Location, speciation, and quantification of carbon in silica phytoliths using synchrotron scanning transmission X-ray microspectroscopy.

Phytoliths of biogenic silica play a vital role in the silicon biogeochemical cycle and occlude a fraction of organic carbon. The location, chemical speciation, and quantification of this carbon within phytoliths have remained elusive due to limited direct experimental evidence. In this work, phytoliths (bilobate morphotype) from the sugarcane stalk epidermis are sectioned with a focused ion beam to produce lamellas (≈10 × 10 µm2 size, <500 nm thickness) and probed by synchrotron scanning transmission X-ray microspectroscopy (≈100-200 nm pixel size; energies near the silicon and carbon K-absorption edges). Analysis of the spectral image stacks reveals the complementarity of the silica and carbon spatial distributions, with carbon found at the borders of the lamellas, in islands within the silica, and dispersed in extended regions that can be described as a mixed silica-carbonaceous matrix. Carbon spectra are assigned mainly to lignin-like compounds as well as to proteins. Carbon contents of 3-14 wt.% are estimated from the spectral maps of four distinct phytolith lamellas. The results provide unprecedented spatial and chemical information on the carbon in phytoliths obtained without interference from wet-chemical digestion.

Histomorphometric evaluation, SEM, and synchrotron analysis of the biological response of biodegradable and ceramic hydroxyapatite-based grafts: from the synthesis to the bed application.

This study aimed to analyze the physicochemical and histological properties of nanostructured hydroxyapatite and alginate composites produced at different temperatures with and without sintering and implanted in rabbit tibiae. Hydroxyapatite-alginate (HA) microspheres (425-600 µm) produced at 90 and 5 °C without (HA90 and HA5) or with sintering at 1000 °C (HA90S and HA5S) were characterized and applied to evaluate thein vitrodegradation; also were implanted in bone defects on rabbit's tibiae (n= 12). The animals were randomly divided into five groups (blood clot, HA90S, HA5S, HA90, and HA5) and euthanized after 7 and 28 d. X-ray diffraction and Fourier-transform infrared analysis of the non-sintered biomaterials showed a lower crystallinity than sintered materials, being more degradablein vitroandin vivo. However, the sinterization of HA5 led to the apatite phase's decomposition into tricalcium phosphate. Histomorphometric analysis showed the highest (p< 0.01) bone density in the blood clot group, similar bone levels among HA90S, HA90, and HA5, and significantly less bone in the HA5S. HA90 and HA5 groups presented higher degradation and homogeneous distribution of the new bone formation onto the surface of biomaterial fragments, compared to HA90S, presenting bone only around intact microspheres (p< 0.01). The elemental distribution (scanning electron microscope and energy dispersive spectroscopy andµXRF-SR analysis) of Ca, P, and Zn in the newly formed bone is similar to the cortical bone, indicating bone maturity at 28 d. The synthesized biomaterials are biocompatible and osteoconductive. The heat treatment directly influenced the material's behavior, where non-sintered HA90 and HA5 showed higher degradation, allowing a better distribution of the new bone onto the surface of the biomaterial fragments compared to HA90S presenting the same level of new bone, but only on the surface of the intact microspheres, potentially reducing the bone-biomaterial interface.

Monte Carlo simulations of synchrotron X-ray dose affecting root growth during in vivo tomographic imaging.

Synchrotron X-ray computed tomography (XCT) has been increasingly applied to study the in vivo dynamics of root growth and rhizosphere processes. However, minimizing radiation-induced damage to root growth warrants further investigation. Our objective was to develop a robust approach for modeling and evaluating ways to reduce synchrotron X-ray dose effects on root growth during in vivo imaging. Wheat roots growing in soil were exposed to X-rays during XCT experiments resolved in space (3D) plus time (4D). The dose rate and cumulative absorbed dose in roots were modelled using the Monte Carlo code FLUKA for different experimental conditions of polychromatic and quasi-monochromatic X-ray beam configurations. The most impactful factors affecting damage to roots were incident X-ray energy spectrum, stored current in the accelerator machine, position of the root in the soil, and possibly the number of exposures during the 4D XCT experiments. Our results imply that radiation dose during in vivo imaging of plant roots can be diminished by using monochromatic radiation at the highest energy suitable for a given sample thickness and field of view, and by controlling the rotation axis of off-centered roots to increase attenuation of radiation by the soil matrix.

Characterization of Systemic Disease Development and Paw Inflammation in a Susceptible Mouse Model of Mayaro Virus Infection and Validation Using X-ray Synchrotron Microtomography.

Mayaro virus (MAYV) is an emerging arthropod-borne virus endemic in Latin America and the causative agent of arthritogenic febrile disease. Mayaro fever is poorly understood; thus, we established an in vivo model of infection in susceptible type-I interferon receptor-deficient mice (IFNAR-/-) to characterize the disease. MAYV inoculations in the hind paws of IFNAR-/- mice result in visible paw inflammation, evolve into a disseminated infection and involve the activation of immune responses and inflammation. The histological analysis of inflamed paws indicated edema at the dermis and between muscle fibers and ligaments. Paw edema affected multiple tissues and was associated with MAYV replication, the local production of CXCL1 and the recruitment of granulocytes and mononuclear leukocytes to muscle. We developed a semi-automated X-ray microtomography method to visualize both soft tissue and bone, allowing for the quantification of MAYV-induced paw edema in 3D with a voxel size of 69 µm3. The results confirmed early edema onset and spreading through multiple tissues in inoculated paws. In conclusion, we detailed features of MAYV-induced systemic disease and the manifestation of paw edema in a mouse model extensively used to study infection with alphaviruses. The participation of lymphocytes and neutrophils and expression of CXCL1 are key features in both systemic and local manifestations of MAYV disease.

Conjunctive use of synchrotron X-ray diffraction and Rietveld refinement in Fe-oxide clays for forensic applications.

Soils have distinctive chemical, physical, mineralogical and biological properties, which make it possible to differentiate them in different environments and also to test for an association of a particular questioned item with a crime scene. Mineral compositions and structures in the soil clay fraction can reflect a distinct characteristic when analyzed by powerful techniques. In this way, the aim of this study was to compare the discriminative power of Fe-oxides concentrated clay samples from the same soil class with and without differences in parent material when analyzed using the Rietveld method and two sources of X-ray diffraction: conventional and high-resolution synchrotron radiations. Clay samples were Fe concentrated (kaolinite and gibbsite removal) to clarify mineralogical composition of 12 samples from three simulated crime scenes, developed under claystone and granite. By Rietveld refinement, detailed crystallographic data were obtained to discriminate samples according their provenance. By synchrotron radiation, mineral data demonstrated the power of quantitative (crystallite size and minerals contents) and qualitative (identification of majoritarian and accessory minerals) analysis by Rietveld refinement, which provides well-resolved data able to discriminate samples from different and same geology. The techniques can be applied in other criminal investigations given their potential of discrimination.

Pressure-induced phase transitions in MBANP crystal: A study by synchrotron radiation X-ray diffraction and Raman spectroscopy.

Raman spectroscopy and synchrotron radiation X-ray diffraction have been used to study the effect of pressure on 2-(-α-methylbenzylamino)-5-dinitropyridine (MBANP). Several changes are observed with increasing pressure in the Raman spectra of this system, such as splitting of various bands and disappearance of bands. Discontinuous shifts in wavenumber vs pressure plot indicate that a conformational phase transition takes place around 0.5 GPa. The behavior of the Raman spectra indicates that MBANP molecules present conformational phase transition at high-pressure. X-ray diffraction, performed with synchrotron radiation, confirms the conformational changes observed by Raman experiments around 0.5 GPa. The pressure provokes a rotational movement of the benzene ring which can be associated with the conformational phase transition.

Knowledge-Based Atomic Polarizabilities Used to Model Circular Dichroism Spectra of Proteins.

We present a model of circular dichroism for proteins, which is mainly based on both the classical theory of optical activity and a series of effective atomic polarizabilities. Such polarizabilities are extracted from the analysis of a set of synchrotron radiation circular dichroism spectra and their corresponding three-dimensional structures from the Protein Data Bank. Each modeled spectrum is obtained from the protein atomic coordinates and the identification of its secondary structure elements. The resulting spectra are in good agreement with additional experimental data and also with the predictions of some other models. Among them, only our approach is able to describe the effect of d-amino acids. Moreover, our model is also utilized to evaluate protein reconstructions as well as structural changes.

Cubosomal lipid nanoassemblies with pH-sensitive shells created by biopolymer complexes: A synchrotron SAXS study.

We report a strategy for sustainable development of pH-responsive cubic liquid crystalline nanoparticles (cubosomes), in which the structure-defining lyotropic nonlamellar lipid and the eventually encapsulated guest molecules can be protected by pH-sensitive polyelectrolyte shells with mucoadhesive properties. Bulk non-lamellar phases as well as pH-responsive polyelectrolyte-modified nanocarriers were formed by spontaneous assembly of the nonlamellar lipid monoolein and two biopolymers tailored in nanocomplexes with pH-dependent net charge. The mesophase particles involved positively charged N-arginine-modified chitosan (CHarg) and negatively charged alginate (ALG) chains assembled at different biopolymer concentrations and charge ratios into a series of pH-responsive complexes. The roles of Pluronic F127 as a dispersing agent and a stabilizer of the nanoscale dispersions were examined. Synchrotron small-angle X-ray scattering (SAXS) investigations were performed at several N-arginine-modified chitosan/alginate ratios (CHarg/ALG with 10, 15 and 20 wt% ALG relative to CHarg) and varying pH values mimicking the pH conditions of the gastrointestinal route. The structural parameters characterizing the inner cubic liquid crystalline organizations of the nanocarriers were determined as well as the particle sizes and stability on storage. The surface charge variations, influencing the measured zeta-potentials, evidenced the inclusion of the CHarg/ALG biopolymer complexes into the lipid nanoassemblies. The polyelectrolyte shells rendered the hybrid cubosome nanocarriers pH-sensitive and influenced the swelling of their lipid-phase core as revealed by the acquired SAXS patterns. The pH-responsiveness and the mucoadhesive features of the cubosomal lipid/polyelectrolyte nanocomplexes may be of interest for in vivo drug delivery applications.

Acoustic levitation and high-resolution synchrotron X-ray powder diffraction: A fast screening approach of niclosamide amorphous solid dispersions.

The levitation of samples in an acoustic field has been of interest in the preparation and study of amorphous solid dispersions (ASD). Here, niclosamide-polymer solutions were levitated in a multi-emitter single-axis acoustic levitator and analyzed for 10 min at a High-resolution synchrotron X-ray powder diffraction beamline. This assembly enabled high-quality and fast time-resolved measurements with microliter sample size and measurement of solvent evaporation and recrystallization of niclosamide (NCL). Polymers HPMCP-55S, HPMCP-50, HPMCP-55, Klucel®, and poloxamers were not able to form amorphous dispersions with NCL. Plasdone® and Soluplus® demonstrated excellent properties to form NCL amorphous dispersions, with the last showing superior solubility enhancement. Furthermore, this fast levitation polymer screening showed good agreement with results obtained by conventional solvent evaporation screening evaluated for five days in a stability study, carried out at 40 °C/75% RH. The study showed that acoustic levitation and high-resolution synchrotron combination opens up a new horizon with great potential for accelerating ASD formulation screening and analysis.

Arabidopsis thaliana zinc accumulation in leaf trichomes is correlated with zinc concentration in leaves.

Zinc (Zn) is a key micronutrient for plants and animals, and understanding Zn homeostasis in plants can improve both agriculture and human health. While root Zn transporters in plant model species have been characterized in detail, comparatively little is known about shoot processes controlling Zn concentrations and spatial distribution. Previous work showed that Zn hyperaccumulator species such as Arabidopsis halleri accumulate Zn and other metals in leaf trichomes. To date there is no systematic study regarding Zn accumulation in the trichomes of the non-accumulating, genetic model species A. thaliana. Here, we used Synchrotron X-Ray Fluorescence mapping to show that Zn accumulates at the base of trichomes of A. thaliana. Using transgenic and natural accessions of A thaliana that vary in bulk leaf Zn concentration, we demonstrate that higher leaf Zn increases total Zn found at the base of trichome cells. Our data indicates that Zn accumulation in trichomes is a function of the Zn status of the plant, and provides the basis for future studies on a genetically tractable plant species to understand the molecular steps involved in Zn spatial distribution in leaves.

Synchrotron microtomography applied to the volumetric analysis of internal structures of Thoropa miliaris tadpoles.

Amphibians are models for studying applied ecological issues such as habitat loss, pollution, disease, and global climate change due to their sensitivity and vulnerability to changes in the environment. Developmental series of amphibians are informative about their biology, and X-ray based 3D reconstruction holds promise for quantifying morphological changes during growth-some with a direct impact on the possibility of an experimental investigation on several of the ecological topics listed above. However, 3D resolution and discrimination of their soft tissues have been difficult with traditional X-ray computed tomography, without time-consuming contrast staining. Tomographic data were initially performed (pre-processing and reconstruction) using the open-source software tool SYRMEP Tomo Project. Data processing and analysis of the reconstructed tomography volumes were conducted using the segmentation semi-automatic settings of the software Avizo Fire 8, which provide information about each investigated tissues, organs or bone elements. Hence, volumetric analyses were carried out to quantify the development of structures in different tadpole developmental stages. Our work shows that synchrotron X-ray microtomography using phase-contrast mode resolves the edges of the internal tissues (as well as overall tadpole morphology), facilitating the segmentation of the investigated tissues. Reconstruction algorithms and segmentation software played an important role in the qualitative and quantitative analysis of each target structure of the Thoropa miliaris tadpole at different stages of development, providing information on volume, shape and length. The use of the synchrotron X-ray microtomography setup of the SYRMEP beamline of Elettra Synchrotron, in phase-contrast mode, allows access to volumetric data for bone formation, eye development, nervous system and notochordal changes during the development (ontogeny) of tadpoles of a cycloramphid frog Thoropa miliaris. As key elements in the normal development of these and any other frog tadpole, the application of such a comparative ontogenetic study, may hold interest to researchers in experimental and environmental disciplines.

Characterization of Schistosoma mansoni Dihydrofolate Reductase (DHFR).

Dihydrofolate reductase (DHFR) is an essential enzyme for nucleotide metabolism used to obtain energy and structural nucleic acids. Schistosoma mansoni has all the pathways for pyrimidine biosynthesis, which include the thymidylate cycle and, consequentially, the DHFR enzyme. Here, we describe the characterization of Schistosoma mansoni DHFR (SmDHFR) using isothermal titration calorimetry for the enzymatic activity and thermodynamic determination, also the folate analogs inhibition. Moreover, X-ray crystallography was used to determine the enzyme atomic model at 1.95 Å.

Uncommon chemical species in PM2.5 and PM10 and its potential use as industrial and vehicular markers for source apportionment studies.

Chemical characterization of PM2.5 and PM10 is important to identify potential compounds that induce biological responses that translate into cardio-respiratory health problems. This study shows the reliability of the use of crystalline phases, identified in samples from receptor sites, as source markers, helping researchers to infer the main sources of air pollution, even without the use of receptor models. PM2.5 and PM10 samples were collected at two sites in an urban industrialized region located at southeast of Brazil and analyzed by Synchrotron X-ray Diffraction to identify crystalline compounds. Results show 5 PM10 and PM2.5 species not previously reported in the literature. We propose reaction mechanisms for these species and identify specific sources for each crystalline phase found: BaTiO3 was found in PM10 receptor samples and proved to be a vehicular marker formed during brake action; maghemite (γ-Fe2O3), pyracmonite [(NH4)3Fe(SO4)3], ammonium perchlorate (NH3OHClO4) and potassium ferrate (K2Fe2O4) were found in PM2.5 proved to be markers of industrial activities. The crystalline phases found in PM samples from receptor sites and the mechanisms of reactions showed the reliability of the use of crystalline phases as source markers in the identification of potential sources of air pollution without misinterpretation of the likely source.

Synchrotron infrared nanospectroscopy on a graphene chip.

A recurring goal in biology and biomedicine research is to access the biochemistry of biological processes in liquids that represent the environmental conditions of living organisms. These demands are becoming even more specific as microscopy techniques are fast evolving in the era of single cell analysis. In the modality of chemical probes, synchrotron infrared spectroscopy (µ-FTIR) is a technique that is extremely sensitive to vibrational responses of materials; however, the classical optical limits prevent the technique to access the biochemistry of specimens at the subcellular level. In addition, due to the intricate environmental requirements and strong infrared absorption of water, µ-FTIR of bioprocesses in liquids remains highly challenging. In phase with these challenges, on-chip liquid cells emerge as a versatile alternative to control the water thickness while providing a biocompatible chemical environment for analytical analyses. In this work we report the development of a liquid platform specially designed for nanoscale infrared analysis of biomaterials in wet environments. A key advantage of our designed platform is the use of graphene as an optical window that interfaces wet and dry environments in the liquid cell. By combining near-field optical microscopy and synchrotron infrared radiation, we measure the nanoscale fingerprint IR absorbance of a variety of liquids often used in biological studies. Further, we demonstrate the feasibility of the platform for the chemical analysis of protein clusters immersed in water with a clear view of the proteins' secondary structure signatures. The simplicity of the proposed platform combined with the high quality of our data makes our findings a template for future microfluidic devices targeting dynamic nanoscale-resolved chemical analysis.

Topsoil pollution in highway medians in the State of São Paulo (Brazil): determination of potentially toxic elements using synchrotron radiation total reflection X-ray fluorescence.

The presence of metals in vehicle emissions is due to several factors, such as the composition of fuels and lubricating oils, the wear of engine components, and their use in catalytic converters. It is known that the soil near highways is greatly affected by heavy vehicle traffic, since the use of highways is of fundamental importance for the flow of goods and public transport, especially in Brazil, street transport being the main form of transport in the country. Considering the scenario described, the main objective of this study was to monitor the concentration of potentially toxic elements in surface soils located on the medians of the main access highways in the city of Campinas (SP-Brazil) and classify the soils through geoaccumulation index. Using SR-TXRF it was possible to detect and determine the concentrations of 5 elements of toxic-environmental interest (Cr, Ni, Cu, Zn, and Pb) and 11 natural soil composition elements (Al, Si, S, Cl, K, Ca, Ti, Mn, Fe, Rb, and Sr). To evaluate the influence of highway distance on elementary concentrations, ANOVA and Tukey statistical tests were applied. Nickel, Cu, and Zn showed a decrease in their concentrations moving away from the highway, indicating their relation with vehicular emissions. Applying principal components analysis (PCA), it was possible to identify four groups of the quantified elements: those mainly related to the soil itself, those produced by traffic of automotive vehicles, and those emitted by industrial activities.

Challenging identification of polymorphic mixture: Polymorphs I, II and III in olanzapine raw materials.

Olanzapine (OLZ), a drug for the treatment of schizophrenia, presents in more than 60 crystal forms. Polymorphs I, II and III were reported, however, the preparation conditions for pure II and III have not been reported. Polymorph IV was reported but this form is actually polymorph II described at different temperature. The diversity of solid forms of OLZ, the change in the nomenclature found in the literature and the presence of polymorphic mixture in samples, increase the difficulty for a correct solid state characterization. Therefore, the goal was the polymorphic identification of three OLZ raw materials, highlighting the limitation of conventional techniques (typically used in analytical control) and the necessity to use a combination of advanced ones to solve this challenge. The samples were studied by conventional techniques such as powder X-ray diffraction, thermoanalytical techniques, infrared spectroscopy. In apart from that, synchrotron powder X-ray diffraction (SPXRD) and solid state nuclear magnetic resonance (ss-NMR) were used. All samples were in accordance with the pharmacopoeia criteria. However, the conventional techniques were not specific for the complete polymorphic identification. Therefore, a combination of advanced techniques (SPXRD and ss-NMR) was necessary to identify the mixture of polymorphs (I, II and III) in all samples.

Location of water in fresh sugarcane bagasse observed by synchrotron X-ray microtomography.

Sugarcane bagasse is a vast lignocellulosic byproduct generated in the industry with ~50% humidity (1 kg dry matter associated with 1 kg water). Although the presence of water brings deleterious consequences for combustion, storage and sugar extraction, the location of water in fresh bagasse remains unknown. In this work, we use synchrotron X-ray microtomography for non-invasive 3D imaging of fresh bagasse particles, which allows the visualization of intraparticle water. The sclerified fiber cells in the sheaths surrounding xylem vessels are often found full of water. We suggest this can be juice preserved from the native stalks as many sclerified fibers seem to keep their structural integrity despite the mechanical action during sugarcane crushing. The microtomograms of fresh bagasse also shows mineral particles adhered to biomass surfaces, with adhesion presumably favored by the presence of water. In summary, this work unveils the location of water in fresh bagasse, solving an old mystery of sugarcane technology.

Resonant Synchrotron X-ray Diffraction determines markers for iron-rich atmospheric particulate matter in urban region.

Particulate matter driven health problems are strongly associated with its chemical composition. Despite the benefits of using source apportionment models for air quality management, limitations such as collinearity effects, restrict their application or compromise the accurate separation of sources, particularly for particulate matter with similar chemical profiles. Receptors models also depend on the operator expertise to appropriately classified sources, a subjective process that can lead to biased results. For highly correlated sources, the identification of specific markers is still the best way to achieve proper source apportionment. In this study, Resonant Synchrotron X-ray Diffraction has been applied to the analysis of atmospheric particles to determine markers for industrial and vehicular sources in the Region of Greater Vitória, Brazil. Total suspended particulate matter, PM10, and PM2.5 samples were analyzed by Resonant Synchrotron X-ray Diffraction showing high levels of iron-based crystalline phases. In comparison to the use of chemical elemental species, the identification of the crystalline phases provided an enhanced approach to classify specific iron-based source markers. For this study, α-Fe2O3 was identified with iron-based sources such as iron ore, pelletizing, and sintering; metallic Fe was inferred with blast furnaces and steelmaking; FeS2 was correlated with coal deposits; and K2Fe2O4 was associated to sintering emissions. Elemental carbon with different X-ray diffraction patterns enabled the differentiation of industrial and vehicular sources. The attribution of crystal rather than elemental composition in the identification of sources improves the accuracy of source apportionment studies.

High-resolution synchrotron-based X-ray microtomography as a tool to unveil the three-dimensional neuronal architecture of the brain.

The assessment of neuronal number, spatial organization and connectivity is fundamental for a complete understanding of brain function. However, the evaluation of the three-dimensional (3D) brain cytoarchitecture at cellular resolution persists as a great challenge in the field of neuroscience. In this context, X-ray microtomography has shown to be a valuable non-destructive tool for imaging a broad range of samples, from dense materials to soft biological specimens, arisen as a new method for deciphering the cytoarchitecture and connectivity of the brain. In this work we present a method for imaging whole neurons in the brain, combining synchrotron-based X-ray microtomography with the Golgi-Cox mercury-based impregnation protocol. In contrast to optical 3D techniques, the approach shown here does neither require tissue slicing or clearing, and allows the investigation of several cells within a 3D region of the brain.