In vivo detection of carnosine and its derivatives using chemical exchange saturation transfer.

PURPOSE: To detect carnosine, anserine and homocarnosine in vivo with chemical exchange saturation transfer (CEST) at 17.2 T. METHODS: CEST MR acquisitions were performed using a CEST-linescan sequence developed in-house and optimized for carnosine detection. In vivo CEST data were collected from three different regions of interest (the lower leg muscle, the olfactory bulb and the neocortex) of eight rats. RESULTS: The CEST effect for carnosine, anserine and homocarnosine was characterized in phantoms, demonstrating the possibility to separate individual contributions by employing high spectral resolution (0.005 ppm) and low CEST saturation power (0.15 µ$$ \mu $$ T). The CEST signature of these peptides was evidenced, in vivo, in the rat brain and skeletal muscle. The presence of carnosine and anserine in the muscle was corroborated by in vivo localized spectroscopy (MRS). However, the sensitivity of MRS was insufficient for carnosine and homocarnosine detection in the brain. The absolute amounts of carnosine and derivatives in the investigated tissues were determined by liquid chromatography-electrospray ionization-tandem mass spectrometry using isotopic dilution standard methods and were in agreement with the CEST results. CONCLUSION: The robustness of the CEST-linescan approach and the favorable conditions for CEST at ultra-high magnetic field allowed the in vivo CEST MR detection of carnosine and related peptides. This approach could be useful to investigate noninvasively the (patho)-physiological roles of these molecules.

Study of syn and anti Xenon-Cryptophanes Complexes Decorated with Aromatic Amine Groups: Chemical Platforms for Accessing New Cryptophanes.

We report the synthesis of C3-symmetric cryptophanes decorated with three aromatic amine groups on the same CTB cap and their interaction with xenon. The relative stereochemistry of these two stereoisomers syn and anti was assessed thanks to the determination of the X-ray structure of an intermediate compound. As previously observed with the tris-aza-cryptophanes analogs anti-1 and syn-2 (J. Org. Chem. 2021, 86, 11, 7648-7658), both compounds anti-5 and syn-6 show a slow in-out exchange dynamics of xenon at 11.7 T. Our work supports the idea that the presence of nitrogen atoms grafted directly onto the cryptophane backbone has a strong impact on the in-out exchange dynamics of xenon whatever their stereochemistry. This result contrasts with the case of other cryptophanes decorated solely with methoxy substituents. Finally, we demonstrate that these new derivatives can be used to design new anti/syn cryptophanes bearing suitable ligands in order to constitute potent 129Xe NMR-based sensors. An example is reported here with the synthesis of the tris-iodo derivatives anti-13 and syn-14 from compounds anti-5 and syn-6.

Are the Physical Properties of Xe@Cryptophane Complexes Easily Predictable? The Case of syn- and anti-Tris-aza-Cryptophanes.

We report the synthesis and optical resolution of C3-symmetrical tris-aza-cryptophanes anti-3 and syn-4, as well as the study of their interaction with xenon via hyperpolarized 129Xe NMR. These molecular cages are close structural analogues of the two well-known cryptophane-A (1; chiral) and cryptophane-B (2; achiral) diastereomers since these new compounds differ only by the presence of three nitrogen atoms grafted onto the same cyclotribenzylene unit. The assignment of their relative (syn vs anti) and absolute configurations was made possible, thanks to the combined use of quantum calculations at the density functional theory level and vibrational circular dichroism spectroscopy. More importantly, our results show that despite the large structural similarities with cryptophane-A (1) and -B (2), these two new compounds show a very different behavior in the presence of xenon in organic solutions. These results demonstrate that prediction of the physical properties of the xenon@cryptophane complexes, only based on structural parameters, remains extremely difficult.

Effects of Low Doses of Herbicides on Different Endpoints in the Life Cycle of Nontarget Terrestrial Plants.

Herbicide drift may cause adverse effects on natural and seminatural plant communities, and it has been debated whether the current ecological risk assessments are adequate to protect nontarget terrestrial plant species. In the present study, 9 nontarget terrestrial plant species with different lifespans (3 annual/6 perennial) belonging to 6 different plant families were exposed to 4 herbicides with different modes of action at the vegetative (6-8 leaf) and reproductive (bud) stages separately. The plant tests were conducted under controlled conditions in 2 greenhouses, 1 located in Denmark and 1 in Canada. For both growth stages, effects were recorded on vegetative (above-ground biomass 3 wk after treatment) and reproductive endpoints (number and germinability of seeds). In most cases, responses following exposure at the juvenile stage were greater than responses following exposure at the reproductive stage. For the combinations of herbicides and plant species included in the present study, we found that the sensitivities of vegetative and reproductive endpoints were equal, or else vegetative endpoints were more sensitive than reproductive endpoints. We also found that annual species were more sensitive than perennial species. The overall conclusions cover many different response patterns, and it is evident that some effects may not be found in the currently used standard tests. Generally, more pronounced effects were obtained in Denmark compared with Canada, highlighting the fact that even under standardized test conditions and following common guidelines, several uncontrollable factors can still induce variable results. Environ Toxicol Chem 2021;40:1389-1404. © 2021 SETAC.

129Xe ultra-fast Z spectroscopy enables micromolar detection of biosensors on a 1 T benchtop spectrometer.

The availability of a benchtop nuclear magnetic resonance (NMR) spectrometer, of low cost and easily transportable, can allow detection of low quantities of biosensors, provided that hyperpolarized species are used. Here we show that the micromolar threshold can easily be reached by employing laser-polarized xenon and cage molecules reversibly hosting it. Indirect detection of caged xenon is made via chemical exchange, using ultra-fast Z spectroscopy based on spatio-temporal encoding. On this non-dedicated low-field spectrometer, several ideas are proposed to improve the signal.

Use of dissolved hyperpolarized species in NMR: Practical considerations.

Hyperpolarization techniques that can transiently boost nuclear spin polarization are generally carried out at low temperature - as in the case of dynamic nuclear polarization - or at high temperature in the gaseous state - as in the case of optically pumped noble gases. This review aims at describing the various issues and challenges that have been encountered during dissolution of hyperpolarized species, and solutions to these problems that have been or are currently proposed in the literature. During the transport of molecules from the polarizer to the NMR detection region, and when the hyperpolarized species or a precursor of hyperpolarization (e.g. parahydrogen) is introduced into the solution of interest, several obstacles need to be overcome to keep a high level of final magnetization. The choice of the magnetic field, the design of the dissolution setup, and ways to isolate hyperpolarized compounds from relaxation agents will be presented. Due to the non-equilibrium character of the hyperpolarization, new NMR pulse sequences that perform better than the classical ones will be described. Finally, three applications in the field of biology will be briefly mentioned.

Effects of Herbicides on Flowering.

Herbicides have been shown to reduce flower production and to delay flowering, with results varying among herbicides and tested plant species. We investigated the effects of herbicides on flowering in an extensive greenhouse study conducted in Canada and Denmark. The effects of low doses of 5 different herbicides (bromoxynil, ioxynil + bromoxynil, metsulfuron-methyl, clopyralid, and glyphosate), simulating realistic drift scenarios (1 and 5% recommended field rates), on plant flowering were examined using 9 wild plant species exposed at either the seedling (6- to 8-leaf) or flower bud stage. Following herbicide exposure, initial flowering date as well as flower production over time were recorded over the growing period. The effect of herbicides on cumulative flower numbers and flowering time were modeled using Gompertz growth models. Significant delays to peak flowering and/or reductions in flower production were observed in at least one plant species for all tested herbicides, with glyphosate often exhibiting the greatest negative effects, that is, plant death. Except for ioxynil + bromoxynil, there was no clear evidence of either the seedling or the flower bud stage being more sensitive. Overall, 58% of all species × life stage × herbicide treatments resulted in either a statistically significant or a strong decline in flower production with herbicide application rates up to 5% of recommended field rates, whereas significant or strong delays in peak flowering were also detected but were slightly less common. Effects at 1% label rates were minimal. Simultaneous delays to peak flowering and reductions in total flower production occurred in approximately 25% of all cases, indicating that herbicide application rates simulating realistic drift scenarios would likely have negative effects on wild floral communities. Environ Toxicol Chem 2020;39:1244-1256. © 2020 SETAC.

Synthesis of Cryptophane-223-Type Derivatives with Dual Functionalization.

In this article, we present the synthesis of new cryptophane-type hosts capable of binding xenon in aqueous media and that may be useful for the development of xenon-based magnetic resonance imaging derivatives. The synthetic route proposed was chosen to facilitate both the introduction of water-solubilizing substituents and the functionalization of the host with a single arm showing recognition properties that constitute two crucial steps. This was made possible by preparing new cryptophane-223 derivatives bearing two different chemical functions that can be easily modified at a later stage. Thus, subsequent reactions allowed the design of a new cryptophane host able to bind zinc or nickel cations. The ability of this molecule to bind cationic species was assessed by calorimetric titration experiments and hyperpolarized 129Xe NMR. The advantages and disadvantages of this approach are discussed.

Effects of Sublethal Doses of Herbicides on the Competitive Interactions Between 2 Nontarget Plants, Centaurea cyanus L. and Silene noctiflora L.

Plant competitive interactions influence the effect of herbicides, and the effect of competitive interactions on plant responses may be important to include in the ecological risk assessment of herbicides. In the present study the effect of competitive interactions and sublethal doses of 2 herbicides on plant species was investigated in competition experiments and fitted to empirical competition models. Two nontarget species commonly found in agroecosystems (Centaurea cyanus L. and Silene noctiflora L.) and 2 herbicides (glyphosate and metsulfuron methyl) were used in separate experiments. Plants were sprayed at the 6- to 8-leaf stage. Effects of herbicide treatments and plant density were modeled by generalization of a discrete hyperbolic competition model. The 10% effective dose (ED10) was calculated for C. cyanus. All experiments showed that as density increased, plants were negatively affected. Furthermore, in all cases, C. cyanus remained a better competitor than S. noctiflora. Nevertheless, the density of S. noctiflora (competitor) was an influential element in determining the ED10 of C. cyanus measured at the mature stage. With herbicide exposure, the competitive interactions were further altered; C. cyanus was less affected by glyphosate when S. noctiflora increased to high density. In contrast, at the young stage, conspecific density was important in determining the sensitivity of C. cyanus to metsulfuron methyl, whereas the density of the competitor S. noctiflora had a limited influence. Overall, the results demonstrate the importance of integrating the effect of herbicide and species interactions measured at the reproductive stage into the ecological risk assessments of pesticides. Environ Toxicol Chem 2019;38:2053-2064. © 2019 SETAC.

Ecological risk assessment of cerium for tropical agroecosystems.

Cerium (Ce) is present in high technology materials and in mineral P fertilizers and the use and discharge of such resources may change the natural status of Ce in the soil environment. Brazilian soils in farming areas are significantly exposed to increased levels of unintentionally-added Ce through intensive input of phosphate fertilizers. The aims of this study were to evaluate the ecotoxicological risk to plants growing in tropical soils contaminated with Ce, as well as to create a database to support future legislation regulating the limits of this element in Brazilian and conceivably other tropical soils. Eight crop species (corn, sorghum, rice, wheat, soybeans, sunflower, radish, and beans) were exposed to a Ce concentration gradient in two typical tropical soils (Oxisol and Inceptsol), and an artificial soil. Our findings showed that among the endpoints measured, Ce phytotoxicity was more pronounced on shoot dry matter than on percent germination and germination speed index. Sensitivity of plants is species specific and our data showed that sunflower and radish exposed to Ce were the most sensitive crop species. Soil properties such as pH, cation exchange capacity, and organic carbon may have influenced the severity of Ce phytotoxicity. Because of that, the Oxisol contaminated with this element caused higher phytotoxicity than the other soils tested. Our risk assessment results (hazardous concentration, HC5 = 281.6 mg Ce kg-1) support the idea that unintentional Ce input through P fertilizers does not pose a risk to soils of Brazilian agroecosystems.

Bimodal Detection of Proteins by 129 Xe NMR and Fluorescence Spectroscopy.

A full understanding of biological phenomena involves sensitive and noninvasive detection. Herein, we report the optimization of a probe for intracellular proteins that combines the advantages of fluorescence and hyperpolarized 129 Xe NMR spectroscopy detection. The fluorescence detection part is composed of six residues containing a tetracysteine tag (-CCXXCC-) genetically incorporated into the protein of interest and of a small organic molecule, CrAsH. CrAsH becomes fluorescent if it binds to the tetracysteine tag. The part of the biosensor that enables detection by means of 129 Xe NMR spectroscopy, which is linked to the CrAsH moiety by a spacer, is based on a cryptophane core that is fully suited to reversibly host xenon. Three different peptides, containing the tetracysteine tag and four organic biosensors of different stereochemistry, are benchmarked to propose the best couple that is fully suited for the in vitro detection of proteins.

Bimodal fluorescence/129Xe NMR probe for molecular imaging and biological inhibition of EGFR in Non-Small Cell Lung Cancer.

Although Non-Small Cell Lung Cancer (NSCLC) is one of the main causes of cancer death, very little improvement has been made in the last decades regarding diagnosis and outcomes. In this study, a bimodal fluorescence/129Xe NMR probe containing a xenon host, a fluorescent moiety and a therapeutic antibody has been designed to target the Epidermal Growth Factor Receptors (EGFR) overexpressed in cancer cells. This biosensor shows high selectivity for the EGFR, and a biological activity similar to that of the antibody. It is detected with high specificity and high sensitivity (sub-nanomolar range) through hyperpolarized 129Xe NMR. This promising system should find important applications for theranostic use.

Role of the Methoxy Groups in Cryptophanes for Complexation of Xenon: Conformational Selection Evidence from 129 Xe-1 H NMR SPINOE Experiments.

We report the laser-polarized 129 Xe and 1 H NMR spectra of a series of cryptophane derivatives that differ only by the number of methoxy groups attached on their benzene rings and the syn or anti arrangement of the linkers (compounds 6 a-s, 9 a-s, 12 a-s). All these compounds bind xenon even though the characteristic signal of the gas encapsulated in the cavity of the cage-molecule cannot always be detected. Interestingly, the exchange dynamics of xenon strongly depends on the degree of substitution and is different from that of the cryptophane derivatives studied previously. In solution, the 1 H NMR spectra of these derivatives show the presence of different conformations in a slow exchange regime that can be explained by a decrease of the flexibility of their skeleton. Thanks to 129 Xe-1 H dipolar cross-relaxation (SPINOE) spectra, we demonstrate that a single conformation present in solution can bind xenon.

Predicting bee community responses to land-use changes: Effects of geographic and taxonomic biases.

Land-use change and intensification threaten bee populations worldwide, imperilling pollination services. Global models are needed to better characterise, project, and mitigate bees' responses to these human impacts. The available data are, however, geographically and taxonomically unrepresentative; most data are from North America and Western Europe, overrepresenting bumblebees and raising concerns that model results may not be generalizable to other regions and taxa. To assess whether the geographic and taxonomic biases of data could undermine effectiveness of models for conservation policy, we have collated from the published literature a global dataset of bee diversity at sites facing land-use change and intensification, and assess whether bee responses to these pressures vary across 11 regions (Western, Northern, Eastern and Southern Europe; North, Central and South America; Australia and New Zealand; South East Asia; Middle and Southern Africa) and between bumblebees and other bees. Our analyses highlight strong regionally-based responses of total abundance, species richness and Simpson's diversity to land use, caused by variation in the sensitivity of species and potentially in the nature of threats. These results suggest that global extrapolation of models based on geographically and taxonomically restricted data may underestimate the true uncertainty, increasing the risk of ecological surprises.

Uptake and Effects of Six Rare Earth Elements (REEs) on Selected Native and Crop Species Growing in Contaminated Soils.

Rare earth elements (REEs) have become increasingly important metals used in modern technology. Processes including mining, oil refining, discarding of obsolete equipment containing REEs, and the use of REE-containing phosphate fertilizers may increase the likelihood of environmental contamination. However, there is a scarcity of information on the toxicity and accumulation of these metals to terrestrial primary producers in contaminated soils. The objective of this work was to assess the phytotoxicity and uptake from contaminated soil of six REEs (chloride forms of praseodymium, neodymium, samarium, terbium, dysprosium, and erbium) on three native plants (Asclepias syriaca L., Desmodium canadense (L.) DC., Panicum virgatum L.) and two crop species (Raphanus sativus L., Solanum lycopersicum L.) in separate dose-response experiments under growth chamber conditions. Limited effects of REEs were found on seed germination and speed of germination. Effects on aboveground and belowground biomass were more pronounced, especially for the three native species, which were always more sensitive than the crop species tested. Inhibition concentrations (IC25 and IC50) causing 25 or 50% reductions in plant biomass respectively, were measured. For the native species, the majority of aboveground biomass IC25s (11 out of 18) fell within 100 to 300 mg REE/kg dry soil. In comparison to the native species, IC25s for the crops were always greater than 400 mg REE/kg, with the majority of results (seven out of 12) falling above 700 mg REE/kg. IC50s were often not detected for the crops. Root biomass of native species was also affected at lower doses than in crops. REE uptake by plants was higher in the belowground parts than in the above-ground plant tissues. Results also revealed that chloride may have contributed to the sensitivity of the native species, Desmodium canadense, one of the most sensitive species studied. Nevertheless, these results demonstrated that phytotoxicity may be a concern in contaminated areas.

Synthesis of Cryptophanes with Two Different Reaction Sites: Chemical Platforms for Xenon Biosensing.

We report the synthesis of new water-soluble cryptophane host molecules that can be used for the preparation of (129)Xe NMR-based biosensors. We show that the cryptophane-223 skeleton can be modified to introduce a unique secondary alcohol to the propylenedioxy linker. This chemical functionality can then be exploited to introduce a functional group that is different from the six chemical groups attached to the aromatic rings. In this approach, the generation of a statistical mixture when trying to selectively functionalize a symmetrical host molecule is eliminated, which enables the efficient large-scale production of new cryptophanes that can be used as chemical platforms ready to use for the preparation of xenon biosensors. To illustrate this approach, two molecular platforms have been prepared, and the ability of these new derivatives to bind xenon has been investigated.

Herbicide impact on the growth and reproduction of characteristic and rare arable weeds of winter cereal fields.

The decline of arable species characteristic of winter cereal fields has often been attributed to different factors related to agricultural intensification but most importantly to herbicide use. Herbicide phytotoxicity is most frequently assessed using short-term endpoints, primarily aboveground biomass. However, short-term sensitivity is usually not sufficient to detect actual effects because plants may or may not recover over time following sublethal herbicide exposures. Therefore, it is important to assess the long-term effects of herbicide applications. Annual species rely on renewable seed production to ensure their persistence; hence, assessment of herbicide sensitivity is more accurately estimated through effects on reproduction. Here we aim to assess the phytotoxicity of two commonly used herbicides: tribenuron and 2,4-D on eight plant species belonging to four families, each with one rare and one more common species. Specifically we examined the pattern of sensitivity using short-term and long-term endpoints (total aboveground biomass, total seed biomass and number of seeds) of these species; we determined the levels of and time to recovery in terms of stem length and fruit number, and assessed whether their rarity relates to their sensitivity to herbicide application. Our results suggest that although differences in herbicide sensitivity are not a direct cause of rarity for all species, it may be an important driver of declining arable plants.

Determining in situ periphyton community responses to nutrient and atrazine gradients via pigment analysis.

Agrochemicals, including fertilizers and herbicides, are significant contributors of non-point source pollution to surface waters and have the potential to negatively affect periphyton. We characterized periphyton communities using pigment markers to assess the effects of nutrient enrichment and the herbicide atrazine with in situ experimental manipulations and by examining changes in community structure along existing agrochemical gradients. In 2008, the addition of nutrients (20 mg/L nitrate and 1.25 mg/L reactive phosphate), atrazine (20 µg/L) and a combination of both nutrients and atrazine had no significant effect on periphyton biomass or community structure in a stream periphytometer experiment. In 2009, similar experiments with higher concentrations of atrazine (200 µg/L) at two stream sites led to some minor effects. In contrast, at the watershed scale (2010) periphyton biomass (mg/m(2) chlorophyll a) increased significantly along correlated gradients of nitrate and atrazine but no direct effects of reactive phosphate were observed. Across the watershed, the average periphyton community was composed of Bacillariophyceae (60.9%), Chlorophyceae (28.1%), Cryptophyceae (6.9%) and Euglenophyceae (4.1%), with the Bacillariophyceae associated with high turbidity and the Chlorophyceae with nitrate enrichment. Overall, effects of nitrate on periphyton biomass and community structure superseded effects of reactive phosphate and atrazine.

Cerium chloride heptahydrate (CeCl3 · 7H2O) induces muscle paralysis in the generalist herbivore, Melanoplus sanguinipes (Fabricius) (Orthoptera: Acrididae), fed contaminated plant tissues.

Of increasing economic importance are the rare earth elements (REEs). Pollution from mining and processing activity is expected to rise with industrial demand. Plants are known to accumulate REEs, although levels vary with species and soil content. However, the effect on wildlife of ingesting REE contaminated vegetation is not well understood. Here we examined the effect of consuming vegetation with elevated levels of cerium on the generalist grasshopper, Melanoplus sanguinipes (Fabricius). Adults excreted a substantial portion of ingested contamination. However, after only four-days of feeding, accumulation in the body occurred at all doses and paralysis of appendages resulted at the highest doses. Short-term toxicity studies may underestimate the impact of ingesting REE contamination. Metals tend to be low in toxicity; however, their persistence in the environment may be better represented by exposure over longer portions of the life cycle.

Rim-functionalized cryptophane-111 derivatives via heterocapping, and their xenon complexes.

Capping of cyclotriphenolene (3a) by the more available cyclotriguaiacylene (3c) or trisbromocyclotriphenolene (3b) gives the first rim-functionalized cryptophane-111 derivatives. Crystal structures of the xenon complexes reveal high cavity packing coefficients and unprecedentedly short Xe···C contacts.