Since the mid-1960s, environmental toxicology focussed on the effects of bioaccumulation of organ... more Since the mid-1960s, environmental toxicology focussed on the effects of bioaccumulation of organochlorine insecticides in organisms, and their consequences for populations of species in the wild. Prompted by the release of Silent Spring [1], scientists sifted focus to the ecological effects of pesticides, thus expanding the narrow field of pesticide toxicology that had been restricted to its effects on pests, weeds and pathogenic fungi since its beginnings. The ensuing decades will witness a tremendous gathering of data related to the toxic impacts that insecticides, herbicides and fungicides have on organisms and ecosystems [
Nuclear Instruments and Methods in Physics Research, Section B: Beam Interactions with Materials and Atoms, 2006
Scanning transmission ion microscopy tomography (STIM-T) was carried out on diatom cells with the... more Scanning transmission ion microscopy tomography (STIM-T) was carried out on diatom cells with the aim of displaying their 3D structure and performing density measurements on their silica skeleton. Two software packages were compared for data reduction: TomoRebuild, based on a simple filtered backprojection algorithm, and DISRA, an iterative program. Silicon carbide microfibres of known density were also analysed as reference
Recent improvements in synchrotron X-ray sources (third generation) and in X-ray focusing element... more Recent improvements in synchrotron X-ray sources (third generation) and in X-ray focusing elements have been realized. This result in delivering highly collimated quasi-monochromatic X-ray beams with tunable energy and beam focused down to sub-micrometer diameter. Preliminary results in the μ-SXRF of single cells were obtained in the hard X-ray range. “Pink”-beam and compound refractive lenses were used resulting in a 1×10 μm 2 (vertical×horizontal) beam size with a flux ∼5×10 10 photon/s / μm 2 . The experiment confirmed that high energy, high intensity X-rays were well suited for microanalysis of sensitive biological specimens such as freeze-dried cells. Results show that the synchrotron microprobe set-up at ESRF allows high sensitivity in trace element measurements for cells treated with pharmacological doses of an iodine-labeled anticancer drug.
Neuronal loss in numerous neurodegenerative disorders has been linked to protein aggregation and ... more Neuronal loss in numerous neurodegenerative disorders has been linked to protein aggregation and oxidative stress. Emerging data regarding overlapping proteinopathy in traditionally distinct neurodegenerative diseases suggest that disease-modifying treatments targeting these pathological features may exhibit efficacy across multiple disorders. Here, we describe proteinopathy distinct from classic synucleinopathy, predominantly comprised of the anti-oxidant enzyme superoxide dismutase-1 (SOD1), in the Parkinson’s disease brain. Significant expression of this pathology closely reflected the regional pattern of neuronal loss. The protein composition and non-amyloid macrostructure of these novel aggregates closely resembles that of neurotoxic SOD1 deposits in SOD1-associated familial amyotrophic lateral sclerosis (fALS). Consistent with the hypothesis that deposition of protein aggregates in neurodegenerative disorders reflects upstream dysfunction, we demonstrated that SOD1 in the Park...
In biological sample microanalysis, a mass-normalisation method is commonly used as a quantitativ... more In biological sample microanalysis, a mass-normalisation method is commonly used as a quantitative index of elemental concentrations determined by particle-induced X-ray emission (PIXE). The organic mass can either be determined using particle backscattering spectrometry (BS) or scanning transmission ion microscopy (STIM). However, the accuracy of quantitative microanalysis in samples such as cultured cells is affected by beam-induced loss of organic mass during analysis. The aim of this paper is to compare mass measurements determined by particle BS or by STIM. In order to calibrate STIM and BS analyses, we measured by both techniques the thickness of standard foils of polycarbonate (3 and 6 μm ) , Mylar® (4 μm ) , Kapton® (7.5 μm ) and Nylon® (15 μm ) , as well as biological samples of mono-layered cultured cells. Non-damaging STIM analysis of samples before PIXE irradiation is certainly one of the most accurate ways to determine the sample mass, however, this requires strong expe...
Two complementary image-based microanalysis methods, digital autoradiography of radioactive eleme... more Two complementary image-based microanalysis methods, digital autoradiography of radioactive elements and X-ray fluorescence microspectroscopy (micro-XRF), were used to study the geochemistry of a speleothem from Eastern Siberia. Our objective was to show the interest of these micro-chemical methods to investigate the distribution of U and some other elements as Ca, Sr, and Si in speleothems. The sample studied was a corallite concretion that formed slowly by the precipitation of minerals from moisture that had condensed underground. Polished sections of the sample revealed alternating laminas of gray calcite and white aragonite. The concretion, which is older than 400 Kyr (U/Th-TIMS), showed no signs of detrital contamination ( 230 Th/ 232 Th > 10,000). Digital autoradiography and XRF analyses indicated exceptionally high uranium contents, ranging from 3–10 to more than 1300 μg ∙ g − 1 in different areas of the sample. Element maps for calcium (Ca), uranium (U), strontium (Sr) an...
This article presents recent research on the sedimentary endokarstic infillings in connection wit... more This article presents recent research on the sedimentary endokarstic infillings in connection with the new contributions of geochemical analysis by autoradiography and X-ray fluorescence (portable analyser, core scanner, micro-XRF, microprobe) in the context of the 'Climanthrope' ANR programme. Coupled with sedimentological analysis (stratigraphy, laser grain size analysis, micromineralogy, and micromorphology), geochemical analysis and imaging refine the study of underground deposits such as carbonated sediments (speleothems), rhythmites, subterranean ice, and prehistoric cave paintings. Formed following a seasonal cycle, stalagmites and rhythmites are likely to provide important data not only on the two main parameters of regional climate, i.e. hydrology and temperature, but also on human impacts (speleothems in urban tunnels) and the rural environment. The microdebris and dusts sampled in subterranean ice record the palaeoenvironment by snow trapping. Parietal paintings c...
Zinc and copper are involved in neuronal differentiation and synaptic plasticity but the molecula... more Zinc and copper are involved in neuronal differentiation and synaptic plasticity but the molecular mechanisms behind these processes are still elusive due in part to the difficulty of imaging trace metals together with proteins at the synaptic level. We correlate stimulated-emission-depletion microscopy of proteins and synchrotron X-ray fluorescence imaging of trace metals, both performed with 40 nm spatial resolution, on primary rat hippocampal neurons. We reveal the co-localization at the nanoscale of zinc and tubulin in dendrites with a molecular ratio of about one zinc atom per tubulin-αβ dimer. We observe the co-segregation of copper and F-actin within the nano-architecture of dendritic protrusions. In addition, zinc chelation causes a decrease in the expression of cytoskeleton proteins in dendrites and spines. Overall, these results indicate new functions for zinc and copper in the modulation of the cytoskeleton morphology in dendrites, a mechanism associated to neuronal plast...
Environmental exposure to neurotoxic metals and metalloids such as arsenic, cadmium, lead, mercur... more Environmental exposure to neurotoxic metals and metalloids such as arsenic, cadmium, lead, mercury, or manganese is a global health concern affecting millions of people worldwide. Depending on the period of exposure over a lifetime, environmental metals can alter neurodevelopment, neurobehavior, and cognition and cause neurodegeneration. There is increasing evidence linking environmental exposure to metal contaminants to the etiology of neurological diseases in early life (e.g., autism spectrum disorder) or late life (e.g., Alzheimer’s disease). The known main molecular mechanisms of metal-induced toxicity in cells are the generation of reactive oxygen species, the interaction with sulfhydryl chemical groups in proteins (e.g., cysteine), and the competition of toxic metals with binding sites of essential metals (e.g., Fe, Cu, Zn). In neurons, these molecular interactions can alter the functions of neurotransmitter receptors, the cytoskeleton and scaffolding synaptic proteins, thereb...
We developed a methodological workflow combining size exclusion chromatography, native isoelectri... more We developed a methodological workflow combining size exclusion chromatography, native isoelectric focusing, and high sensitivity X-ray-based metal detection within electrophoresis gels to analyze the metal content of single proteins purified from minimal amounts (<20 mg) of post-mortem human brain and spinal cord tissue. An important metalloprotein in the human central nervous system is copper-zinc superoxide dismutase (SOD1), an antioxidant enzyme linked to the aetiology of both amyotrophic lateral sclerosis and Parkinson’s disease. Abnormal SOD1 metallation is suspected to play a role in the pathogenic aggregation of SOD1 in both disorders, although data describing SOD1 metal occupancy in human tissues has not previously been reported. Validating our novel approach we demonstrated step-by-step metal preservation, preserved SOD1 activity, and substantial enrichment of SOD1 protein vs confounding metalloproteins. We found Cu and Zn were bound to SOD1 in a ratio of 1.12 ± 0.28 in...
BackgroundAlthough manganese (Mn) is an essential nutrient, recent research has revealed that exc... more BackgroundAlthough manganese (Mn) is an essential nutrient, recent research has revealed that excess Mn in early childhood may have adverse effects on neurodevelopment.MethodsWe estimated daily total Mn intake due to breast milk at average body weights by reviewing reported concentrations of breast milk Mn and measurements of body weight and breast milk intake at 3 weeks, 4.25 months, 7 months, and 18 months. We compared these figures to the Mn content measured in 44 infant, follow-up, and toddler formulas purchased in the United States and France. We calculated Mn content of formula products made with ultra-trace elemental analysis grade water (0 µg Mn/L) and with water containing 250 µg Mn/L, a concentration which is relatively high but less than the World Health Organization Health-based value of 400 µg Mn/L or the United States Environmental Protection Agency Health Advisory of 350 µg Mn/L.ResultsEstimated mean daily Mn intake from breast milk ranged from 1.2 µg Mn/kg/day (3 wee...
Since the mid-1960s, environmental toxicology focussed on the effects of bioaccumulation of organ... more Since the mid-1960s, environmental toxicology focussed on the effects of bioaccumulation of organochlorine insecticides in organisms, and their consequences for populations of species in the wild. Prompted by the release of Silent Spring [1], scientists sifted focus to the ecological effects of pesticides, thus expanding the narrow field of pesticide toxicology that had been restricted to its effects on pests, weeds and pathogenic fungi since its beginnings. The ensuing decades will witness a tremendous gathering of data related to the toxic impacts that insecticides, herbicides and fungicides have on organisms and ecosystems [
Nuclear Instruments and Methods in Physics Research, Section B: Beam Interactions with Materials and Atoms, 2006
Scanning transmission ion microscopy tomography (STIM-T) was carried out on diatom cells with the... more Scanning transmission ion microscopy tomography (STIM-T) was carried out on diatom cells with the aim of displaying their 3D structure and performing density measurements on their silica skeleton. Two software packages were compared for data reduction: TomoRebuild, based on a simple filtered backprojection algorithm, and DISRA, an iterative program. Silicon carbide microfibres of known density were also analysed as reference
Recent improvements in synchrotron X-ray sources (third generation) and in X-ray focusing element... more Recent improvements in synchrotron X-ray sources (third generation) and in X-ray focusing elements have been realized. This result in delivering highly collimated quasi-monochromatic X-ray beams with tunable energy and beam focused down to sub-micrometer diameter. Preliminary results in the μ-SXRF of single cells were obtained in the hard X-ray range. “Pink”-beam and compound refractive lenses were used resulting in a 1×10 μm 2 (vertical×horizontal) beam size with a flux ∼5×10 10 photon/s / μm 2 . The experiment confirmed that high energy, high intensity X-rays were well suited for microanalysis of sensitive biological specimens such as freeze-dried cells. Results show that the synchrotron microprobe set-up at ESRF allows high sensitivity in trace element measurements for cells treated with pharmacological doses of an iodine-labeled anticancer drug.
Neuronal loss in numerous neurodegenerative disorders has been linked to protein aggregation and ... more Neuronal loss in numerous neurodegenerative disorders has been linked to protein aggregation and oxidative stress. Emerging data regarding overlapping proteinopathy in traditionally distinct neurodegenerative diseases suggest that disease-modifying treatments targeting these pathological features may exhibit efficacy across multiple disorders. Here, we describe proteinopathy distinct from classic synucleinopathy, predominantly comprised of the anti-oxidant enzyme superoxide dismutase-1 (SOD1), in the Parkinson’s disease brain. Significant expression of this pathology closely reflected the regional pattern of neuronal loss. The protein composition and non-amyloid macrostructure of these novel aggregates closely resembles that of neurotoxic SOD1 deposits in SOD1-associated familial amyotrophic lateral sclerosis (fALS). Consistent with the hypothesis that deposition of protein aggregates in neurodegenerative disorders reflects upstream dysfunction, we demonstrated that SOD1 in the Park...
In biological sample microanalysis, a mass-normalisation method is commonly used as a quantitativ... more In biological sample microanalysis, a mass-normalisation method is commonly used as a quantitative index of elemental concentrations determined by particle-induced X-ray emission (PIXE). The organic mass can either be determined using particle backscattering spectrometry (BS) or scanning transmission ion microscopy (STIM). However, the accuracy of quantitative microanalysis in samples such as cultured cells is affected by beam-induced loss of organic mass during analysis. The aim of this paper is to compare mass measurements determined by particle BS or by STIM. In order to calibrate STIM and BS analyses, we measured by both techniques the thickness of standard foils of polycarbonate (3 and 6 μm ) , Mylar® (4 μm ) , Kapton® (7.5 μm ) and Nylon® (15 μm ) , as well as biological samples of mono-layered cultured cells. Non-damaging STIM analysis of samples before PIXE irradiation is certainly one of the most accurate ways to determine the sample mass, however, this requires strong expe...
Two complementary image-based microanalysis methods, digital autoradiography of radioactive eleme... more Two complementary image-based microanalysis methods, digital autoradiography of radioactive elements and X-ray fluorescence microspectroscopy (micro-XRF), were used to study the geochemistry of a speleothem from Eastern Siberia. Our objective was to show the interest of these micro-chemical methods to investigate the distribution of U and some other elements as Ca, Sr, and Si in speleothems. The sample studied was a corallite concretion that formed slowly by the precipitation of minerals from moisture that had condensed underground. Polished sections of the sample revealed alternating laminas of gray calcite and white aragonite. The concretion, which is older than 400 Kyr (U/Th-TIMS), showed no signs of detrital contamination ( 230 Th/ 232 Th > 10,000). Digital autoradiography and XRF analyses indicated exceptionally high uranium contents, ranging from 3–10 to more than 1300 μg ∙ g − 1 in different areas of the sample. Element maps for calcium (Ca), uranium (U), strontium (Sr) an...
This article presents recent research on the sedimentary endokarstic infillings in connection wit... more This article presents recent research on the sedimentary endokarstic infillings in connection with the new contributions of geochemical analysis by autoradiography and X-ray fluorescence (portable analyser, core scanner, micro-XRF, microprobe) in the context of the 'Climanthrope' ANR programme. Coupled with sedimentological analysis (stratigraphy, laser grain size analysis, micromineralogy, and micromorphology), geochemical analysis and imaging refine the study of underground deposits such as carbonated sediments (speleothems), rhythmites, subterranean ice, and prehistoric cave paintings. Formed following a seasonal cycle, stalagmites and rhythmites are likely to provide important data not only on the two main parameters of regional climate, i.e. hydrology and temperature, but also on human impacts (speleothems in urban tunnels) and the rural environment. The microdebris and dusts sampled in subterranean ice record the palaeoenvironment by snow trapping. Parietal paintings c...
Zinc and copper are involved in neuronal differentiation and synaptic plasticity but the molecula... more Zinc and copper are involved in neuronal differentiation and synaptic plasticity but the molecular mechanisms behind these processes are still elusive due in part to the difficulty of imaging trace metals together with proteins at the synaptic level. We correlate stimulated-emission-depletion microscopy of proteins and synchrotron X-ray fluorescence imaging of trace metals, both performed with 40 nm spatial resolution, on primary rat hippocampal neurons. We reveal the co-localization at the nanoscale of zinc and tubulin in dendrites with a molecular ratio of about one zinc atom per tubulin-αβ dimer. We observe the co-segregation of copper and F-actin within the nano-architecture of dendritic protrusions. In addition, zinc chelation causes a decrease in the expression of cytoskeleton proteins in dendrites and spines. Overall, these results indicate new functions for zinc and copper in the modulation of the cytoskeleton morphology in dendrites, a mechanism associated to neuronal plast...
Environmental exposure to neurotoxic metals and metalloids such as arsenic, cadmium, lead, mercur... more Environmental exposure to neurotoxic metals and metalloids such as arsenic, cadmium, lead, mercury, or manganese is a global health concern affecting millions of people worldwide. Depending on the period of exposure over a lifetime, environmental metals can alter neurodevelopment, neurobehavior, and cognition and cause neurodegeneration. There is increasing evidence linking environmental exposure to metal contaminants to the etiology of neurological diseases in early life (e.g., autism spectrum disorder) or late life (e.g., Alzheimer’s disease). The known main molecular mechanisms of metal-induced toxicity in cells are the generation of reactive oxygen species, the interaction with sulfhydryl chemical groups in proteins (e.g., cysteine), and the competition of toxic metals with binding sites of essential metals (e.g., Fe, Cu, Zn). In neurons, these molecular interactions can alter the functions of neurotransmitter receptors, the cytoskeleton and scaffolding synaptic proteins, thereb...
We developed a methodological workflow combining size exclusion chromatography, native isoelectri... more We developed a methodological workflow combining size exclusion chromatography, native isoelectric focusing, and high sensitivity X-ray-based metal detection within electrophoresis gels to analyze the metal content of single proteins purified from minimal amounts (<20 mg) of post-mortem human brain and spinal cord tissue. An important metalloprotein in the human central nervous system is copper-zinc superoxide dismutase (SOD1), an antioxidant enzyme linked to the aetiology of both amyotrophic lateral sclerosis and Parkinson’s disease. Abnormal SOD1 metallation is suspected to play a role in the pathogenic aggregation of SOD1 in both disorders, although data describing SOD1 metal occupancy in human tissues has not previously been reported. Validating our novel approach we demonstrated step-by-step metal preservation, preserved SOD1 activity, and substantial enrichment of SOD1 protein vs confounding metalloproteins. We found Cu and Zn were bound to SOD1 in a ratio of 1.12 ± 0.28 in...
BackgroundAlthough manganese (Mn) is an essential nutrient, recent research has revealed that exc... more BackgroundAlthough manganese (Mn) is an essential nutrient, recent research has revealed that excess Mn in early childhood may have adverse effects on neurodevelopment.MethodsWe estimated daily total Mn intake due to breast milk at average body weights by reviewing reported concentrations of breast milk Mn and measurements of body weight and breast milk intake at 3 weeks, 4.25 months, 7 months, and 18 months. We compared these figures to the Mn content measured in 44 infant, follow-up, and toddler formulas purchased in the United States and France. We calculated Mn content of formula products made with ultra-trace elemental analysis grade water (0 µg Mn/L) and with water containing 250 µg Mn/L, a concentration which is relatively high but less than the World Health Organization Health-based value of 400 µg Mn/L or the United States Environmental Protection Agency Health Advisory of 350 µg Mn/L.ResultsEstimated mean daily Mn intake from breast milk ranged from 1.2 µg Mn/kg/day (3 wee...
Nuclear Instruments and Methods in Physics Research B, 2006
Scanning transmission ion microscopy tomography (STIM-T) was carried out on diatom cells with the... more Scanning transmission ion microscopy tomography (STIM-T) was carried out on diatom cells with the aim of displaying their 3D structure and performing density measurements on their silica skeleton. Two software packages were compared for data reduction: TomoRebuild, based on a simple filtered backprojection algorithm, and DISRA, an iterative program. Silicon carbide microfibres of known density were also analysed as reference specimens. Similar results were obtained with both algorithms, demonstrating the ability of STIM-T to provide density measurements at the cell level without requiring any standard calibration samples. This unique feature stresses the interest of STIM-T to accurately normalise X ray emission micro-tomography data from synchrotron radiation (SXRF: synchrotron radiation X-ray fluorescence) or ion beam sources (PIXE: particle induced X-ray emission). Possible enhancements of the DISRA code are discussed in order to facilitate its use for the reconstruction of future PIXE/STIM tomography data. A ''nanoprobe'' coupled to a Singletron Ò accelerator, allowing a spatial resolution of a few tens of nanometers, is going to be built in the coming months at the Centre d'Etudes Nucléaires de Bordeaux Gradignan (CENBG). This new facility will bring promising applications in imaging and analysis at the sub-cellular level.
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Papers by Richard Ortega