Molecular Padlock Assay of Crude Plant Leaf Extracts for Detection of Listeria Monocytogenes

A molecular padlock assay was developed and assessed for detection of Listeria monocytogenes that operated in crude plant extracts. The molecular padlock assay was developed by Liu et al. (1996) and modified by Lizardi et al. (1998). We further modified and described a padlock probe that detected L. monocytogenes oligonucleotide, cDNA and genomic DNA containing a 16s rRNA sequence (GenBank Acc. No. X56 153). This technique was effective in the presence of crude potato leaf extracts in contrast to PCR, which failed to detect the presence of L. monocytogenes targets in crude leaf extracts. Sensitivity of the padlock procedure was determined to be 0.02 ng using L. monocytogenes genomic DNA target templates and 0.0025 nM L. monocytogenes 40 nt oligonucleotide target sequences in an aqueous solution. Results also showed the efficacy of molecular padlocks to detect L. monocytogenes pathogens in a 5 pg potato leaf RNA background and crude leaf extracts, although sensitivity of the assay is insufficient to dispense with a pre-enrichment step for reliable detection of L. monocytogenes. The advantage of the molecular padlock assay over other FDA approved serological and molecular-based assays, is that it may be possible to design the assay to be species or strain specific while still retaining the ability to be performed in crude plant extracts

Selective removal of deletion-bearing mitochondrial DNA in heteroplasmic Drosophila

Mitochondrial DNA (mtDNA) often exists in a state of heteroplasmy, in which mutant mtDNA co-exists in cells with wild-type mtDNA. High frequencies of pathogenic mtDNA result in maternally inherited diseases; maternally and somatically acquired mutations also accumulate over time and contribute to diseases of ageing. Reducing heteroplasmy is therefore a therapeutic goal and in vivo models in post-mitotic tissues are needed to facilitate these studies. Here we describe a transgene-based model of a heteroplasmic lethal mtDNA deletion (mtDNA^Δ) in adult Drosophila muscle. Stimulation of autophagy, activation of the PINK1/parkin pathway or decreased levels of mitofusin result in a selective decrease in mtDNA^Δ. Decreased levels of mitofusin and increased levels of ATPIF1, an inhibitor of ATP synthase reversal-dependent mitochondrial repolarization, result in a further decrease in mtDNA^Δ levels. These results show that an adult post-mitotic tissue can be cleansed of a deleterious genome, suggesting that therapeutic removal of mutant mtDNA can be achieved

A dual-tag microarray platform for high-performance nucleic acid and protein analyses

DNA microarrays serve to monitor a wide range of molecular events, but emerging applications like measurements of weakly expressed genes or of proteins and their interaction patterns will require enhanced performance to improve specificity of detection and dynamic range. To further extend the utility of DNA microarray-based approaches we present a high-performance tag microarray procedure that enables probe-based analysis of as little as 100 target cDNA molecules, and with a linear dynamic range close to 105. Furthermore, the protocol radically decreases the risk of cross-hybridization on microarrays compared to current approaches, and it also allows for quantification by single-molecule analysis and real-time on-chip monitoring of rolling-circle amplification. We provide proof of concept for microarray-based measurement of both mRNA molecules and of proteins, converted to tag DNA sequences by padlock and proximity probe ligation, respectively

Implementation of molecular detection techniques in the field of veterinary virology

This thesis deals with the molecular diagnosis of infectious diseases concerning animal and human health: in particular, with diseases notifiable to the World Organization for Animal Health, OIE (the vesicular complex and avian influenza), as well as with Hepatitis E, representing emerging zoonotic aspects. With the worldwide introduction and use of the polymerase chain reaction (PCR) methodologies, the detection of different pathogens improved significantly - however, these systems have weak points as well. The parallel screening of more than a few pathogens is not resolved and, in general, the multiplexing capacity of most of the methods used in this area is insufficient. In the case of the detection of pathogens causing similar symptoms (like the vesicular complex, involving Foot-and-Mouth Disease (FMD), Swine Vesicular Disease (SVD) and Vesicular Stomatitis (VS), the immediate differential diagnosis is essential, not only regarding the multiplexing, but also because of the high economic risks and the strict legislations. Subtyping of the avian influenza viruses is a broad and extensive task because it needs to differentiate 16 hemagglutinin and 9 neuraminidase types and their variants. The padlock probes, as used in these studies, seem to be optimal to fulfill the multiplexing requirements and to provide novel, high-throughput tools for the improved diagnosis of the vesicular complex and of avian influenza. The general detection and subtyping of Hepatitis E Virus (HEV) is an important and complicated task today, as the virus shows zoonotic potential by causing endemics in humans and persisting infections in different animal species. Thus, there is a high need for sensitive and specific methods and identification of HEV variants. In the frame of this work, a highly specific and sensitive diagnostic assay was developed, based on two types of real-time PCR methods. In addition, a genotyping system was constructed using a simple and quick ligation-based technique

WNT-DEPENDENT REGENERATIVE FUNCTION IS INDUCED IN LEUKEMIA-INITIATING AC133BRIGHT CELLS

The Cancer Stem Cell model supported the notion that leukemia was initiated and maintained in vivo by a small fraction of leukemia-initiating cells (LICs). Previous studies have suggested the involvement of Wnt signaling pathway in Acute Myeloid Leukemia (AML) by the ability to sustain the development of LICs. A novel hematopoietic stem and progenitor cell marker, monoclonal antibody AC133, recognizes the CD34bright CD38- subset of human acute myeloid leukemia cells, suggesting that it may be an early marker for the LICs. During the first part of my phD program we previously evaluated the ability of leukemic AC133+ fraction, to perform engraftment following to xenotransplantation in immunodeficient mouse model Rag2-/-\u3b3c-/-. The results showed that the surface marker AC133 is able to enrich for the cell fraction that contains the LICs. In consideration of our previously reported data, derived from the expression profiling analysis performed in normal (n=10) and leukemic (n=33) human long-term reconstituting AC133+ cells, we revealed that the ligand-dependent Wnt signaling is induced in AML through a diffuse expression and release of WNT10B, a hematopoietic stem cells regenerative-associated molecule. In situ detection performed on bone marrow biopsies of AML patients, showed the activation of the Wnt pathway, through the concomitant presence of the ligand WNT10B and of the active dephosphorylated \u3b2-catenin form, suggesting an autocrine / paracrine-type ligand-dependent activation mechanism. In consideration of the link between hematopoietic regeneration and developmental signaling, we transplanted primary AC133+ AML A46 cells into developing zebrafish. This biosensor model revealed the formation of ectopic structures by activation of dorsal organizer markers that act downstream of the Wnt pathway. These results suggested that the misappropriating Wnt associated functions can promote pathological stem cell-like regeneration responsiveness. The analyses performed in situ retained information on the cellular localization, enabling determination of the activity status of individual cells and allowing the tumor environment view. Taking this issue into consideration, during the second part of my phD program, I set up the application of a new in situ method for localized detection and genotyping of individual transcripts directly in cells and tissues. The mRNA in situ detection technique is based on padlock probes ligation and target priming rolling circle amplification allowing the single nucleotide resolution in heterogenous tissues. The mRNA in situ detection performed on bone marrow biopsies derived from AML patients, showed a diffuse localization pattern of WNT10B molecule in the tissue. Conversely, only the AC133bright cell population shows the Wnt signaling activation signature represented by the cytoplasmatic accumulation and nuclear translocation of the active form of \u3b2-catenin. In spite of this, we previously evidenced that the regenerative function of WNT signaling pathway is defined by the up-regulation of WNT10B, WNT10A, WNT2B and WNT6 loci, we identified the WNT10B as a major locus associated with the regenerative function and over-expressed by all AML patients. By the molecular evaluation of the WNT10B transcript, we isolated an aberrant splicing variant (WNT10BIVS1), that identify Non Core-Binding Factor Leukemia (NCBFL) class and whose potential role is discussed. Moreover, we demonstrate that the function of "leukemia stem cell", present in the cell population enriched for the marker AC133bright, is strictly related to regenerative function associated with WNT signaling, defining the key role of WNT10B ligand as a specific molecular marker for leuchemogenesis. This thesis defines the new suitable approaches to characterize the leukemia-initiating cells (LICs) and suggest the role of WNT10B as a new suitable target for AML

Rolling circle amplification for direct detection of rpoB gene mutations in Mycobacterium tuberculosis directly from clinical specimens

Rapid and accurate detection of multidrug resistance (MDR) inMycobacterium tuberculosisis essential to improve treatment outcomes and reduce global transmission but remains a challenge. Rifampin (RIF) resistance is a reliable marker of MDR tuberculosis (TB) since by far the majority of RIF-resistant strains are also isoniazid (INH) resistant. We have developed a rapid, sensitive, and specific method for detecting the most common mutations associated with RIF resistance, in the RIF resistance determining region (RRDR) ofrpoB, using a cocktail of six padlock probes and rolling circle amplification (RCA). We used this method to test 46 storedM. tuberculosisclinical isolates with known RIF susceptibility profiles (18 RIF resistant, 28 susceptible), a standard susceptible strain (H37Rv, ATCC 27294) and 78M. tuberculosisculture-positive clinical (sputum) samples, 59 of which grew RIF-resistant strains. All stored clinical isolates were correctly categorized, by the padlock probe/RCA method, as RIF susceptible or resistant; the sensitivity and specificity of the method, for direct detection of phenotypically RIF-resistantM. tuberculosisin clinical specimens, were 96.6 and 89.5%, respectively. This method is rapid, simple, and inexpensive and has the potential for high-throughput routine screening of clinical specimens for MDRM. tuberculosis, particularly in high prevalence settings with limited resources.a grant from The National S&T Major Special Project on Major New Drug Innovation (2012ZX09301002-005-003) from the Ministry of Science and Technology of Chin

Chromatin signature of widespread monoallelic expression

In mammals, numerous autosomal genes are subject to mitotically stable monoallelic expression (MAE), including genes that play critical roles in a variety of human diseases. Due to challenges posed by the clonal nature of MAE, very little is known about its regulation; in particular, no molecular features have been specifically linked to MAE. In this study, we report an approach that distinguishes MAE genes in human cells with great accuracy: a chromatin signature consisting of chromatin marks associated with active transcription (H3K36me3) and silencing (H3K27me3) simultaneously occurring in the gene body. The MAE signature is present in ∼20% of ubiquitously expressed genes and over 30% of tissue-specific genes across cell types. Notably, it is enriched among key developmental genes that have bivalent chromatin structure in pluripotent cells. Our results open a new approach to the study of MAE that is independent of polymorphisms, and suggest that MAE is linked to cell differentiation. DOI: http://dx.doi.org/10.7554/eLife.01256.00

A transcriptomic axis predicts state modulation of cortical interneurons

Transcriptomics has revealed that cortical inhibitory neurons exhibit a great diversity of fine molecular subtypes1-6, but it is not known whether these subtypes have correspondingly diverse patterns of activity in the living brain. Here we show that inhibitory subtypes in primary visual cortex (V1) have diverse correlates with brain state, which are organized by a single factor: position along the main axis of transcriptomic variation. We combined in vivo two-photon calcium imaging of mouse V1 with a transcriptomic method to identify mRNA for 72 selected genes in ex vivo slices. We classified inhibitory neurons imaged in layers 1-3 into a three-level hierarchy of 5 subclasses, 11 types and 35 subtypes using previously defined transcriptomic clusters3. Responses to visual stimuli differed significantly only between subclasses, with cells in the Sncg subclass uniformly suppressed, and cells in the other subclasses predominantly excited. Modulation by brain state differed at all hierarchical levels but could be largely predicted from the first transcriptomic principal component, which also predicted correlations with simultaneously recorded cells. Inhibitory subtypes that fired more in resting, oscillatory brain states had a smaller fraction of their axonal projections in layer 1, narrower spikes, lower input resistance and weaker adaptation as determined in vitro7, and expressed more inhibitory cholinergic receptors. Subtypes that fired more during arousal had the opposite properties. Thus, a simple principle may largely explain how diverse inhibitory V1 subtypes shape state-dependent cortical processing