Investigating Membrane-Mediated Antimicrobial Peptide Interactions with Synchrotron Radiation Far-Infrared Spectroscopy.

Synchrotron radiation-based Fourier transform infrared spectroscopy enables access to vibrational information from mid over far infrared to even terahertz domains. This information may prove critical for the elucidation of fundamental bio-molecular phenomena including folding-mediated innate host defence mechanisms. Antimicrobial peptides (AMPs) represent one of such phenomena. These are major effector molecules of the innate immune system, which favour attack on microbial membranes. AMPs recognise and bind to the membranes whereupon they assemble into pores or channels destabilising the membranes leading to cell death. However, specific molecular interactions responsible for antimicrobial activities have yet to be fully understood. Herein we probe such interactions by assessing molecular specific variations in the near-THz 400-40 cm-1 range for defined helical AMP templates in reconstituted phospholipid membranes. In particular, we show that a temperature-dependent spectroscopic analysis, supported by 2D correlative tools, provides direct evidence for the membrane-induced and folding-mediated activity of AMPs. The far-FTIR study offers a direct and information-rich probe of membrane-related antimicrobial interactions.

Rigorous calculations and synchrotron radiation measurements of diffraction efficiencies for tender X-ray lamellar gratings: conical versus classical diffraction.

When reflection gratings are operated at grazing incidence in the extreme off-plane configuration and the incident beam trajectory is parallel to the grooves, the diffraction into the first order can be more efficient than in the classical orientation. This situation is referred to as the conical diffraction case. In the classical configuration the grooves are perpendicular to the incident beam and thus an efficiency-reducing shadowing effect will be observed at very grazing angles. It was recently shown that a laminar grating could provide symmetric and relatively high efficiencies in conical diffraction for diffraction even of photons with large energies of the order of 4 and 6 keV. For photon energies in the tender X-ray range, accurate computing tools for the calculation of diffraction efficiencies from gratings with simple coatings have not been available. Promising results for this spectral range now require the development of tools for modelling the diffraction efficiency expected in optical instrumentation, in which the provision of high efficiency in the indicated spectral range is mandatory. This is the case when weak sources are to be investigated, like in space science. In this study it will be shown that scalar calculations are not appropriate for this purpose, while newly introduced rigorous calculations based on the boundary integral equation method, implemented in the PCGrate® code, can provide predictions that are in agreement with observed diffraction efficiencies. The agreement is achieved by modelling the exact surface profile. This applies for both the conical diffraction configuration and for the classical in-plane configuration, in which a significantly lower efficiency was obtained. Even though the profile of the presented grating was not perfect, but significantly distorted, the calculations show that efficiency-wise the structure provided already more than 75% of the ideally expected efficiency for conical diffraction. This is a very promising result for further optimization of diffraction gratings for use in the tender X-ray range.

Comparison of multiple X-ray fluorescence techniques for elemental analysis of particulate matter collected on air filters.

This work reports on qualitative and semi-quantitative elemental analysis of particulate matter (PM) collected on PTFE membrane filters, for a source apportionment study conducted in Brescia (Italy). Sampling was undertaken in a residential area where an increase in Mn emissions has been highlighted by previous studies. Filters are measured by means of X-ray Fluorescence (XRF) based techniques such as micro-XRF and grazing incidence XRF using synchrotron radiation, Mo or W excitation sources, after applying an automatized sample preparation method. A heterogeneous distribution in PM shape, size and composition was observed, with features typical of anthropogenic sources. XRF measurements performed at various incidence angle, on large areas and different experimental setup were reproducible. The results demonstrate a successful comparison of the various XRF instrumentation, and the decrease in Mn content with the distance away from the identified emission source. This work highlights the potentialities of the presented approach to provide a full quantitative analysis, and ascertain its suitability for providing a direct, fast, simple and sensitive elemental analysis of filters in source apportionment studies and screening purposes.

On amplitude beam splitting of tender X-rays (2-8†keV photon energy) using conical diffraction from reflection gratings with laminar profile.

Conical diffraction is obtained when a radiation beam impinges onto a periodically ruled surface structure parallel or almost parallel to the ruling. In this condition the incident intensity is diffracted through an arc, away from the plane of incidence. The diffracted intensity thus lies on a cone, which leads to the name `conical diffraction'. In this configuration almost no part of the ruled structure will produce any shadowing effect for the incident or the diffracted beam. Then, compared with a grating in the classical orientation, relatively higher diffraction efficiencies will be observed for fewer diffraction orders. When the incident beam is perfectly parallel to the grooves of a rectangular grating profile, the symmetry of the setup causes diffraction of the intensity symmetrically around the plane of incidence. This situation was previously tested experimentally in the VUV spectral range for the amplitude beam splitting of a radiation beam with a photon energy of 25 eV. In this case the ideally expected beam splitting efficiency of about 80% for the diffraction into the two first orders was confirmed for the optimum combination of groove depth and angle of grazing incidence. The feasibility of the amplitude beam splitting for hard X-rays with 12 keV photon energy by use of the same concept was theoretically confirmed. However, no related experimental data are presented yet, not even for lower energy soft X-rays. The present study reports the first experimental data for the conical diffraction from a rectangular grating profile in the tender X-ray range for photon energies of 4 keV and 6 keV. The expected symmetries are observed. The maximum absolute efficiency for beam splitting was measured to be only about 30%. As the reflectivity of the grating coating at the corresponding angle of grazing incidence was found to be only of the order of 50%, the relative beam splitting efficiency was thus 60%. This is to be compared also here with an ideally expected relative efficiency of 80%. It is predicted that a beam splitting efficiency exceeding 50% should be possible by use of more appropriate materials.

On symmetric X-ray beam splitting with high efficiency by use of reflection gratings with rectangular profile in the extreme off-plane configuration.

In order to be reflected or diffracted off a surface structure soft X-rays and hard X-rays need to impinge at grazing angles of incidence onto the surface. In case of a reflection grating of highly symmetric structure with rectangular groove profile these grooves can be oriented parallel to the beam trajectory. In such a symmetric situation the distribution of the diffracted intensity with respect to the plane of incidence is then expected to be symmetric. This is indeed observed with symmetrically oriented diffraction peaks. It can be predicted that for appropriate structure parameters the intensity can be contained mostly in two symmetrically oriented diffraction peaks. This will also be the case for hard X-rays. The diffraction efficiency will be particularly high, when the angle of grazing incidence is chosen in the total reflection regime below the critical angle of the grating coating. These predictions were experimentally verified in this work for hard X-rays with photon energies between 4 keV and 12.4 keV. In the experiment of the order of 30% of the incident intensity was diffracted into the two first orders. This is to be compared to reflectivities of the order of 50% measured at the same coating in an unruled area of the substrate. Consequently the relative structural diffraction efficiency for each first order was about 30%, while ideally it could have been 40%. The presented grating structure will thus be a rather efficient amplitude beam splitter for hard X-rays, e.g. in the coherent beam from a free electron laser. In addition such object could then be used as the first component in Michelson interferometers for the beam characterisation or for introducing a time delay between two coherent beams.

Qualifying label components for effective biosensing using advanced high-throughput SEIRA methodology.

The need for technological progress in bio-diagnostic assays of high complexity requires both fundamental research and constructing efforts on nano-scaled assay recognition elements that can provide unique selectivity and design-enhanced sensitivity features. Nanoparticle induced sensitivity enhancement and its application related to multiplexed capability Surface-Enhanced InfraRed Absorption (SEIRA) assay formats are well suitable for these purposes. The potential of diverse fluorophore-antibody conjugates, being chemisorbed onto low-cost gold nanoparticulate SEIRA substrates, has been explored with respect to their spectral discriminability. These novel biolabels deliver molecular SEIRA fingerprints that have been successfully analyzed by both uni- and multivariate analyzing tools, to discriminate their multiplexing capabilities. We show that this robust spectral encoding via SEIRA fingerprints opens up new opportunities for a fast, reliable and multiplexed high-end screening in biodiagnostics.

Relevance for food sciences of quantitative spatially resolved element profile investigations in wheat (Triticum aestivum) grain.

Bulk element concentrations of whole grain and element spatial distributions at the tissue level were investigated in wheat (Triticum aestivum) grain grown in Zn-enriched soil. Inductively coupled plasma mass spectrometry and inductively coupled plasma optical emission spectrometry were used for bulk analysis, whereas micro-proton-induced X-ray emission was used to resolve the two-dimensional localization of the elements. Soil Zn application did not significantly affect the grain yield, but did significantly increase the grain Ca, Fe and Zn concentrations, and decrease the grain Na, P and Mo concentrations; bulk Mg, S, K, Mn, Cu, Cd and Pb concentrations remained unchanged. These changes observed in bulk element concentrations are the reflection of tissue-specific variations within the grain, revealing that Zn application to soil can lead to considerable alterations in the element distributions within the grain, which might ultimately influence the quality of the milling fractions. Spatially resolved investigations into the partitioning of the element concentrations identified the tissues with the highest element concentrations, which is of utmost importance for accurate prediction of element losses during the grain milling and polishing processes.

Beeswax as dental filling on a neolithic human tooth.

Evidence of prehistoric dentistry has been limited to a few cases, the most ancient dating back to the Neolithic. Here we report a 6500-year-old human mandible from Slovenia whose left canine crown bears the traces of a filling with beeswax. The use of different analytical techniques, including synchrotron radiation computed micro-tomography (micro-CT), Accelerator Mass Spectrometry (AMS) radiocarbon dating, Infrared (IR) Spectroscopy and Scanning Electron Microscopy (SEM), has shown that the exposed area of dentine resulting from occlusal wear and the upper part of a vertical crack affecting enamel and dentin tissues were filled with beeswax shortly before or after the individual's death. If the filling was done when the person was still alive, the intervention was likely aimed to relieve tooth sensitivity derived from either exposed dentine and/or the pain resulting from chewing on a cracked tooth: this would provide the earliest known direct evidence of therapeutic-palliative dental filling.

New insights into globoids of protein storage vacuoles in wheat aleurone using synchrotron soft X-ray microscopy.

Mature developed seeds are physiologically and biochemically committed to store nutrients, principally as starch, protein, oils, and minerals. The composition and distribution of elements inside the aleurone cell layer reflect their biogenesis, structural characteristics, and physiological functions. It is therefore of primary importance to understand the mechanisms underlying metal ion accumulation, distribution, storage, and bioavailability in aleurone subcellular organelles for seed fortification purposes. Synchrotron radiation soft X-ray full-field imaging mode (FFIM) and low-energy X-ray fluorescence (LEXRF) spectromicroscopy were applied to characterize major structural features and the subcellular distribution of physiologically important elements (Zn, Fe, Na, Mg, Al, Si, and P). These direct imaging methods reveal the accumulation patterns between the apoplast and symplast, and highlight the importance of globoids with phytic acid mineral salts and walls as preferential storage structures. C, N, and O chemical topographies are directly linked to the structural backbone of plant substructures. Zn, Fe, Na, Mg, Al, and P were linked to globoid structures within protein storage vacuoles with variable levels of co-localization. Si distribution was atypical, being contained in the aleurone apoplast and symplast, supporting a physiological role for Si in addition to its structural function. These results reveal that the immobilization of metals within the observed endomembrane structures presents a structural and functional barrier and affects bioavailability. The combination of high spatial and chemical X-ray microscopy techniques highlights how in situ analysis can yield new insights into the complexity of the wheat aleurone layer, whose precise biochemical composition, morphology, and structural characteristics are still not unequivocally resolved.

Low-energy X-ray fluorescence microscopy opening new opportunities for bio-related research.

Biological systems are unique matter with very complex morphology and highly heterogeneous chemical composition dominated by light elements. Discriminating qualitatively at the sub-micrometer level the lateral distribution of constituent elements, and correlating it to the sub-cellular biological structure, continues to be a challenge. The low-energy X-ray fluorescence microspectroscopy, recently implemented in TwinMic scanning transmission mode, has opened up new opportunities for mapping the distribution of the light elements, complemented by morphology information provided by simultaneous acquisition of absorption and phase contrast images. The important new information that can be obtained in bio-related research domains is demonstrated by two pilot experiments with specimens of interest for marine biology and food science. They demonstrate the potential to yield important insights into the structural and compositional enrichment, distribution and correlation of essential trace elements in the lorica of Tintinnopsis radix, and the lateral distribution of trace nutrients in the seeds of wheat Triticum aestivum.

Apatite-mediated actin dynamics in resorbing osteoclasts.

The actin cytoskeleton is essential for osteoclasts main function, bone resorption. Two different organizations of actin have been described in osteoclasts, the podosomes belt corresponding to numerous F-actin columns arranged at the cell periphery, and the sealing zone defined as a unique large band of actin. To compare the role of these two different actin organizations, we imaged osteoclasts on various substrata: glass, dentin, and apatite. Using primary osteoclasts expressing GFP-actin, we found that podosome belts and sealing zones, both very dynamic actin structures, were present in mature osteoclasts; podosome belts were observed only in spread osteoclasts adhering onto glass, whereas sealing zone were seen in apico-basal polarized osteoclasts adherent on mineralized matrix. Dynamic observations of several resorption cycles of osteoclasts seeded on apatite revealed that 1) podosomes do not fuse together to form the sealing zone; 2) osteoclasts alternate successive stationary polarized resorption phases with a sealing zone and migration, nonresorption phases without any specific actin structure; and 3) apatite itself promotes sealing zone formation though c-src and Rho signaling. Finally, our work suggests that apatite-mediated sealing zone formation is dependent on both c-src and Rho whereas apico-basal polarization requires only Rho.

Poorly crystalline apatites: evolution and maturation in vitro and in vivo.

Poorly crystalline apatites (PCA) are the major mineral component of mineralized tissues in vertebrates. Their physical-chemical properties are, however, not very well known due to their relative instability and the difficulties to characterize nanocrystalline compounds. Several studies using spectroscopic techniques (Fourier transform infrared [FTIR]; 31P nuclear magnetic resonance [NMR]) have demonstrated the existence, both in precipitated and biological PCA, of labile non-apatitic environments of the mineral ions. These environments are involved in the high surface reactivity and evolution ability of PCA and they are believed to form a hydrated layer at the surface of the nanocrystals in aqueous media. The extent of the hydrated layer may vary considerably depending on the conditions of precipitation and maturation time. As PCA age, the decrease of the non-apatitic environments proportion is associated with a decrease of intracrystalline disorder and an increase of stable apatitic domains. For synthetic and biological apatites, the carbonation rate of the mineral and the uptake of essential or toxic trace elements can be related to the maturation processes. The mineral ions of the hydrated layer can be easily and reversibly substituted by other ions which can either be included in the growing stable apatite lattice during maturation or remain in the hydrated layer. In addition, the non-apatitic environments seem to be involved in the binding of soluble non-collagenic proteins. This phenomenon could be related to calcium phosphate formation; we showed that, at an albumin concentration close to that in human serum, this protein has an inhibitory effect on octacalcium phosphate crystallization on collagen in vitro.