Molecular profiling of lipid droplets inside HuH7 cells with Raman micro-spectroscopy.

Raman imaging has become an attractive technology in molecular biology because of its ability to detect multiple molecular components simultaneously without labeling. Two major limitations in accurately accounting for spectral features, viz., background removal and spectral unmixing, have been overcome by employing a modified and effective routine in multivariate curve resolution (MCR). With our improved strategy, we have spectrally isolated seven structurally specific biomolecules without any post-acquisition spectral treatments. Consequently, the isolated intensity profiles reflected concentrations of corresponding biomolecules with high statistical accuracy. Our study reveals the changes in the molecular composition of lipid droplets (LDs) inside HuH7 cells and its relation to the physiological state of the cell. Further, we show that the accurate separation of spectral components permits analysis of structural modification of molecules after cellular uptake. A detailed discussion is presented to highlight the potential of Raman spectroscopy with MCR in semi-quantitative molecular profiling of living cells.

Universal and Efficient Electroporation Protocol for Genetic Engineering of Gastrointestinal Organoids.

Electroporation is a common method for transfection with different kinds of molecules by electrical permeabilization of the plasma membrane. With the increasing use of organoids as a culturing method for primary patient material in the last years, efficient transfer methods of components for genetic engineering in this 3D culture system are in need. Especially for organoids, the efficiency of genetic manipulations depends on a successful transfection. Thus, this protocol was developed to facilitate the electroporation of organoids and to prove its universal functionality in different entities. Human colorectal, pancreatic, hepatic and gastric cancer organoids were successfully electroporated with small and large plasmids in comparison. Based on GFP encoding vectors, the transfection efficiency was determined by FACS. No extensive preparation of the cells or special, cost-intensive electroporation buffers are necessary, and the protocol can be performed within one day.

Human gastric cancer modelling using organoids.

OBJECTIVE: Gastric cancer is the second leading cause of cancer-related deaths and the fifth most common malignancy worldwide. In this study, human and mouse gastric cancer organoids were generated to model the disease and perform drug testing to delineate treatment strategies. DESIGN: Human gastric cancer organoid cultures were established, samples classified according to their molecular profile and their response to conventional chemotherapeutics tested. Targeted treatment was performed according to specific druggable mutations. Mouse gastric cancer organoid cultures were generated carrying molecular subtype-specific alterations. RESULTS: Twenty human gastric cancer organoid cultures were established and four selected for a comprehensive in-depth analysis. Organoids demonstrated divergent growth characteristics and morphologies. Immunohistochemistry showed similar characteristics to the corresponding primary tissue. A divergent response to 5-fluoruracil, oxaliplatin, irinotecan, epirubicin and docetaxel treatment was observed. Whole genome sequencing revealed a mutational spectrum that corresponded to the previously identified microsatellite instable, genomic stable and chromosomal instable subtypes of gastric cancer. The mutational landscape allowed targeted therapy with trastuzumab for ERBB2 alterations and palbociclib for CDKN2A loss. Mouse cancer organoids carrying Kras and Tp53 or Apc and Cdh1 mutations were characterised and serve as model system to study the signalling of induced pathways. CONCLUSION: We generated human and mouse gastric cancer organoids modelling typical characteristics and altered pathways of human gastric cancer. Successful interference with activated pathways demonstrates their potential usefulness as living biomarkers for therapy response testing.

Astrocytes and oligodendrocytes in grey and white matter regions of the brain metabolize fatty acids.

The grey and white matter regions of the mammalian brain consist of both neurons and neuroglial cells. Among the neuroglia, the two macroglia oligodendrocytes and astrocytes are the most abundant cell types. While the major function of oligodendrocytes is the formation of the lipid-rich myelin structure, the heterogeneous group of astrocytes fulfils a multitude of important roles in cerebral development and homeostasis. Brain lipid homeostasis involves the synthesis of a specific cerebral lipidome by local lipid metabolism. In this study we have investigated the fatty acid uptake and lipid biosynthesis in grey and white matter regions of the murine brain. Key findings were: (i) white matter oligodendrocytes and astrocytes take up saturated and unsaturated fatty acids, (ii) different grey matter regions show varying lipid labelling intensities, (iii) the medial habenula, an epithalamic grey matter structure, and the oligodendrocytes and astrocytes therein are targeted by fatty acids, and (iv) in the medial habenula, the neutral lipid containing lipid droplets are found in cells facing the ventricle but undetectable in the habenular parenchyma. Our data indicate a role for oligodendrocytes and astrocytes in local lipid metabolism of white and grey matter regions in the brain.

Localization of 1-deoxysphingolipids to mitochondria induces mitochondrial dysfunction.

1-Deoxysphingolipids (deoxySLs) are atypical sphingolipids that are elevated in the plasma of patients with type 2 diabetes and hereditary sensory and autonomic neuropathy type 1 (HSAN1). Clinically, diabetic neuropathy and HSAN1 are very similar, suggesting the involvement of deoxySLs in the pathology of both diseases. However, very little is known about the biology of these lipids and the underlying pathomechanism. We synthesized an alkyne analog of 1-deoxysphinganine (doxSA), the metabolic precursor of all deoxySLs, to trace the metabolism and localization of deoxySLs. Our results indicate that the metabolism of these lipids is restricted to only some lipid species and that they are not converted to canonical sphingolipids or fatty acids. Furthermore, exogenously added alkyne-doxSA [(2S,3R)-2-aminooctadec-17-yn-3-ol] localized to mitochondria, causing mitochondrial fragmentation and dysfunction. The induced mitochondrial toxicity was also shown for natural doxSA, but not for sphinganine, and was rescued by inhibition of ceramide synthase activity. Our findings therefore indicate that mitochondrial enrichment of an N-acylated doxSA metabolite may contribute to the neurotoxicity seen in diabetic neuropathy and HSAN1. Hence, we provide a potential explanation for the characteristic vulnerability of peripheral nerves to elevated levels of deoxySLs.

A highly sensitive protocol for microscopy of alkyne lipids and fluorescently tagged or immunostained proteins.

The demand to study the cellular localization of specific lipids has led to recent advances in lipid probes and microscopy. Alkyne lipids bear a small, noninterfering tag and can be detected upon click reaction with an azide-coupled reporter. Fluorescent alkyne lipid imaging crucially depends on appropriate azide reporters and labeling protocols that allow for an efficient click reaction and therefore a sensitive detection. We synthesized several azide reporters with different spacer components and tested their suitability for alkyne lipid imaging in fixed cells. The implementation of a copper-chelating picolyl moiety into fluorescent or biotin-based azide reagents strongly increased the sensitivity of the imaging routine. We demonstrate the applicability and evaluate the performance of this approach using different lipid classes and experimental setups. As azide picolyl reporters allow for reduced copper catalyst concentrations, they also enable coimaging of alkyne lipids with multiple fluorescent proteins including enhanced green fluorescent protein. Alternatively, and as we also show, microscopy of alkyne lipids can be combined with protein detection by immunocytochemistry. In summary, we present a robust, sensitive, and highly versatile protocol for the labeling of alkyne lipids with azide-coupled reporters for fluorescence microscopy that can be combined with different protein detection and imaging techniques.

A novel alkyne cholesterol to trace cellular cholesterol metabolism and localization.

Cholesterol is an important lipid of mammalian cells and plays a fundamental role in many biological processes. Its concentration in the various cellular membranes differs and is tightly regulated. Here, we present a novel alkyne cholesterol analog suitable for tracing both cholesterol metabolism and localization. This probe can be detected by click chemistry employing various reporter azides. Alkyne cholesterol is accepted by cellular enzymes from different biological species (Brevibacterium, yeast, rat, human) and these enzymes include cholesterol oxidases, hydroxylases, and acyl transferases that generate the expected metabolites in in vitro and in vivo assays. Using fluorescence microscopy, we studied the distribution of cholesterol at subcellular resolution, detecting the lipid in the Golgi and at the plasma membrane, but also in the endoplasmic reticulum and mitochondria. In summary, alkyne cholesterol represents a versatile, sensitive, and easy-to-use tool for tracking cellular cholesterol metabolism and localization as it allows for manifold detection methods including mass spectrometry, thin-layer chromatography/fluorography, and fluorescence microscopy.

Alkyne lipids as substrates for click chemistry-based in vitro enzymatic assays.

Click chemistry is evolving as a powerful tool in biological applications because it allows the sensitive and specific detection of compounds with alkyne or azido groups. Here we describe the use of alkyne lipids as substrates for in vitro enzymatic assays of lipid modifying enzymes. The small alkyne moiety is introduced synthetically at the terminus of the hydrocarbon chain of various substrate lipids. After the assay, the label is click-reacted with the azide-bearing fluorogenic dye 3-azido-7-hydroxycoumarin, followed by the separation of the lipid mix by thin-layer chromatography and fluorescence detection, resulting in high sensitivity and wide-range linearity. Kinetic analyses using alkyne-labeled substrates for lysophosphatidic acid acyltransferases, lysophosphatidylcholine acyltransferases, and ceramide synthases resulted in Michaelis-Menten constants similar to those for radiolabeled or natural substrates. We tested additional alkyne substrates for several hydrolases and acyltransferases in lipid metabolism. In this pilot study we establish alkyne lipids as a new class of convenient substrates for in vitro enzymatic assays.

Peptide linkers for the immobilization of bioactive molecules on biphasic calcium phosphate via a modular immobilization system.

Herein we describe the use of peptide linkers to establish a nucleic acid-based immobilization system based on biphasic calcium phosphates (BCP), with which different molecules can be immobilized at the same time in defined ratios. It consists of single-stranded oligonucleotides, anchor strands (AS) which are immobilized to the surface and conjugates of complementary strands (CS) and bioactive molecules that bind to the AS via Watson-Crick base pairing. AS immobilization can be achieved on calcium phosphate ceramics using conjugates of AS and peptides that bind specifically to the ceramic. We successfully immobilized three different peptide sequences on BCP. Among them, we identified Stath (DpSpS EEK FLR RIG RFG, phosphoserine) as the most suitable one and further immobilized Stath-AS conjugates on BCP. This immobilized AS was able to hybridize with CS. Unspecific adsorption of oligonucleotides on the BCP surface was negligible. The stability of the system was proven by short term desorption experiments. The amounts of immobilized peptides, oligonucleotides and peptide-AS conjugates were determined by an enzymatic assay using biotin-streptavidin interactions, and were found to reach surface densities that are of therapeutic relevance (0.03 pmol cm(-2)).

Tracing fatty acid metabolism by click chemistry.

Fatty acids are abundant constituents of all biological systems, and their metabolism is important for normal function at all levels of an organism. Aberrations in fatty acid metabolism are associated with pathological states and have become a focus of current research, particularly due to the interest in metabolic overload diseases. Here we present a click-chemistry-based method that allows tracing of fatty acid metabolism in virtually any biological system. It combines high sensitivity with excellent linearity and fast sample turnover. Since it is free of radioactivity, it can be combined with any other modern analysis technology and can be used in high-throughput applications. Using the new method, we provide for the first time an analysis of cellular fatty metabolism with high time resolution and a comprehensive comparison of utilization of a broad spectrum of fatty acids in hepatoma and adipose cell lines.

Exogenous ether lipids predominantly target mitochondria.

Ether lipids are ubiquitous constituents of cellular membranes with no discrete cell biological function assigned yet. Using fluorescent polyene-ether lipids we analyzed their intracellular distribution in living cells by microscopy. Mitochondria and the endoplasmic reticulum accumulated high amounts of ether-phosphatidylcholine and ether-phosphatidylethanolamine. Both lipids were specifically labeled using the corresponding lyso-ether lipids, which we established as supreme precursors for lipid tagging. Polyfosine, a fluorescent analogue of the anti-neoplastic ether lipid edelfosine, accumulated to mitochondria and induced morphological changes and cellular apoptosis. These data indicate that edelfosine could exert its pro-apoptotic power by targeting and damaging mitochondria and thereby inducing cellular apoptosis. In general, this study implies an important role of mitochondria in ether lipid metabolism and intracellular ether lipid trafficking.