Genexpressionsanalyse der fötalen Wachstumsfuge des Rindes

Erkrankungen des Skelettapparats wie beispielsweise die Osteoporose oder Arthrose gehören neben den Herz-Kreislauferkrankungen und Tumoren zu den Häufigsten Erkrankungen des Menschen. Ein besseres Verständnis der Bildung und des Erhalts von Knochen- oder Knorpelgewebe ist deshalb von besonderer Bedeutung. Viele bisherige Ansätze zur Identifizierung hierfür relevanter Gene, deren Produkte und Interaktionen beruhen auf der Untersuchung pathologischer Situationen. Daher ist die Funktion vieler Gene nur im Zusammenhang mit Krankheiten beschrieben. Untersuchungen, die die Genaktivität bei der Normalentwicklung von knochen- und knorpelbildenden Geweben zum Ziel haben, sind dagegen weit weniger oft durchgeführt worden. rnEines der entwicklungsphysiologisch interessantesten Gewebe ist die Epiphysenfuge der Röhrenknochen. In dieser sogenannten Wachstumsfuge ist insbesondere beim fötalen Gewebe eine sehr hohe Aktivität derjenigen Gene zu erwarten, die an der Knochen- und Knorpelbildung beteiligt sind. In der vorliegenden Arbeit wurde daher aus der Epiphysenfuge von Kälberknochen RNA isoliert und eine cDNA-Bibliothek konstruiert. Von dieser wurden ca. 4000 Klone im Rahmen eines klassischen EST-Projekts sequenziert. Durch die Analyse konnte ein ungefähr 900 Gene umfassendes Expressionsprofil erstellt werden und viele Transkripte für Komponenten der regulatorischen und strukturbildenden Bestandteile der Knochen- und Knorpelentwicklung identifiziert werden. Neben den typischen Genen für Komponenten der Knochenentwicklung sind auch deutlich Bestandteile für embryonale Entwicklungsprozesse vertreten. Zu ersten gehören in erster Linie die Kollagene, allen voran Kollagen II alpha 1, das mit Abstand höchst exprimierte Gen in der fötalen Wachstumsfuge. Nach den ribosomalen Proteinen stellen die Kollagene mit ca. 10 % aller auswertbaren Sequenzen die zweitgrößte Gengruppe im erstellten Expressionsprofil dar. Proteoglykane und andere niedrig exprimierte regulatorische Elemente, wie Transkriptionsfaktoren, konnten im EST-Projekt aufgrund der geringen Abdeckung nur in sehr geringer Kopienzahl gefunden werden. Allerdings förderte die EST-Analyse mehrere interessante, bisher nicht bekannte Transkripte zutage, die detaillierter untersucht wurden. Dazu gehören Transkripte die, die dem LOC618319 zugeordnet werden konnten. Neben den bisher beschriebenen drei Exonbereichen konnte ein weiteres Exon im 3‘-UTR identifiziert werden. Im abgeleiteten Protein, das mindestens 121 AS lang ist, wurden ein Signalpeptid und eine Transmembrandomäne nachgewiesen. In Verbindung mit einer möglichen Glykosylierung ist das Genprodukt in die Gruppe der Proteoglykane einzuordnen. Leicht abweichend von den typischen Strukturen knochen- und knorpelspezifischer Proteoglykane ist eine mögliche Funktion dieses Genprodukts bei der Interaktion mit Integrinen und der Zell-Zellinteraktion, aber auch bei der Signaltransduktion denkbar. rnDie EST-Sequenzierungen von ca. 4000 cDNA-Klonen können aber in der Regel nur einen Bruchteil der möglichen Transkripte des untersuchten Gewebes abdecken. Mit den neuen Sequenziertechnologien des „Next Generation Sequencing“ bestehen völlig neue Möglichkeiten, komplette Transkriptome mit sehr hoher Abdeckung zu sequenzieren und zu analysieren. Zur Unterstützung der EST-Daten und zur deutlichen Verbreiterung der Datenbasis wurde das Transkriptom der bovinen fötalen Wachstumsfuge sowohl mit Hilfe der Roche-454/FLX- als auch der Illumina-Solexa-Technologie sequenziert. Bei der Auswertung der ca. 40000 454- und 75 Millionen Illumina-Sequenzen wurden Verfahren zur allgemeinen Handhabung, der Qualitätskontrolle, dem „Clustern“, der Annotation und quantitativen Auswertung von großen Mengen an Sequenzdaten etabliert. Beim Vergleich der Hochdurchsatz Blast-Analysen im klassischen „Read-Count“-Ansatz mit dem erstellten EST-Expressionsprofil konnten gute Überstimmungen gezeigt werden. Abweichungen zwischen den einzelnen Methoden konnten nicht in allen Fällen methodisch erklärt werden. In einigen Fällen sind Korrelationen zwischen Transkriptlänge und „Read“-Verteilung zu erkennen. Obwohl schon simple Methoden wie die Normierung auf RPKM („reads per kilo base transkript per million mappable reads“) eine Verbesserung der Interpretation ermöglichen, konnten messtechnisch durch die Art der Sequenzierung bedingte systematische Fehler nicht immer ausgeräumt werden. Besonders wichtig ist daher die geeignete Normalisierung der Daten beim Vergleich verschieden generierter Datensätze. rnDie hier diskutierten Ergebnisse aus den verschiedenen Analysen zeigen die neuen Sequenziertechnologien als gute Ergänzung und potentiellen Ersatz für etablierte Methoden zur Genexpressionsanalyse.rn

Identifizierung chemischer Transportinhibitoren zur Modulation (patho)biologischer Genprodukte

Die intrazelluläre Lokalisation von Proteinen und Makromolekülen unterliegt in Eukaryoten einer strengen Regulation. Insbesondere erlaubt die Kompartimentierung eukaryotischer Zellen in Zellkern und Zytoplasma den simultanen Ablauf räumlich getrennter biochemischer Reaktionen, und damit die unabhängige Regulation zellulärer Programme. Da trotz intensiver Forschungsbemühungen bis dato die molekularen Details sowie die (patho)biologische Bedeutung von Kern-Zytoplasma-Transportprozessen noch immer nicht vollkommen verstanden sind, wurde im Rahmen der vorliegenden Arbeit ein Fokus auf die Identifizierung von chemischen Transportinhibitoren gelegt. Das zu diesem Zweck entwickelte Translokations-Biosensor-System basiert auf der Kombination von autofluoreszierenden Proteinen, sowie spezifisch ausgewählten Kernexport- und Kernimportsignalen. Nach Etablierung geeigneter Zellmodelle, die effizient und stabil die Translokations-Biosensoren exprimieren, wurde die 17 000 Substanzen umfassende Bibliothek der ChemBioNet-Initiative nach Kernexportinhibitoren mittels einer Fluoreszenzmikroskopie-basierten Hochdurchsatzanalyse-Plattform durchmustert. Zunächst wurden Translokations-Algorithmen, welche eine zuverlässige automatisierte Erkennung von Zellkern und Zytoplasma erlauben, optimiert. Im Folgenden konnten acht neue niedermolekulare Kernexport-Inhibitoren identifiziert werden, die sich in der Stärke, der Geschwindigkeit, sowie in der Beständigkeit der vermittelten Inhibition unterscheiden. Die Aktivität der Inhibitoren konnte auf den isolierten nukleären Exportsignalen (NES) von HIV-1 Rev und Survivin als auch auf den entsprechenden Volllängeproteinen mittels Mikroinjektionsexperimenten sowie durch umfassende in vitro und biochemische Methoden bestätigt werden. Zur Untersuchung der funktionellen Einheiten der Inhibitoren wurden homologe Substanzen auf Ihre Aktivität hin getestet. Dabei konnten für die Aktivität wichtige chemische Gruppen definiert werden. Alle Substanzen stellen neue Inhibitoren des Crm1-abhängigen Exports dar und zeigen keine nachweisbare NES-Selektivität. Interessanterweise konnte jedoch eine zytotoxische und Apoptose-induzierende Wirkung auf verschiedene Krebszellarten festgestellt werden. Da diese Wirkung unabhängig vom p53-Status der Tumorzellen ist und die Inhibitoren C3 und C5 die Vitalität nicht-maligner humaner Zellen signifikant weniger beeinträchtigen, wurden diese Substanzen zum internationalen Patent angemeldet. Da der nukleäre Export besonders für Tumorzellen einen wichtigen Überlebenssignalweg darstellt, könnte dessen reversible Hemmung ausgenutzt werden, um besonders in Kombination mit gängigen Krebstherapien eine therapeutisch relevante Tumorinhibition zu erzeugen. Eine weitere Anwendungsmöglichkeit der neuen Exportinhibitoren ist auf dem Gebiet der Infektionskrankheiten zu sehen, da auch die Aktivität des essentiellen HIV-1 Rev-Proteins inhibiert wird. Zusätzlich konnte in der Arbeit gezeigt werden, dass der zelluläre Kofaktor des Crm1-abhängigen Exports des HIV-1 Rev-Proteins, die RNA-Helikase DDX3, ein eigenes NES enthält. Der Nachweis einer direkten Interaktion des HIV-1 Rev- mit dem DDX3-Protein impliziert, dass multiple Angriffstellen für chemische Modulatoren hinsichtlich einer antiviralen Therapie gegeben sind. Da die Vielfalt des chemischen Strukturraums es unmöglich macht diesen experimentell vollständig zu durchmustern, wurden im Rahmen dieser Arbeit auch Naturstoffe als vielversprechende Wirkstoffquelle untersucht. Um zukünftig umfassend bioaktive Substanzen aus diesen hochkomplexen Stoffgemischen experimentell identifizieren zu können, wurde eine Fluoreszenzmikroskopie-basierte Hochdurchsatzanalyse-Plattform am Mainz Screening Center (MSC) etabliert. Damit konnte bereits ein weiterer, bisher unbekannter Exportinhibitor aus Cyphellopsis anomala identifiziert werden. Neben einer Anwendung dieser Substanz als chemisches Werkzeug zur Aufklärung der Regulation von Transportvorgängen, stellt sich auch die evolutionsbiologisch relevante Frage, wie es dem Pilzproduzenten gelingt die Blockierung des eigenen Kernexports zu umgehen. Weiterführende Projekte müssen sich neben der Aufklärung der molekularen Wirkmechanismen der gefundenen Substanzen mit der Identifizierung spezifischer chemischer „Funktionseinheiten“ beschäftigen. Neben einem verbesserten mechanistischen Verständnis von Transportvorgängen stellen die erarbeiteten Transportinhibitoren Vorstufen zur Weiterentwicklung möglicher Wirkstoffe dar. Die im Rahmen dieser Arbeit etablierte Technologie-Plattform und molekularen Werkzeuge stellen darüber hinaus eine wichtige Voraussetzung dar, um eine systematische Suche nach möglichen Wirkstoffen im Forschungsfeld der „Chemischen Biomedizin“ voranzutreiben.

Algorithmen zur labelfreien quantitativen Proteomanalyse auf Basis datenunabhängig-akquirierter LC-MS-Daten

Moderne ESI-LC-MS/MS-Techniken erlauben in Verbindung mit Bottom-up-Ansätzen eine qualitative und quantitative Charakterisierung mehrerer tausend Proteine in einem einzigen Experiment. Für die labelfreie Proteinquantifizierung eignen sich besonders datenunabhängige Akquisitionsmethoden wie MSE und die IMS-Varianten HDMSE und UDMSE. Durch ihre hohe Komplexität stellen die so erfassten Daten besondere Anforderungen an die Analysesoftware. Eine quantitative Analyse der MSE/HDMSE/UDMSE-Daten blieb bislang wenigen kommerziellen Lösungen vorbehalten. rn| In der vorliegenden Arbeit wurden eine Strategie und eine Reihe neuer Methoden zur messungsübergreifenden, quantitativen Analyse labelfreier MSE/HDMSE/UDMSE-Daten entwickelt und als Software ISOQuant implementiert. Für die ersten Schritte der Datenanalyse (Featuredetektion, Peptid- und Proteinidentifikation) wird die kommerzielle Software PLGS verwendet. Anschließend werden die unabhängigen PLGS-Ergebnisse aller Messungen eines Experiments in einer relationalen Datenbank zusammengeführt und mit Hilfe der dedizierten Algorithmen (Retentionszeitalignment, Feature-Clustering, multidimensionale Normalisierung der Intensitäten, mehrstufige Datenfilterung, Proteininferenz, Umverteilung der Intensitäten geteilter Peptide, Proteinquantifizierung) überarbeitet. Durch diese Nachbearbeitung wird die Reproduzierbarkeit der qualitativen und quantitativen Ergebnisse signifikant gesteigert.rn| Um die Performance der quantitativen Datenanalyse zu evaluieren und mit anderen Lösungen zu vergleichen, wurde ein Satz von exakt definierten Hybridproteom-Proben entwickelt. Die Proben wurden mit den Methoden MSE und UDMSE erfasst, mit Progenesis QIP, synapter und ISOQuant analysiert und verglichen. Im Gegensatz zu synapter und Progenesis QIP konnte ISOQuant sowohl eine hohe Reproduzierbarkeit der Proteinidentifikation als auch eine hohe Präzision und Richtigkeit der Proteinquantifizierung erreichen.rn| Schlussfolgernd ermöglichen die vorgestellten Algorithmen und der Analyseworkflow zuverlässige und reproduzierbare quantitative Datenanalysen. Mit der Software ISOQuant wurde ein einfaches und effizientes Werkzeug für routinemäßige Hochdurchsatzanalysen labelfreier MSE/HDMSE/UDMSE-Daten entwickelt. Mit den Hybridproteom-Proben und den Bewertungsmetriken wurde ein umfassendes System zur Evaluierung quantitativer Akquisitions- und Datenanalysesysteme vorgestellt.

TILLING for Mutations in Model Plants and Crops

A growing world population, changing climate and limiting fossil fuels will provide new pressures on human production of food, medicine, fuels and feed stock in the twenty-first century. Enhanced crop production promises to ameliorate these pressures. Crops can be bred for increased yields of calories, starch, nutrients, natural medicinal compounds, and other important products. Enhanced resistance to biotic and abiotic stresses can be introduced, toxins removed, and industrial qualities such as fibre strength and biofuel per mass can be increased. Induced and natural mutations provide a powerful method for the generation of heritable enhanced traits. While mainly exploited in forward, phenotype driven, approaches, the rapid accumulation of plant genomic sequence information and hypotheses regarding gene function allows the use of mutations in reverse genetic approaches to identify lesions in specific target genes. Such gene-driven approaches promise to speed up the process of creating novel phenotypes, and can enable the generation of phenotypes unobtainable by traditional forward methods. TILLING (Targeting Induced Local Lesions IN Genome) is a high-throughput and low cost reverse genetic method for the discovery of induced mutations. The method has been modified for the identification of natural nucleotide polymorphisms, a process called Ecotilling. The methods are general and have been applied to many species, including a variety of different crops. In this chapter the current status of the TILLING and Ecotilling methods and provide an overview of progress in applying these methods to different plant species, with a focus on work related to food production for developing nations.

Controlled angiogenesis in the heart by cell-based expression of specific vascular endothelial growth factor levels

Vascular endothelial growth factor (VEGF) can induce normal angiogenesis or the growth of angioma-like vascular tumors depending on the amount secreted by each producing cell because it remains localized in the microenvironment. In order to control the distribution of VEGF expression levels in vivo, we recently developed a high-throughput fluorescence-activated cell sorting (FACS)-based technique to rapidly purify transduced progenitors that homogeneously express a specific VEGF dose from a heterogeneous primary population. Here we tested the hypothesis that cell-based delivery of a controlled VEGF level could induce normal angiogenesis in the heart, while preventing the development of angiomas. Freshly isolated human adipose tissue-derived stem cells (ASC) were transduced with retroviral vectors expressing either rat VEGF linked to a FACS-quantifiable cell-surface marker (a truncated form of CD8) or CD8 alone as control (CTR). VEGF-expressing cells were FACS-purified to generate populations producing either a specific VEGF level (SPEC) or uncontrolled heterogeneous levels (ALL). Fifteen nude rats underwent intramyocardial injection of 10(7) cells. Histology was performed after 4 weeks. Both the SPEC and ALL cells produced a similar total amount of VEGF, and both cell types induced a 50%-60% increase in both total and perfused vessel density compared to CTR cells, despite very limited stable engraftment. However, homogeneous VEGF expression by SPEC cells induced only normal and stable angiogenesis. Conversely, heterogeneous expression of a similar total amount by the ALL cells caused the growth of numerous angioma-like structures. These results suggest that controlled VEGF delivery by FACS-purified ASC may be a promising strategy to achieve safe therapeutic angiogenesis in the heart.

Point-of-care testing in the cardiovascular operating theatre

Point-of-care testing (POCT) remains under scrutiny by healthcare professionals because of its ill-tried, young history. POCT methods are being developed by a few major equipment companies based on rapid progress in informatics and nanotechnology. Issues as POCT quality control, comparability with standard laboratory procedures, standardisation, traceability and round robin testing are being left to hospitals. As a result, the clinical and operational benefits of POCT were first evident for patients on the operating table. For the management of cardiovascular surgery patients, POCT technology is an indispensable aid. Improvement of the technology has meant that clinical laboratory pathologists now recognise the need for POCT beyond their high-throughput areas.

EXPLORATION, NORMALIZATION, AND GENOTYPE CALLS OF HIGH DENSITY OLIGONUCLEOTIDE SNP ARRAY DATA

In most microarray technologies, a number of critical steps are required to convert raw intensity measurements into the data relied upon by data analysts, biologists and clinicians. These data manipulations, referred to as preprocessing, can influence the quality of the ultimate measurements. In the last few years, the high-throughput measurement of gene expression is the most popular application of microarray technology. For this application, various groups have demonstrated that the use of modern statistical methodology can substantially improve accuracy and precision of gene expression measurements, relative to ad-hoc procedures introduced by designers and manufacturers of the technology. Currently, other applications of microarrays are becoming more and more popular. In this paper we describe a preprocessing methodology for a technology designed for the identification of DNA sequence variants in specific genes or regions of the human genome that are associated with phenotypes of interest such as disease. In particular we describe methodology useful for preprocessing Affymetrix SNP chips and obtaining genotype calls with the preprocessed data. We demonstrate how our procedure improves existing approaches using data from three relatively large studies including one in which large number independent calls are available. Software implementing these ideas are avialble from the Bioconductor oligo package.

How reliable and robust are current biomarkers for copper status?

Cu is an essential nutrient for man, but can be toxic if intakes are too high. In sensitive populations, marginal over- or under-exposure can have detrimental effects. Malnourished children, the elderly, and pregnant or lactating females may be susceptible for Cu deficiency. Cu status and exposure in the population can currently not be easily measured, as neither plasma Cu nor plasma cuproenzymes reflect Cu status precisely. Some blood markers (such as ceruloplasmin) indicate severe Cu depletion, but do not inversely respond to Cu excess, and are not suitable to indicate marginal states. A biomarker of Cu is needed that is sensitive to small changes in Cu status, and that responds to Cu excess as well as deficiency. Such a marker will aid in monitoring Cu status in large populations, and will help to avoid chronic health effects (for example, liver damage in chronic toxicity, osteoporosis, loss of collagen stability, or increased susceptibility to infections in deficiency). The advent of high-throughput technologies has enabled us to screen for potential biomarkers in the whole proteome of a cell, not excluding markers that have no direct link to Cu. Further, this screening allows us to search for a whole group of proteins that, in combination, reflect Cu status. The present review emphasises the need to find sensitive biomarkers for Cu, examines potential markers of Cu status already available, and discusses methods to identify a novel suite of biomarkers.

HIGH PERFORMANCE, LOW COST SUBSPACE DECOMPOSITION AND POLYNOMIAL ROOTING FOR REAL TIME DIRECTION OF ARRIVAL ESTIMATION: ANALYSIS AND IMPLEMENTATION

This thesis develops high performance real-time signal processing modules for direction of arrival (DOA) estimation for localization systems. It proposes highly parallel algorithms for performing subspace decomposition and polynomial rooting, which are otherwise traditionally implemented using sequential algorithms. The proposed algorithms address the emerging need for real-time localization for a wide range of applications. As the antenna array size increases, the complexity of signal processing algorithms increases, making it increasingly difficult to satisfy the real-time constraints. This thesis addresses real-time implementation by proposing parallel algorithms, that maintain considerable improvement over traditional algorithms, especially for systems with larger number of antenna array elements. Singular value decomposition (SVD) and polynomial rooting are two computationally complex steps and act as the bottleneck to achieving real-time performance. The proposed algorithms are suitable for implementation on field programmable gated arrays (FPGAs), single instruction multiple data (SIMD) hardware or application specific integrated chips (ASICs), which offer large number of processing elements that can be exploited for parallel processing. The designs proposed in this thesis are modular, easily expandable and easy to implement. Firstly, this thesis proposes a fast converging SVD algorithm. The proposed method reduces the number of iterations it takes to converge to correct singular values, thus achieving closer to real-time performance. A general algorithm and a modular system design are provided making it easy for designers to replicate and extend the design to larger matrix sizes. Moreover, the method is highly parallel, which can be exploited in various hardware platforms mentioned earlier. A fixed point implementation of proposed SVD algorithm is presented. The FPGA design is pipelined to the maximum extent to increase the maximum achievable frequency of operation. The system was developed with the objective of achieving high throughput. Various modern cores available in FPGAs were used to maximize the performance and details of these modules are presented in detail. Finally, a parallel polynomial rooting technique based on Newton’s method applicable exclusively to root-MUSIC polynomials is proposed. Unique characteristics of root-MUSIC polynomial’s complex dynamics were exploited to derive this polynomial rooting method. The technique exhibits parallelism and converges to the desired root within fixed number of iterations, making this suitable for polynomial rooting of large degree polynomials. We believe this is the first time that complex dynamics of root-MUSIC polynomial were analyzed to propose an algorithm. In all, the thesis addresses two major bottlenecks in a direction of arrival estimation system, by providing simple, high throughput, parallel algorithms.

NON-CHROMATOGRAPHIC PURIFICATION OF SYNTHETIC BIO-OLIGOMERS

Synthetic oligonucleotides and peptides have found wide applications in industry and academic research labs. There are ~60 peptide drugs on the market and over 500 under development. The global annual sale of peptide drugs in 2010 was estimated to be $13 billion. There are three oligonucleotide-based drugs on market; among them, the FDA newly approved Kynamro was predicted to have a $100 million annual sale. The annual sale of oligonucleotides to academic labs was estimated to be $700 million. Both bio-oligomers are mostly synthesized on automated synthesizers using solid phase synthesis technology, in which nucleoside or amino acid monomers are added sequentially until the desired full-length sequence is reached. The additions cannot be complete, which generates truncated undesired failure sequences. For almost all applications, these impurities must be removed. The most widely used method is HPLC. However, the method is slow, expensive, labor-intensive, not amendable for automation, difficult to scale up, and unsuitable for high throughput purification. It needs large capital investment, and consumes large volumes of harmful solvents. The purification costs are estimated to be more than 50% of total production costs. Other methods for bio-oligomer purification also have drawbacks, and are less favored than HPLC for most applications. To overcome the problems of known biopolymer purification technologies, we have developed two non-chromatographic purification methods. They are (1) catching failure sequences by polymerization, and (2) catching full-length sequences by polymerization. In the first method, a polymerizable group is attached to the failure sequences of the bio-oligomers during automated synthesis; purification is achieved by simply polymerizing the failure sequences into an insoluble gel and extracting full-length sequences. In the second method, a polymerizable group is attached to the full-length sequences, which are then incorporated into a polymer; impurities are removed by washing, and pure product is cleaved from polymer. These methods do not need chromatography, and all drawbacks of HPLC no longer exist. Using them, purification is achieved by simple manipulations such as shaking and extraction. Therefore, they are suitable for large scale purification of oligonucleotide and peptide drugs, and also ideal for high throughput purification, which currently has a high demand for research projects involving total gene synthesis. The dissertation will present the details about the development of the techniques. Chapter 1 will make an introduction to oligodeoxynucleotides (ODNs), their synthesis and purification. Chapter 2 will describe the detailed studies of using the catching failure sequences by polymerization method to purify ODNs. Chapter 3 will describe the further optimization of the catching failure sequences by polymerization ODN purification technology to the level of practical use. Chapter 4 will present using the catching full-length sequence by polymerization method for ODN purification using acid-cleavable linker. Chapter 5 will make an introduction to peptides, their synthesis and purification. Chapter 6 will describe the studies using the catching full-length sequence by polymerization method for peptide purification.

Infection prevention strategies in a stem cell transplant unit: impact of change of care in isolation practice and routine use of high dose intravenous immunoglobulins on infectious complications and transplant related mortality

OBJECTIVE: Nursing in 'live islands' and routine high dose intravenous immunoglobulins after allogeneic hematopoietic stem cell transplantation were abandoned by many teams in view of limited evidence and high costs. METHODS: This retrospective single-center study examines the impact of change from nursing in 'live islands' to care in single rooms (SR) and from high dose to targeted intravenous immunoglobulins (IVIG) on mortality and infection rate of adult patients receiving an allogeneic stem cell or bone marrow transplantation in two steps and three time cohorts (1993-1997, 1997-2000, 2000-2003). RESULTS: Two hundred forty-eight allogeneic hematopoetic stem cell transplantations were performed in 227 patients. Patient characteristics were comparable in the three cohorts for gender, median age, underlying disease, and disease stage, prophylaxis for graft versus host disease (GvHD) and cytomegalovirus constellation. The incidence of infections (78.4%) and infection rates remained stable (rates/1000 days of neutropenia for sepsis 17.61, for pneumonia 6.76). Cumulative incidence of GvHD and transplant-related mortality did not change over time. CONCLUSIONS: Change from nursing in 'live islands' to SR and reduction of high dose to targeted IVIG did not result in increased infection rates or mortality despite an increase in patient age. These results support the current practice.

The state of in vitro science for use in bioaccumulation assessments for fish

Through the concerted evaluations of thousands of commercial substances for the qualities of persistence, bioaccumulation, and toxicity as a result of the United Nations Environment Program's Stockholm Convention, it has become apparent that fewer empirical data are available on bioaccumulation than other endpoints and that bioaccumulation models were not designed to accommodate all chemical classes. Due to the number of chemicals that may require further assessment, in vivo testing is cost prohibitive and discouraged due to the large number of animals needed. Although in vitro systems are less developed and characterized for fish, multiple high-throughput in vitro assays have been used to explore the dietary uptake and elimination of pharmaceuticals and other xenobiotics by mammals. While similar processes determine bioaccumulation in mammalian species, a review of methods to measure chemical bioavailability in fish screening systems, such as chemical biotransformation or metabolism in tissue slices, perfused tissues, fish embryos, primary and immortalized cell lines, and subcellular fractions, suggest quantitative and qualitative differences between fish and mammals exist. Using in vitro data in assessments for whole organisms or populations requires certain considerations and assumptions to scale data from a test tube to a fish, and across fish species. Also, different models may incorporate the predominant site of metabolism, such as the liver, and significant presystemic metabolism by the gill or gastrointestinal system to help accurately convert in vitro data into representative whole-animal metabolism and subsequent bioaccumulation potential. The development of animal alternative tests for fish bioaccumulation assessment is framed in the context of in vitro data requirements for regulatory assessments in Europe and Canada.

Patient characteristics but not virulence factors discriminate between asymptomatic and symptomatic E. coli bacteriuria in the hospital

Background Escherichia coli is a common cause of asymptomatic and symptomatic bacteriuria in hospitalized patients. Asymptomatic bacteriuria (ASB) is frequently treated with antibiotics without a clear indication. Our goal was to determine patient and pathogen factors suggestive of ASB. Methods We conducted a 12-month prospective cohort study of adult inpatients with E. coli bacteriuria seen at a tertiary care hospital in St. Louis, Missouri, USA. Urine cultures were taken at the discretion of treating physicians. Bacterial isolates were tested for 14 putative virulence genes using high-throughput dot-blot hybridization. Results The median age of the 287 study patients was 65 (19–101) years; 78% were female. Seventy percent had community-acquired bacteriuria. One-hundred ten (38.3%) patients had ASB and 177 (61.7%) had symptomatic urinary tract infection (sUTI). Asymptomatic patients were more likely than symptomatic patients to have congestive heart failure (p = 0.03), a history of myocardial infarction (p = 0.01), chronic pulmonary disease (p = 0.045), peripheral vascular disease (p = 0.04), and dementia (p = 0.03). Patients with sUTI were more likely to be neutropenic at the time of bacteriuria (p = 0.046). Chronic pulmonary disease [OR 2.1 (95% CI 1.04, 4.1)] and dementia [OR 2.4 (95% CI 1.02, 5.8)] were independent predictors for asymptomatic bacteriuria. Absence of pyuria was not predictive of ASB. None of the individual virulence genes tested were associated with ASB nor was the total number of genes. Conclusions Asymptomatic E. coli bacteriuria in hospitalized patients was frequent and more common in patients with dementia and chronic pulmonary disease. Bacterial virulence factors could not discriminate symptomatic from asymptomatic bacteriurias. Asymptomatic E. coli bacteriuria cannot be predicted by virulence screening.

Accident or homicide - virtual crime scene reconstruction using 3D methods

The analysis and reconstruction of forensically relevant events, such as traffic accidents, criminal assaults and homicides are based on external and internal morphological findings of the injured or deceased person. For this approach high-tech methods are gaining increasing importance in forensic investigations. The non-contact optical 3D digitising system GOM ATOS is applied as a suitable tool for whole body surface and wound documentation and analysis in order to identify injury-causing instruments and to reconstruct the course of event. In addition to the surface documentation, cross-sectional imaging methods deliver medical internal findings of the body. These 3D data are fused into a whole body model of the deceased. Additional to the findings of the bodies, the injury inflicting instruments and incident scene is documented in 3D. The 3D data of the incident scene, generated by 3D laser scanning and photogrammetry, is also included into the reconstruction. Two cases illustrate the methods. In the fist case a man was shot in his bedroom and the main question was, if the offender shot the man intentionally or accidentally, as he declared. In the second case a woman was hit by a car, driving backwards into a garage. It was unclear if the driver drove backwards once or twice, which would indicate that he willingly injured and killed the woman. With this work, we demonstrate how 3D documentation, data merging and animation enable to answer reconstructive questions regarding the dynamic development of patterned injuries, and how this leads to a real data based reconstruction of the course of event.

Absolute quantitative real-time polymerase chain reaction for the measurement of human papillomavirus E7 mRNA in cervical cytobrush specimens.

BACKGROUND: Few reports of the utilization of an accurate, cost-effective means for measuring HPV oncogene transcripts have been published. Several papers have reported the use of relative quantitation or more expensive Taqman methods. Here, we report a method of absolute quantitative real-time PCR utilizing SYBR-green fluorescence for the measurement of HPV E7 expression in cervical cytobrush specimens. RESULTS: The construction of a standard curve based on the serial dilution of an E7-containing plasmid was the key for being able to accurately compare measurements between cervical samples. The assay was highly reproducible with an overall coefficient of variation of 10.4%. CONCLUSION: The use of highly reproducible and accurate SYBR-based real-time polymerase chain reaction (PCR) assays instead of performing Taqman-type assays allows low-cost, high-throughput analysis of viral mRNA expression. The development of such assays will help in refining the current screening programs for HPV-related carcinomas.