Interaction Between the Transferrin Protein and Plutonium (and Thorium), What's New?

Transferrin (Tf) is a glycoprotein that transports iron from the serum to the various organs. Several studies have highlighted that Tf can interact with metals other than Fe(III), including actinides that are chemical and radiological toxics. We propose here to report on the behavior of Th(IV) and Pu(IV) in comparison with Fe(III) upon Tf complexation. We considered UV-Vis and IR data of the M2 Tf complex (M=Fe, Th, Pu) and combined experimental EXAFS data with MD models. EXAFS data of the first M-O coordination sphere are consistent with the MD model considering 1 synergistic carbonate. Further EXAFS data analysis strongly suggests that contamination by Th/Pu colloids seems to occur upon Tf complexation, but it seems limited. SAXS data have also been recorded for all complexes and also after the addition of Deferoxamine-B (DFOB) in the medium. The Rg values are very close for apoTf, ThTf and PuTf, but slightly larger than for holoTf. Data suggest that the structure of the protein is more ellipsoidal than spherical, with a flattened oblate form. From this data, the following order of conformation size might be considered:holoTf

Environmental Chemistry of Radionuclides : Open Questions and Perspectives.

Since the discovery of nuclear fission, atomic energy has become for mankind a source of energy, but it has also become a source of consternation. This Perspective presents and discusses the methodological evolution of the work performed in the radiochemistry laboratory that is part of the Institut de Chimie de Nice (France). Most studies in radioecology and environmental radiochemistry have intended to assess the impact and inventory of very low levels of radionuclides in specific environmental compartments. But chemical mechanisms at the molecular level remain a mystery because it is technically impossible (due to large dilution factors) to assess speciation in those systems. Ultra-trace levels of contamination and heterogeneity often preclude the use of spectroscopic techniques and the determination of direct speciation data, thus forming the bottleneck of speciation studies. The work performed in the Nice radiochemistry laboratory underlines this effort to input speciation data (using spectroscopic techniques like X ray Absorption Spectroscopy) in environmental and radioecological metrics.

Interaction of Th(IV), Pu(IV) and Fe(III) with ferritin protein: how similar?

Ferritin is the main protein of Fe storage in eukaryote and prokaryote cells. It is a large multifunctional, multi-subunit protein consisting of heavy H and light L subunits. In the field of nuclear toxicology, it has been suggested that some actinide elements, such as thorium and plutonium at oxidation state +IV, have a comparable `biochemistry' to iron at oxidation state +III owing to their very high tendency for hydrolysis and somewhat comparable ionic radii. Therefore, the possible mechanisms of interaction of such actinide elements with the Fe storage protein is a fundamental question of bio-actinidic chemistry. We recently described the complexation of Pu(IV) and Th(IV) with horse spleen ferritin (composed mainly of L subunits). In this article, we bring another viewpoint to this question by further combining modeling with our previous EXAFS data for Pu(IV) and Th(IV). As a result, the interaction between the L subunits and both actinides appears to be non-specific but driven only by the density of the presence of Asp and Glu residues on the protein shell. The formation of an oxyhydroxide Th or Pu core has not been observed under the experimental conditions here, nor the interaction of Th or Pu with the ferric oxyhydroxide core.

Low doses of uranium and osteoclastic bone resorption: key reciprocal effects evidenced using new in vitro biomimetic models of bone matrix.

Uranium is widely spread in the environment due to its natural and anthropogenic occurrences, hence the importance of understanding its impact on human health. The skeleton is the main site of long-term accumulation of this actinide. However, interactions of this metal with biological processes involving the mineralized extracellular matrix and bone cells are still poorly understood. To get a better insight into these interactions, we developed new biomimetic bone matrices containing low doses of natural uranium (up to 0.85 µg of uranium per cm2). These models were characterized by spectroscopic and microscopic approaches before being used as a support for the culture and differentiation of pre-osteoclastic cells. In doing so, we demonstrate that uranium can exert opposite effects on osteoclast resorption depending on its concentration in the bone microenvironment. Our results also provide evidence for the first time that resorption contributes to the remobilization of bone matrix-bound uranium. In agreement with this, we identified, by HRTEM, uranium phosphate internalized in vesicles of resorbing osteoclasts. Thanks to the biomimetic matrices we developed, this study highlights the complex mutual effects between osteoclasts and uranium. This demonstrates the relevance of these 3D models to further study the cellular mechanisms at play in response to uranium storage in bone tissue, and thus better understand the impact of environmental exposure to uranium on human bone health.

How Does Iron Storage Protein Ferritin Interact with Plutonium (and Thorium)?

The impact of the contamination of living organisms by actinide elements has been a constant subject of attention since the 1950s. But to date still little is understood. Ferritin is the major storage and regulation protein of iron in many organisms, it consists of a protein ring and a ferrihydric core at the center. This work sheds light on the interactions of early actinides (Th, Pu) at oxidation state +IV with ferritin and its ability to store those elements at physiological pH compared to Fe. The ferritin-thorium load curve suggests that ThIV saturates the protein (2840 Th atoms per ferritin) in a similar way that Fe does on the protein ring. Complementary spectroscopic techniques (spectrophotometry, infrared spectroscopy, and X-ray absorption spectroscopy) were combined with molecular dynamics to provide a structural model of the interaction of ThIV and PuIV with ferritin. Comparison of spectroscopic data together with MD calculations suggests that ThIV and PuIV are complexed mainly on the protein ring and not on the ferrihydric core. Indeed from XAS data, there is no evidence of Fe neighbors in the Th and Pu environments. On the other hand, carboxylates from amino acids of the protein ring and a possible additional carbonate anion are shaping the cation coordination spheres. This thorough description from a molecular view point of ThIV and PuIV interaction with ferritin, an essential iron storage protein, is a cornerstone in comprehensive nuclear toxicology.

Interdisciplinary Round-Robin Test on Molecular Spectroscopy of the U(VI) Acetate System.

A comprehensive molecular analysis of a simple aqueous complexing system-U(VI) acetate-selected to be independently investigated by various spectroscopic (vibrational, luminescence, X-ray absorption, and nuclear magnetic resonance spectroscopy) and quantum chemical methods was achieved by an international round-robin test (RRT). Twenty laboratories from six different countries with a focus on actinide or geochemical research participated and contributed to this scientific endeavor. The outcomes of this RRT were considered on two levels of complexity: first, within each technical discipline, conformities as well as discrepancies of the results and their sources were evaluated. The raw data from the different experimental approaches were found to be generally consistent. In particular, for complex setups such as accelerator-based X-ray absorption spectroscopy, the agreement between the raw data was high. By contrast, luminescence spectroscopic data turned out to be strongly related to the chosen acquisition parameters. Second, the potentials and limitations of coupling various spectroscopic and theoretical approaches for the comprehensive study of actinide molecular complexes were assessed. Previous spectroscopic data from the literature were revised and the benchmark data on the U(VI) acetate system provided an unambiguous molecular interpretation based on the correlation of spectroscopic and theoretical results. The multimethodologic approach and the conclusions drawn address not only important aspects of actinide spectroscopy but particularly general aspects of modern molecular analytical chemistry.

How Do Actinyls Interact with Hyperphosphorylated Yolk Protein Phosvitin?

The development of the nuclear industry has raised multiple questions about its impact on the biotope and humans. Proteins are key biomolecules in cell machinery and essential in deciphering toxicological processes. Phosvitin was chosen as a relevant model for phosphorylated proteins because of its important role as an iron, calcium, and magnesium storage protein in egg yolk. A multitechnique spectroscopic investigation was performed to reveal the coordination geometry of two oxocations of the actinide family (actinyl UVI , NpV ) in speciation with phosvitin. IR spectroscopy revealed phosphoryl groups as the main functional groups interacting with UVI . This was confirmed through laser luminescence spectroscopy (U) and UV/Vis absorption spectroscopy (Np). For UVI , X-ray absorption spectroscopy at the LIII edge revealed a small contribution of bidentate binding present, along with predominantly monodentate binding of phosphoryl groups; for NpV , uniquely bidentate binding was revealed. As a perspective to this work, X-ray absorption spectroscopy speciation of UVI and NpV in the extracted yolk of living eggs of the dogfish Scyliorhinus canicula was determined; this corroborated the binding of phosphorous together with a reduction of the actinyl moiety. Such data are essential to pinpoint the mechanisms of heavy metals (actinyls) accumulation and toxicity in oviparous organisms, and therefore, contribute to a shift from descriptive approaches to predictive toxicology.

Uranium Effect on Osteocytic Cells In Vitro.

Once absorbed in the body, natural uranium [U(VI)], a radionucleotide naturally present in the environment, is targeted to the skeleton which is the long-term storage organ. We and others have reported the U(VI) negative effects on osteoblasts (OB) and osteoclasts (OC), the main two cell types involved in bone remodeling. In the present work, we addressed the U(VI) effect on osteocytes (OST), the longest living bone cell type and the more numerous (> 90%). These cells, which are embedded in bone matrix and thus are the more prone to U(VI) long-term exposure, are now considered as the chief orchestrators of the bone remodeling process. Our results show that the cytotoxicity index of OST is close to 730 µM, which is about twice the one reported for OB and OC. However, despite this resistance potential, we observed that chronic U(VI) exposure as low as 5 µM led to a drastic decrease of the OST mineralization function. Gene expression analysis showed that this impairment could potentially be linked to an altered differentiation process of these cells. We also observed that U(VI) was able to trigger autophagy, a highly conserved survival mechanism. Extended X-ray absorption fine structure analysis at the U LIII edge of OST cells exposed to U(VI) unambiguously shows the formation of an uranyl phosphate phase in which the uranyl local structure is similar to the one present in Autunite. Thus, our results demonstrate for the first time that OST mineralization function can be affected by U(VI) exposure as low as 5 µM, suggesting that prolonged exposure could alter the central role of these cells in the bone environment.

Is hydroxypyridonate 3,4,3-LI(1,2-HOPO) a good competitor of fetuin for uranyl metabolism?

Uranium is widespread in the environment, resulting both from natural occurrences and anthropogenic activities. Its toxicity is mainly chemical rather than radiological. In the blood it is transported as uranyl UO22+ cation and forms complexes with small ligands like carbonates and with some proteins. From there it reaches the skeleton, its main target organ for accumulation. Fetuin is a serum protein involved in biomineralization processes, and it was demonstrated to be the main UO22+-binder in vitro. Fetuin's life cycle ends in bone. It is thus suspected to be a key protagonist of U accumulation in this organ. Up to now, there has been no effective treatment for the removal of U from the body and studies devoted to the interactions involving chelating agents with both UO22+ and its protein targets are lacking. The present work aims at studying the potential role of 3,4,3-LI(1,2-HOPO) as a promising chelating agent in competition with fetuin. The apparent affinity constant of 3,4,3-LI(1,2-HOPO) was first determined, giving evidence for its very high affinity similar to that of fetuin. Chromatography experiments, aimed at identifying the complexes formed and quantifying their UO22+ content, and spectroscopic structural investigations (XAS) were carried out, demonstrating that 3,4,3-LI(1,2-HOPO) inhibits/limits the formation of fetuin-uranyl complexes under stoichiometric conditions. But surprisingly, possible ternary complexes stable enough to remain present after the chromatographic process were identified under sub-stoichiometric conditions of HOPO versus fetuin. These results contribute to the understanding of the mechanisms accounting for U residual accumulation despite chelation therapy after internal contamination.

Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis.

Uranium has been shown to interfere with bone physiology and it is well established that this metal accumulates in bone. However, little is known about the effect of natural uranium on the behavior of bone cells. In particular, the impact of uranium on osteoclasts, the cells responsible for the resorption of the bone matrix, is not documented. To investigate this issue, we have established a new protocol using uranyl acetate as a source of natural uranium and the murine RAW 264.7 cell line as a model of osteoclast precursors. Herein, we detailed all the assays required to test uranium cytotoxicity on osteoclast precursors and to evaluate its impact on the osteoclastogenesis and on the resorbing function of mature osteoclasts. The conditions we have developed, in particular for the preparation of uranyl-containing culture media and for the seeding of RAW 264.7 cells allow to obtain reliable and highly reproductive results. Moreover, we have optimized the use of software tools to facilitate the analysis of various parameters such as the size of osteoclasts or the percentage of resorbed matrix.

Polyethyleneimine methylphosphonate: towards the design of a new class of macromolecular actinide chelating agents in the case of human exposition.

The use of uranium and to a minor extent plutonium as fuel for nuclear energy production or as components in military applications is under increasing public pressure. Uranium is weakly radioactive in its natural isotopy but its chemical toxicity, combined with its large scale industrial utilization, makes it a source of concern in terms of health impact for workers and possibly the general population. Plutonium is an artificial element that exhibits both chemical and radiological toxicities. So far, uranium (under its form uranyl, U(vi)) or plutonium (as Pu(iv)) decorporation or protecting strategies based on molecular design have been of limited efficiency to remove the actinide once incorporated after human exposure. In all cases, after human exposure, plutonium and uranium are retained in main target organs (liver, kidneys) as well as skeleton although they exhibit differences in their biodistribution. Polymers could represent an alternative strategy as their tropism for specific target organs has been reported. We recently reported on the complexation properties of methylcarboxylated polyethyleneimine (PEI-MC) with uranyl. In this report we extend our work to methylphosphonated polyethyleneimine (PEI-MP) and to the comparison between actinide oxidation states +IV (thorium) and +VI (uranyl). As a first step, thorium (Th(iv)) was used as a chemical surrogate of plutonium because of the difficulty in handling the latter in the laboratory. For both cations, U(vi) and Th(iv), the uptake curve of PEI-MP was recorded. The functionalized PEI-MP exhibits a maximum loading capacity comprised of between 0.56 and 0.80 mg of uranium (elemental) and 0.15-0.20 mg of thorium (elemental) per milligram of PEI-MP. Complexation sites of U(vi) and Th(iv) under model conditions close to physiological pH were then characterized with a combination of Fourier transform Infra Red (FT-IR) and Extended X-Ray Absorption Fine Structure (EXAFS). Although both cations exhibit different coordination modes, similar structural parameters with phosphonate functions were obtained. For example, the coordination sites are composed of fully monodentate phosphonate functions of the polymer chains. These physical chemical data represent a necessary basic chemistry approach before envisioning further biological evaluations of PEI-MP polymers towards U(vi) and Pu/Th(iv) contamination.

Cyclic Phosphopeptides to Rationalize the Role of Phosphoamino Acids in Uranyl Binding to Biological Targets.

The specific molecular interactions responsible for uranium toxicity are not yet understood. The uranyl binding sites in high-affinity target proteins have not been identified yet and the involvement of phosphoamino acids is still an important question. Short cyclic peptide sequences, with three glutamic acids and one phosphoamino acid, are used as simple models to mimic metal binding sites in phosphoproteins and to help understand the mechanisms involved in uranium toxicity. A combination of peptide design and synthesis, analytical chemistry, extended X-ray absorption fine structure (EXAFS) spectroscopy, and DFT calculations demonstrates the involvement of the phosphate group in the uranyl coordination sphere together with the three carboxylates of the glutamate moieties. The affinity constants measured with a reliable analytical competitive approach at physiological pH are significantly enhanced owing to the presence of the phosphorous moiety. These findings corroborate the importance of phosphoamino acids in uranyl binding in proteins and the relevance of considering phosphoproteins as potential uranyl targets in vivo.

A Combined Spectroscopic/Molecular Dynamic Study for Investigating a Methyl-Carboxylated PEI as a Potential Uranium Decorporation Agent.

Natural uranium has a very limited radioactive dose impact, but its chemical toxicity due to chronic exposure is still a matter of debate. Once inside the human body, the soluble uranium, under its uranyl form (U(VI)), is quickly removed from the blood system, partially excreted from the body, and partially retained in targeted organs, that is, the kidneys and bone matrix essentially. It is then crucial to remove or prevent the incorporation of uranium in these organs to limit the long-term chronic exposure. A lot of small chelating agents such as aminocarboxylates, catecholamides, and hydroxypyridonates have been developed so far. However, they suffer from poor selectivity and targeting abilities. Macromolecules and polymers are known to present a passive accumulation (size related), that is, the so-called enhanced permeability and retention effect, toward the main organs, which can be used as indirect targeting. Very interestingly, the methyl carboxylated polyethylenimine (PEI-MC) derivative has been described as a potent sequestering agent for heavy metals. It would be therefore an interesting candidate to evaluate as a new class of decorporation agents with passive targeting capabilities matching uranium preferential sequestering sites. In the present work, we explored the ability of a highly functionalized (89% rate) PEI-MC to uptake U(VI) close to physiological pH using a combination of analytical and spectroscopic techniques (inductively coupled plasma optical emission spectrometry (ICP-OES); extended X-ray absorption fine structure (EXAFS); and Fourier transformed infrared (FT-IR)) together with molecular dynamics (MD) simulation. A maximum loading of 0.47 mg U(VI) per milligram of PEI-MC was determined by ICP-OES measurements. From FT-IR data, a majority of monodentate coordination of the carboxylate functions of the PEI-MC seems to occur. From EXAFS and MD, a mix of mono and bidentate coordination mode was observed. Note that agreement between the EXAFS metrical parameters and MD radial distribution functions is remarkable. To the best of our knowledge, this is the first comprehensive structural study of a macromolecular PEI-based agent considered for uranium decorporation purposes.

Effect of natural uranium on the UMR-106 osteoblastic cell line: impairment of the autophagic process as an underlying mechanism of uranium toxicity.

Natural uranium (U), which is present in our environment, exerts a chemical toxicity, particularly in bone where it accumulates. Generally, U is found at oxidation state +VI in its oxocationic form [Formula: see text] in aqueous media. Although U(VI) has been reported to induce cell death in osteoblasts, the cells in charge of bone formation, the molecular mechanism for U(VI) effects in these cells remains poorly understood. The objective of our study was to explore U(VI) effect at doses ranging from 5 to 600 µM, on mineralization and autophagy induction in the UMR-106 model osteoblastic cell line and to determine U(VI) speciation after cellular uptake. Our results indicate that U(VI) affects mineralization function, even at subtoxic concentrations (<100 µM). The combination of thermodynamic modeling of U with EXAFS data in the culture medium and in the cells clearly indicates the biotransformation of U(VI) carbonate species into a meta-autunite phase upon uptake by osteoblasts. We next assessed U(VI) effect at 100 and 300 µM on autophagy, a survival process triggered by various stresses such as metal exposure. We observed that U(VI) was able to rapidly activate autophagy but an inhibition of the autophagic flux was observed after 24 h. Thus, our results indicate that U(VI) perturbs osteoblastic functions by reducing mineralization capacity. Our study identifies for the first time U(VI) in the form of meta-autunite in mammalian cells. In addition, U(VI)-mediated inhibition of the autophagic flux may be one of the underlying mechanisms leading to the decreased mineralization and the toxicity observed in osteoblasts.

Competing coexisting phases in 2D water.

The properties of bulk water come from a delicate balance of interactions on length scales encompassing several orders of magnitudes: i) the Hydrogen Bond (HBond) at the molecular scale and ii) the extension of this HBond network up to the macroscopic level. Here, we address the physics of water when the three dimensional extension of the HBond network is frustrated, so that the water molecules are forced to organize in only two dimensions. We account for the large scale fluctuating HBond network by an analytical mean-field percolation model. This approach provides a coherent interpretation of the different events experimentally (calorimetry, neutron, NMR, near and far infra-red spectroscopies) detected in interfacial water at 160, 220 and 250 K. Starting from an amorphous state of water at low temperature, these transitions are respectively interpreted as the onset of creation of transient low density patches of 4-HBonded molecules at 160 K, the percolation of these domains at 220 K and finally the total invasion of the surface by them at 250 K. The source of this surprising behaviour in 2D is the frustration of the natural bulk tetrahedral local geometry and the underlying very significant increase in entropy of the interfacial water molecules.

Thermodynamic and Structural Investigation of Synthetic Actinide-Peptide Scaffolds.

The complexation of uranium and europium, in oxidation states +VI and +III, respectively, was investigated with pertinent bio-inorganic systems. Three aspartate-rich pentapeptides with different structural properties were selected for study to rationalize the structure-affinity relationships. Thermodynamic results, crosschecked by both isothermal titration calorimetry and time-resolved laser fluorescence spectroscopy, showed different affinity depending on the peptide for both Eu(III) and U(VI). The thermodynamic aspects were correlated to structural predictions, which were acquired by density functional theory quantum chemical calculations and from IR and extended X-ray absorption fine structure experiments. The combination of these microscopic properties revealed that carbonyl-metal interactions affected the entropy in the case of europium, while the larger uranyl cation was mostly affected by preorganization and steric effects, so that the affinity was enhanced through enthalpy. The approach described here revealed various microscopic aspects governing peptide actinide affinity. Highlighting these mechanisms should certainly contribute to the rational synthesis of higher affinity biomimetic aspartic ligands.

Actinide(IV) Deposits on Bone: Potential Role of the Osteopontin-Thorium Complex.

In case of a nuclear event, contamination (broad or limited) of the population or of specific workers might occur. In such a senario, the fate of actinide contaminants may be of first concern, in particular with regard to human target organs like the skeleton. To improve our understanding of the toxicological processes that might take place, a mechanistic approach is necessary. For instance, ∼50% of Pu(IV) is known from biokinetic data to accumulate in bone, but the underlining mechanisms are almost unknown. In this context, and to obtain a better description of the toxicological mechanisms associated with actinides(IV), we have undertaken the investigation, on a molecular scale, of the interaction of thorium(IV) with osteopontin (OPN) a hyperphosphorylated protein involved in bone turnover. Thorium is taken here as a simple model for actinide(IV) chemistry. In addition, we have selected a phosphorylated hexapeptide (His-pSer-Asp-Glu-pSer-Asp-Glu-Val) that is representative of the peptidic sequence involved in the bone interaction. For both the protein and the biomimetic peptide, we have determined the local environment of Th(IV) within the bioactinidic complex, combining isothermal titration calorimetry, attenuated total reflectance Fourier transform infrared spectroscopy, theoretical calculations with density functional theory, and extended X-ray absorption fine structure spectroscopy at the Th LIII edge. The results demonstrate a predominance of interaction of metal with the phosphate groups and confirmed the previous physiological studies that have highlighted a high affinity of Th(IV) for the bone matrix. Data are further compared with those of the uranyl case, representing the actinyl(V) and actinyl(VI) species. Last, our approach shows the importance of developing simplified systems [Th(IV)-peptide] that can serve as models for more biologically relevant systems.

Confined H2O molecules as local probes of pressure-induced amorphisation in faujasite.

Confined H2O molecules act as local probes for depressurization phenomena during the pressure induced amorphisation of faujasite NaX at which the OH stretching frequency first decreases and then increases almost to its room pressure value upon further compression. Pair distribution function (PDF) analysis provides evidence that amorphisation corresponds to a collapse of the structure around hydrated sodium cations with strong distortion of the secondary building units (double six-membered rings, sodalite cages). Both the use of guest molecules as local probes in far- and mid-infrared spectroscopy, where we correlate intermolecular water H bonding vibrations and internal mode behaviour under confinement, and PDF analysis could be of great use to study the mechanical behaviour of other hydrated materials.

Osteopontin: a uranium phosphorylated binding-site characterization.

Herein, we describe the structural investigation of one possible uranyl binding site inside a nonstructured protein. This approach couples spectroscopy, thermodynamics, and theoretical calculations (DFT) and studies the interaction of uranyl ions with a phosphopeptide, thus mimicking a possible osteopontin (OPN) hydroxyapatite growth-inhibition site. Although thermodynamical aspects were investigated by using time-resolved laser fluorescence spectroscopy (TRLFS) and isothermal titration calorimetry (ITC), structural characterization was performed by extended X-ray absorption fine structure (EXAFS) at the U LIII -edge combined with attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. From the vibrational and fluorescence spectra, several structural models of a UO2 (2+) /peptide complex were developed and subsequently refined by using theoretical calculations to fit the experimental EXAFS obtained. The structural effect of the pH value was also considered under acidic to moderately acidic conditions (pH 1.5-5.5). Most importantly, the uranyl/peptide coordination environment was similar to that of the native protein.

Self-assembly of bridged silsesquioxanes: modulating structural evolution via cooperative covalent and noncovalent interactions.

The self-assembly of a bis-urea phenylene-bridged silsesquioxane precursor during sol-gel synthesis has been investigated by in situ infrared spectroscopy, optical microscopy, and light scattering. In particular, the evolution of the system as a function of processing time was correlated with covalent interactions associated with increasing polycondensation and noncovalent interactions such as hydrogen bonding. A comprehensive mechanism based on the hydrolysis of the phenylene-bridged organosilane precursor prior to the crystallization of the corresponding bridged silsesquioxane via H-bonding and subsequent irreversible polycondensation is proposed.