The nanoscale-wide 1D contacts allow spininjection both at room and at low temperature, with the latter exhibitingefficiency comparable with 2D tunnel contacts. At low temperature,the spin signals can be enhanced by as much as an order of magnitudeby electrostatic gating, adding new functionality.Spintronicsinvolves the development of low-dimensional electronicsystems with potential use in quantum-based computation. In graphene,there has been significant progress in improving spin transport characteristicsby encapsulation and reducing impurities, but the influence of standardtwo-dimensional (2D) tunnel contacts, via pinholes and doping of thegraphene channel, remains difficult to eliminate. Here, we reportthe observation of spin injection and tunable spin signal in fullyencapsulated graphene, enabled by van der Waals heterostructures withone-dimensional (1D) contacts. This architecture prevents significantdoping from the contacts, enabling high-quality graphene channels,currently with mobilities up to 130 000 cm The useof tunnel barriers has grown out of the need to overcome the so-calledconductivity mismatch problem,20 whichleads to a drastically reduced spin injection efficiency when theresistance of the contact is lower than the spin resistance of thechannel,21Rs = ρλ/W, with W the graphene channel width, λ thespin relaxation length, and ρ the graphene sheet resistance.Nevertheless, standard 2D contacts, even with the use of a tunnelbarrier, are known to introduce strong doping across the channel leadingto inhomogeneity24 and present challenges in the growth of the barrierwithout pinholes, which lead to enhanced spin relaxation.25 On the other hand, fully encapsulated graphene with 1D contacts26 has been shown to produce exceptionally cleandevices and localize the doping within ∼100 nm near the contacts.28 1D contacts have recently enabled spin injection in graphene,29 albeit only at low temperature, with graphenechannel mobilities below 30 000 cm2 V–1 s–1 and contact polarization with multiple inversionsof polarity. Therefore, further study of this architecture is warranted.Full encapsulation by hexagonal boron nitride(hBN) protects graphenefrom direct contact with lithographic polymers, whereas the self-cleaningprocess driven by van der Waals interactions limits any contaminationpresent at the interfaces within small bubbles, ensuring atomicallyclean interfaces in the rest of the heterostructure.2 V–1 s–1, fully encapsulatedby hBN layers, where a spin current is injected via nanoscale-wide1D contacts. Spin precession measurements yield a quantitative understandingby extracting the channel spin relaxation length λ and timeτs and the 1D contacts’ spin injection efficiencyor polarization P. The fabrication process produceshomogeneous graphene channels, including 1D contacts that preventsubstantial charge doping within the channel and offer a gate-tunablecontact resistance. While the spin polarization is comparable to thatfrom standard tunnel 2D contacts, spin transport can be electrostaticallytuned into a mismatch-free spin injection regime. These elements leadto the realization of a ballistic injection process via nanoscale-wide1D contacts and spin transport in graphene with a spin relaxationlength of ∼18 μm and a long mean free path of ∼1μm, opening up possibilities for lateral spintronic elementsthat exploit quantum transport.Here, we report spin transport in high-quality graphene channelswith low-temperature mobility up to ∼130 000 cmSupporting Information, Section 1. Briefly, we use the dry-peel transfertechnique30 to prepare van der Waals heterotructureson a Si/SiO2 substrate. We then use standard electron beamlithography to pattern a hard polymer mask, which defines the channelgeometry by using reactive ion etching.31 Due to different etch rates for hBN and graphene, a ∼10 nmwide step can be seen in the profile of a hBN–graphene–hBNedge  layers of hBN, with ferromagneticcontacts deposited directly onto narrow (∼10 nm wide) stripsof graphene at the sides of the channel . In orderto characterize charge transport, we measured the four-probe resistanceof graphene as a function of its charge carrier density, n, by using a back-gate voltage applied between the highly doped Sisubstrate and the graphene channel /e, as shown in n| ∼ 1 × 1012 cm–2. For device A, the mobility is then45 000 cm2 V–1 s–1 at room temperature and 79 000 cm2 V–1 s–1 at 20 K. The mobilities of our devices, typicallywithin the range of 20 000 to 130 000 cm2 V–1 s–1 , are significantly higherthan previous graphene-based spintronic devices32 that exhibitedmobilities <20 000 cm2 V–1 s–1, the majority of which have used only a partial hBNencapsulation, whereas we ensure this full hBN encapsulation throughoutthe spin transport channel. The charge diffusion coefficient (andcorresponding mean free path) for representative devices is obtainedfrom the graphene sheet resistance via the Einstein relation, D = 1/(ρe2ν), with ν being the density of states for single layer graphene. The contacts exhibit typical Rc values within the range of 3–15 kΩ, whichimplies they should be able to inject efficiently spins into a graphenechannel with Rs < 10 kΩ. Somecontacts had electrical access via their two ends, see Supporting Information, Section 3). The magnetic 1D contacts present charge transportcharacteristics consistent with those of reported nonmagnetic ones26 . Among these are (i) an inverse scaling of Rc with the width of the contact wc , (ii) anegligible temperature dependence of Rc for high carrier density , and (iii) a sizable dependence on carrier density, with a moderateelectron–hole asymmetry and a maximum Rc near the Dirac point . The electron–hole asymmetry of the contact in Rc at n ≲ 0, indicatesthe presence of an n-doped region adjacent to the metal electrode,consistent with the difference between the work functions of the metal(Co) and graphene.33To characterize the 1D contact resistance t wc see S4a, (ii)t wc see S4a, (ii)I into graphene through contacts 1 and 2,and measure a nonlocal voltage VNL betweencontacts 3 and 4. The nonlocal resistance is defined as RNL = VNL/I. The spin valve signal is given by the difference between the twodistinct levels corresponding to the parallel and antiparallel alignmentof the injector and detector electrodes, ΔRNL = RNLP – RNLAP. Spin signals were measuredfor different separations between injector and detector, L, ranging from 2 to 15 μm . An increase of approximately 1 orderof magnitude in ΔRNL from room tolow temperature was observed, as shown in parts a and b of RNL > 1 Ω. Thisstrong temperature dependence is distinct from the weaker dependenceobserved in standard tunnel 2D contacts,5 indicating a different transport mechanism for spin injection in1D contacts.Spin transportis characterized by spin-valve and spin precession(Hanle) measurements in a standard nonlocal geometry, as shown in 34 enablesus to confirm the presence of spin transport and rule out spuriouscontributions which hindered previous efforts in devices with 1D contacts.32 Here RNLP and RNLAP are measuredwhile sweeping an external magnetic field applied perpendicular tothe device plane (Bz).This causes the injected spins, having a polarization along the x-direction (see x–y plane while moving within the channel. The resulting ΔRNL(Bz) , the spin signal is described by a balanceof spin injection and relaxation within the channel, given by34L theinjector–detectordistance and W the channel width. The channel parametersfor charge (σ ∝ D) and spin (τs) transport have a moderate 2-fold increase at low temperature,see 26 Furthermore, P has a 4-foldincrease at low temperature, as confirmed by the parameters extractedfrom Hanle measurements. These observations indicate that the strongtemperature dependence seen in ΔRNL is dominated by the polarization of the contacts, via the scalingΔRNL ∝ P2.The spin signal ΔRc, together with the observation of a more electron–holesymmetric channel resistance, as seen in 38 to the restof the channel. This doped region only exists near the 1D contact,which in our devices has a nominal width wc of 100—350 nm. The n-doped region being localized in thevicinity of the 1D edge is in stark contrast to standard 2D junctionswhich cover the full width of the channel and lead to substantialinhomogeneity.24 Here, the geometry of the 1D contacts plays a keyrole. On the other hand, for the case of the channel sheet resistance,most of the channel is undoped, except for the nanoscale region indirect vicinity of the 1D edge. Therefore, the channel resistance,probed at a length scale L, W ≥1 μm, exhibits a more electron–hole symmetric response.The identification of an n-doped graphene regionnext to our 1Dcontacts from the electron–hole asymmetric response of 38 and is hitherto unaddressed in nonmagnetic28 or magnetic32 1D contacts, is a nonmetallicincrease in resistance at low temperature. All measured 1D contacts and the doped graphene region (∼100 nm); see The descriptionof contact resistance in 2D metal–graphenejunctions involves two processes: carrier transport from the metalto the (doped) graphene region in direct contact with the metal andtransport from the doped graphene region to the rest of the (undoped)graphene channel. The transmission for those two processes gives riseto the observed contact resistance.I (cyandata) has a high electronic quality close to the upper limit of ourmobility range, with μFE = 130 000 cm2 V–1 s–1. Device A (red data) also has a high electronic quality, with μFE = 79 000 cm2 V–1 s–1, whereas device B (black data) has alower quality (μFE = 27 000 cm2 V–1 s–1). As shown in 34 .Parts f–h of s and length λ,shown in parts f and h of s andλ for device I being about twice as large as thosefor device A, while the parameters for device A are ca. three times as great as for device B. Thisobservation indicates spin relaxation dominated by the Elliot–Yafetmechanism,41 where τs increaseswith the diffusion coefficient D . It is clearthat, with our experimental accuracy, it is indistinguishable fromthe fit obtained using s are not significantlyaffected. Further indication for the role of the Elliot–Yafetmechanism is obtained by studying the carrier density dependence.For all our devices, τs and λ increase withincreasing carrier concentration, having a minimum close to the Diracpoint. This behavior, also observed in single layer graphene using2D contacts, has been attributed to the Elliot–Yafet mechanism45 and results in a linear scaling between spin relaxation time τs and momentum relaxation time τp . We have observed spin transport acrossdistances of 15 μm, while crossing several (noninvasive) 1Dcontacts, only limited by device dimensions . Overall, the demonstration of spin transport parametersreaching values up to D ∼ 0.7 m2 s–1 and λ ∼ 18 μm opens upthe exploration of lateral spintronic architectures involving long distance spin transport,42 now within high-quality and homogeneous channels.The spin relaxation time τnt D see 2d. A pris is rather short, in fact shorterthan in many devices with 2D contacts and of lower quality reportedin the literature.12 In the latter case, τs ∼ 2 ns or more have been observed, while the maximumspin lifetime in our device I with the highest mobilityis only ∼0.7 ns. We speculate that the reason is likely tobe due to the specific geometry of spin injection through 1D contacts:here spin-polarized electrons enter from the opposite sides of a graphenechannel which is followed by propagation in the perpendicular directionalong the channel. In this case, spin transport becomes essentially2D and may not be accurately described by the standard 1D Hanle model(where spins are injected uniformly across the channel and continuepropagation in the same direction).We note that, despite the clear indications that Elliot–Yafetmechanism of spin relaxation plays a dominant role in our devices,the observed τRc, to spin resistanceof the graphene channel, Rs, where a ratio Rc/Rs ≫ 1indicates a regime of efficient spin injection.11 Ingraphene spintronic devices, the ratio Rc/Rs has been tuned by using an electrostaticback-gate via the carrier density dependence of ρ and λ,whereas for tunnel junctions Rc typicallyexhibits a weak dependence.25 In our work, not onlythe channel resistance but also the 1D contacts exhibit a dependenceon carrier density (see Rc/Rs > 1 for all carrier densities,increaseswith carrier density, and it is tunable to values Rc/Rs ≫ 1 at high density.We evaluate the degree ofconductivity mismatch via the ratio ofcontact resistance, sity see 2c, whichRNL. As shown in RNL has a minimum at the Diracpoint, consistent with the minimum observed for the spin transportparameters (RNL exhibits a nonmonotonic behavior: in the hole regime,the signal increases and reaches a maximum for n ∼−0.8 × 1012 cm–2, after whichit decreases. For the electron regime, this behavior is much lesspronounced and the spin signal remains comparatively low. This overallbehavior is understood by considering two distinct regimes of conductivitymatching.25 First, at high carrier density,where Rc/Rs ∼ 10, there is a regime free of conductivity mismatch. Here,ΔRNL is insensitive to the valueof Rc, and it scales as ΔRNL ∝ ρ (see Rc/Rs ∼ 3;see Rc. In this case ΔRNL exhibits a scaling similar to that in themismatch regime46 ∝ Rc2/Rs. As indicated by the cyan line in RNL11 at low carrier density and in the electron regime.To further evaluate the spin injection efficiency and its tunability,we discuss the spin valve signal, Δ ∝ ρ see 2. This i32 and tunable spinsignal and achieves a mismatch-free regime at moderate carrier density.The spin signal shows a consistent behavior across all devices, witha minimum near the Dirac point. This observation is compatible withthe proposal of magnetic proximity at the Co–graphene interface,33 where a reversal in the polarity of the spin polarization is expectednear the Dirac point, implying no spin polarization and thus no spinsignal near the Dirac point. The fact that our devices do not showany inversion of the spin signal as a function of carrier densityis consistent with a homogeneous potential profile within the graphenechannel, where both injector and detector contacts would reverse theirpolarizations at similar carrier density. The demonstration of spintransport in graphene with a large mean free path of ∼1 μmat low temperature, comparable to device dimensions, while ensuringsizable spin relaxation lengths, is a key advance in the developmentof low-dimensional spintronic systems approaching47 the high electronic mobility of state of the art charge-baseddevices.48This device architecture demonstrates efficient spin injectionin graphene using nanoscale-wide 1D contacts, reproducibly acrossseveral devices. The ballistic spin injection process allows for observationof the Hanle effect


Salmonella Typhimurium to Escherichia coli in six physiologically relevant environments. We found that the fitness effects of individual genes were highly dependent on the environment, while the distributions of fitness effects across genes were not, with all tested environments resulting in distributions of same shape and spread. Furthermore, the extent to which the fitness effects of a gene varied between environments depended on the average fitness effect of that gene across all environments, with nearly neutral and nearly lethal genes having more consistent fitness effects across all environments compared to deleterious genes. Put together, our results reveal the unpredictable nature of how environmental conditions impact the fitness effects of each individual gene. At the same time, distributions of fitness effects across environments exhibit consistent features, pointing to the generalizability of factors that shape horizontal gene transfer of orthologous genes.Gene-by-environment interactions play a crucial role in horizontal gene transfer by affecting how the transferred genes alter host fitness. However, how the environment modulates the fitness effect of transferred genes has not been tested systematically in an experimental study. We adapted a high-throughput technique for obtaining very precise estimates of bacterial fitness, in order to measure the fitness effects of 44 orthologs transferred from  Horizontal gene transfer (HGT), or the transfer of genetic material between species rather than the vertical transfer from parent to offspring, is an important source of novel genetic variation and adaptive traits. HGT can play a critical role in adaptation and organismal evolution by alleviating the disadvantages of asexual reproduction both in prokaryotes and eukaryotes . For exaThe outcome of a horizontal transfer event—whether the gene is eliminated or fixed in the population—is determined largely by selection and its interplay with the genetic drift . In otheBioinformatics studies identified environmental conditions as a major factor determining the fitness effect of a transferred gene . In factSalmonella enterica subsp. enterica serovar Typhimurium to Escherichia coli recipient cells. The 44 orthologs used in this study were randomly selected from the Salmonella genome, avoiding genes that are known to be related to mobile genetic elements. These genes were used previously by us to test for the factors that determine their fitness effects in a single environment    and measE. coli in six physiologically relevant environments: two standard laboratory growth conditions (M9 and LB media), and four stress conditions that represent ecological conditions potentially experienced by Salmonella Typhimurium and E. coli—low oxygen (LO2), low pH (pH5) and antibiotics chloramphenicol (CAM), and trimethoprim (TMP). To determine the effects of the transferred genes we controlled for the effect of the environmental stress by competing each “mutant” strain carrying the transferred gene against the “wild type” in a pooled competition assay. Instead of the transferred gene, wild type carried a random DNA sequence that did not have a measurable effect on fitness . Competition assays are traditionally performed through mixing of mutant and the wild type strains in 1:1 ratio at the beginning of the competition and the subsequent quantification of their relative frequencies at the end of competition, for each mutant strain individually; the selection coefficients (s) were then estimated from those frequencies (Δs ≈ 0.005), we employed flow cytometry for the quantification of mutant and wild type frequencies, where the two were genetically labeled with different fluorescent markers , x̄ = 0.007, σ = 0.005; t57.83 = 0.702, P = 0.486). Specifically, we performed pooled competition experiments and employed HTS to estimate relative frequencies of pooled strains. To evaluate the reliability of this approach, we performed a pooled competition assay under one of our environmental conditions—M9, which we also used in our previous study . To emphasize the benefit of this approach, one HTS competition experiment with 6 replicates allowed us to compare the fitness of all 44 mutant strains in one environment simultaneously and without a loss in precision, which would otherwise have required 44 flow cytometry competition assays (each mutant vs. the wild type) done in 32 replicates each. Resulting selection coefficients of all replicates are given as part of We adapted a technique from  to combiquencies . Previou markers . Here, wus study . The strF215, 1276 = 82, P < 0.001). In fact, all possible pairwise comparisons of the gene-by-environment interaction between the six environments were significant  involved in multidrug resistance  have a less consistent fitness effect that is more likely to vary between environments  (s̅ > − 0.1); 2) “highly deleterious” (− 0.1 > s̅ > − 0.4); and 3) “nearly lethal” (s̅ < − 0.4)  . This re< 0.001)  online. < − 0.4) . Only thuvrC, outer membrane lipoprotein carrier protein lolA, and DNA topoisomerase III topB) likely arose from the vital role these genes play in the cell. Similarly, some of the “highly deleterious” genes are beneficial in some of the tested environments because of their function, such as the energy transducing Ton system subunit exbB, DNA-binding transcriptional dual regulator HU proteins hupA and hupB that contribute to antibiotic resistance .We attempted to better understand the potential differences between these three groups of genes. Specifically, we examined if the genes in the groups differed based on the number of interaction partners, length of the coding sequence, level of divergence between orthologs, difference in the codon bias between orthologs, and the change in the expression level of the endogenous copy of the gene across all environments. The genes in the three tested groups did not differ significantly based on any of these factors  online. The distribution of fitness effects across all genes (DFE) is a fundamental property in genetics that determines how a population might respond to selection . The shaA, Kolmogorov–Smirnov test, fig. 4B, fig. 4B) while maintaining the same overall shape and spread, environments must amplify the cost of introduced genes in a systematic manner. One possible explanation is that under more stressful conditions cells may be less tolerant to the additional stress of an acquired gene. We did not find evidence for this hypothesis, as the central tendency of DFEs did not scale with the severity of the environmental stress  were in part determined by their length . Here, wMore broadly, trying to explain HGT only by considering the barriers to transfer, as is often done , runs a In this study, we examined how environment shaped the fitness effects of genes transferred to a closely related organism where an orthologous copy is already present in their new host. This is a specific scenario that does not capture all the complexity of HGT. While this scenario might be common , an orthDespite the fitness effects of genes being highly environment-dependent, overall DFEs are consistent in shape and spread across all environments. This finding enables and justifies more robust modeling of HGT, as modeling and predicting evolution requires assuming an underlying DFE . In otheE. coli K12 MG1655 (DSM18039) strain with the chromosomal insertion of a tetR cassette  pZS* plasmid backbone of Lutz & Bujard, which carried Ampicillin resistance cassette and hence all the environments were supplemented with Ampicillin 50 µg/mL , 1% CAA , 0.4% glucose, 2 mM MgSO4, 0.1 mM CaCl2. To estimate gene expression, we inserted gfp under PLtetO-1promoter. Gene expression was induced using anhydrotetracycline (aTc), and using flow cytometry, we have adjusted the aTc concentration to reach similar levels of GFP fluorescence in each environment. All 250 mL cultures for sequencing were grown in 500 mL flasks at 37° C and 180 rpm (except for the LO2 condition) in a water bath and supplemented with ampicillin 50 µg/mL as the pZS* plasmid backbone had Ampicillin resistance gene. We used rich M9 supplemented with ampicillin 50 µg/mL, at pH 7 with aTc 5 ng/mL as the standard medium “M9.” The wild type has a reciprocal time (μ) of 0.0173 min−1 in this standard medium. Other tested growth conditions are described as: “CAM”—M9 rich medium supplemented with ampicillin 50 µg/mL and CAM 1.2 µg/mL and aTc 5 ng/mL, μCAM = 0.0087 min−1; “LB”—Lennox broth and aTc 12 ng/mL, μLB = 0.0289 min−1; “LO2”—M9 rich medium supplemented with ampicillin 50 µg/mL and overlaid with paraffin oil to create a LO2 condition and aTc 5 ng/mL, μLO2 = 0.0116 min−1; “pH5”—M9 rich medium at pH5 supplemented with ampicillin 50 µg/mL and aTc 5 ng/mL, μpH5 = 0.0116 min−1; “TMP”− M9 rich medium supplemented with ampicillin 50 µg/mL, TMP 0.3 µg/mL and aTc 4 ng/mL, μTMP = 0.0087 min−1  was cloned into the pZS* plasmid without a promoter to be used as the “wild type” in competition assays. We checked whether the addition of this fragment altered fitness by conducting a competition assay using BD FACSCanto II flow cytometer as described previously .The set of genes, plasmid constructs, and bacterial strains used in this study are derived from a previous study . Briefly006468.1 ) were cheviously  (s = 0.0E. coli MG1655 att-λ::[tetR-SpR]+att-p21::[CFP/YFP-KnR]), was used as the “wild type” during these competition assays. In total 6 replicate competitions were performed across 3 different days for each environment. 1:1000 dilutions of separate overnight cultures of 45 E. coli clones each carrying a different plasmid were grown in 20 mL M9 rich medium to OD600 0.2 and then mixed at equal volumes. A concentrated stock culture was prepared in 1 × M9 salts with 10% DMSO-Dimethyl sulfoxide. Cell concentration was verified by colony forming uint (CFU) counting of the dilutions of frozen stock. Aliquots of this “mixed stock” of 45 different clones was stored at −80°C to be used only once as inoculum for each competition. Two larger samples of this mixed stock are used to estimate the starting frequencies.An additional genotype, carrying a phenotypically neutral and unique sequence in the pZS* plasmid but otherwise identical to the host cell .For the competition assays, 250 mL of corresponding medium was inoculated with 10www.vbcf.ac.at, Vienna, Austria). In addition, quality checking and de-multiplexing of the raw data was also provided by the VBCF.DNA was sheared with S220 AFA™ Focused-ultrasonicator (Covaris®) to obtain a fragmentation size of 300–700 bp. The DNA library of 6 different environments  and the initial stock  were prepared and sequenced on Illumina HiSeq 2500 (100 bp SE) by the sequencing company Vienna Biocenter Core Facilities (VBCF) NGS Unit  against a personalized reference containing FASTA files of 44 accuracy . Mapped accuracy .In order to obtain depth per gene, bedtools (version: 2.30.0) was used with the following parameters: bedtools genomecov -d −ibam . As the s) were estimated for each replicate by using the regression model ln(1 + s) = (ln t—Rln 0R)/t, where R is the ratio of the frequencies of mutant (depth of gene) to wild type (depth of tetA fragment) and t is the number of generations  and the sequences of cfp and gfp genes are very similar making it difficult to distinguish in sequence data. Therefore, we replaced gfp with mCherry for the RNA-seq experiment. Cells were grown under the same conditions as competition assays, where the starter stock (inoculation of 107 cells) only included mCherry containing strain instead of the mixed stock of 45 strains. Growth was stopped by adding Qiagen RNA protect Bacteria Reagent (cat no. 76506) onto 20 mL of cultures at OD600 0.4. Total RNA preparation after this point was performed as described previously (www.vbcf.ac.at). The data is deposited in NCBI BioProject repository with accession number PRJNA866657, and abundance data is given as We performed RNA-seq to determine the expression level of the inserted genes and of their endogenous orthologs that are already present in the host within each environment, as well as to confirm whether the potential interaction partners of each gene were expressed in each environment. The host strain contained eviously . LibraryE. coli strain MG1655 from NCBI (genome sequence GenBank ID U00096.3).Briefly, RNA-Seq data quality control was done using the Adapter Removal program  of the 44 selected genes were expressed under our experimental conditions. Interacting gene partners for each gene were identified as previously described . Brieflyα = 0.05 on selection coefficients of genes in each pairwise comparison of six environments. In order to test if the shapes of distributions of environments were different, two-sample and two-sided Kolmogorov–Smirnov tests were performed  test was performed by using selection coefficients of the genes composed of 5 or 6 replicates for each environment, with the formula: Selection Coefficients ∼ Environment * Gene + within replicates (replicates). We applied this test on each pairwise comparison of six environments, and final R method .2. We observed a significant linear relationship between the mean and standard deviation of fitness effects of genes across the six environments . A quadratic model, however, explains the data better , suggesting a bell-shaped relationship between mean fitness effects of the genes and variance of them across all environments ” + “PPI level” + “Deviation in GC% between orthologs” + “Deviation in codon usage between orthologs” + “Gene length in nucleotides.” Furthermore, genes were split into three groups: 1) “nearly neutral” (s > − 0.1); 2) “highly deleterious” (− 0.1 > s > − 0.4); and 3) “nearly lethal” (s < − 0.4), based on the mean fitness effects of genes between environments. We inspected if these three groups could be separated from each other by the means of several intrinsic properties of the transferred genes  ∼ genegroup . A statistically significant difference was considered at All statistical analyses were performed in the R software package (version 4.1.2) and RStudio (version 2022.02.1).msac220_Supplementary_DataClick here for additional data file.


Younger samples document a decrease and greater heterogeneity of δ18O due to the development and progression of plate tectonics and subduction. We posit that δ18O in the oldest Archean samples provides the best δ18O estimate for the Earth of 5.37‰ for olivine and 5.57‰ for bulk peridotite, values that are comparable to lunar rocks as the moon did not have plate tectonics. Given the large volume of the continental lithospheric mantle, even small decreases in its δ18O may explain the increasing δ18O of the continental crust since oxygen is progressively redistributed by fluids between these reservoirs via high-δ18O sediment accretion and low-δ18O mantle in subduction zones.Oxygen isotopic ratios are largely homogenous in the bulk of Earth’s mantle but are strongly fractionated near the Earth’s surface, thus these are robust indicators of recycling of surface materials to the mantle. Here we document a subtle but significant ~0.2‰ temporal decrease in δ The 18 O/16 O ratio of the subcontinental mantle has decreased by 0.2‰, while crustal values increased by 4‰ via fluid transfer since the Archean due to the initiation of plate tectonics and subduction, in line with the crust-upper mantle mass balance Furthermore, the temporal evolution of the mantle may exhibit large impacts on the lithosphere, hydrosphere, and atmosphere however this topic remains unexplored.Earth’s peridotitic mantle is the predominant reservoir of the planet, largely controlling elemental and isotopic mass balances of lithophilic elements and their isotopes planet-wide2. Perhaps the late and most significant episode of Earth’s accretion was the Giant Impact at 4.44 Ga when a Mars-sized object Thea collided with a differentiated Earth to expel material in the close gravitational proximity of the Earth forming the moon4. The composition of Thea with respect to its major elements and isotopes  was not dramatically different from that of the Earth6. The magma oceans that followed the collision further homogenized the mantles of both the Earth and the moon. Their subsequent geologic histories, however, were dramatically different as the moon is a smaller body that finished its geological evolution early and did not have plate tectonics or abundant hydrosphere that interacted with rocks on the surface.The formation of the mantle is due to the accretion of Earth from different types of meteorites and planetesimals at 4.56 Ga, followed by its quick segregation from the iron-rich core1, and shallow basalts and mantle fragments (xenoliths) in kimberlites that sample the uppermost boundary layer: the 100–200 km-thick continental lithospheric mantle (CLM). As Earth cools and develops thicker crust and continents, the CLM too is expected to grow and evolve.We have only limited information about the Earth’s mantle provided by basaltic magmas sampling the convective deeper mantle, all the way down to the lower core-mantle boundary layer10. Only zircons are available in the 4.4–4.05 Ga time frame on Earth to shed light on these early processes and the earliest continental terrestrial rocks, such as 4.05 Ga Acasta Gneiss11. However, 4.1–4.3 Ga basalts and zircons are available on the moon12 and their isotopic values may provide a common point of comparison with Earth.Early Earth after crystallization of the magma ocean soon after 4.44 Ga developed its first crust at 4.4–4.3 Ga which preserves evidence of hydrosphere-lithosphere interaction as early as the Hadean4. Even small δ18O variations in global reservoirs are significant because oxygen is by far the most abundant chemical element on Earth. Estimates range from 5.1 to 5.9‰, depending on whether the value is derived from data on meteorites or terrestrial samples13, while the debate on Δ′17O values, centers around the initial difference and the degree of mixing between Thea, moon, and the Earth after the Giant Impact5.Oxygen is the main element on terrestrial planets. The exact O isotope composition of the Bulk Silicate Earth (BSE) is essential to understanding the Earth’s accretion from different types of parental meteoritic reservoirs, including Thea, subsequent differentiation into crust and CLM, yet these processes remain to be well-constrained8. As the basaltic crust was largely submarine, it interacted with seawater both at low and high temperatures10. Oxygen isotopes may be the best and most straightforward tool in addressing the effects of water–rock interaction and recycling of surface-exposed materials15. In the modern world, oceanic crust, created at mid-ocean ridges (MOR), undergoes pervasive hydrothermal exchange with seawater, leading to heavy δ18O (+7–13‰) at the top in pillow basalts and lower δ18O (+2–5‰) at the bottom in gabbros and peridotites14. Оlivine with higher and lower δ18O than the average olivine phenocrysts from mantle-equilibrated MOR basalts has been found in many island arcs around the world16 and attributed to the dehydration or melting of this altered oceanic crust during subduction. Eclogites that represent metamorphosed basalts in subducting slabs display a similar range of δ18O values, suggesting that hydrothermally altered slabs were subducted in the past at least since 2.5 Ga18 and perhaps as early as 3.0–3.2 Ga19, a time window when eclogitic diamonds became prevalent over peridotitic.It is abundantly clear that the surface of the Earth was covered by oceans since the 4.4 Ga20, leads to a higher degree of mantle melting and precludes modern-style plate tectonics from a physical point of view, with its subduction and crustal recycling driven by the transformation of basaltic crust into dense eclogites21. The time of initiation of plate tectonics on Earth is a topic of current debate12. The transition likely happened in the mid-to-late Archean24 but the style of the earliest Archean tectonics may have been very different from that of today. It could have been on-off, with limited subduction surrounding big mantle swells24 or dominated by “sagduction” or vertical lithospheric delamination22 due to the predominance of plume-related activity26. In such regimes, progressive accretion of basaltic lavas on the surface and delamination of the deep lithosphere results in a limited amount of recycling of the surface-altered material  into the convecting mantle. Shallow and flat subduction or plate stacking would also result in limited exchange26.The higher temperature of the Hadean and early Archean mantle27 comes evidence of large lateral continental migrations and continental collisions, and modern-style subduction under the continents. Subduction is capable of accreting sediments and efficient delivery of surface-altered materials via melts and/or fluids, with both low- and high-δ18O, into the mantle and directly under the continental lithosphere. It follows, therefore, that the early Archean subcontinental mantle could have been less affected by subducting slabs, i.e., be more similar to lunar rocks, thus more closely reflecting the BSE. Finally, the earliest CLM (at least pre-3.8 Ga) may not be preserved because the Earth’s earliest lithosphere went through a major resurfacing process in Eoarchean as indicated by Hf isotopes in zircons24.With documented evidence of accretion of the first supercontinent, Kenorland by 2.5 Ga28, degrees of shallow mantle melting were greater (up to 40%) leaving behind extremely melt-depleted harzburgites30. As these highly refractory residues were Fe-depleted, they were less dense and accumulated to form the rigid and stagnant keels of buoyant Archean continental lithosphere30. This difference can be seen, for example, in the distinct composition ) of Archean and modern peridotites29. Formation and subsequent growth of the CLM and continental keels involve imbrication upon collisions of depleted and buoyant subducted oceanic lithosphere (including melt depletion directly at subduction settings followed by slab delamination) and thickening by compression around existing continental margins or subarc settings32. It can thus be expected that CLM rocks, like Earth’s surface rocks, have different ages and details of chemical and isotopic composition related to their age and mode of origin. However, only recently have we started paying attention to this heterogeneity with respect to many elements and isotopic systems. Oxygen, the most abundant element on Earth should in theory reflect these changes.Of independent and particular importance is the question of potential differences between the Earth’s earliest mantle, represented by xenoliths (fragments) of Archean age in volcanic rocks, and younger mantle samples. As the Archean mantle was 100–200 °C hotter than today18O values of peridotite xenoliths from many tectonic settings have been inferred to be roughly uniform  irrespective of age, tectonic setting, bulk composition, and mineralogy  ever since new laser fluorination methods to generate an expansive dataset of global peridotites were first employed13. Later studies of even strongly metasomatized, hydrous (amphibole-bearing) peridotites reported values within the canonical mantle ranges33. Previous studies, including those employing secondary ionization mass spectrometry (SIMS) (within ≥0.2‰ precision), found a range from 5.0 to 5.5‰ for olivine from different peridotite xenoliths, attributed to a subtle diversity of depletion/enrichment processes, but detected no covariation of δ18O with modal or chemical composition, settings, or age35.Nonetheless, for the past ~30 years, the δ18O. The analytical effort and a dataset presented here for 104 individual mantle xenoliths span 13 years but are based on a single set of standards, analytical methodologies, and normalization procedures  as well as equilibration temperatures41, and references in the supplementary). These samples are not uniformly distributed geographically, because of explosive volcanic eruptions: kimberlites and alkali basalts that can fragment the CLM and can carry mantle xenoliths to the surface require volatile-rich magmas. These, however, span many tectonic settings and regions on Earth, and occur above hot spots, at continental plate boundaries, and rift zones where explosive eruptions rapidly carry pieces of the mantle to the surface at rates of meters per second (e.g. ref. 42). This rapid transport prevents extensive chemical and isotopic exchange between the host melt and the xenoliths, especially for major elements including oxygen. Finally, CLM fragments are much easier to find in and extract from, loose pyroclastic facies of volcanic eruptions than in massive (and slower cooled) lavas. This is why much work on mantle xenoliths is done on the best available samples at each locality worldwide and in specific regions. In such cases, the work is typically performed on the largest, freshest, or least altered mantle xenoliths from several such sites. Comparison with smaller, partially altered, but more abundant xenoliths from broader regions usually indicate that the selected best samples are representative of larger areas in a specific lithospheric block and tectonic setting (refs. 42 and references therein). This statement characterizes the suite of xenoliths we present here.Laser fluorination, with its error on the order of 0.01–0.08‰, continues to be the most precise method to resolve small 0.1–0.3‰ differences in δ18O trends for our worldwide peridotite xenolith suite from cratonic, off-craton, and mantle wedge settings spanning ~3 Ga. The formation (melt extraction) ages for cratonic xenoliths were estimated from Re-Os isotope data41 (Supplementary Table RD) estimates that presume complete Re extraction from residues of BSE melting and thus are only valid for highly refractory rocks (harzburgites and dunites). The TRD values are slightly lower than ideally more correct model Re-Os ages (TMA) yet allow to account for common post-melting Re-enrichments in xenoliths due to metasomatism and host magma infiltration responsible for common aberrant TMA values40. For consistency, we attribute the same age to coeval rock types from each locality, e.g., 2.9 Ga and 2.6 Ga for dunites vs. 2.0 Ga for harzburgites from Udachnaya kimberlite pipe in the Siberian craton. These estimates match the main stages of cratonic crust formation supporting their validity40.Figures RD ages cannot be obtained. We presume that the off-craton CLM formed during the most recent crustal growth or accretion event in a given lithospheric domain43. Harzburgite xenoliths from the west Pacific represent residual oceanic mantle, likely melted first at mid-ocean ridges, then in the mantle wedge. The Kamchatka samples are presumed to be ~0.1–0.3 Ga old 44. TRD estimates (0.1–1.1 Ga) are used for the West Bismarck samples because some of them may come from older microcontinents45.Re-Os ages cannot be systematically used to date off-craton peridotites that range broadly in composition, but are generally more fertile than in cratons, hence valid T18O values of olivine exhibit a temporal ~0.2‰ gradual decrease from 5.38‰ in the Mesoarchean to 5.21‰ in the Paleoproterozoic and 5.16‰ in the Phanerozoic. The δ18OOpx decreases as well. The bulk δ18O values  show a sub-parallel trend of comparable magnitude. When the dataset is organized by locality, to avoid overrepresentation of localities with more analyses, this leaves only 15 individual mantle domains sampled by each kimberlite or basalt  Fig. 37,40. OlTo explore the validity of the discovered trends further, we performed thermodynamic calculations of the effects of melt depletion during mantle melting and its effects on residual harzburgite assemblage Fig. .18O than the bulk15, due to the predominant removal of higher δ18O pyroxenes upon basalt production during peridotite melting, fractional melting with the removal of 30% of melt leads to a 0.12‰ decrease in the remaining bulk harzburgitic residue δ18O values. The 45% melt removal and production of dunitic residue, carries its 0.19‰ δ18O depletion. In addition, the noticeable and counter-intuitive feature of the peridotite melting process that has not been discussed previously to our knowledge is that the δ18OOlivine value increases during the formation of these melting residues (harzburgite and dunites), as δ18OOlivine becomes closer to the bulk values while its modal proportion increases. This simple behavior of oxygen isotope repartitioning during peridotite melting accounts for small 0.05–0.1‰ corrections due to the melt removal effects allowing reconstruction of the original peridotitic bulk before melting.Performed modeling of bulk, fractional, and fluxed melting of original lherzolitic peridotite reveals small associated oxygen isotopic effects on coexisting minerals, melt, and remaining residue bulk solid Fig. . As the 2O3, MgO, and Mg# by using the experimental data of ref. 29. It leads to ~45% melt extraction estimates for dunites and lowest-Al (<0.5) harzburgites, 40% for low-Al, high-Mg# (>0.925) harzburgites, 30–40% for Proterozoic harzburgites with 0.5–1% Al2O3, 20–25% for moderate-Al harzburgites, 30% to all Cenozoic Avacha harzburgites (28% to those with 0.8% Al2O3). Although estimates of the melting degrees are likely accurate to ±5–7%, we consider them sufficiently robust because associated effects on Δ18Odepleted-original, and on δ18OOlivine or δ18Obulk are simple and small . Applying this melt depletion correction leads to 0.03–0.05‰ additionally higher δ18Ooriginal Archean peridotite, and the overall estimate of temporal δ18Obulk peridotite difference between the Archean and Phanerozoic samples is further increased to 0.18–0.2‰  is heavier by 0.04‰ than that for data presented in ref. 13, which we attribute to different sample suites and small differences in standardization. The computed average bulk values of 5.34 ± 0.13‰ are identical to ref. 13. We thus consider the average values of the two datasets as compatible and provide the best current estimates of the δ18O in the average lithospheric mantle  recorded in the Archean olivine would stay the same and may thus reflect the best estimate for olivine in the BSE.The 5.37‰ value for Archean olivine and 5.44‰ bulk value for mean Archean refractory peridotite of our dataset with the average 79% modal olivine and 16% modal orthopyroxene enables correction for a decrease in δrce Fig.  that cha17O0.5305 = −0.047 ± 0.011‰ (n = 9) for Archean vs −0.051 ± 0.010‰ (n = 12) for the early-Paleoproterozoic samples. The value for San Carlos Olivine run as standard, likely sampling younger off-craton Phanerozoic (≤1.8 Ga) mantle of N America46, measured in the University of Oregon and two other labs are −0.051‰47. Vein orthopyroxenes with minor amphibole from the Kamchatka arc, most likely formed due to subduction fluid/melt percolation48, yield a mantle-like value −0.052 ± 0.005‰. Modeling demonstrates that fluxing with fluids with any reasonable δ18O (0 to +10‰) or Δ′17O (+0.02 to −0.02‰) values will have a far greater impact on δ18O than on Δ′17O, resulting in largely constant mantle-like Δ′17O. Our data confirm the recent result in ref. 49 on limited Δ′17O mantle ranges.Triple oxygen isotope analyses of a selection of olivines Fig.  yield an17O differences between the Earth and the moon, pertinent to the original Δ′17O composition of Thea and the degree of homogenization of magma ocean, as well as the late-veneer Δ′17O composition49. However, the δ18O value of the earth’s mantle and its predominant rock on earth, peridotite has not been revisited since 199413. This work fills this gap in an attempt to reconstruct the original BSE oxygen isotopic values, and we show here that it is higher than commonly assumed and is closer to that of the moon at 5.57‰ . For example, Kaapvaal craton zircons of the ~3.2 Ga Archean age exhibit the range of 5.32 ± 0.17‰12. Lunar zircon of presumably 4.4–4.3 Ga age has δ18O of 5.61‰12; these values are 0.3‰ heavier than typical terrestrial mantle zircons (5.3‰51), most of which are Phanerozoic. The δ18O composition of typical 4.3–3.8 Ga lunar basalts and zircons can be used to estimate the δ18O value of the lunar  mantle by a simple isotopic mass balance approach involving computing the CIPW mineral norm of basalts, taking a weighted average of Δ18OCIPW mineral – zircon fractionations using published fractionation factors . There are no coeval and unaltered 4.3–3.8 Ga terrestrial basalts to precisely compare with lunar basalts, but lunar basalts can be used to suggest that coeval terrestrial basalts of an equivalent degree o mantle melting of the Hadean age, were also higher in δ18O.Another way to resolve these small but important differences in lunar and terrestrial peridotites is to directly consider terrestrial and lunar basalts derived from their respective mantles. Typical 4.3–3.8 Ga low-Ti lunar basalts are 5.69‰, 0.12‰ heavier than the mode of modern terrestrial MORB  evolved temporally to lower δ18O values, and thus the signature of basalts is directly inherited from peridotites. Both planets had magma oceans after the Giant impact of 4.44 Ga and thus likely had initially similar upper mantles, and basaltic upper crusts with respect to major element oxygen5. Triple oxygen isotopic effort to find differences between the earth and the moon resulted in almost complete overlap6.The higher δ4. These heavier values agree with our extrapolated estimates of δ18OBSE = 5.57‰±0.07‰ (based on peridotites with propagated errors) very well.As the Moon has not had plate tectonics, it thus provides a benchmark for the pre-plate tectonics on Earth since both planets re-accreted after the Giant Impact (4.44 Ga) and had contemporaneous magma oceans18OBSE is of course related to the timing of initiation of plate tectonics, which is a matter of significant debate26. If significant subduction started much earlier than 3 Ga, and TRD ages were also reset, then the pre-plate tectonic δ18OCLM would have been slightly higher than we estimate here. For example, extrapolation of the linear trend for CLM in Fig. 18O in the Archean samples from 3.0 to 2.6 Ga is less steep than the change between the Archean and Proterozoic and Archean vs all post-Archean samples  and the oxygen isotope effects that we describe below, were likely less prominent. Perhaps more importantly, the oldest peridotite samples in our collection are ~3 Ga and worldwide there are no CLM peridotite xenoliths with robust bulk-rock Re-Os ages significantly more than 3 Ga41. This perhaps reflects that there may not be much left of surviving earliest Eoarchean and Hadean CLM as it was recycled back into the convecting mantle immediately after the Hadean , annihilating the effects of early subduction (if any) on crust-CLM oxygen isotope repartition. Thus, if the plate tectonic started at 3.8–4.1 Ga (the earliest suggested estimates24) the δ18O of the CLM was not significantly changed.Our estimate of the δles Fig. , and so 18O trend that we first observed by assigning age to studied collections  but only its more or less melted residues of the Earth’s upper boundary layer of different age, which form 100–200-km-thick continental lithospheric mantle. The decreasing δns Figs. . We demo18O hydrospheric fluid interaction with the peridotites and their time-integrated effects, as well as recycling of hydrothermally altered lithospheric materials  of the shallow mantle in areas accessible to seawater percolation occurs today, in Cenozoic ophiolites  addition of such fluids corresponded to greater (15–25%) degree of mantle melting. Our sample collection has two Phanerozoic arc-related xenolith suites: harzburgites from the Kamchatka44 and the West Bismarck arcs45. Both occupy the low-δ18O end of our sample array, supporting the proposed scenario of fluxing by low-δ18O fluids. In other, shallower areas of the arc, fluids can be high-δ18O and normal (mantle-like)-δ18O, contributing to higher δ18O values of accretionary sediments and the crust. Besides these two localities studied in this work, strongly re-worked mantle peridotites elsewhere with deformed and modally metasomatized at high temperature and pressure textures, show shifts to lower (not higher) δ18O values (down by 1‰55). The preentrainment metasomatism is invoked to occur by deep subduction fluids shortly before magmatic entrainment, which thus also appears low-δ18O.Second, such low-δ18O value of peridotites, dominated by continental harzburgite, is likely due to a billion-year record of seawater-peridotite interaction in areas of spreading and plate bending, and the subduction-accretion of these materials to the CLM  due to the breakdown of serpentine and chlorite ~100–200 km below the mantle wedge56. This estimate is likely robust within a factor of 2–3 depending on the choice of water content, its isotopic composition, and the total volume of the mantle affected. Perhaps more importantly, only a certain fraction of the mantle under volcanic fronts may be affected, compared to neighboring areas, or by different fluids. Besides subduction, as is mentioned above, direct seawater-peridotite interaction at spreading and plate bending zones, followed by plate stacking and imbrication would potentially produce a range of δ18O for xenoliths from a single locality. This depends on the mechanisms and timescales of fluid migration/fluid-rock reaction and subsequent recrystallization of fluxed peridotites. This explains resolvable δ18O heterogeneity observed among xenoliths from a single kimberlite pipe  or xenoliths from variously fluxed mantle wedges in the Phanerozoic Kamchatka or West Bismarck arcs46. This is an important observation that can also explain moderate degrees of δ18O diversity in basaltic melts of a single locality that we call “natural δ18O noise” generated from a moderately laterally diverse δ18O shallow  mantle source.Here reported a temporal decrease in δCLM Fig. . If we caya Fig. 38–40 or 18O sediments, and upper basaltic portions of the subducting slabs at shallow crustal levels, and supplying low-δ18O peridotitic material and fluids into the CLM. We here suggested that the described process of 18O separation may also explain the contemporaneous increase of δ18O in the continental crust  at the University of Oregon, and the effort spanned 13 years  calibrated relative to the other two were used to calibrate the data on VSMOW scale. Each session included analyses of 4–6 standards, and correction for day-to-day variability was 0 to 0.2‰. Standard CO2 gas was used as a working standard and it was periodically rerun against OZTECH CO2 gas. Errors for standards in individual sessions ranged from ±0.01 to ±0.11‰ and on average are ±0.06‰, 1 s.d. Samples from sessions with worse precision on standards were rerun and most data reported in Supplementary Table 18O values for the same samples run on different sessions is a preferred way to recognize differences between samples.Oxygen isotope measurements for δ2 in sessions with SCO standard  of the entire dataset and split (location-based) sub-datasets Figs.  and 2. NSupplementary InformationPeer Review File


Spinal muscular atrophy (SMA) is a childhood motor neuron disease caused by anomalies in the SMN1 gene. Although therapeutics have been approved for the treatment of SMA, there is a therapeutic time window, after which efficacy is reduced. Hallmarks of motor unit pathology in SMA include loss of motor-neurons and neuromuscular junction (NMJs). Following an increase in Smn levels, it is unclear how much damage can be repaired and the degree to which normal connections are re-established. Here, we perform a detailed analysis of motor unit pathology before and after restoration of Smn levels. Using a Smn-inducible mouse model of SMA, we show that genetic restoration of Smn results in a dramatic reduction in NMJ pathology, with restoration of innervation patterns, preservation of axon and endplate number and normalized expression of P53-associated transcripts. Notably, presynaptic swelling and elevated Pmaip levels remained. We analysed the effect of either early or delayed treated of an antisense oligonucleotide (ASO) targeting SMN2 on a range of differentially vulnerable muscles. Following ASO administration, the majority of endplates appeared fully occupied. However, there was an underlying loss of axons and endplates, which was more prevalent following a delay in treatment. There was an increase in average motor unit size following both early and delayed treatment. Together this work demonstrates the remarkably regenerative capacity of the motor neuron following Smn restoration, but highlights that recovery is incomplete. This work suggests that there is an opportunity to enhance neuromuscular junction recovery following administration of Smn-enhancing therapeutics.  Spinal muscular atrophy (SMA) is a childhood motor neuron disease caused by mutations and deletions within the SMN1 gene  with a SMN-inducing ASO by intracerebral injection P1 (associated with a significant extension in lifespan) or P6 (associated with a limited extension in lifespan). Both early and late treatment with ASO led to very low levels of denervation. However, since there was also a reduction in total endplate number, the reduction in the number of denervated endplates can be partly attributed to the loss of these endplates rather than reinnervation. In moderately and severely affected muscles, there was also a loss of intramuscular axons and an increase in average motor unit size, which was more widespread following P6 treatment.Here, we have undertaken a detailed study of motor unit recovery following restoration of Smn levels. Using an Smn inducible mouse model of SMA, we show that at P4 [a time point where treatment with tamoxifen causes > 50% of mice to live over 200 days ], there Collectively, this work highlights that rescue of the neuromuscular system is possible following restoration of SMN levels. However, it is incomplete and dependent on the method of treatment and the time point at which treatment is initiated. The long-term effects of incomplete rescue need to be further investigated to better understand how to support patients undergoing treatment for SMA.Res/Res; SMN2; SMNΔ7; CreERTSmn) have been described previously  in spinal cord of P4 SmnRes/Res mice. This revealed a significant increase in all four transcripts . Res/Res;SMN2;SMNΔ7Smn mice were used as non-induced SMA mice (herein referred to as ‘SMA’) and Smn+/−;SMN2;SMNΔ7;Cre-ER+/− or Smn+/+;SMN2;SMNΔ7;Cre-ER+/− were used as non-SMA controls (herein referred to as ‘Control’). Assessment of body weight revealed a trend towards an increase at P6 and P8, which was statistically significantly compared with ‘SMA’ mice, and indistinguishable from ‘Control’ mice by P10  to induce full length expression of e by P10 . A signie by P10 . Analysie by P10 –F. Over e by P10 .Cdkn1a and Fas in ‘Rescue’ mice compared with ‘SMA’, although Pmaip levels remained elevated . In the moderately affected AAL muscle, although P1 treatment preserved axon and endplate number, following P6 treatment, there was a significant decrease in axon number , suggesting they are progressively disassembled following loss of presynaptic input. Previous work has often reported that although there is a significant increase in denervated endplates in the early symptomatic phases in mouse models of SMA, the severity of denervation does not increase as pathology progresses ,17. The et al. also showed that persistent clusters of AChRs remain over 20 days after denervation. The loss and fading of AChR clusters in this study appear to occur at a much more rapid rate. This could be due to the comparatively immature status of endplates in mouse models of SMA  were purchased from Charles River and maintained as +/Res;SMN2;SMNΔ7;Cre-ERT+/−Smn × +/Res;SMN2;SMNΔ7Smn breeding pairs at the animal facilities at the University of Edinburgh. +/−;SMN2+/+;SMNΔ7+/+Smn  were maintained as breeding pairs at the University of Missouri. All genotyping was performed using standard PCR protocols on DNA extracted from an ear punch (for breeding pairs), tail tip  or tail sample (taken before tamoxifen treatment for tamoxifen treated mice). Mice over P10 were sacrificed by overdose of inhalation anaesthetic or rising CO2 and death was confirmed by exsanguination from the carotid artery. Mice under P10 were decapitated under terminal anaesthesia and death was confirmed by exsanguination from the carotid artery. All experiments were performed in accordance with the regulations set out by the UK Home Office  or approved by the IACUC at the University of Missouri.Res/Res;SMN2;SMNΔ7) were used as SMA model mice, as no recombination will take place, and mice carrying one copy of the Cre transgene  were used as ‘Rescue’ mice.Tamoxifen (Sigma-Aldrich) was dissolved in corn oil to a concentration of 20 mg/ml. Tamoxifen solution was administered to litters of mice at P4 using oral gavage at a dose of 75 mg/kg. For v1.11, a Phosphordiamidate morpholino oligomer (PMO) ASO  forward (5′-tctgtgttcgtgcgtggtgacttt-3′) and mSmn-WT reverse (5′-cccaccacctaagaaagcctcaat-3′) and for the Smn knockout, SMN1-KO forward (5′-ccaacttaatcgccttgcagcaca-3′) and SMN1-KO reverse (5′-aagcgagtggcaacatggaaatcg-3′). intracerebroventricular (ICV) injections were performed on P1 and the injection site was P1 SMNΔ7 mice were injected with 4 μL of a 20 nM solution of E1PMO) ASO . The folThe ‘time to right’ test was carried out on mice younger than P10. Mice were placed on their back and the time it took for them to right themselves  was recorded, with a maximum of 30 s and an average of three attempts.Muscles were immediately dissected from recently sacrificed mice and fixed in 4% paraformaldehyde (Electron Microscopy Science) in PBS for 15 min. Post-synaptic AChRs were labelled with α-BTX  for 2 h. Muscles were permeabilized in 2% Triton X-100 in phosphate buffered saline (PBS) for 30 min, then blocked in 4% bovine serum albumin/1% Triton X-100 in PBS for 30 min before incubation overnight in primary antibodies  and visualized with secondary antibodies . Muscles were then whole-mounted in Mowiol (Sigma).The percentage of fully occupied endplates was determined by classifying each endplate in a given field of view either fully occupied [presynaptic terminal (SV2 and NF) completely overlies endplate (BTX)], partially occupied [presynaptic terminal only partially covers endplate (BTX)] or vacant (no presynaptic label overlies endplate). For presynaptic swelling, all NMJs were classified as having no swelling, mild , moderate  or severe . Endplate size was quantified using ImageJ software. At least three fields of view were analysed per muscle totalling > 50 endplates per muscle. Endplate number was assessed by counting the number of AChR clusters within an anatomically consistent area of the muscle . NMJ numbers were assessed by counting the number of fully or partially innervated endplates in an anatomically distinct area of the muscle. Only muscles which were present in their entirety with no signs of damage or disruption to innervation patterns were used for these measurements. Axon number was counted in a consistent region of each muscle (the point at which axons cross in the AS and 400 μm from the main endplate band in the AAL). Average motor unit size was calculated by dividing the total number of innervated NMJs in a given muscle by the number of axons in the same muscle. All quantification was performed with researcher blind to genotype of material.2 First Strand kit (Qiagen). SYBR green-based Q-RT-PCR was performed using preoptimized primers purchased from Qiagen. Amplification was performed using KAPA SYBR fast universal PCR mastermix as per manufacturer instructions on a BioRad CFX connect real-time PCR detection system. Relative gene expression was calculated using the 2−ΔΔcT formula (RNA was extracted using a micro RNeasy kit (Qiagen) and 1 μg of RNA was used to perform reverse transcriptase using the RT formula . RelativSupp_figure_1_ddac097Click here for additional data file.Supp_figure_2_ddac097Click here for additional data file.


Breastfeeding in the early postpartum period is expected to have mental benefits for mothers; however, the underlying psychobiological mechanisms remain unclear. Previously, we hypothesized that the release of oxytocin in response to the suckling stimuli during breastfeeding would mediate a calming effect on primiparous mothers, and we examined salivary oxytocin measurements in primiparous mothers at postpartum day 4 using saliva samples without extraction, which was erroneous. Thus, further confirmation of this hypothesis with a precise methodology was needed.We collected saliva samples at three time points  to measure oxytocin in 24 primiparous mothers on postpartum day 2 (PD2) and 4 (PD4) across the breastfeeding cycle. Salivary oxytocin levels using both extracted and unextracted methods were measured and compared to determine the qualitative differences. State and trait anxiety and clinical demographics were evaluated to determine their association with oxytocin changes.Breastfeeding elevated salivary oxytocin levels; however, it was not detected to a significant increase in the extraction method at PD4. We found a weak but significant positive correlation between changes in extracted and unextracted oxytocin levels during breastfeeding (feeding minus baseline); there were no other significant positive correlations. Therefore, we used the extracted measurement index for subsequent analysis. We showed that the greater the increase in oxytocin during breastfeeding, the lower the state anxiety, but not trait anxiety. Mothers who exclusively breastfed at the 1-month follow-up tended to be associated with slightly higher oxytocin change at PD2 than those who did not.Breastfeeding in early postpartum days could be accompanied by the frequent release of oxytocin and lower state anxiety, potentially contributing to exclusive breastfeeding.The online version contains supplementary material available at 10.1186/s12884-022-05026-x. Breastfeeding in the early postpartum period not only promotes the recovery of the reproductive organs of the mother but also enhances the immunity of the newborn and protects against infections and allergies –3. SinceThe neuroendocrine basis of breastfeeding is driven by prolactin and oxytocin (OT), which are secreted by the pituitary gland. Prolactin is involved in milk production, and OT stimulates milk ejection by contracting the myoepithelium surrounding the breast. In recent years, OT has attracted more attention as a neurotransmitter than as a hormone for its classical properties . TherefoIt has been well recognized that mothers’ plasma OT levels rise in response to the suckling stimulation during breastfeeding . A studyIn the present study, to test our hypothesis, we used a more sophisticated extraction method to measure salivary OT and assessed state and trait anxiety using a standard questionnaire State-Trait Anxiety Inventory (STAI) at two time points, postpartum days 2 and 4.Twenty-four Japanese mothers hospitalized in the Miyamura Obstetrics and Gynecology Clinic, Nagasaki, participated in this study on postpartum days 2 (PD2) and 4 (PD4). They were recruited during pregnancy and were eligible if they met the following criteria : (i) priAll participants provided written informed consent after the purpose of the experiment was explained. The experimental protocol was in accordance with the Declaration of Helsinki (2013) and was approved by the Ethics Committee of the Nagasaki University Graduate School of Biomedical Sciences.n = 3/22, PD4; n = 8/22) were crying prior to breastfeeding (Table t-test (P > 0.05). Participants were seated on comfortable chairs, and three samples of saliva were collected during the breastfeeding cycle. The first measurement was conducted 30 min before breastfeeding began (baseline), the second at the initiation of breastfeeding (+ 5 min after initiation of breastfeeding [feeding]), and the third was 30 min after the completion of breastfeeding (post-feeding). After breastfeeding was completed, most of the infants fell asleep, and the mothers placed them in the cot. We defined ΔOT (feeding minus baseline), which captures the OT level change in response to breastfeeding. As shown in Table AUCi) and “Area under the curve with respect to ground” (AUCg), which included the influence of individual duration of breastfeeding. The participants refrained from eating or drinking during the sample collection session.As described previously , breastfng Table , there wg for 15 min at 4 °C. Because of the short half-life of OT, the protease inhibitor aprotinin  was added to each collection tube to inhibit the metabolic breakdown of the peptide. The liquid samples were stored at − 80 °C.Saliva samples were collected from the participants using cotton Salivettes®  at all three time points. Participants were asked to place a Salivette® in their mouth and instructed to chew for 1 min until it was saturated with saliva. The Salivettes® were kept on ice for up to 2 h, before being centrifuged at 1,500 × OT concentrations were measured using an enzyme immunoassay kit . Briefly, saliva samples (500 μL per sample) were extracted using an Oasis PRiME HLB 96-well plate at 30 mg sorbent per well  and evaporated at room temperature using compressed nitrogen. Each evaporated sample was reconstituted in 125 μL of assay buffer before OT measurement to provide a sufficient sample volume (100 μL per well). This practice ensured that the plated samples contained four-fold concentrated OT, which is expected to be sufficiently high to be read above the detection limit (15.0 pg/mL). The samples were assayed using a microplate reader  for the 96-well format according to the manufacturer’s instructions. Because each saliva sample volume was less than 1.5–2 mL, we did not measure the samples using a duplicate assay. The mean intra- and inter-assay coefficients of variation calculated by the duplicated seven standard samples were 2.1% and 6.5%, respectively.To compare the differences between the extracted and unextracted method, we also measured the same samples using the unextracted method. Briefly, we used the lyophilization process instead of the extraction process to concentrate the samples four-fold overnight, reconstitute them in 125 μL of assay buffer, and store at − 20 °C until they were assayed. The detailed procedures are described in our previous manuscript ) x breastfeeding cycle ). Pairwise comparisons were further made with Bonferroni corrections when significant differences were observed. Additionally, one-way repeated measures ANOVA was used for easy comparison according to a previous study . SignifiF  = 4.66; P = 0.04, ηG2 = 0.07]) and breastfeeding cycle  = 4.56; P = 0.02, ηG2 = 0.07]). There was no significant interaction between postpartum time point and breastfeeding cycle . Pairwise comparisons across the breastfeeding cycle revealed that OT levels were significantly higher at feeding than at baseline  irrespective of the postpartum time point. No other combinations of pairwise comparisons were statistically significant . One-way repeated-measures ANOVA for breastfeeding cycle at each postpartum time point revealed a significant difference in OT changes across the breastfeeding cycle at PD2  but not at PD4 . OT levels at PD2 increased significantly during feeding compared with baseline  and decreased between feeding and post-feeding; however, this decrease was not statistically significant . OT levels at post-feeding were not significantly higher than those at baseline .OT levels at baseline, feeding, and post-feeding are shown in Fig. F  = 3.52; P = 0.07, ηG2 = 0.02]) and breastfeeding cycle  = 10.92; P = 1.5e-4, ηG2 = 0.17]). There was no significant interaction between postpartum time point and breastfeeding cycle . Pairwise comparisons across the breastfeeding cycle revealed that OT levels were significantly higher at feeding than at baseline  and post-feeding  irrespective of the postpartum time point. Further, no significant difference was observed between baseline and post-feeding . One-way repeated-measures ANOVA for breastfeeding cycle at each postpartum time point revealed a significant difference in OT changes across the breastfeeding cycle at PD2  and PD4 . OT levels at PD2 and 4 significantly increased during feeding compared with during baseline  and decreased between feeding and post-feeding ; however, this decrease was not statistically significant at PD4 . OT levels at post-feeding were not significantly higher than those at baseline .OT levels at baseline, feeding, and post-feeding are shown in Fig. R = -0.38, P = 0.01) and ΔOT . No other pairwise correlations were significant (P > 0.05).As shown in Fig. ΔOT and AUCi) at PD2 were significantly negatively correlated with STAI-S at PD2   are shown in Supplementary Figure SΔOT and AUCi at PD4 were negatively correlated with “Duration of labor”  and “Induction medicine during labor” . “Duration of labor” and “Induction medicine during labor” were also well correlated with each other ; as induction medicine was practically used in cases with a long duration of labor, it can be interpreted to reflect the difficulty of labor. This implies that participants with difficult labor would be less likely to show an OT increase by breastfeeding at PD4. In addition, “Induction medicine during labor” was negatively correlated with the daily number of breastfeeding cycles on PD 1  and 2 ; however, this possibility should be carefully interpreted considering that induction medicine was used in only six cases among the present study population. Although the differences were not statistically significant, participants who were exclusively breastfeeding at the 1-month postpartum follow-up (n = 14) had only a slightly higher trend for OT change indices at PD2 than those who were not exclusively breastfeeding (n = 8) .All the combinations of Pearson’s correlation analyses or ΔOT) was significantly positively correlated with each other. Hence, we subsequently analyzed OT data measured via the extraction method to investigate whether increased OT associated with breastfeeding may associate with lower maternal anxiety. The results showed that the higher the OT change indices at PD2, the lower the state anxiety at PD2. Although they were not statistically significant after the multiple corrections, the OT change indices at PD2 tended to correlate significantly with state anxiety at PD4. In contrast, trait anxiety showed no correlations with those indices.In the present study, we investigated salivary OT changes following the breastfeeding cycle at PD2 and 4 in primiparous mothers who delivered vaginally and who experienced no abnormal symptoms such as heavy bleeding or fever during the postpartum period. We also utilized both extracted and unextracted methods for OT measurements and examined the correlation between the methods. The results showed that the extraction method showed a significant increase in OT concentration with breastfeeding. The unextracted method also showed a significant OT increase during breastfeeding. However, when we examined the correlation between the OT concentrations of the extracted and unextracted methods, we observed a correlation between the baseline values and the inverse direction, but no correlations were found at the other time points or in the total data. However, the difference between feeding and baseline   encouragN = 11 [N = 10 [N = 4 [The present study had four major limitations. First, the recruitment sample size was small. Since our previous study  had a saN = 11 , N = 10  [N = 10  (with so0 [N = 4 . Althoug0 [N = 4 , 34; howBreastfeeding in the early postpartum period resulted in an accompanying increase in endogenous OT, associated with lower maternal state anxiety, and potentially contributed to the exclusive breastfeeding rate at 1-month postpartum. This suggests that early postpartum breastfeeding could be the key to successful breastfeeding, as recommended by the WHO.Additional file 1:Supplementary Figure S1. Pearson’scorrelation coefficients matrix.The size and color of the circles reflect correlation coefficients for eachcombination of the correlation analysis.


Single-molecule techniques such as optical tweezers and fluorescence imaging are powerful tools for probing the biophysics of DNA and DNA-protein interactions. The application of these methods requires efficient approaches for creating designed DNA structures with labels for binding to a surface or microscopic beads. In this paper, we develop a simple and fast technique for making a diverse range of such DNA constructs by combining PCR amplicons and synthetic oligonucleotides using golden gate assembly rules. We demonstrate high yield fabrication of torsionally-constrained duplex DNA up to 10 kbp in length and a variety of DNA hairpin structures. We also show how tethering to a cross-linked antibody substrate significantly enhances measurement lifetime under high force. This rapid and adaptable fabrication method streamlines the assembly of DNA constructs for single molecule biophysics. Single-molecule methods have enabled deep insights into the biophysics of DNA and DNA–protein interactions. Force spectroscopy techniques such as optical and magnetic tweezers enable the investigation of the effects of tension and torsion in DNA processes . ControlThe generation of defined DNA substrates is an important pre-requisite for all single molecule techniques using either force spectroscopy or imaging. For many applications these substrates must be on the order of kilobases in length in order to create space between the coupling surfaces or to enable fluorescence tracking of isolated molecules. λ-DNA is frequently used since it provides an inexpensive source of long (48.5 kb) and pure double-stranded DNA with 12 base overhangs for straightforward attachment of labelled oligonucleotides. Nicking endonuclease based methods can be used to introduce modification at specific sites on λ-DNA ,16. HoweIn this paper, we develop a high-yield fabrication method based on golden gate assembly for the construction of long DNA constructs, which combine PCR amplicons and synthetic oligonucleotides. We leverage recent profiling of ligation accuracy by T4 DNA ligase  to efficBiotin and digoxigenin labelled DNA  were synthesized by incorporation of modified dUTPs using Taq polymerase . The PCR mixture was setup according to the manufacturer's instructions. For synthesizing biotin labelled DNA, biotin-11-dUTP (Jena Bioscience) was added to the PCR mixture at a ratio of 1:8 of biotin-dUTP:dTTP. For synthesizing digoxigenin labelled DNA, dig-11-dUTP (Jena Bioscience) was added to the PCR mixture at a ratio of 1:8 of dig-dUTP:dTTP. The PCR for handle synthesis amplified an 860 bp section of λ-DNA (NEB). Unlabelled PCR sections were synthesized using a Taq based master mix . After the PCR amplification, samples were purified by a spin column  according to the manufacturer's instructions. The primers used for each structure described in this study are given in the SI . The PCRAll oligonucleotides were purchased from Integrated DNA Technologies (IDT). Each oligonucleotide part was separately assembled by mixing the component oligos to a final concentration of 10 μM in IDT duplex buffer  and annealing from 70°C to room temperature over 30 min. The specific sequences of each oligonucleotide part are given in the SI .DNA construct designs were assembled by mixing PCR parts and oligonucleotide parts together with BsaI  and T4 DNA ligase (NEB) in 1× T4 DNA ligase buffer (NEB). The assembly was carried out in a single tube of 20–40 μl volume with final concentrations of 1 U/μl BsaI-HFv2 and 20 U/μl T4 DNA ligase. The total DNA concentration in the golden gate reaction was 30–100 ng/μl and was kept as high as possible to maximize the ligation efficiency. The parts were mixed such that the stoichiometry was an increasing ratio from the central part in the design to the outside parts (see SI for full details). The mixture was incubated for 3 h cycling between 37°C and 16°C every 5 min.Assembly products were analysed on a 1% agarose gel with 1xTAE and run at 80 V. For imaging gels, the DNA was post-stained with GelRed (Biotium) and visualized with a UV illuminator. Three hours run time was sufficient for separating the main band at 10 kb from the side product at 9 kb . ExtensiMicrofludic flow cells with gravity controlled perfusion rates were constructed as previously described . Briefly3 μm latex spheres with sulfate surface groups  were initially introduced into the flow cell in a buffer of 1 mM Tris pH 7.5 and allowed to bind non-specifically to the nitrocellulose surface. These latex spheres act as fiducials for drift correction during an experiment. Antibody solution at 0.7 mg/ml in PBS was then added and incubated for 5 minutes before washing with ∼20 channel volumes of PBS (the channel volume was 5 μl). For Figure For combined flow stretching and TIRF imaging, single molecule tethers were prepared as described above with 1 μm magnetic beads  and the DNA was stretched perpendicular to the optical axis using a flow rate of ∼0.5 μl/s. The imaging buffer contained 20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mg/ml BSA, 1 mg/ml β-casein, 20 nM sytox orange (ThermoFisher). The sample was illuminated with a 532 nm laser and imaged with an Andor Ixon EMCCD (exposure time was 50 ms).In Figure Initially we tested the achievable yield for a long duplex DNA construct constructed from multiple parts using golden gate assembly. We designed a DNA construct which was 10.1 kb in total length, combining four 2.1 kb PCR amplicons and two 0.9 kb PCR amplicons which were labelled with either biotin or digoxigenin nucleotides incorporated during the amplification. These six parts were designed with five connection points represented by the different colours in Figure n = 180 total tethers tested) comparable to previous literature reports using a variety of methods    and formM–1 s–1) . Digoxigassembly . Howeverassembly . Covalenassembly ,42. TherTethering via anti-digoxigenin bound to a nitrocellulose surface relies on non-covalent bonds at two levels: (i) between the nitrocellulose and anti-digoxigenin antibody and (ii) between the binding sites of the antibody and digoxigenin labels on the DNA. Nitrocellulose binding to an antibody is widely used in western blotting and is thought to rely mainly on hydrophobic contacts with the protein . We reasWe selected single DNA tethers by checking for the absence of DNA braiding upon rotation of the magnets . We founHaving demonstrated the efficient assembly of long, end-labelled dsDNA using golden gate assembly, we tested the possibility of constructing DNA structures which accommodate synthetic oligonucleotide parts. Specifically, we made a design which featured a short synthetic DNA hairpin positioned in the centre of the DNA. Such hairpin structures are particularly useful for studying the thermodynamics of DNA duplexes using magnetic and optical tweezers . Figure We also synthesized a DNA construct which uses a larger 1.5 kb PCR amplicon as the duplex hairpin structure—Figure Figure in vitro for genome assembly has led to the development of several techniques such as Gibson assembly and golden gate assembly (The recent drive to assemble long double-stranded DNA assembly ,52. Gibsassembly . The lacassembly . Anotherin vitro assembly and genetic selection (Golden gate assembly has proven useful for large scale assemblies of DNA plasmids and genomes. By improving the efficiency of Type IIs restriction enzymes and T4 DNA ligase activity, several recent advances have been made including the generation of genomes from up to 52 parts by combining election . We antiAll relevant data is available in the paper or the Supplementary Information.gkac300_Supplemental_FileClick here for additional data file.


On the other side, the FE spectra of TPPS have been quenched by both Ag and Au NPs. Via applying quenching calculations, Ag NPs showed only traditional static fluorescence quenching of TPPS with linear Stern–Volmer (SV) plots. On the contrary, quenching of TPPS emission by Au NPs shows composed models. One model is the sphere of action static quenching model that prevails at high quencher concentrations leading to non-linear SV plots with positive deviation. However, at low Au NPs concentrations, traditional dynamic quenching occurs with linear SV plots. The quantum calculations for TPPS structure have been obtained using Gaussian 09 software: in which the TPPS optimized molecular structure was achieved using DFT/B3LYP/6-311G (d) in a gaseous state. Also, the calculated electronic absorption spectra for the same molecule in water as a solvent are obtained using TD/M06/6-311G +  + . Furthermore, the theoretical and experimental results comparable to UV–Vis spectra have been investigated.Colloidal metallic nanoparticles have attracted a lot of interest in the last two decades owing to their simple synthesis and fascinating optical properties. In this manuscript, a study of the effect of both gold nanoparticles (Au NPs) and silver nanoparticles (Ag NPs) on the fluorescence emission (FE) of TPPS has been investigated utilizing steady-state fluorescence spectroscopy and UV–Vis spectrophotometry. From the observed electronic absorption spectra, there is no evidence of the ground state interaction between metallic Au NPs or Ag NPs with TPPS B-band or “Soret band” found around 400 nm is characteristic of macrocyclic conjugation. Additionally, in the Soret band, there are between two and four weaker bands called “Q-bands” situated between 480–700 nm  − ions into metallic Au NPs is explained in detail in the literature  is the molar concentration of the quencher, and r is the radius of quenching sphere volume. Figure Io /I) versus [Q] with a slope equal to VNo. Accordingly, the volume and radius of the quenching sphere were 3.85 × 10–14 cm3 and 209.5 nm, respectively.The Perrin model was valid for the quenching process of TPPS by Ag NPs. The Perrin relationship  is given2O at room temperature. When the concentration of Au NPs was increased gradually, the fluorescence emission intensity decreased significantly as a consequence of TPPS fluorescence quenching without any appreciable change in position and shape of the emission band, Fig. The fluorescence emission of TPPS was studied in the presence of variable concentrations of Au NPs as a quencher in two different media containing 0% and 40% EG/water by volume in H2O and 40% EG solutions, by applying the traditional SV equation  has been calculated as kq = 1.663 × 1016 M−1 s−1  at room temperature.Upward curvatures were observed, as a result of the quenching process of TPPS by Au NPs in Htion Eq. , Fig. 4bin water . This vaThe nonlinear plots showed positive deviation and similar behaviors were observed earlier . The pos‘V’ is the static quenching constant that represents an active volume element surrounding the fluorophore in its excited state. As W depends on the quencher concentration [Q], the SV plots for a quencher with a high quenching ability generally deviate from linearity. Thus, it is worth rewriting Eq. , was obtained by the least-square fit method determining the slope. The intercept of the plot was used to calculate W values for each quencher concentration [Q]. Plotting of lnW against [Q] gave a linear correlation with a negative slope that equals the static quenching constant (V = -6.56 × 107), as in Eq.  and the quencher (RQ) molecules were determined by using the additive model [R is the sum of both molecular radii is named encounter distance R. As seen, the value of kinetic distance r was greater than the encounter distance R. Therefore, the static effect takes place irrespective of the ground state complex formation provided the reactions are limited by diffusion indicating that the sphere of action model holds well [The calculated ve model . R is thlds well . Furtherlds well .Table 1Qo) computed for TPPS in the ground (S0) and first excited states (S1) via applying DFT/B3LYP/6-311G(d) method in the gaseous state are presented in Table 1) for TPPS in the gaseous phase. Various comments can be concluded from Table 2 hybridization over the entire TPPS backbone GS. (4) As presented in Table o to S1. (5) Upon excitation from So to S1, the Mulliken charge of all atoms in TPPS GS is decreased except for the charge of C71, C46 and C2 atoms (See Table 3− group) in the So and S1 states have a large Mulliken charge compared to the nitrogen atoms in the middle ones. These findings could lead to the binding of the excited state near oxygen atoms in SO3− to the Au or Ag NPs [Utilizing the DFT method, the electronic Geometrical structure (GS) for the ground state of TPPS was computed. The electronic GS optimization was investigated at the DFT/B3LYP/6–311G (d) method in a gaseous state; the result is obtained in Fig. g), H -1 and L + 1 (Eg1) and H -2 and L + 2 (Eg2) for TPPS GS have been calculated: the results are collected in Fig. *-antibonding anti-bonding characters distributed over the whole target molecule. (4) Orbital energy level analysis and the resultant H – L energy gap (Eg) referring to kinetic stability of a molecule [g indicates high kinetic stability and low chemical reactivity because it is energetically unfavorable to add electrons to a high-lying L or to remove electrons from a low-lying H [The molecular orbitals (MOs) graphical representation of the highest occupied molecular orbital (HOMO (H)), lowest unoccupied molecular orbital (LUMO (L)), HOMO – 1 (H – 1), LUMO + 1 (L + 1), HOMO – 2 (H – 2), LUMO + 2 (L + 2) and energy gaps between the following: H and L , oscillator strength (f), coefficient, and calculated maximum absorption wavelength (Th. λmax) parameters are listed in Table The electronic absorption spectra of TPPS GS were obtained experimentally in water; the obtained electronic absorption spectra were presented in Fig. The behavior of water-soluble TPPS absorption and fluorescence emission spectra has been studied in the presence of aqueous colloidal citrate capped Ag NPs and Au NPs. The observed UV–Vis absorption spectra of TPPS indicate the absence of interaction between TPPS with Ag NPs and Au NPs. But fluorescence quenching studies revealed a positive deviation from SV upon replacing Ag NPs with Au NPs. The latter behavior can be interpreted in terms of action sphere static quenching models. Various rate parameters for the fluorescence quenching process were determined by using a modified SV equation. The optimized TPPS GS using DFT/B3LY6-311G(d) was obtained successfully. The calculated electronic absorption spectra of TPPS GS in water appear as six transitions. The electronic transitions for TPPS at 591, 559, 411, 405, 362, and 363 nm. the third electronic transitions corresponding to the experimental peak at 411 nm (f = 1.571) arises from transition of MO258—> 262 (coefficient = 45%), MO260—> 263 (coefficient = 56%) and MO261—> 262 (coefficient = 33%).


Hydrolyzed hyaluronic acid high-resolution fine microneedles of 13 µm in diameter and 24 µm in height were fabricated from hydrolyzed hyaluronic acid gels made in mixtures of water using vacuum environment imprint lithography processes with a water permeable mold. The gas traps of water and volatile solvents in the imprint materials cause transfer failure in the conventional water impermeable molds of quartz and metal. However, the water permeable mold allows the use of 67 wt% dilution water with high solubility to increase the fluidity of the hydrolyzed hyaluronic acid during the patterning of high-resolution fine microneedles for cosmetics and pharmaceuticals. This demonstration sets a new paradigm of functional pure gels for high-resolution nano-patterning applications with various cosmetic and pharmaceutical materials containing dilution water using a water permeable mold. Many beauty ingredients, such as hyaluronic acid and collagen gels, are expected to bring significant demand to the global market for cosmetology products . HyaluroThe skin is composed of an epidermal layer, a dermal layer, and subcutaneous tissue, with a stratum corneum on the surface of the epidermal layer. Since the stratum corneum has a barrier function to prevent foreign substances from entering the body and moisture evaporation from the body ,9, high-Currently, technologies such as nano-emulsions, liposomes, nanoparticles, and other nanotechnologies have been developed to penetrate the stratum corneum with active ingredients in cosmetics ,16. ThesConventional microneedles for cosmetic applications are 100–800 µm in diameter and 200–800 µm in height. Conversely, the epidermal layer is approximately 200 µm thick. Consequently, the microneedles reach the dermis layer, which exists below the epidermis layer, leading to a painful injection because the dermal layer contains sensory receptors .However, the microfabrication process involves problems such as gas generation from water and solvents during heating, molding defects, and mold breakage ,27,28. FThe high-resolution fine microneedles of approximately 13 µm in diameter and 24 µm in height, with hydrolyzed hyaluronic acid and water as their two components, were fabricated using the newly vacuum environment imprint lithography processes using a water permeable mold for cosmetic and pharmaceutical applications. The prepared water permeable surface material in the water permeable mold with titanium dioxide and silicon dioxide were cross-linked by the conditions to generate the cross-link density needed to obtain water permeability. The possibility of shortening the molding time by further improving the filling speed is expected to be realized in the next study.In addition, −1 (OH bond), 2880 cm−1 (CH bond), 1740 cm−1 (ester), 1649 cm−1 (amide type I), 1554 cm−1 (amide type II), and 1045 cm−1 (CO bond) peaks were observed before thermal deposition, and 3375 cm−1 (OH bond), 2885 cm−1 (CH bond), 1735 cm−1 (ester), 1640 cm−1 (amide type I), 1551 cm−1 (amide type II), and 1033 cm−1 (CO bond) peaks after thermal deposition also indicated the absorption of hydrolyzed hyaluronic acid and water, and these peaks were somewhat coincident before and after thermal deposition at 80 °C for 90 min.A more accurate evaluation of the biocompatibility such as vivo penetration experiments on mice and skin penetration is the subject of a future study. The result that one can nanoimprint functional gel solutions containing high volatile dilution solvents of over 60% is expected to be a breakthrough for various biomedical applications.The high-resolution fine microneedle pattern failure of hydrolyzed hyaluronic acid gels containing 67 wt% water as a dilution solvent was solved using the water permeable mold in the vacuum environment imprint lithography processes. This demonstration of the high-resolution fine microneedles of 13 µm in diameter and 24 µm in height sets a new paradigm of functional pure gels for high-resolution fine patterning applications with various cosmetic and pharmaceutical materials containing dilution water using a water permeable mold for a skin care cosmetic patch and transdermal drug delivery system.The water permeable porous substrates under the water permeable surface material were fabricated by using a hybrid metal 3D printer  and the standard maraging steel powders with an average particle size of 20–30 μm. The powders were baked and hardened by irradiating them with a 400 W Yb fiber laser using the 3D printer ,40. ThisThe hydrolyzed hyaluronic acid  shown in In addition, to confirm the dissolution behavior and basic properties of the hydrolyzed hyaluronic acid before and after the vacuum environment imprint lithography processes, three types of analysis of the hydrolyzed hyaluronic acid fine microneedles were carried out as follows.1.Measurement of the dissolution behavior using environmental test equipment The evaluation of the dissolution behavior was performed for 35 min at 31 °C and 90–95% humidity using an incubator .2.Measurement using a FT-IR −1, the number of integrations was 4, and the resolution was 4 cm−1. The measurement was performed on films prepared by dropping hydrolyzed hyaluronic acid onto a glass slide and placing the slide at room temperature for 24 h. FT-IR measurements were carried out for component comparison before and after baking the films of hydrolyzed hyaluronic acid using a hot plate at 80 °C for 90 min.The measurements were performed using an FT-IR . The measurement range was 450–4000 cm3.Measurement using a Tg-DTA The measurement in nitrogen was performed using a Tg-DTA  in the temperature range of 30 to 250 °C (temperature gradient: 10 °C /min). The weight of the analyzed sample was 5 mg.