Genome deletions to overcome the directed loss of gene function in Leishmania

With the global reach of the Neglected Tropical Disease leishmanaisis increasing, coupled with a tiny armoury of therapeutics which all have problems with resistance, cost, toxicity and/or administration, the validation of new drug targets in the causative insect vector borne protozoa Leishmania spp is more important than ever. Before the introduction of CRISPR Cas9 technology in 2015 genetic validation of new targets was carried out largely by targeted gene knockout through homologous recombination, with the majority of genes targeted (~70%) deemed non-essential. In this study we exploit the ready availability of whole genome sequencing technology to reanalyse one of these historic cell lines, a L. major knockout in the catalytic subunit of serine palmitoyltransferase (LCB2), which causes a complete loss of sphingolipid biosynthesis but remains viable and infective. This revealed a number of Single Nucleotide Polymorphisms, but also the complete loss of several coding regions including a gene encoding a putative ABC3A orthologue, a putative sterol transporter. Hypothesising that the loss of such a transporter may have facilitated the directed knockout of the catalytic subunit of LCB2 and the complete loss of de novo sphingolipid biosynthesis, we re-examined LCB2 in a L. mexicana line engineered for straightforward CRISPR Cas9 directed manipulation. Strikingly, LCB2 could not be knocked out indicating essentiality. However, simultaneous deletion of LCB2 and the putative ABC3A was possible. This indicated that the loss of the putative ABC3A facilitated the loss of sphingolipid biosynthesis in Leishmania, and suggested that we should re-examine the many other Leishmania knockout lines where genes were deemed non-essential

Stabilin-1 is required for the endothelial clearance of small anionic nanoparticles

Clearance of nanoparticles (NPs) after intravenous injection - mainly by the liver - is a critical barrier for the clinical translation of nanomaterials. Physicochemical properties of NPs are known to influence their distribution through cell-specific interactions; however, the molecular mechanisms responsible for liver cellular NP uptake are poorly understood. Liver sinusoidal endothelial cells and Kupffer cells are critical participants in this clearance process. Here we use a zebrafish model for liver-NP interaction to identify the endothelial scavenger receptor Stabilin-1 as a non-redundant receptor for the clearance of small anionic NPs. Furthermore, we show that physiologically, Stabilin-1 is required for the removal of bacterial lipopolysaccharide (LPS/endotoxin) from circulation and that Stabilin-1 cooperates with its homolog Stabilin-2 in the clearance of larger (~100 nm) anionic NPs. Our findings allow optimization of anionic nanomedicine biodistribution and targeting therapies that use Stabilin-1 and -2 for liver endothelium-specific delivery.Drug Delivery Technolog

An integrative identification guide to the Hydrozoa (Cnidaria) of Bocas del Toro, Panama

This work is the first attempt to assess the biodiversity of the Hydrozoa in the Archipiélago de Bocas del Toro (Panamá, Caribbean Sea) using morphology and molecular taxonomy, and to produce field identification tools to help future identification and monitoring efforts in the area. We sampled, identified, vouchered, and barcoded 112 specimens of Hydrozoa from shallow coastal waters (0–22 m depth) in the Archipiélago de Bocas del Toro. The specimens belong to 70 taxa, of which 53 were identified at the species level, and 17 were identified at the genus or family level. We produced 64 sequences of the large ribosomal subunit of the mitochondrial RNA (mt lsu-rRNA, 16S), the genetic marker generally used for barcoding Hydrozoa. We updated the local checklist that now comprises 118 species, and produced 87 detailed taxon identification tables that display species descriptions augmented with pictures, geographic distribution (worldwide and in Bocas del Toro), GenBank accession numbers for the 16S mitochondrial gene, and a synopsis of the families they belong to

Amphotericin B resistance in Leishmania mexicana: Alterations to sterol metabolism and oxidative stress response

Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. Sterol profiling revealed that, in each resistant line, the predominant wild-type sterol, ergosta-5,7,24-trienol, was replaced by other sterol intermediates. Broadly, two different profiles emerged among the resistant lines. Whole genome sequencing then showed that these distinct profiles were due either to mutations in the sterol methyl transferase (C24SMT) gene locus or the sterol C5 desaturase (C5DS) gene. In three lines an additional deletion of the miltefosine transporter gene was found. Differences in sensitivity to amphotericin B were apparent, depending on whether cells were grown in HOMEM, supplemented with foetal bovine serum, or a serum free defined medium (DM). Metabolomic analysis after exposure to AmB showed that a large increase in glucose flux via the pentose phosphate pathway preceded cell death in cells sustained in HOMEM but not DM, indicating the oxidative stress was more significantly induced under HOMEM conditions. Several of the lines were tested for their ability to infect macrophages and replicate as amastigote forms, alongside their ability to establish infections in mice. While several AmB resistant lines showed reduced virulence, at least two lines displayed heightened virulence in mice whilst retaining their resistance phenotype, emphasising the risks of resistance emerging to this critical drug

Policy design for the Anthropocene

This is the author accepted manuscript. The final version is available from Nature Research via the DOI in this recordToday, more than ever, ‘Spaceship Earth’ is an apt metaphor as we chart the boundaries for a safe planet1. Social scientists both analyse why society courts disaster by approaching or even overstepping these boundaries and try to design suitable policies to avoid these perils. Because the threats of transgressing planetary boundaries are global, long-run, uncertain and interconnected, they must be analysed together to avoid conflicts and take advantage of synergies. To obtain policies that are effective at both international and local levels requires careful analysis of the underlying mechanisms across scientific disciplines and approaches, and must take politics into account. In this Perspective, we examine the complexities of designing policies that can keep Earth within the biophysical limits favourable to human life.Stockholm Resilience CentreBECC - Biodiversity and Ecosystem services in a Changing ClimateMistra Carbon Exi

Assessing the impact of shipping emissions on air pollution in the Canadian Arctic and northern regions: current and future modelled scenarios

A first regional assessment of the impact of shipping emissions on air pollution in the Canadian Arctic and northern regions was conducted in this study. Model simulations were carried out on a limited-area domain (at 15&thinsp;km horizontal resolution) centred over the Canadian Arctic, using the Environment and Climate Change Canada's on-line air quality forecast model, GEM-MACH (Global Environmental Multi-scale – Modelling Air quality and CHemistry), to investigate the contribution from the marine shipping emissions over the Canadian Arctic waters (at both present and projected future levels) to ambient concentrations of criteria pollutants (O3, PM2.5, NO2, and SO2), atmospheric deposition of sulfur (S) and nitrogen (N), and atmospheric loading and deposition of black carbon (BC) in the Arctic. Several model upgrades were introduced for this study, including the treatment of sea ice in the dry deposition parameterization, chemical lateral boundary conditions, and the inclusion of North American wildfire emissions. The model is shown to have similar skills in predicting ambient O3 and PM2.5 concentrations in the Canadian Arctic and northern regions, as the current operational air quality forecast models in North America and Europe. In particular, the model is able to simulate the observed O3 and PM components well at the Canadian high Arctic site, Alert. The model assessment shows that, at the current (2010) level, Arctic shipping emissions contribute to less than 1&thinsp;% of ambient O3 concentration over the eastern Canadian Arctic and between 1 and 5&thinsp;% of ambient PM2.5 concentration over the shipping channels. Arctic shipping emissions make a much greater contributions to the ambient NO2 and SO2 concentrations, at 10&thinsp;%–50&thinsp;% and 20&thinsp;%–100&thinsp;%, respectively. At the projected 2030 business-as-usual (BAU) level, the impact of Arctic shipping emissions is predicted to increase to up to 5&thinsp;% in ambient O3 concentration over a broad region of the Canadian Arctic and to 5&thinsp;%–20&thinsp;% in ambient PM2.5 concentration over the shipping channels. In contrast, if emission controls such as the ones implemented in the current North American Emission Control Area (NA ECA) are to be put in place over the Canadian Arctic waters, the impact of shipping to ambient criteria pollutants would be significantly reduced. For example, with NA-ECA-like controls, the shipping contributions to the population-weighted concentrations of SO2 and PM2.5 would be brought down to below the current level. The contribution of Canadian Arctic shipping to the atmospheric deposition of sulfur and nitrogen is small at the current level, &lt;&thinsp;5&thinsp;%, but is expected to increase to up to 20&thinsp;% for sulfur and 50&thinsp;% for nitrogen under the 2030 BAU scenario. At the current level, Canadian Arctic shipping also makes only small contributions to BC column loading and BC deposition, with &lt;&thinsp;0.1&thinsp;% on average and up to 2&thinsp;% locally over the eastern Canadian Arctic for the former, and between 0.1&thinsp;% and 0.5&thinsp;% over the shipping channels for the latter. The impacts are again predicted to increase at the projected 2030 BAU level, particularly over the Baffin Island and Baffin Bay area in response to the projected increase in ship traffic there, e.g., up to 15&thinsp;% on BC column loading and locally exceeding 30&thinsp;% on BC deposition. Overall, the study indicates that shipping-induced changes in atmospheric composition and deposition are at regional to local scales (particularly in the Arctic). Climate feedbacks are thus likely to act at these scales, so climate impact assessments will require modelling undertaken at much finer resolutions than those used in the existing radiative forcing and climate impact assessments.</p