Raman Spectroscopy and Cystic Fibrosis Disease: An Alternative Potential Tool for Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Modulator Response Differentiation-A Pilot Study Based on Serum Samples.

Cystic fibrosis (CF) is a genetic disorder that alters chloride transport in mucous membranes. Recent studies have demonstrated that treatment with modulators of the chloride channel reduces inflammatory markers, restoring, among others, the imbalance of lipids. In this study, we analyzed the serum samples of treated and non-treated patients with modulators with Raman spectroscopy. Nineteen (eight treated an eleven non-treated) patients were considered. The main difference between the two groups appeared in the 3020-2800 cm-1 range. A Voigt deconvolution fit was performed, and nine sub-bands were identified. To distinguish between treated and non-treated patients, the area ratio between the CH3 and CH2 vibration modes was calculated for each patient. The results were validated using statistical analyses. In particular, receiver operating characteristic (ROC) curves and Youden index (Y) were calculated (Area Under Curve (AUC): 0.977; Y: 3.30). An ROC curve represents the performance of the classification, illustrating the diagnostic ability of Raman spectroscopy. It was demonstrated that Raman spectroscopy is able to highlight peculiar differences between elexacaftor/tezacaftor/ivacaftor (ETI)-treated and non-treated patients, in relation with lipids biomarkers.

Central and Peripheral Fatigue Evaluation during Physical Exercise in Athletic Horses by Means of Raman Spectroscopy.

The evaluation of the performance levels in athletic horses is of major importance to prevent sports injuries. Raman spectroscopy is an innovative technique that allows for a rapid evaluation of biomolecules in biological fluids. It also permits qualitative and quantitative sample analyses, which lead to the simultaneous determination of the components of the examined biological fluids. On the basis of this, the Raman spectroscopy technique was applied on serum samples collected from five Italian Saddle horses subjected to a standardized obstacle course preceded by a warm-up to evaluate the applicability of this technique for the assessment of central and peripheral fatigue in athletic horses. Blood samples were collected via jugular venipuncture in a vacutainer tube with a clot activator before exercise, immediately after exercise, and 30 min and 1 h after the end of the obstacle course. Observing the obtained Raman spectra, the major changes due to the experimental conditions appeared in the (1300-1360) cm-1 and (1385-1520) cm-1 bands. In the (1300-1360) cm-1 band, lipids and tryptophan were identified; in the (1385-1520) cm-1 band, leucine, glycine, isoleucine, lactic acid, tripeptide, adenosine, and beta carotene were identified. A significant effect of exercise was recorded on all the sub-bands. In particular, a change immediately after exercise versus before exercise was found. Moreover, the mean lactic concentration was positively correlated with the Raman area of the sub-band assigned to lactic acid. In this context, the application of Raman spectroscopy on blood serum samples represents a useful technique for secondary-structure protein identification to investigate the metabolic changes that occur in athletic horses during physical exercise.

Comparison of clinical efficacy of three different dentin matrix biomaterials obtained from different devices.

AIM: The aim of the present study was to propose the clinical efficacy of the different dentin matrix obtained from three devices (BonMaker, Tooth Transformer, and Smart Dentin Grinder) and to show their morphological, physical, and biochemical characteristics using scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) spectroscopy, and Raman spectroscopy. RESEARCH DESIGN AND METHODS: The study included 70 patients who underwent bone augmentation using the BonMaker, Tooth Transformer, and Smart Dentin Grinder devices. In addition, 84 implants were placed. Furthermore, four samples, one for each device and one non-demineralized control, were analyzed with scanning electron microscopy (SEM), energy-dispersive X-ray analysis, and Raman spectroscopy. RESULTS: In all patients, augmentation of bone defects with ground dentin matrix was successful, and implants showed correct osseointegration. The morphological organization, the chemical composition, and the presence of organic molecules in the dentin samples processed by the three different devices were demonstrated using SEM, energy-dispersive X-ray analysis, and Raman spectroscopy. CONCLUSIONS: Comparing BonMaker, Tooth Transformer, and Smart Dentin Grinder devices in our practice, we concluded that these systems, even with different structural and chemical differences of the dentin granules, have a comparable potential for obtaining regenerative material from the patient's own teeth.

Occupational Exposure Assessment of the Static Magnetic Field Generated by Nuclear Magnetic Resonance Spectroscopy: A Case Study.

Magnetic resonance (MR) systems are used in academic research laboratories and industrial research fields, besides representing one of the most important imaging modalities in clinical radiology. This technology does not use ionizing radiation, but it cannot be considered without risks. These risks are associated with the working principle of the technique, which mainly involves static magnetic fields that continuously increase-namely, the radiofrequency (RF) field and spatial magnetic field gradient. To prevent electromagnetic hazards, the EU and ICNIRP have defined workers' exposure limits. Several studies that assess health risks for workers and patients of diagnostic MR are reported in the literature, but data on workers' risk evaluation using nuclear MR (NMR) spectroscopy are very poor. Therefore, the aim of this research is the risk assessment of an NMR environment, paying particular attention to workers with active implantable medical devices (AIMDs). Our perspective study consisted of the measurement of the static magnetic field around a 300 MHz (7 T) NMR research spectrometer and the computation of the electric field induced by the movements of an operator. None of the calculated exposure parameters exceeded the threshold limits imposed by legislation for protection against short-term effects of acute occupational exposure, but our results revealed that the level of exposure exceeded the action level threshold limit for workers with AIMD during the execution of tasks requiring the closest proximity to the spectrometer. Moreover, the strong dependence of the induced electric field results from the walking speed models is shown. This case study represents a snapshot of the NMR risk assessment with the specific goal to increase the interest in the safety of NMR environments.

Raman Spectroscopic Study of Amyloid Deposits in Gelatinous Drop-like Corneal Dystrophy.

The genetic and histopathological features of the cornea of a Polish patient with Gelatinous Drop-like Corneal Dystrophy (GDCD) and the molecular composition with Raman spectroscopy of corneal deposits were examined. A 62 year-old Polish woman was diagnosed with GDCD and underwent penetrating corneal transplant. A blood sample was collected, and genetic analysis was performed. The cornea was processed for light microscopy and Raman analysis. The genetic exam revealed a previously undescribed homozygous 1-base pair deletion in exon 1 of TACSTD2 gene (c.185delT), resulting in a frame shift causing a premature stop codon. When compared with a control cornea, in GDCD cornea stained with PAS evident deposits were present over the anterior stroma, with apple green birefringence under polarized light. Raman spectroscopy showed peculiar differences between normal and GDCD cornea, consisting in peaks either of different height or undetectable in the normal cornea and related to amyloid. The possible causative role of the novel mutation was discussed and Raman spectroscopy as a further morphological tool in the evaluation of corneal dystrophies, characterized by the deposition of abnormal materials, was suggested.

Quantifying Serum Total Lipids and Tryptophan Concentrations by Raman Spectroscopy During Standardized Obstacle Course in Horses.

Raman spectroscopy is an inelastic light-scattering phenomenon that provides vibrational spectrum that contains information relative to chemical bonds and symmetry of a specific molecule, allowing the quali-quantitative simultaneous determination of several components in the biological fluids. Raman spectroscopy measurement returns a spectrum over a wavenumber range constituted by several bands representing biomarkers according to investigated biological matrices. In literature, it has been reported that at the frequencies inside the (1,300-1,360) cm-1 total lipids, and tryptophan have been identified. On the basis of that, the aim of the present study was to compare the serum concentration of total lipids and tryptophan in horses subjected to a standardized obstacle course, in comparison with the data obtained in the (1,300-1,360) cm-1 band. At this purpose, five clinically healthy and regularly trained Italian Saddle horses aged between 8, and 10 years old performed with the same rider an obstacle course of 350 m/min with twelve 1.30 m high jumps preceded by warm-up. Blood samples were collected by means of jugular venipuncture into a vacutainer tubes with clot activator at rest, after the exercise, and 30 minutes after the end of exercise. A high correlation was observed between the area of total lipids and tryptophan in the (1,300-1,360) cm-1 band and their serum concentrations in all experimental conditions. Our preliminary results give a hint to study the exact correspondence between the area that identify these parameters in Raman spectrum and their serum concentration in athletic horses.

Raman Spectroscopy Technique: A Non-Invasive Tool in Celiac Disease Diagnosis.

Celiac disease (CD) is diagnosed by a combination of specific serology and typical duodenal lesions. The histological confirmation of CD, mandatory in the majority of patients with suspected CD, is based on invasive and poorly tolerated procedures, such as upper gastrointestinal endoscopy. In this study we propose an alternative and non-invasive methodology able to confirm the diagnosis of CD based on the analysis of serum samples using the Raman spectroscopy technique. Three different bands centered at 1650, 1450 and 1003 cm-1 have been considered and the A1450/A1003 and A1650/A1003 ratios have been computed to discriminate between CD and non-CD subjects. The reliability of the methodology was validated by statistical analysis using receiver operating characteristic (ROC) curves. The Youden index was also determined to obtain optimal cut-off points. The obtained results highlighted that the proposed methodology was able to distinguish between CD and non-CD subjects with 98% accuracy. The optimal cut-off points revealed, for both the A1450/A1003 and A1650/A1003 ratios, high values of sensitivity and specificity (>95.0% and >92.0% respectively), confirming that Raman spectroscopy may be considered a valid alternative to duodenal biopsy and demonstrates spectral changes in the secondary structures of the protein network.

A Novel Phantom and a Dedicated Developed Software for Image Quality Controls in X-Ray Intraoral Devices.

BACKGROUND: Periodic quality control (QC) procedures are important in order to guarantee the image quality of radiological equipment and are also conducted using phantoms simulating human body. OBJECTIVE: To perform (QC) measurements in intraoral imaging devices, a new and simple phantom was manufactured. Besides, to simplify QC procedures, computerized LabView-based software has been devised, enabling determination of image quantitative parameters in real time or during post processing. MATERIAL AND METHODS: In this experimental study, the novel developed phantom consists of a Polymethyl methacrylate (PMMA) circular insert. It is able to perform a complete QC image program of X-ray intraoral equipment and also causes the evaluation of image uniformity, high and low contrast spatial resolution, image linearity and artefacts, with only two exposures. RESULTS: Three raters analyzed the images using the LabView dedicated software and determined the quantitative and qualitative parameters in an innovative and accurate way. Statistical analysis evaluated the reliability of this study. Good accuracy of the quantitative and qualitative measurements for the different intraoral systems was obtained and no statistical differences were found using the inter-rater analysis. CONCLUSION: The achieved results and the related statistical analysis showed the validity of this methodology, which could be proposed as an alternative to the commonly adopted procedures, and suggested that the novel phantom, coupled with the LabView based software, could be considered as an effective tool to carry out a QC image program in a reproducible manner.

Rutin-Loaded Solid Lipid Nanoparticles: Characterization and In Vitro Evaluation.

This study was aimed at preparing and characterizing solid lipid nanoparticles loading rutin (RT-SLNs) for the treatment of oxidative stress-induced diseases. Phospholipon 80H® as a solid lipid and Polysorbate 80 as surfactant were used for the SLNs preparation, using the solvent emulsification/diffusion method. We obtained spherical RT-SLNs with low sizes, ranging from 40 to 60 nm (hydrodynamic radius) for the SLNs prepared starting from 2% and 5% (w/w) theoretical amount. All prepared formulations showed negative zeta-potential values. RT was efficiently encapsulated within SLNs, obtaining high encapsulation efficiency and drug content percentages, particularly for SLNs prepared with a 5% theoretical amount of RT. In vitro release profiles and analysis of the obtained data applying different kinetic models revealed Fickian diffusion as the main mechanism of RT release from the SLNs. The morphology of RT-SLNs was characterized by scanning electron microscopy (SEM), whereas the interactions between RT and the lipid matrix were investigated by Raman spectroscopy, evidencing spectral modifications of characteristic bands of RT due to the establishment of new interactions. Finally, antioxidant activity assay on human glioblastoma astrocytoma (U373) culture cells showed a dose-dependent activity for RT-SLNs, particularly at the highest assayed dose (50 µM), whereas the free drug showed the lesser activity.

Application of Raman Spectroscopy for the Evaluation of Metabolomic Dynamic Analysis in Athletic Horses.

Raman spectroscopy is a rapid qualitative and quantitative technique that allows the simultaneous determination of several components, both biomolecules both chemical compounds, in the biological fluids to assess the metabolic status. In this study, the serum composition was evaluated in regularly trained athletic horses using Raman spectroscopy to identify biomarkers of sports performance. Five clinically healthy and regularly trained Italian Saddle horses were subjected to a standardized obstacle course (350 m/minute; eleven 1.25 high jumps) preceded by a warm-up. On the collected sera, at rest, immediately after exercise, 30 minutes, and 1 hour after the end of the exercise Raman measurements were performed using a diode laser with the excitation wavelength of 785 nm. The analysis of the obtained spectra allowed the identification of peaks and bands different in position and intensity among the experimental conditions. The acquired spectra, obtained from horse sera collected during the experimental protocol, were visually similar, except for the large band detected in the 1,250-1,800 cm-1 range. The spectral intensity of the Raman spectrum decreased after training and 30 minutes after the end of exercise respect to the before exercise value, to come to the basal value after 60 minutes the end of the exercise. In conclusion, we can claim the ability of Raman spectroscopy to reveal the metabolic status of horses after physical exercise.

Irradiation with Polychromatic Incoherent Low Energy Radiation of Human Peripheral Blood Mononuclear Cells In Vitro: Effects on Cytokine Production.

(1) Background: Physical stimuli may activate peripheral blood mononuclear cells (PBMCs) to secrete cytokines, which may favor pro-inflammatory responses or trigger reparative phenomena. The purpose of this study is to evaluate the action of Polarized Polychromatic Incoherent Low Energy Radiation (PILER) on human in vitro PBMCs, by detection of the possible effects on cytokine production; (2) Methods: isolated PBMCs were irradiated with a PILER lamp at different exposure times, at a distance of 10 cm, before incubation. The supernatants were collected after 24 h and 48 h and cytokines evaluated by ELISA; (3) Results: Our results showed a decrease in the levels of pro-inflammatory IL-12p70, IL-17A, IFN-γ, and TNF-α cytokines, whereas IL-10 and TGF-ß1 with regulatory activity increased; (4) Conclusions: PILER irradiation affected the cytokine production by isolated PBMCs driving the immune response toward an anti-inflammatory/reparative profile.

Physicochemical Characterization and Antioxidant Activity Evaluation of Idebenone/Hydroxypropyl-ß-Cyclodextrin Inclusion Complex †.

Idebenone (IDE) is an antioxidant drug active at the level of the central nervous system (CNS), whose poor water solubility limits its clinical application. An IDE/2-hydroxypropyl-ß-cyclodextrin (IDE/HP-ß-CD) inclusion complex was investigated by combining experimental methods and theoretical approaches. Furthermore, biological in vitro/ex vivo assays were performed. Phase solubility studies showed an AL type diagram, suggesting the presence of a 1:1 complex with high solubility. Scanning electron microscopy (SEM) allowed us to detect the morphological changes upon complexation. The intermolecular interactions stabilizing the inclusion complex were experimentally characterized by exploring the complementarity of Fourier-transform infrared spectroscopy in attenuated total reflectance geometry (FTIR-ATR) with mid-infrared light, Fourier-transform near-infrared (FT-NIR) spectroscopy, and Raman spectroscopy. From the temperature evolution of the O-H stretching band of the complex, the average enthalpy ΔHHB of the hydrogen bond scheme upon inclusion was obtained. Two-dimensional (2D) rotating frame Overhauser effect spectroscopy (ROESY) analysis and computational studies involving molecular modeling and molecular dynamics (MD) simulation demonstrated the inclusion of the quinone ring of IDE inside the CD ring. In vitro/ex vivo studies evidenced that complexation produces a protective effect of IDE against the H2O2-induced damage on human glioblastoma astrocytoma (U373) cells and increases IDE permeation through the excised bovine nasal mucosa.

Evaluation of US and MRI techniques for carotid stenosis: a novel phantom approach.

Carotid atherosclerosis is very important in the pathogenesis of cerebral ischemia. Ultrasonography (US) and magnetic resonance imaging (MRI) are the predominant noninvasive techniques capable to identify the presence and stage of intra-plaque hemorrage. In this work, we propose a novel dedicated phantom that can be used for both US and MRI scanners to evaluate carotid atherosclerotic lesions. The phantom consists of a polymethyl metacrylate (PMMA) diagonally crossed by a PMMA hollow cylinder simulating a blood vessel. To simulate a stenosis, we inserted a plastic hollow tube inside the cylinder. Quantitative image analysis, based on accuracy measurements, was performed on two US and two MRI scanners. The accuracy measurements have highlighted the use of the 3.0 T MRI scanner to characterize the vessel stenosis. However, no significant difference between US and MRI techniques was found in Fisher exact test and inter-rater agreement. The concordance correlation coefficient showed a moderate agreement between some methods. Agreement between 3.0 T and other methods results poor, and this could be due to the fact that the 3.0 T has a better resolution compared to a US and MR 1.5 T. These methods seem to have similar efficacies for the evaluation of vessel stenosis, legitimizing the use of the developed phantom as a versatile and reproducible instrument that could be used during quality controls programs.

dB/dt Evaluation in MRI Sites: Is ICNIRP Threshold Limit (for Workers) Exceeded?

The Directive 2013/35/EU establishes standards for workers exposed to static and time varying magnetic fields. These limits are based on ICNIRP guidelines expressed in terms of the electric field induced in the body. The complexity of this measurement led to theoretical models being developed. In this study, the experimental evaluation included varying magnetic field exposures for two classes of MRI workers. The measurements are conducted on four different MRI Systems including one 0.35 T, two 1.5 T, and one 3.0 T. Pocket magnetic dosimeters were used and it was carried out during routine conditions, emergency conditions, and cold-head maintenance/substitution. The acquired data has been processed and the corresponding dB/dt curves have been computed as the first time derivative of the dataset. The weighted peak approach was also implemented for the compliance assessment with regulatory limits. The dB/dt peak values have been compared with the reference level (RL) proposed by ICNIRP. The results show that the RL always exceeds during measurements on the 3.0 T scanner and sometimes on 1.5 T. In light of the foregoing, the diffusion of ultra-high field MRI scanners involves the introduction of behavioral rules that could be more useful than a numerical action level.

CT and MRI slice separation evaluation by LabView developed software.

The efficient use of Computed Tomography (CT) and Magnetic Resonance Imaging (MRI) equipment necessitates establishing adequate quality-control (QC) procedures. In particular, the accuracy of slice separation, during multislices acquisition, requires scan exploration of phantoms containing test objects. To simplify such procedures, a novel phantom and a computerised LabView-based procedure have been devised, enabling determination the midpoint of full width at half maximum (FWHM) in real time while the distance from the profile midpoint of two progressive images is evaluated and measured. The results were compared with those obtained by processing the same phantom images with commercial software. To validate the proposed methodology the Fisher test was conducted on the resulting data sets. In all cases, there was no statistically significant variation between the commercial procedure and the LabView one, which can be used on any CT and MRI diagnostic devices.

In vitro assessment of neurotoxicity and neuroinflammation of homemade MWCNTs.

Multi walled carbon nanotubes (MWCNTs) activate pathways involved in cytotoxicity, genotoxicity and inflammation. Inhaled MWCNTs are translocated to extra pulmonary organs and their hydrophobicity allows them to cross the blood-brain barrier (BBB). Further exposure of central nervous system (CNS) occurs via olfactory neurons. Using differentiated SH-SY5Y, we studied the neurotoxicity and neuroinflammation of pristine and functionalised MWCNTs. ROS overproduction was dose- and time-dependent (P<0.01) and was related to mitochondrial impairment, DNA damage and decreased viability (P<0.05). Transcript levels of TNFα, IL-1ß and IL-6 increased, as confirmed by an ELISA test. Raman spectra were acquired to assess MWCNT-cells interactions. The almost superimposable pro-oxidant activity of both CNTs could be imputable to excessive lengths with regard to the pristine MWCNTs and to the eroded surface, causing increased reactivity, with regard to functionalised MWCNTs. Considering the ease with which lightweight MWCNTs aerosolize and the increased production, the results underlined the potential onset of neurodegenerative diseases, due to unintentional MWCNT exposure.

Evaluation of occupational exposure in magnetic resonance sites.

PURPOSE: In an attempt to evaluate the exposure level of magnetic resonance imaging (MRI) workers to static magnetic fields, the isotropic magnetic flux density values were integrated over time to produce the cumulative exposure. To protect occupational staff a further precautionary step is proposed by introducing a weighting function incorporating the limits imposed by the Italian legislation. The results obtained should be reported, at the end of each working day, on a special dose card, in order to record each worker's exposure to the static magnetic field. Moreover, this dose card could be an important tool if long-term effects occur because it provides a complete history of the occupational exposure in an MRI site. MATERIALS AND METHODS: To conduct measurements, three Hall-sensor probes were used. The consistency of experimental data, tools and methodologies used was evaluated by performing the Kruskal-Wallis test. Finally, the weighted magnitude of the magnetic flux density was integrated over time to obtain global exposure. RESULTS: Measurements were performed on different MRI scanners ranging from 0.25 up to 3.0 T. The results obtained were compared with the 200 mT·h, which represents the upper limit of the Italian regulation. In no case was the 200 mT·h per day exposure exceeded: however, when the strength of the magnetic field was >200 mT the weighted function overestimated the exposure, so that it represents a highly precautionary measure taking into account possible acute and long-term effects. In addition, from the data recorded during patient positioning operations by MRI staff the dB/dt curve was obtained. CONCLUSIONS: The areas obtained from the integral of the weighted static magnetic field strength over time can be indicative of the global exposure of the occupational staff. These values should be reported on a special dose card that could be considered as an important tool if long-term effects occur because it provides a complete history of the occupational exposure in an MRI site.