CHARACTERIZATION AND MEASUREMENT OF THE HIV-1 LATENT RESERVOIR USING SINGLE GENOME ANALYSIS AND DROPLET DIGITAL PCR

Although antiretroviral therapy (ART) suppresses viral replication to clinically undetectable levels, HIV-1 persists in CD4+ T cells in a latent form not targeted by the immune system or ART (Chun et al., 1997b; Finzi et al., 1997; Ruelas and Greene, 2013; Siliciano et al., 2003; Wong et al., 1997a). This latent reservoir is a major barrier to cure. Many individuals initiate ART during chronic infection, and in this setting, most proviruses are defective (Ho et al., 2013a). However, the dynamics of the accumulation and persistence of defective proviruses during acute HIV-1 infection are largely unknown. Here we show that defective proviruses accumulate rapidly within the first few weeks of infection to make up over 93% of all proviruses, regardless of how early ART is initiated. Using an unbiased method to amplify near full-length proviral genomes from HIV-1 infected adults treated at different stages of infection, we demonstrate that early ART initiation limits the size of the reservoir but does not profoundly impact the proviral landscape. This analysis allows us to revise our understanding of the composition of proviral populations and estimate the true reservoir size in individuals treated early vs. late in infection. Additionally, we demonstrate that common assays for measuring the reservoir significantly overestimate or underestimate the size of the latent reservoir and no assay we tested correlates with the number of intact proviruses. Using our analysis of full-genome sequences, we identify regions and features of the HIV-1 genome that, when interrogated simultaneously, specifically distinguish intact HIV from defective genomes. We describe here a novel intact proviral DNA assay (IPDA) using multiplex droplet digital PCR that allows us to accurately quantify the number of intact proviruses, which are likely the closest estimate to the true size of the latent reservoir. In preliminary results from matched patient samples, the IPDA strongly correlates with full-genome sequencing results. Many defective proviruses contain defects that likely preclude elimination by eradication strategies and could obscure the measurement of real changes in the rarer intact proviruses. By eliminating 90-95% of all defective proviruses and measuring primarily intact proviruses, we anticipate the IDPA will better assess the impact of eradication strategies on the true reservoir of virus that must be eliminated to achieve an HIV-1 cure

Triple Helix, Fall 2018

Table of Contents: Science Agenda: The Politics of Grant Writing / by Kavya Rajesh (p. 4) -- From the Experts / by Katherine Bruner (p. 5) -- 3D Printed Drugs: The Future of Pharmaceuticals / by Ethan Wang (p. 6) -- Computerized Markets: Wall Street Takeover / by James Kiraly (p. 10) -- The Evolution of Fear / by Alisha Ahmed (p. 14) -- ADDing Up / by Victor Liaw (p. 18) -- The Clone Wars / by Jina Zhou (p. 22) -- Physician-Assisted Suicide: Drawing the Line / by Haley Wolf (p. 26) -- Supervised Injection Sites / by Alex Gajewski (p. 30) -- On Emerging Medicalization and Health Care / by Patrick Lee (p. 33) -- The Future of Human Gene Modifications / by Elizabeth Robinson (p. 36)College of Natural SciencesUT LibrariesLiberal Art

A quantitative approach for measuring the reservoir of latent HIV-1 proviruses.

A stable latent reservoir for HIV-1 in resting CD4+ T cells is the principal barrier to a cure1-3. Curative strategies that target the reservoir are being tested4,5 and require accurate, scalable reservoir assays. The reservoir was defined with quantitative viral outgrowth assays for cells that release infectious virus after one round of T cell activation1. However, these quantitative outgrowth assays and newer assays for cells that produce viral RNA after activation6 may underestimate the reservoir size because one round of activation does not induce all proviruses7. Many studies rely on simple assays based on polymerase chain reaction to detect proviral DNA regardless of transcriptional status, but the clinical relevance of these assays is unclear, as the vast majority of proviruses are defective7-9. Here we describe a more accurate method of measuring the HIV-1 reservoir that separately quantifies intact and defective proviruses. We show that the dynamics of cells that carry intact and defective proviruses are different in vitro and in vivo. These findings have implications for targeting the intact proviruses that are a barrier to curing HIV infection

Relational legacies impacting on veteran transition from military to civilian life: trajectories of acquisition, loss and re-formulation of a sense of belonging

The veteran cohort has been inextricably linked in the general public's mind by media generated perceptions of high risk and fear of crime, echoed in wider contemporary debates linking issues of place, social identity, social exclusion (Pain 2000) and a loss of belonging in wider communities (Walklate 1998). Despite the growing interest in the longer term outcomes of transition from military to civilian life from policy-makers, practitioners and academics, few qualitative studies explore the social and relational impacts of this transitional experience on those who have experienced it. Tensions and frustrations expressed by ex-forces personnel, engaging in addictions services with a history of engagement in the criminal justice sector, are explored through the lens of belongingness, loss and related citizenship frameworks to expose temporal impacts on the acquisition, loss and reformulation of a sense of belonging across the life course. The relevance of a significant loss of belonging in the transition from military to civilian life is useful, given the widely accepted damaging consequences of having this need thwarted. This paper concludes that a broader understanding of this largely disenfranchised grief (Doka, 2002) can enable more informed reflexive opportunities to facilitate a valued military veteran citizenship status and thereby contribute to the formulation of current policy debates concerning the veteran question

Gender differences in conversation topics, 1922–1990

Gender differences in conversation topics were first systematically studied in 1922 by Henry Moore, who theorized that the gender differences in topic choice he observed in a field observation study would persist over time, as they were manifestations of men's and women's “original natures.” In this paper, I report a 1990 replication of Moore's study, in which similar but smaller gender differences in topic choice are found. In order to explore further the apparent trend toward smaller gender differences, reports of quantitative observation studies conducted between 1922 and 1990 are examined. Other explanations besides change over time—such as variations in conversation setting and audience, target populations, and researcher's intentions—may account for the decline in gender differences in topic choice. Social influences are seen more clearly in the discourse about gender differences in conversation than in gender differences in conversation topics themselves.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45599/1/11199_2004_Article_BF00289744.pd

Depositional environments, petrology, and diagenesis of the Oriskany Sandstone in the subsurface in West Virginia.

The Oriskany Sandstone in West Virginia is a hybrid sandstone and exhibits a high degree of variability in the amount of quartz and carbonate framework constituents. Fluctuating energy conditions and sedimentation rates, distance from the source area, and changes in the depositional environment are responsible for the lithologic variation. Four facies have been identified in the Oriskany. Facies 1 is a very fine-grained, bioturbated calcareous sandstone to sandy limestone, and contains a diverse marine fauna. It was deposited in a quiet water environment, near wave base, and represents the beginning of clastic influx into the shallow Oriskany sea. Facies 2 is a medium-grained, well-sorted, crossbedded quartz arenite that was deposited as a submarine sand ridge by storm or tidal currents. Facies 3 is a medium- to fine-grained, bioturbated and laminated fossiliferous sandstone with interbeds of sandy limestone. It records fair-weather deposition in an environment characterized by variable wave and current energy. Facies 4 is coarse-grained to pebbly, fossiliferous sandstone overlain by medium- to fine-grained laminated sandstone. The facies typically exhibits an erosional base and infiltration fabrics, and represents storm events that periodically interrupted normal shelf sedimentation. The Oriskany shows an overall regressive pattern of sedimentation. Best illustrated in Greenbrier 6, quartz grain size coarsens upward as does the amount of quartz. Lateral changes are also recorded in fair-weather and storm deposits. The Oriskany in eastern cores is more quartzose and storm deposits are thicker, reflecting close proximity to the eastern source area. The sandstone in the east-central cores is thinner and more calcareous. In the western cores storm deposits thin to several millimeters in western cores suggesting deposition in more distal regions of the basin. Lithology and the diagenetic processes of cementation and dissolution have exerted the greatest influence on porosity. In eastern cores all interstitial porosity has been occluded by secondary quartz and late stage Fe-calcite cement. In western cores there is a significant increase in interstitial porosity. Pore types have been identified as (1) primary intergranular pores, (2) solution pores, (3) recrystallization/silicification pores, (4) intercrystalline pores, and (5) combination pores. Marion 321 and Pocahontas 23 showed less than 3% porosity formed by incomplete cementation (intergranular) with minor amounts of other pore types. Samples from Kanawha County, with an average of 12% porosity, showed a predominance of primary intergranular porosity. In Wood 1266, with an average of 7% porosity, secondary pores account for most of the porosity, except near the top where primary intergranular porosity dominates. Porosity in the sandy dolomitic Oriskany in Johnson 31305, averages 12%, and is dominated by intercrystalline pores occurring between replacement and cement crystals of calcite and dolomite

Use of a High-Fidelity Patient Simulator to Introduce an Evidence-Based Emergency Manual into Certified Registered Nurse Anesthetist Practice

Certified Registered Nurse Anesthetists (CRNAs) administer anesthesia care to thousands of patients each year. Despite increased anesthetic safety, low-frequency, high-risk perioperative critical events still occur. Although CRNAs have been expected to rely on memory alone to manage the spectrum of these critical events, the use of an emergency manual (EM) may improve CRNA performance. At one Army community hospital, recent experiences with similar critical events led to the development of the following question: Will the use of high-fidelity simulation training with the staff CRNAs on the use of EMs produce both increased performance in key tasks and positive satisfaction scores related to the use of the emergency manual during critical events now and in the future? Twenty staff CRNAs participated in high-fidelity simulated scenarios involving low-frequency, high-risk critical events before and after training on the use of an EM based on a change strategy developed by Goldhaber-Fiebert and Howard in 2012. Changes in performance after training were evaluated using a tool designed by Arriaga et al. Before EM training, CRNAs completed 46.05% of key tasks in the simulated scenario. After EM training, CRNAs completed up to 94.02% of key tasks in simulated scenarios. The observed increase in the completion of key tasks by CRNAs during simulated critical events after EM training and increase in satisfaction scores demonstrates how a well-constructed training program facilitates the implementation of an evidence-based EM into practice and can improve the quality and consistency of CRNA performance during low-frequency, high-risk perioperative events