. The virus possessing Glu at 627 of PB2 exhibited only a modest decrease in virulence in mice and was highly virulent in ferrets, indicating that for this virus pair, the K627E PB2 difference did not have a prevailing effect on virulence in mice or ferrets. Our results demonstrate the general equivalence of mouse and ferret models for assessment of the virulence of 2003 and 2004 H5N1 viruses. However, the apparent enhancement of virulence of these viruses in humans in 2004 was better reflected in the ferret.     

Above-ground space sequestration determines competitive success in juvenile beech and spruce trees

A 2-yr phytotron study was conducted to investigate the intra- and inter-specific competitive behaviour of juvenile beech (Fagus sylvatica) and spruce (Picea abies). Competitiveness was analysed by quantifying the resource budgets that occur along structures and within occupied space of relevance for competitive interaction. Ambient and elevated CO(2) and ozone (O(3)) regimes were applied throughout two growing seasons as stressors for provoking changes in resource budgets, growth and allocation to facilitate the competition analysis. The hypothesis tested was that the ability to sequester space at low structural cost will determine the competitive success. Spruce was a stronger competitor than beech, as displayed by its higher above-ground biomass increments in mixed culture compared with monoculture. A crucial factor in the competitive success of spruce was its ability to enlarge crown volume at low structural costs, supporting the hypothesis. Interspecific competition with spruce resulted in a size-independent readjustment of above-ground allocation in beech (reduced leaf : shoot biomass ratio). The efficient use of resources for above-ground space sequestration proved to be a parameter that quantitatively reflects competitiveness.     

Evaluation of macrocyclic Grb2 SH2 domain-binding peptide mimetics prepared by ring-closing metathesis of C-terminal allylglycines with an N-terminal beta-vinyl-substituted phosphotyrosyl mimetic

Preferential binding of ligands to Grb2 SH2 domains in beta-bend conformations has made peptide cyclization a logical means of effecting affinity enhancement. This is based on the concept that constraint of open-chain sequences to bend geometries may reduce entropy penalties of binding. The current study extends this approach by undertaking ring-closing metathesis (RCM) macrocyclization between i and i+3 residues through a process involving allylglycines and beta-vinyl-functionalized residues. Ring closure in this fashion results in minimal macrocyclic tetrapeptide mimetics. The predominant effects of such macrocyclization on Grb2 SH2 domain binding affinity were increases in rates of association (from 7- to 16-fold) relative to an open-chain congener, while decreases in dissociation rates were less pronounced (approximately 2-fold). The significant increases in association rates were consistent with pre-ordering of solution conformations to near those required for binding. Data from NMR experiments and molecular modeling simulations were used to interpret the binding results. An understanding of the conformational consequences of such i to i+3 ring closure may facilitate its application to other systems where bend geometries are desired.