Quantifying the HIV reservoir with dilution assays and deep viral sequencing.

People living with HIV on antiretroviral therapy often have undetectable virus levels by standard assays, but "latent" HIV still persists in viral reservoirs. Eliminating these reservoirs is the goal of HIV cure research. The quantitative viral outgrowth assay (QVOA) is commonly used to estimate the reservoir size, that is, the infectious units per million (IUPM) of HIV-persistent resting CD4+ T cells. A new variation of the QVOA, the ultra deep sequencing assay of the outgrowth virus (UDSA), was recently developed that further quantifies the number of viral lineages within a subset of infected wells. Performing the UDSA on a subset of wells provides additional information that can improve IUPM estimation. This paper considers statistical inference about the IUPM from combined dilution assay (QVOA) and deep viral sequencing (UDSA) data, even when some deep sequencing data are missing. Methods are proposed to accommodate assays with wells sequenced at multiple dilution levels and with imperfect sensitivity and specificity, and a novel bias-corrected estimator is included for small samples. The proposed methods are evaluated in a simulation study, applied to data from the University of North Carolina HIV Cure Center, and implemented in the open-source R package SLDeepAssay.

Penetration of laser light at 808 and 980 nm in bovine tissue samples.

OBJECTIVE: The purpose of this study was to compare the penetration of 808 and 980 nm laser light through bovine tissue samples 18-95 mm thick. BACKGROUND DATA: Low-level laser therapy (LLLT) is frequently used to treat musculoskeletal pathologies. Some of the therapeutic targets are several centimeters deep. METHODS: Laser light at 808 and 980 nm (1 W/cm(2)) was projected through bovine tissue samples ranging in thickness from 18 to 95 mm. Power density measurements were taken for each wavelength at the various depths. RESULTS: For 808 nm, 1 mW/cm(2) was achieved at 3.4 cm, but for 980 nm, 1 mW/cm(2) was achieved at only 2.2 cm depth of tissue. CONCLUSIONS: It was determined that 808 nm of light penetrates as much as 54% deeper than 980 nm light in bovine tissue.

Effects of transplacental 17-α-ethynyl estradiol or bisphenol A on the developmental profile of steroidogenic acute regulatory protein in the rat testis.

Previous research from our laboratory has determined the transcript profiles for developing fetal rat female and male reproductive tracts following transplacental exposure to estrogens. Prenatal exposure to bisphenol A (BPA) or 17-α-ethynyl estradiol (EE) significantly affects steroidogenic acute regulatory (StAR) protein transcript levels in the developing male rat reproductive tract. The purpose of this study was to establish the intratesticular distribution and temporal expression pattern of StAR, a key gene involved in steroidogenesis. Beginning on gestation day (GD) 11, pregnant Sprague-Dawley rats were exposed daily to 10µg/kg/day EE and fetal testes were harvested at GD16, 18, or 20. Quantitative reverse transcriptase PCR (QRT-PCR) demonstrated no significant difference in StAR transcript levels present at GD16. However, at GD18, StAR transcripts were significantly decreased following exposure. Immunohistochemistry demonstrated similar StAR protein levels in interstitial region of GD16 testes and an obvious decrease in StAR protein levels in the interstitial region of GD18 testes. Moreover, starting at GD11 additional dams were dosed with 0.001 or 0.1 µg/kg/day EE or 0.02, 0.5, 400 mg/kg/day BPA via subcutaneous injections. QRT-PCR validated previous microarray dose-related decreases in StAR transcripts at GD20, whereas immunohistochemistry results demonstrated decreases in StAR protein levels in the interstitial region at the highest EE and BPA doses only. Neither EE nor BPA exposure caused morphological changes in the developing seminiferous cords, Sertoli cells, gonocytes, or the interstitial region or Leydig cells at GD16-20. High levels of estrogens decrease StAR expression in the fetal rat testis during late gestation.

Uterine temporal response to acute exposure to 17alpha-ethinyl estradiol in the immature rat.

The rat uterus responds to acute estrogen treatment with a series of well-characterized physiological responses; however, the gene expression changes required to elicit these responses have not been fully characterized. In order to understand early events induced by estrogen exposure in vivo, we evaluated the temporal gene expression in the uterus of the immature rat after a single dose of 17 alpha-ethinyl estradiol (EE) by microarray analysis, evaluating the expression of 15,923 genes. Immature 20-day-old rats were exposed to a single dose of EE (10 microg/kg), and the effects on uterine histology, weight, and gene expression were determined after 1, 2, 8, 24, 48, 72, and 96 h. EE induced changes in the expression of 3867 genes, at least at one time point (p < or = 0.0001), and at least 1.5-fold (up- or downregulated). Specifically, the expression of 8, 116, 3030, 2076, 381, 445, and 125 genes was modified at 1, 2, 8, 24, 48, 72, or 96 h after exposure to EE, respectively (p < or = 0.0001, t-test). At the tissue and organ level, a clear uterotrophic response was elicited by EE after only 8 h, reaching a maximum after 24 h and remaining detectable even after 96 h of exposure. The uterine phenotypic changes were induced by sequential changes in the transcriptional status of a large number of genes, in a program that involves multiple molecular pathways. Using the Gene Ontology to better understand the temporal response to estrogen exposure, we determined that the earliest changes were in the expression of genes whose products are involved in transcriptional regulation and signal transduction, followed by genes implicated in protein synthesis, energy utilization, solute transport, cell proliferation and differentiation, tissue remodeling, and immunological responses among other pathways. The compendium of genes here presented represents a comprehensive compilation of estrogen-responsive genes involved in the uterotrophic response.

Design of a microsphere-based high-throughput gene expression assay to determine estrogenic potential.

Recently gene expression studies have been multiplied at an accelerated rate by the use of high-density microarrays. By assaying thousands of transcripts at a time, microarrays have led to the discovery of dozens of genes involved in particular biochemical processes, for example, the response of a tissue/organ to a given chemical with therapeutic or toxic properties. The next step in these studies is to focus on the response of a subset of relevant genes to verify or refine potential therapeutic or toxic properties. We have developed a sensitive, high-throughput gene expression assay for this purpose. In this assay, based on the Luminex xMAP system, carefully selected oligonucleotides were covalently linked to fluorescently coded microspheres that are hybridized to biotinylated cRNA followed by amplification of the signal, which results in a rapid, sensitive, multiplexed assay platform. Using this system, we have developed an RNA expression profiling assay specific for 17 estrogen-responsive transcripts and three controls. This assay can evaluate up to 100 distinct analytes simultaneously in a single sample, in a 96-well plate format. This system has improved sensitivity versus existing microsphere-based assays and has sensitivity and precision comparable with or better than microarray technology. We have achieved detection levels down to 1 amol, detecting rare messages in complex cRNA samples, using as little as 2.5 microg starting cRNA. This assay offers increased throughput with decreased costs compared with existing microarray technologies, with the trade-off being in the total number of transcripts that can be analyzed.

Gene expression changes induced in the testis by transplacental exposure to high and low doses of 17{alpha}-ethynyl estradiol, genistein, or bisphenol A.

The purpose of this study was to determine (1) the transcriptional program elicited by exposure to three estrogen receptor (ER) agonists: 17 alpha-ethynyl estradiol (EE), genistein (Ges), and bisphenol A (BPA) during fetal development of the rat testis and epididymis; and (2) whether very low dosages of estrogens (evaluated over five orders of magnitude of dosage) produce unexpected changes in gene expression (i.e., a non-monotonic dose-response curve). In three independently conducted experiments, Sprague-Dawley rats were dosed (sc) with 0.001-10 microg EE/kg/day, 0.001-100 mg Ges/kg/day, or 0.002-400 mg BPA/kg/day. While morphological changes in the developing reproductive system were not observed, the gene expression profile of target tissues were modified in a dose-responsive manner. Independent dose-response analyses of the three studies identified 59 genes that are significantly modified by EE, 23 genes by Ges, and 15 genes by BPA (out of 8740), by at least 1.5 fold (up- or down-regulated). Even more genes were observed to be significantly changed when only the high dose is compared with all lower doses: 141, 46, and 67 genes, respectively. Global analyses aimed at detecting genes consistently modified by all of the chemicals identified 50 genes whose expression changed in the same direction across the three chemicals. The dose-response curve for gene expression changes was monotonic for each chemical, with both the number of genes significantly changed and the magnitude of change, for each gene, decreasing with decreasing dose. Using the available annotation of the gene expression changes induced by ER-agonist, our data suggest that a variety of cellular pathways are affected by estrogen exposure. These results indicate that gene expression data are diagnostic of mode of action and, if they are evaluated in the context of traditional toxicological end-points, can be used to elucidate dose-response characteristics.

Isolation and characterization of the human syncytin gene promoter.

Syncytin, a protein encoded by an envelope gene of a human endogenous retrovirus-W (HERV-W), plays a critical role in trophoblast differentiation. We isolated the 5'-flanking region of the syncytin gene from human genomic DNA by PCR and identified cis-acting elements on the promoter that are important for transcription. The major transcription initiation site identified by mung bean nuclease protection assays is 56 base pairs (bp) downstream from a putative CCAAT box. Deletion analysis of the 5'-flanking region of the syncytin gene indicated that the proximal 148 bp are essential for minimal promoter activity and that regions of the promoter from nt -1519 to -984 and nt -294 to -148 are required for maximal expression in normal trophoblast cells. DNase I footprint analysis of the region between nt -252 and +110 revealed three protected regions, FP1-FP3. Mutagenesis of a hepatocyte-specific nuclear protein-1 (HAPF1) binding site in FP1 and a TATA box in FP3 had no effects on basal promoter activity. However, mutation of the CCAAT motif and the octamer protein (Oct) binding site in FP2 decreased promoter activity by 88% and 76%, respectively. Mutation of the ecdysone receptor (EcR) response element in FP2, which may bind a nuclear hormone receptor, increased basal promoter activity by 2-fold. Gel shift and supershift assays indicated that CCAAT-binding factor (CBF) binds to the CCAAT motif and that Oct binds to the Oct binding site. Taken together, these findings indicate that the syncytin promoter is located in the 5' long terminal repeat (LTR) of the HERV-W gene and that binding sites for CBF and Oct in the proximal promoter are critical for transcriptional regulation of the gene in trophoblast cells.

Gene expression profile induced by 17 alpha-ethynyl estradiol in the prepubertal female reproductive system of the rat.

The profound effects of 17beta-estradiol on cell growth, differentiation, and general homeostasis of the reproductive and other systems, are mediated mostly by regulation of temporal and cell type-specific expression of different genes. In order to understand better the molecular events associated with the activation of the estrogen receptor (ER), we have used microarray technology to determine the transcriptional program and dose-response characteristics of exposure to a potent synthetic estrogen, 17 alpha-ethynyl estradiol (EE), during prepubertal development. Changes in patterns of gene expression were determined in the immature uterus and ovaries of Sprague-Dawley rats on postnatal day (PND) 24, 24 h after exposure to EE, at 0.001, 0.01, 0.1, 1 and 10 micro g EE/kg/day (sc), for four days (dosing from PND 20 to 23). The transcript profiles were compared between treatment groups and controls using oligonucleotide arrays to determine the expression level of approximately 7000 annotated rat genes and over 1740 expressed sequence tags (ESTs). Quantification of the number of genes whose expression was modified by the treatment, for each of the various doses of EE tested, showed clear evidence of a dose-dependent treatment effect that follows a monotonic response, concordant with the dose-response pattern of uterine wet-weight gain and luminal epithelial cell height. The number of genes whose expression is affected by EE exposure increases according to dose. At the highest dose tested of EE, we determined that the expression level of over 300 genes was modified significantly (p < or = 0.0001). A dose-dependent analysis of the transcript profile revealed a set of 88 genes whose expression is significantly and reproducibly modified (increased or decreased) by EE exposure (p < or = 0.0001). The results of this study demonstrate that, exposure to a potent estrogenic chemical during prepubertal maturation changes the gene expression profile of estrogen-sensitive tissues. Furthermore, the products of the EE-regulated genes identified in these tissues have a physiological role in different intracellular pathways, information that will be valuable to determine the mechanism of action of estrogens. Moreover, those genes could be used as biomarkers to identify chemicals with estrogenic activity.