Diagnostic application of padlock probes—multiplex detection of plant pathogens using universal microarrays

Padlock probes (PLPs) are long oligonucleotides, whose ends are complementary to adjacent target sequences. Upon hybridization to the target, the two ends are brought into contact, allowing PLP circularization by ligation. PLPs provide extremely specific target recognition, which is followed by universal amplification and microarray detection. Since target recognition is separated from downstream processing, PLPs enable the development of flexible and extendable diagnostic systems, targeting diverse organisms. To adapt padlock technology for diagnostic purposes, we optimized PLP design to ensure high specificity and eliminating ligation on non-target sequences under real-world assay conditions. We designed and tested 11 PLPs to target various plant pathogens at the genus, species and subspecies levels, and developed a prototype PLP-based plant health chip. Excellent specificity was demonstrated toward the target organisms. Assay background was determined for each hybridization using a no-target reference sample, which provided reliable and sensitive identification of positive samples. A sensitivity of 5 pg genomic DNA and a dynamic range of detection of 100 were observed. The developed multiplex diagnostic system was validated using genomic DNAs of characterized isolates and artificial mixtures thereof. The demonstrated system is adaptable to a wide variety of applications ranging from pest management to environmental microbiology

Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays™

<p>Abstract</p> <p>Background</p> <p>Diagnostics and disease-management strategies require technologies to enable the simultaneous detection and quantification of a wide range of pathogenic microorganisms. Most multiplex, quantitative detection methods available suffer from compromises between the level of multiplexing, throughput and accuracy of quantification. Here, we demonstrate the efficacy of a novel, high-throughput, ligation-based assay for simultaneous quantitative detection of multiple plant pathogens. The ligation probes, designated Plant Research International-lock probes (PRI-lock probes), are long oligonucleotides with target complementary regions at their 5' and 3' ends. Upon perfect target hybridization, the PRI-lock probes are circularized via enzymatic ligation, subsequently serving as template for individual, standardized amplification via unique probe-specific primers. Adaptation to OpenArrays™, which can accommodate up to 3072 33 nl PCR amplifications, allowed high-throughput real-time quantification. The assay combines the multiplex capabilities and specificity of ligation reactions with high-throughput real-time PCR in the OpenArray™, resulting in a flexible, quantitative multiplex diagnostic system.</p> <p>Results</p> <p>The performance of the PRI-lock detection system was demonstrated using 13 probes targeting several significant plant pathogens at different taxonomic levels. All probes specifically detected their corresponding targets and provided perfect discrimination against non-target organisms with very similar ligation target sites. The nucleic acid targets could be reliably quantified over 5 orders of magnitude with a dynamic detection range of more than 10⁴. Pathogen quantification was equally robust in single target versus mixed target assays.</p> <p>Conclusion</p> <p>This novel assay enables very specific, high-throughput, quantitative detection of multiple pathogens over a wide range of target concentrations and should be easily adaptable for versatile diagnostic purposes.</p

Alternative propulsor for mobile transportation and technological machines wood complex

Лесные машины, оборудованные альтернативным движителем, способны передвигаться по любым типам поверхностей (подготовленным дорогам, пахоте, болоту, песку, заснеженной местности и т.д.) с минимальным негативным воздействием.Forestry machines equipped alternative propulsors are capable to move on any types of land surfaces (the prepared roads, plowed land, bog, the sand, snow-covered land and etc.) with the minimal negative influence

A Wnt-BMP4 signaling axis induces MSX and NOTCH proteins and promotes growth suppression and differentiation in neuroblastoma

The Wnt and bone morphogenetic protein (BMP) signaling pathways are known to be crucial in the development of neural crest lineages, including the sympathetic nervous system. Surprisingly, their role in paediatric neuroblastoma, the prototypic tumor arising from this lineage, remains relatively uncharacterised. We previously demonstrated that Wnt/β-catenin signaling can have cell-type-specific effects on neuroblastoma phenotypes, including growth inhibition and differentiation, and that BMP4 mRNA and protein were induced by Wnt3a/Rspo2. In this study, we characterised the phenotypic effects of BMP4 on neuroblastoma cells, demonstrating convergent induction of MSX homeobox transcription factors by Wnt and BMP4 signaling and BMP4-induced growth suppression and differentiation. An immunohistochemical analysis of BMP4 expression in primary neuroblastomas confirms a striking absence of BMP4 in poorly differentiated tumors, in contrast to a high expression in ganglion cells. These results are consistent with a tumor suppressive role for BMP4 in neuroblastoma. RNA sequencing following BMP4 treatment revealed induction of Notch signaling, verified by increases of Notch3 and Hes1 proteins. Together, our data demonstrate, for the first time, Wnt-BMP-Notch signaling crosstalk associated with growth suppression of neuroblastoma

Transcriptomic analyses of MYCN-regulated genes in anaplastic Wilms' tumour cell lines reveals oncogenic pathways and potential therapeutic vulnerabilities

The MYCN proto-oncogene is deregulated in many cancers, most notably in neuroblastoma, where MYCN gene amplification identifies a clinical subset with very poor prognosis. Gene expression and DNA analyses have also demonstrated overexpression of MYCN mRNA, as well as focal amplifications, copy number gains and presumptive change of function mutations of MYCN in Wilms’ tumours with poorer outcomes, including tumours with diffuse anaplasia. Surprisingly, however, the expression and functions of the MYCN protein in Wilms’ tumours still remain obscure. In this study, we assessed MYCN protein expression in primary Wilms’ tumours using immunohistochemistry of tissue microarrays. We found MYCN protein to be expressed in tumour blastemal cells, and absent in stromal and epithelial components. For functional studies, we used two anaplastic Wilms’ tumour cell-lines, WiT49 and 17.94, to study the biological and transcriptomic effects of MYCN depletion. We found that MYCN knockdown consistently led to growth suppression but not cell death. RNA sequencing identified 561 MYCN-regulated genes shared by WiT49 and 17.94 cell-lines. As expected, numerous cellular processes were downstream of MYCN. MYCN positively regulated the miRNA regulator and known Wilms’ tumour oncogene LIN28B, the genes encoding methylosome proteins PRMT1, PRMT5 and WDR77, and the mitochondrial translocase genes TOMM20 and TIMM50. MYCN repressed genes including the developmental signalling receptor ROBO1 and the stromal marker COL1A1. Importantly, we found that MYCN also repressed the presumptive Wilms’ tumour suppressor gene REST, with MYCN knockdown resulting in increased REST protein and concomitant repression of RE1-Silencing Transcription factor (REST) target genes. Together, our study identifies regulatory axes that interact with MYCN, providing novel pathways for potential targeted therapeutics for poor-prognosis Wilms’ tumour

Correction: LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/β-catenin signalling in neuroblastoma

A Correction on: LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/β-catenin signalling in neuroblastoma Gabriella Cunha Vieira, S. Chockalingam, Zsombor Melegh, Alexander Greenhough, Sally Malik, Marianna Szemes, Ji Hyun Park, Abderrahmane Kaidi, Li Zhou, Daniel Catchpoole, Rhys Morgan, David O. Bates, Peter J. Gabb and Karim Malik Original article: Oncotarget. 2015; 6:40053-67. DOI: 10.18632/oncotarget.5548. The originally Figure 5 contains duplicate total-ERK panels. The proper Figure 5 is attached. The authors sincerely apologize for this error

Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells

© 2014 The Authors. Approximately half of poor prognosis neuroblastomas (NBs) are characterized by pathognomonic MYCN gene amplification and MYCN over-expression. Here we present data showing that short-interfering RNA mediated depletion of the protein arginine methyltransferase 5 (PRMT5) in cell-lines representative of NBs with MYCN gene amplification leads to greatly impaired growth and apoptosis. Growth suppression is not apparent in the MYCN-negative SH-SY5Y NB cell-line, or in two immortalized human fibroblast cell-lines. Immunoblotting of NB cell-lines shows that high PRMT5 expression is strongly associated with MYCN-amplification (P < 0.004, Mann-Whitney U-test) and immunohistochemical analysis of primary NBs reveals that whilst PRMT5 protein is ubiquitously expressed in the cytoplasm of most cells, MYCN-amplified tumours exhibit pronounced nuclear PRMT5 staining. PRMT5 knockdown in MYCN-overexpressing cells, including the SHEP-21N cell-line with inducible MYCN expression leads to a dramatic decrease in MYCN protein and MYCN-associated cell-death in SHEP-21N cells. Quantitative gene expression analysis and cycloheximide chase experiments suggest that PRMT5 regulates MYCN at a post-transcriptional level. Reciprocal co-immunoprecipitation experiments demonstrated that endogenous PRMT5 and MYCN interact in both SK-N-BE(2)C and NGP cell lines. By using liquid chromatography - tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated MYCN protein, we identified several potential sites of arginine dimethylation on the MYCN protein. Together our studies implicate PRMT5 in a novel mode of MYCN post-translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis. Small-molecule inhibitors of PRMT5 may therefore represent a novel therapeutic strategy for neuroblastoma and other cancers driven by the MYCN oncogene

Wnt signaling is a major determinant of neuroblastoma cell lineages

© 2019 Szemes, Greenhough and Malik. The neural crest (NC), which has been referred to as the fourth germ layer, comprises a multipotent cell population which will specify diverse cells and tissues, including craniofacial cartilage and bones, melanocytes, the adrenal medulla and the peripheral nervous system. These cell fates are known to be determined by gene regulatory networks (GRNs) acting at various stages of NC development, such as induction, specification, and migration. Although transcription factor hierarchies and some of their interplay with morphogenetic signaling pathways have been characterized, the full complexity of activities required for regulated development remains uncharted. Deregulation of these pathways may contribute to tumorigenesis, as in the case of neuroblastoma, a frequently lethal embryonic cancer thought to arise from the sympathoadrenal lineage of the NC. In this “Hypothesis and Theory” article, we utilize the next generation sequencing data from neuroblastoma cells and tumors to evaluate the possible influences of Wnt signaling on NC GRNs and on neuroblastoma cell lineages. We propose that Wnt signaling is a major determinant of regulatory networks that underlie mesenchymal/neural crest cell (NCC)-like cell identities through PRRX1 and YAP/TAZ transcription factors. Furthermore, Wnt may also co-operate with Hedgehog signaling in driving proneural differentiation programmes along the adrenergic (ADRN) lineage. Elucidation of Signaling Regulatory Networks can augment and complement GRNs in characterizing cell identities, which may in turn contribute to the design of improved therapeutics tailored to primary and relapsing neuroblastoma

PRMT1 promotes neuroblastoma cell survival through ATF5

Aberrant expression of protein arginine methyltransferases (PRMTs) has been implicated in a number of cancers, making PRMTs potential therapeutic targets. But it remains not well understood how PRMTs impact specific oncogenic pathways. We previously identified PRMTs as important regulators of cell growth in neuroblastoma, a deadly childhood tumor of the sympathetic nervous system. Here, we demonstrate a critical role for PRMT1 in neuroblastoma cell survival. PRMT1 depletion decreased the ability of murine neuroblastoma sphere cells to grow and form spheres, and suppressed proliferation and induced apoptosis of human neuroblastoma cells. Mechanistic studies reveal the prosurvival factor, activating transcription factor 5 (ATF5) as a downstream effector of PRMT1-mediated survival signaling. Furthermore, a diamidine class of PRMT1 inhibitors exhibited anti-neuroblastoma efficacy both in vitro and in vivo. Importantly, overexpression of ATF5 rescued cell apoptosis triggered by PRMT1 inhibition genetically or pharmacologically. Taken together, our findings shed new insights into PRMT1 signaling pathway, and provide evidence for PRMT1 as an actionable therapeutic target in neuroblastoma