A Sphingolipidomic Profiling Approach for Comparing X-ray-Exposed and Unexposed HepG2 Cells.

An analytical method based on tandem mass spectrometry-shotgun is presently proposed to obtain sphingolipidomic profiles useful for the characterization of lipid extract from X-ray-exposed and unexposed hepatocellular carcinoma cells (HepG2). To obtain a targeted lipidic profile from a specific biological system, the best extraction method must be identified before instrumental analysis. Accordingly, four different classic lipid extraction protocols were compared in terms of efficiency, specificity, and reproducibility. The performance of each procedure was evaluated using the Fourier-transform infrared spectroscopic technique; subsequently, the quality of extracts was estimated using electrospray ionization tandem mass spectrometry. The selected procedure based on chloroform/methanol/water was successfully used in mass spectrometry-based shotgun sphingolipidomics, allowing for evaluation of the response of cells to X-ray irradiation, the most common anticancer therapy. Using a relative quantitative approach, the changes in the sphingolipid profiles of irradiated cell extracts were demonstrated, confirming that lipidomic technologies are also useful tools for studying the key sphingolipid role in regulating cancer growth during radiotherapy.

Vibrational spectroscopies for biochemical investigation of X-ray exposure effects on SH-SY5Y human neuroblastoma cells.

Neuroblastoma is the most recurring cancer in childhood and adolescence. The SH-SY5Y neuroblastoma cell line is generally adopted for elaborating new therapeutical approaches and/or elaborating strategies for the prevention of central nervous system disturbances. In fact, it represents a valid model system for investigating in vitro the effects on the brain of X-ray exposure using vibrational spectroscopies that can detect early radiation-induced molecular alterations of potential clinical usefulness. In recent years, we dedicated significant efforts in the use of Fourier-transform and Raman microspectroscopy techniques for characterizing such radiation-induced effects on SH-SY5Y cells by examining the contributions from different cell components (DNA, proteins, lipids, and carbohydrates) to the vibrational spectra. In this review, we aim at revising and comparing the main results of our studies to provide a wide outlook of the latest outcomes and a framework for future radiobiology research using vibrational spectroscopies. A short description of our experimental approaches and data analysis procedures is also reported.

Self-esterified hyaluronan hydrogels: Advancements in the production with positive implications in tissue healing.

Hyaluronan-(HA) short half-life in vivo limits its benefits in tissue repair. Self-esterified-HA is of great interest because it progressively releases HA, promoting tissue-regeneration longer than the unmodified-polymer. Here, the 1-ethyl-3-(3-diethylaminopropyl)carbodiimide(EDC)-hydroxybenzotriazole(HOBt) carboxyl-activating-system was evaluated for self-esterifying HA in the solid state. The aim was to propose an alternative to the time-consuming, conventional reaction of quaternary-ammonium-salts of HA with hydrophobic activating-systems in organic media, and to the EDC-mediated reaction, limited by by-product formation. Additionally, we aimed to obtain derivatives releasing defined molecular-weight(MW)-HA that would be valuable for tissue renewal. A 250 kDa-HA(powder/sponge) was reacted with increasing EDC/HOBt amounts. HA-modification was investigated through Size-Exclusion-Chromatography-Triple-Detector-Array-analyses, FT-IR/1H NMR and the products(XHAs) extensively characterized. Compared to conventional protocols, the set procedure is more efficient, avoids side-reactions, allows for an easier processing to diverse clinically-usable 3D-forms, leads to products gradually releasing HA under physiological conditions with the possibility to tune the MW of the biopolymer-released. Finally, the XHAs exhibit sound stability to Bovine-Testicular-Hyaluronidase, hydration/mechanical properties suitable for wound-dressings, with improvements over available matrices, and prompt in vitro wound-regeneration, comparably to linear-HA. To the best of our knowledge, the procedure is the first valid alternative to conventional protocols for HA self-esterification with advances in the process itself and in product performance.

Different experimental approaches for Fourier-transform infrared spectroscopy applications in biology and biotechnology: A selected choice of representative results.

Fourier transform infrared (FTIR) spectroscopy is a powerful tool for analyzing the biochemical properties of biological samples such as proteins, cellular materials, and tissues. It provides objective information on samples and has been adopted in many research areas of biomedical and biotechnological interest. FTIR spectroscopy can be performed using different approaches at the macro and micro levels allowing the examination of an incredibly broad class of materials. However, it has become evident that the choice of proper spectra acquisition geometries and the modalities of sample preparation in FTIR spectroscopy analysis require special consideration, especially for certain classes of materials such as cells and tissues. In the present paper, we described the different procedures used for preparing and analyzing different types of biological and biotechnological samples when the more largely available approaches are employed using a commercial FTIR spectrometer. Some basic aspects of data analysis procedures are presented in an Appendix. A certain number of our previous experimental results are reported for demonstrating once more the versatility and the potentiality of FTIR spectroscopy.

µ-FTIR, µ-Raman, and SERS Analysis of Amide I Spectral Region in Oral Biofluid Samples during Orthodontic Treatment.

Gingival crevicular fluid (GCF) is a site-specific exudate deriving from the epithelium lining of the gingival sulcus. GCF analysis provides a simple and noninvasive diagnostic procedure to follow-up periodontal and bone remodeling in response to diseases or mechanical stimuli such as orthodontic tooth movement (OTM). In recent years, the use of vibrational spectroscopies such as Fourier Transform Infrared and Raman microspectroscopy and Surface-Enhanced Raman spectroscopy contributed to characterizing changes in GCF during fixed orthodontic treatment. Amide I band plays a relevant role in the analysis of these changes. The aim of this study was to investigate the spectroscopy response of Amide I depending on the OTM process duration. A model based on Gaussian-Lorentzian curves was used to analyze the infrared spectra, while only Lorentzian functions were used for Raman and SERS spectra. Changes induced by the OTM process in subcomponents of the Amide I band were determined and ascribed to secondary structure modification occurring in proteins. The vibrational spectroscopies allow us to efficiently monitor the effects of the orthodontic force application, thus gaining increasing attention as tools for individual patient personalization in clinical practice.

Gelatin-biofermentative unsulfated glycosaminoglycans semi-interpenetrating hydrogels via microbial-transglutaminase crosslinking enhance osteogenic potential of dental pulp stem cells.

Gelatin hydrogels by microbial-transglutaminase crosslinking are being increasingly exploited for tissue engineering, and proved high potential in bone regeneration. This study aimed to evaluate, for the first time, the combination of enzymatically crosslinked gelatin with hyaluronan and the newly developed biotechnological chondroitin in enhancing osteogenic potential. Gelatin enzymatic crosslinking was carried out in the presence of hyaluronan or of a hyaluronan-chondroitin mixture, obtaining semi-interpenetrating gels. The latter proved lower swelling extent and improved stiffness compared to the gelatin matrix alone, whilst maintaining high stability. The heteropolysaccharides were retained for 30 days in the hydrogels, thus influencing cell response over this period. To evaluate the effect of hydrogel composition on bone regeneration, materials were seeded with human dental pulp stem cells and osteogenic differentiation was assessed. The expression of osteocalcin (OC) and osteopontin (OPN), both at gene and protein level, was evaluated at 7, 15 and 30 days of culture. Scanning electron microscopy (SEM) and two-photon microscope observations were performed to assess bone-like extracellular matrix (ECM) deposition and to observe the cell penetration depth. In the presence of the heteropolysaccharides, OC and OPN expression was upregulated and a higher degree of calcified matrix formation was observed. Combination with hyaluronan and chondroitin improved both the biophysical properties and the biological response of enzymatically crosslinked gelatin, fastening bone deposition.

Application of Vibrational Spectroscopies in the Qualitative Analysis of Gingival Crevicular Fluid and Periodontal Ligament during Orthodontic Tooth Movement.

Optical vibrational techniques show a high potentiality in many biomedical fields for their characteristics of high sensitivity in revealing detailed information on composition, structure, and molecular interaction with reduced analysis time. In the last years, we have used these techniques for investigating gingival crevicular fluid (GCF) and periodontal ligament (PDL) during orthodontic tooth treatment. The analysis with Raman and infrared signals of GCF and PDL samples highlighted that different days of orthodontic force application causes modifications in the molecular secondary structure at specific wavenumbers related to the Amide I, Amide III, CH deformation, and CH3/CH2. In the present review, we report the most relevant results and a brief description of the experimental techniques and data analysis procedure in order to evidence that the vibrational spectroscopies could be a potential useful tool for an immediate monitoring of the individual patient's response to the orthodontic tooth movement, aiming to more personalized treatment reducing any side effects.

Surface-enhanced Raman spectroscopy of tears: toward a diagnostic tool for neurodegenerative disease identification.

SIGNIFICANCE: A noninvasive method based on surface-enhanced Raman spectroscopy (SERS) of tears was proposed as a support for diagnosing neurodegenerative pathologies, including different forms of dementia and Alzheimer's disease (AD). In this field, timely and reliable discrimination and diagnosis are critical aspects for choosing a valid medical therapy, and new methods are highly required. AIM: The aim is to evince spectral differences in SERS response of human tears from AD affected, mild cognitive impaired (MCI), and healthy control (Ctr) subjects. APPROACH: Human tears were characterized by SERS coupled with multivariate data analysis. Thirty-one informed subjects (Ctr, MCI, and AD) were considered. RESULTS: Average SERS spectra from Ctr, MCI, and AD subjects evidenced differences related to lactoferrin and lysozyme protein components. Quantitative changes were also observed by determining the intensity ratio between selected bands. We also constructed a classification model that discriminated among AD, MCI, and Ctr subjects. The model was built using the scores obtained by performing principal component analysis on specific spectral regions (i-PCA). CONCLUSIONS: The results are very encouraging with interesting perspectives for medical applications as support of clinical diagnosis and discrimination of AD from other forms of dementia.

Graphene-Based and Surface-Enhanced Raman Spectroscopy for Monitoring the Physio-Chemical Response of Thermophilic Bacterial Spores to Low Temperatures Exposure.

Monitoring the spore life cycle is one of the main issues in several fields including environmental control, sustainable ecosystems, food security, and healthcare systems. In this framework, the study of the living organism resistance to extreme conditions like those mimicking space environments is particularly interesting. The assessment of the local change of the pH level can be extremely useful for this purpose. An optical physiometer method based on the Raman response of the graphene, which is able to locally sense pH of a fluid on a micrometric scale, has been recently proposed. Due to the presence of π -bonds at the surface, the electronic doping of graphene is determined by the external conditions and can be electrochemically controlled or altered by the contact with an acid or alkaline fluid. The doping level affects the vibrational energies of the graphene that can be monitored by conventional Raman spectroscopy. In addition, Surface-Enhanced Raman Spectroscopy (SERS) can give direct information on the biochemical changes occurring in spore components. In this work, we propose the joint use of Graphene-Based Raman Spectroscopy (GbRS) and SERS for the monitoring of the response of spores to exposure to low temperatures down to 100 K. The spores of the thermophilic bacterium Parageobacillus thermantarcticus isolated from an active volcano of Antarctica (Mt. Melbourne) were investigated. These spores are particularly resistant to several stressing stimuli and able to adapt to extreme conditions like low temperatures, UV irradiation, and γ -rays exposure. The results obtained showed that the joint use of GbRS and SERS represents a valuable tool for monitoring the physio-chemical response of bacterial spores upon exposure to stressing stimuli.

An insight into pH-induced changes in FAD conformational structure by means of time-resolved fluorescence and circular dichroism.

Optical properties of flavin adenine dinucleotide (FAD) moiety are widely used nowadays for biotechnological applications. Given the fundamental role played by FAD, additional structural information about this enzymatic cofactor can be extremely useful in order to obtain a greater insight into its functional role in proteins. For this purpose, we have investigated FAD behaviour in aqueous solutions at different pH values by a novel approach based on the combined use of time-resolved fluorescence and circular dichroism spectroscopies. The results showed that pH strongly affects time-resolved fluorescence emission and the analysis allowed us to detect a three-component decay for FAD in aqueous solution with pH-depending lifetimes and relative amplitudes. Circular dichroism data were analyzed by a multi-Gaussian fitting procedure and the trends of properly chosen parameters confirmed pH-depending changes. The comparison between the results obtained by these two optical techniques allowed us to improve the significance of the outcome of circular dichroism. This combined approach may provide a useful tool for biotechnological investigation.

Characterization of Human Tear Fluid by Means of Surface-Enhanced Raman Spectroscopy.

Tears are exceptionally rich sources of information on the health status of the eyes, as well as of whole body functionality, due to the presence of a large variety of salts and organic components whose concentration can be altered by pathologies, eye diseases and/or inflammatory processes. Surface enhanced Raman spectroscopy (SERS) provides a unique method for analyzing low concentrations of organic fluids such as tears. In this work, a home-made colloid of gold nanoparticles has been used for preparing glass substrates able to efficiently induce an SERS effect in fluid samples excited by a He⁻Ne laser ( λ = 633 nm). The method has been preliminary tested on Rhodamine 6G aqueous solutions at different concentrations, proving the possibility to sense substance concentrations as low as few µ M, i.e., of the order of the main tear organic components. A clear SERS response has been obtained for human tear samples, allowing an interesting insight into tear composition. In particular, aspartic acid and glutamic acid have been shown to be possible markers for two important human tear components, i.e., lactoferrin and lysozyme.

A novel experimental approach for liver analysis in rats exposed to Bisphenol A by means of LC-mass spectrometry and infrared spectroscopy.

An innovative complementary approach using a liquid chromatographic-mass spectrometer method and infrared spectroscopy is proposed for measuring internal biological exposure to dangerous chemical contaminants and for monitoring biochemical changes in target organs. The proposed methodologies were validated and applied in the case of rats exposed to low-doses of Bisphenol A (BPA). A liquid chromatographic method coupled to a tandem mass spectrometer was used in order to measure BPA concentration in rat livers. BPA was detected at different levels in all liver samples from BPA-treated rats, although the exposure dose was the same in all treated animals, and also from control rats, highlighting the difficulties in eliminating external uncontrolled exposure and the need for internal biological monitoring. Fourier Transform Infrared analysis was applied to detect structural changes occurring in several molecules (lipids, proteins, carbohydrates and nucleic acids) as well as the presence of specific metabolic processes. The spectroscopic analyses clearly demonstrated a different lipid composition more than an evident lipid accumulation and a glycogen accumulation decrease, revealing a metabolic disturbance in livers with a normal histological aspect. These results demonstrated the potential of an integrated approach based on mass spectrometry and infrared spectroscopy to evaluate at an early stage the hepatotoxic effect of BPA exposure in an animal model. This approach can be usefully exploited in all the investigations aimed to provide better information concerning the interrelationships between contaminant exposure, dose, and health effects.

Study of SH-SY5Y Cancer Cell Response to Treatment with Polyphenol Extracts Using FT-IR Spectroscopy.

Plant polyphenols are important components of human diet and a number of them are considered to possess chemo-preventive and therapeutic properties against cancer. They are recognized as naturally occurring antioxidants, but also as pro-oxidant, pro-apoptotic, or chromosomal aberrations inducers, depending on their concentration and/or the stage of cell-cycle of the cells with which they interact. For these reasons, particular interest is devoted to knowing the total effects of polyphenols on the cell cycle and metabolism. Fourier-Transform Infrared (FT-IR) spectroscopy thanks to its ability in analyzing cells at a molecular level can be particularly useful in investigating the biochemical changes induced in protein, nucleic acid, lipid, and carbohydrate content of cells by means of polyphenols administration. Spectroscopic analysis was performed on in vitro human SH-SY5Y neuroblastoma cells that were exposed to different doses of a cherry derived polyphenol extract. The infrared spectra that were obtained from unexposed and exposed cells show significant differences that can be helpful in order to understand the cells-polyphenols interaction.

Insights into Insulin Fibril Assembly at Physiological and Acidic pH and Related Amyloid Intrinsic Fluorescence.

Human insulin is a widely used model protein for the study of amyloid formation as both associated to insulin injection amyloidosis in type II diabetes and highly prone to form amyloid fibrils in vitro. In this study, we aim to gain new structural insights into insulin fibril formation under two different aggregating conditions at neutral and acidic pH, using a combination of fluorescence, circular dichroism, Fourier-transform infrared spectroscopy, and transmission electron miscroscopy. We reveal that fibrils formed at neutral pH are morphologically different from those obtained at lower pH. Moreover, differences in FTIR spectra were also detected. In addition, only insulin fibrils formed at neutral pH showed the characteristic blue-green fluorescence generally associated to amyloid fibrils. So far, the molecular origin of this fluorescence phenomenon has not been clarified and different hypotheses have been proposed. In this respect, our data provide experimental evidence that allow identifying the molecular origin of such intrinsic property.

Monitoring early phases of orthodontic treatment by means of Raman spectroscopies.

Gingival crevicular fluid (GCF) is a site-specific exudate in the gingival sulcus. GCF composition changes in response to diseases or mechanical stimuli, such as those occurring during orthodontic treatments. Raman microspectroscopy (µ-RS) and surface-enhanced Raman spectroscopy (SERS) were adopted for a GCF analysis during different initial phases of orthodontic force application. GCF samples were pooled from informed patients using paper cones. SERS spectra were obtained from GCF extracted from these cones, whereas µ-RS spectra were directly acquired on paper cones without any manipulation. The spectral characteristics of the main functional groups and the changes in cytochrome, amide III, and amide I contributions were highlighted in the different phases of orthodontic treatment with both SERS and µ-RS analysis. µ-RS directly performed on the paper cones together with proper statistical methods can offer an effective approach for the development of a tool for monitoring the processes occurring during orthodontic treatments, which may help the clinician in the choice of type of treatment individually for each patient and accelerate and improve the orthodontic therapy.

Hyaluronan hydrogels with a low degree of modification as scaffolds for cartilage engineering.

In the field of cartilage engineering, continuing efforts have focused on fabricating scaffolds that favor maintenance of the chondrocytic phenotype and matrix formation, in addition to providing a permeable, hydrated, microporous structure and mechanical support. The potential of hyaluronan-based hydrogels has been well established, but the ideal matrix remains to be developed. This study describes the development of hyaluronan sponges-based scaffolds obtained by lysine methyl-ester crosslinking. The reaction conditions are optimized with minimal chemical modifications to obtain materials that closely resemble elements in physiological cellular environments. Three hydrogels with different amounts of crosslinkers were produced that show morphological, water-uptake, mechanical, and stability properties comparable or superior to those of currently available hyaluronan-scaffolds, but with significantly fewer hyaluronan modifications. Primary human chondrocytes cultured with the most promising hydrogel were viable and maintained lineage identity for 3 weeks. They also secreted cartilage-specific matrix proteins. These scaffolds represent promising candidates for cartilage engineering.

Fructose and Pectin Detection in Fruit-Based Food Products by Surface-Enhanced Raman Spectroscopy.

Surface-Enhanced Raman Spectroscopy (SERS) enables the investigation of samples with weak specific Raman signals, such as opaque samples, including fruit juices and pulp. In this paper, biological apple juices and apple/pear pulp have been studied in order to evidence the presence of fructose and pectin, which are components of great relevance for quality assessment of these kinds of products. In order to perform SERS measurements a low-cost home-made substrate consisting of a glass slide decorated with 30-nm-sized gold nanoparticles has been designed and used. By employing a conventional micro-Raman spectroscopy set-up and a suitable data treatment based on "wavelet" denoising algorithms and background subtraction, spectra of pectin and fructose with clear Raman features have been obtained. The results have confirmed the potential of SERS in the food industry for product characterization, also considering the low-cost and the relative ease of the fabrication process of the employed SERS substrate.

Optical detection of different phenolic compounds by means of a novel biosensor based on sol-gel immobilized laccase.

A novel sol-gel-based biosensor exploiting the optical absorption properties of sol-gel immobilized laccase has been constructed to increase enzyme specificity toward different phenolic substrates. Laccase from Trametes versicolor has been immobilized in optically transparent sol-gel matrices. Using Fourier transform infrared spectroscopy and data analysis based on a wavelet algorithm, a successful enzyme immobilization has been determined. The changes in the optical absorption spectra of laccase reaction products at 425, 375, and 400 nm have been used to determine hydroquinone, resorcinol, and catechol concentrations, respectively. Owing to the slow response time of the hydroquinone-laccase reaction, our optical biosensor has been tested with resorcinol and catechol. Linear ranges up to 1.4 and 0.2 mM, limit-of-detection (LOD) of 4.5 and 0.6 µΜ, have been evidenced for resorcinol and catechol, respectively. Data for determining the resorcinol concentration have been particularly interesting since no other biosensor device has been reported in the literature. In comparison with other biosensors using laccase from the same native source, our biosensor has been characterized by larger linear ranges, significant sensitivities, and good LODs. To challenge our biosensor with real samples, tap water samples spiked with known amount of catechol and resorcinol have been employed.

Removal of methylparaben from synthetic aqueous solutions using polyacrylonitrile beads: kinetic and equilibrium studies.

The removal of methylparaben (MP), a well-known endocrine disruptor, from aqueous solutions using polyacrylonitrile (PAN) beads has been studied under batch conditions, at room temperature and at different initial MP concentrations. The kinetic and equilibrium results have been analyzed. Kinetic modeling analysis has been carried out with three different types of adsorption models: pseudo-first-order, pseudo-second-order, and Elovich model. Kinetic data analysis indicated that the adsorption was a second-order process. The MP adsorption by PAN was also quantitatively evaluated by using the equilibrium adsorption isotherm models of Langmuir, Freundlich, Dubinin-Radushkevich (D-R), and Temkin and the applicability of the respective isotherm equations has been compared through the correlation coefficients. Adsorption data resulted well fitted by the Freundlich isotherm model. Data of MP adsorption have also been used to test different adsorption diffusion models. The diffusion rate equations inside particulate of Dumwald-Wagner and the intraparticle diffusion model have been used to calculate the diffusion rate. The actual rate-controlling step involved in the MB adsorption process was determined. The kinetic expression by Boyd gave the right indications. All together, our results indicate that PAN beads are a useful tool to remediate water bodies polluted by endocrine disruptors.

Migration of bisphenol A into canned tomatoes produced in Italy: dependence on temperature and storage conditions.

A method based on solid-phase extraction followed by liquid chromatography, coupled to UV-visible and fluorescence spectrophotometry, has been developed for determination of bisphenol A (BPA) in canned tomatoes. The limit of quantification (LOQ) of the procedure used is 0.03 µM (0.26 µg BPA/kg tomato). For each of three different tomato based products (peeled, cherry and concentrated paste), 16 samples belonging to six commercial brands, retailed in Italian markets, were tested for migration of BPA epoxy-coating cans. All the tomato samples exhibited migration levels below 0.4 µg/kg, while samples subjected to heating process and/or can's damage by denting, exhibited a significant increase in the migration levels. In any case, no sample contained BPA exceeding the European Union limit for migration, set at 600 µg/kg of food. By comparing the results for each brand, no relevant difference in BPA concentration was found depending on the kind of tomato products.