Mapping the world's free-flowing rivers.

Free-flowing rivers (FFRs) support diverse, complex and dynamic ecosystems globally, providing important societal and economic services. Infrastructure development threatens the ecosystem processes, biodiversity and services that these rivers support. Here we assess the connectivity status of 12 million kilometres of rivers globally and identify those that remain free-flowing in their entire length. Only 37 per cent of rivers longer than 1,000 kilometres remain free-flowing over their entire length and 23 per cent flow uninterrupted to the ocean. Very long FFRs are largely restricted to remote regions of the Arctic and of the Amazon and Congo basins. In densely populated areas only few very long rivers remain free-flowing, such as the Irrawaddy and Salween. Dams and reservoirs and their up- and downstream propagation of fragmentation and flow regulation are the leading contributors to the loss of river connectivity. By applying a new method to quantify riverine connectivity and map FFRs, we provide a foundation for concerted global and national strategies to maintain or restore them.

Author Correction: Mapping the world's free-flowing rivers.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Determination of the Lung Clearance Index (LCI) in a Paediatric Cystic Fibrosis Cohort.

The pathogenesis of CF lung disease may start in infancy. Therefore, it is important to monitor the early stages of its progress. The Exhalyzer D is the first commercially available device designed to measure lung ventilation inhomogeneity at any age. This study was conducted to assess the performance and feasibility of using the Exhalyzer D in a paediatric CF clinic. A total of 91 subjects were recruited (23 controls, and 68 patients with CF). The majority of CF patients (79%) and controls (78%) completed at least two successful washouts. A strong linear correlation was noted between LCI and FEV1. Children with CF under six years of age struggled to perform the washout in a technically correct manner. A clear learning effect was observed, with improved technique and shorter testing times on repeated visits.

Gene expression in heart, kidney, and liver identifies possible mechanisms underpinning fetal resistance and susceptibility to in utero PRRSV infection.

Porcine reproductive and respiratory syndrome (PRRS) is one of the costliest diseases to pork producers worldwide. We tested samples from the pregnant gilt model (PGM) to better understand the fetal response to in-utero PRRS virus (PRRSV) infection. Our goal was to identify critical tissues and genes associated with fetal resilience or susceptibility. Pregnant gilts (N=22) were infected with PRRSV on day 86 of gestation. At 21 days post maternal infection, the gilts and fetuses were euthanized, and fetal tissues collected. Fetuses were characterized for PRRS viral load in fetal serum and thymus, and preservation status (viable or meconium stained: VIA or MEC). Fetuses (N=10 per group) were compared: uninfected (UNIF; <1 log/µL PRRSV RNA), resilient (HV_VIA, >5 log virus/µL but viable), and susceptible (HV_MEC, >5 log virus/µL with MEC). Gene expression in fetal heart, kidney, and liver was investigated using NanoString transcriptomics. Gene categories investigated were hypothesized to be involved in fetal response to PRRSV infection: renin- angiotensin-aldosterone, inflammatory, transporter and metabolic systems. Following PRRSV infection, CCL5 increased expression in heart and kidney, and ACE2 decreased expression in kidney, each associated with fetal PRRS susceptibility. Liver revealed the most significant differential gene expression: CXCL10 decreased and IL10 increased indicative of immune suppression. Increased liver gene expression indicated potential associations with fetal PRRS susceptibility on several systems including blood pressure regulation (AGTR1), energy metabolism (SLC16A1 and SLC16A7), tissue specific responses (KL) and growth modulation (TGFB1). Overall, analyses of non-lymphoid tissues provided clues to mechanisms of fetal compromise following maternal PRRSV infection.

Optimized quantification of intra-host viral diversity in SARS-CoV-2 and influenza virus sequence data.

High error rates of viral RNA-dependent RNA polymerases lead to diverse intra-host viral populations during infection. Errors made during replication that are not strongly deleterious to the virus can lead to the generation of minority variants. However, accurate detection of minority variants in viral sequence data is complicated by errors introduced during sample preparation and data analysis. We used synthetic RNA controls and simulated data to test seven variant-calling tools across a range of allele frequencies and simulated coverages. We show that choice of variant caller and use of replicate sequencing have the most significant impact on single-nucleotide variant (SNV) discovery and demonstrate how both allele frequency and coverage thresholds impact both false discovery and false-negative rates. When replicates are not available, using a combination of multiple callers with more stringent cutoffs is recommended. We use these parameters to find minority variants in sequencing data from SARS-CoV-2 clinical specimens and provide guidance for studies of intra-host viral diversity using either single replicate data or data from technical replicates. Our study provides a framework for rigorous assessment of technical factors that impact SNV identification in viral samples and establishes heuristics that will inform and improve future studies of intra-host variation, viral diversity, and viral evolution. IMPORTANCE When viruses replicate inside a host cell, the virus replication machinery makes mistakes. Over time, these mistakes create mutations that result in a diverse population of viruses inside the host. Mutations that are neither lethal to the virus nor strongly beneficial can lead to minority variants that are minor members of the virus population. However, preparing samples for sequencing can also introduce errors that resemble minority variants, resulting in the inclusion of false-positive data if not filtered correctly. In this study, we aimed to determine the best methods for identification and quantification of these minority variants by testing the performance of seven commonly used variant-calling tools. We used simulated and synthetic data to test their performance against a true set of variants and then used these studies to inform variant identification in data from SARS-CoV-2 clinical specimens. Together, analyses of our data provide extensive guidance for future studies of viral diversity and evolution.

Development of a novel ArcCHECK(™) insert for routine quality assurance of VMAT delivery including dose calculation with inhomogeneities.

PURPOSE: To design a versatile, nonhomogeneous insert for the dose verification phantom ArcCHECK(™) (Sun Nuclear Corp., FL) and to demonstrate its usefulness for the verification of dose distributions in inhomogeneous media. As an example, we demonstrate it can be used clinically for routine quality assurance of two volumetric modulated arc therapy (VMAT) systems for lung stereotactic body radiation therapy (SBRT): SmartArc(®) (Pinnacle(3), Philips Radiation Oncology Systems, Fitchburg, WI) and RapidArc(®) (Eclipse(™), Varian Medical Systems, Palo Alto, CA). METHODS: The cylindrical detector array ArcCHECK(™) has a retractable homogeneous acrylic insert. In this work, we designed and manufactured a customized heterogeneous insert with densities that simulate soft tissue, lung, bone, and air. The insert offers several possible heterogeneity configurations and multiple locations for point dose measurements. SmartArc(®) and RapidArc(®) plans for lung SBRT were generated and copied to ArcCHECK(™) for each inhomogeneity configuration. Dose delivery was done on a Varian 2100 ix linac. The evaluation of dose distributions was based on gamma analysis of the diode measurements and point doses measurements at different positions near the inhomogeneities. RESULTS: The insert was successfully manufactured and tested with different measurements of VMAT plans. Dose distributions measured with the homogeneous insert showed gamma passing rates similar to our clinical results (∼99%) for both treatment-planning systems. Using nonhomogeneous inserts decreased the passing rates by up to 3.6% in the examples studied. Overall, SmartArc(®) plans showed better gamma passing rates for nonhomogeneous measurements. The discrepancy between calculated and measured point doses was increased up to 6.5% for the nonhomogeneous insert depending on the inhomogeneity configuration and measurement location. SmartArc(®) and RapidArc(®) plans had similar plan quality but RapidArc(®) plans had significantly higher monitor units (up to 70%). CONCLUSIONS: A versatile, nonhomogeneous insert was developed for ArcCHECK(™) for an easy and quick evaluation of dose calculations with nonhomogeneous media and for comparison of different treatment planning systems. The device was tested for SmartArc(®) and RapidArc(®) plans for lung SBRT, showing the uncertainties of dose calculations with inhomogeneities. The new insert combines the convenience of the ArcCHECK(™) and the possibility of assessing dose distributions in inhomogeneous media.

Optimized Quantification of Intrahost Viral Diversity in SARS-CoV-2 and Influenza Virus Sequence Data.

High error rates of viral RNA-dependent RNA polymerases lead to diverse intra-host viral populations during infection. Errors made during replication that are not strongly deleterious to the virus can lead to the generation of minority variants. However, accurate detection of minority variants in viral sequence data is complicated by errors introduced during sample preparation and data analysis. We used synthetic RNA controls and simulated data to test seven variant calling tools across a range of allele frequencies and simulated coverages. We show that choice of variant caller, and use of replicate sequencing have the most significant impact on single nucleotide variant (SNV) discovery and demonstrate how both allele frequency and coverage thresholds impact both false discovery and false negative rates. We use these parameters to find minority variants in sequencing data from SARS-CoV-2 clinical specimens and provide guidance for studies of intrahost viral diversity using either single replicate data or data from technical replicates. Our study provides a framework for rigorous assessment of technical factors that impact SNV identification in viral samples and establishes heuristics that will inform and improve future studies of intrahost variation, viral diversity, and viral evolution. IMPORTANCE: When viruses replicate inside a host, the virus replication machinery makes mistakes. Over time, these mistakes create mutations that result in a diverse population of viruses inside the host. Mutations that are neither lethal to the virus, nor strongly beneficial, can lead to minority variants that are minor members of the virus population. However, preparing samples for sequencing can also introduce errors that resemble minority variants, resulting in inclusion of false positive data if not filtered correctly. In this study, we aimed to determine the best methods for identification and quantification of these minority variants by testing the performance of seven commonly used variant calling tools. We used simulated and synthetic data to test their performance against a true set of variants, and then used these studies to inform variant identification in data from clinical SARS-CoV-2 clinical specimens. Together, analyses of our data provide extensive guidance for future studies of viral diversity and evolution.

Localization of major histocompatibility complex class I and II mRNA in human first-trimester chorionic villi by in situ hybridization.

Maternal immune recognition of pregnancy occurs despite the nonexpression of classical major histocompatibility complex (MHC) antigenic determinants by chorionic villous trophoblast, which comprise the major surface area where maternal blood contacts fetal-derived cells. cDNA-mRNA in situ hybridization was used to probe expression of transcripts corresponding to nonpolymorphic MHC determinants in first-trimester chorionic villus samples. The HLA-B7 probe hybridization signals were localized to syncytiotrophoblast and to cells of the mesenchyme but not to villous cytotrophoblast. HLA-G mRNA was found only in syncytiotrophoblast. A DR beta clone hybridized to both villous cytotrophoblast and syncytiotrophoblast. The results suggest that expression of trophoblast class I and class II determinants early in gestation (10 wk) may be regulated by posttranscriptional events. This also suggests the potential for maternal antifetal alloimmune responses.

Protective effects of sialylated oligosaccharides in immune complex-induced acute lung injury.

Using sialyl Lewisx (SLX) oligosaccharides derived from fucosyl transferase-expressing cells or generated synthetically, the ability of these compounds to protect against acute lung damage after deposition of immunoglobulin (Ig)G or IgA immune complexes has been determined. The synthetic compounds were tetra- and pentasaccharide derivates of SLX as well as the nonfucosylated forms of SLX as controls. In the IgG immune complex model of lung injury, which is E-selectin dependent, SLX preparations provided dose-dependent protective effects, as assessed by changes in lung vascular permeability and hemorrhage. Protective effects were associated with diminished tissue accumulation of neutrophils in lungs (as assessed by myeloperoxidase). Morphological assessment revealed reduced physical contact of neutrophils with the pulmonary vascular endothelium and reduced tissue accumulation of neutrophils. In the model of IgA immune complex-induced lung injury, which does not involve participation of neutrophils and is independent of the requirement for E-selectin, SLX preparations were not protective. These data suggest that, in neutrophil-mediated and E-selectin-dependent lung injury, SLX preparations provide significant, protective effects against inflammatory vascular injury. The ability to achieve antiinflammatory outcomes in vivo with appropriate oligosaccharides suggests a new approach to the blocking of acute inflammatory responses.

Immunomodulatory effects of mixed hematopoietic chimerism: immune tolerance in canine model of lung transplantation.

Long-term survival after lung transplantation is limited by acute and chronic graft rejection. Induction of immune tolerance by first establishing mixed hematopoietic chimerism (MC) is a promising strategy to improve outcomes. In a preclinical canine model, stable MC was established in recipients after reduced-intensity conditioning and hematopoietic cell transplantation from a DLA-identical donor. Delayed lung transplantation was performed from the stem cell donor without pharmacological immunosuppression. Lung graft survival without loss of function was prolonged in chimeric (n = 5) vs. nonchimeric (n = 7) recipients (p < or = 0.05, Fisher's test). There were histological changes consistent with low-grade rejection in 3/5 of the lung grafts in chimeric recipients at > or =1 year. Chimeric recipients after lung transplantation had a normal immune response to a T-dependent antigen. Compared to normal dogs, there were significant increases of CD4+INFgamma+, CD4+IL-4+ and CD8+ INFgamma+ T-cell subsets in the blood (p < 0.0001 for each of the three T-cell subsets). Markers for regulatory T-cell subsets including foxP3, IL10 and TGFbeta were also increased in CD3+ T cells from the blood and peripheral tissues of chimeric recipients after lung transplantation. Establishing MC is immunomodulatory and observed changes were consistent with activation of both the effector and regulatory immune response.

Protection against vaginal SIV transmission with microencapsulated vaccine.

Although protection in animal models against intravenous challenges with simian immunodeficiency virus (SIV) has been reported, no previous vaccines have protected against a heterosexual route of infection. In this study, five of six macaques were protected against vaginal challenge when immunized with formalin-treated SIV in biodegradable microspheres by the intramuscular plus oral or plus intratracheal route. Oral immunization alone did not protect. After a second vaginal challenge, three of four intramuscularly primed and mucosally boosted macaques remained protected. The data suggest that protection against human immunodeficiency virus vaginal transmission could be provided by microsphere-based booster vaccines when used to immunize women who are systemically primed.

Evaluating the impacts of protected areas on human well-being across the developing world.

Protected areas (PAs) are fundamental for biodiversity conservation, yet their impacts on nearby residents are contested. We synthesized environmental and socioeconomic conditions of >87,000 children in >60,000 households situated either near or far from >600 PAs within 34 developing countries. We used quasi-experimental hierarchical regression to isolate the impact of living near a PA on several aspects of human well-being. Households near PAs with tourism also had higher wealth levels (by 17%) and a lower likelihood of poverty (by 16%) than similar households living far from PAs. Children under 5 years old living near multiple-use PAs with tourism also had higher height-for-age scores (by 10%) and were less likely to be stunted (by 13%) than similar children living far from PAs. For the largest and most comprehensive socioeconomic-environmental dataset yet assembled, we found no evidence of negative PA impacts and consistent statistical evidence to suggest PAs can positively affect human well-being.

Heart and lung transplantation in the United States, 1997-2006.

This article highlights trends in heart and lung transplantation between 1997 and 2006, drawing on data from the OPTN and SRTR. The total number of candidates actively awaiting heart transplantation declined by 45% over the last decade, dropping from 2414 patients in 1997 to 1327 patients in 2006. The overall death rates among patients awaiting heart transplantation declined over the same period. The distribution of recipients among the different status groups at the time of heart transplantation changed little between the inception of the new classification system in 1999 and 2005. Deaths in the first year after heart transplantation have steadily decreased. At the end of 2006, 2885 candidates were awaiting a lung transplant, up 10% from the 1997 count. The median time-to-transplant for listed patients decreased by 87% over the decade, dropping from 1053 days in 1997 to 132 days in 2006. Selection for listing and transplantation has shifted toward more urgent patients since the May 2005 implementation of a new lung allocation system based on survival benefit and urgency rather than waiting time. Only 31 heart-lung transplants were performed in 2006, down from a high of 62 in 1997.

Post-genomic behavioral genetics: From revolution to routine.

What was once expensive and revolutionary-full-genome sequence-is now affordable and routine. Costs will continue to drop, opening up new frontiers in behavioral genetics. This shift in costs from the genome to the phenome is most notable in large clinical studies of behavior and associated diseases in cohorts that exceed hundreds of thousands of subjects. Examples include the Women's Health Initiative (www.whi.org), the Million Veterans Program (www. RESEARCH: va.gov/MVP), the 100 000 Genomes Project (genomicsengland.co.uk) and commercial efforts such as those by deCode (www.decode.com) and 23andme (www.23andme.com). The same transition is happening in experimental neuro- and behavioral genetics, and sample sizes of many hundreds of cases are becoming routine (www.genenetwork.org, www.mousephenotyping.org). There are two major consequences of this new affordability of massive omics datasets: (1) it is now far more practical to explore genetic modulation of behavioral differences and the key role of gene-by-environment interactions. Researchers are already doing the hard part-the quantitative analysis of behavior. Adding the omics component can provide powerful links to molecules, cells, circuits and even better treatment. (2) There is an acute need to highlight and train behavioral scientists in how best to exploit new omics approaches. This review addresses this second issue and highlights several new trends and opportunities that will be of interest to experts in animal and human behaviors.

Small extracellular particles with big potential for horizontal gene transfer: membrane vesicles and gene transfer agents.

Bacteria are known to release different types of particles that serve various purposes such as the processing of metabolites, communication, and the transfer of genetic material. One of the most interesting aspects of the production of such particles is the biogenesis and trafficking of complex particles that can carry DNA, RNA, proteins or toxins into the surrounding environment to aid in bacterial survival or lead to gene transfer. Two important bacterial extracellular complexes are membrane vesicles and gene transfer agents. In this review, we will discuss the production, contents and functions of these two types of particles as related to their abilities to facilitate horizontal gene transfer.

Neutrophil-dependent acute lung injury. Requirement for P-selectin (GMP-140).

Rapid translocation of P-selectin (GMP-140) from cytoplasmic granules to the cell membrane of endothelial cells promotes adhesive interactions with neutrophils which, when activated, damage the endothelium. The role of P-selectin in lung vascular endothelial injury in rats after systemic activation of complement by intravenous infusion of cobra venom factor has been assessed. Within 5-10 min after cobra venom factor infusion, the pulmonary vasculature demonstrated immunohistochemical expression of an epitope that reacts with anti-human P-selectin. Monoclonal antibody to human P-selectin blocked in vitro adherence of rat or human platelets (activated with thrombin) to neutrophils and was demonstrated to react with thrombin-activated rat platelets. The antibody did not react with rat neutrophils. In vivo, the antibody had strongly protective effects against cobra venom factor-induced pulmonary vascular injury as determined by permeability changes and hemorrhage. In parallel, lung myeloperoxidase content was greatly reduced and, by transmission electron microscopy, there was markedly diminished adherence of neutrophils to the pulmonary vascular endothelium and much diminished injury of endothelial cells, as defined by hemorrhage. These data indicate that anti-human P-selectin reacts with a pulmonary vascular antigen in rats and that this antigen is essential for the full expression of lung injury.

Requirements for leukocyte adhesion molecules in nephrotoxic nephritis.

Requirements for leukocyte adhesion molecules as well as cytokines have been determined in the rat model of acute nephrotoxic nephritis. Proteinuria (at 24 h) and neutrophil accumulation in renal glomeruli (at 6 h) have been used as the endpoints. For full accumulation in glomeruli of neutrophils as well as full development of proteinuria, requirements have been demonstrated for TNF alpha, (but not IL-1), CD11b (but not CD11a), very late arising-4 (CD49d/CD29), and intercellular adhesion molecule-1 but not endothelial leukocyte adhesion molecule-1 (E-selectin). By immunohistochemical approaches, infusion of antibody to glomerular basement membrane induced glomerular upregulation of intercellular adhesion molecule-1, endothelial leukocyte adhesion molecule-1, and vascular adhesion molecule-1. Treatment of rats with anti-TNF alpha or soluble recombinant human TNF receptor-1 blocked this expression. Renal arterial infusion of TNF alpha induced glomerular expression of all three endothelial adhesion molecules, but infusion of IL-1 beta did not. These data suggest that, in neutrophil and complement-dependent anti-glomerular basement membrane-induced acute nephritis in rats, there are selective requirements for cytokines, beta 1 and beta 2 integrins, and endothelial adhesion molecules. These requirements contrast with those found in other vascular beds in which complement and neutrophil-induced vascular injury has been induced by deposition of immune complexes.

Role of endothelial-leukocyte adhesion molecule 1 (ELAM-1) in neutrophil-mediated lung injury in rats.

Two murine monoclonal antibodies (CL-3 and CL-37, both F(ab')2) to human endothelial-leukocyte adhesion molecule-1 (ELAM-1) were found to react immunohistochemically with rat pulmonary artery endothelial cells that had been pretreated with tumor necrosis factor (TNF alpha). CL-3, but not CL-37, blocked in vitro adherence of neutrophils to TNF alpha-treated endothelial cells and the killing of TNF alpha-treated rat endothelial cells by phorbol ester activated neutrophils. In rats treated systemically with CL-3, there was a 70% reduction in accumulation of neutrophils in glycogen-induced peritoneal exudates. Treatment of animals with CL-37 anti-ELAM-1 did not reduce neutrophil accumulation under the same conditions. When IgG immune complex deposition was induced in dermis and in lungs of rats, treatment with CL-3 anti-ELAM-1 markedly reduced vascular injury as measured by changes in vascular permeability (leakage of 125I-albumin) and hemorrhage (extravasation of 51Cr-red blood cells). The protective effects of CL-3 anti-ELAM-1 were related to greatly diminished recruitment of neutrophils (as assessed morphologically, by tissue extraction of myeloperoxidase, and by retrieval, via bronchoalveolar lavage, of neutrophils from lung). CL-37 had no protective effects in vivo after deposition of immune complexes in lung. Using either CL-3 or CL-37 anti-ELAM-1, immunohistochemical analysis of lungs undergoing IgG immune complex-induced injury revealed a striking upregulation of ELAM-1 in the lung vasculature (venules and interstitial capillaries), with a peak intensity developing between 3 and 4 h after deposition of immune complexes in lung. Vascular beds of spleen, liver, and kidney failed to show upregulation of ELAM-1 under these same conditions. The immunohistochemical reactivity of rat lung was abolished if the anti-ELAM-1 preparation was first absorbed with monolayers of human umbilical vein endothelial cells that had been pretreated with TNF alpha. Untreated human endothelial cells failed to cause loss of lung reactivity of the anti-ELAM-1 preparation. These data indicate that ELAM-1 is upregulated in the pulmonary vasculature of rats during deposition of immune complexes and that ELAM-1 appears to play an obligate role in the recruitment of neutrophils.

Requirement and role of C5a in acute lung inflammatory injury in rats.

The complement activation product, C5a, may play a key role in the acute inflammatory response. Polyclonal antibody to rat C5a was used to define the requirements for C5a in neutrophil-dependent inflammatory lung injury after systemic activation of complement by cobra venom factor (CVF) or after intrapulmonary deposition of IgG immune complexes. In the CVF model, intravenous infusion (but not intratracheal instillation) of anti-C5a produced a dose-dependent reduction in lung permeability and in lung content of myeloperoxidase. In C6-deficient rats, CVF infusion caused the same level of lung injury (measured by leak of 125I-albumin) as found in C6-sufficient rats. In the IgG immune complex model of lung injury, anti-C5a administered intratracheally (but not intravenously) reduced in a dose-dependent manner both the increase in lung vascular permeability as well as the buildup of lung myeloperoxidase. Treatment with anti-C5a greatly suppressed upregulation of lung vascular intercellular adhesion molecule-1 (ICAM-1). This was correlated with a substantial drop in levels of TNFalpha in bronchoalveolar fluids. These data demonstrate the requirement for C5a in the two models of injury. In the IgG immune complex model, C5a is required for the full production of TNFalpha and the corresponding upregulation of lung vascular ICAM-1.