Solid-Phase Extraction of Aquatic Organic Matter: Loading-Dependent Chemical Fractionation and Self-Assembly

Dissolved organic matter (DOM) is an important component in marine and freshwater environments and plays a fundamental role in global biogeochemical cycles. In the past, optical and molecular-level analytical techniques evolved and improved our mechanistic understanding about DOM fluxes. For most molecular chemical techniques, sample desalting and enrichment is a prerequisite. Solid-phase extraction has been widely applied for concentrating and desalting DOM. The major aim of this study was to constrain the influence of sorbent loading on the composition of DOM extracts. Here, we show that increased loading resulted in reduced extraction efficiencies of dissolved organic carbon (DOC), fluorescence and absorbance, and polar organic substances. Loading-dependent optical and chemical fractionation induced by the altered adsorption characteristics of the sorbent surface (styrene divinylbenzene polymer) and increased multilayer adsorption (DOM self-assembly) can fundamentally affect biogeochemical interpretations, such as the source of organic matter. Online fluorescence monitoring of the permeate flow allowed to empirically model the extraction process and to assess the degree of variability introduced by changing the sorbent loading in the extraction procedure. Our study emphasizes that it is crucial for sample comparison to keep the relative DOC loading (DOCload [wt %]) on the sorbent always similar to avoid chemical fractionation

Health care worker vaccination against Ebola: Vaccine acceptance and employment duration in Sierra Leone.

Health care workers (HCW) are at high risk of Ebola virus disease (EVD) infection during epidemics and may contribute to onward transmission, and therefore HCW-targeted prophylactic vaccination strategies are being considered as interventions. To assess the feasibility of preventive HCW vaccination, we conducted a pilot survey on staff turnover and vaccine acceptance amongst 305 HCW in Freetown and Kambia districts of Sierra Leone. Multivariable logistic regression demonstrated which demographic and behavioural factors were associated with acceptance of a hypothetical new vaccine. We quantified the duration of employment of HCW, and used multivariable gamma regression to detect associations with duration of employment in current or any health care position. Finally, we simulated populations of HCW, to determine the likely future immunisation coverage amongst HCW based on our estimates of vaccine acceptance and employment duration. Most HCW we surveyed had a positive opinion of EVD vaccination (76.3%). We found that being a volunteer HCW (vs being on the government payroll) was associated with increased vaccine acceptance. We found that HCW have stable employment, with a mean duration of employment in the health sector of 10.9 years (median 8.0 years). Older age and being on the government payroll (vs volunteer HCW) were associated with a longer duration of employment in the health sector. Assuming a single vaccine campaign, with 76.3% vaccine acceptance, 100% vaccine efficacy and no waning of vaccine-induced protection, immunisation coverage was sustained over 50% until 6 years after a vaccination campaign. If vaccine-induced immunity wanes at 10% per year, then the immunisation coverage among HCW would fall below 50% after 3 years. Vaccinating HCW against EVD could be feasible as employment appeared stable and vaccine acceptance high. However, even with high vaccine efficacy and long-lasting immunity, repeated campaigns or vaccination at employment start may be necessary to maintain high coverage

Stoichiometry, polarity, and organometallics in solid-phase extracted dissolved organic matter of the Elbe-Weser estuary

Dissolved organic matter (DOM) is ubiquitous in natural waters and plays a central role in the biogeochemistry in riverine, estuarine and marine environments. This study quantifies and characterizes solid-phase extractable DOM and trace element complexation at different salinities in the Weser and Elbe River, northern Germany, and the North Sea. Dissolved organic carbon (DOC), total dissolved nitrogen (TDN), Co and Cu concentrations were analyzed in original water samples. Solid-phase extracted (SPE) water samples were analyzed for DOC (DOCSPE), dissolved organic nitrogen (DONSPE), sulfur (DOSSPE) and trace metal (51V, 52Cr, 59Co, 60Ni, 63Cu, 75As) concentrations. Additionally, different pre-treatment conditions (acidification vs. non-acidification prior to SPE) were tested. In agreement with previous studies, acidification led to generally higher recoveries for DOM and trace metals. Overall, higher DOM and trace metal concentrations and subsequently higher complexation of trace metals with carbon and sulfur-containing organic complexes were found in riverine compared to marine samples. With increasing salinity, the concentrations of DOM decreased due to estuarine mixing. However, the slightly lower relative decrease of both, DOCSPE and DONSPE (~77%) compared to DOSSPE (~86%) suggests slightly faster removal processes for DOSSPE. A similar distribution of trace metal and carbon and sulfur containing DOM concentrations with salinity indicates complexation of trace metals with organic ligands. This is further supported by an increase in Co and Cu concentration after oxidation of organic complexes by UV treatment. Additionally, the complexation of metals with organic ligands (analyzed by comparing metal/DOCSPE and metal/DOSSPE ratios) decreased in the order Cu > As > Ni > Cr > Co and thus followed the Irving-Williams order. Differences in riverine and marine trace metal containing DOMSPE are summarized by their average molar ratios of (C107N4P0.013S1)1000V0.05Cr0.33Co0.19Ni0.39Cu3.41As0.47 in the riverine endmember and (C163N7P0.055S1)1000V0.05Cr0.47Co0.16Ni0.07Cu4.05As0.58 in the marine endmember

Comparative and Functional Genomics of Rhodococcus opacus PD630 for Biofuels Development

The Actinomycetales bacteria Rhodococcus opacus PD630 and Rhodococcus jostii RHA1 bioconvert a diverse range of organic substrates through lipid biosynthesis into large quantities of energy-rich triacylglycerols (TAGs). To describe the genetic basis of the Rhodococcus oleaginous metabolism, we sequenced and performed comparative analysis of the 9.27 Mb R. opacus PD630 genome. Metabolic-reconstruction assigned 2017 enzymatic reactions to the 8632 R. opacus PD630 genes we identified. Of these, 261 genes were implicated in the R. opacus PD630 TAGs cycle by metabolic reconstruction and gene family analysis. Rhodococcus synthesizes uncommon straight-chain odd-carbon fatty acids in high abundance and stores them as TAGs. We have identified these to be pentadecanoic, heptadecanoic, and cis-heptadecenoic acids. To identify bioconversion pathways, we screened R. opacus PD630, R. jostii RHA1, Ralstonia eutropha H16, and C. glutamicum 13032 for growth on 190 compounds. The results of the catabolic screen, phylogenetic analysis of the TAGs cycle enzymes, and metabolic product characterizations were integrated into a working model of prokaryotic oleaginy.Cambridge-MIT InstituteMassachusetts Institute of Technology. (Seed Grant program)Shell Oil CompanyNational Institute of Allergy and Infectious Diseases (U.S.)United States. National Institutes of HealthNational Institutes of Health. Department of Health and Human Services (Contract No. HHSN272200900006C

Safety and immunogenicity of the two-dose heterologous Ad26.ZEBOV and MVA-BN-Filo Ebola vaccine regimen in children in Sierra Leone: a randomised, double-blind, controlled trial

Background—Children account for a substantial proportion of cases and deaths from Ebola virus disease. We aimed to assess the safety and immunogenicity of a two-dose heterologous vaccine regimen, comprising the adenovirus type 26 vector-based vaccine encoding the Ebola virus glycoprotein (Ad26.ZEBOV) and the modified vaccinia Ankara vectorbased vaccine, encoding glycoproteins from the Ebola virus, Sudan virus, and Marburg virus, and the nucleoprotein from the Tai Forest virus (MVA-BN-Filo), in a paediatric population in Sierra Leone. Methods—This randomised, double-blind, controlled trial was done at three clinics in Kambia district, Sierra Leone. Healthy children and adolescents aged 1–17 years were enrolled in three age cohorts (12–17 years, 4–11 years, and 1–3 years) and randomly assigned (3:1), via computer-generated block randomisation (block size of eight), to receive an intramuscular injection of either Ad26.ZEBOV (5 × 1010 viral particles; first dose) followed by MVA-BN-Filo (1 × 108 infectious units; second dose) on day 57 (Ebola vaccine group), or a single dose of meningococcal quadrivalent (serogroups A, C, W135, and Y) conjugate vaccine (MenACWY; first dose) followed by placebo (second dose) on day 57 (control group). Study team personnel (except for those with primary responsibility for study vaccine preparation), participants, and their parents or guardians were masked to study vaccine allocation. The primary outcome was safety, measured as the occurrence of solicited local and systemic adverse symptoms during 7 days after each vaccination, unsolicited systemic adverse events during 28 days after each vaccination, abnormal laboratory results during the study period, and serious adverse events or immediate reportable events throughout the study period. The secondary outcome was immunogenicity (humoral immune response), measured as the concentration of Ebola virus glycoprotein-specific binding antibodies at 21 days after the second dose. The primary outcome was assessed in all participants who had received at least one dose of study vaccine and had available reactogenicity data, and immunogenicity was assessed in all participants who had received both vaccinations within the protocol-defined time window, had at least one evaluable post-vaccination sample, and had no major protocol deviations that could have influenced the immune response. This study is registered at ClinicalTrials.gov, NCT02509494. Findings—From April 4, 2017, to July 5, 2018, 576 eligible children or adolescents (192 in each of the three age cohorts) were enrolled and randomly assigned. The most common solicited local adverse event during the 7 days after the first and second dose was injection-site pain in all age groups, with frequencies ranging from 0% (none of 48) of children aged 1–3 years after placebo injection to 21% (30 of 144) of children aged 4–11 years after Ad26.ZEBOV vaccination. The most frequently observed solicited systemic adverse event during the 7 days was headache in the 12–17 years and 4–11 years age cohorts after the first and second dose, and pyrexia in the 1–3 years age cohort after the first and second dose. The most frequent unsolicited adverse event after the first and second dose vaccinations was malaria in all age cohorts, irrespective of the vaccine types. Following vaccination with MenACWY, severe thrombocytopaenia was observed in one participant aged 3 years. No other clinically significant laboratory abnormalities were observed in other study participants, and no serious adverse events related to the Ebola vaccine regimen were reported. There were no treatment-related deaths. Ebola virus glycoprotein-specific binding antibody responses at 21 days after the second dose of the Ebola virus vaccine regimen were observed in 131 (98%) of 134 children aged 12–17 years (9929 ELISA units [EU]/mL [95% CI 8172–12 064]), in 119 (99%) of 120 aged 4–11 years (10 212 EU/mL [8419–12 388]), and in 118 (98%) of 121 aged 1–3 years (22 568 EU/mL [18 426–27 642]). Interpretation—The Ad26.ZEBOV and MVA-BN-Filo Ebola vaccine regimen was well tolerated with no safety concerns in children aged 1–17 years, and induced robust humoral immune responses, suggesting suitability of this regimen for Ebola virus disease prophylaxis in children

Safety and long-term immunogenicity of the two-dose heterologous Ad26.ZEBOV and MVA-BN-Filo Ebola vaccine regimen in adults in Sierra Leone: a combined open-label, non-randomised stage 1, and a randomised, double-blind, controlled stage 2 trial

Background The Ebola epidemics in west Africa and the Democratic Republic of the Congo highlight an urgent need for safe and effective vaccines to prevent Ebola virus disease. We aimed to assess the safety and long-term immunogenicity of a two-dose heterologous vaccine regimen, comprising the adenovirus type 26 vector-based vaccine encoding the Ebola virus glycoprotein (Ad26.ZEBOV) and the modified vaccinia Ankara vector-based vaccine, encoding glycoproteins from Ebola virus, Sudan virus, and Marburg virus, and the nucleoprotein from the Tai Forest virus (MVA-BN-Filo), in Sierra Leone, a country previously affected by Ebola. Methods The trial comprised two stages: an open-label, non-randomised stage 1, and a randomised, double-blind, controlled stage 2. The study was done at three clinics in Kambia district, Sierra Leone. In stage 1, healthy adults (aged ≥18 years) residing in or near Kambia district, received an intramuscular injection of Ad26.ZEBOV (5×1010 viral particles) on day 1 (first dose) followed by an intramuscular injection of MVA-BN-Filo (1×108 infectious units) on day 57 (second dose). An Ad26.ZEBOV booster vaccination was offered at 2 years after the first dose to stage 1 participants. The eligibility criteria for adult participants in stage 2 were consistent with stage 1 eligibility criteria. Stage 2 participants were randomly assigned (3:1), by computer-generated block randomisation (block size of eight) via an interactive web-response system, to receive either the Ebola vaccine regimen (Ad26.ZEBOV followed by MVA-BN-Filo) or an intramuscular injection of a single dose of meningococcal quadrivalent (serogroups A, C, W135, and Y) conjugate vaccine (MenACWY; first dose) followed by placebo on day 57 (second dose; control group). Study team personnel, except those with primary responsibility for study vaccine preparation, and participants were masked to study vaccine allocation. The primary outcome was the safety of the Ad26.ZEBOV and MVA-BN-Filo vaccine regimen, which was assessed in all participants who had received at least one dose of study vaccine. Safety was assessed as solicited local and systemic adverse events occurring in the first 7 days after each vaccination, unsolicited adverse events occurring in the first 28 days after each vaccination, and serious adverse events or immediate reportable events occurring up to each participant’s last study visit. Secondary outcomes were to assess Ebola virus glycoprotein-specific binding antibody responses at 21 days after the second vaccine in a per-protocol set of participants (ie, those who had received both vaccinations within the protocol-defined time window, had at least one evaluable post-vaccination sample, and had no major protocol deviations that could have influenced the immune response) and to assess the safety and tolerability of the Ad26.ZEBOV booster vaccination in stage 1 participants who had received the booster dose. This study is registered at ClinicalTrials.gov, NCT02509494. Findings Between Sept 30, 2015, and Oct 19, 2016, 443 participants (43 in stage 1 and 400 in stage 2) were enrolled; 341 participants assigned to receive the Ad26.ZEBOV and MVA-BN-Filo regimen and 102 participants assigned to receive the MenACWY and placebo regimen received at least one dose of study vaccine. Both regimens were well tolerated with no safety concerns. In stage 1, solicited local adverse events (mostly mild or moderate injection-site pain) were reported in 12 (28%) of 43 participants after Ad26.ZEBOV vaccination and in six (14%) participants after MVA-BN-Filo vaccination. In stage 2, solicited local adverse events were reported in 51 (17%) of 298 participants after Ad26.ZEBOV vaccination, in 58 (24%) of 246 after MVA-BN-Filo vaccination, in 17 (17%) of 102 after MenACWY vaccination, and in eight (9%) of 86 after placebo injection. In stage 1, solicited systemic adverse events were reported in 18 (42%) of 43 participants after Ad26.ZEBOV vaccination and in 17 (40%) after MVA-BN-Filo vaccination. In stage 2, solicited systemic adverse events were reported in 161 (54%) of 298 participants after Ad26.ZEBOV vaccination, in 107 (43%) of 246 after MVA-BN-Filo vaccination, in 51 (50%) of 102 after MenACWY vaccination, and in 39 (45%) of 86 after placebo injection. Solicited systemic adverse events in both stage 1 and 2 participants included mostly mild or moderate headache, myalgia, fatigue, and arthralgia. The most frequent unsolicited adverse event after the first dose was headache in stage 1 and malaria in stage 2. Malaria was the most frequent unsolicited adverse event after the second dose in both stage 1 and 2. No serious adverse event was considered related to the study vaccine, and no immediate reportable events were observed. In stage 1, the safety profile after the booster vaccination was not notably different to that observed after the first dose. Vaccine-induced humoral immune responses were observed in 41 (98%) of 42 stage 1 participants (geometric mean binding antibody concentration 4784 ELISA units [EU]/mL [95% CI 3736–6125]) and in 176 (98%) of 179 stage 2 participants (3810 EU/mL [3312–4383]) at 21 days after the second vaccination. Interpretation The Ad26.ZEBOV and MVA-BN-Filo vaccine regimen was well tolerated and immunogenic, with persistent humoral immune responses. These data support the use of this vaccine regimen for Ebola virus disease prophylaxis in adults

Unraveling the Function of the Rhodospirillum rubrum Activator of Polyhydroxybutyrate (PHB) Degradation: the Activator Is a PHB-Granule-Bound Protein (Phasin)

Efficient hydrolysis of native poly(3-hydroxybutyrate) (nPHB) granules in vitro by soluble PHB depolymerase of Rhodospirillum rubrum requires pretreatment of nPHB with an activator compound present in R. rubrum cells (J. M. Merrick and M. Doudoroff, J. Bacteriol. 88:60-71, 1964). Edman sequencing of the purified activator (17.4 kDa; matrix-assisted laser desorption ionization—time of flight mass spectrometry) revealed identity to a hypothetical protein deduced from a partially sequenced R. rubrum genome. The complete activator gene, apdA (activator of polymer degradation), was cloned from genomic DNA, expressed as a six-His-tagged protein in recombinant Escherichia coli (M(r), 18.3 × 10(3)), and purified. The effect of ApdA on PHB metabolism was studied in vitro and in vivo. In vitro, the activity of the activator could be replaced by trypsin, but recombinant ApdA itself had no protease activity. Comparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the protein patterns of trypsin- and ApdA-treated nPHB granules isolated from different PHB-accumulating bacteria showed that trypsin activated nPHB by removing proteins of the surface layer of nPHB regardless of the origin of nPHB, but ApdA bound to and interacted with the surface layer of nPHB in a nonproteolytic manner, thereby transforming nPHB into an activated form that was accessible to the depolymerase. In vivo, expression of ApdA in E. coli harboring the PHB biosynthetic genes, phaCBA, resulted in significant increases in the number and surface/volume ratio of accumulated PHB granules, which was comparable to the effect of phasin proteins, such as PhaP in Ralstonia eutropha. The amino acid sequence of ApdA was 55% identical to the amino acid sequence of Mms16, a magnetosome-associated protein in magnetotactic Magnetospirillum species. Mms16 was previously reported to be a GTPase with an essential function in magnetosome formation (Y. Okamura, H. Takeyama, and T. Matsunaga, J. Biol. Chem. 276:48183-48188, 2001). However, no GTPase activity of ApdA could be demonstrated. We obtained evidence that Mms16 of Magnetospirillum gryphiswaldense can functionally replace ApdA in R. rubrum. Fusions of apdA and mms16 to gfp or yfp were functionally expressed, and both fusions colocalized with PHB granules after conjugative transfer to R. rubrum. In conclusion, ApdA in vivo is a PHB-bound, phasin-like protein in R. rubrum. The function of Mms16 in magnetotactic bacteria requires further clarification

Adaptation to Chronic-Cycling Hypoxia Renders Cancer Cells Resistant to MTH1-Inhibitor Treatment Which Can Be Counteracted by Glutathione Depletion

Tumor hypoxia and hypoxic adaptation of cancer cells represent major barriers to successful cancer treatment. We revealed that improved antioxidant capacity contributes to increased radioresistance of cancer cells with tolerance to chronic-cycling severe hypoxia/reoxygenation stress. We hypothesized, that the improved tolerance to oxidative stress will increase the ability of cancer cells to cope with ROS-induced damage to free deoxy-nucleotides (dNTPs) required for DNA replication and may thus contribute to acquired resistance of cancer cells in advanced tumors to antineoplastic agents inhibiting the nucleotide-sanitizing enzyme MutT Homologue-1 (MTH1), ionizing radiation (IR) or both. Therefore, we aimed to explore potential differences in the sensitivity of cancer cells exposed to acute and chronic-cycling hypoxia/reoxygenation stress to the clinically relevant MTH1-inhibitor TH1579 (Karonudib) and to test whether a multi-targeting approach combining the glutathione withdrawer piperlongumine (PLN) and TH1579 may be suited to increase cancer cell sensitivity to TH1579 alone and in combination with IR. Combination of TH1579 treatment with radiotherapy (RT) led to radiosensitization but was not able to counteract increased radioresistance induced by adaptation to chronic-cycling hypoxia/reoxygenation stress. Disruption of redox homeostasis using PLN sensitized anoxia-tolerant cancer cells to MTH1 inhibition by TH1579 under both normoxic and acute hypoxic treatment conditions. Thus, we uncover a glutathione-driven compensatory resistance mechanism towards MTH1-inhibition in form of increased antioxidant capacity as a consequence of microenvironmental or therapeutic stress