Cessation of Nightly Voluntary Wheel Running Activity Following Exposure to a Mouse Model of Posttraumatic Stress

Regular physical activity (PA) is well known to positively impact physical and mental health outcomes. In our work to examine cardiovascular benefits of PA in a mouse model of posttraumatic stress, we stumbled upon the reciprocal relationship between PA and stress exposure, wherein stress significantly reduced healthy levels of routine PA. The aim of the present studies was to define the parameters of our paradigm. C67BL/6J male mice were divided into four groups (n=8/group): sedentary/control, voluntary running/control, sedentary/stress, and voluntary running/stress. Voluntary running groups were given unlimited access to a running wheel for 9 weeks. Stress groups were then exposed to a 5-day resident-intruder social stress that models human posttraumatic stress. Running behavior essentially ceased following stress. Habituation to stress occurred, as running distance increased by the 5th day of stress but remained significantly low. A separate study examined a single exposure to resident-intruder social stress. Plasma corticosterone significantly increased while nightly running dropped significantly but returned to normal by the 3rd night post-stress. These studies show the sensitivity of habitual running behavior to stress exposure and suggest the utility of this mouse model in exploring the means by which stress negatively impacts routine PA

The Tudor SND1 protein is an m6A RNA reader essential for replication of Kaposi’s sarcoma-associated herpesvirus

N6-methyladenosine (m6A) is the most abundant internal RNA modification of cellular mRNAs. m6A is recognised by YTH domain-containing proteins, which selectively bind to m6A-decorated RNAs regulating their turnover and translation. Using an m6A-modified hairpin present in the Kaposi’s sarcoma associated herpesvirus (KSHV) ORF50 RNA, we identified seven members from the ‘Royal family’ as putative m6A readers, including SND1. RIP-seq and eCLIP analysis characterised the SND1 binding profile transcriptome-wide, revealing SND1 as an m6A reader. We further demonstrate that the m6A modification of the ORF50 RNA is critical for SND1 binding, which in turn stabilises the ORF50 transcript. Importantly, SND1 depletion leads to inhibition of KSHV early gene expression showing that SND1 is essential for KSHV lytic replication. This work demonstrates that members of the ‘Royal family’ have m6A-reading ability, greatly increasing their epigenetic functions beyond protein methylation

Use of CRISPR-Cas9 Gene Editing System to Characterize the Human CXCL10 Viral Response Gene

Given its ease of use and accurate functionality, the CRISPR-Cas9 gene editing system has been at the forefront of recent genetic and biochemical research ranging from the development of powerful gene therapies to the characterization of novel genes. Here we attempt to characterize the human CXCL10 gene, a secreted chemokine ligand associated with cellular response to viruses, by directed gene mutation accomplished by CRISPR-Cas9. Use of this system requires the design and synthesis of a complete plasmid, containing DNA that codes for all necessary components of CRISPR-Cas9, and amplification of this plasmid in E. coli. Following characterization tests, the amplified plasmid could be transfected into an immortal human lung cell line sample and possibly effect a targeted knockdown of the CXCL10 gene. In this process, we observed evidence of correct CRISPR-Cas9 plasmid construction by PCR amplification followed by gel electrophoresis analysis. We also observed successful transfection of E. coli with our plasmid by ampicillin screening followed by sequencing analysis of the amplified product. Current efforts lie in optimizing lung cell transfection, though recent observations of our puromycin screen indicate some level of desired transfection. After establishing a transfected cell population, our future work will include identification of successful gene knockdown by sequencing CXCL10 where we expect to see a mutation made by CRISPR-Cas9, in addition to performing Western blots to detect possible changes in the CXCL10 protein product. The creation of a CXCL10 knockdown cell line will aid in future tests regarding the functionality of this gene in immune response, as well as demonstrate the exciting potential of CRISPR-Cas9 as both an accessible learning tool and a powerful instrument for new discovery