Clinical Evaluation of an Affordable Qualitative Viral Failure Assay for HIV Using Dried Blood Spots in Uganda

Background

WHO recommends regular viral load (VL) monitoring of patients on antiretroviral therapy (ART) for timely detection of virological failure, prevention of acquired HIV drug resistance (HIVDR) and avoiding unnecessary switching to second-line ART. However, the cost and complexity of routine VL testing remains prohibitive in most resource limited settings (RLS). We evaluated a simple, low–cost, qualitative viral–failure assay (VFA) on dried blood spots (DBS) in three clinical settings in Uganda.

Methods

We conducted a cross–sectional diagnostic accuracy study in three HIV/AIDS treatment centres at the Joint Clinical Research Centre in Uganda. The VFA employs semi-quantitative detection of HIV–1 RNA amplified from the LTR gene. We used paired dry blood spot (DBS) and plasma with the COBASAmpliPrep/COBASTaqMan, Roche version 2 (VL_ref) as the reference assay. We used the VFA at two thresholds of viral load, (>5,000 or >1,000 copies/ml).

Results

496 paired VFA and VL_ref results were available for comparative analysis. Overall, VFA demonstrated 78.4% sensitivity, (95% CI: 69.7%–87.1%), 93% specificity (95% CI: 89.7%–96.4%), 89.3% accuracy (95% CI: 85%–92%) and an agreement kappa = 0.72 as compared to the VL_ref. The predictive values of positivity and negativity among patients on ART for >12 months were 72.7% and 99.3%, respectively.

Conclusions

VFA allowed 89% of correct classification of VF. Only 11% of the patients were misclassified with the potential of unnecessary or late switch to second–line ART. Our findings present an opportunity to roll out simple and affordable VL monitoring for HIV–1 treatment in RLS.     

Peptidylarginine Deiminase 3 (PAD3) Is Upregulated by Prolactin Stimulation of CID-9 Cells and Expressed in the Lactating Mouse Mammary Gland

Peptidylarginine deiminases (PADs) post-translationally convert arginine into neutral citrulline residues. Our past work shows that PADs are expressed in the canine and murine mammary glands; however, the mechanisms regulating PAD expression and the function of citrullination in the normal mammary gland are unclear. Therefore, the first objective herein was to investigate regulation of PAD expression in mammary epithelial cells. We first examined PAD levels in CID-9 cells, which were derived from the mammary gland of mid-pregnant mice. PAD3 expression is significantly higher than all other PAD isoforms and mediates protein citrullination in CID-9 cells. We next hypothesized that prolactin regulates PAD3 expression. To test this, CID-9 cells were stimulated with 5 μg/mL of prolactin for 48 hours which significantly increases PAD3 mRNA and protein expression. Use of a JAK2 inhibitor and a dominant negative (DN)-STAT5 adenovirus indicate that prolactin stimulation of PAD3 expression is mediated by the JAK2/STAT5 signaling pathway in CID-9 cells. In addition, the human PAD3 gene promoter is prolactin responsive in CID-9 cells. Our second objective was to investigate the expression and activity of PAD3 in the lactating mouse mammary gland. PAD3 expression in the mammary gland is highest on lactation day 9 and coincident with citrullinated proteins such as histones. Use of the PAD3 specific inhibitor, Cl4-amidine, indicates that PAD3, in part, can citrullinate proteins in L9 mammary glands. Collectively, our results show that upregulation of PAD3 is mediated by prolactin induction of the JAK2/STAT5 signaling pathway, and that PAD3 appears to citrullinate proteins during lactation.     

A comparative study of <Emphasis Type="Italic">k</Emphasis>-spectrum-based error correction methods for next-generation sequencing data analysis

Background

Innumerable opportunities for new genomic research have been stimulated by advancement in high-throughput next-generation sequencing (NGS). However, the pitfall of NGS data abundance is the complication of distinction between true biological variants and sequence error alterations during downstream analysis. Many error correction methods have been developed to correct erroneous NGS reads before further analysis, but independent evaluation of the impact of such dataset features as read length, genome size, and coverage depth on their performance is lacking. This comparative study aims to investigate the strength and weakness as well as limitations of some newest k-spectrum-based methods and to provide recommendations for users in selecting suitable methods with respect to specific NGS datasets.

Methods

Six k-spectrum-based methods, i.e., Reptile, Musket, Bless, Bloocoo, Lighter, and Trowel, were compared using six simulated sets of paired-end Illumina sequencing data. These NGS datasets varied in coverage depth (10× to 120×), read length (36 to 100 bp), and genome size (4.6 to 143 MB). Error Correction Evaluation Toolkit (ECET) was employed to derive a suite of metrics (i.e., true positives, false positive, false negative, recall, precision, gain, and F-score) for assessing the correction quality of each method.

Results

Results from computational experiments indicate that Musket had the best overall performance across the spectra of examined variants reflected in the six datasets. The lowest accuracy of Musket (F-score?=?0.81) occurred to a dataset with a medium read length (56 bp), a medium coverage (50×), and a small-sized genome (5.4 MB). The other five methods underperformed (F-score?<?0.80) and/or failed to process one or more datasets.

Conclusions

This study demonstrates that various factors such as coverage depth, read length, and genome size may influence performance of individual k-spectrum-based error correction methods. Thus, efforts have to be paid in choosing appropriate methods for error correction of specific NGS datasets. Based on our comparative study, we recommend Musket as the top choice because of its consistently superior performance across all six testing datasets. Further extensive studies are warranted to assess these methods using experimental datasets generated by NGS platforms (e.g., 454, SOLiD, and Ion Torrent) under more diversified parameter settings (k-mer values and edit distances) and to compare them against other non-k-spectrum-based classes of error correction methods.

     

Expansion of Islet-Resident Macrophages Leads to Inflammation Affecting β Cell Proliferation and Function in Obesity

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A nuclear lamina‐chromatin‐Ran GTPase axis modulates nuclear import and DNA damage signaling

The Ran GTPase regulates nuclear import and export by controlling the assembly state of transport complexes. This involves the direct action of RanGTP, which is generated in the nucleus by the chromatin‐associated nucleotide exchange factor, RCC1. Ran interactions with RCC1 contribute to formation of a nuclear:cytoplasmic (N:C) Ran protein gradient in interphase cells. In previous work, we showed that the Ran protein gradient is disrupted in fibroblasts from Hutchinson–Gilford progeria syndrome (HGPS) patients. The Ran gradient disruption in these cells is caused by nuclear membrane association of a mutant form of Lamin A, which induces a global reduction in heterochromatin marked with Histone H3K9me3 and Histone H3K27me3. Here, we have tested the hypothesis that heterochromatin controls the Ran gradient. Chemical inhibition and depletion of the histone methyltransferases (HMTs) G9a and GLP in normal human fibroblasts reduced heterochromatin levels and caused disruption of the Ran gradient, comparable to that observed previously in HGPS fibroblasts. HMT inhibition caused a defect in nuclear localization of TPR, a high molecular weight protein that, owing to its large size, displays a Ran‐dependent import defect in HGPS. We reasoned that pathways dependent on nuclear import of large proteins might be compromised in HGPS. We found that nuclear import of ATM requires the Ran gradient, and disruption of the Ran gradient in HGPS causes a defect in generating nuclear γ‐H2AX in response to ionizing radiation. Our data suggest a lamina–chromatin–Ran axis is important for nuclear transport regulation and contributes to the DNA damage response.     

Evaluation of treatment options for well water contaminated with perfluorinated alkyl substances using life cycle assessment

Purpose

As knowledge grows of the potentially harmful effects of chemicals in widespread use, emerging contaminants have become a major source of concern and uncertainty for public health officials and water quality managers. Perfluorinated alkyl substances, often referred to as perfluorinated compounds, have come under recent scrutiny and are present in groundwater at many sites across the USA. We examine the life cycle impacts of treating drinking water at one such site.

Methods

We assembled life cycle models for groundwater treatment and bottled water delivery to residents of Wright-Patterson Air Force Base, Ohio, where wells were recently taken out of service due to concerns related to perfluoroalkyl and polyfluoroalkyl substance (PFAS) contamination. Two treatment methods, granular activated carbon filtration and ion-exchange columns, were modeled under a range of contaminant concentrations covering three orders of magnitude: 0.7, 7.0, and 70 μg/L PFAS. On-site infrastructure, operations, and adsorbent cycling were included in models. Impacts of bottled water production and supply were assessed using two data sets reflecting a range of production and supply chain assumptions. Uncertainty in input data was captured using Monte Carlo simulations.

Results and discussion

Results show that for contaminant concentrations below 70 μg/L, the dominant contributor to life cycle impacts is electricity use at the treatment facility. Production, reactivation, and disposal of treatment media become major sources of impact only at very high PFAS concentrations. Though the life cycle impacts of bottled water are up to three orders of magnitude higher than remediated groundwater on a volumetric basis, supplementing a contaminated water supply with bottled drinking water may result in lower life cycle human health impacts when only a small proportion of the total population is vulnerable.

Conclusions

These results provide quantitative data and proposed scenarios for water quality managers and risk management officials in developing plans to address PFAS contamination and emerging contaminants in general. However, more information on the direct human health effects of these poorly understood pollutants is needed before the trade-offs in life cycle health impacts can be comprehensively assessed.

     

Beta-amino acid analogs of an insect neuropeptide feature potent bioactivity and resistance to peptidase hydrolysis

Insect neuropeptides of the insect kinin class share a common C-terminal pentapeptide sequence F(1)X(1)(2)X(2)(3)W(4)G(5)-NH(2) (X(2)(3) = P, S, A) and regulate such critical physiological processes as water balance and digestive enzyme release. Analogs of the insect kinin class, in which the critical residues of F(1), P(3), and W(4) were replaced with beta(3)-amino acid or their beta(2)-homo-amino acid variants, have been synthesized by the solid phase peptide strategy. The resulting single- and double-replacement analogs were evaluated in an insect diuretic assay and enzyme digestion trials. Analogs modified in the core P(3) position produce a potent and efficacious diuretic response that is not significantly different from that obtained with the endogenous achetakinin peptides. The analogs also demonstrate enhanced resistance to hydrolysis by ACE and NEP, endopeptidases that inactivate the natural insect neuropeptides. This paper describes the first instance of beta-amino acids analogs of an arthropod peptide that demonstrate significant bioactivity and resistance to peptidase degradation.     

Oxidation and S-nitrosylation of cysteines in human cytosolic and mitochondrial glutaredoxins: effects on structure and activity

Glutathione (GSH) is the major intracellular thiol present in 1-10-mm concentrations in human cells. However, the redox potential of the 2GSH/GSSG (glutathione disulfide) couple in cells varies in association with proliferation, differentiation, or apoptosis from -260 mV to -200 or -170 mV. Hydrogen peroxide is transiently produced as second messenger in receptor-mediated growth factor signaling. To understand oxidation mechanisms by GSSG or nitric oxide-related nitrosylation we studied effects on glutaredoxins (Grx), which catalyze GSH-dependent thiol-disulfide redox reactions, particularly reversible glutathionylation of protein sulfhydryl groups. Human Grx1 and Grx2 contain Cys-Pro-Tyr-Cys and Cys-Ser-Tyr-Cys active sites and have three and two additional structural Cys residues, respectively. We analyzed the redox state and disulfide pairing of Cys residues upon GSSG oxidation and S-nitrosylation. Cytosolic/nuclear Grx1 was partly inactivated by both S-nitrosylation and oxidation. Inhibition by nitrosylation was reversible under anaerobic conditions; aerobically it was stronger and irreversible, indicating inactivation by nitration. Oxidation of Grx1 induced a complex pattern of disulfide-bonded dimers and oligomers formed between Cys-8 and either Cys-79 or Cys-83. In addition, an intramolecular disulfide between Cys-79 and Cys-83 was identified, predicted to have a profound effect on the three-dimensional structure. In contrast, mitochondrial Grx2 retains activity upon oxidation, did not form disulfide-bonded dimers or oligomers, and could not be S-nitrosylated. The dimeric iron sulfur cluster-coordinating inactive form of Grx2 dissociated upon nitrosylation, leading to activation of the protein. The striking differences between Grx1 and Grx2 reflect their diverse regulatory functions in vivo and also adaptation to different subcellular localization.     

Human disease classification in the postgenomic era: A complex systems approach to human pathobiology

Contemporary classification of human disease derives from observational correlation between pathological analysis and clinical syndromes. Characterizing disease in this way established a nosology that has served clinicians well to the current time, and depends on observational skills and simple laboratory tools to define the syndromic phenotype. Yet, this time-honored diagnostic strategy has significant shortcomings that reflect both a lack of sensitivity in identifying preclinical disease, and a lack of specificity in defining disease unequivocally. In this paper, we focus on the latter limitation, viewing it as a reflection both of the different clinical presentations of many diseases (variable phenotypic expression), and of the excessive reliance on Cartesian reductionism in establishing diagnoses. The purpose of this perspective is to provide a logical basis for a new approach to classifying human disease that uses conventional reductionism and incorporates the non-reductionist approach of systems biomedicine.     

Middle Pleistocene Steppe Lion Remains from Grotte de la Carrière (Têt Valley,Eastern Pyrenees)

Journal of Mammalian Evolution - Late Pleistocene cave lions are one of the most iconic species of Northern Hemisphere Quaternary taphocoenoses. Despite their often-scarce record in cave...