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Drug properties of antisense oligonucleotides (ASOs) differ significantly from those of traditional small-molecule therapeutics. In this review, we focus on ASO disposition, mainly as characterized by distribution and biotransformation, of nonconjugated and conjugated ASOs. We introduce ASO chemistry to allow the following in-depth discussion on bioanalytical methods and determination of distribution and elimination kinetics at low concentrations over extended periods of time. The resulting quantitative data on the parent oligonucleotide, and the identification and quantification of formed metabolites define the disposition. Proper quantitative understanding of disposition is pivotal for nonclinical to clinical predictions, supports communication with health agencies, and increases the probability of delivering optimal ASO therapy to patients.

In this study a 3D printed capsule designed to break from the physiological pressures in the antropyloric region was evaluated for its ability to deliver the synthetic octapeptide octreotide in beagle dogs when co-formulated with the permeation enhancer sodium caprate. The pressure sensitive capsules were compared to traditional enteric coated hard gelatin capsules and enteric coated tablets. Paracetamol, which is completely absorbed in dogs, was included in the formulations and used as an absorption marker to give information about the in vivo performance of the dosage forms. The pressure sensitive capsules released drug in 50% of the dogs. In the cases where drug was released, there was no difference in octreotide bioavailability or Cmax compared to the enteric coated dosage forms. When comparing all dosage forms, a correlation was seen between paracetamol Cmax and octreotide bioavailability, suggesting that a high drug release rate may be beneficial for peptide absorption when delivered together with sodium caprate.

We investigated the effects of mineral oil on statin pharmacokinetics and inflammatory markers in animal models. A new synthesis strategy produced regioisomers that facilitated the characterization of the main metabolite (M1) of atorvastatin, a lipophilic statin, in C57BL/6NCrl mice. The chemical structure of M1 in mice was confirmed as ortho-hydroxy β-oxidized atorvastatin. Atorvastatin and M1 pharmacokinetics and inflammatory markers were assessed in C57BL6/J mice given atorvastatin 5 mg/kg/day or 10 mg/kg/day, as a single dose or for 21 days, with or without 10 µL or 30 µL mineral oil. No consistent differences in plasma exposure of atorvastatin or M1 were observed in mice after single or repeat dosing of atorvastatin with or without mineral oil. However, mice administered atorvastatin 10 mg/kg with 30 µL mineral oil for 21 days had significantly increased plasma levels of serum amyloid A (mean 9.6 µg/mL vs 7.9 µg/mL without mineral oil; p0.01) and significantly increased proportions of C62L

3D cultures of primary human hepatocytes (PHH) are emerging as a more in vivo-like culture system than previously available hepatic models. This work describes the characterisation of drug metabolism in 3D PHH spheroids. Spheroids were formed from three different donors of PHH and the expression and activities of important cytochrome P450 enzymes (CYP1A2, 2B6, 2C9, 2D6, and 3A4) were maintained for up to 21 days after seeding. The activity of CYP2B6 and 3A4 decreased, while the activity of CYP2C9 and 2D6 increased over time (P 0.05). For six test compounds, that are metabolised by multiple enzymes, intrinsic clearance (CL

Prematurity is a severe pathophysiological condition, however, little is known about the gestational age-dependent development of the neonatal metabolome.Using an untargeted liquid chromatography-tandem mass spectrometry metabolomics protocol, we measured over 9000 metabolites in 298 neonatal residual heel prick dried blood spots retrieved from the Danish Neonatal Screening Biobank. By combining multiple state-of-the-art metabolome mining tools, we retrieved chemical structural information at a broad level for over 5000 (60%) metabolites and assessed their relation to gestational age.A total of 1459 (~16%) metabolites were significantly correlated with gestational age (false discovery rate-adjusted P 0.05), whereas 83 metabolites explained on average 48% of the variance in gestational age. Using a custom algorithm based on hypergeometric testing, we identified compound classes (617 metabolites) overrepresented with metabolites correlating with gestational age (P 0.05). Metabolites significantly related to gestational age included bile acids, carnitines, polyamines, amino acid-derived compounds, nucleotides, phosphatidylcholines and dipeptides, as well as treatment-related metabolites, such as antibiotics and caffeine.Our findings elucidate the gestational age-dependent development of the neonatal blood metabolome and suggest that the application of metabolomics tools has great potential to reveal novel biochemical underpinnings of disease and improve our understanding of complex pathophysiological mechanisms underlying prematurity-associated disorders.A large variation in the neonatal dried blood spot metabolome from residual heel pricks stored at the Danish Neonatal Screening Biobank can be explained by gestational age. While previous studies have assessed the relation of selected metabolic markers to gestational age, this study assesses metabolome-wide changes related to prematurity. Using a combination of recently developed metabolome mining tools, we assess the relation of over 9000 metabolic features to gestational age. The ability to assess metabolome-wide changes related to prematurity in neonates could pave the way to finding novel biochemical underpinnings of health complications related to preterm birth.

It remains a formidable challenge to characterize the diverse complexes of RNA binding proteins and their targets. While crosslink and immunoprecipitation (CLIP) methods are powerful techniques that identify RNA targets on a global scale, the resolution and consistency of these methods is a matter of debate. Here we present a comparative analysis of LIN28-pre-let-7 UV-induced crosslinking using a tandem mass spectrometry (MS/MS) and deep sequencing interrogation of in vitro crosslinked complexes. Interestingly, analyses by the two methods diverge in their identification of crosslinked nucleotide identity – whereas bioinformatics and sequencing analyses suggest guanine in mammalian cells, MS/MS identifies uridine. This work suggests the need for comprehensive analysis and validation of crosslinking methodologies.

Origins of life hypotheses often invoke a transitional phase of nonenzymatic template-directed RNA replication prior to the emergence of ribozyme-catalyzed copying of genetic information. Here, using NMR and ITC, we interrogate the binding affinity of guanosine 5′-monophosphate (GMP) for primer–template complexes when either another GMP, or a helper oligonucleotide, can bind downstream. Binding of GMP to a primer–template complex cannot be significantly enhanced by the possibility of downstream monomer binding, because the affinity of the downstream monomer is weaker than that of the first monomer. Strikingly, GMP binding affinity can be enhanced by ca. 2 orders of magnitude when a helper oligonucleotide is stably bound downstream of the monomer binding site. We compare these thermodynamic parameters to those previously reported for T7 RNA polymerase-mediated replication to help address questions of binding affinity in related nonenzymatic processes.

Early protocells are likely to have arisen from the self-assembly of RNA, peptide, and lipid molecules that were generated and concentrated within geologically favorable environments on the early Earth. The reactivity of these components in a prebiotic environment that supplied sources of chemical energy could have produced additional species with properties favorable to the emergence of protocells. The geochemically plausible activation of amino acids by carbonyl sulfide has been shown to generate short peptides via the formation of cyclic amino acid N-carboxyanhydrides (NCAs). Here, we show that the polymerization of valine-NCA in the presence of fatty acids yields acylated amino acids and peptides via a mixed anhydride intermediate. Notably, Nα-oleoylarginine, a product of the reaction between arginine and oleic acid in the presence of valine-NCA, partitions spontaneously into vesicle membranes and mediates the association of RNA with the vesicles. Our results suggest a potential mechanism by which activated amino acids could diversify the chemical functionality of fatty acid membranes and colocalize RNA with vesicles during the formation of early protocells.

Primary human hepatocytes (PHH), HepaRG™, HepG2, and two sources of induced pluripotent stem cell (iPSC) derived hepatocytes were characterized regarding gene expression and function of key hepatic proteins, important for the metabolic fate of drugs. The gene expression PCA analysis showed a distance between the two iPSC derived hepatocytes as well as the HepG2 and HepaRG™ cells to the three PHH donors and PHH pool, which were clustered more closely together. Correlation-based hierarchical analysis clustered HepG2 close to the stem cell derived hepatocytes both when the expression of 91 genes related to liver function or only cytochrome P450 (P450) genes were analyzed indicating the non-liver feature and a similar low P450 profile in these cell models. The specific P450 activities and the metabolic pattern of well-characterized drug substances in the cell models demonstrated that iPSC derived hepatocytes had modest levels of CYP3A and CYP2C9, while CYP1A2, 2B6, 2C8, 2C9, 2C19, and 2D6 were barely detectable. High expression of several extrahepatic P450s such as CYP1A1 and 1B1 detected in the stem cell derived hepatocytes may have significant effects on metabolite profiles. However, one of the iPSC derived hepatocytes demonstrated significant combined P450 and conjugating enzyme activity of certain drugs. HepaRG™ cells showed many metabolic properties similar to PHHs and will in many respects be a good model in studies of metabolic pathways and induction of drug metabolism whereas there is still ground to cover before iPSC derived hepatocytes will be seen as a substitute to PHH in drug metabolism studies.

Mass spectrometry (MS) is a powerful technique for characterizing noncanonical nucleobases and other chemical modifications in small RNAs, yielding rich chemical information that is complementary to high-throughput indirect sequencing. However, mass spectra are often prohibitively complex when fragment ions are analyzed following either solution phase hydrolysis or gas phase fragmentation. For all but the simplest cases, ions arising from multiple fragmentation events, alternative fragmentation pathways, and diverse salt adducts frequently obscure desired single-cut fragment ions. Here we show that it is possible to take advantage of predictable regularities in liquid chromatographic (LC) separation of optimized RNA digests to greatly simplify the interpretation of complex MS data. A two-dimensional analysis of extracted compound chromatograms permits straightforward and robust de novo sequencing, using a novel Monte Carlo algorithm that automatically generates bidirectional paired-end reads, pinpointing the position of modified nucleotides in a sequence. We demonstrate that these advances permit routine LC-MS sequencing of RNAs containing noncanonical nucleotides, and we furthermore examine the applicability of this approach to the study of oligonucleotides containing artificial modifications as well as those commonly observed in post-transcriptionally modified RNAs.

High affinity RNA-protein interactions are critical to cellular function, but directly identifying the determinants of binding within these complexes is often difficult. Here, we introduce a stable isotope mass labeling technique to assign specific interacting nucleotides in an oligonucleotide-protein complex by photo-cross-linking. The method relies on generating site-specific oxygen-18-labeled phosphodiester linkages in oligonucleotides, such that covalent peptide-oligonucleotide cross-link sites arising from ultraviolet irradiation can be assigned to specific sequence positions in both RNA and protein simultaneously by mass spectrometry. Using Lin28A and a let-7 pre-element RNA, we demonstrate that mass labeling permits unambiguous identification of the cross-linked sequence positions in the RNA-protein complex.

UV-driven photoredox processing of cyanocuprates can generate simple sugars necessary for prebiotic synthesis. We investigate the wavelength dependence of this process from 215 to 295 nm and generally observe faster rates at shorter wavelengths. The most efficient wavelengths are accessible to a range of potential prebiotic atmospheres, supporting the potential role of cyanocuprate photochemistry in prebiotic synthesis on the early Earth.

We prepared cholesteryl phosphocholine (CholPC) by chemical synthesis and studied its interactions with small (ceramide and cholesterol) and large headgroup (sphingomyelin (SM) and phosphatidylcholine) colipids in bilayer membranes. We established that CholPC could form bilayers (giant uni- and multilamellar vesicles, as well as extruded large unilamellar vesicles) with both ceramides and cholesterol (initial molar ratio 1:1). The extruded bilayers appeared to be fluid, although highly ordered, even when the ceramide had an N-linked palmitoyl acyl chain. In binary systems containing CholPC and either palmitoyl SM or 1,2-dipalmitoyl-sn-glycero-3-phospholine, CholPC markedly destabilized the gel phase of the respective large headgroup lipid. In 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers, CholPC was much less efficient than cholesterol in ordering the acyl chains. In complex bilayers containing POPC and cholesterol or palmitoyl ceramide, CholPC appeared to prefer interacting with the small headgroup lipids over POPC. When the degree of order in CholPC/PCer bilayers was compared to Chol/PSM bilayers, CholPC/PCer bilayers were more disordered (based on DPH anisotropy). This finding may result from different headgroup orientation and dynamics in CholPC and PSM. Our results overall can be understood if one takes into account the molecular shape of CholPC (large polar headgroup and modest size hydrophobic part) when interpreting molecular interactions between small and large headgroup colipids. The results are also consistent with the proposed umbrella model" for explaining cholesterol/colipid interactions.

We have prepared palmitoyl sphingomyelin (PSM) analogs in which either the 2-NH was methylated to NMe, the 3-OH was methylated to OMe, or both were methylated simultaneously. The aim of the study was to determine how such modifications in the membrane interfacial region of the molecules affected interlipid interactions in bilayer membranes. Measuring DPH anisotropy in vesicle membranes prepared from the SM analogs, we observed that methylation decreased gel-phase stability and increased fluid phase disorder, when compared to PSM. Methylation of the 2-NH had the largest effect on gel-phase instability (T(m), was lowered by similar to 7 degrees C). Atomistic molecular dynamics simulations showed that fluid phase bilayers with methylated SM analogs were more expanded but thinner compared to PSM bilayers. It was further revealed that 3-OH methylation dramatically attenuated hydrogen bonding also via the amide nitrogen, whereas 2-NH methylation did not similarly affect hydrogen bonding via the 3-OH. The interactions of sterols with the methylated SM analogs were markedly affected. 3-OH methylation almost completely eliminated the capacity of the SM analog to form sterol-enriched ordered domains, whereas the 2-NH methylated SM analog formed sterol-enriched domains but these were less thermostable (and thus less ordered) than the domains formed by PSM. Cholestatrienol affinity to bilayers containing methylated SM analogs was also markedly reduced as compared to its affinity for bilayers containing PSM. Molecular dynamics simulations revealed further that cholesterol's bilayer location was deeper in PSM bilayers as compared to the location in bilayers made from methylated SM analogs. This study shows that the interfacial properties of SMs are very important for interlipid interactions and the formation of laterally ordered domains in complex bilayers. (C) 2011 Elsevier B.V. All rights reserved.

Inositol phosphorylceramides (IPCs) are a class of anionic sphingolipids with a single inositol-phosphate head group coupled to ceramide. IPCs and more complex glycosylated IPCs have been identified in fungi, plants and protozoa but not in mammals. IPCs have also been identified in detergent resistant membranes in several organisms. Here we report on the membrane properties of the saturated N-palmitoyl-IPC (P-IPC) in one component bilayers as well as in complex bilayers together with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and cholesterol. The membrane properties of P-IPC were shown to be affected by calcium. According to anisotropy changes reported by DPH, the gel-to-liquid transition temperature (T(m)) of P-IPC was 48 degrees C. Addition of 5 mM CaCl(2) during vesicle preparation markedly increased the T(m) (65 degrees C). According to fluorescence quenching experiments in complex lipid mixtures, P-IPC formed sterol containing domains in an otherwise fluid environment. The P-IPC containing domains melted at a lower temperature and appeared to contain less sterol as compared to domains containing N-palmitoyl-sphingomyelin. Calcium further reduced the sterol content of the ordered domains and also increased the thermal stability of the domains. Calcium also induced vesicle aggregation of unilamellar vesicles containing P-IPC, as was observed by 4D confocal microscopy and dynamic light scattering. We believe that IPCs and the calcium induced effects could be important in numerous membrane associated cellular processes such as membrane fusion and in membrane raft linked processes.

We have characterized the molecular properties and membrane behavior of synthetically modified sphingomyelin analogues, modified on the oxygen connecting the phosphocholine group to the ceramide backbone. The oxygen was replaced with an S-atom (S-PSM), an NH-group (NH-PSM) or a CH(2)-group (CH(2)-PSM). Diphenylhexatriene and Laurdan anisotropy experiments showed that an S-linkage increased and NH- and CH(2)-linkages decreased the stability of PSM-analogue bilayer membranes as compared to PSM. When the polarity of the interface was probed using Laurdan, S-PSM appeared to have a lower polarity as compared to PSM whereas NH-PSM and CH(2)-PSM had higher polarities of their respective interfaces. Fluorescence quenching-studies with cholestatrienol showed that all compounds formed SM/cholesterol-rich domains. The S-PSM/cholesterol and PSM/cholesterol domains displayed a similar thermostability, whereas NH-PSM/cholesterol and CH(2)-PSM/cholesterol domains were less thermostable. DSC on vesicles containing the PSM-analogues showed a more complex melting behavior as compared to PSM, whereas equimolar mixtures of the PSM-analogues and PSM showed almost ideal mixing with PSM for NH- and S-PSM. Our data show that the properties of the bond linking the phosphocholine head group to the 1-hydroxyl on the ceramide molecule is important for the stability of SM/SM and SM/cholesterol interactions.

Elaidic acid is a trans-fatty acid found in many food products and implicated for having potentially health hazardous effects in humans. Elaidic acid is readily incorporated into membrane lipids in vivo and therefore affects processes regulating membrane physical properties. In this study the membrane properties of sphingomyelin and phosphatidylcholine containing elaidic acid (N-E-SM and PEPC) were determined in bilayer membranes with special emphasis on their interaction with cholesterol and participation in ordered domain formation. In agreement with previous studies the melting temperatures were found to be about 20 °C lower for the elaidoyl than for the corresponding saturated lipids. The trans-unsaturation increased the polarity at the membrane–water interface as reported by Laurdan fluorescence. Fluorescence quenching experiments using cholestatrienol as a probe showed that both N-E-SM and PEPC were incorporated in lateral membrane domains with sterol and saturated lipids. At low temperatures the elaidoyl lipids were even able to form sterol-rich domains without any saturated lipids present in the bilayer. We conclude from this study that the ability of N-E-SM and PEPC to form ordered domains together with cholesterol and saturated phospho- and sphingolipids in model membranes indicates that they might have an influence on raft formation in biological membranes.

Targeting of the Hsp function in tumor cells is currently being assessed as potential anticancer therapy. An improved understanding of the molecular signals that trigger or attenuate the stress protein response is essential for advances to be made in this field. The present study provides evidence that the membrane fluidizer benzyl alcohol (BA), a documented nondenaturant, acts as a chaperone inducer in B16(F10) melanoma cells. It is demonstrated that this effect relies basically on heat shock transcription factor 1 (HSF1) activation. Under the conditions tested, the BA-induced Hsp response involves the up-regulation of a subset of hsp genes. It is shown that the same level of membrane fluidization (estimated in the core membrane region) attained with the closely analogous phenethyl alcohol (PhA) does not generate a stress protein signal. BA, at a concentration that activates heat shock genes, exerts a profound effect on the melting of raft-like cholesterol-sphingomyelin domains in vitro , whereas PhA, at a concentration equipotent with BA in membrane fluidization, has no such effect. Furthermore, through the in vivo labeling of melanoma cells with a fluorescein labeled probe that inserts into the cholesterol-rich membrane domains [fluorescein ester of polyethylene glycol-derivatized cholesterol (fPEG-Chol)], we found that, similarly to heat stress per se , BA, but not PhA, initiates profound alterations in the plasma membrane microdomain structure. We suggest that, apart from membrane hyperfluidization in the deep hydrophobic region, a distinct reorganization of cholesterol-rich microdomains may also be required for the generation and transmission of stress signals to activate hsp genes.

Sphingomyelins (SMs) and sterols are important constituents of the plasma membrane and have also been identified as major lipid components in membrane rafts. Using SM analogs with decreasing headgroup methylation, we systemically analyzed the effect of headgroup size on membrane properties and interactions with cholesterol. An increase in headgroup size resulted in a decrease in the main phase transition. Atom-scale molecular-dynamics simulations were in agreement with the fluorescence anisotropy experiments, showing that molecular areas increased and acyl chain order decreased with increasing headgroup size. Furthermore, the transition temperatures were constantly higher for SM headgroup analogs compared to corresponding phosphatidylcholine headgroup analogs. The sterol affinity for phospholipid bilayers was assessed using a sterol-partitioning assay and an increased headgroup size increased sterol affinity for the bilayer, with a higher sterol affinity for SM analogs as compared to phosphatidylcholine analogs. Moreover, the size of the headgroup affected the formation and composition of cholesterol-containing ordered domains. Palmitoyl-SM (the largest headgroup) seemed to attract more cholesterol into ordered domains than the other SM analogs with smaller headgroups. The ordering and condensing effect of cholesterol on membrane lipids was also largest for palmitoyl-SM as compared to the smaller SM analogs. The results show that the size of the SM headgroup is crucially important for SM-SM and SM-sterol interactions. Our results further emphasize that interfacial electrostatic interactions are important for stabilizing cholesterol interactions with SMs.

Calcium entry is one of the main regulators of intracellular signaling. Here, we have described the importance of sphingosine, sphingosine kinase 1 (SK1), and sphingosine 1-phosphate (S1P) in regulating calcium entry in thyroid FRTL-5 cells. In cells incubated with the phosphatase inhibitor calyculin A, which evokes calcium entry without mobilizing sequestered intracellular calcium, sphingosine inhibited calcium entry in a concentration-dependent manner. Furthermore, inhibiting SK1 or the ATP-binding cassette ABCC1 multidrug transporter attenuated calcium entry. The addition of exogenous S1P restored calcium entry. Neither sphingosine nor inhibition of SK1 attenuated thapsigargin-evoked calcium entry. Blocking S1P receptor 2 or phospholipase C attenuated calcium entry, whereas blocking S1P receptor 3 did not. Overexpression of wild-type SK1, but not SK2, enhanced calyculin-evoked calcium entry compared with mock-transfected cells, whereas calcium entry was decreased in cells transfected with the dominant-negative G82D SK1 mutant. Exogenous S1P restored calcium entry in G82D cells. Our results suggest that the calcium entry pathway is blocked by sphingosine and that activation of SK1 and the production of S1P, through an autocrine mechanism, facilitate calcium entry through activation of S1P receptor 2. This is a novel mechanism by which the sphingosine-S1P rheostat regulates cellular calcium homeostasis.

Sphingomyelins (SMs) are important constituents of the plasma membrane and have, together with other sphingolipids and sterols, been identified as lipid components in membrane rafts. Interactions between sphingolipids and sterols have been shown to be important for the formation of ordered domains in model systems and also suggested to be a driving force in the formation of membrane rafts. The structure of sphingomyelins is important for interactions with sterols and in this study we have investigated the importance of the methyl groups in the head group of SMs upon membrane properties and interactions with cholesterol. Using specifically synthesized SM-analogues, having a stepwise decreasing number of methyl groups in the phosphocholine head group, we are able to systematically study how the size of the head group affects membrane properties and sterol interactions. The sphingomyelin analogues were composed of sphingosine having palmitic acid in the N-linked position. Using the anisotropy of 1,6-diphenylhexatriene we have determined the transition temperature between gel and liquid crystalline phases (Tm). Decreased methylation of the head group was shown to increase the Tm from 42°C (PSM), 53°C (dimethyl), 61°C (monomethyl) to 65 °C for ceramide-phoshoethanolamine (no methyl groups). Initial experiments using fluorescence quenching of cholestatrienol in complex model systems, shows that the ability to form sterol containing ordered domains also is affected by the degree of methylation in the head group. We are currently analyzing sterol interaction for the SM-analogues using DPH anisotropy and further prospects include determination sterol partitioning to vesicles containing each of the SM-analogues and differential scanning calorimetry studies.

We have characterized the membrane properties of inositol phosphorylceramide (IPC) in model membranes with emphasis on calcium induced effects. IPCs are a class of anionic sphingolipids with a single phosphoinositol head group coupled to ceramide. IPCs have been identified in eukaryotic species and also observed in detergent resistant membranes in Leishmania major. Here we report on the properties of N-palmitoyl-IPC (PIPC) in one component bilayers as well as in complex bilayers together with POPC and sterols. According to anisotropy changes reported by DPH and initial DSC experiments, the gel-to-liquid main transition temperature (Tm) of PIPC is about 50 °C. Addition of 5 mM Ca2+ during vesicle preparation markedly increased the Tm to about 65 °C. DPH anisotropy also showed that cholesterol (at 20 mol %) was able to decrease the order in the gel-phase and increase the order in the liquid disordered-phase. Fluorescence quenching studies using the fluorescent sterol analogues cholestatrienol or dehydroergosterol and the doxyl labeled lipid 7SLPC as quencher, showed that PIPC was able to form sterol-enriched ordered domains in the otherwise fluid bilayer environment. Dynamic light scattering studies indicate a clustering of PIPC containing SUVs (PSM:PIPC, 8:2) due to addition of Ca2+ to a final concentration of 5 mM. The increase in Tm and apparent clustering of PIPC containing vesicles shows that PIPC/Ca2+ interactions have substantial effects on the behavior of PIPC in model membranes. IPCs and the calcium induced effects could be important in membrane associated cellular processes such as membrane fusion and membrane raft linked processes.

Some ceramides have been shown to displace cholesterol from liquid ordered domains rich in sphingomyelin or saturated phosphatidylcholine in model membranes. The addition of ceramide will stabilize the formed ceramide/sphingomyelin domains and the subsequent partitioning of cholesterol to the disordered phase will also increase order and stabilization in this phase. The displacement of cholesterol may be due to more favorable interactions of ceramide with the ordered phase lipids, mainly sphingomyelin, compared to cholesterol. The ceramide could also interact less favorably with the disordered phase lipids compared to cholesterol, driving it into the ordered domains. It would also be of interest to better understand how cholesterol and ceramide can interact with each other in bilayer membranes. To study these questions, we have prepared a cholesterol analog with a phosphocholine head group on the 3-oxygen. We have studied the distribution and behavior of chol-PC and ceramides in bilayer systems on their own, and together with saturated and monounsaturated phospholipids. Chol-PC together with ceramide was able to form stable bilayer phases (extruded LUVs, electroformed GUVs). Phosphatidylcholine acyl chain order measurements showed that chol-PC was less efficient in inducing acyl chain order compared to cholesterol. Fluorescence quenching measurements in POPC/ceramide/sterol mixtures further showed a decrease in ordered domain stability of saturated ceramide in the presence of cholesterol or chol-PC. We have also examined the possibility to use chol-PC:ceramide mixtures to load ceramide into cultured cells. Proliferation experiments on HeLa and FRTL-5 cells indicates that chol-PC:ceramide bilayer mixtures are more efficient in inducing apoptosis than DMSO:ceramide mixtures. Our results suggest that chol-PC and ceramide interact with each other under specific conditions, and that the binary bilayers could constitute an interesting formulation for providing cells and tissues with solvent-free ceramides.

BackgroundBirth by caesarean section is linked to an increased risk of developing asthma, but the underlying mechanisms are unclear.ObjectiveTo elucidate the link between birth by caesarean section and asthma using newborn metabolomic profiles and integrating early-life gut microbiome data and cord blood immunology.MethodsWe investigated the influence of caesarean section on liquid chromatography mass spectrometry metabolomic profiles of dried blood spots from newborns of the two independent Copenhagen Prospective Studies on Asthma in Childhood cohorts, i.e. COPSAC2010 (n=677) and COPSAC2000 (n=387). We assessed the associations between the caesarean section metabolic profile, gut microbiome data and frequency of cord blood regulatory T-cells (Tregs) at 1 week of age.ResultsIn COPSAC2010, a partial least square discriminant analysis model showed that children born by caesarean section versus natural delivery had different metabolic profiles (area under the curve (AUC)=0.77, p=2.2×10−16), which was replicated in COPSAC2000 (AUC=0.66, p=1.2×10−5). The metabolic profile of caesarean section was significantly associated with an increased risk of asthma at school age in both COPSAC2010 (p=0.03) and COPSAC2000 (p=0.005). Caesarean section was associated with lower abundance of tryptophan, bile acid and phenylalanine metabolites, indicative of a perturbed gut microbiota. Furthermore, gut bacteria dominating after natural delivery, i.e. Bifidobacterium and Bacteroides were correlated with caesarean section-discriminative microbial metabolites, suggesting maternal microbial transmission during birth regulating the newborn's metabolism. Finally, the caesarean section metabolic profile was associated with frequency of cord blood Tregs.ConclusionsThese findings propose that caesarean section programmes the risk of childhood asthma through perturbed immune responses and gut microbial colonisation patterns reflected in the blood metabolome at birth.

The polymorphic phase behavior of different phospholipids depends strongly on fine details of their head group and backbone structures. Studies of phospholipid head group modification offers an attractive strategy in elucidating how structural elements of the head group effect membrane properties. We are investigating phosphatidyl alcohols with saturated fatty acyl chains and their interactions with phospholipids and sterols. Molecules of interest are dipalmitoyl glycerophospholipids with methanol, ethanol, propanol and butanol as different headgroups. These molecules can be formed from e.g., corresponding phosphatidylcholines due to the transphosphatidylation activity of phospholipase D. Phosphatidyl ethanol is of physiological relevance since it is increasingly formed in cells of chronic alcoholics. Thermodynamic properties such as phase transition temperatures, heat capacity of transitions and transition cooperativity have been studied with high sensitivity differential scanning calorimetry (DSC). The miscibility and effects of cholesterol on mixtures of these negatively charged phospholipids with high Tm and low Tm phospholipids have been determined using DSC, and with fluorescence spectroscopy using domain-selective lipid probes. The relative acyl-chain order in the bilayer membranes has been assessed using steady-state DPH anisotropy. We will report on the phase behavior of these molecules in simple and more complex bilayer systems. The formation, by the dipalmitoyl phosphatidyl alcohols, of sterol-enriched or ordered domains in fluid bilayer membranes will also be determined. Comparison of the properties of these phosphatidyl alcohols with molecules having closely related structures will help in understanding the relationships between the chemical structures, physical properties and functional properties of the phospholipid molecules in model as well as biological membranes.