ABSTRACT
To improve detection sensitivity, molecular diagnostics require preconcentration of low concentrated samples followed by rapid nucleic acid extraction. This is usually achieved by multiple centrifugation, lysis and purification steps, for instance, using chemical reagents, spin columns or magnetic beads. These require extensive infrastructure as well as time consuming manual handling steps and are thus not suitable for point of care testing (POCT). To overcome these challenges, we developed a microfluidic chip combining free-flow electrophoretic (FFE) preconcentration (1 ml down to 5 µl) and thermoelectric lysis of bacteria as well as purification of nucleic acids by gel-electrophoresis. The integration of these techniques in a single chip is unique and enables fast, easy and space-saving sample pretreatment without the need for laboratory facilities, making it ideal for the integration into small POCT devices. A preconcentration efficiency of nearly 100% and a lysis/gel-electrophoresis efficiency of about 65% were achieved for the detection of E. coli. The genetic material was analyzed by RT-qPCR targeting the superfolder Green Fluorescent Protein (sfGFP) transcripts to quantify mRNA recovery and qPCR to determine DNA background.
ABSTRACT
[This corrects the article DOI: 10.1039/C8RA02177E.].
ABSTRACT
Polo-like kinase 1 (PLK1) is frequently overexpressed in cancer, which correlates with poor prognosis. Therefore, we investigated PLK1 as therapeutic target using rhabdomyosarcoma (RMS) as a model. Here, we identify a novel synthetic lethal interaction of PLK1 inhibitors and microtubule-destabilizing drugs in preclinical RMS models and elucidate the underlying molecular mechanisms of this synergism. PLK1 inhibitors (i.e., BI 2536 and BI 6727) synergistically induce apoptosis together with microtubule-destabilizing drugs (i.e., vincristine (VCR), vinblastine (VBL) and vinorelbine (VNR)) in several RMS cell lines (combination index <0.9) including a patient-derived primary RMS culture. Importantly, PLK1 inhibitors and VCR cooperate to significantly suppress RMS growth in two in vivo models, including a mouse xenograft model, without causing additive toxicity. In addition, no toxicity was observed in non-malignant fibroblast or myoblast cultures. Mechanistically, BI 2536/VCR co-treatment triggers mitotic arrest, which initiates mitochondrial apoptosis by inactivation of antiapoptotic BCL-2 family proteins, followed by BAX/BAK activation, production of reactive oxygen species (ROS) and activation of caspase-dependent or caspase-independent effector pathways. This conclusion is supported by data showing that BI 2536/VCR-induced apoptosis is significantly inhibited by preventing cells to enter mitosis, by overexpression of BCL-2 or a non-degradable MCL-1 mutant, by BAK knockdown, ROS scavengers, caspase inhibition or endonuclease G silencing. This identification of a novel synthetic lethality of PLK1 inhibitors and microtubule-destabilizing drugs has important implications for developing PLK1 inhibitor-based combination treatments.
Subject(s)
Apoptosis/drug effects , Cell Cycle Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/toxicity , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Pteridines/toxicity , Tubulin Modulators/toxicity , Animals , Cell Cycle Checkpoints/drug effects , Cell Cycle Proteins/metabolism , Disease Models, Animal , Drug Synergism , Humans , Mice , Mitochondria/drug effects , Mitochondria/metabolism , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Reactive Oxygen Species/metabolism , Rhabdomyosarcoma/metabolism , Rhabdomyosarcoma/pathology , Signal Transduction/drug effects , Transplantation, Heterologous , Tumor Cells, Cultured , bcl-2 Homologous Antagonist-Killer Protein/antagonists & inhibitors , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2 Homologous Antagonist-Killer Protein/metabolism , Polo-Like Kinase 1ABSTRACT
With a rapid and simple actuation protocol electrophoretic nucleic acid extraction is easy automatable, requires no moving parts, is easy to miniaturize and furthermore possesses a size dependent cut-off filter adjustable by the pore size of the hydrogel. However electrophoretic nucleic acid extraction from bacteria has so far been applied mainly for short RNA targets. One of the reasons is that electrophoretic processing of unfragmented genomic DNA strands is time-consuming, because of the length. Here DNA fragmentation would accelerate extraction and isolation. We introduce on-chip lysis and non-enzymatic DNA cleavage directly followed by a purifying step for receiving amplifiable DNA fragments from bacteria in less than 25 min. In contrast to restriction enzymes the Fenton reaction is known to cleave DNA without nucleotide specificity. The reaction mix contains iron(II) EDTA, sodium ascorbate, hydrogen peroxide and lysozyme. The degree of fragmentation can be adjusted by the concentration of reagents. The results enable electrophoretic extraction methods to unspecifically process long genomic DNA in a short time frame, e.g. for pathogen detection in a lab-on-a-chip format.