The molecules of life were created by a continuous physicochemical process on an early Earth. In this hadean environment, chemical transformations were driven by fluctuations of the naturally given physical parameters established for example by wet–dry cycles. These conditions might have allowed for the formation of (self)-replicating RNA as the fundamental biopolymer during chemical evolution. The question of how a complex multistep chemical synthesis of RNA building blocks was possible in such an environment remains unanswered. Here we report that geothermal fields could provide the right setup for establishing wet–dry cycles that allow for the synthesis of RNA nucleosides by continuous synthesis. Our model provides both the canonical and many ubiquitous non-canonical purine nucleosides in parallel by simple changes of physical parameters such as temperature, pH and concentration. The data show that modified nucleosides were potentially formed as competitor molecules. They could in this sense be considered as molecular fossils. How RNA building blocks have formed on an early Earth by a continuous process is still a mystery awaiting its solution. Here, the authors report that fluctuations of physical parameters like temperature and pH could have been enough to facilitate nucleoside formation from simple starting materials. Molecules such as nucleic acids and amino acids must have formed by a continuous physicochemical process, in which greater structural complexity was generated based on fluctuations of the naturally given physical parameters3. Geothermal fields, for example, could have established such fluctuations by wet–dry cycles that may have driven chemical transformations, which ultimately allowed the emergence of life8. The appearance of (self)-replicating RNA was certainly of central importance for the transition from an abiotic world to biology11. We need to consider, however, that an early genetic polymer might have been structurally different from contemporary RNA. This involves differences regarding the sugar configuration  or the presence of other nucleobases13. Selection pressure led in this scenario to the chemical evolution of RNA. Contemporary RNA molecules contain four canonical nucleosides , which establish the sequence information. In addition, >120 non-canonical nucleosides are present, which govern a diverse set of properties such as correct folding, e.g., to enable catalysis14. In fact, the genetic system of all known life is dependent on modified nucleosides. Many of these non-canonical nucleosides are found today in all three domains of life, which indicates that they were present early on during the development of life. For the ubiquitous non-canonical nucleosides we may assume that they were already formed as competitors in parallel with the canonical ones on the early Earth15. So far, however, a geochemical scenario that would allow for the parallel formation of canonical and non-canonical RNA building blocks by a continuous process is not known. All reported multistep chemical models so far rely on tightly controlled laboratory conditions and the isolation and purification of central reaction intermediates by sophisticated methods19.The molecules of life originated around 4 billion years ago under conditions governed by the composition of the Earth’s crust and atmosphere at that time2A, m2A, DA, G, m2G, m22G, m1G). Since chemical evolution depended on those molecules that were available on early Earth, these non-canonical nucleosides may be considered to be molecular fossils, which maintained their essential life-supporting character until the present day.Herein, we report a robust synthetic pathway, which is purely based on fluctuations of physicochemical parameters such as pH, concentration, and temperature, driven by wet–dry cycles. These fluctuations enable the direct enrichment or purification of all reaction intermediates that are directly used for the next synthetic steps. As such, a continuous synthesis is established. Our results show that RNA building blocks can indeed be formed in a prebiotically plausible geochemical environment without sophisticated isolation and purification procedures. The chemical scenario presented here supports the hypothesis that life may have originated in a hydrothermal milieu on land rather than in a deep sea environment. The key assembly step in our pathway is the formation of variously substituted 5-nitroso-pyrimidines (nitrosoPys) that can be converted into formamidopyrimidines (FaPys) in the presence of formic acid and elementary metals (Ni or Fe). When combined with ribose, the FaPy compounds react to give a set of purine nucleosides. This chemical pathway delivers not only the canonical purine nucleosides but concomitantly many of the ubiquitously present non-canonical relatives, suggesting their origin as prebiotic competitor nucleosides , both recognized prebiotic compounds18. In addition, sodium nitrite and acetic acid are present to establish a slightly acidic pH of around 4. Under these conditions the amidine molecules (2a-d) are protonated, which leads to their chemical deactivation. This allows selective nitrosation of malononitrile 1 to give (hydroxyimino)malononitrile 3 in situ. Slow evaporation of water under ambient conditions, followed by gentle cooling to 8–10 °C resulted in crystallization of a salt from the ca. 1 M amidinium solution. This crystallization is very robust and resembles naturally occurring concentration processes. The resultant crystals had excellent quality for X-ray analysis, which showed that the salts are formed from the amidinium cations 2a-d and the (hydroxyimino)malononitrile anion of 3  was applied to simulate soil that would slowly heat up. Subsequent 1H-NMR analysis indicated successful formation of the anticipated nitroso-pyrimidines 4a-d  in excellent purities and good chemical yields between 60 and 85%  and its 2-thiomethyl derivative (ms2A) are derived later, whereas 4e gives rise to guanosine derivatives  or 2-methyl (4d) substituted nitroso-pyrimidines, we noted that the 2-thiomethyl functionality in 4b and 4e was prone to undergo selective nucleophilic substitution. Reaction of 4e with different amines leads to efficient formation of the nitroso-pyrimidines 4g-i with the concomitant release of methanethiol. Due to its insolubility under basic conditions, nucleophilic substitutions of 4b are very inefficient. To confirm this, we partially hydrolyzed 4b to 4e in the presence of methylamine (300 mM) and dimethylamine (100 mM). The pH was carefully adjusted with Na2CO3 to about pH 10. Compound 4b precipitated, while 4e stayed in solution, consequently protecting 4b from further reactions. It is in this context interesting that nucleosides that would form via aminolysis of 4b have not yet been found in nature. In contrast, 4e reacts efficiently and after 3–4 days at room temperature 4e is almost completely converted into 4g and 4h, which are direct precursors to the ubiquitous non-canonical RNA bases m2G and m22G , all of which possess the right substitution pattern for the synthesis of naturally occuring canonical and non-canonical RNA nucleosides. Because all the formed nitroso-pyrimidines are poorly soluble in water at neutral pH, neutralizing the solutions leads to their efficient precipitation, providing a naturally occurring purification step  are soluble in water under acidic conditions, while the nitroso-compounds that are converted into guanosine-derived nucleosides  are soluble under basic pH conditions. These properties allow for potentially divergent chemical pathways leading to A-derived and G-derived nucleosides  the latter compounds precipitate as insoluble carbonate or hydroxide salts. The FaPys 5a-h are thus washed away, while the transition metal compounds sediment out. Final evaporation of water concentrates the reaction mixture, leading to the crystallization of the FaPy molecules. This third physical enrichment step, involving a wet–dry cycle, leads to the NMR-clean formation of FaPy-derivatives 5a-h   H2 Fig. . In the a-h Fig. . The wata-h Fig. .4b) gives after treatment with formic acid and elementary Ni two different FaPy products depending on the reaction conditions. One of the products (5b) contains a thiomethyl group, while the other (5c) is desulfurated. The desulfurization reaction is simply controlled by time and can be promoted when H2 is bubbled through the solution prior to reaction. Compound 5c is always generated in a stepwise reaction cascade via compound 5b which was confirmed by reacting 4b for 2 h and isolating the only product formed , which is today found in all three domains of life.The 2-(methylthio)-5-nitrosopyrimidine-4,6-diamine  engage in a rapid reaction with ribose to give the corresponding FaPy-ribosides. Upon dissolution in water and subsequent heating under basic conditions, all four expected purine α/β-ribofuranosides (α/β−f) and α/β-pyranosides (α/β−p) are obtained  m2G, m22G, m1G, ms2A and m2A  other regioisomers were found as well. These isomers were not formed when pure FaPy starting materials were used that were not derived from our continuous synthesis. We believe that these isomers might be the N3-connected nucleosides, previously proposed by Wächtershäuser for homo-purine RNA25. Despite the presence of these side products, we observe efficient N9-nucleoside formation with remarkable yields of up to 60% for the canonical and the non-canonical nucleosides. This work demonstrates that the non-canonical compounds could plausibly have formed as companion and potential competitor compounds in parallel to the canonical nucleosides.All of the prepared FaPy compounds undergo rapid and regioselective condensations with ribose when they are present in the same dry-state environment Fig. . We do nLife on earth certainly did not start in a chemists’ laboratory, where the relevant compounds are assembled in a step-by-step process from pure starting materials under tightly controlled conditions. Even if the individual reaction steps are performed under plausibly prebiotic conditions, the controlled assembly over many steps with sophisticated isolation and purification procedures of reaction intermediates is an unlikely scenario for chemical synthesis under early Earth conditions. For the process of chemical evolution on the early Earth, we may rather envision a more continuous synthesis, in which small organic molecules, initially formed by volcanic action or lightning, reacted to give increasingly more complex structures Fig. . Here, c18 and pyrimidine 16 RNA building blocks has been previously demonstrated in aqueous environments. It is questionable, however, if these multistep synthesis pathways are able to provide all four canonical bases at the same time, which fuels the development of new prebiotically plausible nucleoside formation reactions27. Recently, all four canonical nucleosides  were accessed in low yields via a one-pot procedure from pure formamide28. However, in order to establish a functional genetic system a number of non-canonical nucleosides is required as well that provide other functions related to folding and catalysis34. Since many of these non-canonical bases are present in all three domains of life, it is likely that they have been early on part of the abiotic chemical selection process15. We report here the discovery of a continuous synthesis pathway that enables the efficient production of canonical and non-canonical purine bases in parallel. Our data show that formation of the many nucleosides needed to establish a functional genetic system is in fact an unavoidable event if we assume the presence of simple starting materials such as formic acid, acetic acid, sodium nitrite, malononitrile (1), amidinium compounds (2a-d), as well as transition metals like Ni or Fe. These simple compounds react in several successive wet–dry phases, leading to physical enrichment  of reaction intermediates to finally give RNA building blocks. Wet–dry cycles have already been shown to be a plausible geological scenario especially for polymerization reactions36. Our reported chemical scenario shows now that such a geological setup can also result in a diverse set of purine nucleosides by continuous synthesis 40. Based on the continuous synthesis pathway reported here, we hypothesize that the canonical and at least these non-canonical nucleosides could have formed side by side, dating the formation of the first non-canonical nucleosides back to the origin of chemical evolution around 4 billion years ago. As such, they could have been part of the chemical evolution process that established the putative RNA world41. Our chemistry invokes that methylated and thiomethylated nucleosides could particularly have been integral components of the first instructional (pre)-RNA molecules, likely to stabilize folded structures in order to accelerate catalytic processes34. We therefore propose that these nucleosides could be vestiges and molecular fossils of an early Earth, as it was suggested for cofactors42.Importantly, all here-reported non-canonical bases are known to exist in the three domains of life. Many are postulated components of the early genetic system of the last universal common ancestor (LUCA), suggesting that they were indeed present already at the onset of biological evolution2a) hydrochloride salt  and malononitrile (1)  was dissolved in H2O  in a 500 mL beaker. A solution of NaNO2  was slowly added at room temperature. After stirring at room temperature for 2 h the reaction mixture was kept at 45 °C in an oil bath for 3–4 days open to the air until the mixture was concentrated to about 100 mL. The reaction mixture was placed in a fridge overnight at 8–10 °C. The formed yellow crystals were filtered off to give the desired product .1-methylguanidine  δ = 7.23 , 2.72 . 13C-NMR  δ = 157.85, 119.50, 113.31, 107.18, 28.23. IR (cm−1): 3405 (m), 3351 , 3197 (m), 2977 , 2229 (s), 2218 (s) 1675 (s), 1635 (s), 1465 (w) 1428 (m), 1344 (s), 1269 (s) 1226 (s), 1172 (w), 1098 (m), 915 (m), 765 (m).S-Methylisothiourea (2b) hemisulfate salt  and malononitrile (1)  was dissolved in H2O  in a 600 mL beaker. A solution of NaNO2  was slowly added at room temperature. After stirring at room temperature for 2 h the reaction mixture was kept at 45 °C in an oil bath for 3–4 days open to the air until the mixture was concentrated to about 200 mL. The reaction mixture was placed in a fridge overnight at 8–10 °C. The formed yellow crystals were filtered off to give the desired product .Mp.: 126 °C. 1H-NMR  δ 8.90 , 2.56 . 13C-NMR  δ 171.62, 119.46, 113.29, 107.17, 13.71. IR (cm−1): 3282(m), 3146 , 2742 , 2530 (w), 2222 (s), 2213 (s) 1698 (m), 1663 (s), 1643 (s) 1549 (m), 1450 (m), 1424 (s) 1375 (w), 1335 (s), 1269 (s), 1223 (s), 1180 (w), 1099 (m), 1076 (w), 982 (m), 970 (w), 960 (w), 897 , 801 (m), 736 (m).2c) hydrochloride salt  and malononitrile (1)  was dissolved in H2O  in a 500 mL beaker. A solution of NaNO2  was slowly added at room temperature. After stirring at room temperature for 2 h the reaction mixture was kept at 45 °C in an oil bath for 3–4 days open to the air until the mixture was concentrated to about 100 mL. The reaction mixture was placed in a fridge overnight at 8–10 °C. The formed yellow crystals were filtered off to give the desired product .Guanidine  δ = 6.91 . 13C-NMR  δ = 158.32, 119.52, 113.32, 107.18. IR (cm−1): 3473 (m), 3373 (m), 3172 (w), 3087 (w), 2815 , 2223 (s), 2217 (s), 1668 (m), 1641 (s), 1578 (w), 1552 (w), 1369 (w), 1343 (s), 1294 (w), 1264 (s), 1220 (s), 1140 (w), 975 (w), 792 (w), 757 (w).2d) hydrochloride salt  and malononitrile (1)  was dissolved in H2O  in a 500 mL beaker. A solution of NaNO2  was slowly added at room temperature. After stirring at room temperature for 2 h the reaction mixture was kept at 45 °C in an oil bath for 3–4 days open to the air until the mixture was concentrated to about 100 mL. The reaction mixture was placed in a fridge overnight at 8–10 °C. The formed yellow crystals were filtered off to give the desired product .Acetamidine  δ = 8.60 , 2.11 . 13C-NMR  δ = 168.11, 119.50, 113.31, 107.18, 18.72. IR (cm−1): 3282 (m), 3140 , 2781 (m), 2395 (w), 2236 (s), 2217 (s) 1708 (s), 1661 (s), 1592  1513 (m), 1373 (s), 1351 (s) 1259 (s), 1200 (s), 1191 (s), 1160 (w), 1125 (m), 969 (w), 907 (w), 883 (w), 857 (w), 790 (s), 691 (s), 684 (s).4a-d depend on the crystal size of the organic salts and the heating source. Usually conversions were done in a beaker open to the air in an oil bath to simulate hot soil. Large crystals sometimes already convert into nitroso-pyrimidines by a solid-state reaction without melting. Then much longer reaction times of up to 7 days are required because of unequally distributed temperature. Alternatively, the organic salt can be converted by heating in an oven where temperature is equally distributed within the sample.The reaction time of the following procedures for the formation of 2a) salt of (hydroxyimino)malononitrile (3)  was heated slowly to its melting temperature of 108 °C and kept overnight. The compound melts suddenly but becomes a dark red solid again after leaving it overnight. The quantitative reaction mixture can be directly used for the next step without purification.1-methylguanidine (2a) salt of (hydroxyimino)malononitrile (3)  was reacted as described above. The reaction mixture was suspended well in water (2–3 mL) and the dark red solid was filtered off to give 6-imino-1-methyl-5-nitroso-1,6-dihydropyrimidine-2,4-diamine .For analytical reasons a small batch of 1-methylguanidine  δ = 11.44 , 8.41 , 8.08 , 7.67 , 7.49 , 3.26 . 13C-NMR  δ = 165.44, 157.64, 146.95, 137.22, 27.64. HRMS (ESI + ): calc. for [C5H9N6O]+ 169.0832, found: 169.0832 [M + H]+2b) salt of (hydroxyimino)malononitrile (3)  was heated slowly to its melting temperature of 126 °C and kept overnight. Caution: if the product is heated too quickly above the melting temperature it decomposes with the release of MeSH! The compound melts suddenly but becomes a dark green solid again. The quantitative reaction mixture can be directly used for the next step without purification.Methylthioamidine (2b) salt of (hydroxyimino)malononitrile (3)  was reacted as described above. The reaction mixture was suspended in water (2–3 mL) and the dark green solid was filtered off to give 2-(methylthio)-5-nitrosopyrimidine-4,6-diamine .For analytical reasons a small batch of methylthioamidine  δ 10.18 , 9.00 , 8.42 , 8.02 , 2.46 . 13C-NMR  δ 179.05, 164.73, 146.22, 139.43, 14.08. HRMS (ESI+): calc. for [C5H8N5OS]+ 186.0444, found: 186.0444 [M + H]+2c) salt of (hydroxyimino)malononitrile (3)  was heated slowly to its melting temperature of 159 °C and kept overnight. The compound melts suddenly but becomes a red/pinkish solid again after leaving it overnight. The quantitative reaction mixture can be directly used for the next step without purification.Guanidine (2c) salt of (hydroxyimino)malononitrile (3)  was reacted as described above. The reaction mixture was suspended in water (2–3 mL) and the solid was filtered off to give NMR clean 5-nitrosopyrimidine-2,4,6-triamine .For analytical reasons a small batch of guanidine  δ = 10.26 , 8.15 , 7.75 , 7.35 , 7.19 . 13C-NMR  δ = 166.52, 165.32, 151.43, 138.04. HRMS (ESI+): calc. for [C4H7N6O]+ 155.0676, found: 155.0676 [M + H]+2d) salt of (hydroxyimino)malononitrile (3)  was heated slowly to its melting temperature of 142 °C and kept overnight. The compound melts suddenly but becomes a red/pinkish solid again after leaving it overnight. The quantitative reaction mixture can be directly used for the next step without purification.Acetamidine (2d) salt of (hydroxyimino)malononitrile (3)  was reacted as described above. After suspension in H2O (2–3 mL) the solid is filtered off to give NMR clean 2-methyl-5-nitrosopyrimidine-4,6-diamine .For analytical reasons a small batch of acetamidine  δ = 10.04 , 8.97 , 8.36 , 7.96 , 2.20 . 13C-NMR  δ = 175.20, 166.38, 146.76, 139.94, 26.83. HRMS (ESI-): calc. for [C5H6N5O]− 152.0578, found: 152.0578 [M-H]−2a) salt of (hydroxyimino)malononitrile (3); 1574223 for methylthioamidine (2b) salt of (hydroxyimino)malononitrile (3); 1574225 for guanidine (2c) salt of (hydroxyimino)malononitrile (3); 1574224 for acetamidine (2d) salt of (hydroxyimino)malononitrile (3). These can be obtained free of charge from CCDC via www.ccdc.cam.ac.uk/data_request/cif.All data generated or analyzed during this study are presented in this article and its Supplementary Information File, or are available from the corresponding author upon reasonable request. X-ray crystallographic data were also deposited at the Cambridge Crystallographic Data Centre (CCDC) under the following CCDC deposition numbers: 1574226 for 1-methylguanidine (Supplementary InformationPeer Review File


Cells of the Th1 and Th17 responses cause renal tissue damage, while Tregs mediate protection. Recently, a high degree of plasticity among these T cell lineages was proposed. During inflammation, Th17 cells were shown to have the potential of transdifferentiation into Th1, Th2, or alternatively anti-inflammatory Tr1 cells. Currently available data from studies in GN, however, do not indicate relevant Th17 to Th1 or Th2 conversion, leaving the Th17 cell fate enigmatic. Tregs, on the other hand, were speculated to transdifferentiate into Th17 cells. Again, data from GN do not support this concept. Rather, it seems that previously unrecognized subspecialized effector Treg lineages exist. These include Th1 specific Treg1 as well as Th17 directed Treg17 cells. Furthermore, a bifunctional Treg subpopulation was recently identified in GN, which secrets IL-17 and coexpresses Foxp3 together with the Th17 characteristic transcription factor RORγt. Similarities between these different and highly specialized effector Treg subpopulations with the corresponding T helper effector cell lineages might have resulted in previous misinterpretation as Treg transdifferentiation. In summary, Th17 cells have a relatively stable phenotype during GN, while, in the case of Tregs, currently available data suggest lineage heterogeneity rather than plasticity.Multiple studies have identified CD4 These include primary forms, for example, IgA nephropathy or membranous GN, as well as secondary forms developing as a consequence of systemic diseases as lupus nephritis and ANCA-associated vasculitides . Howeverms of GN .It has been shown that especially cells of the Th1 and Th17 responses are highly nephritogenic –8. Dysre+ T cell subtypes follow a concerted time course. Th17 cells were shown to be involved very early in inflammation [Given the central roles of T helper effector and T regulatory cells in GN, it is well worthwhile to study their biology and function in detail. Interestingly, increasing evidence suggests that systemic expansion and renal infiltration of different CD4ammation –17. Afteammation . Finallyammation , 17.+ Tregs.This defined time course of renal infiltration, which is initially dominated by Th17 cells, followed by Th1 cells and Tregs at later stages, has led to speculations of possible transdifferentiation of Th17 cells into another cell type. One possibility, which has been suggested, is reprogramming of Th17 cells to acquire a Th1 phenotype , 19. Alt+ T cells, that is, plasticity, has been addressed using different methods over the past decade in human and mouse. In vitro approaches as well as transfer experiments using highly purified populations of CD4+ T cell subsets have established the view that CD4+ T cells can change their polarity under certain conditions [+ T cells, lineage-tracing systems using Cre-recombinase expression under the control of key cytokines or transcription factors and subsequent permanent fluorochrome expression have been established [ in vitro polarized T cell subsets.The change in characteristics of single CD4nditions , 20–26. ablished , 27–29.  in vitro culture [+ T cells, on the other hand, can be addressed by using T cell receptors (TCR) as an endogenous barcoding system. Sequencing of TCR revealed a great diversity in the phenotype of cells that presumably descend from a single CD4+ T, cell indicating potential transdifferentiation [+ T cells have been reviewed recently in detail by DuPage and Bluestone [In very elegant studies with IL-17A-Cre fate reporter mice, Hirota et al. have established the concept that encephalitogenic Th17 cells have a high degree of plasticity into the Th1 phenotype in experimental autoimmune encephalomyelitis (EAE), the mouse model for multiple sclerosis . Further culture , 33. Plantiation , 35. Stuluestone .+ Tregs [+ T cell plasticity with a particular focus on glomerulonephritis.In summary, increasing data suggest instability or plasticity, especially, of Th17 cells. However, to complicate things, numerous studies have also postulated a diametrically opposite concept; namely, Th17 cells might derive from transdifferentiation of Foxp3+ Tregs , 37–40.  in vitro polarized Th1 and Th17 cells into T cell deficient RAG1 knockout mice and analyzed the pathogenicity of these cell types in a planted-antigen model of GN [γ but no IL-17 or IL-4 was produced by splenocytes after the transfer of Th1 cells. In contrast, some IFNγ was also produced by spleen cells after the transfer of Th17 cells, indicating that some Th17 cells might have adopted a Th1 phenotype. It is, however, important to note that T cell pathogenicity rather than plasticity was the primary focus of this study. As a result, certain restrictions limit the interpretation of the results. In particular, the in vitro polarized Th17 cells contained a relevant fraction of IFNγ producing Th1 cells even before transfer, which clearly limits analysis. Furthermore, only systemic but not organ specific T cell responses in the kidney were addressed. In summary, this study indicates stability of splenic Th1 cells, without significant Th1 to Th17 or Th2 plasticity but suggests some degree of Th17 cell transdifferentiation into cells of the Th1 type during GN. More recent data, however, do no support this latter concept. Tulone et al. traced the fate of in vitro Th17 polarized cells in another planted-antigen model of GN [Given the high nephritogenic potential of Th17 cells , 41, theel of GN . Analysiel of GN . These a in vitro polarized Th17 cells from fluorescence reporter mice [In a recent study, we have addressed the plasticity of renal Th17 cells in more detail . After tter mice  and subster mice , these fter mice , leavingter mice . This inSince Th17 cells are pathogenic in crescentic glomerulonephritis , 41, we + Tregs. However, available data from renal disease do not support this notion either. In a recent study, we have transferred Treg depleted spleen cells into RAG1-deficient mice and induced a model of accelerated crescentic glomerulonephritis [+ cells at the end of the experiment. This finding indicates that there is no transdifferentiation of non-Treg T cell subsets, including Th17 cells, into FoxP3+ Tregs in this model. Furthermore, in another study, we transferred highly purified Th17 cells into lymphocyte intact mice and traced their fate during GN, using a congenic marker system. Interestingly, most of the transferred Th17 cells had lost their RORγt expression. Importantly, though, none of these ex-Th17 cells had upregulated Foxp3 in kidneys or spleens, which excludes transdifferentiation into Tregs [Since renal Th17 cells do not seem to acquire either Th1 or Th2 phenotypes, an alternative scenario would be Th17 transdifferentiation into anti-inflammatory Foxp3to Tregs . An over+ T cell plasticity is the hypothesis that Foxp3+ Tregs might possess the capacity to transdifferentiate into T effector cells. This notion was supported by early experiments, in which Tregs were highly purified from Foxp3 fluorescence reporter mouse strains and transferred into different T cell deficient recipients [γ. Further rounds of experiments, however, made clear that this observed high degree of instability was overestimated due to the lymphopenic environment in the recipient mice. When Tregs were cotransferred with T helper effector cells or transferred into lymphocyte intact recipients, only few Tregs lost their phenotype [γt [Another much discussed aspect of CD4cipients . Surprishenotype , 40, 47.henotype , 48–50. henotype . Anotherhenotype –54. Fourhenotype , 55, 56.henotype –60. In ahenotype , 61–63. henotype , 60 and henotype , 57, 58.type [γt , 65. TheLandmark studies by Chaudhry et al.'s group have suggested that Tregs can coactivate the Th17 characteristic transcription factor Stat3 . This pr+ Tregs express large amounts of the Th1-type cytokine IFNγ. However, the opposite was the case. Elaborate transfer studies, using Tregs from T-bet pan knockout mice, showed that T-bet confers Tregs with the capacity to effectively downregulate type-1 immunity. Absence of Treg1 cells resulted in overshooting Th1 responses, underlining their regulatory rather than proinflammatory function [+ Tregs as effector Treg population, specialized for the control of pathogenic Th1-type inflammation. Taken together the available data from studies of GN suggest Treg lineage heterogeneity and specialization during organ inflammation rather than instability or plasticity. An illustration of this concept is shown in Similar to activation of the Th17 characteristic transcription factor Stat3, pioneering studies by Koch et al. have noted coexpression of Foxp3 with the Th1 master inducer T-bet . Again, function . In analfunction . Importafunction . These oγt [γt+ Tregs were found to be present not only in mice but also in healthy humans as well as in many inflammatory conditions [The observation that Tregs can coactivate certain transcription factors of T helper effector cell lineages has led to the discovery of cells, expressing Foxp3 together with the Th17 master transcription factor RORγt . These Rnditions –68.γt likely candidates [γt+Foxp3+ T cells. During different models of GN, we found that RORγt+ Tregs massively expanded systemically and also in the nephritic kidneys, early during the course of inflammation [γt+ Tregs cytokine profile revealed production of both, Treg characteristic cytokines TGF-β, IL-35/EBI-3, and IL-10 and the Th17 hallmark cytokine IL-17A. Since this observation indeed suggested that RORγt+ Tregs are cells undergoing transdifferentiation, either from Th17 into Tregs or the other way around, we performed transfer studies. Transfer of highly purified RORγt+Foxp3− Th17 cells or RORγt− Foxp3+ conventional Tregs into congenic lymphocyte intact recipients revealed that none of the single transcription factor positive cells upregulated the other transcription factor during the course of experimental GN. This observation surprisingly indicated that RORγt+ Tregs did not derive from Treg/Th17 transdifferentiation but rather represent an independent cell lineage. However, a second hypothesis, explaining the origin of RORγt+ Tregs, might be transient and promiscuous upregulation of Foxp3 during early activation of Th17 cells [γt+ Th17 cells, as event leading to generation of RORγt+Foxp3+ Tregs [γt+ Tregs, however, remained. Since RORγt is a downstream target of Stat3, we wanted to evaluate whether RORγt+ Tregs might possibly resemble Treg17 cells, which we had previously shown to be induced by activation of Stat3 [γt expression in Tregs in both spleens and nephritic kidneys, excluding this possibility and further underlining the independent nature of RORγt+ Tregs. Next, we decided to generate mice with selectively impaired RORγt activation in Foxp3+ Tregs. If RORγt+ Tregs were Treg to Th17 transdifferentiating cells, this transdifferentiation would be impaired in these mice and they should progressively accumulate Tregs, while Th17 cell numbers should decline over time. In case that bi-Tregs resembled Treg17 cells, Th17 responses should be overshooting in mice with deficient RORγt activation in Tregs. Analyses at different time points in different models of GN, however, revealed unchanged Treg and Th17 cell homeostasis. Again, this observation indicated an independent nature of RORγt+ Tregs, with no signs of Th17 transdifferentiation or Treg17 specialization [γt+ Tregs. For this purpose, they were highly purified by flow cytometric sorting and subsequently transferred into congenic recipient mice. 10 days after induction of GN, the transferred cells were reanalyzed in spleens and nephritic kidneys. Our results showed that the transferred RORγt+ Tregs had massively expanded in both organs during inflammation. Much to our surprise, the vast majority had lost both transcription factors, RORγt and Foxp3, to become double negative (ex-RORγt+ Tregs). Importantly, however, we did not observe relevant transdifferentiation of RORγt+ Tregs into either RORγt+Foxp3− Th17 cells or conventional RORγt−Foxp3+ Tregs, leaving the fate of ex-RORγt+ Tregs unknown [γt+ Tregs are not Tregs transdifferentiating into Th17 cells, (2) RORγt+ Tregs are not Th17 cells on their way to become Tregs, (3) RORγt+ Tregs are not Th17 cells transiently expressing Foxp3 during activation, and (4) RORγt+ Tregs do not resemble Th17 specific Treg17 cells. Rather, they represent an independent effector Treg lineage, which rapidly expands during inflammation and subsequently retracts by as to yet unknown mechanisms. Based on currently available data, we thus propose the concept outlined in Several authors had suggested before that some Treg subpopulations might transdifferentiate into Th17 cells, making cells that coexpress Foxp3 with RORndidates –40, 48. 17 cells , 55, 56.of Stat3 , 58. Anaγt+ Tregs has recently been confirmed by three highly ranked studies [γt+ Tregs showed a high degree of stability with no relevant transdifferentiation into Th17 cells or conventional Tregs [The independent nature of ROR studies , 65, 69.γt+ Tregs, concepts are, however, still evolving. Our studies have shown that exogenously transferred RORγt+ Tregs were highly protective in a model of acute GN. However, endogenous RORγt+ Tregs had additional proinflammatory functions. Selective inhibition of RORγt activation in Tregs resulted in complete abrogation of their IL-17 production and ameliorated renal tissue damage in acute GN [γt expression in Tregs is crucial for suppression of Th2 immunity [γt+ Tregs might thus be potent regulators of Th2 responses and could be important to protect from allergies [γt coexpressing Tregs are not Treg/Th17 transdifferentiating or Th17 specialized Treg17 cells but represent a unique, stable, and independent T cell lineage with both regulatory and proinflammatory functions.With respect to the function of RORimmunity . Our ownllergies . In summ+ T cell plasticity is of high clinical relevance in the light of newly emerging T helper cell directed therapies. While Th1 cells display a relatively high degree of stability in GN, Th17 cells may have different fates. In several experimental models of nonrenal inflammatory disease, most Th17 cells transdifferentiate into a Th1 phenotype. In GN, however, a significant proportion of Th17 cells seems to be stable and maintains the differentiation status. Another fraction, in contrast, appears to undergo functional changes and loses Th17 characteristics. Nevertheless, currently available data do not support Th17 conversion into Th1, Th2, or Treg cells during the course of GN, leaving the nature of ex-Th17 cells unknown. Interestingly, however, Th17 cells can be pushed into a tolerogenic phenotype by anti-CD3 treatment, which represents a promising therapeutic strategy. In the case of Tregs, data from GN underline a very high degree of stability and show no hint for transdifferentiation. Rather, a new paradigm is emerging, suggesting activation and diversification of naive Tregs into different effector Treg lineages as RORγt+ Tregs, T-bet+ Treg1, and Stat3+ Treg17 cells.A thorough understanding of CD4


Wound healing represents a dynamic process involving directional migration of different cell types. Chemokines, a family of chemoattractive proteins, have been suggested to be key players in cell-to-cell communication and essential for directed migration of structural cells. Today, the role of the chemokine network in cutaneous wound healing is not fully understood. Unraveling the chemokine-driven communication pathways in this complex process could possibly lead to new therapeutic strategies in wound healing disorders.We performed a systematic, comprehensive time-course analysis of the expression and function of a broad variety of cytokines, growth factors, adhesion molecules, matrixmetalloproteinases and chemokines in a murine cutaneous wound healing model.Strikingly, chemokines were found to be among the most highly regulated genes and their expression was found to coincide with the expression of their matching receptors. Accordingly, we could show that resting and activated human primary keratinocytes , dermal fibroblasts  and dermal microvascular endothelial cells  express a distinct and functionally active repertoire of chemokine receptors. Furthermore, chemokine ligand–receptor interactions markedly improved the wound repair of structural skin cells in vitro.Taken together, we here present the most comprehensive analysis of mediators critically involved in acute cutaneous wound healing. Our findings suggest therapeutic approaches for the management of wound closure by targeting the chemokine network.The online version of this article (10.1186/s40001-017-0299-0) contains supplementary material, which is available to authorized users. Wound healing is a complex phenomenon that requires an integrated network of repair mechanisms, including cell migration, proliferation and cellular production of structural molecules essential for tissue regeneration. The tissue response to injury is characterized by three overlapping phases: (a) Inflammation, (b) granulation and (c) matrix remodeling . These pChemokines are small, secreted proteins which mediate directional migration and regulate leukocyte trafficking –21. NotaThe aim of this work was to investigate the complex interactions of chemokines and their receptors in cutaneous wound healing. We systematically measured cytokines, growth factors, adhesion molecules, matrixmetalloproteinases and chemokines in a murine model for acute cutaneous wound healing. Strikingly, a subset of chemokines proved to be among the most highly regulated genes. The chemokine expression is highly orchestrated and coincides with the appearance of matching receptors. Accordingly, we demonstrate that structural cells, such as human keratinocytes, dermal fibroblasts and dermal microvascular endothelial cells, express a distinct and functionally active repertoire of chemokine receptors. Furthermore, migration assays suggest that chemokines and their cognate receptors accelerate wound healing via chemotaxis of structural cells.4 cells for the culture and let it grow up to 80% confluence. Cells were treated with TNF-α (10 ng/ml)/IL-1β (5 ng/ml)  for 10 to 12 h or left untreated. RNA was extracted from cells immediately, after 8 and 18 h as described later.Human primary epidermal keratinocytes, dermal fibroblasts, melanocytes and dermal microvascular endothelial cells were purchased from Clonetics  and cultured in keratinocyte (KGM-2), fibroblast (FGM-2), or endothelial cell (EGM-2) growth medium  . We have® universal master mix , 0.625 μl of gene-specific TaqMan® probe, 0.5 μl of gene-specific forward and reverse primers, and 0.5 μl of water. As an internal positive control, 0.125 μl of 18S RNA-specific TaqMan® probe and 0.125 μl of 18S RNA-specific forward and reverse primers were added to each reaction. Gene-specific probes used FAM™ as reporter dye, whereas probes for the internal positive control (18S RNA) were associated with the VIC™ reporter dye. Alternatively, 25 ng of cDNA was amplified using target-specific primer combinations and SYBR green master mix . Chemokine ligand- and receptor-specific primers and target-specific probes were obtained from Applied Biosystems . Gene-specific PCR products were measured by means of an ABI PRISM® 7700 or 5700 Sequence Detection Systems  continuously during 40 cycles. Target gene expression was normalized between different samples based on the values of the expression of the internal positive control (18S RNA) or ubiquitin.Quantitative real-time PCR analyses were performed as previously described , 24, 25.6 cells were stained with anti-chemokine receptor mAb or isotype and analyzed using a FACScan and CELLQuest software .To analyze chemokine receptor expression of non-hematopoietic cells, cultured primary epidermal keratinocytes, dermal fibroblasts and dermal microvascular endothelial cells from different donors were analyzed using flow cytometry and the following monoclonal antibodies (mAb): PE-conjugated anti-CCR1 , CCR2 , CCR3 , CCR7 , CCR9 , CXCR4 , CXCR5 , CXCR6/STRL33  all R&D Systems, Mineapolis, MN. CCR4 , CCR5 , CCR6  CXCR1 , CXCR2 , CXCR3  all Pharmingen, San Diego, CA, CCR8  Alexis Biochemicals, Grünberg, Germany, CCR10  DNAX Research Inc., Palo Alto CA . Briefly, 104 human primary dermal fibroblasts, 2 × 104 dermal microvascular endothelial cells and 2 × 104 epidermal keratinocytes were cultured in 24-well plates in FGM-2, EBM-2 and KGM-2  medium and grown to confluency analogously to the previously described cultivation. The medium was removed and a single path wound was made with a sterile pipette tip through the intact cell layer. The width of the wound was 200 µm. Medium supplemented with or without CCL1/I-309, CCL11/eotaxin, CCL17/TARC, CCL20/MIP-3α, CXCL8/IL-8, CXCL9/MIG, CXCL10/IP-10 for keratinocytes, CCL1/I-309, CCL11/eotaxin, CCL17/TARC, CCL27/CTACK for dermal fibroblasts and CCL1/I-309, CCL11/eotaxin, CCL17/TARC, CCL20/MIP-3α, CCL27/CTACK, CXCL8/IL-8, CXCL9/MIG, CXCL10/IP-10 for dermal microvascular endothelial cells was added to each well. To confirm the involvement of G(i) alpha protein-coupled receptor signaling in chemokine-induced wound repair responses, structural cells were pre-incubated with or without pertussis toxin  10–12 h before injury and chemokine stimulation. The status of the single path wound was determined immediately as well as 8 and 12 h after the wounding using a digital camera system , or was monitored for 12–18 h by time lapse video microscopy system .2 × 102 inhalation 6, 12, 18 h, 1, 2, 3, 5, 7, and 10 days after incisions were done. At the time of harvest, dorsal pelts surrounding the incisions were removed and normal skin was trimmed to 1–2 mm of incision. One half of each sample was placed in 10% buffered formalin and processed for paraffin embedding. The other half of each sample was snap frozen in liquid nitrogen and processed for RNA extraction. Each cohort consisted of three animals and the subsequent experiment was repeated twice.A total of 27 (3 mice per defined time point) female BALB/c mice were anesthetized with an intraperitoneal injection of ketamine (120 mg/kg bw) and xylazine (16 mg/kg bw). The lumbar region was shaved and the surgical area was disinfected with betadine and isopropanol. Full-thickness 2 cm dorsal incisions were made with a scalpel through the epidermis and dermis leaving the subcutaneous muscle layer undisturbed. Wounds were closed using a single 5 W surgical staple. Mice were euthanized by COAll experiments with mice were carried out after examination by the local ethics committee of the University of Düsseldorf and after an internal identification number was given. The tests were carried out by trained personnel, who respect the internal and international guidelines for animal research (e.g. ARRIVE Guidelines).This study was approved by the local ethics committee and informed consent was secured from all patients participating in this study. A total of 4 patients were included in the study. Skin biopsies were taken from either healthy individuals or patients undergoing surgery and secondary wound healing. Prior, 24 h, 2, 3, 5, 7, 9 and 12 days after radical surgery for acne inversa, biopsies were taken from the non-lesional wound edges. Skin samples were immediately frozen in liquid nitrogen and stored at – 80 °C.2O2 followed by an avidin and biotin blocking step (VECTOR Blocking Kit). Sections were stained with monoclonal antibodies against CCR2α, CCR2β, CCR3, , CCR4 (rabbit IgG), CCL1/I-309, CCL11/eotaxin, CCL24/eotaxin-2 (goat IgG) , CD31 , CD68  , CCL4/MIP-1β, CCL17/TARC, CCL18/DC-CK1, CCL20/MIP-3α, CCL27/ALP , CXCL9/MIG, CXCL10/IP-10 , CXCL12/SDF-1α , CCR1 , CCR5 , CCR6 , CXCR1 , CXCR2 , CXCR3 , CXCR4 , CXCL10/IP-10, CXCL9/MIG, CCL17/TARC  all R&D Systems, Minneapolis, MN), CXCL8/IL-8  , CCR8 and CCR10  at 4oC. The staining was performed with an ABC-Kit (VECTOR Vectastain ABC-Kit) and an ACE-Kit (VECTOR). Sections were counterstained with hematoxylin. The immunohistochemical examinations were conducted by three independent pathologists. If there were differences in the evaluation, a joint result was finally established.Frozen skin samples, cut to a thickness of 6 µm on a cryomicrotome, were used for immunohistochemical analyses of human secondary wound healing, skin sections were fixed with 3% acetone and preprocessed with HTo elucidate the dynamic network of mediators controlling the highly orchestrated process of cutaneous wound healing, we performed comprehensive real-time PCR analyses of more than 100 genes including cytokines, growth factors, adhesion molecules, matrix-metalloproteinases and chemokines in a murine model of cutaneous wound healing   of structural cells of the human skin in further detail , CCL17 (CCR4 ligand) and CCL27 (CCR10 ligand) induced significant migration of cells into single path wounds leading to enhanced wound repair while ‘irrelevant’ chemokines such as CCL3 (CCR1 and CCR5 ligand) failed to induce responses  among structural cells of the skin presented markedly enhanced wound repair responses after stimulation with CCL1 (CCR8 ligand), CCL11 (CCR3 ligand) and CCL27 (CCR10 ligand)  alpha protein-coupled receptor signaling for chemokine-induced wound repair in vitro.To confirm findings in mouse and in vitro observations with human structural cells, we studied the kinetics of human secondary wound healing in vivo Fig. ; Table 3−/CXCR4+ suprabasal keratinocytes in healthy skin , CCL17 , CCL18/DC-CK1 , CXCL8 , and CXCL10  Table . Intereskin Fig. q. Similakin Fig. . Other ckin Fig. .Next to keratinocytes, dermal endothelial cells also showed abundant homeostatic chemokine ligand  and receptor  expression confirming our previous in vitro results.After cutaneous injury, monocytes infiltrate the skin, get activated, and differentiate into macrophages and dendritic cells expressing CCR1, CCR2β, CCR6, CCR8, and CXCR4 Fig. ; Table 3Two days after wounding, keratinocytes of the wound edge migrate into the wound bed and start to form a neo-epidermis. Basal keratinocytes of the neo-epidermis are strong producers of CCL27 throughout the re-epithelialization period. Next to CCL27, basal keratinocytes of the neo-epidermis also show low levels of CCL1 expression Fig. l, n. Ove+ leukocytes were seen at perivascular pockets delimiting the wound bed and in close proximity to the developing neo-epidermis. This population of skin infiltrating monocytes, activated macrophages and dendritic cells was an abundant source of CCL18, CXCL9, and CXCL12 , vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), angiogenin, angiotrofin, angiopoietin-1, thrombospondin, low oxygen and elevated lactic acid, have been linked to the induction of angiogenesis , 34–39. Bernardini et al. and Haque et al. demonstrated that the CCR8 ligand CCL1 binds to endothelial cells, stimulates chemotaxis and invasion of these cells, enhances human umbilical vein endothelial cell differentiation into capillary-like structures in vitro and induces angiogenesis in vivo –37. PrevTaken together, chemokines and chemokine receptors are expressed in a distinct temporal and spatial manner during wound healing and play many different functions during wound healing processes. Our findings elucidate an important role for chemokines in the complex wound healing cascade and underscore the central paradigm that cutaneous injury begins with proinflammatory cytokine and chemokine production which results in leukocyte recruitment and subsequent growth factor production. In turn, growth factor production by leukocytes and structural cells combined with chemokine signalling will orchestrate the dynamic process of migration, invasion, and proliferation of parenchymal cells for tissue repair. Not much has been done in the clinic regarding the use of chemokines and their receptors as a basis for treatment. Chronic inflammation and alteration in angiogenesis can potentially be reduced or eliminated by interfering with proinflammatory or angiogenic chemokines and their receptors using small molecules that interfere with receptor function. To our knowledge, this study is the most comprehensive analysis of chemokine and chemokine receptor expression both in vivo and in vitro. In addition to the extensive analysis of the central structural and soluble network partners in wound healing, our work was able to elucidate previously unknown interactions between chemokine ligands and chemokine receptors. We were able to gain information on a faster wound healing through the use of specific chemokines and we revealed patterns of expression that have not been described so far. Especially ligand–receptor signaling through CCR6 can enhance keratinocyte wound repair in vitro. CCL27 markedly enhanced wound repair of endothelial cell and fibroblast indicating an important role for CCL27–CCR10 interactions in tissue repair.Nevertheless, there are some limitations to this study, which are based on the fact that a very special patient collective of the so-called Acne inversa was investigated. To support the results of our work, the number of samples should be increased and different patient groups should be examined in subsequent studies. The selection of the test mice can also contribute to the results. We have only examined female BALB/c mice in our work. However, recently published studies suggest that wound healing in female BALB/c mice may differ from male mice . To achiIn conclusion, our findings enhance the understanding of the underlying mechanisms for cutaneous wound healing and may impact the development of novel therapeutic strategies for the treatment of wound healing disorders such as chronic ulcers.Additional file 1: Figure S1. Human primary keratinocytes expressing CCR4, CCR6, CCR9, CXCR1 and CXCR3 on their surface. Flow cytometric analysis of chemokine receptor repertoire in cultured human primary keratinocytes. Representative results from one of at least three different donors.Additional file 2: Figure S2. Human primary dermal fibroblasts expressing CCR3, CCR4 and CCR10 on their surface. Flow cytometric analysis of chemokine receptor repertoire in cultured human primary dermal fibroblasts. Representative results from one of at least three different donors.Additional file 3: Figure S3. Human primary dermal microvascular endothelial cells expressing CCR3, CCR4, CCR6, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3 and CXCR4 on their surface. Flow cytometric analysis of chemokine receptor repertoire in cultured human primary dermal microvascular endothelial cells. Representative results of one of at least three different donors.


Immunoblot analysis indicated that nicotine increased cortical protein levels of caspase-1, apoptosis-associated speck-like protein containing a CARD (ASC) and pro-inflammatory cytokines interleukin (IL)-1β by 88% (p < 0.05), 48% (p < 0.05) and 149% (p < 0.05), respectively, when compared to the saline-treated group. Next, using an in vitro model of ischemia in organotypic slice cultures, we tested the hypothesis that inhibition of nicotine-induced inflammasome activation improves post-ischemic neuronal survival. Accordingly, slices were exposed to nicotine  or saline, followed by treatment with the inflammasome inhibitor isoliquiritigenin  prior to oxygen-glucose deprivation . Quantification of neuronal death demonstrated that inflammasome inhibition significantly decreased nicotine-induced ischemic neuronal death. Overall, this study shows that chronic nicotine exposure exacerbates ischemic brain damage via activation of the inflammasome in the brain of female rats.Smoking is a preventable risk factor for stroke and smoking-derived nicotine exacerbates post-ischemic damage via inhibition of estrogen receptor beta (ER-β) signaling in the brain of female rats. ER-β regulates inflammasome activation in the brain. Therefore, we hypothesized that chronic nicotine exposure activates the inflammasome in the brain, thus exacerbating ischemic brain damage in female rats. To test this hypothesis, adult female Sprague-Dawley rats (6–7 months old) were exposed to nicotine (4.5 mg/kg/day) or saline for 16 days. Subsequently, brain tissue was collected for immunoblot analysis. In addition, another set of rats underwent transient middle cerebral artery occlusion  with or without nicotine exposure. One month after tMCAO, histopathological analysis revealed a significant increase in infarct volume in the nicotine-treated group (64.24 ± 7.3 mm Cigarette smoking is a preventable risk factor for stroke, and smoking-ingested nicotine exacerbates post-stroke brain damage ,2. StrokAlthough we know that women are more susceptible to stroke, we have a limited understanding of the underlying mechanisms for increased stroke severity and possible sex-specific prevention and treatment of stroke. Sex differences in stroke are highly complicated, and sex-specific risk factors in part account for epidemiological findings in stroke incidence, prevalence, and mortality. As stated in a review by Girijala et al., there are a number of modifiable risk factors such as cardiac conditions, hypertension, diabetes mellitus type 2, metabolic syndrome, alcohol consumption, physical inactivity, and cigarette smoke exposure, which are common for both sexes ,6,7. At 2; a potent estrogen) levels [2-induced ischemic protection, suggesting a key role of ER-α and/or ER-β-activation [2-mediated decrease in caspase-1, ASC and IL-1β [p < 0.05), as compared to the vehicle-treated, reproductively senescent rats [Smoking-attributed nicotine is known to inhibit aromatase enzyme activity, which catalyzes the conversion of androgens into estrogens . Consequ) levels . Estrogetivation ,21,22. Otivation . The inftivation . In a pund IL-1β . On the ent rats . Studiesent rats ,25. Thern = 8; p < 0.05) and 31%  reduction in cortex and hippocampus, respectively, as compared with saline .Because our previous results demonstrated that nicotine reduced the level of membrane-bound and mitochondrial ER-β in the hippocampus, in this study, we investigated protein expression of ER-β in the cortex, the main brain area vulnerable after transient middle cerebral artery occlusion (tMCAO). Our results demonstrated that nicotine significantly reduced cortical ER-β protein levels as compared with the saline group . ER-β prp < 0.05), 48% (p < 0.05) and 149% (p < 0.05) respectively in the cortex, as compared to the saline-treated group  compared to the saline-treated group   A. Histoln = 10) and 70 ± 1.2% (n = 10) in the saline and nicotine + vehicle-control groups respectively (p < 0.01). Nicotine and saline exposed slices were treated with the inflammasome inhibitor isoliquiritigenin . Inflammasome inhibition at 10 µM ILG concentration showed a significant decrease in nicotine-induced ischemic neuronal death when compared to the nicotine + vehicle group. The PI fluorescence values of the saline + ILG, nicotine + ILG, and vehicle treated nicotine groups were 44 ± 6.2% (n = 8), 27 ± 1.5% (n = 8), and 70 ± 1.2% , respectively. The PI fluorescence values of the nicotine + 1 µM ILG and nicotine + 40 µM ILG were 55 ± 5.3% (n = 8) and 61 ± 2.5% (n = 8), which were not significantly different as compared to PI values of saline + 1 µM ILG and saline + 40 µM ILG groups  region of the hippocampus as compared to saline. The propidium iodide (PI) fluorescence values were 56 ± 5.5% -like receptor 3 (NLRP3) activation ,27. In oIt has been shown that inflammasome activation in the brain is regulated by sex hormones . In a prInflammasome activation occurs after the detection of pro-inflammatory molecules by pattern recognition receptors, such as a toll-like receptor or a NOD-like receptor (NLR) . The mecFinally, relinquishing the smoking habit reduces the risk for stroke; however, impact of smoking cessation on stroke outcome remains unknown. Because it is difficult to stop smoking, more tobacco users have switched to the “fashionable” e-Cigarette (electronic nicotine delivery systems) as an alternative to tobacco smoking or even as an aid for smoking cessation. However, the safety of e-Cigarettes remains questionable and as demonstrated in the current study, negative effects of nicotine on brain will persist, which makes current research in this area timely and of a high impact.All animal procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. Public Health and procedures involving animal subjects and were approved by the Animal Care and Use Committee  of the University of Miami.Adult female Sprague–Dawley rats  were used for this study. The stages of estrous cycle were monitored as described  and onlyn = 21) or saline (n = 20) were divided into two groups. One group of eight rats treated with either nicotine or saline was used for brain tissue collection, while the remaining rats in both treatment groups underwent transient middle cerebral artery occlusion (tMCAO) or sham surgery. Since we have previously demonstrated that the higher endogenous estrogen levels seen during proestrus protect CA1 neurons against cerebral ischemia [To obtain sustained nicotine delivery, osmotic pumps  containing nicotine or saline were implanted in rats for 16–21 days as described . The pumischemia , tMCAO o2, pO2, pH, HCO3 and arterial blood pressure were maintained within normal limits prior to and after tMCAO  using an occluding intraluminal suture inserted past the internal carotid artery to occlude the middle cerebral artery as described previously ,47. In pThe neurological score was monitored at an hour, 1, 7, 15 and 30 days after tMCAO. A standardized neurobehavioral test battery was conducted as described previously , which iRats exposed to nicotine (16–21 days) or saline were anesthetized using 5% isoflurane, decapitated, and the hippocampal and cortical tissues were collected, flash frozen, and stored at −80 °C. At the time of immunoblotting, hippocampal and cortical tissues were homogenized, protein content was analyzed, and proteins were separated by 12% SDS-PAGE as described . ProteinN-methyl-d-aspartate (NMDA) treatment to assess maximum damage to neuronal cells. The hippocampal CA1 subfield was chosen as the region of interest, and quantification was performed using Scion Image software [Hippocampal organotypic slice cultures were prepared from female neonatal (9–11 days old) Sprague–Dawley rats and the details of slice culture are as described previously ,52,53,54software ,52,53,54software ,52,53,54t test and p < 0.05 was considered statistically significant.The data are presented as mean value ± SEM and results were analyzed by a two-tailed Student’s


Triticum aestivum L.) grains biofortified with Zn. Effects of application of three rates of nitrogen (N) fertilizer  to soil in combination with three foliar applications  on grain yield, yield components, grain Zn concentration, protein, phytic acid (PA), phosphorus (P), calcium (Ca), and carbon (C), as well as on Zn bioavailability, were investigated in four wheat cultivars  under field conditions. Enhanced N increased Zn and protein concentrations as well as bioavailability; excessive N input did not result in further improvements. Zinc spraying was more effective than soil fertilizer N application, the spray of Zn (with or without sucrose) increased grain Zn concentrations by 11.1–15.6 mg·kg−1 (27.1–38.1%), and increased grain Zn bioavailability, estimated using total daily absorbed Zn (TAZ) and molar ratios of PA/Zn) and PA × Ca/Zn, by 0.4–0.6 mg d−1 (28.6–42.9%), 23.1–27.4% and 24.0–28.0%, respectively. Remarkably, increases caused by ‘Zn + sucrose’ were higher than spraying Zn alone. Grain Zn bioavailability was more sensitive to the selection of cultivar than Zn concentrations. Among cultivars, the higher the grain yields and concentrations of antinutritional compounds, the lower the grain Zn nutritional quality would be. 200 kg N ha−1 application rate in combination with foliar spraying of “Zn + sucrose” maximized grain Zn concentrations of “Jinan 17,” “Jimai 20,” “Jimai 22,” and “Luyuan 502” to be 59.4, 56.9, 55.8, and 60.9 mg kg−1, respectively, achieving the target value for biofortification. Additionally, PA/Zn and PA × Ca/Zn of “Jinan 17,” “Jimai 20,” and “Luyuan 502” were <15 and 200, and TAZ was maximized to be 2.2, 2.0, and 2.1 mg d−1, respectively, indicating higher bioavailability. Therefore, optimal soil N and foliar Zn management together with suitable cultivars maintained high grain yield with lower N input and could substantially increase grain Zn nutritional quality simultaneously.To alleviate human zinc (Zn) deficiency, it is worthy to develop rational agronomic managements to achieve high yielding and high resource-use efficiency wheat ( Zinc (Zn) is an essential micronutrient for the survival of plants, animals and human beings. Zinc deficiency, however, as one of the most widespread nutritional disorders, is prevalent in a lot of regions worldwide, especially in developing countries Cakmak, . Zinc de−1 with an average of 23.3 mg kg−1 in seven major wheat production provinces of China. However, this averaged concentration is much lower than the biofortification target concentration 38 mg kg−1 set by the HarvestPlus program  in grain Zn was achieved by foliar Zn spraying alone, while soil Zn fertilization was less effective  than by traditional breeding or genetic engineering methods  in grains  to quantify the effects of soil fertilizer N and foliar Zn (with or without sucrose) application on grain yields and yield components; (2) to quantify their efforts on Zn nutritional qualities including Zn concentrations and bioavailability for humans in whole flours; (3) to quantify their effects on protein, total and phytate P, Ca, and carbon (C) concentrations, and on the ratios of C/N and phyate-P/total P in whole flours; and (4) to elucidate relationships among the abovementioned grain Zn-related nutritional traits across different wheat cultivars. The differences in Zn nutritional quality-related parameters among different kinds of wheat genotypes were also evaluated.w/v in water). The top 20 cm of the soil contained 19 g kg−1 organic matter analyzed using the Walkley-Black method , Olsen P (24 mg kg−1) and exchangeable K (162 mg kg−1) were analyzed by extracting 5.0 g soil with 50 mL 2.0 mol L−1 KCl, 100 ml 0.5 mol L−1 NaHCO3 and 50 mL 1.0 mol L−1 NH4OAc, respectively  solution , Shandong Academy of Agricultural Sciences, China. The area has a typical continental and warm climate, with an annual mean temperature of 13.6°C and a long-term mean annual rainfall of 625 mm. The soil at the site was classified as sandy loam, with a pH of 7.8  cultivars were “Jinan 17,” “Jimai 20,” “Jimai 22,” and “Luyuan 502,” respectively. “Jinan 17” is a high-quality strong gluten cultivar, suitable for making bread. “Jimai 20” is suitable for making both bread and noodles. “Jimai 22” is a high-yielding wheat cultivar and is sown over the largest area in contemporary China. “Luyuan 502” is also a high-yielding wheat cultivar, which is widely cultivated in northern China. The three foliar treatments were: (1) foliar spray of deionized water as a control; (2) spray of ZnSO4·7H2O ; and (3) a combination spray of ZnSO4·7H2O  and sucrose . All solutions, containing 0.01% (v/v) Tween 20 as a surfactant, were foliar-applied at 900 L ha−1 after sunset. Foliar applications were conducted four times at 5-days intervals, starting from 5 days after wheat flowering. The area of the main plot was 4 × 20 m = 80 m2, that of the subplot was 4 × 5 m = 20 m2, and that of the split-split plot was 2 × 1 m = 2 m2.The experiment was a split-split-plot design with three factors consisting of three foliar spray treatments (split-split plot), four cultivars (subplot), and three N application rates (main plot) in four replicates. The N application rates were 75, 200, and 275 kg of N ha2O5 ha−1, supplied as calcium superphosphate), K fertilizer , and Zn fertilizer (30 kg of ZnSO4·7H2O ha−1) were evenly distributed and incorporated into the upper 20 cm of the soil prior to wheat planting. The remaining half of the N was top-dressed with irrigation at the jointing stage. All plots were adequately irrigated at stages of pre-wintering, stem elongation and flowering, and weeded manually. There were no fungicides applied during the growth period. At the booting stage, omethoate (2-dimethoxyphosphinoylthio-N-methylacetamide)  was sprayed to control aphids.A half amount of each N application rate (supplied as urea), and all of the P fertilizer . The concentrations of nutrients  in the digests were determined by inductively coupled plasma atomic emission spectroscopy . A reference grain sample IPE556 from Wageningen University was included in each batch to ensure analytical quality. Phytate-P concentration was analyzed according to the method of Haug and Lantzsch . Ground samples were digested with HNOLantzsch . The carLantzsch .The molar ratio of PA/Zn has been widely used as a simplified indicator of Zn bioavailability in the human diet  was based on reference adults consuming wheat flour (0.3 kg d−1) as a sole daily source of Zn and phytate  = Zn concentration in wheat flour (mg kg−1) × reference adults consuming wheat flour (0.3 kg d−1) ÷ the relative atomic mass of Zn (65 g mol−1); KR = equilibrium dissociation constant of the Zn-receptor binding reaction; TDP  = PA concentration in wheat flour (mg kg−1) × reference adults consuming wheat flour (0.3 kg d−1) ÷ the relative molecular mass (660.04 g mol−1); and KP = equilibrium dissociation constant of the Zn–PA binding reaction  was used for calculating Pearson correlation coefficients.Data were subjected to ANOVA using SAS software  and means were compared by Fisher's protected least significant difference (LSD) at −1 and from 200 to 275 kg ha−1, decreased grain yields gradually from the initial 7719.7 kg ha−1 to the final 7226.6 kg ha−1, and decreased spike numbers from 449.1 to 415.5 104 ha −1 (Table −1), the 1,000 kernel weight was significantly increased to its maximum value of 45.6 g by 200 kg N ha−1. No significant effects of N on the kernel number per spike were observed. Among different wheat cultivars, grain yields varied from 7073.3 to 7703.0 kg ha−1. ‘Luyuan’ 502 had the lowest grain yield, which was significantly lower than that of “Jimai 20,” and “Jimai 22” had the maximum yield. Spike numbers are in the order of “Jinan 17” > “Jimai 20” > “Jimai 22” > “Luyuan 502,” and varied dramatically from 503.2 to 345.2 104 ha−1, while the kernel per spike and the 1,000 grain weight were the reverse of the previous order, varying from 32.5 to 40.2 g and from 41.5 to 48.9 g, respectively. As expected, foliar treatments had non-significant impacts on grain yields and yield components (Table Increasing the N application rate from 75 to 200 kg ha−1) significantly increased grain Zn concentration by 9.9%, from 47.5 to 52.2 mg kg−1. It also increased Zn yield and Zn bioavailability as compared with low N application (75 kg N ha−1), estimated by TAZ, PA/Zn, and PA × Ca/Zn (Table −1) led to slightly lower grain Zn concentration and yield, and did not make an extra contribution to grain Zn bioavailability.Adequate N application (200 kg N ha−1), TAZ (1.4–2.0 mg d−1), and molar ratios of PA/Zn (15.1–20.8) and PA × Ca/Zn (168.6–234.2) , total P (3.33–4.01 g kg−1), and Ca (379.9–482.5 mg kg−1), as well as on the ratios of C/N (17.5–18.2) and phytate-P/total P (63.3–71.5%). “Luyuan 502” had a significantly lower protein concentration than the other three wheat cultivars; in addition, the protein concentration of “Jinan 17” was significantly lower than “Jimai 22.” “Jinan 17” had relatively higher C concentration than “Jimai 20,” “Jimai 22,” and “Luyuan 502.” The ratios of C/N of “Jinan 17” and “Luyuan 502” were significantly higher than those of “Jimai 20” and “Jimai 22.” “Jinan 17,” as a high-quality strong gluten wheat cultivar, had the lowest concentrations of phytate-P, total P, and Ca; in contrast, the high-yielding “Jimai 22” had the highest concentrations of phytate-P, total P, and Ca, and the highest ratio of phytate-P/total P , C 42.7–43.0%), phytate-P , while the relationship between TAZ and PA × Ca/Zn can be described by another negative power function (R2 = 0.85****). According to the fitted curves, the critical value of 15 for PA/Zn and 200 for PA × Ca/Zn corresponded with TAZ values of 1.9 and 1.6 mg d−1, respectively.Considering all 144 data points in this study, the molar ratios of PA/Zn and PA × Ca/Zn varied from 6.9 to 30.5 and from 62.7 to 357.2, respectively, and the TAZ ranged from 0.93 to 4.0 mg dThere were also significant and negative correlations between spike numbers and kernel numbers per spike, 1,000 kernel weights, phytate-P, total P, Ca concentrations, or molar ratios of PA × Ca/Zn; however, correlations between spike numbers and grain yields, Zn yields, C concentrations, or C/N ratios were all positive. Kernel numbers per spike were also negatively correlated with protein concentrations, but positively correlated with 1,000 kernel weights and Ca concentrations. One thousand kernel weights were also negatively correlated with C concentration, and positively correlated with phytate-P, total P, Ca concentrations, and molar ratios of PA/Zn and PA × Ca/Zn Table .Grain yields were positively correlated with Zn yields and C/N ratios, and Zn yields were negatively correlated with molar ratios of PA/Zn and PA × Ca/Zn. C concentrations were also negatively correlated with phytate-P concentrations, and concentrations of protein, Ca, and phytate-P were all negatively correlated with C/N ratios. In addition, positive correlations were observed among concentrations of Ca, phytate-P and total P, phytate-P/total P and molar ratios of PA/Zn and PA × Ca/Zn, except for a negative correlation between phytate-P/total P and total P Table .−1), excessive N application (275 kg ha−1) did not make an extra contribution to wheat growth, grain Zn concentration, Zn yield, bioavailability, or protein concentration, but resulted in a slight decline in grain yield, Zn concentration, and Zn yield at maturity (Tables −1 in the first year and 195 kg ha−1 in the second year) to excessive N supply  caused non-significant increases in grain Zn concentration and content. However, N deficiency is still prevalent in many other developing countries, which may not only limit grain yield increase, but also lead to low grain Zn concentrations  use by 2020  caused by foliar Zn application alone would have a measurable impact on improving dietary intake of Zn by human beings and human health. In addition to bran, the increase in wheat grain Zn concentration also occurs in endosperm, where Zn has higher bioavailability  a longer drying time of the Zn solution; (2) improved leaf cuticle penetration; and (3) improved translocation rates of Zn from the site of absorption to grains, as suggested by Zhao et al. . FernándMany studies showed that there is a contradiction between grain yield and nutrition quality in crop breeding. For example, although the low phytate wheat lines have been used in breeding to improve grain Zn bioavailability, research along these lines has so far failed because of the associated 8–25% yield reduction  in combination with soil application of 30 kg ZnSO4·7H2O ha−1. In the location of Samsun with 1.59 mg kg−1 DTPA-Zn and Adana with 0.49 mg kg−1, at the rate of 50 kg ha−1 of ZnSO4·7H2O application to soil, Cakmak et al.  should be considered and managed in whole and in a proper way.Wang et al.  found Zno et al.  reporteddy Table  is much dy Table  and Gomedy Table , properlk et al.  found grk et al. , of 44.8k et al. , of 39.5k et al. , of abouk et al. , of abouk et al. , varying−1 is set by the HarvestPlus program  in the controls of this study without foliar Zn and foliar “Zn + sucrose” supply are adequate , and increased grain Zn bioavailability, estimated using TAZ, PA/Zn, and PA × Ca/Zn, by 0.4–0.6 mg d−1 (28.6–42.9%), 23.1–27.4%, and 24.0–28.0%, respectively. Increases caused by N application were only 2.1–4.7 mg kg−1 (4.4–9.9%), 0.2 mg d−1 (12.5%), 9.7–10.3%, and 6.9–7.4%, respectively. Among different wheat cultivars, grain Zn concentrations only varied from 47.0–52.9 mg kg−1, while TAZ, PA/Zn, and PA × Ca/Zn dramatically varied from 1.4 to 2.0 mg d−1, 14.7–20.7, and 140.1–249.1, respectively, suggesting that grain Zn bioavailability was more likely to be influenced by cultivar.In this study, the strategy of foliar Zn spraying was more effective than soil fertilizer N application to biofortify wheat with Zn. Compared with no Zn application, Zn spraying (with or without sucrose) increased grain Zn concentrations by 11.1–15.6 mg kg−1  saved) of biofortified wheat flour and its reduction of the current health burden by Liu et al. (−1) in whole flours reduced the health burden (DALYs) caused by Zn deficiency by 6.58–18.21%. Therefore, optimal N and foliar Zn management combined with suitable cultivars will eventually and substantially increase the Zn concentration and bioavailability, and the health impact of whole wheat flours, and contribute to mitigate the health burden of Zn deficiency among infants and children.Here, we propose an integrated strategy to improve/maximize the grain Zn nutritional quality while ensuring high yields and protecting the environment. At least three factors above should be managed in coordination in wheat production: (i) adoption of biofortified cultivars with low phytate, high grain Zn concentration and/or bioavailability, high yield, and high resistance to stresses; (ii) optimization of a N fertilizer application amount that ensures higher grain yield, better Zn nutritional quality and lower N loss; and (iii) creation of an adequate available Zn pool in wheat shoots for its re-translocation to grains by efficient and/or economic foliar Zn application after anthesis in combination with soil Zn application according to the initial soil DTPA-Zn status before sowing. Under such a scenario, the target for biofortification will be rapidly achieved by combining agronomic and genetic strategies. The target value for wheat grain Zn biofortification is set to be 38–50 mg kgu et al. , who fouIn the current study, enhanced N application rate increased both the Zn concentration and bioavailability in whole wheat flours; excessive fertilizer N input did not improve either of them further. The strategy of foliar Zn spraying was more effective than soil fertilizer N application to biofortify wheat with Zn, especially for foliar Zn supply combined with sucrose. Compared to small variations in grain Zn concentrations, the grain Zn bioavailability was more sensitive to the selection of cultivar. Among different wheat cultivars, the higher the grain yields and concentrations of antinutritional compounds , the lower the grain Zn nutritional quality will be. All of the results indicated that Zn biofortification of wheat through optimal soil N and foliar Zn management combined with suitable cultivars can maintain high grain yield with lower N input and simultaneously increase the Zn concentration, bioavailability, and thus the health impact of whole flours.HX and Yanfang Xue: conceived and designed the experiments; WK, Yanhui Xue, YT, JL, DoL, and PM: performed the experiments; HX, Yanfang Xue, and DuL: analyzed the data and wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We believe this is an innovative sight to design new generation of transition-metal-based multifunctional coatings. Besides, our results are applicable for industrial application because it can be realized by simple manufacturing approaches, e.g. magnetron sputtering technology.Either hardness or toughness has been the core interest in scientific exploration and technological pursuit for a long time. However, it is still a big challenge to enhance the hardness and toughness at the same time, since the improvement of one side is always at the expense of the other one. Here, we have succeeded in dealing with this pair of conflict based on tungsten (W) coating by doping boron (B) via magnetron co-sputtering. The results reveal that the introduction of low concentrations of B (6.3 at. %), in the doping regime, leads to the formation of W(B) supersaturated solid solution with refined grains. Meanwhile, the doping-induced higher compressive stress, higher H/E Nevertheless, it is of great importance for engineering application if a material exhibits both high hardness and toughness. Consequently, the work associated with simultaneously increasing these two properties is filled with challenge but significance. To date, this task has been well accomplished by embedding carbon nanotubes (CNTs) into brittle ceramics like silicon carbide6 and alumina9. In this way, as-synthesized composite materials exhibited both improved hardness and toughness, wherein the intrinsic attributes of CNTs dominates the strengthening and toughening effect. Besides, rare data for simultaneous investigation in terms of hardness and toughness has been available on alloying elements, instead, most efforts have been focused on improving the single aspect, either hardness or toughness. Just as the reports about the metal tungsten (W), which is not as hard as ceramics but exhibits brittle behavior11. To improve its toughness, enormous attentions have been put on alloying W with transition metals like rhenium (Re)13, iridium14, vanadium15 and tantalum16, etc. In which, Re attracts much attention since the tough W can be obtained on account of “Re ductilizing effect”17, this theory is based on optimized dislocation-core structure, along with lower Peierls stress. Nevertheless, its application is subject to a certain limitation when taking account of cost. In addition to the toughness enhancement, another important subject for W is to improve its hardness. Aiming at this point, some researchers employed the same method of alloying with other transition metals like Lu18 and V19. However, the strengthening efficiency is limited because the substitutional atoms can only cause slight lattice distortion and weak solid solution strengthening.Hardness and toughness seem as a pair of conflicts20. Similarly, Shang et al.21 have employed the same method to deposit Al film alloyed by 1.89 at. % B, and found that the film exhibited nearly 2-fold microhardness relative to pure Al. Besides, more phenomena relevant to the strengthening effect of suchlike alloying small atoms into metallic films could be traced to refs 23 have presented a beautiful work, they first reported the N-doped bcc-Cr films exhibiting the unique combination of high hardness and excellent toughness at low N concentrations, ~5 at. %. Particularly, the film was synthesized in virtue of specific high-power pulsed magnetron sputtering (HIPIMS)24. Through this deposited technology, synchronized Cr-ions rather than gas-ions irradiation can be monitored and predominantly selected to arrive at the growth surface, thus dense and atomically-smooth film with less lattice defect can be prepared. Inspired by the works above, for the transition metal W, two questions remain: (1) whether the hardness could be enhanced by addition of unexpectedly low level B content just through simple magnetron co-sputtering method? (2) As W is much brittle than Cr, could the small quantity of B atoms also improve the toughness?Recently, limited reports concerning metal films doped by few small atoms attract much attention. For instance, just incorporation of a small concentrations of B (5 at. %) by magnetron radio frequency sputtering into Cr film to form solid solute Cr(B) film, 2.6× increase in hardness can be obtainedbcc-W (α-W(B)) coating via magnetron co-sputtering technology. Besides, pure W and W2B were prepared as references for comparison. Our studies revealed that the W film reinforced by 6.3 at. % B showed promising results: in comparison with pure W, 2-fold increase in hardness and drastically enhanced toughness can be achieved. Moreover, it is worth mentioning that such foreign-element doping is an effective approach not only for enhancing mechanical properties, but also for improving electrical26, optical28 and magnetic features30 of materials, which are equally important for high technical products. Hence, our findings possess a certain scientific and applied values, and could help develop a new group of transition-metal-based multifunctional coatings.Taking account of above questions, we deposited low-concentration B doped 7/2 and W4f5/2 can be observed, by means of Gaussian functions, they were deconvoluted into four peaks. Wherein two peaks located at 33.3 eV and 31.1 eV are considered as metallic W-W bonds31, while two other peaks centered at 33.5 and 31.5 eV can be assigned to W-B bonds, since a shift to high-energy side occurs when charges transfer from W to B. We note that only W-W bonds but no W-B bonds exist in the 6.3 at. % B-containing coating, and it can be further confirmed by the high noise level of the corresponding B1s spectrum, implying that all the B atoms occupy the interstitial sites of W lattice. When B content is 24.4 at. %, B1s spectrum exhibits a symmetrical binding energy peak at 187.7 eV, indicating that the single chemical state exists between W and B atoms, which is attributed to W-B bonds, while no B-B bonds exist in the film. Moreover, the 24.4 at. % B atoms in the coating can only to form sub-stoichiometric W2B phase, so it can be concluded that no interstitial B atoms exists in the coating with 24.4 at. % B.According to pre-etching XPS results, we obtained 0, 6.3 and 24.4 at. % B content by regulating B target power to 0, 70 and 400 W, while W target power was kept constant at 70 W Table . To furtθ = 35.62° and 40.16° are related to (200) and (210) plane of body-centered cubic α-W (JCPDF:65-6453), respectively. With B content increasing to 6.3 at. %, α-W (200) peak disappears and only α-W (210) remains. Combined with its XPS result, we conclude that the single peak should be assigned to B doped α-W (α-W(B)), and the solute B atoms expand α-W lattice to make it shift towards lower Bragg-angle relative to the standard diffraction peak of α-W (210). It’s worth noting that this coating comprises supersaturated solid solution of W, as the solute B content (6.3 at. %) is far larger than the equilibrium solid solubility limit of W solid solution (<1 at. % B32). The formation of such supersaturated solid solution is due to the non-equilibrium characteristic of magnetron sputtering21. As B content increases to 24.4 at. %, the XRD pattern gives two new broad peaks at 2θ = 37.52° and 40.61°, which can be identified as (002) and (211) reflections of tetragonal γ-W2B (JCPDF: 25-0990), respectively, showing a good consistency with the XPS result. Notably, as-deposited W2B coating possesses extremely poor crystallinity. Here, grain sizes calculated roughly by Debye-Scherrer’s formula33 are depicted in Table 2B coating was obtained.Figure d) of 0.130 nm from the HRTEM image, and the SAED pattern also confirm that the sample consists of α-W phase. When B content is 6.3 at. %, the inset still shows a typical α-W SAED pattern, without any sign of B or borides phase, meanwhile, the grain size identified by HRTEM image is around 10 nm, consistent with XRD result that the grain size is 8.6 nm. In contrast, the d211 increases to 0.137 nm for the film with 6.3 at. % B, in agreement with the peak shift toward low angle in XRD, proving that a certain lattice expansion occurs due to the interstitial sites of W lattice are partially occupied by B atoms. As B content increases to 24.4 at. %, the diffraction ring  is 0.194 nm, slightly lower than the theoretical value at 0.204 nm (JCPDF: 25-0990). This is ascribed to the presence of large amount of B vacancies in W2B phase due to B deficiency.To acquire more information about microstructure, we employed TEM analysis, as displayed in Fig. ing Fig.  correspo2B, respectively. The deduced grain size  coating exhibits the highest compressive stress at 1.23 ± 0.06 GPa, because all B atoms implant into the subsurface of film to occupy the interstitial positions of W, resulting in significant lattice expansion. For the W2B coating, a dropped stress value to 0.04 ± 0.04 GPa can be ascribed to the poor crystallinity caused by phase transformation from α-W(B) to W2B. Here, the compressive stress may provide partial contribution to the variation in hardness35. In comparison with pure W, the α-W(B) coating exhibits 2-fold increase in hardness. Based on above analysis of grain size, microstructure, and coating growth, it is rational to conclude that such high hardness is caused by the synergistic effect of finer grains of ~8.6 nm (fine-grain strengthening), supersaturated solid solution (solid solution hardening36), non-columnar growth and higher residual compressive stress. As B content further increases to 24.4 at. %, a dropped hardness to 20.1 ± 1.29 GPa can be obtained. And the intrinsically low hardness (lower than 20 GPa) estimated by Xiao et al.37 together with its defective lattice structure govern the lower hardness, although similar non-columnar features can be observed.As we known, internal stress (IS) is an important factor affecting the hardness of coating, it was calculated and displayed in the insert of Fig. 2B coating with 24.4 at. % B  if the grain size reduces40, that is the crack initiation becomes difficult. This concept may work in this work, wherein the mean grain size of α-W(B) coating is 8.6 nm, only half as many as that of W (~15.3 nm). Comparatively speaking, larger grains make pure W coating prefers to form cracks when applied the same load (~200 mN), besides, the representative columnar structure  and denser microstructure are obtained, which is because that the formation of cracks is a complex process relating to not only the grain size and microstructure, but also other factors like mechanical properties  and internal stress41, and the cracks result from a combined action of all factors.As far as we know, the cracks can only nucleate when the stress at the tip of pre-existing flaw beyond crack formation stress of the material, the qualitative comparation can refer to following equation45. To investigate the different fracture mode between pure W and W2B coating, we further calculated the H/E* (E* = E/(1−υ2), υ = 0.25), as summarized in Table 2B coating, relative to pure W, tend to prevent the formation of radial cracks, since the two samples obtain similar H/E* value and larger than 0.1. Together with their denser microstructure  coating keep perfect indention without any cracks, but abundant circumferential cracks arise in the W2B coating. As shown in the XRD and HRTEM of Fig.  could prevent radial cracks could prure Fig. , the rad of Fig. , the poobcc-W coating doped by 6.3 at. % B (α-W(B)) via magnetron co-sputtering W and B targets. Besides, the other coatings including W, and W2B were synthesized further for comparison. The combined experiments of XPS, XRD and TEM confirmed the existence of α-W(B) single phase, and revealed its supersaturated solid solution feature. The addition of B atoms refines the W grains, decreasing the average grain size from 15.3 nm for pure W to 8.6 nm for α-W(B), and leads to higher compressive stress. Accordingly, the coating microstructure changes from columnar to featureless, along with relatively smooth surface. Based on the nanoindentation and SEM measurement, our studies reveal promising results: the incorporation of B in W film could remarkably improve its hardness from 14.1 ± 0.93 to 28.1 ± 0.79 GPa (2-fold increment than pure W), the synergetic contribution of fine-grain strengthening, solid solution hardening, denser microstructure and higher compressive stress governs the strengthening mechanism. The W2B coating possesses intrinsically low hardness (~20 GPa). Simultaneously, in comparison with pure W and W2B, the α-W(B) coating exhibits dramatically enhanced toughness (perfect impression without any crack), which can be mainly attributed to the higher crack formation threshold induced by finer grains, and the higher H/E* (>0.1) improving crack resistance. To sum up, such integrate of higher hardness and superior toughness could be realized by amazingly small (6.3 at. %) addition of B to W.Here, we successfully prepared −4 Pa, then the working pressure was controlled at 0.8 Pa with Ar flow rate of 32 sccm. During deposition, to tailor different B content in coatings, the radio frequency power inserted B target ranged in 0–400 W, while the direct current power linked to W target was kept constant at 70 W. The substrate bias, temperature and rotated speed was fixed at −60 V, 200 °C and 10 rpm, respectively.Deposition of α-W(B) with a thickness of around 700 nm was conducted on (100) oriented Si wafers by magnetron co-sputtering W (99.95%) and B (99.95%) targets in Ar. Prior to deposition, the substrates were successively ultrasonic cleaned in acetone, alcohol, and deionized water for 20 min respectively, and then mounted at 8 cm from targets. The background pressure was pumped down to 1.0 × 1046. The composition and chemical structure in all coatings were investigated by X-ray photoelectron spectroscopy , where sputtering cleaned by Ar+ for 500 s was carried out prior to XPS test. X-ray diffractometer  in θ–2θ configuration together with transmission electron microscopy  operated at 200 kV were employed to study their phase structures. The samples on the Cu grids for TEM analysis were spread with pieces of film peeled off from the surface slightly. To explore the growth condition of films when alloyed by B atoms, cross-section scanning electron microscope  under 1-kV accelerate voltage and 8-cm working distance was carried out. Subsequently, the surface morphology was characterized by atomic force microscope using ScanAsyst mode in air . To determine the hardness for all samples, we introduced nanometer-scale indents (nanoindentation) into the material using MTS Nanoindenter XP in continuous stiffness mode42 with the maximum load of 200 mN, each test was performed on nine testing position to avoid data deviation, and the penetration depth of every indentation was set at 1000 nm. Meanwhile, the accurate hardness was guaranteed by standardizing the fused silica standard sample. And the impression overviews were acquired by SEM.The thickness and radius curvature of coatings were measured by a surface profiler (AMBIOS XP-2). Particularly, the reliable data about curvatures of all samples were got by averaging the minimum value of each eight measurements along two orthogonal surface directions, and outlier data points were rejected, subsequently, the total residual stress was calculated using the Stoney equation