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Environmentalcontaminationduetohumanactivitiesisaworldwideproblemthathas ledtothedevelopmentofdifferentremediationtechniques,includingbiotechnologicalapproachessuchasphytoextractionandphytostabilization.Thesetechniquestakeadvantageofpioneerplants that naturally develop tolerance mechanisms to survive in extreme environments. A multi-techniqueandmulti-disciplinaryapproachwasappliedfortheinvestigationofHelichrysum microphyllum subsp. tyrrhenicum samples, bulk soil, and rhizospheres collected from a metal-extremeenvironment(Zn-PbmineofCampoPisano,SWSardinia,Italy).Zinc,Pb,andCd are the most abundant metals, with Zn attaining 3 w/w% in the rhizosphere solid materials, inducing oxidative stress in the roots as revealed by infrared microspectroscopy (IR). X-ray diffraction (XRD), scanning electron microscopy (SEM), and chemical analysis coupled with synchrotron radiation-based (SR) techniques demonstrate that quartz, dolomite, and weddellite biomineralsprecipitateinroots,stems,andleaves,likelyasaresponsetoenvironmentalstress.In therhizosphere,ZnchemicalspeciationismainlyrelatedtotheZnoreminerals(smithsoniteandhydrozincite) whereas, in plant tissues, Zn is primarily bound to organic compounds such as malate,cysteine,andhistidinemoleculesthatactasmetalbindersand,eventually,detoxification agents for the Zn excess. These findings suggest that H. microphyllum subsp. tyrrhenicum has developed its own adaptation strategy to survive in polluted substrates, making it a potential candidateforphytostabilizationaimedatmitigatingthedispersionofmetalsinthesurrounding areas.

To understand the fate of plastic in oceans and the interaction with marine organisms, we investigated the incorporation of (bio)polymers and microplastics in selected benthic foraminiferal species by applying FTIR (Fourier Transform Infrared) microscopy. This experimental methodology has been applied to cultured benthic foraminifera Rosalina globularis, and to in situ foraminifera collected in a plastic remain found buried into superficial sediment in the Mediterranean seafloor, Rosalina bradyi, Textularia bocki and Cibicidoides lobatulus. In vitro foraminifera were treated with bis-(2-ethylhexyl) phthalate (DEHP) molecule to explore its internalization in the cytoplasm. Benthic foraminifera are marine microbial eukaryotes, sediment-dwelling, commonly short-lived and with reproductive cycles which play a central role in global biogeochemical cycles of inorganic and organic compounds. Despite the recent advances and investigations into the occurrence, distribution, and abundance of plastics, including microplastics, in marine environments, there remain relevant knowledge gaps, particularly on their effects on the benthic protists. No study, to our knowledge, has documented the molecular scale effect of plastics on foraminifera. Our analyses revealed three possible ways through which plastic-related molecules and plastic debris can enter a biogeochemical cycle and may affect the ecosystems: 1) foraminifera in situ can grow on plastic remains, namely C. lobatulus, R. bradyi and T. bocki, showing signals of oxidative stress and protein aggregation in comparison with R. globularis cultured in negative control; 2) DEHP can be incorporated in the cytoplasm of calcareous foraminifera, as observed in R. globularis; 3) microplastic debris, identified as epoxy resin, can be found in the cytoplasm and the agglutinated shell of T. bocki. We hypothesize that plastic waste and their associated additives may produce modifications related to the biomineralization process in foraminifera. This effect would be added to those induced by ocean acidification with negative consequences on the foraminiferal biogenic carbon (C) storage capacity.

Extracellular vesicles (EVs) are small vesicles ensuring transport of molecules between cells and throughout the body. Their small size and biological and physical functions make them optimal candidates as therapeutic agents in several medical frameworks. The elucidation of biophysical and biochemical characteristics of EVs is crucial for the understanding of their interaction with recipient cells and their functional activity. In particular, for therapeutic applications understanding how the process of manufacturing affects the biological function of EVs is mandatory before going to the clinical testing. The absence of standardized methodologies and technologies to establish reliable criteria has been the main hurdle for real therapeutic applications of EVs. Here we exploited a combination of different biophysical techniques (Fourier Transform Infrared Spectroscopy, Ultraviolet Resonant Raman Spectroscopy, Atomic Force Microscopy and Small Angle X-Ray Scattering) to screen EVs derived from umbilical cord-mesenchymal stem cells with different isolation procedures, evidencing subtle differences in the biophysical properties of EVs. The results underline the need of a multiparametric analysis to address size, stability and purity of different preparations and to correlate phenotypic parameters of EVs with their functional activity.

Cytosine plays a preeminent role in DNA methylation, an epigenetic mechanism that regulates gene expression, the misregulation of which can lead to severe diseases. Several methods are nowadays employed for assessing the global DNA methylation levels, but none of them combines simplicity, high sensitivity, and low operating costs to be translated into clinical applications. Ultraviolet (UV) resonant Raman measurements at excitation wavelengths of 272 nm, 260 nm, 250 nm, and 228 nm have been carried out on isolated deoxynucleoside triphosphates (dNTPs), on a dNTP mixture as well as on genomic DNA (gDNA) samples, commercial from salmon sperm and non-commercial from B16 murine melanoma cell line. The 228 nm excitation wavelength was identified as the most suitable energy for enhancing cytosine signals over the other DNA bases. The UV Raman measurements performed at this excitation wavelength on hyper-methylated and hypo-methylated DNA from Jurkat leukemic T-cell line have revealed significant spectral differences with respect to gDNA isolated from salmon sperm and mouse melanoma B16 cells. This demonstrates how the proper choice of the excitation wavelength, combined with optimized extraction protocols, makes UV Raman spectroscopy a suitable technique for highlighting the chemical modifications undergone by cytosine nucleotides in gDNA upon hyper- and hypo-methylation events.

Biofilms are communities of bacteria living embedded in a highly hydrated matrix composed of polysaccharides, proteins, and extracellular DNA. This life style confers numerous advantages to bacteria including protection against external threats. However, they also contribute to increase bacterial resistance against antimicrobials, an issue particularly relevant in dangerous infections. Due to the complexity of the matrix, few information is present in the literature on details of its architecture including the spatial distribution of the macromolecular components which might give hints on the way the biofilm scaffold is built up by bacteria. In this study, we investigated the possibility to combine well-established microbiological procedures with advanced microscopies to get information on composition and distribution of the macromolecular components of biofilm matrices. To this, confocal microscopy, diffraction-limited infrared (IR) spectral imaging, and atomic force microscopy (AFM) were used to explore biofilm produced by a clinical strain of Klebsiella pneumoniae. IR imaging permitted to have clues on how the biofilm grows and spreads on surfaces, and the local distribution of the components within it. Through the analysis of the pure component spectra, it was possible to assess the chemical and structural composition of the saccaridic matrix, confirming the data obtained by NMR. It was also possible to follow the time course of biofilm from 6 up to 48 h when the biofilm grew into a 3-dimensional multi-layered structure, characteristic of colonies of bacteria linked together by a complex matrix. In addition, nanoFTIR and AFM investigations allowed the estimation of biofilm growth in the vertical direction and the morphological analysis of bacterial colonies at different time points and the evaluation of the chemical composition at the nanoscale.

Environmental and occupational inhalants may induce a large number of pulmonary diseases, with asbestos exposure being the most risky. The mechanisms are clearly related to chemical composition and physical and surface properties of materials. A combination of X-ray fluorescence (μXRF) and Fourier Transform InfraRed (μFTIR) microscopy was used to chemically characterize and compare asbestos bodies versus environmental particulates (anthracosis) in lung tissues from asbestos exposed and control patients. μXRF analyses revealed heterogeneously aggregated particles in the anthracotic structures, containing mainly Si, K, Al and Fe. Both asbestos and particulates alter lung iron homeostasis, with a more marked effect in asbestos exposure. μFTIR analyses revealed abundant proteins on asbestos bodies but not on anthracotic particles. Most importantly, the analyses demonstrated that the asbestos coating proteins contain high levels of β-sheet structures. The occurrence of conformational changes in the proteic component of the asbestos coating provides new insights into long-term asbestos effects.

Infrared Microspectroscopy (IRMS) has been proposed as a powerful diagnostic tool in biology, due to the rich molecular, structural and conformational information contained in IR spectra of cells and tissues. In particular, IRMS of live cells in microfluidic devices has to cope with the strong water absorption in the medium infrared spectral region and the scarce knowledge about fabrication protocols suitable for micro-structuring infrared-transparent materials. Based on these motivations we are developing and testing a class of microfluidic devices consisting of a patterned photoresist sandwiched between two CaF 2 optical windows. In this paper we propose solutions to a few specific issues, namely, (i) the poor resist adhesion during micro-fabrication processes due to the low surface energy of CaF 2, (ii) the potentially harmful effects of CaF 2 dissolution on interesting cellular lines (such as neurons or stem cells), (iii) the sealing of the devices. Specifically, we modified the surface properties of CaF 2 substrates by sputtering a thin layer of Si, as to obtain the following advantages: (a) all lithographic steps can be performed as if they were carried out on silicon wafers; (b) the chemical functionalization and nanostructuring of the surface in contact with cells can be obtained by usual protocols used for Si; (c) the deposited silicon separates living cells and their environment from CaF 2. A device sealing process is discussed, based on a polymer bonding protocol, in order to tune the content of residual solvent. Finally, we present IR hyperspectral images acquired on MCF-7 living cells, cultured inside our devices for 48 h. © 2012 Elsevier B.V. All rights reserved.

First experiments demonstrating the suitability of novel microfabricated fluidic devices for measuring living cells in physiological environment by synchrotron radiation (SR) Fourier Transform Infrared microspectroscopy (μ-FTIR) are presented. The devices were fabricated on CaF 2 windows, using the photoresist XARP 3100/10 to define the liquid cell lay-out. Therefore, the sample holder is transparent to both visible and infrared light, robust, completely recyclable and with a precise spacing. Using prototype devices of thicknesses 9, 5 and 3 μm, we studied the response of the U937 monocytic cell line to mechanical compression. The temporal evolution of the FTIR spectra, characteristic for the status of living cells, was used to monitor the cellular system stability in time. Protein biosynthesis and lipid metabolism alterations upon deformation have been identified by monitoring specific cell band ratios such as amide I to amide II, amide I to lipids, methylene to methyl and asymmetric to symmetric stretching of phosphates. Taking advantage of the high brilliance of the synchrotron radiation, chemical maps of monocyte cells were collected, demonstrating the versatility of the device.

This work aims to assess the feasibility of studying the distribution of drugs and diagnostic agents in biological specimens by means of XRF detected with high-resolution and high sensitivity Silicon Drift Detectors. In the present study we have employed as diagnostic agent a gadolinium complex (Gadocoletic acid trisodium salt - B22956/1). The primary excitation beam was the light of the SYRMEP beamline of the Elettra synchrotron light source. For calibration purposes we analyzed solutions of the Gd complex in water at different dilutions. The minimum level of Gd detected is 5 mug/ml. We measured the Gd complex distribution in different organs (liver, kidney, spleen, lung) and in blood of mice at different times after a single injection of B22956 (250 mg/Kg body weight) and the Gd complex uptake in human liver-derived cells. This technique will allow the measure of pharmacokinetics and the study of the homogeneity of drug distribution inside organs providing a new tool in the development, follow-up and tracking of drug delivery in vitro.

The three-dimensional structures of the native cytochrome c(2) from Rhodopseudomonas palustris and of its ammonia complex have been obtained at pH 4.4 and pH 8.5, respectively. The structure of the native form has been refined in the oxidized state at 1.70 A and in the reduced state at 1.95 A resolution. These are the first high-resolution crystal structures in both oxidation states of a cytochrome c(2) with relatively high redox potential (+350 mV). The differences between the two oxidation states of the native form, including the position of internal water molecules, are small. The unusual six-residue insertion Gly82-Ala87, which precedes the heme binding Met93, forms an isolated 3(10)-helix secondary structural element not previously observed in other c-type cytochromes. Furthermore, this cytochrome shows an external methionine residue involved in a strained folding near the exposed edge of the heme. The structural comparison of the present cytochrome c(2) with other c-type cytochromes has revealed that the presence of such a residue, with torsion angles phi and psi of approximately -140 and -130 degrees, respectively, is a typical feature of this family of proteins. The refined crystal structure of the ammonia complex, obtained at 1.15 A resolution, shows that the sulphur atom of the Met93 axial ligand does not coordinate the heme iron atom, but is replaced by an exogenous ammonia molecule. This is the only example so far reported of an X-ray structure with the heme iron coordinated by an ammonia molecule. The detachment of Met93 is accompanied by a very localized change in backbone conformation, involving mainly the residues Lys92, Met93, and Thr94. Previous studies under typical denaturing conditions, including high-pH values and the presence of exogenous ligands, have shown that the detachment of the Met axial ligand is a basic step in the folding/unfolding process of c-type cytochromes. The ammonia adduct represents a structural model for this important step of the unfolding pathway. Factors proposed to be important for the methionine dissociation are the strength of the H-bond between the Met93 and Tyr66 residues that stabilizes the native form, and the presence in this bacterial cytochrome c(2) of the rare six-residue insertion in the helix 3(10) conformation that increases Met loop flexibility.