Search

The precise measurement of the amount fraction of atmospheric nitrous oxide (N2O) is required to understand global emission trends. Analysis of the site-specific stable isotopic composition of N2O provides a means to differentiate emission sources. The availability of accurate reference materials of known N2O amount fractions and isotopic composition is critical for achieving these goals. We present the development of nitrous oxide gas reference materials for underpinning measurements of atmospheric composition and isotope ratio. Uncertainties target the World Metrological Organisation Global Atmosphere Watch (WMO-GAW) compatibility goal of 0.1 nmol mol−1 and extended compatibility goal of 0.3 nmol mol−1, for atmospheric N2O measurements in an amount fraction range of 325–335 nmol mol−1. We also demonstrate the stability of amount fraction and isotope ratio of these reference materials and present a characterisation study of the cavity ring-down spectrometer used for analysis of the reference materials.

BACKGROUND The negative consequences associated with alcohol misuse remain a concern on college campuses nationwide. Alcohol protective behavioral strategies are important factors in mitigating college students' experiences of alcohol-related negative consequences. Overparenting, or "helicopter parenting," is a form of over-involved parenting which has been associated with a range of substance use concerns in college students. The mechanism by which overparenting is associated with alcohol use and/or consequences is unclear; however, it may be partially explained by failure to engage in the use of protective behavioral strategies. Purpose/objectives: The current study explored the degree to which alcohol protective behavioral strategies mediated the relationship between overparenting and alcohol-related negative consequences when accounting for alcohol consumption and if these associations were invariant across race and gender. Methods: Participants were 625 traditional age college students who consumed alcohol within the 30 days prior to completing measures of alcohol consumption, overparenting, alcohol-related negative consequences, and alcohol protective behavioral strategies. Results: Alcohol protective behavioral strategies, specifically manner of drinking, partially mediated the relationship between overparenting and alcohol-related negative consequences when accounting for alcohol misuse. There were no differences noted across gender, but the models were found to be invariant across White and African American participants in relation to the protective value of alcohol protective behavioral strategies. Conclusion/Importance: Overparenting may relate to negative alcohol outcomes in part through less use of harm reduction strategies. More investigation into the value of alcohol protective behavioral strategies across race is needed.

Silicon (Si) fertilization has been established as a sustainable technology, with great potential for reducing the use of agrochemicals and increasing productivity. The objective of this study was ...

Cytochrome P450 enzymes have tremendous potential as industrial biocatalysts, including in biological lignin valorization. Here, we describe P450s that catalyze the O-demethylation of lignin-derived guaiacols with different ring substitution patterns. Bacterial strains Rhodococcus rhodochrous EP4 and Rhodococcus jostii RHA1 both utilized alkylguaiacols as sole growth substrates. Transcriptomics of EP4 grown on 4-propylguaiacol (4PG) revealed the up-regulation of agcA, encoding a CYP255A1 family P450, and the aph genes, previously shown to encode a meta-cleavage pathway responsible for 4-alkylphenol catabolism. The function of the homologous pathway in RHA1 was confirmed: Deletion mutants of agcA and aphC, encoding the meta-cleavage alkylcatechol dioxygenase, grew on guaiacol but not 4PG. By contrast, deletion mutants of gcoA and pcaL, encoding a CYP255A2 family P450 and an ortho-cleavage pathway enzyme, respectively, grew on 4-propylguaiacol but not guaiacol. CYP255A1 from EP4 catalyzed the O-demethylation of 4-alkylguaiacols to 4-alkylcatechols with the following apparent specificities (k(cat)/K(M)): propyl > ethyl > methyl > guaiacol. This order largely reflected AgcA’s binding affinities for the different guaiacols and was the inverse of GcoA(EP4)’s specificities. The biocatalytic potential of AgcA was demonstrated by the ability of EP4 to grow on lignin-derived products obtained from the reductive catalytic fractionation of corn stover, depleting alkylguaiacols and alkylphenols. By identifying related P450s with complementary specificities for lignin-relevant guaiacols, this study facilitates the design of these enzymes for biocatalytic applications. We further demonstrated that the metabolic fate of the guaiacol depends on its substitution pattern, a finding that has significant implications for engineering biocatalysts to valorize lignin.

Globally, tropical forests are assumed to be an important source of atmospheric nitrous oxide (N2O) and sink for methane (CH4). Yet, although the Congo Basin comprises the second largest tropical forest and is considered the most pristine large basin left on Earth, in situ N2O and CH4 flux measurements are scarce. Here, we provide multi-year data derived from on-ground soil flux (n = 1558) and riverine dissolved gas concentration (n = 332) measurements spanning montane, swamp, and lowland forests. Each forest type core monitoring site was sampled at least for one hydrological year between 2016 - 2020 at a frequency of 7-14 days. We estimate a terrestrial CH4 uptake (in kg CH4-C ha−1 yr−1) for montane (−4.28) and lowland forests (−3.52) and a massive CH4 release from swamp forests (non-inundated 2.68; inundated 341). All investigated forest types were a N2O source (except for inundated swamp forest) with 0.93, 1.56, 3.5, and −0.19 kg N2O-N ha−1 yr−1 for montane, lowland, non-inundated swamp, and inundated swamp forests, respectively., Nature Communications, 13, ISSN:2041-1723

Bile salts are amphiphilic steroids with a C5 carboxylic side chain with digestive functions in vertebrates. Upon excretion, they are degraded by environmental bacteria. Degradation of the bile-salt steroid skeleton resembles the well-studied pathway for other steroids like testosterone, while specific differences occur during side-chain degradation and the initiating transformations of the steroid skeleton. Of the latter, two variants via either Δ1,4- or Δ4,6-3-ketostructures of the steroid skeleton exist for 7-hydroxy bile salts. While the Δ1,4- variant is well-known from many model organisms, the Δ4,6-variant involving a 7-hydroxysteroid dehydratase as key enzyme has not been systematically studied. Here, combined proteomic, bioinformatic and functional analyses of the Δ4,6-variant in Sphingobium sp. strain Chol11 were performed. They revealed a degradation of the steroid rings similar to the Δ1,4-variant except for the elimination of the 7-OH as key difference. In contrast, differential production of the respective proteins revealed a putative gene cluster for side-chain degradation encoding a CoA-ligase, an acyl-CoA dehydrogenase, a Rieske monooxygenase, and an amidase, but lacking most canonical genes known from other steroid-degrading bacteria. Bioinformatic analyses predicted the Δ4,6-variant to be widespread among the Sphingomonadaceae, which was verified for three type strains which also have the predicted side-chain degradation cluster. A second amidase in the side-chain degradation gene cluster of strain Chol11 was shown to cleave conjugated bile salts while having low similarity to known bile-salt hydrolases. This study signifies members of the Sphingomonadaceae remarkably well-adapted to the utilization of bile salts via a partially distinct metabolic pathway.ImportanceThis study highlights the biochemical diversity of bacterial degradation of steroid compounds, in particular bile salts. Furthermore, it substantiates and advances knowledge of a variant pathway for degradation of steroids by sphingomonads, a group of environmental bacteria that are well-known for their broad metabolic capabilities. Biodegradation of bile salts is a critical process due to the high input of these compounds from manure into agricultural soils and wastewater treatment plants. In addition, these results may also be relevant for the biotechnological production of bile salts or other steroid compounds with pharmaceutical functions.

The valorization of lignin, a major component of plant-derived biomass, is essential to sustainable biorefining. We identified the major monoaromatic compounds present in black liquor, a lignin-rich stream generated in the kraft pulping process, and investigated their bacterial transformation. Among tested solvents, acetone extracted the greatest amount of monoaromatic compounds from softwood black liquor, with guaiacol, vanillin, and acetovanillone, in an approximately 4:3:2 ratio, constituting ~90% of the total extracted monoaromatic content. 4-Ethanol guaiacol, vanillate, and 4-propanol guaiacol were also present. Bacterial strains that grew on minimal media supplemented with the BL extracts at 1mM total aromatic compounds included Pseudomonas putida KT2442, Sphingobium sp. SYK-6, and Rhodococcus rhodochrous EP4. By contrast, the extracts inhibited the growth of Rhodococcus jostii RHA1 and Rhodococcus opacus PD630, strains extensively studied for lignin valorization. Of the strains that grew on the extracts, only R. rhodochrous GD01 and GD02, isolated for their ability to grow on acetovanillone, depleted the major extracted monoaromatics. Genomic analyses revealed that EP4, GD01, and GD02 share an average nucleotide identity (ANI) of 98% and that GD01 and GD02 harbor a predicted three-component carboxylase not present in EP4. A representative carboxylase gene was upregulated ~100-fold during growth of GD02 on a mixture of the BL monoaromatics, consistent with the involvement of the enzyme in acetovanillone catabolism. More generally, quantitative RT-PCR indicated that GD02 catabolizes the BL compounds in a convergent manner via the β-ketoadipate pathway. Overall, these studies help define the catabolic capabilities of potential biocatalytic strains, describe new isolates able to catabolize the major monoaromatic components of BL, including acetovanillone, and facilitate the design of biocatalysts to valorize under-utilized components of industrial lignin streams.

Familial exudative vitreoretinopathy (FEVR) is a human disease characterized by defective retinal angiogenesis and associated complications that can result in vision loss. Defective Wnt/β-catenin signaling is an established cause of FEVR, whereas other molecular alterations contributing to the disease remain insufficiently understood. Here, we show that integrin-linked kinase (ILK), a mediator of cell-matrix interactions, is indispensable for retinal angiogenesis. Inactivation of the murine Ilk gene in postnatal endothelial cells results in sprouting defects, reduced endothelial proliferation and disruption of the blood-retina barrier, resembling phenotypes seen in established mouse models of FEVR. Retinal vascularization defects are phenocopied by inducible inactivation of the gene for α-parvin (Parva), an interactor of ILK. Screening genomic DNA samples from exudative vitreoretinopathy patients identifies three distinct mutations in human ILK, which compromise the function of the gene product in vitro. Together, our data suggest that defective cell-matrix interactions are linked to Wnt signaling and FEVR., Integrin-linked kinase (ILK) is an important mediator of integrin signaling. Here Park et al. show that mice with endothelial-specific deletion of Ilk develop vascular defects that resemble familial exudative vitreoretinopathy, and identify mutations in ILK in patients with exudative vitreoretinopathy suggesting a potential role in human pathogenesis.

This study investigated the short- and long-term effects of dental adhesives doped with nano-sized bioactive glass 45S5 (BAG) on the resin–dentin interfacial bond strength. Two etch-and-rinse adhesives (Adper Scotchbond Multi-Purpose (ASB) and Solobond Plus (SB)) and one self-etch adhesive (Clearfil SE Bond (CF)) were doped with different concentrations of BAG (5, 10, and 20 wt%). The unmodified (0 wt% BAG) commercial adhesives served as control groups. Dentin of 120 molars (n = 10 per group) was treated with the different adhesives, followed by buildups with a conventional composite restorative material. From each tooth, 14 sticks were prepared for micro-tensile bond strength (µTBS) testing. The sticks were stored in simulated body fluid at 37 °C and tested after 24 h or six months for µTBS and failure mode. Data were analyzed using Kruskal–Wallis tests in combination with post-hoc Conover-tests and Wilcoxon signed-rank tests at a level of significance of a = 0.05. After 24 h and six months, both etch-and-rinse adhesives with a low BAG content (up to 10 wt% for ASB and 5 wt% for SB) showed similar µTBSs as their respective control groups (0 wt% BAG). CF showed a significant decrease in µTBS even after addition of 5 wt% BAG. At a high concentration of added BAG (20 wt%), all three adhesives showed a significant decrease in µTBS compared to the unmodified controls. The CF control group showed significantly lower µTBS after 6 months of storage than after 24 h. In contrast, the µTBS of all CF groups modified with BAG was unaffected by aging. In conclusion, the tested etch-and-rinse adhesives can be modified with up to 5 wt% (SB), or 10 wt% (ASB) of BAG without reducing their short- and long-term dentin bond strength. Moreover, the addition of nano-sized BAG may prevent long-term bond strength deterioration of a self-etch adhesive., Nanomaterials, 11 (8), ISSN:2079-4991

Summary Adrenoleukodystrophy is a peroxisomal X-linked recessive disease caused by mutations in the ABCD1 gene, located on the X-chromosome (Xq28). Gene mutations in patient with adrenoleukodystrophy induce metabolic alterations characterized by impaired peroxisomal beta-oxidation and accumulation of very long chain fatty acid (VLCFA) in plasma and in all tissues. Although nutritional intervention associated with a various mixture of oil prevents the accumulation of VLCFA, to date no causal treatment is available. Therefore, haematopoietic stem cell transplantation (HSCT) and gene therapy are allowed only for very early stages of cerebral forms diagnosed during childhood.We reported a case series describing five family members affected by X-linked adrenoleukodystrophy caused by a novel mutation of the ABCD1 gene. Particularly, three brothers were affected while the sister and mother carried the mutation of the ABCD1 gene. In this family, the disease was diagnosed at different ages and with different clinical pictures highlighting the wide range of phenotypes related to this novel mutation. In addition, these characteristics stress the relevant role of early diagnosis to properly set a patient-based follow-up. Learning points We report a novel mutation in the ABCD1 gene documented in a family group associated to an X-ALD possible Addison only phenotype. All patients present just Addison disease but with different phenotypes despite the presence of the same mutations. Further follow-up is necessary to complete discuss the clinical development. The diagnosis of ALD needs to be included in the differential diagnosis in all patients with idiopathic PAI through accurate evaluation of VLCFA concentrations and genetic confirmation testing. Early diagnosis of neurological manifestation is important in order to refer timely to HSCT. Further follow-up of these family members is necessary to characterize the final phenotype associated with this new mutation.

SummaryThe COVID-19 pandemic caused by SARS-CoV-2 has been socially and economically devastating. Despite an unprecedented research effort, effective therapeutics are still missing to limit severe disease and mortality. Using high-throughput screening, we identified acriflavine as a potent papain-like protease (PLpro) inhibitor. NMR titrations and a co-crystal structure confirm that acriflavine blocks the PLprocatalytic pocket in an unexpected binding mode. We show that the drug inhibits viral replication at nanomolar concentration in cellular models,in vivoin mice andex vivoin human airway epithelia, with broad range activity against SARS-CoV-2 and other betacoronaviruses. Considering that acriflavine is an inexpensive drug approved in some countries, it may be immediately tested in clinical trials and play an important role during the current pandemic and future outbreaks.

Anthropogenic activities, particularly fertilisation, have resulted in significant increases in reactive nitrogen (rN) in soils globally, leading to eutrophication, acidification, poor air quality, and emissions of the important greenhouse gas N2O. Understanding the partitioning of rN losses into different environmental compartments is critical to mitigate negative impacts, however, loss pathways are poorly quantified, and potential changes driven by climate warming and societal shifts are highly uncertain. We present a coupled soil-atmosphere isotope model (IsoTONE; ISOtopic Tracing Of Nitrogen in the Environment) to partition rN losses into leaching, harvest, NH3 volatilization, and production of NO, N2 and N2O based on a global dataset of soil δ15N, as well as numerous other geoclimatic and experimental datasets. The model was optimized in a Bayesian framework using a time series of N2O mixing ratios and isotopic compositions since the preindustrial era, as well as a global dataset of N2O emission factors (EF). The posterior model results showed that the total anthropogenic flux in 2020 (7.8 Tg N2O-N a-1) was dominated by indirect emissions resulting from N deposition, while the growth rate and trend in anthropogenic N2O was driven by both direct N fertilisation and deposition inputs. In contrast, inputs from fixation N drive natural N2O emissions, and were responsible for subdecadal interannual variability in total emissions.Total N gas (N2O + NO + N2) production and N2O losses were strongly dependent on geoclimate and thus spatially variable, therefore the spatial pattern of N inputs strongly impacted resulting EFs and total N2O emissions. The area-weighted global EF for N2O was 1% of anthropogenic N inputs in 2020, similar to the current IPCC default of 1.4%, however the N input-weighted global EF was 4.3%. Shifts in fertilisation inputs from the temperate Northern hemisphere towards warmer regions with higher EFs such as India and China have led to accelerating N2O emissions (1.02±0.7 Tg N2O-N a-1). In addition, N2O emissions have increased over the past decades due to climate warming (0.76±0.4 Tg N2O-N a-1). Predicted increases in fertilisation in India and Africa in the coming decades could further accelerate N2O-driven climate warming, unless mitigation measures are implemented to increase fertiliser N use efficiency and reduce N2O emission factors.

The precise measurement of the amount fraction of atmospheric nitrous oxide (N2O) is required to understand global emission trends. Analysis of the site-specific stable isotopic composition of N2O provides a means to differentiate emission sources. The availability of accurate reference materials of known N2O amount fractions and isotopic composition is critical for achieving these goals. We present the development of nitrous oxide gas reference materials for underpinning measurements of atmospheric composition and isotope ratio. Uncertainties target the World Metrological Organisation Global Atmosphere Watch (WMO-GAW) compatibility goal of 0.1 nmol mol−1 and extended compatibility goal of 0.3 nmol mol−1, for atmospheric N2O measurements in an amount fraction range of 325–335 nmol mol−1. We also demonstrate the stability of amount fraction and isotope ratio of these reference materials and present a characterisation study of the cavity ring down spectrometer used for analysis of the reference materials.

Introduction Hydrogen peroxide (H2O2) and sodium hypochlorite (NaOCl) solutions are similar in that they contain oxidizing agents with a bleaching effect. NaOCl solutions are stable at high pH, at which they also exert increased cleansing/proteolysis. H2O2 solutions, on the other hand, are natively acidic, yet gain bleaching power on organic stains when alkalized. It was investigated whether alkalizing a H2O2 solution would also let it dissolve soft tissue or increase its bleaching power on blood-stained dentin. Methods The stability of alkalized H2O2 solutions was assessed by iodometric titration. Soft tissue dissolution was investigated on porcine palatal mucosa. The bleaching effect (ΔL*) after 60 min of exposure was monitored in blood-stained human dentin using a calibrated spectrophotometer. To compare similar molarities, 2.5% H2O2 solutions were used here, and 5.0% NaOCl was used as the positive control, while non-buffered saline solution served as the negative control. Results Adding alkali (NaOH) to the H2O2 solutions rendered them unstable in a dose-dependent manner. A H2O2 solution of pH 11.2 was chosen for the main experiments (tissue dissolution and bleaching effect) and compared to a native counterpart (pH 4.7). Alkalizing the H2O2 solution had no discernible effect on its soft tissue dissolution or bleaching power (P = .75 compared to the native H2O2 solution). The NaOCl solution of similar strength had a considerably (P < .001) higher tissue dissolving and bleaching effect under current conditions. Conclusions The proteolytic/bleaching effects of NaOCl solutions are unique, and cannot be achieved by altering the pH of peroxide solutions., Journal of endodontics, 47 (2), ISSN:0099-2399, ISSN:1878-3554

Isotope ratio measurements and scale definitions are typically related to mass specectroscopy. This work discusses the challenges of optical isotope ratio spectroscopy and its prospects to significantly complement isotope ratio mass spectrometry.

Methods and techniques are described for automated measurements of greenhouse gases (GHGs) in both the laboratory and the field. Robotic systems are currently available to measure the entire range of gases evolved from soils including dinitrogen (N2). These systems usually work on an exchange of the atmospheric N2with helium (He) so that N2 fluxes can be determined. Laboratory systems are often used in microbiology to determine kinetic response reactions via the dynamics of all gaseous N species such as nitric oxide (NO), nitrous oxide (N2O), and N2. Latest He incubation techniques also take plants into account, in order to study the effect of plant–soil interactions on GHGsand N2 production. The advantage of automated in-field techniques is that GHG emission rates can be determined at a high temporal resolution. This allows, for instance, to determine diurnal response reactions (e.g. with temperature) and GHG dynamics over longer time periods.

Primary tropical forests generally exhibit large gaseous nitrogen (N) losses, occurring as nitric oxide (NO), nitrous oxide (N2O) or elemental nitrogen (N2). The release of N2O is of particular concern due to its high global warming potential and destruction of stratospheric ozone. Tropical forest soils are predicted to be among the largest natural sources of N2O; however, despite being the world’s second-largest rainforest, measurements of gaseous N-losses from forest soils of the Congo Basin are scarce. In addition, long-term studies investigating N2O fluxes from different forest ecosystem types (lowland and montane forests) are scarce. In this study we show that fluxes measured in the Congo Basin were lower than fluxes measured in the Neotropics, and in the tropical forests of Australia and South East Asia. In addition, we show that despite different climatic conditions, average annual N2O fluxes in the Congo Basin’s lowland forests (0.97 ± 0.53 kg N ha−1 year−1) were comparable to those in its montane forest (0.88 ± 0.97 kg N ha−1 year−1). Measurements of soil pore air N2O isotope data at multiple depths suggests that a microbial reduction of N2O to N2 within the soil may account for the observed low surface N2O fluxes and low soil pore N2O concentrations. The potential for microbial reduction is corroborated by a significant abundance and expression of the gene nosZ in soil samples from both study sites. Although isotopic and functional gene analyses indicate an enzymatic potential for complete denitrification, combined gaseous N-losses (N2O, N2) are unlikely to account for the missing N-sink in these forests. Other N-losses such as NO, N2 via Feammox or hydrological particulate organic nitrogen export could play an important role in soils of the Congo Basin and should be the focus of future research., The ISME Journal, 15, ISSN:1751-7362, ISSN:1751-7370

The incidence of amputation worldwide is 1.5 per 1000 habitants and the total number of amputees is about 10 million...

The source (emitter) contact of MOSFETs (IGBTs) is one of the most sensitive joints of power semiconductor modules. Due to limitations with regards to current density and power cycling reliability, state-of-the-art aluminum wire bonding is replaced, for example, by copper wire or ribbon bonding. In this study, a novel laser micro welding technology has been implemented to connect the source contact of 1.2 kV SiC MOSFETs reinforced by silver-sintered copper bond buffer top plates. The feasibility of laser bonding of 0.3 mm thick copper ribbons or 0.15 mm thick copper clips has been investigated on bond buffer top plates of 0.1 mm or 0.15 mm thickness.The laser bonding process was optimized by optical inspection of the weld seam, shear strength measurements of the bonds, analysis of polished micrograph sections, and electrical characterization of the power MOSFETs. Shear strength values exceeding 200 MPa have been repeatedly achieved for all material combinations. Results of electrical characterization are promising, and optically impeccable welding seams strongly correlate with electrically functional semiconductor devices. Power cycling of encapsulated power modules is currently ongoing, and results will be presented.An outlook is given on future measures to further improve the laser bonding process, and we discuss how laser bonding compares to other advanced topside interconnection methods.

Thermal swamps are unique ecosystems where geothermally warmed waters mix with decomposing woody biomass, hosting novel biogeochemical-cycling and lignin-degrading microbial consortia. Assembly of shotgun metagenome libraries resolved 351 distinct genomes from hot-spring (30–45 °C) and mesophilic (17 °C) sediments. Annotation of 39 refined draft genomes revealed metabolism consistent with oligotrophy, including pathways for degradation of aromatic compounds, such as syringate, vanillate, p-hydroxybenzoate, and phenol. Thermotolerant Burkholderiales, including Rubrivivax ssp., were implicated in diverse biogeochemical and aromatic transformations, highlighting their broad metabolic capacity. Lignin catabolism was further investigated using metatranscriptomics of sediment incubated with milled or Kraft lignin at 45 °C. Aromatic compounds were depleted from lignin-amended sediment over 148 h. The metatranscriptomic data revealed upregulation of des/lig genes predicted to specify the catabolism of syringate, vanillate, and phenolic oligomers in the sphingomonads Altererythrobacter ssp. and Novosphingobium ssp., as well as in the Burkholderiales genus, Rubrivivax. This study demonstrates how temperature structures biogeochemical cycling populations in a unique ecosystem, and combines community-level metagenomics with targeted metatranscriptomics to identify pathways with potential for bio-refinement of lignin-derived aromatic compounds. In addition, the diverse aromatic catabolic pathways of Altererythrobacter ssp. may serve as a source of thermotolerant enzymes for lignin valorization.

Rationale Unravelling the biogeochemical cycle of the potent greenhouse gas nitrous oxide (N2 O) is an underdetermined problem in environmental sciences due to the multiple source and sink processes involved, which complicate mitigation of its emissions. Measuring the doubly isotopically substituted molecules (isotopocules) of N2 O can add new opportunities to fingerprint and constrain its cycle. Methods We present a laser spectroscopic technique to selectively and simultaneously measure the eight most abundant isotopocules of N2 O, including three doubly substituted species - so called "clumped isotopes". For the absolute quantification of individual isotopocule abundances, we propose a new calibration scheme that combines thermal equilibration of a working standard gas with a direct mole fraction-based approach. Results The method is validated for a large range of isotopic composition values by comparison with other established methods (laser spectroscopy using conventional isotopic scale and isotope ratio mass spectrometry). Direct intercomparison with recently developed ultrahigh-resolution mass spectrometry shows clearly the advantages of the new laser technique, especially with respect to site specificity of isotopic substitution in the N2 O molecule. Conclusions Our study represents a new methodological basis for the measurements of both singly substituted and clumped N2 O isotopes. It has a high potential to stimulate future research in the N2 O community by establishing a new class of reservoir-insensitive tracers and molecular-scale insights.

The isotopic composition of nitrous oxide (N2 O) provides useful information for evaluating N2 O sources and budgets. Due to the co-occurrence of multiple N2 O transformation pathways, it is, however, challenging to use isotopic information to quantify the contribution of distinct processes across variable spatiotemporal scales. Here, we present an overview of recent progress in N2 O isotopic studies and provide suggestions for future research, mainly focusing on: analytical techniques; production and consumption processes; and interpretation and modelling approaches. Comparing isotope-ratio mass spectrometry (IRMS) with laser absorption spectroscopy (LAS), we conclude that IRMS is a precise technique for laboratory analysis of N2 O isotopes, while LAS is more suitable for in situ/inline studies and offers advantages for site-specific analyses. When reviewing the link between the N2 O isotopic composition and underlying mechanisms/processes, we find that, at the molecular scale, the specific enzymes and mechanisms involved determine isotopic fractionation effects. In contrast, at plot-to-global scales, mixing of N2 O derived from different processes and their isotopic variability must be considered. We also find that dual isotope plots are effective for semi-quantitative attribution of co-occurring N2 O production and reduction processes. More recently, process-based N2 O isotopic models have been developed for natural abundance and 15 N-tracing studies, and have been shown to be effective, particularly for data with adequate temporal resolution. Despite the significant progress made over the last decade, there is still great need and potential for future work, including development of analytical techniques, reference materials and inter-laboratory comparisons, further exploration of N2 O formation and destruction mechanisms, more observations across scales, and design and validation of interpretation and modelling approaches. Synthesizing all these efforts, we are confident that the N2 O isotope community will continue to advance our understanding of N2 O transformation processes in all spheres of the Earth, and in turn to gain improved constraints on regional and global budgets.

The present study evaluated the effects of linseed supplementation on CH4 emission and milk fatty acid composition in dairy cows measured at the group level in an experimental dairy loose housing using a tracer gas technique and individually in tied stalls and respiration chambers. Cows (2 &times, 20) were maintained in two separate sections under loose-housing conditions and received a diet supplemented with extruded linseed (L) lipids (29 g&middot, kg&minus, 1 dry matter) or a control (C) diet containing corn flour. Subsequently, 2 &times, 6 cows per dietary group were investigated in a tied-housing system and respiration chambers. Substantially higher proportions of favorable milk fatty acids were recovered in L cows when compared with C cows at the group level, making the analysis of bulk milk a suitable control instrument for retailers. Linseed supplementation resulted in a slightly lower diurnal course of CH4 emission intensity than the control at the group and individual levels. However, we found no more than a trend for a CH4 mitigating effect, unlike in other studies supplementing similar linseed lipid levels. Feed supplements in concentrations that lead to a significant reduction in CH4 emissions must show whether the reduction potential determined at the group and individual levels is comparable.

Nitrous oxide (N2O) has been for long a major focus of all greenhouse gas accounting agreements. Understanding the mechanisms of its formation and clarifying its sources and sinks are highly important for mitigating N2O emissions. In this context, measuring the doubly substituted isotopocules of N2O can add new and unique opportunities to fingerprint and constrain the biogeochemical N2O cycle, similar to other atmospheric species such as CO2, CH4, and O2.We address this challenging research field by developing and validating a laser spectroscopic technique for selective analysis of the eight most abundant N2O isotopic species including the doubly substituted isotopocules 14N15N18O, 15N14N18O, and 15N15N16O. This method is based on quantum cascade laser absorption spectroscopy (QCLAS) and reaches a precision of 0.01 – 0.20 ‰ with 1 – 2 min spectral averaging on samples of 4 μmol of N2O in N2 at 4 hPa.Furthermore, we have established a new reference frame combining two independent approaches: (1) clumped N2O isotopocule abundances were linked to stochastic distribution by equilibrating the N–O bond in the N2O molecule over activated Al2O3 at 100 and 200 °C, and (2) individual isotopocule concentrations were calibrated using a set of high-accuracy gravimetric N2O-in-N2 gas mixtures. The latter approach, applied for the first time to clumped isotope measurements, has a particular potential in realizing regular multi-point calibration for species like 15N15N16O, because no procedure for equilibration of the N–N bond has been successful yet.Results of the validation measurements, using the QCLAS method and calibration approach, are presented for a large range of δ values (approx. 100 ‰ for d15N and d18O). Inter-comparison measurements with high-resolution mass spectrometry show compatible results for bulk isotopic composition (d15N, d(458+548)), but superior performance of QCLAS for determining site-selectivity (SP, SP18). In summary, this work provides new methodological basis for the measurements of clumped N2O isotopes and has a high potential to stimulate future research in the N2O community by establishing a new class of reservoir-insensitive tracers and molecular-scale insights.

Objectives Childhood obesity is an important cause of end-stage renal disease. To date, available markers do not characterize kidney changes, especially in the early stages. kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) are already detected before the onset of proteinuria or alterations of glomerular filtration rate and thus might represent biomarkers that directly reflect kidney injury. Methods We characterize kidney injury in a group of 40 obese-prepubertal children compared to 29-healthy age- and gender matched-peers. Anthropometric measurements and body composition were determined. Fasting blood samples were collected for measurement of insulin, glucose, lipid profile, transaminases, cystatin C and creatinine. Urine samples were collected to assess urinary NGAL, KIM-1 and urinary isoprostanes. Kidney length was measured with ultrasound evaluation. Differences between the two groups were evaluated by Mann–Whitney U test, and Spearman correlation analysis was used to explore relationship between variables. Results Triglycerides, alanine transaminase (ALT), glucose, insulin, homeostasis model assessment insulin resistance, triglycerides/high-density lipoprotein (HDL)-cholesterol ratio and cystatin C values were significantly higher in obese children than normal weight peers. Creatinine values were normal and similar between the two groups, while isoprostanes were higher in obese. Obese children had larger kidney sizes, indicating organ hypertrophy. NGAL and KIM-1 were increased in obese children compared to controls. A significant association between NGAL and KIM-1 with adiposity indices, insulin status and markers of oxidative stress postulated a possible effect of obesity in inducing kidney abnormalities. KIM-1 and NGAL are directly related respectively to cystatin C and isoprostanes, supporting the ability of these biomarkers in reflecting early kidney damages in obese subjects. Conclusions These findings suggest that obese subjects exhibit a certain degree of renal damage before kidney function loss.

Stable isotope measurements of nitrogen and oxygen in nitrogen-containing molecules provide important constraints on the sources, sinks and pools of these molecules in the environment. Anammox is one of two known biological processes for converting fixed nitrogen to N2, and through its consumption of ammonium and nitrite and production of nitrate, it impacts the supply of a wide variety of fixed N molecules. Nevertheless, the isotope fractionations associated with the various anammox-associated redox reactions remain poorly constrained. We have measured the isotope effects of anammox in microbial communities enriched for the purpose of nitrogen removal from wastewater by anammox. In this system, we can replicate the ecological complexity exhibited in environmental settings, while also performing controlled experiments. We find that under a variety of conditions, the nitrogen isotope effect for the anaerobic oxidation of ammonium in this system (NH4+ to N2) is between 19‰ and 32‰, that for the reduction of nitrite (NO2– to N2) is between 7‰ and 18‰, and that for the production of nitrate (NO2– to NO3–) is between -16‰ and -43‰. We propose that these ranges reflect both (1) a mixture of signals from different anammox-performing species and (2) variation of the isotope effect associated with the anammox process within a given microbial community under different conditions. We seek to understand further what factors control this variability to better interpret stable isotope measurements of N-bearing molecules in environmental settings.

Carotenoids are orange, yellow, and red lipophilic pigments present in many fruit and vegetables, as well as other food groups. Some carotenoids contribute to vitamin A requirements. The consumption and blood concentrations of specific carotenoids have been associated with reduced risks of a number of chronic conditions. However, the interpretation of large, population-based observational and prospective clinical trials is often complicated by the many extrinsic and intrinsic factors that affect the physiologic response to carotenoids. Extrinsic factors affecting carotenoid bioavailability include food-based factors, such as co-consumed lipid, food processing, and molecular structure, as well as environmental factors, such as interactions with prescription drugs, smoking, or alcohol consumption. Intrinsic, physiologic factors associated with blood and tissue carotenoid concentrations include age, body composition, hormonal fluctuations, and variation in genes associated with carotenoid absorption and metabolism. To most effectively investigate carotenoid bioactivity and to utilize blood or tissue carotenoid concentrations as biomarkers of intake, investigators should either experimentally or statistically control for confounding variables affecting the bioavailability, tissue distribution, and metabolism of carotene and xanthophyll species. Although much remains to be investigated, recent advances have highlighted that lipid co-consumption, baseline vitamin A status, smoking, body mass and body fat distribution, and genetics are relevant covariates for interpreting blood serum or plasma carotenoid responses. These and other intrinsic and extrinsic factors are discussed, highlighting remaining gaps in knowledge and opportunities for future research. To provide context, we review the state of knowledge with regard to the prominent health effects of carotenoids.

Agriculture, and in particular dairy farming, is an important source of ammonia (NH3) and non-carbon dioxide greenhouse gas (GHG) emissions. This calls for the development and quantification of effective mitigation strategies. Our study presents the implementation of a dual tracer ratio method in a novel experimental dairy housing with two identical, but spatially separated housing areas. Modular design and flexible floor elements allow the assessment of structural, process engineering and organisational abatement measures at practical scale. Thereby, the emission reduction potential of specific abatement measures can be quantified in relation to a reference system. Emissions in the naturally ventilated housing are determined by continuous dosing of two artificial tracers (sulphur hexafluoride SF6, trifluoromethylsulphur pentafluoride SF5CF3) and their real-time detection in the ppt range with an optimized GC-ECD method. The two tracers are dosed into different experimental sections, which enables the independent assessment of both housing areas. Mass flow emissions of NH3 and GHGs are quantified by areal dosing of tracer gases and multipoint sampling as well as real-time analysis of both tracer and target gases. Validation experiments demonstrate that the technique is suitable for both areal and point emission sources and achieves an uncertainty of less than 10% for the mass emissions of NH3, methane (CH4) and carbon dioxide (CO2), which is superior to other currently available methods. Comparative emission measurements in this experimental dairy housing will provide reliable, currently unavailable information on emissions for Swiss dairy farming and demonstrate the reduction potential of mitigation measures for NH3, GHGs and potentially other pollutants.

Basic helix–loop–helix (bHLH) transcription factors are evolutionarily conserved and structurally similar proteins important in development. The temporospatial expression of atonal bHLH transcription factor 7 (ATOH7) directs the differentiation of retinal ganglion cells and mutations in the human gene lead to vitreoretinal and/or optic nerve abnormalities. Characterization of pathogenic ATOH7 mutations is needed to understand the functions of the conserved bHLH motif. The published ATOH7 in-frame deletion p.(Arg41_Arg48del) removes eight highly conserved amino acids in the basic domain. We functionally characterized the mutant protein by expressing V5-tagged ATOH7 constructs in human embryonic kidney 293T (HEK293T) cells for subsequent protein analyses, including Western blot, cycloheximide chase assays, Förster resonance energy transfer fluorescence lifetime imaging, enzyme-linked immunosorbent assays and dual-luciferase assays. Our results indicate that the in-frame deletion in the basic domain causes mislocalization of the protein, which can be rescued by a putative dimerization partner transcription factor 3 isoform E47 (E47), suggesting synergistic nuclear import. Furthermore, we observed (i) increased proteasomal degradation of the mutant protein, (ii) reduced protein heterodimerization, (iii) decreased DNA-binding and transcriptional activation of a reporter gene, as well as (iv) inhibited E47 activity. Altogether our observations suggest that the DNA-binding basic domain of ATOH7 has additional roles in regulating the nuclear import, dimerization, and protein stability., International Journal of Molecular Sciences, 23 (3), ISSN:1422-0067

Purpose: Students with social anxiety are vulnerable to hazardous drinking patterns due to their social evaluative fears and tendency to perceive alcohol use as a socially-approved, normative behav...

The nature and the amount of secondary compounds produced by plant species depends on environmental factors, which can act directly on the synthesis of the secondary metabolites. Hydrocotyle umbellata L. var. bonariensis (Lam.) Mr. Spreng has been traditionally used for medicinal purposes, including for antinociceptive, anti-inflammatory and anxiolytic-like effects, and phytochemical studies revealed its bioactive compounds. This work aimed to evaluate the effects of chemical and organic fertilization, and the soil base saturation correction in the H. umbellata crop in dystrophic yellow latosol soil in the production of the secondary metabolites (total phenolic, total flavonoid and hibalactone). The plant was cultivated in the soil of a rural property in the municipality of Anápolis (Goiás state). The experimental design was completely randomized in a controlled environment. The experiment with fertilization had five treatments (control; cattle manure; poultry manure; chemical fertilization; chemical and organic fertilization) and the experiment with liming included the correction of soil base saturation to 20%, 40%, 60% and 80%. The results in most of the two experiments were not statistically significant in the content of the metabolites studied. In the fertilization experiment, the control and manure treatments were statistically better in the content of total phenols in aerial mass analysis. Regarding the saturation correction experiment, the treatment without liming afforded higher levels of hibalactone content (considering the whole plant) and total phenolic content (considering the air mass). The treatment with correction of saturation to 40% afforded higher levels of total phenolic (considering the whole plant). La naturaleza y cantidad de compuestos secundarios producidos por especies vegetales dependen de factores ambientales, que pueden actuar directamente en la síntesis de metabolitos secundarios. Hydrocotyle umbellata var. bonariensis (Lam.) Spreng se ha utilizado tradicionalmente con fines medicinales, incluso para efectos antinociceptivos, antiinflamatorios y ansiolíticos, y los estudios fitoquímicos han revelado sus compuestos bioactivos. Este trabajo tuvo como objetivo evaluar los efectos de la fertilización química y orgánica y la corrección de la saturación de bases del suelo en el cultivo de H. umbellata en latosol amarillo distrófico sobre la producción de metabolitos secundarios (fenólico total, flavonoide total e hibalactona). La planta se cultivó en el suelo de una propiedad rural de la ciudad de Anápolis (GO). El diseño experimental fue completamente aleatorio en un ambiente controlado. El experimento de fertilización tuvo cinco tratamientos (control; estiércol de ganado; estiércol de aves de corral; fertilización química; fertilización química y orgánica) y el experimento de encalado incluyó la corrección de la saturación de la base del suelo para 20%, 40%, 60% y 80%. Los resultados en la mayoría de los dos experimentos no fueron estadísticamente significativos en el contenido de los metabolitos estudiados. En el experimento de fertilización, los tratamientos de control y estiércol fueron estadísticamente mejores en el contenido total de fenol en el análisis de masa aérea. En relación al experimento de corrección de saturación, el tratamiento sin encalado proporcionó niveles más altos de hibalactona (considerando toda la planta) y contenido fenólico total (considerando la masa de aire). El tratamiento con corrección de saturación al 40% proporcionó niveles más altos de fenoles totales (considerando toda la planta). A natureza e a quantidade de compostos secundários produzidos pelas espécies vegetais dependem de fatores ambientais, que podem atuar diretamente na síntese dos metabólitos secundários. Hydrocotyle umbellata L. var. bonariensis (Lam.) Spreng tem sido tradicionalmente usado para fins medicinais, incluindo para efeitos antinociceptivos, antiinflamatórios e ansiolíticos, e estudos fitoquímicos revelaram seus compostos bioativos. Este trabalho teve como objetivo avaliar os efeitos da fertilização química, orgânica e da correção da saturação por bases do solo na cultura de H. umbellata em latossolo amarelo distrófico na produção dos metabólitos secundários (fenól total, flavonóide total e hibalactona). A planta foi cultivada no solo de uma propriedade rural no município de Anápolis (GO). O delineamento experimental foi inteiramente casualizado em ambiente controlado. O experimento com adubação teve cinco tratamentos (controle; esterco bovino; esterco aviário; adubação química; adubação química e orgânica) e o experimento com calagem incluiu a correção da saturação por bases do solo para 20%, 40%, 60% e 80%. Os resultados na maioria dos dois experimentos não foram estatisticamente significativos no conteúdo dos metabólitos estudados. No experimento de fertilização, os tratamentos controle e esterco foram estatisticamente melhores no teor de fenóis totais na análise da massa aérea. Em relação ao experimento de correção da saturação, o tratamento sem calagem proporcionou maiores teores de hibalactona (considerando toda a planta) e teor de fenólicos totais (considerando a massa de ar). O tratamento com correção da saturação para 40% proporcionou maiores teores de fenólicos totais (considerando toda a planta).

Methane (CH4) emissions from dairy housings, mainly originating from enteric fermentation of ruminating animals, are a significant source of greenhouse gases. The quantification of emissions from naturally ventilated dairy housings is challenging due to the spatial distribution of sources (animals, housing areas) and variable air exchange. The inverse dispersion method (IDM) is a promising option, which is increasingly used to determine gaseous emissions from stationary sources, as it offers high flexibility in the application at reasonable costs. We used a backward Lagrangian stochastic model combined with concentration measurements by open-path tunable diode laser spectrometers placed up- and downwind of a naturally ventilated housing with 40 dairy cows to determine the CH4 emissions. The average emissions per livestock unit (LU) were 317 (±44) g LU−1 d−1 and 267 (±43) g LU−1 d−1 for the first and second campaign, in September – October and November – December, respectively. For each campaign, inhouse tracer ratio measurements (iTRM) were conducted in parallel during two subperiods. For simultaneous measurements, IDM showed average emissions which were lower by 8% and 1% than that of iTRM, respectively, for the two campaigns. The differences are within the uncertainty range of any of the two methods. The IDM CH4 emissions were further analysed by wind direction and atmospheric stability and no differences in emissions were found. Overall, IDM showed its aptitude to accurately determine CH4 emissions from dairy housings or other stationary sources if the site allows adequate placement of sensors up- and downwind in the prevailing wind direction. To acquire reliable emission data, depending on the data loss during measurements due to quality filtering or instrument failure, a measuring time of at least 10 days is required.

Lutein, a yellow xanthophyll carotenoid found in egg yolks and many colorful fruits and vegetables, has gained public health interest for its putative role in visual performance and reducing the risk of age-related macular degeneration. The National Academies of Sciences, Engineering and Medicine’s recommended Dietary Reference Intakes (DRIs) focus on preventing deficiency and toxicity, but there is a budding interest in establishing DRI-like guidelines for non-essential bioactives, like lutein, that promote optimal health and/or prevent chronic diseases. Lupton et al. developed a set of nine criteria to determine whether a bioactive is ready to be considered for DRI-like recommendations. These criteria include: (1) an accepted definition; (2) a reliable analysis method; (3) a food database with known amounts of the bioactive; (4) cohort studies; (5) clinical trials on metabolic processes; (6) clinical trials for dose–response and efficacy; (7) safety data; (8) systematic reviews and/or meta-analyses; (9) a plausible biological rationale. Based on a review of the literature supporting these criteria, lutein is ready to be considered for intake recommendations. Establishing dietary guidance for lutein would encourage the consumption of lutein-containing foods and raise public awareness about its potential health benefits.

Finding the appropriate cues to trigger the desired differentiation is a challenge in tissue engineering when stem cells are involved. In this regard, three-dimensional environments are often compared to cells' two-dimensional culture behaviour (plastic culture dish). Here, we compared the gene expression pattern of human adipose-derived stem cells (ASC) seeded in a three-dimensional (3D) electrospun mesh and on a two-dimensional (2D) film - both of exactly the same material. Additionally, we conducted experiments with a scaffold floating above a film to investigate two-way paracrine effects (co-system). Electrospun meshes (3D scaffolds) and films (2D), consisting either of pristine poly-lactic-co-glycolic acid (PLGA) or of PLGA-containing dispersed amorphous calcium phosphate nanoparticles (PLGA/aCaP), were seeded with ASCs and cultured either in Dulbecco Minimum Essential Medium (DMEM) or in osteogenic medium. After two weeks, minimum stem cell criteria markers as well as typical markers for osteogenesis, endothelial cell differentiation, adipogenesis and chondrogenesis were analysed by quantitative real-time PCR. Interestingly, mostly osteogenic genes of cells seeded on 3D meshes were upregulated compared to those on 2D films, while stem cell markers seemed to be only slightly affected. Runx2 and osteocalcin showed an especially strong upregulation under all conditions, while most other factors analysed for 2D/3D changes were highly dependent on the material composition, the culture medium and on paracrine signalling effects. The beneficial 3D environment for stem cells found in many studies has therefore not to be attributed to the third dimension alone and should carefully be compared to 2D films fabricated of the same material. Furthermore, paracrine interactions triggering differentiation are not negligible.

Understanding and quantifying the biogeochemical cycle of N2O is essential to develop effective N2O emission mitigation strategies. This study presents a novel, fully automated measurement techniqu...

Epidemiological studies suggest that consumption of lutein-rich foods may be of benefit in promoting cognitive health. Among the carotenoids, lutein and it isomer, zeaxanthin, are the only 2 that cross the blood-retina barrier to form macular pigment (MP) in the retina. Lutein also preferentially accumulates in the human brain across multiple life stages. Lutein concentrations in the retina of both human and nonhuman primates are significantly correlated with their levels in matched brain tissues, allowing for the use of MP density, which can be measured noninvasively in humans, as a biomarker of lutein in the brain. This has important implications for intervention studies involving lutein given that MP density, such as brain lutein, has been reported to be significantly related to cognitive function in adults. Although less is known about infants, cross-sectional studies have shown that breast milk lutein content enriches lutein in brain tissue and is related to infant recognition memory scores. Intervention studies in adults indicate that lutein may positively affect cognitive performance, and this effect may be influenced by the omega-3 fatty acid, docosahexaenoic acid. Although lutein is not an essential nutrient, efforts may be warranted to establish age-specific recommended intakes for this dietary bioactive for promotion of cognitive health.

Introduction A clinically useful all-in-one endodontic irrigant with combined proteolytic and decalcifying properties is still elusive. In this study, the chemical effects of dissolving the tetrasodium salts of 1-hydroxyethane 1,1-diphosphonic acid (Na 4 HEDP) or Na 4 EDTA directly in sodium hypochlorite (NaOCl) irrigants in polypropylene syringes were assessed during the course of 1 hour. Methods The solubility of the salts in water was determined. Their compatibility with 1% and 5% NaOCl was measured by iodometric titration and in a calcium complexation experiment by using a Ca 2+ -selective electrode. Results The salts dissolved within 1 minute. The dissolution maximum of Na 4 HEDP in water (wt/total wt) was 44.6% ± 1.6%. The corresponding dissolution maximum of Na 4 EDTA was 38.2% ± 0.8%. Na 4 HEDP at 18% in 5% NaOCl caused a mere loss of 16% of the initially available chlorine during 1 hour. In contrast, a corresponding mixture between NaOCl and the Na 4 EDTA salt caused 95% reduction in available chlorine after 1 minute. Mixtures of 3% Na 4 EDTA with 1% NaOCl were more stable, but only for 30 minutes. Na 4 HEDP lost 24% of its calcium complexation capacity after 60 minutes. The corresponding loss for Na 4 EDTA was 34%. Conclusions The compatibility and solubility of particulate Na 4 HEDP with/in NaOCl solutions are such that these components can be mixed and used for up to 1 hour. In contrast, short-term compatibility of the Na 4 EDTA salt with NaOCl solutions was considerably lower, decreasing at higher concentrations of either compound. Especially for Na 4 HEDP but also for Na 4 EDTA, the NaOCl had little effect on calcium complexation.