Extracorporeal membrane oxygenation and effects on tendon tissue: A vibrational spectroscopy study.

Extracorporeal membrane oxygenation (ECMO) is an invasive medical technique used to provide life support in persons with insufficient cardiac and respiratory functionalities, or to preserve, postmortem, and organ function addressing organ/tissue transplant. Although a lot of information is available about organs in their entirety, the safety and effectiveness of allogeneic tissues collected from ECMO donors have not been fully elucidated. In this preliminary study, samples of tibial and peroneal human tendons were analyzed along their length with Raman microspectroscopy and attenuated total reflection-Fourier transform infrared micro-imaging. Both techniques evidenced a different chemical composition in the terminal with respect to the central part of the tendon. Thus, a differentiated analysis was performed depending on the specific position with respect to the bone or the muscle junctions. Spectroscopic analyses showed significant differences in the characteristics of the extracellular matrix between tendons from ECMO and non-ECMO donors, suggesting changes in the amino acid (proline and hydroxyproline) content and protein structure.

FTIR Analysis of Renal Tissue for the Assessment of Hypertensive Organ Damage and proANP31-67 Treatment.

The kidneys are one of the main end organs targeted by hypertensive disease. Although the central role of the kidneys in the regulation of high blood pressure has been long recognized, the detailed mechanisms behind the pathophysiology of renal damage in hypertension remain a matter of investigation. Early renal biochemical alterations due to salt-induced hypertension in Dahl/salt-sensitive rats were monitored by Fourier-Transform Infrared (FTIR) micro-imaging. Furthermore, FTIR was used to investigate the effects of proANP31-67, a linear fragment of pro-atrial natriuretic peptide, on the renal tissue of hypertensive rats. Different hypertension-induced alterations were detected in the renal parenchyma and blood vessels by the combination of FTIR imaging and principal component analysis on specific spectral regions. Changes in amino acids and protein contents observed in renal blood vessels were independent of altered lipid, carbohydrate, and glycoprotein contents in the renal parenchyma. FTIR micro-imaging was found to be a reliable tool for monitoring the remarkable heterogeneity of kidney tissue and its hypertension-induced alterations. In addition, FTIR detected a significant reduction in these hypertension-induced alterations in the kidneys of proANP31-67-treated rats, further indicating the high sensitivity of this cutting-edge imaging modality and the beneficial effects of this novel medication on the kidneys.

Brillouin-Raman micro-spectroscopy and machine learning techniques to classify osteoarthritic lesions in the human articular cartilage.

In this study, Brillouin and Raman micro-Spectroscopy (BRamS) and Machine Learning were used to set-up a new diagnostic tool for Osteoarthritis (OA), potentially extendible to other musculoskeletal diseases. OA is a degenerative pathology, causing the onset of chronic pain due to cartilage disruption. Despite this, it is often diagnosed late and the radiological assessment during the routine examination may fail to recognize the threshold beyond which pharmacological treatment is no longer sufficient and prosthetic replacement is required. Here, femoral head resections of OA-affected patients were analyzed by BRamS, looking for distinctive mechanical and chemical markers of the progressive degeneration degree, and the result was compared to standard assignment via histological staining. The procedure was optimized for diagnostic prediction by using a machine learning algorithm and reducing the time required for measurements, paving the way for possible future in vivo characterization of the articular surface through endoscopic probes during arthroscopy.

Methodology for clinical research.

A clinical research requires a systematic approach with diligent planning, execution and sampling in order to obtain reliable and validated results, as well as an understanding of each research methodology is essential for researchers. Indeed, selecting an inappropriate study type, an error that cannot be corrected after the beginning of a study, results in flawed methodology. The results of clinical research studies enhance the repertoire of knowledge regarding a disease pathogenicity, an existing or newly discovered medication, surgical or diagnostic procedure or medical device. Medical research can be divided into primary and secondary research, where primary research involves conducting studies and collecting raw data, which is then analysed and evaluated in secondary research. The successful deployment of clinical research methodology depends upon several factors. These include the type of study, the objectives, the population, study design, methodology/techniques and the sampling and statistical procedures used. Among the different types of clinical studies, we can recognize descriptive or analytical studies, which can be further categorized in observational and experimental. Finally, also pre-clinical studies are of outmost importance, representing the steppingstone of clinical trials. It is therefore important to understand the types of method for clinical research. Thus, this review focused on various aspects of the methodology and describes the crucial steps of the conceptual and executive stages.

Ethical considerations regarding animal experimentation.

Animal experimentation is widely used around the world for the identification of the root causes of various diseases in humans and animals and for exploring treatment options. Among the several animal species, rats, mice and purpose-bred birds comprise almost 90% of the animals that are used for research purpose. However, growing awareness of the sentience of animals and their experience of pain and suffering has led to strong opposition to animal research among many scientists and the general public. In addition, the usefulness of extrapolating animal data to humans has been questioned. This has led to Ethical Committees' adoption of the 'four Rs' principles (Reduction, Refinement, Replacement and Responsibility) as a guide when making decisions regarding animal experimentation. Some of the essential considerations for humane animal experimentation are presented in this review along with the requirement for investigator training. Due to the ethical issues surrounding the use of animals in experimentation, their use is declining in those research areas where alternative in vitro or in silico methods are available. However, so far it has not been possible to dispense with experimental animals completely and further research is needed to provide a road map to robust alternatives before their use can be fully discontinued.

Hydration Dynamics of Model Peptides with Different Hydrophobic Character.

The multi-scale dynamics of aqueous solutions of the hydrophilic peptide N-acetyl-glycine-methylamide (NAGMA) have been investigated through extended frequency-range depolarized light scattering (EDLS), which enables the broad-band detection of collective polarizability anisotropy fluctuations. The results have been compared to those obtained for N-acetyl-leucinemethylamide (NALMA), an amphiphilic peptide which shares with NAGMA the same polar backbone, but also contains an apolar group. Our study indicates that the two model peptides induce similar effects on the fast translational dynamics of surrounding water. Both systems slow down the mobility of solvating water molecules by a factor 6-8, with respect to the bulk. Moreover, the two peptides cause a comparable far-reaching spatial perturbation extending to more than two hydration layers in diluted conditions. The observed concentration dependence of the hydration number is explained considering the random superposition of different hydration shells, while no indication of solute aggregation phenomena has been found. The results indicate that the effect on the dynamics of water solvating the amphiphilic peptide is dominated by the hydrophilic backbone. The minor impact of the hydrophobic moiety on hydration features is consistent with structural findings derived by Fourier transform infrared (FTIR) measurements, performed in attenuated total reflectance (ATR) configuration. Additionally, we give evidence that, for both systems, the relaxation mode in the GHz frequency range probed by EDLS is related to solute rotational dynamics. The rotation of NALMA occurs at higher timescales, with respect to the rotation of NAGMA; both processes are significantly slower than the structural dynamics of hydration water, suggesting that solute and solvent motions are uncoupled. Finally, our results do not indicate the presence of super-slow water (relaxation times in the order of tens of picoseconds) around the peptides investigated.

Early cardiac-chamber-specific fingerprints in heart failure with preserved ejection fraction detected by FTIR and Raman spectroscopic techniques.

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) is a matter of investigation and its diagnosis remains challenging. Although the mechanisms that are responsible for the development of HFpEF are not fully understood, it is well known that nearly 80% of patients with HFpEF have concomitant hypertension. We investigated whether early biochemical alterations were detectable during HFpEF progression in salt-induced hypertensive rats, using Fourier-transformed infrared (FTIR) and Raman spectroscopic techniques as a new diagnostic approach. Greater protein content and, specifically, greater collagen deposition were observed in the left atrium and right ventricle of hypertensive rats, together with altered metabolism of myocytes. Additionally, Raman spectra indicated a conformational change, or different degree of phosphorylation/methylation, in tyrosine-rich proteins. A correlation was found between tyrosine content and cardiac fibrosis of both right and left ventricles. Microcalcifications were detected in the left and right atria of control animals, with a progressive augmentation from six to 22 weeks. A further increase occurred in the left ventricle and right atrium of 22-week salt-fed animals, and a positive correlation was shown between the mineral deposits and the cardiac size of the left ventricle. Overall, FTIR and Raman techniques proved to be sensitive to early biochemical changes in HFpEF and preceded clinical humoral and imaging markers.

Brillouin and Raman Micro-Spectroscopy: A Tool for Micro-Mechanical and Structural Characterization of Cortical and Trabecular Bone Tissues.

Human bone is a specialized tissue with unique material properties, providing mechanical support and resistance to the skeleton and simultaneously assuring capability of adaptation and remodelling. Knowing the properties of such a structure down to the micro-scale is of utmost importance, not only for the design of effective biomimetic materials but also to be able to detect pathological alterations in material properties, such as micro-fractures or abnormal tissue remodelling. The Brillouin and Raman micro-spectroscopic (BRmS) approach has the potential to become a first-choice technique, as it is capable of simultaneously investigating samples' mechanical and structural properties in a non-destructive and label-free way. Here, we perform a mapping of cortical and trabecular bone sections of a femoral epiphysis, demonstrating the capability of the technique for discovering the morpho-mechanics of cells, the extracellular matrix, and marrow constituents. Moreover, the interpretation of Brillouin and Raman spectra merged with an approach of data mining is used to compare the mechanical alterations in specimens excised from distinct anatomical areas and subjected to different sample processing. The results disclose in both cases specific alterations in the morphology and/or in the tissue chemical make-up, which strongly affects bone mechanical properties, providing a method potentially extendable to other important biomedical issues.

Amyloid Self-Assembly of Lysozyme in Self-Crowded Conditions: The Formation of a Protein Oligomer Hydrogel.

A method is designed to quickly form protein hydrogels, based on the self-assembly of highly concentrated lysozyme solutions in acidic conditions. Their properties can be easily modulated by selecting the curing temperature. Molecular insights on the gelation pathway, derived by in situ FTIR spectroscopy, are related to calorimetric and rheological results, providing a consistent picture on structure-property correlations. In these self-crowded samples, the thermal unfolding induces the rapid formation of amyloid aggregates, leading to temperature-dependent quasi-stationary levels of antiparallel cross ß-sheet links, attributed to kinetically trapped oligomers. Upon subsequent cooling, thermoreversible hydrogels develop by the formation of interoligomer contacts. Through heating/cooling cycles, the starting solutions can be largely recovered back, due to oligomer-to-monomer dissociation and refolding. Overall, transparent protein hydrogels can be easily formed in self-crowding conditions and their properties explained, considering the formation of interconnected amyloid oligomers. This type of biomaterial might be relevant in different fields, along with analogous systems of a fibrillar nature more commonly considered.

Effect of DMSO on the Mechanical and Structural Properties of Model and Biological Membranes.

Dimethyl sulfoxide (DMSO) is widely used in a number of biological and biotechnological applications, mainly because of its effects on the cell plasma membrane, but the molecular origins of this action are yet to be fully clarified. In this work, we used two- and three-component synthetic membranes (liposomes) and the plasma membrane of human erythrocytes to investigate the effect of DMSO when added to the membrane-solvating environment. Fourier transform infrared spectroscopy and thermal fluctuation spectroscopy revealed significant differences in the response of the two types of liposome systems to DMSO in terms of the bilayer thermotropic behavior, available free volume of the bilayer, its excess surface area, and bending elasticity. DMSO also alters the mechanical properties of the erythrocyte membrane in a concentration-dependent manner and is capable of increasing membrane permeability to ATP at even relatively low concentrations (3% v/v and above). Taken in its entirety, these results show that DMSO is likely to have a differential effect on heterogeneous biological membranes, depending on their local composition and structure, and could affect membrane-hosted biological functions.

Natural small molecules as inhibitors of coronavirus lipid-dependent attachment to host cells: a possible strategy for reducing SARS-COV-2 infectivity?

BACKGROUND: Viral infectivity depends on interactions between components of the host cell plasma membrane and the virus envelope. Here we review strategies that could help stem the advance of the SARS-COV-2 epidemic. METHODS AND RESULTS: We focus on the role of lipid structures, such as lipid rafts and cholesterol, involved in the process, mediated by endocytosis, by which viruses attach to and infect cells. Previous studies have shown that many naturally derived substances, such as cyclodextrin and sterols, could reduce the infectivity of many types of viruses, including the coronavirus family, through interference with lipid-dependent attachment to human host cells. CONCLUSIONS: Certain molecules prove able to reduce the infectivity of some coronaviruses, possibly by inhibiting viral lipid-dependent attachment to host cells. More research into these molecules and methods would be worthwhile as it could provide insights the mechanism of transmission of SARS-COV-2 and, into how they could become a basis for new antiviral strategies.

Free volume and dynamics in a lipid bilayer.

The lateral diffusion of lipids and of small molecules inside a membrane is strictly related to the arrangement of acyl chains and to their mobility. In this study, we use FTIR and time resolved 2D-IR spectroscopic techniques to characterize the structure and dynamics of the hydrophobic region of palmitoyl-oleylphosphatidylcholine/cholesterol vesicles dispersed in water/dimethylsulfoxide solutions. By means of a non-polar probe, hexacarbonyl tungsten, we monitor the distribution of free volumes inside the bilayer and the conformational dynamics of hydrophobic tails in relation to the different compositions of the membrane or the different compositions of the solvent. Despite the important structural changes induced by the presence of DMSO in the solvating medium, the picosecond dynamics of the membrane is preserved under the different conditions.

Solvation properties of raft-like model membranes.

Dimethyl sulfoxide (DMSO) is a universal water-soluble solvent widely used in many biotechnological and medical applications, such as cells cryopreservation, and for the treatment of different human diseases (e.g. amyloidosis). Despite the great number of reported studies, the effects of DMSO on the physico-chemical properties of biological membranes are poorly understood. Often, these studies are limited to model membranes composed of phosphatidylcholines (PCs) and cholesterol (Chol). In this work, we explored the effect of DMSO on liposomes composed of the natural egg sphingomyelin (ESM) and Chol as raft-like model membranes. With a multi-technique approach we probe the structure and the thermal stability of ESM/Chol bilayer at different Chol mole fractions. In particular, we investigate the ESM-solvent interactions to clarify the role of DMSO in perturbing the solvating conditions of lipid vesicles and show that the addition of DMSO increases the thermal stability of vesicles. An increase of transition temperature, a decrease of both enthalpy and entropy as well as a decrease of the cooperativity of the gel to liquid phase transition are observed at 0.1 DMSO mole fraction. Fluorescence experiments with the probe Laurdan and FTIR spectra strongly indicate that DMSO exerts a dehydration effect on the membrane. Besides, FTIR measurements with tungsten hexacarbonyl, in combination with fluorescence data of the probe NBD-PE, indicate that DMSO promotes the formation of a highly packed membrane by reducing the thickness of the membrane.

Trehalose-induced slowdown of lysozyme hydration dynamics probed by EDLS spectroscopy.

We use extended depolarized light scattering spectroscopy to study the dynamics of water in a lysozyme-trehalose aqueous solution over a broad time scale, from hundreds to fractions of picoseconds. We provide experimental evidence that the sugar, present in the ternary solution in quantity relevant for biopreservation, strongly modifies the solvation properties of the protein. By comparing aqueous solutions of lysozyme with and without trehalose, we show that the combined action of sugar and protein produces an exceptional dynamic slowdown of a fraction of water molecules around the protein, which become more than twice slower than in the absence of trehalose. We speculate that this ultraslow water may be caged between the sugar and protein surface, consistently with a water entrapment scenario. We also demonstrate that the dynamics of these water molecules gets slower and slower upon cooling. On the basis of these findings, we believe such ultraslow water close to the lysozyme is likely to be involved in the mechanism of bioprotection.

Influence of Dimethyl Sulfoxide on the Low-Temperature Behavior of Cholesterol-Loaded Palmitoyl-oleyl-phosphatidylcholine Membranes.

The properties of lipid membranes at low temperature are important for a number of biomedical and biotechnological applications, and the success of these applications depends on understanding the effects of temperature changes on intermolecular lipid-lipid and lipid-water interactions. Here, we use Fourier transform infrared spectroscopy to study lipid suspensions in water/dimethyl sulfoxide (DMSO) solutions in the -60 to 30 °C range. DMSO is a cryopreservative agent of cellular systems, and its action is largely related to its interaction with the lipid membrane, especially in the low-temperature regime. In the present work, we analyze the effects of solvent composition on the structural and thermotropic properties of cholesterol (chol)-loaded liposomes of palmitoyl-oleylphosphatidylcholine (POPC) because POPC/chol liposomes are suitable models of the plasmatic membrane. To this extent, we compare the properties of lipid vesicles suspended in water and water/DMSO solution at 0.10 DMSO mole fraction and we observe that the gel phase of the membrane has an increased thermal stability on DMSO addition. We estimate that the amount of unfrozen water at T = -60 °C is much reduced by the presence of DMSO, both in the gel- and the liquid-ordered phase of the membrane. Interestingly, we also evidence a reduced hydration of the lipid heads in the presence of DMSO when the vesicles are dispersed in a liquid solution, whereas the addition of DMSO does not alter the hydration state of phosphate and carbonyl groups in the frozen state of the membrane.

Glioblastoma single-cell microRaman analysis under stress treatments.

Glioblastoma multiforme (GBM) is the most frequent malignant brain tumor characterized by highly heterogeneous subpopulations. In order to reveal the heterogeneous cell response, single cell analysis is an essential requirement. In this study, optical microscopy and Raman microspectroscopy were used to follow the stress response of U251 single cells adherent on a silicon substrate. Cultured cells on silicon substrate were treated with hydrogen peroxide to promote apoptosis. Under these conditions expected changes occurred after a few hours and were revealed by the reduction of cytochrome c, lipid, nucleic acid and protein Raman signals: this ensured the possibility to analyse U251 cell line as grown on Si substrate, and to monitor the response of single cells to stress conditions. As a consequence, we used microRaman to monitor the effects induced by nutrient depletion: a fast change of Raman spectra showed two different sub-populations of sensible and resistant U251 cells. Furthermore, spectral variations after DMSO addition were associated to volume changes and confirmed by morphological analysis. Thus, our results highlight the sensitivity of Raman microspectroscopy to detect rapid variations of macromolecule concentration due to oxidative stress and/or cell volume changes at the single cell level.

DMSO-induced perturbation of thermotropic properties of cholesterol-containing DPPC liposomes.

Dimethyl sulfoxide (DMSO) is a universal solvent widely used in many fields, from basic research to industrial applications. At low concentration, it is the most important cryoprotectant agent against cellular damage caused during a freeze-thaw cycle. Although the effects of this cosolvent on the physico-chemical properties of a lipid bilayer have been extensively studied with both in vitro and in vivo experiments, the molecular mechanism of cryopreservation is not completely understood. Cholesterol (Chol) represents one of the essential cell membrane component and is fundamental to maintain the integrity and fluidity of the membrane. Here we report a study on the effect of DMSO on the stability of Chol-containing model membranes. We investigated the effect of DMSO on thermal stability of model membranes formed by dipalmitoylphospatidylcholine (DPPC) and DPPC/Chol by means of Fourier Transform Infrared Spectroscopy (FT-IR) and Differential Scanning Calorimetry (DSC) measurements. It is well known that cholesterol reduces the thermal stability of DPPC vesicles and also the pre-transition is abolished. Our results show that DMSO induces a stabilization of the lipid bilayer of DPPC liposomes increasing both the pre- and main transition temperatures. In DPPC/Chol liposomes a similar thermal stabilization was observed for the main transition indicating that DMSO is capable to stabilize the lipid bilayer even in the presence of the sterol. Moreover, by direct inspection of the hydration degree of the lipid bilayers, we evidenced the role played by DMSO on the thermal stability of the membrane as connected to the hydration of the polar head groups.

Spectroscopic and Microscopic Studies of Aggregation and Fibrillation of Lysozyme in Water/Ethanol Solutions.

The thermal aggregation of lysozyme has been analyzed in water/ethanol solutions at low pH to induce the specific protein aggregation pathway which leads to fibrillar structures in a few hours. In this solvating medium, the protein undergoes a conformational rearrangement promoting the formation of fibrils that are structurally similar to amyloid ones. As the process evolves with different steps, a multitechnique approach has been used by means of analytical probes that can be selectively sensitive in the detection of the different stages of protein association. Fourier transform infrared spectroscopy, intrinsic fluorescence, stationary fluorescence anisotropy, transmission electron microscopy (TEM), and atomic force microscopy (AFM) measurements have been carried out at different times to access and characterize the whole aggregation pathway. The data recorded with different experimental setups revealed different sensitivity to different stages of protein assembling. The whole set of data together with the direct visualization of different aggregate structures by use of TEM and AFM imaging enable to discuss a possible mechanism of fibrillation.

Concentration dependence of hydration water in a model peptide.

The molecular dynamics of aqueous solutions of a model amphiphilic peptide is studied as a function of concentration by broad-band light scattering experiments. Similarly to protein aqueous solutions, a considerable retardation, of about a factor 6-8, of hydration water dynamics with respect to bulk water is found, showing a slight dependence on solute concentration. Conversely, the average number of water molecules perturbed by the presence of peptide, i.e. the hydration number, appears to be strongly modified by adding solute. Its behaviour, decreasing upon increasing concentration, can be interpreted considering the random close-to-contact condition experienced by solute particles. Overall, the present findings support the view of a "long range" effect of peptides on the surrounding water, extending beyond the first two hydration shells.

Complex dynamical aspects of organic electrolyte solutions.

Molecular dynamics of acetone-alkali metal halide (LiBr, LiI) solutions were investigated using depolarized Rayleigh scattering (DRS) and low-frequency Raman spectroscopy in the frequency range from ~0.5 to 200 cm(-1) (~20 GHz to 6 THz). These experiments probe fast dynamical fluctuations of the polarizability anisotropy at picosecond and sub-picosecond time scales that are mainly driven by acetone orientational dynamics. Two distinct contributions were revealed: a fast process (units of picosecond, ps) related to the essentially unperturbed bulk solvent and a slow one (tens of ps) assigned to acetone molecules forming Li(+) solvation shells, decelerated by the motional constraint imposed by the cation. The increase of LiBr and LiI concentration significantly slows down the overall solvent relaxation as a consequence of the increased fraction of acetone molecules involved in the ion solvation shells. The global retardation is larger in LiI than LiBr solutions consistently with viscosity trends. This is explained in terms of ion association (at least ion pairing) more favorably promoted by Br(-) than I(-), with reduced Li(+)-acetone interactions in LiBr than LiI solutions. Anion-induced modulation of the Li(+)···O═C contacts, largely responsible for electrostriction phenomena, also affects the reduced THz-Raman spectral density, ascribed to ultrafast librational motions of acetone molecules. Overall, these findings enlighten the interplay between ion-dipole and ion-ion interactions on the fast solvation dynamics in electrolyte solutions of a typical polar aprotic solvent.