A data-driven,high-throughput methodology to determine tissue-specific differentially methylated regions able to discriminate body fluids

Tissue-specific differentially methylated regions (tDMRs) are regions of the genome with methylation patterns that modulate gene expression in those tissue types. The detection of tDMRs in forensic evidence can permit the identification of body fluids at trace levels. In this report, we have performed a bioinformatic analysis of an existing array dataset to determine if new tDMRs could be identified for use in body fluid identification from forensic evidence. Once these sites were identified, primers were designed and bisulfite modification was performed. The relative methylation level for each body fluid at a given locus was then determined using qPCR with high-resolution melt analysis (HRM). After screening 127 tDMR's in multiple body fluids, we were able to identify four new markers able to discriminate blood (2 markers), vaginal epithelia (1 marker) and buccal cells (1 marker). One marker for each target body fluid was also tested with pyrosequencing showing results consistent with those obtained by HRM. This work successfully demonstrates the ability of in silico analysis to develop a novel set of tDMRs capable of being differentiated by real time PCR/HRM. The method can rapidly determine the body fluids left at crime scenes, assisting the triers of fact in forensic casework.     

Multidimensional Raman spectroscopic signature of sweat and its potential application to forensic body fluid identification

This proof-of-concept study demonstrated the potential of Raman microspectroscopy for nondestructive identification of traces of sweat for forensic purposes. Advanced statistical analysis of Raman spectra revealed that dry sweat was intrinsically heterogeneous, and its biochemical composition varies significantly with the donor. As a result, no single Raman spectrum could adequately represent sweat traces. Instead, a multidimensional spectroscopic signature of sweat was built that allowed for the presentation of any single experimental spectrum as a linear combination of two fluorescent backgrounds and three Raman spectral components dominated by the contribution from lactate, lactic acid, urea and single amino acids.     

A method of identifying the blood contributor in mixture stains through detecting blood-specific mRNA polymorphism

In the past decades, messenger RNA (mRNA) biomarkers have been employed to identify the origin of body fluids in forensic medicine. We hypothesized that the polymorphism of mRNA could be applied to identify individuals in mixture samples composed of two body fluids. In this study, we selected five blood-specific mRNA biomarkers of venous blood (SPTB, CD3G, AMICA1, ANK1, and GYPA) that encompass 16 SNPs to identify the mixture contributor(s). Five specific gene markers for menstrual blood, semen, skin, saliva, and vaginal secretions were amplified and typed as body-fluid positive controls. We established the system of multiplex PCR and single base extension (SBE) reaction followed by CE. The amplicon size was between 90bp and 294bp. The peripheral blood specificity was examined against other human body fluids, including saliva, semen, skin, menstrual blood, and vaginal secretion. The 16 SNPs were peripheral blood specific and could be successfully typed in homemade mixtures which are composed of different body fluids with 1 ng peripheral blood mRNA added. This system showed a supersensitivity (1:100) in detecting the trace amount of peripheral blood mixed in other body fluids and a combined discrimination power (CDP) of 0.99929 in Chinese population. It was the first time to establish a method for identifying the blood donors and deconvoluting mixtures through detecting mRNA polymorphism with SNaPshot assay. This peripheral blood specific SNP typing system showed high sensitivity to the typing of blood source specific markers regardless of other body fluids in the mixture.     

Expression profile analysis and stability evaluation of 18 small RNAs in the Chinese Han population

In recent years, differentially expressed small RNAs have been widely used to identify the compositions of forensically relevant biological samples, and a vast number of such RNA candidates have been proposed. Nevertheless, when assessing the expression levels of target small RNAs using relative quantitative analysis methods, credible internal controls are usually required for reliable data normalization. Therefore, the identification of optimal reference genes is an important task. In this study, the expression profile of 18 small RNA reference genes was characterized in the Chinese Han population using TaqMan real-time quantitative PCR. Systematic evaluations of these candidate genes were performed based on their expression levels and stability in several common types of body fluids (i.e., venous blood, menstrual blood, saliva, semen, and vaginal secretions). Analysis results from the ΔCq method, BestKeeper, NormFinder, and geNorm were integrated by RefFinder for ranking and comparing the candidates in each type of body fluid. Among all the candidates, miR-191 was identified as the most suitable reference gene because it had a favorable ranking value in all tested samples. In addition, miR-423, miR-93, miR-484, and let-7i were also shown to be applicable reference genes. Overall, this study provides detailed assessment results of these candidate genes in different body fluids; thus, it could be used as a guide for the selection of reference genes according to their performance in the sample of choice.     

Changes of trace element concentrations of human tissues and body fluids due to therapeutic and diagnostic treatment

Summary The normal levels of more than 30 trace elements in human tissues and body fluids are changed as a consequence of medical treatments. In this work, a survey is given on the occurrences of such effects, the acting mechanisms and the medical implications, the latter ranging from pathological signs of deficiency to severe intoxications. Moreover, they can cause long-term disturbances of the trace element balance, the consequences of which cannot even be estimated up to now. The changes of trace element levels by therapeutic treatment originate from intense medical treatment, from medication, from medical implants, as well as from medical and everydays body and skin care and from the so-called healthy style of living. Also diagnostic treatment which mostly has no medical implications can strongly alter the trace element composition of tissues and body fluids by interaction of the samples with medicaments and materials used during diagnostic interventions. All these effects demonstrate that the investigation of normal human tissues can be heavily biased by medical treatment and that this source of variability of trace elements has to be properly taken into account.

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Veränderungen der Spurenelement-Konzentrationen in menschlichen Geweben und Körperflüssigkeiten als Folge von therapeutischen und diagnostischen Maßnahmen

Zusammenfassung Für mehr als 30 Spurenelemente werden die normalen Konzentrationen in menschlichen Geweben und Körperflüssigkeiten durch medizinische Behandlung verändert. In dieser Arbeit wird ein Überblick gegeben über derartige Effekte, die zugrundeliegenden Mechanismen und die medizinischen Implikationen. Letztere reichen von pathologischen Mangelerscheinungen bis zu schwersten Vergiftungen. Darüber hinaus können sie Langzeitveränderungen des Spurenelementhaushaltes bewirken, deren Konsequenzen derzeit noch nicht abgeschätzt werden können. Veränderungen der Spurenelementkonzentrationen durch therapeutische Maßnahmen werden beobachtet bei intensiver medizinischer Behandlung, als Folge von Medikation und bei Anwendung von Implantaten. Sie treten aber auch bei medizinischer und normaler Körper- und Hautpflege und als Folge sog. gesunder Lebensführung auf. Diagnostische Maßnahmen, die im allgemeinen keine medizinischen Implikationen nach sich ziehen, können die Spurenelementzusammensetzung von Geweben und Körperflüssigkeiten extrem verändern durch Wechselwirkung des Probenmaterials mit Medikamenten und Materialien, die während diagnostischer Eingriffe verwendet werden. Alle diese Effekte belegen, daß die Spurenelementanalyse normaler menschlicher Gewebe durch medizinische Behandlung beeinflußt wird und daß diese Ursache für Variationen normaler Spurenelementkonzentrationen stärker als bisher in Rechnung gestellt werden muß.

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Dedicated to Prof. Dr. W. Fresenius on the occasion of his 70th birthday     

Proteomics of human cancer tissues and cells

Proteomic analysis of cancer tissues and cells provides valuable information to identify promising targets for cancer diagnosis, prognosis and therapy. Novel strategies have emerged to optimize the workflow of tissue procurement, and tissue and cell selection, and to improve protocols for the extraction of protein from fresh, frozen and paraffin-embedded tissue. Moreover, in the context of advanced approaches to proteomics, mass spectrometry and array-based technologies strongly contribute to protein profiling of cancer tissues and cells.The focus of this review is the methods by which all the steps of a proteomic investigation on human-cancer tissue (from choice of the experimental model to validation of candidate biomarkers) should be performed, paying particular attention to recently developed strategies. The review also presents an overview of the most recent high-throughput proteomic studies in cancer research.     

Neutron activation analysis of human tissues,organs and body fluids to describe the interaction of orthopaedic implants made of cobalt-chromium alloy with the patients organisms

In order to describe the impact of corrosion of medical implants on the trace element balance of man samples of blood, serum and of a variety of tissues and organs were analysed for their trace element composition using instrumental neutron activation techniques. By the analysis of blood and serum the trace element status after long-term implantation as well as its dependence on time after implantation was investigated. Using autopsy samples of human organs such as heart, spleen, liver, of aorta and of lymphatic tissue from the lower pelvis transport and storage of the corrosion products was studided. These investigations were supplemented by a comprehensive study of normal human blood, serum, tissues and organs from patients without implants. The results demonstrate that there are high enrichments of corrosion products in several tissues and organs and that also blood and serum reveal the presence of the metal implants in the trace element levels, increasing shortly after implantation and pertaining during the entire implantation time. Thus the corrosion of metallic implants is a process not only affecting tissues from the vicinity of the implants but also influencing the trace element balance of the entire organism.     

HPLC determination of safflor yellow A and three active isoflavones from TCM Naodesheng in rat plasma and tissues and its application to pharmacokinetic studies

A high-performance liquid chromatographic method was developed for the simultaneous determination and pharmacokinetic studies of safflor yellow A, puerarin, 3'-methoxyl-puerarin, and puerarinapioside in the plasma and tissues of rats that had been administered with the traditional Chinese medicine (TCM) preparation Naodesheng via the caudal vein. Samples taken from rats were subjected to protein precipitation with acetone. Separation of these four compounds was accomplished on a Kromisil C18 stationary phase using a mobile phase of acetonitrile-0.1% phosphoric acid-tetrahydrofuran (8:92:2, v/v/v) at a flow-rate of 1.0 mL/min. The detection wavelength was set at 250 nm. The calibration curves of the four components were linear in the given concentration ranges. The intra- and inter-day precisions in plasma and tissues were less than 15% and the extraction recoveries were higher than 60%. The lower limits of quantitation of four components were low enough to determine the four components. These four components all exhibited kinetics that fitted a two-compartment model in rats. The elimination half-life was 1.19 h for safflor yellow A, 2.69 h for puerarin, 2.94 h for 3'-methoxyl-puerarin, and 0.87 h for puerarinapioside, respectively. Following administration of a single injection of Naodesheng, the concentration (C) of the four components in the tissues showed C(kidney) > C(lung), C(liver) > C(spleen), C(stomach), C(heart), approximately. The method is a reliable tool for performing studies of safflor yellow A and three puerarin isoflavones in different biological material.     

Determination of the neuropharmacological drug nodakenin in rat plasma and brain tissues by liquid chromatography tandem mass spectrometry: Application to pharmacokinetic studies

A rapid and sensitive liquid chromatography tandem mass spectrometry detection using selected reaction monitoring in positive ionization mode was developed and validated for the quantification of nodakenin in rat plasma and brain. Pareruptorin A was used as internal standard. A single step liquid–liquid extraction was used for plasma and brain sample preparation. The method was validated with respect to selectivity, precision, accuracy, linearity, limit of quantification, recovery, matrix effect and stability. Lower limit of quantification of nodakenin was 2.0 ng/mL in plasma and brain tissue homogenates. Linear calibration curves were obtained over concentration ranges of 2.0–1000 ng/mL in plasma and brain tissue homogenates for nodakenin. Intra‐day and inter‐day precisions (relative standard deviation, RSD) were <15% in both biological media. This assay was successfully applied to plasma and brain pharmacokinetic studies of nodakenin in rats after intravenous administration.     

A novel liquid chromatography Orbitrap mass spectrometry method with full scan for simultaneous determination of multiple bioactive constituents of Shenkang injection in rat tissues: Application to tissue distribution and pharmacokinetic studies

Shenkang injection is a traditional Chinese formula with good curative effect on chronic renal failure. In this paper, a novel, rapid and sensitive ultra‐high‐performance liquid chromatography coupled with Q Exactive hybrid quadrupole Orbitrap high‐resolution accurate mass spectrometry was developed and validated for simultaneous determination of seven bioactive constituents of Shenkang injection in rat plasma and tissues after intravenous administration. Acetonitrile was used as a protein precipitation agent in biological samples disposal with carbamazepine as internal standard. The chromatographic separation was carried out on a C₁₈ column with a gradient mobile phase consisting of acetonitrile and water (containing 0.1% formic acid). The MS analysis was performed in the full‐scan positive and negative ion mode. The lower limits of quantification for the seven analytes in rat plasma and tissues were 0.1–10 ng/mL. The validated method was successfully applied to tissue distribution and pharmacokinetic studies of Shenkang injection after intravenous administration. The results of the tissue distribution study showed that the high concentrations of seven constituents were primarily in the kidney tract. This is the first report of the application of Q‐Orbitrap with full‐scan mass spectrometry in tissue distribution and pharmacokinetic studies of Shenkang injection.     

Validated UPLC‐MS/MS method for quantification of seven compounds in rat plasma and tissues: Application to pharmacokinetic and tissue distribution studies in rats after oral administration of extract of Eclipta prostrata L.

A rapid, sensitive and specific ultra‐high‐performance liquid chromatography coupled with tandem mass spectrometry (UPLC‐MS/MS) method was developed to investigate the pharmacokinetics and tissue distribution of Eclipta prostrata extract. Rats were orally administrated the 70% ethanol extract of E. prostrata, and their plasma as well as various organs were collected. The concentrations of seven main compounds, ecliptasaponin IV, ecliptasaponin A, apigenin, 3′‐hydroxybiochanin A, luteolin, luteolin‐7‐O‐glucoside and wedelolactone, were quantified by UPLC‐MS/MS through multiple reactions monitoring method. The precisions (RSD) of the analytes were all <15.00%. The extraction recoveries ranged from 74.65 to 107.45% with RSD ≤ 15.36%. The matrix effects ranged from 78.00 to 118.06% with RSD ≤ 15.04%. To conclude, the present pharmacokinetic and tissue distribution studies provided useful information for the clinical usage of Eclipta prostrata L.