Diffusion MRI Microstructural Abnormalities at Term-Equivalent Age Are Associated with Neurodevelopmental Outcomes at 3 Years of Age in Very Preterm Infants.

BACKGROUND AND PURPOSE: Microstructural white matter abnormalities on DTI using Tract-Based Spatial Statistics at term-equivalent age are associated with cognitive and motor outcomes at 2 years of age or younger. However, neurodevelopmental tests administered at such early time points are insufficiently predictive of mild-moderate motor and cognitive impairment at school age. Our objective was to evaluate the microstructural antecedents of cognitive and motor outcomes at 3 years' corrected age in a cohort of very preterm infants. MATERIALS AND METHODS: We prospectively recruited 101 very preterm infants (<32 weeks' gestational age) and performed DTI at term-equivalent age. The Differential Ability Scales, 2nd ed, Verbal and Nonverbal subtests, and the Bayley Scales of Infant and Toddler Development, 3rd ed, Motor subtest, were administered at 3 years of age. We correlated DTI metrics from Tract-Based Spatial Statistics with the Bayley Scales of Infant and Toddler Development, 3rd ed, and the Differential Ability Scales, 2nd ed, scores with correction for multiple comparisons. RESULTS: Of the 101 subjects, 84 had high-quality DTI data, and of these, 69 returned for developmental testing (82%). Their mean (SD) gestational age was 28.4 (2.5) weeks, and birth weight was 1121.4 (394.1) g. DTI metrics were significantly associated with Nonverbal Ability in the corpus callosum, posterior thalamic radiations, fornix, and inferior longitudinal fasciculus and with Motor scores in the corpus callosum, internal and external capsules, posterior thalamic radiations, superior and inferior longitudinal fasciculi, cerebral peduncles, and corticospinal tracts. CONCLUSIONS: We identified widespread microstructural white matter abnormalities in very preterm infants at term that were significantly associated with cognitive and motor development at 3 years' corrected age.

Selective flowers to attract and enhance Telenomus laeviceps (Hymenoptera: Scelionidae): a released biocontrol agent of Mamestra brassicae (Lepidoptera: Noctuidae).

The importance of the right food source for the survival and reproduction of certain insect species is well documented. In the case of biocontrol agents, this is even more important in order to reach a high predation or parasitation performance. The egg parasitoid Telenomus laeviceps (Förster, 1861) (Hymenoptera: Scelionidae) is a promising candidate for mass release as a biological control agent of the cabbage moth Mamestra brassicae (Linnaeus, 1758) (Lepidoptera: Noctuidae). However, adult T. laeviceps need a sugar-rich food source to increase their parasitation performance and produce a good amount of female offspring. Released biocontrol agents were shown to benefit from conservation biocontrol, which includes the provision of selected flowers as nectar resources for beneficial insects. In Switzerland, Centaurea cyanus L. (Asteraceae), Fagopyrum esculentum Moench (Polygonaceae) and Vicia sativa L. (Fabaceae) are successfully implemented in the field to attract and promote natural enemies of different cabbage pests. In this study, we investigated the potential of these selected flowers to attract and promote T. laeviceps under laboratory conditions. In Y-tube olfactometer experiments, we first tested whether the three nectar providing plant species are attractive to T. laeviceps. Furthermore, we assessed their effects on survival and parasitation performance of adult T. laeviceps. We found that flowers of F. esculentum and C. cyanus were attractive in contrast to V. sativa. Also fecundity and the number of female offspring produced were higher for females kept on F. esculentum and C. cyanus than on V. sativa. In contrast, survival was similar on all treatments. Our findings present a further key step towards the implementation of T. laeviceps as a biocontrol agent.

Bioavailability of detomidine administered sublingually to horses as an oromucosal gel.

The objective of the study was to determine the absorption, bioavailability and sedative effect of detomidine administered to horses as an oromucosal gel compared to intravenous and intramuscular administration of detomidine injectable solution. The study was open and randomized, with three sequences crossover design. Nine healthy horses were given 40 µg/kg detomidine intravenously, intramuscularly or administered under the tongue with a 7-day wash-out period between treatments. Blood samples were collected before and after drug administration for the measurement of detomidine concentrations in serum. The effects of the route of administration on heart rate and rhythm were evaluated and the depth of sedation assessed. Mean (±SD) bioavailability of detomidine was 22% (±5.3%) after sublingual administration and 38.2% (±7.9%) after intramuscular administration. The sedative effects correlated with detomidine concentrations regardless of the route of administration. We conclude that less detomidine is absorbed when given sublingually than when given intramuscularly, because part of it does not reach the circulation. Sublingual administration of detomidine oromucosal gel at 40 µg/kg produces safe sedation in horses. Slow absorption leads to fewer and less pronounced adverse effects than the more rapid absorption after intramuscular injection.

Comparison of fMRI activation at 3 and 1.5 T during perceptual, cognitive, and affective processing.

Previous studies comparing fMRI data acquired at 1.5 T and higher field strengths have focused on examining signal increases in the visual and motor cortices. No information is, however, available on the relative gain, or the comparability of data, obtained at higher field strengths for other brain regions such as the prefrontal and other association cortices. In the present study, we investigated fMRI activation at 1.5 and 3 T during visual perception, visuospatial working memory, and affect-processing tasks. A 23% increase in striate and extrastriate activation volume was observed at 3 T compared with that for 1.5 T during the visual perception task. During the working memory task significant increases in activation volume were observed in frontal and parietal association cortices as well as subcortical structures, including the caudate, globus pallidus, putamen, and thalamus. Increases in working memory-related activation volume of 82, 73, 83, and 36% were observed in the left frontal, right frontal, left parietal, and right parietal lobes, respectively, for 3 T compared with 1.5 T. These increases were characterized by increased activation at 3 T in several prefrontal and parietal cortex regions that showed activation at 1.5 T. More importantly, at 3 T, activation was detected in several regions, such as the ventral aspects of the inferior frontal gyrus, orbitofrontal gyrus, and lingual gyrus, which did not show significant activation at 1.5 T. No difference in height or extent of activation was detected between the two scanners in the amygdala during affect processing. Signal dropout in the amygdala from susceptibility artifact was greater at 3 T, with a 12% dropout at 3 T compared with a 9% dropout at 1.5 T. The spatial smoothness of T2* images was greater at 3 T by less than 1 mm, suggesting that the greater extent of activation at 3 T beyond these spatial scales was not due primarily to increased intrinsic spatial correlations at 3 T. Rather, the increase in percentage of voxels activated reflects increased sensitivity for detection of brain activation at higher field strength. In summary, our findings suggest that functional imaging of prefrontal and other association cortices can benefit significantly from higher magnetic field strength.

Peptide mimics of SNARE transmembrane segments drive membrane fusion depending on their conformational plasticity.

SNARE proteins are essential for different types of intracellular membrane fusion. Whereas interaction between their cytoplasmic domains is held responsible for establishing membrane proximity, the role of the transmembrane segments in the fusion process is currently not clear. Here, we used an in vitro approach based on lipid mixing and electron microscopy to examine a potential fusogenic activity of the transmembrane segments. We show that the presence of synthetic peptides representing the transmembrane segments of the presynaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) synaptobrevin II (also referred to as VAMP II) or syntaxin 1A, but not of an unrelated control peptide, in liposomal membranes drives their fusion. Liposome aggregation by millimolar Ca(2+) concentrations strongly potentiated the effect of the peptides; this indicates that juxtaposition of the bilayers favours their fusion in the absence of the cytoplasmic SNARE domains. Peptide-driven fusion is reminiscent of natural membrane fusion, since it was suppressed by lysolipid and involved both bilayer leaflets. This suggests transient presence of a hemifusion intermediate followed by complete membrane merger. Structural studies of the peptides in lipid bilayers performed by Fourier transform infrared spectroscopy indicated mixtures of alpha-helical and beta-sheet conformations. In isotropic solution, circular dichroism spectroscopy showed the peptides to exist in a concentration-dependent equilibrium of alpha-helical and beta-sheet structures. Interestingly, the fusogenic activity decreased with increasing stability of the alpha-helical solution structure for a panel of variant peptides. Thus, structural plasticity of transmembrane segments may be important for SNARE protein function at a late step in membrane fusion.

Membrane structure and fusion-triggering conformational change of the fusion domain from influenza hemagglutinin.

The N-terminal domain of the influenza hemagglutinin (HA) is the only portion of the molecule that inserts deeply into membranes of infected cells to mediate the viral and the host cell membrane fusion. This domain constitutes an autonomous folding unit in the membrane, causes hemolysis of red blood cells and catalyzes lipid exchange between juxtaposed membranes in a pH-dependent manner. Combining NMR structures determined at pHs 7.4 and 5 with EPR distance constraints, we have deduced the structures of the N-terminal domain of HA in the lipid bilayer. At both pHs, the domain is a kinked, predominantly helical amphipathic structure. At the fusogenic pH 5, however, the domain has a sharper bend, an additional 3(10)-helix and a twist, resulting in the repositioning of Glu 15 and Asp 19 relative to that at the nonfusogenic pH 7.4. Rotation of these charged residues out of the membrane plane creates a hydrophobic pocket that allows a deeper insertion of the fusion domain into the core of the lipid bilayer. Such an insertion mode could perturb lipid packing and facilitate lipid mixing between juxtaposed membranes.

Reconstituted syntaxin1a/SNAP25 interacts with negatively charged lipids as measured by lateral diffusion in planar supported bilayers.

According to the soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein (SNAP) receptor hypothesis (SNARE hypothesis), interactions between target SNAREs and vesicle SNAREs (t- and v-SNAREs) are required for membrane fusion in intracellular vesicle transport and exocytosis. The precise role of the SNAREs in tethering, docking, and fusion is still disputed. Biophysical measurements of SNARE interactions in planar supported membranes could potentially resolve some of the key questions regarding the mechanism of SNARE-mediated membrane fusion. As a first step toward this goal, recombinant syntaxin1A/SNAP25 (t-SNARE) was reconstituted into polymer-supported planar lipid bilayers. Reconstituted t-SNAREs in supported bilayers bound soluble green fluorescent protein/vesicle-associated membrane protein (v-SNARE), and the SNARE complexes could be specifically dissociated by NSF/alpha-SNAP in the presence of ATP. The physiological activities of SNARE complex formation were thus well reproduced in this reconstituted planar model membrane system. A large fraction (~75%) of the reconstituted t-SNARE was laterally mobile with a lateral diffusion coefficient of 7.5 x 10(-9) cm(2)/s in a phosphatidylcholine lipid background. Negatively charged lipids reduced the mobile fraction of the t-SNARE and the lipids themselves. Phosphatidylinositol-4,5-bisphosphate was more effective than phosphatidylserine in reducing the lateral mobility of the complexes. A model of how acidic lipid-SNARE interactions might alter lipid fluidity is discussed.

Structure of outer membrane protein A transmembrane domain by NMR spectroscopy.

We have determined the three-dimensional fold of the 19 kDa (177 residues) transmembrane domain of the outer membrane protein A of Escherichia coli in dodecylphosphocholine (DPC) micelles in solution using heteronuclear NMR. The structure consists of an eight-stranded beta-barrel connected by tight turns on the periplasmic side and larger mobile loops on the extracellular side. The solution structure of the barrel in DPC micelles is similar to that in n-octyltetraoxyethylene (C(8)E(4)) micelles determined by X-ray diffraction. Moreover, data from NMR dynamic experiments reveal a gradient of conformational flexibility in the structure that may contribute to the membrane channel function of this protein.

Biophysical approaches to membrane protein structure determination.

Recently, there have been several technical advances in the use of solution and solid-state NMR spectroscopy to determine the structures of membrane proteins. The structures of several isolated transmembrane (TM) helices and pairs of TM helices have been solved by solution NMR methods. Similarly, the complete folds of two TM beta-barrel proteins with molecular weights of 16 and 19 kDa have been determined by solution NMR in detergent micelles. Solution NMR has also provided a first glimpse at the dynamics of an integral membrane protein. Structures of individual TM helices have also been determined by solid-state NMR. A combination of NMR with site-directed spin-label electron paramagnetic resonance or Fourier transform IR spectroscopy allows one to assemble quite detailed protein structures in the membrane.

pH-dependent self-association of influenza hemagglutinin fusion peptides in lipid bilayers.

We have recently designed a host-guest peptide system that allows us to quantitatively measure the energetics of interaction of viral fusion peptides with lipid bilayers. Here, we show that fusion peptides of influenza hemagglutinin reversibly associate with one another at membrane surfaces above critical surface concentrations, which range from one to five peptides per 1000 lipids in the systems that we investigated. It is further demonstrated by using circular dichroism and Fourier transform infrared spectroscopy that monomeric peptides insert into the bilayers in a predominantly alpha-helical conformation, whereas self-associated fusion peptides adopt predominantly antiparallel beta-sheet structures at the membrane surface. The two forms are readily interconvertible and the equilibrium between them is determined by the pH and ionic strength of the surrounding solution. Lowering the pH favors the monomeric alpha-helical conformation, whereas increasing the ionic strength shifts the equilibrium towards the membrane-associated beta-aggregates. The binding data are interpreted in terms of a cooperative binding model that yields free energies of insertion and free energies of self-association for each of the peptides studied at pH 7.4 and pH 5. At pH 5 and 35 mM ionic strength, the insertion energy of the 20 residue influenza hemagglutinin fusion peptide is -7.2 kcal/mol and the self-association energy is -1.9 kcal/mol. We propose that self-association of fusion peptides could be a major driving force for recruiting a small number of hemagglutinin trimers into a fusion site.

A host-guest system to study structure-function relationships of membrane fusion peptides.

We designed a host-guest fusion peptide system, which is completely soluble in water and has a high affinity for biological and lipid model membranes. The guest sequences are those of the fusion peptides of influenza hemagglutinin, which are solubilized by a highly charged unstructured C-terminal host sequence. These peptides partition to the surface of negatively charged liposomes or erythrocytes and elicit membrane fusion or hemolysis. They undergo a conformational change from random coil to an obliquely inserted ( approximately 33 degrees from the surface) alpha-helix on binding to model membranes. Partition coefficients for membrane insertion were measured for influenza fusion peptides of increasing lengths (n = 8, 13, 16, and 20). The hydrophobic contribution to the free energy of binding of the 20-residue fusion peptide at pH 5.0 is -7.6 kcal/mol (1 cal = 4.18 J). This energy is sufficient to stabilize a "stalk" intermediate if a typical number of fusion peptides assemble at the site of membrane fusion. The fusion activity of the fusion peptides increases with each increment in length, and this increase strictly correlates with the hydrophobic binding energy and the angle of insertion.

Tethered polymer-supported planar lipid bilayers for reconstitution of integral membrane proteins: silane-polyethyleneglycol-lipid as a cushion and covalent linker.

There is increasing interest in supported membranes as models of biological membranes and as a physiological matrix for studying the structure and function of membrane proteins and receptors. A common problem of protein-lipid bilayers that are directly supported on a hydrophilic substrate is nonphysiological interactions of integral membrane proteins with the solid support to the extent that they will not diffuse in the plane of the membrane. To alleviate some of these problems we have developed a new tethered polymer-supported planar lipid bilayer system, which permitted us to reconstitute integral membrane proteins in a laterally mobile form. We have supported lipid bilayers on a newly designed polyethyleneglycol cushion, which provided a soft support and, for increased stability, covalent linkage of the membranes to the supporting quartz or glass substrates. The formation and morphology of the bilayers were followed by total internal reflection and epifluorescence microscopy, and the lateral diffusion of the lipids and proteins in the bilayer was monitored by fluorescence recovery after photobleaching. Uniform bilayers with high lateral lipid diffusion coefficients (0.8-1.2 x 10(-8) cm(2)/s) were observed when the polymer concentration was kept slightly below the mushroom-to-brush transition. Cytochrome b(5) and annexin V were used as first test proteins in this system. When reconstituted in supported bilayers that were directly supported on quartz, both proteins were largely immobile with mobile fractions < 25%. However, two populations of laterally mobile proteins were observed in the polymer-supported bilayers. Approximately 25% of cytochrome b(5) diffused with a diffusion coefficient of approximately 1 x 10(-8) cm(2)/s, and 50-60% diffused with a diffusion coefficient of approximately 2 x 10(-10) cm(2)/s. Similarly, one-third of annexin V diffused with a diffusion coefficient of approximately 3 x 10(-9) cm(2)/s, and two-thirds diffused with a diffusion coefficient of approximately 4 x 10(-10) cm(2)/s. A model for the interaction of these proteins with the underlying polymer is discussed.

Secondary structure, orientation, oligomerization, and lipid interactions of the transmembrane domain of influenza hemagglutinin.

Influenza virus hemagglutinin (HA), the viral envelope glycoprotein that mediates fusion between the viral and cellular membranes, is a homotrimer of three subunits, each containing two disulfide-linked polypeptide chains, HA(1) and HA(2). Each HA(2) chain spans the viral membrane with a single putative transmembrane alpha-helix near its C-terminus. Fusion experiments with recombinant HAs suggest that this sequence is required for a late step of membrane fusion, as a glycosylphosphatidylinositol-anchored analogue of HA only mediates "hemifusion" of membranes, i.e., the merging of the proximal, but not distal, leaflets of the two juxtaposed lipid bilayers [Kemble et al. (1994) Cell 76, 383-391]. To find a structural explanation for the function of the transmembrane domain of HA(2) in membrane fusion, we have studied the secondary structure, orientation, oligomerization, and lipid interactions of a synthetic peptide representing the transmembrane segment of X:31 HA (TMX31) by circular dichroism and attenuated total reflection Fourier transform infrared spectroscopy and by gel electrophoresis. The peptide was predominantly alpha-helical in detergent micelles and in phospholipid bilayers. The helicity was increased in lipid bilayers composed of acidic lipids compared to pure phosphatidylcholine bilayers. In planar lipid bilayers, the helices were oriented close to the membrane normal. TMX31 aggregated into small heat-resistant oligomers composed of two to five subunits in SDS micelles. Amide hydrogen exchange experiments indicated that a large fraction of the helical residues were accessible to water, suggesting the possibility that TMX31 forms pores in lipid bilayers. Finally, the peptide increased the acyl chain order in lipid bilayers, which may be related to the preferential association of HA with lipid "rafts" in the cell surface and which may be an important prerequisite for complete membrane fusion.

Refolded outer membrane protein A of Escherichia coli forms ion channels with two conductance states in planar lipid bilayers.

Outer membrane protein A (OmpA), a major structural protein of the outer membrane of Escherichia coli, consists of an N-terminal 8-stranded beta-barrel transmembrane domain and a C-terminal periplasmic domain. OmpA has served as an excellent model for studying the mechanism of insertion, folding, and assembly of constitutive integral membrane proteins in vivo and in vitro. The function of OmpA is currently not well understood. Particularly, the question whether or not OmpA forms an ion channel and/or nonspecific pore for uncharged larger solutes, as some other porins do, has been controversial. We have incorporated detergent-purified OmpA into planar lipid bilayers and studied its permeability to ions by single channel conductance measurements. In 1 M KCl, OmpA formed small (50-80 pS) and large (260-320 pS) channels. These two conductance states were interconvertible, presumably corresponding to two different conformations of OmpA in the membrane. The smaller channels are associated with the N-terminal transmembrane domain, whereas both domains are required to form the larger channels. The two channel activities provide a new functional assay for the refolding in vitro of the two respective domains of OmpA. Wild-type and five single tryptophan mutants of urea-denatured OmpA are shown to refold into functional channels in lipid bilayers.

Viral fusion peptides: a tool set to disrupt and connect biological membranes.

The structure and function of viral fusion peptides are reviewed. The fusion peptides of influenza virus hemagglutinin and human immunodeficiency virus are used as paradigms. Fusion peptides associated with lipid bilayers are conformationally polymorphic. Current evidence suggests that the fusion-promoting state is the obliquely inserted alpha-helix. Fusion peptides also have a tendency to self-associate into beta-sheets at membrane surfaces. Although the conformational conversion between alpha- and beta-states is reversible under controlled conditions, its physiological relevance is not yet known. The energetics of peptide insertion and self-association could be measured recently using more soluble "second generation" fusion peptides. Fusion peptides have been reported to change membrane curvature and the state of hydration of membrane surfaces. The combined results are built into a model for the mechanism by which fusion peptides are proposed to assist in biological membrane fusion.

Outer membrane protein A of E. coli folds into detergent micelles, but not in the presence of monomeric detergent.

Outer membrane protein A (OmpA) of Escherichia coli is a beta-barrel membrane protein that unfolds in 8 M urea to a random coil. OmpA refolds upon urea dilution in the presence of certain detergents or lipids. To examine the minimal requirements for secondary and tertiary structure formation in beta-barrel membrane proteins, folding of OmpA was studied as a function of the hydrophobic chain length, the chemical structure of the polar headgroup, and the concentration of a large array of amphiphiles. OmpA folded in the presence of detergents only above a critical minimal chain length of the apolar chain as determined by circular dichroism spectroscopy and a SDS-PAGE assay that measures tertiary structure formation. Details of the chemical structure of the polar headgroup were unimportant for folding. The minimal chain length required for folding correlated with the critical micelle concentration in each detergent series. Therefore, OmpA requires preformed detergent micelles for folding and does not adsorb monomeric detergent to its perimeter after folding. Formation of secondary and tertiary structure is thermodynamically coupled and strictly dependent on the interaction with aggregated amphiphiles.

Interaction of mutant influenza virus hemagglutinin fusion peptides with lipid bilayers: probing the role of hydrophobic residue size in the central region of the fusion peptide.

The amino-terminal region of the membrane-anchored subunit of influenza virus hemagglutinin, the fusion peptide, is crucial for membrane fusion of this virus. The peptide is extruded from the interior of the protein and inserted into the lipid bilayer of the target membrane upon induction of a conformational change in the protein by low pH. Although the effects of several mutations in this region on the fusion behavior and the biophysical properties of the corresponding peptides have been studied, the structural requirements for an active fusion peptide have still not been defined. To probe the sensitivity of the fusion peptide structure and function to small hydrophobic perturbations in the middle of the hydrophobic region, we have individually replaced the alanine residues in positions 5 and 7 with smaller (glycine) or bulkier (valine) hydrophobic residues and measured the extent of fusion mediated by these hemagglutinin constructs as well as some biophysical properties of the corresponding synthetic peptides in lipid bilayers. We find that position 5 tolerates a smaller and position 7 a larger hydrophobic side chain. All peptides contained segments of alpha-helical (33-45%) and beta-strand (13-16%) conformation as determined by CD and ATR-FTIR spectroscopy. The order parameters of the peptide helices and the lipid hydrocarbon chains were determined from measurements of the dichroism of the respective infrared absorption bands. Order parameters in the range of 0.0-0.6 were found for the helices of these peptides, which indicate that these peptides are most likely aligned with their alpha-helices at oblique angles to the membrane normal. Some (mostly fusogenic) peptides induced significant increases of the order parameter of the lipid hydrocarbon chains, suggesting that the lipid bilayer becomes more ordered in the presence of these peptides, possibly as a result of dehydration at the membrane surface.

Time-resolved distance determination by tryptophan fluorescence quenching: probing intermediates in membrane protein folding.

The mechanism of insertion and folding of an integral membrane protein has been investigated with the beta-barrel forming outer membrane protein A (OmpA) of Escherichia coli. This work describes a new approach to this problem by combining structural information obtained from tryptophan fluorescence quenching at different depths in the lipid bilayer with the kinetics of the refolding process. Experiments carried out over a temperature range between 2 and 40 degrees C allowed us to detect, trap, and characterize previously unidentified folding intermediates on the pathway of OmpA insertion and folding into lipid bilayers. Three membrane-bound intermediates were found in which the average distances of the Trps were 14-16, 10-11, and 0-5 A, respectively, from the bilayer center. The first folding intermediate is stable at 2 degrees C for at least 1 h. A second intermediate has been isolated at temperatures between 7 and 20 degrees C. The Trps move 4-5 A closer to the center of the bilayer at this stage. Subsequently, in an intermediate that is observable at 26-28 degrees C, the Trps move another 5-10 A closer to the center of the bilayer. The final (native) structure is observed at higher temperatures of refolding. In this structure, the Trps are located on average about 9-10 A from the bilayer center. Monitoring the evolution of Trp fluorescence quenching by a set of brominated lipids during refolding at various temperatures therefore allowed us to identify and characterize intermediate states in the folding process of an integral membrane protein.