Neuroligin dependence of social behaviour in Caenorhabditis elegans provides a model to investigate an autism-associated gene.

Autism spectrum disorder (ASD) is characterized by a triad of behavioural impairments including social behaviour. Neuroligin, a trans-synaptic adhesion molecule, has emerged as a penetrant genetic determinant of behavioural traits that signature the neuroatypical behaviours of autism. However, the function of neuroligin in social circuitry and the impact of genetic variation to this gene is not fully understood. Indeed, in animal studies designed to model autism, there remains controversy regarding the role of neuroligin dysfunction in the expression of disrupted social behaviours. The model organism, Caenorhabditis elegans, offers an informative experimental platform to investigate the impact of genetic variants on social behaviour. In a number of paradigms, it has been shown that inter-organismal communication by chemical cues regulates C. elegans social behaviour. We utilize this social behaviour to investigate the effect of autism-associated genetic variants within the social domain of the research domain criteria. We have identified neuroligin as an important regulator of social behaviour and segregate the importance of this gene to the recognition and/or processing of social cues. We also use CRISPR/Cas9 to edit an R-C mutation that mimics a highly penetrant human mutation associated with autism. C. elegans carrying this mutation phenocopy the behavioural dysfunction of a C. elegans neuroligin null mutant, thus confirming its significance in the regulation of animal social biology. This highlights that quantitative behaviour and precision genetic intervention can be used to manipulate discrete social circuits of the worm to provide further insight into complex social behaviour.

Neuroligin tuning of pharyngeal pumping reveals extrapharyngeal modulation of feeding in Caenorhabditis elegans.

The integration of distinct sensory modalities is essential for behavioural decision making. In Caenorhabditiselegans, this process is coordinated by neural circuits that integrate sensory cues from the environment to generate an appropriate behaviour at the appropriate output muscles. Food is a multimodal cue that impacts the microcircuits to modulate feeding and foraging drivers at the level of the pharyngeal and body wall muscle, respectively. When food triggers an upregulation in pharyngeal pumping, it allows the effective ingestion of food. Here, we show that a Celegans mutant in the single gene orthologous to human neuroligins, nlg-1, is defective in food-induced pumping. This was not due to an inability to sense food, as nlg-1 mutants were not defective in chemotaxis towards bacteria. In addition, we found that neuroligin is widely expressed in the nervous system, including AIY, ADE, ALA, URX and HSN neurons. Interestingly, despite the deficit in pharyngeal pumping, neuroligin was not expressed within the pharyngeal neuromuscular network, which suggests an extrapharyngeal regulation of this circuit. We resolved electrophysiologically the neuroligin contribution to the pharyngeal circuit by mimicking food-dependent pumping and found that the nlg-1 phenotype is similar to mutants impaired in GABAergic and/or glutamatergic signalling. We suggest that neuroligin organizes extrapharyngeal circuits that regulate the pharynx. These observations based on the molecular and cellular determinants of feeding are consistent with the emerging role of neuroligin in discretely impacting functional circuits underpinning complex behaviours.

Thermal acclimation and subspecies-specific effects on heart and brain mitochondrial performance in a eurythermal teleost (Fundulus heteroclitus).

Mitochondrial performance may play a role in setting whole-animal thermal tolerance limits and their plasticity, but the relative roles of adjustments in mitochondrial performance across different highly aerobic tissues remain poorly understood. We compared heart and brain mitochondrial responses to acute thermal challenges and to thermal acclimation using high-resolution respirometry in two locally adapted subspecies of Atlantic killifish (Fundulus heteroclitus). We predicted that 5°C acclimation would result in compensatory increases in mitochondrial performance, while 33°C acclimation would cause suppression of mitochondrial function to minimize the effects of high temperature on mitochondrial metabolism. In contrast, acclimation to both 33 and 5°C decreased mitochondrial performance compared with fish acclimated to 15°C. These adjustments could represent an energetic cost-saving mechanism at temperature extremes. Acclimation responses were similar in both heart and brain; however, this effect was smaller in the heart, which might indicate its importance in maintaining whole-animal thermal performance. Alternatively, larger acclimation effects in the brain might indicate greater thermal sensitivity compared with the heart. We detected only modest differences between subspecies that were dependent on the tissue assayed. These data demonstrate extensive plasticity in mitochondrial performance following thermal acclimation in killifish, and indicate that the extent of these responses differs between tissues, highlighting the importance and complexity of mitochondrial regulation in thermal acclimation in eurytherms.

Metabotropic Glutamate Receptors: MODULATORS OF CONTEXT-DEPENDENT FEEDING BEHAVIOUR IN C. ELEGANS.

Glutamatergic neurotransmission is evolutionarily conserved across animal phyla. A major class of glutamate receptors consists of the metabotropic glutamate receptors (mGluRs). In C. elegans, three mGluR genes, mgl-1, mgl-2, and mgl-3, are organized into three subgroups, similar to their mammalian counterparts. Cellular reporters identified expression of the mgls in the nervous system of C. elegans and overlapping expression in the pharyngeal microcircuit that controls pharyngeal muscle activity and feeding behavior. The overlapping expression of mgls within this circuit allowed the investigation of receptor signaling per se and in the context of receptor interactions within a neural network that regulates feeding. We utilized the pharmacological manipulation of neuronally regulated pumping of the pharyngeal muscle in the wild-type and mutants to investigate MGL function. This defined a net mgl-1-dependent inhibition of pharyngeal pumping that is modulated by mgl-3 excitation. Optogenetic activation of the pharyngeal glutamatergic inputs combined with electrophysiological recordings from the isolated pharyngeal preparations provided further evidence for a presynaptic mgl-1-dependent regulation of pharyngeal activity. Analysis of mgl-1, mgl-2, and mgl-3 mutant feeding behavior in the intact organism after acute food removal identified a significant role for mgl-1 in the regulation of an adaptive feeding response. Our data describe the molecular and cellular organization of mgl-1, mgl-2, and mgl-3. Pharmacological analysis identified that, in these paradigms, mgl-1 and mgl-3, but not mgl-2, can modulate the pharyngeal microcircuit. Behavioral analysis identified mgl-1 as a significant determinant of the glutamate-dependent modulation of feeding, further highlighting the significance of mGluRs in complex C. elegans behavior.

Identification of double-bond positions in isomeric alkenones from a lacustrine haptophyte.

RATIONALE: Measurements of alkenone unsaturation ratios are widely used for paleotemperature reconstructions in ocean and lake environments. Previously, we reported the discovery of a series of tri-unsaturated alkenone positional isomers (Δ(14, 21, 28) ) from oligosaline and freshwater lakes in Greenland and Alaska. In this work we provide a detailed analysis of the structures and isotopic compositions (δ(13) C and δ(2) H) of the alkenones produced by the "Greenland haptophyte". METHODS: Alkenones were extracted from sediments of Lake BrayaSø, Greenland. Alkenone double-bond positions were determined by GC/EI-MS analysis of alkenone dimethyl disulfide and cyclobutylimine derivatives. Alkenones were purified by semi-preparative HPLC using a silver(I) thiolate stationary phase. Carbon and hydrogen isotope analysis was performed by gas chromatography/isotope ratio mass spectrometry (GC/IRMS). RESULTS: A series of novel tri-unsaturated alkenone positional isomers were identified among four alkenone homologues (i.e. C37 Me , C38 Me , C38 Et , and C39 Et ) with double-bond positions at Δ(14, 21, 28) . The hydrogen isotope compositions (δ(2) H, VSMOW) of the tri-unsaturated positional isomers from C37 Me and C38 Et were slightly depleted (~ -11 ‰) relative to the common tri-unsaturated alkenone. The carbon isotope composition (δ(13) C, VPDB) of the tri-unsaturated positional isomers from the C37 Me , C38 Me , C38 Et , and C39 Et alkenones were significantly enriched (~ +4 ‰) relative to the common alkenones (di-, tri-, and tetra-unsaturated). CONCLUSIONS: The novel tri-unsaturated alkenone positional isomers produced by the Greenland haptophyte possess Δ(14, 21, 28) double-bond positions, instead of the common Δ(7, 14, 21) double-bond positions. The hydrogen isotope values suggest the novel tri-unsaturated positional isomers could be biosynthetic precursors to the tetra-unsaturated alkenones (Δ(7, 14, 21, 28) ). However, the significantly higher carbon isotope values of the tri-unsaturated positional isomers relative to the common di-, tri- and tetra-unsaturated alkenones suggest these positional isomers may have different/additional biosynthetic precursors.

Discovery of alkenones with variable methylene-interrupted double bonds: implications for the biosynthetic pathway.

Alkenones (C37 -C40 ) are highly specific biomarkers produced by certain haptophyte algae in ocean and lacustrine environments and have been widely used for paleoclimate studies. Unusual shorter-chain alkenones (SCA; e.g., C35 and C36 ) have been found in environmental and culture samples, but the origin and structure of these compounds are much less understood. The marine alkenone producer, Emiliania huxleyi CCMP2758 strain, was reported with abundant C35:2 Me (∆12, 19 ) alkenones when cultured at 15°C (Prahl et al. 2006). Here we show, when this strain is cultured at 4°C-10°C, that CCMP2758 produces abundant C35:3 Me, C36:3 Me, and small amounts of C36:3 Et alkenones with unusual double-bond positions of ∆7, 12, 19 . We determine the double-bond positions of the C35:3 Me and C36:3 Me alkenones by GC-MS analysis of the dimethyl disulfide and cyclobutylamine derivatives, and we provide the first temperature calibrations based on the unsaturation ratios of the C35 and C36 alkenones. Previous studies have found C35:2 Me (∆14, 19 ) and C36:2 Et (∆14, 19 ) alkenones with three-methylene interruption in the Black Sea sediments, but this is the first reported instance of alkenones with a mixed three- and five-methylene interruption configuration in the double-bond positions. The discovery of these alkenones allows us to propose a novel biosynthetic scheme, termed the SCA biosynthesis pathway, that simultaneously rationalizes the formation of both the C35:3 Me (∆7, 12, 19 ) alkenone in our culture and the ∆14, 19 Black Sea type alkenones without invoking new desaturases for the unusual double-bond positions.

Non-enzymatic glycation of α-crystallin as an in vitro model for aging, diabetes and degenerative diseases.

Alpha crystallin, a small heat-shock protein, has been studied extensively for its chaperone function. Alpha crystallin subunits are expressed in stress conditions and have been found to prevent apoptosis by inhibiting the activation of caspase pathway. Non-enzymatic glycation of protein leads to the formation of advanced glycation end-products (AGEs). These AGEs bind to receptors and lead to blocking the signaling pathways or cause protein precipitation as observed in aggregation-related diseases. Methylglyoxal (MGO) is one of the major glycating agents expressed in pathological conditions due to defective glycolysis pathway. MGO reacts rapidly with proteins, forms AGEs and finally leads to aggregation. The goal of this study was to understand the non-enzymatic glycation-induced structural damage in alpha crystallin using biophysical and spectroscopic characterization. This will help to develop better disease models for understanding the biochemical pathways and also in drug discovery.

Measuring the viscosity of whole bovine lens using a fiber optic oxygen sensing system.

PURPOSE: To obtain a better understanding of oxygen and nutrient transport within the lens, the viscosity of whole lenses was investigated using a fiber optic oxygen sensor (optode). The diffusion coefficient of oxygen was calculated using the Stokes-Einstein equation at the slip boundary condition. METHODS: The optode was used to measure the oxygen decay signal in samples consisting of different glycerol/water solutions with known viscosities. The oxygen decay signal was fitted to a double exponential decay rate equation, and the lifetimes (tau) were calculated. It was determined that the tau-viscosity relationship is linear, which served as the standard curve. The same procedure was applied to fresh bovine lenses, and the unknown viscosity of the bovine lens was calculated from the tau-viscosity relationship. RESULTS: The average viscosity in a whole bovine lens was determined to be 5.74 ± 0.88 cP by our method. Using the Stokes-Einstein equation at the slip boundary condition, the diffusion coefficient for oxygen was calculated to be 8.2 × 10(-6) cm(2)/s. CONCLUSIONS: These data indicate a higher resistance to flow for oxygen and nutrients in the lens than what is currently assumed in the literature. Overall, this study allows a better understanding of oxygen transport within the lens.

Distinct molecular targets including SLO-1 and gap junctions are engaged across a continuum of ethanol concentrations in Caenorhabditis elegans.

Ethanol (alcohol) interacts with diverse molecular effectors across a range of concentrations in the brain, eliciting intoxication through to sedation. Invertebrate models including the nematode worm Caenorhabditis elegans have been deployed for molecular genetic studies to inform on key components of these alcohol signaling pathways. C. elegans studies have typically employed external dosing with high (>250 mM) ethanol concentrations: A careful analysis of responses to low concentrations is lacking. Using the C. elegans pharyngeal system as a paradigm, we report a previously uncharacterized continuum of cellular and behavioral responses to ethanol from low (10 mM) to high (300 mM) concentrations. The complexity of these responses indicates that the pleiotropic action of ethanol observed in mammalian brain is conserved in this invertebrate model. We investigated two candidate ethanol effectors, the calcium-activated K(+) channel SLO-1 and gap junctions, and show that they contribute to, but are not sole determinants of, the low- and high-concentration effects, respectively. Notably, this study shows cellular and whole organismal behavioral responses to ethanol in C. elegans that directly equate to intoxicating through to supralethal blood alcohol concentrations in humans and provides an important benchmark for interpretation of paradigms that seek to inform on human alcohol use disorders.

Fluensulfone is a nematicide with a mode of action distinct from anticholinesterases and macrocyclic lactones.

Plant parasitic nematodes infest crops and present a threat to food security worldwide. Currently available chemical controls e.g. methyl bromide, organophosphates and carbamates have an unacceptable level of toxicity to non-target organisms and are being withdrawn from use. Fluensulfone is a new nematicide of the fluoroalkenyl thioether group that has significantly reduced environmental impact with low toxicity to non-target insects and mammals. Here, we show that the model genetic organism Caenorhabditis elegans is susceptible to the irreversible nematicidal effects of fluensulfone. Whilst the dose required is higher than that which has nematicidal activity against Meloidogyne spp. the profile of effects on motility, egg-hatching and survival is similar to that reported for plant parasitic nematodes. C. elegans thus provides a tractable experimental paradigm to analyse the effects of fluensulfone on nematode behaviour. We find that fluensulfone has pleiotropic actions and inhibits development, egg-laying, egg-hatching, feeding and locomotion. In the case of feeding and locomotion, an early excitation precedes the gross inhibition. The profile of these effects is notably distinct from other classes of anthelmintic and nematicide: the inhibition of motility caused by fluensulfone is not accompanied by the hypercontraction which is characteristic of organophosphates and carbamates and C. elegans mutants that are resistant to the carbamate aldicarb and the macrocyclic lactone ivermectin retain susceptibility to fluensulfone. These data indicate fluensulfone's mode of action is distinct from currently available nematicides and it therefore presents a promising new chemical entity for crop protection.

Identification of a role for CLASP2 in insulin action.

Insulin stimulates the mobilization of glucose transporter 4 (GLUT4) storage vesicles to the plasma membrane, resulting in an influx of glucose into target tissues such as muscle and fat. We present evidence that CLIP-associating protein 2 (CLASP2), a protein previously unassociated with insulin action, is responsive to insulin stimulation. Using mass spectrometry-based protein identification combined with phosphoantibody immunoprecipitation in L6 myotubes, we detected a 4.8-fold increase of CLASP2 in the anti-phosphoserine immunoprecipitates upon insulin stimulation. Western blotting of CLASP2 immunoprecipitates with the phosphoantibody confirmed the finding that CLASP2 undergoes insulin-stimulated phosphorylation, and a number of novel phosphorylation sites were identified. Confocal imaging of L6 myotubes revealed that CLASP2 colocalizes with GLUT4 at the plasma membrane within areas of insulin-mediated cortical actin remodeling. CLASP2 is responsible for directing the distal end of microtubules to the cell cortex, and it has been shown that GLUT4 travels along microtubule tracks. In support of the concept that CLASP2 plays a role in the trafficking of GLUT4 at the cell periphery, CLASP2 knockdown by siRNA in L6 myotubes interfered with insulin-stimulated GLUT4 localization to the plasma membrane. Furthermore, siRNA mediated knockdown of CLASP2 in 3T3-L1 adipocytes inhibited insulin-stimulated glucose transport. We therefore propose a new model for CLASP2 in insulin action, where CLASP2 directs the delivery of GLUT4 to cell cortex landing zones important for insulin action.

The unique liquid chromatographic properties of Group 11 transition metals for the separation of unsaturated organic compounds.

Silver(I) and copper(I) are known to form reversible complexes with π bonds, which have been exploited in LC for separating unsaturated organic compounds. Prominent examples include the use of AgNO3-impregnated silica gel in LC, and the use of copper(I) salts for selective extraction of alkenes from hydrocarbon mixtures. The Dewar-Chatt-Duncanson model is often invoked to explain the interaction between Ag(I) and Cu(I) and π bonds. However, it is unclear if such a reversible interaction is directly related to their d(10) outer electronic configurations. Particularly, Au(I) has not been reported to separate olefins with different numbers of double bonds in LC. Also, there has not been a systematic comparison of the liquid chromatographic properties of other d(10) transition metal salts (e.g., Zn(II), Cd(II)), making it difficult to fully understand the observed reversible interactions of Ag(I) and Cu(I) with π bonds. We demonstrate for the first time that silica gel impregnated with all three Group 11 transition metals with 1+ oxidation state strongly and similarly retain olefin compounds in LC, while transition metals from Groups 10 and 12 do not. We also tested a range of functionalized silica gels to improve the stability of Cu(I) and Au(I) ions on the surface of the silica.

Changes in neuropsychological and behavioral functioning in children with and without obstructive sleep apnea following Tonsillectomy.

The most common treatment for sleep disordered breathing (SDB) is adenotonsillectomy (AT). Following AT, SDB resolves in most cases, and gains in cognitive and behavior scores are consistently reported, although persistent neuropsychological deficits or further declines also have been noted. This study presents results of the comprehensive 1-year follow-up neuropsychological examinations for children in the Washtenaw County Adenotonsillectomy Cohort I (95% return rate). After adjusting for normal developmental and practice-effect related changes in control children, significant improvements 1 year following AT were noted in polysomnography and sleepiness, as well as parental reports of behavior, although cognitive outcomes were mixed. Children undergoing AT with and without polysomnography-confirmed obstructive sleep apnea improved across a range of academic achievement measures, a measure of delayed visual recall, short-term attention/working memory, and executive functioning, along with parental ratings of behavior. On the other hand, measures of verbal abstraction ability, arithmetic calculations, visual and verbal learning, verbal delayed recall, sustained attention, and another measure of visual delayed recall demonstrated declines in ability, while other measures did not improve over time. These findings call into question the expectation that AT resolves most or all behavioral and cognitive difficulties in children with clinical, office-based diagnoses of SDB.

Confounds of using the unc-58 selection marker highlights the importance of genotyping co-CRISPR genes.

Multiple advances have been made to increase the efficiency of CRISPR/Cas9 editing using the model genetic organism Caenorhabditis elegans (C. elegans). Here we report on the use of co-CRISPR 'marker' genes: worms in which co-CRISPR events have occurred have overt, visible phenotypes which facilitates the selection of worms that harbour CRISPR events in the target gene. Mutation in the co-CRISPR gene is then removed by outcrossing to wild type but this can be challenging if the CRISPR and co-CRISPR gene are hard to segregate. However, segregating away the co-CRISPR modified gene can be less challenging if the worms selected appear wild type and are selected from a jackpot brood. These are broods in which a high proportion of the progeny of a single injected worm display the co-CRISPR phenotype suggesting high CRISPR efficiency. This can deliver worms that harbour the desired mutation in the target gene locus without the co-CRISPR mutation. We have successfully generated a discrete mutation in the C. elegans nlg-1 gene using this method. However, in the process of sequencing to authenticate editing in the nlg-1 gene we discovered genomic rearrangements that arise at the co-CRISPR gene unc-58 that by visual observation were phenotypically silent but nonetheless resulted in a significant reduction in motility scored by thrashing behaviour. This highlights that careful consideration of the hidden consequences of co-CRISPR mediated genetic changes should be taken before downstream analysis of gene function. Given this, we suggest sequencing of co-CRISPR genes following CRISPR procedures that utilise phenotypic selection as part of the pipeline.

Silver (I)-dimercaptotriazine functionalized silica: A highly selective liquid chromatography stationary phase targeting unsaturated molecules.

Silver(I)-mercaptopropyl (Ag-MP) functionalized silica gel has demonstrated its effectiveness in separating various unsaturated organic compounds including unsaturated fatty acid ethyl esters (FAEEs), triglycerols (TAGs) and long-chain alkyl ketones (alkenones). While Ag-MP stationary phase displays many advantages over the conventional silver ion-impregnated silica gel (e.g., stability, high recovery, etc.), potential drawbacks of Ag-MP include relatively low retentions for unsaturated molecules, which could limit chromatographic resolutions under certain circumstances. In this study, we evaluate a new silver-thiolate stationary phase: silver(I)-dimercaptotriazine (Ag-DMT) functionalized silica gel targeting the separation of unsaturated compounds. We show Ag-DMT affords substantially higher retention factors, peak resolutions and capacities for TAGs and FAEEs than Ag-MP does. Ag-DMT also yields higher purity eicosapentaenoic acid (EPA) from fish oil FAEE mixtures than Ag-MP. In addition, Ag-DMT resolves double bond positional and cis/trans-isomers of C18:1 fatty acid methyl esters (FAMEs) as well as unsaturated methyl/ethyl alkenones with different number of double bonds. Based on van't Hoff plots, enthalpy changes during the adsorption of unsaturated FAEEs onto Ag-DMT are ~2 times higher than those on Ag-MP. Such difference may be attributed to the stronger electron-withdrawing effect of the thiol group on DMT, which results in more positively charged silver ions hence greater interactions with unsaturated molecules. The stronger interaction between double bonds and Ag-DMT is further corroborated by density-functional theory (DFT) calculations. Ag-DMT shows its high stability for repeated uses in the separation of TAGs over 319 runs, with peak resolutions decreasing by < 3%. Collectively, our data demonstrate the exceptionally high efficiency of Ag-DMT column for separating unsaturated molecules.

Investigating autism associated genes in C. elegans reveals candidates with a role in social behaviour.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterised by a triad of behavioural impairments and includes disruption in social behaviour. ASD has a clear genetic underpinning and hundreds of genes are implicated in its aetiology. However, how single penetrant genes disrupt activity of neural circuits which lead to affected behaviours is only beginning to be understood and less is known about how low penetrant genes interact to disrupt emergent behaviours. Investigations are well served by experimental approaches that allow tractable investigation of the underpinning genetic basis of circuits that control behaviours that operate in the biological domains that are neuro-atypical in autism. The model organism C. elegans provides an experimental platform to investigate the effect of genetic mutations on behavioural outputs including those that impact social biology. Here we use progeny-derived social cues that modulate C. elegans food leaving to assay genetic determinants of social behaviour. We used the SAFRI Gene database to identify C. elegans orthologues of human ASD associated genes. We identified a number of mutants that displayed selective deficits in response to progeny. The genetic determinants of this complex social behaviour highlight the important contribution of synaptopathy and implicates genes within cell signalling, epigenetics and phospholipid metabolism functional domains. The approach overlaps with a growing number of studies that investigate potential molecular determinants of autism in C. elegans. However, our use of a complex, sensory integrative, emergent behaviour provides routes to enrich new or underexplored biology with the identification of novel candidate genes with a definable role in social behaviour.

Physical properties of the lipid bilayer membrane made of cortical and nuclear bovine lens lipids: EPR spin-labeling studies.

The physical properties of membranes derived from the total lipids extracted from the lens cortex and nucleus of a 2-year-old cow were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in membranes made from cortical lipids. Properties of these membranes are very similar to those reported by us for membranes made of the total lipid extract of 6-month-old calf lenses (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta 1768 (2007) 1454-1465). However, in membranes made from nuclear lipids, two domains were detected by the EPR discrimination by oxygen transport method using the cholesterol analogue spin label and were assigned to the bulk phospholipid-cholesterol domain (PCD) and the immiscible cholesterol crystalline domain (CCD), respectively. Profiles of the order parameter, hydrophobicity, and the oxygen transport parameter are practically identical in the bulk PCD when measured for either the cortical or nuclear lipid membranes. In both membranes, lipids in the bulk PCD are strongly immobilized at all depths. Hydrophobicity and oxygen transport parameter profiles have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. The permeability coefficient for oxygen, estimated at 35 degrees C, across the bulk PCD in both membranes is slightly lower than across the water layer of the same thickness. However, the evaluated upper limit of the permeability coefficient for oxygen across the CCD (34.4 cm/s) is significantly lower than across the water layer of the same thickness (85.9 cm/s), indicating that the CCD can significantly reduce oxygen transport in the lens nucleus.

Characterization of lipid domains in reconstituted porcine lens membranes using EPR spin-labeling approaches.

The physical properties of membranes derived from the total lipid extract of porcine lenses before and after the addition of cholesterol were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves indicate that the spin labels detect a single homogenous environment in membranes before the addition of cholesterol. After the addition of cholesterol (when cholesterol-to-phospholipid mole to mole ratio of 1.55-1.80 was achieved), two domains were detected by the discrimination by oxygen transport method using a cholesterol analogue spin label. The domains were assigned to a bulk phospholipid-cholesterol bilayer made of the total lipid mixture and to a cholesterol crystalline domain. Because the phospholipid analogue spin labels cannot partition into the pure cholesterol crystalline domain, they monitor properties of the phospholipid-cholesterol domain outside the pure cholesterol crystalline domain. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are identical within experimental error in this domain when measured in the absence and presence of a cholesterol crystalline domain. This indicates that both domains, the phospholipid-cholesterol bilayer and the pure cholesterol crystalline domain, can be treated as independent, weakly interacting membrane regions. The upper limit of the oxygen permeability coefficient across the cholesterol crystalline domain at 35 degrees C had a calculated value of 42.5 cm/s, indicating that the cholesterol crystalline domain can significantly reduce oxygen transport to the lens center. This work was undertaken to better elucidate the major factors that determine membrane resistance to oxygen transport across the lens lipid membrane, with special attention paid to the cholesterol crystalline domain.

Profiling emergency department presentations of 14-15-year-olds in modern Ireland.

BACKGROUND: Mid-adolescence, that twilight era when the human child transitions to adulthood, is an often overlooked developmental age yet harbours a subpopulation of patients with their own myriad of medical problems somewhat unique to their age group. AIMS: Our study is aimed at reviewing the typical presentations to a paediatric emergency department of modern Irish teenagers in mid-adolescence, the profile of which has changed significantly over the past 10 years. METHODS: Hospital electronic databases were used to conduct a retrospective review of the paediatric emergency department presentations of patients aged 14-15 years during the year of 2017. We collated data on the presenting complaint, background history, admission rate and medical specialities involved in each patient's care while in our Emergency Department. RESULTS: A total of 1485 presentations were made, with 1363 being eligible for inclusion in this study. The results highlight the varied and challenging presentations (Table 1) and the high number of specialities required within emergency medicine to care for this unique population (Table 2). CONCLUSION: The results highlight the most common presentations of this subgroup of patients, with trauma, in keeping with recent international data, being the most common presentation. The noted high frequency in the number of mental health/intoxication/self-harm presentations among the Irish teenagers in our region is consistent with trends reported in world literature and serves to emphasise one of the main challenges facing those working in paediatrics in Ireland over the next 10 years.