TRIM46 Organizes Microtubule Fasciculation in the Axon Initial Segment.

Selective cargo transport into axons and dendrites over the microtubule network is essential for neuron polarization. The axon initial segment (AIS) separates the axon from the somatodendritic compartment and controls the microtubule-dependent transport into the axon. Interestingly, the AIS has a characteristic microtubule organization; it contains bundles of closely spaced microtubules with electron dense cross-bridges, referred to as microtubule fascicles. The microtubule binding protein TRIM46 localizes to the AIS and when overexpressed in non-neuronal cells forms microtubule arrays that closely resemble AIS fascicles in neurons. However, the precise role of TRIM46 in microtubule fasciculation in neurons has not been studied. Here we developed a novel correlative light and electron microscopy approach to study AIS microtubule organization. We show that in cultured rat hippocampal neurons of both sexes, TRIM46 levels steadily increase at the AIS during early neuronal differentiation and at the same time closely spaced microtubules form, whereas the fasciculated microtubules appear at later developmental stages. Moreover, we localized TRIM46 to the electron dense cross-bridges and show that depletion of TRIM46 causes loss of cross-bridges and increased microtubule spacing. These data indicate that TRIM46 has an essential role in organizing microtubule fascicles in the AIS.SIGNIFICANCE STATEMENT The axon initial segment (AIS) is a specialized region at the proximal axon where the action potential is initiated. In addition the AIS separates the axon from the somatodendritic compartment, where it controls protein transport to establish and maintain neuron polarity. Cargo vesicles destined for the axon recognize specialized microtubule tracks that enter the AIS. Interestingly the microtubules entering the AIS form crosslinked bundles, called microtubule fascicules. Recently we found that the microtubule-binding protein TRIM46 localizes to the AIS, where it may organize the AIS microtubules. In the present study we developed a novel correlative light and electron microscopy approach to study the AIS microtubules during neuron development and identified an essential role for TRIM46 in microtubule fasciculation.

Cinnamaldehyde, Carvacrol and Organic Acids Affect Gene Expression of Selected Oxidative Stress and Inflammation Markers in IPEC-J2 Cells Exposed to Salmonella typhimurium.

Essential oils and organic acids are used as feed additives to improve health status and reduce colonization with pathogens. Although bactericidal in vitro, concentrations achieved in the animal gut are probably not lethal to pathogens. The aim of this study was to investigate the effects of cinnamaldehyde, carvacrol and cinnamic, lactic and propionic acids on the ability of Salmonella typhimurium ATCC 14028 (ST) to invade intestinal epithelial cells (IPEC-J2) and on the expression levels of immune related genes in the cells. The minimum inhibitory concentration (MIC) and non-inhibitory concentration (NIC) were determined and influence on the invasion capacity of ST was investigated. The structure of fimbriae and flagella was analysed by electron microscopy, and expression levels of HSP70, IkBa, IL-8 and IL-10 in the IPEC-J2 cells were carried out by q-PCR. Cinnamaldehyde, carvacrol and cinnamic and propionic acids inhibited ST invasion but not cell viability, bacterial viability and motility or the development of flagella. Propionic acid and cinnamaldehyde in combination with cinnamic acid caused structural impairment of fimbriae. Cinnamaldehyde up-regulated expression of HSP70 irrespective of the presence of organic acids or ST; exposure to carvacrol induced HSP70 only in the presence of propionic acid and ST. © 2016 The Authors. Phytotherapy Research published by John Wiley & Sons Ltd.

RNA Sequencing Analysis of Intracranial Aneurysm Walls Reveals Involvement of Lysosomes and Immunoglobulins in Rupture.

BACKGROUND AND PURPOSE: Analyzing genes involved in development and rupture of intracranial aneurysms can enhance knowledge about the pathogenesis of aneurysms, and identify new treatment strategies. We compared gene expression between ruptured and unruptured aneurysms and control intracranial arteries. METHODS: We determined expression levels with RNA sequencing. Applying a multivariate negative binomial model, we identified genes that were differentially expressed between 44 aneurysms and 16 control arteries, and between 22 ruptured and 21 unruptured aneurysms. The differential expression of 8 relevant and highly significant genes was validated using digital polymerase chain reaction. Pathway analysis was used to identify enriched pathways. We also analyzed genes with an extreme pattern of differential expression: only expressed in 1 condition without any expression in the other. RESULTS: We found 229 differentially expressed genes in aneurysms versus controls and 1489 in ruptured versus unruptured aneurysms. The differential expression of all 8 genes selected for digital polymerase chain reaction validation was confirmed. Extracellular matrix pathways were enriched in aneurysms versus controls, whereas pathways involved in immune response and the lysosome pathway were enriched in ruptured versus unruptured aneurysms. Immunoglobulin genes were expressed in aneurysms, but showed no expression in controls. CONCLUSIONS: For rupture of intracranial aneurysms, we identified the lysosome pathway as a new pathway and found further evidence for the role of the immune response. Our results also point toward a role for immunoglobulins in the pathogenesis of aneurysms. Immune-modifying drugs are, therefore, interesting candidate treatment strategies in the prevention of aneurysm development and rupture.

TRIM46 Controls Neuronal Polarity and Axon Specification by Driving the Formation of Parallel Microtubule Arrays.

Axon formation, the initial step in establishing neuronal polarity, critically depends on local microtubule reorganization and is characterized by the formation of parallel microtubule bundles. How uniform microtubule polarity is achieved during axonal development remains an outstanding question. Here, we show that the tripartite motif containing (TRIM) protein TRIM46 plays an instructive role in the initial polarization of neuronal cells. TRIM46 is specifically localized to the newly specified axon and, at later stages, partly overlaps with the axon initial segment (AIS). TRIM46 specifically forms closely spaced parallel microtubule bundles oriented with their plus-end out. Without TRIM46, all neurites have a dendrite-like mixed microtubule organization resulting in Tau missorting and altered cargo trafficking. By forming uniform microtubule bundles in the axon, TRIM46 is required for neuronal polarity and axon specification in vitro and in vivo. Thus, TRIM46 defines a unique axonal cytoskeletal compartment for regulating microtubule organization during neuronal development.

Visualization of the aneurysm wall: a 7.0-tesla magnetic resonance imaging study.

BACKGROUND: Risk prediction of rupture of intracranial aneurysms is poor and is based mainly on lumen characteristics. However, characteristics of the aneurysm wall may be more informative predictors. The limited resolution of currently available imaging techniques and the thin aneurysm wall make imaging of wall thickness challenging. OBJECTIVE: To introduce a novel protocol for imaging wall thickness variation using ultra--high-resolution 7.0-Tesla (7.0-T) magnetic resonance imaging (MRI). METHODS: We studied 33 unruptured intracranial aneurysms in 24 patients with a T1-weighted 3-dimensional magnetization-prepared inversion-recovery turbo-spin-echo whole-brain sequence with a resolution of 0.8 × 0.8 × 0.8 mm. We performed a validation study with a wedge phantom and with 2 aneurysm wall biopsies obtained during aneurysm treatment using ex vivo MRI and histological examination and correlating variations in MRI signal intensity with variations in actual thickness of the aneurysm wall. RESULTS: In vivo, the aneurysm wall was visible in 28 of the 33 aneurysms. Variation in signal intensity was observed in all visible aneurysm walls. Ex vivo MRI showed variation in signal intensity across the wall of the biopsies, similar to that observed on the in vivo images. Signal intensity and actual thickness in both biopsies had a linear correlation, with Pearson correlation coefficients of 0.85 and 0.86. CONCLUSION: Unruptured intracranial aneurysm wall and its variation in thickness can be visualized with 7.0-T MRI. Aneurysm wall thickness variation can now be further studied as a risk factor for rupture in prospective studies.

A novel permeabilization protocol to obtain intracellular 3D immunolabeling for electron tomography.

Electron tomography (ET) is a very important high-resolution tool for 3D imaging in cell biology. By combining the technique with immunolabeling, ET can provide essential insights into both cellular architecture and dynamics. We recently developed a protocol to achieve 3D immunolabeling of intracellular antigens without the need for uncontrolled permeabilization steps that cause random, extensive cell membrane disruption. Here we describe this novel method based on well-controlled permeabilization by targeted laser cell perforation. Mechanical permeabilization of the plasma membrane can be applied at specific sites without affecting other parts of the plasma membrane and intracellular membranes. Despite the relatively small opening created in the plasma membrane, the method allows specific 3D immunolocalization of cytoplasmic antigens in cultured cells by a pre-embedment protocol. The approach is unique and leads to a superior ultrastructural preservation for transmission electron microscopy and electron tomography.

Amyotrophic lateral sclerosis (ALS)-associated VAPB-P56S inclusions represent an ER quality control compartment.

BACKGROUND: Protein aggregation and the formation of intracellular inclusions are a central feature of many neurodegenerative disorders, but precise knowledge about their pathogenic role is lacking in most instances. Here we have characterized inclusions formed in transgenic mice carrying the P56S mutant form of VAPB that causes various motor neuron syndromes including ALS8. RESULTS: Inclusions in motor neurons of VAPB-P56S transgenic mice are characterized by the presence of smooth ER-like tubular profiles, and are immunoreactive for factors that operate in the ER associated degradation (ERAD) pathway, including p97/VCP, Derlin-1, and the ER membrane chaperone BAP31. The presence of these inclusions does not correlate with signs of axonal and neuronal degeneration, and axotomy leads to their gradual disappearance, indicating that they represent reversible structures. Inhibition of the proteasome and knockdown of the ER membrane chaperone BAP31 increased the size of mutant VAPB inclusions in primary neuron cultures, while knockdown of TEB4, an ERAD ubiquitin-protein ligase, reduced their size. Mutant VAPB did not codistribute with mutant forms of seipin that are associated with an autosomal dominant motor neuron disease, and accumulate in a protective ER derived compartment termed ERPO (ER protective organelle) in neurons. CONCLUSIONS: The data indicate that the VAPB-P56S inclusions represent a novel reversible ER quality control compartment that is formed when the amount of mutant VAPB exceeds the capacity of the ERAD pathway and that isolates misfolded and aggregated VAPB from the rest of the ER. The presence of this quality control compartment reveals an additional level of flexibility of neurons to cope with misfolded protein stress in the ER.

In-situ integrity control of frozen-hydrated, vitreous lamellas prepared by the cryo-focused ion beam-scanning electron microscope.

Recently a number of new approaches have been presented with the intention to produce electron beam transparent cryo-sections (lamellas in FIB-SEM terminology) from hydrated vitreously frozen cryo samples with a Focused Ion Beam (FIB) system, suitable for cryo-Transmission Electron Microscopy (cryo-TEM). As the workflow is still challenging and time consuming, it is important to be able to determine the integrity and suitability (cells vs. no cells; vitreous vs. crystalline) of the lamellas. Here we present an in situ method that tests both conditions by using the cryo-Scanning Electron Microscope (cryo-SEM) in transmission mode (TSEM; Transmission Scanning Electron Microscope) once the FIB-made lamella is ready. Cryo-TSEM imaging of unstained cells yields strong contrast, enabling direct imaging of material present in the lamellas. In addition, orientation contrast is shown to be suitable for distinguishing crystalline lamellas from vitreous lamellas. Tilting the stage a few degrees results in changes of contrast between ice grains as a function of the tilt angle, whereas the contrast of areas with vitreous ice remains unchanged as a function of the tilt angle. This orientation contrast has subsequently been validated by cryo-Electron BackScattered Diffraction (EBSD) in transmission mode. Integration of the presented method is discussed and the role it can play in future developments for a new and innovative all-in-one cryo-FIB-SEM life sciences instrument.

A new, rapid and reproducible method to obtain high quality endothelium in vitro.

Human umbilical vein endothelial cells (HUVECs) cultured in vitro are a commonly used experimental system. When properly differentiated they acquire the so-called cobblestone phenotype; thereby mimicking an endothelium in vivo that can be used to shed light on multiple endothelial-related processes. In the present paper we report a simple, flexible, fast and reproducible method for an efficient isolation of viable HUVECs. The isolation is performed by sequential short trypsinization steps at room temperature. As umbilical cords are often damaged during labor, it is noteworthy that this new method can be applied even to short pieces of cord with success. In addition, we describe how to culture HUVECs as valid cobblestone cells in vitro on different types of extracellular matrix (basement membrane matrix, fibronectin and gelatin). We also show how to recognize mature cobblestone HUVECs by ordinary phase contrast microscopy. Our HUVEC model is validated as a system that retains important features inherent to the human umbilical vein endothelium in vivo. Phase contrast microscopy, immuno-fluorescence and electron microscopy reveal a tight cobblestone monolayer. Therein cells show Weibel-Palade bodies, caveolae and junctional complexes (comparable to the in vivo situation, as also shown in this study) and can internalize human low density lipoprotein. Isolation and culture of HUVECs as reported in this paper will result in an endothelium-mimicking experimental model convenient for multiple research goals.

Sub-lethal levels of carvacrol reduce Salmonella Typhimurium motility and invasion of porcine epithelial cells.

The European ban on the use of antibiotic growth promotors has increased the search for new alternatives to prevent pig intestinal microbial diseases and to stimulate growth. The addition of essential oils or components thereof, such as carvacrol, to pig feed is a promising alternative. In this report we determined the effect of sub-lethal concentrations of carvacrol on Salmonella Typhimurium. At concentrations where growth of Salmonella was not inhibited, carvacrol completely inhibited motility of the bacterium. This loss of motility was not due to the loss of the flagellum or to ATP shortage upon carvacrol treatment. Adhesion of Salmonella to IPEC-J2, porcine intestinal epithelial cells, was not affected by carvacrol but invasion was significantly reduced. In addition, the epithelial gene expression of porcine ß-defensin 2, an innate immune response to Salmonella infection, was reduced when Salmonella was exposed to carvacrol. This indicates that invasion but not adhesion of Salmonella triggers the porcine ß-defensin 2 expression of porcine epithelial cells.

The porosity, acidity, and reactivity of dealuminated zeolite ZSM-5 at the single particle level: the influence of the zeolite architecture.

A combination of atomic force microscopy (AFM), high-resolution scanning electron microscopy (HR-SEM), focused-ion-beam scanning electron microscopy (FIB-SEM), X-ray photoelectron spectroscopy (XPS), confocal fluorescence microscopy (CFM), and UV/Vis and synchrotron-based IR microspectroscopy was used to investigate the dealumination processes of zeolite ZSM-5 at the individual crystal level. It was shown that steaming has a significant impact on the porosity, acidity, and reactivity of the zeolite materials. The catalytic performance, tested by the styrene oligomerization and methanol-to-olefin reactions, led to the conclusion that mild steaming conditions resulted in greatly enhanced acidity and reactivity of dealuminated zeolite ZSM-5. Interestingly, only residual surface mesoporosity was generated in the mildly steamed ZSM-5 zeolite, leading to rapid crystal coloration and coking upon catalytic testing and indicating an enhanced deactivation of the zeolites. In contrast, harsh steaming conditions generated 5-50 nm mesopores, extensively improving the accessibility of the zeolites. However, severe dealumination decreased the strength of the Brønsted acid sites, causing a depletion of the overall acidity, which resulted in a major drop in catalytic activity.

Spiral coating of the endothelial caveolar membranes as revealed by electron tomography and template matching.

Caveolae are invaginations of the plasma membrane involved in multiple cellular processes, including transcytosis. In this paper we present an extensive 3-D electron tomographic study of the endothelial caveolar system in situ. Analysis of large cellular volumes of (high-pressure frozen, freeze-substituted and epon-embedded) human umbilical vein endothelial cells (HUVECs) provided a notable view on the architecture of the caveolar system that comprises--as confirmed by 3-D immunolabeling for caveolin of 'intact' cells--bona fide caveolae, free plasmalemmal vesicles, racemose invaginations and free multi-caveolar bodies. Application of template matching to tomograms allowed the 3-D localization of caveolar membrane coatings in a robust manner. In this way we observed that bona fide endothelial caveolae, cryofixed and embedded in their cellular context, show a spiral organization of the coating as shown in the past for chemically fixed and freeze-etched caveolae from fibroblasts. Meticulous 3-D analysis further revealed that the coatings are distributed in triads of spirals over the caveolar bulb and neck. Remarkably, this coating distribution is consistently present over the membranes of the other members of the caveolar system in HUVECs. The novel observations that we present clarify the ultrastructural complexity of the 'intact' caveolar system, setting a detailed morphological basis for its functional diversity.

Tannic acid-mediated osmium impregnation after freeze-substitution: a strategy to enhance membrane contrast for electron tomography.

In this technical note we report a tannic acid-mediated osmium impregnation method that, applied after freeze-substitution, increases membrane contrast in cells for transmission electron microscopy and tomography studies. The general staining that is achieved allows visualization of organelles, plasma membrane and associated specializations (e.g. caveolae) in non-post-stained plastic sections by conventional transmission electron microscopy. In combination with electron tomography it results in membranes with a proper contrast and equal staining pattern through the depth of the tomograms. The protocol that we contribute can serve as starting point for those willing to improve the membrane contrast of their specimens or to make 3D studies on the architecture of membranous compartments by electron tomography.

Selection and characterization of KDEL-specific VHH antibody fragments and their application in the study of ER resident protein expression.

Several diseases are caused by defects in the protein secretory pathway of the cell, particularly in the endoplasmic reticulum (ER). These defects are manifested by the activation of the unfolded protein response (UPR) that involves the transcriptional up-regulation of several ER resident proteins, the down-regulation of protein translation and up-regulation of ER associated degradation (ERAD). Although this transcriptional up-regulation of ER resident proteins during ER stress has been well described, data on differential protein expression of these same proteins are hardly available. Tools that would enable the simultaneous analysis of this set of proteins would be of high importance. Since the C-terminal KDEL sequence is a conserved epitope present in a large set of ER resident proteins, an antibody directed against this sequence would be such a tool. Using a carefully designed selection strategy, VHH antibody fragments from a non-immune phage display library were isolated that recognize the KDEL sequence at the C-terminus of proteins, irrespective of the protein context. In an accepted in vitro model for ER stress, this antibody was shown to be an excellent tool to study differences in ER resident protein expression. Furthermore, the application of this antibody showed differences in ER resident protein levels during replicative senescence of human umbilical vein endothelial cells (HUVECs), underlining its significance in biological research. The selection strategy used to obtain these KDEL-specific antibodies opens up ways to select antibodies to other conserved epitopes, such as the nuclear localization signal (NLS) or the peroxisomal targeting sequence, permitting the simultaneous analysis of specific groups of proteins.

Anti-beta1 integrin antibody reduces surgery-induced adhesion of colon carcinoma cells to traumatized peritoneal surfaces.

OBJECTIVE: To study the mechanisms behind surgery-induced augmentation of tumor outgrowth. SUMMARY BACKGROUND DATA: Surgery provides the best chance of cure for most primary intra-abdominal carcinomas. Effective treatment is however relatively frequent complicated by peritoneal recurrences, which often originate from free-floating intraperitoneal tumor cells that implant on peritoneal surfaces. We previously reported that surgical trauma promotes development of peritoneal metastases. METHODS: Evaluation of adhesion of CC531s rat colon carcinoma cell line intraperitoneally after laparotomy using in vivo, ex vivo, and in vitro models. Also, human ex vivo models were used to study peritoneal tumor cell adhesion. RESULTS: Peritoneal imprints of operated rats showed that direct damaging of the peritoneum resulted in enhanced adhesion of rat CC531 colon carcinoma cells to submesothelial extracellular matrix (ECM) proteins in vivo, which was confirmed by electron microscopy. Additionally, the inflammatory reaction of the peritoneal cavity led to retraction of mesothelial cells, hereby also exposing ECM at peritoneal surfaces that had not been traumatized directly. Furthermore, we demonstrated that beta1 integrin subunits represented the primary mediators involved in adherence to either isolated ECM components or excised traumatized rat and human peritoneum. Importantly, incubation of CC531s cells with anti-beta1 integrin antibodies resulted in a significant decrease of tumor cell adhesion in vivo. CONCLUSIONS: Surgical trauma results in exposure of ECM at directly and nondirectly damaged peritoneal surfaces, leading to increased beta1 integrin-dependent tumor cell adhesion. Perioperative therapies, which aim to block beta1 integrin subunits, might therefore serve as new clinical tools for the prevention of peritoneal recurrences.

Improved anti-IgG and HSA affinity ligands: clinical application of VHH antibody technology.

Large scale, highly specific purification of valuable proteins from blood and removal of undesirable components promise to have wide therapeutic applications. Moreover, depletion of bulk proteins from blood is a prerequisite for clinical proteomics. Here we describe the development of specific, high affinity Camelid antibody fragments (VHH) derived from immune libraries for purification and depletion of the bulk protein HSA and IgG from human serum and plasma for therapeutic and research purposes. The anti-IgG VHH substantially improved depletion of IgGs from blood over the classical method based on protein A. To demonstrate the improved performance of VHH based IgG depletion, we analyzed the presence of auto-antibodies in human plasma before and after depletion from two groups of patients with auto-immune disease: Goodpasture syndrome (GP) and systemic lupus erythematosus (SLE). VHHs can be produced efficiently and cost effectively in Saccharomyces cerevisiae, a genetically regarded as safe (GRAS) microorganism. A good manufacturing process (GMP) for purification of these VHHs has also been developed. Moreover, as VHHs are single protein chains, they can be coupled relatively easily to solid matrices. These three factors are important for developing affinity purification medication.

Protein expression dynamics during replicative senescence of endothelial cells studied by 2-D difference in-gel electrophoresis.

Endothelial senescence contributes to endothelium dysfunctionality and is thereby linked to vascular aging. A dynamic proteomic study on human umbilical vein endothelial cells, isolated from three umbilical cords, was performed. The cells were cultured towards replicative senescence and whole cell lysates were subjected to 2-D difference gel electrophoresis (DIGE). Despite the biological variability of the three independent isolations, a set of proteins was found that showed senescence-dependent expression patterns in all isolations. We focused on those proteins that showed significant changes, with a paired analysis of variance (RM-ANOVA) p-value of < or =0.05. Thirty-five proteins were identified with LC-Fourier transform MS, and functional annotation revealed that endothelial replicative senescence is accompanied by increased cellular stress, protein biosynthesis and reduction in DNA repair and maintenance. Nuclear integrity becomes affected and cytoskeletal structure is also changed. Such important changes in the cell infrastructure might accelerate endothelium dysfunctionality. This study provides biological information that will initiate studies to further unravel endothelial senescence and gain more knowledge about the consequences of this process in the in vivo situation.

Mass spectrometric characterization of the oxidation of the fluorescent lipid peroxidation reporter molecule C11-BODIPY(581/591).

C11-BODIPY(581/591) is a fluorescent lipid peroxidation reporter molecule that shifts its fluorescence from red to green when challenged with oxidizing agents, i.e., reactive oxygen species (ROS) or reactive nitrogen species (RNS). To understand the molecular mechanism responsible for this shift, we studied the molecular rearrangements leading to the shift in fluorescence in C11-BODIPY(581/591). Furthermore, we aimed to determine if these rearrangements were dependent on the nature of the applied ROS, in homogenous solution, bilayer vesicles, and living cells. C11-BODIPY(581/591) was challenged with various ROS- or RNS-generating systems, including peroxynitrite, NO(2)(?), peroxides, and hydroxyl, alkoxyl, tyrosyl, and peroxyl radicals. The reaction products were subsequently analyzed by means of mass spectrometry. Our results show that the initial target for free radical-mediated oxidation is the conjugated diene interconnection between the BODIPY core and the terminal phenyl moiety, which already explains the shift in fluorescence properties of the probe. After oxidative challenge, three different stable products were identified, one of which was specific for oxidation by peroxynitrite. The two other stable end products had lost the entire phenyl moiety, irrespective of the type of radical generating system used. These products were also recovered from Rat-1 fibroblasts stressed either by GSH depletion/serum starvation or by exposure to peroxynitrite, and were the only C11-BODIPY(581/591) oxidation products detectable in these cells.

Attenuation of lipid peroxidation by antioxidants in rat-1 fibroblasts: comparison of the lipid peroxidation reporter molecules cis-parinaric acid and C11-BODIPY(581/591) in a biological setting.

Lipid peroxidation is a major factor in the pathogenesis of many disease states. To detect the initial stages of lipid peroxidation or evaluate antioxidant efficacy, cis-parinaric acid (cis-PnA) has been successfully used and thoroughly validated. However, cis-PnA is not very well suited for medium throughput screening of antioxidants in living cells. We recently introduced and validated a lipid peroxidation reporter molecule, C11-BODIPY(581/591). To further explore this probe, we evaluated the protective effect of 12 natural antioxidants in rat-1 fibroblasts subjected to 50 microM cumene-hydroperoxide using both probes. The same pecking order for the individual antioxidant efficacies was obtained: alpha-tocopherol approximately gamma-tocopherol > quercetin approximately lycopene > kaempferol > palm oil > hydroxy-tyrosol > > alpha-carotene = beta-carotene = lutein = tyrosol = chlorogenic acid. This validates the accuracy of the C11-BODIPY(581/591) method and shows that this assay is an accurate and highly flexible method for indexing lipid peroxidation or determining antioxidant efficacy in living cells in a medium throughput scenario. The antioxidant efficacy was compared with their one-electron reduction potential, hydrophobicity and Trolox C equivalent antioxidant capacity. Our results show that although these parameters are valuable for determining structure-function relationships, they have limited predictive value for antioxidant efficacy in vivo.