Entwicklung eines diagnostischen Mikroarrays zum Nachweis von Beta-Laktamasen mit erweitertem Wirkungsspektrum für den Einsatz in der klinischen Mikrobiologie

Among the most important types of resistances to be detected are the extended spectrum beta-lactamases (ESBLs). ESBLs are found in many different species of the family Enterobacteriacae. Most ESBLs are mutants of TEM- or SHV-type beta-lactamases. The TEM- and SHV- subtypes are derived from parental sequences (TEM-1, SHV-1) and differ from them by a variable number of amino acid substitutions. These mutations lead to an extended spectrum of activity against newer lactams, especially against 3rd generation cephalosporins. Other derivatives of the classical TEM or SHV enzymes also show an inhibitor resistant TEM (IRT) phenotype conferring resistance to beta-lactamase inhibitors. ESBL producing organisms are difficult to detect in standard phenotypic screening tests, mainly because of their widely varying levels of activity against various cephalosporins. For an improved accuracy confirmatory susceptibility tests have to be performed resulting in a response time of three days until the ESBL phenotype can be identified unequivocally. Since infections with ESBL producing organisms are registered with increased prevalence and are associated with significantly longer hospital stays and higher costs, more accurate tests to detect ESBLs in clinical isolates are necessary. The microarray technology allows the genotypic identification of resistance traits in less than one day of analysis time. Furthermore, the identification of the beta-lactamase variant on a molecular level will define for most ESBL isolates a specific substrate pattern, which can be considered for the determination of the appropriate antibiotic treatment. Additionally, the genotyping of resistances can be used for the reliable surveillance of multiresistant bacteria in wards or hospitals. In the present study a diagnostic microarray was developed for the rapid identification of mutations of the majority of the currently known TEM or SHV beta-lactamase variants, which are related to the ESBL and/or IRT phenotype. The assay enabled the detection and identification of 99 % of the relevant polymorphisms for TEM beta-lactamases and 100 % of the mutations of SHV beta-lactamases. This allows the detection of 96 % of the currently known TEM-variants and 100 % of the known SHV-variants. Consensus primers were developed and used for target amplification covering the majority of the known variant sequences. The sensitivity, reproducibility and identification capability of the developed arrays was determined with a set of reference samples. Furthermore, the TEM-array was validated by testing 72 clinical isolates collected in diverse institutions in Germany, Croatia and Russia. The SHV-array was validated by testing 30 clinical isolates collected in Croatia. The simultaneous detection of an extended spectrum-variant in presence of a narrow spectrum-variant was shown in a model system for TEM up to a ratio of 1:10, as well as in clinical isolates for SHV. Starting from the isolated DNA, the assay could be performed in less than 3.5 hours. The discrimination level, the sensitivity and the reproducibility were enhanced by automation of the hybridization procedure. The development of a marketable diagnostic ESBL microarray based on the presented prototypes and the extension of the developed system towards the detection of other relevant beta-lactamase families is in progress. In conclusion, the diagnostic test developed in this study offers a promising approach for the rapid identification and epidemiologic monitoring of TEM or SHV ESBL and IRT beta-lactamases.Das Auftreten von Resistenzen gegen Beta-Laktam Antibiotika ist ein Problem von zunehmender Bedeutung weltweit. Das Vorkommen von Beta-Laktamasen mit erweitertem Wirkungsspektrum (Extended Spectrum Beta-Lactamases, ESBL) ist hierbei besonders problematisch. ESBLs treten in vielen verschiedenen Spezies der Gattung Enterobacteriacae auf. Die am häufigsten vertretenen Enzyme dieser Art in klinischen Isolaten sind Varianten von TEM-1 oder SHV-1 Beta-Laktamasen. Aufgrund von Aminosäureaustauschmutationen, die zu einer Erweiterung des Substratspektrums führen, vermitteln diese Enzyme auch Resistenzen gegen neuere Generationen der Beta-Laktam Antibiotika (besonders Cephalosporine der 3. und 4. Gruppe). Es treten auch Varianten der TEM- oder SHV-Familie auf, die Resistenzen gegen Inhibitoren (z. B. Clavulansäure) vermitteln. Diese Enzyme werden als IRTs (ursprünglich für Inhibitor Resistant TEM) bezeichnet. Da die einzelnen ESBL Enzymvarianten stark unterschiedliche Substratspektren aufweisen, ist ihr Nachweis mit herkömmlichen phänotypischen Screening-Methoden schwierig. Um die Zuverlässigkeit der Ergebnisse des ESBL Screenings zu erhöhen, ist die Durchführung von zusätzlichen phänotypischen Tests zur Bestätigung des ESBL-Verdachts unerlässlich. Es kann daher bis zu drei Tagen dauern, bis der Verdacht auf eine Infektion mit einem ESBL-produzierenden Organismus bestätigt wird. Solche Infektionen werden mit längeren Krankenhausaufenthalten und höheren Behandlungskosten in Verbindung gebracht. Daher ist die Entwicklung präziserer und schnellerer Testmethoden zum Nachweis dieser Resistenzen notwendig. Die Mikroarray Technologie erlaubt die genotypische Identifizierung von Resistenzen in einer Analysezeit von weniger als einem Tag. Hierbei können, im Gegensatz zur phänotypischen Diagnostik und auch zu vielen anderen genotypischen Nachweisverfahren, alle relevanten Mutationspositionen erkannt und somit auch verschiedene ESBL-Typen differenziert nachgewiesen werden. Für viele ESBL-Varianten wurde bereits ein spezifisches Substratspektrum definiert, welches nach der eindeutigen Identifizierung auf der molekularen Ebene zu Rate gezogen werden kann, um eine spezialisierte, prediktive Diagnostik zu ermöglichen. Weiterhin kann die genotypische Identifizierung, im Gegensatz zum phänotypischen Nachweis, für eine zuverlässige Überwachung der Verbreitung bestimmter Resistenzmerkmale oder multiresistenter Bakterien im klinischen Umfeld eingesetzt werden. In dieser Arbeit wurde ein diagnostischer Mikroarray entwickelt für den schnellen und spezifischen Nachweis der Mutationen der TEM und SHV Beta-Laktamase Varianten, die verantwortlich für die Ausprägung eines ESBL oder IRT Phänotyps sind. Die Allel-spezifische Hybridisierung auf dem DNS-Mikroarray erlaubt den Nachweis von 41 Mutationspositionen der TEM Beta-Laktamase Familie (entspricht 99 % der veröffentlichten Positionen) und 37 Mutationspositionen (entspricht 100 % der veröffentlichten Positionen) relevant für SHV. Dies ermöglicht die Identifizierung von 96 % der bis jetzt bekannten TEM Varianten und 100 % der SHV Varianten. Die Vervielfältigung der Resistenzgene erfolgte per PCR über Konsensus-Primer. Die Sensitivität, Reproduzierbarkeit und die Spezifität der Identifizierung der unterschiedlichen Varianten wurde anhand der Mikroarrayanalyse von Referenzstämmen nachgewiesen. Weiterhin wurde der TEM-Array durch die Analyse von 72 klinischen Isolaten, die aus unterschiedlichen Institutionen in Deutschland, Kroatien und Russland stammten, validiert. Der SHV-Array wurde mit 30 klinischen Isolaten aus Kroatien getestet. Der Nachweis von zwei Varianten der gleichen Laktamase-Familie innerhalb eines Isolats, wurde für TEM in einem Modell-System bis zu einem Verhältnis von 1 zu 10 und für SHV in realen klinischen Proben gezeigt. Die Mikroarrayanalyse konnte nach der DNS-Isolation innerhalb von 3.5 Stunden durchgeführt werden. Die Sensitivität und die Reproduzierbarkeit der Analyse wurden durch Automatisierung der Hybridisierung und des Waschvorgangs gesteigert. Die Entwicklung eines marktreifen Systems aus den in dieser Studie entwickelten Prototypen, sowie die Ergänzung der bisher entwickelten TEM und SHV-Arrays zu einem Komplettsystem zum Nachweis aller Merkmale klinisch relevanter Laktamasefamilien ist derzeit in Arbeit. Die hier entwickelten DNS-Mikroarrays stellen ein viel versprechendes System zur schnellen Identifizierung von TEM und SHV ESBL und IRT Varianten dar und ermöglichen die Überwachung der Verbreitung dieser Resistenzen im klinischen Umfeld

Plasmalogens Inhibit APP Processing by Directly Affecting γ-Secretase Activity in Alzheimer's Disease

Lipids play an important role as risk or protective factors in Alzheimer's disease (AD). Previously it has been shown that plasmalogens, the major brain phospholipids, are altered in AD. However, it remained unclear whether plasmalogens themselves are able to modulate amyloid precursor protein (APP) processing or if the reduced plasmalogen level is a consequence of AD. Here we identify the plasmalogens which are altered in human AD postmortem brains and investigate their impact on APP processing resulting in Aβ production. All tested plasmalogen species showed a reduction in γ-secretase activity whereas β- and α-secretase activity mainly remained unchanged. Plasmalogens directly affected γ-secretase activity, protein and RNA level of the secretases were unaffected, pointing towards a direct influence of plasmalogens on γ-secretase activity. Plasmalogens were also able to decrease γ-secretase activity in human postmortem AD brains emphasizing the impact of plasmalogens in AD. In summary our findings show that decreased plasmalogen levels are not only a consequence of AD but that plasmalogens also decrease APP processing by directly affecting γ-secretase activity, resulting in a vicious cycle: Aβ reduces plasmalogen levels and reduced plasmalogen levels directly increase γ-secretase activity leading to an even stronger production of Aβ peptides

Intracellular APP Domain Regulates Serine-Palmitoyl-CoA Transferase Expression and Is Affected in Alzheimer's Disease

Lipids play an important role as risk or protective factors in Alzheimer's disease (AD), a disease biochemically characterized by the accumulation of amyloid beta peptides (Aβ), released by proteolytic processing of the amyloid precursor protein (APP). Changes in sphingolipid metabolism have been associated to the development of AD. The key enzyme in sphingolipid de novo synthesis is serine-palmitoyl-CoA transferase (SPT). In the present study we identified a new physiological function of APP in sphingolipid synthesis. The APP intracellular domain (AICD) was found to decrease the expression of the SPT subunit SPTLC2, the catalytic subunit of the SPT heterodimer, resulting in that decreased SPT activity. AICD function was dependent on Fe65 and SPTLC2 levels are increased in APP knock-in mice missing a functional AICD domain. SPTLC2 levels are also increased in familial and sporadic AD postmortem brains, suggesting that SPT is involved in AD pathology

High-quality draft genome sequence of Bifidobacterium longum E18, isolated from a healthy adult

Bifidobacteria are important gastrointestinal commensals of a number of animals, including humans, and various beneficial effects on host health have been attributed to them. Here, we announce the noncontiguous finished genome sequence of Bifidobacterium longum E18, isolated from a healthy adult, which reveals traits involved in its interaction with the host

Evaluation of a Novel Thiol–Norbornene-Functionalized Gelatin Hydrogel for Bioprinting of Mesenchymal Stem Cells

Introduction: Three-dimensional bioprinting can be considered as an advancement of the classical tissue engineering concept. For bioprinting, cells have to be dispersed in hydrogels. Recently, a novel semi-synthetic thiolene hydrogel system based on norbornene-functionalized gelatin (GelNB) and thiolated gelatin (GelS) was described that resulted in the photoclick hydrogel GelNB/GelS. In this study, we evaluated the printability and biocompatibility of this hydrogel system towards adipose-tissue-derived mesenchymal stem cells (ASCs). Methods: GelNB/GelS was synthesized with three different crosslinking densities (low, medium and high), resulting in different mechanical properties with moduli of elasticity between 206 Pa and 1383 Pa. These hydrogels were tested for their biocompatibility towards ASCs in terms of their viability, proliferation and differentiation. The extrusion-based bioprinting of ASCs in GelNB/GelS-high was performed to manufacture three-dimensional cubic constructs. Results: All three hydrogels supported the viability, proliferation and chondrogenic differentiation of ASCs to a similar extent. The adipogenic differentiation of ASCs was better supported by the softer hydrogel (GelNB/GelS-low), whereas the osteogenic differentiation was more pronounced in the harder hydrogel (GelNB/GelS-high), indicating that the differentiation fate of ASCs can be influenced via the adaption of the mechanical properties of the GelNB/GelS system. After the ex vivo chondrogenic differentiation and subcutaneous implantation of the bioprinted construct into immunocompromised mice, the production of negatively charged sulfated proteoglycans could be observed with only minimal inflammatory signs in the implanted material. Conclusions: Our results indicate that the GelNB/GelS hydrogels are very well suited for the bioprinting of ASCs and may represent attractive hydrogels for subsequent in vivo tissue engineering applications

mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy

Large genes including several CRISPR-Cas modules like gene activators (CRISPRa) require dual adeno-associated viral (AAV) vectors for an efficient in vivo delivery and expression. Current dual AAV vector approaches have important limitations, e.g., low reconstitution efficiency, production of alien proteins, or low flexibility in split site selection. Here, we present a dual AAV vector technology based on reconstitution via mRNA trans-splicing (REVeRT). REVeRT is flexible in split site selection and can efficiently reconstitute different split genes in numerous in vitro models, in human organoids, and in vivo. Furthermore, REVeRT can functionally reconstitute a CRISPRa module targeting genes in various mouse tissues and organs in single or multiplexed approaches upon different routes of administration. Finally, REVeRT enabled the reconstitution of full-length ABCA4 after intravitreal injection in a mouse model of Stargardt disease. Due to its flexibility and efficiency REVeRT harbors great potential for basic research and clinical applications

New Alzheimer Amyloid β Responsive Genes Identified in Human Neuroblastoma Cells by Hierarchical Clustering

Alzheimer's disease (AD) is characterized by neuronal degeneration and cell loss. Aβ42, in contrast to Aβ40, is thought to be the pathogenic form triggering the pathological cascade in AD. In order to unravel overall gene regulation we monitored the transcriptomic responses to increased or decreased Aβ40 and Aβ42 levels, generated and derived from its precursor C99 (C-terminal fragment of APP comprising 99 amino acids) in human neuroblastoma cells. We identified fourteen differentially expressed transcripts by hierarchical clustering and discussed their involvement in AD. These fourteen transcripts were grouped into two main clusters each showing distinct differential expression patterns depending on Aβ40 and Aβ42 levels. Among these transcripts we discovered an unexpected inverse and strong differential expression of neurogenin 2 (NEUROG2) and KIAA0125 in all examined cell clones. C99-overexpression had a similar effect on NEUROG2 and KIAA0125 expression as a decreased Aβ42/Aβ40 ratio. Importantly however, an increased Aβ42/Aβ40 ratio, which is typical of AD, had an inverse expression pattern of NEUROG2 and KIAA0125: An increased Aβ42/Aβ40 ratio up-regulated NEUROG2, but down-regulated KIAA0125, whereas the opposite regulation pattern was observed for a decreased Aβ42/Aβ40 ratio. We discuss the possibilities that the so far uncharacterized KIAA0125 might be a counter player of NEUROG2 and that KIAA0125 could be involved in neurogenesis, due to the involvement of NEUROG2 in developmental neural processes

Optical imaging of the peri-tumoral inflammatory response in breast cancer

<p>Abstract</p> <p>Purpose</p> <p>Peri-tumoral inflammation is a common tumor response that plays a central role in tumor invasion and metastasis, and inflammatory cell recruitment is essential to this process. The purpose of this study was to determine whether injected fluorescently-labeled monocytes accumulate within murine breast tumors and are visible with optical imaging.</p> <p>Materials and methods</p> <p>Murine monocytes were labeled with the fluorescent dye DiD and subsequently injected intravenously into 6 transgenic MMTV-PymT tumor-bearing mice and 6 FVB/n control mice without tumors. Optical imaging (OI) was performed before and after cell injection. Ratios of post-injection to pre-injection fluorescent signal intensity of the tumors (MMTV-PymT mice) and mammary tissue (FVB/n controls) were calculated and statistically compared.</p> <p>Results</p> <p>MMTV-PymT breast tumors had an average post/pre signal intensity ratio of 1.8+/- 0.2 (range 1.1-2.7). Control mammary tissue had an average post/pre signal intensity ratio of 1.1 +/- 0.1 (range, 0.4 to 1.4). The p-value for the difference between the ratios was less than 0.05. Confocal fluorescence microscopy confirmed the presence of DiD-labeled cells within the breast tumors.</p> <p>Conclusion</p> <p>Murine monocytes accumulate at the site of breast cancer development in this transgenic model, providing evidence that peri-tumoral inflammatory cell recruitment can be evaluated non-invasively using optical imaging.</p

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements