Next-Generation Phylogeography: A Targeted Approach for Multilocus Sequencing of Non-Model Organisms

The field of phylogeography has long since realized the need and utility of incorporating nuclear DNA (nDNA) sequences into analyses. However, the use of nDNA sequence data, at the population level, has been hindered by technical laboratory difficulty, sequencing costs, and problematic analytical methods dealing with genotypic sequence data, especially in non-model organisms. Here, we present a method utilizing the 454 GS-FLX Titanium pyrosequencing platform with the capacity to simultaneously sequence two species of sea star (Meridiastra calcar and Parvulastra exigua) at five different nDNA loci across 16 different populations of 20 individuals each per species. We compare results from 3 populations with traditional Sanger sequencing based methods, and demonstrate that this next-generation sequencing platform is more time and cost effective and more sensitive to rare variants than Sanger based sequencing. A crucial advantage is that the high coverage of clonally amplified sequences simplifies haplotype determination, even in highly polymorphic species. This targeted next-generation approach can greatly increase the use of nDNA sequence loci in phylogeographic and population genetic studies by mitigating many of the time, cost, and analytical issues associated with highly polymorphic, diploid sequence markers

Adeno-Associated Viral Vector-Mediated Transgene Expression Is Independent of DNA Methylation in Primate Liver and Skeletal Muscle

Recombinant adeno-associated viral (rAAV) vectors can support long-term transgene expression in quiescent tissues. Intramuscular (IM) administration of a single-stranded AAV vector (ssAAV) in the nonhuman primate (NHP) results in a peak protein level at 2–3 months, followed by a decrease over several months before reaching a steady-state. To investigate transgene expression and vector genome persistence, we previously demonstrated that rAAV vector genomes associate with histones and form a chromatin structure in NHP skeletal muscle more than one year after injection. In the mammalian nucleus, chromatin remodeling via epigenetic modifications plays key role in transcriptional regulation. Among those, CpG hyper-methylation of promoters is a known hallmark of gene silencing. To assess the involvement of DNA methylation on the transgene expression, we injected NHP via the IM or the intravenous (IV) route with a recombinant ssAAV2/1 vector. The expression cassette contains the transgene under the transcriptional control of the constitutive Rous Sarcoma Virus promoter (RSVp). Total DNA isolated from NHP muscle and liver biopsies from 1 to 37 months post-injection was treated with sodium bisulfite and subsequently analyzed by pyrosequencing. No significant CpG methylation of the RSVp was found in rAAV virions or in vector DNA isolated from NHP transduced tissues. Direct de novo DNA methylation appears not to be involved in repressing transgene expression in NHP after gene transfer mediated by ssAAV vectors. The study presented here examines host/vector interactions and the impact on transgene expression in a clinically relevant model

Outcomes of C1–2 joint injections

Christopher M Aiudi,1 W Michael Hooten,2 Rebecca A Sanders,2 James C Watson,2 Susan M Moeschler,2 Halena M Gazelka,2 Bryan C Hoelzer,2 Jason S Eldrige,2 Wenchun Qu,3 Tim J Lamer2 1Mayo Clinic School of Medicine, 2Division of Pain Medicine, Department of Anesthesia and Perioperative Medicine, Mayo Clinic, 3Department of Physical Medicine and Rehabilitation, Mayo Clinic, Rochester, MN, USA Objective: Intra-articular injections of the C1–2 joint are an effective therapeutic option for pain generated from degenerative and inflammatory conditions affecting the joint. Limited information exists about the adverse events (AEs) associated with these injections. The primary aim of this study is to describe the frequency and type of AEs associated with C1–2 joint injections. The secondary aim is to identify clinical factors associated with the occurrence of AEs of C1–2 joint injections. Design/methods: A retrospective chart review was conducted on all C1–2 joint injections performed at the Mayo Pain Medicine Clinic in Rochester, MN, from January 1, 2005 through July 31, 2015. AE data were extracted from procedural and post-procedural clinical notes. Analysis was conducted to determine correlations between any AE and demographic and clinical characteristics. Using univariate and multivariate logistic regression analyses, associations were determined. Results: From January 1, 2005 to July 31, 2015, 135 C1–2 injections were performed on 72 patients. Overall, at least 1 AE was reported in 18.5% of the injections. The most common AEs were post-procedural increase in pain and procedural vascular contrast uptake. There was a significant association between AE occurrence and greater pre-procedural maximum pain score. Conclusions: AEs from C1–2 joint injections occurred commonly, but there were no persistent or serious AEs associated with these injections. The data also demonstrate that patients with higher pre-procedural maximum pain scores are more likely to experience an AE. Keywords: C1–2 joint, facet, injection, adverse even