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Chenopodium rubrum L. , is a suitable model plant for studying ontogenesis in vitro as an early flowering species. Culture of intact plants in vitro and antioxidative enzymes detection were performed. Growth pattern to the end of ontogenesis, flowering and seed development are all determined by the photoperiod seedlings experience during induction and evocation of flowering. Different phases of vegetative and reproductive development are characterized by changes in antioxidative enzymes activities. We showed sequential expression of antioxidative enzymes during seed germination. Prior to radicule protrusion, CAT and SOD showed maximal activity, while POD activity appeared later. The highest catalase (CAT) activity was measured at the time of flowering while peroxidases (PODs) are involved in determination of growth and development in accordance with the environmental clues. The absence of some superoxide dismutase (SOD) isoforms could be the indicator of senescence. Seed ageing affect changes in antioxidative status of seeds, germination, seedling growth and flowering.

We studied changes in the concentrations of ascorbate and glutathione, composition of soluble phenolics, and activity of oxalate oxidase in 75-day-old Serbian spruce plants after exposure to 5 μM and 50μM cadmium for 6-48 h. The presence of OxOxactivity in a conifer species is here demonstrated for the first time. Both Cd concentrations induced a decrease of OxOxactivity in treated plants in comparison with the control at all sampling dates. The concentrations of reduced glutathione, its oxidized form, and reduced ascorbate in the plants decreased during 48-h treatment with cadmium. Among simple phenolics, only catechin increased significantly during Cd treatment.

Picea omorika (Panč) Purkyně (Serbian spruce) is a Balkan endemic coniferous species. We studied soluble peroxidase, catalase, polyphenol oxidase, and superoxide-dismutase activity in the needles of five omorika lines grown in a generative seed orchard. The peroxidase and polyphenol oxidase isoenzyme patterns were also investigated. Activity of the studied enzymes varied among different lines. The highest activity of peroxidase, catalase and polyphenol oxidase was found in the A3 ("borealis") and B5 ("semidichotomous") lines. Four acidic and two basic peroxidase isoenzymes and one polyphenol oxidase isoenzyme were detected. There was no variation in either the peroxidase or the polyphenol oxidase isoenzyme pattern among the different lines.

We predicted the protein therapeutic targets specific to a Ru-based potential drug and its combination with pristine and N-doped carbon dot drug delivery systems, denoted as RuCN/CDs and RuCN/N-CDs. Synchrotron-based FTIR microspectroscopy (µFTIR) in addition to bioinformatics data on drug structures and protein sequences were applied to assess changes in the protein secondary structure of A2780 cancer cells. µFTIR revealed the moieties of the target proteins' secondary structure changes only after the treatment with RuCN and RuCN/N-CDs. A higher content of α-helices and a lower content of β-sheets appeared in A2780 cells after RuCN treatment. Treatment with RuCN/N-CDs caused a substantial increase in parallel β-sheet numbers, random coil content, and tyrosine residue numbers. The results obtained suggest that the mitochondrion-related proteins NDUFA1 and NDUFB5 are affected by RuCN either via overexpression or stabilisation of helical structures. RuCN/N-CDs either induce overexpression of the β-sheet-rich protein NDUFS1 and affect its random coil structure or interact and stabilise its structure via hydrogen bonding between -NH2 groups from N-CDs with protein C=O groups and -OH groups of serine, threonine, and tyrosine residues. The N-CD nanocarrier tunes this drug's action by directing it toward a specific protein target, changing this drug's coordination ability and inducing changes in the protein's secondary structures and function.

The literature data emphasize that nanoparticles might improve the beneficial effects of near-infrared light (NIR) on wound healing. This study investigates the mechanisms of the synergistic wound healing potential of NIR light and silver metal-organic frameworks combined with nitrogen- and sulfur-doped carbon dots (AgMOFsN-CDs and AgMOFsS-CDs, respectively), which was conducted by testing the fibroblasts viability, scratch assays, biochemical analysis, and synchrotron-based Fourier transform infrared (SR-FTIR) cell spectroscopy and imaging. Our findings reveal that the combined treatment of AgMOFsN-CDs and NIR light significantly increases cell viability to nearly 150% and promotes cell proliferation, with reduced interleukin-1 levels, suggesting an anti-inflammatory response. SR-FTIR spectroscopy shows this combined treatment results in unique protein alterations, including increased α-helix structures and reduced cross-β. Additionally, protein synthesis was enhanced upon the combined treatment. The likely mechanism behind the observed changes is the charge-specific interaction of N-CDs from the AgMOFsN-CDs with proteins, enhanced by NIR light due to the nanocomposite's optical characteristics. Remarkably, the complete wound closure in the in vitro scratch assay was achieved exclusively with the combined NIR and AgMOFsN-CDs treatment, demonstrating the promising application of combined AgMOFsN-CDs with NIR light photodynamic therapy in regenerative nanomedicine and tissue engineering.

Nanoparticle-based nanocarriers represent a viable alternative to conventional direct administration in cancer cells. This advanced approach employs the use of nanotechnology to transport therapeutic agents directly to cancer cells, thereby reducing the risk of damage to healthy cells and enhancing the efficacy of treatment. By approving nanoparticle-based nanocarriers, the potential for targeted, effective treatment is greatly increased. The so-called carbon-based nanoparticles, or carbon dots, have been hydrothermally prepared and initiated by a polymerization process. We synthesized and characterized nanoparticles of 2-acrylamido-2-methylpropanesulfonic acid, which showed biocompatibility with glioblastoma cells, and further, we tested them as a carrier for the drug riluzole. The obtained nanoparticles have been extensively characterized by techniques to obtain the exact composition of their surface by using Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), and nuclear magnetic resonance (NMR) spectroscopy, as well as cryo-transmission electron microscopy. We found that the surface of the synthesized nanoparticles (NPs) is covered mainly by sulfonated, carboxylic, and substituted amide groups. These functional groups make them suitable as carriers for drug delivery in cancer cells. Specifically, we have successfully utilized the NPs as a delivery system for the drug riluzole, which has shown efficacy in treating glioblastoma cancer cells. The effect of nanoparticles as carriers for the riluzole system on glioblastoma cells was studied using live-cell synchrotron-based FTIR microspectroscopy to monitor in situ biochemical changes. After applying nanoparticles as nanocarriers, we have observed changes in all biomacromolecules, including the nucleic acids and protein conformation. These findings provide a strong foundation for further exploration into the development of targeted treatments for glioblastoma., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Fourier transform infrared (FTIR) spectroscopy is a rapid, non-destructive and label-free technique for identifying subtle changes in all bio-macromolecules, and has been used as a method of choice for studying DNA conformation, secondary DNA structure transition and DNA damage. In addition, the specific level of chromatin complexity is introduced via epigenetic modifications forcing the technological upgrade in the analysis of such an intricacy. As the most studied epigenetic mechanism, DNA methylation is a major regulator of transcriptional activity, involved in the suppression of a broad spectrum of genes and its deregulation is involved in all non-communicable diseases. The present study was designed to explore the use of synchrotron-based FTIR analysis to monitor the subtle changes in molecule bases regarding the DNA methylation status of cytosine in the whole genome. In order to reveal the conformation-related best sample for FTIR-based DNA methylation analysis in situ, we used methodology for nuclear HALO preparations and slightly modified it to isolated DNA in HALO formations. Nuclear DNA-HALOs represent samples with preserved higher-order chromatin structure liberated of any protein residues that are closer to native DNA conformation than genomic DNA (gDNA) isolated by the standard batch procedure. Using FTIR spectroscopy we analyzed the DNA methylation profile of isolated gDNA and compared it with the DNA-HALOs. This study demonstrated the potential of FTIR microspectroscopy to detect DNA methylation marks in analyzed DNA-HALO specimens more precisely in comparison with classical DNA extraction procedures that yield unstructured whole genomic DNA. In addition, we used different cell types to assess their global DNA methylation profile, as well as defined specific infrared peaks that can be used for screening DNA methylation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Amyloid-β (Aβ) plaques from Alzheimer's Disease (AD) can be visualized ex vivo in label-free brain samples using synchrotron X-ray phase-contrast tomography (XPCT). However, for XPCT to be useful as a screening method for amyloid pathology, it is essential to understand which factors drive the detection of Aβ plaques. The current study was designed to test the hypothesis that Aβ-related contrast in XPCT could be caused by Aβ fibrils and/or by metals trapped in the plaques. Fibrillar and elemental compositions of Aβ plaques were probed in brain samples from different types of AD patients and AD models to establish a relationship between XPCT contrast and Aβ plaque characteristics. XPCT, micro-Fourier-Transform Infrared spectroscopy and micro-X-Ray Fluorescence spectroscopy were conducted on human samples (one genetic and one sporadic case) and on four transgenic rodent strains (mouse: APPPS1, ArcAβ, J20; rat: TgF344). Aβ plaques from the genetic AD patient were visible using XPCT, and had higher β-sheet content and higher metal levels than those from the sporadic AD patient, which remained undetected by XPCT. Aβ plaques in J20 mice and TgF344 rats appeared hyperdense on XPCT images, while they were hypodense with a hyperdense core in the case of APPPS1 and ArcAβ mice. In all four transgenic strains, β-sheet content was similar, while metal levels were highly variable: J20 (zinc and iron) and TgF344 (copper) strains showed greater metal accumulation than APPPS1 and ArcAβ mice. Hence, a hyperdense contrast formation of Aβ plaques in XPCT images was associated with biometal entrapment within plaques. STATEMENT OF SIGNIFICANCE: The role of metals in Alzheimer's disease (AD) has been a subject of continuous interest. It was already known that amyloid-β plaques (Aβ), the earliest hallmark of AD, tend to trap endogenous biometals like zinc, iron and copper. Here we show that this metal accumulation is the main reason why Aβ plaques are detected with a new technique called X-ray phase contrast tomography (XPCT). XPCT enables to map the distribution of Aβ plaques in the whole excised brain without labeling. In this work we describe a unique collection of four transgenic models of AD, together with a human sporadic and a rare genetic case of AD, thus exploring the full spectrum of amyloid contrast in XPCT., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

The reprogramming of human somatic cells to induced pluripotent cells (iPSCs) has become a milestone and a paradigm shift in the field of regenerative medicine and human disease modeling including drug testing and genome editing. However, the molecular processes occurring during reprogramming and affecting the pluripotent state acquired remain largely unknown. Of interest, different pluripotent states have been described depending on the reprogramming factors used and the oocyte has emerged as a valuable source of information for candidate factors. The present study investigates the molecular changes occurring in somatic cells during reprogramming with either canonical (OSK) or oocyte-based (AOX15) combinations using synchrotron-radiation Fourier transform infrared (SR FTIR) spectroscopy. The data acquired by SR FTIR indicates different representation and conformation of biological relevant macromolecules (lipids, nucleic acids, carbohydrates and proteins) depending on the reprogramming combination used and at different stages during the reprogramming process. Association analysis based on cells spectra suggest that pluripotency acquisition trajectories converge at late intermediate stages while they diverge at early stages. Our results suggest that OSK and AOX15 reprogramming operates through differential mechanisms affecting nucleic acids reorganization and day 10 comes out as a candidate hinge point to further study the molecular pathways involved in the reprogramming process. This study indicates that SR FTIR approach contribute unpaired information to distinguish pluripotent states and to decipher pluripotency acquisition roadmaps and landmarks that will enable advanced biomedical applications of iPSCs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

The mode of action toward gastric cancer cells of brominated Coelenteramine, an analogue of a metabolic product of a marine bioluminescent reaction, was investigated by synchrotron radiation-based Fourier Transform Infrared spectrocopy (FTIR). This method revealed that the anticancer activity of brominated Coelenteramine is closely connected with cellular lipids, by affecting their organization and composition. More specifically, there is an increasing extent of oxidative stress, which results in changes in membrane polarity, lipid chain packing and lipid composition. However, this effect was not observed in a noncancer cell line, helping to explain its selectivity profile. Thus, synchrotron radiation-based FTIR helped to identify the potential of this Coelenteramine analogue in targeting membrane lipids, while proving to be a powerful technique to probe the mechanism of anticancer drugs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, with the most common adult-onset neurodegenerative disorder affecting motoneurons. Although disruptions in macromolecular conformation and homeostasis have been described in association with ALS, the underlying pathological mechanisms are still not completely understood, and unambiguous biomarkers are lacking. Fourier Transform Infrared Spectroscopy (FTIR) of cerebrospinal fluid (CSF) is appealing to extensive interest due to its potential to resolve biomolecular conformation and content, as this approach offers a non-invasive, label-free identification of specific biologically relevant molecules in a few microliters of CSF sample. Here, we analyzed the CSF of 33 ALS patients compared to 32 matched controls using FTIR spectroscopy and multivariate analysis and demonstrated major differences in the molecular contents. A significant change in the conformation and concentration of RNA is demonstrated. Moreover, significantly increased glutamate and carbohydrates are found in ALS. Moreover, key markers of lipid metabolism are strongly altered; specifically, we find a decrease in unsaturated lipids and an increase in peroxidation of lipids in ALS, whereas the total amount of lipids compared to proteins is reduced. Our study demonstrates that FTIR characterization of CSF could represent a powerful tool for ALS diagnosis and reveals central features of ALS pathophysiology.

Metabolism of metals in microalgae and adaptation to metal excess are of significant environmental importance. We report a three-step mechanism that the green microalga Chlorella sorokiniana activates during the acquisition of and adaptation to manganese (Mn), which is both an essential trace metal and a pollutant of waters. In the early stage, Mn2+ was mainly bound to membrane phospholipids and phosphates in released mucilage. The outer cell wall was reorganized and lipids were accumulated, with a relative increase in lipid saturation. Intracellular redox settings were rapidly altered in the presence of Mn excess, with increased production of reactive oxygen species that resulted in lipid peroxidation and a decrease in the concentration of thiols. In the later stage, Mn2+ was chelated by polyphosphates and accumulated in the cells. The structure of the inner cell wall was modified and the redox milieu established a new balance. Polyphosphates serve as a transient Mn2+ storage ligand, as proposed previously. In the final stage, Mn was stored in multivalent Mn clusters that resemble the structure of the tetramanganese-calcium core of the oxygen-evolving complex. The present findings elucidate the bioinorganic chemistry and metabolism of Mn in microalgae, and may shed new light on water-splitting Mn clusters., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Synchrotron radiation-based Fourier Transform Infrared (SR-FTIR) microspectroscopy is a non-destructive and chemically sensitive technique for the rapid detection of changes in the different components of the cell's biomacromolecular profile. Reactive oxygen species and oxidative stress may cause damage to the DNA, RNA, and proteins in the retinal pigment epithelium (RPE), which can further lead to age-related macular degeneration (AMD) and visual loss in the elderly. In this study, human primary RPEs (hRPEs) were used to study AMD pathogenesis by using an established in vitro cellular model of the disease. Autophagy-a mechanism of intracellular degradation, which is altered during AMD, was studied in the hRPEs by using the autophagy inducer rapamycin and treated with the autophagy inhibitor bafilomycin A1. In addition, oxidative stress was induced by the hydrogen peroxide (H 2 O 2 ) treatment of hRPEs. By using SR-FTIR microspectroscopy and multivariate analyses, the changes in the phosphate groups of nucleic acids, Amide I and II of the proteins, the carbonyl groups, and the lipid status in the hRPEs showed a significantly different pattern under oxidative stress/autophagy induction and inhibition. This biomolecular fingerprint can be evaluated in future drug discovery studies affecting autophagy and oxidative stress in AMD.

Hydroxyapatite nanoparticles are popular tools in bone regeneration, but they have also been used for gene delivery and as anticancer drugs. Understanding their mechanism of action, particularly for the latter application, is crucial to predict their toxicity. To this end, we aimed to elucidate the importance of nanoparticle membrane interactions in the cytotoxicity of MG-63 cells using two different types of nanoparticles. In addition, conventional techniques for studying nanoparticle internalisation were evaluated and compared with newer and less exploited approaches. Hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles were used as suspensions or compacted as specular discs. Comparison between cells seeded on the discs and those supplemented with the nanoparticles allowed direct interaction of the cell membrane with the material to be ruled out as the main mechanism of toxicity. In addition, standard techniques such as flow cytometry were inconclusive when used to assess nanoparticles toxicity. Interestingly, the use of intracellular calcium fluorescent probes revealed the presence of a high number of calcium-rich vesicles after nanoparticle supplementation in cell culture. These structures could not be detected by transmission electron microscopy due to their liquid content. However, by using cryo-soft X-ray imaging, which was used to visualise the cellular ultrastructure without further treatment other than vitrification and to quantify the linear absorption coefficient of each organelle, it was possible to identify them as multivesicular bodies, potentially acting as calcium stores. In the study, an advanced state of degradation of the hydroxyapatite and magnesium-doped hydroxyapatite nanoparticles within MG-63 cells was observed. Overall, we demonstrate that the combination of fluorescent calcium probes together with cryo-SXT is an excellent approach to investigate intracellular calcium, especially when found in its soluble form., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

S and N-doped carbon dots (S-CDs and N-CDs) and their cisplatin (cis-Pt) derivatives. (S-CDs@cis-Pt and N-CDs@cis-Pt) were tested on two ovarian cancer cell lines: A2780 and A2780 cells resistant to cis-Pt (A2780R). Several spectroscopic techniques were employed to check S-CDs@cis-Pt and N-CDs@cis-Pt: solid- and solution-state nuclear magnetic resonance, matrix-assisted laser desorption, ionization time-of-flight mass spectrometry, and X-ray photoelectron spectroscopy. In addition, synchrotron-based Fourier Transformed Infrared spectro-microscopy was used to evaluate the biochemical changes in cells after treatment with cis-Pt, S-CDs, N-CDs, or S-CDs@cis-Pt and N-CDs@cis-Pt, respectively. Computational chemistry was applied to establish the model for the most stable bond between S-CDs and N-CDs and cis-Pt. The results revealed the successful modification of S-CDs and N-CDs with cis-Pt and the formation of a stable composite system that can be used for drug delivery to cancer cells and likewise to overcome acquired cis-Pt resistance. Nanoparticle treatment of A2780 and A2780R cells led to the changes in their structure of lipids, proteins, and nucleic acids depending on the treatment. The results showed the S-CDs@cis-Pt and N-CDs@cis-Pt might be used in the combination with cis-Pt to treat the adenocarcinoma, thus having a potential to be further developed as drug delivery systems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Carbon dots (CDs) and N-carbon dots (N-CDs) loaded with Ru-complex (CDs@RuCN, N-CDs@RuCN, respectively) were investigated as media imposing biochemical changes induced by UV illumination of ovarian cancer, A2780, and osteosarcoma, CAL72, cells. Synchrotron radiation-based Fourier Transform Infrared Spectroscopy was performed, and the spectra were subjected to a Principal Component Analysis. The CDs@RuCN and N-CDs@RuCN effects on cancer cells were analyzed by the theoretical modelling of the stability of the composite systems and a protein database search. Moreover, a detailed evaluation of surface and optical properties of CDs@RuCN and N-CDs@RuCN was carried out. Results demonstrated selective action of the CDs@RuCN and N-CDs@RuCN-based photodynamic therapy, with N-CDs@RuCN being the most active in inducing changes in A2780 and CDs@RuCN in CAL72 cells. We assume that different surface charges of nanoparticles led to direct interactions of N-CDs@RuCN with a Wnt signalling pathway in A2780 and those of CDs@RuCN with PI3-K/Akt in CAL72 cells and that further biochemical changes occurred upon light illumination., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In the last decade, targeting membrane lipids in cancer cells has been a promising approach that deserves attention in the field of anticancer drug development. To get a comprehensive understanding of the effect of the drug [Ru(η 5 -Cp)(PPh 3 ) 2 CN] (RuCN) on cell lipidic components, we combine complementary analytical approaches, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI TOF MS) and synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectroscopy. Techniques are used for screening the effect of potential metallodrug, RuCN, without and with drug carriers (carbon dots (CDs) and nitrogen-doped carbon dots (N-CDs)) on the lipids of the human ovarian cancer cell line A2780. MALDI TOF MS results revealed that the lysis of ovarian cancer membrane lipids is promoted by RuCN and not by drug carriers (CDs and N-CDs). Furthermore, SR-FTIR results strongly suggested that the phospholipids of cancer cells undergo oxidative stress after the treatment with RuCN that was accompanied by the disordering of the fatty acid chains. On the other hand, using (N-)CDs as RuCN nanocarriers prevented the oxidative stress caused by RuCN but did not prevent the disordering of the fatty acid chain packing. Finally, we demonstrated that RuCN and RuCN/(N-)CDs alter the hydration of the membrane surface in the membrane-water interface region.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor, characterized by short median survival and an almost 100% tumor-related mortality. The standard of care treatment for newly diagnosed GBM includes surgical resection followed by concomitant radiochemotherapy. The prevention of disease progression fails due to the poor therapeutic effect caused by the great molecular heterogeneity of this tumor. Previously, we exploited synchrotron radiation-based soft X-ray tomography and hard X-ray fluorescence for elemental microimaging of the shock-frozen GBM cells. The present study focuses instead on the biochemical profiling of live GBM cells and provides new insight into tumor heterogenicity. We studied bio-macromolecular changes by exploring the live-cell synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy in a set of three GBM cell lines, including the patient-derived glioblastoma cell line, before and after riluzole treatment, a medicament with potential anticancer properties. SR-FTIR microspectroscopy shows that GBM live cells of different origins recruit different organic compounds. The riluzole treatment of all GBM cell lines mainly affected carbohydrate metabolism and the DNA structure. Lipid structures and protein secondary conformation are affected as well by the riluzole treatment: cellular proteins assumed cross β-sheet conformation while parallel β-sheet conformation was less represented for all GBM cells. Moreover, we hope that a new live-cell approach for GBM simultaneous treatment and examination can be devised to target cancer cells more specifically, i.e., future therapies can develop more specific treatments according to the specific bio-macromolecular signature of each tumor type.

Toxic metals such as cadmium (Cd) and mercury (Hg) represent a threat to photosynthetic organisms of polluted aquatic ecosystems, and knowledge about mechanisms of toxicity is essential for appropriate assessment of environmental risks. We used Synchrotron Radiation-Fourier Transformed Infrared microspectroscopy (μSR-FTIR) to characterise major changes of biomolecules caused by Cd and Hg in the model green microalga Chlamydomonas reinhardtii. μSR-FTIR showed several metabolic alterations in different biochemical groups such as carbohydrates, proteins, and lipids in a time-dose dependent manner, with the strongest changes occurring at concentrations above 10 μM Cd and 15 μM Hg after short-term (24 h) treatments. This occurred in a context where metals triggered intracellular oxidative stress and chloroplast damage, along with autophagy induction by overexpressing AUTOPHAGY-RELATED PROTEIN 8 (ATG8). Thin layer chromatography analysis confirmed that toxic metals promoted remarkable changes in lipid profile, with higher degree of esterified fatty acid unsaturation as detected by gas chromatography coupled with mass spectrometry. Under Cd stress, there was specifically higher unsaturation of free fatty acids, while Hg led to stronger unsaturation in monogalactosyldiacylglycerol. μSR-FTIR spectroscopy proved as a valuable tool to identify biochemical alterations in microalgae, information that could be exploited to optimise approaches for metal decontamination., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Carbon dots are biocompatible and non-toxic nanoparticles with chemical affinity to some heavy metals. Human activities increase soil pollution with copper. Cu is an essential microelement in plants, but excess can induce a harmful effects. In plant response to Cu, the cell wall plays an important role. This study aims to estimate possible amelioration effects of folic acid based CDs on Cu toxicity by studying the intracellular and cell wall compounds in maize (Zea mays L.) roots and leaves after 7 day-treatment in hydroponics. The sub-cellular compartmentalization and bio-macromolecular changes induced by 5 μM Cu applied alone or with CDs (167 and 500 mg/L) were studied using the Synchrotron-based Fourier transformmicro-spectroscopy (SR-FTIR) combined with X-Ray photoelectron spectroscopy (XPS). Cu induced changes in content of cell wall polysaccharides, proteins, and lipids. The XPS detected CDs transport throughout the plants. The Cu/167CDs treatment reduced Cu concentration in the roots, possibly by complexation/trapping between the functional groups on CDs surface and Cu 2+ . Principal component analysis of FTIR spectra confirmed that Cu/500CDs treatment increased Cu adverse effects in most tissues but alleviated adverse Cu effects on cell wall polysaccharides in the root xylem, and on polysaccharides and proteins in leaf phloem and mesophyll., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Ultraviolet (UV) irradiation is an important risk factor in cataractogenesis. Lens epithelial cells (LECs), which are a highly metabolically active part of the lens, play an important role in UV-induced cataractogenesis. The purpose of this study was to characterize cell compounds such as nucleic acids, proteins, and lipids in human UV C-irradiated anterior lens capsules (LCs) with LECs, as well as to compare them with the control, non-irradiated LCs of patients without cataract, by using synchrotron radiation-based Fourier transform infrared (SR-FTIR) micro-spectroscopy. In order to understand the effect of the UV C on the LC bio-macromolecules in a context of cataractogenesis, we used the SR-FTIR micro-spectroscopy setup installed on the beamline MIRAS at the Spanish synchrotron light source ALBA, where measurements were set to achieve a single-cell resolution with high spectral stability and high photon flux. UV C irradiation of LCs resulted in a significant effect on protein conformation with protein formation of intramolecular parallel β-sheet structure, lower phosphate and carboxyl bands in fatty acids and amino acids, and oxidative stress markers with significant increase of lipid peroxidation and diminishment of the asymmetric CH 3 band.

Objects of the present study are improved fullerene C 60 drug carrier properties trough encapsulation by microbial polysaccharides, levan (LEV), pullulan (PUL), and their hydrophobized cholesterol-derivatives (CHL and CHP), that show better interaction with cancer cells. The zeta potential, polydispersity index, and the diameter of particles were determined, and their cytotoxicity against three cancer cell lines were tested. Biochemical changes in HeLa cells are analyzed by synchrotron radiation (SR) FTIR spectro-microscopy combined with the principal component analysis (PCA). The most significant changes occur in HeLa cells treated with LEV-C 60 and correspond to the changes in the protein region, i.e. Amide I band, and the changes in the structure of lipid bodies and membrane fluidity are evident. The highest cytotoxicity was also induced by LEV-C 60 . In HeLa cells, cytotoxicity could not be strictly associated with biochemical changes in lipids, proteins and nucleic acids, but these findings are significant contribution to the study of the mechanism of interaction of C 60 -based nanoparticles with cellular biomolecules. In conclusion, LEV, PUL, CHL, and CHP enhanced fullerene C 60 potential to be used as target drug delivery system with the ability to induce specific intracellular changes in HeLa cancer cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Microalgae have evolved mechanisms to respond to changes in copper ion availability, which are very important for normal cellular function, to tolerate metal pollution of aquatic ecosystems, and for modulation of copper bioavailability and toxicity to other organisms. Knowledge and application of these mechanisms will benefit the use of microalgae in wastewater processing and biomass production, and the use of copper compounds in the suppression of harmful algal blooms. Here, using electron microscopy, synchrotron radiation-based Fourier transform infrared spectroscopy, electron paramagnetic resonance spectroscopy, and X-ray absorption fine structure spectroscopy, we show that the microalga Chlorella sorokiniana responds promptly to Cu2+ at high non-toxic concentration, by mucilage release, alterations in the architecture of the outer cell wall layer and lipid structures, and polyphosphate accumulation within mucilage matrix. The main route of copper detoxification is by Cu2+ coordination to polyphosphates in penta-coordinated geometry. The sequestrated Cu2+ was accessible and could be released by extracellular chelating agents. Finally, the reduction in Cu2+ to Cu1+ appears also to take place. These findings reveal the biochemical basis of the capacity of microalgae to adapt to high external copper concentrations and to serve as both, sinks and pools of environmental copper., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, causing death of motor neurons controlling voluntary muscles. The pathological mechanisms of the disease are only partially understood. The hSOD1-G93A ALS rat model is characterized by an overexpression of human mutated SOD1, causing increased vulnerability by forming intracellular protein aggregates, inducing excitotoxicity, affecting oxidative balance and disturbing axonal transport. In this study we followed the bio-macromolecular organic composition and compartmentalization together with trace metal distribution in situ in single astrocytes from the ALS rat model and compared them to the control astrocytes from nontransgenic littermates by simultaneous use of two synchrotron radiation-based methods: Fourier transform infrared microspectroscopy (SR-FTIR) and hard X-ray fluorescence microscopy (XRF). We show that ALS cells contained more Cu, which colocalized with total lipids, increased carbonyl groups and oxidized lipids, thus implying direct involvement of Cu in oxidative stress of lipidic components without direct connection to protein aggregation in situ., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cataract is the leading cause of blindness worldwide but the mechanisms involved in the process of cataractogenesis are not yet fully understood. Two most prevalent types of age-related cataracts are nuclear (N) and cortical (C) cataracts. A common environmental factor in most age-related cataracts is believed to be oxidative stress. The lens epithelium, the first physical and biological barrier in the lens, is build from lens epithelial cells (LECs). LECs are important for the maintenance of lens transparency as they control energy production, antioxidative mechanisms and biochemical transport for the whole lens. The purpose of this study is to characterize compounds in LECs originated from N and C cataracts, by using the synchrotron radiation-based Fourier Transform Infrared (SR-FTIR) microspectroscopy, in order to understand the functional importance of their different bio-macromolecules in cataractogenesis. We used the SR-FTIR microspectroscopy setup installed on the beamline MIRAS at the Spanish synchrotron light source ALBA, where measurements were set to achieve single cell resolution, with high spectral stability and high photon flux. The results showed that protein aggregation in form of fibrils was notably pronounced in LECs of N cataracts, while oxidative stress and the lipids peroxidation were more pronounced in LECs of C cataracts.

Schizophrenia (SCZ) is a neurodevelopmental disorder with a high genetic component, but the presence of environmental stressors can be important for its onset and progression. Cannabis use can be a major risk factor for developing SCZ. However, despite the available data on the neurobiological underpinnings of SCZ, there is an important lack of studies in human neuronal tissue and living cells addressing the effects of cannabis in SCZ patients. In this study, we analysed the most relevant bio-macromolecular constituents in olfactory neuroepithelium (ON) cells of healthy controls non-cannabis users, healthy cannabis users, SCZ patients non-cannabis users, and SCZ patients cannabis users using Synchrotron Radiation-Fourier Transform Infrared (SR-FTIR) spectrometry and microscopy. Our results revealed that SCZ patients non-cannabis users, and healthy cannabis users exhibit similar alterations in the macromolecular profile of ON cells, including disruption in lipid composition, increased lipid membrane renewal rate and lipid peroxidation, altered proteins containing more β-sheet structures, and showed an increase in DNA and histone methylation. Notably, these alterations were not observed in SCZ patients who use cannabis regularly. These data suggest a differential effect of cannabis in healthy controls and in SCZ patients in terms of the macromolecular constituents of ON cells.

The phosphates (Pi) are nowadays recognized as pollutants. We studied the effect of Pi (0.625-12.500 mM KH 2 PO 4 ) in the culture medium on in vitro grown 2-month-old Populus alba trees. The levels of sugar phosphates and vacuolar and cytoplasmic Pi in cell compartments of roots and stems were determined using 31 P NMR, while tissue-specific micro- and macroelements mapping on stem cross-sections were performed using synchrotron-based X-ray microfluorescence. Plants grown on 0.625 mM Pi (MS/2 medium) showed a survival rate of 70%. With the increase in Pi concentrations up to 6.250 mM, plant growth and survival increased, without changes in total P content per mass or in the levels of cytoplasmic and vacuolar phosphates, in both stems and roots, while the levels of Fe, Cu, Zn, Ca and Mn in stems increased. Further increase in Pi to 9.375 and 12.500 mM in the medium resulted in inhibited growth comparable with plants grown on MS/2, with the increase in total P content per mass up to 50%, in both stems and roots, but with no changes in cytoplasmic and vacuolar phosphates; 12.500 mM Pi affected even plant survival (70%) and thus might be considered as mildly toxic. 31 P NMR results indicate that the high tolerance of P. alba to increased Pi could result from its ability to maintain an intracellular P homeostasis, despite P accumulation up to 50%, in both stems and roots, indicating P. alba as a promising wood species for dendroremediation.

The outcome of radiotherapy can be further improved by combining radiotherapy with nanoparticles. Previous biological studies showed a significant amplification of the biological damage in cells charged with nanoparticles prior to radiotherapy treatments. The rationale has been based on the physical dose enhancement. However, this subject is still a matter of controversy and there are clear indications that biochemical effects may play a key role in the radiosensitization effects of nanoparticles. Within this context, the main goal of our study was to provide new insights into the radiosensitization effects of F98 glioma cells exposed to gadolinium nanoparticles combined with clinical megavoltage beams, and compare them with respect to kilovoltage radiotherapy (commonly used in combination with nanoparticles). For this purpose, we used synchrotron-based Fourier transform infrared microspectroscopy (SR-FTIRM) to provide relevant information on the treatment-induced biochemical changes of the main cell biomolecules. Biochemical differences were evaluated after the treatments to assess cellular damage. Multivariate analysis revealed nanoparticle-dependent changes in megavoltage treated cells. The main spectral variations were related to conformational changes in the protein secondary structures, which might be induced by radiation damage and by changes or rearrangements in the nucleic acid structures due to the initiation of DNA repair mechanisms. We also observed significant changes in the phosphate I and II bands, which concerns DNA damage, while few changes were detected in the lipid region. Spectroscopic data showed that these changes increased as a function of the dose. Finally, PCA analysis did not discriminate clearly between megavoltage and kilovoltage groups treated with nanoparticles, indicating that megavoltage radiosensitization effects might not differ significantly from those in kilovoltage radiotherapy.

Pathological mechanisms in amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease, are still poorly understood. One subset of familial ALS cases is caused by mutations in the metallo-enzyme copper-zinc superoxide dismutase (SOD1), increasing the susceptibility of the SOD1 protein to form insoluble intracellular aggregates. Here, we employed synchrotron radiation-based Fourier transform infrared spectroscopy and microscopy to investigate brainstem cross-sections from the transgenic hSOD1 G93A rat model of ALS that overexpresses human-mutated SOD1. We compared the biomacromolecular organic composition in brainstem tissue cross-sections of ALS rats and their non-transgenic littermates (NTg). We demonstrate that the proteins and especially their antiparallel β-sheet structure significantly differed in all three regions: the facial nucleus (FN), the gigantocellular reticular nucleus (GRN) and the trigeminal motor nucleus (TMN) in the brainstem tissue of ALS rats. The protein levels varied between different brainstem areas, with the highest concentration observed in the region of the FN in the brainstem tissue of NTg animals. Furthermore, the concentration of lipids and esters was significantly decreased in the TMN and FN of ALS animals. A similar pattern was detected for choline and phosphate assigned to nucleic acids with the highest concentrations in the FN of NTg animals. The spectroscopic analysis showed significant differences in phosphates, amide and lipid structure in the FN of NTg animals in comparison with the same area of ALS rats. These results show that the hG93A SOD1 mutation causes metabolic cellular changes and point to a link between bioorganic composition and hallmarks of protein aggregation.

Amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease, is the most common adult onset neurodegenerative disorder affecting motor neurons. Disruptions in metal ion homeostasis have been described in association with ALS, but the pathological mechanisms are still poorly understood. One of the familial ALS cases is caused by mutations in the metallo-enzyme copper-zinc superoxide dismutase (SOD1). In this study, we employed orthogonal cellular synchrotron radiation based spectro-microscopies to investigate the astrocytes of an ALS animal model: the rat hSOD1 G93A that overexpresses human mutated SOD1, which is known to increase the susceptibility of the SOD1 protein to form insoluble intracellular aggregates. Specifically, we applied soft X-ray transmission tomography and hard X-ray fluorescence microscopy in situ, Fourier transform infrared spectro-microscopy to detect and analyze aggregates, as well as to determine the alterations in the cellular ultrastructure and the elemental and the organic composition of ALS model astrocytes with respect to the control astrocytes isolated from nontransgenic littermates (NTg). The present study demonstrates that large aggregates in the form of multivesicular inclusions form exclusively in the ALS model astrocytes and not in the NTg counterpart. Furthermore, the number of mitochondria, the cellular copper concentration, and the amount of antiparallel β-sheet structures were significantly changed within the cells of the ALS model as well as the lipid localization and composition. Also, our data indicate that choline was decreased in the ALS model astrocytes, which could explain their higher sensitivity to oxidative stress that we observed. These results show that the hG93A SOD1 mutation causes metabolic and ultrastructural cellular changes and point to a link between an increased copper concentration and aggregation: the most probable that the aggregation of G93A hSOD1 may perturb its binding to Cu, thus directly or indirectly affecting Cu homeostasis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting motor and cognitive domains of the CNS. Mutations in the Cu,Zn-superoxide dismutase (SOD1) cause 20% of familial ALS and provoke formation of intracellular aggregates and copper and zinc unbinding, leading to glial activation and neurodegeneration. Therefore, we investigated glial cell morphology, intracellular SOD1 distribution, and elemental composition in the brainstem and hippocampus of the hSOD1 G93A transgenic rat model of ALS. Immunostaining for astrocytes, microglia and SOD1 revealed glial proliferation and progressive tissue accumulation of SOD1 in both brain regions of ALS rats starting already at the presymptomatic stage. Glial cell morphology analysis in the brainstem of ALS rats revealed astrocyte activation occurring before disease symptoms onset, followed by activation of microglia. Hippocampal ALS astrocytes exhibited an identical reactive profile, while microglial morphology was unchanged. Additionally, ALS brainstem astrocytes demonstrated progressive SOD1 accumulation in the cell body and processes, while microglial SOD1 levels were reduced and its distribution limited to distal cell processes. In the hippocampus both glial cell types exhibited SOD1 accumulation in the cell body. X-ray fluorescence imaging revealed decreased P and increased Ca, Cl, K, Ni, Cu and Zn in the brainstem, and higher levels of Cl, Ni and Cu, but lower levels of Zn in the hippocampus of symptomatic ALS rats. These results bring new insights into the glial response during disease development and progression in motor as well as in non-motor CNS structures, and indicate disturbed tissue elemental homeostasis as a prominent hallmark of disease pathology., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

The glioblastoma (GBM) is characterized by a short median survival and an almost 100% tumor related mortality. GBM cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores application of X-ray and visible light microscopy to display the elemental and structural images of cells from 3 patient derived GMB samples and an established GMB cell line. Slight differences in elemental concentrations, in actin cytoskeleton organization and cell morphology were noted between all cells types by X-ray fluorescence and full field soft X-ray microscopy, as well as the Structured Illumination Super-resolution Microscope (SIM). Different sample preparation approaches were used to match each imaging technique. While preparation for SIM included cell fixation and staining, intact frozen hydrated cells were used for the trace element imaging by hard X-ray fluorescence and exploration of the structural features by soft X-ray absorption tomography. Each technique documented differences between samples with regard to morphology and elemental composition and underscored the importance of use of multiple patient derived samples for detailed GBM study.

Background: Closed reduction and percutaneous pinning are the preferred treatment of displaced supracondylar humeral fractures in children. The purpose of this study is to evaluate the non-standard Dorgan's method and compare its results with those of the standard percutaneous cross pinning method in treatment of unstable or irreducible Gartland type II and III supracondylar humeral fractures in children., Patients and Methods: This was a prospective evaluation of 138 consecutive patients with Gartland type II or III extension supracondylar humeral fractures referred to University Children's Hospital during a four-year period. The patients were randomized into two groups: the first group, comprised of 71 patients, was treated with standard pin configuration and the second group, comprised of 67 patients, underwent Dorgan's method. The study included 88 boys and 50 girls aged 1.5-11.4 years (mean 6.5±2). At initial presentation 8.7% (n-12) fractures were classified as Gartland type IIa, 25.4% (n-35) as Gartland type IIb and 65.9% (n-91) as Gartland type III., Results: Flynn's criteria were used to evaluate the results. An excellent clinical outcome was reported in about 90% of patients (n-90) treated with standard pin configuration and 89.5% (n-60) of patients treated with Dorgan's method. There were no statistically significant differences in outcomes between the groups in terms of their gender, age, fracture types, function and cosmetics. Neurological lesions were observed in 9.9% of patients (n=7) who were treated using the standard configuration Kirschner pins, while in those treated by Dorgan's method neurological complications were not observed. However, the procedure time was longer (mean 36.54±5.65min) and radiation exposure significantly higher (mean 10.19±2.70 exposures) in the group that was treated using Dorgan's method, compared to the conventional method (mean 28.66±3.76min and 7.54±1.63 exposures)., Conclusion: Two laterally inserted crossed pins provide adequate stability with good functional and cosmetic outcome for most unstable paediatric supracondylar humeral fractures with no risk of iatrogenic ulnar nerve injury., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Parkinson's disease is the most common α-synucleinopathy, and increased levels of iron are found in the substantia nigra of Parkinson's disease patients, but the potential interlink between both molecular changes has not been fully understood. Metal to protein binding assays have shown that α-synuclein can bind iron in vitro; therefore, we hypothesized that iron content and iron distribution could be modified in cellulo, in cells over-expressing α-synuclein. Owing to particle-induced X-ray emission and synchrotron X-ray fluorescence chemical nano-imaging, we were able to quantify and describe the iron distribution at the subcellular level. We show that, in neurons exposed to excess iron, the mere over-expression of human α-synuclein results in increased levels of intracellular iron and in iron redistribution from the cytoplasm to the perinuclear region within α-synuclein-rich inclusions. Reproducible results were obtained in two distinct recombinant expression systems, in primary rat midbrain neurons and in a rat neuroblastic cell line (PC12), both infected with viral vectors expressing human α-synuclein. Our results link two characteristic molecular features found in Parkinson's disease, the accumulation of α-synuclein and the increased levels of iron in the substantia nigra.

Manganese (Mn) may foster aggregation of alpha-synuclein (αSyn) contributing to the pathogenesis of PD. Here, we examined the influence of αSyn overexpression on distribution and oxidation states of Mn in frozen-hydrated primary midbrain neurons (PMNs) by synchrotron-based X-ray fluorescence (XRF) and X-ray absorption near edge structure spectroscopy (XANES). Overexpression of αSyn increased intracellular Mn levels, whereas levels of Ca, Zn, K, P, and S were significantly decreased. Mn oxidation states were not altered. A strong correlation between Cu-/Mn-levels as well as Fe-/Mn-levels was observed in αSyn-overexpressing cells. Subcellular resolution revealed a punctate or filament-like perinuclear and neuritic distribution of Mn, which resembled the expression of DMT1 and MnSOD. While overexpression of αSyn did not significantly alter the expression patterns of the most-expressed Mn transport proteins (DMT1, VGCC, Fpn1), it attenuated the Mn release from Mn-treated neurons. Thus, these data suggest that αSyn may act as an intracellular Mn store. In total, neurotoxicity in PD could be mediated via regulation of transition metal levels and the metal-binding capacity of αSyn, which could represent a promising therapeutic target for this neurodegenerative disorder.

Vanadium speciation in the fungus Phycomyces blakesleeanus was examined by X-ray absorption near-edge structure (XANES) spectroscopy, enabling assessment of oxidation states and related molecular symmetries of this transition element in the fungus. The exposure of P. blakesleeanus to two physiologically important vanadium species (V(5+) and V(4+)) resulted in the accumulation of this metal in central compartments of 24 h old mycelia, most probably in vacuoles. Tetrahedral V(5+), octahedral V(4+), and proposed intracellular complexes of V(5+) were detected simultaneously after addition of a physiologically relevant concentration of V(5+) to the mycelium. A substantial fraction of the externally added V(4+) remained mostly in its original form. However, observable variations in the pre-edge-peak intensities in the XANES spectra indicated intracellular complexation and corresponding changes in the molecular coordination symmetry. Vanadate complexation was confirmed by (51)V NMR and Raman spectroscopy, and potential binding compounds including cell-wall constituents (chitosan and/or chitin), (poly)phosphates, DNA, and proteins are proposed. The evidenced vanadate complexation and reduction could also explain the resistance of P. blakesleeanus to high extracellular concentrations of vanadium.

Background: The aim of this study was to assess whether high-density lipoprotein (HDL) labeled with superparamagnetic iron oxide nanoparticles (SPIOs) and quantum dots was able to detect atherosclerotic lesions in mice after intravenous and intraperitoneal injection by multimodal imaging., Methods and Results: Nanoparticle-labeled HDLs (NP-HDLs) were characterized in vitro by dynamic light scattering and size exclusion chromatography with subsequent cholesterol and fluorescence measurements. For biodistribution and blood clearance studies, NP-HDL(SPIOs) radiolabeled with (59)Fe (NP-HDL(59Fe-SPIOs)) were injected intravenously or intraperitoneally into ApoE knockout mice (n=6), and radioactivity was measured using a gamma counter. NP-HDL accumulation within atherosclerotic plaques in vivo and ex vivo was estimated by MRI at 7 Tesla, ex vivo confocal fluorescence microscopy, x-ray fluorescence microscopy, and histological analysis (n=3). Statistical analyses were performed using a 2-tailed Student t-test. In vitro characterization of NP-HDL confirmed properties similar to endogenous HDL. Blood concentration time curves showed a biexponential decrease for the intravenous injection, whereas a slow increase followed by a steady state was noted for intraperitoneal injection. Radioactivity measurements showed predominant accumulation in the liver and spleen after both application approaches. NP-HDL(59Fe-SPIOs) uptake into atherosclerotic plaques increased significantly after intraperitoneal compared with intravenous injection (P<0.01). In vivo MRI showed an increased uptake of NP-HDL into atherosclerotic lesions after intraperitoneal injection, which was confirmed by ex vivo MRI, x-ray fluorescence microscopy, confocal fluorescence microscopy, and histological analysis., Conclusions: In vivo MRI and ex vivo multimodal imaging of atherosclerotic plaque using NP-HDL is feasible, and intraperitoneal application improves the uptake within vessel wall lesions compared with intravenous injection.

The aim of this study was to investigate the feasibility of noninvasive monitoring of plaque burden in apolipoprotein E-deficient (ApoE-/-) mice by Gadospin F (GDF)-enhanced magnetic resonance imaging (MRI). Gadolinium uptake in plaques was controlled using transmission electron microscopy (TEM) and x-ray fluorescence (XRF) microscopy. To monitor the progression of atherosclerosis, ApoE-/- (n  =  5) and wild-type (n  =  2) mice were fed a Western diet and imaged at 5, 10, 15, and 20 weeks. Contrast-enhanced MRI was performed at 7 T Clinscan (Bruker, Ettlingen, Germany) before and 2 hours after intravenous injection of GDF (100 μmol/kg) to determine the blood clearance. Plaque size and contrast to noise ratio (CNR) were calculated for each time point using region of interest measurements to evaluate plaque progression. Following MRI, aortas were excised and GDF uptake was cross-validated by TEM and XRF microscopy. The best signal enhancement in aortic plaque was achieved 2 hours after application of GDF. No signal differences between pre- and postcontrast MRI were detectable in wild-type mice. We observed a gradual and considerable increase in plaque CNR and size for the different disease stages. TEM and XRF microscopy confirmed the localization of GDF within the plaque. GDF-enhanced MRI allows noninvasive and reliable estimation of plaque burden and monitoring of atherosclerotic progression in vivo.

Synchrotron-based X-ray microfluorescence (µSXRF) is an analytical method suitable for in situ investigation of the distribution of micronutrient and macronutrient elements in several-micrometres-thick unstained biological samples, e.g. single cells and tissues. Elements are mapped and quantified at sub-p.p.m. concentrations. In this study the quantity, distribution and grouping/co-localization of various elements have been identified in straight and twisted internodes of the stems of the monocotyledonous climber D. balcanica Košanin. Three different statistical methods were employed to analyse the macronutrient and micronutrient distributions and co-localization. Macronutrient elements (K, P, Ca, Cl) are distributed homogeneously in both straight and twisted internodes. Micronutrient elements are mostly grouped in the vasculature and in the sclerenchyma cell layer. In addition, co-localization of micronutrient elements is much more prominent in twisted than in straight internodes. These image analyses and statistical methods provided very similar outcomes and could be applied to various types of biological samples imaged by µSXRF.

Transition metals have been suggested to play a pivotal role in the pathogenesis of Parkinson's disease. X-ray microscopy combined with a cryogenic setup is a powerful method for elemental imaging in low concentrations and high resolution in intact cells, eliminating the need for fixation and sectioning of the specimen. Here, we performed an elemental distribution analysis in cultured primary midbrain neurons with a step size in the order of 300 nm and ~ 0.1 ppm sensitivity under cryo conditions by using X-ray fluorescence microscopy. We report the elemental mappings on the subcellular level in primary mouse dopaminergic (DAergic) and non-DAergic neurons after treatment with transition metals. Application of Fe(2+) resulted in largely extracellular accumulation of iron without preference for the neuronal transmitter subtype. A quantification of different Fe oxidation states was performed using X-ray absorption near edge structure analysis. After treatment with Mn(2+) , a cytoplasmic/paranuclear localization of Mn was observed preferentially in DAergic neurons, while no prominent signal was detectable after Mn(3+) treatment. Immunocytochemical analysis correlated the preferential Mn uptake to increased expression of voltage-gated calcium channels in DAergic neurons. We discuss the implications of this differential elemental distribution for the selective vulnerability of DAergic neurons and Parkinson's disease pathogenesis., (© 2012 International Society for Neurochemistry.)

Acidification of forest ecosystems leads to increased plant availability of the micronutrient manganese (Mn), which is toxic when taken up in excess. To investigate whether ectomycorrhizas protect against excessive Mn by improving plant growth and nutrition or by retention of excess Mn in the hyphal mantle, seedlings of two populations of Douglas fir (Pseudotsuga menziesii), two varieties, one being menziesii (DFM) and the other being glauca (DFG), were inoculated with the ectomycorrhizal fungus Rhizopogon subareolatus in sand cultures. Five months after inoculation, half of the inoculated and non-inoculated seedlings were exposed to excess Mn in the nutrient solution for further 5 months. At the end of this period, plant productivity, nutrient concentrations, Mn uptake and subcellular compartmentalisation were evaluated. Non-inoculated, non-stressed DFM plants produced about 2.5 times more biomass than similarly treated DFG. Excess Mn in the nutrient solution led to high accumulation of Mn in needles and roots but only to marginal loss in biomass. Colonisation with R. subareolatus slightly suppressed DFM growth but strongly reduced that of DFG (-50%) despite positive effects of mycorrhizas on plant phosphorus nutrition. Growth reductions of inoculated Douglas fir seedlings were unexpected since the degree of mycorrhization was not high, i.e. ca. 30% in DFM and 8% in DFG. Accumulation of high Mn was not prevented in inoculated seedlings. The hyphal mantle of mycorrhizal root tips accumulated divalent cations such as Ca, but not Mn, thus not providing a barrier against excessive Mn uptake into the plants associated with R. subareolatus.

Manganese (Mn) is an essential micronutrient in all organisms but may become toxic when present in excess. To investigate whether interior and coastal varieties of Douglas fir [Pseudotsuga menziesii (Mirbel) Franco, var. glauca (DFG) and var. viridis (DFV)] differed in Mn tolerance, seedlings were exposed to excess Mn in hydroponic solutions. Root growth, biomass production, Mn concentrations in different tissues and Mn subcellular localisation were determined. Both varieties showed similar whole-plant Mn accumulation and biomass reduction in response to increases in Mn in the nutrient solution. Since excess Mn inhibited root elongation growth more strongly in DFG than in DFV, biomass allocation in DFV was shifted towards a relative increase in root biomass and a relative decrease in DFG. X-ray microanalysis showed that Mn enrichment in cell walls and vacuoles of DFV root cortex and epidermis cells was higher than in DFG. In roots, precipitates were observed in which Mn concentrations correlated with phosphorus (P) and to a minor extent with calcium. We suggest that the higher persistence of root growth under Mn stress in DFV was caused by greater entrapment in the vacuole and in the apoplast and by more efficient Mn detoxification in insoluble complexes with P than in DFG. To investigate whether P supply affected Mn uptake and toxicity, Douglas fir seedlings were exposed to excess Mn under P deficiency. In P-limited seedlings of both varieties, roots growth was less sensitive to excess Mn than under sufficient P-supply, although in planta Mn concentrations were not diminished. In DFG, which maintained P homeostasis under limited P supply, the negative effect of Mn stress was partially reversed, showing that Mn susceptibility is affected by P metabolism.