Metal dyshomeostasis in the substantia nigra of patients with Parkinson's disease or multiple sclerosis.

Abnormal metal distribution in vulnerable brain regions is involved in the pathogenesis of most neurodegenerative diseases, suggesting common molecular mechanisms of metal dyshomeostasis. This study aimed to compare the intra- and extra-neuronal metal content and the expression of proteins related to metal homeostasis in the substantia nigra (SN) from patients with Parkinson's disease (PD), multiple sclerosis (MS), and control subjects. Metal quantification was performed via ion-beam micro-analysis in neuromelanin-positive neurons and the surrounding tissue. For proteomic analysis, SN tissue lysates were analyzed on a nanoflow chromatography system hyphenated to a hybrid triple-quadrupole time-of-flight mass spectrometer. We found increased amounts of iron in neuromelanin-positive neurons and surrounding tissue in patients with PD and MS compared to controls (4- to 5-fold higher) that, however, also showed large inter-individual variations. Copper content was systematically lower (-2.4-fold) in neuromelanin-positive neurons of PD patients compared with controls, whereas it remained unchanged in MS. Protein-protein interaction (PPI) network analyses revealed clusters related to Fe and Cu homeostasis among PD-deregulated proteins. An enrichment for the term "metal homeostasis" was observed for MS-deregulated proteins. Important deregulated hub proteins included hemopexin and transferrin in PD, and calreticulin and ferredoxin reductase in MS. Our findings show that PD and MS share commonalities in terms of iron accumulation in the SN. Concomitant proteomics experiments revealed PPI networks related to metal homeostasis, substantiating the results of metal quantification.

Altered SOD1 maturation and post-translational modification in amyotrophic lateral sclerosis spinal cord.

Aberrant self-assembly and toxicity of wild-type and mutant superoxide dismutase 1 (SOD1) has been widely examined in silico, in vitro and in transgenic animal models of amyotrophic lateral sclerosis. Detailed examination of the protein in disease-affected tissues from amyotrophic lateral sclerosis patients, however, remains scarce. We used histological, biochemical and analytical techniques to profile alterations to SOD1 protein deposition, subcellular localization, maturation and post-translational modification in post-mortem spinal cord tissues from amyotrophic lateral sclerosis cases and controls. Tissues were dissected into ventral and dorsal spinal cord grey matter to assess the specificity of alterations within regions of motor neuron degeneration. We provide evidence of the mislocalization and accumulation of structurally disordered, immature SOD1 protein conformers in spinal cord motor neurons of SOD1-linked and non-SOD1-linked familial amyotrophic lateral sclerosis cases, and sporadic amyotrophic lateral sclerosis cases, compared with control motor neurons. These changes were collectively associated with instability and mismetallation of enzymatically active SOD1 dimers, as well as alterations to SOD1 post-translational modifications and molecular chaperones governing SOD1 maturation. Atypical changes to SOD1 protein were largely restricted to regions of neurodegeneration in amyotrophic lateral sclerosis cases, and clearly differentiated all forms of amyotrophic lateral sclerosis from controls. Substantial heterogeneity in the presence of these changes was also observed between amyotrophic lateral sclerosis cases. Our data demonstrate that varying forms of SOD1 proteinopathy are a common feature of all forms of amyotrophic lateral sclerosis, and support the presence of one or more convergent biochemical pathways leading to SOD1 proteinopathy in amyotrophic lateral sclerosis. Most of these alterations are specific to regions of neurodegeneration, and may therefore constitute valid targets for therapeutic development.

Native Separation and Metallation Analysis of SOD1 Protein from the Human Central Nervous System: a Methodological Workflow.

Studies of the metal content of metalloproteins in tissues from the human central nervous system (CNS) can be compromised by preparative techniques which alter levels of, or interactions between, metals and the protein of interest within a complex mixture. We developed a methodological workflow combining size exclusion chromatography, native isoelectric focusing, and either proton or synchrotron X-ray fluorescence within electrophoresis gels to analyze the endogenous metal content of copper-zinc superoxide dismutase (SOD1) purified from minimal amounts (<20 mg) of post-mortem human brain and spinal cord tissue. Abnormal metallation and aggregation of SOD1 are suspected to play a role in amyotrophic lateral sclerosis and Parkinson's disease, but data describing SOD1 metal occupancy in human tissues have not previously been reported. Validating our novel approach, we demonstrated step-by-step metal preservation, preserved SOD1 activity, and substantial enrichment of SOD1 protein versus confounding metalloproteins. We analyzed tissues from nine healthy individuals and five CNS regions (occipital cortex, substantia nigra, locus coeruleus, dorsal spinal cord, and ventral spinal cord). We found that Cu and Zn were bound to SOD1 in a ratio of 1.12 ± 0.28, a ratio very close to the expected value of 1. Our methodological workflow can be applied to the study of endogenous native SOD1 in a pathological context and adapted to a range of metalloproteins from human tissues and other sources.

Isotopic variations of copper at the protein fraction level in neuronal human cells exposed in vitro to uranium.

The study of isotopic variations of endogenous and toxic metals in fluids and tissues is a recent research topic with an outstanding potential in biomedical and toxicological investigations. Most of the analyses have been performed so far in bulk samples, which can make the interpretation of results entangled, since different sources of stress or the alteration of different metabolic processes can lead to similar variations in the isotopic compositions of the elements in bulk samples. The downscaling of the isotopic analysis of elements at the sub-cellular level, is considered as a more promising alternative. Here we present for the first time the accurate determination of Cu isotopic ratios in four main protein fractions from lysates of neuron-like human cells exposed in vitro to 10 µM of natural uranium for seven days. These protein fractions were isolated by Size Exclusion Chromatography and analysed by Multi-Collector Inductively Coupled Plasma Mass Spectrometry to determine the Cu isotopic variations in each protein fraction with regard to the original cell lysate. Values obtained, expressed as δ65Cu, were -0.03 ± 0.14 ‰ (Uc, k = 2), -0.55 ± 0.20 ‰ (Uc, k = 2), -0.32 ± 0.21 ‰ (Uc, k = 2) and +0.84 ± 0.21 ‰ (Uc, k = 2) for the four fractions, satisfying the mass balance. The results obtained in this preliminary study pave the way for dedicated analytical developments to identify new specific disease biomarkers, to gain insight into stress-induced altered metabolic processes, as well as to decipher metabolic pathways of toxic elements.

Manganese Mapping Using a Fluorescent Mn2+ Sensor and Nanosynchrotron X-ray Fluorescence Reveals the Role of the Golgi Apparatus as a Manganese Storage Site.

Elucidating dynamics in transition-metal distribution and localization under physiological and pathophysiological conditions is central to our understanding of metal-ion regulation. In this Forum Article, we focus on manganese and specifically recent developments that point to the relevance of the Golgi apparatus in manganese detoxification when this essential metal ion is overaccumulated because of either environmental exposure or mutations in manganese efflux transporters. In order to further evaluate the role of the Golgi apparatus as a manganese-ion storage compartment under subcytotoxic manganese levels, we use a combination of confocal microscopy using a sensitive "turn-on" fluorescent manganese sensor, M1, and nanosynchrotron X-ray fluorescence imaging to show that manganese ions are stored in the Golgi apparatus under micromolar manganese exposure concentrations. Our results, along with previous reports on manganese accumulation, now indicate a central role of the Golgi apparatus in manganese storage and trafficking under subcytotoxic manganese levels and hint toward a possible role of the Golgi apparatus in manganese storage even under physiological conditions.

Deciphering the uranium target proteins in human dopaminergic SH-SY5Y cells.

Uranium (U) is the heaviest naturally occurring element ubiquitously present in the Earth's crust. Human exposure to low levels of U is, therefore, unavoidable. Recently, several studies have clearly pointed out that the brain is a sensitive target for U, but the mechanisms leading to the observed neurological alterations are not fully known. To deepen our knowledge of the biochemical disturbances resulting from U(VI) toxicity in neuronal cells, two complementary strategies were set up to identify the proteins that selectively bind U(VI) in human dopaminergic SH-SY5Y cells. The first strategy relies on the selective capture of proteins capable of binding U(VI), using immobilized metal affinity chromatography, and starting from lysates of cells grown in a U(VI)-free medium. The second strategy is based on the separation of U-enriched protein fractions by size-exclusion chromatography, starting from lysates of U(VI)-exposed cells. High-resolution mass spectrometry helped us to highlight 269 common proteins identified as the urano-proteome. They were further analyzed to characterize their cellular localization and biological functions. Four canonical pathways, related to the protein ubiquitination system, gluconeogenesis, glycolysis, and the actin cytoskeleton proteins, were particularly emphasized due to their high content of U(VI)-bound proteins. A semi-quantification was performed to concentrate on the ten most abundant proteins, whose physico-chemical characteristics were studied in particular depth. The selective interaction of U(VI) with these proteins is an initial element of proof of the possible metabolic effects of U(VI) on neuronal cells at the molecular level.

Evidence of isotopic fractionation of natural uranium in cultured human cells.

The study of the isotopic fractionation of endogen elements and toxic heavy metals in living organisms for biomedical applications, and for metabolic and toxicological studies, is a cutting-edge research topic. This paper shows that human neuroblastoma cells incorporated small amounts of uranium (U) after exposure to 10 µM natural U, with preferential uptake of the 235U isotope with regard to 238U. Efforts were made to develop and then validate a procedure for highly accurate n(238U)/n(235U) determinations in microsamples of cells. We found that intracellular U is enriched in 235U by 0.38 ± 0.13‰ (2σ, n = 7) relative to the exposure solutions. These in vitro experiments provide clues for the identification of biological processes responsible for uranium isotopic fractionation and link them to potential U incorporation pathways into neuronal cells. Suggested incorporation processes are a kinetically controlled process, such as facilitated transmembrane diffusion, and the uptake through a high-affinity uranium transport protein involving the modification of the uranyl (UO22+) coordination sphere. These findings open perspectives on the use of isotopic fractionation of metals in cellular models, offering a probe to track uptake/transport pathways and to help decipher associated cellular metabolic processes.

Amyotrophic lateral sclerosis-like superoxide dismutase 1 proteinopathy is associated with neuronal loss in Parkinson's disease brain.

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 Parkinson's disease brain exhibits evidence of misfolding and metal deficiency, similar to that seen in mutant SOD1 in fALS. Our data suggest common mechanisms of toxic SOD1 aggregation in both disorders and a potential role for SOD1 dysfunction in neuronal loss in the Parkinson's disease brain. This shared restricted proteinopathy highlights the potential translation of therapeutic approaches targeting SOD1 toxicity, already in clinical trials for ALS, into disease-modifying treatments for Parkinson's disease.

Heterogeneous intratumoral distribution of gadolinium nanoparticles within U87 human glioblastoma xenografts unveiled by micro-PIXE imaging.

Metallic nanoparticles have great potential in cancer radiotherapy as theranostic drugs since, they serve simultaneously as contrast agents for medical imaging and as radio-therapy sensitizers. As with other anticancer drugs, intratumoral diffusion is one of the main limiting factors for therapeutic efficiency. To date, a few reports have investigated the intratumoral distribution of metallic nanoparticles. The aim of this study was to determine the quantitative distribution of gadolinium (Gd) nanoparticles after direct intratumoral injection within U87 human glioblastoma tumors grafted in mice, using micro-PIXE (Particle Induced X-ray Emission) imaging. AGuIX (Activation and Guiding of Irradiation by X-ray) 3 nm particles composed of a polysiloxane network surrounded by gadolinium chelates were used. PIXE results indicate that the direct injection of Gd nanoparticles in tumors results in their heterogeneous diffusion, probably related to variations in tumor density. All tumor regions contain Gd, but with markedly different concentrations, with a more than 250-fold difference. Also Gd can diffuse to the healthy adjacent tissue. This study highlights the usefulness of mapping the distribution of metallic nanoparticles at the intratumoral level, and proposes PIXE as an imaging modality to probe the quantitative distribution of metallic nanoparticles in tumors from experimental animal models with micrometer resolution.

Reduced net charge and heterogeneity of pI isoforms in familial amyotrophic lateral sclerosis mutants of copper/zinc superoxide dismutase.

Familial cases of amyotrophic lateral sclerosis (fALS) are related to mutations of copper/zinc superoxide dismutase 1 (SOD1). Aggregation of SOD1 plays a central role in the pathogenesis of fALS and altered metallation of SOD1 mutants could be involved in this process. Using IEF gel electrophoresis under non-denaturating conditions and particle induced X-ray emission (PIXE) analysis, we studied the pI distribution and metallation status of fALS SOD1 mutants (A4V, G93A, D125H) compared to human wild-type (hWT). SOD1 fALS mutants are characterized by a variable number of isoforms and higher pI compared to hWT, reflecting a reduced net charge that might explain their greater propensity to precipitation and aggregation. Cu/Zn ratios were slightly different for the predominant expressed isoforms of A4V, G93A, and D125H mutants compared to hWT. Differences in metallation were observed within each genotype, the more basic isoforms exhibiting lower Cu/Zn ratios. Moreover, we revealed the existence of a pool of fALS mutants SOD1 pI isoforms, slightly expressed (<10%), with a low Cu/Zn ratio and high pI values. Overall, IEF-PIXE results suggest that the toxicity of SOD1 mutants should be studied at the pI isoform level with a particular attention to the species with the lowest charges.

Stratigraphic analysis of the Sterkfontein StW 573 Australopithecus skeleton and implications for its age.

StW 573, Little Foot, is the most complete Australopithecus skeleton yet discovered, with many of its bones found in their correct anatomical position. Since the discovery of the in situ skeleton in the Silberberg Grotto in 1997, several teams have attempted to date the fossil. This appeared a simple process because several flowstones are inter-bedded in the breccia above and below StW 573. Dating of these flowstones, using U-Pb (uranium-lead) isotope decay techniques, gave younger results than expected from the fauna and stratigraphic position, around 2.2 Ma (millions of years). Our recent stratigraphic, micromorphological and geochemical studies revealed that the stratigraphy is much more complicated than was previously thought, with localized post-depositional processes leading to the creation of voids within the breccia around the skeleton. These voids were then filled by multiple generations of flowstone growth. The research we present here demonstrates that the proposed dates based on the flowstone deposition can give only a minimum age for StW 573 and that the flowstone formation came after, and probably long after, the breccia deposition. If one takes account of the long evolution of these karst fillings, StW 573 appears to be significantly older than 2.2 Ma.

Correlative organelle fluorescence microscopy and synchrotron X-ray chemical element imaging in single cells.

X-ray chemical element imaging has the potential to enable fundamental breakthroughs in the understanding of biological systems because chemical element interactions with organelles can be studied at the sub-cellular level. What is the distribution of trace metals in cells? Do some elements accumulate within sub-cellular organelles? What are the chemical species of the elements in these organelles? These are some of the fundamental questions that can be addressed by use of X-ray chemical element imaging with synchrotron radiation beams. For precise location of the distribution of the elements, identification of cellular organelles is required; this can be achieved, after appropriate labelling, by use of fluorescence microscopy. As will be discussed, this approach imposes some limitations on sample preparation. For example, standard immunolabelling procedures strongly modify the distribution of the elements in cells as a result of the chemical fixation and permeabilization steps. Organelle location can, however, be performed, by use of a variety of specific fluorescent dyes or fluorescent proteins, on living cells before cryogenic fixation, enabling preservation of element distribution. This article reviews the methods used for fluorescent organelle labelling and X-ray chemical element imaging and speciation of single cells. Selected cases from our work and from other research groups are presented to illustrate the potential of the combination of the two techniques.

Low-solubility particles and a Trojan-horse type mechanism of toxicity: the case of cobalt oxide on human lung cells.

BACKGROUND: The mechanisms of toxicity of metal oxide particles towards lung cells are far from being understood. In particular, the relative contribution of intracellular particulate versus solubilized fractions is rarely considered as it is very challenging to assess, especially for low-solubility particles such as cobalt oxide (Co3O4). METHODS: This study was possible owing to two highly sensitive, independent, analytical techniques, based on single-cell analysis, using ion beam microanalysis, and on bulk analysis of cell lysates, using mass spectrometry. RESULTS: Our study shows that cobalt oxide particles, of very low solubility in the culture medium, are readily incorporated by BEAS-2B human lung cells through endocytosis via the clathrin-dependent pathway. They are partially solubilized at low pH within lysosomes, leading to cobalt ions release. Solubilized cobalt was detected within the cytoplasm and the nucleus. As expected from these low-solubility particles, the intracellular solubilized cobalt content is small compared with the intracellular particulate cobalt content, in the parts-per-thousand range or below. However, we were able to demonstrate that this minute fraction of intracellular solubilized cobalt is responsible for the overall toxicity. CONCLUSIONS: Cobalt oxide particles are readily internalized by pulmonary cells via the endo-lysosomal pathway and can lead, through a Trojan-horse mechanism, to intracellular release of toxic metal ions over long periods of time, involving specific toxicity.

TauSTED super-resolution imaging of labile iron in primary hippocampal neurons.

Iron dyshomeostasis is involved in many neurological disorders, particularly neurodegenerative diseases where iron accumulates in various brain regions. Identifying mechanisms of iron transport in the brain is crucial for understanding the role of iron in healthy and pathological states. In neurons, it has been suggested that iron can be transported by the axon to different brain regions in the form of labile iron; a pool of reactive and exchangeable intracellular iron. Here we report a novel approach to imaging labile ferrous iron, Fe(II), in live primary hippocampal neurons using confocal and TauSTED (stimulated emission depletion) microscopy. TauSTED is based on super-resolution STED nanoscopy, which combines high spatial resolution imaging (<40 nm) with fluorescence lifetime information, thus reducing background noise and improving image quality. We applied TauSTED imaging utilizing biotracker FerroFarRed Fe(II) and found that labile iron was present as submicrometric puncta in dendrites and axons. Some of these iron-rich structures are mobile and move along neuritic pathways, arguing for a labile iron transport mechanism in neurons. This super-resolution imaging approach offers a new perspective for studying the dynamic mechanisms of axonal and dendritic transport of iron at high spatial resolution in living neurons. In addition, this methodology could be transposed to the imaging of other fluorescent metal sensors.

Multimodal and multiscale correlative elemental imaging: From whole tissues down to organelles.

Chemical elements, especially metals, play very specific roles in the life sciences. The implementation of correlative imaging methods, of elements on the one hand and of molecules or biological structures on the other hand, is the subject of recent developments. The most commonly used spectro-imaging techniques for metals are synchrotron-induced X-ray fluorescence, mass spectrometry and fluorescence imaging of metal molecular sensors. These imaging methods can be correlated with a wide variety of other analytical techniques used for structural imaging (e.g., electron microscopy), small molecule imaging (e.g., molecular mass spectrometry) or protein imaging (e.g., fluorescence microscopy). The resulting correlative imaging is developed at different scales, from biological tissue to the subcellular level. The fields of application are varied, with some major research topics, the role of metals in the aetiology of neurodegenerative diseases and the use of metals for medical imaging or cancer treatment.

Combined use of hard X-ray phase contrast imaging and X-ray fluorescence microscopy for sub-cellular metal quantification.

Hard X-ray fluorescence microscopy and magnified phase contrast imaging are combined to obtain quantitative maps of the projected metal concentration in whole cells. The experiments were performed on freeze dried cells at the nano-imaging station ID22NI of the European Synchrotron Radiation Facility (ESRF). X-ray fluorescence analysis gives the areal mass of most major, minor and trace elements; it is validated using a biological standard of known composition. Quantitative phase contrast imaging provides maps of the projected mass and is validated using calibration samples and through comparison with Atomic Force Microscopy and Scanning Transmission Ion Microscopy. Up to now, absolute quantification at the sub-cellular level was impossible using X-ray fluorescence microscopy but can be reached with the use of the proposed approach.

Plant cell nucleolus as a hot spot for iron.

Many central metabolic processes require iron as a cofactor and take place in specific subcellular compartments such as the mitochondrion or the chloroplast. Proper iron allocation in the different organelles is thus critical to maintain cell function and integrity. To study the dynamics of iron distribution in plant cells, we have sought to identify the different intracellular iron pools by combining three complementary imaging approaches, histochemistry, micro particle-induced x-ray emission, and synchrotron radiation micro X-ray fluorescence. Pea (Pisum sativum) embryo was used as a model in this study because of its large cell size and high iron content. Histochemical staining with ferrocyanide and diaminobenzidine (Perls/diaminobenzidine) strongly labeled a unique structure in each cell, which co-labeled with the DNA fluorescent stain DAPI, thus corresponding to the nucleus. The unexpected presence of iron in the nucleus was confirmed by elemental imaging using micro particle-induced x-ray emission. X-ray fluorescence on cryo-sectioned embryos further established that, quantitatively, the iron concentration found in the nucleus was higher than in the expected iron-rich organelles such as plastids or vacuoles. Moreover, within the nucleus, iron was particularly accumulated in a subcompartment that was identified as the nucleolus as it was shown to transiently disassemble during cell division. Taken together, our data uncover an as yet unidentified although abundant iron pool in the cell, which is located in the nuclei of healthy, actively dividing plant tissues. This result paves the way for the discovery of a novel cellular function for iron related to nucleus/nucleolus-associated processes.

Coupling of native IEF and extended X-ray absorption fine structure to characterize zinc-binding sites from pI isoforms of SOD1 and A4V pathogenic mutant.

Extended X-ray absorption fine structure (EXAFS) has already provided high-resolution structures of metal-binding sites in a wide variety of metalloproteins. Usually, EXAFS is performed on purified metalloproteins either in solution or crystallized form but purification steps are prone to modify the metallation state of the protein. We developed a protocol to couple EXAFS analysis to metalloprotein separation using native gel electrophoresis. This coupling opens a large field of applications as metalloproteins can be characterized in their native state avoiding purification steps. Using native isoelectric focusing, the method enables the EXAFS analysis of metalloprotein pI isoforms. We applied this methodology to SOD1, wild-type, and Ala4Val mutant (A4V), a mutation found in amyotrophic lateral sclerosis (ALS) because decreased Zn affinity to SOD1 mutants is suggested to be involved in the pathogenesis of this neurodegenerative disease. We observed similar coordination structures for Zn in wild-type and mutant proteins, in all measured pI isoforms, demonstrating the feasibility of EXAFS on electrophoresis gels and suggesting that the Zn-binding site is not structurally modified in A4V SOD1 mutant.

Neurotoxicity of Environmental Metal Toxicants: Special Issue.

Environmental exposure to metallic neurotoxicants is a matter of growing concern, since it may have very significant consequences for human health, from impairing neurodevelopment in children to the neurodegeneration processes involved in aging [...].

Correlative nano-imaging of metals and proteins in primary neurons by synchrotron X-ray fluorescence and STED super resolution microscopy: Experimental validation.

BACKGROUND: It is becoming increasingly clear that biological metals such as iron, copper or zinc are involved in synaptic functions, and in particular in the mechanisms of synaptogenesis and subsequent plasticity. Understanding the role of metals on synaptic functions is a difficult challenge due to the very low concentration of these elements in neurons and to the submicrometer size of synaptic compartments. NEW METHOD: To address this challenge we have developed a correlative nano-imaging approach combining metal and protein detection. First, stimulated emission depletion (STED) microscopy, a super resolution optical microscopy technique, is applied to locate fluorescently labeled proteins. Then, synchrotron radiation induced X-ray fluorescence (SXRF) is performed on the same regions of interest, e.g. synaptic compartments. RESULTS: We present here the principle scheme that allows this correlative nano-imaging and its experimental validation. We applied this correlative nano-imaging to the study of the physiological distribution of metals in synaptic compartments of primary rat hippocampal neurons. We thus compared the nanometric distribution of metals with that of synaptic proteins, such as PSD95 or cytoskeleton proteins. COMPARISON WITH EXISTING METHOD(S): Compared to correlative imaging approaches currently used to characterize synaptic structures, such as electron microscopy correlated with optical fluorescence, our approach allows for ultra-sensitive detection of trace metals using highly focused synchrotron radiation beams. CONCLUSION: We provide proof-of-principle for correlative imaging of metals and proteins at the synaptic scale and discuss the present limitations and future developments in this area.