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

Unfolding and aggregation of lysozyme: A thermodynamic and kinetic study by FTIR spectroscopy

International audienceThe unfolding of hen egg-white lysozyme dissolved both in DO and CHCHOD/DO was studied by Fourier Transform Infrared (FTIR) absorption spectroscopy at different protein concentrations. A detailed description of the local and global rearrangement of the secondary structure upon a temperature increase, in the range 295 to 365K, was obtained through the analysis of the amide I band. Thermodynamic parameters for the melting, and the effect of the co-solvent in determining a change in thermal stability of the protein were evaluated. The protein-protein interactions were also followed as a function of temperature: a strong dependence of the cluster stability and aggregation yield on the solvent composition was observed. Finally, FTIR spectra taken at successive time steps of the aggregation enabled intermolecular contacts to be monitored as a function of time, and kinetic information to be obtained showing that both unfolded and folded states of lysozyme act as reactants for the clustering event

More Is Different: Experimental Results on the Effect of Biomolecules on the Dynamics of Hydration Water

Biological interfaces characterized by a complex mixture of hydrophobic, hydrophilic, or charged moieties interfere with the cooperative rearrangement of the hydrogen-bond network of water. In the present study, this solute-induced dynamical perturbation is investigated by extended frequency range depolarized light scattering experiments on an aqueous solution of a variety of systems of different nature and complexity such as small hydrophobic and hydrophilic molecules, amino acids, dipeptides, and proteins. Our results suggest that a reductionist approach is not adequate to describe the rearrangement of hydration water because a significant increase of the dynamical retardation and extension of the perturbation occurs when increasing the chemical complexity of the solute

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

Denaturation and Preservation of Globular Proteins: The Role of DMSO

The thermal denaturation of hen egg white lysozyme (HEWL) in D₂O was followed by IR absorption after addition of dimethyl sulfoxide (DMSO) at different molar fractions. Amide I intensity and position revealed that DMSO reduces the thermal stability of the native protein and favors the formation of ordered aggregates. The comparison with ethanol/water solutions evidenced that ethanol (partially deuterated ethanol EtOD) has a stronger effect on the thermal stability of HEWL: the same down-shift of melting temperature was measured at 0.18 and 0.30 molar fraction of ethanol and DMSO, respectively. This is probably due to lower polarity of EtOD/D₂O with respect to DMSO/D₂O solutions. A kinetic study of protein assembling at 0.30 DMSO molar fraction, was also performed at different temperatures. The high viscosity of the solvent was observed to cause a sensitive slowing down of aggregation rate in comparison to that of water/alcohol solutions. The evidence of a retarded self-assembling put forward a possible explanation for the use of dimethyl sulfoxide as a protectant of protein structure. In fact, for both organic solvents a nonspecific interaction with the protein and a water-mediated action is deduced, but the addition of DMSO reduces the irreversible denaturation due to kinetic effects and this can be exploited for lessening one of the main degradation routes of globular proteins during freezing-thawing cycles

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