Ex-vivo models of the Retinal Pigment Epithelium (RPE) in long-term culture faithfully recapitulate key structural and physiological features of native RPE

The Retinal Pigment Epithelium (RPE) forms the primary site of pathology in several blinding retinopathies. RPE cultures are being continuously refined so that dynamic disease processes in this important monolayer can be faithfully studied outside the eye over longer periods. The RPE substrate, which mimics the supportive Bruch’s membrane (BrM), plays a key role in determining how well in-vitro cultures recapitulate native RPE cells. Here, we evaluate how two different types of BrM substrates; (1) a commercially-available polyester transwell membrane, and (2) a novel electrospun scaffold developed in our laboratory, could support the generation of realistic RPE tissues in culture. Our findings reveal that both substrates were capable of supporting long-lasting RPE monolayers with structural and functional specialisations of in-situ RPE cells. These cultures were used to study autofluorescence and barrier formation, as well as activities such as outer-segment internalisation/trafficking and directional secretion of key proteins; the impairment of which underlies retinal disease. Hence, both substrates fulfilled important criteria for generating authentic in-vitro cultures and act as powerful tools to study RPE pathophysiology. However, RPE grown on electrospun scaffolds may be better suited to studying complex RPE-BrM interactions such as the formation of drusen-like deposits associated with early retinal disease.     

Associations of maize protein bodies with cytoskeleton, membranes, and ribosomes in the endosperm of wild type and opaque-2 mutant

Maize endosperm was homogenized in a cytoskeleton-stabilizing buffer, filtered and layered on gradients of 20–80% sucrose and analyzed by monitoring their UV absorbance. A major peak of UV-light absorbing material was detected on the gradient, at about 60–65% sucrose (density of approximately 1.3 g·ml⁻¹). Biochemical, fluorescence microscopic, and immunoblot analyses of this peak showed that it consisted of protein bodies associated with actin, membranes, and RNA (ribosomes). Seeds of wild type and opaque-2 mutant were then homogenized, the homogenate was modified using detergents and/or cytoskeleton-disrupting agents, and centrifuged on sucrose gradients. In wild type maize endosperm, detergent treatment caused the major peak (protein bodies) to increase in density so that they sediment further down the gradient. However, in opaque-2 the protein bodies formed a broader, but smaller peak which, upon treatment with detergent, generated protein bodies which pelleted to the bottom of the gradient. Analysis of gradient fractions by gel electrophoresis and immuno-blotting showed that both the wild type and the mutant had cytoskeleton proteins in the upper regions (soluble, non-polymerized microfilaments and microtubules) as well as in the peak regions. Comparisons of both the UV-absorbance profiles and the immunoblot data suggest that the protein bodies from the two maize types associate differently with the membranes and the cytoskeleton.     

Uptake and distribution of S-sulfate in needles and roots of spruce seedlings as affected by exposure to SO2 and H2S

The interaction between pedospheric and atmospheric sulfur nutrition was studied in seedlings of Norway spruce. Spruce was grown on a 25% Hoagland nutrient solution containing ³⁵S-sulfate and simultaneously exposed to 250 nl l⁻¹ atmospheric SO₂ or H₂S. A 6-day exposure to SO₂ and H₂S resulted in a substantial increase in the total sulfur concentration of the needles. This increase could be ascribed to increased needle concentrations of sulfate, water-soluble non-protein thiols and organic sulfur. SO₂ and H₂S exposure resulted in slight but significant increases in the concentration of sulfur compounds in roots. In all sulfur fractions, except sulfate, there was a substantial decrease in the level of ³⁵S in needle and root sulfur fractions upon SO₂ and H₂S exposure, demonstrating that spruce was able to switch from pedospheric sulfate to atmospheric sulfur as a source for growth. In needles, the amount of ³⁵S decreased in total organic S and glutathione fraction, whereas it increased in sulfate. This supports continued import of S taken up by the roots into the needles in spite of a decreased channeling of ³⁵S into synthesis in needles. A greater part of total sulfate increase was due to unlabeled S, which points towards metabolic oxidation of H₂S and SO₂ to sulfate. Increased concentrations of S compounds (including sulfate) in roots were mainly due to unlabeled S, indicating an import of sulfur from the foliage. The significance of glutathione in the translocation of reduced sulfur from the needles to the roots is discussed.     

Safety of Oral Amphetamine Administered during Positron Emission Tomography Scans in Medically Screened Humans

Changes in endogenous dopamine levels can be detected in humans using positron emission tomography scans by measuring the amount by which a specific D_2/3 radioligand is displaced. In some cases, a challenge drug such as amphetamine is introduced to increase the amount of dopamine released into the synaptic cleft. Although intravenous amphetamine is often utilized, oral amphetamine has been shown to be just as effective in increasing endogenous dopamine levels. Based on our own use of oral amphetamine as a challenge drug, we have retroactively reviewed our study charts to determine the cardiovascular safety of 0.5 mg kg^-1 oral d-amphetamine. Of 172 amphetamine administrations in 144 individuals, only 2.8% of subjects experienced any transient adverse effects. In addition, we found no clinically relevant differences in increases of vital signs between healthy controls and patients. We therefore reaffirm the safety of 0.5 mg kg^-1 oral amphetamine in subjects previously screened for cardiovascular risk factors.     

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.     

Effect of treadmill exercise testing on serum enzymes and the resting electrocardiogram.

Fifty-three adult male patients with chest pain underwent treadmill exercise stress testing according to the Bruce protocol. The resting 12-lead electrocardiogram (ECG) and serum concentrations of glutamic oxaloacetic transaminase, lactic dehydrogenase creatine phosphokinase and alpha-hydroxybutyrate dehydrogenase were evaluated before, and at 1 and 20 hours after exercise. Twenty-eight subjects (53 percent) had a normal test result, 10 (19 percent) had ischemic ST -segment changes and anginal pain, and 15 (28 percent) were considered to have equivocal results because of an abnormal baseline ECG or the concurrent administration of cardioactive medication. In contrast to earlier reports, no significant changes in the serum enzyme values were seen in any of the three groups orin any individual subject, nor were ECG changes detected after recovery from exercise. The diagnostic evaluation of the exercise ECG must depend upon the demonstration of ischemic ST -segment changes and not upon changes in concentrations of serum enzymes.     

Early Morphogenesis of Glugea-lnduced Xenomas in Laboratory-Reared Flounder

Glugea stephani-infecled submucosal cells of laboratory-reared winter flounder, Pseudopleuronectes americanus, change to unique giant cell xenomas. These cells hypertrophy while the intracellular Glugea propagate to high numbers in the cytoplasm and eventually overrun the host cell. The xenomas slowly reach 20-25 muml;m (at 17°C) by day 10 after parasite invasion. These xenomas (eight often examined by electron microscopy) are coated with a mucus-like envelope onto which is attached a layer of endothelial ceils. The 10-day xenomas display (a) host cell endonuclear polyploidization and amitosis, (b) limited parasite growth and reproduction, and (c) a host cell cytoplasm structure similar to that seen in undifferentiated phagocytes. Glugea parasites do not induce obvious cell degenerative effects in 10-day xenomas; the 20-day xenomas are hypertrophied to 70-100 m?m. These cells are characterized by (a) a host cell component denuded of endoplasmic reticulum and phagosome membrane, (b) a reduction in host cell ribosomes and the disappearance of host cell cytoskeletal assemblages, including microtubules, and (c) a significant increase in vegetative Glugea within xenomas. Evidence indicates cytoplasmic calcium of the host cell xenoma is perturbed by the intracellular Glugea; the alterations in the host cell calcium domains may be a big factor in the induction of the block of mitosis in the host cell and in the disruption in its cytoskeletal controls.     

Aortography in the Diagnosis of Cancer

The role of angiography in the diagnosis of cancer includes the direct visualization of the tumor, the demonstration of its size, extent and possible metastasis. The criteria which are used to judge the prognosis of a tumor include the size, angiographic appearance, relationship to adjacent organs and tissues, growth or extension into draining veins and the presence or absence of metastasis. Chemotherapeutic agents can be instilled through the same catheters that are used for angiography and these catheters may be left in place from five to eight days.