Atomically resolvedvisualization of individual N2 dissociation events elucidatesthe fundamental reactive dynamics of the N2/Ru(0001) systemby providing a detailed understanding of the on-surface dissociationdynamics: the distance and angle between nitrogen atoms from the samedissociated N2 molecule, site specificity and coordinationof binding on terrace sites, and the local evolution of surroundingnanoscopic areas. These properties are precisely measured over a rangeof impinging N2 kinetic energies and angles, revealingpreviously unattainable information about the energy dissipation channelsthat govern the reactivity of the system. The experimental resultspresented in this paper provide insight into the fundamental N2 dissociation mechanism that, in conjunction with ongoingtheoretical modeling, will help determine the role of dynamical processessuch as energy transfer to surface phonons and nonadiabatic excitationof electron–hole pairs (ehps). These results will not onlyhelp uncover the underlying chemistry and physics that give rise tothe unique behavior of this activated dissociative chemisorption systembut also represent an exciting approach to studying reaction dynamicsby pairing the angstrom-level spatiotemporal resolution of an in situ STM with nonequilibrium fluxes of reactive gasesgenerated in a supersonic molecular beam to access highly activatedchemical dynamics and observe the results of individual reaction events.This paper examines the reactive surface dynamics ofenergy- andangle-selected N A fundamentalunderstanding of N2 dissociation onto ruthenium surfacesis therefore of great fundamental, technological, and economic importance.12The interaction of gaseous species onruthenium surfaces has beenstudied extensively due to the importance of ruthenium as a catalystfor a wide variety of applications.2 dissociation onto Ru(0001) is a prototypicalactivated dissociativechemisorption process, and understanding the mechanistic featuresof this would have a considerable impact on the field of heterogeneouscatalysis. Compared to other activated dissociation benchmark systems—namelyH2 on Cu15 or CH4 on transition metals—thathave dissociation probabilities (s0) thatapproach unity at normal incident kinetic energies (EN) much greater than the potential barrier(V*),18 N2 exhibits substantially different adsorptionbehavior, demonstrating s0 ≪ 1at EN ≫ V*.21 Nonadiabatic coupling/tunneling mechanisms28 and energy transfer to surface phonons,30 along with theoretical formulations using only adiabatic treatments,33 have all been proposed to describe the unusual dissociation behaviorof N2 on Ru(0001) with increasing number of degrees offreedom explicitly treated in forming the potential energy surface—someformulations treating several degrees25 and some formulationstreating all six degrees of freedom for the nitrogen molecule.36 Neural networkshave been used to treat all degrees of freedom explicitly using ahigh-dimensional fit of molecule–surface interactions, allowingfor less computationally expensive ab initio molecular dynamics simulationsfor a system in which nonreactive scattering dominates (s0 ≪ 1), making reactivity more arduous to sample.30 Neural networks have also beenused to sample the free energy surface of nitrogen dissociation onRu(0001) showing vibrational entropy of surface atoms add appreciablyto the reaction barrier.37 Further experimentalwork is required to answer questions that remain about the fundamentalreactive surface dynamics of this important system.In addition to its relevance to ammoniasynthesis, N2 on a clean Ru(0001) surface held at room temperatureand 262 K. Atomically resolved visualization of individual dissociationevents at different impinging energies and angles provide a detailedunderstanding of the spacing and angle of nitrogen atoms from thesame dissociated N2 molecule, site specificity of terracebinding, and local, nanoscopic information about the reactive evolutionof the Ru(0001) surface in the low-coverage regime. By monitoringthe nanoscopic evolution of the Ru(0001) surface during exposure toenergy- and angle-selected N2, this work elucidates newinformation about the mechanisms of energy dissipation into the surfaceof this important gas–surface interface and more generallyshowcases how nonequilibrium fluxes of reactant molecules from a supersonicmolecular beam paired with an in-line insitu STM can capture the fundamental dynamics of individualreaction events. In conjunction with ongoing and future theoreticalstudies, observation of individual dissociation events will revealinsight into how energy is dissipated into the surface and may divulgethe role surface phonons and nonadiabatic coupling to electron–holepairs (ehps) play in transferring energy during and immediately afterthe activated dissociation event.In thispaper, we present results detailing the reactive dynamicsof N2 on a clean Ru(0001)surface under ambient conditions is very low (s0 ≈ 10–12)40 due to a highactivation barrier that occurs late in the dissociation process andrequires significant stretching of the N2 bond.41 Thermal sticking occurs exclusively at crystal steps due to a 1.5eV difference between the activation barrier at terrace (∼1.9eV) and step (∼0.4 eV) sites,44 illustrating the role step edges and defects can play in adsorptionon single-crystal model systems.46 Molecularbeam studies47 demonstrate that high impingingkinetic energies of N2 activate dissociation on terracesites, with no dependence on surface temperature. The dissociationprobability increases slowly with increasing N2 kineticenergy and plateaus at s0 ≈ 10–2 for kinetic energies much higher than the activationbarrier;21 while molecular beams of N2 seeded inHe and H2 carrier gases demonstrate how vibrational excitationof the impinging N2 increases the dissociation probabilitymarkedly,19 nonthermal plasmas have beenused to populate vibrational and electronic excited states, therebyincreasing reactivity of N2 with Ru-based catalysts.50 Additionally, measured isotope effects for N2 dissociation24 and hydrogenation51 of atomic nitrogen make N/Ru(0001) a suitable system for the studyof nonadiabatic tunneling mechanisms.The dissociation probabilityof N53 in conjunction with theoreticalcalculations,43 have helped elucidate the spatial properties, adsorbate–adsorbateinteractions, and binding structures of adsorbed nitrogen atoms withatomic resolution. At low temperatures, molecular N2 adsorbsto Ru(0001) binding perpendicularly at on-top sites, and molecularlybound N2 desorbs from Ru(0001) at temperatures greaterthan 128 K54—ensuring that adsorbedmolecular N2 will not be found in STM images within thisstudy. STM visualization depicts adsorbed nitrogen atoms as triangulardepressions 5 Å wide in topographical scans.53 N adsorbates (Nad) occupy the hcp 3-fold hollow siteon terraces52 and bridge site on steps43 of the Ru(0001) surface. Interactions betweenNad are repulsive at nearest-neighbor and second-nearest-neighborsites and slightly attractive at third-nearest-neighbor sites, resultingin an approximate pair potential of hard spheres that blocks the first-and second-nearest-neighbor sites in the low coverage regime at roomtemperature.52 Experimental53 and theoretical1 barriersto diffusion of 0.9 and 1.1 eV, respectively, have been observed forNad on the Ru(0001) surface. This allows adsorbate movementto be frozen out kinetically at moderate temperatures after the dissociationevent occurs.STM studies,24 and inelastic scattering47 of N2 onRu(0001); however, the techniques used heretofore are not directlysuited to investigate energy dissipation during/after dissociativeadsorption. Inelastic scattering provides information about the translationalenergy and quantum vibrational and rotational states of reflectedmolecules, but inelastic scattering notably looks at molecules thathave been scattered from the surface and does not directly probe thereaction dynamics of molecules that adsorb to the surface. Temperatureprogrammed desorption (TPD) provides initial sticking coefficientsfor various N2 beam energies, but each value only representsthe ensemble average of a Boolean value—whether the moleculeadsorbs or not—convoluting a multistep process into one scalar value. Instead of measuring ensemble values, our studycaptures the results of individual dissociation events of a processthat demonstrates an atypical sticking coefficient (s0 ≪ 1 at EN ≫ V*).Molecular beam studies have investigated adsorption2 impinging on Ru(0001) terrace sites.STM images from this study will serve as a benchmark for future computationalmodels to provide insight into how energy is transferred in the highlyactivated dissociation of N2 on Ru(0001). This work providesa deeper understanding of N2 dissociation on rutheniumand in collaboration with theoretical exploration can contribute amore fundamental understanding of activated dissociative adsorptionsystems.The results in this paperrepresent the visualization of individualdissociation events resulting from nonequilibrium fluxes of energy-and angle-selected N57 the instrument is composed of a triply differentiallypumped beamline, a surface characterization/preparation chamber thatcontains Auger electron spectroscopy (AES) and low-energy electrondiffraction (LEED) capabilities, and a scanning probe microscope (SPM)chamber that holds the variable temperature SPM based on the ultrastabledesign of Shuheng Pan, built in collaboration with RHK. The custom-builtPAN STM allows the Ru(0001) surface to be exposed to the supersonicmolecular beam at variable polar angles of incidence with the abilityto move the STM tip micrometers away from the area of interest toavoid blocking the molecular beam and then return the tip after thesurface has been exposed to the molecular beam to reveal the morphologyof the same nanoscopic area both before and after exposure to nonequilibriumfluxes of reactive gases.The results reported in this paper were acquiredutilizing a newUHV instrument that contains both a supersonic molecular beam andSTM/AFM techniques. As reported in previous publications,2/97% He gas mixture through a 30 μm molybdenumpinhole at pressures from 20 to 100 psi and nozzle temperatures rangingfrom 300 to 1150 K (±5%). The translational kinetic energy ofthe molecular beam at each nozzle temperature was measured using time-of-flight(TOF), and values of 0.8 ± 0.3, 1.1 ± 0.4, and 1.3 ±0.6 eV were found for nozzle temperatures of 730, 1000, and 1150 K,respectively. The uncertainty values in these energies represent theFWHM of each energy distribution. The molecular beam flux at the crystalfor all beam conditions was on the order of 1013 N2 molecules cm–2 s–1. Thekinetic energy values reported represent only translational kineticenergy, and the role of vibrational excitation on spatial distributionsis not directly quantified in this study. A Boltzmann distributionwith nozzle temperatures of 730, 1000, and 1150 K indicates, assuming,with no relaxation during expansion, populations of vibrationallyexcited impinging N2 molecules are 0.97%, 3.4%, and 5.5%,respectively.Supersonic molecular beams were generatedby the expansion of a3% N2 molecular beam exposures ontothe Ru(0001) surfacewere performed with the sample in the SPM chamber, which correspondsto a 4 mm diameter beam spot on the crystal. The sample was eitherheld at room temperature or cryocooled with liquid nitrogen duringimaging/exposures; the temperature of the sample was monitored usinga cryostat thermocouple attached to the STM assembly. The surfacetemperature was held constant between exposure and imaging for eachexperiment. The surface plane could be oriented to achieve an incidentpolar angle from 0 to 45° during exposures to the N2 molecular beam.All N2 molecular beam and not as a result of trace thermalizedN2 reflected from the tip/chamber, which was confirmedby the fact that only areas of the surface with direct line of sightto the N2 beam were reacted.Reactive evolution of the surface only arosedue to exposure tothe N–10 Torr base pressure)by multiple sputter/anneal cycles, similar to those reported previously.59 The Ru(0001) surface was sputtered at room temperature using 0.5keV Ar+ ions generated by a PHI 04-150 ion gun resultingin a current of 0.1–0.5 μA cm–2 onthe sample; the sample was flash annealed by electron beam bombardmentto 1500 K for 10 s after sputtering cycles. The temperature was monitoredusing a Mikron infrared pyrometer (ε = 0.35) during annealing.Hundreds of cleaning cycles were necessary to produce a clean andordered Ru(0001) surface. An Omicron NGL 10 SPECTALEED with both LEEDand AES capabilities was used to determine if the Ru(0001) surfacewas ordered and free of impurities.The Ru(0001) crystalsused in all experiments were cleaned in the characterization/preparationchamber  at slightly lower temperatures(<1400 K) were used and produced a partially clean surface. Localareas of clean and ordered Ru(0001) surface were produced from theseprocedures, but subsequent STM imaging revealed the presence of Moirépatterns indicative of monolayer and bilayer graphene formation.60 Subsequent higher energy (3 keV) Ar+ sputtering cycles followed by shorter (5 s) and higher temperature(1500 K) annealing cycles produced a clean, ordered Ru(0001) surface,characterized by AES, LEED, and STM visualization in 0.8Ir0.2 tips.Initially, lower energy(0.5 keV) Ar2. As shown in 53 the 3-fold symmetry of the adsorbate suggests binding at eitherthe fcc or hcp sites, with previous experimental and theoretical studiesindicating binding at hcp sites is most favorable.61Once a clean Ru(0001) surfacewas achieved and characterized, thesurface was exposed to N12 Contraryto a previous STM study,53 these “diffusion”experiments were performed multiple times on the room temperatureRu(0001) surface, and no observable diffusion was observed over thecourse of hours. The results can be explained most likely by the useof a tunneling current in this study that is <100 times that usedpreviously,53 thereby minimizing tip interactionswith the adsorbates that can influence their movement upon the surface.To further elaborate that room temperature was sufficient to freezediffusion and rule out small fluctuations in room temperature generatingmarkedly different adsorbate distributions on the surface, the Ru(0001)surface was held at 262 K to probe the effect of temperature changeson adsorbate distributions. As expected, the lower surface temperatureshowed no effects on spatial distributions of adsorbates. Annealingabove 1500 K provides an atomically clean surface for N2 molecular beam exposures, but coadsorbed species such as oxygenwere observed after several hours of STM imaging from residual backgrounddosing. Oad was not observed to promote Nad diffusionat this low coverage limit and is thus not expected to impact thespatial distributions of Nad measured in this study. Notably,the lack of diffusion enables the N2 dissociation eventto be directly investigated at room temperature by measuring the distancebetween and location of the resulting nitrogen atom adsorbates. STMimages of atomic nitrogen adsorbate pairs thus provide direct insightinto the energy transfer of the impinging N2 with the surfaceand the liberated energy during the dissociation event and do not show the effects of random thermal diffusion.STM imaging of the same nanoscopic area revealedno observablediffusion of the nitrogen atom adsorbates 3.12 Cont2 impinging 45° to the surface normal, where 1 langmuircorresponds to one impinging N2 for every one Ru surfaceatom. In the low coverage regime, the reactivity of 1.1 eV N2 impinging normal to the Ru(0001) surface does not significantlydiffer from the reactivity of the same energy N2 impinging45° with respect to the surface plane, suggesting that reactivityscales with total N2 energy, corroborating results froma previous study that showed molecular N2 adsorption onRu(0001) is only weakly affected by incident angle when N2 translational kinetic energies are >0.4 eV.62 N2 molecules impinging normal and 45° tothe surfacewith 1.1 eV translational kinetic energy were measured to have anapproximately 1 in 10000 chance of sticking, which is in qualitativeagreement with previous molecular beam studies.47Shown in 2 kinetic energy and angleaffect these values, is crucial to elucidating how the N2 interacts with the surface and what energy transfer mechanisms governthe reaction. Determination of the intrapair distances and relativeangles of nitrogen pairs was accomplished by reacting at extremelylow coverages (<0.08%) to reduce the total number of nitrogen atompairs such that the identity of the nitrogen atom pairs could be uniquelyassigned. Representative STM topography of the low coverage exposuresis shown in 2 impinging normal to the Ru(0001) surfacewhich clearly highlights individual nitrogen pairs formedfrom singular dissociation events. Misidentification of adsorbatespairs is avoided, as clean surfaces were always observed before reactingwith N2 fluxes (as demonstrated in Uncovering the pair distance(s) and the relativeangle(s) betweentwo adsorbates and the azimuthal direction of the incident molecularbeam, and whether the impinging N2 on Ru(0001) wereperformed with varied translational kinetic energies and impingingangles. Polar coordinates  are used to describethe relative position of two nitrogen adsorbates from the same dissociatedN2 molecule on the Ru(0001) lattice, where φ is theazimuthal angle between two adsorbates relative to the incident azimuthaldirection of the molecular beam. 2 translational kinetic energies and impinging anglesused in this study.Extremely low coverage exposures of NN = 318.The uncertainty of pair distances is reported as standard deviationthroughout this paper. Intrapair spacing did not change significantlyupon increasing the translational kinetic energy of N2 impingingnormal to the surface from 0.85 eV  to 1.3 eV , indicatingthat there is not a strong coupling of impinging translational energyto intrapair distance for the energies explored herein. The distancesmeasured across all molecular beam energies are extremely large consideringthat values are over 20 times larger than the lattice constant ofruthenium.A scatter plot 6A demons2 on Pt(111),63 Al(111),64 Cu(110),65 Rh(110),66 TiO2(110),67 and RuO2(110)68 leaves oxygenatoms from the same dissociated O2 molecule within severallattice spacings on the surface, i.e., <1 nm apart. An early studyof O2 on Al(111)69 indicateddissociative products may be spaced >80 Å apart, developingthenotion of hot adatom motion, and more STM work followed showing “lowtransient mobility” after dissociation.64 Plasmon-induced dissociation of O2 on Ag(110)resulted in oxygen atoms within several lattice constants as well.70 The large intrapair distances observed for N2/Ru(0001) are not without precedent. Notable systems for far-rangedtransient motion following dissociative chemisorption include O2 on Ag(001),71 in which intrapairdistances were found to be either 2 or 4 nm, and Cl2 onTiO2(110),72 which demonstratesan average intrapair distance of 2.6 nm. A “cannonball mechanism”was proposed for Cl2/TiO2 in which one atomis ballistically propelled from the surface, thereby overcoming surfacecorrugation to explain the large distances observed; this ballisticmotion that avoids corrugation in the potential energy surface couldsimilarly explain the large intrapair distances observed for N2/Ru(0001) despite large barriers to diffusion and providesdirection for future computational work. However, hundreds of identifiedNad pairs identified in this study and a lack of lone Natoms discourage promoting an abstractive mechanism where one atomdesorbs entirely from the surface. STM tip-induced dissociation ofsmall molecules on single crystalline surfaces has also yielded productsat nonadjacent binding sites with products from CH2I2/Cu(110),73m-iodopyridine/Cu(110),74 and O2/Ag(110)75 separated by several lattice constants and F and CF2 up to 5.3 nm apart after CF3 dissociates on Cu(110).76 These previously studied model systems demonstratethat relatively large intrapair distances do occur for specific systems.Comparisons to other dissociative chemisorption systemshighlightthe significance of these results. Background dosing of Oab initio density functionaltheory (DFT) calculations by Prof. Hua Guo’s research groupare getting underway. Mechanistically, how these large intrapair distancesare achieved despite a large barrier (0.9–1.1 eV)53 to diffusion poses interesting questions for future computationalmodeling. Does a purely adiabatic picture describe the N2/Ru(0001) system in which a nonthermalized hot adatom diffuses onthe surface until phonons dissipate energy from the adsorbate intothe bulk? Or do the dynamics sample excited neutral and ionic potentialenergy surfaces leading to changed overall dynamics? Beyond identifyingthe average distance between adsorbates, STM images provide a richspatial understanding of the N2/Ru(0001) system.Note that such large distances observed provide important insightinto elucidating the operative energy dissipation channels for a givensystem. Adiabatic processes will contribute dissipation primarilyvia phonon channels, while nonadiabatic processes on excited statesand including ehps when appropriate can account for interesting dynamics.Preliminary results from STM images showed that nitrogen adsorbate pairs were not observedto preferentially align themselves in any direction on the Ru(0001)lattice. We observe that points are isotopically distributed between−90° and 90° in the scatter plot 6 A, and 2 angles and energies found in 2 molecular beam with respect to the Ru(0001)lattice—confirmed by STM imaging of the nitrogen adsorbates77—critical to determining correlations between the incidentangle of N2 and the resulting nitrogen pair spacing, relativeangle, and binding sites.Analysisthus far has focused on the aggregated results of allimpinging N2 impinging 90° and 45° to the Ru(0001)surface isshown in The distribution of adatom pairs following exposureto 1.3 eV Nnot an obvious memory function in which the relativeangle (φ) between nitrogen adsorbates is affected by the non-normalcomponent of momentum of incident N2 impinging at 45°to the surface. Similarly, pairs that were more aligned with the azimuthalincident beam direction  in The scatter plots shown in 2 with 1.3 eV total translationalkinetic energy impinging at both 45° and 90° to the surface,and average intrapair distances resulting from 1.3 eV impinging normal and impinging at 45° are similar as well.This result indicates that under these conditions, momentum alongthe azimuthal direction of the impinging N2 is not stronglycoupled into motion of one Nad along the Ru(0001) surfacefollowing dissociation. Results summarized in 2 impinging at non-normal angles does not resultin an obvious memory function for adsorbate spacings or angular distributions.These results are made more compelling by the fact that the angledN2 exposure  was performed below roomtemperature (262 K) so that any potential random isotropic motionfollowing dissociation would be frozen out.Analogously, 2 impinging 45° to the surfacenormal did not demonstrate a markedly different trend in adsorbatedistributions on the surface from other room temperature runs surface with 1.1 eV N2 impinging at 45° did not producemarkedly different results from 1.1 eV impinging at 90° to thesurface—in either the spacing or the relative angle of thepaired nitrogen atoms.  surfaceto 262 K duringexposure to 1.3 eV Nure runs 8 and 9, 2 activated dissociative adsorption on Ru(0001).STM visualization of individual dissociation events identifies thesite and location of the resulting nitrogen adsorbates with angstrom-levelprecision. The spacing of the adsorbates correlates to the energytransfer mechanisms involved in the dissociation process and willhelp uncover the importance of nonadiabatic excitation of ehps inthese reactive events. Paired with theoretical insight from ongoingcollaborations, these previously inaccessible experimental resultswill help to answer many of the unsolved questions about this unusualactivated dissociative adsorption system.The results of this paper represent deep insight into the fundamentaldynamics of N2 from a supersonic molecular beam impinging on a Ru(0001)single crystal. The insights gathered herein are only possible dueto the prescient pairing of a supersonic molecular beam of highlyenergetic molecules and the angstrom-level visualization of an in situ in-line STM to provide a rich spatial understandingof this highly activated dissociative adsorption system. Our workuniquely characterizes single adsorption events to capture the reactivedynamics of this industrially relevant process. We present distributionsof nitrogen adsorbate pairs resulting from highly energetic N2 impinging onto Ru(0001), resulting in an average spacingof 6.6 ± 2.8 nm between previously bonded nitrogen atoms. Thesedistances are more than 20 times the lattice constant of Ru(0001)and pose a novel opportunity for theoretical formulations to investigatethe importance of electronic friction, adiabatic phonon coupling,multielectronic surface dynamics, and ballistic motion in energy dissipationfollowing activated dissociation events. When discussing the effectof N2 impinging energy and angle on the reactivity in alow coverage limit, our preliminary results suggest there is not anobvious memory function for incident momenta/kinetic energy of impingingN2 on the distance between resulting nitrogen adsorbates.Additionally, the non-normal component of impinging N2 momentumdoes not impact spatial distributions on the surface. Our resultsprovide direct experimental insight into the energy transfer mechanismsand detailed aspects of the dissociation process and help addressimportant and unresolved questions surrounding the fundamentally andtechnologically important activated dissociative adsorption processof N2 dissociation on Ru(0001).We present STM images depicting the productsof individual dissociationevents following nonequilibrium fluxes of energy- and angle-selectedN


Sox9 expression pattern that translates into the cartilage rings has remained elusive. Here, we review the molecular regulatory interactions that have been elucidated, and discuss possible patterning mechanisms. Understanding the principles of self-organisation is important, both to define biomedical interventions and to enable tissue engineering.The trachea is a long tube that enables air passage between the larynx and the bronchi. C-shaped cartilage rings on the ventral side stabilise the structure. On its esophagus-facing dorsal side, deformable smooth muscle facilitates the passage of food in the esophagus. While the symmetry break along the dorsal-ventral axis is well understood, the molecular mechanism that results in the periodic  The trachea is a long , almost cylindrical tube that serves as a passage of air to the bronchial system Nkx2.1-expressing lung field on the ventral side of the mouse foregut Bmp4) expression is restricted to the ventral foregut from early stages (E8.5) Sox)2 expression in the ventral foregut Efnb2, which establishes an EPH/EPHRIN boundary that results in the physical separation of tracheal and esophageal cells Nkx2.1 null mice, and endodermal mutants for the BMP type I receptor genes Bmpr1a and Bmpr1b upregulate Sox2 and form a continuous ring of smooth muscle and no cartilage rings Bmp4 from the foregut endoderm from E8.5 and from the mesenchyme by E9.5 does not prevent the ventral expression of Nkx2.1 at E9.5, but by E12.5 Nkx2.1 is absent, and expression of the cartilage marker Collagen Type II Alpha 1 Chain (Col2a1) is not observed The separation of cartilage and smooth muscles domains follows the already established dorsal-ventral polarity. Fibroblastic growth factor (FGF) from the cardiac mesoderm induces the Nkx2.1 expression restricted to the ventral side, and Sox2 and Sonic Hedgehog (Shh) expression higher on the dorsal side (Noggin null) that fail to split the tubes Sox9 as early as E10.5 Acta2, a smooth muscle marker Sox9 or a key smooth muscle gene does not alter the expression domain of the other in the trachea Wls conditional mutants blocks Sox9 expression and results in smooth muscle formation also on the ventral side β-catenin in either the epithelium (Shh-Cre driven) or mesenchyme (Dermo1-Cre driven) results in loss of mesenchymal expression of the chondrogenic factor Tbx4Shh null mice, the ventral restriction of Sox9 expression is lost, and until E13.5, Sox9 is transiently weakly expressed in a circumferential expression pattern on both the dorsal and ventral sides Shh is expressed more strongly dorsally, overexpression of Shh does not affect the relative cartilage and smooth muscle domains Sox9 and Bmp4 expression around the entire tracheal epithelium Once the trachea has split from the future esophagus, it maintains the dorsal-ventral polarity, with sal side . This poIn summary, the separation of the smooth muscle and cartilage domains along the dorsal-ventral axis is controlled by the already existing embryonic dorsal-ventral polarity. The dorsal-ventral polarity is first induced along the epithelial tube, and later translated to the mesenchyme via diffusible morphogens.Sox9 and type II collagen (Col2a1) expression Col2a1-expressing cells do not transdifferentiate into non-cartilage cells Col2a1-expressing cells condense in the cartilage rings, and the intervening space becomes filled by other mesenchymal cells. The Col2a1 gene encodes the pro-alpha1 (II) chain component of type II collagen, which is primarily found in cartilage. At E11.5, collagen type II is restricted to the lamina propria on the ventral side of the trachea Ptch1 appears to be restricted to the nascent cartilage condensations from E13.5 Shh assumes a periodic pattern on the ventral, but not on the dorsal side of the tracheal epithelial tube Tbx5 disappears from the cartilage condensations Fgf10 expression becomes restricted in between the nascent cartilage condensations, but its receptor Fgfr2b remains uniformly expressed in the epithelial tube Bmp4 emerges.The positions of the future cartilage rings in the ventral tracheal mesenchyme first become apparent between embryonic day (E)12.75 and E13 as periodic patterns in Sox9/Col2a1 expression patterns or tracheal cartilage ring formation, even though the trachea forms with correct dorsal-ventral polarity. This analysis thus necessarily excludes potential core components that are involved also in processes upstream of periodic pattern formation as their contribution to periodic patterning cannot be evaluated by this approach. The following mouse mutants have so far been reported that lack tracheal cartilage rings, even though the trachea forms with correct dorsal-ventral polarity: Shh null Sox9 null Mek1/Mek2 removal Wls removal Rspo2Tg/Tg;Lrp6−/−) tracheal rings were absent on the shortened tracheal structure Dermo1-Cre-driven conditional removal of β-catenin, a core component of canonical WNT signalling, result in loss of mesodermal Tbx4, impaired mesenchymal growth, and lack of cartilage rings at E16.5 Fgfr2b and Fgf10 null mice have a different phenotype from that reported for human FGFR2 (S351C) in that they develop shorter tracheas with 6–8 distorted cartilage rings FgfR2b in FgfR2c heterozygous mouse mutants results in overgrowth of the tracheal rings and absence of noncartilaginous mesenchyme Functional genetics can help to identify the components of the core mechanism as their null mutations should result in the loss of cartilage rings. In the following, we will focus on mutants that do not show any periodic Sox9 and Bmp4 expression in Shh null lung explants, but cartilage formation is then no longer restricted to the ventral side; it has not been reported whether periodic patterns are obtained Bmp4 conditional mutants, Nkx2.1 is restricted to the ventral side at E9.5, but is lost by E12.5, and no Col2a1 expression and cartilage ring formation is observed Bmpr1b and Sox2, and a SHH-driven endodermal conditional knockout of Bmpr1a develop a ventral NKX2.1 domain that forms disorganized isolated cartilage pieces/nodules, but not rings at later stages Bmpr1a and Bmpr1b has so far not been reported.BMP4 and its antagonist NOGGIN can both rescue cartilage formation as well as While perturbations in many other pathways affect tracheal ring formation or tracheal growth, no other pathway has been described that is necessary for cartilage ring formation once the tracheal mesenchyme has emerged Sox9 knockout mice Sox9 removal is stopped at E13.5, then some cartilage nodules are observed by E18.5 in the most proximal part Sox9 expression Sox9 expression appears strongly reduced or absent in Rspo2Tg/Tg mutant tracheal mesenchyme β-catenin in the mesenchyme of the developing trachea to influence expression of chondrogenic factors including Tbx4, Tbx5, Msx1, Msx2, Sox9, and Col2a1Wnt5a and its receptor Ror2, and ablation of Wnt5a or its receptor Ror2 results in shorter trachea with fewer cartilage rings Wnt7b, expressed by the respiratory epithelium and known to mediate Wnt/β-catenin signaling, does not affect trachea length or width, but results in incomplete cartilaginous rings Wnt4 does not affect tracheal length, but results in 12 distorted rather than 14 tracheal rings, and results in reduced Sox9 and increased Fgf10 expression at 13.5 SOX9 controls all steps of the cartilage differentiation process, and is a necessary factor for cartilage ring formation such that cartilage rings are absent in mesenchymal Col2a1 expression , while regulating SOX9 transcription factor activity via pSMAD1/5/8 and p38 Sox9 and Col2a expression and cartilage formation Despite its importance for cartilage ring formation, the upstream regulators of the MEK/ERK cascasde have remained elusive. FGFs signal via ERK, and overexpression of Interestingly, upon conditional removal of Myorcardin, the cartilage rings fail to expand towards the dorsal side, and the trachael lumen is reduced Sox9 expression, and SOX9 is essential for cartilage formation. Sox9 is still expressed weakly in Shh and mesenchymal Mek1/Mek2 mutants, but fails to organise into rings. As such, SHH and MEK1/2 are either part of the core mechanism that results in periodic Sox9 patterning, or periodic patterning fails because Sox9 expression is too weak in those mutants. Myocardin, a master regulatory of smooth muscle differentiation, is necessary for the dorsal expansion of the nascent cartilage rings to their characteristic C-shape. But what leads to the periodic Sox9 pattern?In summary, WNT signalling  is essential for mesenchymal Drosophila blastoderm, which were initially accounted to a Turing mechanism, but have since been shown to result from cross-repressive transcription factor cascades downstream of opposing morphogen gradients β and either the extracellular matrix (ECM) or TGF-β antagonists β-catenin appears necessary also for tracheal Sox9 expression Bmpr1a and Bmpr1b are not known. Unlike in lung branching morphogenesis Fgf10 mice, if delayed and less uniformly shaped compared to the wildtype Shh expression levels Fgf10 null mice Shh expression. Apart from chemical signalling, cell-cell interactions can also result in Turing instabilities Col2a1-expressing cells EEpi and thickness h relative to a thick, incompressible substrate with modulus EMes results in buckling with wavelength λ, and epithelial thickness, h, that is observed in the trachea EEpi, and mesenchyme, EMes, would need to be similar. However, even if the epithelial folds arise from epithelial buckling, they may well be a consequence rather than a driver of mesenchymal condensations. After all, mesenchymal condensations reduce spatial expansion. Given this wide range of possibilities, more quantitative experimental studies and mathematical modelling are required to delineate the mechanism by which the cartilage rings form.A wide range of chemical and/or mechanical instabilities can result in biological pattern formation. The Swift-Hohenberg equation has been shown to recapitulate the complex tracheal cartilage pattern also at the tracheobranchial juncture, if coupled with a gradient to achieve the correct stripe orientation Despite the simplicity of the pattern and the importance of the structure, tracheal cartilage ring formation remains poorly understood. Conditional mutants in combination with explant cultures, organoids, quantitative imaging, and mathematical modelling may help to unravel this patterning mechanism.


The corrected mCherry and Gad1, Gad2 mRNA in the VTA and SN in cells with and without active Pax5 expression (Pax5 expression also originate from Pax5-expressing progenitor cells.” The errors appear in print and in PDFs downloaded before December 1, 2022.The authors regret that in the original version of pression , demonst


Defects in the iridocorneal angle tissues, including the trabecular meshwork (TM) and Schlemm's canal (SC), impair aqueous humor flow and increase the intraocular pressure (IOP), eventually resulting in glaucoma. Activation of endothelial tyrosine kinase receptor Tie2 by angiopoietin-1 (Angpt1) has been demonstrated to be essential for SC formation, but roles of the other two Tie2 ligands, Angpt2 and Angpt4, have been controversial or not yet characterized, respectively.Angpt2 and Angpt4 and tamoxifen-inducible deletion of Angpt1 in mice were used to study the effects of Angpt4 deletion alone and in combination with the other angiopoietins. SC morphology was examined with immunofluorescent staining. IOP measurements, electron microscopy, and histologic evaluation were used to study glaucomatous changes.Angpt4 expression was investigated using genetic cell fate mapping and reporter mice. Congenital deletion of Angpt4 was postnatally expressed in the TM. While Angpt4 deletion alone did not affect SC and Angpt4 deletion did not aggravate Angpt1 deletion phenotype, absence of Angpt4 combined with Angpt2 deletion had detrimental effects on SC morphology in adult mice. Consequently, Angpt2−/−;Angpt4−/− mice displayed glaucomatous changes in the eye. Mice with Angpt2 deletion alone showed only moderate SC defects, but Angpt2 was necessary for proper limbal vasculature development. Mechanistically, analysis of Tie2 phosphorylation suggested that Angpt2 and Angpt4 cooperate as agonistic Tie2 ligands in maintaining SC integrity.Our results indicated an additive effect of Angpt4 in SC maintenance and Tie2 activation and a spatiotemporally regulated interplay between the angiopoietins in the mouse iridocorneal angle. Impaired AH outflow increases the intraocular pressure (IOP), which is a significant risk factor for glaucoma, a heterogenous group of ocular diseases that are the major cause of irreversible blindness worldwide.,,,,––The main exit route for AH, produced by the ciliary processes, drains from the anterior chamber via the trabecular meshwork (TM) to Schlemm's canal (SC) and from there to the aqueous and episcleral veins.–The vascular angiopoietin (Angpt)/Tie signaling pathway consists of three ligands, Angpt1, Angpt2, and Angpt4; the primary Angpt receptor tyrosine kinase Tie2; and an orphan regulatory receptor Tie1.Tie2 deletion as well as Angpt1;Angpt2 double deletion in mice lead to complete absence of SC, highly elevated IOP, and severe glaucoma.,–Tie2 heterozygosity or Angpt1 deletion is sufficient to partially degenerate SC in mice,,,–TIE2 and ANGPT1 loss-of-function mutations who have been diagnosed with glaucoma.,,TIE2 and ANGPT1 variants have been associated with increased IOP and glaucoma in genome-wide association studies (GWASs).–,Compelling evidence from recent years shows that Tie2 activation is essential for both SC development and its maintenance. Angpt2 deletion mice, and ANGPT2 variant loci have been associated with increased IOP in glaucoma patient GWAS analyses.–Angpt2 deletion mice, and administration of Angpt2-blocking antibody has been reported to have no effect on IOP in monkeys or on SC area in mice.However, among the Angpt ligands, the importance of Angpt4 in the iridocorneal angle has not been investigated, and the individual role of Angpt2 has remained somewhat controversial. Kim et al.Angpt4 deletion alone did not affect the SC, its deletion combined with Angpt2 deletion had detrimental effects on both SC morphology and AH outflow, causing glaucomatous changes in the eye. Furthermore, being the first to use congenital Angpt2 deletion in this context, we show that Angpt2 is indeed necessary for normal SC morphology and also for the corneolimbal vasculature. Collectively, our data establish a novel role for Angpt4 in the iridocorneal angle and reveal cooperation between Angpt2 and Angpt4 necessary for full Tie2 activation and SC maintenance.Here, we used genetic mouse models to study the importance of Angpt4 in AH drainage. We show that Angpt4 expression is spatiotemporally distinct form the other Angpts in the iridocorneal angle, and while Angpt2 deletion mice were generated by CRISPR/Cas9-based genome editing.–All animal experiments were performed with the approval of the Finnish Project Authorization Board following national and European Union (EU)–level legislation (EU directive 2010/63/EU) and adhering to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Angpt1 allele (referred as Angpt1fl throughout the text) was ordered from The Jackson Laboratory  under a strain name Angpt1tm2.1Sjm/J (#028925).Rosa26CreERT2 mice expressing tamoxifen-inducible Cre under ubiquitous Rosa26 promoter were kindly provided by the GIE-CERBM . Generation of the Angpt4 knockout allele  has been described previously.Angpt1fl/fl;Angpt4−/−;Rosa26CreERT2 mouse line, and the mice were administered tamoxifen  by oral gavaging starting from 4 weeks of age and continued at a 1-week interval until 8 weeks of age to delete Angpt1 and create an Angpt1del;Angpt4−/− mouse line. Littermates received the same tamoxifen administrations and were used as controls. Angpt4Cre/+;Rosa26mTmG/+ cell fate mapping and Angpt4LacZ reporter mice have been introduced before.,Floxed ,,Primers used for genotyping the herein introduced mouse lines are presented in the Detailed protocols for immunofluorescence whole-mount and section stainings, X-gal staining, hematoxylin and eosin staining, confocal microscopy, transmission electron microscopy, all image analyses, single-cell RNA sequencing (scRNAseq) data analysis, and quantitative PCR (qPCR) are given in the ,,,,IOP was measured using a Tonolab rebound tonometer . Mice were accustomed to handling prior to the measurements. While measuring (consistently around noon), mice were restrained by an experienced animal caretaker, and IOP values were obtained from two to three sets of six recordings, which were finally averaged from both eyes to obtain a single value for each mouse.t-test was used for comparing two groups after confirming normal distribution and equal variance. Statistical significances are marked in figures as following: *P < 0.05, **P < 0.01, and ***P < 0.001, and all figures represent individual data points and mean ± SD.Normal distribution of the data was confirmed with the Shapiro–Wilk test for normality performed in Prism 9 software , and equal variance was tested with Levene's test for homogeneity of variance using Origin Pro software . When both assumptions were met, comparisons between multiple groups were performed with one-way ANOVA followed by the Tukey post hoc test, and when variances were unequal (indicated in the figure legends), Welch's ANOVA followed by Dunnett's T3 multiple comparisons test was performed using Prism 9 software. Unpaired two-tailed Student's Rosa26mTmG with Angpt4Cre mice,,Angpt4 promoter by Cre-mediated conversion of continuous tdTomato expression to membrane-targeted green fluorescence protein (GFP) expression. We observed that the Angpt4-driven GFP signal started postnatally: at P5  and P10, flat-mounted corneal limbus samples had only very few, faint GFP+ cells, while at P13, their number and signal intensity were beginning to multiply  directly adjacent to the SC inner wall, as well as into the uveal and corneoscleral meshworks next to the anterior chamber , and Ptprb (protein tyrosine phosphatase receptor type B encoding VE-PTP) were all expressed in the Angpt4-negative vascular and SC endothelial cells  and were thereafter downregulated, in line with previously reported results.,Angpt2 expression was downregulated more gradually than Angpt1  was used to determine an individual expression profile over time for the and Tie2 E–H. Tie2n Angpt1 F–G whileh of age H. This wCollectively, spatiotemporal gene expression data from the cell fate tracing and reporter mice, scRNAseq, and qPCR analyses indicated that Angpt4 is expressed at relatively low levels in the TM cells close to the SC. In our analyses, Angpt4 was not enriched during the postnatal SC development and was partly produced in the same cell types than Angpt1 in adult mice, and the cellular sources of Angpt4 were markedly different from those of Angpt2.Angpt4 deletion on the SC. Since prominent Angpt4 expression appeared postnatally at a time point when the SC is already mostly developed ,–,,+ SC area in 1-year-old Angpt4Cre/Cre (hereafter Angpt4−/−) mice , existence of circulating limbal arteries and veins used as a measure of complexity of the limbal vasculature,+ corneolimbal LV area were changed in any of the genotypes  mice A, 2B, inf 1 year A. qPCR cficiency B. As exp2 allele C, S4D, benotypes C, 4E–G. deletion H. Hence,ANGPT2 has been identified as a risk locus associated with glaucoma,–Angpt2 deletion model has been used in any of the previous mouse studies.,,Angpt2 allele (Angpt2−/− mice), we utilized the CRISPR/Cas9 methodology to induce a 35-bp deletion in the Angpt2 exon 1. This resulted in a codon frameshift and decreased Angpt2 mRNA expression, and Angpt2 antibody staining confirmed the absence of Angpt2 protein , and no circulating veins were found when 8 Angpt2−/− and 5 Angpt2−/−;Angpt4−/− samples were analyzed .To investigate the possible Tie2-dependent mechanism of cooperative action of Angpt2 and Angpt4 on the SC, we performed antibody staining using phospho-Tie2 (pTie2) antibody that has previously been used to indicate Tie2 activation state in the SC.n the SC A, 5B. Sirylation A, 5B, budeletion A, 5B. WhAngpt2 deletion by comparing Angpt2+/− and Angpt2+/−;Angpt4−/− mice. While at 12 weeks of age, these mice did not have major differences in the SC morphology , or corneolimbal LVs were seen . Despite the reduction in SC area, aged Angpt2+/−;Angpt4−/− mice did not display major signs of glaucoma ,Angpt1 is deleted at 8 weeks of age.Angpt1 deletion was induced at 4 weeks of age. This was sufficient to cause a hypomorphic SC and an increase in IOP, but the defects were not as severe as in the abovementioned earlier time-point deletions by others (25% decrease in SC area in our model). Altogether, these studies indicate that SC maintenance is less dependent on Angpt1 and that other Tie2 ligands may be more important for that. In this regard, it was interesting to find that both Angpt1 (our data and previously reported),,+ JCT cells, 10.5% simultaneously expressed Angpt4, being 50% of Angpt4+ JCT cells). The lack of any SC defect in the Angpt1fl/fl;Angpt4Cre/Cre mice, in which Angpt4 promoter–driven Cre expression (starting at P10) would prevent Angpt1 expression from the floxed allele if occurring in the same cells, also supports this observation.An outstanding question regarding the Angpt/Tie2-dependent development and maintenance of the SC is the evident presence of three Angpt ligands to activate the Tie2 receptor in the same vascular structure. According to our data presented in this study and the results of others, Angpt1 is highly expressed at the time of SC development and at lower levels later in life,Angpt4 deletion exacerbated the phenotype of Angpt2 deletion but not Angpt1 deletion might be explained by the result that Angpt2 and Angpt4 are produced in different cellular compartments, making their combination deletion more harmful and difficult to compensate by the remaining ligand Angpt1. In addition, as Angpt1 is the high-affinity and strongly Tie2-phosphorylating agonistic ligand, combination deletion with Angpt4, which is a weaker ligandIn contrast to Angpt1 and Angpt4, Angpt2 was in our studies rather steadily present throughout the lifetime in almost all iridocorneal angle structures, but Angpt2 expression was detected only in the JCT but not in the other TM compartments. We showed that Angpt2 is expressed and essential in the limbal blood, lymphatic, and SC endothelium, suggesting that Angpt2 may also activate Tie2 in an autocrine manner, thereby mechanistically differing from the other Angpt ligands in the iridocorneal angle vascular structures. The notion that Angpt2 deficiency on AH drainage have been conflicting, either showing no effect at all,Angpt1 deficiency.,,Angpt2 deletion alone is sufficient to cause defects in the SC in mice, an observation that goes hand in hand with GWAS reports associating ANGPT2 genetic variants with patients who have increased IOP and glaucoma.–Angpt2−/− mice are also known to have abnormal retinal vasculature with sprouting defects and persistent hyaloid vessels,,,Angpt4−/− mice have impaired retinal vein development, leading to inner nuclear layer swelling.Angpt2−/−;Angpt4−/− mice.So far, reports of the effect of Angpt2−/−;Angpt4−/− mice than in the Angpt2−/− mice, correlating with the slightly diminished Tie2 phosphorylation state in the SC endothelium. Strikingly, also Angpt2+/−;Angpt4−/− mice, unlike Angpt2+/− or Angpt4−/− mice, had a decreased SC area and Tie2 phosphorylation state, providing further evidence of Angpt4 as a regulator of the SC. The herein proposed model of Angpt2 and Angpt4 acting as Tie2 agonists in the SC endothelium correlates with the established role of Angpt2 as a Tie2 agonist in the lymphatic endothelium,Angpt2 deletion dramatically affected certain areas while other portions were unaffected, suggesting that local changes in the availability of the relatively weaker Tie2 ligands Angpt2 and Angpt4 could also fine-tune Tie2 activation state, providing homeostatic support for the SC after its developmental phase mainly mediated by the strongest Tie2 ligand Angpt1. Considering previous publications,,Angpt2−/−;Angpt4−/− mice, it can be concluded that Tie2-dependent AH homeostasis is dependent on a sufficiently high amount of Angpt ligands.Our data intriguingly showed that certain SC phenotypes were more severe in the double-deficient Angpt2+/−;Angpt4−/− mice, the SC was continuously slightly narrower than in WT, Angpt2+/−, or Angpt4−/− mice, but it did not have as much such extremely narrow convolutions as the SC in Angpt2−/− and Angpt2−/−;Angpt4−/− mice had, suggesting that Angpt2 is essential for adequate SC morphogenesis, but also Angpt4 contributes to the maintenance of optimal width of the mature canal. Despite the reduction in total SC area and pTie2/Tie2 ratio comparable to Angpt2−/− mice, Angpt2+/−;Angpt4−/− mice did not have elevated IOP or significant RGC damage. Likely, the numerous SC narrowing points and convolutions seen in Angpt2−/− and Angpt2−/−;Angpt4−/− mice are obstructive and inhibit the proper flow of AH in the SC more than a steadily narrow but otherwise normal SC does.Intriguingly, our data also revealed qualitative differences between the genotypes investigated. In Tie1 and Tie2 and double Angpt1;Angpt2 deletion mice,,,,Angpt1del mice did not show any apparent defects in the limbal vasculature maintenance, and limbal BV defects have not been reported in the early Angpt1 deletion models by others, implying that specifically, Angpt2/Tie/Svep1 interplay would be needed in the corneolimbal vasculature. We also found that Angpt2−/− mice completely lacked corneolimbal lymphatics, in line with published data on early postnatal deletion of Angpt2 or Tie2 resulting in significant but not complete absence of corneolimbal LVs,Angpt1;Angpt2 deletion mice have also been reported to lack corneolimbal LVs,Angpt1 deletion alone has only minor–Angpt2−/−;Angpt4−/−) or presence (Angpt1del) of both corneolimbal BVs and LVs had no obvious additional effects on IOP in models with comparable reduction in SC area.As another novel finding, we showed that Angpt2 is required for the formation of proper corneolimbal blood vasculature . Interestingly, defects in the limbal vasculature have also been described in ,In conclusion, this is the first study to indicate a role for Angpt4 in the SC maintenance. In addition, we provide evidence of Angpt2 as an essential SC and corneolimbal vasculature regulator. All three angiopoietins, Angpt1, Angpt2, and Angpt4, seem to be derived from varying cellular sources in the iridocorneal angle. Previous studies comparing the primary structures of human and mouse Angpt1, Angpt2, and Angpt4 have revealed that Angpt4 is the least conserved ligand among the angiopoietins.


Computational clusters were further validated by immunohistochemistry with accuracy in a small subset of neurons. Thus, unbiased cluster analysis using electrophysiological properties is a tool that can enhance current interneuronal subclassifications and can complement groupings based on transcription factor and molecular expression.Spinal cord neurons integrate sensory and descending information to produce motor output. The expression of transcription factors has been used to dissect out the neuronal components of circuits underlying behaviors. However, most of the canonical populations of interneurons are heterogeneous and require additional criteria to determine functional subpopulations. Neurons expressing the transcription factor Shox2 can be subclassified based on the co-expression of the transcription factor Chx10 and each subpopulation is proposed to have a distinct connectivity and different role in locomotion. Adult Shox2 neurons have recently been shown to be diverse based on their firing properties. Here, in order to subclassify adult mouse Shox2 neurons, we performed multiple analyses of data collected from whole-cell patch clamp recordings of visually-identified Shox2 neurons from lumbar spinal slices. A smaller set of Chx10 neurons was included in the analyses for validation. We performed k-means and hierarchical unbiased clustering approaches, considering electrophysiological variables. Unlike the categorizations by firing type, the clusters displayed electrophysiological properties that could differentiate between clusters of Shox2 neurons. The presence of clusters consisting exclusively of Shox2 neurons in both clustering techniques suggests that it is possible to distinguish Shox2 The spinal neuronal circuitry participates in the control of a wide variety of movements, ranging from reflexes to highly sophisticated motor skills  transgenic mice  was sectioned transversely using a vibrating microtome (Leica Microsystems). Slices were next transferred to an artificial cerebrospinal fluid, containing the following (in mM): 111 NaCl, 3 KCl, 11 glucose, 25 NaHCO3, 1.3 MgSO4, 1.1 KH2PO4, and 2.5 CaCl2 at 37°C for 30 min and then passively equilibrated to room temperature for another 30 min before recording. Dissecting and recording solutions were continuously aerated with 95%/5% O2/CO2.For terminal electrophysiology experiments, adult mice  were anesthetized with ketamine (150 mg/kg) and xylazine (15 mg/kg), decapitated, and eviscerated. Spinal cords were then removed in ice-cold dissecting solution containing the following (in mM): 222 glycerol, 3 KCl, 11 glucose, 25 NaHCO2, 1 glucose, 4 NaCl, 5 ATP, and 0.3 GTP, with pH adjusted to 7.4. In some experiments, biocytin  was included in the patch electrode. Data were collected with a Multiclamp 700B amplifier (Molecular Devices) and Clampex software . Signals were digitized at 20 kHz and filtered at 10 kHz.All recordings were performed at room temperature. Fluorescently labeled (tdTomato) Shox2 and (eGFP) Chx10 neurons were visualized with a 63x objective lens on a BX51WI scope (Olympus) using LED illumination (Andor Mosaic System or Lumen Dynamics X-Cite 120 LED). Patch electrodes were pulled to tip resistances of 5–8 MΩ using a multistage puller (Sutter Instruments) and were filled with intracellular solution, which contained the following (in mM): 128 K-gluconate, 10 HEPES, 0.0001 CaClm) was recorded shortly after entering whole-cell mode. Input resistance (Rin) was calculated from the current/voltage slope in response to hyperpolarizing voltage steps in which no voltage-gated current activation was evident. Membrane time constant (τ) was calculated as the time to reach 63% of the maximum depolarization in response to a subthreshold depolarizing current step. Membrane capacitance (Cm) was calculated from the time constant and input resistance (Cm = Rin/τ). Rheobase was the minimal current step required to generate an action potential, applied in intervals of 2–3pA. Additionally, we recorded action potential (AP) properties from the first AP observed in the depolarizing current at rheobase  and MATLAB. All results are presented as mean ± SD. Statistical significance was set at p < 0.05 unless otherwise stated. The distribution of the data was determined by Shapiro–Wilk normality test. The statistical comparisons between Shox2 and Chx10 neurons were performed by Mann-Whitney test or unpaired t-test. Comparisons between clusters were performed by Kruskal-Wallis with Dunn's post-hoc test or repeated-measures one-way ANOVA with Bonferroni post-hoc test. Comparisons between percentages were performed by chi-square test.Data analysis was performed with Clampfit (Molecular Devices) and MATLAB (MathWorks). Statistical tests and p < 0.01 to be correlated. Principal component analysis (PCA) and multidimensional cluster analyses were performed on the 6 parameters obtained that were not highly correlated. PCA was used to reduce dimensionality of the number of variables recorded  Hierarchical clusters were determined by the cosine distance between pairs of observations and consideration of an average for the distance between clusters. This combination was used because it resulted in the highest cophenetic correlation coefficient. The number of clusters was determined considering the cutoff below the maximum inconsistency coefficient for each link. For the k-means and hierarchical cluster analyses, we applied MATLAB algorithms, initially standardizing the data to set a mean of 0 and standard deviation of 1 to be able to compare variables with different units.To determine correlations in between the 12 properties obtained for each cell, we performed a Pearson's linear correlation test and considered values of recorded . To visuThe slices containing biocytin-filled neurons were fixed overnight , and subsequently placed in PBS at 4°C. To visualize biocytin, the slices were incubated for 2 h at room temperature in DyLight 633 conjugated streptavidin . Then, the slices were washed in PBS (3 x 10 min), permeated with 1% Bovine Serum Albumin (BSA), 5% Donkey Serum (NGS), 0.1% Fish Gelatin, and 0.2% Triton x-100 for 1 h and incubated in sheep anti-Chx10 antibody 1:100  at room temperature for 48 hrs. Slices were washed in PBS (3 x 10 min) and incubated for 2 h at room temperature with rabbit anti-sheep Dylight 488 1:400  followed by a final wash in PBS (3 x 10 min). Slices were then placed on slides within tissue spacers and coverslipped with a Vectashield mounting medium (Vector labs). Sections were scanned using a laser scanning confocal microscope  in stacks of 10 optical sections across approximately 50 μm at 20x magnification. Images were condensed into maximum projections using the Leica collection software and brightness and contrast were adjusted in ImageJ.p < 0.001) between some passive and active cellular properties (m), capacitance (Cm), fast afterhyperpolarization (fAHP) duration, fAHP amplitude, activation voltage of the persistent inward current (PICon) and frequency-current (F/I) slope  slope . To visuI) slope . The firn = 143) with those of Chx10 neurons (n = 28) (in), larger capacitance (Cm), shorter AP half width, shorter fAHP duration, and more depolarized PIC activation voltages (PIC on) than Chx10 neurons. Resting membrane potential, time constant, rheobase, action potential threshold, the sAHP duration, fAHP amplitude, and F/I slope were similar between the groups. This comparison shows that Shox2 neurons and Chx10 neurons share some electrophysiological characteristics, however, differences are observed between subpopulations.Graphical display of the three first PCAs shows that Shox2 and Chx10 neurons partially overlap. We compared the initial 12 electrophysiological properties of adult Shox2 neurons ((n = 28) . Shox2 nn = 88, 51.5%). Initial doublet neurons  fire a doublet of action potentials at the start of the step  and continue firing throughout the step but at a lower frequency (steady frequency of 3.9 ± 5.1 Hz). Neurons with burst of action potentials at the start of the current step are called initial burst firing neurons . These neurons displayed three or more initial spikes at high frequency (>25 Hz) and were either silent or fired action potentials later in the step but these action potentials were not a regular frequency like the tonic neurons. Lastly, a small number of neurons, delay neurons  fired action potentials after a delay from the beginning of the current step. We next looked at the prevalence of firing types in Shox2 and Chx10 neuronal populations. Of the 143 Shox2 neurons, tonic firing neurons were most common , followed by initial doublet  and initial burst  firing, with delayed firing neurons  being rare , over one third were tonic firing , and few neurons fired with an initial burst  or were delayed firing . Chx10 neurons preferentially displayed initial doublet firing and Shox2 neurons were mostly tonic firing.To identify differences in active and passive cellular properties based on firing type, we classified the 171 Shox2 and Chx10 neurons based on the response to suprathreshold depolarizing current steps. We found four types of responses . NeuronsTo identify the electrophysiological characteristics that corresponded to the different firing types, we performed statistical analyses on both groups considered as a single population , higher capacitance , shorter AP half width , and shorter fast  but longer slow  AHPs. We did not find differences between Shox2 and Chx10 neurons with initial doublet firing patterns. The numbers of delayed and initial burst firing Chx10 neurons were low and precluded from statistical analysis. The electrophysiological properties between neurons classified by type of firing were expected to be different since action potential features and spike frequency rely on these properties. Although firing types were differentially distributed in Shox2 and Chx10 populations, it is not possible to separate Shox2 and Chx10 neuronal populations by firing type since the types are common to both.We also compared properties of Shox2 and Chx10 neurons within each firing group . Considek-means cluster analysis (see Methods) on the set of 171 neurons, which included mainly Shox2 neurons and a small sampling of Chx10 neurons to serve as comparison with a known subdivision with the Shox2 population. The cluster analysis considered the six electrophysiological parameters that were not highly correlated, as described above . Chx10 neurons appear more prevalent in k2 cluster than Shox2 neurons, with 14% of Chx10 neurons but only <5% of Shox2 neurons belonging to that cluster, but this is not significant . The proportion of Chx10 neurons in each of the k3 and k4 clusters (39.2% and 39.9%) is similar to that of Shox2 neurons . The silhouette values for each of the 171 neurons in the k-clusters (n = 8) have negative values which indicate that the neuron is close to its cluster centroid but also close to other cluster centroids. Together, this shows that the k-means analysis . We did not find differences in k3 cluster  between Shox2 and Chx10 populations. The k4 also had equal proportions of Shox2 and Chx10 neurons and the only differences between the Shox2 and Chx10 neurons in that cluster were in AP halfwidth  and fast AHP amplitude  = 67, p = 0.04). Notably, there were less differences between Shox2 and Chx10 neurons from individual k clusters than there were differences in the whole populations of Shox2 and Chx10 neurons. This demonstrates similarity within clusters regardless of the transcription factor expressed.We next performed statistical analysis of the k clusters considering Shox2 and Chx10 neurons separately . In k2, The number of clusters in the k-means analysis here was defined by the elbow rule on a graph of the silhouette values  with more neurons in H6 (n = 39). The 28 Chx10 neurons (n = 2–13) but there are no Chx10 neurons in H1 and H2 clusters. The difference in the distribution of the 6 H-clusters was statistically significant when comparing Shox2 and Chx10 populations (p = 0.01). Note that H1 and H2 clusters were composed of Shox2 neurons exclusively and these two clusters were most similar to each other, as seen by the height of the next branch point in the analysis. Although clusters H3-H6 contained both Shox2 and Chx10 neurons, the uneven numbers of Shox2 and Chx10 input neurons (143 vs. 28) should be considered  with depolarized voltage thresholds are characteristic of Shox2+Chx10− neurons.To identify the electrophysiological characteristics of each H-cluster, we performed statistical analysis on these clusters considering all 171 neurons . Neuronsk-means clusters, there were few differences between Shox2 and Chx10 neurons within any of the hierarchical clusters. These were outnumbered by differences between the clusters, suggesting relatively homogeneous populations within each cluster.We also performed statistical analysis of the H clusters considering Shox2 and Chx10 neurons separately . There wk-means and hierarchical algorithms  and 6 Shox2 neurons expressed Chx10 (Shox2+Chx10+). We next determined which clusters each of the 8 neurons belonged to. Considering k clusters (+Chx10− were classified in k1 (1 neuron) and k3 (1 neuron) clusters, while Shox2+Chx10+ neurons were classified in k2 (1 neuron), k3 (2 neurons), and k4 (1 neuron) clusters. This matches rather well with predictions from the clustering results (+Chx10− where classified in H1 (1 neuron) and H2 (1 neuron), the clusters devoid of Chx10 neurons. The Shox2+Chx10+ neurons were in H3 (1 neuron), H4 (2 neurons), H5 (1 neuron) and H6 (2 neurons), all clusters which contain both Shox2 and Chx10 neurons. Thus, these results further support the ability of the H clustering method to distinguish populations of Shox2 neurons.A subset of the Shox2 neurons used for this analysis were recorded with biocytin in the electrode and were recovered for labeling with an antibody to Chx10. In total, we stained 8 neurons to determine Shox2identity . Of thosclusters , we foun results  because  results , we founk-means algorithm was run for 4 populations of neurons while hierarchical clustering analysis defined 6 populations. Interestingly, in each of the two clustering analyses, we found clusters containing exclusively Shox2 neurons, suggesting possible definition of Shox2+Chx10− populations by electrophysiological properties. Finally, as preliminary validation, Chx10 antibody staining of a small subset of biocytin-filled and recovered Shox2 neurons showed that all (8/8) of the filled and post-processed neurons were appropriately found in the expected hierarchical (H) clusters based on Chx10 presence/absence. The k-means clustering corresponded to the expected in 7/8 cases. Taken together, the data demonstrate that it is possible to classify neurons expressing Shox2 in at least six different subpopulations based on active and passive membrane properties. These subpopulations correspond well with Chx10 absence/presence and may provide further divisions between neurons currently defined with intersectional genetics.In the present study, electrophysiological properties were used to define populations of adult spinal Shox2 interneurons. Since Shox2 neurons overlap partly with Chx10-expressing neurons . One may expect that electrophysiological properties are related to transcription factor expression and inferred function. Thus, there would be clusters that contain each of the possible combinations. If this is the case, the distribution of Shox2 and Chx10 neurons in the clusters identified by the k-means and hierarchical algorithms could potentially be used to predict the type of neurons (Shox2+Chx10− or Shox2+Chx10+) that correspond to each cluster. For example, the lack of Chx10 neurons in k1, H1 and H2 clusters suggest that these clusters are composed of Shox2+Chx10− neurons. H5 cluster could also be considered as a putative Shox2+Chx10− cluster, since 93% of H5 cluster is comprised by Shox2 neurons, suggesting higher influence of Shox2 features than Chx10. As k3, k4, H3, H4 and H6 clusters have similar distributions of Shox2 neurons  as the total proportion of Shox2 neurons sampled (83%), we suggest that these clusters are composed of Shox2+Chx10+ neurons. In contrast, Shox2 neurons constitute 64% of the neurons in k2. Even though this percentage is lower than the total distribution of Shox2, this is not significantly different  and therefore we cannot classify them as Shox2−Chx10+ neurons.A main motivation for the analyses was to determine if there was a way to identify subpopulations within a class of interneurons using electrophysiological properties rather than combinatorial genetics. The neurons expressing Shox2 can be divided into 2 subpopulations based on the expression of Chx10, Shox2−Chx10+ neurons. This is most likely because there is a low number of Chx10 neurons in our analysis which resulted in an underrepresentation of the population of Shox2−Chx10+ neurons. Our primary focus here was the division of the Shox2 population and how it would match up with the subgroupings by transcription factor (Chx10) expression. A future analysis may include a more balanced sample of Chx10 neurons, and we expect that would generate clusters consisting of only Chx10 neurons.We do not have any clusters of Shox2k-mean algorithms, previous studies have compared the output of both clusters and then corrected one or both algorithms  and longer fAHP durations in neurons in the k2 cluster (Shox2−Chx10+ enriched), which corresponds well with the proportion of initial doublet firing neurons in each group. Taken together, the computational clustering analysis performed here exposed potential novel differences between Shox2 populations that allow for hypotheses to be made regarding the characteristics of these populations. It is important to note that the electrophysiological recordings were performed at room temperature, as with most recordings of adult locomotor-related neurons in vitro , the Edward Jekkal Muscular Dystrophy Association Fellowship (DG-R), and NIH T32 NS121768 (SS).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


L-valine is one of the essential branched-chain amino acids (BCAAs) required for synthesis of proteins in human body. It promotes muscle growth and tissue repair and is important for immune function. Recent data indicate that BCAAs can activate sirtuins expression and elevate mitochondrial biogenesis and fatty acid oxidation in both adipocytes and myotubes thereby increasing life span. Sirtuins are a conserved family of proteins, play a critical role in maintaining metabolic health by deacetylating many target proteins in numerous tissues, and regulate mitochondrial function and the aging process. Due to multiple effect of sirtuins on aging, we sought to determine whether the addition of valine might enhance sirtuin gene expression. We utilized the C2C12 skeletal muscle cell line grown on physiological normal glucose (100mg/dL) media. The cells were treated with two different concentrations of valine (0.5 and 1.0mM) for different time intervals (18 and 24). Gene expression of sirtuin 1 (SIRT1) and sirtuin 2 (SIRT2) isoforms were determined by RT-PCR. The results showed increased expression of the sirtuin gene isoforms after treatment with valine. Relative expression varies with in different isoforms of SIRT1 (v1 and v2) and SIRT2 . Among all, SIRT1 v1 and SIRT2 v1 showed maximum expression as compared to the other isoforms used in the study. Our study showed that adequate supplementation of L-valine enhanced sirtuin gene expression, which may promote healthy muscles and healthy aging.


Following the publication of this article , the UniThe Fig 6A Control and TM+TUDCA panels  partially overlap.○ Fig 5A GRP78 and NCK1 panels.○ Fig 7A PTP1B and GRP78 panels.Areas indicating splicing and inappropriate image manipulation were detected in the following panels:In addition to these concerns, editorial reassessment of the article raised further concerns with results presented in Figs 5, 6, 8, and 9. Specifically,Additional irregularities were detected in the background of the Fig 5A PTP1B panel.○ The Fig 6A TM+NAC panels  also appear to partially overlap with the respective Fig 6A Control and TM+TUDCA panels .○ Fig 8A, lanes 1–4 of the Cyt. NFκB p65 panel appear similar to lanes 1–4 of the Cyt. GAPDH panel.○ Fig 9C, lane 4 and lane 9 of the Cyt. Lamin A panel appear similar.Additional similarities were detected between and within panels:The underlying data for this article have not been submitted to the journal for editorial review.PLOS ONE Editors retract this article.In light of the concerns affecting multiple figure panels that question the integrity of these data, the SN agreed with the retraction and apologizes for the issues with the article. EP and JR either did not respond directly or could not be reached.


Gene variability related to carcinogen activation and detoxification may interfere with susceptibility to head and neck cancer.GSTT1 and GSTM1 null polymorphisms and the risk of head and neck squamous cell carcinoma in cigarette smokers.To investigate the relation between GSTM1 and GSTT1 null genotype frequencies were evaluated by multiplex PCR in A case-control study conducted at the Sao Jose do Rio Preto Medical School, Brazil. GSTT1 null genotype was found in 33.3% of the Experimental Group and 23.3% of the Control Group (p= 0.311). Experimental and Control Groups had GSTM1 null genotype frequencies of 35% and 48.3% (p=0.582). No association between alcohol consumption and GSTT1 and GSTMI null genotypes was found in these groups . There were more men, and alcohol consumption was prevalent in both groups.The oral cavity was the most prevalent tumor site for squamous cell carcinoma. The GSTM1 and GSTT1 genotypes and the development of head and neck squamous cell carcinomas in cigarette smokers.In this study we were unable to show a correlation between  Head and neck neoplasms are responsible for many deaths worldwide, being the sixth cause of death by câncer.GSTT1 and GSTM1 - that code phase II enzymes belonging to the glutathione S-transferases (GSTs) family seem relevant for susceptibility to head and neck squamous cell carcinoma; they detoxify carcinogenic tobacco smoke reactive metabolites.23Various polymorphic genes that code enzymes involved in carcinogen biotransformation have been associated with cancer development.GSTM1 gene is polymorphic in humans, including a null-activity allele (GSTM1-) due to a major genic deletion, and two functional alleles (GSTM1A and GSTM1B). The GSTT1 gene is also polymorphic in humans, and may have a deletion null genotype.The Thus, individual gene variability in the metabolic activation and detoxification process appears to be crucial to head and neck cancer susceptibility.GSTT1 and GSTM1 gene null genotypes in smokers with head and neck squamous cell carcinoma and to compare these frequencies with those seen in smokers without a history of cancer, to identify possible susceptibility biomarkers for head and neck cancer.This study aimed to identify Individuals diagnosed with squamous cell head and neck carcinomas confirmed by histopathology came from the Department of Otorhinolaryngology and Head and Neck Surgery of the Navy Hospital / Medical School of São José do Rio Preto and the Arnaldo Vieira de Carvalho Institute, SP. The control group included individuals with no history of neoplastic disease, paired by gender, age, ethnic group and use of alcoholic beverages. All subjects (patients and controls) were smokers. Individuals were included in the study after signing a free and informed consent form, and all information was obtained using a confidential standardized data-collection questionnaire . Information on smoking and alcohol-drinking was limited to a definition of user or non-user. The study was approved by the Research Ethics Committee of the Sao Jose do Rio Preto Medical School (CEP-FAMERP - 5639/2002) and the National Research Ethics Council .Genomic DNA was extracted from peripheral blood according to the Abdel-Rahman et al technique.GSTT1 and GSTM1 genes was simultaneously done by the chain reaction (PCR) according to Abdel-Rahman et al.CYP1A1 gene exon 7 sequence was coamplified to serve as an internal amplification control. PCR products were analyzed in agarose gel at 1.5%, stained with ethydium bromide, and the null genotype  for the GSTT1 and GSTM1 genes was identified by the absence of amplification fragments of 480 base pairs (bp) and 219 bp respectively. Presence of the 312 bp fragment corresponded to the amplified CYP1A1 gene sequence, which was evidence of a successful amplification reaction.The analysis of Demographics were presented as mean ± standard deviation (SD) or proportions. For the statistical analysis of the genotype frequencies obtained we used the exact Fisher test, with a significance level below 5%.Demography data: 120 individuals were recruited, of which 60 had head and neck squamous cell carcinoma (average age 54.6 ± 8 years) and 60 had no history of neoplastic disease (average age 54 ± 9 years). Men predominated (90% men vs. 10% women) and there were more alcohol drinkers .Primary sites: all head and neck squamous cell carcinoma cases were diagnosed and confirmed by pathology exams. The distribution of the primary tumor site is shown on GSTT1 null genotype [-] was found in 33.3% (20 of 60) of patients and in 23.3% (14 of 60) of controls (P = 0.311). The GSTM1 null genotype [-] was found in 21 (35%) of patients and 29 (48.3 %) of controls (p = 0.582). The combined GSTT1 and GSTM1 gene null genotype was seen in 10% (6 of 60) of patients and in 8.3% (5 of 60) of controls (p = 1.0). The most frequent genotypic combination, considering the presence of an unfavorable genotype (null GST), was GSTT1 [+] / GSTM1 [-] in 31.6% (19 of 60) of patients and 40% (24 of 60) of controls (p = 0.353) (Frequency of polymorphisms: The = 0.353) .Table 2GGSTT1 and GSTM1 null genotypes.Genotypes were grouped by tumor site  and therGSTT1 and GSTM1 null genotypes [-] when we compared alcohol-drinking patients and controls .Statistical analysis did not reveal any relation between alcohol use and Epidemiological data has suggested that alcohol use and smoking are the main risk factors for malignant transformation in head and neck cancers.Studies have shown an association between alcohol drinking and the development of head and neck tumors when taking into account exposure time and the amount of alcohol consumed.The higher number of men in this study corroborates findings by Drummond et al,The most frequent tumor site in our patients was the mouth, which is also the most frequent site for head and neck tumors reported in literature.GSTT1 and GSTM1 polymorphism studies done in Brazilian sub-populations reveal similar frequencies for both genotypes. Rossit et al,GSTT1 and GSTM1 gene null genotypes, respectively. Rossini et al,GSTT1 [-] and 42.1% for GSTM1 [-] in the state of Rio de Janeiro. Our study showed similar frequencies for these genotypes . Higher GSTT1 [-] and GSTM1 [-] polymorphism frequencies were observed by Drummond et al.GSTT1 [-] and 70.5% for GSTM1 [-]).GSTT1 and GSTM1 polymorphism studies done in head and neck carcinomas are contradictory. Various authors have demonstrated an association with the GSTM1 null genotype [-],GSTT1 null genotype [-] was also shown in some studies,GSTM1-enzyme activity was significantly reduced in patients with head and neck carcinoma compared to controls, although it did not depend on the unfavorable GSTM1 genotype, which may suggest that other enzymes participate as regulators.GSTT1 genotype [+] is associated with an increased risk of head and neck squamous cell carcinoma in smokers  and that the GSTT1 null genotype [-] may protect individuals against the development of this cancer. Although usually GSTs are considered detoxification enzymes, for certain specific chemical substrates such as dichloromethane (DCM), conjugation of glutathione with the GSTT1 enzyme may activate an electrophilic component, resulting in mutagenic potential.Interestingly, Evans et alGSTT1 and GSTM1 null genotypes and the development of head and neck squamous cell carcinoma in smokers.This study did not allow us to establish any correlation between


F is incorrect, and the correct definition is RF = –CF(CF3)(OCF2CF(CF3))nOCF2CF2CF3. The correct versions of The authors regret that a consistent structure error appears in The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.


The tensile property and electrical conductivity of monolayer membranes were explored. The results showed that PVA with 2 wt.% MWNTs nanofibre membrane has the best conductivity (1.0 × 10−5 S/cm) and tensile strength (29.36 MPa) compared with other fillers. Meanwhile, the combination of multilayer membrane ZnO/Fe3O4/Fe3O4/MWNTs/ZnO showed the highest conductivity (1.39 × 10−5 S/cm). The parallel circuit and calculation of parallel resistance were attempted to demonstrate the conductive mechanism of multilayer membranes, which can predict the conductivity of other multilayer films. The production of multilayer composites that enhance electrical conductivity and improve conductive predictions was successfully explored.Advanced research on improving the performance of conductive polymer composites is essential to exploring their potential in various applications. Thus, in this study, the electrical conductivity of multilayer nanofibre membranes composed of polyvinyl alcohol (PVA) with different electroconductive fillers content including zinc oxide (ZnO), multiwalled carbon nanotubes (MWNTs), and Ferro ferric oxide (Fe Electrospinning is an efficient method of producing nanosized or micro-sized fibres using an electrical field. When millions of fibres are collected on the collector device or rotating disk collector, dry polymer fibres of submicron diameter were deposited and formed nanofiber membranes ,2. In thTextile material that contributes to the electrostatic effect due to friction is not suitable to be worn or used near explosive surroundings, such as at gas stations or explosive handling areas because there could be a high risk of explosion . Therefo3O4 into the polymer matrix [PVA is water-soluble and is easy to form nanofibers by electrospinning, and it has extensive applications due to its biocompatible and biodegradable properties . It is wr matrix ,14,15,16r matrix . With thHowever, most of the electrospun nanofibre membrane studies focus on the production of monolayer and single-added particles or monolayer and multicomponent fillers. Multilayer nanofibre membranes are a common form of arrangement in composites that contribute to outstanding material performance compared with monolayer composites . MultilaIn this paper, we present the electrical conductivity of multivariate and multilayer nanofibre membranes using an improved formula to calculate in parallel circuits by considering the interfacial effects between the adjacent films. Before that, the optimization of different filler contents, tensile properties, and electrical conductivity of the monolayer nanofibre membranes were investigated. A hot-pressing process then prepared the multivariate and multilayer nanofibre membranes with three optimal nanofibre membrane contents (wt.%) following a particular combination.−3 centipoises) was obtained from Guangzhou Feng Bai Shun Trade Co., Ltd., Guangzhou, China; nanosized zinc oxide (ZnO) (100 nm) was supplied by Shijiazhuang Baisheng Chemical Co., Ltd., Shijiazhuang, China; nanoscale magnetite or ferro ferric oxide (Fe3O4) (80 nm) were provided by Qinhuangdao Taiji Ring Nanoscale Co., Ltd., Qinhuangdao, China); multiwalled carbon nanotubes (MWNTs) (9.4∼16.4 nm) purchased from Qinhuangdao Taiji Ring Nanoscale Co., Ltd., China. Polyvinyl alcohol (PVA)  and mass fractions (0 to 5 wt.%) were prepared, as shown in The PVA polymer was dissolved in distilled water (PVA: distilled water = 1:9), and the mixture was stirred for 12 h at room temperature. The PVA solution was defoamed and loaded into a 15 mL syringe (needle number 21) for the electrospinning. Different electroconductive fillers . The mechanical properties (tensile strength and modulus) of the membrane were then measured with a universal strength testing machine . Five tests for each group of samples (90 mm (length) × 10 mm (width)) were performed under stoke control at a constant speed of 10 mm/min, and the width of clamps was 30 mm. The optical images for all fabricated nanofibre membranes of PVA with different fillers were observed under a scanning electron microscope (SEM) .3O4  of Fe3O4 compared with ZnO (100 nm). Wang and co-workers explored the in situ composite approach of electrospun PVA/Fe3O4 and found that PVA acted as a stabilizer during the coprecipitation [3O4 by making it compatible and embedded together within the fibre. Hence, less precipitation formed on the matrix fibre. However, an agglomeration of fillers appeared on the nanofibre surface at 4 wt.%, uniformly attached to the matrix fibre as shown in 3O4 particles were agglomerated and had attached to the nanofibre surface [3O4  were used, and the nanofibre membrane micrographs are shown in 0.5 wt.% a. It canThe tensile properties of PVA/ZnO nanofibre membrane with varying mass fractions are shown in 3O4 nanofibre membrane are exhibited in 3O4 and a sudden drop at 5 wt.%. This tensile pattern occurred probably due to the cohesion force that weakened and slippage of Fe3O4 particle among fibres PVA matrix; thus, decrement of tensile strength was observed at the first 1 to 3 wt.%. Meanwhile, at 4 wt.% Fe3O4, the particle cohesion force increased suddenly, similar to the tensile strength and tensile modulus with 15.03 MPa and 187.50 GPa, which increased by 20.26 and 125.01%, respectively, in comparison with the pure PVA membrane [3O4 fillers (more than 5 wt.%) resulted in particle agglomeration, which led to the decrease of its tensile properties. This result is in accordance with previous research, which reported at 1 to 3 wt.% Fe3O4 loading exhibit no significant difference on tensile stress and ultimate strain; however higher value was observed if more than 5 wt.% [The fluctuation results for the tensile properties of the PVA/Fen 5 wt.% . The nonInterestingly, a different pattern of tensile strength and modulus of PVA/MWNTs membrane was observed with continuous increment as the mass fraction of MWNTs fillers (0 to 2 wt.%) increased. Jeong and co-researchers also reported the increment of tensile strength at 1 to 2.5 wt.% MWNTs, which attributed to a higher degree of filler orientation in the wrap nanofibre . The pre2 to 107 Hz) are shown in 2 to 105 Hz. Meanwhile, at approximately 106 Hz, the increment was more obvious where the conductivity exponentially increased until 107 Hz for all samples. Thus, from this finding, the conductivity of monolayer nanofibre membrane of PVA/ZnO was fixing at 107 Hz frequency to investigate the effects of different wt.% of ZnO (1 to 5 wt.%) as interpreted in 7 Hz, the coincident conductivity increased when the filler content was at 1 wt.% and decreased for higher filler contents. However, when the ZnO filler was at 5 wt.%, the conductivity was as higher as 1wt.%. In combination with the SEM micrograph, the uniform distribution of 1 wt.% ZnO in PVA fibrous membranes contributing to the possibility of forming a conductive pathway among the particles. The higher amount of ZnO fillers caused severe agglomeration, owing to the higher content and large specific surface area and interaction force of nanosized particles. In contrast, filler at 5 wt.% was enough to contribute to the conductive pathway. The optimization of ZnO contents in the PVA matrix was at 1wt.% because of its sharp efficiency effect; thus, it was referred to as threshold. In particular, the relative conductivity effect should enormously increase when the concentration of filler particles approaches the conductive threshold [The conductivities of monolayer nanofibre membranes of PVA/ZnO with different wt.% of ZnO (1 to 5 wt.%) at different frequencies (10hreshold . Therefo3O4 mass fractions at different frequencies (102 to 107 Hz) are illustrated in 7 Hz are displayed in 3O4 fillers was the highest from 106 to 107 Hz. The curve of all samples in 3O4 filler played an important role in forming the conductive network. Hence, with prominent particle cohesion force at 4 wt.% Fe3O4, it can be determined that the conductivity threshold and the optimized content for PVA/Fe3O4 were at 4 wt.%.The conductivities of monolayer nanofibre membranes with different Fe2 to 107 HZ). Similar to PVA/ZnO and PVA/Fe3O4 monolayer membranes as showed in 6 Hz until 107 Hz. The continuous increasing trend with the increase wt.% of MWNTs is portrayed in −6). The PVA membrane obtained conductive property at 2 wt.% MWNTs due to the conductive nature of carbon nanotubes also contributed to a decrease in internal resistance [3O4, and MWNT in films were 1, 4, and 2 wt.%, respectively.sistance . Finally7 Hz frequency of membranes among different fillers are presented in 3O4 particles in the PVA matrix. Despite this, the lowest result in tensile strength was observed when Fe3O4 filler was used in the membrane. The electrical conductivity at 107 Hz frequency showed a similar trend as tensile strength, which is that the sequence from higher to lower conductivity at 2 wt.% MWNTs, 1 wt.% ZnO and 4 wt.% Fe3O4 membrane. Therefore, the PVA membrane with ZnO was selected to be the surface layer. The MWNTs membrane with the highest conductivity and the lowest Fe3O4 membrane was utilized as the interlayers for more effective comparison.The tensile strength and electrical conductivity comparisons at 103O4/ZnO due to the contribution of higher electrical conductivity of PVA/MWNTs. The four-layer membranes were prepared with two interlayers (Fe3O4 and MWNTs). Finally, the additional Fe3O4 was added to the four-layer membranes. 3O4 membrane possessed lower conductivity, however, the effect on the conductivity of the multilayer membrane was greater than that of the single membrane itself.Multivariate and multilayer nanofibre membranes were produced by different univariate and monolayer electrospun nanofibre membranes through the heat and press process, as presented in n is the resistance of different monolayer membrane. According to R = ρL/S and ρ = 1/σ, the total electrical conductivity of multilayer membranes could be calculated by Equation (2).n is the electrical conductivity of different monolayer membrane. The parallel circuit was drawn into a multilayer membrane, shows in n−1⋅n is the lost conductivity due to the incompatibility of between adjacent membrane. If the two adjacent films have similar conductive particles, the σn−1⋅n is assumed to be zero. The lost conductivity of each adjacent film could be calculated. Finally, the σZF,σZM, and σFM were worked out through the theoretical value and actual value. The predicted actual electrical conductivity of the ZnO/Fe3O4/Fe3O4/MWNTs/ZnO membrane based on Equation (3) was 1.335 × 10−5 S/cm, and the error value was 3.96%, lower than 0.05. Therefore, Equation (3) was considered able to predict the multivariate and multilayer membranes. However, incompatibility or interaction between adjacent layers was inferred from the interfacial effects between different conductive films and the diverse conductive mechanisms of different conductive particles. The result from the calculation showed the conductivity of the actual multilayer films was considerably lower than the value calculated by Equation (2). The revised equation (Equation (3)) for the total electrical conductivity of the multilayer membrane was put forward, which revealed the regularity of interfaces.3O4, so the electrical conductivity of ZnO/MWNTs/ZnO is better than ZnO/Fe3O4/ZnO because the decreasing degree of resistance is related to the added resistance in parallel circuits [3O4 has electromagnetic shielding effectiveness [Finally, according to the revised equation, the conductivity of multivariate and multilayer conductive membranes relies on the number of layers, the conductivity of different single films, and the interfacial effects between adjacent films. The characteristic of parallel circuits is that the total resistance is smaller than the minimum parallel resistance. The total resistance decreases when the new resistance is added ,34, thuscircuits . Howevertiveness , and thetiveness ,37. The 3O4 and MWNTs) were successfully produced by electrospinning. A PVA/MWNTs monolayer membrane particularly for 2 wt.% was well dispersed and uniformly embedded within PVA fibre, resulting in the highest tensile strength (30 MPA) and revealing great electrical conductivity (1.0 × 10−5 S/cm). The multilayer film conductivity was successfully estimated using the modified conductivity formula based on parallel circuits. However, the accuracy of the lost conductivity of each adjacent film mentioned in this paper still needs to be investigated.Conductive PVA-based nanofibre membranes with different electroconductive fillers (ZnO, Fe


Arabidopsis thaliana seedlings exposed to elevated ambient temperatures. The transcription factor PHYTOCHROME INTERACTING FACTOR 4 (PIF4) functions as a central hub in this response and promotes hypocotyl growth by transcriptionally inducing auxin biosynthesis genes . Measurements of hypocotyl length at different time points during the night revealed that hypocotyls of seedlings transferred from 10°C to 28°C at the beginning of the night grew less during the night than those of seedlings continuously kept at 28°C. Conversely, seedlings transferred from 28°C to 10°C showed enhanced nighttime hypocotyl growth compared with seedlings that were maintained at 10°C during both day and night  and its consequence (PIF4 levels) not only depends on the size of the change but also its direction.To further unravel how daytime temperature affects nighttime growth, responses of hypocotyl elongation to different combinations of day and night temperatures were monitored in different mutant backgrounds. Short-term temperature memory was disturbed in mutants of several known thermomorphogenic regulators including PIF4 and another transcription factor, ELONGATED HYPOCOTYL 5 (HY5). Confocal imaging of plants expressing PIF4 and HY5 reporter constructs indicated that nuclear levels of both proteins accounted for 80% of the observed growth rate variability. Using luminescence assays, the authors demonstrated that temperature-induced effects on nuclear PIF4 protein levels were largely driven by changes in PIF4 expression, the authors investigated the involvement of this clock protein in the observed temperature memory. ELF3 undergoes a phase transition in response to warm temperatures, forming speckles in the nucleus via its predicted prion-like domain (elf3 mutant background, indicating that ELF3 functions upstream of PIF4 in establishing short-term temperature memory. This was further supported by the observation that ELF3 speckle formation also showed a hysteretic pattern, with a high sensitivity to increasing temperatures and a much lower responsivity to cooling.As ELF3 is a known regulator of e domain . Murcia Taken together,


It was determined via STR profiling that cell line UMSCC2 should have been labeled as HSC3 cells. Like UMSCC2 cells, HSC3 cells are squamous cell carcinoma cells that express wild type EGFR, the primary substrate of PTPRJ/DEP1 investigated in this publication. Therefore, this correction does not affect the results and conclusions of the paper.