Methods in molecular biology
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Exon-skipping therapy is an emerging approach that uses synthetic DNA-like molecules called antisense oligonucleotides (ASOs) to splice out frame-disrupting parts of mRNA, restore the reading frame, and produce truncated yet functional proteins. Phosphorodiamidate morpholino oligomer (PMO) is one of the safest among therapeutic ASOs for patients and has recently been approved under the accelerated approval program by the US Food and Drug Administration (FDA) as the first ASO-based drug for Duchenne muscular dystrophy (DMD). Multi-exon skipping utilizing ASOs can theoretically treat 80-90% of patients with DMD. Here, we describe the systemic delivery of a cocktail of ASOs to skip exon 51 and exons 45-55 in the mdx52 mouse, an exon 52 deletion model of DMD produced by gene targeting, and the evaluation of their efficacies in vivo.
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The diversity and huge omics data take biology and biomedicine research and application into a big data era, just like that popular in human society a decade ago. They are opening a new challenge from horizontal data ensemble (e.g., the similar types of data collected from different labs or companies) to vertical data ensemble (e.g., the different types of data collected for a group of person with match information), which requires the integrative analysis in biology and biomedicine and also asks for emergent development of data integration to address the great changes from previous population-guided to newly individual-guided investigations. Data integration is an effective concept to solve the complex problem or understand the complicate system. ⋯ Current integration approaches on biological data have two modes: one is "bottom-up integration" mode with follow-up manual integration, and the other one is "top-down integration" mode with follow-up in silico integration. This paper will firstly summarize the combinatory analysis approaches to give candidate protocol on biological experiment design for effectively integrative study on genomics and then survey the data fusion approaches to give helpful instruction on computational model development for biological significance detection, which have also provided newly data resources and analysis tools to support the precision medicine dependent on the big biomedical data. Finally, the problems and future directions are highlighted for integrative analysis of omics big data.
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Reactive oxygen species (ROS) are a group of unstable and highly reactive molecules or free radicals typically generated as by-products of cellular processes involving molecular oxygen. In vascular cells, the excessive ROS generation results in the initiation and progression of cardiovascular diseases (CVD). Therefore, a dynamic, robust, and accurate ROS detection method in the blood vessels is essential for pathophysiological research studies of the cardiovascular system. ⋯ The protocol includes preparation of frozen aortic tissue sections, monitoring DHE oxidation-derived fluorescence by fluorescence microscopy, and high-performance liquid chromatograph-based analysis of MitoSOX and its oxidation products. For studying the role of AMP-activated protein kinase (AMPK) in the redox regulation, we employed AMPKα2 knockout mice and observed increased superoxide and mitochondrial superoxide levels in the aorta of AMPK knockout mice relative to the wild-type group. This novel ROS detection method will be valuable for investigating the roles of cellular and/or mitochondrial ROS in the pathogenesis of CVDs.
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Antisense oligonucleotides (AONs) hold great promise for therapeutic splice-switching correction in many genetic diseases and in particular for Duchenne muscular dystrophy (DMD), where AONs can be used to reframe the dystrophin transcript and give rise to a partially deleted but yet functional dystrophin protein. Many different chemistries of AONs can be used for splice switching modulation, and some of them have been evaluated in clinical trials for DMD. ⋯ Here, we describe the methods to evaluate the potency of antisense oligonucleotides, and in particular of tricyclo-DNA (tcDNA)-AONs, a novel class of AONs which displays unique pharmacological properties and unprecedented uptake in many tissues after systemic administration. We focus on the most widely used mouse model for DMD, the mdx mouse and detail methods to analyze the skipping of the mouse exon 23 both in vitro in H2K mdx cells and in vivo in the mdx mouse model.
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Studies in psychoneuroimmunology (PNI) would provide better insights into the "whole mind-body system." Systems biology models of the complex adaptive systems (CASs), such as a conceptual framework of "Yin-Yang dynamics," may be helpful for identifying systems-based biomarkers and targets for more effective prevention and treatment. The disturbances in the Yin-Yang dynamical balance may result in stress, inflammation, and various disorders including insomnia, Alzheimer's disease, obesity, diabetes, cardiovascular diseases, skin disorders, and cancer. At the molecular and cellular levels, the imbalances in the cytokine pathways, mitochondria networks, redox systems, and various signaling pathways may contribute to systemic inflammation. ⋯ The studies of cancer have revealed the importance of the Yin-Yang dynamics in the tumoricidal and tumorigenic activities of the immune system. Stress-induced neuroimmune imbalances are also essential in chronic skin disorders including atopic dermatitis and psoriasis. With the integrative framework, the restoration of the Yin-Yang dynamics can become the objective of dynamical systems medicine.