Added Value of Next Generation over Sanger Sequencing in Kenyan Youth with Extensive HIV-1 Drug Resistance.

HIV-1 drug resistance testing in children and adolescents in low-resource settings is both important and challenging. New (more sensitive) drug resistance testing technologies may improve clinical care, but evaluation of their added value is limited. We assessed the potential added value of using next-generation sequencing (NGS) over Sanger sequencing for detecting nucleoside reverse transcriptase inhibitor (NRTI) and nonnucleoside reverse transcriptase inhibitor (NNRTI) drug resistance mutations (DRMs). Participants included 132 treatment-experienced Kenyan children and adolescents with diverse HIV-1 subtypes and with already high levels of drug resistance detected by Sanger sequencing. We examined overall and DRM-specific resistance and its predicted impact on antiretroviral therapy and evaluated the discrepancy between Sanger sequencing and six NGS thresholds (1%, 2%, 5%, 10%, 15%, and 20%). Depending on the NGS threshold, agreement between the two technologies was 62% to 88% for any DRM, 83% to 92% for NRTI DRMs, and 73% to 94% for NNRTI DRMs, with more DRMs detected at low NGS thresholds. NGS identified 96% to 100% of DRMs detected by Sanger sequencing, while Sanger identified 83% to 99% of DRMs detected by NGS. Higher discrepancy between technologies was associated with higher DRM prevalence. Even in this resistance-saturated cohort, 12% of participants had higher, potentially clinically relevant predicted resistance detected only by NGS. These findings, in a young, vulnerable Kenyan population with diverse HIV-1 subtypes and already high resistance levels, suggest potential benefits of more sensitive NGS over existing technology. Good agreement between technologies at high NGS thresholds supports their interchangeable use; however, the significance of DRMs identified at lower thresholds to patient care should be explored further. IMPORTANCE HIV-1 drug resistance in children and adolescents remains a significant problem in countries facing the highest burden of the HIV epidemic. Surveillance of HIV-1 drug resistance in children and adolescents is an important public health strategy, particularly in resource-limited settings, and yet, it is limited due mostly to cost and infrastructure constraints. Whether newer and more sensitive next-generation sequencing (NGS) adds substantial value beyond traditional Sanger sequencing in detecting HIV-1 drug resistance in real life settings remains an open and debatable question. In this paper, we attempt to address this issue by performing a comprehensive comparison of drug resistance identified by Sanger sequencing and six NGS thresholds. We conducted this study in a well-characterized, vulnerable cohort of children and adolescents living with diverse HIV-1 subtypes in Kenya and, importantly, failing antiretroviral therapy (ART) with already extensive drug resistance. Our findings suggest a potential added value of NGS over Sanger even in this unique cohort.

Evaluation of the robustness of polarization attraction for 10.7-GBaud NRZ-BPSK after long-haul 100-GHz DWDM transmission.

An investigation was carried out on the polarization attraction (PA) of a polarization-scrambled 10.7-GBaud NRZ-BPSK signal in a 1-km-long highly nonlinear fiber (HNLF). For the back-to-back case, PA on an ASE-loaded signal yielded a receiver sensitivity penalty of ≈ 14.5 dB at the ITU-T G.975.1.I3 FEC threshold of 3.5 × 10-3, relative to matched-filter reception theory. After long-haul 100-GHz DWDM transmission in a recirculating loop, PA on the output signal was found to achieve approximately the same receiver sensitivity performance, as that of the back-to-back case. From these experiments, it is concluded that the Gordon-Mollenauer effect due to propagation in the HNLF during PA dominates other impairments including those arising from the long-haul 100-GHz DWDM recirculating loop transmission.

Polarization attraction of 10-Gb/s NRZ-BPSK signal in a highly nonlinear fiber.

Polarization attraction of a 10-Gb/s non-return-to-zero binary phase-shift keyed (NRZ-BPSK) signal has been successfully demonstrated for the first time in a counter-propagating beam configuration using a continuous-wave pump, in a highly nonlinear fiber, by utilizing the Kerr nonlinear cross-polarization process inherent to that fiber. The efficacy of mitigating polarization-dependent loss across polarization-sensitive devices was emulated with a linear polarizer located before the receiver. The receiver sensitivity penalty at 10-9 bit-error-rate relative to the baseline NRZ-BPSK signal was < 0.5 dB, when polarization attraction was employed for a polarization-scrambled signal (after achieving a degree of polarization > 90%). The results confirm that polarization attraction is independent of modulation format.

HemaSpot, a Novel Blood Storage Device for HIV-1 Drug Resistance Testing.

HemaSpot, a novel dried-blood storage filter device, was used for HIV-1 pol resistance testing in 30 fresh United States blood samples and 54 previously frozen Kenyan blood samples. Genotyping succeeded in 79% and 58% of samples, respectively, improved with shorter storage and higher viral load, and had good (86%) resistance mutation concordance to plasma.

Effect of HSV-2 suppressive therapy on genital tract HIV-1 RNA shedding among women on HAART: a pilot randomized controlled trial.

BACKGROUND: The role of suppressive HSV therapy in women coinfected with HSV-2 and HIV-1 taking highly active antiretroviral therapy (HAART) is unclear. METHODS: 60 women with HIV-1/HSV-2 coinfection on HAART with plasma HIV-1 viral load (PVL) ≤75 copies/mL were randomized to receive acyclovir (N = 30) or no acyclovir (N = 30). PVL, genital tract (GT) HIV-1, and GT HSV were measured every 4 weeks for one year. RESULTS: Detection of GT HIV-1 was not significantly different in the two arms (OR 1.23, P = 0.67), although this pilot study was underpowered to detect this difference. When PVL was undetectable, the odds of detecting GT HIV were 0.4 times smaller in the acyclovir arm than in the control arm, though this was not statistically significant (P = 0.07). The odds of detecting GT HSV DNA in women receiving acyclovir were significantly lower than in women in the control group, OR 0.38, P < 0.05. CONCLUSIONS: Chronic suppressive therapy with acyclovir in HIV-1/HSV-2-positive women on HAART significantly reduces asymptomatic GT HSV shedding, though not GT HIV shedding or PVL. PVL was strongly associated with GT HIV shedding, reinforcing the importance of HAART in decreasing HIV sexual transmission.

Acute hepatitis C virus in an HIV clinic: a screening strategy, risk factors, and perception of risk.

Acute hepatitis C virus (HCV) infection is being acquired undetected among HIV-infected individuals. A practical way to regularly screen HIV-infected patients for acute HCV irrespective of perceived risk or symptoms is needed. We piloted implementation of an acute HCV screening strategy using routine HIV clinical care schedules and the least costly blood tests, in a Rhode Island HIV care center. Study participants had ongoing HCV risk, completed questionnaires encompassing risk behaviors and perception of risk, and were screened with quarterly alanine aminotransferase (ALT). ALT rise triggered HCV RNA testing, with pooled rather than individual specimen HCV RNA testing for underinsured participants. Participants were primarily older, college-educated men who have sex with men (MSM) with history of sexually transmitted infection other than HIV. One of 58 participants developed acute HCV in 50 person-years of observation for an annual incidence of 2.0% per year (95% confidence interval [CI] 0.05-11.1%). The majority (54%) of MSM did not perceive that traumatic sexual and drug practices they were engaging in put them at risk for HCV. Unprotected sex often occurred under the influence of drugs or alcohol. Self-reported HCV risk and participation in several risk behaviors declined during the study. It was possible to collect frequent ALTs in a busy HIV clinic with 71% of total projected ALTs obtained and 88% of participants having at least one ALT during the 9-month follow-up period. All instances of ALT rise led to reflexive HCV RNA testing. Tracking quarterly ALT for elevation to systematically prompt HCV RNA testing before seroconversion is a promising approach to screen for acute HCV in a real-world HIV clinical setting.

Polar auxin transport: controlling where and how much.

Auxin is transported through plant tissues, moving from cell to cell in a unique polar manner. Polar auxin transport controls important growth and developmental processes in higher plants. Recent studies have identified several proteins that mediate polar auxin transport and have shown that some of these proteins are asymmetrically localized, paving the way for studies of the mechanisms that regulate auxin transport. New data indicate that reversible protein phosphorylation can control the amount of auxin transport, whereas protein secretion through Golgi-derived vesicles and interactions with the actin cytoskeleton might regulate the localization of auxin efflux complexes.

Genetic and chemical reductions in protein phosphatase activity alter auxin transport, gravity response, and lateral root growth.

Auxin transport is required for important growth and developmental processes in plants, including gravity response and lateral root growth. Several lines of evidence suggest that reversible protein phosphorylation regulates auxin transport. Arabidopsis rcn1 mutant seedlings exhibit reduced protein phosphatase 2A activity and defects in differential cell elongation. Here we report that reduced phosphatase activity alters auxin transport and dependent physiological processes in the seedling root. Root basipetal transport was increased in rcn1 or phosphatase inhibitor-treated seedlings but showed normal sensitivity to the auxin transport inhibitor naphthylphthalamic acid (NPA). Phosphatase inhibition reduced root gravity response and delayed the establishment of differential auxin-induced gene expression across a gravity-stimulated root tip. An NPA treatment that reduced basipetal transport in rcn1 and cantharidin-treated wild-type plants also restored a normal gravity response and asymmetric auxin-induced gene expression, indicating that increased basipetal auxin transport impedes gravitropism. Increased auxin transport in rcn1 or phosphatase inhibitor-treated seedlings did not require the AGR1/EIR1/PIN2/WAV6 or AUX1 gene products. In contrast to basipetal transport, root acropetal transport was normal in phosphatase-inhibited seedlings in the absence of NPA, although it showed reduced NPA sensitivity. Lateral root growth also exhibited reduced NPA sensitivity in rcn1 seedlings, consistent with acropetal transport controlling lateral root growth. These results support the role of protein phosphorylation in regulating auxin transport and suggest that the acropetal and basipetal auxin transport streams are differentially regulated.

Phytobilin biosynthesis: the Synechocystis sp. PCC 6803 heme oxygenase-encoding ho1 gene complements a phytochrome-deficient Arabidopsis thalianna hy1 mutant.

The phytobilin chromophores of phycobiliproteins and phytochromes are biosynthesized from heme in a pathway that begins with the opening of the tetrapyrrole macrocycle of protoheme to form biliverdin IXalpha, in a reaction catalyzed by heme oxygenase. An Arabidopsis thaliana hy1 mutant was previously shown to be deficient in phytochrome responses, and these responses were regained when the plants were administered biliverdin IXalpha. A heme oxygenase-encoding gene, ho1, was recently cloned from the cyanobacterium Synechocystis sp. PCC 6803. When ho1 was expressed in Escherichia coli, the cells produced active ferredoxin-dependent soluble heme oxygenase. The open reading frame of ho1 was fused in frame with a chloroplast transit peptide-encoding sequence from the oli gene of Antirrhinum majus. This construct was placed in a binary plasmid vectorcontaining a kanamycin resistance marker and a cauliflower mosaic virus 35S promoter to control expression of the chimeric oli-ho1 gene and used to transform A. thaliana hy1 plants. Two independent transformed lines were obtained that had the phenotype of the parental Landsberg erecta line and expressed the chimeric gene, as indicated by detection of its mRNA by reverse transcriptase-polymerase chain reaction. The results indicate that Synechocystis sp. PCC 6803 heme oxygenase encoded by ho1 can substitute for the defective HY1 gene product and that the only required enzyme activity of the HY1 gene product is heme oxygenase.

The RCN1-encoded A subunit of protein phosphatase 2A increases phosphatase activity in vivo.

Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic C subunit and two distinct regulatory subunits, A and B. The RCN1 gene encodes one of three A regulatory subunits in Arabidopsis thaliana. A T-DNA insertion mutation at this locus impairs root curling, seedling organ elongation and apical hypocotyl hook formation. We have used in vivo and in vitro assays to gauge the impact of the rcn1 mutation on PP2A activity in seedlings. PP2A activity is decreased in extracts from rcn1 mutant seedlings, and this decrease is not due to a reduction in catalytic subunit expression. Roots of mutant seedlings exhibit increased sensitivity to the phosphatase inhibitors okadaic acid and cantharidin in organ elongation assays. Shoots of dark-grown, but not light-grown seedlings also show increased inhibitor sensitivity. Furthermore, cantharidin treatment of wild-type seedlings mimics the rcn1 defect in root curling, root waving and hypocotyl hook formation assays. In roots of wild-type seedlings, RCN1 mRNA is expressed at high levels in root tips, and accumulates to lower levels in the pericycle and lateral root primordia. In shoots, RCN1 is expressed in the apical hook and the basal, rapidly elongating cells in etiolated hypocotyls, and in the shoot meristem and leaf primordia of light-grown seedlings. Our results show that the wild-type RCN1-encoded A subunit functions as a positive regulator of the PP2A holoenzyme, increasing activity towards substrates involved in organ elongation and differential cell elongation responses such as root curling.

Functional expression of human and Arabidopsis protein phosphatase 2A in Saccharomyces cerevisiae and isolation of dominant-defective mutants.

Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic subunit and two distinct regulatory subunits, A and B. The primary sequence of the catalytic (C) subunit is highly conserved in evolution, and its function has been shown to be essential in yeast, Drosophila and mice. In many eukaryotes, the C subunit is encoded by at least two nearly identical genes, impeding conventional loss-of-function genetic analysis. We report here the development of a functional complementation assay in S. cerevisiae that has allowed us to isolate dominant-defective alleles of human and Arabidopsis C subunit genes. Wild-type human and Arabidopsis C subunit genes can complement the lethal phenotype of S. cerevisiae PP2A-C mutations. Site-directed mutagenesis was used to create two distinct, catalytically impaired C subunit mutants of the human and Arabidopsis genes. In both cases, expression of the mutant subunit in yeast prevented growth, even in the presence of functional C subunit proteins. This dominant growth defect is consistent with a dominant-interfering mode of action. Thus, we have shown that S. cerevisiae provides a rapid system for the functional analysis of heterologous PP2A genes, and that two mutations that abrogate phosphatase activity exhibit dominant-defective phenotypes in S. cerevisiae.

A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis.

The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.

High-performance liquid chromatographic assay for xanomeline, a specific M-1 agonist, and its metabolite in human plasma.

A reversed-phase high-performance liquid chromatographic assay (HPLC) was utilized for monitoring xanomeline (LY246708/NNC 11-0232) and a metabolite, desmethylxanomeline, in human plasma. Xanomeline, desmethylxanomeline and internal standard were extracted from plasm with hexane at basic pH. The organic solvent extract was evaporated to dryness with nitrogen and the dried residue was reconstituted with 0.2 M HCl-methanol (50:50, v/v). A Zorbax CN 150 x 4.6 mm I.D., 5-microns column and mobile phase consisting of 0.5% (5 ml/l) triethylamine (TEA) adjusted to pH 3.0 with concentrated orthophosphoric acid-tetrahydrofuran (THF) (70:30, v/v) produced consistent resolution of analytes from endogenous co-extracted plasma components. Column effluent was monitored at 296 nm/0.008 a.u.f.s. and the assay limit of quantification was 1.5 ng/ml. A linear response of 1.5 to 20 ng/ml was sufficient to monitor plasma drug/metabolite concentrations during clinical trials. HPLC assay validation as well as routine assay quality control (QC) samples indicated assay precision/accuracy was better than +/- 15%.

Determination of xanomeline and active metabolite, N-desmethylxanomeline, in human plasma by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

We have developed a method for the determination of xanomeline and its pharmacologically active N-desmethyl metabolite. The validated method uses hexane to extract xanomeline and its N-desmethyl metabolite from basified plasma. The hexane extract is dried, reconstituted, and analyzed using a liquid chromatographic-atmospheric pressure chemical ionization tandem mass spectrometry system. The method was developed to support phase II clinical trials and has proven to be extremely sensitive, fast, and rugged. The method has a limit of quantitation of 75 and 200 pg/ml plasma for xanomeline and the N-desmethyl metabolite, respectively. Sample analysis times were less than 3 min from one injection to the next.

Determination of xanomeline in human plasma by ion-spray tandem mass spectrometry.

Xanomeline is a muscarinic receptor agonist currently in phase II clinical trials for the treatment of Alzheimer's disease. A fast, sensitive and specific assay has been developed to determine xanomeline plasma concentrations using ion-spray tandem mass spectrometry. Xanomeline and a structural analog, LY282122, were extracted from basifed plasma into hexane. The dried hexane extracts were reconstituted and injected onto a 10 x 1 mm C18 reversed-phase column. A mobile phase of 33 mM ammonium acetate and 0.33% acetic acid in 30/70 (v/v) water-acetonitrile was pumped through the column at 50 microliters min-1. The mobile phase eluant was introduced directly into the ion-spray interface. The mass spectrometer was operated in the positive ion mode for specific detection of the product ions of xanomeline and the internal standard. The method has a linear range of 0.075-5.0 ng xanomeline per milliliter of plasma. Sample run times were 2.5 min from one injection to the next.

Sex determination gene TASSELSEED2 of maize encodes a short-chain alcohol dehydrogenase required for stage-specific floral organ abortion.

Maize produces separate unisexual flowers through programmed abortion of preformed organ primordia. In the male inflorescence (tassel), stamen primordia develop to sexual maturity, while gynoecia (pistil primordia) are aborted. In tasselseed2 (ts2) mutant plants, floral structures in the tassel adopt a female developmental program. Here we report the transposon tagging and cloning of the TS2 gene, which plays a late but pivotal role in determining the sexual fate of floral meristems. Shortly before abortion of the gynoecium, Ts2 mRNA is expressed subepidermally in that primordium. Phenotypic instability of the Activator (Ac)-induced allele ts2-m1 indicates that late restoration of TS2 action in somatic tissues, which is correlated with Ac excision, reactivates the male developmental program. The predicted amino acid sequence of the Ts2 protein shows significant similarity to short-chain alcohol dehydrogenases, particularly hydroxysteroid dehydrogenases.

Isomazole disposition in man as a function of dose and route of administration.

The disposition of a new cardiotonic agent (isomazole (ISO] was evaluated in healthy volunteers after various single oral (p.o.), intravenous (i.v.), and multiple p.o. doses. Blood samples were collected after dosing in all studies, with urine collected in the single i.v. and multiple dose studies. All biological samples were measured for ISO. In the multiple dose study, samples were collected for analysis after the first and last doses administered. In addition to ISO, several known metabolites (hydroxyisomazole (OHISO), sulfone (SULF), and hydroxysulfone (OHSULF) analogs) were measured after the first and last doses given in the multiple dose study. Pharmacokinetic values compared between doses suggested no saturable processes existed over the entire dose range. The single i.v. dose data showed ISO experienced some extravascular distribution (mean V beta = 1.82 l kg-1), with a high clearance (mean Cls = 18.8 ml min-1 kg-1) and a short half-life (mean t 1/2 = 1.1 h). Elimination was primarily nonrenal (Clr = 3.5 ml min-1 kg-1). Single p.o. data supported these findings and further suggested rapid absorption. ISO data from the first dose of the multiple dose study was in agreement with these data; however, the last dose showed a higher Cls (33.0 ml min-1 kg-1) (p = 0.055). Although not statistically significant, metabolite plasma data and and urinary excretion patterns changed. An increase was observed in plasma AUC and metabolite excretion of SULF and OHSULF, while a decrease was observed in the same parameters for OHISO. These results suggest that multiple dosing of ISO produces autoinduction of ISO metabolism through selective metabolic routes.

14C-isomazole disposition in man after oral administration.

A 50-mg dose containing 50 microCi 14C-isomazole was administered orally to five healthy male volunteers. Blood, plasma, urine, feces, and saliva were collected and measured for total 14C; in addition, all collections except feces were measured for parent drug (ISO) and three metabolites: hydroxyisomazole (OHISO) and sulfone (SULF) and hydroxysulfone (OHSULF) analogues. Urine and fecal recoveries accounted for 97.0% of the drug administered, with 62.6% excreted in urine and 32.4% in feces. Only 47% of the drug recovered in urine could be identified, with ISO the largest constituent. Total plasma 14C peaked at 1.5 hr, indicating rapid absorption, and produced a mean half-life of 3.7 hr. This was similar to the total 14C half-life found in blood (3.1 hr) but longer than in red blood cells (1.8 hr) or saliva (1.4 hr), suggesting that different ISO-related compounds contributed to the results found in each fluid or tissue. An unidentified metabolite(s) composed a large portion of circulating plasma 14C and produced the longer half-life encountered in plasma. ISO exhibited a short half-life (1.35 hr), a high oral clearance (Cls/F; 24.2 ml/min/kg), and some extravascular distribution (V beta; 3.07 L/kg). Total 14C in red blood cells and saliva related very well to plasma ISO disposition, suggesting preferential distribution of parent drug across cellular membranes. The estimated RBC:plasma ISO ratio (1.79) confirmed this hypothesis. Saliva may be used as a noninvasive means to monitor ISO disposition.

Trans-acting transposase mutant from Tn5.

Transposition of Tn5 and of its component insertion sequence IS50R is regulated through the action of two proteins it encodes: a cis-acting transposase, Tnp, and a trans-acting inhibitor of transposition, Inh. The mechanism of the cis-acting Tnp and the relevance of inhibition to cis action have been addressed in the current study. A specific colony morphology assay for transposition of Tn5 was shown to be sensitive to Inh produced in trans and was used to screen for mutants in Inh and/or Tnp with altered regulation. A dominant mutant in IS50R that promotes transposition in trans was isolated and characterized. The mutant (449F) carries a Leu----Phe mutation at position 449 in Tnp. This mutation reduces the frequency of Tn5 or IS50R transposition in cis but allows Tnp-449F to act as efficiently in trans as it does in cis. Tnp-449F is sensitive to inhibition and, furthermore, Inh-449F is a competent inhibitor in trans. These results show that Tnp-449F is a trans-acting transposase, unlike wild-type Tnp, which is cis-acting.

Cell lineage analysis of the gynoecium of maize using the transposable element Ac.

The maize gynoecium develops from a primordium comprising two distinct cell lineages: an epidermal LI and a subepidermal LII lineage. We have analyzed somatic gynoecial sectors marked with red flavonoid pigments by excision of Ac from the P locus. Somatic sector analysis indicates the epidermal lineage starts as a single cell layer at the base of the ovary and thickens into several cell layers, presumably by periclinal divisions, near the silk attachment point. The silk attachment point appears to be mainly an epidermal outgrowth of two of three fused carpels and presumably the silk contains only LI, or mostly LI, with only traces of subepidermal LII cells. The third carpel, covering the germinal face of the kernel, retains a multicellular LII and unicellular LI organization but fails to contribute substantially to stylar outgrowth. Ac transpositions in subepidermal somatic sectors are transmitted to the archesporial cell lineage. Ac transpositions that occur in epidermal sectors are not transmitted to offspring. These results demonstrate that the female megasporocyte is derived from subepidermal (LII) cells.