¹⁵N stable isotope probing of pulp and paper wastewaters

Stable isotope probing (SIP) is an established technique that can be applied to identify the metabolically active micro-organisms within a microbial population. The SIP method utilises an isotopically-labelled substrate and PCR techniques to discern the members of a microbial community that incorporate the isotope into their DNA or RNA. The current literature gap around using ¹⁵N isotopes with RNA-SIP offers real potential and advantages for targeting and identifying active members from mixed communities involved in global biogeochemical nitrogen cycling. This study specifically investigated whether nitrogen based compounds ncan be used as substrates in RNA-SIP methodologies and whether they can in turn be used to probe mixed community environments known to be actively fixing nitrogen. The nitrogen-limited systems targeted represented an ideal opportunity to assess the suitability of ¹⁵N-RNA-SIP approaches due to their known high nitrogen fixation rates. Identifying these nitrogen-fixing bacteria could provide a better representation analysis of the community, leading to an improved prediction on how to manage and optimise the treatment performance of target waste systems and to exploit the unique bioconversion properties of these types of organisms. Initially, the project undertook methodological proof of concept by using a soluble nitrogen source, ¹⁵NH₄Cl, to label the RNA of Novosphingobium nitrogenifigens and a mixed microbial community. Successful separation of the ¹⁴N- (control) and ¹⁵N-RNA was achieved for both pure and mixed communities using isopycnic caesium trifluoroacetate (CsTFA) gradients in an ultracentrifuge. The usefulness of this technique to identify active diazotrophs in real environmental samples was tested using a nitrogen-fixing community from a pulp and paper wastewater treatment system. After growing the mixed culture with ¹⁵N₂ as the sole nitrogen source, the labelled RNA was extracted and fractionated using isopycnic centrifugation in CsTFA gradients. The community composition of the active nitrogen-fixing community in the ¹⁵N₂ enriched fraction was analysed by establishing a 16S rRNA gene clone library containing over 200 members. These were analysed by comparison with published sequences and by phylogenetic analysis. It was found that the more isotopic label substrate incorporated, the further the buoyant density (BD) separation between ¹⁵N- and ¹⁴N-RNA. Novosphingobium nitrogenifigens gave an average BD shift of 0.03 + 0.004 g ml⁻¹ (95.0 atom % ¹⁵N) with ¹⁵NH₄Cl. For mixed communities the average BD shift was 0.02 + 0.004 g ml⁻¹ (80.0 atom % ¹⁵N) with 15NH4Cl and 0.013 + 0.002 g ml⁻¹ (32.6 atom % ¹⁵N) when using ¹⁵N₂. Clone library analysis of 16S rRNA genes present in the enriched ¹⁵N-RNA fraction of the mixed community was shown to consist of a diverse population of bacteria as indicated by a Shannon Weaver index value of >2.8. Three dominant genera (Aeromonas, Pseudomonas and Bacillus) were identified by comparison with published sequences and phylogenetic analysis. Many other groups not known as archetypal nitrogen-fixing bacteria were also identified, demonstrating that ¹⁵N₂-RNA-SIP provides a useful tool for the identification of important and previously unknown contributors to nitrogen fixation in a range of environments. Overall, this project has established that nitrogen based RNA-SIP is a powerful tool that can be used successfully and reproducibly with both pure and complex mixed microbial communities to study active diazotrophs in environmental samples

Stable isotope probing: Technical considerations when resolving ¹⁵N-labeled RNA in gradients

RNA based stable isotope probing (SIP) facilitates the detection and identification of active members of microbial populations that are involved in the assimilation of an isotopically labeled compound. ¹⁵N-RNA-SIP is a new method that has been discussed in recent literature but has not yet been tested. Herein, we define the limitations to using ¹⁵N-labeled substrates for SIP and propose modifications to compensate for some of these shortcomings. We have used ¹⁵N-RNA-SIP as a tool for analysing mixed bacterial populations that use nitrogen substrates. After incubating mixed microbial communities with ¹⁵N-ammonium chloride or ¹⁵N₂ we assessed the fractionation resolution of ¹⁵N-RNA by isopycnic centrifugation in caesium trifluoroacetate (CsTFA) gradients. We found that the more isotopic label incorporated, the further the buoyant density (BD) separation between ¹⁵N- and ¹⁴N-RNA, however it was not possible to resolve the labeled from unlabeled RNA definitively through gradient fractionation. Terminal-restriction fragment length polymorphism (T-RFLP) analysis of the extracted RNA and fluorescent in situ hybridisation (FISH) analysis of the enrichment cultures provided some insight into the organisms involved in nitrogen fixation. This approach is not without its limitations and will require further developments to assess its applicability to other nitrogen-fixing environments

Mutations in Known Monogenic High Bone Mass Loci Only Explain a Small Proportion of High Bone Mass Cases.

High bone mass (HBM) can be an incidental clinical finding; however, monogenic HBM disorders (eg, LRP5 or SOST mutations) are rare. We aimed to determine to what extent HBM is explained by mutations in known HBM genes. A total of 258 unrelated HBM cases were identified from a review of 335,115 DXA scans from 13 UK centers. Cases were assessed clinically and underwent sequencing of known anabolic HBM loci: LRP5 (exons 2, 3, 4), LRP4 (exons 25, 26), SOST (exons 1, 2, and the van Buchem's disease [VBD] 52-kb intronic deletion 3'). Family members were assessed for HBM segregation with identified variants. Three-dimensional protein models were constructed for identified variants. Two novel missense LRP5 HBM mutations ([c.518C>T; p.Thr173Met], [c.796C>T; p.Arg266Cys]) were identified, plus three previously reported missense LRP5 mutations ([c.593A>G; p.Asn198Ser], [c.724G>A; p.Ala242Thr], [c.266A>G; p.Gln89Arg]), associated with HBM in 11 adults from seven families. Individuals with LRP5 HBM (∼prevalence 5/100,000) displayed a variable phenotype of skeletal dysplasia with increased trabecular BMD and cortical thickness on HRpQCT, and gynoid fat mass accumulation on DXA, compared with both non-LRP5 HBM and controls. One mostly asymptomatic woman carried a novel heterozygous nonsense SOST mutation (c.530C>A; p.Ser177X) predicted to prematurely truncate sclerostin. Protein modeling suggests the severity of the LRP5-HBM phenotype corresponds to the degree of protein disruption and the consequent effect on SOST-LRP5 binding. We predict p.Asn198Ser and p.Ala242Thr directly disrupt SOST binding; both correspond to severe HBM phenotypes (BMD Z-scores +3.1 to +12.2, inability to float). Less disruptive structural alterations predicted from p.Arg266Cys, p.Thr173Met, and p.Gln89Arg were associated with less severe phenotypes (Z-scores +2.4 to +6.2, ability to float). In conclusion, although mutations in known HBM loci may be asymptomatic, they only account for a very small proportion (∼3%) of HBM individuals, suggesting the great majority are explained by either unknown monogenic causes or polygenic inheritance.This study was supported by The Wellcome Trust and NIHR CRN (portfolio number 5163). CLG was funded by a Wellcome Trust Clinical Research Training Fellowship (080280/Z/06/Z), the EU 7th Framework Programme under grant agreement number 247642 (GEoCoDE), a British Geriatric Society travel grant, and is now funded by Arthritis Research UK (grant ref 20000). SH acknowledges Arthritis Research UK support (grant ref 19580). KESP acknowledges the support of Cambridge NIHR Biomedical Research Centre. KAW is supported by the core programme of the MRC Nutrition and Bone Health group at MRC Human Nutrition Research, funded by the UK Medical Research Council (Grant code U10590371). EM acknowledges support of the Sheffield Teaching Hospitals Foundation Trust Clinical Research Facility. The SGC is a registered charity (no. 1097737) that receives funds from AbbVie, Bayer, Boehringer Ingelheim, Genome Canada (Ontario Genomics Institute OGI- 055), GlaxoSmithKline, Janssen, Lilly Canada, Novartis Research Foundation, Ontario Ministry of Economic Development & Innovation, Pfizer, Takeda, and Wellcome Trust (092809/Z/10/Z).This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1002/jbmr.270

TOI-836 : a super-Earth and mini-Neptune transiting a nearby K-dwarf

Funding: TGW, ACC, and KH acknowledge support from STFC consolidated grant numbers ST/R000824/1 and ST/V000861/1, and UKSA grant ST/R003203/1.We present the discovery of two exoplanets transiting TOI-836 (TIC 440887364) using data from TESS Sector 11 and Sector 38. TOI-836 is a bright (T = 8.5 mag), high proper motion (∼200 mas yr−1), low metallicity ([Fe/H]≈−0.28) K-dwarf with a mass of 0.68 ± 0.05 M⊙ and a radius of 0.67 ± 0.01 R⊙. We obtain photometric follow-up observations with a variety of facilities, and we use these data-sets to determine that the inner planet, TOI-836 b, is a 1.70 ± 0.07 R⊕ super-Earth in a 3.82 day orbit, placing it directly within the so-called ‘radius valley’. The outer planet, TOI-836 c, is a 2.59 ± 0.09 R⊕ mini-Neptune in an 8.60 day orbit. Radial velocity measurements reveal that TOI-836 b has a mass of 4.5 ± 0.9 M⊕, while TOI-836 c has a mass of 9.6 ± 2.6 M⊕. Photometric observations show Transit Timing Variations (TTVs) on the order of 20 minutes for TOI-836 c, although there are no detectable TTVs for TOI-836 b. The TTVs of planet TOI-836 c may be caused by an undetected exterior planet.Publisher PDFPeer reviewe

TOI-836: A super-Earth and mini-Neptune transiting a nearby K-dwarf

We present the discovery of two exoplanets transiting TOI-836 (TIC 440887364) using data from TESS Sector 11 and Sector 38. TOI-836 is a bright ($T = 8.5$ mag), high proper motion ($\sim\,200$ mas yr$^{-1}$), low metallicity ([Fe/H]$\approx\,-0.28$) K-dwarf with a mass of $0.68\pm0.05$ M$_{\odot}$ and a radius of $0.67\pm0.01$ R$_{\odot}$. We obtain photometric follow-up observations with a variety of facilities, and we use these data-sets to determine that the inner planet, TOI-836 b, is a $1.70\pm0.07$ R$_{\oplus}$ super-Earth in a 3.82 day orbit, placing it directly within the so-called 'radius valley'. The outer planet, TOI-836 c, is a $2.59\pm0.09$ R$_{\oplus}$ mini-Neptune in an 8.60 day orbit. Radial velocity measurements reveal that TOI-836 b has a mass of $4.5\pm0.9$ M$_{\oplus}$ , while TOI-836 c has a mass of $9.6\pm2.6$ M$_{\oplus}$. Photometric observations show Transit Timing Variations (TTVs) on the order of 20 minutes for TOI-836 c, although there are no detectable TTVs for TOI-836 b. The TTVs of planet TOI-836 c may be caused by an undetected exterior planet

TOI-836: A super-Earth and mini-Neptune transiting a nearby K-dwarf

peer reviewe

Identifying diazotrophs by incorporation of nitrogen from ¹⁵N₂ into RNA

The diversity and abundance of active diazotrophs was investigated in a New Zealand pulp and paper wastewater by enrichment with ¹⁵N₂. Purified ¹⁵N-RNA was analysed by reverse transcription, molecular cloning and sequence analysis of 16S rRNA to reveal a diverse community of bacteria as indicated by a Shannon Weaver Index value of > 2.8. The major class represented in the enriched culture were the γ-Proteobacteria at 85% with a secondary group of the phylum Firmicutes present at 8.2%, the remaining sequences were affiliated with the α- and β-Proteobacterial classes (1.4% and 4.3%, respectively). Three dominant genera, Aeromonas, Pseudomonas and Bacillus, were identified by comparison with published sequences and phylogenetic analysis. To confirm that representatives of the taxonomic groups identified from the active enriched nitrogen-fixing community were capable of fixing nitrogen Aeromonas and Pseudomonas species were cultivated and shown to possess nifH genes. In wastewater, fluorescence in situ hybridisation probing revealed that the dominant nitrogen-fixing population identified in this study were present in the population, but at lower levels. The population is, therefore, reliant on a small sub-population of diazotrophs to supply the community's nitrogen needs above that already present in the wastewater

Identifying diazotrophs by incorporation of nitrogen from ¹⁵N₂ into RNA

The diversity and abundance of active diazotrophs was investigated in a New Zealand pulp and paper wastewater by enrichment with ¹⁵N₂. Purified ¹⁵N-RNA was analysed by reverse transcription, molecular cloning and sequence analysis of 16S rRNA to reveal a diverse community of bacteria as indicated by a Shannon Weaver Index value of > 2.8. The major class represented in the enriched culture were the γ-Proteobacteria at 85% with a secondary group of the phylum Firmicutes present at 8.2%, the remaining sequences were affiliated with the α- and β-Proteobacterial classes (1.4% and 4.3%, respectively). Three dominant genera, Aeromonas, Pseudomonas and Bacillus, were identified by comparison with published sequences and phylogenetic analysis. To confirm that representatives of the taxonomic groups identified from the active enriched nitrogen-fixing community were capable of fixing nitrogen Aeromonas and Pseudomonas species were cultivated and shown to possess nifH genes. In wastewater, fluorescence in situ hybridisation probing revealed that the dominant nitrogen-fixing population identified in this study were present in the population, but at lower levels. The population is, therefore, reliant on a small sub-population of diazotrophs to supply the community's nitrogen needs above that already present in the wastewater

Synthesis and structure of mono-, bi- and trimetallic amine-bis(phenolate) cobalt(II) complexes

A series of cobalt(II) amine-bis(phenolate) complexes has been prepared and characterized. The protonated tripodal tetradentate ligand precursors; dimethylaminoethylamino-N,N-bis(2-methylene-4-tert-butyl-6-methylphenol), H2[O2NN¢]BuMeNMe2, dimethylaminoethylamino-N,N-bis(2-methylene-4,6-di-tert-butylphenol), H2[O2NN¢]BuBuNMe2, diethylaminoethylamino-N,N-bis(2-methylene-4,6-di-tert-amylphenol), H2[O2NN¢]AmAmNEt2 and 2-pyridylamino-N,N-bis(2-methylene-4,6-di-tert-amylphenol), H2[O2NN¢]AmAmPy; were reacted with cobaltous acetate tetrahydrate under varying conditions to afford a range of monometallic, bimetallic and trimetallic species. An unusual four coordinate complex Co[O2NN¢]AmAmNEt2 containing CoII in a trigonal monopyramidal environment was structurally characterized, whereas using a less sterically demanding ligand a series of five coordinate complexes Co[O2NN¢]BuBuNMe2(L) (L = H2O, CH3OH, (CH3)2C=O, propylene oxide) containing CoII in a trigonal bipyramidal environment was prepared. A new angular structural parameter related to t is defined, where t¢ may be used to compare complexes with trigonal monopyramidal structures. In contrast, ligands containing a pendant pyridyl donor afford dimeric species including {Co(m-CH3OH)[O2NN¢]AmAmPy}2. In the absence of base and in the presence of excess cobaltous acetate, trimetallic complexes were isolated containing a central CoII in an octahedral environment coordinated to four CH3OH and two bridging acetate ligands between two Co[O2NN¢] fragments with CoII in a trigonal bipyramidal setting. The paramagnetic CoII complexes reported were also characterized by UV-vis spectroscopy, mass spectrometry, cyclic voltammetry and magnetic measurements

Spinning up a Daze: TESS Uncovers a Hot Jupiter orbiting the Rapid-Rotator TOI-778

NASA's Transiting Exoplanet Survey Satellite (TESS) mission, has been uncovering a growing number of exoplanets orbiting nearby, bright stars. Most exoplanets that have been discovered by TESS orbit narrow-line, slow-rotating stars, facilitating the confirmation and mass determination of these worlds. We present the discovery of a hot Jupiter orbiting a rapidly rotating (vsin(i)=35.1±1.0km/s) early F3V-dwarf, HD115447 (TOI-778). The transit signal taken from Sectors 10 and 37 of TESS's initial detection of the exoplanet is combined with follow-up ground-based photometry and velocity measurements taken from Minerva-Australis, TRES, CORALIE and CHIRON to confirm and characterise TOI-778b. A joint analysis of the light curves and the radial velocity measurements yield a mass, radius, and orbital period for TOI-778b of 2.76+0.24−0.23Mjup, 1.370±0.043Rjup and ∼4.63 days, respectively. The planet orbits a bright (V=9.1mag) F3-dwarf with M=1.40±0.05Msun, R=1.70±0.05Rsun, and logg=4.05±0.17. We observed a spectroscopic transit of TOI-778b, which allowed us to derive a sky-projected spin-orbit angle of 18∘±11∘, consistent with an aligned planetary system. This discovery demonstrates the capability of smaller aperture telescopes such as Minerva-Australis to detect the radial velocity signals produced by planets orbiting broad-line, rapidly rotating stars