The self-organizational properties of DNA have been used to realize synthetic hosts for protein encapsulation. However, current strategies of DNA–protein conjugation still limit true emulation of natural host–guest systems, whose formation relies on non-covalent bonds between geometrically matching interfaces. Here we report one of the largest DNA–protein complexes of semisynthetic origin held in place exclusively by spatially defined supramolecular interactions. Our approach is based on the decoration of the inner surface of a DNA origami hollow structure with multiple ligands converging to their corresponding binding sites on the protein surface with programmable symmetry and range-of-action. Our results demonstrate specific host–guest recognition in a 1:1 stoichiometry and selectivity for the guest whose size guarantees sufficient molecular diffusion preserving short intermolecular distances. DNA nanocontainers can be thus rationally designed to trap single guest molecules in their native form, mimicking natural strategies of molecular recognition and anticipating a new method of protein caging. Current strategies for protein encapsulation in DNA vessels for controlled enzymatic catalysis or therapeutic delivery rely on formation of covalent complexes. Here, the authors design a system that mimics natural reversible non-covalent host–guest interactions between a DNA host and the protein DegP. Through the physical separation of molecular species into specialized compartments, nature achieves control of matter distribution both in space and time1246131415172122232627293031Despite notable progresses in this field, full exploitation of synthetic DNA nanostructures as programmable protein hosts is still limited by the chemical strategies used for DNA–protein conjugation. Indeed, protein loading into DNA nanocontainers mostly relies on the chemical cross-linking of functionalized DNA strands to reactive cysteine or lysine side chains exposed on the protein surface and further hybridization of the resulting DNA–protein conjugate to complementary handles appended to the DNA–host surface. This method presents at least two disadvantages: first, a lack in the regioselectivity and stoichiometric control of protein modification, which leads to a heterogeneous mixture of products; and second, a permanent modification of the protein surface, which may interfere with molecular recognition events. Although alternative strategies have been reported26343536Here we show an architectural strategy for the selective encapsulation of a protein guest into a synthetic DNA host, using supramolecular interactions of programmable symmetry and range-of-action. In our approach, the collective and convergent action of multiple ligands pre-oriented towards a common protein target is exploited to strengthen the binding affinity, circumventing the need for covalent protein tagging and favouring the formation of a 1:1 host–guest complex. Inspired by natural strategies of molecular recognition, DNA shells can be thus engineered to specifically identify the chemistry and geometry of a guest surface for its controlled caging with minimal human intervention.6; ca. 250 kDa) to the 12-mer  till the highest 24-mer . Each DegP monomer consists of three domains (We chose DegP as the guest of our DNA origami host. DegP is a serine protease38 domains : a serin domains . This en domains . The strKd=ca. 5 μM), which acts as a specific substrate mimics , an inner radius of 20 nm (Rin) and a free accessible room of ca. 10 nm radius (Rfree). The prism measured 49 nm in length, except for two opposite faces with slightly longer edges (56 nm), which offered a useful topographical feature for atomic force microscopy (AFM) characterization , a 240° angle will be instead associated to PAs directed outwards (6p240), and finally, edge-to-edge connections by flexible T-hinges (180°) will refer to a structure with an undefined direction of the arms (6p180). In addition, the number of protruding arms available for attachment of peptide ligands will be indicated as 0cA1, 6cA1 or 18cA1, corresponding, respectively, to zero, one or three PAs per origami face  through out-of-plane crossovers between the helices of adjacent edges . This alami face . Successami face . To bettami face , having 6, this arrangement places the PDZ1 domains pointing towards the solvent. The PDZ1 domains in DegP12 are instead located at the entrance of the central hole and are grouped in units of three arranged in a tetrahedral orientation. Conversely, DegP24 features a cube with six possible entrances, formed each by four PDZ1 domains. In both DegP12 and DegP24 the binding sites of the PDZ1 domains are oriented towards the inner cavity of the protein. Our Molecular Dynamics (MD) simulations suggest that up to three DNA helices can point into a quartet of PDZ1 domains without compromising the binding of the incoming peptide  of protein particles traversing the host in a typical experiment  is given by I=166 Rfree/rprot  and the DNA host, functionalized with 18 protruding arms in its inner cavity (6p120-18cA1), occurs only in presence of the TAMRA-labelled peptide ligands  and the wild-type form DegP6A488WT  microscopy , AFM Fi and tranRA donor  and the RA donor  followinRA donor , thus clca. twofold the height of a monolayer of double helices  in order to induce the release of the partially complementary A1-peptide linkers after addition of fully complementary A1 sequences . The reaConsistent with the model recently proposed by Yan and co-authors43In this study, we have shown that merging DNA nanotechnology and supramolecular chemistry, a spatially defined envelope of weak non-covalent interactions can be placed around the surface of a single protein to drive its encapsulation in a programmable way. The arrangement of a distinct number of ligands in the vicinity of their corresponding binding sites on the protein surface can be geometrically and stoichiometrically controlled, thus allowing to modulate local concentration effects and multivalent short-range interactions of the host–guest system. This method results in the ligand-specific and size-selective encapsulation of one protein molecule on the basis of a fine compromise between optimal host–guest intermolecular distance and sufficient molecular diffusion of the guest through the host. In other words, both the chemical affinity and the geometric compatibility of the interacting species are taken into consideration for efficient binding and, most importantly, without any previous covalent modification of the protein. One can easily envisage using such structures as molecular sieves for trapping single guest molecules from a mixture of compounds of similar binding affinity, avoiding chemical conjugation strategies and thus preserving the native state of the target protein. Interestingly, we observed that the partial suppression of the DNA–protein electrostatic attractions through lysine-selective molecular tweezers enhances the specificity of host–guest recognition thus confirming the important role of the molecular shell surrounding the protein surface for DNA-caging purposesIn principle, the method proposed here is applicable to every protein-recognition motif and enables to overcome the low affinity of ligand binding with a multiplicity of recognition events occurring in a confined space at predictable distances. Finally, one can foresee that modifying the inner cavity of the host with distinct ligands, multiple enzymes can be arranged into synthetic protein scaffolds, allowing, for example, to improve metabolic pathways in a programmable mannerwww.cadnano.org) and assembled using a 1:10 molar ratio between the M13mp18 ssDNA scaffold (20 nM) and each of the staple strands, in 1 × TEMg buffer . Thermal annealing was performed on a Thermocycler Mastercycler nexus gradient (Eppendorf) by decreasing the temperature from 90 to 20 °C at −1 °C min−1 for the 6prism construct. The 3prism structure was folded instead using the following gradient: from 90 to 45 °C at −1 °C min−1 and from 44 to 20 °C with −1 °C every 2.5 h. The DNA origami structures were decorated in their inner cavity with a distinct number of DNA strands of identical sequence (cA1: 5′-CTTCACGATTGCCACTTTCCAC-3′) and hybridized with A1-tagged peptides . Purified peptide-modified origami structures were reacted with 25 molar equivalents of DegP protein for at least 3 h (till max 12 h) at room temperature. The reaction mixtures were then analysed by agarose gel electrophoresis and desired bands were excised with a clean scalpel from an identical gel lacking ethidium bromide . The DNA ladder used was 1 kbp (Roth). Products were recovered using a Freeze ‘N Squeeze spin column' and directly used for AFM, TEM and TIRF microscopy characterization. All DNA sequences are provided as DNA origami structures were designed with caDNAno  for further spectroscopic tracking .degP with a C-terminal His tagAll plasmids were derivatives of pCS20 expressing wild-type The missing parameters for the MD simulations were generated using Swissparam502 and supplemented with an oxygen-scavenging system composed of 2 mM Trolox (Sigma-Aldrich), glucose oxidase , catalase  and β-D-(+) glucose . Data analysis was performed by the home-made software package iSMSThe sample was immobilized on a BSA–biotin streptavidin-coated quartz coverslip for prism-based TIRF microscopy. Fluorescence was measured using an inverted wide-field optical microscope and alternate laser excitation at 514 and 630 nm of the donor and acceptor fluorophore, respectively. Fluorescence was divided into a red and blue spectral channel  and movies were recorded with an EMCCD camera  using a 0.5 s integration time per image and a total length of 100 s. Measurements were performed in TAE (pH 8.0) buffer (20 mM Tris-acetate and 1 mM EDTA) containing 12.5 mM MgClMalvern Zetasizer Nano ZS. A disposable cuvette ZEN0040 was used for the measurements. Parameters were set to a refractive index of 2.1 and an absorption of 0.184 for the sample. For the TEMg buffer a refractive index of 1.331 was entered to the software (Zetasizer Software 7.03). Samples were equilibrated for 30 s at 25 °C and the measurement, done using a 173° backscatter angle, was set to an automatic duration. Measurements were repeated three times for each sample.Size measurements based on DLS were carried out using a 2O, the sample was dried under gentle argon flow and scanned in ScanAsyst Mode using a MultiMode microscope (Bruker) equipped with a Nanoscope V controller. Force (0.4 N m−1) constant cantilevers with sharpened pyramidal tips  were used for scanning. After engagement the peak force setpoint was typically 0.02 volt and the scan rates ∼1 Hz. All images were analysed using the Gwyddion software (more details are given in the The sample was deposited on freshly cleaved mica surface (Plano GmbH) and adsorbed for 3 min at room temperature. After washing with ddH6 cathode at an operation voltage of 120 kV. Digital micrographs were recorded with a 4 k × 4 k CMOS Camera F416 (TVIPS) using low-dose conditions. Single particles were boxed out manually using e2boxerProtein and protein–DNA origami samples were prepared for electron microscopy (EM) characterization as previously described54The data that support the findings of this study are available from the corresponding author on request.How to cite this article: Sprengel, A. et al. Tailored protein encapsulation into a DNA host using geometrically organized supramolecular interactions. Nat. Commun.8, 14472 doi: 10.1038/ncomms14472 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables, Supplementary Discussion, Supplementary Methods and Supplementary References.Supplementary dataset one.


A cable model that includes polarization-induced capacitive current is derived for modeling the solitonic conduction of electrotonic potentials in neuronal branchlets with microstructure containing endoplasmic membranes. A solution of the nonlinear cable equation modified for fissured intracellular medium with a source term representing charge ‘soakage’ is used to show how intracellular capacitive effects of bound electrical charges within mitochondrial membranes can influence electrotonic signals expressed as solitary waves. The elastic collision resulting from a head-on collision of two solitary waves results in localized and non-dispersing electrical solitons created by the nonlinearity of the source term. It has been shown that solitons in neurons with mitochondrial membrane and quasi-electrostatic interactions of charges held by the microstructure  have a slower velocity of propagation compared with solitons in neurons with microstructure, but without endoplasmic membranes. When the equilibrium potential is a small deviation from rest, the nonohmic conductance acts as a leaky channel and the solitons are small compared when the equilibrium potential is large and the outer mitochondrial membrane acts as an amplifier, boosting the amplitude of the endogenously generated solitons. These findings demonstrate a functional role of quasi-electrostatic interactions of bound electrical charges held by microstructure for sustaining solitons with robust self-regulation in their amplitude through changes in the mitochondrial membrane equilibrium potential. The implication of our results indicate that a phenomenological description of ionic current can be successfully modeled with displacement current in Maxwell’s equations as a conduction process involving quasi-electrostatic interactions without the inclusion of diffusive current. This is the first study in which solitonic conduction of electrotonic potentials are generated by polarization-induced capacitive current in microstructure and nonohmic mitochondrial membrane current. Although the H-H model portrays the nerve as an electrical analogue in terms of capacitors and conductors, it does not incorporate a physico-chemical understanding of ionic diffusion within the excitable membranes. The Frankenhaeuser and Huxley (F-H) model developed in 1964 ),ρ) and the current density (J) in a volume (B) is given as [A/cm2) and J = JC+JD is the current density flowing along the cable in the x-direction (A/cm2). The conductivity current density JC = σE = (Ohm’s law) where the electric conductivity σ(S/cm) is constant, neglects ionic concentration gradients in the electrolytes and the nature of the different ionic species, and therefore represents ionic homogeneity within the microstructure. The electric displacement field is D = ε0E + P once differentiated with respect to time yields the displacement current density x and letting Δx → 0 yieldsπr2ρbound = q is the surface bound charge per unit length of cable in the positive x—direction (C/cm).The equation of continuity for the charge density (given as :-∫∫∫B∂ρM is the membrane thickness. Changes in capacitance due to compressive forces acting on the membrane (electrostriction) are electromechanical. One such example is the voltage-dependent longitudinal  capacitance Ci(Vi) characterized by a quadratic dependence on the voltage , Vi is the intracellular membrane potential (mV), and ξ is fraction increase in capacitance per square millivolt (mV−2).The voltage-dependent charge transfer in the squid axon without microstructure, but due to electrocompression, follows a quadratic dependence see , since tage (see ):Ci(Vi)Vi in the range  determined from the electrical charge stored in the capacitance of the microstructure. The parameter represents the capacity to hold more charge or electrical energy than a linear capacitor.Mitochondrial membrane potential is regulated between −158mV and −108mV , which mange see . This is2αVi0.73  or as shnonlinear capacitor represented by a voltage-dependent longitudinal capacitance Ci(Vi) = CiC(Vi) is defined as charge transfer C(Vi) is the linearized polarization capacitance-voltage characteristic (dimensionless) generated by the voltage-dependent charge transfer in the microstructure. Upon integration of C/cm):α = 0 implies there is no charge stored due to the absence of microstructure.The Vi is the intracellular potential, ci is the axial capacitance across unit length ci = 2ε0πr2(Fcm), and ri is the intracellular resistance per unit length rm becomesVe = 0) then effect of the external potential on the mitochondrial membrane potential  is negligible. Hence letting V = Vm − Er be the depolarization (mV) and Er be the resting mitochondrial membrane potential (mV), together with mitochondrial membrane as shown in Vrev = Va − Er is the reversal potential (mV). Va is the equilibrium potential (mV). im is the total membrane current per unit length (A/cm), Im is the total membrane current density (A/cm2), ga(V) is the mitochondrial membrane conductance (S/cm), cm is the membrane capacitance per unit length of cable (F/cm), and rm is the membrane resistance across a unit length of passive membrane cable (Ωcm). Note that Er = −139mV [ga(V) = 2πrGa(V) where Ga(V) is the mitochondrial membrane conductance per unit area (S/cm2), Rm = 2πrrm is the membrane resistivity or resistance across a unit area of passive membrane (Ωcm2), F/cm2), F/cm), and Ri = 0.5σ is the intracellular resistivity (Ω cm). The intracellular resistance per unit length ri(Ω/cm) differs from the intracellular resistivity Ri = 0.5σ(Ω cm) or volume resistivity of the intracellular medium also referred to as specific resistance, which is σ is the electrical conductivity (S/cm).Since the current density is = −139mV , ga(V) =Electrical conductivity neglects ionic concentration gradients in the electrolyte solution and the nature of different ionic species and therefore represents ionic homogeneity within the microstructure. However, in our model in addition to bound charge on charged proteins there are inactive membranes encasing the branchlet corresponding to mitochondrial membranes, which also contribute to the displacement current, depending on the equilibrium potential (when the nonohmic conductance is non-zero). The model membrane reverts to a passive neuronal plasma membrane when the mitochondrial conductance is zero. This implies that the mitochondrial membrane channel activity is simultaneous with the opening of plasma membrane channels. This ignores the dependence of the mitochondrial channel on second messengers during synaptic transmission .S/cm), ‘a’ (dimensionless) and ‘b’ (mV−1).The total ionic membrane current per unit length is assumed to be a quadratic nonlinearity as depicted in red (cf. ). The eqτm = cmrm (passive membrane time-constant in msec), cm), Δ = cirm, from Q = C(V)VCi is the total surface charge per unit length of cable (C/cm) and from C(V) = 2αV is a polarization capacitance-voltage characteristic (dimensionless), substituting τρ = ciri the dimensionless form of the nonlinear cable equation is:U → ακV such that η = 0 and δ = 0 then Let ga(V) = 0). The third-order term counters the steepness of the voltage gradient due to the nonlinear terms. Given that γ is small; the longitudinal polarization current will be conducted with a steep voltage gradient giving grounds for the existence of solitary waves, although, its presence prevents a sharp change in the voltage, so quasi-electrostatic conditions prevail.The right-hand side of ζ = (X − Xp − vT) where Xp is the initial location of the electrotonic signal positioned along the cable and v is its velocity (dimensionless). The electrotonic signal is moving towards ζ → ∞. The electrotonic signal moving in the other direction ζ → −∞ we would use ζ = (X − Xp + vT). For convenience, we use the ansatz U* = Ω(ζ) where U* is the free-space version of U on an infinite interval , with the following identities:Nonlinear cable equations may admit solitary wave solutions and if they do they are either severely restricted or approximated . Solitarζ) → f(y) and f(±1) → 0. Substituting the above new variables into We obtain solitary wave solutions in free-space using the tanh-function expansion method by introducing a new independent variable :y=tanh → 0 yields a1 = 1 and the solution takes the form:y → 1 then upon substituting f(1) → 0 yields the dimensionless velocity of the electrotonic signal:η < 3 for positive velocity. Now substitution of y = tanh(ζ) into v:a0 > 0 is the dimensionless amplitude determined in quasi-soliton reflecting on the electrotonic signal propagating at a constant velocity v > 0 The solitary-wave solution is only an approximate solution of The tanh-function expansion method admits the use of a finite expansion of the form U* in non-dimensional terms along an infinite cable (in free-space) with passive membrane  in The results presented in membrane  or mitocmembrane . The ele.2mV cf. . In this.2mV cf. . While fcase cf. . This inX1, T1) = . The co-ordinate for the last point is  =  for passive membrane. Thus slope is X2, T2) =  for mitochondrial membrane with η = 0.5 and δ = 10. The co-ordinate for the last point is  =  for mitochondrial membrane with η = 2.5 and δ = 10. The co-ordinate for last point is  =  for mitochondrial membrane with η = 2.9 and δ = 8. The slope is η = 0.5 and δ = 10. The slope is η = 2.5 and δ = 10. The slope is η = 2.9 and δ = 8.As shown in T → ∞. In T → ∞ that the linearized quasi-soliton dissipates as it propagates. The quasi-soliton is self-generating due to the reservior of electrical charge stored in the capacitance and dissipates only in the absence of microstructure. The Boussinesq paradigm which states that the balance between the steepening effect of the nonlinearity and the flattening effect of the dispersion maintains the shape of the soliton [If the interaction between two quasi-solitons is robust (i.e. preserves their shape and velocities during the interaction) then quasi-solitons reappear after collision. Amazingly, this is the major property of solitons . Quasi-s soliton . This cl soliton . The quasummation of localized traveling waves that can interact without changing their shapes, amplitudes and velocities since linear superposition of quasi-solitons is assumed:Considering the approximate solitonic interactions based on the U* = Ω(ζ) are shown to satisfy the following conditions [ζ) = Ω″(ζ) = Ω‴(ζ) = 0 where prime denotes differentiation with respect to ζ, further reinforcing that quasi-solitons are solitons.The collision between two quasi-solitons with identical velocities is illustrated in nditions : Ω′(ζ) =Gonzalez-Perez and his colleagues performed an experimental study showing head-on collision between two nerve pulses of less than 5mV undergoing an elastic interaction instead of annihilating upon contact (see ). Our moIntracellular capacitive effects entail self-excitability due to charge ‘soakage’ held by the endogenous membrane capacitor which results in electrotonic signals propagating as solitary waves due to the energy stored in the microstructure . SolitarThe crucial test for solitary waves to be solitons is robustness to collision . ElectriThere are mechanical models of soliton propagation in nerve, but none that specifically address electrical solitons. Aizawa and colleagues  pointed W in a subsidiary linear rate equation:The source term containing charge ‘soakage’ in the cable equation resulted in solitary traveling waves instead of traveling fonts as one would expect in solutions of a cable equation without recovery processes. The inclusion of recovery processes, which is designed to model the slower membrane response of potassium activation and sodium inactivation based on H-H kinetics  entails . Huxley  had obseEndogenous electric field is a term used to infer on the absence of any externally applied electric fields. The endogenous electrical field effects are considered to be extracellular fields induced ephaptically, which have been shown to affect spike timing of a neuron , but areD is the electric flux density and J is the current density. Furthermore, for a nonlinear dissipative neural media, it also requires the susceptibility (χ) to be a function of time and location:P is the polarization field in the longitudinal direction . The consequence of this re-evaluation renders the model to include dispersive capacitive effects  seen both theoretically [The implications of a model for understanding the intracellular capacitive effects of macroscopic polarization on membrane potential and on the excitability process in general needs to be developed further. The present formalism can be further extended through inclusion of inhomogeneous linear dissipative neural media of a more general form where the electric conductivity and permittivity are no longer constants, but functions that depend on location and time. In such a circumstance, there is a need to reconsider more realistic cases were inhomogeneities in the conductivity and permittivity is present. One such example is a more general form where the electric conductivity and permittivity are no longer constants, but functions that depend on location and time:etically  and expeetically .In this paper, we provided a phenomenological description of the electrolytic microenvironment that assumes electrodiffusion of ions to be reflected by electrically charged homogenous core-conductor, where charge densities are continuous distributions reflecting displacement current in an electrolytic cable with polarized microstructure.The nonlinear cable equation with a source term representing charge ‘soakage’ in a linearly dissipative medium was derived from Maxwell’s equations under quasi-electrostatic conditions (slow moving electric field) and solved to describe traveling wave solutions as solitary waves. The head-on collision between two oppositely traveling solitary waves produced an elastic interaction confirming the existence of electrical solitons. The charge ‘soakage’ produced non-dispersing effects that sustained the amplitudes of the solitons from dissipating. The results are consistent with solitonic conduction of electrotonic potentials based on spatial and ionic homogeneity, negligible concentration gradients, and extracellular isopotentiality. The effect of polarized microstructure in a cable model with mitochondrial membranes has confirmed that electrotonic signals can be conducted as solitons. In principle, the confirmation of electrotonic signals as electrical solitons complements the 65 year standing H-H model at the subcellular scale.S1 Appendix(PDF)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.S3 Appendix(PDF)Click here for additional data file.


We study the sensitivity and selectivity of the developed chip at operating temperatures up to 400 °C towards organic vapors in the ppm range. The results indicate that the titania nanotubes are a promising material platform for novel cost-effective and powerful gas-analytical multisensor units.Reliable environmental monitoring requires cost effective but highly sensitive and selective gas sensors. While the sensitivity of the sensors is improved by reducing the characteristic dimensions of the gas-sensing material, the selectivity is often approached by combining the sensors into multisensor arrays. The development of scalable methods to manufacture such arrays based on low-dimensional structures offers new perspectives for gas sensing applications. Here we examine an approach to produce multisensor array chips based on the TiO The most remarkable success in that field is expected in material science of gas-sensitive elements primarily matured from technologies which allow one designing nano-dimensional structures4. The targeted sensor characteristics are higher sensitivity, stability and selectivity6 which should be combined with low production cost and low energy consumption of the final units8.The development of gas sensors with advanced characteristics is driven by the necessity of fast, reliable and cheap monitoring of the environment9. The general approach to greatly enhance their sensitivity is a reduction of the characteristic dimensions of the crystallites/grains composing the material10. This enhances the impact of surface processes on the total conductivity of the material as the ratio between Debye length and structure size increases11. However, 3D-nanosized oxide structures might be rather unstable especially at elevated temperatures which are conventionally employed to activate the sensors, particularly chemiresistors12, what questions their stability. In order to address this issue, quasi-1D structures such as nanowires, nanobelts or nanotubes (NTs) are considered to be the promising candidates for a new generation of gas sensors (see recent reviews15 and references therein). Still, the selectivity remains to be the most challenging aspect because metal oxide semiconductors are fundamentally unselective for chemisorbed gas species. Therefore, the most powerful way is to combine various sensors16, including those based on quasi 1D structures17, into arrays and process the obtained vector signal by pattern recognition techniques.So far, metal oxide semiconductors are recognized as materials which fully meet the requirements of commercial gas sensors and have a great potential to advance further, mainly through application of nanotechnologies21. The second one is thought to be more suitable for mass production because of lower cost and better control the surface area to define the thickness at the nanoscale22. Among quasi-1-D structures, NTs are distinguished by their high specific surface due to their hollow structure with inner and outer surfaces that are exposed to the gases which makes it easier to fully deplete the oxide from free carriers due to surface localization under chemisorption and catalytic processes. That favors a “transduction function” in this material for sensor applications while a “receptor function” is still defined by a preferential oxide crystalline phase23.The quasi 1D oxide structures can be synthesized via gas-based or solution-based techniques24. The most cost effective and well established technology is anodization of titanium in F−containing electrolytes which is appropriate for mass-scaling20. By properly choosing the anodization bias one can tune fabricating oxide NT layers with precise control of the diameter, height, wall thickness of the tubes, inter-tube distance, thickness of the barrier layer, etc.25. This renders the material to be promising for chemiresistors using a conventional two-electrode design30. Although, a realization of these sensors is challenged by a negative influence of titanium underlayer which shunts NTs and leads to poor electrical contacts which reduces the reliability of the devices31.In this report, we consider NTs of titanium dioxide which can be reliably fabricated using high yield protocols2 NT-based sensors, the selectivity of gas identification remains unresolved. Even the approaches to functionalize the NTs by various dopants have not allowed one obtaining absolute sensitivity to only one gas33. Therefore, here we consider employing a multisensor approach34 to be realized possibly in the cheapest design35 able in frames of microelectronic technologies by segmenting the pristine NT array by multiple co-planar electrodes. In particular, we demonstrate the capability of the chip to selectively detect few exemplary organic vapors at ppm concentrations.Despite some success in enhancing the sensitivity of the TiO37. The as-anodized samples are further etched for removing the remained metal in absolute methanol/ Br2 solution38 in order to extract the pristine TiOx NT array. The full details of the processes are given in Methods. Then, the TiOx NT array is cleaned in pure alcohols and placed for floating over the distillated water surface. This array is then picked up and thoroughly positioned over the Si/SiO2 multielectrode chip substrate39. The as-prepared chip is dried for few hours at room temperature in air.The process to employ the titanium dioxide NTs in the multisensor chip includes Fig.  primaril2 chip substrate equipped with multiple Pt strip electrodes provides a number (up to 38) of NT array segments whose electrical/chemiresistive properties are measured independently.The Si/SiOx NT array possesses a structural integrity as evidenced by scanning electron microscopy (SEM) inspection  and X-ray photoelectron spectroscopy (XPS) techniques; the details are given in Methods.x NTs are closely packed into an array where adjacent NTs have a distance of a few nm, which favors good gas access . In the temperature range of 200–400 °C, the I-V curves measured for the NT array segments have been found to be nearly linear .Following the chip exposure towards isopropanol, ethanol and acetone vapors, 5–10 ppm concentrations, in mixture with laboratory air at 30–35 rel. % humidity, we have observed a clear chemiresistive effect in NTs at all operating temperatures in 200–400 °С range. The maximum gas response as a relative resistance change,  °C Fig. . At thisgly Fig.  which isx NT array when exposed to isopropanol vapors at 350 °C which is optimal one to observe the chemiresistive effect. The similar data have been observed in case of other test vapors at different temperatures.These remarkable differences in responses to these gases are caused by variations in chemical adsorption and dissociation of these molecules at the NT surface that provides a background to selective recognition of these vapors by multisensor chip array as further discussed. Here, it is worth noting that the change of the median resistance exceeds 8% to 5 ppm of isopropanol already at 200 °C what indicates rather low gas detection limit for the developed multisensor. Figure n-type semiconductor response of non-stoichiometric TiO2. Still, the spatial inhomogeneous local morphology of the NT array across the chip segments results in the variation of both the resistance and gas sensitivity. It allows one employing it to build a vector signal whose components are constitued by signals from the gas-sensitive segments in the chip and approach the gas selectivity in the framework of known multisensor concept16. In order to process the NT-based multisensor vector response to the test vapors we have applied the Linear Discriminant Analysis (LDA) which maximizes the vector variations induced by different vapors and minimizes the vector scatters under exposure to certain vapor51. The confidence probability to build the LDA clusters related to gases has been installed to be equal to 0.95. By these means, we have tested all the multisensor chip vector signals recorded at operating temperatures in 200–400 °C range , μ the electron mobility, which we consider equal to approx. 0.1 cm2/Vs, characteristic for polycrystalline TiO256. The geometry coefficient D and d the estimated value of g is equal to approx. 5.1. For instance, at operating temperature of 350 °C one gets n equal to approx. 3.3 ± 0.4·1013 cm−3 that is lower by 3–4 orders of magnitude when compared to concentrations reported for a single anatase NT measured in vacuum conditions57. The corresponding Debye length, k is Boltzmann constant, ε0 the electric constant, ε the dielectric permittivity of anatase 59, T the operating temperature, at these conditions goes to micrometer range that obviously exceeds the NT wall. Therefore, under air conditions the Fermi level in the NTs is pinned at the local states induced by oxidizing/hydroxyl adspecies in the oxide gap that is ordinarily described as a flat-band approximation60. It results in remarkable reduction both the free electron concentration in the NT wall and the electron mobility due to enhancing the potential barrier, ψB, between NTs61. Consequently, this originates a high NT array resistance observed. So, the electron transport in such structures is mainly governed by carrier-depleted NT walls contacting each other like in the case of grains in polycrystalline samples54.While the electron exchange between the chemisorbed species and semiconducting oxide surface depends mostly on the material itself, the micro- and mesostructure of the layer, its porosity and characteristic geometry substantially contribute to the overall NT array resistance measured at different environmentsψB55. We consider the only electronic component of the oxide conductance because the operating temperatures below 400 °C are still not enough to stimulate a significant exchange between surface and bulk of NTs by intrinsic defects58. So, under application of electric field the electrons go through NT adjacent walls whose potential barrier height and “tunneling density” depend on a gas environment  foil of 127 µm thickness. Prior to anodization the titanium foil has been immersed into acetone and ethanol, both for 10 min, in sequence under sonification and then rinsed in deionized water (18.2 MΩcm). The titanium foil is characterized with a roughness less than 0.7 µm measured with surface profiler (Veeco DekTak 150). The electrochemical anodization has been performed in a Teflon® cell in two-electrode configuration with a platinum mesh as cathode biased at 30 V for 1 hour at room temperature using electrolyte based on glycerol/water 3:1 solution with addition of 0.8% NH4F  connected to a digital multimeter (Keithley 2700 multimeter /data acquisition system) interfaced to personal computer (PC). The TiOx NT array is afterwards rinsed in ethanol and is dried at room temperature for 0.5–1 h. The Ti sublayer remains have been selectively dissolved in an absolute methanol/Br2 solution, 1:9 vol., at 20 °С for 2 hours according to ref. 2 chip equipped with multiple planar electrodes. Thermal treatment of titania NTs has been performed in PEO-601 oven (ATV Technologie GmbH) in air atmosphere at 400 °C during 24 h. Following the annealing the samples have been rinsed by distilled water and dried in air.TiOx NTs from the substrate, they have been dispersed in n-heptane by sonification and deposited on a carbon coated Cu grid (Quantifoil) for the TEM analysis. For an electron tomographic characterization in HAADF-STEM mode, 15 nm gold nanoparticles (University of Utrecht) were added for alignment and a tilt-series recorded from −60° to 60° in 2° steps. The tilt-series alignment was performed using IMOD (University of Colorado) and the 3D volume reconstructed using the SIRT algorithm implemented in Inspect3D (FEI Company). The Supplementary video shows a volume rendering of the 3D structure of the NT array morphology and a surface rendering of a selected part of the 3D reconstruction prepared in Amira (FEI Company).The morphological characterization of the NT array has been carried out using a scanning electron microscope  equipped with an energy dispersive X-ray (EDX) detector. The NT structure has been studied by transmission electron microscopy (TEM) using an aberration (image) corrected Titan 80–300 (FEI Company) equipped with a US1000 (Gatan) CCD camera. After mechanical removing the TiO62. We have utilized Si substrate with sputtered Au layer, approx. 1 µm thick, to place the titania NT array after the metallic Ti is removed. All samples have been measured using a microfocused monochromated Al Kα X-ray source of 30–400 µm spot size. The K-Alpha charge compensation system is employed during analysis using electrons of 8 eV energy and low-energy argon ions to prevent any localized charge build-up. The spectra are fitted with one or more Voigt profiles; the binding energy uncertainty is ±0.2 eV. The analyzer transmission function, Scofield sensitivity factors63 and effective attenuation lengths (EALs) for photoelectrons are applied for quantification. EALs are calculated using the standard TPP-2M formalism64. All spectra have been referenced to the C 1s peak of hydrocarbon at 285.0 eV binding energy controlled by means of the well-known photoelectron peaks of metallic Cu, Ag, and Au.The X-ray photoelectron spectroscopy (XPS) measurements have been conducted using a K-Alpha+ XPS spectrometer . Data acquisition and processing using the @Thermo Avantage software are described elsewhere2 substrate equipped with 39 Pt strip electrodes which define, if necessary, 38 segments of the NT array as a maximum number39. The inter-electrode distance is about 80 µm, electrode width is 100 µm, and the electrodes are 1 μm thick. All the electrodes have a length of about 4 mm that defines the surface area of about 4 × 8 mm2 whose part or total is covered by NT array. The frontside of the substrate has two meander-shaped Pt thermoresistiors at the edges to monitor the operating temperature which is adjusted by four meander heaters located at the backside. The operating temperature is maintained by corresponding PC-driven electronics unit with accuracy of 1 °С while the temperature distribution at isothermal chip operation can be of ±10 °C52.We have utilized multielectrode chip based on Si/SiOWe have tested a number of chips in preliminary studies, the reported data belong to one exemplary chip.x NT array towards different organic vapors has been studied using a setup which includes home-made data acquisition unit, test gas generator, gas delivery tubes and electronic unit to maintain and monitor the chip operating temperature. Data acquisition module  together with current pre-amplifier  has provided the resistivity measurements up to tens GOhms of each segment in the chip. To read out the total array resistances we have employed a multiplexing card for switching between the segments. Test vapor mixtures with laboratory air have been obtained with gas generator  which employs heated permeation tubes containing a test liquid to yield vapors of several ppm of concentration under equilibrium conditions. The vapors of the same reducing origin, alcohols and acetone, have been utilized as test ones. The vapors have fed the chip by series of 10 min duration interrupted by pulses of pure laboratory air for 10–25 minutes to ensure the recovery of the chip segment resistances. The concentration of vapors in mixture with air has been maintained to be 5, 7, 10 ppm for each one. The laboratory air is delivered by an oil-free compressor as is to be of normal humidity level in range of approx. 30–35 rel. %. The all gases have been supplied in a flow mode with 100 sccm rate. We have utilized only the data for NT array segments with resistivity less than 10 GOhm at maximum operating temperature of 400 °C. Therefore, 16 segments of the multielectrode chip have been considered in the reported data.The response of multisensor chip equipped with TiOSupplementary InfoVideo 1Video 2


Interestingly, we found that the apterous neurons required for female receptivity are neither doublesex nor fruitless positive suggesting that apterous neurons are not specified by the sex-determination cascade. Overall, these findings identify a neuronal substrate underlying female response to courtship and highlight the central role of walking speed in the receptivity behaviour.Courtship behaviours allow animals to interact and display their qualities before committing to reproduction. In fly courtship, the female decides whether or not to mate and is thought to display receptivity by slowing down to accept the male. Very little is known on the neuronal brain circuitry controlling female receptivity. Here we use genetic manipulation and behavioural studies to identify a novel set of neurons in the brain that controls sexual receptivity in the female without triggering the postmating response. We show that these neurons, defined by the expression of the transcription factor  During courtship, typically the male does the displays and the female decides whether or not to mateIn fruit flies, courtship represents a complex set of behaviours22446744510The behaviour of the female changes dramatically once she has copulated12121314161719202123fruitless2930spinster mutant mosaics revealed that loss of spinster function in VA1v olfactory projection neurons also leads to receptivity reductionFemale receptivity is thought to be dependent on the assessment of the male’s quality. In this respect, the detection of the male-specific pheromone cisVaccenyl acetate (cVA) and the male courtship song are particularly relevant for the female to evaluate the courting male. The courtship song is perceived in a subset of mechanosensory neurons of the auditory system that converge onto the antennal mechanosensory and motor centre (AMMC). More recently, a specific set of second order neurons dedicated to sensing the courtship song have been identified2526doublesex-expressing clusters of neurons, pC1 and pCd, were shown to be required for female receptivityAbdominal-B-expressing neurons controls pausing, which was identified as a discrete component of the female behaviour that signal a receptive state5Classic mosaic studies identified a dorsal region of the brain that needs to be genetically female for the animal to be receptiveapterous is required for female receptivity. The behavioural alterations associated with manipulation of apterous neurons are distinct from those observed in postmating responses, suggesting that they may be a component of virgin female receptivity circuit. We found that apterous neurons in the brain are required for the appropriate reduction of the walking speed in the presence of a courting male but not for the recognition of the presence of the male. We show that the lack of reduction in walking speed is a consequence rather then a cause of low receptivity. The apterous neurons in the brain involved in the phenotype are neither doublesex nor fruitless positive indicating that they are not part of a sexually differentiated circuit.Here we show that activity in neurons that are defined by the expression of apterous gene have low sexual receptivity3637apterous-positive neurons could play a role in female sexual behaviours. For this, we used an apterous enhancer trap line, apmd544 (hereafter referred to as apGAL4), which was previously shown to accurately report expression of the apterous geneKir2.1GAL80 (GAL80ts), that blocks Kir2.1 expression at lower temperature, 18 °C, but allows expression at higher temperatures (above 29 °C)apGAL4 neurons  mutant flies, that have no phototransductionKir2.1 in the MB using the OK107-GAL4 driver line and observed a wild-type phenotype  specifically in the brainapterous brain neurons silenced copulate less than controls . We observed that upon silencing of the apterous neurons outside of the brain, the flies remain receptive . The apterous-silenced virgin females did not show an increase in egg laying .To further dissect the velocity phenotype, we examined the progression of the velocity of the females in 100 s leading to copulationapterous-silenced females not copulating because they cannot reduce their speed or do they not reduce their speed because they are not receptive? To address this question we performed mating experiments in arenas where flies do not have room to walk away. The arenas are 4 × 5 × 6 mm  and doublesex (dsx) establish the neuronal circuits of sexually dimorphic behavioursapterous neurons in the central brain are fruitless or doublesex positive. We performed Doublesex immunostaining in female adult brains where GFP expression is driven by apGAL4 (apterous and Doublesex markers (n = 3). We also compared expression of apGAL4 and Doublesex in female pupal brains but no colocalization was observed (data not shown). Sex-specific Fruitless protein is only produced in males (FruM) as a consequence of sex-specific splicing48M but the P1 promoter involved in the expression of the sex-specific fru transcript, is transcriptionally active in females50fruGAL4 and fruLexA driver lines allow access to these cells5051fruLexA and apGAL4 neurons (Kir2.1 expression only in the neurons that coexpress fruLexA and apGAL4 (apterous neurons is not part of the circuitry in which fru P1 promoter is expressed.The transcription factors y apGAL4 . Surpris neurons . We saw d apGAL4 . This maapterous neurons. The splicing factors transformer (tra) and transformer-2 (tra2) work in conjunction to control female specific splicing of fruitless and doublesextra-2 gene exhibit male-like morphology and behaviour5455apterous neurons in the female .We further investigated the possibility of the sexual differentiation cascade specifying the e female . In otheapterous neurons are not directly part of a sexually differentiated circuit.Altogether these experiments indicate that Drosophila melanogaster, as in many other species, the decision of whether or not to mate is made by the female. However, we still have a very poor understanding about the behaviour displayed by the virgin female fly and the neuronal circuits underlying it. We set out to identify neurons involved in the response of virgin females to courting males. We show that female virgin flies with compromised activity in apterous neurons in the brain display a substantial reduction in copulation. What is specifically changed in the premating behaviour of apterous-silenced females? apterous-silenced females do not slowdown during courtship even though they do recognize they are in the presence of a courting male because they extrude the ovipositor. Ovipositor extrusion is a display that occurs exclusively in the context of courtship. A caveat of this work is the large number of neurons that are affected by this manipulation. Is the low copulation rate a result of an issue created by silencing large numbers of neurons? The capacity to recognize the male partner does not seem to be affected and the phenotypes we observed are revealed only in the context of courtship which strongly suggests that we specifically affected parts of the natural receptivity circuit.Reproductive behaviours are essential for the survival and fitness of the species. In apterous neurons affect female velocity during courtship? The best understood cue from the male that shapes female velocity is the songapterous-silenced females have impaired hearing. apGAL4 labels very faintly the region that is innervated by first and second order auditory neurons, AMMC. Third order auditory neurons innervate the ventral lateral protocerebrum (VLP). We silenced the VLP neurons (and a few other neurons) that express apterous and did not observe a receptivity phenotype (apterous and glutamatergic neurons). Moreover, flies with impaired hearing do not copulate within the time of our analysisapterous neurons are involved in integrating sensory cues provided by the male that would lead to a receptive state of the female and over time result in a decrease of female velocity.How can changes in activity of apterous neurons in controlling egg laying, another critical aspect of reproductive behaviour. Females that have mated and then have their apterous neurons in the brain inhibited lay very few eggs unlike control females. Classic gynandromorph studies pointed to an egg laying suppression focus in the braindoublesex-positive and female-specific PMN2 descending neurons in oviposition behaviourapterous neurons are neither doublesex positive nor descending, so it is reasonable to assume that we identified a novel set of neurons.We have also uncovered a role for apterous clusters and reveal how they interact with the sexual specification circuits.In conclusion, our findings contribute to understanding female receptivity and highlight the central role of female speed modulation during courtship. It will be interesting to elucidate in the future the role of the different apmd544 UAS-Kir2.1tub-GAL80TS UAS-CD8-GFP20xUAS-CD8-GFPUAS-AUG-DsRed ; Otd-nls:FLPo; UAS > stop > CD8-GFP63; UAS > stop > Kir2; fruLexA ; LexAop-CD2-GFP8xLexAop2-FLPLUAS-dcr-2UAS-tra-2IR elav-GAL80repo-GAL80; OK107-GAL4norpA36tub > stop > GAL80actin5C-GAL4VGlutMI04979-LexA:QFAD, GAD1MI09277-LexA:QFAD, ChaMI04508-LexA:QFADWild-type: Canton-S (CS) and Dickinson Lab (DL). Detailed genotypes can be found in the Flies were raised in standard cornmeal-agar medium at 25 °C and 70% relative humidity in a 12 h:12 h dark:light cycle, unless otherwise indicated. Fly strains and sources are as follows: For temporally restricted neuronal silencing experiments, female flies were raised at 18 °C for 6 to 12 days. Tester flies were subsequently incubated at 30 °C for 24 h, whereas control flies were maintained at 18 °C. Both control and tester flies were shifted to 25 °C, 24 h before the behavioural assay for acclimation and to prevent the effect of temperature treatment in the behaviour. Target males were wild-type CS flies aged for 3 to 8 days at 25 °C. Silencing experiments without temporal restriction were performed in female flies raised at 25 °C for 3 to 12 days.norpA mutant females, flies were aged for 3 to 6 days at 25 °C. Wild-type DL flies were used as background control in these experiments. Target males were CS flies aged for 3 to 5 days at 25 °C.For experiments with apterous neurons was achieved by knocking down endogenous Tra-2, which is required for the establishment of female specific morphology and neural circuitryactin5C-GAL4 driver. The females exhibited male-like external sexual morphology (data not shown). To improve the knockdown efficiency, the UAS-dcr-2 transgene was co-expressed with the short hairpin RNA. Flies were raised at 25 °C for 4 to 8 days. Target males were CS flies aged for 4 to 8 days at 25 °C.‘Masculinization’ of Females in groups of 5 were aged at 18 °C for 6 to13 days before the assay. Females were mated by adding 3 males to each group during ~7 h. Virgin and mated tester flies were subsequently incubated at 30 °C for 24 h, whereas control flies were kept at 18 °C. Both control and tester flies were incubated overnight at 25 °C for acclimation, before being transferred to a plate containing apple medium. Groups of five females were kept in the plate at 25 °C, with 70% relative humidity, for 24 h before eggs were counted.In plexiglass chambers: Virgin females and males were collected soon after eclosion and kept isolated in individual vials until behavioural experiments. Single females were gently aspirated into circular plexiglass chambers (16 mm in diameter ×4 mm height) and subsequently paired with the male. Individual pairs were recorded for 30 min using SONY HDR-CX570E, HDRSR10E or HDR-XR520VE video cameras . A white LED backlight was used as light source . A custom made software was developed to track the flies and automatically compute the time to copulation, whenever it occurred. All behavioural experiments were performed at 25 °C, with 70% relative humidity.Small arenas: The plexiglass chambers were adapted to generate the small arenas. A mold with the dimensions 4 × 5 × 6 mm was inserted in the chamber that was then filled with 2% agarose. Removal of the mold generated the small arena. The experiments followed the pexiglass chamber protocol above. The position of the flies provided by the custom made tracking software was used to calculate velocity.Conical shaped arena: We built an experimental setup consisting of a camera ) mounted above a conical-shaped arenaVirgin females were collected as soon as they ecloded, housed individually, and aged for 6–12 days at 18 °C. Temperature treatment was performed as stated above . Mated flies were treated as control virgin flies (kept at 18 °C). They were mated 24 h prior to the experiment, by incubating them with males for 1 hr. Immature virgin females were collected by aspiration and aged for 3 to 4 h at 25 °C before testing. Target males were naïve wild-type CS, aged for 4–8 days at 25 °C. For the experiments with paired females, target females were also wild-type CS, aged for 4–8 days at 25 °C.We used the Caltech FlyTrackerIn-house developed software was used to manually annotate the starting and ending time of each observed event of male courtship behaviour. We considered courtship behaviour moments when the male is either orienting toward the female, chasing the female, extending the wing, taping the female, licking the female or attempting copulation. The annotator was blind to the treatment of the video being annotated.The courtship index is defined as the fraction of time the male spent courting the female from the moment he initiates the courtship and up to 10 minutes or until copulation. Percentage of copulation is the percentage of flies that mated within the duration of the video .In-house annotation software was used to annotate the moments when the female extrudes the ovipositor. The annotator was blind to the treatment of the video being annotated. Ovipositor extrusion is the time the female spent extruding the ovipositor over the total courtship duration.Female mean speed was calculated by averaging the speed within courtship and outside of courtship periods for each video. For the analysis we used the videos in which the courtship and non-courtship periods totalled at least 30 seconds in each one of them. Considering that the bulk of the female pausing bouts are less than one second long , 30 seco2, as reported previouslyPausing frames were defined as the frames in which the fly speed was less than 4 mm/s and the angular acceleration less than 15 rad/sThe speed before copulation was normalized by subtracting the mean speed of the 120–100 s period before copulation and dividing by the standard deviation within the final 100 s, as reported previouslyWe classified as wing extension wing angles above 60 degrees relative to the body axis. Wing extension index is the total number of frames where wing extension occurred over number of frames of courtship.Tissues were dissected in phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde in PBL (PBS and 0.12 M Lysine) for 30 min at room-temperature, washed 3 times for 5 min in PBT (PBS and 0.5% Triton X-100) and blocked for 20 min at room temperature in 10% normal goat serum in PBT . Subsequently, tissues were incubated with the primary antibodies in blocking solution, for 72 h at 4 °C. The following primary antibodies were used: rabbit anti-GFP ; mouse anti-GFP 3E6 ; mouse anti-nc82 ; mouse anti-Dsx  and SciPy 0.18. The statistical analysis for the locomotor and annotated behaviours was performed with SciPy 0.18.How to cite this article: Aranha, M. M. et al. apterous brain neurons control receptivity to male courtship in Drosophila melanogaster females. Sci. Rep.7, 46242; doi: 10.1038/srep46242 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Background and Aims Fluctuations in [CO2] have been widely studied as a potential driver of plant evolution; however, the role of a fluctuating [O2]:[CO2] ratio is often overlooked. The present study aimed to investigate the inherent physiological plasticity of early diverging, extant species following acclimation to an atmosphere similar to that across the Triassic–Jurassic mass extinction interval , a time of major ecological change.Methods Mature plants from two angiosperm (Drimys winteri and Chloranthus oldhamii), two monilophyte  and one gymnosperm (Ginkgo biloba) species were grown for 2 months in replicated walk-in Conviron BDW40 chambers running at TJB treatment conditions of 16 % [O2]–1900 ppm [CO2] and ambient conditions of 21 % [O2]–400 ppm [CO2], and their physiological plasticity was assessed using gas exchange and chlorophyll fluorescence methods.Key Results TJB acclimation caused significant reductions in the maximum rate of carboxylation (VCmax) and the maximum electron flow supporting ribulose-1,5-bisphosphate regeneration (Jmax) in all species, yet this downregulation had little effect on their light-saturated photosynthetic rate (Asat). Ginkgo was found to photorespire heavily under ambient conditions, while growth in low [O2]:[CO2] resulted in increased heat dissipation per reaction centre (DIo/RC), severe photodamage, as revealed by the species’ decreased maximum efficiency of primary photochemistry (Fv/Fm) and decreased in situ photosynthetic electron flow (situJ).Conclusions It is argued that the observed photodamage reflects the inability of Ginkgo to divert excess photosynthetic electron flow to sinks other than the downregulated C3 and the diminished C2 cycles under low [O2]:[CO2]. This finding, coupled with the remarkable physiological plasticity of the ferns, provides insights into the underlying mechanism of Ginkgoales’ near extinction and ferns’ proliferation as atmospheric [CO2] increased to maximum levels across the TJB. The end of the Triassic marked the beginning of a period of geological and ecological upheaval known as the Triassic–Jurassic mass extinction event (approx. 200 Mya). Although several studies have questioned the high rates . Plant c2 to be tracked at high resolution across the TJB, revealing a 2·5- to 4-fold increase at the end of the Triassic , every change in the relative abundance of :[CO2] air mixture.CO2 partial pressure (A–Ci curves) were recorded within the chambers with a CIRAS-2 gas analyser (PP-Systems) attached to a PLC6(U) cuvette fitted with a 4·5 cm2 measurement window and a red/white light LED unit. Theoretically, measuring within the TJB chambers at 16 % O2 could introduce error in our measurements due to band-broadening effects; however, it has been shown that these effects are negligible even when using O2-free air  and intercellular COfree air . MeasureO. claytoniana, C. australis and C. oldamii) or 1600 (G. biloba and D. winteri) μmol m–2 s–1 until full photosynthetic induction, as judged from three consecutive stable readings of CO2 assimilation (A) and stomatal conductance (gs), usually within 30 min. The CO2 and O2 concentrations used were identical to the corresponding growth values for each treatment . Light levels were then adjusted from 1200/1600 to 20 μmol m–2 s–2 in nine/ten descending steps, each witha 3 min duration, which was always adequate to obtain stable A readings. The light-saturated photosynthetic rate (Asat) and saturating light intensity were then calculated according to For the light response curves, tagged leaves from 3–4 plants per species and treatment were enclosed in the cuvette and illuminated at either 1200  and saturating light intensity  for 30 min. CO2 concentration in the cuvette (Ca) was then stepwise decreased from 400 to 50 μmol mol–1  and then increased from 50 to 2000 μmol mol–1 . Relative stability of Ca and A values at each step typically took 4 min, while a close agreement between the two measurements taken at 400 μmol mol–1 indicated that exposure to low Ca had not affected the activation state of Rubisco (VCmax) and the maximum electron flow rate supporting RuBP regeneration (Jmax). Respiration in the light (Rd) was also calculated as the y-axis intercept of the A–Ci response curve. We have to note here that the VCmax and Jmax values reported in our study are adjusted at 25ºC using the temperature functions of A–Ci curves obtained using dried leaves of each species were used to correct all measurements for CO2 leakages (For the  Rubisco . The resQ) for each species was measured with an MQ-200 quantum sensor  and was then used to calculate the in situ electron transport rates from the non-rectangular hyperbola that describes the relationship between photon flux and electron transport  and is calculated as:Q is the incident photosynthetically active radiation, abs is the leaf absorptance and ΦPSIImax is the maximum efficiency of primary photochemistry. A common absorptance value of 0·85 was used in our calculations . Average values of Q, Fv/Fm and Jmax were used for the calculation of situJ at the species and treatment level.Incident growth light intensity (ransport  as:(1)Je of 0·7 , Jmax isImax×0·5 where Q ulations , and ΦPSIn situ rates of ribulose 1,5-bisphosphate (RuBP) oxygenation (VO) and carboxylation (VC) were calculated from the corresponding light curve recordings (i.e. the step of the light curve with an intensity close to that received by each species in situ) according to 2 concentrations (Cc) were calculated from the corresponding Ci values as:gm) was calculated using the constant J modelling method of Ci values between 50 and 120 Pa) of each A–Ci response curve were used to calculate the photosynthetic linear electron flow rate (J) as:J is constant when A is limited by the regeneration rate of RuBP, the gm value that minimizes the variance in J was calculated iteratively using the Solver Microsoft Excel add-in  and depends on the temperature-sensitive relative affinity of Rubisco for CO2 and O2. Γ* is considered to be relatively conserved among C3 species and is linearly correlated with O2 concentration; thus, we assigned standard values of 3·16/2·41 Pa at 20 °C and 21 %/16 % O2, respectively, for all five species in our study (a fluorescence transients. Saturating light (approx. 3500 μmol m–2 s–1) was provided by a single high intensity red LED (peak at 627 nm), and chlorophyll fluorescence values were recorded from 10 μs to 1 s. Data acquisition rates were 105, 104, 103, 102 and 10 readings per second in the time intervals of 10–300 μs, 0·3–3 ms, 3–30 ms, 30–300 ms and 0·3–1 s, respectively. The cardinal points of the recorded polyphasic fluorescence kinetics  were then used to derive the following parameters according to the JIP-test  was used to capture their fast chlorophyll JIP-test :Fv/Fm = (Fm – Fo)/Fm or maximum quantum yield of primary photochemistry. Fv/Fm is a sensitive indicator of stress conditions with typical values of around 0·83 for healthy plants ((1) DIo/RC = (ABS/RC) – (TRo/RC) is the heat dissipation per reaction centre at time zero.(2) ABS/RC = (Mo/Vj)/(1 – Fo/Fm) is the absorption energy flux per PSII reaction centre.TRo/RC = Mo/Vj is the trapping per reaction centre at time zero.andMo = 4 × (F300 ms – Fo)/ (Fm – Fo) is the slope at the origin of the fluorescence rise.Vj = (F2 ms/Fo)/Fm/Fo) is the relative variable fluorescence at 2 ms.where:Statistical analysis was performed in R (v·3·1·1). Data were tested for normality and equal variance, and analysed using mixed effects models. Chamber identity was treated as a random effect to identify a possible chamber effect (no chamber effect was detected). Multiple models were run with random and interaction effects. Models were compared using analysis of variance (ANOVA) comparison, and the best fit model was determined using the Akaike information criterion (AIC). One-way ANOVA and Tuckey post-hoc analysis were performed to assess the significance level of the differences between all fixed effects (i.e. species and treatments) from the best fit model.Asat of the test species did not acclimate uniformly to low [O2]:[CO2] . We should note here that Ginkgo exhibited a decrease in its light-saturated photosynthetic rate despite the fact that light curves for all species were taken at growth CO2 concentration, i.e. nearly 5-fold higher CO2 concentration for the TJB treatment plants compared with controls.Light response curves indicated that the 2]:[CO2] . Even th2]:[CO2] was confirmed by the results of the A–Ci curves  did not display significant changes, despite the decrease in VCmax, due to the much higher growth CO2 concentration of the TJB treatment (in situ oxygenation rates of Rubisco (VO) diminished due to the very low [O2]:[CO2] ratio of the growth air mixture in combination with the altered absolute concentrations of both gases  and relative (VO/VC) rates of oxygenation primarily as a result of its extraordinarily high rates of Rubisco oxygenation under ambient conditions (gt) from the measured values of gs and gm revealed that under ambient atmospheric CO2G. biloba poses substantially higher resistances to CO2 diffusion and its photosynthetic machinery operates under significantly lower Cc compared with the rest of the test species . Indeed, situJ values followed a similar pattern, showing small, non-significant changes between control and TJB plants in ferns and C. oldhamii, moderate decrease in D. winteri, a substantial decrease in G. biloba /VOamb] and the decreases of Rubisco content-dependent VCmax when plants are exposed to the TJB atmospheric treatment  (2]:[CO2]-acclimated plants.Exposure to the TJB treatment led to plant group-specific changes in Fv/Fm . Compareu and VO . Further(DIo/RC) . This clGinkgo biloba diverts an extraordinarily high percentage of the ATP and NADPH produced during the photosynthetic light reactions to photorespiratory metabolism when grown under ambient CO2 and O2 concentrations  compared with the rest of the species, with ferns and angiosperms only showing negligible changes  and the maximum efficiency of primary photochemistry (Fv/Fm) under nearly non-photorespiratory conditions further highlights their ability to acclimate their physiology effectively to low [O2]:[CO2] (Our results do not allow us to identify the increased rate of photorespiration in and Jmax  were cout, trend ; Fig. 1.2]:[CO2]  and 7.2-induced decreased transpiration of the broadleaved Ginkgoales must have played a role in their decline across the TJB (2 levels, gymnosperms still display relatively low values of stomatal and mesophyll conductance, even though higher values would have enabled them to maintain higher photosynthetic rates and/or higher photosynthetic nitrogen use efficiency (G. biloba to a TJB treatment are likely to resemble those of Ginkgoales present during the TJB. Overall, our study stresses the importance of differences in the physiological plasticity of different plant groups in shaping evolutionary patterns under fluctuating atmospheric O2 and CO2. Thus, in light of ongoing rapid increases in atmospheric CO2 levels, we argue that further investigation of the innate physiological characteristics and constraints of different plant groups are of considerable significance as we are likely to be on the verge of major shifts in the composition of plant communities.We acknowledge that an increase in global temperature and high atmospheric COhttps://academic.oup.com/aob and consist of the following. Table S1: classification, size of pots, compost mix and fertilizer used for each of the studies. Table S2: mean values ± s.d. of growth conditions in the chambers used in the study.Supplementary DataClick here for additional data file.


This, in turn, has serious consequences for speleothem-based fluid inclusion research as closed system transformation of ACC to calcite may induce a negative oxygen isotope shift in fluid inclusion water, resulting in deterioration of the original compositions. ACC formation increases the speleothems’ sensitivity to alteration as its interaction with external solutions may result in the partial loss of original proxy signals. Mineralogical analysis of freshly precipitating carbonate at the studied speleothem site is suggested in order to determine the potential influence of ACC formation.Speleothem deposits are among the most valuable continental formations in paleoclimate research, as they can be dated using absolute dating methods, and they also provide valuable climate proxies. However, alteration processes such as post-depositional mineralogical transformations can significantly influence the paleoclimatic application of their geochemical data. An innovative sampling and measurement protocol combined with scanning and transmission electron microscopy, X-ray diffraction and Fourier transform infrared spectroscopy is presented, demonstrating that carbonate precipitating from drip water in caves at ~10 °C contains amorphous calcium carbonate (ACC) that later transforms to nanocrystalline calcite. Stable oxygen isotope fractionations among calcite, ACC and water were also determined, proving that ACC is  Speleothems (cave-hosted carbonate deposits) are among the most important climate archives, as their formation age, texture and geochemical composition provide information on temperature, amount of precipitation, and even seasonality, i.e. changes in the relative amount of precipitation during cold and warm seasons234et al.et al.18O values) of water trapped in the fluid inclusions of selected speleothems of the Baradla cave system shows that, whereas hydrogen isotope values reflect the composition of the cave drip water from which the carbonate precipitated, the oxygen isotope data of the inclusion-hosted water display increasingly negative shifts. The observed negative δ18O(water) shift starts from the stalagmite’s surface and increase inwards, towards the older layers. Although Demény et al.7818O shiftA recent study2 and NaCO3124)2CO3Detection of ACC is rather difficult in cave deposits, as ACC can undergo transformation to calcite in minutes in a hydrous environment, and even stabilizing compounds like Mg or organic matter101213vs. calcite precipitation was approached via the application of an innovative sample collection method in which transmission electron microscopy (TEM) grids and silica wool are placed directly under the dripping water in the studied caves. These sample holders were able to collect enough carbonate precipitate for mineralogical and isotopic analyses in relatively short times . This had the effect of allowing direct analyses without further sample concentration. Evidence is presented for ACC formation in natural cave environments and an estimate for calcite-ACC oxygen isotope fractionation at the studied cave temperature  is provided.In the present paper, the issue of ACC 2 content of the cave air. These are listed in 3 was occasionally deposited in globular forms . The wide peak centered on 1635 cm−1 indicates molecular water−1 is consistent with spectra obtained for clay mineralsFTIR spectroscopy, micro-focus XRD and TEM were used in order to characterize the structure of the carbonate precipitate. The FTIR spectra  obtainedThe same grid that yielded the characteristic ACC FTIR spectrum was also analysed by means of micro-focus XRD. A 2Θ range of 20 to 50° was selected because this range is uniquely capable of defining ACC113 film consisting of 10–30 nm size rounded clusters . Lime may act as a proxy for ACC as interaction between the electron beam and ACC may release CO2, resulting in the formation of limeTEM analysis of the fresh precipitates showed either a thin (10–40 nm), commonly hollow, CaCOclusters  or 20–40clusters , similar3, while this form had disappeared after 3 weeks, being replaced by porous, crystalline carbonate  exceeds the sum of molar volumes of calcite (36 cm−3 mol−1) and water (18 cm−3 mol−1)3 polymorphs (line vaterite) may also be formed during the transformation process, depending on pH and Mg concentration in the parent solutionFTIR analyses of the stored grid with recrystallized carbonate match with that of calcite, no ACC-like spectra were observed. The formation of porous calcite is in accordance with the transformation of ACC to calcite. Since the ACC contains up to one H−1 deposition rates, i.e., deposition of 800 to 2700 nm thick carbonate layers per day. At such deposition rates 10–30 nm sized ACC particles can be trapped in the precipitated carbonate layer that precludes interaction with the ambient solution resulting in closed system ACC-calcite transformation. However, heterogeneous carbonate precipitation on the rugged stalagmite surface and temporal variation of the deposition rate produces spatially variable carbonate formation and hence can leave a part of the ACC uncovered. Hence, the most plausible assumption is that the ACC-calcite transformation during stalagmite formation at the study site takes place both in closed and open systems.In general, during stalagmite formation, ACC-calcite transformation may occur in a closed or open system. If the ACC particles are on the stalagmite surface in contact with drip water during the transformation, then we can assume a fully open system, and hence the calcite attains equilibrium with the ambient water through a dissolution-reprecipitation process. If the precipitated ACC particles are covered by the subsequent carbonate layers , then the transformation may proceed in a closed system, through solid phase recrystallization. The possibility of ACC entrapment can be evaluated in the light of ACC particle sizes and the rate of carbonate deposition. If the thickness of the carbonate layer formed during the period of ACC transformation significantly exceeds the size of ACC particles, then the latter can be – at least partially – embedded. As ACC was detected in the freshly precipitated carbonate several days after the collection, we can assume that the transformation period is extended to days. Carbonate deposition rates can be estimated from the sizes (5 and 7 cm in height) and formation ages  of actively forming stalagmites (NU-1 and NU-2) at the sampling site2 degassing and H2O evaporation related to strong ventilation can significantly affect the stable C and O isotope compositions of the precipitating carbonate18O values. The potential influence of degassing and evaporation at the study site can be determined by conducting the so-called Hendy test18O shifts. This observation does not imply that the carbonate precipitated in thermodynamic equilibrium with the drip water, but it shows that the degassing/evaporation effect may be considered negligible at the site and hence its influence on the carbonate–water system can be excluded.In order to evaluate the influence of ACC formation on the use of stable oxygen isotope compositions in speleothem research, calcite-ACC oxygen isotope fractionation was determined by measuring the carbonate content of grids and silica wool placed directly beneath dripping water. Since the carbonate precipitated on the copper grids and silica wool is a mixture of calcite and ACC, the oxygen isotope composition of the calcite component should be determined along with the measurement of water composition. Hence, the stable isotope composition of the outer, ~3 mm thick part of a recently forming stalagmite (sample NU-2) collected at the Baradla Cave19202118O values of the NU-2 stalagmite calcite (23.4‰) and the drip water (−9.5‰) yields a site-specific fractionation value of 32.7 for the longer term (about 2 years) cave temperature (9.7 °C). Slight variations in cave temperature and drip water composition at the sampling site, however, may influence the oxygen isotope composition of the precipitating carbonate, hence current conditions prevailing during sample collection should be used for the evaluation. The theoretical oxygen isotope composition of the calcite component of the carbonate sample can be calculated from formation temperature and drip water composition, provided that the calcite-water oxygen isotope fractionation equation is known. Although a pioneering experimental study determined the temperature-fractionation relationship for the calcite-water system almost 50 years ago2625281930313222The δThe other selected study18O/16Occ/18O/16Owater, by definition:Since the α fractionation factor is the ratio of where “cc” stands for calcite.18Occ values of 23.8‰, 23.7‰ and 23.1‰ for the Baradla Cave grids, silica wool and the Pál-völgyi Cave silica wool samples, respectively. The composition of the ACC component in the sampled carbonate could be determined by simple mass balance calculation as follows, provided that the ACC amount in the precipitating carbonate is known.Weighting et al.cc), whereas the 55 nm average size (Savg) at the stalagmite surface would be determined by the size and the amount of ACC-derived nanocrystalline calcite  according to a simple mass balance equation:The amount of the ACC component can be estimated on the basis of crystallinity data of actively forming stalagmites. Demény cc = 70 nm, Savg = 55 nm, SACC = 10–40 nm) determine the amount of the nanocrystalline fraction (XACC) that ranges from 0.25 to 0.5. Since a part of this size fraction can be precipitated originally as nanocrystalline calcite, the 25–50% range represents an upper limit to the ACC amount estimation. The ACC amounts and the theoretical calcite composition (δ18Occ) can be used to calculate the isotopic composition of the ACC fraction (δ18OACC) using the equationThe size estimations (SACC = 0.25 to 0.5) in 18O difference of 2.4 ± 0.8‰ is obtained. As a part of the nanocrystalline fraction may also be precipitated originally as calcite, the XACC values are maximum estimations, and lower XACC values would yield larger calcite-ACC δ18O differences. An additional source of uncertainty derives from the partial transformation of ACC to calcite during the storage period between sample collection and isotope analyses. Although the low temperature in the refrigerator (2–5 °C) precludes solid-phase carbonate-water oxygen isotope exchange, structural reorganization during calcitization may also be associated with isotope exchange between the newly formed calcite and ambient water (containing the released hydration water and the ambient H2O vapor). At such low temperatures, calcite-water isotope fractionation is elevated relative to the studied cave’s temperature (~10 °C)18O-enrichment in the calcite. The presence of such newly formed calcite would mean that the original carbonate sample should have had lower δ18O value than measured, and consequently the calculated calcite-ACC difference should be larger. Transformation of ACC to calcite during sample storage also leads to the assumption that the calcite-ACC difference (~2.4‰) represents a lower limit.For the Baradla grid and silica wool samples, the calcite end-member composition calculations  yielded 23.8 and 23.7‰, whereas the Pál-völgyi Cave site yielded 23.1‰. Substituting the 25–50% ACC amount in the carbonate mixture  ACC rapidly transforms to calcite during the preparation and (ii) laboratory ACC synthesis requires physical and chemical conditions distinctly different from those found in a cave environment. Available estimations of δ dolomite. These e18O-depleted ACC and its subsequent transformation into calcite can also exercise an especially significant effect in fluid inclusion research, inducing oxygen isotope re-equilibration between the host carbonate and water trapped in fluid inclusions18O values in order to achieve the larger calcite-water fractionation relative to the ACC-water value. As discussed by Demény et al.18O values for inclusion-hosted water, whereas the oxygen isotope compositions of inclusion-hosted water obtained for samples from cold/temperate regions are generally not appropriate for paleotemperature calculations18O shifts in the inclusion water18O-depleted ACC formation in caves at about 10 °C. Since the δ18O value of inclusion-hosted water may carry significant paleoclimatic/paleohydrological information, it is important to note that its use is limited by the cave environment.As a general rule, speleothems with well developed lamination and compact texture  are selected for paleoclimate analyses362342− and organic compounds in the solution101115383912131417A number of experimental studies have shown that the formation and stability of ACC may be influenced by the physical parameters of the ambient environment and the chemical compositions of the parent solutions. In natural cave environments the most important factors might be the cave temperature, drip water pH, as well as concentrations of Mg, SOThe formation of ACC was detected in carbonate samples precipitated from drip water in cave environments monitored for temperature, drip water pH, conductivity and stable isotope compositions. In order to capture and analyse very small amounts of carbonate without further sample concentration, Cu grids used for TEM and quartz wool were placed under drip water for times ranging between 24 hours and two weeks at two cave sites . Globular forms of carbonate were observed on the copper grids using SEM; these then transformed into porous carbonate after several weeks. FTIR spectra, X-Ray powder diffraction and TEM analyses of freshly precipitated carbonates collected on TEM Cu grids showed the characteristic features reported for synthetic ACC precipitates. ACC occurred as films containing clusters 10–30 nm in size and as particles 10–40 nm in size. It may be assumed that this material is capable of serving as a precursor of nanocrystalline calcite found in the outermost layer of recently formed speleothemsThe stable oxygen isotope compositions of the freshly precipitating carbonate, stalagmite calcite and host drip water were used to infer oxygen isotope fractionation between calcite and ACC. The calcite-ACC isotope fractionation estimation obtained in this study >2.4 ± 0.8‰) is in agreement with those obtained from the data contained in earlier publications‰ is in a2 content in cave air were monitored at both locations and water samples were collected. Cave air and water temperatures were measured using a GMH-3710 type Pt-100 thermometer digital puncture  with a precision of ±0.03 °C. CO2 contents in cave air at 150 cm from the ground were determined using different NDIR instruments for low  and high  levels. Electrical conductivity and pH values of dripping water and cave river waters were measured within 1 minute after sampling using a COMBO PH & EC instrument  with precisions of 2% and 0.05, respectively. Plastic funnels holding Cu grids and silica wool over a filter were placed under the dripwater at both caves. Grids and silica wool samples were collected from 24 hours to two weeks. The samples were immediately placed in a cold box and transported to the laboratory, where they were stored in a refrigerator at 2–5 °C. According to literature data, low temperature can help stabilizing ACCFresh carbonate precipitate was collected in two caves, the Baradla Cave in the northeastern part of Hungary, and the Pál-völgyi Cave in Budapest, central Hungary. Temperature, pH, conductivity, and COX-ray powder diffraction (XRD) analysis of stalagmite carbonate (sample NU-2/1) was performed using a Philips PW-1730 diffractometer (PW-1820/00 goniometer) equipped with a graphite monochromator using Cu-Kα radiation at 45 kV and 35 mA with 1° divergence slit and 1° receiving slit. Scanning rate was 0.05° 2Θ per minute from 3° to 70°. Micro-focus X-ray diffraction measurements were made by using D/MAX RAPIDII type diffractometer and RINTRAPID software at the Institute for Geological és Geochemical Research (IGGR), Budapest. Measurement conditions were 50 kV accelerating voltage, 0.6 mA current, using a 800 micrometer beam collimator. The analysed Cu grid (grid #2) was held in a fixed position during measurement. The 2DP software was applied to convert the Debye-rings into 2theta intensity values with 0.01 steps.−1 spectral range with a resolution of 4 cm−1.FT-IR (Fourier-transformation infrared) analyses were carried out using a Bruker Vertex 70 IR spectrometer equipped with a Hyperion 2000 microscope with video-controlled visualization and automatized sample stage operated by OPUS 7.2 software, at the IGGR, Budapest. The measurements were performed in transmittance mode using a 15X objective. For each sample 32 scans were recorded in the 4000–400 cmTEM studies of the samples collected on the Cu grids were carried out in a Philips CM20  type transmission electron microscopes at Institute of Technical Physics and Materials Science of the Centre for Energy Research, Budapest. Bright-field images and selected area diffraction (SAED) patterns were recorded on CCD camera (Morgagni microscope) and image plates (Philips microscope). Radial distributions of intensities were generated from SAED patterns following the method described by Lábár3PO4 at 70 °C in an automated GASBENCH II preparation unit2 yields were variable. The sample signals were monitored and samples below 50 μg and above 200 μg were excluded in order to match the standards’ signal  and to avoid linearity related isotope shifts. Stable carbon and oxygen isotope compositions were determined on the evolved CO2 and are expressed as δ13C and δ18O in ‰ relative to V-PDB and V-SMOW, respectively. Precision and accuracy of stable carbon and oxygen isotope compositions were tested by analysing international standards that yielded the following compositions. NBS-18: δ13C = −5.03 ± 0.06‰, δ18O = −23.29 ± 0.07‰ (n = 16); NBS-19: δ13C = −1.92 ± 0.07‰, δ18O = −2.22 ± 0.04‰ (n = 16); LSVEC: δ13C = −46.60 ± 0.16‰, δ18O = −26.60 ± 0.08‰ (n =17), all of the values within 0.1‰ from the certified compositions reported by the International Atomic Energy Agency. Stable oxygen isotope compositions of drip water samples  were determined using a liquid water isotope analyser (LWIA) manufactured by Los Gatos Research Ltd, model LWIA-24d with an analytical precision of 0.1‰Carbonate-bearing Cu grids and silica wool samples were divided to several sub-samples and placed in borosilicate exetainers, flushed with helium and reacted with pure HHow to cite this article: Demény, A. et al. Formation of amorphous calcium carbonate in caves and its implications for speleothem research. Sci. Rep.6, 39602; doi: 10.1038/srep39602 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.