Selected Reaction Monitoring (SRM) frente a Western Blot

We propose the SRM technology as a complementary method to the Western Blot for the detection and quantification of proteins in a sample. The technique Western Blot has its own limitations: i) only a protein-of-choice is detected, ignoring any non-relevant proteins, ii) the sensitivity of the technique depends on the specificity of the antibody and iii) Western Blot is expensive and time-consuming. The advantages of SRM with respect Western Blot are remarkable: i) you can detect up to hundreds of different proteins in a sample, ii) SRM is more sensitive, because just 50 copies of the target protein per cell are enough for the detection and iii) once it has been made an investment in the necessary machinery to develop this technique, the detection of proteins in a sample turns into a cheaper, faster, more specific and full-quantitative procedure, without the need of using antibodies. First of all, SRM requires the identification of little peptides, obtained by tryptic digestion, whose sequence must be unique for a single protein or isoform. There is software for that aim. Then, it’s necessary to create isotope-labeled peptides of that identified for acting as internal standards. That sample is introduced in a triple quadrupole mass spectrometer: it passes through a first quadrupole, which functions as a filter, where the fragments are selected, previously ionized, attending to the mass/charge (m/z) relation that correspond to that unique fragments of the protein of interest. In this first selection may be other peptides from other proteins, with the same m/z but with different sequence. To select those that are exclusive from the target protein, the fragments are moved to a second quadrupole, where they are fragmented again with a physical method, and so new smaller fragments are generated. All the new fragments are conduced to the third quadrupole, where just those which come from the protein of interest are selected, attending at their m/z again. The target peptide concentration is determined by measuring the observed signal response for the target peptide relative to that of the isotopic-labeled peptide, the concentration of which is calculated from a pre-determined calibration-response curve. Calibration curves have to be generated for each target peptide in the sample. Because SRM technology is increasing its use, there have been developed databases where the scientific community upload information about protocols and standards for each protein with the aim to facilitate the work to other researchers.

Magnetic and Acoustic Investigations of Turbulent Spherical Couette Flow

Title of dissertation: MAGNETIC AND ACOUSTIC INVESTIGATIONS OF TURBULENT SPHERICAL COUETTE FLOW Matthew M. Adams, Doctor of Philosophy, 2016 Dissertation directed by: Professor Daniel Lathrop Department of Physics This dissertation describes experiments in spherical Couette devices, using both gas and liquid sodium. The experimental geometry is motivated by the Earth's outer core, the seat of the geodynamo, and consists of an outer spherical shell and an inner sphere, both of which can be rotated independently to drive a shear flow in the fluid lying between them. In the case of experiments with liquid sodium, we apply DC axial magnetic fields, with a dominant dipole or quadrupole component, to the system. We measure the magnetic field induced by the flow of liquid sodium using an external array of Hall effect magnetic field probes, as well as two probes inserted into the fluid volume. This gives information about possible velocity patterns present, and we extend previous work categorizing flow states, noting further information that can be extracted from the induced field measurements. The limitations due to a lack of direct velocity measurements prompted us to work on developing the technique of using acoustic modes to measure zonal flows. Using gas as the working fluid in our 60~cm diameter spherical Couette experiment, we identified acoustic modes of the container, and obtained excellent agreement with theoretical predictions. For the case of uniform rotation of the system, we compared the acoustic mode frequency splittings with theoretical predictions for solid body flow, and obtained excellent agreement. This gave us confidence in extending this work to the case of differential rotation, with a turbulent flow state. Using the measured splittings for this case, our colleagues performed an inversion to infer the pattern of zonal velocities within the flow, the first such inversion in a rotating laboratory experiment. This technique holds promise for use in liquid sodium experiments, for which zonal flow measurements have historically been challenging.

Measurement of picosecond lifetimes in neutron-rich Xe isotopes

Background: Lifetimes of nuclear excited states in fission fragments have been studied in the past following isotope separation, thus giving access mainly to the fragments' daughters and only to long-lived isomeric states in the primary fragments. For the first time now, short-lived excited states in the primary fragments, produced in neutron-induced prompt fission of U-235 and Pu-241, were studied within the EXILL&FATIMA campaign at the intense neutron-beam facility of the Institute Laue-Langevin in Grenoble. Purpose: We aim to investigate the quadrupole collective properties of neutron-rich even-even Xe-138,Xe-140,Xe-142 isotopes lying between the double shell closure N = 82 and Z = 50 and a deformed region with octupole collectivity. Method: The gamma rays emitted from the excited fragments were detected with a mixed array consisting of 8 HPGe EXOGAM Clover detectors (EXILL) and 16 LaBr3(Ce) fast scintillators (FATIMA). The detector system has the unique ability to select the interesting fragment making use of the high resolution of the HPGe detectors and determine subnanosecond lifetimes using the fast scintillators. For the analysis the generalized centroid difference method was used. Results: We show that quadrupole collectivity increases smoothly with increasing neutron number above the closed N = 82 neutron shell. Our measurements are complemented by state-of-the-art theory calculations based on shell-model descriptions. Conclusions: The observed smooth increase in quadrupole collectivity is similar to the evolution seen in the measured masses of the xenon isotopic chain and is well reproduced by theory. This behavior is in contrast to higher Z even-even nuclei where abrupt change in deformation occurs around N = 90.

Liquid chromatographic-mass spectrometric method for determination of drug content uniformity of two commonly used dermatology medications in a split-tablet dosage form

Purpose: To develop and validate a simple, efficient and reliable Liquid chromatographic-mass spectrometric (LC-MS/MS) method for the quantitative determination of two dermatological drugs, Lamisil® (terbinafine) and Proscar® (finasteride), in split tablet dosage form. Methods: Thirty tablets each of the 2 studied medications were randomly selected. Tablets were weighed and divided into 3 groups. Ten tablets of each drug were kept intact, another group of 10 tablets were manually split into halves using a tablet cutter and weighed with an analytical balance; a third group were split into quarters and weighed. All intact and split tablets were individually dissolved in a water: methanol mixture (4:1), sonicated, filtered and further diluted with mobile phase. Optimal chromatographic separation and mass spectrometric detection were achieved using an Agilent 1200 HPLC system coupled with an Agilent 6410 triple quadrupole mass spectrometer. Analytes were eluted through an Agilent eclipse plus C8 analytical column (150 mm × 4.6 mm, 5 μm) with a mobile phase composed of solvent A (water) containing 0.1% formic acid and 5mM ammonium formate pH 7.5, and solvent B (acetonitrile mixed with water in a ratio A:B 55:45) at a flow rate of 0.8 mL min-1 with a total run time of 12 min. Mass spectrometric detection was carried out using positive ionization mode with analyte quantitation monitored by multiple reaction monitoring (MRM) mode. Results: The proposed analytical method proved to be specific, robust and adequately sensitive. The results showed a good linear fit over the concentration range of 20 - 100 ng mL-1 for both analytes, with a correlation coefficient (r2) ≥ 0.999 and 0.998 for finasteride and terbinafine, respectively. Following tablet splitting, the drug content of the split tablets fell outside of the proxy USP specification for at least 14 halves (70 %) and 34 quarters (85 %) of FIN, as well as 16 halves (80 %) and 37 quarters (92.5 %) of TBN. Mean weight loss, after splitting, was 0.58 and 2.22 % for FIN half- and quarter tablets, respectively, and 3.96 and 4.09 % for TBN half- and quarter tablets,respectively. Conclusion: The proposed LC-MS/MS method has successfully been used to provide precise drug content uniformity of split tablets of FIN and TBN. Unequal distribution of the drug on the split tablets is indicated by the high standard deviation beyond the accepted value. Hence, it is recommended not to split non-scored tablets especially, for those medications with significant toxicity

Assessment oxidative stress biomarkers –neuroprostanes and dihomo-isoprostanes- in elite triathletes urine after two weeks of moderate altitude training

This randomized and controlled trial investigated whether the increase in elite training at different altitudes altered the oxidative stress biomarkers of the nervous system. This is the first study to investigate four F4-neuroprostanes and four F2-dihomo-isoprostanes quantified in 24-hour urine. The quantification was carried out by Ultra High Pressure Liquid Chromatography-triple Quadrupole-Tandem Mass Spectrometry (UHPLC-QqQ-MS/MS). Sixteen elite triathletes agreed to participate in the project. They were randomized in two groups, a group submitted to Altitude Training (n=8) and a group submitted to Sea Level Training (n=8), with a Control group of non-athletes (n=8). After experimental period, the Altitude Training group triathletes gave significant data: 17-epi-17-F2t-dihomo-IsoP (from 5.2 ± 1.4 µg/mL 24 h-1 to 6.6 ± 0.6 µg/mL 24 h-1), ent-7(RS)-7-F2t-dihomo-IsoP (from 6.6 ± 1.7 µg/mL 24 h-1 to 8.6 ± 0.9 µg /mL 24 h-1), and ent-7-epi-7-F2t-dihomo-IsoP (from 8.4 ± 2.2 µg/mL 24 h-1 to 11.3 ± 1.8 µg/mL 24 h-1) increased, while, of the neuronal degeneration-related compounds, only 10-epi-10-F4t-NeuroP (8.4 ± 1.7 µg/mL 24 h-1) and 10-F4t-NeuroP (5.2 ± 2.9 µg/mL 24 h-1) were detected in this group. For the control group and sea level training groups, no significant changes had occurred at the end of the 2-weeks experimental period. Therefore, and as the main conclusion, the training at moderate altitude increased the F4-NeuroPs- and F2-dihomo-isoPs-related oxidative damage of the central nervous system (CNS) compared to similar training at sea level.

Laser cooling and trapping of argon metastable atomic beam

The high velocity of free atoms associated with the thermal motion, together with the velocity distribution of atoms has imposed the ultimate limitation on the precision of ultrahigh resolution spectroscopy. A sample consisting of low velocity atoms would provide a substantial improvement in spectroscopy resolution. To overcome the problem of thermal motion, atomic physicists have pursued two goals; first, the reduction of the thermal motion (cooling); and second, the confinement of the atoms by means of electromagnetic fields (trapping). Cooling carried sufficiently far, eliminates the motional problems, whereas trapping allows for long observation times. In this work the laser cooling and trapping of an argon atomic beam will be discussed. The experiments involve a time-of-flight spectroscopy on metastable argon atoms. Laser deceleration or cooling of atoms is achieved by counter propagating a photon against an atomic beam of metastable atoms. The solution to the Doppler shift problem is achieved using spatially varying magnetic field along the beam path to Zeeman shift the atomic resonance frequency so as to keep the atoms in resonance with a fixed frequency cooling laser. For trapping experiments a Magnetooptical trap (MOT) will be used. The MOT is formed by three pairs of counter-propagating laser beams with mutual opposite circular polarization and a frequency tuned slightly below the center of the atomic resonance and superimposed on a magnetic quadrupole field.

Development of advanced analytical methodologies for Alzheimer’s disease drug discovery

Advanced analytical methodologies were developed to characterize new potential active MTDLs on isolated targets involved in the first stages of Alzheimer’s disease (AD). In addition, the methods investigated drug-protein bindings and evaluated protein-protein interactions involved in the neurodegeneration. A high-throughput luminescent assay allowed the study of the first in class GSK-3β/ HDAC dual inhibitors towards the enzyme GSK-3β. The method was able to identify an innovative disease-modifying agent with an activity in the micromolar range both on GSK-3β, HDAC1 and HDAC6. Then, the same assay reliably and quickly selected true positive hit compounds among natural Amaryllidaceae alkaloids tested against GSK-3β. Hence, given the central role of the amyloid pathway in the multifactorial nature of AD, a multi-methodological approach based on mass spectrometry (MS), circular dichroism spectroscopy (CD) and ThT assay was applied to characterize the potential interaction of CO releasing molecules (CORMs) with Aβ1-42 peptide. The comprehensive method provided reliable information on the different steps of the fibrillation process and regarding CORMs mechanism of action. Therefore, the optimal CORM-3/Aβ1−42 ratio in terms of inhibitory effect was identified by mass spectrometry. CD analysis confirmed the stabilizing effect of CORM-3 on the Aβ1−42 peptide soluble form and the ThT Fluorescent Analysis ensured that the entire fibrillation process was delayed. Then the amyloid aggregation process was studied in view of a possible correlation with AD lipid brain alterations. Therefore, SH-SY5Y cells were treated with increasing concentration of Aß1-42 at different times and the samples were analysed by a RP-UHPLC system coupled with a high-resolution quadrupole TOF mass spectrometer in comprehensive data-independent SWATH acquisition mode. Each lipid class profiling in SH-SY5Y cells treated with Aß1-42 was compared to the one obtained from the untreated. The approach underlined some peculiar lipid alterations, suitable as biomarkers, that might be correlated to Aß1-42 different aggregation species.

Study of charge, spin, and structural orderings in quantum materials using nuclei and muons as local probes

Quantum Materials are many body systems displaying emergent phenomena caused by quantum collective behaviour, such as superconductivity, charge density wave, fractional hall effect, and exotic magnetism. Among quantum materials, two families have recently attracted attention: kagome metals and Kitaev materials. Kagome metals have a unique crystal structure made up of triangular lattice layers that are used to form the kagome layer. Due to superconductivity, magnetism, and charge ordering states such as the Charge Density Wave (CDW), unexpected physical phenomena such as the massive Anomalous Hall Effect (AHE) and possible Majorana fermions develop in these materials. Kitaev materials are a type of quantum material with a unique spin model named after Alexei Kitaev. They include fractional fluctuations of Majorana fermions and non-topological abelian anyons, both of which might be used in quantum computing. Furthermore, they provide a realistic framework for the development of quantum spin liquid (QSL), in which quantum fluctuations produce long-range entanglements between electronic states despite the lack of classical magnetic ordering. In my research, I performed several nuclear magnetic resonance (NMR), nuclear quadrupole resonance (NQR), and muon spin spectroscopy (µSR) experiments to explain and unravel novel phases of matter within these unusual families of materials. NMR has been found to be an excellent tool for studying these materials’ local electronic structures and magnetic properties. I could use NMR to determine, for the first time, the structure of a novel kagome superconductor, RbV3Sb5, below the CDW transition, and to highlight the role of chemical doping in the CDW phase of AV3Sb5 superconductors. µSR has been used to investigate the effect of doping on kagome material samples in order to study the presence and behaviour of an anomalous phase developing at low temperatures and possibly related to time-reversal symmetry breaking.

Study of the urinary biomarkers of synthetic cannabinoid receptor agonists (SCRAs) for the forensic unequivocal proof of consumption: medico-legal implications for drug abuse prevention

Introduction. Synthetic cannabinoid receptor agonists (SCRAs) represent the widest group of New Psychoactive Substances (NPS) and, around 2021-2022, new compounds emerged on the market. The aims of the present research were to identify suitable urinary markers of Cumyl-CB-MEGACLONE, Cumyl-NB-MEGACLONE, Cumyl-NB-MINACA, 5F-EDMB-PICA, EDMB-PINACA and ADB-HEXINACA, to present data on their prevalence and to adapt the methodology from the University of Freiburg to the University of Bologna. Materials and methods. Human phase-I metabolites detected in 46 authentic urine samples were confirmed in vitro with pooled human liver microsomes (pHLM) assays, analyzed by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-qToF-MS). Prevalence data were obtained from urines collected for abstinence control programs. The method to study SCRAs metabolism in use at the University of Freiburg was adapted to the local facilities, tested in vitro with 5F-EDMB-PICA and applied to the study of ADB-HEXINACA metabolism. Results. Metabolites built by mono, di- and tri-hydroxylation were recommended as specific urinary biomarkers to monitor the consumption of SCRAs bearing a cumyl moiety. Monohydroxylated and defluorinated metabolites were suitable proof of 5F-EDMB-PICA consumption. Products of monohydroxylation and amide or ester hydrolysis, coupled to monohydroxylation or ketone formation, were recognized as specific markers for EDMB-PINACA and ADB-HEXINACA. The LC-qToF-MS method was successfully adapted to the University of Bologna, as tested with 5F-EDMB-PICA in vitro metabolites. Prevalence data showed that 5F-EDMB-PINACA and EDMB-PINACA were more prevalent than ADB-HEXINACA, but for a limited period. Conclusion. Due to undetectability of parent compounds in urines and to shared metabolites among structurally related compounds, the identification of specific urinary biomarkers as unequivocal proofs of SCRAs consumption remains challenging for forensic laboratories. Urinary biomarkers are necessary to monitor SCRAs abuse and prevalence data could help in establishing tailored strategies to prevent their spreading, highlighting the role for legal medicine as a service to public health.