Substantial contribution of tree canopy nitrifiers to nitrogen fluxes in European forests

Human activities have greatly increased the reactive nitrogen in the biosphere, thus profoundly altering global nitrogen cycling. The large increase in nitrogen deposition over the past few decades has led to eutrophication in natural ecosystems, with negative effects on forest health and biodiversity. Recent studies, however, have reported oligotrophication in forest ecosystems, constraining their capacity as carbon sinks. Here we demonstrate the widespread biological transformation of atmospheric reactive nitrogen in the canopies of European forests by combining nitrogen deposition quantification with measurements of the stable isotopes in nitrate and molecular analyses across ten forests through August–October 2016. We estimate that up to 80% of the nitrate reaching the soil via throughfall was derived from canopy nitrification, equivalent to a flux of up to 5.76 kg N ha−1 yr−1. We also document the presence of autotrophic nitrifiers on foliar surfaces throughout European forests. Canopy nitrification thus consumes deposited ammonium and increases nitrate inputs to the soil. The results of this study highlight widespread canopy nitrification in European forests and its important contribution to forest nitrogen cycling

Contributions of mean and shape of blood pressure distribution to worldwide trends and variations in raised blood pressure: A pooled analysis of 1018 population-based measurement studies with 88.6 million participants

© The Author(s) 2018. Background: Change in the prevalence of raised blood pressure could be due to both shifts in the entire distribution of blood pressure (representing the combined effects of public health interventions and secular trends) and changes in its high-blood-pressure tail (representing successful clinical interventions to control blood pressure in the hypertensive population). Our aim was to quantify the contributions of these two phenomena to the worldwide trends in the prevalence of raised blood pressure. Methods: We pooled 1018 population-based studies with blood pressure measurements on 88.6 million participants from 1985 to 2016. We first calculated mean systolic blood pressure (SBP), mean diastolic blood pressure (DBP) and prevalence of raised blood pressure by sex and 10-year age group from 20-29 years to 70-79 years in each study, taking into account complex survey design and survey sample weights, where relevant. We used a linear mixed effect model to quantify the association between (probittransformed) prevalence of raised blood pressure and age-group- and sex-specific mean blood pressure. We calculated the contributions of change in mean SBP and DBP, and of change in the prevalence-mean association, to the change in prevalence of raised blood pressure. Results: In 2005-16, at the same level of population mean SBP and DBP, men and women in South Asia and in Central Asia, the Middle East and North Africa would have the highest prevalence of raised blood pressure, and men and women in the highincome Asia Pacific and high-income Western regions would have the lowest. In most region-sex-age groups where the prevalence of raised blood pressure declined, one half or more of the decline was due to the decline in mean blood pressure. Where prevalence of raised blood pressure has increased, the change was entirely driven by increasing mean blood pressure, offset partly by the change in the prevalence-mean association. Conclusions: Change in mean blood pressure is the main driver of the worldwide change in the prevalence of raised blood pressure, but change in the high-blood-pressure tail of the distribution has also contributed to the change in prevalence, especially in older age groups

A safety evaluation of the intravitreal use of a beta-2 agonist in rabbit eyes

status: publishe

Long-read sequencing assay allows accurate characterization of the HIV-1 reservoir

Background The advent of near full-length (NFL) HIV-1 proviral genome sequencing greatly expanded our understanding of the quality of the viral reservoir, revealing that only 2-5% of the persistent proviruses in ART-treated individuals can be considered genome-intact. However, current NFL assays are based on labor-intensive and costly principles of repeated PCRs at limiting dilution, restricting their scalability. We developed a long-read sequencing assay to characterize many proviral genomes in parallel from bulk DNA. Methods The sensitivity of the long-read assay was determined on a DNA dilution series of J-Lat in uninfected Jurkat ranging from 80,000 to <8 HIV-1 copies. Next, the assay was performed on 15 chronic ART-suppressed individuals, using a fixed input of 500 ng DNA extracted from peripheral blood CD4 T cells (reservoir sizes ranging from 321 to 6581 total HIV-1 DNA copies/million CD4 T cells). Individual proviruses were tagged with a different unique molecular identifier (UMI) at each end during a single reaction, followed by NFL PCR amplification and long-read sequencing on an Oxford Nanopore MinION. UMI-based demultiplexing allowed for the construction of highly accurate consensus genomes, while excluding aberrant chimeric PCR artefacts. In addition, Full-Length Individual Provirus Sequencing (FLIPS) was performed on 2 individuals. Data from both assays were compared through phylogenetic analyses. Results The lower limit of the long-read assay was found to be <8 HIV-1 copies. The long-read assay yielded an average of 14 distinct HIV-1 proviruses per participant (range: 3-42). Across all participants, 213 distinct proviruses were retrieved of which 8% were considered putatively intact. In terms of reservoir composition, data obtained with FLIPS showed an overall agreement with data obtained with the long-read assay. In an individual with limited clonality (6% clonality of FLIPS data, n=1 clone) only 1 overlapping provirus was found, while an overlap of 3 proviruses was observed in an individual with higher clonality (91% clonality of FLIPS data, n=4 clones). Comparing the 4 overlapping proviral consensus genomes to their matching FLIPS counterparts showed an average sequence accuracy of 99,97%. Conclusion The long-read assay offers a high-throughput NFL sequencing method which enables an accurate characterization of the proviral landscape while retaining sequencing accuracy comparable to current gold standard NFL assays

HIV-PULSE : a long-read sequencing assay for high-throughput near full-length HIV-1 proviral genome characterization

A deep understanding of the composition of the HIV-1 reservoir is necessary for the development of targeted therapies and the evaluation of curative efforts. However, current near full-length (NFL) HIV-1 proviral genome sequencing assays are based on labor-intensive and costly principles of repeated PCRs at limiting dilution, restricting their scalability. To address this, we developed a high-throughput, long-read sequencing assay called HIV-PULSE (HIV Proviral UMI-mediated Long-read Sequencing). This assay uses unique molecular identifiers (UMIs) to tag individual HIV-1 genomes, allowing for the omission of the limiting dilution step and enabling long-range PCR amplification of many NFL genomes in a single PCR reaction, while simultaneously overcoming poor single-read accuracy. We optimized the assay using HIV-infected cell lines and then applied it to blood samples from 18 individuals living with HIV on antiretroviral therapy, yielding a total of 1308 distinct HIV-1 genomes. Benchmarking against the widely applied Full-Length Individual Proviral Sequencing assay revealed similar sensitivity (11 vs 18%) and overall good concordance, although at a significantly higher throughput. In conclusion, HIV-PULSE is a cost-efficient and scalable assay that allows for the characterization of the HIV-1 proviral landscape, making it an attractive method to study the HIV-1 reservoir composition and dynamics

HIV-PULSE: A long-read sequencing assay for high-throughput near full-length HIV-1 proviral genome characterization

A deep understanding of the composition of the HIV-1 reservoir is necessary for the development of targeted therapies and the evaluation of curative efforts. However, current near full-length (NFL) HIV-1 proviral genome sequencing assays are based on labor-intensive and costly principles of repeated PCRs at limiting dilution, restricting their scalability. To address this, we developed a high-throughput, long-read sequencing assay called HIV-PULSE (HIV Proviral UMI-mediated Long-read Sequencing). This assay uses unique molecular identifiers (UMIs) to tag individual HIV-1 genomes, allowing for the omission of the limiting dilution step and enabling long-range PCR amplification of many NFL genomes in a single PCR reaction, while simultaneously overcoming poor single-read accuracy. We optimized the assay using HIV-infected cell lines and then applied it to blood samples from 18 individuals living with HIV on antiretroviral therapy, yielding a total of 1,308 distinct HIV-1 genomes. Benchmarking against the widely applied Full-Length Individual Proviral Sequencing assay revealed similar sensitivity (11% vs 18%) and overall good concordance, though at a significantly higher throughput. In conclusion, HIV-PULSE is a cost-efficient and scalable assay that allows for the characterization of the HIV-1 proviral landscape, making it an attractive method to study the HIV-1 reservoir composition and dynamics.</jats:p