Isolated total RNA and protein are preserved after thawing for more than twenty-four hours

OBJECTIVE: The preservation of biological samples at a low temperature is important for later biochemical and/or histological analyses. However, the molecular viability of thawed samples has not been studied sufficiently in depth. The present study was undertaken to evaluate the viability of intact tissues, tissue homogenates, and isolated total RNA after defrosting for more than twenty-four hours. METHODS: The molecular viability of the thawed samples (n = 82) was assessed using the A260/A280 ratio, the RNA concentration, the RNA integrity, the level of intact mRNA determined by reverse transcriptase polymerase chain reaction, the protein level determined by Western blotting, and an examination of the histological structure. RESULTS: The integrity of the total RNA was not preserved in the thawed intact tissue, but the RNA integrity and level of mRNA were perfectly preserved in isolated defrosted samples of total RNA. Additionally, the level of beta-actin protein was preserved in both thawed intact tissue and homogenates. CONCLUSION: Isolated total RNA does not undergo degradation due to thawing for at least 24 hours, and it is recommended to isolate the total RNA as soon as possible after tissue collection. Moreover, the protein level is preserved in defrosted tissues.

A LED Based Photometer for Solid Phase Photometry: Zinc Determination in Pharmaceutical Preparation Employing a Multicommuted Flow Analysis Approach

In this work, a LED (light emitting diode) based photometer for solid phase photometry is described. The photometer was designed to permit direct coupling of a light source (LED) and a photodiode to a flow cell with an optical pathlength of 4 mm. The flow cell was filled with adsorbing solid phase material (C-18), which was used to immobilize the chromogenic reagent 1-(2-thiazolylazo)-2-naphthol (TAN). Aiming to allow accuracy assessment, samples were also analyzed employing ICP OES (inductively coupled plasma optical emission spectrometry) methodology. Applying the paired t-test at the 95% confidence level, no significant difference was observed. Other useful features were also achieved: linear response ranging from 0.05 to 0.85 mg L-1 Zn, limit of detection of 9 mu g L-1 Zn (3 sigma criterion), standard deviation of 1.4% (n = 10), sampling throughput of 36 determinations per h, and a waste generation and reagent consumption of 1.7 mL and of 0.03 mu g per determination, respectively.

Processing of high resolution magic angle spinning spectra of breast cancer cells by the filter diagonalization method

Proton nuclear magnetic resonance (H-1 NMR) spectroscopy for detection of biochemical changes in biological samples is a successful technique. However, the achieved NMR resolution is not sufficiently high when the analysis is performed with intact cells. To improve spectral resolution, high resolution magic angle spinning (HR-MAS) is used and the broad signals are separated by a T-2 filter based on the CPMG pulse sequence. Additionally, HR-MAS experiments with a T-2 filter are preceded by a water suppression procedure. The goal of this work is to demonstrate that the experimental procedures of water suppression and T-2 or diffusing filters are unnecessary steps when the filter diagonalization method (FDM) is used to process the time domain HR-MAS signals. Manipulation of the FDM results, represented as a tabular list of peak positions, widths, amplitudes and phases, allows the removal of water signals without the disturbing overlapping or nearby signals. Additionally, the FDM can also be used for phase correction and noise suppression, and to discriminate between sharp and broad lines. Results demonstrate the applicability of the FDM post-acquisition processing to obtain high quality HR-MAS spectra of heterogeneous biological materials.

Septic shock in older people: a prospective cohort study

Abstract Background Septic shock is the first cause of death in Intensive Care Units. Despite experimental data showing increased inflammatory response of aged animals following infection, the current accepted hypothesis claims that aged patients are immunocompromised, when compared to young individuals. Results Here, we describe a prospective cohort study designed to analyze the immune profile of this population. Conclusion Older people are as immunocompetent as the young individual, regarding the cytokines, chemokines and growth factors response to devastating infection.

Monte Carlo Simulations of Novel Scintillator Detectors and Dosimetry Calculations

Monte Carlo (MC) simulation techniques are becoming very common in the Medical Physicists community. MC can be used for modeling Single Photon Emission Computed Tomography (SPECT) and for dosimetry calculations. 188Re, is a promising candidate for radiotherapeutic production and understanding the mechanisms of the radioresponse of tumor cells "in vitro" is of crucial importance as a first step before "in vivo" studies. The dosimetry of 188Re, used to target different lines of cancer cells, has been evaluated by the MC code GEANT4. The simulations estimate the average energy deposition/per event in the biological samples. The development of prototypes for medical imaging, based on LaBr3:Ce scintillation crystals coupled with a position sensitive photomultiplier, have been studied using GEANT4 simulations. Having tested, in the simulation, surface treatments different from the one applied to the crystal used in our experimental measurements, we found out that the Energy Resolution (ER) and the Spatial Resolution (SR) could be improved, in principle, by machining in a different way the lateral surfaces of the crystal. We have then studied a system able to acquire both echographic and scintigraphic images to let the medical operator obtain the complete anatomic and functional information for tumor diagnosis. The scintigraphic part of the detector is simulated by GEANT4 and first attempts to reconstruct tomographic images have been made using as method of reconstruction a back-projection standard algorithm. The proposed camera is based on slant collimators and LaBr3:Ce crystals. Within the Field of View (FOV) of the camera, it possible to distinguish point sources located in air at a distance of about 2 cm from each other. In particular conditions of uptake, tumor depth and dimension, the preliminary results show that the Signal to Noise Ratio (SNR) values obtained are higher than the standard detection limit.

Anwendung der konfokalen Laser-Scanning-Mikroskopie zur Charakterisierung der Struktur von Zähnen und von oberflächenmodifiziertem Titan für Knochenimplantate

Zusammenfassung Mittels Fluoreszenzfarbstoffen können Strukturen sichtbar gemacht werden, die auf kon-ventionellem Weg nicht, oder nur schwer darzustellen sind. Besonders in Kombination mit der Konfokalen Laser Scanning Mikroskopie eröffnen sich neue Wege zum spezifischen Nachweis unterschiedlichster Komponenten biologischer Proben und gegebenenfalls deren dreidimensionale Widergabe.Die Visualisierung des Proteinanteils des Zahnhartgewebes kann mit Hilfe chemisch kopplungsfähiger Fluorochrome durchgeführt werden. Um zu zeigen, daß es sich bei dieser Markierung nicht um unspezifische Adsorption des Farbstoffes handelt, wurde zur Kontrolle die Proteinkomponente der Zahnproben durch enzymatischen Verdau beseitigt. Derartig behandelte Präparate wiesen eine sehr geringe Anfärbbarkeit auf.Weiterführend diente diese enzymatische Methode als Negativkontrolle zum Nachweis der Odontoblastenfortsätze im Dentin bzw. im Bereich der Schmelz-Dentin-Grenze. Hiermit konnte differenziert werden zwischen reinen Reflexionsbildern der Dentinkanäle und den Zellausläufern deren Membranen gezielt durch lipophile Fluoreszenzfarbstoffe markiert wurden.In einem weiteren Ansatz konnte gezeigt werden, daß reduzierte und daher nichtfluoreszente Fluoresceinabkömmlinge geeignet sind, die Penetration von Oxidationsmitteln (hier H2O2) in den Zahn nachzuweisen. Durch Oxidation dieser Verbindungen werden fluoreszierende Produkte generiert, die den Nachweis lieferten, daß die als Zahnbleichmittel eingesetzten Mittel rasch durch Schmelz und Dentin bis in die Pulpahöhle gelangen können.Die Abhängigkeit der Fluoreszenz bestimmter Fluorochrome von deren chemischer Um-gebung, im vorliegenden Fall dem pH-Wert, sollte eingesetzt werden, um den Säuregrad im Zahninneren fluoreszenzmikroskopisch darzustellen. Hierbei wurde versucht, ein ratio-metrisches Verfahren zu entwickeln, mit dem die pH-Bestimmung unter Verwendung eines pH-abhängigen und eines pH-unabhängigen Fluorochroms erfolgt. Diese Methode konnte nicht für diese spezielle Anwendung verifiziert werden, da Neutralisationseffekte der mineralischen Zahnsubstanz (Hydroxylapatit) die pH-Verteilung innerhalb der Probe beeinflußen. Fluoreszenztechniken wurden ebenfalls ergänzend eingesetzt zur Charakterisierung von kovalent modifizierten Implantatoberflächen. Die, durch Silanisierung von Titantestkörpern mit Triethoxyaminopropylsilan eingeführten freien Aminogruppen konnten qualitativ durch den Einsatz eines aminspezifischen Farbstoffes identifiziert werden. Diese Art der Funktionalisierung dient dem Zweck, Implantatoberflächen durch chemische Kopplung adhäsionsvermittelnder Proteine bzw. Peptide dem Einheilungsprozeß von Implantaten in den Knochen zugänglicher zu machen, indem knochenbildende Zellen zu verbessertem Anwachsverhalten stimuliert werden. Die Zellzahlbestimmung im Adhäsionstest wurde ebenfalls mittels Fluoreszenzfarbstoffen durchgeführt und lieferte Ergebnisse, die belegen, daß die durchgeführte Modifizierung einen günstigen Einfluß auf die Zelladhäsion besitzt.

Analytical challenges in the fields of Nanobioscience and Nanobiotecnology: integrated methods for separation and characterization

Nano(bio)science and nano(bio)technology play a growing and tremendous interest both on academic and industrial aspects. They are undergoing rapid developments on many fronts such as genomics, proteomics, system biology, and medical applications. However, the lack of characterization tools for nano(bio)systems is currently considered as a major limiting factor to the final establishment of nano(bio)technologies. Flow Field-Flow Fractionation (FlFFF) is a separation technique that is definitely emerging in the bioanalytical field, and the number of applications on nano(bio)analytes such as high molar-mass proteins and protein complexes, sub-cellular units, viruses, and functionalized nanoparticles is constantly increasing. This can be ascribed to the intrinsic advantages of FlFFF for the separation of nano(bio)analytes. FlFFF is ideally suited to separate particles over a broad size range (1 nm-1 μm) according to their hydrodynamic radius (rh). The fractionation is carried out in an empty channel by a flow stream of a mobile phase of any composition. For these reasons, fractionation is developed without surface interaction of the analyte with packing or gel media, and there is no stationary phase able to induce mechanical or shear stress on nanosized analytes, which are for these reasons kept in their native state. Characterization of nano(bio)analytes is made possible after fractionation by interfacing the FlFFF system with detection techniques for morphological, optical or mass characterization. For instance, FlFFF coupling with multi-angle light scattering (MALS) detection allows for absolute molecular weight and size determination, and mass spectrometry has made FlFFF enter the field of proteomics. Potentialities of FlFFF couplings with multi-detection systems are discussed in the first section of this dissertation. The second and the third sections are dedicated to new methods that have been developed for the analysis and characterization of different samples of interest in the fields of diagnostics, pharmaceutics, and nanomedicine. The second section focuses on biological samples such as protein complexes and protein aggregates. In particular it focuses on FlFFF methods developed to give new insights into: a) chemical composition and morphological features of blood serum lipoprotein classes, b) time-dependent aggregation pattern of the amyloid protein Aβ1-42, and c) aggregation state of antibody therapeutics in their formulation buffers. The third section is dedicated to the analysis and characterization of structured nanoparticles designed for nanomedicine applications. The discussed results indicate that FlFFF with on-line MALS and fluorescence detection (FD) may become the unparallel methodology for the analysis and characterization of new, structured, fluorescent nanomaterials.

Method development using ICP-MS and LA-ICP-MS and their application in environmental and material science

Within this PhD thesis several methods were developed and validated which can find applicationare suitable for environmental sample and material science and should be applicable for monitoring of particular radionuclides and the analysis of the chemical composition of construction materials in the frame of ESS project. The study demonstrated that ICP-MS is a powerful analytical technique for ultrasensitive determination of 129I, 90Sr and lanthanides in both artificial and environmental samples such as water and soil. In particular ICP-MS with collision cell allows measuring extremely low isotope ratios of iodine. It was demonstrated that isotope ratios of 129I/127I as low as 10-7 can be measured with an accuracy and precision suitable for distinguishing sample origins. ICP-MS with collision cell, in particular in combination with cool plasma conditions, reduces the influence of isobaric interferences on m/z = 90 and is therefore well-suited for 90Sr analysis in water samples. However, the applied ICP-CC-QMS in this work is limited for the measurement of 90Sr due to the tailing of 88Sr+ and in particular Daly detector noise. Hyphenation of capillary electrophoresis with ICP-MS was shown to resolve atomic ions of all lanthanides and polyatomic interferences. The elimination of polyatomic and isobaric ICP-MS interferences was accomplished without compromising the sensitivity by the use of a high resolution mode as available on ICP-SFMS. Combination of laser ablation with ICP-MS allowed direct micro and local uranium isotope ratio measurements at the ultratrace concentrations on the surface of biological samples. In particular, the application of a cooled laser ablation chamber improves the precision and accuracy of uranium isotopic ratios measurements in comparison to the non-cooled laser ablation chamber by up to one order of magnitude. In order to reduce the quantification problem, a mono gas on-line solution-based calibration was built based on the insertion of a microflow nebulizer DS-5 directly into the laser ablation chamber. A micro local method to determine the lateral element distribution on NiCrAlY-based alloy and coating after oxidation in air was tested and validated. Calibration procedures involving external calibration, quantification by relative sensitivity coefficients (RSCs) and solution-based calibration were investigated. The analytical method was validated by comparison of the LA-ICP-MS results with data acquired by EDX.

From lipid bilayers to synaptic vesicles: atomic force microscopy on lipid-based systems

The aim of this thesis was to apply the techniques of the atomic force microscope (AFM) to biological samples, namely lipid-based systems. To this end several systems with biological relevance based on self-assembly, such as a solid-supported membrane (SSM) based sensor for transport proteins, a bilayer of the natural lipid extract from an archaebacterium, and synaptic vesicles, were investigated by the AFM. For the characterization of transport proteins with SSM-sensors proteoliposomes are adsorbed that contain the analyte (transport protein). However the forces governing bilayer-bilayer interactions in solution should be repulsive under physiological conditions. I investigated the nature of the interaction forces with AFM force spectroscopy by mimicking the adsorbing proteoliposome with a cantilever tip, which was functionalized with charged alkane thiols. The nature of the interaction is indeed repulsive, but the lipid layers assemble in stacks on the SSM, which expose their unfavourable edges to the medium. I propose a model by which the proteoliposomes interact with these edges and fuse with the bilayer stacks, so forming a uniform layer on the SSM. Furthermore I characterized freestanding bilayers from a synthetic phospholipid with a phase transition at 41°C and from a natural lipid extract of the archaebacterium Methanococcus jannaschii. The synthetic lipid is in the gel-phase at room temperature and changes to the fluid phase when heated to 50°C. The bilayer of the lipid extract shows no phase transition when heated from room temperature to the growth temperature (~ 50°C) of the archeon. Synaptic vesicles are the containers of neurotransmitter in nerve cells and the synapsins are a family of extrinsic membrane proteins, that are associated with them, and believed to control the synaptic vesicle cycle. I used AFM imaging and force spectroscopy together with dynamic light scattering to investigate the influence of synapsin I on synaptic vesicles. To this end I used native, untreated synaptic vesicles and compared them to synapsin-depleted synaptic vesicles. Synapsin-depleted vesicles were larger in size and showed a higher tendency to aggregate compared to native vesicles, although their mechanical properties were alike. I also measured the aggregation kinetics of synaptic vesicles induced by synapsin I and found that the addition of synapsin I promotes a rapid aggregation of synaptic vesicles. The data indicate that synapsin I affects the stability and the aggregation state of synaptic vesicles, and confirm the physiological role of synapsins in the assembly and regulation of synaptic vesicle pools within nerve cells.

Il corpo e l'individuo tra proprietà ed autodeterminazione. Con particolare riferimento al fenomeno delle biobanche

La bioetica è il luogo ideale per cercare risposte ai grandi interrogativi concernenti la vita, la morte e la cura dell'essere umano. I recenti dibattiti sull'uso, ed il temuto abuso, del corpo umano in medicina hanno messo in evidenza la necessità di una discussione approfondita sul potere di scelta che l'individuo può esercitare sulla propria mente e sul proprio corpo. Spinta dal desidero di indagare l'estensione di tale potere di scelta ho voluto analizzare le tematiche riguardanti “il corpo”, “l'individuo”, “la proprietà” e “l'autodeterminazione”. L'analisi è stata condotta individuando alcuni dei differenti significati che questi termini assumono nei diversi ambiti che la bioetica lambisce e mostrando, in particolare, la visione di tale realtà attraverso le lenti del giurista. A chi appartiene il corpo? Chi ha il potere di decidere su di esso? Il potere di scelta valica gli antichi i confini legati al corpo del paziente e coinvolge tessuti, organi e cellule staccati dal corpo umano, parti che un tempo erano considerati scarti operatori sono oggi divenuti tesori inestimabili per la ricerca. L'importanza assunta dai campioni biologici ha portato alla creazione di biobanche nelle quali sono raccolti, catalogati e il DNA studiato in campioni biologici Le biobanche riflettono le tensioni della bioetica e del biodititto. Lo studio delle biobanche riguarda, tra l'altro, la riceca dell'equilibrio tra le diverse esigenze meritevoli di tutela: in primo luogo il diritto alla privacy, diritto a che le “proprie informazioni” non vengano divulgate ed il diritto a non essere discriminato ed in secondo luogo le necessità dettate dalla ricerca e dalla scienza medica. Nel 2009 la rivista Times messo biobanche tra le 10 idee in grado di cambiare il mondo anche in considarazione della medicina personalizzata e del fatto che costituiscono una la speranza per la ricerca contro le malattie attuali e future.

Evolutionary epigenetics of modern human populations

Epigenetic variability is a new mechanism for the study of human microevolution, because it creates both phenotypic diversity within an individual and within population. This mechanism constitutes an important reservoir for adaptation in response to new stimuli and recent studies have demonstrated that selective pressures shape not only the genetic code but also DNA methylation profiles. The aim of this thesis is the study of the role of DNA methylation changes in human adaptive processes, considering the Italian peninsula and macro-geographical areas. A whole-genome analysis of DNA methylation profile across the Italian penisula identified some genes whose methylation levels differ between individuals of different Italian districts (South, Centre and North of Italy). These genes are involved in nitrogen compound metabolism and genes involved in pathogens response. Considering individuals with different macro-geographical origins (individuals of Asians, European and African ancestry) more significant DMRs (differentially methylated regions) were identified and are located in genes involved in glucoronidation, in immune response as well as in cell comunication processes. A "profile" of each ancestry (African, Asian and European) was described. Moreover a deepen analysis of three candidate genes (KRTCAP3, MAD1L and BRSK2) in a cohort of individuals of different countries (Morocco, Nigeria, China and Philippines) living in Bologna, was performed in order to explore genetic and epigenetic diversity. Moreover this thesis have paved the way for the application of DNA methylation for the study of hystorical remains and in particular for the age-estimation of individuals starting from biological samples (such as teeth or blood). Noteworthy, a mathematical model that considered methylation values of DNA extracted from cementum and pulp of living individuals can estimate chronological age with high accuracy (median absolute difference between age estimated from DNA methylation and chronological age was 1.2 years).

Original analytical methods for the determination of psychoactive compounds in complex matrices

This thesis work aims to develop original analytical methods for the determination of drugs with a potential for abuse, for the analysis of substances used in the pharmacological treatment of drug addiction in biological samples and for the monitoring of potentially toxic compounds added to street drugs. In fact reliable analytical techniques can play an important role in this setting. They can be employed to reveal drug intake, allowing the identification of drug users and to assess drug blood levels, assisting physicians in the management of the treatment. Pharmacological therapy needs to be carefully monitored indeed in order to optimize the dose scheduling according to the specific needs of the patient and to discourage improper use of the medication. In particular, different methods have been developed for the detection of gamma-hydroxybutiric acid (GHB), prescribed for the treatment of alcohol addiction, of glucocorticoids, one of the most abused pharmaceutical class to enhance sport performance and of adulterants, pharmacologically active compounds added to illicit drugs for recreational purposes. All the presented methods are based on capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) coupled to various detectors (diode array detector, mass spectrometer). Biological samples pre-treatment was carried out using different extraction techniques, liquid-liquid extraction (LLE) and solid phase extraction (SPE). Different matrices have been considered: human plasma, dried blood spots, human urine, simulated street drugs. These developed analytical methods are individually described and discussed in this thesis work.

Entwicklung eines Screeningverfahrens zum simultanen Nachweis von Opioiden in menschlichen Körperflüssigkeiten, Geweben und Haaren für forensisch-toxikologische und klinische Untersuchungen

In den letzten Jahren stieg in Deutschland der Gebrauch bzw. Missbrauch von Opioid-Analgetika zunehmend an. Das entwickelte Verfahren sollte unter Einbeziehung neuer Substanzen möglichst viele verschiedene Opioide und auch ihre pharmakologisch aktiven Stoffwechselprodukte berücksichtigen.rnVor Analyse wurden Blut-, Serum- oder Urinproben mit Phosphatpuffer versetzt und mittels Festphasenextraktion an C18-Säulenmaterial aufgearbeitet. Post-Mortem-Gewebematerial wurde mit isotonischer Kochsalzlösung versetzt, homogenisiert und anschließend durch eine Festphasenextraktion aufgereinigt. Haarproben wurden nach Zerkleinerung mit Methanol unter Ultrabeschallung extrahiert. Die Flüssigchromatographie gekoppelt mit Tandem-Massenspektrometrie (Elektrosprayionisation im positiven Modus) erwies sich als geeignetes Verfahren für die simultane Bestimmung der Opioide in biologischem Probenmaterial (Körperflüssigkeiten, Gewebe und Haaren). Der Multi-Analyt Assay erlaubt die quantitative Analyse von 35 verschiedenen Opioiden. Die Analyten wurden durch eine Phenyl-Hexyl Säule und einen Wasser/Acetonitril Gradienten durch eine UPLC 1290 Infinity gekoppelt mit einem 6490 Triple Quadrupol von Agilent Technologies separiert.rnDie LC/MS Methode zur simultanen Bestimmung von 35 Opioiden in Serum und Haaren wurde nach den Richtlinien der Gesellschaft für Toxikologische und Forensische Chemie (GTFCh) validiert. Im Fall der Serumvalidierung lagen die Nachweisgrenzen zwischen 0.02 und 0.6 ng/ml und die Bestimmungsgrenzen im Bereich von 0.1 bis 2.0 ng/ml. Die Kalibrationskurven waren für die Kalibrationslevel 1 bis 6 linear. Wiederfindungsraten lagen für alle Verbindungen zwischen 51 und 88 %, außer für Alfentanil, Bisnortiliidn, Pethidin und Morphin-3-Glucuronid. Der Matrixeffekt lag zwischen 86 % (Ethylmorphin) und 105 % (Desomorphin). Für fast alle Analyten konnten akzeptable Werte bei der Bestimmung der Genauigkeit und Richtigkeit nach den Richtlinien der GTFCh erhalten werden. Im Fall der Validierung der Haarproben lagen die Nachweisgrenzen zwischen 0.004 und 0.6 ng/Probe und die Bestimmungsgrenzen zwischen 0.1 ng/Probe und 2.0 ng/Probe. Für die Kalibrationslevel 1 bis 6 waren alle Kalibrationsgeraden linear. Die Wiederfindungsraten lagen für die Opioide im Bereich von 73.5 % (Morphin-6-Glucuronid) und 114.1 % (Hydrocodon). Die Werte für die Bestimmung der Richtigkeit lagen zwischen - 6.6 % (Methadon) und + 11.7 % (Pholcodin). Präzisionsdaten wurden zwischen 1.0 % für Dextromethorphan und 11.5 % für Methadon ermittelt. Die Kriterien der GTFCh konnten bei Ermittlung des Matrixeffekts für alle Substanzen erfüllt werden, außer für 6-Monoacetylmorphin, Bisnortilidin, Meperidin, Methadon, Morphin-3-glucuronid, Morphin-6-glucuronid, Normeperidin, Nortilidin und Tramadol.rnZum Test des Verfahrens an authentischem Probenmaterial wurden 206 Proben von Körperflüssigkeiten mit Hilfe der simultanen LC/MS Screening Methode untersucht. Über 150 Proben wurden im Rahmen von forensisch-toxikologischen Untersuchungen am Instituts für Rechtsmedizin Mainz analysiert. Dabei konnten 23 der 35 Opioide in den realen Proben nachgewiesen werden. Zur Untersuchung der Pharmakokinetik von Opioiden bei Patienten der anästhesiologischen Intensivstation mit Sepsis wurden über 50 Blutproben untersucht. Den Patienten wurde im Rahmen einer klinischen Studie einmal täglich vier Tage lang Blut abgenommen. In den Serumproben wurde hauptsächlich Sufentanil (0.2 – 0.8 ng/ml in 58 Fällen) und Piritramid (0.4 – 11 ng/ml in 56 Fällen) gefunden. Außerdem wurden die Proben von Körperflüssigkeiten und Gewebe von 13 verschiedenen Autopsiefällen mit Hilfe des Multi-Analyt Assays auf Opioide untersucht.rnIn einem zweiten Schritt wurde die Extraktions- und Messmethode zur Quantifizierung der 35 Opioide am Forensic Medicine Center in Ho Chi Minh City (Vietnam) etabliert. Insgesamt wurden 85 Herzblutproben von Obduktionsfällen mit Verdacht auf Opiatintoxikation näher untersucht. Der überwiegende Teil der untersuchten Fälle konnte auf eine Heroin- bzw. Morphin-Vergiftung zurückgeführt werden. Morphin wurde in 68 Fällen im Konzentrationsbereich 1.7 – 1400 ng/ml und der Heroinmetabolit 6-Monoactetylmorphin in 34 Fällen (0.3 – 160 ng/ml) nachgewiesen werden.rnSchließlich wurden noch 15 Haarproben von Patienten einer psychiatrischen Klinik, die illegale Rauschmittel konsumiert hatten, mit Hilfe der simultanen Opioid-LC/MS Screeningmethode gemessen. Die Ergebnisse der Untersuchung wurden mit früheren Auswertungen von gaschromatographischen Analysen verglichen. Es zeigte sich eine weitgehende Übereinstimmung der Untersuchungsergebnisse für die Opioide 6-Monoacetylmorphin, Morphin, Codein, Dihydrocodein und Methadon. Mit der LC/MS Methode konnten weitere Substanzen, wie zum Beispiel Bisnortilidin, Dextromethorphan und Tramadol in den Haarproben gefunden werden, die bislang nicht entdeckt worden waren.rn

Rapidly excised and cryofixed rat tissue

All preparation efforts of biological samples in electron microscopy are focused to preserve structures as close as possible to the native state. To achieve this goal with tissues, it is of advantage to have a very short time between excision and fixation. The most common approach is chemical fixation: cross-linking of the tissue samples with aldehydes followed by postfixation with osmium tetroxide. Here, the fastest approach for tissue samples is perfusion. However, the diffusion of the fixation solution from blood vessels into the depth of the tissue is still slow and does not allow an overall instant fixation of a single cell. As a result, osmotic effects become evident (swelling or shrinkage of cell organelles). Another possibility is to take a tissue sample from the experimental animal. Excision of tissue can last quite some time, which results in even more pronounced autolytic induced osmotic effects. Furthermore, the animal does not survive the procedure in most cases. Alternatively, microbiopsies are an elegant technique to rapidly excise small quantities of tissue. Some tissues, such as liver and muscle, may be obtained using a non-lethal approach. To avoid the artifacts introduced by chemical fixation, high-pressure freezing of microbiopsies (brain, liver, kidney, and muscle) is a powerful alternative to chemical fixation. Here, we describe the microbiopsy method, and high-pressure freezing/freeze-substitution (HPF/FS) as a follow-up procedure. Cryosectioning of high-pressure frozen samples is optimally preserving the ultrastructure; however, it is not considered to be a routine approach yet.

Eliminating Adhesion Errors in Nanoindentation of Compliant Polymers and Hydrogels

Nanoindentation is a valuable tool for characterization of biomaterials due to its ability to measure local properties in heterogeneous, small or irregularly shaped samples. However, applying nanoindentation to compliant, hydrated biomaterials leads to many challenges including adhesion between the nanoindenter tip and the sample. Although adhesion leads to overestimation of the modulus of compliant samples when analyzing nanoindentation data using traditional analysis techniques, most studies of biomaterials have ignored its effects. This paper demonstrates two methods for managing adhesion in nanoindentation analysis, the nano-JKR force curve method and the surfactant method, through application to two biomedically-relevant compliant materials, poly(dimethyl siloxane) (PDMS) elastomers and poly(ethylene glycol) (PEG) hydrogels. The nano-JKR force curve method accounts for adhesion during data analysis using equations based on the Johnson-Kendall-Roberts (JKR) adhesion model, while the surfactant method eliminates adhesion during data collection, allowing data analysis using traditional techniques. In this study, indents performed in air or water resulted in adhesion between the tip and the sample, while testing the same materials submerged in Optifree Express() contact lens solution eliminated tip-sample adhesion in most samples. Modulus values from the two methods were within 7% of each other, despite different hydration conditions and evidence of adhesion. Using surfactant also did not significantly alter the properties of the tested material, allowed accurate modulus measurements using commercial software, and facilitated nanoindentation testing in fluids. This technique shows promise for more accurate and faster determination of modulus values from nanoindentation of compliant, hydrated biological samples. Copyright 2013 Elsevier Ltd. All rights reserved.