Search

The recent upgrading of synchrotron radiation (SR) sources has favored, in the last few years, the construction and design of beamlines optimized for the study of cultural heritage materials, which may require ad hoc setups, specific spatial resolutions, and detection limits. In the field of cultural heritage, integrated approaches combining different techniques are often required, even at large facilities, where some beamlines offer the possibility of performing different types of measurements at the same point of analysis, complementing preliminary information usually obtained by conventional laboratory and/or portable in situ methods. An overview of the last ten years of synchrotron applications for the study of pigments is given, with discussion of upstream and downstream challenges to methods and techniques. The possibilities offered by the synchrotron techniques are illustrated by a case study of a particular class of painted ceramics, as an example of different research questions that are solved by a combination of SR-based methods. [ABSTRACT FROM AUTHOR]

Even though measurements in cryogenic conditions are the preferred choice for single-cell and plant analyses to reduce sample preparation artefacts, preserve the integrity of samples and prevent loss of some chemical elements, cryo-fixation of human or animal soft tissue for X-ray fluorescence analysis still remains challenging. Here we report, compare and discuss analyses of bovine ovarian tissues performed in cryogenic or room temperature conditions, the latter both chemically fixed or freeze-dried. [ABSTRACT FROM AUTHOR]

Computational techniques allow breaking the limits of traditional imaging methods, such as time restrictions, resolution, and optics flaws. While simple computational methods can be enough for highly controlled microscope setups or just for previews, an increased level of complexity is instead required for advanced setups, acquisition modalities or where uncertainty is high; the need for complex computational methods clashes with rapid design and execution. In all these cases, Automatic Differentiation, one of the subtopics of Artificial Intelligence, may offer a functional solution, but only if a GPU implementation is available. In this paper, we show how a framework built to solve just one optimisation problem can be employed for many different X-ray imaging inverse problems. [ABSTRACT FROM AUTHOR]

An improved understanding of an ovary's structures is highly desirable to support advances in folliculogenesis knowledge and reproductive medicine, with particular attention to fertility preservation options for prepubertal girls with malignant tumors. Although currently the golden standard for structural analysis is provided by combining histological sections, staining, and visible 2D microscopic inspection, synchrotron radiation phase-contrast microtomography is becoming a new challenge for three-dimensional studies at micrometric resolution. To this aim, the proper use of contrast agents can improve the visualization of internal structures in ovary tissues, which normally present a low radiopacity. In this study, we report a comparison of four staining protocols, based on iodine or tungsten containing agents, applied to bovine ovarian tissues fixed in Bouin's solution. The microtomography (microCT) analyses at two synchrotron facilities under different set-ups were performed at different energies in order to maximize the image contrast. While tungsten-based agents allow large structures to be well identified, Iodine ones better highlight smaller features, especially when acquired above the K-edge energy of the specific metal. Further scans performed at lower energy where the setup was optimized for overall quality and sensitivity from phase-contrast still provided highly resolved visualization of follicular and intrafollicular structures at different maturation stages, independent of the staining protocol. The analyses were complemented by X-ray Fluorescence mapping on 2D sections, showing that the tungsten-based agent has a higher penetration in this type of tissues. [ABSTRACT FROM AUTHOR]

Scanning transmission X-ray microscopy (STXM) provides the imaging of biological specimens allowing the parallel collection of localized spectroscopic information by X-ray fluorescence (XRF) and/or X-ray Absorption Near Edge Spectroscopy (XANES). The complex metabolic mechanisms which can take place in biological systems can be explored by these techniques by tracing even small quantities of the chemical elements involved in the metabolic pathways. Here, we present a review of the most recent publications in the synchrotrons' scenario where soft X-ray spectro-microscopy has been employed in life science as well as in environmental research. [ABSTRACT FROM AUTHOR]

The range of materials analysed at Elettra, the Italian synchrotron facility, in the area of cultural heritage (CH) is very broad and goes from inorganic materials such as metals, stone, glass, ceramics and pigments, to organic-based materials such as wood, paper, leather, fabrics and biological samples. Several applications are involved, such as restoration and conservation, analysis of alteration products, identification of manufacturing techniques used by ancient communities, as well as the study of human and animal evolution. Synchrotron radiation offers a large portfolio of analytical techniques and several advantages, such as the unique non-destructive capabilities of most analytical approaches and the possibility of using synergic way more X-ray techniques in the same environment. In this paper, we will describe the Elettra source and the beamlines routinely dedicated to CH experiments, as well as the office designed to facilitate users with little or no experience in the use of large research infrastructures. [ABSTRACT FROM AUTHOR]

Fibrous erionite is the only zeolite classified as Group 1 carcinogen by the International Agency for Research on Cancer (IARC). Carcinogenesis induced by erionite is thought to involve several factors as biopersistence, the iron role and cation exchange processes. To better understand these mechanisms, a detailed investigation at the micro scale was performed, collecting elemental information on iron and cation release and their distribution in biological systems by synchrotron micro-X-ray fluorescence mapping (SR-micro-XRF) and synchrotron micro-X-ray absorption spectroscopy (SR-micro-XANES) at the TwinMic beamline (Elettra synchrotron) and at the ID21 beamline of the European Synchrotron Radiation Facility (ESRF). By microscopy and chemical mapping, highly detailed maps of the chemical and morphological interaction of biological systems with fibres could be produced. In detail, THP-1 cell line derived macrophages, used as in vitro model, were analysed during erionite-Na phagocytosis at different time intervals, after single dose exposure. For comparison, cellular fluorescent probes were also used to evaluate the intracellular free sodium and calcium concentrations. Synchrotron analyses visualised the spatial distribution of both fibre and mineral particle associated metals during the phagocytosis, describing the mechanism of internalisation of erionite-Na and its accessory mineral phases. The intracellular distribution of metals and other cations was mapped to evaluate metal release, speciation changes and/or cation exchange during phagocytosis. The fluorescent probes complemented microchemical data clarifying, and confirming, the cation distribution observed in the SR-micro-XRF maps. The significant cytoplasmic calcium decrease, and the concomitant sodium increase, after the fibre phagocytosis seemed due to activation of plasma membrane cations exchangers triggered by the internalisation while, surprisingly, the ion-exchange capacity of erionite-Na could play a minor role in the disruption of the two cations intracellular homeostasis. These results help to elucidate the role of cations in the toxicity of erionite-treated THP-1 macrophages and add knowledge to its carcinogenicity process. [Display omitted] • Synchrotron XRF microscopy used to study fibrous erionite-Na phagocytosis for 96 h. • No metal release from the fibres was observable inside the macrophage cells. • No extraframework cation exchange was observable on the fibres inside the cells. • Intracellular imbalance of Na+ and Ca2+ concentrations was observed inside the cells. • The cation imbalance is the result of an influx/efflux from the extracellular space. [ABSTRACT FROM AUTHOR]

Photobiomodulation therapy (PBMT) is a form of treatment commonly used for routine clinical applications, such as wound healing of the skin and reduction of inflammation. Additionally, PBMT has been explored for its potential in pain relief. In this work, we investigated the effect of PBMT on ion content within the 50B11 sensory neurons cell line in vitro using X-Ray fluorescence (XRF) and atomic force microscope (AFM) analysis. Two irradiation protocols were selected utilizing near-infrared laser lights at 800 and 970 nm, with cell fixation immediately following irradiation. Results showed a decrease in Calcium content after irradiation with both protocols, and with lidocaine, used as an analgesic control. Furthermore, a reduction in Potassium content was observed, particularly evident when normalized to cellular volume. These findings provide valuable insights into the molecular impact of PBMT within 50B11 sensory neurons under normal conditions. Such understanding may contribute to the wider adoption of PBMT as a therapeutic approach. [Display omitted] • Photobiomodulation therapy (PBMT) is a form of treatment, employed for pain relief. • The effect of PBMT on ions in 50B11 sensory neurons using XRF and AFM was studied. • a decrease in Calcium and Potassium content after irradiation was observed. • these findings confirmed a molecular impact of PBMT on 50B11 sensory neurons. [ABSTRACT FROM AUTHOR]

As the prospect of human beings exposed to new nanomaterials increases, there are growing concerns about the foetal exposure and effects of such nanomaterials during pregnancy. Among others, due to their ample usage, it becomes urgent to address the vulnerability of the human placental barrier to carbon nanotubes (CNTs). Recently, by applying a combination of advanced synchrotron‐based X‐ray microscopy and X‐ray fluorescence (XRF) techniques, we demonstrated that raw single‐walled CNTs (SWCNTs) may cause an asbestos‐like iron‐related toxicity in mesothelial (MeT5A) cells. This work shows the results obtained with the same approach and experimental conditions in human choriocarcinoma (BeWo) cells as a placental model. XRF microscopy in mild and soft X‐ray regimes was used to map the distribution of P, S, Ca, and Fe in exposed and control cells, whereas absorption and phase‐contrast imaging (soft X‐ray microscopy) provided simultaneous morphological information. The findings show that exposure to SWCNTs affects cell viability and causes a small increase in intracellular iron content, further confirmed by the stimulation of the ferritin protein. It is interesting to note that the iron changes are negligible when highly purified SWCNTs are used. In all tested conditions, including exposure to iron sulfate, the iron accumulation or ferritin increase was much lower in BeWo cells than in MeT5A ones, suggesting a different iron metabolism and possibly a lower vulnerability of the placental barrier to iron‐rich nanomaterials. [ABSTRACT FROM AUTHOR]

The approval and granting of marketing authorization for a putative biosimilar is based on strong comparability studies with its biological reference product. This is due to the complexity of the structure and nature of the manufacturing process of biological drugs. Hence, a rigorous analytical workflow for chemical characterization and clinical trials to evaluate the efficacy and safety is required to demonstrate their high similarities to the reference drug. This work is focused on the comparison of the originator of filgrastim with three of its biosimilars by evaluating their structural similarity and biological activity. Qualiquantitative analyses were performed by MALDI-TOF/TOF-MS and RP-HPLC-UV. An innovative functional assay using zebrafish as the animal model was developed to evaluate the biological activities of the drugs. The different analyses performed in this study highlighted the structural similarity of biosimilar drugs and their originator. This result was further confirmed by a similar in vivo biological activity. [ABSTRACT FROM AUTHOR]

It is well recognized that a large number of pulmonary diseases are induced by the effects of inhaled particulates. Anthracosis is defined as an asymptomatic, mild form of pneumoconiosis caused by the accumulation of “black carbon” in the lungs due to repeated exposure to air pollution or inhalation of smoke or coal dust particles. Since the human population is progressively exposed to an increasing number and doses of anthropogenic micro and nano particles/compounds, there is a pressing urgency to explore toxicological impact arising from these exposures and the molecular mechanisms driving the body defense or possible related diseases. The toxicity mechanisms are clearly related to chemical composition and physical and surface properties of materials. A combination of synchrotron radiation-based (SR-based) nano X-ray fluorescence (XRF) imaging and soft X-ray microscopy was used to chemically characterize environmental particulates (anthracosis) in lung tissues from urban subjects with the aim of better understanding the complex nature of related lungs' deposits. High-resolution XRF analyses performed at ESRF and Elettra synchrotrons allowed discriminating single particles in the heterogeneous aggregates found in the lung tissue. The small particles have variable composition resulting from the different combinations of Ti with O, K and Si, Al and Si, or Zn and Fe with O. Interestingly, simultaneous absorption and phase contrast images showed the particulate morphology and allowed to predict the presence of very dense nanoparticles or high concentration of heavy elements. [ABSTRACT FROM AUTHOR]

X‐ray fluorescence (XRF) spectroscopy is a widely used technique in microscopy, spanning from biology to cultural heritage applications. Its purpose is to characterize qualitatively and quantitatively, the presence of elemental species in a sample. This is accomplished through fitting the acquired data to a Gaussian model, identifying which XRF lines and associated elements are present. As a result, 2D images of cumulative count‐rate maps associated with each element are produced. This procedure is not trivial to apply efficiently in a workflow, as it requires the user to be able to set a series of parameters (e.g., beam energy, background subtraction, etc.) on top of selecting XRF lines under investigation. Furthermore, users should easily and swiftly be able to change setup parameters and evaluate the effects on the results. In the present work, we introduce a web‐based application that allows users to load the XRF data, setup a fit and inspect the results interactively within a simple graphical user interface (GUI) that enables easily going back and forth from setup to result inspection. In particular, it is possible to quickly view the count‐rate maps and curve fitting simultaneously, on any single pixel spectra present in the images. The web‐application can be accessed locally by a web‐browser, but runs remotely on a cloud, freeing from the need of installing any software and will be made publicly available in the near future. At present, it has been designed to work on both conventional and sparse XRF data such as Compressive Sensing, in an embarrassingly parallel manner. [ABSTRACT FROM AUTHOR]

In spite of the constant technological improvements in the field of detector development, X-ray fluorescence (XRF) in the soft X-ray regime remains a challenge. The low intrinsic fluorescence yield for energies below 2 keV indeed renders the applicability of low-energy XRF still difficult. Here, we report on a new multi-element multi-tile detection system currently under development, designed to be integrated into a soft X-ray microscopy end station. The system will be installed at the TwinMic beamline of Elettra synchrotron (Trieste, Italy) in order to increase the detected count rate by up to an order of magnitude. The new architecture is very versatile and can be adapted to any XRF experimental setup. Even though the first results of the previous version of such a multi-element system were encouraging, several issues still needed to be addressed. The system described here represents a further step in the detector evolution. It is based on four trapezoidal-shaped monolithic silicon drift detector tiles (matrices) with six hexagonal elements each equipped with a custom ultra-low noise application-specific integrated circuit readout. The whole signal processing chain has been improved leading to an overall increase in performances, namely, in terms of energy resolution and acquisition rates. The design and development of this new detection system will be described, and recent results obtained at the TwinMic beamline at Elettra will be presented. Future perspectives and improvements will also be discussed. Copyright © 2017 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Nanotoxicology and nanomedicine investigations often require the probing of nano-objects such as fibres and particles in biological samples and cells, whilst internalization and intracellular destiny are the main issues for in vitro cellular studies. Various high resolution microscopy techniques are well suited for providing this highly sought-after information. However, sample preparation, nanomaterial composition and sectioning challenges make it often difficult to establish whether the fibres or particles have been internalized or they are simply overlaying or underlying the biological matter. In this paper we suggest a novel suitable combination of two different microscopic techniques to reveal in intact cells the uptake of asbestos fibres by mesothelial cells. After exposure to asbestos fibres and fixation, cells were first analysed under the AFM instrument and then imaged under the TwinMic soft X-ray microscope at Elettra Sincrotrone. The suggested approach combines standard soft X-ray microscopy imaging and AFM microscopy, with a common non-invasive sample preparation protocol which drastically reduces the experimental uncertainty and provides a quick and definitive answer to the nanoparticle cellular and tissue uptake. [ABSTRACT FROM AUTHOR]

The current status of the TwinMic beamline at Elettra synchrotron light source, that hosts the European twin X-ray microscopy station, is reported. The X-ray source, provided by a short hybrid undulator with source size and divergence intermediate between bending magnets and conventional undulators, is energy-tailored using a collimated plane-grating monochromator. The TwinMic spectromicroscopy experimental station combines scanning and full-field imaging in a single instrument, with contrast modes such as absorption, differential phase, interference and darkfield. The implementation of coherent diffractive imaging modalities and ptychography is ongoing. Typically, scanning transmission X-ray microscopy images are simultaneously collected in transmission and differential phase contrast and can be complemented by chemical and elemental analysis using across-absorption-edge imaging, X-ray absorption near-edge structure or low-energy X-ray fluorescence. The lateral resolutions depend on the particular imaging and contrast mode chosen. The TwinMic range of applications covers diverse research fields such as biology, biochemistry, medicine, pharmacology, environment, geochemistry, food, agriculture and materials science. They will be illustrated in the paper with representative results. [ABSTRACT FROM AUTHOR]

The last decades have witnessed substantial efforts in the development of several detector technologies for X-ray fluorescence (XRF) applications. In spite of the increasing trend towards performing, cost-effective and reliable XRF systems, detectors for soft X-ray spectroscopy still remain a challenge, requiring further study, engineering and customization in order to yield effective and efficient systems. In this paper we report on the development, first characterization and tests of a novel multielement detector system based on low leakage current silicon drift detectors (SDD) coupled to ultra low noise custom CMOS preamplifiers for synchrotron-based low energy XRF. This new system exhibits the potential for improving the count rate by at least an order of magnitude resulting in ten-fold shorter dwell time at an energy resolution similar to that of single element silicon drift detectors. [ABSTRACT FROM AUTHOR]

Background: Authorization to market a biosimilar product by the appropriate institutions is expected based on biosimilarity with its originator product. The analogy between the originator and its biosimilar(s) is assessed through safety, purity, and potency analyses. Objective: In this study, we proposed a useful quality control system for rapid and economic primary screening of potential biosimilar drugs. For this purpose, chemical and functional characterization of the originator rhEPO alfa and two of its biosimilars was discussed. Methods: Qualitative and quantitative analyses of the originator rhEPO alfa and its biosimilars were performed using reversed-phase high-performance liquid chromatography (RP-HPLC). The identification of proteins and the separation of isoforms were studied using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and two-dimensional gel electrophoresis (2D-PAGE), respectively. Furthermore, the biological activity of these drugs was measured both in vitro, evaluating the TF-1 cell proliferation rate, and in vivo, using the innovative experimental animal model of the zebrafish embryos. Results: Chemical analyses showed that the quantitative concentrations of rhEPO alfa were in agreement with the labeled claims by the corresponding manufacturers. The qualitative analyses performed demonstrated that the three drugs were pure and that they had the same amino acid sequence. Chemical differences were found only at the level of isoforms containing N-glycosylation; however, functional in vitro and in vivo studies did not show any significant differences from a biosimilar point of view. Conclusion: These rapid and economic structural and functional analyses were effective in the evaluation of the biosimilarity between the originator rhEPO alfa and the biosimilars analyzed. [ABSTRACT FROM AUTHOR]

In this paper, we report on the use of high-space resolution soft X-ray fluorescence microspectroscopy for the study of electrodeposited composites containing catalytic ternary metal nanostructures. X-ray fluorescence maps are interpreted in terms of a dynamic mathematical model of the electrode morphology and metal space distribution, allowing to reproduce the observed space patterns and electrochemical transients by assigning an appropriate set of electrokinetic parameters. The discussed materials-science case is the electrochemical growth of a Mn-Mg-Cu-polypyrrole nanocomposite electrocatalyst material - free of expensive Pt and environmentally unfriendly Co - with promising performance for fuel-cell oxygen electrodes. The synergy of high-resolution compositional mapping with electrokinetic modelling not only provides the general rationale for quantitative use of potentially large compositional distribution datasets but also yields unprecedented insight into the specific catalyst synthesis process. The expounded application is just a prototypical case study of a more general approach, which can be employed for the understanding of electrochemical material science processes, both in situ and ex situ, as well as for the characterisation of the corresponding products, with no other limitations in principle than X-ray transmission and beam damage. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

In this paper we report on the electrochemical reconstruction of a Tarentum hemiobolus Ag coin, severely corroded in marine environment. As assessed by conventional analytical tools, most of the initially metallic Ag coin had been converted to AgCl by exposure to the aggressive coastal burial conditions. X-ray computed microtomography proved that only small portions of the artefact had preserved their metallic nature. Since the engraving was preserved partly in the corrosion product bulk and partly in the metallic rests, electrodeposition of Ag from the AgCl layer, under controlled conditions ensuring shape preservation, resulted in the reconstruction of the coin surface with full recovery of the original engraving. Such optimal electrodeposition conditions were identified by a combination of electrochemical and quasi-in situ X-ray microtomography experiments, carried out with artificially corrored engraved Ag wires. Microtomography of the reconstructed coin confirmed the compaction of the external Ag layer and disclosed that the central core of the coin still contains unconverted AgCl. The presence of such a mineralised core does not however impact the numismatic use of the coin and the safeguard of the original engraving. [ABSTRACT FROM AUTHOR]

It has been recently demonstrated that the internal jugular vein may exhibit abnormalities classified as truncular venous malformations (TVMs). The investigation of possible morphological and biochemical anomalies at jugular tissue level could help to better understand the link between brain venous drainage and neurodegenerative disorders, recently found associated with jugular TVMs. To this end we performed sequential X-ray Fluorescence (XRF) analyses on jugular tissue samples from two TVM patients and two control subjects, using complementary energies at three different synchrotrons. This investigation, coupled with conventional histological analyses, revealed anomalous micro-formations in the pathological tissues and allowed the determination of their elemental composition. Rapid XRF analyses on large tissue areas at 12.74 keV showed an increased Ca presence in the pathological samples, mainly localized in tunica adventitia microvessels. Investigations at lower energy demonstrated that the high Ca level corresponded to micro-calcifications, also containing P and Mg. We suggest that advanced synchrotron XRF micro-spectroscopy is an important analytical tool in revealing biochemical changes, which cannot be accessed by conventional investigations. Further research on a larger number of samples is needed to understand the pathogenic significance of Ca micro-depositions detected on the intramural vessels of vein walls affected by TVMs. [ABSTRACT FROM AUTHOR]

Cobalt/polypyrrole ORR electrocatalysts has been electrochemically synthesized by a potentiostatic anodic/cathodic pulse-plating procedure. The deposition electrochemistry has been studied by cyclic voltammetry and in-situ Micro Raman Spectroscopy complemented with scanning X-ray microscopy and micro-spot X-ray absorption spectroscopy (µ-XAS). Linear sweep voltammetry (LSV) under oxygen reduction has been used to assess the electrocatalytic effect of as-electrodeposited Co/PPy. The obtained results have provided new information about the concomitant electropolymerisation and metal incorporation processes occurring under different pulsed co-electrodeposition conditions, important for optimising the electrosynthesis procedure. The colocation of O and Co, evidenced by the O and Co XRF maps indicates that the Co in the formed composite is in oxidized state and for composites formed at low currents using shorter Co pulses coexistence of both CoO and Co3O4is evidenced by the Co L3XAS spectra. Bimodal Co distribution as micro-grains on a background of nano-grains and co-nulcleation of PPy and Co is evidenced by correlation of Co and N XRF maps. [ABSTRACT FROM AUTHOR]

In this paper we report on the fabrication and testing of a novel concept of sealed electrochemical microcell for in situ soft X-ray microspectroscopy in transmission, dedicated for nonvacuum compatible electrolytes. The microcell, fabricated using ultraviolet lithography, at variance with previous versions of electrochemical wet cells, that featured an optical window glued on top of the electrode system and a very limited electrolyte volume, the device presented here is a single solid block based around a microfabricated channel with fixed optical windows and apt for microfluidic work. Moreover, this cell allows to employ an advanced electrodic geometry developed in our group - so far used only in open electrochemical cells for work with vacuum-compatible electrolytes - also with low-vapor pressure liquids, possibly saturated with the required gases. The cell optimal electrode design allows three-electrode electrochemical control typical of traditional electrochemical experiments. The first electrochemical experiments with this new cell explore the electrochemical growth of a Co-polypyrrole, a composite electrocatalyst material with promising performance to replace the expensive Pt catalyst in fuel-cell oxygen electrodes. Morphological and chemical-state distributions of Co codeposited with polypyrrole has been followed as a function of time and position, yielding unprecedented information on the processes relevant to the synthesis of this catalyst. [ABSTRACT FROM AUTHOR]

Highlights: [•] Electrodeposited AuMn is the starting material for functionalised nanoporous gold. [•] Nanoporous-Au/MnO2-nanowire hybrids were fabricated from AuMn precursor alloys. [•] Submicrometric Mn distribution monitored quasi-in situ by scanning X-ray microscopy. [•] Mn chemical state followed by quasi-in situ by micro-X-ray absorption spectroscopy. [•] Ideal nanoporous-Au/Mn-oxide electronic contact achieved by controlled AuMn oxidation. [Copyright &y& Elsevier]

This study aimed to assess the potential of a multidimensional approach to differentiate body hairs based on their physico-chemical properties and whether body hairs can replace the use of scalp hair in studies linked to forensic and systemic intoxication. This is the first case report controlling for confounding variables to explore the utility of multidimensional profiling of body hair using synchrotron synchrotron microbeam X-ray fluorescence (SR-XRF) for longitudinal and hair morphological region mapping) and benchtop methods, including attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) (complemented with chemometrics analysis), energy dispersive X-ray analysis (EDX) (complemented with heatmap analysis), differential scanning calorimetry (DSC), and scanning electron microscopy (SEM) analysis (complemented by descriptive statistics) to profile different body hairs in terms of their elemental, biochemical, thermal, and cuticle properties. This multidimensional approach provided supportive information to emphasize the intricate and rather complex interplay between the organization and levels of elements and biomolecules within the crystalline and amorphous matrix of different body hairs responsible for the differences in physico-chemical properties between body hairs that are predominantly affected by the growth rate, follicle or apocrine gland activity, and external factors such as cosmetic use and exposure to environmental xenobiotics. The data from this study may have important implications for forensic science, toxicology and systemic intoxication, or other studies involving hair as a research matrix. [ABSTRACT FROM AUTHOR]

Abstract: In this work, we describe a novel approach to the fabrication of MnO2, consisting in Mn electrodeposition from a choline chloride/urea eutectic ionic liquid bath followed by controlled electrochemical oxidation in an aqueous solution. Both electrodeposition and electro-oxidation processes have been studied by electrochemical and in situ spectroelectrochemical approaches. Synchrotron-based soft X-ray scanning microscopy and absorption spectroscopy were used to measure in situ the Mn valence distribution on the electrodes and within the cell. The oxidation process was followed in situ by visible reflectivity measurements: the different stages of the oxidation process could be discriminated dynamically on the basis of relative reflectivity changes. The capacitance of the electrochemically fabricated films has been measured with cyclic voltammetry and electrochemical impedance spectrometry. [Copyright &y& Elsevier]

Abstract: A series of novel iodinated benzimidazoles have been prepared by iodination of respective benzimidazole with iodine and periodic acid in sulfuric acid solution. Additionally several 2-substituted- and N-1-carboxymethyl-substituted derivatives of 4,5,6,7-tetraiodobenzimidazole (TIBI) were obtained. For sake of comparison, some new 4,5,6,7-tetrabromobenzimidazoles were also synthesized. The ability of the new compounds to inhibit protein kinase CK2 has been evaluated. The results show that 4,5,6,7-tetraiodobenzimidazoles are more powerful inhibitors of CK2 than their tetrabrominated analogs. Molecular modeling supports the experimental data showing that tetraiodobenzimidazole moiety fills better the binding pocket than respective tetrabromo and tetrachlorocompounds. To note that 4,5,6,7-tetraiodobenzimidazole (TIBI) is one of the most efficient CK2 inhibitors (K i =23nM) described to date. [Copyright &y& Elsevier]

Abstract: In eukaryotes, protein phosphorylation of serine, threonine or tyrosine residues by protein kinases plays an important role in many cellular processes. Members of the protein kinase CK1 family usually phosphorylate residues of serine that are close to other phosphoserine in a consensus motif of pS-X-X-S, and they are implicated in the regulation of a variety of physiological processes as well as in pathologies like cancer and Alzheimer’s disease. Using a structure-based virtual screening (SBVS) approach we have identified two anthraquinones as novel CK1δ inhibitors. These amino-anthraquinone analogs (derivatives 1 and 2) are among the most potent and selective CK1δ inhibitors known today (IC50 =0.3 and 0.6μM, respectively). [Copyright &y& Elsevier]

The corrosion control of WC-Co type hardmetals is steadily gaining momentum for novel crucial applications in the Oil & Gas and food industries. The corrosion rate of the Co-based binder, and the extent to which coupling to WC speeds it up, are strongly influenced by alloying. In this paper, we investigate the impact of Cr on the corrosion-product film formation of Co- and Co Ni based hardmetal binders in acidic, neutral and alkaline aqueous solutions. We centred our study on the role of Cr in driving the distribution of oxidized Co and Ni at the micrometre scale, thanks to synchrotron-based soft-X ray microspectroscopy. We have investigated morphochemical distributions for the following grades: Co 96 Cr 4 , Co 48 Ni 48 Cr 4 , Co 50 Cr 50. Chemical-state mapping has been complemented by electrochemical measurements and metallographic observations. Amounts of Cr and Ni of ca. 50% notably increase the corrosion resistance in all ambients, with a stronger beneficial effect of the former element. 4% addition of Cr results in slight positive effects, with the exception of the Co Ni system in alkaline ambient, that, together with Co 50 Cr 50 , outperforms the other grades. In all investigated alloy-ambient combinations, a continuous oxidized metal film grows, onto which micrometric island form of shape and dimensions that depend on the specific grade and aggressive conditions. Quantitative descriptors of chemical-state maps and their theoretical interpretation in terms of electrochemical phase-formation by oxy-hydroxide precipitation, allow to correlate the island patterns with the degree of pseudopassivation. Unlabelled Image • Role of Cr in corrosion resistance of Co- and Co-Ni-based hardmetal model binders. • Corrosion of Co Cr based grades studied in aerated aqueous solutions in pH range 1–10. • Soft-X ray microspectroscopy yields chemical-state maps of pseudopassivation films. • Pseudopassivation layers form oxidized metal patterns, depending on attack conditions. • Corrosion resistance correlates with morphochemical details of oxide film patterns. [ABSTRACT FROM AUTHOR]

On the basis of the knowledge from traditional herbal and folk medicine, flavonoids are among the most studied chemical classes of natural compounds for their potential activity as phosphodiesterase 5 (PDE5) inhibitors. We here describe the preparation of a semi-synthetic hydrazone derivative of quercetin, 2-(3,4-dihydroxyphenyl)-4-(2-(4-nitrophenyl)hydrazono)-4H-chromene-3,5,7-triol, that was obtained via a single-step modification of the natural compound. The product was characterized by NMR, mass spectrometry and HPLC. Preliminary molecular modeling studies suggest that this compound could efficiently interact with PDE5. [ABSTRACT FROM AUTHOR]

In this report, we discuss the synthesis of a compound obtained from the derivatization of the natural compound celastrol. This derivative is connected to PEG azide moiety through an amide linkage. The linkage was achieved through the activation of the carboxylic acid using HOBt/EDC. The compound was fully characterized by proton (1H), carbon-13 (13C), heteronuclear single quantum coherence (HSQC), correlation spectroscopy (1H-1H-COSY), and distortionless enhancement by polarization transfer (DEPT) NMR. Ultraviolet (UV), Fourier-transform infrared (FTIR), and high-resolution mass spectrometry (HRMS) were also adopted. Computational investigations were conducted to forecast the binding mode between the synthesized compound and sarco-endoplasmic reticulum (SR) Ca2+ transport ATPase (SERCA), a known target for the development of novel therapeutics for rheumatoid arthritis. Additionally, the drug-likeness of the synthesized compound was assessed by predicting its pharmacokinetic properties. [ABSTRACT FROM AUTHOR]

Background: Most patients with testicular germ cell tumors (GCTs) are treated with cisplatin (CP)-based chemotherapy. However, some of them may develop CP resistance and therefore represent a clinical challenge. Cyclin-dependent kinase 5 (CDK5) is involved in chemotherapy resistance in different types of cancer. Here, we investigated the possible role of CDK5 and other CDKs targeted by dinaciclib in nonseminoma cell models (both CP-sensitive and CP-resistant), evaluating the potential of the CDK inhibitor dinaciclib as a single/combined agent for the treatment of advanced/metastatic testicular cancer (TC). Methods: The effects of dinaciclib and CP on sensitive and resistant NT2/D1 and NCCIT cell viability and proliferation were evaluated using MTT assays and direct count methods. Flow cytometry cell-cycle analysis was performed. The protein expression was assessed via Western blotting. The in vivo experiments were conducted in zebrafish embryos xenografted with TC cells. Results: Among all the CDKs analyzed, CDK5 protein expression was significantly higher in CP-resistant models. Dinaciclib reduced the cell viability and proliferation in each cell model, inducing changes in cell-cycle distribution. In drug combination experiments, dinaciclib enhances the CP effect both in vitro and in the zebrafish model. Conclusions: Dinaciclib, when combined with CP, could be useful for improving nonseminoma TC response to CP. [ABSTRACT FROM AUTHOR]

Multimodal imaging and spectroscopy like concurrent scanning transmission X-ray microscopy (STXM) and X-ray fluorescence (XRF) are highly desirable as they allow retrieving complementary information. This paper reports on the design, development, integration and field testing of a novel in situ atomic force microscopy (AFM) instrument for operation under high vacuum in a synchrotron soft X-ray microscopy STXM–XRF end-station. A combination of μXRF and AFM is demonstrated for the first time in the soft X-ray regime, with an outlook for the full XRF–STXM–AFM combination. [ABSTRACT FROM AUTHOR]

Capacitation makes spermatozoa competent to fertilize oocytes and can be induced in vitro before Artificial Reproductive Technology (ART) procedures to select the best quality and motile spermatozoa. The sperm quality is routinely measured under light microscopy. In the present study we tested the possibility of adding quality information by evaluating the content of some light elements and metals by XRF imaging, similarly to what we previously performed for female primordial gametes. With this approach we identified some major differences in elemental concentration between capacitated and not capacitated semen, indicating that Mg and Cu sperm contents are greatly reduced by capacitation, while there is an apparent Na increase. [ABSTRACT FROM AUTHOR]

Scanning microscopies and spectroscopies like X-ray Fluorescence (XRF), Scanning Transmission X-ray Microscopy (STXM), and Ptychography are of very high scientific importance as they can be employed in several research fields. Methodology and technology advances aim at analysing larger samples at better resolutions, improved sensitivities and higher acquisition speeds. The frontiers of those advances are in detectors, radiation sources, motors, but also in acquisition and analysis software together with general methodology improvements. We have recently introduced and fully implemented an intelligent scanning methodology based on compressive sensing, on a soft X-ray microscopy beamline. This demonstrated sparse low energy XRF scanning of dynamically chosen regions of interest in combination with STXM, yielding spectroimaging data in the megapixel-range and in shorter timeframes than were previously not feasible. This research has been further developed and has been applied to scientific applications in biology. The developments are mostly in the dynamic triggering decisional mechanism in order to incorporate modern Machine Learning (ML) but also in the suitable integration of the method in the control system, making it available for other beamlines and imaging techniques. On the applications front, the method was previously successfully used on different samples, from lung and ovarian human tissues to plant root sections. This manuscript introduces the latest methodology advances and demonstrates their applications in life and environmental sciences. Lastly, it highlights the auxiliary development of a mobile application, designed to assist the user in the selection of specific regions of interest in an easy way. [ABSTRACT FROM AUTHOR]

Laboratory and synchrotron X‐ray tomography are powerful tools for non‐invasive studies of biological samples at micrometric resolution. In particular, the development of phase contrast imaging is enabling the visualization of sample details with a small range of attenuation coefficients, thus allowing in‐depth analyses of anatomical and histological structures. Reproductive medicine is starting to profit from these techniques, mainly applied to animal models. This study reports the first imaging of human ovarian tissue where the samples consisted of surgically obtained millimetre fragments, properly fixed, stained with osmium tetroxide and included in epoxydic resin. Samples were imaged by the use of propagation phase contrast synchrotron radiation micro‐computed tomography (microCT), obtained at the SYRMEP beamline of Elettra light source (Trieste, Italy), and X‐ray absorption microCT at the Theoretical Biology MicroCT Imaging Laboratory in Vienna, Austria. The reconstructed microCT images were compared with the soft X‐ray absorption and phase contrast images acquired at the TwinMic beamline of Elettra in order to help with the identification of structures. The resulting images allow the regions of the cortex and medulla of the ovary to be distinguished, identifying early‐stage follicles and visualizing the distribution of blood vessels. The study opens to further application of micro‐resolved 3D imaging to improve the understanding of human ovary's structure and support diagnostics as well as advances in reproductive technologies. [ABSTRACT FROM AUTHOR]

We herein report the synthesis of a derivative of the natural compound celastrol linked to the antimalarial drug primaquine through an amide obtained by the activation of the carboxylic acid with HOBt/EDC. The chemical structure of the new molecule was fully characterized by proton nuclear magnetic resonance (1H-NMR), carbon-13 nuclear magnetic resonance (13C-NMR), heteronuclear single quantum coherence (HSQC), correlation spectroscopy (1H-1H-COSY), distortionless enhancement by polarization transfer (DEPT), mass spectrometry, Fourier-transform infrared (FTIR), and ultraviolet (UV) spectroscopies. Computational studies were enrolled to predict the interaction of the synthesized compound with sarco-endoplasmic reticulum (SR) Ca2+ transport ATPase (SERCA), a target of relevance for developing new therapeutics against arthritis. The drug-likeness of the compound was also investigated by predicting its pharmacokinetic properties. [ABSTRACT FROM AUTHOR]

1,2,3-triazole pharmacophore is a widely recognized motif used for a variety of applications, including drug discovery, chemical biology, and materials science. We herein report the synthesis of a derivative of azidothymidine (AZT), which was combined with the 7-chloro quinoline scaffold through a 1,4-disubstituted 1,2,3-triazole. The chemical structure of the new molecule was fully characterized by Fourier transform infrared (FTIR) spectroscopy, proton nuclear magnetic resonance (1H-NMR), carbon-13 nuclear magnetic resonance (13C-NMR), heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond correlation (HMBC) distortionless enhancement by polarization transfer (DEPT), correlation spectroscopy (1H-1H-COSY), ultraviolet (UV) spectroscopy, and high-resolution mass spectrometry (HRMS). Computational studies were used to predict the interaction of the synthesized compound with HIV reverse transcriptase, a target of relevance for developing new therapeutics against AIDS. The drug-likeness of the compound was also investigated by computing the physico-chemical properties that are important for the pharmacokinetic profile. [ABSTRACT FROM AUTHOR]

Research question Does synchrotron X-ray fluorescence (XRF) provide novel chemical information for the evaluation of human ovarian tissue cryopreservation protocols? Design Tissues from five patients undergoing laparoscopic surgery for benign gynaecological conditions were fixed for microscopic analysis either immediately or after cryopreservation. After fixation, fresh and slowly frozen samples were selected by light microscopy and transmission electron microscopy, and subsequently analysed with synchrotron XRF microscopy at different incident energies. Results The distributions of elements detected at 7.3 keV (S, P, K, Cl, Fe, and Os) and 1.5 keV (Na and Mg) were related to the changes revealed by light microscopy and transmission electron microscopy analyses. The light elements showed highly informative findings. The S distribution was found to be an indicator of extracellular component changes in the stromal tissues of the freeze–stored samples, further revealed by the transmission electron microscopy analyses. Low-quality follicles, frequent in the freeze–thawed tissues, showed a high Na level in the ooplasm. On the contrary, good-quality follicles were detected by a homogeneous Cl distribution. The occurrence of vacuolated follicles increased after cryopreservation, and the XRF analyses showed that the vacuolar structures contained mainly Cl and Na. Conclusions The study demonstrates that elemental imaging techniques, particularly revealing the distribution of light elements, could be useful in establishing new cryopreservation protocols. [ABSTRACT FROM AUTHOR]

Crucial for the advancement of electrochemical materials science is understanding the lateral variations in the elemental and chemical state of constituents induced by electrochemical reactions at nanoscales. This requires in situ studies to provide observables that contribute to both modeling beyond the phenomenological level and transducing exactly the functionally relevant quantities A range of X-ray coherent diffraction imaging (CDI) approaches has recently been proposed for imaging beyond the diffraction limit with potentially dramatic improvements of time resolution with chemical sensitivity. In this paper we report a selection of ptychography results obtained in situ after successive steps of electrochemically driven growth, complemented with absorption and phase spectroscopy at high lateral resolution. We demonstrate the onset of morphological instability feature formation and correlate the chemical state of Mn with local growth rate controlled by the current density distribution resulting from morphological evolution. [ABSTRACT FROM AUTHOR]

The growing interest on the therapeutic potential against neurodegeneration of Cannabis sativa extracts, and of phytocannabinoids in particular, is paralleled by a limited understanding of the undergoing biochemical pathways in which these natural compounds may be involved. Computational tools are nowadays commonly enrolled in the drug discovery workflow and can guide the investigation of macromolecular targets for such molecules. In this contribution, in silico techniques have been applied to the study of C. sativa constituents at various extents, and a total of seven phytocannabinoids and four terpenes were considered. On the side of ligand‐based virtual screening, physico‐chemical descriptors were computed and evaluated, highlighting the phytocannabinoids possessing suitable drug‐like properties to potentially target the central nervous system. Our previous findings and literature data prompted us to investigate the interaction of these molecules with phosphodiesterases (PDEs), a family of enzymes being studied for the development of therapeutic agents against neurodegeneration. Among the compounds, structure‐based techniques such as docking and molecular dynamics (MD), highlighted cannabidiol (CBD) as a potential and selective PDE9 ligand, since a promising calculated binding energy value (−9.1 kcal/mol) and a stable interaction in the MD simulation timeframe were predicted. Additionally, PDE9 inhibition assay confirmed the computational results, and showed that CBD inhibits the enzyme in the nanomolar range in vitro, paving the way for further development of this phytocannabinoid as a therapeutic option against neurodegeneration. [ABSTRACT FROM AUTHOR]

The development of inhibitors that target the papain‐like protease (PLpro) has the potential to counteract the spread of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), the agent causing coronavirus disease 2019 (COVID‐19). Based on a consideration of its several downstream effects, interfering with PLpro would both revert immune suppression exerted by the virus and inhibit viral replication. By following a repurposing strategy, the current study evaluates the potential of antimalarial drugs as PLpro inhibitors, and thereby the possibility of their use for treatment of SARS‐CoV‐2 infection. Computational tools were employed for structural analysis, molecular docking, and molecular dynamics simulations to screen antimalarial drugs against PLpro, and in silico data were validated by in vitro experiments. Virtual screening highlighted amodiaquine and methylene blue as the best candidates, and these findings were complemented by the in vitro results that indicated amodiaquine as a μM PLpro deubiquitinase inhibitor. The results of this study demonstrate that the computational workflow adopted here can correctly identify active compounds. Thus, the highlighted antimalarial drugs represent a starting point for the development of new PLpro inhibitors through structural optimization. [ABSTRACT FROM AUTHOR]

An important computational operation in a set of spectra is that of aligning them to a reference spectrum. In X-ray fluorescence, this is referred to as energy calibration and may be necessary for fitting low-count acquisitions. It may be useful for multi-element detector systems. Typically, this is carried out in a linear manner, and sometimes requires user feedback. Automated methods exist but are often focused on specific type of data. We propose a new automated method that is based on a nonlinear approach and is specialised for X-ray fluorescence data. The initial application in two different multi-element detector systems in the TwinMic beamline (Elettra - Sincrotrone Trieste) yielded promising results. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Ovarian cancer represents a major health concern for the female population: there is no obvious cause, it is frequently misdiagnosed, and it is characterized by a poor prognosis. Additionally, patients are inclined to recurrences because of metastasis and poor treatment tolerance. Combining innovative therapeutic techniques with established approaches can aid in improving treatment outcomes. Because of their multi-target actions, long application history, and widespread availability, natural compounds have particular advantages in this connection. Thus, effective therapeutic alternatives with improved patient tolerance hopefully can be identified within the world of natural and nature-derived products. Moreover, natural compounds are generally perceived to have more limited adverse effects on healthy cells or tissues, suggesting their potential role as valid treatment alternatives. In general, the anticancer mechanisms of such molecules are connected to the reduction of cell proliferation and metastasis, autophagy stimulation and improved response to chemotherapeutics. This review aims at discussing the mechanistic insights and possible targets of natural compounds against ovarian cancer, from the perspective of medicinal chemists. In addition, an overview of the pharmacology of natural products studied to date for their potential application towards ovarian cancer models is presented. The chemical aspects as well as available bioactivity data are discussed and commented on, with particular attention to the underlying molecular mechanism(s). [ABSTRACT FROM AUTHOR]

Background: Although X-ray fluorescence microscopy is becoming a widely used technique for single-cell analysis, sample preparation for this microscopy remains one of the main challenges in obtaining optimal conditions for the measurements in the X-ray regime. The information available to researchers on sample treatment is inadequate and unclear, sometimes leading to wasted time and jeopardizing the experiment's success. Many cell fixation methods have been described, but none of them have been systematically tested and declared the most suitable for synchrotron X-ray microscopy. Methods: The HEC-1-A endometrial cells, human spermatozoa, and human embryonic kidney (HEK-293) cells were fixed with organic solvents and cross-linking methods: 70% ethanol, 3.7%, and 2% paraformaldehyde; in addition, HEK-293 cells were subjected to methanol/ C3H6O treatment and cryofixation. Fixation methods were compared by coupling low-energy X-ray fluorescence with scanning transmission X-ray microscopy and atomic force microscopy. Results: Organic solvents lead to greater dehydration of cells, which has the most significant effect on the distribution and depletion of diffusion elements. Paraformaldehyde provides robust and reproducible data. Finally, the cryofixed cells provide the best morphology and element content results. Conclusion: Although cryofixation seems to be the most appropriate method as it allows for keeping cells closer to physiological conditions, it has some technical limitations. Paraformaldehyde, when used at the average concentration of 3.7%, is also an excellent alternative for X-ray microscopy. [ABSTRACT FROM AUTHOR]

The pharmacological approach to adrenocortical carcinoma (ACC) is based on mitotane with/without etoposide, doxorubicin, and cisplatin, according to the disease stage. Considering the limited efficacy and toxicity of this treatment, new strategies are required. Trabectedin is a marine-derivated antitumoral agent that inhibits oncogenic transcription. We have already demonstrated trabectedin cytotoxic activity at subnanomolar concentrations in ACC cells. Here, we expanded the investigation of trabectedin effect on ACC preclinical models, evaluating whether trabectedin could affect ACC cells' invasiveness and metastasis formation. NCI-H295R, MUC-1, and TVBF-7 cell lines were used. Cell tumor xenografts in Danio rerio embryos w ere performed. The tumor mass areas and the number of embryos with metastasis were evaluated. The in vitro invasiveness of cells was evaluated. Effects of trabectedin of MMP2, TIMP1, and TIMP2 were evaluated at gene level qRT-PCR. MMP2 secreted in the cel l medium was evaluated by Western blot and by zymography. Xenograft experiments demonstrated that trabectedin significantly reduced the tumor area in each ACC cell model and metastasis formation in embryos injected with metastasis-derived cell lines. Trabectedin treatment reduced the invasiveness of ACC cells across the matrix, which was greater at baseline for the metastatic models. In metastatic cell models, protein analysis demonstrated a reduction of MMP2 secretion and activity in the culture medium after treatment. Our results indicate that trabectedin interferes with invasiveness and metastasis processes, both dramatic features of ACC. Furthermore, these results support those previously published in providing the rationale for a clinical evaluation of the efficacy of trabectedin in ACC patients. [ABSTRACT FROM AUTHOR]

The need to identify effective therapies for the treatment of psychiatric disorders is a particularly important issue in modern societies. In addition, difficulties in finding new drugs have led pharmacologists to review and re-evaluate some past molecules, including psychedelics. For several years there has been growing interest among psychotherapists in psilocybin or lysergic acid diethylamide for the treatment of obsessive-compulsive disorder, of depression, or of post-traumatic stress disorder, although results are not always clear and definitive. In fact, the mechanisms of action of psychedelics are not yet fully understood and some molecular aspects have yet to be well defined. Thus, this review aims to summarize the ethnobotanical uses of the best-known psychedelic plants and the pharmacological mechanisms of the main active ingredients they contain. Furthermore, an up-to-date overview of structural and computational studies performed to evaluate the affinity and binding modes to biologically relevant receptors of ibogaine, mescaline, N,N-dimethyltryptamine, psilocin, and lysergic acid diethylamide is presented. Finally, the most recent clinical studies evaluating the efficacy of psychedelic molecules in some psychiatric disorders are discussed and compared with drugs already used in therapy. [ABSTRACT FROM AUTHOR]

The work focused on the analysis of two cultivars of tomato (Solanum lycopersicum L.), Aragon and Gladis, under two different treatments of silicon, Low, 2 L of 0.1 mM CaSiO3, and High, 0.5 mM CaSiO3, weekly, for 8 weeks, under stress-free conditions. We subsequently analyzed the morphology, chemical composition, and elemental distribution using synchrotron-based µ-XRF techniques, physiological, and molecular aspects of the response of the two cultivars. The scope of the study was to highlight any significant response of the plants to the Si treatments, in comparison with any response to Si of plants under stress. The results demonstrated that the response was mainly cultivar-dependent, also at the level of mitochondrial-dependent oxidative stress, and that it did not differ from the two conditions of treatments. With Si deposited mainly in the cell walls of the cells of fruits, leaves, and roots, the treatments did not elicit many significant changes from the point of view of the total elemental content, the physiological parameters that measured the oxidative stress, and the transcriptomic analyses focalized on genes related to the response to Si. We observed a priming effect of the treatment on the most responsive cultivar, Aragon, in respect to future stress, while in Gladis the Si treatment did not significantly change the measured parameters. [ABSTRACT FROM AUTHOR]