Project description:Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated protein 13a (Cas13a) has been described as a superior tool to short-interfering (si) RNAs for specific gene silencing by targeting RNAs through Watson-Crick binding of the target and activation of nuclease activity. However, we observed that activation of LwaCas13a in mammalian HEK293T cells leads to unspecific targeting RNAs, which led to cell toxicity and death.
Project description:Details of the experiment are available within the Nucleic Acid Research, 2004 manuscript "Genomic profiling by DNA amplification of laser capture microdissected tissues and array CGH", by Cardoso J. et al. Keywords: other
Project description:To evaluate targeted MinION next generation sequencing as a diagnostic method for detection of pathogens in human blood and plasma, human blood or plasma samples were spiked with measured amounts of viruses, bacteria, protozoan parasites or tested pathogen-free as negative controls. Nucleic acid was extracted from samples and PCR amplification performed in multiplex primer pools with a procedure described in ArrayExpress experiment submission ID 18379. The PCR products were used for library preparation. The libraries sequenced on an Oxford Nanopore MinION. The passed reads aligned with a custom reference file to determine the identity of the pathogen in the sample.
Project description:Free extracellular DNA (exDNA) accumulation within the cytoplasm is closely associated with chronic inflammation in intervertebral disc degeneration (IVDD). While numerous minimally invasive treatments targeting the various etiologies of IVDD have been developed, there remains a lack of systematic approaches to address the chronic inflammation caused by both intracellular and extracellular nucleic acids. In this study, we propose a novel strategy for treating IVDD that involves dual nucleic acid clearance. This approach targets and regulates mitochondrial DNA (mtDNA) release while clearing exDNA from the microenvironment, working synergistically to block the inflammatory cascade. We first analyzed the mechanism of chronic inflammation mediated by intracellular and extracellular nucleic acids during the progression of IVDD using single-cell sequencing and clinical sample analysis. Next, we developed a composite delivery system consisting of engineered nanovesicles with mitochondrial targeting functions (BNPMT) and a nucleic acid clearance hydrogel (H-P gel). BNPMT achieves targeted delivery to nucleus pulposus cells via membrane modification with triphenylphosphonium (TPP), while encapsulated BAI1, a small molecule drug, specifically inhibits mitochondrial outer membrane permeabilization (miMOMP). This reduces mitochondrial DNA (mtDNA) leakage into the cytoplasm, thereby blocking the mtDNA-cGAS/STING signaling pathway-mediated inflammatory response in nucleus pulposus cells. The H-P gel, crosslinked by Schiff base reactions and using G3-PAMAM dendrimers as functional crosslinkers, efficiently captures exDNA from the intervertebral disc microenvironment. This inhibits cfDNA-induced M1 macrophage polarization and the release of inflammatory cytokines. In an IVDD rat model, this dual-modal system demonstrated synergistic therapeutic advantages, significantly reversing the progression of IVDD over 8 weeks. This targeted nucleic acid clearance strategy provides a novel approach for treating IVDD and offers a theoretical framework for addressing other nucleic acid-related inflammatory diseases.
Project description:Details of the experiment are available within the Nucleic Acid Research, 2004 manuscript "Genomic profiling by DNA amplification of laser capture microdissected tissues and array CGH", by Cardoso J. et al.
Project description:Interventions: Gold Standard:Colonoscopy and tissue case biopsy;Index test:EVs nucleic acid combination (miRNA, lncRNA, circRNA) detection system.
Primary outcome(s): RNA;SEN, SPE, ACC, AUC of ROC
Study Design: Factorial
Project description:In our original grant we proposed to use the NR3B-null mouse model to study the role of NR3B subunit in motor neuron function. We have now successfully generated NR3B null mice. Interestingly, NR3B-null mice invariably die at age P4-P8. Our preliminary examination indicates that the motor strength of these mice is severely impaired prior to death. As we continue to explore the cause of death in NR3B null mice, we propose to conduct gene profiling experiments to search for transcription changes in the brain related to ablation of the NR3B gene. We have previously used the UCLA array facility provided by the NINDS/NIMH Microarray Consortium to identify genes that show abnormal expression patterns in these mice using whole brain samples. Now, we would like to use the same approach to analyze pure motor neuron samples obtained by Laser Capture Microdisection (LCM). As these samples are of small size we need to use RNA amplification method. We would like to compare probes from two amplification methods to see which one gives the best result. We will collect the cDNA probe obtained by RNA-based single primer isothermal amplification developed by Nugen, Inc. and the aRNA probe obtained from T7-based RNA amplification. Both the hybridization intensity and the percentage of the present call of these two types of probe will be analyzed. The cDNA probe obtained by RNA-based single primer isothermal amplification developed by Nugen, Inc. may have some advantage over the aRNA probe obtained from T7-based RNA amplification. Two samples will be processed by either RNA-based single primer isothermal amplification developed by Nugen or T7-based RNA amplification to obtain cDNA or aRNA probes, respectively. For each sample we will use 600 motor neurons obtained by LCM. Both types of probe will be labeled by biotin and hybridize to Affymetrix GeneChip Mouse Genome 430 2.0 Array. Data analysis will be performed by array facility. Keywords: dose response