Structural Analysis of Uranyl Complexation by the EF-Hand Motif of Calmodulin: Effect of Phosphorylation.

Better understanding of uranyl-protein interactions is a prerequisite to predict uranium chemical toxicity in cells. The EF-hand motif of the calmodulin site I is about thousand times more affine for uranyl than for calcium, and threonine phosphorylation increases the uranyl affinity by two orders of magnitude at pH 7. In this study, we confront X-ray absorption spectroscopy with Fourier transform infrared (FTIR) spectroscopy, time-resolved laser-induced fluorescence spectroscopy (TRLFS), and structural models obtained by molecular dynamics simulations to analyze the uranyl coordination in the native and phosphorylated calmodulin site I. For the native site I, extended X-ray absorption fine structure (EXAFS) data evidence a short U-Oeq distance, in addition to distances compatible with mono- and bidentate coordination by carboxylate groups. Further analysis of uranyl speciation by TRLFS and thorough investigation of the fluorescence decay kinetics strongly support the presence of a hydroxide uranyl ligand. For a phosphorylated site I, the EXAFS and FTIR data support a monodentate uranyl coordination by the phosphoryl group and strong interaction with mono- and bidentate carboxylate ligands. This study confirms the important role of a phosphoryl ligand in the stability of uranyl-protein interactions. By evidencing a hydroxide uranyl ligand in calmodulin site I, this study also highlights the possible role of less studied ligands as water or hydroxide ions in the stability of protein-uranyl complexes.

Structural Environment and Stability of the Complexes Formed Between Calmodulin and Actinyl Ions.

Because of their presence in the nuclear fuel cycle, neptunium and uranium are two actinides of main interest in case of internal contamination. Complexation of U(VI) and Np(V) by the target protein calmodulin (CaM(WT)) was therefore studied herein. Both actinides have two axial oxygen atoms, which, charge aside, makes them very similar structurally wise. This work combines spectroscopy and theoretical density functional theory (DFT) calculations. Structural characterization was performed by extended X-ray absorption fine structure (EXAFS) at the L(III)-edge for each studied actinide. Models for the binding site of the protein were developed and then refined by using DFT to fit the obtained experimental EXAFS data. The effect of hydrolysis was also considered for both actinides (the uranyl experiment was performed at pH 3 and 6, while the neptunyl experiment was conducted at pH 7 and 9). The effect of the pH variation was apparent on the coordination sphere of the uranyl complexes, while the neptunyl complex characteristics remained stable under both studied conditions. The DFT calculations showed that at near physiological pH the complex formed by CaM(WT) with the neptunium ion is more stable than the one formed with uranyl.

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.

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.

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.

Validity and Reliability of the Arabic Version of the Three-Factor Eating Questionnaire-R18.

The present study translated and validated an Arabic version of the Three-Factor Eating Questionnaire-R18 (TFEQ-R18), which assesses eating behaviour traits among Arabic speakers in the three domains: cognitive restraint (CR), uncontrolled eating (UE), and emotional eating (EE). Three bilingual experts first translated the English questionnaire into Arabic, and the questionnaire's comprehension was tested among 298 Arabic speakers. Confirmatory factor analysis (CFA) tested the model fit and flagged four items in the questionnaire with low consistency. These items were modified, and the revised Arabic version of TFEQ-R18 was validated in a population of 513 individuals. Validity was evaluated using CFA and Pearson's correlation coefficients, internal consistency using Cronbach's α, and the reproducibility of the questionnaire was confirmed with Bland-Altman analysis and T-test. The construct validity of the Arabic TFEQ-R18 was evaluated by comparing the response score in the population based on gender, BMI and age group. CFA confirmed that the model fit is good and strongly agrees with the collected data. Except for item 15 in the CR domain, all questions showed moderate to high correlation within their respective domains. The Cronbach's α for UE, EE, and CR domains recorded was 0.778, 0.784 and 0.588, respectively. Item 15 had the lowest consistency in the Arabic-TFEQ-R18, and Cronbach's α increased to 0.608 with its removal. There was no significant difference between the first and second attempts of the Arabic TFEQ-R18, indicating good test-retest reliability. Moreover, UE and EE were positively correlated to the BMI of the participants (r = 0.159, p = 0·000; r = 0.158, p = 0·000, respectively). The study concludes that the Arabic TFEQ is a valid and reliable tool for studying the three psychometric domains of UE, EE and CR among Arabic speakers.

The attitudes and practices of United Arab Emirates consumers towards food waste: A nationwide cross-sectional study.

Background: Reducing global food waste is an international environmental, health, and sus-tainability priority. Although significant reductions have been achieved across the food chain, progress by UAE households and consumers remain inadequate. This study seeks to understand the association between consumer attitudes, knowledge, and awareness relating to food waste practice of residents living in the UAE. to help inform policy and action for addressing this national priority. Methods: A cross-sectional study was conducted using a validated semi-structured online questionnaire through stratified sampling (n =1052). The Spearman correlation coefficient was performed to determine the correlations. Two independent regression analysis were used to determine the association between food waste practice with: 1) knowledge and awareness and attitude subdomains, and 2) sociodemographic characteristics. Respondents (n=1072) largely reflect the socio-demographic characteristics and population distribution across the seven Emirates. Results: As expected, a significant and negative correlation was found between food waste practice knowledge and awareness and overall attitude. The regression models showed reduced food waste practice was associated with better knowledge, personal attitude, financial attitude (first model), older age and fewer adults in the household (second model). We found a significant and negative association of personal attitude (a commitment, intention), financial attitude (cost-saving motivation), and (existing) knowledge of Food waste (FW) with practice of food waste, indicating that better knowledge about FW, personal attitude or financial attitude was associated with reduction in undesirable food waste practice. While awareness and emotional attitude (moral concerns) were positively and significantly associated with food waste practice (undesirable behaviour). Conclusions: Food waste poses significant challenges in the UAE, and addressing it requires a comprehensive understanding of the multifaceted factors influencing consumer behavior. By promoting knowledge, fostering positive attitudes, and considering socio-cultural factors, policymakers can develop effective strategies to reduce food waste in households and contribute to sustainable development goals.

Interfacing capillary-based separations to mass spectrometry using desorption electrospray ionization.

The powerful hybrid analysis method of capillary-based separations followed by mass spectrometric analysis gives substantial chemical identity and structural information. It is usually carried out using electrospray ionization. However, the salts and detergents used in the mobile phase for electrokinetic separations suppress ionization efficiencies and contaminate the inlet of the mass spectrometer. This report describes a new method that uses desorption electrospray ionization (DESI) to overcome these limitations. Effluent from capillary columns is deposited on a rotating Teflon disk that is covered with paper. As the surface rotates, the temporal separation of the eluting analytes (i.e., the electropherogram) is spatially encoded on the surface. Then, using DESI, surface-deposited analytes are preferentially ionized, reducing the effects of ion suppression and inlet contamination on signal. With the use of this novel approach, two capillary-based separations were performed: a mixture of the rhodamine dyes at milligram/milliliter levels in a 10 mM sodium borate solution was separated by capillary electrophoresis, and a mixture of three cardiac drugs at milligram/milliliter levels in a 12.5 mM sodium borate and 12.5 mM sodium dodecyl sulfate solution was separated by micellar electrokinetic chromatography. In both experiments, the negative effects of detergents and salts on the MS analyses were minimized.

Design and characterization of immunogens for raising antibodies directed towards chelated alkali metals.

The properties of immunogens synthesized from a calix[4]crown-6 were investigated with the aim of generating specific antibodies towards caesium chelates. Affinity capillary electrophoresis was successfully used to determine the stability parameters with caesium of both the hapten and the corresponding immunogens after coupling to bovine serum albumin (BSA). Unfortunately, the stability of the caesium chelate was shown to decrease drastically as the polarity of the medium increased. Nevertheless, the selectivity was proved to be rather stable, with a clear preference of caesium over potassium. The binding mechanism for the protein conjugates proved to be complex, as revealed by isotherms, whatever the number of calixarene groups coupled to BSA. Immunization of mice with caesium loaded immunogen resulted in the production of polyclonal antibodies able to distinguish between free calixarene and its complexes with either potassium or caesium.

Characterization of protein conjugates using capillary electrophoresis.

With the aim of generating antibodies, a calix[4]arene-crown-6 was coupled to bovine serum albumin. For that purpose, a complete procedure to optimize and characterize the coupling of hydrophobic haptens based on capillary electrophoresis (CE) was developed. We demonstrated the existence of a polynomial relationship between the electrophoretic mobility (mu(ep)) and the hapten density. This correlation was used not only to study the coupling reaction in terms of optimization and kinetics but also to determine the average coupling molar ratio of any given conjugate. An estimation of the heterogeneity of these conjugates by simulation of experimental peaks was also proposed.

Enzymatic activity of the CaM-PDE1 system upon addition of actinyl ions.

The threat of a dirty bomb which could cause internal contamination has been of major concern for the past decades. Because of their high chemical toxicity and their presence in the nuclear fuel cycle, uranium and neptunium are two actinides of high interest. Calmodulin (CaM) which is a ubiquitous protein present in all eukaryotic cells and is involved in calcium-dependent signaling pathways has a known affinity for uranyl and neptunyl ions. The impact of the complexation of these actinides on the physiological response of the protein remains, however, largely unknown. An isothermal titration calorimetry (ITC) was developed to monitor in vitro the enzymatic activity of the phosphodiesterase enzyme which is known to be activated by CaM and calcium. This approach showed that addition of actinyl ions (AnO2n+), uranyl (UO22+) and neptunyl (NpO2+), resulted in a decrease of the enzymatic activity, due to the formation of CaM-actinide complexes, which inhibit the enzyme and alter its interaction with the substrate by direct interaction. Results from dynamic light scattering rationalized this result by showing that the CaM-actinyl complexes adopted a specific conformation different from that of the CaM-Ca2+ complex. The effect of actinides could be reversed using a hydroxypyridonate actinide decorporation agent (5-LIO(Me-3,2-HOPO)) in the experimental medium demonstrating its capacity to efficiently bind the actinides and restore the calcium-dependent enzyme activation.

In vitro evaluation of the anti-proliferative activities of the wood essential oils of three Cedrus species against K562 human chronic myelogenous leukaemia cells.

There are four kinds of Cedar: Cedrus libani naturally occurring in Lebanon, Syria and Turkey, Cedrus atlantica in Morocco and Algeria, Cedrus brevefolia in Cyprus Island and Cedrus deodara which is distributed in Himalayan Mountains. Wood essential oils obtained from C. libani, C. atlantica and C. deodara were tested for the inhibition of K562 cell proliferation and for the induction of erythroid differentiation. The wood essential oils of C. libani, C. atlantica and C. deodara inhibited the proliferation of the K562 cell line exhibiting IC(50) values 23.38 ± 1.7, 59.37 ± 2.6 and 37.09 ± 1.4 µg mL(-1), respectively. Meanwhile, C. libani wood oils induced a percentage of erythroid differentiation of 15 ± 2% at concentration 5 µg mL(-1). Cedrus deodara wood oil indicated a percentage of erythroid differentiation of 20 ± 2% at concentration 25 µg mL(-1) and C. atlantica wood oils showed a percentage of erythroid differentiation of 12 ± 1.8% at concentration 10 µg mL(-1).

Synthesis and characterization of immunogens based on calix[4]arene-crown-6 for the generation of antibodies directed towards cesium ions.

With the aim of generating antibodies directed towards cesium, a calix[4]arene-crown-6 in the 1,3-alternate conformation bearing an N-hydroxysuccinimide function was synthesized. The calixarene was coupled to bovine serum albumin using different calixarene/protein ratios, and cesium was loaded. Conjugates were characterized using trinitrobenzenesulfonic acid derivatization, matrix assisted laser desorption ionisation-mass spectrometry (MALDI-TOF-MS), and inductively coupled plasma-mass spectrometry (ICP-MS). Targeted lysines were determined after enzymatic cleavage followed by MALDI-TOF-MS, and a progressive targeting related to lysines accessibility was observed.