Photosynthesis in the surface ocean produces approximately 100 gigatonnes of organic carbon per year, of which 5 to 15 per cent is exported to the deep ocean. The rate at which the sinking carbon is ...converted into carbon dioxide by heterotrophic organisms at depth is important in controlling oceanic carbon storage. It remains uncertain, however, to what extent surface ocean carbon supply meets the demand of water-column biota; the discrepancy between known carbon sources and sinks is as much as two orders of magnitude. Here we present field measurements, respiration rate estimates and a steady-state model that allow us to balance carbon sources and sinks to within observational uncertainties at the Porcupine Abyssal Plain site in the eastern North Atlantic Ocean. We find that prokaryotes are responsible for 70 to 92 per cent of the estimated remineralization in the twilight zone (depths of 50 to 1,000 metres) despite the fact that much of the organic carbon is exported in the form of large, fast-sinking particles accessible to larger zooplankton. We suggest that this occurs because zooplankton fragment and ingest half of the fast-sinking particles, of which more than 30 per cent may be released as suspended and slowly sinking matter, stimulating the deep-ocean microbial loop. The synergy between microbes and zooplankton in the twilight zone is important to our understanding of the processes controlling the oceanic carbon sink.
Copper is controlled by a sophisticated network of transport and storage proteins within mammalian cells, yet its uptake and efflux occur with rapid kinetics. Present as Cu(I) within the reducing ...intracellular environment, the nature of this labile copper pool remains elusive. While glutathione is involved in copper homeostasis and has been assumed to buffer intracellular copper, we demonstrate with a ratiometric fluorescent indicator, crisp-17, that cytosolic Cu(I) levels are buffered to the vicinity of 1 aM, where negligible complexation by glutathione is expected. Enabled by our phosphine sulfide-stabilized phosphine (PSP) ligand design strategy, crisp-17 offers a Cu(I) dissociation constant of 8 aM, thus exceeding the binding affinities of previous synthetic Cu(I) probes by four to six orders of magnitude. Two-photon excitation microscopy with crisp-17 revealed rapid, reversible increases in intracellular Cu(I) availability upon addition of the ionophoric complex CuGTSM or the thiol-selective oxidant 2,2′-dithiodipyridine (DTDP). While the latter effect was dramatically enhanced in 3T3 cells grown in the presence of supplemental copper and in cultured Menkes mutant fibroblasts exhibiting impaired copper efflux, basal Cu(I) availability in these cells showed little difference from controls, despite large increases in total copper content. Intracellular copper is thus tightly buffered by endogenous thiol ligands with significantly higher affinity than glutathione. The dual utility of crisp-17 to detect normal intracellular buffered Cu(I) levels as well as to probe the depth of the labile copper pool in conjunction with DTDP provides a promising strategy to characterize perturbations of cellular copper homeostasis.
Maintaining the chemical identity of DNA depends on ribonucleotide exclusion by DNA polymerases. However, ribonucleotide exclusion during DNA synthesis in vitro is imperfect. To determine whether ...ribonucleotides are incorporated during DNA replication in vivo, we substituted leucine or glycine for an active-site methionine in yeast DNA polymerase ϵ (Pol ϵ). Ribonucleotide incorporation in vitro was three-fold lower for M644L and 11-fold higher for M644G Pol ϵ compared to wild-type Pol ϵ. This hierarchy was recapitulated in vivo in yeast strains lacking RNase H2. Moreover, the pol2-M644G rnh201Δ strain progressed more slowly through S phase, had elevated dNTP pools and generated 2-5-base-pair deletions in repetitive sequences at a high rate and in a gene orientation-dependent manner. The data indicate that ribonucleotides are incorporated during replication in vivo, that they are removed by RNase H2-dependent repair and that defective repair results in replicative stress and genome instability via DNA strand misalignment.
DNA polymerase δ (Pol δ) and DNA polymerase ɛ (Pol ɛ) are both required for efficient replication of the nuclear genome, yet the division of labor between these enzymes has remained unclear for many ...years. Here we investigate the contribution of Pol δ to replication of the leading and lagging strand templates in Saccharomyces cerevisiae using a mutant Pol δ allele (pol3-L612M) whose error rate is higher for one mismatch (e.g., T•dGTP) than for its complement (A•dCTP). We find that strand-specific mutation rates strongly depend on the orientation of a reporter gene relative to an adjacent replication origin, in a manner implying that >90% of Pol δ replication is performed using the lagging strand template. When combined with recent evidence implicating Pol ɛ in leading strand replication, these data support a model of the replication fork wherein the leading and lagging strand templates are primarily copied by Pol ɛ and Pol δ, respectively.
Summary Background Intradermal MVA85A, a candidate vaccine against tuberculosis, induces high amounts of Ag85A-specific CD4 T cells in adults who have already received the BCG vaccine, but aerosol ...delivery of this vaccine might offer immunological and logistical advantages. We did a phase 1 double-blind trial to compare the safety and immunogenicity of aerosol-administered and intradermally administered MVA85A Methods In this phase 1, double-blind, proof-of-concept trial, 24 eligible BCG-vaccinated healthy UK adults were randomly allocated (1:1) by sequentially numbered, sealed, opaque envelopes into two groups: aerosol MVA85A and intradermal saline placebo or intradermal MVA85A and aerosol saline placebo. Participants, the bronchoscopist, and immunologists were masked to treatment assignment. The primary outcome was safety, assessed by the frequency and severity of vaccine-related local and systemic adverse events. The secondary outcome was immunogenicity assessed with laboratory markers of cell-mediated immunity in blood and bronchoalveolar lavage samples. Safety and immunogenicity were assessed for 24 weeks after vaccination. Immunogenicity to both insert Ag85A and vector modified vaccinia virus Ankara (MVA) was assessed by ex-vivo interferon-γ ELISpot and serum ELISAs. Since all participants were randomised and vaccinated according to protocol, our analyses were per protocol. This trial is registered with ClinicalTrials.gov , number NCT01497769. Findings Both administration routes were well tolerated and immunogenic. Respiratory adverse events were rare and mild. Intradermal MVA85A was associated with expected mild local injection-site reactions. Systemic adverse events did not differ significantly between the two groups. Three participants in each group had no vaccine-related systemic adverse events; fatigue (11/24 46%) and headache (10/24 42%) were the most frequently reported symptoms. Ag85A-specific systemic responses were similar across groups. Ag85A-specific CD4 T cells were detected in bronchoalveolar lavage cells from both groups and responses were higher in the aerosol group than in the intradermal group. MVA-specific cellular responses were detected in both groups, whereas serum antibodies to MVA were only detectable after intradermal administration of the vaccine. Interpretation Further clinical trials assessing the aerosol route of vaccine delivery are merited for tuberculosis and other respiratory pathogens. Funding The Wellcome Trust and Oxford Radcliffe Hospitals Biomedical Research Centre.
Understanding the surface reactivity of CO, which is a key intermediate during electrochemical CO2 reduction, is crucial for the development of catalysts that selectively target desired products for ...the conversion of CO2 to fuels and chemicals. In this study, a custom-designed electrochemical cell is utilized to investigate planar polycrystalline copper as an electrocatalyst for CO reduction under alkaline conditions. Seven major CO reduction products have been observed including various hydrocarbons and oxygenates which are also common CO2 reduction products, strongly indicating that CO is a key reaction intermediate for these further-reduced products. A comparison of CO and CO2 reduction demonstrates that there is a large decrease in the overpotential for C–C coupled products under CO reduction conditions. The effects of CO partial pressure and electrolyte pH are investigated; we conclude that the aforementioned large potential shift is primarily a pH effect. Thus, alkaline conditions can be used to increase the energy efficiency of CO and CO2 reduction to C–C coupled products, when these cathode reactions are coupled to the oxygen evolution reaction at the anode. Further analysis of the reaction products reveals common trends in selectivity that indicate both the production of oxygenates and C–C coupled products are favored at lower overpotentials. These selectivity trends are generalized by comparing the results on planar Cu to current state-of-the-art high-surface-area Cu catalysts, which are able to achieve high oxygenate selectivity by operating at the same geometric current density at lower overpotentials. Combined, these findings outline key principles for designing CO and CO2 electrolyzers that are able to produce valuable C–C coupled products with high energy efficiency.
There is an urgent need for an effective tuberculosis (TB) vaccine. Heterologous prime-boost regimens induce potent cellular immunity. MVA85A is a candidate TB vaccine. This phase I clinical trial ...was designed to evaluate whether alternating aerosol and intradermal vaccination routes would boost cellular immunity to the Mycobacterium tuberculosis antigen 85A (Ag85A).
Between December 2013 and January 2016, 36 bacille Calmette-Guérin-vaccinated, healthy UK adults were randomised equally between 3 groups to receive 2 MVA85A vaccinations 1 month apart using either heterologous (Group 1, aerosol-intradermal; Group 2, intradermal-aerosol) or homologous (Group 3, intradermal-intradermal) immunisation. Bronchoscopy and bronchoalveolar lavage (BAL) were performed 7 days post-vaccination. Adverse events (AEs) and peripheral blood were collected for 6 months post-vaccination. The laboratory and bronchoscopy teams were blinded to treatment allocation. One participant was withdrawn and was replaced. Participants were aged 21-42 years, and 28/37 were female. In a per protocol analysis, aerosol delivery of MVA85A as a priming immunisation was well tolerated and highly immunogenic. Most AEs were mild local injection site reactions following intradermal vaccination. Transient systemic AEs occurred following vaccination by both routes and were most frequently mild. All respiratory AEs following primary aerosol MVA85A (Group 1) were mild. Boosting an intradermal MVA85A prime with an aerosolised MVA85A boost 1 month later (Group 2) resulted in transient moderate/severe respiratory and systemic AEs. There were no serious adverse events and no bronchoscopy-related complications. Only the intradermal-aerosol vaccination regimen (Group 2) resulted in modest, significant boosting of the cell-mediated immune response to Ag85A (p = 0.027; 95% CI: 28 to 630 spot forming cells per 1 × 106 peripheral blood mononuclear cells). All 3 regimens induced systemic cellular immune responses to the modified vaccinia virus Ankara (MVA) vector. Serum antibodies to Ag85A and MVA were only induced after intradermal vaccination. Aerosolised MVA85A induced significantly higher levels of Ag85A lung mucosal CD4+ and CD8+ T cell cytokines compared to intradermal vaccination. Boosting with aerosol-inhaled MVA85A enhanced the intradermal primed responses in Group 2. The magnitude of BAL MVA-specific CD4+ T cell responses was lower than the Ag85A-specific responses. A limitation of the study is that while the intradermal-aerosol regimen induced the most potent cellular Ag85A immune responses, we did not boost the last 3 participants in this group because of the AE profile. Timing of bronchoscopies aimed to capture peak mucosal response; however, peak responses may have occurred outside of this time frame.
To our knowledge, this is the first human randomised clinical trial to explore heterologous prime-boost regimes using aerosol and systemic routes of administration of a virally vectored vaccine. In this trial, the aerosol prime-intradermal boost regime was well tolerated, but intradermal prime-aerosol boost resulted in transient but significant respiratory AEs. Aerosol vaccination induced potent cellular Ag85A-specific mucosal and systemic immune responses. Whilst the implications of inducing potent mucosal and systemic immunity for protection are unclear, these findings are of relevance for the development of aerosolised vaccines for TB and other respiratory and mucosal pathogens.
ClinicalTrials.gov NCT01954563.
Naive CD8+ T cells differentiating into effector T cells increase glucose uptake and shift from quiescent to anabolic metabolism. Although much is known about the metabolism of cultured T cells, how ...T cells use nutrients during immune responses in vivo is less well defined. Here, we combined bioenergetic profiling and 13C-glucose infusion techniques to investigate the metabolism of CD8+ T cells responding to Listeria infection. In contrast to in vitro-activated T cells, which display hallmarks of Warburg metabolism, physiologically activated CD8+ T cells displayed greater rates of oxidative metabolism, higher bioenergetic capacity, differential use of pyruvate, and prominent flow of 13C-glucose carbon to anabolic pathways, including nucleotide and serine biosynthesis. Glucose-dependent serine biosynthesis mediated by the enzyme Phgdh was essential for CD8+ T cell expansion in vivo. Our data highlight fundamental differences in glucose use by pathogen-specific T cells in vivo, illustrating the impact of environment on T cell metabolic phenotypes.
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•Developed 13C-infusion method for studying T cell metabolism in vivo•T cell glucose use and bioenergetics differ between cell culture and mouse models•Glucose metabolism in T cells changes dynamically over an immune response•Glucose-dependent serine biosynthesis supports T cell proliferation in vivo
Although much is known about the metabolism of cultured T cells, how T cells use nutrients during immune responses in vivo is not well defined. Using 13C-glucose isotope tracing in mice, Ma et al. demonstrate prominent use of glucose for anabolic metabolism by T cells in vivo. This work highlights the importance of studying T cell metabolism in a physiological environment.
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