Getting to know carotenoids.

More than 600 different carotenoids have been isolated from natural sources and fully or partly characterized. Fortunately, although they are all different individual compounds, the carotenoids may be considered as a group in terms of many of their basic properties. The aim of this chapter is to help newcomers and others that are not carotenoid specialists to understand the peculiarities of carotenoids, and how best to work with them. The basic principles of structure and nomenclature of carotenoids are described (especially the IUPAC semi-systematic rules) and the chemical and physical properties on which the biological functions depend are discussed. Guidance is given on how to handle and work with carotenoids safely and reliably and reduce the risk of forming artifacts and introducing impurities. Recommendations are made about their isolation, identification by various physical methods, and spectrophotometric quantitative analysis. Structural features that determine the shape of the carotenoid molecule are discussed, especially the conjugated polyene system. This also determines the light absorption properties of carotenoids and their chemical reactivity, especially toward oxidising agents. The properties of carotenoids in situ are strongly influenced by aggregation in the aqueous environment and by interactions with other molecules in their vicinity, notably proteins and membrane lipids. Many substances that look like fragments of carotenoids have been isolated from natural sources. This chapter does not provide comprehensive coverage of these apocarotenoids, but does consider their nomenclature and numbering according to the IUPAC rules.

cytoNet: Spatiotemporal network analysis of cell communities.

We introduce cytoNet, a cloud-based tool to characterize cell populations from microscopy images. cytoNet quantifies spatial topology and functional relationships in cell communities using principles of network science. Capturing multicellular dynamics through graph features, cytoNet also evaluates the effect of cell-cell interactions on individual cell phenotypes. We demonstrate cytoNet's capabilities in four case studies: 1) characterizing the temporal dynamics of neural progenitor cell communities during neural differentiation, 2) identifying communities of pain-sensing neurons in vivo, 3) capturing the effect of cell community on endothelial cell morphology, and 4) investigating the effect of laminin α4 on perivascular niches in adipose tissue. The analytical framework introduced here can be used to study the dynamics of complex cell communities in a quantitative manner, leading to a deeper understanding of environmental effects on cellular behavior. The versatile, cloud-based format of cytoNet makes the image analysis framework accessible to researchers across domains.

Human neural tube morphogenesis in vitro by geometric constraints.

Understanding human organ formation is a scientific challenge with far-reaching medical implications1,2. Three-dimensional stem-cell cultures have provided insights into human cell differentiation3,4. However, current approaches use scaffold-free stem-cell aggregates, which develop non-reproducible tissue shapes and variable cell-fate patterns. This limits their capacity to recapitulate organ formation. Here we present a chip-based culture system that enables self-organization of micropatterned stem cells into precise three-dimensional cell-fate patterns and organ shapes. We use this system to recreate neural tube folding from human stem cells in a dish. Upon neural induction5,6, neural ectoderm folds into a millimetre-long neural tube covered with non-neural ectoderm. Folding occurs at 90% fidelity, and anatomically resembles the developing human neural tube. We find that neural and non-neural ectoderm are necessary and sufficient for folding morphogenesis. We identify two mechanisms drive folding: (1) apical contraction of neural ectoderm, and (2) basal adhesion mediated via extracellular matrix synthesis by non-neural ectoderm. Targeting these two mechanisms using drugs leads to morphological defects similar to neural tube defects. Finally, we show that neural tissue width determines neural tube shape, suggesting that morphology along the anterior-posterior axis depends on neural ectoderm geometry in addition to molecular gradients7. Our approach provides a new route to the study of human organ morphogenesis in health and disease.

Fate-Patterning of 2D Gastruloids and Ectodermal Colonies Using Micropatterned Human Pluripotent Stem Cells.

In the developing mammalian embryo, intercellular signaling allows cells to self-organize to create spatial patterns of different cell fates. This process is challenging to study because of the difficulty of observing or manipulating embryos on the spatial and temporal scales required. In vitro models can provide a complement to in vivo systems for addressing these issues. These models are also the only windows we have into early human development. Here we provide protocols for two systems based on differentiating human pluripotent stem cells in micropatterned colonies on defined size and shape. The first model replicates the patterning of the germ layers at gastrulation, while the second replicates the medial-lateral patterning of the ectoderm. These systems allow study of how signaling underlies self-organized patterning at stages of development which are otherwise inaccessible.

Carotenoid research: History and new perspectives for chemistry in biological systems.

The history of carotenoid research as this progressed from chemistry to biochemistry and biology is outlined. Proposed roles of carotenoids in eye health, as antioxidants, and in protection against cancer and other degenerative diseases, as well as stimulatory effects on the immune system and metabolism are covered. Proposed biological actions must be consistent with the chemistry of carotenoids in the largely aqueous biological systems, which may differ from the known chemistry of carotenoids in organic solvents. In particular, carotenoids tend to form aggregates. The effects of this aggregation and of other molecular interactions in vivo are likely to be crucial to biological activity. These perspectives of the chemistry of carotenoids and carotenoid free radicals are examined and the need for carotenoid samples used in experimental work to be pure and free from breakdown products and pro-oxidant peroxides is emphasised. This article is part of a Special Issue entitled Carotenoids recent advances in cell and molecular biology edited by Johannes von Lintig and Loredana Quadro.

A novel self-organizing embryonic stem cell system reveals signaling logic underlying the patterning of human ectoderm.

During development, the ectoderm is patterned by a combination of BMP and WNT signaling. Research in model organisms has provided substantial insight into this process; however, there are currently no systems in which to study ectodermal patterning in humans. Further, the complexity of neural plate border specification has made it difficult to transition from discovering the genes involved to deeper mechanistic understanding. Here, we develop an in vitro model of human ectodermal patterning, in which human embryonic stem cells self-organize to form robust and quantitatively reproducible patterns corresponding to the complete medial-lateral axis of the embryonic ectoderm. Using this platform, we show that the duration of endogenous WNT signaling is a crucial control parameter, and that cells sense relative levels of BMP and WNT signaling in making fate decisions. These insights allowed us to develop an improved protocol for placodal differentiation. Thus, our platform is a powerful tool for studying human ectoderm patterning and for improving directed differentiation protocols.This article has an associated 'The people behind the papers' interview.

Ultrasound-enhanced bevacizumab release from echogenic liposomes for inhibition of atheroma progression.

CONTEXT: Bevacizumab (BEV) is a monoclonal antibody to vascular endothelial growth factor (VEGF) that ameliorates atheroma progression by inhibiting neovascularization. OBJECTIVE: We aimed to determine whether BEV release from echogenic liposomes (BEV-ELIP) could be enhanced by color Doppler ultrasound (US) and whether the released BEV inhibits VEGF expression by endothelial cells in vitro. MATERIALS AND METHODS: BEV-ELIP samples were subjected to 6 MHz color Doppler ultrasound (MI = 0.4) for 5 min. We assessed release of BEV with a direct ELISA and with fluoresceinated BEV (FITC-BEV) loaded into ELIP by the same method. Human umbilical vein endothelial cell (HUVEC) cultures were stimulated to express VEGF by 10 nM phorbol-12-myristate 13-acetate (PMA). Cell-associated VEGF levels were determined using a cell-based ELISA. RESULTS: Overall, US caused an additional 100 µg of BEV to be released or exposed per BEV-ELIP aliquot within 60 min BEV-ELIP treated with US inhibited VEGF expression by 90% relative to non-treated controls and by 70% relative to BEV-ELIP without US. Also, US-treated BEV-ELIP inhibited HUVEC proliferation by 64% relative to untreated controls and by 45% relative to BEV-ELIP without US. DISCUSSION AND CONCLUSION: We have demonstrated that BEV-ELIP retains its VEGF-binding activity in a liposomal formulation and that clinical Doppler US can significantly increase that activity, both by releasing free BEV and by enhancing the surface exposure of the immunoreactive antibody.

Nitric oxide-loaded echogenic liposomes for treatment of vasospasm following subarachnoid hemorrhage.

Delayed cerebral vasospasm following subarachnoid hemorrhage causes severe ischemic neurologic deficits leading to permanent neurologic dysfunction or death. Reduced intravascular and perivascular nitric oxide (NO) availability is a primary pathophysiology of cerebral vasospasm. In this study, we evaluated NO-loaded echogenic liposomes (NO-ELIP) for ultrasound-facilitated NO delivery to produce vasodilation for treatment of vasospasm following subarachnoid hemorrhage. We investigated the vasodilative effects of NO released from NO-ELIP both ex vivo and in vivo. Liposomes containing phospholipids and cholesterol were prepared, and NO was encapsulated. The encapsulation and release of NO from NO-ELIP were determined by the syringe/vacuum method and ultrasound imaging. The ex vivo vasodilative effect of NO-ELIP was investigated using rabbit carotid arteries. Arterial vasodilation was clearly observed with NO-ELIP exposed to Doppler ultrasound whereas there was little vasodilative effect without exposure to Doppler ultrasound in the presence of red blood cells. Penetration of NO into the arterial wall was determined by fluorescent microscopy. The vasodilative effects of intravenously administered NO-ELIP in vivo were determined in a rat subarachnoid hemorrhage model. NO-ELIP with ultrasound activation over the carotid artery demonstrated effective arterial vasodilation in vivo resulting in improved neurologic function. This novel methodology for ultrasound-controlled delivery of NO has the potential for therapeutic treatment of vasospasm following subarachnoid hemorrhage. This ultrasound-controlled release strategy provides a new avenue for targeted bioactive gas and therapeutic delivery for improved stroke treatment.

Therapeutic time window and dose dependence of xenon delivered via echogenic liposomes for neuroprotection in stroke.

AIMS: Neurologic impairment following ischemic injury complicates the quality of life for stroke survivors. Xenon (Xe) has favorable neuroprotective properties to modify stroke. Xe delivery is hampered by a lack of suitable administration strategies. We have developed Xe-containing echogenic liposomes (Xe-ELIP) for systemic Xe delivery. We investigated the time window for Xe-ELIP therapeutic effect and the most efficacious dose for neuroprotection. Molecular mechanisms for Xe neuroprotection were investigated. METHODS: Xenon-containing echogenic liposomes were created by a previously developed pressurization-freezing method. Following right middle cerebral artery occlusion (2 h), animals were treated with Xe-ELIP at 2, 3, or 5 h to determine time window of therapeutic effect. The neuroprotectant dosage for optimal effect was evaluated 3 h after stroke onset. Expression of brain-derived neurotrophic factor (BDNF), protein kinase B (Akt), and mitogen-activated protein kinases (MAPK) was determined. RESULTS: Xenon-containing echogenic liposomes administration for up to 5 h after stroke onset reduced infract size. Treatment groups given 7 and 14 mg/kg of Xe-ELIP reduced infarct size. Behavioral outcomes corresponded to changes in infarct volume. Xe-ELIP treatment reduced ischemic neuronal cell death via activation of both MAPK and Akt. Elevated BDNF expression was shown following Xe-ELIP delivery. CONCLUSION: This study demonstrates the therapeutic efficacy of Xe-ELIP administered within 5 h after stroke onset with an optimal dosage range of 7-14 mg/kg for maximal neuroprotection.

Sapotexanthin, an A-provitamin carotenoid from red mamey (Pouteria sapota).

From the ripe fruits of red mamey (Pouteria sapota) sapotexanthin, a new carotenoid, was identified as (all-E,5'R)-ß,κ-caroten-6'-one.

In vivo therapeutic gas delivery for neuroprotection with echogenic liposomes.

BACKGROUND: Ischemia-related neurological injury is a primary cause of stroke disability. Studies have demonstrated that xenon (Xe) may have potential as an effective and nontoxic neuroprotectant. Xe delivery is, however, hampered by lack of suitable administration methods. We have developed a pressurization-freeze method to encapsulate Xe into echogenic liposomes (Xe-ELIP) and have modulated local gas release with transvascular ultrasound exposure. METHODS AND RESULTS: Fifteen microliters of Xe were encapsulated into each 1 mg of liposomes (70% Xe and 30% argon). Xe delivery from Xe-ELIP into cells and consequent neuroprotective effects were evaluated with oxygen/glucose-deprived and control neuronal cells in vitro. Xe-ELIP were administered into Sprague-Dawley rats intravenously or intra-arterially after right middle cerebral artery occlusion. One-megahertz low-amplitude (0.18 MPa) continuous wave ultrasound directed onto the internal carotid artery triggered Xe release from circulating Xe-ELIP. Effects of Xe delivery on ischemia-induced neurological injury and disability were evaluated. Xe-ELIP delivery to oxygen/glucose-deprived neuronal cells improved cell viability in vitro and resulted in a 48% infarct volume decrease in vivo. Intravenous Xe-ELIP administration in combination with the ultrasound directed onto the carotid artery enhanced local Xe release from circulating Xe-ELIP and demonstrated 75% infarct volume reduction. This was comparable to the effect after intra-arterial administration. Behavioral tests on limb placement and grid and beam walking correlated with infarct reduction. CONCLUSIONS: This novel methodology may provide a noninvasive strategy for ultrasound-enhanced local therapeutic gas delivery for cerebral ischemia-related injury while minimizing systemic side effects.

HPLC analysis of geometrical isomers of lutein epoxide isolated from dandelion (Taraxacum officinale F. Weber ex Wiggers).

Lutein epoxide has been isolated from petals of dandelion (Taraxacum officinale F. Weber ex Wiggers) by thin-layer chromatography (TLC) on silica to be used for the accurate identification of this carotenoid in other sources. The extract was analyzed by high-performance liquid chromatography (HPLC) using a C(30) column, as a result of which six geometrical isomers were separated. The identification of these isomers was performed on the basis of their UV/vis spectroscopic features in the mobile phase. In quantitative terms, it was observed that all-E-lutein epoxide was the major carotenoid and that there were also high amounts of the (9Z)- and (9'Z)-isomers, although the latter may be an artifact.

Identification of isolutein (lutein epoxide) as cis-antheraxanthin in orange juice.

The carotenoid profile of orange juice is very complex, a common characteristic for citrus products in general. This fact, along with the inherent acidity of the product, which promotes the isomerization of some carotenoids, makes the correct identification of some of these pigments quite difficult. Thus, one of the carotenoids occurring in orange juice has been traditionally identified as isolutein, a term used to refer to lutein epoxide, although enough evidence to support that identification has not been given. In this study, the carotenoid previously identified as isolutein/lutein epoxide in orange juice has been isolated and identified as a 9 or 9'-cis isomer of antheraxanthin as a result of different tests. To support this identification, a mixture of geometrical isomers of lutein epoxide isolated from petals of dandelions was analyzed under the same conditions used for orange juice carotenoids to check that neither their retention times nor their spectroscopic features matched with those of the orange juice carotenoid now identified as a cis isomer of antheraxanthin.

Identification of zeinoxanthin in orange juices.

The monohydroxycarotenoid fraction of orange juice has been isolated by TLC and studied to determine whether the carotenoid accompanying beta-cryptoxanthin was alpha-cryptoxanthin or zeinoxanthin. The provitamin A carotenoid alpha-cryptoxanthin has been widely reported in orange juice, although its identification has been carried out mainly on the basis of its spectral features, which are virtually identical with those of its non-provitamin A isomer, zeinoxanthin. As a result of a study of the UV-vis and mass spectra of the monohydroxycarotenoid fraction and of the methylation test, it was concluded that the carotenoid accompanying beta-cryptoxanthin was the non-provitamin A carotenoid zeinoxanthin.