Quantifying three-dimensional morphology and RNA from individual embryos.

Quantitative analysis of morphogenesis aids our understanding of developmental processes by providing a method to link changes in shape with cellular and molecular processes. Over the last decade, many methods have been developed for 3D imaging of embryos using microCT scanning to quantify the shape of embryos during development. These methods generally involve a powerful, cross-linking fixative such as paraformaldehyde to limit shrinkage during the CT scan. However, the extended time frames that these embryos are incubated in such fixatives prevent use of the tissues for molecular analysis after microCT scanning. This is a significant problem because it limits the ability to correlate variation in molecular data with morphology at the level of individual embryos. Here we outline a novel method that allows RNA, DNA, or protein isolation following CT scan while also allowing imaging of different tissue layers within the developing embryo. We show shape differences early in craniofacial development (E11.5) between common mouse genetic backgrounds, and demonstrate that we are able to generate RNA from these embryos after CT scanning that is suitable for downstream real time PCR (RT-PCR) and RNAseq analyses. Developmental Dynamics 246:431-436, 2017. © 2017 Wiley Periodicals, Inc.

Understanding the delay in starting antiretroviral therapy despite recent guidelines for HIV patients retained in care.

Despite strong evidence of a clinical benefit from initiating antiretroviral therapy (ART) immediately after diagnosis some patients remain ART naïve. We examined explanations, over a four-year period in a centralized HIV clinical cohort under universal health care, for newly diagnosed patients, while being fully engaged and retained in HIV care, delaying ART initiation for >180 days following their HIV diagnosis. All patients followed at the Southern Alberta Clinic, Calgary, Canada between 1 January 2010 and 1 January 2014 were included and followed until they moved, were lost to follow-up, died or until 1 January 2015. Of 269 patients, 56 (21.8%) deferred ART >180 days; 26 (9.7%) remained ART naïve until the end of the study. Patients delaying or deferring ART were younger, Canadian-born, and with higher CD4 counts (p < .01). "No clinical urgency" especially for patients with higher CD4 counts, was most often listed for deferring ART, however when ART was offered "patient not ready", "unstable substance use", "difficulties adjusting" or "wanting to wait" were often cited regardless of CD4 levels. At times ART, when offered, was adamantly declined by the patient. The physician's assessment of a patient's ability to adhere to lifelong ART was an issue in some cases. While structural or financial issues may impact ART initiation, our results suggest that, even in an environment of free and easy access to ART, many challenges still exist at the implementation stage. Intense efforts in both patient and physician education will be required if the benefits of early ART as recommended by the WHO in their recent guidelines, are to be achieved at the individual and population level.