We wished to examine whether mRNA would be destabilized in the edc3∆ lsm4∆C mutant as compared to the wild-type in response to osmotic stress, when P bodies are intense and numerous. Our results show that the edc3∆ lsm4∆C mutant limits the mRNA stability in response to osmotic stress, while the magnitude of stabilization was similar as compared to the wild-type. The reduced mRNA stability in the edc3∆ lsm4∆C mutant was correlated with a shorter PGK1 poly(A) tail. Similarly, the MFA2 mRNA was more rapidly deadenylated as well as significantly stabilized in the ccr4∆ deadenylation mutant in the edc3∆ lsm4∆C background. These results suggest a role for these decapping factors in stabilizing mRNA and may implicate P bodies as sites of reduced mRNA degradation.Yeast is exposed to changing environmental conditions and must adapt its genetic program to provide a homeostatic intracellular environment. An important stress for yeast in the wild is high osmolarity. A key response to this stress is increased mRNA stability primarily by the inhibition of deadenylation. We previously demonstrated that mutations in decapping activators ( Saccharomyces cerevisiae must adapt rapidly to environmental changes to maintain intracellular osmotic homeostasis. An example of these rapid changes in extracellular osmolarity includes the transition from an isotonic to a hypo-osmotic environment by being washed off a fruit in the rain. Alternatively, yeast can be exposed to high osmolarity environments arising from, for example, the high sugar content of ruptured fruit. These types of changes require rapid sensing and adaptation to maintain intracellular osmotic homeostasisA change in environmental osmolarity is a fundamental stress for cells as they strive to maintain consistent intracellular osmolarity to allow proper cellular functioning. Single-celled organisms that possess motility can avoid unfavourable osmotic conditions by altering their location, whereas multicellular organisms often maintain constant osmolarity in their bodily fluids. For example, osmolarity is maintained at a constant range in humans by the kidneys, maintaining a normal variation of less than three per centYeast has several key responses to changes in osmolarity. An initial response to a hyper-osmotic shock results in the inhibition of translation within 10–15 minutes, which can persist for an hour or more depending on its intensity34578911121314111213111314edc3∆ lsm4∆C) reduces mRNA stability without additional stressedc3∆ lsm4∆C mutant would contribute to mRNA stability in response to osmotic stress. Our results demonstrate that the edc3∆ lsm4∆C mutant has reduced mRNA stabilization in response to osmotic stress for all of the mRNAs we examined. However, the fold stabilization upon hyper-osmotic stress was similar in both the wild-type and edc3∆ lsm4∆C mutant. By examining mRNA degradation mutants and poly(A) length, these results suggest that mRNA deadenylation can be affected in the edc3∆ lsm4∆C mutant, which results in more rapid mRNA degradation. Finally, although we did not observe a significant role for the 3′-to-5′ mRNA degradation pathway in the edc3∆ lsm4∆C mutant in response to osmotic stress our results suggest that it could play an important role in the degradation of mRNA found in P bodies.P bodies contain factors involved in deadenylation, the initial stage of mRNA degradation, as well as the components of the decapping-dependent 5′-to-3′ mRNA decay pathway16171819edc3∆ lsm4∆C mutant has been reported previously as being defective in P body assembly under stress19edc3∆ lsm4∆C mutant were quantified for area, intensity and number per cell to provide a consistent assessment of P body levelsedc3∆ lsm4∆C mutant did not  ADH1 and (2) RPL3 and (3) CYH2/RPL28. Again, we observed the same trend in the edc3∆ lsm4∆C mutant  tail, the shortening of which is generally a prerequisite for subsequent degradation17PGK1 had a shorter poly(A) tail in the edc3∆ lsm4∆C mutant under both unstressed and hyper-osmotic conditions  tail lengths (data not shown). To more rigorously examine this mRNA, we performed a transcriptional shut-off experiment under osmotic stress  and an exosome (ski2∆) mutant. However, we did not use a mutant from the decapping-dependent pathway, xrn1∆, as it already displays exceptionally long half-lives for the mRNAs we are examining without osmotic stressPGK1 and MFA2 poly(A) tails were shorter at steady-state or had faster deadenylation, we examined whether the Ccr4/Not complex was acting to limit hyper-osmotic shock-induced mRNA stabilization. Ccr4 is the primary enzyme for cytoplasmic deadenylation in yeast and is required for catalytic activity of the Ccr4/Not complexedc3∆ lsm4∆C strain when compared to ccr4∆, MFA2 was significantly stabilized, whereas PGK1 surprisingly exhibited the opposite effect  had a fragment to full length ratio suggestive of slower decapping (33edc3∆ and lsm4∆C) is consistent with the longer half-life observed in the mRNAs examined for the lsm4∆C and edc3∆ strains1921edc3∆ lsm4∆C mutant has a significantly reduced half-life . Finally, under osmotic stress the apparent in vivo mRNA decapping is similar in all strains (These results taken together suggest that under unstressed conditions the single mRNA decapping mutants’ apparent  strains  consiste strains .in vivo mRNA decapping assay (fragment to full-length mRNA ratio) revealed that the edc3∆ lsm4∆C strain displays slower apparent decapping  when the exosome activity is removed. We used the ski2∆ mutant, a component of the Ski2/3/8 complex, which is defective in cytoplasmic exosome-mediated degradationski2∆ mutantMFA2 mRNA as the degradation of the decay intermediate by the exosome has been calculated to be an order of magnitude slower than the decapping and 5′-to-3′ degradation that generated itTo examine this possibility, we first determined the effect on apparent MFA2 mRNA ratio for ski2∆ mutants in the wild-type and edc3∆ lsm4∆C backgrounds, we found two major results  tails are more consistent with faster deadenylation. This model is supported by the mRNA stability of both the wild-type yeast and the edc3∆ lsm4∆C mutant combined with the exosome mutant ski2∆  tracts in their 3′UTR  and hygromycin B (hphMX4) antibiotic resistance genes as described previouslyYeast genomic knock-out strains were generated using homologous recombination with regions of homology approximately 50 nucleotides upstream of the ATG and 50 nucleotides downstream of the stop codon. The gene of interest was replaced with the nourseothricin  with an Olympus 60 × 1.4NA objective without binning. Microscopic images were deconvolved using the classical maximum likelihood estimation algorithm in Huygens Essential 4.4 , except those deconvolved using the Deltavision standard settings as noted in the figure legends. Each of the resulting images was depicted by means of sum intensity projections from a Z series of 20 0.25-μm thickness slices displayed with Fiji−1 thiolutin or by the use of an inducible GAL promoter integrated into the genome, which controls the MFA2pG and PGK1pG mRNAs. The optimal thiolutin concentration was obtained by titration and half-life determinationGAL promoter was repressed by the addition of 4% dextrose to the cell medium after washing as indicated. Yeast were grown in SDC-Trp for the unstressed half-life determinations for ADH1, RPL3 and CYH2), otherwise in YEP with the indicated carbon source. The quantification of the northern blot bands was performed with Quantity One (Bio-Rad). The mRNA half-lives were determined by the best linear fits of the mRNA band intensities normalized to the SCR1 loading control. The mRNA half-lives were subjected to two-tailed unpaired Student’s t tests. Statistical significance was determined with a p-value cut-off of 0.05. The oligonucleotides used in this study are indicated in RNA was purified by lysing yeast cells with glass beads; this was followed by a phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. Northern blots were probed with the oligonucleotides listed in How to cite this article: Huch, S. and Nissan, T. An mRNA decapping mutant deficient in P body assembly limits mRNA stabilization in response to osmotic stress. Sci. Rep.7, 44395; doi: 10.1038/srep44395 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


This study investigates the mixed convection flow of Jeffrey liquid by an impermeable inclined stretching cylinder. Thermal radiation and non-uniform heat source/sink are considered. The convective boundary conditions at surface are imposed. Nonlinear expressions of momentum, energy and concentration are transformed into dimensionless systems. Convergent homotopic solutions of the governing systems are worked out by employing homotopic procedure. Impact of physical variables on the velocity, temperature and concentration distributions are sketched and discussed. Numerical computations for skin friction coefficient, local Nusselt and Sherwood numbers are carried out. It is concluded that velocity field enhances for Deborah number while reverse situation is observed regarding ratio of relaxation to retardation times. Temperature and heat transfer rate are enhanced via larger thermal Biot number. Effect of Schmidt number on the concentration and local Sherwood number is quite reverse. Flow analyses of non-Newtonian liquids have significantly attracted the attention of researchers and scientists during the past few decades –5. It isThe features of stretched surface within frame of mixed convection flow have abundant applications in engineering and industry. Free convection has significance within the light of gravitational force. Heat and mass transfer in flow is also affected by both buoyancy forces and stretching. Such phenomena is important in solar energy systems, cooling of electronic devices, heat exchangers set in a low velocity situation, boilers, atomic reactors cooling during emergency shutdown, cooling of combustion chamber wall in a gas turbine, defroster system, automobile demister and flows in the ocean and atmosphere. Ashraf et al.  examinedHeat transfer radiation is very essential in the design of dependable equipment, gas turbines and several propulsion gadgets for aircraft missiles, nuclear plants, space vehicles and satellites. Likewise the impacts of thermal radiation on the free and forced/constrained convection flows are significant in space technology and procedures including high temperature . AnalysiEffects of heat source/sink have importance in issues managing with chemical reactions geonuclear repositions and those stressed with dissociating liquids. Heat source/sink can be presumed constant, temperature-dependent or space-dependent. Unsteady magnetohydrodynamic (MHD) flow of pseudoplastic nanofluid in a finite thin film towards a stretching surface with internal heat generation is explored analytically by Lin et al. . Ramesh The purpose of present article is three fold. Firstly to examine non-uniform heat source and sink effects in mixed convection flow of Jeffrey fluid. Secondly to investigate convectively heated cylinder. For this purpose the convective conditions of both heat and mass transfer are considered. Thirdly to address thermal radiation. Therefore our main purpose is to investigate such salient features in the mixed convection flow of Jeffrey liquid by an inclined stretching cylinder. This article is prepared as follows. Mathematical modeling is given in section two. Sections three and four deal with the development and convergence of series solutions. Discussion is arranged in section five. The velocity, temperature and concentration are calculated through homotopy analysis approach –40. Solux–axis is along the axial direction of cylinder and r–axis is normal to the cylinder. The convective boundary conditions for a cylinder are employed. The conservation laws after using the boundary layer approximations are reduced into the forms:We analyze the steady and axisymmetric mixed convection radiative flow of an incompressible Jeffrey fluid due to an inclined cylinder. The physical sketch for the flow is shown in The associated boundary conditions areu, v) the components of velocity in the  directions, ν = (μ/ρ) the fluid kinematic viscosity, μ the dynamic viscosity of fluid, λ1 the relaxation to retardation times ratio, g the gravitational acceleration, λ2 the retardation time, ρ the fluid density, α the angle of inclination, βT the coefficient of thermal expansion and βC the coefficient of concentration expansion, cp the specific heat, k the thermal conductivity, q‴ the non-uniform heat source/sink, uw(x) the linear stretching velocity, u0 the reference velocity, qr the radiative heat flux, Tf the convective fluid temperature, Cf the convective fluid concentration, ht the coefficient of convective heat transfer, hc the coefficient of convective mass transfer, T and T∞ the fluid and ambient temperatures, D the mass diffusivity, C and C∞ the fluid and ambient concentrations respectively and l the characteristic length.Here we see that second and third terms on the right hand side (R. H. S) in Eq  show theqr through Rosseland approximation is given as follows [σ* denotes the Stefan-Boltzmann constant and k* shows mean absorption coefficient. We assume that the difference of temperature inside the flow is such that the term T4 might be characterized as a linear function of temperature. This is achieved by expanding T4 in a Taylor series about T∞ as follows:Radiative heat flux  follows :qr=−4σ*Neglecting higher order terms we obtainFrom Eqs  and 6)  6) one hFurther Eqs  and 9)  9) yieldq‴ satisfies [A is taken regarding space-dependent coefficient and B for temperature-dependent coefficient of the heat source/sink. Note that the case  relates to internal heat generation and  for internal heat absorption.Non-uniform heat source/sink atisfies :q‴=kuw  1) is trη, γ for curvature parameter, λ1 for relaxation to retardation times ratio, β for Deborah number (in view of retardation time), λT for mixed convection  parameter, N for ratio of concentration to thermal buoyancy forces, α for angle of inclination, R for radiation parameter, Pr for Prandtl number, Sc for Schmidt number, Bi1 and Bi2 for Biot numbers due to heat and mass transfer, Gr for temperature Grashof number and Gr* for mass Grashof number. These parameters are defined byHere prime shows differentiation with respect to Bi1 → ∞ and Bi2 → ∞. The skin friction coefficient Cf, local Nusselt Nux and Sherwood Shx numbers are defined in dimensional forms as follows:τw (surface shear stress), qw (surface heat flux) and jw (surface mass flux) are given byNote that the conditions in Eq  are reduCf, local Nusselt number Nux and local Sherwood number Shx areSkin friction coefficient f0, θ0, ϕ0) and auxiliary linear operators  for the momentum, energy and concentration equations in the formsAi (i = 1–7) are the arbitrary constants. The zeroth-order and mth-order deformation systems are constructed as follows:Here we take initial approximations , θ0(η) and ϕ0(η) to the desired solutions f(η), θ(η) and ϕ(η). Using Taylor expansion and considering the convergence of the series solutions at p = 1 we haveWhen fm, θm, ϕm) of Eqs  areUsing the boundary conditions  on velocity is plotted in λ1. Since λ1 is the relaxation and retardation times ratio so for larger values of λ1 the relaxation time enhances which provides additional resistance to the fluid motion. Hence velocity field decreases. Effect of Deborah number β on the velocity distribution is sketched in β increases. With larger Deborah number β the retardation time enhances. It leads to an enhancement in velocity. Variation of mixed convection parameter λT on velocity is sketched in λT correspond to an increase in velocity and thickness of momentum layer. Physically we observed that an increase in mixed convection parameter λT corresponds to an enhancement of thermal buoyancy force which is responsible for higher velocity. Characteristics of N i.e., concentration to thermal buoyancy forces ratio on the velocity are plotted in N. Since N is the concentration and thermal buoyancy forces ratio so with the increment of N the concentration buoyancy force enhances which results in upgradation of velocity. α on velocity field. Larger values of α results in the reduction of velocity field. In fact through higher α the gravity affect decreases which results in the decrease of velocity profile.This section aims to investigate the behavior of various pertinent quantities on the velocity, temperature and concentration fields. γ on temperature distribution is expressed in λ1 on temperature. Here we see that temperature field enhances for larger values of λ1. Since λ1 is the relaxation to retardation times ratio so for higher values of λ1 the relaxation time increases. It produces heat due to additional resistance to the fluid motion. Hence temperature distribution increases. Impact of Deborah number β on the temperature profile is shown in β. Influence of thermal radiation R on temperature distribution is examined in R. Physically it is verified as heat is produced due to radiation process in the working fluid so temperature field enhances. For various values of Prandtl number Pr the temperature is depicted in A and B on the temperature distribution. It is analyzed that temperature and thickness of thermal boundary layer increase with larger values of heat . It is because of the fact that more heat is delivered due to heat source which is responsible in the enhancement of temperature. Bi1 on temperature distribution. Both temperature and thickness of thermal boundary layer have increasing behavior of thermal Biot number Bi1. In fact Biot number involves the heat transfer coefficient ht so with the increase of ht thermal Biot number enhances. Thus higher values of heat transfer coefficient lead to enhancement of temperature and thickness of thermal boundary layer.Variation of curvature parameter γ. λ1 on concentration. Higher values of λ1 results in the enhancement of concentration field and thickness of associated boundary layer. Characteristics of Schmidt number Sc on the concentration are captured in Sc. Larger values of Schmidt number Sc lead to lower mass diffusivity which results in the reduction of concentration field. Analysis of mass Biot number Bi2 on the concentration is sketched in Bi2.th order of approximation is enough for the convergence of velocity and 18th order of approximation is enough for the convergence of temperature and concentration. γ, Deborah number β and angle of inclination α the skin friction coefficient enhances while it shows decreasing behavior for larger relaxation to retardation times ratio λ1, mixed convection parameter λT, ratio of concentration to thermal buoyancy forces N and Biot number Bi1. Behavior of various quantities on Nusselt number is explored in γ, mixed convection parameter λT, radiation parameter R, Prandtl number Pr, Deborah number β and Biot number Bi1. Nusselt number decreases when non-uniform heat source/sink parameter (A > 0 and B > 0) is increased. γ, Deborah number β, mixed convection parameter λT, Schmidt number Sc and Biot number Bi2 while it decreases with increasing values of relaxation to retardation times ratio λ1 and angle of inclination α.A > 0 and B > 0) lead to an enhancement in temperature field. The temperature and concentration increases for larger biot numbers. Larger Schmidt number Sc result in the reduction of concentration field. Larger values of heat source (A > 0 and B > 0) result in the enhancement of temperature profile. However Nusselt number decreases in this case. Variation of mixed convection  parameter λT results in the enhancement of velocity field while skin friction coefficient decays. The presence of curvarure parameter leads to an enhance the skin friction coefficient, local Nusselt and Sherwood numbers.Behaviors of non-uniform heat source/sink and thermal radiation in mixed convection flow of Jeffrey fluid due to an inclined stretching cylinder with convective conditions are addressed. We have see that velocity, temperature and concentration far away from the cylinder increase for larger curvature parameter. Larger non-uniform heat source/sink parameter (


Escherichia coli transcription open complexes. Our results revealed the existence of two stable states within RNAP–DNA complexes in which the promoter DNA appears to adopt closed and partially open conformations, and we observed large-scale transitions in which the transcription bubble fluctuated between open and closed states; these transitions, which occur roughly on the 0.1 s timescale, are distinct from the millisecond-timescale dynamics previously observed within diffusing open complexes. Mutational studies indicated that the σ70 region 3.2 of the RNAP significantly affected the bubble dynamics. Our results have implications for many steps of transcription initiation, and support a bend-load-open model for the sequence of transitions leading to bubble opening during open complex formation.Transcription initiation is a major step in gene regulation for all organisms. In bacteria, the promoter DNA is first recognized by RNA polymerase (RNAP) to yield an initial closed complex. This complex subsequently undergoes conformational changes resulting in DNA strand separation to form a transcription bubble and an RNAP-promoter open complex; however, the series and sequence of conformational changes, and the factors that influence them are unclear. To address the conformational landscape and transitions in transcription initiation, we applied single-molecule Förster resonance energy transfer (smFRET) on immobilized  The most abundant σ factor in Escherichia coli is σ70, which uses its conserved regions 2 and 4 to enable RNAP to recognize the −10 and −35 promoter elements respectively (C), which subsequently undergoes large conformational changes that trigger promoter DNA strand separation over 12–14 bp, resulting in the formation of the catalytically competent RNAP–promoter open complex (RPO) , a multi-subunit enzyme consisting of a catalytic core αββ’ω and O formation has emerged through structural, chemical footprinting, and rapid mixing kinetic approaches  strand at the active site  is very unstable  studies on diffusing transcription complexes using a variant of the lacUV5 promoter showed that the template DNA within RPO exhibits dynamic heterogeneity (The stability of RPr lacUV5  and 11 h at 37°C ), whereaf 1 min; ). Howeveogeneity . Furtherogeneity ,25.O undergoes large-scale structural transitions between different conformations, and identified two long-lived heparin-resistant complexes with transcription-bubble conformations distinct from the open transcription bubble expected for RPO. We also show that region σ3.2 stabilizes the open complex conformation. Our results have implications for the mechanisms of promoter opening, and provide a starting point for correlative measurements that monitor the coordination of conformational transitions during promoter recognition and bubble formation.Here, we use real-time smFRET and alternating laser excitation ,27 on imEscherichia coli wild-type (wt) σ70, wt RNAP core and ΔJaw RNAP core were prepared as described previously (Escherichia coli σ70 with residues 513–519 deleted (Δ3.2) was purified as described previously (70 for 30 min at 33°C.Oligonucleotides labelled with Cy3B and ATTO647N were purchased from IBA (Germany) and purified using gel electrophoresis. The purified single-stranded DNA (ssDNA) were annealed in annealing buffer . All DNA sequences and labelling schemes are shown in eviously . Escherieviously , whereasO) were formed as described previously , 1 mM dithiothreitol, 5% glycerol) giving a final volume of 20 μl. The mixture was incubated at 37°C for 15 min, after which 1 mg/ml heparin sepharose  was added to disrupt non-specific RNAP–DNA complexes. The mixture was incubated at 37°C for 30 s and then centrifuged to remove sepharose beads. The supernatant was then removed and transferred to a pre-warmed Eppendorf tube and incubated for a further 20 min at 37°C.RNAP-promoter DNA open complexes  experiments were performed on a custom-built objective type TIRF microscope. A green (532-nm Cobolt Samba) and a red (635-nm Cube Coherent) laser were combined using a dichroic mirror and coupled into a fibre optic cable. The fibre output was focused into the back focal plane of the objective  and displaced perpendicular to the optical axis such that laser light was incident at the slide–solution interface at an angle greater than the critical angle, thus creating an evanescent excitation field. Alternating laser excitation (ALEX) was implemented by directly modulating the lasers, and data was acquired using either a 100-Hz or 10-Hz alternation rate (indicated where appropriate). Excitation powers of 3 mW (green) and 1.5 mW (red) were used for 100-Hz experiments, while excitation powers of 1 mW (green) and 0.5 mW (red) were used for 10-Hz experiments. Fluorescence emission was collected by the objective and separated from the excitation light by a dichroic  and cleanup filters . The emission signal was focused on a rectangular slit to crop the image and then spectrally separated using a dichroic  into two emission channels. The channels were focused side-by-side onto an EMCCD camera (Andor iXon 897). The EMCDD was set to an EM gain of 300, corresponding to an approximate real gain of 4.55 counts per photon.O was then incubated on the surface for 5 min followed by washing to remove excess unbound complexes. Imaging buffer containing 50 mM Tris–HCl, 100 mM KCl, 10 mM MgCl2, 100 μg·ml−1 BSA, 1 mM DTT, 5% glycerol and 2 mM UV-treated Trolox was added to the observation chamber, which was then sealed using a glass coverslip as a lid. An enzymatic oxygen scavenging system consisting of 1 mg·ml−1 glucose oxidase, 40 μg·ml−1 catalase and 1.0% (wt/vol) glucose was added prior to image acquisition just before sealing the sample. Experiments were performed at 22°C unless indicated otherwise.Neutravidin-coated glass coverslip chambers were prepared as described . A 10 nMFluorescence intensities were extracted from images using previously described TwoTone software . The appWe manually classified the intensity time trajectories of individual molecules into groups. Static closed bubble signals were defined as time trajectories showing a stable E*∼0.2 signal with no fluctuations to higher FRET states during our observation period. Static open bubble signals exhibited a stable E*∼0.45 signal with no fluctuations to lower FRET states. Dynamic bubble signals were defined as time-trajectories showing any anti-correlated E* changes during our observation periods.open) and the mean closed dwell times (τclosed).Hidden Markov Modelling (HMM) analysis was performed on time-trajectories displaying dynamic FRET fluctuations using custom-written MATLAB software ,40. EachO  of our previous work hindered further study of the heterogeneity, especially at longer timescales (from seconds to minutes).In previous work, we used solution-based smFRET to study the dynamics of RNAP–promoter DNA complexes . Using aO . The widO dynamics in the 10-ms to 10-sec timescale by using fluorescent substrate DNA and TIRF microscopy to observe immobilized RNAP–promoter DNA complexes for extended periods. Our measurements were performed using a full σ70-consensus promoter (lacCONS) based on α lac promoter derivative extending from positions −39 to +25 relative to the transcription start site. As in . Our measurements resulted in a FRET histogram showing a major population (∼75%) with a FRET efficiency centred at E*∼0.45, and a smaller population (∼25%) centred at E*∼0.2 . The loO while using 100 μg/ml heparin in the observation chamber. The addition of heparin did not change the FRET histogram significantly  revealed the presence of additional heterogeneity, with complexes exhibiting four characteristic behaviors. Three behaviors involved single FRET states that appeared ‘static’ : 50% of the molecules exhibited a stable E*∼0.45 state  with dyes positioned at +15 and −15 . By placO state ,45. For O state ; howeverO state . The smaComparison of time-traces of pmDNA at 22°C Figure  and E wiIncreasing the temperature from 22 to 37°C for the pmDNA produced a single population centred at E*∼0.45, which showed an increased width compared to the 0.45 population at 22°C Figure ; the inc70 region 3.2 (σ3.2) and the β’ jaw, influence the profile of transcription complexes. Both regions play important roles in transcription initiation. For example, crystal structures of Thermus thermophilus RNAP–promoter complexes showed that σ3.2 forms a loop that protrudes towards the RNAP active centre, where it interacts with the template strand ;,46;70 red Figure ,47, and d Figure . The β’ d Figure  and stabromoters , and sta70 variant that lacks amino acids 513–519 (Δ3.2), a region that corresponds to the tip of the σ3.2 loop. We analyzed RPO complexes made using fully complementary DNA and recovered a bimodal E* distribution with peaks similar to that of wt RNAP–promoter dsDNA complexes (centred at E*∼0.2 and ∼0.45) , which showed a similar FRET distribution compared to that for wt RNAP Figure  revealedNext, we studied the FRET distributions of Δ3.2 RNAP complexes formed using pmDNA Figure . Here, tTo measure the rates for the transitions between the low- and high-FRET states for the dynamic complexes, we analyzed individual time-traces for wt, Δ3.2 data and ΔJaw complexes. After obtaining dwell time distributions for the low- and high-FRET states using HMM analysis, we fitted them to exponential functions, and quantified the interconversion rates, which we interpret as bubble opening and closing rates (see ‘Discussion’ section).open of 6.4 s−1  exhibited a mean lifetime (dwell time) of ∼150 ms, which corresponds to an opening rate kO, was found to have little effect on the kinetics of interconversion for the dynamic molecules , the latter studies could not distinguish between free DNA and transcription complexes with a similar FRET signature. Here, all DNA molecules observed on the surface are retained via long-lived interactions with surface-immobilized RNAP molecules, and thus our experiments do not contain any free DNA.To our knowledge, this is the first study that identifies three distinct heparin-resistant RNAP–promoter DNA complexes existing as a heterogeneous mixture at a olecules ; specifiUsing the FRET signatures as a guide, and supported by the dependence of the states on temperature, competitors and mutations, we assigned the observed structural states to initiation complexes exhibiting different degrees of transcription bubble opening: a first state in which the bubble is likely to be either closed or in the very early stages of opening (E*∼0.2), a second where the bubble is likely to be fully open (E*∼0.45), and a third with a FRET signature intermediate to those of the open- and closed-bubble states (E*∼0.35).i intermediate reported in lacUV5 .Our static E*∼0.45 complex is likely to represent a stable RPl identified at the lacUV5 promoter: the degree of promoter unwinding in Ol was different from that of the Oh complex, which was the more abundant species at 37°C and presumably has a fully open transcription bubble. Alternatively, the E*∼0.35 state could represent scrunched or anti-scrunched promoter conformations that shift the transcription start site in OC more downstream or upstream, respectively  and the large decrease of the isomerization rate k2 for RPo formation. Specifically for lacUV5 (2 decreased 10- to 50-fold (relative to full-length fragments) for short fragments ending at −42 or −45, and the equilibrium constant K1 for the formation of RPc increased by 10-fold (for native RNAP). Both effects are expected to stabilize a heparin-resistant closed complex that we assign to our static 0.2 FRET species at 22°C. The work by Ross et al.  and to a complex conformational landscape which contains slow conformational transitions (slower than 10 s) inaccessible to our current FRET rulers. The absence of larger differences in the transition kinetics between the ΔJaw mutant and wtRNAP suggest that the observed FRET transitions are not due to the repositioning of the ‘downstream mobile element’ .downstream end of the transcription bubble (i.e. melting of the −3/+2 region). This means that the low-FRET dynamic state in both dsDNA and pmDNA is partially open, whereas the high-FRET dynamic state is the first complex with a fully open bubble. The latter is often referred to as intermediate I2  and reqbend-load-open and open-bend-load , rendering the open-bend-load model less likely .An alternative explanation of our results along the lines of the he clamp  will reqThe fact that the Δ3.2 RNAP–DNA complexes exhibit faster bubble dynamics supports proposals based on structural studies that σ3.2 stabilizes the template strand at the active site, and raises the intriguing possibility that the altered opening and closing kinetics might influence later steps in initiation (see next section). Our results do not show a significant role for the β’ jaw in controlling bubble dynamics; however, they do suggest that the jaw influences the degree of transcription bubble opening, in agreement with earlier studies .l intermediate was found to produce more abortive products and be less efficient at promoter escape than an Ou complex. Further, in  that showed the presence of open complex subpopulations associated with different properties in terms of initiation transcription and promoter escape (R) produced longer abortive products than short-lived open complexes (e.g. on T7A1), which instead produced shorter abortive products.The fact that the initiating dinucleotide ApA—which is expected to stabilize the template strand in one particular register at the active site —does notther, in , RNAP–prther, in . Since sr escape ; specifiO formation pathway, to determine whether the initial heterogeneity persists in later stages (such as in abortive initiation and promoter escape), and to identify additional DNA and RNAP determinants of transcription complex heterogeneity and dynamics.In future work, we will use our single-molecule FRET assay to monitor transcription initiation from the initial DNA binding event to promoter escape. Such studies will allow us to determine whether the conformational states we observe are sequential intermediates along the RPSupplementary DataClick here for additional data file.


The tyrosine and serine site-specific recombination systems of bacteriophages HK022 and ΦC31 are widely used for stable directional exchange and relocation of DNA sequences, making them valuable tools in these contexts. We have developed site-specific recombination tools that allow the direct selection of recombination events by embedding the attB sites from each system into the signal peptide cleavage site coding sequence of bla. All possible open reading frames (ORFs) were inserted and tested for recombination efficiency and bla activity. Efficient recombination was observed for all tested ORFs  as shown through a cointegrate formation assay. The bla gene with the embedded attB site was functional for eight of the nine constructs tested.The HK and ΦC31 tools were developed by placing the att-bla system offers a simple way to directly select recombination events, thus enhancing the use of site-specific recombination systems for carrying out precise, large-scale DNA manipulation, and adding useful tools to the genetics toolbox. We further show the power and flexibility of bla to be used as a reporter for recombination.The HK/ΦC31  In each family, DNA exchange requires host-encoded proteins for recombination that differ between systems. These systems are attractive due to their directionality and stability, and both systems are functional in prokaryotic and eukaryotic organisms [The ability to precisely and directly manipulate DNA is important for functional studies and the synthetic assembly of large genetic constructs. Site-specific recombinase (SSR) systems are widely used as tools to rearrange, insert, remove, and join DNA with virtually no upper limit in size. For biotechnology purposes, this can include the insertion of exogenous DNA into chromosomes, the fusing of DNA molecules, or the construction of synthetic gene networks . The tyrrganisms –5.attB and attP integrative recombination forms attL and attR sites. The reverse attL x attR excisive reaction also requires Int as well as a recombination directionality factor (RDF), named Xis in the HK system and gp3 in the ΦC31 system [in trans from a helper plasmid, a non-replicating DNA molecule, or as mRNA [att sites differ in size, with the HK attB sites being generally shorter than the HK attP sites, 21 base pairs (bp) vs 234 bp [attP contains binding sites for Int and Xis along with host-encoded proteins Fis and IHF [attB and attP sites are similar in size (~50 bp) and do not require additional proteins to carry out recombination [Mechanistically, 1 system , typical as mRNA . Structus 234 bp , 9; in a and IHF –11. ΦC31bination .CRE/loxP [bla) gene is an attractive marker, as it is a useful reporter gene for both pro- and eukaryotic applications [bla has also been developed to discover directed evolution-modified SSR enzymes capable of recombining designer sequences [bla signal peptide is an attractive region for peptide insertion [attB of HK and ΦC31 inserted in frame with bla, allowing expression of the gene and enabling the direct selection of recombination events. The selective agent is not expressed when the att sites are in attL and attR form, as the reporter gene fragments are physically separated , resulting in large-scale chromosomal rearrangements [lacZ reporter allows the observation of recombination events but not to select for them, we developed a reporter system for HK recombination based on antibiotic selection. We have used an HK attB site placed in-frame within the β-lactamase (bla) gene to carry out relocation of the S10-spec-α ribosomal locus in V. cholerae in order to study the consequences of essential gene positioning as it relates to dosage [bla to carry out large-scale genome inversions around the origin region (ori) of V. cholerae chromosome one (Chr1) to shift the timing of the initiation of chromosome two (Chr2) replication relative to Chr1 in order to study the mechanisms involved in bacterial chromosome replication timing [This approach has been used to explore the physical structure of the i genome , 22. Genbacteria . We havetem from  to exchangements . Becauseo dosage . We furtn timing .bla and a similar tool using the serine ΦC31 att system (ΦC31-bla). We placed attB sites from each system immediately downstream of the bla signal peptide coding sequence, which directs transport of β-lactamase to the periplasm and is removed in the mature protein. β-lactamase is generally tolerant of insertions into this region. When each system is present as attL and attR sites, they are associated with fragment sequences bla’  and ‘bla (the 3′ region comprising the mature protein sequence), respectively  signal peptide sequence for protein transmembrane transport that is cleaved during protein maturation . We inseein Fig. , as thisattBHK site comprises a 7 bp core, or overlap, (O) region where strand exchange occurs, and flanking B and B′ arm regions of 7 bp each that are recognized by Int monomers to form a synaptic complex, although sites shorter than this 21 bp have been shown to be functional but with low efficiency [attB and attP must perfectly overlap, and the arm regions must share similarity. Flanking the core minimal region, there are homologous nucleotides that may play an additional role in recombination efficiency [attB into bla extends the gene and could affect either transport through the membrane or mature enzyme function. It is therefore necessary to test different open reading frames encoded by the attBHK sequence to avoid unwanted interference with bla. The native attBHK sequence encodes two open reading frames (ORFs) that do not have stop codons. As we wished to increase the potential sequences we could test within bla, we added a third potential ORF by mutating one bp just outside of the B′ region  of each to test and measure bla function. All ORFs provided resistance to ampicillin at an MIC >256 μg/ml  followed by a post-hoc Tukey-Kramer test (p < 0.001). Additionally, all six ΦC31 ORF constructions recombined at a higher rate than HK ORFs 1–3  was used. Plasmid minipreps were performed using the GeneJET Plasmid Miniprep kit (Life Technologies). All PCR reactions for plasmid construction were performed using the Phusion High-Fidelity PCR Master Mix (Life Technologies), and all diagnostic PCR reactions were performed using DreamTaq DNA Polymerase (Life Technologies). Oligonucleotides were synthesized by Sigma-Aldrich and Eurofins Genomics. Oligonucleotides were phosphorylated by T4 polynucleotide kinase (NEB). DNA was sequenced by GATC Biotech and Eurofins Genomics.attB sequences into pSW23T was performed by annealing phosphorylated oligos containing the respective att sequence with overhangs overlapping with BamHI and EcoRI restriction sites, followed by cloning of these sequences into the pSW23T fragment. Insertion of attP sequences into pHK11-Amp was similarly performed. The various attB ORFs for both HK and ΦC31 were inserted into the β-lactamase (bla) by overlapping PCR, in which the 5′ region of bla was amplified from pMP58 using oligos MV26 and the appropriate reverse attB oligo, and the 3′ bla region amplified using a forward attB oligo and JB13. These products were gel purified and co-amplified using oligos MV26 – JB13 to form a DNA fragment containing bla with the inserted attB. This product was digested with EagI and EcoRI and cloned into pSW23T and transformed into MFDpir. The pMP58 bla gene comes from pUC19.Insertion of To make plasmid pPhiC31-Int, we first deleted the XbaI site in pZJ7 (a kind gift of Jia Zhao and Sean Colloms) by digestion with SpeI-XbaI followed by religation to make plasmid pZJ7ΔXbaI. The ΦC31 integrase gene was amplified using oligos PhiC31 IntF and PhiC31 IntR. The pHK-Int backbone was amplified using oligos JB485 and JB486. These oligos produce DNA fragments with overlapping ends, which were then joined by Gibson assembly . PlasmidoriTRP4 transfer region and oriVR6K π-controlled replication origin were transferred from the π+/DAP- donor strain MFDpir to a recipient strain containing an attP plasmid and a helper plasmid expressing the appropriate integrase gene under control of the temperature-sensitive CI857 promoter. Prior to conjugation, strains were diluted 1/100 from an overnight starter culture and grown to OD600 = 0.3. Conjugations were performed by two techniques: for the attB HKWT/MUT strains, 0.5 ml of donor was mixed with 4.5 ml of recipient and applied to a 0.45 μm filter (Millipore) by vacuum-filtration through a glass column. The attB ORF insertions into bla were performed by mixing 0.2 ml of donor with 1.8 ml of recipient, and following centrifugation at 6000 RPM for 5 min, ~1.8 ml of supernatant was removed, the pellet resuspended in the remaining liquid media, and similarly placed onto a 0.45 μm filter. For both techniques, the filters were then incubated on an LB-DAP plate for approx. 16 h prior to resuspension and plating. Recombinants were recovered by selecting for Cm resistance in DAP-free media, and recombination frequencies were measured as the ratio of recovered recombinants over donor CFUs. Each att site was tested three times.Recombination frequencies were tested by performing a conjugation assay in which the plasmid pSW23T containing the E. coli strains containing plasmids with either attB inserted into bla, or bla fragments associated with attL and attR were performed by plating and aspirating 2 ml of a 1/100 dilution of an overnight culture onto an LB/DAP agar petri dish. An Etest (bioMérieux) ampicillin antibiotic strip was placed onto the plate and incubated overnight at 37 °C, and the level of antibiotic resistance was scored the following day.The MICs of Recombination frequencies were analyzed for statistical significance using MATLAB software . 1 and 2-way analysis of variance (ANOVA) tests were performed using the anova1 and anova2 functions. Tukey-Kramer post-hoc tests were performed using the multcompare function.Secondary DNA structures were analyzed using the mfold software . Protein


The present study has demonstrated the unique and unpredicted simultaneous enhancement in toughness and strength with very high flaw tolerance of zirconia/Ta composites. In addition to their excellent static mechanical properties, these composites also have exceptional resistance to fatigue loading. It has been shown that the major contributions to toughening are the resulting crack bridging and plastic deformation of the metallic particles, together with crack deflection and interfacial debonding, which is compatible with the coexistence in the composite of both, strong and weak ceramic/metal interfaces, in agreement with predictions of ab-initio calculations. Therefore, these materials are promising candidates for designing damage tolerance components for aerospace industry, cutting and drilling tools, biomedical implants, among many others.Dense (>98 th%) and homogeneous ceramic/metal composites were obtained by spark plasma sintering (SPS) using ZrO Cermets are ideally designed to combine the optimal properties of both, high wear resistant ceramics and ductile metals, which possess the ability to reduce crack propagation and prevent catastrophic failure.However, these improvements are often achieved at the expense of strength. In these structural materials strength and toughness are usually considered mutually exclusive2-based cermets reported by diverse scientific studies are substantially scattered. Exemplarily, the flexural strength (σf) and fracture toughness (KIc) of zirconia/metal composites described in the literature reach values up to 1200 MPa and 5.9 MPa∙m1/2 for ZrO2/Ni cermet1/2 for ZrO2/Ti cermet141618192021Several studies on zirconia reinforced with metals such as nickel3457810This suggests that a more refined understanding of the interrelations between different strengthening and toughening mechanisms is needed to optimize ceramic/metal composite microstructures for structural applications.Additionally, embedding a ductile metallic phase into a brittle matrix increases not only their fracture toughness and damage tolerance, but also influences the fatigue performance232526272830311/2 and 16 MPa∙m1/2, respectively. In the present work, we focus on the interplay between the damage tolerance and fatigue resistance phenomena within zirconia/Nb and zirconia/Ta composites.Recently, we have demonstrated that wet mixing route and hot press sintering tailored suits the fabrication of zirconia/Nb2/metal composites are shown in 2/Ta and ZrO2/Nb interfaces are well bonded and no microcracks are observed. It has only been observed that a solid solution of Nb2O5 and Ta2O5 in ZrO2 takes place332O5 and Nb2O5 that is always present on the particle surfaces of the metal starting powder is dissolved in a solid solution in the zirconia matrix after sintering. On the other hand, it may well be that the oxygen is dissolved and distributed statistically in the metalScanning electron micrographs corresponding to ZrO2/Ta interface is shown in A representative high resolution transmission electron micrograph corresponding to ZrOThe results related to the mechanical evaluation of the composites are enclosed in 2, ZrO2/Ta and ZrO2/Nb were found to be 1217 ± 10 MPa, 970 ± 18 MPa and 850 ± 20 MPa, respectively. As a direct consequence of the smaller critical grain size in ZrO2, the bending strength of the monolithic ceramic is higher than the strength corresponding to both zirconia/metal composites. The Young’s moduli of the composites (≈200 GPa) were found to be very close to the values predicted by the rule of mixtures by Voigt and Reuss models. The average fracture toughness of ZrO2/Nb and ZrO2/Ta composites was found to be 15 ± 1 and 16 ± 0.9 MPa·m1/2, respectively; much higher than the value obtained for the monolithic zirconia (6 ± 0.3 MPa·m1/2).The mean biaxial flexural strength values corresponding to ZrOIn N). The tests were interrupted at N = 107 cycles, for the unfailed samples, which are marked with an arrow symbol. Three maximum stress levels (σmax) were selected in relation to the initial strength obtained under static tests /ZrO2(3–30). This interface has a mismatch of 16% and has not been previously observed in the similar system ZrO2/Nb2 crystallographic orientations form interfaces with the Ta (100) surface, as it occurs in ZrO2/Nb composites2/Ta interfaces indicate that the experimentally observed (3–30)/(1-1-2) interface is thermodynamically stable and its bonding strength, measured by the work of separation (Wsep)372/(100) Ta polar interface presents the largest bonding strength, although its stability is lower. The Wsep of the representative investigated interfaces ranges from high (~10 J/m2) to medium values (~2 J/m2)  sintered in a C free atmosphere (Ar atmosphere)292 and C impurities doesn’t affect significantly the ductility of Ta and Nb flakes.The increase in toughness must be due to the ductile metallic phase, which can absorb the crack propagation energy during fracture, and could enhance crack deflection and bridging as well as stress relaxation near the crack tip. Moreover, this increase has occurred due to the presence of solid solution of pentavalent oxides such as Nb−6 °C−1 and 7.3 × 10−6 °C−1 for Ta and Nb, respectively; while for 3Y-TZP is 12 × 10−6 °C−1. Thus, when these composites cool down from the sintering temperature, the reinforcement contracts less than the matrix; the metal particles are subjected to compressive stress and residual tensile stresses are accumulated in the zirconia matrix when cooling from the sintering temperature. When the residual tensile stress is imposed on t-ZrO2 grains, phase transformation can occur easily. Therefore, the phase transformation is promoted by an applied stress and the transformation rate increases with its magnitude.Additionally, the effects of the residual stresses related to the CTE mismatch between zirconia and the metals case of study have to be taken into consideration. The average thermal expansion coefficient, in the 20–1000 °C range, is 6.73 × 102 only about 2 vol.% of the tetragonal zirconia transformed to the monoclinic phase during the failure. Moreover, the analysis of XRD data proved that the fraction of zirconia transformation considerably increases for zirconia/metal composites. The enhanced transformability can be related to the alloying effect on the tetragonality, i.e., the cell parameter ratio c/a, of stabilized t-ZrO2 as well39Analyses of the fracture surfaces of both ceramic/metal composites after bending tests indicate two distinct failure modes of the metal particles: i) the tantalum particles fracture surface bears little resemblance to typical ductile necking associated with void coalescence, such as those shown for niobium flakes , and ii)2/Ta composites with indentation crack propagation, FIB technique was used. In order to investigate microstructural evolution of ZrO2/Ta and ZrO2/Nb composites always showed higher values of fracture strength, which means that the damage tolerances of these composites are higher than for monolithic zirconia ceramic. Although un-indented ZrO2 ceramic exhibits the highest value of the fracture strength, the failure strength of the indented ceramic/metal composites is higher than in the monolithic zirconia.The lower slopes of the ceramic/metal composites in σmax vs cycles to failure N (2/Nb composite exhibits fatigue behaviour in contrast to ZrO2/Ta composite and monolithic zirconia. This can be deduced from the fact that in the S–N plots the maximum stress (σmax) for ZrO2/Nb decreases with the cycles to failure N, while in case of ZrO2/Ta composite it slightly changes; and it is practically constant for ZrO2. Furthermore, the slope of the discussed dependency log σmax vs log N is similar for the zirconia when compared to the ZrO2/Ta composites. Therefore, the sensitivity of ZrO2/Nb to cyclic stresses is higher than for the monolithic zirconia and for the ZrO2/Ta composites. Higher slope in 4431The first conclusion that can be drawn from the obtained dependencies of stress amplitude ailure N  is that Ra), are presented in 2/Ta composites  can be attributed to crack-particle interaction intrinsic mechanisms, including crack re-nucleation (in the matrix and/or particle), crack branching, crack blunting, limited interface debonding and discontinuous (out-of-plane) crack deflection. On the other hand, niobium and tantalum metallic particles showed lower level of plastic deformation under fatigue load than under bending stress and hence, lower roughness . Therefore, crack particle interactions under cyclic loading are different than those observed under monotonic loading. Since fatigue crack growth occurs at stress intensities lower than critical crack growth, the toughening contribution from metals deformation would naturally be smaller than under monotonic loads. But the absence of significant plastic deformation of Ta particles in the ZrO2/Ta composite under cyclic loading is unexpected as metallic materials are known to be susceptible to fatigue. Thus, the presence of Ta reinforcements, while providing a potent influence on toughening zirconia ceramics under monotonic loading, is also effective in improving fatigue crack growth resistance. In order to predict fatigue life of indented materials with equivalent initial flaw size, indentation load of 98 N and 294 N were chosen for monolithic ceramic and for ceramic/metal composites, respectively. The initial flaw size was about 335 μm for all studied compositions. The indentation fatigue life for disc specimens are presented in semi logarithmic form as peak stress versus cycles to failure  and yield strength (240 MPa vs 380 MPa)mposites  are lowematerial . Ra valu failure . The ind2/Ta, ZrO2/Nb and ZrO2 is 500 ± 25 MPa, 270 ± 10 MPa and 130 ± 40 MPa, respectively.It has been shown that fatigue life of well-polished ceramic/metal composites without any artificial produced flaw is reduced when compared with monolithic zirconia . HoweverIn 2/Ta materials, allows to visualize the growth of defects before reaching critical size and prevent sudden fracture. In order to observe crack propagation, the experiment was stopped at various stages of cycling tests. At each stop the test specimen was dismounted from the jig for SEM analysis. The combination of this information with the test load is showed in graphs of crack lengths as a function of the number of cycles  of flexural strength value of un-indented specimens cracks start to growth. For load levels as high as ~41% (400 MPa) of flexural strength value of un-indented specimens, the ZrO2/Ta composites show subcritical crack growth resistance and the cracks do not propagate. Moreover, fatigue crack length propagation for maximum stress under which the specimens never fracture (fatigue limit) is ~550 μm and ~398 μm for ZrO2/Nb and ZrO2, respectively. However, ZrO2/Ta composites show infinite fatigue life even in a severely damaged state (crack length of ~3500 μm) and therefore excellent flaw tolerance fatigue behaviour.The strength and reliability of monolithic zirconia are significantly reduced when compared with composites. Meanwhile, less sensitivity of ceramic/metal composites to defects under cyclic loading, especially ZrOf cycles . In this2/Ta composites, there is a way to avoid the conflicts between mutually exclusive properties of toughness and fatigue resistance, through the presence of multiple mechanisms acting at different length scales, decreasing locally stresses through limited plastic deformation to provide intrinsic toughness and further extrinsic mechanisms, such as elastic bridging of the tantalum particles, with about double elastic modulus and yield strength value than Nb particles.It was established that in contrast to fracture toughness, fatigue crack growth values do not exhibit a monotonic correlation with microstructural aspects. Zirconia/metal cermets reinforced with more ductile metal (Nb), were found to be more fatigue sensitive in terms of crack growth resistance. Moreover, these materials are more fatigue susceptible with artificial induced flaws (indentation) under cyclic loading. The experimental fact that failure under cyclic loading of zirconia/metal cermets is controlled by subcritical growth of preexisting flaws shows that a damage tolerance analysis for structural design involving these materials is difficult to apply in practice. In the case of ZrO2/Ta composites, considering the fact that materials with the more ductile metal are more fatigue sensitive. Cracks propagate only when the crack tip energy reaches a threshold that corresponds to the bond failure energy. If the metal particle-matrix bonds are both, strong and weak or the interface cleaves easily, the crack tip surface is multiplied (for instance by a particle circumference) and propagation stops since the stress intensity falls instantaneously below the above-mentioned threshold. In the case of Ta particle embedded in a zirconia matrix this threshold is much higher than in the case of more ductile Nb particles.The results obtained in this investigation suggest that ductile phases are associated with multiple and competing mechanisms operating under fatigue loading and being sensitive to elastic and plastic properties (yield stress and elastic modulus) of the reinforcement. These properties and the interlocking operative mechanisms play a very important and critical role in the unexpected fatigue resistance of these ZrOTo our knowledge, the ability of tantalum metal to dramatically improve the fatigue resistance and damage tolerance of a ceramic material has not been previously reported in the literature, and was discovered here only because the particular microstructural feature of this ceramic/metal composite. These unprecedented properties presented in this work could, in principle, stimulate multidisciplinary applied research on ceramic/tantalum composites, which are attractive for various fields such as thermoelectric power generation, functionally graded materials, biomaterials, strain-tolerant and thermal-shock-resistant multifunctional ceramics, static-charge dissipation devices, electric-discharge manufacturing, and many more, and that open the doors for massive and sustainable utilization of cyclic fatigue resistance structures.2O3; TZ-3YE, Tosoh Corp.), with an average particle size of d50 = 0.26 ± 0.05 μm, a BET specific surface area of 16 ± 3 m2/g. (2) Tantalum  with an average particle size d50 = 44 μm. (3) Niobium  with an average particle size of d50 = 35 μm.The following commercially available powders have been used as raw materials: (1) Tetragonal zirconia polycrystals  working at 40 kV and 30 mA in a step-scanning mode from 27° to 33° with a step size of 0.01° and a scan speed of 0.06°/min. The amount of m-ZrOThe microstructure of the sintered specimens was studied by Scanning Electron Microscopy , Focused Ion Beam Scanning Electron Microscope (FIB-SEM) AURIGA 60 CrossBeam Workstation , and by transmission electron microscopy . The TEM samples were prepared by diamond cutting and mechanical polishing to a thickness of 100 μm, followed by dimpling to a thickness of 20 μm and ion milling at 6.0 KeV and 0.5 mA. In order to determine the content of tantalum and niobium oxide solid solution in zirconia, scanning electron microscopy  with energy dispersive X-ray spectroscopy (EDX) was using for chemical microanalysis of sintered samples. One hundred random points were chosen to obtain a semi-quantitative anlysis for the tantala and niobia distribution in the ceramic matrix.Ic), flexural strength (σf) and Young modulus (E), formulas and calculation procedures have been reported in previous publications2341The density of the compacts was measured according to the Archimedes technique. Methods to measure Vickers hardness (Hv), fracture toughness , operating under load control at 20 Hz in order to generate a stress number curve. A sinusoidal cyclic load was applied to the specimens. The stress ratio was equal to 0.1 and the fatigue cycle was limited at 10Indentation fatigue life was analyzed following the same protocol that in the case of the flaw tolerance measurements. Indentation prints have been introduced in the tensile surfaces of all of the test discs. In order to obtain equivalent initial flaw size load of 196 N and 294 N was selected for ceramic and in ceramic/metal composites, respectively. The crack length was measured discontinuously by means of an SEM microscope .2 (×550). Three samples of each material were scanned to evaluate the average surface roughness (Ra) of the fracture surfaces at five different locations.Roughness reconstruction and coloured height map of the composites fracture surface after biaxial and fatigue tests were carried out by using SEM  with the 3D Roughness Reconstruction application. The field of view area for the height map calculation was 490 μmHow to cite this article: Smirnov, A. et al. Unprecedented simultaneous enhancement in damage tolerance and fatigue resistance of zirconia/Ta composites. Sci. Rep.7, 44922; doi: 10.1038/srep44922 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Genome-wide methylation of lung tumors in adult offspring was determined using methylated DNA immunoprecipitation (MeDIP) with the NimbleGen mouse DNA methylation CpG island array. This data article refers to the research article “DNA methylation in lung tissues of mouse offspring exposed in utero to polycyclic aromatic hydrocarbons,” Data in this article depict patterns of methylation in lung tissues obtained from the offspring of B6129SF1/J dams and 129S1/SvImJ sires exposed  Then, a one-sided Kolmogorov-Smirnov test was applied to identify probes with apparent high methylation within a 750 bp window, yielding a −log10p-value. NimbleScan detected peaks by identifying at least two probes with a −log10p-value>2, and peaks within 500 bp of each other were merged. Finally, the peak score was calculated as the average −log10p-values from probes within that peak. Gene lists for clustering and gene ontology analyses were generated by selecting features with peak score>2 (indicative of methylated DNA) in all three samples from the treatment group of interest and by excluding all features that were not methylated (peak score<2) in at least two of the three samples from the comparison treatment group(s). Unsupervised, bi-directional hierarchical cluster analysis was performed using TM4 Multi-Experiment Viewer [6]P<0.05 and the minimum number of mapping entries set at five genes. The gene ontology type performed was a generic GO slim (Gene Ontology Consortium).A complete description of methylation data processing and analyses performed is provided in the accompanying paper


The obtained results revealed that high coulombic efficiency (97.5%) can be achieved at high current density, additionally showing the strong influence of charge capacity on the above redox reactions. Moreover, AFM imaging indicated the occurrence of solvent cointercalation during the first reduction, as reflected by the presence of hills and blisters on the basal plane of highly oriented pyrolytic graphite subjected to the above process.Interfacial reactions strongly influence the performance of lithium-ion batteries, with the main interfacial reaction between graphite and propylene carbonate- (PC-) based electrolytes corresponding to solvent cointercalation. Herein, the redox reactions of solvated lithium ions occurring at the graphite interface in 1 M·LiClO Graphite is extensively used for the fabrication of lithium-ion batteries (LIBs) –4 due to+/Li, hindering the lowering of the electrode potential to values corresponding to lithium intercalation (0.25–0.0 V versus Li+/Li) and thus inducing graphite exfoliation prior to lithium intercalation during the first charging. Thus, solvent cointercalation significantly degrades the performance of LIBs and should be suppressed to allow reversible intercalation/deintercalation of lithium ions at graphite-negative electrodes.Despite its remarkable ionic conductivity at low temperature (melting point = −49°C), propylene carbonate (PC), widely used as a main organic solvent in primary lithium batteries, has not been applied to LIBs due to undergoing ceaseless cointercalation into graphite during the first charging –16. PC cThe effective suppression of solvent cointercalation at graphite-negative electrode requires a deep understanding of numerous factors of influence. Commonly, the irreversible reduction of solvated lithium ions at ∼1 V is the main interfacial reaction in PC-based electrolyte solutions, indicating the importance of understanding the redox behavior of PC-solvated lithium ions. Herein, we probed the above behavior by chronopotentiometry, characterizing the potential change upon application of negative/positive currents and investigated the effects of current density on the redox reactions of solvated lithium ions. Moreover, in situ atomic force microscopy (AFM) and in situ Raman spectroscopy were used to clarify certain aspects of solvent cointercalation during the first reduction reaction.4 in PC  was used as an electrolyte.Natural graphite powder  was used as an active electrode material for chronopotentiometry. A composite working electrode was prepared by coating copper foil (Nilaco Co.) with a 9 : 1 (w/w) mixture of NG-7 and poly(vinylidene difluoride) and drying it at 80°C in a vacuum oven  for 12 h. Highly oriented pyrolytic graphite  was used as a model electrode for in situ AFM imaging, which was carried out for freshly cleaved HOPG surfaces. Lithium foil  was used as reference and counter electrodes in all electrochemical measurements, and a 1 M solution of LiClO−1) to understand the effect of current density on the redox behavior of solvated lithium ions.Chronopotentiometric measurements were performed using a battery test system , with 2032 coin cells tested at various C-rates (1 C = 372 mA·g4/PC. AFM images (5 μm × 5 μm) were automatically captured at each potential during cyclic voltammetry (CV) scans between 3.0 and 0.0 V (scan rate = 2 mV·s−1) using an AFM imaging system  equipped with a potentiostat . All AFM characterizations were performed at room temperature in an argon-filled glove box .The basal plane of HOPG was imaged in contact mode using a pyramidal silicon nitride tip  in 1 M·LiClOAn electrochemical (quartz) cell for in situ Raman spectroscopy was assembled in an argon-filled glove box, sealed, and removed from the glove box into ambient atmosphere. The 514.5 nm line of an argon-ion laser was scattered on HOPG during the first reduction by applying a constant current of 1 C. Raman spectra were collected using a triple monochromator  equipped with a multichannel charge-coupled device detector.4/PC. During these measurements, the graphite-negative electrode was charged to 60 mAh·g−1 and instantly discharged without any rest time, providing insights into the redox behavior of solvated lithium ions above the lithium intercalation potential, with a different discharge capacity observed in each cycle reflecting the oxidation of solvated lithium ions. In general, solvent cointercalation (corresponding to the reduction of solvated lithium ions) takes place at ∼1 V in PC-based electrolytes [trolytes –16. Howe−1) was observed as the charge capacity increased from 20 to 500 mAh·g−1  and E2g2(boundary) bands at 1583 and 1597 cm−1, respectively, were observed during the first reduction reaction, indicating the absence and presence of intercalates within graphite layers, respectively (We also employed in situ Raman spectroscopy to clarify the nature of the intercalate formed during the first reduction of graphite in constant-current mode in a PC-based electrolyte. In this case, the electrode potential did not drop to the lithium intercalation potential while the graphite electrode was charged to 20 mAh·gectively . Moreove4/PC by chronopotentiometry, in situ AFM, and in situ Raman spectroscopy, revealing that these reactions were reversible at high current density and thus highlighting the key role of the latter parameter. Moreover, the discharge capacity related to the oxidation of solvated lithium ions increased with increasing current density, and improved coulombic efficiency was observed at high current densities of 10, 15, and 20 C in the case of charging to 20 mAh·g−1. AFM imaging revealed the appearance of hills and blisters on the otherwise smooth electrode surface during the first reduction, indicating the occurrence of solvent cointercalation and decomposition at graphite. Thus, the redox reactions of solvated lithium ions were shown to be the main interfacial reactions taking place at the graphite-negative electrode, with their control being possible by appropriate variation of current density and charge capacity.Herein, we investigated the redox reactions of solvated lithium ions at graphite in 1 M·LiClO


In the brain, learning is achieved through the ability of synapses to reconfigure the strength by which they connect neurons (synaptic plasticity). In promising solid-state synapses called memristors, conductance can be finely tuned by voltage pulses and set to evolve according to a biological learning rule called spike-timing-dependent plasticity (STDP). Future neuromorphic architectures will comprise billions of such nanosynapses, which require a clear understanding of the physical mechanisms responsible for plasticity. Here we report on synapses based on ferroelectric tunnel junctions and show that STDP can be harnessed from inhomogeneous polarization switching. Through combined scanning probe imaging, electrical transport and atomic-scale molecular dynamics, we demonstrate that conductance variations can be modelled by the nucleation-dominated reversal of domains. Based on this physical model, our simulations show that arrays of ferroelectric nanosynapses can autonomously learn to recognize patterns in a predictable way, opening the path towards unsupervised learning in spiking neural networks. et al. establish a model that enables learning and retrieving patterns in a neural system.Accurate modelling of memristor dynamics is essential for the development of autonomous learning in artificial neural networks. Through a combined theoretical and experimental study of the polarization switching process in ferroelectric memristors, Boyn  Cortical information flows from neuron to neuron through synapses of variable connection strength. The overall distribution of the synaptic strengths provides the neural network with memory, while learning is achieved through the synapses' reconfiguration 2  tunnel barriers combined with MnO3 (CCMO) bottom and Co top electrodes give rise to OFF/ON resistance ratios up to 104 (ref. We work with purely electronic memristors based on ferroelectric tunnel junctions (FTJs), in which an ultrathin ferroelectric film is sandwiched between two electrodes23104 ref.  paired wVth, so that a single spike cannot induce a change in resistance.t, their superposition produces the waveforms (Vpre−Vpost) displayed in the inset of G>0, synapse strengthening) or a decrease  of the FTJ conductance (G), depending on the sign of Δt. As can be seen from the experimental STDP curve in When both pre- and post-neuron spikes reach the memristor with a delay ΔModelling the shape of the STDP curve requires understanding the physical process underlying the time-dependent variation of the FTJ conductance while the waveform is applied. For this purpose, we image the pseudo real-time evolution of the ferroelectric domain configuration by means of stroboscopic piezoresponse force microscopy (PFM)S from the phase images and plot it as a function of the cumulated pulse duration in R and this normalized reversed area S  and centred at log(tmean(V))—the normalized reversed area S can be approximated as a function of time t and applied voltage V , in agreement with previous results obtained on thick ferroelectric capacitorstmean and Γ with the magnitude of the electric field. tmean) as a function of the inverse electric field, extracted from transport measurements (tmean∝exp(α/E)) where the activation field α is of the order of 3.0 V nm−1. Due to the idealized nature of BFO during MD simulations , the timescale for polarization switching is much shorter than in experiments, while the electric field is larger. Nevertheless, Merz's law can be applied to the MD simulation results  and Γ(V).Because of the direct relationship between This full description for each memristor device makes it possible to predict the conductance changes driving STDP learning in ferroelectric synapses. In We now use this physical model to simulate unsupervised learning in a spiking neural network with ferroelectric synapses. These simulations serve as a test bench to investigate the influence of the STDP waveform shape on the ability of the network to recognize patterns in images /Ca0.96Ce0.04MnO3 (20 nm) thin films are grown on YAlO3 (YAO) (001) substrates by pulsed laser deposition using a Nd:YAG laser (details of the growth can be found in ref. 3 (BFO) stabilizes the supertetragonal polymorph of BFO with giant tetragonality and a potentially large polarization pointing initially towards the bottom electrode of Ca0.96Ce0.04MnO3 (CCMO)The BiFeOFTJs are then fabricated by defining Pt (10−90 nm)/Co (5−10 nm) nanopillar electrodes (with diameters of 180–500 nm) on top of the unpatterned BFO/CCMO heterostructure, combining conventional electron-beam lithography and lift-off processes. For experiments involving electron transport and ferroelectric domain imaging experiments as in , arrays Vac=0.6 V, Vdc=−0.17 V, f=14 kHz). The bias voltage is applied to the tip while the bottom electrode is grounded.Combined transport and PFM measurements are performed using solid-state FTJs with diameters of 180 nm, as detailed in ref. t<0 and to an intermediate resistance state between ON and OFF for Δt>0. The exact values of initial resistance states in each figure areThe resistance–voltage cycles and STDP curves were obtained on fully processed FTJs with diameters of 400 and 500 nm, described in ref. Rinit(Δt<0)=6 × 105 Ω, Rinit(Δt>0)=1.2 × 107 Ω,Rinit=1.2 × 107 Ω,Rinit(Δt<0)=6 × 105 Ω, Rinit(Δt>0)=1.2 × 107 Ω.−1, which illustrate the (intrinsic) inhomogeneous switching of polarization in bulk-like supertetragonal BFO. The switched area as function of time is plotted in −2 (it reaches 100 μC cm−2 when being fully switched) in the The switching process under dc electric fields is simulated using a recently developed schemet  that integrate them weighted by the corresponding connection strength, that is, by the normalized conductance value of the respective FTJ. Once an output neuron .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures


TNXB was also associated with CKD attributed to type 1 diabetes (p = 2.5 × 10−7), a membranous nephropathy-associated variant at HLA-DQA1 was also associated with CKD attributed to systemic lupus erythematosus (p = 5.9 × 10−6), and an IgA risk variant at HLA-DRB1 was associated with both CKD attributed to granulomatosis with polyangiitis (p = 2.0 × 10−4) and to type 1 diabetes (p = 4.6 × 10−11). Associations were independent of additional risk variants in the respective genetic regions. Evaluation of CKD stage showed a significant association of the UMOD risk variant, previously identified in population-based studies for association with kidney function, for advanced (stage ≥G3b) compared to early-stage CKD (≤stage G2). Shared genetic associations across CKD etiologies and stages highlight the role of the immune response in CKD. Association studies with detailed information on CKD etiology can reveal shared genetic risk variants.Chronic kidney disease (CKD) is a global health problem with a genetic component. Genome-wide association studies have identified variants associated with specific CKD etiologies, but their genetic overlap has not been well studied. This study examined SNP associations across different CKD etiologies and CKD stages using data from 5,034 CKD patients of the German Chronic Kidney Disease study. In addition to confirming known associations, a systemic lupus erythematosus-associated risk variant at  Its genetic architecture is complex and incompletely understood. Genome-wide association studies (GWAS) have helped to gain insight into complex disease genetics3 by identifying single-nucleotide polymorphisms (SNPs) in >70 independent risk loci associated with the estimated glomerular filtration rate (eGFR), CKD disease risk and microalbuminuria (MA)6 as well as specific kidney diseases such as IgA8 or membranous nephropathy9 in case control studies. Because many of these specific kidney diseases are individually rare, only very few studies have collected sufficient numbers of patients with CKD attributed to various of these specific etiologies using one study design and protocol. Consequently, genetic risk variants identified in association with a specific etiology of CKD have so far not been examined for their association with CKD attributed to other etiologies. Capitalizing on data from the large German Chronic Kidney Disease (GCKD) study, we therefore aimed to systematically examine whether risk loci discovered for specific etiologies of CKD, especially for autoimmune conditions10, are associated with other CKD etiologies as well. Additionally, we aimed to examine whether risk loci discovered in the general population are also associated with advanced stages of CKD and with CKD in patients for whom the leading cause of disease was hypertension or diabetes, the most common causes of CKD.The prevalence of chronic kidney disease (CKD) is high with >10% of the adult population affected in many countries12 is an ongoing prospective observational study of 5,217 patients under nephrological care, followed for up to 10 years. At enrolment, all patients had CKD defined as an estimated glomerular filtration rate (eGFR) of 30–60 mL/min/1.73 m2 or either a urinary albumin-to-creatinine ratio (UACR) >300 mg/g or a protein-to-creatinine ratio >500 mg/g when eGFR was >60 mL/min/1.73 m2. The GCKD study was approved by local ethics committees and registered in the national registry of clinical studies (DRKS 0003971). All methods were carried out in accordance with relevant guidelines and regulations. Written informed consent was obtained from all subjects. Case groups for all analyses of specific CKD etiologies were derived based on the leading cause of CKD, which was determined by using standardized case report forms by the treating nephrologist. Serum creatinine was measured using an IDMS traceable gold-standard method. eGFR was calculated using the CKDepi equation13. Regardless of CKD etiology, two case groups of “advanced CKD” status were defined and included all patients with eGFR <45 ml/min/1.73 m²  or UACR ≥ 300 mg/g , respectively. As controls, 1,655 GCKD patients for whom CKD etiology was assigned to a cause that could reasonably be assumed to differ from the case groups  were used as the control group for the etiology-specific analyses. For the examination of stage G3b+ and A3 CKD, GCKD patients with eGFR ≥ 45 ml/min/1.73 m² (n = 1006) and UACR < 30 mg/g (n = 2117) were selected as control groups, respectively.The GCKD study15. Plink v1.90, R programming language and custom shell scripts were used during cleaning. Individual-level data were filtered for missingness (<3%) and mean heterozygosity (>2 SD). Sex checks were performed. These checks resulted in 57 individuals being filtered. Identity-by-descent (IBD) allele sharing measure calculations were used to check for unrecognized and cryptic relatedness. Across 1.28 × 107 evaluated pairs, we detected 11 with the proportion of alleles shared IBD of >0.1875 (between second and third degree relatives). For these pairs, we removed one individual prior to data analysis. Principal component analyses (PCA) using the software Eigenstrat SmartPCA16 were conducted to examine and account for population stratification. Outliers were removed in terms of genetic ancestry . SNPs were filtered for callrate (>96%), minor allele frequency (>1%) and for deviation from the Hardy-Weinberg equilibrium (p > 1 × 10−5). Genotype imputation using the 1000 Genomes Phase 3 ALL reference panel17 was conducted according to standard protocols15. Imputation quality was assessed using the Impute2 “info” measure and was >0.8 for all SNPs. The final dataset after QC contained 5,034 individuals with genotypes for 2,337,794 SNPs.In the GCKD study, 5,123 participants were genotyped for 2,612,357 markers at the Helmholtz Center Munich using the Illumina Infinium Omni 2.5 Exome-8 microarray . Data cleaning was performed according to standard protocols−8) and with evidence for replication3.A literature search was performed to assemble a table of all previously reported SNPs that were associated with either the kidney function measures eGFR and UACR, or with general or etiology-specific CKD risk in populations of European ancestry at genome-wide significance  = 4.5 × 10−4. Power calculations have been performed using the software package Quanto18.Thresholds of statistical significance were defined for each analysis using a Bonferroni correction to account for multiple testing and set to p < 0.05/n for two-sided hypothesis testing, with n as the number of tested SNPs multiplied by the number of phenotypes. For the analyses comparing etiologies, we used α = 0.05/(38*5) = 2.6 × 10Multivariable adjusted logistic regression analyses was used to evaluate the association between CKD etiology and genotype dosage assuming an additive genetic model, and accounted for sex and age as covariates. Conditional analyses accounted for further SNPs in addition. We did not adjust the logistic models for principal components as the genetic ancestry of the study population was very homogenous because of the study design and data cleaning. Sensitivity analyses adjusting for prinicipal components were carried out to verify that results were robust. STATA 13.0  was used to perform all analyses.3 as the exposure (Subjects and Methods). All GCKD patients were of European ancestry, 60% of them were male. Mean age at study entrance was 60 ± 12 years, and mean eGFR was 49.5 ± 18.1 ml/min/1.73 m² , STAT4 was associated with SLE (p = 9.7 × 10−5) and HLA-DPB1 with GPA , membranous nephropathy MN, n = 147), systemic lupus erythematosus , granulomatosis with polyangiitis  and type 1 diabetes mellitus , but also with MN .Interestingly, several SNPs previously reported as associated with one specific CKD-etiology were associated with one or more additional etiologies of CKD. While es Table . For insHLA-DQA1, suggesting that new associations between HLA risk variants for other CKD etiologies and MN were observed because of linkage disequilibrium with a known MN risk variant. Conversely, the association between CKD attributed to T1DM and rs1150754 was independent of previously reported risk variants for other CKD etiologies in the HLA-region . In addition, the IgA risk locus at HLA-DRB1 was associated independently with GPA conditioned on the known GPA risk variant in HLA-DPB1  and HLA-DRB1  showed independent associations with CKD attributed to T1DM. These associations remained robust in effect size and direction when adjusting for additional previously reported risk variants for T1DM27 in the HLA region  with CKD stage 3b  and the second as UACR ≥ 300 mg/g . These cases were compared to a GCKD control group of 2,245 patients with eGFR > 60 ml/min/1.73 m² and 2,117 patients with UACR < 30 mg/g, respectively. Of the risk loci discovered in the general population, In a second set of analyses, the association between the 55 population-based risk loci and CKD for which the treating nephrologists had determined diabetes (n = 653) or hypertension n = 1086) as the leading cause were examined because they represent the majority of CKD cases in population-based studies. No significant association was detected for both hypertensive nephropathy and diabetic kidney disease  or the MN risk variant rs2187668 and an association with SLE nephropathy (90%). Nevertheless, power was moderate for other combinations, such as the association between the IgA risk variant rs660895 and GPA (50%). A priori power calculations were complicated by the fact that it is unclear whether the effect size of a genetic risk variant is the same or similar for different CKD etiologies. Power calculations were therefore provided across a range of case numbers, allele frequencies and effect sizes  to detect some true-positive associations such as the ones between MN nephropathy and the validated risk alleles at 9% to detThe HLA region is known for containing various risk loci for CKD of different etiologies. Several of them could be examined across CKD etiologies available in the GCKD study, and showed evidence of novel, additional associations that had not been identified at genome-wide significance in genome-wide association studies of these additional etiologies. The associations across CKD etiologies highlight the shared role of the adaptive immune response, and suggest some overlap between etiologies. For example, known risk variants for MN were independently associated with CKD attributed to SLE, which is interesting in light of the histopathological appearance of membranous nephropathy in lupus nephritis class V.28. In this report, SLE and T1DM co-exist more often than expected based on the prevalence of both diseases. Several case reports describe different overlapping auto-immune diseases that affect the kidney, such as MN and IgA nephropathy29 or MN and further extra-renal autoimmune diseases such as colitis ulcerosa)30. More detailed and better powered studies focusing on SNP associations across sub-types of different autoimmune diseases are required to address these important questions in more detail. Furthermore, studies with larger case groups of the respective disease could examine co-incidences of two diseases with overlapping genetic risk factors such as MN/SLE and T1DM/GPA.The shared genetic risk variant for CKD attributed to SLE and to T1DM is supported by a report that examined the co-existence of auto-immune diseasesThe T1DM risk loci found in our study can be interpreted in two ways: either as risk loci for T1DM itself, or as risk loci for diabetic nephropathy resulting from T1DM, or a combination of both. To resolve this question, a control group of T1DM patients without CKD would be required.UMOD locus known to be associated with kidney function in the general population was also associated with advanced CKD (stage G3b). Experimental evidence links genotype at the UMOD risk variant identified in GWAS to altered gene expression and salt-sensitive hypertensive CKD31. In support, the risk allele at the UMOD variant was significantly associated with stage G3b + CKD in our study, and the association with CKD attributed to hypertension was direction-consistent to the UMOD variant reported in previous GWAS of hypertension32. This finding illustrates associations across a spectrum of renal function from small changes in eGFR in population-based studies over advanced CKD in the GCKD study to severe renal phenotypes in monogenic diseases caused by loss of function mutations in UMOD33.We additionally found that the Strengths of our study include the availability of CKD of different stages and from different etiologies in one study using the same standardized recruitment procedures, and the availability of genome-wide genotype data that allows for carrying out conditional analyses. Other CKD cohorts do not have access to comparable numbers of carefully phenotyped subgroups of patients with as many specific CKD etiologies. Nevertheless, because of limited sample size within subgroups, analyses in our study were restricted to the examination of a predefined number of candidate SNPs and the study of large and moderate genetic effects, rigorously accounting for multiple comparisons. Limitations include the absence of an internal control group of patients without CKD who suffer from the specific diseases that can give risk to CKD, such as T1DM patients without nephropathy.UMOD locus that is associated with CKD in the general population to also associate with advanced stage CKD (G3b+) in the GCKD study, supporting the presence of genetic risk across the spectrum of kidney function.In conclusion, genetic risk variants for specific etiologies of CKD were shared across some etiologies, suggesting a common mechanism by which the adaptive immune system may contribute to the shared etiologies. In addition, we found the known risk variant at the Supplementary Material