Synergistic Enhancement of Targeted Wound Healing by Near-Infrared Photodynamic Therapy and Silver Metal-Organic Frameworks Combined with S- or N-Doped Carbon Dots.

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-ß. 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.

Multifunctionalized Carbon Dots as an Active Nanocarrier for Drug Delivery to the Glioblastoma Cell Line.

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.

Virtual histology of Alzheimer's disease: Biometal entrapment within amyloid-ß plaques allows for detection via X-ray phase-contrast imaging.

Amyloid-ß (Aß) 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 Aß plaques. The current study was designed to test the hypothesis that Aß-related contrast in XPCT could be caused by Aß fibrils and/or by metals trapped in the plaques. Fibrillar and elemental compositions of Aß plaques were probed in brain samples from different types of AD patients and AD models to establish a relationship between XPCT contrast and Aß 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, ArcAß, J20; rat: TgF344). Aß plaques from the genetic AD patient were visible using XPCT, and had higher ß-sheet content and higher metal levels than those from the sporadic AD patient, which remained undetected by XPCT. Aß 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 ArcAß mice. In all four transgenic strains, ß-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 ArcAß mice. Hence, a hyperdense contrast formation of Aß 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-ß plaques (Aß), 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 Aß plaques are detected with a new technique called X-ray phase contrast tomography (XPCT). XPCT enables to map the distribution of Aß 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.

Synchrotron-based FTIR microspectroscopy reveals DNA methylation profile in DNA-HALO structure.

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.

Synchrotron-based FTIR evaluation of biochemical changes in cancer and noncancer cells induced by brominated marine coelenteramine.

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.

Synchrotron-Based Fourier-Transform Infrared Micro-Spectroscopy of Cerebrospinal Fluid from Amyotrophic Lateral Sclerosis Patients Reveals a Unique Biomolecular Profile.

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.

Monitoring oocyte-based human pluripotency acquisition using synchrotron-based FTIR microspectroscopy reveals specific biomolecular trajectories.

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.

Biomacromolecular Profile in Human Primary Retinal Pigment Epithelial Cells-A Study of Oxidative Stress and Autophagy by Synchrotron-Based FTIR Microspectroscopy.

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 (H2O2) 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.

A three-step process of manganese acquisition and storage in the microalga Chlorella sorokiniana.

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.

Hydroxyapatite nanoparticles-cell interaction: New approaches to disclose the fate of membrane-bound and internalised nanoparticles.

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.

S, N-doped carbon dots-based cisplatin delivery system in adenocarcinoma cells: Spectroscopical and computational approach.

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.

Biochemical changes in cancer cells induced by photoactive nanosystem based on carbon dots loaded with Ru-complex.

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.

Lipid Status of A2780 Ovarian Cancer Cells after Treatment with Ruthenium Complex Modified with Carbon Dot Nanocarriers: A Multimodal SR-FTIR Spectroscopy and MALDI TOF Mass Spectrometry Study.

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(η5-Cp)(PPh3)2CN] (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.

Live-Cell Synchrotron-Based FTIR Evaluation of Metabolic Compounds in Brain Glioblastoma Cell Lines after Riluzole Treatment.

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 ß-sheet conformation while parallel ß-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.

Synchrotron Radiation-Fourier Transformed Infrared microspectroscopy (µSR-FTIR) reveals multiple metabolism alterations in microalgae induced by cadmium and mercury.

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.

UV Effect on Human Anterior Lens Capsule Macro-Molecular Composition Studied by Synchrotron-Based FTIR Micro-Spectroscopy.

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 ß-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 CH3 band.

Estimation of carbon dots amelioration of copper toxicity in maize studied by synchrotron radiation-FTIR.

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 Cu2+. 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.

Mechanisms of detoxification of high copper concentrations by the microalga Chlorella sorokiniana.

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.

Synchrotron-based FTIR microspectroscopy of protein aggregation and lipids peroxidation changes in human cataractous lens epithelial cells.

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.

Lipids status and copper in a single astrocyte of the rat model for amyotrophic lateral sclerosis: Correlative synchrotron-based X-ray and infrared imaging.

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.