Correction to: Wnt3a induces exosome secretion from primary cultured rat microglia.

Following the publication of this article [1], it has been noted by the authors that an image of the same cell nuclei has been used in error twice, in Fig. 8, parts A and B. These images are redundant in this figure as the images in parts D and E show Wnt3a treated and control cells stained with both Hoechst 33342 (as in parts A and B) and fluorescein diacetate. The data from multiple repetitions of the Hoechst 33342 stain experiment are presented in graph C. Thus, the duplicated images (in Fig. 8A and B) add no additional data and do not change the results or conclusions reached in the article. The authors apologize for any confusion this may have caused.

in vivo localization of the neuronal ceroid lipofuscinosis proteins, CLN3 and CLN7, at endogenous expression levels.

The neuronal ceroid lipofuscinoses are a group of recessively inherited, childhood-onset neurodegenerative conditions. Several forms are caused by mutations in genes encoding putative lysosomal membrane proteins. Studies of the cell biology underpinning these disorders are hampered by the poor antigenicity of the membrane proteins, which makes visualization of the endogenous proteins difficult. We have used Drosophila to generate knock-in YFP-fusions for two of the NCL membrane proteins: CLN7 and CLN3. The YFP-fusions are expressed at endogenous levels and the proteins can be visualized live without the need for overexpression. Unexpectedly, both CLN7 and CLN3 have restricted expression in the CNS of Drosophila larva and are predominantly expressed in the glia that form the insect blood-brain-barrier. CLN7 is also expressed in neurons in the developing visual system. Analogous with murine CLN3, Drosophila CLN3 is strongly expressed in the excretory and osmoregulatory Malpighian tubules, but the knock-in also reveals unexpected localization of the protein to the apical domain adjacent to the lumen. In addition, some CLN3 protein in the tubules is localized within mitochondria. Our in vivo imaging of CLN7 and CLN3 suggests new possibilities for function and promotes new ideas about the cell biology of the NCLs.

In vivo assessment of aortic aneurysm wall integrity using elastin-specific molecular magnetic resonance imaging.

BACKGROUND: The incidence of abdominal aortic aneurysms (AAAs) has increased during the last decades. However, there is still controversy about the management of medium-sized AAAs. Therefore, novel biomarkers, besides aneurysmal diameter, are needed to assess aortic wall integrity and risk of rupture. Elastin is the key protein for maintaining aortic wall tensile strength and stability. The progressive breakdown of structural proteins, in particular, medial elastin, is responsible for the inability of the aortic wall to withstand intraluminal hemodynamic forces. Here, we evaluate the usefulness of elastin-specific molecular MRI for the in vivo characterization of AAAs. METHODS AND RESULTS: To induce AAAs, ApoE(-/-) mice were infused with angiotensin-II. An elastin-specific magnetic resonance molecular imaging agent (ESMA) was administered after 1, 2, 3, and 4 weeks of angiotensin-II infusion to assess elastin composition of the aorta (n=8 per group). The high signal provided by ESMA allowed for imaging with high spatial resolution, resulting in an accurate assessment of ruptured elastic laminae and the compensatory expression of elastic fibers. In vivo contrast-to-noise ratios and R1-relaxation rates after ESMA administration were in good agreement with ex vivo histomorphometry (Elastica van Gieson stain) and gadolinium concentrations determined by inductively coupled plasma mass spectroscopy. Electron microscopy confirmed colocalization of ESMA with elastic fibers. CONCLUSIONS: Changes in elastin content could be readily delineated and quantified at different stages of AAAs by elastin-specific molecular magnetic resonance imaging. ESMA-MRI offers potential for the noninvasive detection of the aortic rupture site prior to dilation of the aorta and the subsequent in vivo monitoring of compensatory repair processes during the progression of AAAs.

Nanohydroxyapatite shape and its potential role in bone formation: an analytical study.

Bone cells (osteoblasts) produce a collagen-rich matrix called osteoid, which is mineralized extracellularly by nanosized calcium phosphate (CaP). Synthetically produced CaP nanoparticles (NPs) have great potential for clinical application. However few studies have compared the effect of CaP NPs with different properties, such as shape and aspect ratio, on the survival and behaviour of active bone-producing cells, such as primary human osteoblasts (HOBs). This study aimed to investigate the biocompatibility and ultrastructural effects of two differently shaped hydroxyapatite [Ca10(PO4)6(OH)2] nanoparticles (HA NPs), round- (aspect ratio 2.12, AR2) and rice-shaped (aspect ratio 3.79, AR4). The ultrastructural response and initial extracellular matrix (ECM) formation of HOBs to HA NPs were observed, as well as matrix vesicle release. A transmission electron microscopy (TEM)-based X-ray microanalytical technique was used to measure cytoplasmic ion levels, including calcium (Ca), phosphorus (P), sodium (Na) and potassium (K). K/Na ratios were used as a measure of cell viability. Following HA NP stimulation, all measured cytoplasmic ion levels increased. AR2 NPs had a greater osteogenic effect on osteoblasts compared with AR4 NPs, including alkaline phosphatase activity and matrix vesicle release. However, they produced only a moderate increase in intracellular Ca and P levels compared with AR4. This suggests that particular Ca and P concentrations may be required for, or indicative of, optimal osteoblast activity. Cell viability, as measured by Na and K microanalysis, was best maintained in AR2. Initial formation of osteoblast ECM was altered in the presence of either HA NP, and immuno-TEM identified fibronectin and matrilin-3 as two ECM proteins affected. Matrilin-3 is here described for the first time as being expressed by cultured osteoblasts. In summary, this novel and in-depth study has demonstrated that HA NP shape can influence a range of different parameters related to osteoblast viability and activity.

CD73 expression on extracellular vesicles derived from CD4+ CD25+ Foxp3+ T cells contributes to their regulatory function.

CD4(+)CD25(+)Foxp3(+) Treg cells maintain immunological tolerance. In this study, the possibility that Treg cells control immune responses via the production of secreted membrane vesicles, such as exosomes, was investigated. Exosomes are released by many cell types, including T cells, and have regulatory functions. Indeed, TCR activation of both freshly isolated Treg cells and an antigen-specific Treg-cell line resulted in the production of exosomes as defined morphologically by EM and by the presence of tetraspanin molecules LAMP-1/CD63 and CD81. Expression of the ecto-5-nucleotide enzyme CD73 by Treg cells has been shown to contribute to their suppressive function by converting extracellular adenosine-5-monophosphate to adenosine, which, following interaction with adenosine receptors expressed on target cells, leads to immune modulation. CD73 was evident on Treg cell derived exosomes, accordingly when these exosomes were incubated in the presence of adenosine-5-monophosphate production of adenosine was observed. Most importantly, CD73 present on Treg cell derived exosomes was essential for their suppressive function hitherto exosomes derived from a CD73-negative CD4(+) T-cell line did not have such capabilities. Overall our findings demonstrate that CD73-expressing exosomes produced by Treg cells following activation contribute to their suppressive activity through the production of adenosine.

Pathophysiological consequences of TAT-HKII peptide administration are independent of impaired vascular function and ensuing ischemia.

RATIONALE: We have shown that partial dissociation of hexokinase II (HKII) from mitochondria in the intact heart using low-dose transactivating transcriptional factor (TAT)-HKII (200 nmol/L) prevents the cardioprotective effects of ischemic preconditioning, whereas high-dose TAT-HKII (10 µmol/L) administration results in rapid myocardial dysfunction, mitochondrial depolarization, and disintegration. In this issue of Circulation Research, Pasdois et al argue that the deleterious effects of TAT-HKII administration on cardiac function are likely because of vasoconstriction and ensuing ischemia. OBJECTIVE: To investigate whether altered vascular function and ensuing ischemia recapitulate the deleterious effects of TAT-HKII in intact myocardium. METHODS AND RESULTS: Using a variety of complementary techniques, including mitochondrial membrane potential (ΔΨm) imaging, high-resolution optical action potential mapping, analysis of lactate production, nicotinamide adenine dinucleotide epifluorescence, lactate dehydrogenase release, and electron microscopy, we provide direct evidence that refutes the notion that acute myocardial dysfunction by high-dose TAT-HKII peptide administration is a consequence of impaired vascular function. Moreover, we demonstrate that low-dose TAT-HKII treatment, which abrogates the protective effects of ischemic preconditioning, is not associated with ischemia or ischemic injury. CONCLUSIONS: Our findings challenge the notion that the effects of TAT-HKII are attributable to impaired vascular function and ensuing ischemia, thereby lending further credence to the role of mitochondria-bound HKII as a critical regulator of cardiac function, ischemia-reperfusion injury, and cardioprotection by ischemic preconditioning.

Wnt3a induces exosome secretion from primary cultured rat microglia.

BACKGROUND: Microglia, the immune effector cells of the CNS and the signaling molecule Wnt, both play critical roles in neurodevelopment and neurological disease. Here we describe the inducible release of exosomes from primary cultured rat microglia following treatment with recombinant carrier-free Wnt3a. RESULTS: Wnt3a was internalised into microglia, being detectable in early endosomes, and secreted in exosomes through a GSK3-independent mechanism. Electron microscopy demonstrated that exosomes were elliptical, electron-dense (100 nm) vesicles that coalesced with time in vitro. In contrast to microglia, primary cortical neurons released exosomes constitutively and the quantity of exosomes released was not altered by Wnt3a treatment. The proteomic profile of the microglial-derived exosomes was characterised using liquid chromatography-tandem mass spectrometry (LC/MS/MS) and the vesicles were found to be associated with proteins involved in cellular architecture, metabolism, protein synthesis and protein degradation including ß-actin, glyceraldehyde-3-phosphate dehydrogenase, ribosomal subunits and ubiquitin (45 proteins in total). Unlike lipopolysaccharide, Wnt3a did not induce a neurotoxic, pro-inflammatory phenotype in primary microglia. CONCLUSION: These findings reveal a novel mechanism through which Wnt3a signals in microglia resulting in the release of exosomes loaded with proteinaceous cargo.

Noninvasive magnetic resonance imaging evaluation of endothelial permeability in murine atherosclerosis using an albumin-binding contrast agent.

BACKGROUND: Endothelial dysfunction promotes atherosclerosis and precedes acute cardiovascular events. We investigated whether in vivo magnetic resonance imaging with the use of an albumin-binding contrast agent, gadofosveset, could detect endothelial damage associated with atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice. Furthermore, we tested whether magnetic resonance imaging could noninvasively assess endothelial function by measuring the endothelial-dependent vasodilation in response to acetylcholine. METHODS AND RESULTS: ApoE(-/-) mice were imaged at 4, 8, and 12 weeks after commencement of a high-fat diet. Statin-treated ApoE(-/-) mice were scanned after 12 weeks of a high-fat diet. Wild-type mice were imaged before and 48 hours after injection of Russell's viper venom, an endothelial toxin. Delayed enhancement magnetic resonance imaging and T1 mapping of the brachiocephalic artery, 30 minutes after injection of gadofosveset, showed increased vessel wall enhancement and relaxation rate (R(1)) with progression of atherosclerosis in ApoE(-/-)(R(1) [s(-1)]: R(4 weeks) 2.42±0.35, R(8 weeks) 3.45±0.54, R(12 weeks) 3.83±0.52) and Russell's viper venom-injected wild-type mice (R(1)=4.57±0.86). Conversely, wild-type (R(1)=2.15±0.34) and statin-treated ApoE(-/-) (R(1)=3.0±0.65) mice showed less enhancement. Uptake of gadofosveset correlated with Evans blue staining, morphological changes of endothelial cells, and widening of the cell-cell junctions, suggesting that uptake occurs in regions of increased vascular permeability. Endothelial-dependent vasomotor responses showed vasoconstriction of the arteries of the ApoE(-/-) (-22.22±7.95%) and Russell's viper venom-injected (-10.37±17.60%) mice compared with wild-type mice (32.45±12.35%). Statin treatment improved endothelium morphology and function (-8.12±8.22%). CONCLUSIONS: We demonstrate the noninvasive assessment of endothelial permeability and function with the use of an albumin-binding magnetic resonance contrast agent. Blood albumin leakage could be a surrogate marker for the in vivo evaluation of interventions that aim to restore the endothelium.

Three-dimensional imaging of the aortic vessel wall using an elastin-specific magnetic resonance contrast agent.

OBJECTIVE: The aim of this study was to demonstrate the feasibility of high-resolution 3-dimensional aortic vessel wall imaging using a novel elastin-specific magnetic resonance contrast agent (ESMA) in a large animal model. MATERIALS AND METHODS: The thoracic aortic vessel wall of 6 Landrace pigs was imaged using a novel ESMA and a nonspecific control agent. On day 1, imaging was performed before and after the administration of a nonspecific control agent, gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA; Bayer Schering AG, Berlin, Germany). On day 3, identical scans were repeated before and after the administration of a novel ESMA (Lantheus Medical Imaging, North Billerica, Massachusetts). Three-dimensional inversion recovery gradient echo delayed-enhancement imaging and magnetic resonance (MR) angiography of the thoracic aortic vessel wall were performed on a 1.5-T MR scanner (Achieva; Philips Medical Systems, the Netherlands). The signal-to-noise ratio and the contrast-to-noise ratio of arterial wall enhancement, including the time course of enhancement, were assessed for ESMA and Gd-DTPA. After the completion of imaging sessions, histology, electron microscopy, and inductively coupled plasma mass spectroscopy were performed to localize and quantify the gadolinium bound to the arterial vessel wall. RESULTS: Administration of ESMA resulted in a strong enhancement of the aortic vessel wall on delayed-enhancement imaging, whereas no significant enhancement could be measured with Gd-DTPA. Ninety to 100 minutes after the administration of ESMA, significantly higher signal-to-noise ratio and contrast-to-noise ratio could be measured compared with the administration of Gd-DTPA (45.7 ± 9.6 vs 13.2 ± 3.5, P < 0.05 and 41.9 ± 9.1 vs 5.2 ± 2.0, P < 0.05). A significant correlation (0.96; P < 0.01) between area measurements derived from ESMA scans and aortic MR angiography scans could be found. Electron microscopy and inductively coupled plasma mass spectroscopy confirmed the colocalization of ESMA with elastic fibers. CONCLUSION: We demonstrate the feasibility of aortic vessel wall imaging using a novel ESMA in a large animal model under conditions resembling a clinical setting. Such an approach could be useful for the fast 3-dimensional assessment of the arterial vessel wall in the context of atherosclerosis, aortic aneurysms, and hypertension.

In vivo assessment of intraplaque and endothelial fibrin in ApoE(-/-) mice by molecular MRI.

OBJECTIVE: Molecular magnetic resonance imaging (MRI) has emerged as a promising non-invasive modality to characterize atherosclerotic vessel wall changes on a morphological and molecular level. Intraplaque and endothelial fibrin has recently been recognized to play an important role in the progression of atherosclerosis. This study aimed to investigate the feasibility of intraplaque and endothelial fibrin detection using a fibrin-targeted contrast-agent, FTCA (EPIX Pharmaceuticals, Lexington, MA), in a mouse model of atherosclerosis. METHODS: Male apolipoproteinE-knockout mice (ApoE(-/-)) were fed a high fat diet (HFD) for one to three months. MRI of the brachiocephalic artery was performed prior to and 90 min after the administration of FTCA (n=8 per group). Contrast to noise ratios (CNR) and longitudinal relaxation rates (R1) of plaques were determined and compared to ex vivo fibrin density measurements on immunohistological sections stained with a fibrin-specific antibody and gadolinium concentrations measured by inductively coupled mass spectroscopy (ICP-MS). RESULTS: Molecular MRI after FTCA administration demonstrated a significant increase (p<0.05) in contrast agent uptake in brachiocephalic artery plaques. In vivo CNR measurements were in good agreement with ex vivo fibrin density measurements on immunohistochemistry (y=2.4x+11.3, R(2)=0.82) and ICP-MS (y=0.95x+7.1, R(2)=0.70). Late stage atherosclerotic plaques displayed the strongest increase in CNR, R1, ex vivo fibrin staining and gadolinium concentration (p<0.05). CONCLUSION: This study demonstrated the feasibility of intraplaque and endothelial fibrin imaging using FTCA. Direct in vivo fibrin detection and quantification could be useful for characterization and staging of coronary and carotid atherosclerotic lesions, which may aid diagnosis and intervention.

VAPB interacts with the mitochondrial protein PTPIP51 to regulate calcium homeostasis.

A proline to serine substitution at position 56 in the gene encoding vesicle-associated membrane protein-associated protein B (VAPB) causes some dominantly inherited familial forms of motor neuron disease including amyotrophic lateral sclerosis (ALS) type-8. VAPB is an integral endoplasmic reticulum (ER) protein whose amino-terminus projects into the cytosol. Overexpression of ALS mutant VAPBP56S disrupts ER structure but the mechanisms by which it induces disease are not properly understood. Here we show that VAPB interacts with the outer mitochondrial membrane protein, protein tyrosine phosphatase-interacting protein 51 (PTPIP51). ER and mitochondria are both stores for intracellular calcium (Ca(2+)) and Ca(2+) exchange between these organelles occurs at regions of ER that are closely apposed to mitochondria. These are termed mitochondria-associated membranes (MAM). We demonstrate that VAPB is a MAM protein and that loss of either VAPB or PTPIP51 perturbs uptake of Ca(2+) by mitochondria following release from ER stores. Finally, we demonstrate that VAPBP56S has altered binding to PTPIP51 and increases Ca(2+) uptake by mitochondria following release from ER stores. Damage to ER, mitochondria and Ca(2+) homeostasis are all seen in ALS and we discuss the implications of our findings in this context.

Disruption of hexokinase II-mitochondrial binding blocks ischemic preconditioning and causes rapid cardiac necrosis.

RATIONALE: Isoforms I and II of the glycolytic enzyme hexokinase (HKI and HKII) are known to associate with mitochondria. It is unknown whether mitochondria-bound hexokinase is mandatory for ischemic preconditioning and normal functioning of the intact, beating heart. OBJECTIVE: We hypothesized that reducing mitochondrial hexokinase would abrogate ischemic preconditioning and disrupt myocardial function. METHODS AND RESULTS: Ex vivo perfused HKII(+/-) hearts exhibited increased cell death after ischemia and reperfusion injury compared with wild-type hearts; however, ischemic preconditioning was unaffected. To investigate acute reductions in mitochondrial HKII levels, wild-type hearts were treated with a TAT control peptide or a TAT-HK peptide that contained the binding motif of HKII to mitochondria, thereby disrupting the mitochondrial HKII association. Mitochondrial hexokinase was determined by HKI and HKII immunogold labeling and electron microscopy analysis. Low-dose (200 nmol/L) TAT-HK treatment significantly decreased mitochondrial HKII levels without affecting baseline cardiac function but dramatically increased ischemia-reperfusion injury and prevented the protective effects of ischemic preconditioning. Treatment for 15 minutes with high-dose (10 µmol/L) TAT-HK resulted in acute mitochondrial depolarization, mitochondrial swelling, profound contractile impairment, and severe cardiac disintegration. The detrimental effects of TAT-HK treatment were mimicked by mitochondrial membrane depolarization after mild mitochondrial uncoupling that did not cause direct mitochondrial permeability transition opening. CONCLUSIONS: Acute low-dose dissociation of HKII from mitochondria in heart prevented ischemic preconditioning, whereas high-dose HKII dissociation caused cessation of cardiac contraction and tissue disruption, likely through an acute mitochondrial membrane depolarization mechanism. The results suggest that the association of HKII with mitochondria is essential for the protective effects of ischemic preconditioning and normal cardiac function through maintenance of mitochondrial potential.

MRI of coronary wall remodeling in a swine model of coronary injury using an elastin-binding contrast agent.

BACKGROUND: The extracellular matrix (ECM) plays an important role in the pathogenesis of atherosclerosis and in-stent restenosis. Elastin is an essential component of the ECM. ECM degradation can lead to plaque destabilization, whereas enhanced synthesis typically leads to vessel wall remodeling resulting in arterial stenosis or in-stent restenosis after stent implantation. The objective of this study was to demonstrate the feasibility of MRI of vascular remodeling using a novel elastin-binding contrast agent (BMS-753951). METHODS AND RESULTS: Coronary injury was induced in 6 pigs by endothelial denudation and stent placement. At day 28, delayed-enhancement MRI coronary vessel wall imaging was performed before and after injection of gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA). Two days later, DE-MRI was repeated after administration of BMS-753951. Contrast-to-noise-ratio and areas of enhancement were determined. Delayed-enhancement MRI with BMS-753951 caused strong enhancement of the aortic, pulmonary artery, and injured coronary artery walls, whereas Gd-DTPA did not. Delayed-enhancement MRI of the stented coronary artery with BMS-753951 yielded a 3-fold higher contrast-to-noise-ratio when compared with the balloon-injured and control coronary artery (21±6 versus 7±3 versus 6±4; P<0.001). The area of enhancement correlated well with the area of remodeling obtained from histological data (R(2)=0.86, P<0.05). CONCLUSIONS: We demonstrate the noninvasive detection and quantification of vascular remodeling in an animal model of coronary vessel wall injury using an elastin-specific MR contrast agent. This novel approach may be useful for the assessment of coronary vessel wall remodeling in patients with suspected coronary artery disease. Further studies in atherosclerotic animal models and degenerative ECM disease are now warranted.

X-ray microanalysis investigation of the changes in Na, K, and hemoglobin concentration in plasmodium falciparum-infected red blood cells.

Plasmodium falciparum is responsible for severe malaria. During the ∼48 h duration of its asexual reproduction cycle in human red blood cells, the parasite causes profound alterations in the homeostasis of the host red cell, with reversal of the normal Na and K gradients across the host cell membrane, and a drastic fall in hemoglobin content. A question critical to our understanding of how the host cell retains its integrity for the duration of the cycle had been previously addressed by modeling the homeostasis of infected cells. The model predicted a critical contribution of excess hemoglobin consumption to cell integrity (the colloidosmotic hypothesis). Here we tested this prediction with the use of electron-probe x-ray microanalysis to measure the stage-related changes in Na, K, and Fe contents in single infected red cells and in uninfected controls. The results document a decrease in Fe signal with increased Na/K ratio. Interpreted in terms of concentrations, the results point to a sustained fall in host cell hemoglobin concentration with parasite maturation, supporting a colloidosmotic role of excess hemoglobin digestion. The results also provide, for the first time to our knowledge, comprehensive maps of the elemental distributions of Na, K, and Fe in falciparum-infected red blood cells.

Noninvasive assessment of atherosclerotic plaque progression in ApoE-/- mice using susceptibility gradient mapping.

BACKGROUND: Macrophages have been identified as a major contributor to plaque development and destabilization in atherosclerosis. The aim of this study was to noninvasively assess uptake of citrate coated very small iron oxide particles at different stages of plaque development in the brachiocephalic artery of apoE(-/-) mice. Susceptibility gradient mapping (SGM) was applied to generate positive contrast images and to quantify iron oxide uptake. METHODS AND RESULTS: ApoE(-/-) mice were fed a high-fat diet for 4, 8, or 12 weeks; 300 µmol Fe/kg was injected 24 and 48 hours before final MRI. Increasing very small iron oxide particle uptake was observed over the course of atherosclerotic plaque development. Simultaneous administration of pravastatin led to a significant decrease in very small iron oxide particle uptake, assessed by mass spectroscopy and histology. SGM-MRI allowed the generation of positive contrast images, and magnitudes (mT/m) of contrast enhancement in SG parameter maps significantly correlated with the absolute iron oxide content (R(2)=0.70, P<0.05) and the macrophage density (R(2)=0.71, P<0.05). CONCLUSIONS: This study shows an increase in iron oxide uptake (measured by in vivo SGM-MRI, histology, and mass spectroscopy) with the progression of plaque development in an apoE(-/-) mouse model of accelerated atherosclerosis. Positive contrast provided by SGM-MRI allowed for a clear visualization of intraplaque iron oxide depositions, and magnitudes (mT/m) of contrast enhancement in SG parameter maps allowed for the quantification of intraplaque iron oxide particles.

Assessment of atherosclerotic plaque burden with an elastin-specific magnetic resonance contrast agent.

Atherosclerosis and its consequences remain the main cause of mortality in industrialized and developing nations. Plaque burden and progression have been shown to be independent predictors for future cardiac events by intravascular ultrasound. Routine prospective imaging is hampered by the invasive nature of intravascular ultrasound. A noninvasive technique would therefore be more suitable for screening of atherosclerosis in large populations. Here we introduce an elastin-specific magnetic resonance contrast agent (ESMA) for noninvasive quantification of plaque burden in a mouse model of atherosclerosis. The strong signal provided by ESMA allows for imaging with high spatial resolution, resulting in accurate assessment of plaque burden. Additionally, plaque characterization by quantifying intraplaque elastin content using signal intensity measurements is possible. Changes in elastin content and the high abundance of elastin during plaque development, in combination with the imaging properties of ESMA, provide potential for noninvasive assessment of plaque burden by molecular magnetic resonance imaging (MRI).

Isolation of nano-meso scale detergent resistant membrane that has properties expected of lipid 'rafts'.

This review assesses problems that confound attempts to isolate 'raft' domains from cell membranes, focusing in particular upon the isolation of detergent resistant membrane (DRM). Despite its widespread use, this technique is rightly viewed with skepticism by many membrane biochemists and biophysics for reasons that include the inability to isolate DRMs at 37°C, the temperature at which their lipids are supposed to be ordered and so exclude detergents. If solubilization is done in an ionic buffer that preserves the lamellar phase of the metastable inner leaflet lipids, DRMs can readily be isolated at 37°C, and these have many properties expected of lipid rafts. However, to date these DRMs have remained somewhat larger than current concepts of rafts. We describe an adaptation of this method that purifies nano-meso scale DRMs, and could be a significant step towards purifying the membrane of individual 'rafts'.

Biophotonic techniques for the study of malaria-infected red blood cells.

Investigation of the homeostasis of red blood cells upon infection by Plasmodium falciparum poses complex experimental challenges. Changes in red cell shape, volume, protein, and ion balance are difficult to quantify. In this article, we review a wide range of optical techniques for quantitative measurements of critical homeostatic parameters in malaria-infected red blood cells. Fluorescence lifetime imaging and tomographic phase microscopy, quantitative deconvolution microscopy, and X-ray microanalysis, are used to measure haemoglobin concentration, cell volume, and ion contents. Atomic force microscopy is briefly reviewed in the context of these optical methodologies. We also describe how optical tweezers and optical stretchers can be usefully applied to empower basic malaria research to yield diagnostic information on cell compliance changes upon malaria infection. The combined application of these techniques sheds new light on the detailed mechanisms of malaria infection providing potential for new diagnostic or therapeutic approaches.

Magnetic conjugated polymer nanoparticles as bimodal imaging agents.

Hybrid nanoparticles which incorporate multiple functionalities, such as fluorescence and magnetism, can exhibit enhanced efficiency and versatility by performing several tasks in parallel. In this study, magnetic-fluorescent semiconductor polymer nanospheres (MF-SPNs) have been synthesized by encapsulation of hydrophobic conjugated polymers and iron oxide nanoparticles in phospholipid micelles. Four fluorescent conjugated polymers were used, yielding aqueous dispersions of nanoparticles which emit across the visible spectrum. The MF-SPNs were shown to be magnetically responsive and simultaneously fluorescent. In MRI studies, they were seen to have a shortening effect on the transverse T(2)* relaxation time, which demonstrates their potential as an MR contrast agent. Finally, successful uptake of the MF-SPNs by SH-SY5Y neuroblastoma cells was demonstrated, and they were seen to behave as bright and stable fluorescent markers. There was no evidence of toxicity or adverse affect on cell growth.