Considering the pivotal role that creative ideas play in human societies, and creativity's contribution to multiple aspects of human life, understanding the cognitive components underlying creativity has become increasingly fundamental. Since the Five-Stages Model of the creative process proposed by Wallas , creativZedelius and Schooler); imagination of activities over a long period of time, relevant especially for actual creative achievements in science and writing ; as well as ‘looking at nothing’ and blinking behaviors, that do not necessarily involve visual imagery . In addition, we explored the relationships between different creative objects' production and artistic drawings with different mental imaging processes .In the present Frontiers in Cognition Research Topic 11 novel publications were collected: 8 Original Research Articles, 1 Review, and 2 Perspective Articles. From the beginning, the Research Topic was planned as a collection of studies exploring the relationships between creativity and mental imagery. Mental imagery is a representational medium for providing researchers access to thoughts, symbolization, and combination of elements, possibly facilitating the emergence of new ideas and creativity. In this direction, different aspects of mental imagery were considered which could increase or explain the emergence of creativity: daydreaming styles ; or the relationships between flexibility of divergent thinking (the number of categories encompassing the relevant responses) and attentive processing . Interestingly, the relationships between convergent thinking involving insight and intelligence , and working memory updating . In addition, neural correlates of creativity were investigated. Chavez highlighted the key role of brain areas involved in motor imagery on highly creative individuals, whereas Boccia et al. showed that general creativity relies on multi-componential neural networks supporting executive functions, whereas domain-specific creativity  roughly depends on different functional specialized brain regions.We also collected studies that investigated distinct and peculiar aspects of creativity and its cognitive components, such as: the equal-odds rule of divergent thinking, also known as the relationships between fluency (the number of responses) and creativity as assessed by independent judges (Jankowska and Karwowski), aimed at assessing three components of creative imagination: vividness of imagery, originality of responses, and transformative imagery ability; and the Artistic Creativity Domains Compendium (Lunke and Meier), aimed at measuring artistic creativity in visual arts, performing arts, literature and music.Finally, two different tests recently developed have been reported: the Test of Creative Imagery Abilities  might help to better understand the cognitive and neural correlates of creativity and the relationships among creativity and other cognitive domains, such as mental imagery, attention, and problem solving. We hope that the papers included in this Research Topic can help to stimulate more studies on these topics and in increasing research in the field of creativity.MP: Planned the topic and edited the most of papers included in the topic. LaP: Edited some papers included in the topic. RN: Edited some papers included in the topic. LiP: Edited some papers included in the topic. CS: Edited some papers included in the topic. CG: Edited some papers included in the topic.This work was supported by NIH [grant number T32 NS047987] to CS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We investigate the formation and evolution of Au–CO single-molecule break junctions. The conductance histogram exhibits two distinct molecular configurations, which are further investigated by a combined statistical analysis. According to conditional histogram and correlation analysis these molecular configurations show strong anticorrelations with each other and with pure Au monoatomic junctions and atomic chains. We identify molecular precursor configurations with somewhat higher conductance, which are formed prior to single-molecule junctions. According to detailed length analysis two distinct types of molecule-affected chain-formation processes are observed, and we compare these results to former theoretical calculations considering bridge- and atop-type molecular configurations where the latter has reduced conductance due to destructive Fano interference. The break junction method is widely used to establish single-molecule nanowires –2. DurinDuring the rupture conductance traces are recorded, i.e., the conductance of the breaking wire is measured as a function of electrode displacement. By repeating the break-junction measurement several thousand times a statistical ensemble of conductance traces are collected, from which a conductance histogram can be plotted. Peaks in the histogram reflect the conductance of typical junction configurations, such as single-atom contacts. After the addition of molecules the formation of single-molecule nanowires is signalled by the appearance of new peaks in the histograms –2. HowevIn this paper we investigate the formation of Au–CO–Au molecular junctions and show that in the presence of CO two new molecular configurations are formed. To identify them we carry out a statistical analysis going far beyond the simple conductance histogram approach. By combining conditional conductance histograms, two-dimensional correlation histograms (2DCH) –27 and tT = 4.2 K is shown in G0, with smaller peaks at higher conductance, typical for low-temperature measurements on Au junctions [18G0 corresponds both to monoatomic junctions and to atomic chains.The conductance histogram of clean gold junctions at unctions ,32–33. Iunctions –1834–36.G0 at 0.17G0 and 0.67G0. This histogram shows similarities to former measurements on Au–CO junctions [G0), and in [G0 are reported, whereas in our measurement these two peaks coexist.After characterizing clean Au junctions CO molecules are dosed to the junction through a heated metallic tube . The conductance histogram of Au–CO junctions is shown in unctions –38, thouunctions  only theG0, brown) and M2  regions correspond to the two molecular configurations. The region of monoatomic Au junctions and chains is marked by A1 , whereas configurations with a little higher conductance  are labeled by A2. The latter region is attributed to precursor molecular configurations, as discussed later and in [To simplify further analysis we define separate conductance regions marked by different colors in r and in . To gainr and in ,25–28.The two dimensional correlation histogram (2DCH) investigates the correlations between different conductance regions. Configurations that tend to appear together during the breaking process induce positive correlations in the 2DCH, whereas configurations excluding each other along a contact rupture induce negative correlations. It can also happen, that both configurations appear, but the lengths of the plateaus within the studied conductance bins exhibit distinct correlations. More details on the technique can be found in –26.The 2DCH for Au–CO–Au junctions is shown in G0 (A2), shows positive correlation with both molecular configurations M1–M2. Between the two molecular configurations M1 and M2 an anticorrelation is observed.The 2DCH in Whereas 2DCHs are useful to make a two dimensional map of the relevant correlation effects, to gain a more quantitative picture about the nature and strength of correlations between different regions it is also useful to investigate conditional histograms. Conditional conductance histograms are conductance histograms for breaking traces selected according to a predefined condition. Here, we focus on conductance traces, which are long enough in a predefined conductance region. To construct these histograms, we have determined the average length of breaking traces in the predefined region and selected the conductance traces longer than average. Conditional histograms are normalized to the number of traces included, therefore direct comparison of them is possible. We note here, that the selected data sets obtained by this technique are not distinct, because the length of a given trace can be longer than the average in more regions (positive correlation). More details on the method can be found in ,26–27.G0 are observed. According to the conditional histogram these traces do not show molecular configurations, i.e., the corresponding negative correlation in the 2DCH between A1 and M1–M2, is a strong feature: The A1 and M1–M2 configurations practically exclude each other. A similar feature is seen from the other side as well: The conditional histogram selected for the molecular region M1–M2 (green line) exhibits the almost complete suppression of the peak at 1G0. This conditional histogram shows an additional interesting feature, namely the enhanced weight in the A2 region compared to the total histogram, which was also reflected by the positive correlation between M1–M2 and A2 in the 2DCH . All these imply that Au–CO–Au single-molecule junctions are usually not preceded by clean Au monoatomic contacts or atomic chains. There is rather a so-called precursor configuration with higher conductance (A2 region), similarly to our previous report on Ag–CO junctions [Conditional conductance histograms selected for different conductance regions are shown in unctions . We inteTo investigate the relation of the two molecular configurations to each other we have selected the opening traces, where the molecular configurations have been formed  and on one trace only one out of these three configurations is pronounced with a length up to 5–6 Au–Au unit. This means that three distinct chain formation processes are observed: (i) pure Au monoatomic chains, (ii) molecule affected chains with M2 conductance, (iii) molecule affected chains with M1 conductance. According to our analysis there is no significant transition between these three types of chains.G0), where the CO molecule sits between two neighboring Au chain atoms in a perpendicular direction with respect to the contact axis  [As an alternative we consider the so-called atop geometry reported in –30, wherG0) –30 agreeSimilarly to our previous study on Ag–CO junctions the A2 precursor configuration is considered as a CO molecule bound to the side of a dimer Au junction see 4.We also note that the M2 configuration exhibits a clear positive slope of the conductance plateau see , which aIn this paper we have investigated the formation and evolution of Au–CO single-molecule junctions. To get more insight into the junction formation we used different statistical analysis methods, such as correlational analysis, 2D conductance–displacement histograms, and conditional histograms. The combination of these techniques can reveal information far beyond simple conductance histogram measurements. We have observed the formation of two molecular configurations, which can stretch over several atom-atom distances, forming molecule-decorated atomic chains. The evolution of Au–CO atomic chains is in clear contrast to our previous report on Pt–CO molecule-affected chain formation. In the latter case the molecular configuration with higher conductance (perpendicular CO) is transformed to the molecular configuration with lower conductance  along the chain formation. In the present experiments, however, no transition is observed between the two distinct types of molecule-decorated chains. This behaviour is naturally explained if the low conductance molecular configuration is not a derivative of the high conductance one, rather it is a completely different structural arrangement. The two simulated molecular configurations in –30 mightThe measurements were performed with a custom-built MCBJ setup at liquid helium temperature and the CO molecules were dosed with a custom-built vacuum system from a high purity container through a heated tube. The detailed description of our experimental technique is introduced in our previous publication .We have performed our measurements on one break-junctions sample that was measured for several weeks as follows. In order to exclude unwanted contamination we have a very strict protocol for this type of measurement. First we bake out and pump the sample holder for approximately one week. Afterwards we cool down the sample holder and record clean histograms. As a next step we precisely mimic all steps of molecule dosing  except that the gas container remains closed and we check that the clean metal histogram is preserved. After the real dosing of the molecules, new peaks appeared and were present at moderate bias voltage (approx. 30–50 mV) for a long time  without further dosing. Then we could permanently desorb the molecules through a high bias voltage (≥100 mV) histogram measurement. With this step, a clean Au histogram was reestablished, and no molecular peaks were observed even if the bias voltage was reduced back (approx. 30–50 mV), so a new dosing was necessary to see molecular peaks again. In the present study three independent data sets were acquired by permanent desorption of the CO molecules and repeated dosing, which show reproducible data.The displacement of the electrodes was calibrated prior to molecule dosing using both plateau-length histograms and conductance traces in the tunneling region. To ensure proper cleanliness during the molecular measurements the turbomolecular pump was continuously running. This has introduced vibrations on a scale below 1 Å to the junction, which resulted in the appearance of false peaks in the plateaus-length histograms of molecular junctions, thus we have avoided drawing conclusions from the plateau-length histograms of the molecules.


We present a theoretical analysis aimed at understanding electrical conduction in molecular tunnel junctions. We focus on discussing the validity of coherent versus incoherent theoretical formulations for single-level tunneling to explain experimental results obtained under a wide range of experimental conditions, including measurements in individual molecules connecting the leads of electromigrated single-electron transistors and junctions of self-assembled monolayers (SAM) of molecules sandwiched between two macroscopic contacts. We show that the restriction of transport through a single level in solid state junctions (no solvent) makes coherent and incoherent tunneling formalisms indistinguishable when only one level participates in transport. Similar to Marcus relaxation processes in wet electrochemistry, the thermal broadening of the Fermi distribution describing the electronic occupation energies in the electrodes accounts for the exponential dependence of the tunneling current on temperature. We demonstrate that a single-level tunnel model satisfactorily explains experimental results obtained in three different molecular junctions (both single-molecule and SAM-based) formed by ferrocene-based molecules. Among other things, we use the model to map the electrostatic potential profile in EGaIn-based SAM junctions in which the ferrocene unit is placed at different positions within the molecule, and we find that electrical screening gives rise to a strongly non-linear profile across the junction. The last decade has led to substantial advances in the understanding of electrical conduction through molecular systems, mostly due to an improved control of the different measuring techniques employed to study molecular junctions, including electromigrated single-electron transistors (SETs)12345679111213141516171819221234567i.e., they need to be tailored to the specific junction under study) and may require very long calculation times even on high-performance computing platforms. Alternatively, analytical models dealing mainly with the energetics of the junction, such as the Landauer formalism for coherent tunneling (or the rate equations for incoherent tunneling), are easy to employ but rely on rough approximations of some critical parameters and may miss some of the physics involved in the process, particularly when conduction is driven or assisted by interactions with the molecular surroundings .Despite the increasing number of experimental reports, and to a lesser extent theoretical works, dedicated to the topic of electrical conduction through molecular junctions  Three-terminal SET experiments performed at different temperatures on individual S(CH2)4Fc(CH2)4S molecules; and (ii) experiments in SAM-based EGaIn junctions of S(CH2)11Fc2 molecules where two distinct molecular levels participate in charge transport2)nFcC13−n molecules where the ferrocene (Fc) unit is placed at different positions (n) within the alkyl chainJ(V) curves, the shape of the electrostatic potential profile and the temperature dependence of the transport excitations. Finally, we use this model to discuss in detail the range of validity of coherent versus incoherent theoretical formulations that have been used to rationalise some recent experimental results.There are ongoing theoretical efforts focused on solving the problem by integrating different effects to formulate a non-equilibrium theory in terms of molecular states that includes many-body correlations when computing electron transport through molecular junctions  then the process will become incoherent, with an associated loss of phase in the case of elastic tunneling, and loss of both phase and energy in the case of inelastic tunneling. In wet electrochemical molecular charge-transfer processes, energy relaxation is usually facilitated by intramolecular vibrational modes and polaron excitations in the surrounding solvent molecules. In terms of Marcus theoryConduction through a molecule can, in principle, be understood in terms of sequential tunneling (either coherent or incoherent) involving one molecular level. In such a process the electron tunnels from the source into the molecule at a rate kBT = 13–86 meV in the range 100–1000 K). Thus, when only one level participates in the conduction through the junction (which may be the case in many molecular junctions), the vibrational modes can be formally incorporated into an effective temperature-dependent level broadening , which means that it is reasonable to still treat the transport process with simple single-level transport models, as we do in the present work. Note the different causes of level broadening, which we can separate into two main contributions: (i) broadening due to the coupling of the molecule to the electrodes, with tunneling rate γ = γL + γR, and, (ii) broadening arising from the coupling of the electron to other degrees of freedom of the molecule, γ0. Of course, this is just a simplification  and should be taken as such. However, this picture can be justified from a more fundamental calculation involving a non-equilibrium quantum mechanical treatment of the response of the molecule to its coupling to leads. When a single energy level is active in the molecule, and the coherent interactions between electrons in the molecule and those in the leads are negligible, the non-equilibrium expression for the current reduces to an expression similar to the Landauer-Büttiker formulaWe expect that inner-sphere reorganization will always be important, and can be related to intra-molecular vibrational modes with energies usually below a few tens of millielectronvolts <30 meV), which is smaller than or comparable to other energy scales relevant to the conduction process, such as thermal broadening at high temperatures  = 1/(ex + 1), μL and μR are the chemical potential at the leads, γL(E) and γR(E) are the partial level widths due to the coupling to right and left leads, respectively, and Gr is the molecule’s single-particle retarded Green’s function, which contains a self-energy that takes into account the coupling to leads and to other degrees of freedom as an imaginary part, namely, a total level width. In fact, this has been the starting point of several successful theoretical descriptions of molecular electronic transport experiments . The capacitive couplings of the frontier orbital with the electrodes determine the electric potential drops at both sides of the active conduction unit (VL and VR) and provide asymmetry to the junctions when LL ≠ LR. This asymmetry is parameterized by the dimensionless division parameter η = VR/(VL + VR), which gives the ratio of the voltage drop between the molecule and the right electrode with respect to the total voltage drop in the junction.As mentioned before, the transition between coherent and incoherent tunneling is determined by the tunneling rates in and out of the molecule (when compared with the intrinsic molecular relaxation rates). As explained by Moth-Poulsen and Bjørnholm in their review articleital see , and areEm = μR(V) + ε + ηqV, where ε is the zero-bias energy offset between the molecular orbital and the electrodes, q is the electron charge, and μR is the electrochemical potential of the right electrode. The behaviour of Em, μL and μR with respect to the bias potential V is shown in η. For η ~ 0.5 the energy of the level remains constant as the bias potential is increased, while for η ~ 0 or ~1, the molecular level closely follows the right or left potentials, respectively.The molecular frontier orbital follows the average effective potential within the molecule and its energy can be expressed as Finally, thermal broadening of the energy of the electrons in the leads also greatly affects conduction through the junction and needs to be taken into account. When a bias potential is applied, two distinct Fermi distributions need to be used, one for each lead:In the case of a single conduction level, the electrical current can be calculated by solving the rate equations for each side of the molecule , from which one can extract an expression for N:where N in Substituting this functional for i.e., within the conductance window). Of course, this approximation fails when the level width surpasses such energy separation, since it will lie partially out of the conductance window. In other words, the current will not increase indefinitely by simply increasing the level width . On the other hand, the resistance has a quantum mechanical limit, which for a single transport channel translates into a universal quantum of conductance, G0, as originally predicted by LandauerNote that this treatment accounts for sequential tunneling of the electron from one of the leads into the molecule and further into the opposite lead and is incoherent in nature. In other words, the rate equations formulation assumes that the electron’s phase memory is completely lost once it tunnels into the molecule. That can only happen through relaxation processes —purely elastic in the case of a single level (no other levels to transit into), but possibly even inelastic. Apart from this, the first conclusion that one can extract from this simple expression is that current will only flow through the junction if a molecular level lies between the electrochemical potentials of the left and right electrodes . In the derivation based on rate equations  one can introduce an additional source of broadening unrelated to leakage, namely, γ = γR + γL + γ0. Other than that, the two results are formally identical at any temperature or bias voltageThis expression matches exactly the one derived by Jauho, Wingreen, and Meir in 1994Landauer derived an equation similar to where the level width associated to the level lifetime is given byIn the linear regime and when the molecular level falls within the bias voltage window, Thus, it is our claim that μL < E < μR, = 0 otherwise) and low bias (μL ~ μR), the maximum conductance  will beIt is easy to see from γL = γR) [for the spinless case].this expression coincides with the universal conductance As a side note, one can break up the Landauer conductance into two contributions, sincewith M representing the number of channels and the characteristics of the junction see  when . TAn interesting exercise within the context of the discussion in this article is to solve the asymptotic limit of ε being the offset between the energy level in the molecule and the Fermi energy in the leads. Note that a similar behaviour is expected from classical Marcus charge transfer processes, for which the activation energy has a completely different origin, as described above. We note that the activation energies arising from these two very different mechanisms may display different dependencies on the electric potentials applied to the junction, which therefore become powerful diagnostic tools to discern charge transport mechanisms. From ε would decrease with increasing bias voltage, as the electrostatic potential of the lead approaches the molecular level. This does not need to be the case in, e.g., polaron-assisted conduction, where polarization of neighbouring molecules may be affected by the applied bias in intricate ways, resulting from its direct effect on the polaron dynamics.The current in this case shows an activation behavior, with the “activation energy” ε = 0.4 eV, η = 0.5, VG = 0, and γL = γR = 1 meV  depends on the bias voltage: the lower the bias the higher the transition temperature . In addition, one can observe that increasing the level width smears out the difference between the two regimes 4-Fc-(CH2)4-S molecule placed in between the leads of an electromigrated three-terminal Al2O3/Au SET  of the molecule, when no current is allowed to flow at low bias . Details of the transport results and the synthesis of the molecule are given in ref. I vs. V − Vg measurements, the actual separation between these two charge points is 160 meV, from which we extract a gate capacitance factor  of c = 0.08. The data in ε1 = −25 meV, ε2 = 135 meV (ε1 − ε2 = 0.160 eV), η = 0.5, and γL1 = γR1 = 0.21 meV and γL2 = γR2 = 0.06 meV.T = 220 K. The behavior of the tunnel current with temperature is shown in Vg = −1.5, −0.7, −0.3, and 0.9 V) characteristic of the different conduction regimes in this junction, as indicated by the arrows in Vg = −1.5 and +0.9 V) or in the transport regime (Vg = −0.7 and −0.3 V).The measurement in The inset to We note that the same temperature behaviour has been observed in several other studies of this family of ferrocene-based molecules in EGaIn junctions2)11Fc2 molecules (with Fc2 representing a biferrocenylene (Fc = Fc) head group) sandwiched in between 2 lies at the end of a long insulating alkyl chain, providing a non-covalent contact  to the top electrode. This results in a highly asymmetric drop of electric potential at both sides of the Fc2  enabling large rectification ratios. In particular, the Fc2 presents two distinct conduction levels |/|I(V)|) as a function of the bias voltage applied to the junction. Apart from the high rectification ratio of 1.1 × 103 reached at V = 0.875 V, the R(V) plot shows two clear kinks at around 0.35 and 0.75 volts which were ascribed to the HOMO and HOMO-1 levels subsequently entering the conduction window defined by the difference in electrochemical potential of the top and bottom electrodesLet us focus now on some very recent experiments in SAM-based molecular junctions where a record-high rectification ratio of three orders of magnitude has been achieved2 levels, i.e., the HOMO and the HOMO-1. Specifically, the addition of two different Lorentzian functions, as given in ε1 = 0.35 eV, ε2 = 0.75 eV, η = 0.95, and γL1, = 3 meV, γR1, = 1 meV, γL2, = 5 meV, γR2, = 2 meV. Note: the same results are obtained by 2 group, as well as the overall value of the rectification for most of the bias voltage range, including the maximum rectification of three orders of magnitude. As discussed in ref. These results can be easily explained by incorporating a second level into the single-level transport model to account for the two FcGiven the fact that molecules are not all identically placed with respect to the electrodes and that their disposition may actually change during the course of a complete measurement, we have used Gaussian distributions for the energies of the HOMO and HOMO-1 levels to account for the degree of dispersion in the system. To do so, we modify where,W = 0.04 eV for each level (green line in The best results are obtained for a width of the Gaussian distribution γ ~ 1–10 meV) is at the theoretical maximum limit expected from a single-level transport mechanism for characteristic positions of the frontier orbital with respect to the Fermi energy (ε ~ 0.5–1 eV) and operating voltages (V ~ 1 volt). This is easy to deduce by looking in i.e., derived from experimental data). In particular, the calculations have been obtained for a single-level positioned at ε = 0.5 eV, and with a maximally asymmetric voltage divider parameter η = 0, to maximize the rectification ratio. Obviously, the rectification will be maximum for a bias voltage larger than −0.5 V, which is the forward voltage needed to bring the molecular level into resonance when η = 0  for both polarities of the bias voltage (V = ±1 volt). As expected, the difference between the forward and reverse currents increases as the tunneling rates (and consequently the level widths) decrease. Both currents reach the same saturation value at sufficiently high tunneling rates , when the level width is larger than the separation between electrochemical potentials in the leads and the conductance becomes constant, as discussed above. The corresponding R for these voltages is shown in −5 < γi ≲ 1 eV the electrical rectification decreases exponentially at room-temperature and below, saturating at R = 1 (no rectification) for γI ≳ 1 eV, corresponding to the behaviour of the current in γi takes values typically above 1 meV, the rectification ratio will be limited to three orders of magnitude when a single electrostatic molecular level contributes to the conductance through the junction, which is the value found in the experiments reported in ref. i.e., η = 0) but with an experimentally relevant energy location of the frontier orbital (ε = 0.5 eV) and typical values of γi. Larger ε values and, consequently, larger voltages will increase this limit, but not substantially. Evidently, if more than one molecular level is involved in charge transport the rectification can be improved by a factor proportional to the total number of levels involved. However, since it is unlikely to find molecular junctions with more than a few levels in the vicinity of the electrochemical potential of the leads, it is unwise to expect substantially higher electrical rectification ratios originating from the molecular level structure. Imaginative ways should be explored in order to overcome this limit in practice, perhaps by engineering junctions that mix high single-level rectification properties with conformational changes in the molecule that can build up to increase the overall rectification of the junction.We want to stress again that this result is found for voltage conditions that maximize the rectification efficiency , with n = 0 to 13η), since the electric potential at both sides of the conduction unit  will strongly depend on its position along the chain. This enables sampling of the electrostatic potential profile along the junction gap, which was found to be highly non-linear in this particular molecular junctionSome of the authors in this article have recently reported a study of electrical rectification in EGaIn SAM-based junctions where the Fc unit was placed at 14 different positions within the SAM. The molecules have the form of S-CR as a function of n measured at room-temperature and calculated by comparing the electrical currents at V = ±1 V. As can be clearly observed, the rectification behavior is highly non-linear, with several distinct areas, and with the maximum rectification values achieved when the Fc unit is placed close to one of the electrodes , as expected from the asymmetry in the potential drops at both sides of the Fc. For n < 3 the strong hybridization between the Fc and the bottom electrode (chemisorbed contact) leads to large level broadenings and leakage currents, which in turn results in lower rectification ratios , although weaker, arises for n > 10 due to proximity to the top electrode, where the coupling between the molecule and the lead is non-covalent . The intermediate range of Fc positions (4 < n < 11) is the most interesting in the context of the analysis of the electrostatic potential profile in this article, since coupling between the Fc unit and the electrodes is non-covalent, and the molecular level responsible for conduction is well defined, with a width that can be considered constant, as we discuss below.R for intermediate n values, and particularly its small slope for n = 5–9  = 0.75 eV . Therefore, for n > 5 the energy offset ε(n) is expected to be constant . Departures from linearity (as observed for n < 5) are ascribed to a real change in the distance between the molecular level and the Fermi energy of the electrode, which in this case decreases. In the following we describe the analytical models that have been employed to fit the rectification data in γL(n) and γR (n), and consequently the HOMO level width γ(n) = γL(n) + γR(n), and ε(n) in order to account for the effect of the coupling to the respective electrodes. γ(n) and ε(n) used in the calculations (solid curves). The broadening of the HOMO level in proximity with the electrode is expected to increase exponentially with decreasing Fc-electrode distance. Fermi’s golden rule gives L,R is the effective coupling matrix and DL,R the electron bandwidth in the electrodesk is the tunneling attenuation coefficient, proportional to L and γR are not purely exponential with n close to the electrodes but saturate for distances below two CH2 units (n < 2 and n > 12) from the respective electrodes. This fitting is based on the spectroscopic dataL(n < 2) = 360 meV (due to strong hybridization with the Ag electrode) and γR(n > 11) = 40 meV, and a base value of γ = 30 meV (with γL = γR = 15 meV) for intermediate distances, as obtained from the fitting of the rectification curves (see below). Note that taking the full width at half maximum from UPS data to estimate the molecular level broadening results in an overestimation of γ due to limitations of the technique (see ref. Distance-dependence functions have been chosen to represent the coupling energies η(n). We have used two different models to obtain this parameter: (i) A simple linear electrostatic potential profile; and, (ii) a correction to account for electrical screening in the alkyl chain.According to i.e., VL α LR), one can rewrite η = VR/(VL + VR) as η ≈ LR/(LL + LR). To account for the voltage drop at the Fc, the expression above can be rewritten assuming a correction LFc for the respective lengthsAssuming a linear dependence of the voltage drop as a function of the distance between the Fc and the respective electrodes  observed in equivalent alkyl chains without the Fc unit, or when the Fc is placed in the middle of the chain. The corresponding η in the case of our molecule is given in LFc/(LA + 2 LFc)] of the voltage drops in the Fc and remains unavailable for use in the rectification process. We note that the strength of this correction is proportional to the size of the active unit employed to sample the electrostatic potential profile, which in our case is substantially larger than the adjacent CH2 units. An ideal unit to sample the electrostatic profile would be one of negligible size . That would also guarantee that the sampled molecule would remain undistorted when moving the active unit within the chain.where The resulting rectification curve obtained using the linear model defined by e.g., molecule) for sufficiently long electric screening lengths, λ, which is the case in semiconductors and molecular insulators ), which is taken as the effective diameter of the alkyl chain, the electrostatic potential profiles for different screening lengths  are shown in λ → 0, the electrostatic potential will follow the electrochemical potential and drop entirely within the vicinity of the contacts.Using σ = 2.38 Å (=0.125(λ = 1.30 Å (black line), λ = 1.50 Å (blue line) and λ = 1.90 Å (green line). From the good quantitative agreement with the experimental results, where the small slope in the rectification ratio can be well accounted for at intermediated values of n, one can estimate the characteristic screening length to be approximately λ = 1.50 ± 0.2 Å. These values of the screening length are comparable to the molecular diameter σ, indicating that the electric field lines from vacuum penetrate well into the molecule.The results of the corresponding fittings of the rectification data are shown in I(V) curves that can be experimentally obtained , and the zero-bias energy offset between the molecular orbital and the electrodes ε). This model was tested against a well-characterized molecular diode and faithfully recovered the rectification ratio and the temperature dependent behavior of the junctions, from which we conclude that it is applicable to describe transport data from both single molecule and SAM-based junctions where intra-molecular collective behavior does not significantly affect the electronic structure of the junction.We have shown the validity of a simple but formal analytical single-level tunneling model that uses input parameters to calculate λ = 1.50 ± 0.2 Å, for ferrocene-alkanethiol). Fitting our equations to the experimental results shows that the electrostatics of the molecular junction may be sampled simply by placing the redox Fc unit at different positions along the junction. These results confirm the expectation that potential profiles in insulating molecules are neither linear (as in vacuum) nor do they follow the electrochemical potential profile, which would lead to flat profiles within the entire length of the molecule and would prevent the attainment of high rectification ratios by capacitive coupling with the active transport unit. It is important thus to identify molecular ligands that provide long screening lengths in order to achieve linear-like potential profiles and extend the range of lengths for which the molecular diode would be an efficient rectifier of electrical current.One of the main conclusions from our theoretical treatment of the rectification ratio is that the highly non-linear electrostatic potential profile in typical molecular tunneling junctions is the result of a sizeable screening length in the molecule  can account for the typical temperature dependencies of the conductance in solid state junctions and provide a powerful analysis tool to extract the characteristic parameters governing tunneling in these junctions in a consistent way.How to cite this article: Garrigues, A. R. et al. A Single-Level Tunnel Model to Account for Electrical Transport through Single Molecule- and Self-Assembled Monolayer-based Junctions. Sci. Rep.6, 26517; doi: 10.1038/srep26517 (2016).


Streptococcus pyogenes  is an etiological agent for pharyngitis, pyoderma, and invasive infections in humans. Pharyngitis and pyoderma may lead to serious immune sequelae such as rheumatic heart disease and post-streptococcal glomerulonephritis (PSGN). Streptococcal Inhibitor of Complement (SIC) and its orthologue, distantly related to SIC (DRS), are virulence factors expressed by only four of more than 100 M types of GAS. These four types  are among the M types, which are associated with PSGN. In several populations PSGN has been shown to be a risk factor for chronic kidney disease (CKD) and end-stage renal disease (ESRD). Previous studies showed SIC or DRS antibody-prevalence was associated with PSGN, and seroprevalence of SIC antibodies is significantly high among CKD and ESRD patients in Mumbai.Streptococcal isolates recovered from GAS pyoderma cases were typed. Seropositivity for SIC and DRS antibodies in subjects with pyoderma, PSGN pediatric cases, age matched healthy controls and non-GAS pyoderma cases were determined.We confirm in this study an association between seroprevalence to SIC and DRS antibodies, and PSGN in Mumbai population despite low point prevalence of M1, M12, M55 and M57. In addition we extended the study to GAS-pyoderma and non-GAS pyoderma cases. To our surprise, we found a positive association between the seroprevalence to SIC and DRS antibodies, and GAS-pyoderma owing to infection with diverse M types. The mechanism of increased predisposition to pyoderma owing to infection by diverse GAS among SIC or DRS antibody-positive population is not clear. Nonetheless, our findings could be explained by a phenomenon akin to antibody-dependent enhancement (ADE).This is the first report showing a small number of GAS M types conferring predisposition to pyoderma by diverse types. Implications of this ADE-like phenomenon are discussed in the light of evolutionary advantage to GAS, vaccine design and control of renal diseases. Streptococcus pyogenes , a human-specific pathogen, is responsible for diverse diseases such as pharyngitis, pyoderma, cellulitis, necrotising faciitis, toxic shock syndrome and life-threatening immune sequelae including rheumatic heart disease and post-streptococcal glomerulonephritis (PSGN) [emm types) are associated with PSGN, of which emm1, 12, 55, 57 are among the major ones. These emm types express a major secretory antigen called Streptococcal Inhibitor of Complement  or its orthologue called distantly related to SIC  [emm1-2 type which was claimed to be distinct from emm1 subtypes, isolates of both emm1 and emm1-2 are clonally related. Both SIC and DRS elicit high antibody responses during natural infections and the antibodies are persistent [s (PSGN) ,2. Pharyd emm55) -5. Althod emm55)  reportedrsistent .emm types in these two geographical regions, to the differences in study design or both. Although the prognosis of PSGN is generally excellent, many studies suggest that PSGN is a strong risk factor for chronic kidney disease (CKD) and end-stage renal disease (ESRD) [Serological reactions to several GAS antigens have been observed subsequent to PSGN . In an ee (ESRD) -14. Intee (ESRD)  that SICemm types were not overly represented among the isolates from the GAS pyoderma cases. This startling finding clearly highlights increased predisposition to GAS pyoderma in Mumbai region among the subjects seropositive to SIC or DRS antibodies due to past infection with emm types expressing these antigens. We attribute this to a phenomenon akin to antibody-dependent enhancement (ADE) of skin infection. However, only a limited number of GAS strains seem to confer ADE of infection by diverse GAS types. Such ADE may have a role in the evolution of GAS as a highly successful human pathogen. We discuss implications of our findings in relation to the management of CKD patients and vaccine strategy.We now show that acute PSGN pediatric patients have high seroprevalence for SIC and DRS antibodies. We also extended this study to pyoderma patients attending outpatient wards of the same hospital. To our surprise we found that significantly greater proportion of GAS pyoderma patients are positive to SIC and DRS antibodies than those with non-GAS pyoderma patients, or age-matched healthy control subjects. Despite this observation, SIC or DRS positive Staphylococcus) were age matched to that of GAS pyoderma cases (mainly adult population).Informed consent was obtained from all participants and guardians. The study was conducted under ethics committee approval from Seth G.S. Medical College & KEM Hospital . All the patients and control subjects are of similar demography. All the subjects included were from low socio-economic strata living in vicinity of KEM Hospital and the study was conducted in 2 years between June 2011 and June 2013. They were therefore temporally and geographically matched populations. The control 1 subjects were age-matched to that of PSGN patients; they are paediatric population. The control 2 subjects and the cases of non-GAS pyoderma  the above 150 GAS pyoderma subjects  at the time of their visit to the hospital’s outpatient department, and b) 25 children with PSGN  admitted to the pediatric ward of KEM hospital diagnosed as acute nephritic syndrome and having ASO titres > 300 IU (see below).Blood samples were also collected from 50 non-GAS pyoderma cases  and two age-matched control groups . Sera samples from patients and healthy individuals were stored at −80°C until used.emm typing according to the protocol described at www.cdc.gov/ncidod/biotech/strep/strepblast.htm.The isolates were characterized by Anti-streptolysin O (ASO) titers in sera were determined according to Karmarkar et al. . Both coSerum antibody titres for SIC and DRS were measured by Enzyme-Linked Immunosorbent Assay (ELISA) as described by Karmarkar et al. . BrieflySerum samples  were pre-incubated at 37°C with competitor antigens for 1 hour before adding to plates coated with SIC or DRS. The competing antigens were: a) no antigen (control), b) 10 μg of SIC, DRS or both, c) 50 μg of SIC, DRS or both. The rest of ELISA conditions were as described above.Urine was collected for all PSGN cases and the control group 1. Proteinuria, protein-creatinine ratio, blood cells in urine were determined as described earlier ,18.Cutoff values to assess whether or not a serum sample should be regarded as positive for SIC or DRS antibody were determined as before  and wereemm typed. In all, the isolates belonged to 57 emm types or sub-types over the 2 year study period, and each type is represented by 1–5 isolates in a year  were of the same type for each patient. Taken together our results show it is unlikely that SIC- or DRS-positive types per se are main contributors to pyoderma in these subjects; instead diverse emm types circulating in the community are responsible for skin infection.Of the 701 patients, GAS isolates were recovered from 150 pyoderma cases. Multiple isolates from the same skin sore for each subject were analyzed for 20 pyoderma patients, and in an additional 17 pyoderma cases isolates from multiple skin sores were Sera from a) the 150 GAS pyoderma subjects, b) 25 children with PSGN, c) 50 non-GAS pyoderma cases, d) two age-matched control groups; control 1 for PSGN and control 2 for GAS pyoderma were analysed for various parameters. Both the control groups and the non-GAS pyoderma cases had no known history of streptococcal infections, and ASO titres were <300 IU. This suggested no recent GAS infection in these individuals. Furthermore, urinalysis of the control 1 showed no proteinuria, protein-creatinine ratio <0.2, and absence RBCs or pus cells suggesting no subclinical PSGN or CKD in this control group. Figure emm1 and emm12) among the pyoderma cases in the 2 years  may simply be a reflection of low prevalence of these types circulating in the KEM hospital catchment-community. Since SIC and DRS antibodies are persistent [The low recovery rates of SIC- and DRS-positive types . The fact that some patients exhibit seroreaction to one or the other antigen, suggests that double seropositivity is not due to possible cross-reactions of the antibodies with the antigens. Moreover, mature SIC and DRS proteins have only limited homology; mostly confined to the proline-rich region [Since seroprevalence to these antibodies in non-GAS pyoderma cases, like in the control group 2, is significantly lower than in GAS pyoderma cases (p < 0.0001 for SIC and DRS), we believe this phenomenon of high SIC and DRS antibody seroprevalence is specific to GAS pyoderma cases.emm types [emm types expressing these antigens. We attribute this to a possible role of SIC and DRS antibodies in a mechanism akin to antibody-dependent enhancement (ADE) of infection by GAS. While ADE of infection is common occurrences in viruses, bacteria too could deploy such strategies. For instance, ADE of infection has been reported in S. pneumoniae [S. pneumoniae to respiratory epithelial cells. More recently ADE-like phenomenon was observed in Pseudomonas aeruginosa wherein increased severity of respiratory infections was found to be associated with elevated levels of IgG2 against the bacterial O antigen [SIC and DRS antibodies are persistent, and overwhelming evidence show that the distribution of the SIC and DRS genes is restricted to mainly four mm types ,8,19. Thmm types . Given teumoniae ,21. Clea antigen . The ADEThe seroprevalence rate is 38.7% for SIC antibodies in GAS pyoderma cases, and that for DRS antibodies is 37.3% Figure D. Therefemm 1, 12, 55 and 57) are “core types” and the rest “non-core types”. Global distributions of emm types clearly show, whereas in most regions one or more of the 4 “core types” are recovered, the distributions of the “non-core types” vary considerably with some types predominating in many places and others are rarely found or not at all [emm types. Should the differences persist, it is possible that contributions to the GAS genetic pool by the low-infective types would be depleted at the expense of a few expanding types. ADE by the “core” types may partly correct this imbalance. Such a scenario may offer a possible novel strategy for efficient control of GAS diseases by targeting future vaccine candidates against the “core types”. By such intervention, a decrease in overall GAS infection may be achieved.Further work is warranted, and being planned, to demonstrate the SIC and DRS mediated ADE-like phenomenon in pharyngeal infections. Also, demonstration of the ADE of infection in cell culture or in animal models may provide further support to this phenomenon. If ADE of GAS infections occur at both the major tissue sites, it may offer continued stability for the host-pathogen interaction in evolutionary terms, and may be important factor in GAS’s success as a human-specific pathogen. We suggest that the four SIC and DRS positive types (t at all ,24. ThisIn many populations PSGN is an independent risk factor for CKD -14. We rThe role of SIC and DRS is not well understood. It is intriguing that only four types express these antigens. They elicit persistent antibody response. Therefore, antibody prevalence in a population is cumulative result of past infections by one of these four “core” types. Intuitively, one would expect that the rate of prevalence would be the same in a given population among various subjects; namely subjects with pyoderma, non-GAS pyoderma, PSGN cases or healthy controls. However, contrary to this expectation we found that the rate of seroprevalence is high specifically in GAS pyoderma cases. Since the pyoderma is resultant of diverse GAS strains not necessarily expressing these antigens, we interpret the results to mean that antibodies to SIC or DRS predispose subjects to skin infection by GAS. This is a unique phenomenon of ADE of infection rendered by a small number of GAS strains on a wide number of strains. Such a mechanism could have implications to maintenance of GAS gene pool despite possible inherent variations in infectivity.


P < 0.01), and the R-values were −0.065 and −0.152, respectively. The correlation of TT4, FT4, TT3, and FT3 with TSH was statistically significant in patients with hyperthyroidism, and the R-values were −0.241, −0.225, −0.195, and −0.176, respectively. The correlation of TT4, FT4, TT3, and FT3 with TSH was statistically significant in patients with hypothyroidism, and the R-values were −0.322, −0.262, −0.179, and −0.136, respectively. In our opinion, TSH and FT4 are the most valuable indicators in assessing thyroid function in a healthy population, and TSH and TT4 are the most meaningful in hyperthyroidism and hypothyroidism.To clarify the clinical value of serums total triiodothyronine (TT3), total thyroxine (TT4), free triiodothyronine (FT3), and free thyroxine (FT4) and provide a more eligible and economic strategy to assess thyroid function. A total of 2,673 participants  were involved in our study. Serums TT3, TT4, FT3, and FT4 and thyrotropin (TSH) were measured with VIDAS fluorescent enzyme immunoassay. The Pearson correlation between TT3, TT4, FT3, and FT4 and TSH was determined to identify the most important indicator for thyroid function besides TSH. The correlation of TT4, and FT4 with TSH was statistically significant in healthy individuals ( The thyroid is an important gland involved in the metabolism, growth, development, and maintenance of the internal environment , 2. ThyrA total of 2,673 participants  were enrolled from Shanghai Tenth People's Hospital from Jan. 2010 to Dec. 2013. Three groups were analysed: (1) healthy population: 20–60 y, 489 males, and 1184 females; all the subjects were enrolled from the medical centre and presented with no thyroid diseases as evaluated through history, blood testing, and thyroid ultrasound; (2) hyperthyroidism : 20–75 y, 136 males, and 364 females; (3) hypothyroidism : 20–75 y, 155 males, and 345 females. All patients with hyperthyroidism or hypothyroidism were from the outpatient department of endocrinology and had been confirmed to be in good general health by history, physical examination, screening blood tests, X-ray, electrocardiogram, and so forth. The clinical characteristics of the included participants are shown in Venous blood (2 mL) was collected from participants using a vacutainer. Serums TT3, TT4, FT3, FT4, and TSH were measured by chemiluminescent immunometric assay (Roche Diagnostics GmbH). The manufacturer's reference limits are TT3 (1.2–3.4 nmol/L), TT4 (54–174 nmol/L), FT3 (3.5–6.5 pmol/L), FT4 (10.2–31 pmol/L), and TSH (0.35–5.5 mIU/L).P < 0.05 and the absolute value of the R-value was close to 1.We used SPSS software version 15.0 for data analysis . The results are presented as the mean ± SD. The Pearson correlation was adopted to identify the most important indicator besides TSH, excluding the interference of aging at the same time. The correlation was considered statistically significant and more relevant when The baseline data of the individuals and concentrations of TT4, TT3, FT4, FT3, and TSH from the three groups are presented in Tables P < 0.01) in 1673 healthy people, and the R-values were −0.065 and −0.152, respectively. The correlation between FT4 and TSH was the maximum. The correlations between TT4, TT3, FT4, FT3, and TSH were all statistically significant (P < 0.01) in 500 patients diagnosed with hyperthyroidism. The R-values were −0.241, −0.225, −0.195, and −0.176, respectively. The correlation between TT4 and TSH was the maximum. The correlations between TT4, FT4, TT3, FT3, and TSH were all statistically significant (P < 0.01) in 500 patients with hypothyroidism. The R-values were −0.322, −0.262, −0.179, and −0.136, respectively. The correlation between TT4 and TSH was the maximum , serum T3 is usually maintained at a stable level, which suggests that energy metabolism is changed by a T4 dependent pathway . TherefoIt is commonly known that the severity of hyperthyroidism is only partially correlated with serum FT4 and FT3 levels, and a report of 25 patients diagnosed with Graves' disease showed that there was no strong correlation between thyroid symptom spectrum and serum free thyroxine levels . StudiesThyroid disease can always be ruled out when the serum TSH level is normal without drug administration or in the absence of obvious hypothalamic-pituitary disease . HoweverIn summary, we recommend TSH and FT4 as measuring indicators to assess thyroid function in healthy people and TT4 and TSH as measuring indicators to assess hypothyroidism and hypothyroidism. We think such a permutation is an accurate, convenient, and economic recommendation for clinicians. The significance of properly assessing thyroid function is that it can effectively guide the dose adjustments of antithyroid drugs or hormone replacement therapy.


Background. Previous data suggest that (i) dopamine modulates the ability to implement nonroutine schemata and update operations (flexibility processes) and that (ii) dopamine-related improvement may be related to baseline dopamine levels in target pathways (inverted U-shaped hypothesis). Objective. To investigate above hypotheses in individuals with Parkinson's disease (PD). Methods. Twenty PD patients were administered tasks varying as to flexibility load in two treatment conditions: (i) “off” condition, about 18 hours after dopamine dose and (ii) “on” condition, after dopamine administration. PD patients were separated into two groups: low performers  and high performers . Twenty healthy individuals performed the tasks in two sessions without taking drugs. Results. Passing from the “off” to the “on” state, only low performer PD patients significantly improved their performance on high-flexibility measures ; no significant effect was found on low-flexibility tasks. Conclusions. These findings document that high-flexibility processes are sensitive to dopamine neuromodulation in the early phases of PD. This is in line with the hypothesis that striatal dopamine pathways, affected early by PD, are precociously implicated in the expression of cognitive disorders in these individuals. Dopamine is a brain catecholamine originating from subcortical neurons. It supplements the activity of several neural circuitries belonging to both subcortical and neocortical structures. At this level, dopamine acts as a neuromodulator of synapses involved in the mediation of various aspects of human functioning and behaviour . The rolA focus has been posed on the study of dopamine neuromodulation of mental operations underpinned by the activity of prefrontal neurons. Indeed, the prefrontal cortex has abundant dopamine receptors and receives important dopamine fibre projections from the striatum and ventral-tegmental regions . In factn-back working memory performance) in a subgroup of PD subjects who exhibited the lowest working memory baseline scores ; in all cases the explicative factors were the digit span backward score and the order of administration of the experimental conditions. The model was significant only when applied to the performance changes on the interference condition of the Stroop test (response times). In this case, the variable entering the regression equation was the score subjects achieved on the Digit Span Backward  = 5.06; β = 0.47; P = .037), while the order of experimental conditions administration did not . This indicates that, in PD patients, lower scores on the Digit Span Backward significantly predict response times decrease on the Stroop test  passing from the off to the on condition. Results of regression analyses involving performance changes on the other cognitive measures administered document that no independent variable enters in the equation.To investigate the effect of dopamine administration/withdrawal on the Beck Depression Inventory and STAY-S scores of PD patients, two ANOVAs with Clinical Group (PD patients versus NCs) and Working Memory Group (low versus high performers) as between factors, Treatment  as within factor, were performed.F = 2.96; P > .09), Working Memory Group  = 2.82; P > .10), Treatment  = 0.22; P > .10), and the interaction between the main factors  show that depressive symptoms are comparably severe in low performer PD patients , high performer  PD patients, low performer NCs , and in high performer NCs , and that they do not vary as a function of the dopamine therapy condition in the PD group .Beck Depression Inventory Score. The absence of the main effects of Clinical Group  = 4.34; P = .0044). However, Tukey HSD tests performed to qualify this interaction failed to evidence significant between groups difference . This indicates that, although high performer NCs tended to show the lowest STAY-S scores , these appear not to be significantly different from those obtained by low performer PD patients , high performer PD patients , and by low performer NCs . Moreover, the absence of significant interactions involving the factor Treatment  evidences that there was no difference between off  and on  states in the PD group.STAY-S Score. In this case, the Clinical Group*Working Memory Group Interaction was significant  and high performers PD patients. The reduced response latency cannot simply be attributed to increased impulsivity after taking the dopamine compound, because low performer PD patients did not obtain a worse accuracy score on this task passing from the on to the off condition. Instead, in the high performer PD group no significant effect of pharmacological manipulation was found on high flexibility tasks. Above effects are confirmed by results of regression analyses performed on the PD sample taken as a whole. Indeed, these results document that lower scores on the variable here used to classify PD patients as low-and high performers (Digit Span Backward) significantly predict a greater improvement after dopamine therapy intake selectively on the Stroop test .Compared with the four tasks we can consider as low flexibility measures , the interference condition of the Stroop test makes high demands on executive processes that require inhibiting the adoption of overriding automatic response to implement nonroutine schemata , 53, 54.The request to inhibiting the adoption of overriding automatic response also differentiates the interference resistance subtest of the Stroop test from the alternate fluency task that just requires the implementation of shifting abilities without the need to inhibit automated processes. In fact, we found no significant effect of dopamine administration on PD patients' performance on the latter task. It could be argued that the poor performance of our low performer PD patients on the alternate fluency task might be partially related to processes different from shifting, which may be less sensitive to dopamine modulation. In fact, compared with healthy controls these patients show reduced ability to generate words also in the single phonemic and semantic fluency subtests. So, difficulty in accessing the retrieval of stored information and in checking and monitoring mental representations could affect alternate fluency accuracy without implying shifting abilities.However, the differential effect of dopamine therapy withdrawal/administration we found on the interference resistance subtest of the Stroop test and on the alternate fluency task is congruent with results of previous PD studies that failed to evidence a significant effect of dopamine administration on shifting tasks , 56 and Interestingly, it has been proposed that striatal activity, mediated by phasic D2 receptor integrity, allows the flexible modification of mental representations by signalling the need for implementation of nonroutine schemata to prefrontal neurons; conversely, prefrontal cortex activity, mediated by the action of tonic D1 receptors, allows the stable maintenance of mental representations , 22. IndIn conclusion, although the relatively low sample size may represent a limit of the present study, our results lend support to the idea that flexibility processes allowing the adoption of nonroutine schemata in between-response competing conditions are affected early by dopamine dysregulation in PD, thus indicating that cognitive mechanisms involved in these conditions are particularly sensitive to dopamine brain stimulation. The dopamine-related effects we found cannot be explained as due to a general influence of drug administration/withdrawal on the general efficiency of attention processes; furthermore, they are not affected by some potentially confounding factors such as presence of anxiety and depressive symptoms. This information appears to be useful in a clinical perspective. Indeed, dopamine functioning is altered in several medical conditions . In this vein, an integrated therapeutic approach that takes into account the potential effect of dopamine drug manipulation on cognitive functions would likely allow better management of the disease .


E- and Z-configuration. While in isolated oligomers the azobenzene units undergo reversible photoisomerization independently, in the nanofibers they are coupled via intermolecular interactions and switch cooperatively as evidenced by unusual thermal and kinetic behavior. We find that the photoisomerization rate from the Z-isomer to the E-isomer depends on the fraction of Z-azobenzene in the nanofibers, and is increased by more than a factor of 4 in Z-rich fibers when compared to E-rich fibers. This demonstrates the great potential of coupling individual photochromic units for increasing their quantum efficiency in the solid state with potential relevance for actuation and sensing.Next-generation molecular devices and machines demand the integration of molecular switches into hierarchical assemblies to amplify the response of the system from the molecular level to the meso- or macro-scale. Here, we demonstrate that multi-azobenzene oligomers can assemble to form robust supramolecular nanofibers in which they can be switched repeatedly between the  Synthetic photoresponsive molecules can be used as building blocks for a large variety of emerging applications ranging from molecular sensors45678911131516e.g., by supporting switching cascades2122Within these densely packed supramolecular systems, it is important to control and, ideally, leverage the intra- and intermolecular interaction between switching units, because steric interaction, as well as excitonic and electronic coupling between adjacent molecular switches, alter the switching process19e.g., by releasing stored internal energy corresponding to the energetic difference between two switching states. Possible applications include sensing devices, where cooperativity could be used to amplify single-molecule events at the nanoscale to readily measurable effects involving many molecules and thus to increase the sensitivity25In synthetic photoresponsive systems, it is an interesting question, if cooperative interaction between molecular switches could be used to amplify the impact of individual photoisomerization events. Ultimately, an array of cooperatively coupled molecular switches could act as a molecular amplifier that magnifies the power of an input signal at the nanoscale, such as absorption of a single photon, or a molecular recognition event into an output signal at the macroscopic scale, E-isomer to the compact Z-isomer is accompanied by significant changes of geometry and dipole moment, which enables neighboring chromophores to interact during isomerization through steric or electronic coupling. Azobenzene polymers incorporating multiple covalently bonded azobenzenes in their main chain exhibit contractile motion upon UV-irradiation30P1) that incorporates more than 30 azobenzene units in its poly(p-phenylene)s (PPP) backbone3233P1 backbone makes sure that the azobenzenes along the polymeric chain are electronically decoupled and photoisomerize with high efficiencyHowever, there are only very few reports of artificial systems exhibiting cooperative switching, yet2728P1 self-assemble into well-defined nanofibers and quantitatively analyze the switching kinetics of these linear, supramolecular assemblies. We find that the Z → E photoisomerization of azobenzenes in P1 nanofibers is significantly slower at elevated temperatures, in contrast to isolated azobenzene oligomers where both thermal and optical Z → E isomerizations are faster at elevated temperatures. Further, we find that in the nanofibers the effective Z → E photoisomerization rate is increased by up to a factor of 4, depending on the fraction of azobenzenes in the Z-configuration. We attribute this to an increased photoisomerization efficiency due to cooperativity among individual switching unitsIn this work, we show that azobenzene oligomers of type 2 surface is covered by a fibrillar network. Single nanofibers exhibit a characteristic length in the range of 1–2 μm, a height of about 4 nm, and an apparent width of about 20 nm, which, due to the finite radius of the AFM tip (<10 nm)E-configuration is only around 8 nm, the nanofibers are attributed to supramolecular aggregates consisting of many oligomers. Interestingly, only very few of the nanofibers on the SiO2 surface have a loose end, which indicates the formation of higher order aggregates such as in multiple helices38P1 polymers can form coherently ordered nanodomains of interdigitating alkyl side-chains. X-ray diffraction measurements performed on a P1 oligomer nanofiber sample, albeit at a higher nominal thickness, show one of the three Bragg reflections of P1 polymers .After the UV LED is switched off at t = 60 s, the reflectance returns to its initial value before the first UV-irradiation due to the white probe light. The nanofibers persist even after many switching cycles as we checked with AFM see the . Further2 eV see . The incP1 film on silicon covered with native oxide using the transfer matrix method .The photoisomerization kinetics of the azobenzene chromophores can be extracted from the temporal evolution of the differential reflectance. To find the relation between the DRS signal and the photoisomerization kinetics, we simulated the DRS signal of a  see the 41. Our sZ-azobenzene to E-azobenzene can be described by the following rate equationThe conversion process from E] and [Z] denote the fraction of E- and Z-azobenzene, kEZ denotes the rate constant of the E → Z photoisomerization having contributions from both the UV-part of the Xe-lamp spectrum and the UV LED, and Z → E isomerization rate constant of azobenzenes having both thermal contributions and contributions from the Xe-lamp. The thermal contribution to the Z → E isomerization is, however, negligible in comparison with the optical contribution since pure thermal back-switching of azobenzenes typically takes several hours whereas the isomerization process we observe occurs within seconds.Here, [keff can be determined experimentally from the real-time DRS data viaThe solution of Z] are the velocity of the Z → E conversion and the fraction of Z-azobenzene at a certain time and Δ[Z] denotes the absolute difference between the high and low asymptotical level of Z-azobenzene fraction. In the following, we discuss the temperature and light-intensity dependence of the Z → E isomerization rate, that is, the initial decay of the time-dependent Z-azobenzene fraction.where Z → E photoisomerization, we find that the azobenzene chromophores in the nanofibers do not switch independently but exhibit cooperativity and a non-Arrhenius-type thermal switching behavior. The first indication is shown in keff is plotted  in order to avoid aggregation, do not switch cooperatively, that is, the individual azobenzene units within a single isolated macromolecule switch independentlyIn addition to real-time measurements at different temperatures, we also measured the rate of the eratures . At roomZ-azobenzene and the extracted Z-dependent keff that have been used for the fitting are shown in Z-azobenzene corresponding to the differential reflectance during several cycles of UV-irradiation at room temperature with varying intensities. In addition, the sample is exposed to white probe light all the time. When the dark sample gets exposed to white probe light of the Xe-lamp, the Z-fraction increases and reaches a steady state. Additional UV-light from the LED  further increases the Z-fraction. With a higher LED UV-light intensity, a larger fraction of the azobenzenes is switched from E to Z. After the UV LED is switched off, the Z-fraction returns back to the level corresponding to exclusive irradiation with Xe-light.For a quantitative understanding of the cooperativity, we use an intentionally simple model to fit the isomerization kinetics. The real-time kinetics of back-switching from different fractions of Z → E back-isomerization Z → E isomerization rate E → Z isomerization. For example, the Z → E isomerization rate after UV irradiation with 110 mW/cm2 is about 50% faster than the Z → E isomerization rate after UV irradiation with 1.7 mW/cm2. The dependency of the back-switching rate on the Z-fraction cannot be described with the simple rate equation for isolated azobenzene moieties (1) because its solution is a monoexponential function with the time- and Z-independent rate Z-dependency of the rates. In a first approximation, this introduces a dependency on the isomeric state of the microenvironment. We find that a modification of the E → Z isomerization rate alone cannot explain the observed switching behavior; however, both the kinetics and the photostationary levels of the real-time data can be fitted when modifying the back-switching rate as followsFor independent azobenzene chromophores, the effective rate of the c denotes a constant.where Z → E isomerization is faster when there is a higher Z-azobenzene fraction in the film  ≈ 4.1). The red line in E → Z switching direction for five different UV-intensities and for five corresponding Z → E back-switching processes. The good agreement of our simulation with the measured cooperative kinetics indicates that the linear Z-dependence of the Z → E isomerization rate, indeed, is a good approximation.Inserting film see . Note thZ-dependent Z → E isomerization rate in nanofibers, we now discuss the origin of cooperativity, in particular, inter- and intramolecular interactions of the azobenzene chromophores. In principle, intramolecular interactions between azobenzene units can lead to cooperative switching behavior, and other isolated multi-azobenzene derivatives have been shown to exhibit cooperative switching in solution due to π stacking or electronic coupling between azobenzene moietiesP1 the azobenzene chromophores within a single isolated molecule are electronically decoupled. In an earlier study no signs of cooperative switching have been found for isolated P1-type molecules in extremely diluted cyclohexane solution (≈10−5 mol/L), where no supramolecular aggregates are formed−2 mol/L, whose high absorbance made spectroscopic measurements in solution difficult. In this earlier study, the isolated P1-type molecules exhibited Arrhenius-type thermal behavior and their switching kinetics followed a monoexponential function and thus did not depend on the fraction of Z-azobenzene. This clearly demonstrates that the cooperative behavior of P1-oligomers emerges due to their aggregation into nanofibers.Based on the findings of a P1 azobenzenes in nanofibers is possible, either via mechanical strain, changing free volume or dipole-dipole interactions. In the Z-configuration, azobenzene has a static dipole moment of about 3 Debye while azobenzene in the E-configuration has a dipole moment near zeroZ-azobenzene could influence the back-switching kinetics of Z-azobenzene in the nanofibers. However, the interaction between molecular dipoles of Z-azobenzenes is probably not a dominating effect, because the enhancement starts even at the stages where the Z-azobenzene fraction is very low and the reacting Z-form is most likely to be surrounded by the E-form.Intermolecular coupling of Z → E photoisomerization rate at higher Z-azobenzene fraction could be an increased number of structural defects in the nanofibers when there are more azobenzenes in the bulky Z-configuration. A greater amount of free volume available to the switching units in the vicinity of defects could lead to a relative acceleration of the photoisomerization rate for a higher Z-azobenzene concentration and thus for a higher defect concentration. However, this theory cannot explain the finding of accelerated Z → E photoisomerization rates at lower temperatures. At higher temperatures beyond the melting temperature of the alkyl side-chain the number of gauge defects and other structural defects among the side-chain should be higher leading to an increased free volume per azobenzene unit. Thus, also the apparent photoisomerization rate should be larger at higher temperatures, which contradicts our experimental observations  of typically 0.4–0.6 is foundOur second observation of slower back-switching at higher temperatures also can be explained by the temperature dependence of strain. At temperatures above 120–150 °C, roughly corresponding to the melting temperature of polyethylene, ordered alkyl side-chain domains involve more of 8 see . A detaiZ → E photoisomerization beyond unity, that is, to switch more than one azo-benzene unit with a single photon. While strain accelerates the Z → E photoisomerization, the strain thereby is also released and the cooperativity reduced in our system. Further tuning of the coupling between the chromophores, e.g., by introducing external mechanical strain or by using different coupling mechanisms would be needed to enable a switching cascade. This would be an important step towards a molecular amplifier, that is, a synthetic molecular device that converts input signals corresponding to single-molecule events at the nanoscale into a measurable signal involving many molecules at the macroscopic scale, e.g., by leveraging internal energy, stored in the metastable Z-configuration of azobenzenes.Theoretically, in multi-component molecular systems that utilize cooperativity it might be possible to raise the quantum yield of P1-type molecules switch independently, P1 exhibits positive cooperativity when self-assembled into nanofibers. The Z → E isomerization rate depends approximately linearly on the fraction of Z-azobenzene within the film. In contrast to the common Arrhenius-type behavior of isolated molecular switches, the isomerization rate of azobenzenes in the nanofibers is faster at lower temperatures. We attribute this to the temperature dependency of the intermolecular coupling strength of the azobenzene chromophores via side-chain mediated strain. Moreover, we have shown that cooperativity of mechanically interlocked molecular switches can be utilized to increase the efficiency of the Z → E photoisomerization. Our findings are important for next-generation optomechanical devices and highly sensitive molecular sensors based on arrays of molecular switches.In this work, we have shown that azobenzene oligomers form nanofibers with well-defined dimensions and shapes, having lengths of 1–2 μm, a height of 4 nm and a typical width on the same order. Whereas in diluted solution, isolated P1 incorporates azobenzene chromophores in a poly(p-phenylene) backbone and has two dodecyl side-chain per monomer  upon irradiation with UV light. The polymers were synthesized as previously reported. The number average molecular weight Mn was 2900 g mol-1 (corresponding to five repeat units) and the sample had a polydispersity index of 1.45 as determined by GPC vs. polystyrene standards. The azobenzene oligomers can be switched from a thermodynamically stable linear and elongated conformation where the azobenzenes are in the E-configuration to a compact and kinked conformation with azobenzenes in the Z-configuration. Thin films of P1 oligomers were cast from 25 mg/mL solution in toluene onto silicon wafers covered with native silicon oxide. The rotation speed of the bare substrate was set to 1500 rpm before the solution was dispensed.The rigid-rod polymer omer see . A cruciE → Z isomerization of P1 was induced with a 365 nm high power UV LED (Thorlabs) with variable optical output and a FWHM of 12 nm. The light-intensity on the sample surface was measured with a thermal power sensor (Thorlabs).AFM measurements were carried out with a NanoWizard III microscope . The AFM was operated in air at room temperature in tapping mode with silicon cantilevers . For UV/Vis spectroscopy, we used a setup consisting of a 75 W Xenon Lamp , a fiber with a reflectance probe , and a spectrometer consisting of a spectrograph  and a cooled CCD (Andor). All UV/Vis spectroscopy measurements presented in this paper have been performed under atmospheric pressure. How to cite this article: Weber, C. et al. Cooperative Switching in Nanofibers of Azobenzene Oligomers. Sci. Rep.6, 25605; doi: 10.1038/srep25605 (2016).


A 41-year-old Japanese woman was admitted to the emergency department of our hospital due to a seizure. She had been diagnosed with pyoderma gangrenosum 11 years ago without bone marrow disease, rheumatoid arthritis, or inflammatory bowel disease. Pyoderma with painful ulcers had repeatedly appeared on the arms, legs, face, and pubic arch despite treatment with systemic prednisolone and cyclosporine. She also had frequently complained about syncopal episodes, however, there was no evidence of neurological diseases, including epilepsy and endocrine diseases. On admission, the blood test revealed a negative anion gap, hyperchloremia, and neutrophilic leukocytosis. Since one of the major causes of negative anion gaps with hyperchloremia is bromide intoxication, we measured the serum bromide level and found that it was significantly high at 21 mg/dl. Bromide is not normaly detectable in healthy indivisuals. Furthermore, 296 empty sheets of over-the-counter(O-T-C) analgesic tablets containing bromide  were found in her house, suggesting that she had bromide intoxication. Repeated syncopal episodes may have been due to this intoxication. These findings forced us to reconsider the diagnosis of pyoderma gangrenosum in this case, because bromide causes the same clinical symptoms as pyoderma. In addition, we realized neutrophilic leukocytosis and hyperchloremia had been associated with exacerbation of the clinical symptoms for 11 years. Therefore, we examined the bromide level of the serum samples collected from the past 10 years and found that the bromide level had been as high as 112.7 mg/dl. In conclusion, we diagnosed this case with bromoderma caused by O-T-C analgesic mimicking pyoderma gangrenosum. After the patient stop taking the analgesic, the pyoderma rapidly disappeared, and the white blood cell count and the serum chloride level returned to normal levels, with no recurrence, even after the discontinuation of prednisolone and cyclosporine.


Moreover, calcium buffer proteins, such as calbindin D28k (CB-D28k), may play an important role mitigating cellular destruction due to disease processes, and more specifically, in some neurological diseases. We addressed the hypothesis that CB-D28k inhibits WNV replication in cell culture and infected rodents. WNV envelope immunoreactivity (ir) was not readily co-localized with CB-D28k ir in WNV-infected Vero 76 or motor neuron-like NSC34 cells that were either stably or transiently transfected with plasmids coding for CB-D28k gene. This was confirmed in cultured cells fixed on glass coverslips and by flow cytometry. Moreover, WNV infectious titers were reduced in CB-D28k-transfected cells. As in cell culture studies, WNV env ir was not co-localized with CB-D28k ir in the cortex of an infected WNV hamster, or in the hippocampus of an infected mouse. Motor neurons in the spinal cord typically do not express CB-D28k and are susceptible to WNV infection. Yet, CB-D28k was detected in the surviving motor neurons after the initial phase of WNV infection in hamsters. These data suggested that induction of CB-D28k elicit a neuroprotective response to WNV infection.Evidence indicates that West Nile virus (WNV) employs Ca Intracellular Ca2+ is tightly regulated by the integrity of membranes, regulated ion channels, and controlled calcium exchange between mainly the extracellular milieu, endoplasmic reticulum, and sarcoplasmic reticulum 2+ homeostasis while still maintaining a >10,000-fold gradient across plasma membranes.Calcium  infection, a study by Scherbik and Brinton 2+ homeostasis. Examples of these calcium buffer proteins are parvalbumin, calbindin-D9k, calretinin, and relevant to this study, calbindin-D28k (CB-D28k) Evidence suggests that calcium buffer proteins may play an important role mitigating cellular destruction due to disease processes, and more specifically, in some neurological diseases. A subset of calcium-binding proteins is designated as buffer proteins, because they are one component in maintaining Ca+2 via L-type calcium channel activity Another mechanism in which CB-D28k might protect cells is by binding directly to caspase-3 and L-type calcium channel protein. In a cell-free system, CB-D28k inhibits recombinant caspase-3 enzyme activity 2+ buffering 2+-permeable AMPA receptors lacking the GluR2 receptor unit 2+ buffering capacity in a motor neuron is valuable under normal physiological conditions, because the buffer facilitates rapid relaxation times of calcium transients important for normal motor function. However, under pathological conditions, weak Ca2+ buffers are disadvantageous, because this condition accelerates a precarious circle of calcium dysregulation, mitochondrial disruption, and excitotoxic damage Further evidence for the neuroprotective effects is the correlation of the lack of CB-D28k in spinal cord tissue with vulnerability to neuronal injury. A large subset of motor neurons in the spinal cord are especially vulnerable to destruction due to low cytosolic Cain vivo.Considering these published data on the role of calcium in viral replication generally and specific WNV replication, and putative CB-D28k neuroprotective properties, we conducted experiments to determine if CB-D28k in transfected cells inhibits WNV infection or cellular damage. In infected rodents, we determined that WNV immunoreactive (ir) cells were not co-localized with CB-D28k ir cells in the brain, suggesting that CB-D28k protective properties also occur The ability of CB-D28k to inhibit WNV ir was evaluated in Vero 76 and NSC34 motor neuron-like cells transiently and stably transfected with a vector expressing CB-D28k and with the empty vector control. An MOI of 5 in these experiments ensured that a large proportion of cells were exposed to WNV. Despite a high MOI, transiently transfected Vero cells with abundant CB-D28k ir were not co-localized with WNV ir . Since mTo support the observation that transient CB-D28k expression appeared to suppress replication of WNV, stably transfected NSC34 cells were generated and verified by western blot analysis . The NSCTo quantify the apparent resistance of CB-D28k-expressing cells to WNV replication, the intensity of the CB-D28k ir and WNV ir were quantified in individual stably transfected cells using confocal microscopy and ZEN blue software . The expFlow cytometry was used to substantiate the same finding that CB-D28k-expressing NSC34 cells are resilient to WNV replication. NSC34 cells stably transfected with CB-D28k or the vector control were infected with WNV at 5 MOI. At 48 hours after infection, detached and fixed cells were double-immunostained for WNV env (green-Alexa fluor 488) and CB-D28k (red-Alexa fluor 568). Flow cytometry analysis was performed with 10,000 cells for each group . A largeTo investigate the effect of CB-D28k on viral replication, infectious viral titers were measured in the supernatants of stably or transiently transfected NSC34 or Vero cells at 18 or 24 hours after infection with WNV. The cells were infected with WNV at a MOI of 5, allowed to absorb for 1 hour, and washed with medium. As expected among individual transiently transfected cells, the level of CB-D28k expression, as identified by ir, was variable between cells. Unexpectedly, the level of CB-D28k expression was not uniform among individual stably transfected NSC34 cells, as illustrated in To determine if the findings in cell culture had any relevance to WNV infection in rodents, we evaluated sections of neurological tissues from hamsters and mice subcutaneously infected with WNV. Necropsy was performed on a hamster and a mouse that could not right itself, and tissues were inspected for areas containing both WNV env ir, plus CB-D28k ir. Mouse tissues were also immunostained for parvalbumin (PV), a kinetically slow-acting buffer, as compared to CB-D28k, a kinetically fast-acting buffer WNV infects motor neurons to cause poliomyelitis-like disease in hamsters To measure the CB-D28k induction over the course of the experiment, hamsters were infected subcutaneously with WNV or sham. At 6, 8, 11, 16, and 21 days after viral challenge, spinal cord tissues of hamsters were processed for immunoperoxidase detection of WNV env , and wesThe unique findings of this report were that WNV ir was markedly suppressed in Vero cells and NSC34 motor neuron-like cells transfected with CB-D28k and that CB-D28k inhibited virus replication. In hamster and mouse studies, we did not observe WNV env ir co-localized with CB-D28k ir in areas of the brainstem, limbic system, and cerebral cortex. In the spinal cord, WNV infects motor neurons 10 probably because of variable expression of CB-D28k in cloned cells.CB-D28k can theoretically affect WN neurological disease (WNND) by sequestering calcium to inhibit essential stages of viral replication as suggested by Scherbik and Brinton Some of these CB-D28k beneficial effects might be relevant for protecting spinal cord motor neurons of WNV-infected rodents. CB-D28k-neuronal cells might be resilient to WNV replication, as suggested by a lack of co-localization of WNV env ir and CB-D28k ir in the brainstem, limbic system, and cerebral cortex, but a large subset of motor neurons in the spinal cord do not express basal levels of CB-D28k as described previously Modeling the induction of CB-D28k by WNV in cultured motor neurons would verify observations in the spinal cords of infected mice and also facilitate mechanism studies. However, there are two current limitations. The first limitation is that the NSC34 neuronal cells divide and are not terminally differentiated. Consequently, the cells senesce in culture after 5–6 days. WNV-infected NSC34 neuronal cells die before they can induce CB-28k. As such, the cultured cells do not model motor neurons in the spinal cord. Fortunately, we have presumptively terminally differentiated NSC34 cells in differentiation medium. These differentiated NSC34 cells cease to proliferate, survive longer in culture, have increased size, and produce large neurite processes (data not shown). If these differentiated motor neurons are validated, future studies could address questions of induction of CB-D28k by WNV.A study with the infection of borna disease virus (BDV), a neurotropic virus, in submucous and myenteric neurons of the colon offers further insights in the induction of CB-D28k during viral infection 2+, whereas CB-D28k is a kinetically fast-acting buffer The co-localization of PV ir with WNV env ir in cells within the dentate gyrus, stratum pyramidale, and ventral medulla, as compared to no co-localization of CB-D28k ir with WNV env ir, suggest that the anti-WNV effects of CB-D28k are not non-specific for all calcium buffer proteins. PV is a kinetically slow-acting buffer and has a high affinity for MgThe susceptibility and cellular damage by WNV in low-buffer capacity cells may not be unique compared to other diseases. Catecholaminergic neurons in the substantia nigra express low amounts of calcium-binding proteins and are very vulnerable in Parkinson’s disease in human tissues 2D3) increases the expression of calbindin D28k 2D3 and immunoreactivity for the antibodies to vitamin D3 receptor (VDR) have been demonstrated in the CNS, including motor neurons 2D3 in the cerebral ventricles of rats reportedly induced CB-D28k ir in ventral motor neurons 2D3 to induce CB-D28k in neurological tissues and specifically in motor neurons of the spinal cord, and ultimately if 1,25(OH)2D3 can protect against WNV infection through the induction of CB-D28k. Moreover, identification of effectors of the anti-WNV effect of CB-D28k might lead to other strategies for treatment of WNND.The data of this report suggest that CB-D28k may suppress WNV replication to provide neuroprotective benefits to motor neurons of the spinal cord if CB-D28k is rapidly induced ahead of spread of the virus. There might be opportunities to pharmacologically induce CB-D28k to provide therapeutic benefits, or to utilize the effector mediating the anti-WNV effect of CB-D28k to suppress virus replication. One such effector might be an antagonist to an L-type calcium ion channel, because CB-D28k has been shown to decrease L-type calcium channel activity and modulate intracellular calcium in response to K+ depolarization in specific cultured cells This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Committee on the Ethics of Animal Experiments of Utah State University (USU) . All efforts were made to minimize suffering. The work was done in the AAALAC-accredited Laboratory Animal Research Center of USU.7 pfu of a New York WNV (NY WNV) isolate from crow brain 6 pfu of a WN02 isolate designated as KERN 515 from Dr. Robert Tesh . These two strains of WNV were used because of suitable mortality in the two rodent strains used and to demonstrate that the findings of this report are not rodent- or strain-specific.Adult female Syrian golden hamsters or C57BL/6 mice  were used (Charles River Laboratories). Numbered animals were randomize to groups with the use of a random-number generator. WNV was propagated in MA-104 cells. The stock was diluted in minimal essential medium (MEM) immediately prior to subcutaneous (s.c.) injection in the groin area Animals were monitored twice daily for morbidity. Rodents were humanely sacrificed by overdose of pentobartitol  if they did not step forward if prodded, or if they did not right themselves when placed on their backs. Ketamine/xylazine anesthesia and buprenorphine  analgesia were used when spinal cord injections were performed.Virus was assayed using an infectious cell-culture assay A murine motor neuron-like cell line, NSC34 2 incubator. After 48 hours of incubation, cells were infected with WN02 strain of WNV at 5 MOI. Two days after the infection, cells were fixed for immunocytochemistry with freshly prepared 4% paraformaldehyde for 20 minutes.Magnetofection Neuromag  procedure was adopted per the manufacturer’s instructions for transient transfection. NSC34 and Vero cells were seeded onto glass coverslips in 24-well plates. To prepare for transfection, 1 µg of linearized RSV-Neo or RSV-CB-Neo plasmids was incubated with magnetic beads at room temperature for 20 minutes. When cells were 60–70% confluent, 100-µL plasmid-bead complex was laid over each well, and the plates were kept on top of the magnetic bar for 20 minutes in a CO5 cells) were electroporated with 1 µg of linearized RSV-CB-Neo and RSV-Neo plasmids using a Nucleofector kit . Transfected NSC34 cells were selected with G418 (500 µg/mL)  for 14 days. Individual clones were trypsinized cells collected with cloning cylinders under the microscope, and transferred to 96-well plates for further cloning and expansion. Selected clones were verified to have elevated CB-D28k expression by western blot. Clones 1/1, 1/2, 1/3, 1/5, 1/6, 2/1 were obtained from one round of cloning; whereas, clones C20 and C25 were obtained after three rounds of cloning.Electroporation protocol was used to generate stably transfected cells. NSC34 cells . Using the histo option and freehand tools on the software, cells were identified individually based on the morphology to obtain the staining mean intensity for individual cells. Data were plotted as a scatter diagram for both stable and transient transfection experiments.Total cell lysates were obtained from WNV-infected hamster lumbosacral cord tissues at different points after the infection. Fresh tissues were lysed with MPER extraction buffer plus a cocktail of Halt protease inhibitors . Protein concentrations were determined using a Nanodrop spectrophotometer, (Nanodrop ND1000 Spectrophotometer). For each condition, 10 µg of protein per lane were separated on 10–20% SDS-PAGE gel in an Invitrogen Xcell Surelock Electrophoresis Cell  and transferred to PVDF membranes . The membranes were blocked for 1 hour at room temperature with 5% BSA in PBS, and subsequently incubated with the primary antibody against polyclonal CB-D28k  in 5% BSA overnight at 4°C with shaking. The membranes were washed three times with PBS for 10 minutes each. The membranes were incubated with anti-rabbit horseradish peroxidase secondary antibody (1∶2000)  for 1–2 hours at room temperature, and then washed as above. Antibody–antigen complexes were visualized using the Pierce ECL2 Western detection kit  and exposed to GeneMate Blue autoradiography film . Densitometrically, CB-D28k bands were normalized against their respective β-actin using ImageJ software .NSC34 cells stably transfected with CB-D28k- and vector-plasmids were seeded onto 6 well plates. When cells reached 70–80% confluence, they were infected with the WN02 strain of WNV at an MOI of 5. Forty-eight hours later, cells were trypsinized, washed with cold PBS, and fixed with freshly prepared 2% paraformaldehyde for 20 minutes on ice. After washing with FACS buffer , the cells were permeabilized with 0.1% Triton X100 for 5 minutes. After washing, cells were incubated with mouse monoclonal anti-CB-D28k  and humanized monoclonal anti-WNV (MGAWN1)