Chiral DNA sequences as commutable controls for clinical genomics.

Chirality is a property describing any object that is inequivalent to its mirror image. Due to its 5'-3' directionality, a DNA sequence is distinct from a mirrored sequence arranged in reverse nucleotide-order, and is therefore chiral. A given sequence and its opposing chiral partner sequence share many properties, such as nucleotide composition and sequence entropy. Here we demonstrate that chiral DNA sequence pairs also perform equivalently during molecular and bioinformatic techniques that underpin genetic analysis, including PCR amplification, hybridization, whole-genome, target-enriched and nanopore sequencing, sequence alignment and variant detection. Given these shared properties, synthetic DNA sequences mirroring clinically relevant or analytically challenging regions of the human genome are ideal controls for clinical genomics. The addition of synthetic chiral sequences (sequins) to patient tumor samples can prevent false-positive and false-negative mutation detection to improve diagnosis. Accordingly, we propose that sequins can fulfill the need for commutable internal controls in precision medicine.

How to Define the Latent Reservoir: Tools of the Trade.

HIV is a devastating worldwide epidemic that has had substantial social and economic impacts throughout the globe. Due to the presence of a small pool of latently infected cells that persists during antiretroviral therapy (ART), HIV is not curable. Because of the high cost of ART and the lack of reliable accessibility across the globe, life-long ART is unfortunately not a feasible solution for the epidemic. Therefore, new strategies need to be developed and implemented to address HIV-1 infection. Several approaches toward this end are currently under investigation (Ebina et al. in Sci Rep 3:2510, 2013; Archin et al. in Nature 487:482-5, 2012; Elliott et al. in PLoS Pathog 10:e1004473, 2014; Rasmussen et al. in Lancet HIV 1:e13-e21, 2014; Tebas et al. in N Engl J Med 370:901-10, 2014; Archin et al. in Nat Rev Microbiol 12:750-64, 2014; Barton et al. in PLoS One 9:e102684, 2014; Sogaard et al. in PLoS Pathog 11:e1005142, 2015). Initial studies have proven promising, but have highlighted the need for sensitive and accurate assays to detect changes in very low concentrations of virus to allow confident interpretation of the success of curative approaches. This review will focus on assays that are currently available and the advantages and limitations of each.

Selective HDAC inhibition for the disruption of latent HIV-1 infection.

Selective histone deacetylase (HDAC) inhibitors have emerged as a potential anti-latency therapy for persistent human immunodeficiency virus type 1 (HIV-1) infection. We utilized a combination of small molecule inhibitors and short hairpin (sh)RNA-mediated gene knockdown strategies to delineate the key HDAC(s) to be targeted for selective induction of latent HIV-1 expression. Individual depletion of HDAC3 significantly induced expression from the HIV-1 promoter in the 2D10 latency cell line model. However, depletion of HDAC1 or -2 alone or in combination did not significantly induce HIV-1 expression. Co-depletion of HDAC2 and -3 resulted in a significant increase in expression from the HIV-1 promoter. Furthermore, concurrent knockdown of HDAC1, -2, and -3 resulted in a significant increase in expression from the HIV-1 promoter. Using small molecule HDAC inhibitors of differing selectivity to ablate the residual HDAC activity that remained after (sh)RNA depletion, the effect of depletion of HDAC3 was further enhanced. Enzymatic inhibition of HDAC3 with the selective small-molecule inhibitor BRD3308 activated HIV-1 transcription in the 2D10 cell line. Furthermore, ex vivo exposure to BRD3308 induced outgrowth of HIV-1 from resting CD4+ T cells isolated from antiretroviral-treated, aviremic HIV+ patients. Taken together these findings suggest that HDAC3 is an essential target to disrupt HIV-1 latency, and inhibition of HDAC2 may also contribute to the effort to purge and eradicate latent HIV-1 infection.

Expression patterns and cell cycle profiles of PCNA, MCM6, cyclin D1, cyclin A2, cyclin B1, and phosphorylated histone H3 in the developing mouse retina.

A challenge in studying organogenesis is the ability to identify progenitor cell populations. To address this problem, we characterized the expression patterns of cell cycle proteins during mouse retinal development and used flow cytometry to determine the expression profiles in the cell cycle. We found that MCM6 and PCNA are expressed in essentially all retinal progenitor cells throughout the proliferative period and these proteins are readily detectable in all cell cycle phases. Furthermore, their expression levels are downregulated as cells exit the cell cycle and differentiate. We also analyzed the expression of Cyclins D1, A2, and B1, and phosphorylated Histone H3 and found unexpected expression patterns and cell cycle profiles. The combined utilization of the markers tested and the use of flow cytometry should further facilitate the study of stem and progenitor cell behavior during development and in adult tissues.