Assessing eDNA capture method from aquatic environment to optimise recovery of human mt-eDNA.

Previous studies have shown that environmental DNA (eDNA) from human sources can be recovered from natural bodies of water, and the generation of DNA profiles from such environmental samples may assist in forensic investigations. However, fundamental knowledge gaps exist around the factors influencing the probability of detecting human eDNA and the design of optimal sampling protocols. One of these is understanding the particle sizes eDNA signals are most strongly associated with and the most appropriate filter size needed for efficiently capturing eDNA particles. This study assessed the amount of mitochondrial eDNA associated with different particle sizes from human blood and skin cells recovered from freshwater samples. Samples (300 mL) were taken from experimental 10 L tanks of freshwater spiked with 50 µL of human blood or skin cells deposited by vigorously rubbing hands together for two minutes in freshwater. Subsamples were collected by passing 250 mL of experimental water sample through six different filter pore sizes (from 0.1 to 8 µm). This process was repeated at four time intervals after spiking over 72 hours to assess if the particle size of the amount of eDNA recovered changes as the eDNA degrades. Using a human-specific quantitative polymerase chain reaction (qPCR) assay targeting the HV1 mitochondrial gene region, the total amount of mitochondrial eDNA associated with different particle size fractions was determined. In the case of human blood, at 0 h, the 0.45 µm filter pore size captured the greatest amount of mitochondrial eDNA, capturing 42 % of the eDNA detected. The pattern then changed after 48 h, with the 5 µm filter pore size capturing the greatest amount of eDNA (67 %), and 81 % of eDNA at 72 h. Notably, a ten-fold dilution proved to be a valuable strategy for enhancing eDNA recovery from the 8 µm filter at all time points, primarily due to the PCR inhibition observed in hemoglobin. For human skin cells, the greatest amounts of eDNA were recovered from the 8 µm filter pore size and were consistent through time (capturing 37 %, 56 %, and 88 % of eDNA at 0 hours, 48 hours, and 72 hours respectively). There is a clear variation in the amount of eDNA recovered between different cell types, and in some forensic scenarios, there is likely to be a mix of cell types present. These results suggest it would be best to use a 5 µm filter pore size to capture human blood and an 8 µm filter pore size to capture human skin cells to maximize DNA recovery from freshwater samples. Depending on the cell type contributing to the eDNA, a combination of different filter pore sizes may be employed to optimize the recovery of human DNA from water samples. This study provides the groundwork for optimizing a strategy for the efficient recovery of human eDNA from aquatic environments, paving the way for its broader application in forensic and environmental sciences.

Real-world data in retinal diseases treated with anti-vascular endothelial growth factor (anti-VEGF) therapy - a systematic approach to identify and characterize data sources.

BACKGROUND: Real-world data (RWD) has been a valuable addition to the scientific literature regarding treatment pathways, clinical outcomes and characteristics of patients with retinal diseases in recent years. Registries, observational studies and patient databases are often used for real-world research. However, there is limited information for each data source on the design, consistency, data captured, limitations and usability for assessing research questions. Using a systematic approach, we identified RWD sources for patients with retinal diseases and assessed them for completeness of data relating to different outcomes. METHODS: A systematic literature review was carried out to identify RWD sources for patients with retinal disease. Potentially relevant articles published between 2006 and 2016 were screened following electronic searches in Embase and MEDLINE. Congress and supplementary searches were undertaken to identify RWD sources that may not be referenced in full publications. For each data source, availability and quantity of data on baseline status, clinical outcomes, treatment and management, safety, and patient-reported and economic burden were assessed using a bespoke completeness assessment tool based on International Consortium for Health Outcomes Measurement guidelines for macular degeneration. Completeness of data for each area of interest in each data source was assessed and rated using a 'good-moderate-poor' rating system based on availability and quantity of available data. Each data source was then given an overall score based on its score for each of the 7 areas of interest. RESULTS: A total of 128 RWD sources from 32 countries were identified. Of the identified sources, 64 sources from 16 countries of interest were analyzed. Most of these sources provided information on baseline status and clinical outcomes and treatment, but few collected data on economic and patient-reported burden. Of the RWD sources analyzed, 10 scored highly in the overall completeness assessment, collecting data on most or all of the areas of interest; these sources are considered to be robust data sources for performing ophthalmology real-world studies. CONCLUSIONS: The study provides a comprehensive list of RWD sources for patients with retinal disease, many of which will be useful for conducting real-world studies in the field of ophthalmology.

Adherence to HIV treatment regimens: systematic literature review and meta-analysis.

BACKGROUND: Antiretroviral therapy (ART), when taken consistently, reduces morbidity and mortality associated with human immunodeficiency virus and viral transmission. Suboptimal treatment adherence is associated with regimen complexity and high tablet burden. Single-tablet regimens (STRs) provide a complete treatment regimen in a single tablet. This study examined the relationship between STRs (vs multiple-tablet regimens [MTRs]), treatment adherence, and viral suppression. METHODS: A systematic review was conducted to identify studies investigating at least one of the following: (1) STR/MTR use and adherence; (2) levels of adherence and viral suppression; and (3) STR/MTR use and viral suppression. Meta-analysis was performed to assess the relationship between STR vs MTR use and adherence in observational settings at ≥95% and ≥90% adherence thresholds. RESULTS: In total, 29 studies were identified across the three objectives; two studies were relevant for all objectives. STRs were associated with higher treatment adherence than MTRs in 10/11 observational studies: a 63% greater likelihood of achieving ≥95% adherence (95% CI=1.52-1.74; P<0.001) and a 43% increase in the likelihood of achieving ≥90% adherence (95% CI=1.21-1.69; P<0.001). Higher adherence rates were associated with higher levels of viral suppression in 13/18 studies. Results were mixed in five studies investigating the association between STR or MTR use and viral suppression. CONCLUSION: Although the direct effect of STRs vs MTRs on viral suppression remains unclear, this study provided a quantitative estimate of the relationship between STRs and ART adherence, demonstrating that STRs are associated with significantly higher ART adherence levels at 95% and 90% thresholds. Findings from the systematic review showed that improved adherence results in an increased likelihood of achieving viral suppression in observational settings. Future research should utilize similar measures for adherence and evaluate viral suppression to improve assessment of the relationship between pill burden, adherence, and viral suppression.

Epidemiology and disease burden of pathologic myopia and myopic choroidal neovascularization: an evidence-based systematic review.

PURPOSE: To summarize the epidemiology of pathologic myopia and myopic choroidal neovascularization (CNV) and their impact on vision. DESIGN: Systematic literature review of all English-language studies evaluating the epidemiology and visual burden of pathologic myopia or myopic CNV. METHODS: PubMed and EMBASE were searched with no time limits using predefined search strings for English-language studies evaluating the epidemiology and visual burden of pathologic myopia and myopic CNV. RESULTS: In total, 39 relevant publications were identified. Population-based studies reported pathologic myopia to be the first to third most frequent cause of blindness. The prevalence of pathologic myopia was reported to be 0.9%-3.1%, and the prevalence of visual impairment attributable to pathologic myopia ranged from 0.1%-0.5% (European studies) and from 0.2%-1.4% (Asian studies). The prevalence of CNV in individuals with pathologic myopia was reported to be 5.2%-11.3%, and was bilateral in approximately 15% of patients. All studies of visual outcome in patients with myopic CNV (duration ranging from less than 3 months to 21.5 years) reported deterioration in best-corrected visual acuity over time. Older age, subfoveal CNV location, and larger baseline lesion size were predictors of worse visual outcomes. CONCLUSIONS: Pathologic myopia is an important cause of vision loss worldwide, affecting up to 3% of the population. Of these, a substantial proportion of patients develop myopic CNV, which mostly causes a significant progressive decrease in visual acuity. This condition should therefore be a target for new treatment strategies.

HIV-1 infects macrophages by exploiting an endocytic route dependent on dynamin, Rac1 and Pak1.

Recent studies provide compelling evidence that HIV-1 entry in cell lines and lymphocytes proceeds by endocytosis, but these studies are still lacking in macrophages, an important natural target cell for HIV-1. Macrophages exhibit continual and extensive endocytic activity as part of their natural functions, so we investigated the uptake pathways involved in productive HIV-1 entry. We find that caveolae are not utilised by HIV-1, because the main structural proteins, caveolin-1 and 2 are absent from most human leukocytes. We then focused on macropinocytosis; we find that HIV-1 entry into macrophages is sensitive to inhibitors of Na(+)/H(+) exchange, actin rearrangement, dynamin, Rho family GTPases, and Pak1, but not to inhibitors of PI-3 kinase and myosin II. This leads us to conclude that HIV entry into macrophages proceeds by an endocytic pathway that is not classical macropinocytosis. Because of the limitations of a purely pharmacological study such as this, the final elucidation of this pathway awaits the development of reliable forward genetic approaches in authentic macrophages.

Acidic residues in the membrane-proximal stalk region of vaccinia virus protein B5 are required for glycosaminoglycan-mediated disruption of the extracellular enveloped virus outer membrane.

The extracellular enveloped virus (EEV) form of vaccinia virus (VACV) is surrounded by two lipid envelopes. This presents a topological problem for virus entry into cells, because a classical fusion event would only release a virion surrounded by a single envelope into the cell. Recently, we described a mechanism in which the EEV outer membrane is disrupted following interaction with glycosaminoglycans (GAGs) on the cell surface and thus allowing fusion of the inner membrane with the plasma membrane and penetration of a naked core into the cytosol. Here we show that both the B5 and A34 viral glycoproteins are required for this process. A34 is required to recruit B5 into the EEV membrane and B5 acts as a molecular switch to control EEV membrane rupture upon exposure to GAGs. Analysis of VACV strains expressing mutated B5 proteins demonstrated that the acidic stalk region between the transmembrane anchor sequence and the fourth short consensus repeat of B5 are critical for GAG-induced membrane rupture. Furthermore, the interaction between B5 and A34 can be disrupted by the addition of polyanions (GAGs) and polycations, but only the former induce membrane rupture. Based on these data we propose a revised model for EEV entry.

HIV entry in macrophages is dependent on intact lipid rafts.

Macrophages are an important natural target cell for HIV-1, but previous studies of virus entry into these cells are limited, and the involvement of membrane cholesterol and lipid rafts is unknown. Cholesterol disruption of macrophage membranes using four pharmacological agents acting by different mechanisms: methyl-beta cyclodextrin, nystatin, filipin complex and Lovastatin, all significantly inhibited productive HIV entry and reverse transcription. The inhibitory effects of these drugs resulted in decreased virus release from infected cells, and could be substantially reversed by the addition of water-soluble cholesterol. The virus bound equally to cholesterol-disrupted cells even though HIV receptor expression levels were significantly reduced. Macrophage CD4 and CCR5 were found to partition with the detergent-resistant membranes with a typical raft-associating protein flotillin-1. HIV particles were observed co-localising with a marker of lipid rafts (CTB-FITC) early post infection. These data suggest that macrophage membrane cholesterol is essential for HIV entry, and implicate lipid raft involvement.

Ligand-induced and nonfusogenic dissolution of a viral membrane.

Hitherto, all enveloped viruses were thought to shed their lipid membrane during entry into cells by membrane fusion. The extracellular form of Vaccinia virus has two lipid envelopes surrounding the virus core, and consequently a single fusion event will not deliver a naked core into the cell. Here we report a previously underscribed mechanism in which the outer viral membrane is disrupted by a ligand-induced nonfusogenic reaction, followed by the fusion of the inner viral membrane with the plasma membrane and penetration of the virus core into the cytoplasm. The dissolution of the outer envelope depends on interactions with cellular polyanionic molecules and requires the virus glycoproteins A34 and B5. This discovery represents a remarkable example of how viruses manipulate biological membranes, solves the topological problem of how a double-enveloped virus enters cells, reveals a new effect of polyanions on viruses, and provides a therapeutic approach for treatment of poxvirus infections, such as smallpox.

Entry of the vaccinia virus intracellular mature virion and its interactions with glycosaminoglycans.

Vaccinia virus (VACV) produces two distinct enveloped virions, the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV), but the entry mechanism of neither virion is understood. Here, the binding and entry of IMV particles have been investigated. The cell receptors for IMV are unknown, but it was proposed that IMV can bind to glycosaminoglycans (GAGs) on the cell surface and three IMV surface proteins have been implicated in this. In this study, the effect of soluble GAGs on IMV infectivity was reinvestigated and it was demonstrated that GAGs affected IMV infectivity partially in some cells, but not at all in others. Therefore, binding of IMV to GAGs is cell type-specific and not essential for IMV entry. By using electron microscopy, it is demonstrated that IMV from strains Western Reserve and modified virus Ankara enter cells by fusion with the plasma membrane. After an IMV particle bound to the cell, the IMV membrane fused with the plasma membrane and released the virus core into the cytoplasm. IMV surface antigen became incorporated into the plasma membrane and was not left outside the cell, as claimed in previous studies. Continuity between the IMV membrane and the plasma membrane was confirmed by tilt-series analysis to orientate membranes perpendicularly to the beam of the electron microscope. This analysis shows unequivocally that IMV is surrounded by a single lipid membrane and enters by fusion at the cell surface.

Vaccinia virus cores are transported on microtubules.

Infection with Vaccinia virus (VV) produces several distinct virions called intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). In this report, we have investigated how incoming virus cores derived from IMV are transported within the cell. To do this, recombinant VVs (vA5L-EGFP-N and vA5L-EGFP-C) were generated in which the A5L virus core protein was fused with the enhanced green fluorescent protein (EGFP) at the N or C terminus. These viruses were viable, induced formation of actin tails and had a plaque size similar to wild-type. Immunoblotting showed the A5L-EGFP fusion protein was present in IMV particles and immunoelectron microscopy showed that the fusion protein was incorporated into VV cores. IMV made by vA5L-EGFP-N were used to follow the location and movement of cores after infection of PtK(2) cells. Confocal microscopy showed that virus cores were stained with anti-core antibody only after they had entered the cell and, once intracellular, were negative for the IMV surface protein D8L. These cores co-localized with microtubules and moved in a stop-start manner with an average speed of 51.8 (+/-3.9) microm min(-1), consistent with microtubular movement. Treatment of cells with nocodazole or colchicine inhibited core movement, but addition of cytochalasin D did not. These data show that VV cores derived from IMV use microtubules for intracellular transport after entry.