Raman spectroscopic and X-ray diffraction studies of the effect of temperature and Ca2+ on phosphatidylethanolamine dispersions

Raman spectroscopy and X-ray diffraction are used to study the effect of heat and Ca²⁺ on dimyristoylphosphatidylethanolamine dispersions. Unlike phosphatidylcholine dispersions, dimyristoylphosphatidylethanolamine bilayers (at pH 8) require heating above T_m in order for hydration to occur and apparently bind Ca²⁺ at very low levels. These results are related to models for membrane fusion.     

Green synthesis and characterization of Carica papaya leaf extract coated silver nanoparticles through X-ray diffraction,electron microscopy and evaluation of bactericidal properties

The evolution of nanotechnology and the production of nanomedicine from various sources had proven to be of intense value in the field of biomedicine. The smaller size of nanoparticles is gaining importance in research for the treatment of various diseases. Moreover the production of nanoparticles is eco-friendly and cost effective. In the present study silver nanoparticles were synthesized from Carica papaya leaf extract (CPL) and characterized for their size and shape using scanning electron microscopy and transmission electron microscopy, respectively. Fourier transform infrared spectroscopy (FTIR), Energy dispersive X-ray spectroscopy (EDS/EDX) and X-ray diffraction spectroscopy (XRD) were conducted to determine the concentration of metal ions, the shape of molecules. The bactericidal activity was evaluated using Luria Bertani broth cultures and the minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) were estimated using turbidimetry. The data analysis showed size of 50–250 nm spherical shaped nanoparticles. The turbidimetry analysis showed MIC and MBC was >25 μg/mL against both Gram positive and Gram negative bacteria in Luria Bertani broth cultures. In summary the synthesized silver nanoparticles from CPL showed acceptable size and shape of nanoparticles and effective bactericidal activity.     

Calcite-forming Bacillus licheniformis Thriving on Underwater Speleothems of a Hydrothermal Cave

The underwater environment of Grotta Giusti (Monsummano Terme, Italy) is a suggestive setting with different types of speleothems including “leafy” and “cauliflower” concretions along the walls and roof, and conical pseudo-stalagmites on the floor. Very high calcium and dissolved CO₂ levels, and massive calcium carbonate precipitation characterize this cave environment. Yet, life thrives on the leafy concretion surfaces with loads of cultivable heterotrophic microorganisms around 10⁵ colony-forming units per cm². Bacillus licheniformis appeared to be the prevalent cultivable microorganism on a low-nutrient medium that was used for screening. 16S rRNA gene-based polymerase chain reaction–single strand conformation polymorphism profiling indicated that Group VI Bacillaceae species was well represented in the bacterial community of underwater speleothems. Interpretation of X-ray diffraction spectra and Raman spectroscopy data indicated that the B. licheniformis isolate produced in vitro abundant calcite microcrystals that were also characterized by scanning electron microscopy coupled with energy dispersive X-ray spectroscopy. Production of calcite microcrystals was analyzed in different media (Christensen’s urea agar and B4 calcium carbonate precipitation medium) and incubation conditions, and it was found to be enhanced by nitrate supplement in B4 medium under low-oxygen conditions. B4 and B4-nitrate media also stimulated antibiotic production by the B. licheniformis isolate, which was analyzed by microbiological assays.     

Synthesis and characterization of lanthanide orthothiophosphates: LaPS4

The single crystal structure of LaPS₄, (1), is reported. The space group is tetragonal, I4(1)/acd. Unit cell dimensions are a = 10.9641(3) Å and c = 19.4828(9) Å. The far infrared absorption and Raman spectra (100-600 cm⁻¹) are consistent with the groups being in a distorted tetrahedral geometry. The room temperature emission spectrum of LaPS₄ doped with Er³⁺ is also presented. Emission peaks at 529, 534, 549, and 554 nm were observed when the sample was excited at 492 nm. The compound reported here is isomorphous and isostructural to several other lanthanide orthothiophosphates.     

Iron–sulfur repair YtfE protein from <Emphasis Type="Italic">Escherichia coli</Emphasis>: structural characterization of the di-iron center

YtfE was recently shown to be a newly discovered protein required for the recovery of the activity of iron-sulfur-containing enzymes damaged by oxidative and nitrosative stress conditions. The Escherichia coli YtfE purified protein is a dimer with two iron atoms per monomer and the type and properties of the iron center were investigated by using a combination of resonance Raman and extended X-ray absorption fine structure spectroscopies. The results demonstrate that YtfE contains a non-heme dinuclear iron center having mu-oxo and mu-carboxylate bridging ligands and six histidine residues coordinating the iron ions. This is the first example of a protein from this important class of di-iron proteins to be shown to be involved in the repair of iron-sulfur centers.     

An intracellular silver deposition method for targeted detection and chemical analysis of uncultured microorganisms

The vast majority of environmental bacteria remain uncultured, despite two centuries of effort in cultivating microorganisms. Our knowledge of their physiology and metabolic activity depends to a large extent on methods capable of analyzing single cells. Bacterial identification is a key step required by all currently used single-cell imaging techniques and is typically performed by means of fluorescent labeling. However, fluorescent cells cannot be visualized by ion- and electron microscopy and thus only correlative, indirect, cell identification is possible. Here we present a new method of bacterial identification by in situ hybridization coupled to the deposition of elemental silver nanoparticles (silver-DISH). We show that hybridized cells containing silver can be directly visualized by light microscopy, scanning electron microscopy, energy dispersive X-ray spectroscopy, secondary ion mass spectrometry (nanoSIMS), and confocal Raman micro-spectroscopy. Silver-DISH did not alter the isotopic (¹³C) and elemental composition of stable-isotope probed cells more than other available hybridization methods, making silver-DISH suitable for broad applications in stable-isotope labeling studies. Additionally, we demonstrate that silver-DISH can induce a surface-enhanced Raman scattering (SERS) effect, amplifying the Raman signal of biomolecules inside bacterial cells. This makes silver-DISH the only currently available method that is capable of delivering a SERS-active substrate inside specifically targeted microbial cells.     

Mild acid hydrolysis of fucoidan: characterization by electrophoresis and FT-Raman spectroscopy

Along with proteins, lipids, water and minerals, polysaccharides are the main chemical compounds of which macroalgae are built. Among the chemical compounds now widely examined is fucoidan (fucan, fucosan, sulfate fucan or sulfated fucan), a fucose-containing sulfated polysaccharide. Fucoidans isolated from different species have been extensively studied because of their varied biological properties, including anticoagulant and antitumor effects. Methodology based on mild acid hydrolysis can be used as an efficient tool to study the relationship between molecular weight of the sulfated polysaccharides and their biological activities. Anticancer activity of fucoidans can be significantly enhanced by lowering their molecular weight only when they are depolymerized under mild conditions. In this study, fucoidan was identified during extraction with H₂SO₄ and HCl; its presence was confirmed by FT-Raman spectroscopy in aqueous solution. In particular, shifts at 840 cm⁻¹ were analysed, which are due to the presence of sulfate at the axial C-4 position, as were the shifts at about 811–809 cm⁻¹, for which the sulfated fucoidan is responsible. Shifts of electrophoretic bands of fucoidan resulting from mild acid hydrolysis in H₂SO₄ and HCl were also analysed. The analytical procedure was developed using apparatus for cellulose acetate membrane electrophoresis and this was supplemented by semi-quantitative analysis.     

Bladder tissue characterization using probe‐based Raman spectroscopy: Evaluation of tissue heterogeneity and influence on the model prediction

Existing approaches for early‐stage bladder tumor diagnosis largely depend on invasive and time‐consuming procedures, resulting in hospitalization, bleeding, bladder perforation, infection and other health risks for the patient. The reduction of current risk factors, while maintaining or even improving the diagnostic precision, is an underlying factor in clinical instrumentation research. For example, for clinic surveillance of patients with a history of noninvasive bladder tumors real‐time tumor diagnosis can enable immediate laser‐based removal of tumors using flexible cystoscopes in the outpatient clinic. Therefore, novel diagnostic modalities are required that can provide real‐time in vivo tumor diagnosis. Raman spectroscopy provides biochemical information of tissue samples ex vivo and in vivo and without the need for complicated sample preparation and staining procedures. For the past decade there has been a rise in applications to diagnose and characterize early cancer in different organs, such as in head and neck, colon and stomach, but also different pathologies, for example, inflammation and atherosclerotic plaques. Bladder pathology has also been studied but only with little attention to aspects that can influence the diagnosis, such as tissue heterogeneity, data preprocessing and model development. The present study presents a clinical investigative study on bladder biopsies to characterize the tumor grading ex vivo, using a compact fiber probe‐based imaging Raman system, as a crucial step towards in vivo Raman endoscopy. Furthermore, this study presents an evaluation of the tissue heterogeneity of highly fluorescent bladder tissues, and the multivariate statistical analysis for discrimination between nontumor tissue, and low‐ and high‐grade tumor.     

Structural characterization of the 5' untranslated RNA of hepatitis C virus by vibrational spectroscopy

Raman and FTIR spectroscopy have been used to characterize the structure of 5'untranslated region (5'UTR, 342-mer RNA) of the HCV genome. The study of the 750-850 cm(-1) Raman spectral domain of the ribose-phosphate backbone reveals that the percentage of nucleobases involved in double helix-loop junctions is 19+/-1%, which is very close to that of a theoretical secondary structure model (18.7%) proposed on the basis of comparative sequence analysis and thermodynamic modelling. In addition, about 68+/-2% of the bases are helically ordered having C(3')-endo ribofuranose pucker. FTIR-monitored H/D exchange provides the following results: (a) base-paired guanine and cytosine nucleobases show the lowest rate of isotopic exchange, and some synchronous intensity changes of marker bands of A.U pair and single stranded adenine are consistent with the presence of A(*)A.U triplets; (b) the vibrational coupling between the ribose ether C-O stretching and 2'OH bending motions reveals that helical regions of 5'UTR RNA are characterized by hydrogen bonding between the 2'OH ribose groups and the ether oxygen atoms of neighbouring ribose residues.     

Raman confocal microscopy and biophysics multiparametric characterization of the skin barrier evolution with age

Skin aging is a multifactorial phenomenon that involves alterations at the molecular, cellular and tissue levels. Our aim was to carry out a multiparametric biophysical and Raman characterization of skin barrier between individuals of different age groups (<24 and >70 years old). Our results showed a significant decrease of lipids to proteins ratio overall the thickness of the stratum corneum and higher lateral packing in the outer part of the SC for elderly. This can explain the decrease in trans epidermal water loss measured values rather than only SC thickening. Both age groups showed similar water content at SC surface while elderly presented higher water content in deep SC and viable epidermis. Mechanical measurements showed a decrease in the elasticity and an increase in the fatigability with age and were correlated with partially bound water. Highest correlation and anti-correlation values were observed for the deepest part of the SC and the viable epidermis.     

Hydration properties of N-(alpha-hydroxyacyl)-sphingosine: X-ray powder diffraction and FT-Raman spectroscopic studies

The thermotropic properties of N-(alpha-hydroxyacyl)-sphingosine (CER[AS]) in dry and hydrated state were studied by means of X-ray powder diffraction and FT-Raman spectroscopy. The polymorphic states of the CER[AS]/water mixture (lamellar crystalline, lamellar hexagonal gel, liquid crystalline) depend on the thermal pre-treatment of the sample. Only by heating the CER[AS]/water mixture above the melting chain transition can the system be hydrated. At room temperature, both dry and hydrated states form lamellar structures, which differ in their repeat distance and packing of hydrocarbon chains. Above the melting chain transition, hydrated CER[AS] forms a liquid crystalline hexagonal phase, whereas anhydrous CER[AS] forms an isotropic liquid phase. The various phases of hydrated CER[AS] are distinguished on the basis of the corresponding Raman spectra.     

Copper-cobalt oxide ceramic thin films from single source precursors: Preparation and characterization

Two heterobimetallic coordination complexes [Co(acac)Cu₂(bdmap)₂Cl₃]·C₇H₈ (1) and [Co(acac)Cu₂(bdmap)₂Cl₃]·3CH₂Cl₂ (2) [bdmap = 1,3-Bis(dimethylamino)-2-propanol and acac = 2,4-pentanedionate], have been synthesized by simple chemical technique and characterized by their melting points, elemental composition, FT-IR spectroscopy, mass spectrometry and single crystal X-ray analysis. Thermograms of both the precursors indicated their facile decomposition at relatively low temperature of 454 °C to give stable residual mixture of Cu and CoO. Both the precursors are utilized for the deposition of Cu-CoO thin films by aerosol-assisted chemical vapor deposition (AACVD) equipment at 450 °C on glass substrates that were subsequently characterized for their morphology and composition of the ceramic material. The scanning electron microscopy of copper-cobalt oxide films grown from both the precursors describe highly compact and smooth morphology with homogenously dispersed spherical particles with excellent adhesion properties to the substrates. The EDX analysis shows homogeneous distribution of metallic elements with Cu:Co ratio close to 2:1. Powder X-ray diffraction analysis of the films shows that they are composed of Cu/CoO composite and are crystalline in nature having particle size in the range of 0.3-0.8 μm.     

Spectroscopic study of human lung epithelial cells (A549) in culture: living cells versus dead cells

The noninvasive analysis of living cells grown on 3-dimensional scaffold materials is a key point in tissue engineering. In this work we show the capability of Raman spectroscopy for use as a noninvasive method to distinguish cells at different stages of the cell cycle and living cells from dead cells. The spectral differences between cells in different stages of the cell cycle are characterized mainly by variations in DNA vibrations at 782, 788, and 1095 cm(-1). The Raman spectrum of dead human lung derived (A549 line) cells indicates the breakdown of both phosphodiester bonds and DNA bases. The most sensitive peak for identifying dead cells is the 788 cm(-1) peak corresponding to DNA Obond;Pbond;O backbone stretching. The magnitude of this peak is reduced by 80% in the spectrum of dead cells. Changes in protein peaks suggest significant conformational changes; for example, the magnitude of the 1231 cm(-1) peak assigned to random coils is reduced by 63% for dead cells. The sharp peak of phenylalanine at 1005 cm(-1) drops to half, indicating a decrease of stable proteins associated with cell death. The differences in the 1190-1385 cm(-1) spectral region also suggest a decrease in the amount of nucleic acids and proteins. Using curve fitting, we quantify these spectral differences that can be used as markers of cell death.     

Lysozyme fibrillation: deep UV Raman spectroscopic characterization of protein structural transformation

Deep ultraviolet resonance Raman spectroscopy was demonstrated to be a powerful tool for structural characterization of protein at all stages of fibril formation. The evolution of the protein secondary structure as well as the local environment of phenylalanine, a natural deep ultraviolet Raman marker, was documented for the fibrillation of lysozyme. Concentration-independent irreversible helix melting was quantitatively characterized as the first step of the fibrillation. The native lysozyme composed initially of 32% helix transforms monoexponentially to an unfolded intermediate with 6% helix with a characteristic time of 29 h. The local environment of phenylalanine residues changes concomitantly with the secondary structure transformation. The phenylalanine residues in lysozyme fibrils are accessible to solvent in contrast to those in the native protein.     

Microperoxidase 8 adsorbed on a roughened silver electrode as a monomeric high-spin penta-coordinated species: characterization by SERR spectroscopy and electrochemistry

Microperoxidase 8 (MP8), a heme octapeptide obtained by hydrolytic digestion of cytochrome c, was adsorbed at the surface of a roughened silver electrode in order to provide a new supported biomimetic system for hemoproteins. A combination of two techniques was used to study its redox and coordination properties: electrochemistry and surface-enhanced resonance Raman (SERR) spectroscopy. This allowed us to show that MP8 could be adsorbed as a monolayer at the surface of the roughened silver electrode, where it could undergo a reversible electron transfer. Under those conditions, a redox potential of –0.4 V vs. SCE (–0.16 V vs. NHE) was measured for MP8, which was almost identical to that reported for N-acetyl-MP8 in aqueous solution. In addition, whereas MP8 appeared to aggregate in solution, and led to a mixture of high-spin penta-coordinated (5cHS) and low-spin hexa-coordinated (6cLS) iron(III) or iron(II) species, it was recovered almost exclusively as a monomeric high-spin penta-coordinated species at the surface of the electrode, both in the reduced and in the oxidized states. This then allowed a free coordination site on the iron, on the distal face of MP8 accessible to ligands. Accordingly, experiments performed in the presence of potassium cyanide demonstrated that MP8 adsorbed on a silver electrode could be ligated by a sixth CN^– ligand. Thus there is the possibility of binding several kinds of ligands such as O₂ or H₂O₂, which will open the way to biocatalysis of oxidation reactions at the surface of an electrode, or ligands such as drugs which will lead to the design of new biosensors for molecules of biological interest.     

EXAFS reveals a structural zinc binding site in the bovine NADH-Q oxidoreductase

The metal content of bovine NADH-Q oxidoreductase determined by inductively-coupled plasma atomic-emission spectroscopy reveals the presence of about one atom of zinc per molecule of flavin mononucleotide. We applied Zn K-edge extended X-ray absorption fine structure spectroscopy (EXAFS) to investigate the local structure of the bound zinc ion and to identify the nature of the coordinating residues. The EXAFS spectrum is consistent with a structured zinc binding site. By combining information from first-shell analysis and from metalloprotein data bases putative binding clusters have been built and fitted to the experimental spectrum using ab initio simulations. The best fitting binding cluster is formed by two histidine and two cysteine residues arranged in a tetrahedral geometry.     

Molecular characterization of reconstructed skin model by Raman microspectroscopy: comparison with excised human skin

Human skin is directly exposed to different exogenous agents. Many research works have studied the diffusion, interactions, absorption mechanisms, and/or toxicity of these agents toward different cutaneous structures. With the use of living animals for such tests being more and more rejected; and the number of human volunteers being limited; different types of skin models are used. In the last few years, reconstructed epidermis from cell cultures has been frequently employed, and recent changes in the European chemical policy have approved and encouraged the use of these reconstructed models for skin-related research works and assessments. Among the techniques used actually to study the skin, Raman microspectroscopy is a rising and powerful nondestructive technique that detects characteristic molecular vibrations. In this study, we created a spectral database to index the vibration peaks and bands of a well-known reconstructed epidermis model, the Episkin. The comparison with a native epidermis signal enabled us to put in evidence several spectral differences associated with molecular and structural differences between the skin and the reconstructed model, both maintained in living conditions. In addition to that, we have showed the feasibility of tracking the penetration of a pharmaceutical molecule through the Episkin model. (     

Synthesis, characterization and structure of metallocene dicyanamide complexes

The bis(cyclopentadienyl) complexes [Cp₂Ti(dca)]₂O and Cp₂V(dca)₂ (dca = dicyanamide) have been prepared by reaction of sodium dicyanamide with aqueous solution of titanocene dichloride and vanadocene dichloride, respectively. The X-ray structure analyses of both complexes confirmed monodentate coordination of dicyanamide ligand through the terminal nitrogen atom of cyano group.     

Internalization kinetics and cytoplasmic localization of functionalized diatomite nanoparticles in cancer cells by Raman imaging

Porous biosilica nanoparticles obtained from diatomites (DNPs) have been recently demonstrated to be non‐toxic nanovectors of therapeutic agents in cancer cells. In this work, the internalization kinetics and intracellular spatial distribution of functionalized DNPs incubated with human lung epidermoid carcinoma cell line (H1355) up to 72 hours are investigated by Raman imaging. The label‐free Raman results are compared with confocal fluorescence microscopy and photoluminescence (PL) data. Raman bands specifically assigned to DNPs and cellular components provide evidence that the nanovectors are internalized and co‐localize with lipid environments. A considerable DNPs uptake in cells is observed within 6 hours, with equilibrium being achieved after 18 hours. The obtained data show the presence of DNPs up to 72 hours, without damage to cell viability or morphology. The PL measurements performed on DNPs not penetrating the cells at different incubation times are strongly correlated with the results obtained by Raman imaging and confocal microscopy analyses.