Recombinant production of A1S_0222 from Acinetobacter baumannii ATCC 17978 and confirmation of its DNA-(adenine N6)-methyltransferase activity.

Acinetobacter baumannii appears as an often multidrug-resistant nosocomial pathogen in hospitals worldwide. Its remarkable persistence in the hospital environment is probably due to intrinsic and acquired resistance to disinfectants and antibiotics, tolerance to desiccation stress, capability to form biofilms, and is possibly facilitated by surface-associated motility. Our attempts to elucidate surface-associated motility in A. baumannii revealed a mutant inactivated in a putative DNA-(adenine N6)-methyltransferase, designated A1S_0222 in strain ATCC 17978. We recombinantly produced A1S_0222 as a glutathione S-transferase (GST) fusion protein and purified it to near homogeneity through a combination of GST affinity chromatography, cation exchange chromatography and PD-10 desalting column. Furthermore we demonstrate A1S_0222-dependent adenine methylation at a GAATTC site. We propose the name AamA (Acinetobacteradenine methyltransferase A) in addition to the formal names M.AbaBGORF222P/M.Aba17978ORF8565P. Small angle X-ray scattering (SAXS) revealed that the protein is monomeric and has an extended and likely two-domain shape in solution.

Regulatory microRNA networks: complex patterns of target pathways for disease-related and housekeeping microRNAs.

Blood-based microRNA (miRNA) signatures as biomarkers have been reported for various pathologies, including cancer, neurological disorders, cardiovascular diseases, and also infections. The regulatory mechanism behind respective miRNA patterns is only partially understood. Moreover, "preserved" miRNAs, i.e., miRNAs that are not dysregulated in any disease, and their biological impact have been explored to a very limited extent. We set out to systematically determine their role in regulatory networks by defining groups of highly-dysregulated miRNAs that contribute to a disease signature as opposed to preserved housekeeping miRNAs. We further determined preferential targets and pathways of both dysregulated and preserved miRNAs by computing multi-layer networks, which were compared between housekeeping and dysregulated miRNAs. Of 848 miRNAs examined across 1049 blood samples, 8 potential housekeepers showed very limited expression variations, while 20 miRNAs showed highly-dysregulated expression throughout the investigated blood samples. Our approach provides important insights into miRNAs and their role in regulatory networks. The methodology can be applied to systematically investigate the differences in target genes and pathways of arbitrary miRNA sets.

Circulating Biomarker Panels in Alzheimer's Disease.

The early diagnosis of diseases frequently represents an important unmet clinical need supporting in-time treatment of pathologies. This also applies to neurodegenerative diseases such as Alzheimer's disease (AD), the most common form of dementia, estimated to affect millions of individuals worldwide. The respective diagnostic and prognostic markers, especially for the preclinical stages of AD, are expected to improve patients' outcome significantly. In the last decades, many approaches to detecting AD have been developed, including markers to discover changes in amyloid-ß levels [from cerebrospinal fluid (CSF) or using positron emission tomography] or other brain imaging technologies such as structural magnetic resonance imaging (MRI), functional-connectivity MRI or task-related functional MRI. A major challenge is the detection of AD using minimally or even noninvasive biomarkers from body fluids such as plasma or serum. Circulating biomarker candidates based on mRNAs or proteins measured from blood cells, plasma or serum have been proposed for various pathologies including AD. As for other diseases, there is a tendency to use marker signatures obtained by high-throughput approaches, which allow the generation of profiles of hundreds to thousands of biomarkers simultaneously [microarrays, mass spectrometry or next-generation sequencing (NGS)]. Beyond mRNAs and proteins, recent approaches have measured small noncoding RNA (so-called microRNA) profiles in AD patients' blood samples using NGS or array-based technologies. Generally, the development of marker panels is in its early stages and requires further, substantial clinical validation. In this review, we provide an overview of different circulating AD biomarkers, starting with a brief summary of CSF markers and focusing on novel biomarker signatures such as small noncoding RNA profiles.

MicroRNA in vitro diagnostics using immunoassay analyzers.

BACKGROUND: The implementation of new biomarkers into clinical practice is one of the most important areas in medical research. Besides their clinical impact, novel in vitro diagnostic markers promise to have a substantial effect on healthcare costs. Although numerous publications report the discovery of biomarkers, only a fraction of those markers are routinely used. One key challenge is a measurement system that is compatible with clinical workflows. METHODS: We designed a new immunoassay for microRNA (miRNA) quantification. The assay combines streptavidin-linked microparticles, a biotinylated catcher oligonucleotide complementary to a single miRNA species, and finally, a monoclonal antibody to DNA/RNA heterohybrids labeled with acridinium ester. Importantly, our assay runs on standard immunoassay analyzers. After a technical validation of the assay, we evaluated the clinical performance on 4 Alzheimer disease miRNAs. RESULTS: Our assay has an analytical specificity of 99.4% and is at the same time sensitive (concentrations in the range of 1 pmol/L miRNA can be reliably profiled). Because the novel approach did not require amplification steps, we obtained high reproducibility for up to 40 biological replicates. Importantly, our assay prototype exhibited a time to result of <3 h. With human blood samples, the assay was able to measure 4 miRNAs that can detect Alzheimer disease with a diagnostic accuracy of 82% and showed a Pearson correlation >0.994 with the gold standard qRT-PCR. CONCLUSIONS: Our miRNA immunoassay allowed the measurement of miRNA signatures with sufficient analytical sensitivity and high specificity on commonly available laboratory equipment.