Dysmyelination and glycolipid interference caused by phenylalanine in phenylketonuria.

Phenylketonuria (PKU) is a metabolic disorder connected to an excess of phenylalanine (Phe) in the blood and tissues, with neurological consequences. The disease's molecular bases seem to be related to the accumulation of Phe at the cell membrane surface. Radiological outcomes in the brain demonstrate decreased water diffusivity in white matter, involving axon dysmyelination of not yet understood origin. We used a biophysical approach and model membranes to extend our knowledge of Phe-membrane interaction by clarifying Phe's propensity to affect membrane structure and dynamics based on lipid composition, with emphasis on modulating cholesterol and glycolipid components to mimic raft domains and myelin sheath membranes. Phe showed affinity for the investigated membrane mimics, mainly affecting the Phe-facing membrane leaflet. The surfaces of our neuronal membrane raft mimics were strong anchoring sites for Phe, showing rigidifying effects. From a therapeutic perspective, we further investigated the role of doxycycline, known to disturb Phe packing, unveiling its action as a competitor in Phe interactions with the membrane, suggesting its potential for treatment in the early stages of PKU. Our results suggest how Phe accumulation in extracellular fluids can impede normal growth of myelin sheaths by interfering with membrane slipping and by remodulating free water and myelin-associated water contents.

Hybrid polymeric micelles stabilized by gallium ions: Structural investigation.

The addition of gallium ions to a solution of a double-hydrophilic block copolymer, i.e. poly(ethylene oxide)-block-poly(acrylic acid), leads to the spontaneous formation of highly monodisperse micelles with a Hybrid PolyIon Complexes (HPICs) core. By combining several techniques, a precise description of the HPIC architecture was achieved. In particular and for the first time, NMR and anomalous small angle X-ray scattering (ASAXS) enable tracking of the inorganic ions in solution and highlighting the co-localization of the gallium and the poly(acrylic acid) blocks in a rigid structure at the core of the micelle. Such a core has a radius of ca 4.3 nm while the complete nano-object with its poly(ethylene oxide) shell has a total radius of ca 11 nm. The aggregation number was also estimated using the ASAXS results. This comprehensive structural characterization of the Ga HPICs corroborates the assumptions made for HPICs based on other inorganic ions and demonstrates the universality of the HPIC structure leading, for example, to new families of contrast agents in medical imaging.

Assessing Fast Structure Formation Processes in Isotactic Polypropylene with a Combination of Nanofocus X-ray Diffraction and In Situ Nanocalorimetry.

A combination of in situ nanocalorimetry with simultaneous nanofocus 2D Wide-Angle X-ray Scattering (WAXS) was used to study polymorphic behaviour and structure formation in a single micro-drop of isotactic polypropylene (iPP) with defined thermal history. We were able to generate, detect, and characterize a number of different iPP morphologies using our custom-built ultrafast chip-based nanocalorimetry instrument designed for use with the European Synchrotron Radiation Facility (ESRF) high intensity nanofocus X-ray beamline facility. The detected iPP morphologies included monoclinic alpha-phase crystals, mesophase, and mixed morphologies with different mesophase/crystalline compositional ratios. Monoclinic crystals formed from the mesophase became unstable at heating rates above 40 K s-1 and showed melting temperatures as low as ~30 K below those measured for iPP crystals formed by slow cooling. We also studied the real-time melt crystallization of nanogram-sized iPP samples. Our analysis revealed a mesophase nucleation time of around 1 s and the co-existence of mesophase and growing disordered crystals at high supercooling ≤328 K. The further increase of the iPP crystallization temperature to 338 K changed nucleation from homogeneous to heterogeneous. No mesophase was detected above 348 K. Low supercooling (≥378 K) led to the continuous growth of the alpha-phase crystals. In conclusion, we have, for the first time, measured the mesophase nucleation time of supercooled iPP melted under isothermal crystallization conditions using a dedicated experimental setup designed to allow simultaneous ultrafast chip-based nanocalorimetry and nanofocus X-ray diffraction analyses. We also provided experimental evidence that upon heating, the mesophase converts directly into thermodynamically stable monoclinic alpha-phase crystals via perfection and reorganization and not via partial melting. The complex phase behaviour of iPP and its dependence on both crystallization temperature and time is presented here using a time-temperature-transformation (TTT) diagram.

Novel O/W nanoemulsions for nasal administration: Structural hints in the selection of performing vehicles with enhanced mucopenetration.

We propose novel oil-in-water nanoemulsions (O/W NEs) including PEGylated surfactants and chitosan, showing good biocompatibility and optimization for nasal administration of drugs or vaccines. The transmucosal route has been shown to be ideal for a fast and efficient absorption and represents a viable alternative when the oral administration is problematic. The critical structural features in view of optimal encapsulation and transmucosal delivery were assessed by characterizing the NEs with complementary scattering techniques, i.e. dynamic light scattering (DLS), small angle X-ray (SAXS) and neutron scattering (SANS). Combined results allowed for selecting the formulations with the best suited structural properties and in addition establishing their propensity to enter the mucus barrier. To this scope, mucin was used as a model system and the effect of adding chitosan to the NEs, as adjuvant, was investigated. Remarkably, the presence of chitosan had a positive impact on the diffusion of the NE particles through the mucin matrix. We can infer that chitosan-mucin interaction induces density inhomogeneity and an increase in the pore size within the gel matrix that enhances the PEGylated NEs mobility. The coupling of mucoadhesive and mucopenetrating agents is shown to be a promising strategy for innovative transmucosal delivery systems.

Chitosan/glycosaminoglycan scaffolds for skin reparation.

Burns and chronic wounds, often related to chronic diseases (as diabetes and cancer), are challenging lesions, difficult to heal. The prompt and full reconstitution of a functional skin is at the basis of the development of biopolymer-based scaffolds, representing a 3D substrate mimicking the dermal extracellular matrix. Aim of the work was to develop scaffolds intended for skin regeneration, according to: fabrication by electrospinning from aqueous polysaccharide solutions; prompt and easy treatment to obtain scaffolds insoluble in aqueous fluids; best performance in supporting wound healing. Three formulations were tested, based on chitosan (CH) and pullulan (P), associated with glycosaminoglycans (chondroitin sulfate - CS or hyaluronic acid - HA). A multidisciplinary approach has been used: chemico-physical characterization and preclinical evaluation allowed to obtain integrated information. This supports that CS gives distinctive properties and optimal features to the scaffold structure for promoting cell proliferation leading tissue reparation towards a complete skin restore.

Exploring the water/oil/water interface of phospholipid stabilized double emulsions by micro-focusing synchrotron SAXS.

Surfactant stabilized water/oil/water (w/o/w) double emulsions have received much attention in the last years motivated by their wide applications. Among double emulsions, those stabilized by phospholipids present special interest for their imitation of artificial cells, allowing the study of the effect of confining chemical reactions in biomimetic environments. Upon evaporation of the oil shell, phospholipid stabilized double emulsions can also serve as templates for giant vesicles. In this context, general assumptions have been made on the self-assembly and structural organization/arrangement of amphiphilic molecules, at the aqueous/oil liquid interface. However, to the best of our knowledge, no detailed evidence of the interfacial structuring have been reported. In this paper, w/o/w double emulsions formulated using the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a mixture of chloroform and cyclohexane as the oil phase were produced using a microfluidic device. To obtain information on the phospholipid arrangement, the w/o/w interface was investigated by spatially resolved micro-focusing SAXS. We observed that (i) the basic units forming both the w/o and o/w interfaces were oil-swollen DMPC bilayers, arranged into a substantially disordered shell of ∼45 µm thickness surrounding the internal oil phase; (ii) the evaporation process was slow, i.e. in the order of one hour at 50 °C and (iii) oil evaporation led to a shrinkage of the interfacial shell, but not to an increase of the ordering of the lipid bilayers. Interestingly, no stacked DMPC bilayers were observed during the evaporation process, as shown by the absence of Bragg's peaks in the SAXS intensity profiles.

Lipid-Stabilized Water-Oil Interfaces Studied by Microfocusing Small-Angle X-ray Scattering.

Water-in-oil (w/o) simple emulsions are dispersed microconfined systems that find applications in many areas of advanced materials and biotechnology, such as the food industry, drug delivery, and cosmetics, to name but a few. In these systems, the structural and chemical properties of the boundary layer at the w/o interface are of paramount importance in determining functionality and stability. Recently, microfluidic methods have emerged as a valuable tool for fabricating monodisperse emulsion droplets. Because of the intrinsic flexibility of microfluidics, different interfaces can be obtained, and general principles governing their stability are needed to guide the experimental approach. Herein, we investigate the structural characteristics of the region encompassing the liquid/liquid (L/L) interface of w/o emulsions generated by a microfluidic device in the presence of phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and other intercalating amphiphiles (dopants) using microfocused small-angle X-rays scattering (µ-SAXS). We show that phospholipids provide a stable and versatile boundary film of ∼100 µm whose basic units are swollen and uncorrelated DMPC bilayers. The internal arrangement of this interfacial film can be tuned by adding molecules with a different packing parameter, such as cholesterol, which is able to increase the stiffness of the lipid membranes and trigger interbilayer correlation.

Complexation of short ds RNA/DNA oligonucleotides with Gemini micelles: a time resolved SAXS and computational study.

Gene therapy is based on nucleic acid delivery to pathogenic cells in order to modulate their gene expression. The most used non viral vectors are lipid-based nanoaggregates, which are safer than viral carriers and have been shown to assemble easily with both DNA and RNA. However, the transfection efficiency of non viral carriers still needs to be improved before intensive practise in clinical trials can be implemented. For this purpose, the in depth characterization of the complexes formed by nucleic acids and their transporters is of great relevance. In particular, information on the structure and assembly mechanism can be useful to improve our general knowledge of these artificial transfection agents. In this paper, the complexation mechanism of short interfering RNA and DNA molecules (siRNA and siDNA, respectively) with cationic micelles is investigated by combining small angle X-ray scattering experiments and molecular dynamics simulations. Micelles were obtained by Gemini surfactants with different spacer lengths (12-3-12, 12-6-12). The siRNA and siDNA used were double strand molecules characterized by the same length and homologous sequence, in order to perform a close comparison. We showed that complexes appear in solution immediately after mixing and, therefore, the investigation of complex formation requires fast experimental techniques, such as time resolved synchrotron SAXS (Tr-SAXS). The obtained systems had internal arrangement constituted by layers of squeezed micelles alternating the nucleic acids. Both SAXS and MD analyses allowed us to evaluate the mean size of complexes in the range of a few nanometers, with looser and less ordered stacking for the DNA containing aggregates.

Small Angle X-ray and Neutron Scattering: Powerful Tools for Studying the Structure of Drug-Loaded Liposomes.

Nanovectors, such as liposomes, micelles and lipid nanoparticles, are recognized as efficient platforms for delivering therapeutic agents, especially those with low solubility in water. Besides being safe and non-toxic, drug carriers with improved performance should meet the requirements of (i) appropriate size and shape and (ii) cargo upload/release with unmodified properties. Structural issues are of primary importance to control the mechanism of action of loaded vectors. Overall properties, such as mean diameter and surface charge, can be obtained using bench instruments (Dynamic Light Scattering and Zeta potential). However, techniques with higher space and time resolution are needed for in-depth structural characterization. Small-angle X-ray (SAXS) and neutron (SANS) scattering techniques provide information at the nanoscale and have therefore been largely used to investigate nanovectors loaded with drugs or other biologically relevant molecules. Here we revise recent applications of these complementary scattering techniques in the field of drug delivery in pharmaceutics and medicine with a focus to liposomal carriers. In particular, we highlight those aspects that can be more commonly accessed by the interested users.

Superhydrophobic Surfaces Boost Fibril Self-Assembly of Amyloid ß Peptides.

Amyloid ß (Aß) peptides are the main constituents of Alzheimer's amyloid plaques in the brain. Here we report how the unique microfluidic flows exerted by droplets sitting on superhydrophobic surfaces can influence the aggregation mechanisms of several Aß fragments by boosting their fibril self-assembly. Aß(25-35), Aß(1-40), and Aß(12-28) were dried both on flat hydrophilic surfaces (contact angle (CA) = 37.3°) and on nanostructured superhydrophobic ones (CA = 175.8°). By embedding nanoroughened surfaces on top of highly X-ray transparent Si3N4 membranes, it was possible to probe the solid residues by raster-scan synchrotron radiation X-ray microdiffraction (µXRD). As compared to residues obtained on flat Si3N4 membranes, a general enhancement of fibrillar material was detected for all Aß fragments dried on superhydrophobic surfaces, with a particular emphasis on the shorter ones. Indeed, both Aß(25-35) and Aß(12-28) showed a marked crystalline cross-ß phase with varying fiber textures. The homogeneous evaporation rate provided by these nanostructured supports, and the possibility to use transparent membranes, can open a wide range of in situ X-ray and spectroscopic characterizations of amyloidal peptides involved in neurodegenerative diseases and for the fabrication of amyloid-based nanodevices.

On radiation damage in FIB-prepared softwood samples measured by scanning X-ray diffraction.

The high flux density encountered in scanning X-ray nanodiffraction experiments can lead to severe radiation damage to biological samples. However, this technique is a suitable tool for investigating samples to high spatial resolution. The layered cell wall structure of softwood tracheids is an interesting system which has been extensively studied using this method. The tracheid cell has a complex geometry, which requires the sample to be prepared by cutting it perpendicularly to the cell wall axis. Focused ion beam (FIB) milling in combination with scanning electron microscopy allows precise alignment and cutting without splintering. Here, results of a scanning X-ray diffraction experiment performed on a biological sample prepared with a focused ion beam of gallium atoms are reported for the first time. It is shown that samples prepared and measured in this way suffer from the incorporation of gallium atoms up to a surprisingly large depth of 1 µm.

Thermal transformations of self-assembled gold glyconanoparticles probed by combined nanocalorimetry and X-ray nanobeam scattering.

Noble metal nanoparticles with ligand shells are of interest for applications in catalysis, thermo-plasmonics, and others, involving heating processes. To gain insight into the structure-formation processes in such systems, self-assembly of carbohydrate-functionalized gold nanoparticles during precipitation from solution and during further heating to ca. 340 °C was explored by in situ combination of nanobeam SAXS/WAXS and nanocalorimetry. Upon precipitation from solution, X-ray scattering reveals the appearance of small 2D domains of close-packed nanoparticles. During heating, increasing interpenetration of the nanoparticle soft shells in the domains is observed up to ca. 81 °C, followed by cluster formation at ca. 125 °C, which transform into crystalline gold nuclei at around 160 °C. Above ca. 200 °C, one observes the onset of coalescence and grain growth resulting in gold crystallites of average size of about 100 nm. The observed microstructural changes are in agreement with the in situ heat capacity measurements with nanocalorimetry.

A combined small-angle X-ray and neutron scattering study of the structure of purified soluble gastrointestinal mucins.

The structures of purified soluble porcine gastric (Muc5ac) and duodenal (Muc2) mucin solutions at neutral and acidic pH were examined using small-angle X-ray scattering and small-angle neutron scattering experiments. We provide evidence for the morphology of the network above the semidilute overlap concentration and above the entanglement concentration. Furthermore, we investigated the gelation of both types of mucin solutions in response to a reduction in pH, where we observed the formation of large-scale heterogeneities within the polymer solutions, typical of microphase-separated gels. The concentration dependence of the inhomogeneity length scale (Ξ) and the amplitude of the excess scattering intensity [I(ex) (0)] are consistent with previously studied gelled synthetic polymeric systems. The persistence lengths of the chains were found to be similar for both Muc5ac and Muc2 from Kratky plots of the neutron data (8 ± 2 nm).

Oral lymphomatoid granulomatosis, the first sign of a 'rare disease': a case report.

INTRODUCTION: Lymphomatoid granulomatosis is an uncommon Epstein-Barr virus-positive B-cell lymphoma, an angiocentric-destructive process with a predominant T-cell background. Lymphomatoid granulomatosis is listed among rare diseases. Common localization is in the lungs. Lymphomatoid granulomatosis with oral involvement is described in only two reports. In this report, we describe a third case of oral lymphomatoid granulomatosis. CASE PRESENTATION: A 65-year-old Caucasian man with a gingival ulceration underwent a biopsy. The histological pattern was compatible with a grade III lymphomatoid granulomatosis. The staging revealed a nodular lesion in the lower lobe of his right lung. Our patient also presented with hemoptysis, an unusual and not reported clinical sign. Rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone chemotherapy was performed every three weeks for six cycles. CONCLUSIONS: The pulmonary nodule and the gingival lesion disappeared. At eight-month follow-up, our patient is disease-free. We wish to emphasize that the oral manifestation described was the first sign of the disease and allowed for diagnosis. This case report adds to the medical literature for the particular clinical presentation of this rare disease.

Changes of statistical structural fluctuations unveils an early compacted degraded stage of PNS myelin.

Degradation of the myelin sheath is a common pathology underlying demyelinating neurological diseases from Multiple Sclerosis to Leukodistrophies. Although large malformations of myelin ultrastructure in the advanced stages of Wallerian degradation is known, its subtle structural variations at early stages of demyelination remains poorly characterized. This is partly due to the lack of suitable and non-invasive experimental probes possessing sufficient resolution to detect the degradation. Here we report the feasibility of the application of an innovative non-invasive local structure experimental approach for imaging the changes of statistical structural fluctuations in the first stage of myelin degeneration. Scanning micro X-ray diffraction, using advances in synchrotron x-ray beam focusing, fast data collection, paired with spatial statistical analysis, has been used to unveil temporal changes in the myelin structure of dissected nerves following extraction of the Xenopus laevis sciatic nerve. The early myelin degeneration is a specific ordered compacted phase preceding the swollen myelin phase of Wallerian degradation. Our demonstration of the feasibility of the statistical analysis of SµXRD measurements using biological tissue paves the way for further structural investigations of degradation and death of neurons and other cells and tissues in diverse pathological states where nanoscale structural changes may be uncovered.

Time resolved SAXS to study the complexation of siRNA with cationic micelles of divalent surfactants.

The complexation of siRNA (small interfering RNA) with cationic micelles was studied using time dependent synchrotron SAXS. Micelles were formed by two types of divalent cationic surfactants, i.e. Gemini bis(quaternary ammonium) bromide with variable spacer length (12-3-12, 12-6-12, 12-12-12) and a weak electrolyte surfactant (SH14) with triazine head. Immediately after mixing (t < 50 ms), new large aggregates appeared in solution and the scattering intensity at low q increased. Concomitantly, the presence of a quasi-Bragg peak at q ∼ 1.5 nm(-1) indicated core structuring within the complexes. We hypothesize that siRNA and micelles are alternately arranged into "sandwiches", forming domains with internal structural coherence. The process of complex reorganization followed a first-order kinetics and was completed in less than about 5 minutes, after which a steady state was reached. Aggregates containing Geminis were compact globular structures whose gyration radii Rg depended on the spacer length and were in the order of 7-27 nm. Complexes containing SH14 (Rg = 14-16 nm) were less ordered and possessed a looser internal arrangement. The obtained data, joint with previous structural investigation using Dynamic Light Scattering, Zeta Potential and Small Angle Neutron Scattering, are encouraging evidence for using these systems in biological trials. In fact we showed that transfection agents can be obtained by simply mixing a micelle solution of the cationic surfactant and a siRNA solution, both of which are easily prepared and stable.

Submicrometer 3D Structural Evidence of Fuel Cell Membrane Heterogeneous Degradation.

Polymer membranes used in the proton exchange membrane fuel cell (PEMFC) technology are subject to severe chemical and physical degradations during operation. A microscopic diagnosis of the effects of aging on the microstructure of benchmark perfluorinated sulfonic acid (PFSA) membranes is crucial to developing long-lasting devices. We report here the first µSAXS study of membranes aged for 2500 h in a stack. SAXS spectra recorded with submicrometer resolution in-plane and along the membrane thickness provide a 3D mapping of the aging effect. Nanoscale heterogeneities are evidenced and found to depend on the membrane position relative to the electrodes, to the air inlets, and proximity to channels (distributing gas) or ribs (collecting the current). Long-term aging in a fuel cell operating in stationary conditions around 65 °C results in a small voltage degradation rate of 13 µV/h, without any evidence of membrane failure, but to an irreversible over-swelling of the membrane due to polymer relaxation. Regions under the gas distribution channels close to the air inlet are profoundly degraded due to an increased water gradient concentration from the cathode to the anode. These observations provide a novel and unique insight for developing new strategies toward the design of more durable polymers inserted in smart fuel cells.

Evidence of Cybotactic Order in the Nematic Phase of a Main-Chain Liquid Crystal Polymer with Bent-Core Repeat Unit.

We report the synthesis and structural characterization of a main-chain liquid crystal polymer constituted by a 1,2,4-oxadiazole-based bent-core repeat unit. For the first time, a liquid crystal polymer made of bent mesogenic units is demonstrated to exhibit cybotactic order in the nematic phase. Coupled with the chain-bond constraints, cybotaxis results in maximized molecular correlations that make this material of great potential in the search for the elusive biaxial and ferroelectric nematic phases. Indeed, repolarization current measurements in the nematic phase hint at a ferroelectric-like switching response (upon application of an electric field of only 1.0 V µm-1) that, albeit to be definitely confirmed by complementary techniques, is strongly supported by the comparative repolarization current measurements in the nematic and isotropic phases. Finally, the weak tendency of this polymer to crystallize makes it possible to supercool the cybotactic nematic phase down to room temperature, thus, paving the way for a glassy phase in which the biaxial (and possibly polar) order is frozen at room temperature.

High-resolution thermal imaging with a combination of nano-focus X-ray diffraction and ultra-fast chip calorimetry.

A microelectromechanical-systems-based calorimeter designed for use on a synchrotron nano-focused X-ray beamline is described. This instrument allows quantitative DC and AC calorimetric measurements over a broad range of heating/cooling rates (≤100000 K s(-1)) and temperature modulation frequencies (≤1 kHz). The calorimeter was used for high-resolution thermal imaging of nanogram-sized samples subjected to X-ray-induced heating. For a 46 ng indium particle, the measured temperature rise reaches ∼0.2 K, and is directly correlated to the X-ray absorption. Thermal imaging can be useful for studies of heterogeneous materials exhibiting physical and/or chemical transformations. Moreover, the technique can be extended to three-dimensional thermal nanotomography.

Non-radial growth of helical homopolymer crystals: breaking the paradigm of the polymer spherulite microstructure.

Radial symmetry is essential for the conventional view of the polymer spherulite microstructure. Typically it is assumed that, in the course of the spherulite morphogenesis, the lamellar crystals grow radially. Using submicron X-ray diffraction, it is shown that in banded spherulites of poly(propylene adipate) the crystals have the shape of a helix with flat-on crystals winding around a virtual cylinder of about 6 µm in diameter. The helix angle of 30° implies that the crystal growth direction is tilted away from the spherulite radius by this angle. The implications of the helical crystal shape contradict the paradigm of the spherulitic microstructure. The radial growth rate of such spherulites does not correspond to the crystal growth rate, but to the propagation rate of the virtual cylinder the ribbons wind around.