The use of chitosan as an antioxidant in the feed of cultivated P. vannamei shrimp against oxidative stress induced by exposure to microplastics.

Microplastics (MP) are omnipresent in aquaculture and can induce several toxic effects, mainly oxidative stress. Therefore, alternatives to minimize these effects are welcome. In this study, chitosan (1 and 3 g/kg) was supplemented through the feed of farmed shrimp P. vannamei for 30 days. After this period, the shrimp were exposed to MP (0.5 mg/L) for 7 days. The results showed the presence of MP in hepatopancreas, gills and muscle. Hepatopancreas morphological alterations, as well as lipid peroxidation, a decrease in GSH level, and an increase in SOD activity indicated an oxidative stress that was reversed by chitosan. The muscle was also affected by MP, showing decreased CAT activity and increased SOD activity, though no lipid peroxidation was observed. In muscle, chitosan reversed the SOD increase to basal activity. The results obtained showed that chitosan was more effective against oxidative stress than in preventing accumulation and histological damage.

Oxidative effects of consuming microplastics in different tissues of white shrimp Litopenaeus vannamei.

In this study, we evaluated the effect of microplastic (MP, polystyrene, 1.1 µm) exposure through diet at two different levels (40 and 400 µg MP/kg of ration) in the shrimp Litopenaeus vannamei for seven days. After the exposure period, oxidative stress parameters, histological alterations, and MP accumulation in different shrimp tissues (gut, gills, hepatopancreas, and muscle) were also evaluated. The results showed that MP was detected in the gills, muscles, and hepatopancreas. In addition, in the gut, gills, and hepatopancreas, disruption in redox cells was observed. Also, lipid and DNA damage was evident in the hepatopancreas. Histopathological analysis revealed edema in the intestine, hepatopancreas, and in the muscle. Granuloma formation with infiltrated hemocytes occurred in the intestine and hepatopancreas. These results show that MP exposure can affect the health and welfare of L. vannamei and may also affect the final consumers once MP is accumulated.

Multigenerational effects of co-exposure to dimethylarsinic acid and polystyrene microplastics on the nematode Caenorhabditis elegans.

In the current study, we assessed the impact of DMA (dimethylarsinic acid) and MPs (microplastics) interactions in C. elegans over the course of five generations. We found that the redox state of the organisms changed over generations as a result of exposure to both pollutants. From the third generation onward, exposure to MPs reduced GST activity, indicating reduced detoxifying abilities of these organisms. Additionally, dimethylarsinic exposure decreased the growth of organisms in the second, fourth, and fifth generations. In comparison to isolated pollutants, the cumulative effects of co-exposure to DMA and MPs seem to have been more harmful to the organisms, as demonstrated by correlation analysis. These findings demonstrate that DMA, despite being considered less hazardous than its inorganic equivalents, can still have toxic effects on species at low concentrations and the presence of MPs, can worsen these effects.

Biochemical and physiological effects of multigenerational exposure to spheric polystyrene microplastics in Caenorhabditis elegans.

Although studies have already shown the effects of exposure to microplastics (MP) in different species, the effects over generations in these individuals remain poorly understood. Therefore, the present study aimed to evaluate the effect of polystyrene MP (spherical, 1 µm) on the responses of the free-living nematode Caenorhabditis elegans in a multigenerational approach over five subsequent generations. MP concentrations of both 5 and 50 µg/L induced a detoxification response, increasing glutathione S-transferase (GST) activity and inducing the generation of reactive oxygen species (ROS) and lipid peroxidation (TBARS). MP also demonstrated the ability to accumulate in the animal's body during the 96 h of each generational exposure, and possibly, this constant interaction was the main reason for the decreased response in physiological parameters as in the exploratory behavior (body bending) of nematodes, and in the reproduction, being this last parameter most negatively affected during the five exposed generations, with a reduction of almost 50% in the last generation. These results emphasize the importance of multigenerational approaches, highlighting their advantage in the assessment of environmental contaminants.

Arsenite and arsenate toxicity in the earthworm Eisenia andrei (Bouché 1972) in natural soil and tropical artificial soil.

Inorganic forms of As (arsenite - As(III) and arsenate - As(V)) are prevalent in soil and recognized for their high toxicity. Once in the soil, these forms of As can compromise key organisms for ecological processes, such as earthworms. The aim of the study was to evaluate the toxicity of arsenite and arsenate in the Californian earthworm Eisenia andrei exposed in natural soil and tropical artificial soil (TAS). Adverse effects were evaluated using avoidance test, acute toxicity test, and a sublethal concentration test to assess biochemical parameters. LC50 values for arsenite were 21.27 mg/kg in natural soil and 19.0 mg/kg in TAS and for arsenate were 76.18 mg/kg in natural soil and above 120 mg/kg in TAS. In the avoidance test, this behavior was shown to be significantly higher in the natural soil and for earthworms exposed to arsenite, while total antioxidant capacity, glutathione levels, lipid damage, and DNA damage were significantly higher in animals exposed to arsenite, but without differences in relation to the two types of soil tested. Animals exposed to As(V) showed increased activity of enzymes related to glutathione metabolism. The results obtained in the present study show the impact of As exposure on the health of the Californian earthworm E. andrei, especially in the form of arsenite, and alert the public authorities that legal limits should, whenever possible, consider the soil properties and also the different chemical species of the contaminants.

Comparison of the toxic effects of organic and inorganic arsenic in Caenorhabditis elegans using a multigenerational approach.

Although arsenic (As) is a persistent contaminant in the environment, few studies have assessed its effects over generations, as it requires an animal model with a short lifespan and rapid development, such as the nematode Caenorhabditis elegans. Furthermore, few studies have evaluated the effects of As metabolites such as dimethylarsinic acid (DMAV), and several authors have considered DMA as a moderately toxic intermediate of As, although recent studies have shown that this chemical form can be more toxic than inorganic arsenic (iAs) even at low concentrations. In the present study, we compared the toxic effects of arsenate (AsV) and DMAV in C. elegans over 5 subsequent generations. We evaluated biochemical parameters such as reactive oxygen species (ROS) concentration, the activity of antioxidant defense system (ADS) enzymes such as catalase (CAT) and glutathione-S-transferase (GST), and nonenzymatic components of ADS such as reduced glutathione (GSH) and protein-sulfhydryl groups (P-SH). Exposure to 50 µg L-1 of AsV led to an increase in ROS generation and GSH levels together with a decrease in GST activity, while exposure to DMAV led to an increase in ROS levels, with an increase in lipid peroxidation, CAT activity, and a decrease in GSH levels. In addition, both treatments reduced animal growth from the third generation onward and caused disturbances in their reproduction throughout all 5 generations. This study shows that the accumulated effects of DMA need to be considered; it highlights the importance of this type of multigenerational approach for evaluating the effects of organic contaminants considered low or nontoxic.

Co-exposure to nTiO2 impairs arsenic metabolism and affects antioxidant capacity in the marine shrimp Litopenaeus vannamei.

Aquatic animals are vulnerable to arsenic (As) toxicity. However, rarely does a contaminant occur alone in the aquatic environment. For this reason, this study was conducted to evaluate whether titanium dioxide nanoparticles (nTiO2) can interfere with the effects induced by As in Litopenaeus vannamei. Arsenic accumulation and metabolic capacity; expression and enzymatic activity of GSTΩ (glutathione-S-transferase omega isoform); antioxidant responses such as GSH, GR, and GST (reduced glutathione levels, glutathione reductase, and glutathione-S-transferase activity, respectively); and lipid peroxidation in the gills and hepatopancreas of shrimp were evaluated. The results are summarized as follows: (1) higher accumulation of As occurred in both tissues after exposure to As alone; (2) co-exposure to nTiO2 affected the capacity to metabolize As; (3) GSTΩ gene expression was not modified, but its activity was decreased by co-exposure to both contaminants; (4) As alone increased the GSH levels in the hepatopancreas, and co-exposure to nTiO2 reduced these levels in both tissues; (5) a decrease in the GST activity in the gills occurred with all treatments; (6) in the gills, GR activity was increased by As, and nTiO2 reversed this increase, whereas in the hepatopancreas co-exposure inhibited enzyme activity; (7) only in the hepatopancreas lipid damage was observed when animals were exposed to As or nTiO2 but not in co-exposure. The results showed that the As induces toxic effects in both tissues of shrimp and that co-exposure to nTiO2 can potentiate these effects and decrease the capacity to metabolize As, favoring the accumulation of more toxic compounds.

Superficial white matter imaging: Contrast mechanisms and whole-brain in vivo mapping.

Superficial white matter (SWM) contains the most cortico-cortical white matter connections in the human brain encompassing the short U-shaped association fibers. Despite its importance for brain connectivity, very little is known about SWM in humans, mainly due to the lack of noninvasive imaging methods. Here, we lay the groundwork for systematic in vivo SWM mapping using ultrahigh resolution 7 T magnetic resonance imaging. Using biophysical modeling informed by quantitative ion beam microscopy on postmortem brain tissue, we demonstrate that MR contrast in SWM is driven by iron and can be linked to the microscopic iron distribution. Higher SWM iron concentrations were observed in U-fiber-rich frontal, temporal, and parietal areas, potentially reflecting high fiber density or late myelination in these areas. Our SWM mapping approach provides the foundation for systematic studies of interindividual differences, plasticity, and pathologies of this crucial structure for cortico-cortical connectivity in humans.

Leaf development stages and ontogenetic changes in passionfruit (Passiflora edulis Sims.) are detected by narrowband spectral signal.

During shoot development, leaves undergo various ontogenetic changes, including variation in size, shape, and geometry. Passiflora edulis (passionfruit) is a heteroblastic species, which means that it experiences conspicuous changes throughout development, enabling a morphological distinction between the juvenile and adult vegetative phases. Quantification of heteroblasty requires a practical, inexpensive, reliable, and non-destructive method, such as remote sensing. Moreover, relationships among ontogenetic changes and spectral signal at leaf level can be scaled up to support precision agriculture in passion fruit crops. In the present study, we used laboratory spectroscopic measurements (400-2500 nm) and narrowband vegetation indexes (or hyperspectral vegetation indexes - HVIs) to evaluate ontogenetic changes related to development and aging in P. edulis leaves. We also assessed leaf pigment concentration to further support the application of biochemical-related narrowband indexes. We report that 30-d-old leaves can be discriminated into developmental stages through their spectral signals. MSI (Moisture Stress Index) and NDVI750 (Normalized Difference Vegetation Index ρ750) contribute most to the variation of age (15 to 30-d-old leaves) and developmental stage (phytomer positions along the plant axis) in passionfruit leaves. PRI (Photochemical Reflectance Index) played an important role in detecting age and development alterations, including heteroblasty. A biochemical and spectral comparison of pigments revealed that spectroscopy offered potential for diagnosing phenology in P. edulis, as some narrowband indexes correlated strongly with chlorophylls and carotenoids content. Narrowband vegetation indexes are found to be a suitable tool for monitoring passionfruit crops.

Impact of different crystalline forms of nTiO2 on metabolism and arsenic toxicity in Limnoperna fortunei.

Arsenic (As) is a ubiquitous contaminant in the environment and it is known to induce oxidative stress in aquatic organisms. In an attempt to remove As from water, some studies have suggested the titanium dioxide nanomaterial (nTiO2) as a promising alternative. However, it has been observed that nTiO2 can induce toxicity alone or in combination with metals, and this toxicity is dependent on its crystalline form of nanomaterial (mainly rutile as nTiO2R and anatase as nTiO2A, respectively). Considering that both (nTiO2 and As) can occur together, the objective of this study was to evaluate if co-exposure to rutile and anatase may influence accumulation, metabolisation, and toxicity of arsenite (As+3) in the golden mussel Limnoperna fortunei after 48 h of co-exposure to nTiO2 (1 mg/L) and As (50 µg/L). Accumulation and chemical speciation of As in organisms were determined. Also, biochemical analyses, such as the activity of the enzymes glutathione S-transferase omega (GSTΩ), catalase (CAT) and glutathione S-transferase (GST), as well as lipid peroxidation (LPO) were investigated. Results showed that co-exposure to nTiO2A + As changed accumulation pattern of metalloid in gills and digestive gland. Both crystalline forms of nTiO2 affected the metabolisation capacity favoring the accumulation of more toxic As compounds and nTiO2A alone or in combination with As showed induce oxidative stress in gills of L. fortunei. In this way, it has a high potential risk of the co-exposure of these contaminants to aquatic organisms, and it also needs to consider the nanomaterial (nTiO2) properties and their application in the environmental remediation, carefully and judiciously.

Regulatory framework for dietary supplements and the public health challenge.

OBJECTIVE: The new regulatory framework for dietary supplements in Brazil prompted this analysis of the current outlook of these products and the challenges posed by the new guidelines. METHODS: We conducted a qualitative, observational and descriptive study of dietary supplements commercialized in Brazilian online stores with the help of the Google® search tool. We considered the ingredients on the labels, the effects attributed to these products and the commercial claims used as a means of promoting them to assess the necessary changes for the legal framework in the new guidelines. Finally, with the help of a database, we compared the effects declared by the manufacturers and attributed to certain ingredients with the scientific evidence described in literature. RESULTS: In total, we purchased 44 dietary supplements from Brazilian online stores (n = 7). Of the samples studied, 34.2% could not be classified in the category Dietary Supplements, as recommended by the new regulation of the Brazilian Health Regulatory Agency due to the presence of prohibited substances; 16% of products should be commercialized as medicines. Regarding the commercial appeals, 97.7% had banned expressions. Numerous claims of effects attributed to certain products were characterized as consumer fraud because they have no scientific evidence. CONCLUSIONS: The necessary changes represent a major regulatory and production challenge due to the wide range of dietary supplements and markets, an effort that aims to protect the consumers' health. Some previous gaps in the regulatory framework were not fully solved.

A new approach to ion exchange chromatography with conductivity detection for adulterants investigation in dietary supplements.

The extent of adulteration of dietary supplements has significantly increased in recent years. This situation worries health authorities and requires auxiliary analytical tools for the investigation of illegal substances purposely added. Ion exchange chromatography with conductivity detection is a consolidated analytical technique for the determination of inorganic compounds in various matrices. This technique has been applied to the pharmaceutical characterization of mainly impurities and degradation products. This work presents a new approach to ion exchange chromatography as a screening method to investigate the presence of amfepramone, femproporex, sibutramine, bisacodyl and amiloride in dietary supplements advertised for weight loss. The method was optimized and validated using a Metrosep C4 100/4.0 cation exchange column. The mobile phase consisted of 1.8 mm HNO3 containing 2% acetonitrile (v/v), with a flow rate of 0.9 ml min-1 , and nonsuppressed conductivity detection was applied. The limits of detection and quantification varied from 1.01 to 3.62 mg L-1 and from 1.48 to 8.72 mg L-1 , respectively. The proposed method was successful applied to 78 solid dietary supplement samples, in two of which adulterations were found. Moreover, ion exchange chromatography with conductivity detection could be easily used for quality control without prior complex sample pre-treatment.

Genotoxic effect of dimethylarsinic acid and the influence of co-exposure to titanium nanodioxide (nTiO2) in Laeonereis culveri (Annelida, Polychaeta).

Few data are available about the effect of dimethylated forms (DMA) on aquatic organisms. As rarely a contaminant occurs alone, studies evaluating the combined effect of different contaminants in aquatic organisms are needed. In fact, the presence of nanomaterials, such as titanium dioxide nanoparticles (nTiO2), in the aquatic environment is now a reality due to its intensive production and use. So, this study evaluated the toxicological effects of DMA in an acute exposure condition and considered the potential influence of nTiO2 on the effects induced by DMA in the polychaete, Laeonereis culveri. The animals were exposed over 48 h to DMA (50 and 500 µg/l) alone or in combination with nTiO2 (1 mg/l). Biochemical parameters such as concentration of reactive oxygen species (ROS), glutathione-S-transferase (GST) activity, levels of reduced glutathione levels (GSH) and macromolecular (lipid and DNA) damage were evaluated, as well the DNA repair system. In addition, the accumulation of total As and the chemical speciation of the metalloid in the organisms was determined. The results showed that: (1) only the group exposed to 500 µg of DMA/l accumulated As and when co-exposed to nTiO2, this accumulation was not observed. (2) The levels of ROS increased in the group exposed to 50 µg/l of DMA alone and the effect was reversed when this group was co-exposed to nTiO2 (3) None of the treatments showed altered GST activity or GSH levels. (4) All groups that received nTiO2 (alone or in combination with DMA) showed lipid peroxidation. (5) The exposure to DMA (both concentrations) alone or in combination with nTiO2 induced DNA damage in L. culveri. These results showed that DMA exhibits a genotoxic effect and that co-exposure to nTiO2 had an influence on its toxicity. So the occurrence of both contaminants simultaneously can represent a threat to aquatic biota.

An Ultra-High Performance Liquid Chromatography-Electrospray Tandem Mass Spectrometric Method for Screening and Simultaneous Determination of Anorexic, Anxiolytic, Antidepressant, Diuretic, Laxative and Stimulant Drugs in Dietary Supplements Marketed for Weight Loss.

The consumption of dietary supplements is increasing every year all over the world and has been accompanied by an increased frequency of adulteration of these products with synthetic pharmaceuticals. Analytical methods that allow testing for the presence of synthetic drugs in dietary supplements are needed to detect such fraudulent practices. To investigate the adulteration of dietary supplements marketed for weight loss using different commercial appeals, we developed an analytical method using ultra-high-performance liquid chromatography-electrospray tandem mass spectrometry (UHPLC-ESI-MS/MS) for simultaneous determination of 32 drugs, including anorexics, anxiolytics, antidepressants, diuretics, laxatives and stimulants. Separation was accomplished in 19 minutes using a Zorbax SB-C18 column and a gradient elution program with 0.05% formic acid in water/acetonitrile as a mobile phase. Limits of quantification ranged from 0.14 to 3.92 µg L-1, and accuracy ranged from 80.00 to 119.48%. A simple extraction procedure was used in the pretreatment step by dissolving the samples in 100% methanol followed by a 1000 to 10,000-fold dilution in the mobile phase and filtration through a Teflon membrane (0.2 µm). The method was applied to the screening and quantification of the drugs in 108 formulations marketed as food supplements for slimming, weight loss, thermogenics, and supplements for meal replacement. Caffeine and p-synephrine were found as stimulants in 80 samples, listed or not on the label.

Circular Estimate Method (CEM) - a Simple Method to Estimate Caenorhabditis elegans Culture Densities in Liquid Medium.

BACKGROUND: Nematodes are used in many different fields of science, including environmental and biomedical research. Counting and/or estimating nematode numbers is required during research. Although being one of the most common procedures, this apparently simple task is a time-consuming process, prone to errors and concerns regarding procedure, reliability, and accuracy. When an estimate is necessary, there is a traditional manual counting procedure that in this study it will be called as "drop method" (DM). This popular method that extrapolates an animal count from a small drop of fluid shows a high coefficient of variation. To solve this problem, the present study used the free-living nematode Caenorhabditis elegans to develop a new estimation procedure that was based on a relationship between area and volume of a larger sample. RESULTS: The new method showed a low coefficient of variation and a close relationship between estimated and real counts of the total number of nematodes in large C. elegans suspensions. Reactive oxygen concentration was measured as an example of method application and to allow comparison between methods. CONCLUSION: The proposed method is accurate, facile and reproducible, requiring simple, inexpensive materials that make it an excellent alternative to the DM manual counting procedure. Although the DM is faster, its estimates are not as accurate or as precise as those of the new proposed method.

Regulatory framework for dietary supplements and the public health challenge / Marco regulatório dos suplementos alimentares e o desafio à saúde pública

ABSTRACT OBJECTIVE The new regulatory framework for dietary supplements in Brazil prompted this analysis of the current outlook of these products and the challenges posed by the new guidelines. METHODS We conducted a qualitative, observational and descriptive study of dietary supplements commercialized in Brazilian online stores with the help of the Google® search tool. We considered the ingredients on the labels, the effects attributed to these products and the commercial claims used as a means of promoting them to assess the necessary changes for the legal framework in the new guidelines. Finally, with the help of a database, we compared the effects declared by the manufacturers and attributed to certain ingredients with the scientific evidence described in literature. RESULTS In total, we purchased 44 dietary supplements from Brazilian online stores (n = 7). Of the samples studied, 34.2% could not be classified in the category Dietary Supplements, as recommended by the new regulation of the Brazilian Health Regulatory Agency due to the presence of prohibited substances; 16% of products should be commercialized as medicines. Regarding the commercial appeals, 97.7% had banned expressions. Numerous claims of effects attributed to certain products were characterized as consumer fraud because they have no scientific evidence. CONCLUSIONS The necessary changes represent a major regulatory and production challenge due to the wide range of dietary supplements and markets, an effort that aims to protect the consumers' health. Some previous gaps in the regulatory framework were not fully solved.

RESUMO OBJETIVO O novo marco regulatório para os suplementos alimentares no Brasil instigou a presente análise do panorama atual desses produtos e os desafios impostos pelas novas diretrizes. MÉTODOS Foi realizado um estudo qualitativo, observacional e descritivo dos suplementos alimentares comercializados em lojas virtuais brasileiras, com o auxílio da ferramenta de busca Google®. Os ingredientes declarados nos rótulos, bem como os efeitos atribuídos a esses produtos e as alegações comerciais utilizadas como forma de promovê-los foram levados em consideração a fim de avaliarmos as mudanças necessárias para o enquadramento legal nas novas diretrizes. Por fim, com o auxílio de base de dados, foram comparados os efeitos declarados pelos fabricantes e atribuídos a determinados ingredientes com as evidências científicas descritas na literatura. RESULTADOS No total, foram adquiridos 44 suplementos alimentares provenientes de lojas virtuais brasileiras (n = 7). Das amostras estudadas, 34,2% não poderiam ser enquadradas na categoria Suplementos Alimentares, conforme preconiza a nova regulação da Agência Nacional de Vigilância Sanitária, em decorrência de presença de substâncias não permitidas; 16% dos produtos deveriam ser comercializados como medicamentos. Quanto aos apelos comerciais, 97,7% apresentavam expressões não permitidas. Inúmeras alegações de efeitos atribuídos a determinados produtos, por não possuírem comprovação científica, foram caracterizadas como fraude contra o consumidor. CONCLUSÕES Dada a extensa gama de suplementos alimentares e pontos de comercialização, as mudanças necessárias representam um grande desafio regulatório e de produção, esforço este que visa a proteger a saúde dos consumidores. Algumas lacunas previamente existentes ao marco regulatório ainda não foram totalmente solucionadas.

Genetic Polymorphisms of IL17 and Chagas Disease in the South and Southeast of Brazil.

The aim of this study was to investigate possible associations between genetic polymorphisms of IL17A G197A (rs2275913) and IL17F T7488C (rs763780) with Chagas Disease (CD) and/or the severity of left ventricular systolic dysfunction (LVSD) in patients with chronic Chagas cardiomyopathy (CCC). The study with 260 patients and 150 controls was conducted in the South and Southeast regions of Brazil. The genotyping was performed by PCR-RFLP. The A allele and A/A genotype of IL17A were significantly increased in patients and their subgroups (patients with CCC; patients with CCC and LVSD; and patients with CCC and severe LVSD) when compared to the control group. The analysis according to the gender showed that the A/A genotype of IL17A was more frequent in female with LVSD and mild to moderate LVSD and also in male patients with LVSD. The frequency of IL17F T/C genotype was higher in male patients with CCC and severe LVSD and in female with mild to moderate LVSD. The results suggest the possible involvement of the polymorphisms of IL17A and IL17F in the susceptibility to chronic Chagas disease and in development and progression of cardiomyopathy.

Hybrid Imaging Labels: Providing the Link Between Mass Spectrometry-Based Molecular Pathology and Theranostics.

BACKGROUND: Development of theranostic concepts that include inductively coupled plasma mass spectrometry (ICP-MS) and laser ablation ICP-MS (LA-ICP-MS) imaging can be hindered by the lack of a direct comparison to more standardly used methods for in vitro and in vivo evaluation; e.g. fluorescence or nuclear medicine. In this study a bimodal (or rather, hybrid) tracer that contains both a fluorescent dye and a chelate was used to evaluate the existence of a direct link between mass spectrometry (MS) and in vitro and in vivo molecular imaging findings using fluorescence and radioisotopes. At the same time, the hybrid label was used to determine whether the use of a single isotope label would allow for MS-based diagnostics. METHODS: A hybrid label that contained both a DTPA chelate (that was coordinated with either 165Ho or 111In) and a Cy5 fluorescent dye was coupled to the chemokine receptor 4 (CXCR4) targeting peptide Ac-TZ14011 (hybrid-Cy5-Ac-TZ4011). This receptor targeting tracer was used to 1) validate the efficacy of (165Ho-based) mass-cytometry in determining the receptor affinity via comparison with fluorescence-based flow cytometry (Cy5), 2) evaluate the microscopic binding pattern of the tracer in tumor cells using both fluorescence confocal imaging (Cy5) and LA-ICP-MS-imaging (165Ho), 3) compare in vivo biodistribution patterns obtained with ICP-MS (165Ho) and radiodetection (111In) after intravenous administration of hybrid-Cy5-Ac-TZ4011 in tumor-bearing mice. Finally, LA-ICP-MS-imaging (165Ho) was linked to fluorescence-based analysis of excised tissue samples (Cy5). RESULTS: Analysis with both mass-cytometry and flow cytometry revealed a similar receptor affinity, respectively 352 ± 141 nM and 245 ± 65 nM (p = 0.08), but with a much lower detection sensitivity for the first modality. In vitro LA-ICP-MS imaging (165Ho) enabled clear discrimination between CXCR4 positive and negative cells, but fluorescence microscopy was required to determine the intracellular distribution. In vivo biodistribution patterns obtained with ICP-MS (165Ho) and radiodetection (111In) of the hybrid peptide were shown to be similar. Assessment of tracer distribution in excised tissues revealed the location of tracer uptake with both LA-ICP-MS-imaging and fluorescence imaging. CONCLUSION: Lanthanide-isotope chelation expands the scope of fluorescent/radioactive hybrid tracers to include MS-based analytical tools such as mass-cytometry, ICP-MS and LA-ICP-MS imaging in molecular pathology. In contradiction to common expectations, MS detection using a single chelate imaging agent was shown to be feasible, enabling a direct link between nuclear medicine-based imaging and theranostic methods.

LA-ICP-MS Allows Quantitative Microscopy of Europium-Doped Iron Oxide Nanoparticles and is a Possible Alternative to Ambiguous Prussian Blue Iron Staining.

The development of iron oxide nanoparticles for biomedical applications requires accurate histological evaluation. Prussian blue iron staining is widely used but may be unspecific when tissues contain substantial endogenous iron. Here we tested whether microscopy by laser ablation coupled to inductively coupled plasma mass spectrometry (LA-ICP-MS) is sensitive enough to analyze accumulation of very small iron oxide particles (VSOP) doped with europium in tissue sections. For synthesis of VSOP, a fraction of Fe3+ (5 wt%) was replaced by Eu3+, resulting in particles with 0.66 mol% europium relative to iron (Eu-VSOP) but with otherwise similar properties as VSOP. Eu-VSOP or VSOP was intravenously injected into ApoE-/- mice on Western cholesterol diet and accumulated in atherosclerotic plaques of these animals. Prussian blue staining was positive for ApoE-/- mice with particle injection but also for controls. LA-ICP-MS microscopy resulted in sensitive and specific detection of the europium of Eu-VSOP in liver and atherosclerotic plaques. Furthermore, calibration with Eu-VSOP allowed calculation of iron and particle concentrations in tissue sections. The combination of europium-doped iron oxide particles and LA-ICP-MS microscopy provides a new tool for specific and quantitative analysis of particle distribution at the tissue level and allows correlation with other elements such as endogenous iron.

Climatic drivers of leaf traits and genetic divergence in the tree Annona crassiflora: a broad spatial survey in the Brazilian savannas.

The Cerrado is the largest South American savanna and encompasses substantial species diversity and environmental variation. Nevertheless, little is known regarding the influence of the environment on population divergence of Cerrado species. Here, we searched for climatic drivers of genetic (nuclear microsatellites) and leaf trait divergence in Annona crassiflora, a widespread tree in the Cerrado. The sampling encompassed all phytogeographic provinces of the continuous area of the Cerrado and included 397 individuals belonging to 21 populations. Populations showed substantial genetic and leaf trait divergence across the species' range. Our data revealed three spatially defined genetic groups (eastern, western and southern) and two morphologically distinct groups (eastern and western only). The east-west split in both the morphological and genetic data closely mirrors previously described phylogeographic patterns of Cerrado species. Generalized linear mixed effects models and multiple regression analyses revealed several climatic factors associated with both genetic and leaf trait divergence among populations of A. crassiflora. Isolation by environment (IBE) was mainly due to temperature seasonality and precipitation of the warmest quarter. Populations that experienced lower precipitation summers and hotter winters had heavier leaves and lower specific leaf area. The southwestern area of the Cerrado had the highest genetic diversity of A. crassiflora, suggesting that this region may have been climatically stable. Overall, we demonstrate that a combination of current climate and past climatic changes have shaped the population divergence and spatial structure of A. crassiflora. However, the genetic structure of A. crassiflora reflects the biogeographic history of the species more strongly than leaf traits, which are more related to current climate.