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An artificial metalloenzyme is compartmentalized and evolved in vivo for olefin metathesis—an archetypal organometallic reaction without equivalent in nature; the evolved metathase reveals broad substrate scope and compares favourably with commercial catalysts. Directed evolution of an artificial metalloenzyme Artificial metalloenzymes—made by incorporating an abiotic cofactor within a protein scaffold—have the potential to engineer non-natural in vivo reactions. To be of practical use, such catalysts must maintain their activity in a cellular environment, which means overcoming the tendency of introduced metal cofactors to be inhibited by cellular components. This paper demonstrates that directed evolution can overcome this difficulty. Markus Jeschek et al . report on the in vivo evolution of a ruthenium–protein complex that can catalyse olefin metathesis—an archetypal organometallic reaction with no equivalent in nature—in the periplasm of Escherichia coli . The evolved metathase compares favorably with commercial catalysts, shows activity for different metathesis substrates and can be further evolved in different directions by adjusting the protocol. The field of biocatalysis has advanced from harnessing natural enzymes to using directed evolution to obtain new biocatalysts with tailor-made functions 1 . Several tools have recently been developed to expand the natural enzymatic repertoire with abiotic reactions 2 , 3 . For example, artificial metalloenzymes, which combine the versatile reaction scope of transition metals with the beneficial catalytic features of enzymes, offer an attractive means to engineer new reactions. Three complementary strategies exist 3 : repurposing natural metalloenzymes for abiotic transformations 2 , 4 ; in silico metalloenzyme (re-)design 5 , 6 , 7 ; and incorporation of abiotic cofactors into proteins 8 , 9 , 10 , 11 . The third strategy offers the opportunity to design a wide variety of artificial metalloenzymes for non-natural reactions. However, many metal cofactors are inhibited by cellular components and therefore require purification of the scaffold protein 12 , 13 , 14 , 15 . This limits the throughput of genetic optimization schemes applied to artificial metalloenzymes and their applicability in vivo to expand natural metabolism. Here we report the compartmentalization and in vivo evolution of an artificial metalloenzyme for olefin metathesis, which represents an archetypal organometallic reaction 16 , 17 , 18 , 19 , 20 , 21 , 22 without equivalent in nature. Building on previous work 6 on an artificial metallohydrolase, we exploit the periplasm of Escherichia coli as a reaction compartment for the ‘metathase’ because it offers an auspicious environment for artificial metalloenzymes, mainly owing to low concentrations of inhibitors such as glutathione, which has recently been identified as a major inhibitor 15 . This strategy facilitated the assembly of a functional metathase in vivo and its directed evolution with substantially increased throughput compared to conventional approaches that rely on purified protein variants. The evolved metathase compares favourably with commercial catalysts, shows activity for different metathesis substrates and can be further evolved in different directions by adjusting the workflow. Our results represent the systematic implementation and evolution of an artificial metalloenzyme that catalyses an abiotic reaction in vivo , with potential applications in, for example, non-natural metabolism.

This protocol describes a mutagenesis screening method for the optimization of artificial streptavidin-based metalloenzymes. Artificial metalloenzymes (ArMs) based on the incorporation of a biotinylated metal cofactor within streptavidin (Sav) combine attractive features of both homogeneous and enzymatic catalysts. To speed up their optimization, we present a streamlined protocol for the design, expression, partial purification and screening of Sav libraries. Twenty-eight positions have been subjected to mutagenesis to yield 335 Sav isoforms, which can be expressed in 24-deep-well plates using autoinduction medium. The resulting cell-free extracts (CFEs) typically contain >1 mg of soluble Sav. Two straightforward alternatives are presented, which allow the screening of ArMs using CFEs containing Sav. To produce an artificial transfer hydrogenase, Sav is coupled to a biotinylated three-legged iridium pianostool complex Cp*Ir(Biot- p -L)Cl (the cofactor). To screen Sav variants for this application, you would determine the number of free binding sites, treat them with diamide, incubate them with the cofactor and then perform the reaction with your test compound (the example used in this protocol is 1-phenyl-3,4-dihydroisoquinoline). This process takes 20 d. If you want to perform metathesis reactions, Sav is coupled to a biotinylated second-generation Grubbs-Hoveyda catalyst. In this application, it is best to first immobilize Sav on Sepharose-iminobiotin beads and then perform washing steps. Elution from the beads is achieved in an acidic reaction buffer before incubation with the cofactor. Catalysis using your test compound (in this protocol, 2-(4-( N , N -diallylsulfamoyl)phenyl)- N , N , N -trimethylethan-1-aminium iodide) is performed using the formed metalloenzyme. Screening using this approach takes 19 d.

Herein we report on a 96-well plate assay based on the fluorescence resulting from the ring-closing metathesis of two profluorophoric substrates. To demonstrate the validity of the approach, four commercially available ruthenium-metathesis catalysts were evaluated in six different solvents. The results from the fluorescent assay agree well with HPLC conversions, validating the usefulness of the approach.

Nine biotinylated Grubbs–Hoveyda and Grubbs-type metathesis catalysts were synthesized and evaluated in ring closing metathesis reactions of N -tosyl diallylamine and 5-hydroxy-2-vinylphenyl acrylate. Their catalytic activity in organic- and aqueous solvents was compared with the second generation Grubbs–Hoveyda catalyst. The position of the biotin-moiety on the N -heterocyclic carbene was found to critically influence the catalytic activity of the corresponding ruthenium-based catalysts. Graphical Abstract

Accurately characterizing land surface changes with Earth Observation requires geo-located ground truth. In the European Union (EU), a tri-annual surveyed sample of land cover and land use has been collected since 2006 under the Land Use/Cover Area frame Survey (LUCAS). A total of 1351293 observations at 651780 unique locations for 106 variables along with 5.4 million photos were collected during five LUCAS surveys. Until now, these data have never been harmonised into one database, limiting full exploitation of the information. This paper describes the LUCAS point sampling/surveying methodology, including collection of standard variables such as land cover, environmental parameters, and full resolution landscape and point photos, and then describes the harmonisation process. The resulting harmonised database is the most comprehensive in-situ dataset on land cover and use in the EU. The database is valuable for geo-spatial and statistical analysis of land use and land cover change. Furthermore, its potential to provide multi-temporal in-situ data will be enhanced by recent computational advances such as deep learning.

Background The number of obese children is rising worldwide. Many studies have investigated single determinants of children’s body mass index (BMI), yet studies measuring determinants at different potential levels of influence are sparse. The aim of this study is to investigate the independent role of parental socioeconomic position (SEP), additional family factors at the micro level, as well as early childhood education and care (ECEC) centre characteristics at the meso level regarding BMI. Methods Analyses used the baseline data of the PReschool INtervention Study (PRINS) including up to 1,151 children from 53 ECEC centres. Multi-level models first estimated the associations of parental SEP indicators (parental school education, vocational training, and household income) with the children’s standard deviation scores for BMI (SDS BMI, standardised for age and gender). Second, structural (number of siblings), psychosocial (strained family relationships), and nutrition behavioural (soft-drink consumption, frequency of fast-food restaurant visits) family factors at the micro level were included. Third, characteristics of the ECEC centre at the meso level in terms of average group size, the ratio of overweight children in the group, ECEC centre type (all-day care), and the location of the ECEC centre (rural vs urban) were included. All analyses were stratified by gender and adjusted for age, migration background, and parental employment status. Results Estimates for boys and girls appeared to differ. In the full model, for boys the parental SEP indicators were not related to SDS BMI. Factors related to SDS BMI in boys were: two or more siblings; B = -.55; p  = 0.045 [ref.: no sibling]), the characteristics of the ECEC centre in terms of average group size (20 – 25 children; B = -.54; p  = 0.022 [ref.: < 20 children]), and the ratio of overweight children (more overweight children B = -1.39; p  < 0.001 [ref.: few overweight children]). For girls the number of siblings (two and more siblings; B = .67; p  = 0.027 [ref.: no sibling]) and average group size (> 25 children; B = -.52; p  = 0.037 [ref.: < 20 children]) were related to SDS BMI. Conclusions The BMI of preschool children appears to be associated with determinants at the micro and meso level, however with some gender differences. The identified factors at the micro and meso level appear largely modifiable and can inform about possible interventions to reduce obesity in preschool children.

The effective calcineurin inhibitor (CNI) tacrolimus (Tac) is an integral part of the standard immunosuppressive regimen after renal transplantation (RTx). However, as a potent CNI it has nephrotoxic potential leading to impaired renal function in some cases. Therefore, it is of high clinical impact to identify factors which can predict who is endangered to develop CNI toxicity. We hypothesized that the Tac metabolism rate expressed as the blood concentration normalized by the dose (C/D ratio) is such a simple predictor. Therefore, we analyzed the impact of the C/D ratio on kidney function after RTx. Renal function was analyzed 1, 2, 3, 6, 12 and 24 months after RTx in 248 patients with an immunosuppressive regimen including basiliximab, tacrolimus, mycophenolate mofetil and prednisolone. According to keep the approach simple, patients were split into three C/D groups: fast, intermediate and slow metabolizers. Notably, compared with slow metabolizers fast metabolizers of Tac showed significantly lower estimated glomerular filtration rate (eGFR) values at all the time points analyzed. Moreover, fast metabolizers underwent more indication renal biopsies (p = 0.006) which revealed a higher incidence of CNI nephrotoxicity (p = 0.015) and BK nephropathy (p = 0.024) in this group. We herein identified the C/D ratio as an easy calculable risk factor for the development of CNI nephrotoxicity and BK nephropathy after RTx. We propose that the simple C/D ratio should be taken into account early in patient's risk management strategies.

Although health inequalities in adolescence are well documented, the underlying mechanisms remain unclear. Few studies have examined the role of the family in explaining the association between the family's socioeconomic position and adolescents' self-rated health. The current study aimed to explore whether the association between socioeconomic position and self-rated health was mediated by familial determinants. Using data from wave 2 of the\"German Health Interview and Examination Survey for Children and Adolescents\" (KiGGS) (1,838 female and 1,718 male 11- to 17-year-olds), linear regression analyses were conducted to decompose the total effects of income, education, occupational status, socioeconomic position index and adolescents' subjective social status on self-rated health into direct effects and indirect effects through familial determinants (family cohesion, parental well-being, parental stress, parenting styles, parental obesity, smoking and sporting activity). A significant total effect of all socioeconomic position indicators on self-rated health was found, except for income in male adolescents. In female adolescents, more than 70% of the total effects of each socioeconomic position indicator were explained by familial mediators, whereas no significant direct effects remained. The most important mediator was parental well-being, followed by family cohesion, parental smoking and sporting activity. In male adolescents, the associations between income, parental education, the socioeconomic position index and subjective social status were also mediated by familial determinants (family cohesion, parental smoking, obesity and living in a single-mother family). However, a significant direct effect of subjective social status remained. The analysis revealed how a family's position of socioeconomic disadvantage can lead to poorer health in adolescents through different family practices. The family appears to play an important role in explaining health inequalities, particularly in female adolescents. Reducing health inequalities in adolescence requires policy interventions (macro-level), community-based strategies (meso-level) and programs to improve parenting and family functioning (micro-level).

Fast metabolism of immediate-release tacrolimus (IR-Tac) is associated with decreased kidney function after renal transplantation (RTx) compared to slow metabolizers. We hypothesized, by analogy, that fast metabolism of extended-release tacrolimus (ER-Tac) is associated with worse renal function. We analyzed data from patients who underwent RTx at three different transplant centers between 2007 and 2016 and received an initial immunosuppressive regimen with ER-Tac, mycophenolate, and a corticosteroid. Three months after RTx, a Tac concentration to dose ratio (C/D ratio) < 1.0 ng/ml · 1/mL defined fast ER-Tac metabolism and ≥ 1.0 ng/ml · 1/mL slow metabolism. Renal function (estimated glomerular filtration rate, eGFR), first acute rejection (AR), conversion from ER-Tac, graft and patient survival were observed up to 60-months. 610 RTx patients were divided into 192 fast and 418 slow ER-Tac metabolizers. Fast metabolizers showed a decreased eGFR at all time points compared to slow metabolizers. The fast metabolizer group included more patients who were switched from ER-Tac (p < 0.001). First AR occurred more frequently (p = 0.008) in fast metabolizers, while graft and patient survival rates did not differ between groups (p = 0.529 and p = 0.366, respectively). Calculation of the ER-Tac C/D ratio early after RTx may facilitate individualization of immunosuppression and help identify patients at risk for an unfavorable outcome.

Given the gap between patients in need of a renal transplantation (RTx) and organs available, transplantation centers increasingly accept organs of suboptimal quality, e.g. from donors with acute kidney injury (AKI). To determine the outcome of kidney transplants from deceased donors with AKI (defined as ≥ AKIN stage 1), all 107 patients who received a RTx from donors with AKI between August 2004 and July 2014 at our center were compared to their respective consecutively transplanted patients receiving kidneys from donors without AKI. 5-year patient and graft survival, frequencies of delayed graft function (DGF), acute rejections and glomerular filtration rate (eGFR, CKD-EPI) were assessed. Patient survival was similar in both groups, whereas death-censored and overall graft survival were decreased in AKI kidney recipients. AKI kidney recipients showed higher frequencies of DGF and had a reduced eGFR at 7 days, three months and one and three years after RTx. However, mortality was noticeably lower compared to waiting list candidates. Rejection-free survival was similar between groups. In our cohort, both short-term and long-term renal function was inferior in recipients of AKI kidneys, while patient survival was similar. Our data indicates that recipients of donor AKI kidneys should be carefully selected and additional factors impairing short- and long-term outcome should be minimized to prevent further deterioration of graft function.