The proposed formalism is of universal concern and could be applied to any natural water surfactant system .Spatial and temporal variability of natural surfactant sea surface film structural parameters were evaluated from force-area isotherms, film pressure-temperature isochors, dynamic surface tension-time relations performed on samples collected in Baltic Sea shallow coastal waters. The film structure state was postulated as a 10-D dimensionless vector created from the normalized thermodynamic, adsorptive, and viscoelastic film parameters. The normalization procedure is based on the concept of self-corresponding states known in thermodynamics. The values taken by all the reduced parameters indicated a significant deviation from the reference ideal-2D gas behavior. The exhibited deviations of the surface parameters from the background values of the same thermodynamic state of each film were independent on the film-collecting procedure, sample solvent treatment, and temperature. The structural similarity was expressed quantitatively as a  distance between two vectors of the analyzed film and the standard one from the database, and appeared to be related to environmental conditions, surface-active organic matter production, and migration in the studied coastal sea region. The most distinctive parameters differing the films were  The sea surface microlayer (SML) is the thin surface layer of the ocean at the ocean-atmosphere interface that has distinctive physical, chemical, and biological properties compared to the underlying water .The novel classification (quantification) method of natural film structure presented here, based on physical attributes, required to specify a group of the sensitive film parameters originating from commonly available the film surface pressure-area isotherms, surface pressure-temperature isochors, dynamic surface pressure-time plots , surface material concentration , film molecules mobility, their interfacial interaction strength and thermal properties , film material solubility, 3D interfacial architecture, and miscibility of its film-forming components (y) , related to the 2D film molecule aggregation number (~ Naggr = Dmon/Deff), and activation energy barrier Ea/RgT are both attributed to mixed adsorption mechanism at the air/water interface met in natural sea layers T, for natural films, differ significantly from ideal 2D gas behavior πAm = kT, that is why the following quadratic equation of state, e.g., the 2D virial equation was proposed and scaled according to specific area  and surfactant solution, respectively,AmГ; Am = 1/ГNA,the area per film molecule related to the Gibbs’ adsorption kthe Boltzmann constant,Tthe temperature in Kelvins,NAthe Avogadro number,C0, C1, C2are the virial coefficients,A2)is the film-covered area (in cmThe isotherms (π-A)T, can be related to the mean number of moles nm present in the film, specific limiting area Alim, and mean molecular mass Mw of the film-forming surfactant mixture, as described in detail elsewhere  were evaluated using the Clausius-Clapeyron equation applied to the force-area isotherms taken at different temperatures  of the interfacial system.The isotherm hysteresis is a phenomenon reflecting different arrangements of the film-forming molecules in the closed compression-expansion cycles, and is attributed to a structural entropy change R:The isotherm reversibility y)—can be obtained from the relation Eisoth = yπ, by measuring the high-frequency limit of the surface modulus Eisoth as a function of the surface pressure (Pogorzelski y (< 3.5), the interfacial film-water subphase system demonstrates a “good” solvent behavior and one is concerned with a homogeneous monomolecular mixed film. Higher y values (y ≈ 8) lead to less film homogeneity observed as patches or domains (2D micelles) of film-forming components. For highest y values (> 10–16), the system indicates the vertical segregated film structures forming nearly separate horizontally situated layers at the interface (sandwich-like) where the most insoluble (hydrophobic) compound is placed on the outermost surface of this layered system A. dependence which leads to the surface pressure-temperature coefficient βs = (∂π/∂T)A  considered recently in (Boniewicz and Pogorzelski 2016) is related to the thermal translational molecules motion quantified with the kinetic surface pressure πk = βsT  dependence demonstrates the inflection points indicating the presence of particular critical phenomena  not reflected in surface pressure-area isotherms  are derived from the virial coefficients obtained via the best-fit procedure  applied to the registered (π-A)T isotherms within the initial surface pressure interval 0–2 mN m−1 where a surface film demonstrates the ideal 2D gas behavior. The elasticity modulus Eisoth is computed from the initial part of the isotherm (0 < π < 1 mN m−1) according to Eq.  part, as shown in Pogorzelski βs is obtained from a slope of the straight line tangent to the experimental plot computed using a least-squares fitting procedure , and applied to the particular temperature ranges below and above the cusp points evidenced at each isochore plots T and isochore (π-T)A plots are given elsewhere  number defined as the ratio of the film relaxation process time τ to the tobs—“time of observation” /dt]−1), as discussed in , De parameter is less than unity, and the film system is in its quasi-equilibrium thermodynamic state. Any relaxation process in films leads to surface viscoelasticity and may affect the shape of isotherms and consequently the recovered film parameters  and imaginary Ei  parts: E = Ed + iEi. The dilational viscoelasticity modulus can be approximated by its static Gibbs analogue Eisoth, under low film area deformation rates (De < <1).The thermodynamic equilibrium in the film is a serious problem in the light of the phase transitions evidenced therein. The effect can be quantified by means of the dimensionless parameter Deborah (∆t~0.1–0.7 s) relative area deformation (∆A/A0 = 0.1–0.3) was applied to the film, and the surface pressure-time decay curve was analyzed, as described in Pogorzelski and Kogut Ed » Ei). Recent natural seawater film studies revealed a two-step relaxation process with characteristic times τ1 (1.1–2.8) and τ2 (5.6–25.6) seconds  dependences at short (t→0) and long (t→∞) adsorption time intervals, where the saturation film pressure πs, and Gibbs adsorption Γs=πs/RgT are supplementary entering quantities  and 2D interfacial molecular aggregation number Naggr T, (π-T)A, and π(t) dependences have to be evaluated to get the normalization base parameters. All of them considered in our practice are collected in Table Mw(C17), Γ∞; Liss Rg, kT, dγ/dT; Birdi In order to create the multidimensional film structure vector, first the experimentally derived :Cartesian—geometric distance in a A dimension of the film structure vector can be rationalized by excluding the parameters closely correlated to each other since they born no more information on the film properties. A further step is to select a functional form of the distance in a multidimensional space. Exemplary dependences commonly used in multidimensional analyses are (Borg and Groenen 2.Street distance:3.Czebyszew distance suitable for establishing the difference in one particular parameter (dimension):Natural marine surfactant film surface rheology and adsorption studies in shallow off-shore waters of the Baltic Sea  were carried out in April–May, 2013 at selected locations along the coast from Brzeźno to Gdynia  from a rubber boat or a pier (platform).Sea surface microlayer samples were collected mostly under calm sea conditions (wind speed at a10-m height A0 = (1200 cm2) is compressed with an average deformation speed v = 0.6 cm2 s−1  by moving two paraffin wax-coated glass sliders towards each other symmetrically around the film pressure sensor (Wilhelmy plate technique using a piece of filter paper 5 cm wide attached to the force sensor). A detailed description of the measuring procedures and physical conditions adopted in surface pressure-area isotherm, surface pressure-T, and dynamic surface pressure registrations can be found in Pogorzelski and Kogut , film solubility and components miscibility , surface concentration , and film molecules mobility , the particular normalized vector components analyzed alone provide a synthetic characteristics of the film structure. On the basis of the presented data, the following film signatures can be distinguished, as evidenced from the parameters evolution on the large data set in Mazurek et al. −2) did not form saturate surfactant layers  layers  and formed non-uniform surface structures  and R4 (dCartesian = 26.546) sampling times in reference to the initial R1 (dCartesian = 10.358) that could be a result of the nature of hydrodynamic and atmospheric conditions further related to the surface water mixing at shallow sea regions. An increase of the vector length was observed for the spatial evolution starting from R6 (Gdynia) to R10 (Brzeźno). The highest value was noticed at Sopot (dCartesian = 31.317) where the municipal effluents are expected to contribute significantly in surface-active film-forming matter. Generally, dCartesian higher values were evidenced in locations close to town areas and the mouth of a river (R7). It is of interest to consider whether other distance functions operating in a multidimensional vector space can be equally useful or provide additional information. Temporal and spatial evolution of structure vector distance between the reference vectors R1 and R6 versus sampling locations, for different forms of the metric function , are shown in Fig. d(R1→R5) and d(R6→R10) in Fig. dstreet and lowest for dCzebyszew at all the locations and the characteristic points (maxima and minima). Czebyszew distance function allows one to point out the most distinctive parameter for the base and considered vectors (differing the both vectors in question), as it is evident from Eq.  registered at Jelitkowo. The largest vector lengths were observed at R2  of the film structure evolution between vector “In addition, the film structure quantification with a multidimensional approach allows application of multivariate statistical methods. From among many statistical techniques, principal components analysis (PCA) also known as empirical orthogonal function analysis and cluster analyses (CA) are frequently applied to geochemical data sets Meglen . In brieR) of similar chemical nature  if compared to the surfactant films evidenced in rainwater. In fact, the novel film structure classification approach seems to be of general concern applicable to a variety of natural water surfactant systems.It is interesting to compare the structural parameters of atmospheric water surfactant films (on rainwater and snowmelt samples) studied in neighboring sea  coastal areas  and higher miscibility (y = 6.46 ± 1.33), and elasticity modulus (Eisoth = 18.35 ± 2.02 mN m−1) in comparison to structural parameters  obtained for samples collected in a period of lower biological production  as evidenced in experiments with marine surfactant films  features. To support such an assumption, the following examples can be given. It could be learned from monolayer studies and a force-area quantification approach applied for physicochemical characterization of surface-active substances of the sea surface microlayer from Middle Adriatic stations . The variability of the reduced-normalized parameters introduced here rather than their absolute values is postulated to be a useful indicator of the film structural state, and can be used as a tool for natural water surfactant pathways tracing and reflecting the film spatial, temporal, and seasonal signatures.A set of the normalized structural parameters can be treated as coordinates of a multidimensional vector in a 10D space quantifying the film structural state. The structural similarity of the considered films can be expressed quantitatively as Cartesian distance between two vectors of the analyzed film and the standard one from the database, respectively. Application of different distance functions, i.e., street or Czebyszew may result in a better data presentation dynamics or pointing to the most distinctive parameters differing the studied films structure.Quantification of film structure with a multidimensional vector approach allows application of Principal Component Analysis (PCA) and Cluster Analysis (CA) to the data set amended with environmental characteristics.It is a promising starting point to create cost-effective, automatic, and rather simple technique to monitoring and assessment of environmental pollution in oceanographic practice.


Rational design of binder-free materials with high cyclic stability and high conductivity is a great need for high performance supercapacitors. We demonstrate a facile one-step synthesis method of binder-free MnO@C nanofibers as electrodes for supercapacitor applications. The topology of the fabricated nanofibers was investigated using FESEM and HRTEM. The X-ray photoelectron spectroscopy (XPS) and the X-ray diffraction (XRD) analyses confirm the formation of the MnO structure. The electrospun MnO@C electrodes achieve high specific capacitance of 578 F/g at 1 A/g with an outstanding cycling performance. The electrodes also show 127% capacity increasing after 3000 cycles. An asymmetric supercapacitor composed of activated carbon as the negative electrode and MnO@C as the positive electrode shows an ultrahigh energy density of 35.5 Wh/kg with a power density of 1000 W/kg. The device shows a superior columbic efficiency, cycle life, and capacity retention. To this end, supercapacitors (SCs), also known as ultracapacitors or electrochemical capacitors, have gained much attention as the next generation power storage devices, mainly due to their safe operation, outstanding cycling life, higher energy than conventional capacitors, and higher power density than batteries and fuel cells4. However, the bottle nick preventing their efficient use in future applications is their comparatively low energy density7. In this regard, transition metal oxide pseudocapacitive materials are primarily being used in SCs devices due to their high capacitance compared to carbonaceous materials9. While the former stores charge via rapid and reversible faradaic reactions at the electrode surface, capable of storing higher energy, the latter stores energy physically via the formation of electric double layer with lower energy storage but higher cycle life stability13. Among the various oxides used19, manganese oxides (MnxOy) have been widely investigated as promising candidates for SCs23. Recently, manganese monoxide (MnO) has attracted the attention as an active material for supercapacitor applications owing to its abundance, low cost, environmental compatibility, and high theoretical specific capacitance (1360 F g−1) that is higher than that of MnO2 (1110 F g−1) counterpart26. However, MnO suffers from limited cyclic stability, poor conductivity and loss of surface active sites within the synthesis process due to the agglomeration of the particles28. Consequently, the reported specific capacitance is still far from the theoretical specific capacitance. To this end, different approaches have been utilized including nanostructuring the material and/or mixing with conductive materials. Hu et al. reported the fabrication of MnO2 nanoflowers supported by a backbone of nanoporous gold (NPG) wires, which enabled fast ionic/electronic transfer at the electrode surface29. Yu et al. reported the hydrothermal growth of MnO@C directly on carbon cloth and its use as a negative binder-free electrode23. Although carbon coating provides efficient electron transfer pathway, the capacitance dropped to 86% after 5000 cycles30. Moreover, Liao et al. have synthesized MnO nanoparticles anchored to vertically aligned graphene sheets (VAGN), showing 80% capacitance retention after 4000 cycles27. Otherwise, Wang et al. reported that MnO@mesoporous carbon achieved a high cycling stability of 110% of its original value24. However, the electrode material showed low specific capacitance (160 F @ 1 A g−1)31. To this end, the fabrication of MnO-based materials with integrated high properties  is still an open challenge.The development of energy storage devices is indisputably one of the great challenges in the 2133. The resulted binder-free electrode was used as a positive electrode in asymmetric supercapacitor devices. The fabricated composite material system was chosen for the following reasons: (1) carbon matrix not only does enhance the electronic/ionic transfer pathways34, but also provides a robust support for MnO nanoparticles, preventing the agglomeration during the synthesis and utilization28, (2) the binder-free electrode avoids the contact impedance between the current collector and the active film, which simplifies electrode fabrication steps35, allowing for a full utilization of the composite material, and (3) the 3D interconnected matrix provides a high capacity and cycling stability36.Herein, we demonstrate the ability to grow MnO nanoparticles within interconnected carbon matrix via direct electrospinning on graphite substrate followed by thermal treatment directly in one step without incorporation of pre-synthesized manganese oxide nanostructures with polymersAll chemicals were directly used as purchased without further purification. 10% Polyvinylpyrrolidone (PVP Mw = 1300000) solution was prepared in ethanol (99.9%). Then, 2 M manganese chloride solution was slowly added to the solution of PVP with constant stirring for 6 hours. The spinning solution was then transferred into a syringe with fine capillary metallic needle. The electrospinning process was carried out using a conventional electrospinning setup  at a working distance of 15 cm, a working voltage of 15 kV, and a feeding rate of 0.6 ml/h. Samples were collected on graphite sheets and aluminum foils. The dried nanofibers were annealed for 4 h at 650 °C with 1 °C/min uprate in argon gas. For comparison, a pure graphite sheet was annealed under the same conditions and weighed before and after annealing. As no mass loss was observed, the mass of the formed MnO@C was measured by the same way.The microstructure and morphology of the samples were observed using field emission scanning electron microscopy  and high-resolution transmission electron microscope  operating at 200 kV accelerating voltage of. X-ray powder diffraction (PXRD) patterns were recorded on a Panalytical X′pert PRO MPD X-ray Diffractometer with Cu Kα radiation . The elemental composition was investigated using X-ray photoelectron spectroscopy .2) acted as the working electrode, a platinum foil served as the counter electrode, and Ag/AgCl electrode as a reference electrode and 1 M Na2SO4 as the electrolyte. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were carried out using a BioLogic SP-200 potentiostat. Constant current charge/discharge (CCCD) measurements were conducted using a CHI model 700D series electrochemical workstation. Specific capacitance was calculated based on the mass of the MnO@carbon.The three-electrode configuration was adopted to measure the electrochemical behavior of the electrode material. The formed electrode  analysis , as shown in Fig. sis Fig.  demonstr38. No other peaks of impurities are detected, suggesting the successful synthesis of very pure composite electrodes with a manifested crystallinity as confirmed via SAED and HRTEM results. To further investigate the surface and chemical composition of the fabricated electrode materials, XPS analysis was performed. The high-resolution Mn 2p spectrum . The CV plots at low scan rates (<50 mV s−1) appeared as approximately quasi rectangular shape without any battery-like redox peaks, indicating that the cell potential is varying linearly with time. Besides that, the cyclic voltammograms showed symmetric characteristics with respect to voltage axis at zero current. All these observations reflect that MnO@C possess a close-to-ideal pseudocapacitive electrochemical characteristics42. On the other hand, at high scan rates (50–200 mV/s), the CV plots reveal a little deviation from rectangular style owing to the electrode polarization, which can be due to the diffusion difficulty which faced Na+ ions to get intercalated deeper within the bulk of the material at high scan rates43. The charge mechanism at low/high scan rates is based on the intercalation/deintercalation and adsorption/desorption encompassing bulk and surface similar to the other manganese oxides reported in literature45. This behavior is accompanied by oxidation/reduction between Mn+2 and Mn+3 ions at the bulk and surface the of the material27. Figure −1) of MnO@C with the scan rate calculated from the CV plots according to Eq. −1 at a scan rate of 5 mV s−1 and drops to 199.8 F g−1 at a scan rate of 200 mV s−1. Additionally, CCCD curves measured at various current densities (1 to 20 A g−1) are illustrated in Fig. dt is the discharging time (s), I is the discharging current (A), m is the mass of the active material (g) within the electrode and dV is the discharging potential range (V). Figure −1 at 1, 2, 5, 10 and 20 A g−1, respectively. Note the gradual decrease in the specific capacitance with increasing the current density, as the mass efficiency/utilization gets decreased at high current densities. However, MnO@C electrode was capable of maintaining about 42.9% of its initial specific capacitance as the current density was increased from 1 A g−1 to 20 A g−1. Note also that the specific capacitances calculated from both CV and CCCD have very close values. Thus, CV and CCCD methods effectively determine the capacitance of the electrode.The electrochemical characteristics of MnO@C were assessed by cyclic voltammetry (CV), constant current charge and discharge (CCCD) and potentioelectrochemical impedance spectroscopy (PEIS) in a 3-electrode electrochemical cell containing 1 M Na−1), Fig. xOy electrodes47. Upon cycling, the surface wettability increases, which facilitates the continuous diffusion of the electrolyte ions into the electrode microstructure with better pathways reaching more deep particles. This diffusion enhancement leads to an activation effect throughout the electrochemical cycling, resulting in capacitance increase during cycling48. This also could be attributed to the superior architecture design of the electrode material, which provides a conductive matrix (carbon) throughout the MnO particles with smooth electron and ion transport pathways. Without this supporting design, the electrode material would suffer from low cycling stability. This issue can also be corroborative by determining the equivalent series resistance (ESR) of the electrode material as well as the charge transfer resistance. On the other hand, PEIS measurements were executed for the MnO@C electrode material before and after 3000 cycles in the frequency range 100 kHz–10 mHz with an excitation signal of 5 mV.Moreover, as the cycle life is a unique metric parameter of supercapacitors in practical application, the cycling performance of the fabricated MnO@C electrodes was investigated at high current density , indicating a negligible Ohmic loss during cycling. Furthermore, the near-vertical line in the low frequency range alludes to the good capacitive behavior of the electrode material with low electrolyte diffusion impedance and fast electron transfer within the electrode material. Moreover, the observed straight line in the low frequency range became a little steeper after 3000 cycle, indicating easier diffusion, in agreement with the capacitance increase during the cycling stability test51.No semicircles were detected at high frequency Fig.  inset, i2SO4 aqueous electrolyte. Besides, it is well-known that the voltage in an asymmetric capacitor will divide relying on the capacity of each electrode. Consequently, the optimum cell voltage with maximum capacitance and best cycle life would be achieved if the charges at both positive/negative (q+/q−) electrodes are balanced. As the stored charges (q+/−) are relevant to the potential window (ΔE), the specific capacitance (Cs), and the mass (m) of the electrode are relevant according to Eq. 54To further assess the MnO@C electrode for real applications, a two-electrode asymmetric supercapacitor device was assembled by integrating activated carbon as the negative electrode, MnO@C electrode as the positive electrode, and a filter paper as the separator in 1 M Na−1). It is also noticed that the device achieved a maximum operating cell voltage of 2 V. Thus, it would be a positive sign to obtain a high energy density supercapacitor in aqueous medium. The capacitive property of the assembled MnO@C//AC device was further investigated via CCCD tests at different current densities from 2 to 12 A g−1 in a potential window of 0–2 V. Figure Figure −1 for 7000 cycles using the CCCD technique. Figure The stability test of the assembled MnO@C//AC device was performed at a discharging current density of 12 A gPower (P) and energy (E) densities are considered the two substantial performance metrics of supercapacitor devices. Figure −1) in 1 M Na2SO4 at 1 Ag−1 with excellent cycling stability. MnO@C was examined as the positive electrode in asymmetric device; delivering a high energy density of 35.5 Wh kg−1 with a power density of 1000 W kg−1 with very high columbic efficiency and cycling stability.We demonstrate the successful fabrication of MnO@C via electrospinning method. The XRD, XPS and TEM results confirmed the formation of 3D interconnected MnO@C with high crystallinity and purity. The binder-free MnO@C electrodes showed very high specific capacitance (578 Fg


The Stark effect plays a key role in understanding why, against expectation, alkane thiols are not high-performance molecular rectifiers. I–V) results for CP-AFM (conducting probe atomic force microscope) CnT junctions of various molecular lengths  and different metallic contacts  are consistent with conduction dominated by the HOMO, but the measured RR ∼ 1.5 is much smaller than that predicted by the potentiometer rule framework. Further, the linear shift in the HOMO position with applied bias, γ, which gives rise to rectification, is also smaller than expected, and critically, γ has the opposite sign from potentiometer rule predictions. Companion ab initio OVGF  quantum chemical calculations provide important insight. Namely, a linear Stark shift γm is calculated for the HOMO of CnT molecules for electric field strengths (106–107 V cm–1) typical of molecular junctions, and the sign of γm matches the sign of the experimental γ for junctions derived from transport measurements, suggesting that the Stark effect plays an important role. However, the magnitude of the measured γ is only 10–15% of the computed value γm. We propose that this implies that the contacts are far from optimal; they substantially screen the effect of the applied bias, possibly via molecule–electrode interface states. We predict that, with optimized contacts, the rectification ratios in CnT-based junctions can reach reasonably high values (RR ≈ 500). We believe that Stark shifts and limited current rectification due to non-ideal contacts discussed here for the specific case of alkane monothiol junctions are issues of general interest for molecular electronics that deserve further consideration.Many attempts to obtain high current rectification ratios (RRs) in molecular electronics are triggered by a potentiometer rule argument, which predicts that a strongly asymmetric location of the dominant molecular orbital yields large RR-values. Invoking this argument, molecular junctions based on alkane monothiols (CnT) can be expected to exhibit high RRs; the HOMO of these molecules is localized on the thiol terminal group bonded to one electrode. The extensive current–voltage ( Building on the two-parameter model (HOMO offset |ε0| and coupling Γ) utilized earlier for symmetric junctions,γ  method,γ → γm of the Stark effect strength for isolated molecules.Second, by performing accurate γ with a sign that matches the sign of the γ values extracted from transport measurements. Yet the magnitude of the measured γ for junctions is only 10–15% of the computed value γm for isolated molecules. Contact effects are likely the cause that the applied bias (and hence electric field) felt by the molecules in the junction is small, giving rise to a smaller γ. The lower value of γ is in turn responsible for the low RR (second observation).Third, we demonstrate that the CP-AFM CnT junctions diverge from the predictions of the potentiometer framework in two important respects: (1) the sign of the bias-induced HOMO energy shift is opposite to the value one would predict given the spatial location of the HOMO; (2) the junctions exhibit weak rectification (RR ∼ 1.5–2). The first observation is explained in terms of the above mentioned Stark effect. In fact, the potentiometer rule does not hold in CnT systems; instead electric fields in the junction interact with the HOMO to produce Stark shifts γ and γm obtained as outlined above, which is substantial, is likely related to phenomena occurring at molecule–electrode interfaces and demonstrates that the latter are very important for rectification. This finding provides a fresh look at the problem of molecular current rectification and highlights the surprisingly important roles that the Stark effect and contacts (possibly interface states) have in this context.Notwithstanding the very modest measured RR-values, the analysis emerging from the present joint experimental–theoretical study provides significant insight into the understanding of molecular rectification going beyond the specific case of CnT. The difference between the values I–V equation deduced in cf.eqn (2)), and a Lorentzian-shaped transmissionε0 = EMO – EF represents the MO energy offset relative to the Fermi level characterizing the unbiased junction. In general, the MO energy EMO(V) in a biased junction (V ≠ 0) is shifted with respect to its position in the absence of bias EMO ≡ EMO(V = 0) by a quantity proportional to VTo interpret transport measurements on our CP-AFM junctions based on CnT, we employ below the basic γ in eqn (2) should be more properly referred to as the Stark effect strength.22In view of possible screening effects and arguments presented later, rather than the voltage division (potentiometric) factor,γ is in order at this point. We define the bias polarity such that a positive bias (V > 0) corresponds to the tip having a higher electric potential than that of the substrate . In real CnT junctions, methyl terminal groups are coupled to the tip while thiol groups are coupled to the substrate. Adopting this convention, in the quantum chemical calculations presented below, a positive bias corresponds to an electric field , a positive bias  causes a downward shift of the MO energy, whereas a negative bias  yields an upward shift of the MO energy. This means that, in cases where γ < 0 and conduction is dominated by the HOMO , a positive bias takes the HOMO away from the Fermi energy, thereby reducing the current, while a negative bias brings the HOMO closer to the Fermi level, thereby enhancing the current. Therefore, for γ < 0 and HOMO conduction, the HOMO level tracks the tip  for V > 0 and increased (LUMO more distant from the Fermi level) for V < 0; this would result in higher currents at positive biases than at negative biases.A discussion of the sign of tip cf.,  and currG = 1/R of the CP-AFM junction can be expressed as follows: being the geometrical average of the couplings Γs and Γt between the MO and the substrate (s) and tip (t) electrodes, G0 = 2e2/h is the quantum conductance, and N is the number of molecules contributing to the transport through the CP-AFM junction.Returning to the single level model, the zero-bias conductance I–V characteristics, the quantity V2/|I|Vt+ > 0, Vt– = –|Vt–| < 0) and different magnitudes (Vt+ ≠ –Vt–) defining two transition voltages. They can be used to estimate the magnitude of the energy offset of the occupied level (ε0 = –|ε0| = EHOMO – EF < 0) that dominates the charge transport and the Stark effect strength γ as followsIn the case of molecular junctions with asymmetric R-data presented in R exponentially increases with the molecular size n  and contact (Rc) are disentangled. This exponential dependence is well knownvia off-resonant tunneling. Second, R (and Rc) dramatically decreases as the electrode work function Φ increases , this dependence on Φ represents unambiguous evidence that charge transport in CnT junctions is mediated by an occupied level.The measured low bias I–V characteristics of CP-AFM junctions based on CnT  with various metallic electrodes . The semilogarithmic I–V traces are shown in Fig. S4.i.e., fixed n) and bias, currents increase as the electrode work function increases. Again, these two features indicate hole transport via tunneling mediated by an occupied level.I–V characteristics over the interval ±1.6 V (±2.0 V for Ag/Ag junctions), recasting them as curves of V2/|I| versus V (V = Vt) for either bias polarity (Vt → Vt±) where the differential conductance is two times larger than the nominal (pseudo-ohmic) conductance . This approach is an alternative reformulation of transition voltage spectroscopy (TVS),Vt (defined as the bias at the minimum of the Fowler–Nordheim plot) are mathematically identical.I–V curves of alkane dithiol junctions,I–V curves of the present CnT exhibit a significant asymmetry [I(–V) ≠ –I(V)]. Accordingly, the magnitude of the transition voltages at positive (Vt+ > 0) and negative (Vt– < 0) biases for CnT differs (cf.i.e. the tip in contact with the methyl end is positively polarized with respect to the substrate in contact with the thiol anchoring group) Vt+ is ∼0.2 V larger than the magnitude of the average value |Vt–| at negative bias polarity (i.e. the tip in contact with the methyl ends is negatively polarized with respect to the substrate in contact with the thiol anchoring group).For a more comprehensive examination of transport properties as in our previous study on alkane dithiols,versus V . This tyffers cf.. Table 1Vt± and eqn (4) allow us to determine the energy offset of the dominant level |ε0| , this results in n-independent |ε0|-values  allows us to determine the Stark strength γ-histograms of Fig. S7γ obtained in this way is negative. By virtue of eqn (2), this implies that an electric field pointing toward the methyl group  yields an upward shift of the dominant level energy while an electric field pointing toward the thiol group  yields a downward level shift, as shown in γ).In spite of some statistical spread .As we will see below, corroborating the information on ε0|, γ, and Γ determined from the experimental Vt±- and G-values viaeqn (3)–(5) we are able to reproduce well the individual I–V traces measured for CnT junctions. Some representative I–V theoretical curves obtained viaeqn (1) and (2) superimposed on the corresponding measured I–V traces in the bias range (|V| < 1.5|ε0|/e) where eqn (1) appliesI–V curves ,I–V curves are asymmetric . Noteworthy is the fact that, similar to the case of molecular junctions with symmetric I–V characteristics, to determine our model parameters, we need not fit the full I–V curves; we only fix these model parameters as described above. Of course, the fact that the I–V curves calculated in this way excellently reproduce the experimental curves represents a significant self-consistency check for the model based on the bias-driven shifted single level, thereby validating this model for the benchmark case of molecular (CnT) junctions with asymmetric I–V characteristics.With the model parameters |To end this part, the presently employed model provides us with a better understanding of the properties of the CP-AFM junctions investigated here than the Simmons model used earlier by our groupI–V measurements presented above demonstrated that the charge transport in the present CP-AFM molecular junctions based on alkane monothiols can be explained by assuming that a single occupied level is dominant. On this basis, a LUMO-mediated conduction should be ruled out  yielded values of the HOMO energy practically independent of the molecular size n. Because this behavior is similar to our recent report for alkane dithiol junctionsn-independent values of |ε0| deduced from the transport measurements  level. The subscript m denotes the calculated results for isolated molecules.OVGF-calculations for isolated CnT molecules placed in an external electric field on Є see . This lierest cf.6EHOMO,m(ρHOMO(r) ≡ |Ψ(r)|2, where its wave function Ψ is an eigenfunction. Quantum mechanics  in the absence of bias generated with GABEDIT–2 a.u. (atomic units). The right panel visualizes changes in the HOMO distribution ρHOMO at biases close to the highest values used in experiment. If we depicted those HOMO distributions ρHOMO, their very small differences from the left panel at V = 0 would be invisible within any reasonable drawing accuracy. Therefore, to give a flavor of these tiny changes, in the right panels of δρHOMO ≡ ρHOMO – ρHOMO by using a much smaller isovalue of 9.4 × 10–5 a.u.It is worthwhile noting that an applied field/bias acts as a perturbation on the molecule that in principle may cause corrections to both the HOMO energy (eqn (6)) and HOMO spatial density. From the point of view of quantum mechanics, the HOMO energy is an eigenvalue, and the HOMO spatial distribution is expressed by cs e.g.,  tells usi.e., of first order in V). This implies that an applied bias negligibly affects the HOMO wave function. In turn, this means that bias-driven changes to the HOMO spatial density are altogether negligible. For this reason, MO (HOMO or else) spatial distributions are not significantly altered by applied biases in all other cases where quantum chemical calculations yield a linear dependence on the V of the type expressed by eqn (6). In the same vein, improving rectification by a bias-driven enhancement of the asymmetry of MO distributions can hardly be expected.We noted above that the dependence of the HOMO energy (eqn (6)) is found to be linear (The linear dependence of the HOMO energy expressed by eqn (6) is analogous to the linear behavior of the energy of the occupied level found to dominate the transport in CnT junctions expressed by eqn (2). Note also that the calculated linear dependence exhibited in V for junctions or Vm for isolated molecules) means an electric potential at the methyl (tip) end higher than the electric potential at the thiol (substrate) end, this result implies that the energies of the HOMO (for an isolated molecule) and of the occupied level that dominates the transport in a junction exhibit a similar qualitative behavior, namely a linear dependence on bias with a negative slope; γ of eqn (2) and γm of eqn (6) have the same negative sign.Our OVGF-calculations indicated that an electric field directed toward the methyl (thiol) group raises (lowers) the HOMO energy . Noting (iv)Є, longer molecular species exhibit larger HOMO energy shifts than shorter species. However, by recasting the dependence of the HOMO energy on the electric field Є of versus bias (Vm = ЄdHH), we found that the straight line of EHOMOvs. Vm is practically independent of the molecular size n  (and γm of eqn (6)) represents a Stark effect strength.Є (and on bias) is the direct consequence of the strongly inhomogeneous spatial distribution of the relevant level; the linear contribution in Є would vanish and the correction to the level energy would be proportional to Є2 if the spatial distribution was homogeneous, like in the case of the Stark effect for atoms.61So, as anticipated, the results of quantum chemical calculations presented above support the assumption of a HOMO-mediated conduction in CnT junctions. They reveal a series of similarities between the HOMO energies and the values extracted from transport data of the energies of the single level found to dominate the charge transport, which indicates that it is plausible to ascribe the occupied dominant level to the HOMO. Out of these similarities, the fact that both group cf., very clγ of eqn (2) describes the bias-driven shift of the HOMO energy of a molecule embedded in a CP-AFM junction, while γm of eqn (6) refers to the HOMO of an isolated molecule. If we identified the bias Vm with the experimental tip-substrate bias V, the slopes of γm) would be almost one order of magnitude larger than the values of γ extracted from experiment (cf.V) applied on the junction; what the HOMO energy feels is only a small fraction q of the bias applied between the tip and the substrate:More quantitatively, one should note that iment cf., Fig. 7Aq is represented in q ≈ 0.1–0.15 visible in This ratio I–V data demonstrate that nanojunctions based on alkane monothiols act as weak molecular rectifiers: currents at positive biases V  are lower than currents at negative biases: I(+V) < |I(–V)|. The rectification ratios RR ≡ –I(–V)/I(V) are in the range of 1.5–2 at 1.5 V for alkane monothiol junctions. According to eqn (3) this weak current rectification property traces back to the fact that the energy level offset |ε0(V)| at positive tip biases (V > 0) is larger than that at the same bias of opposite polarity (–V < 0); that is, |ε0(–V < 0)| < |ε0(V > 0)|, as depicted in As seen above, 2 or even 105.I–V asymmetry (“rectification”) – current rectification is very sensitive to the values of γ (RR = 1 for γ = 0). To illustrate this point, in γ ranging from γ = –0.05 to γ = –0.3; where, the lowest γ-value corresponds to the experimental situation  comparable to reasonably high values reported in the literature can be reached if improved contact engineering made it possible that the active HOMO level would feel the entire applied bias.Recently, various groups succeeded in fabricating molecular devices exhibiting rectification ratios (RRs) larger than 10ation cf. while the.g., van der Waals interactions) seems to be a meaningful route to pursue in view of the recent rectification improvements achieved in this way (the weak coupling regime).From the foregoing perspective, identifying and implementing appropriate electrically transparent contacts (no screening) appears to be an important problem for molecular rectification. In this vein, it is not at all surprising that significant improvements of molecular rectifiers were obtained with non-traditional approaches of contacting the SAM to electrodes. Attempting to employ platforms wherein covalent coupling between the electrode and the “active” unit (thiol in our case) on which the dominant HOMO orbital is centered is replaced by non-covalent interactions  having work functions Φ varying within a broad range of ∼1.4 eV. An important aspect of the present study, which enables us to propose a coherent picture of the transport, is the validation of the model of a single dominant level that is linearly shifted by the applied bias (linear Stark effect as opposed to quadratic Stark effect in atoms).In this paper, we have reported results of a joint experimental–theoretical investigation of the transport properties of CP-AFM molecular junctions based on alkane monothiols (CnT) of various lengths ab initio quantum chemical calculations based on the outer valence Green's function (OVGF) method for isolated molecules, we are able to understand why this rectification is so unexpectedly weak. Namely, we have demonstrated that it is only a small fraction of the applied voltage that is responsible for the bias driven shift of the dominant level energy, which in turn is responsible for current rectification. This is a clear indication that phenomena occurring at molecule–electrode contacts are responsible for the unexpected weak rectification of CnT junctions. We believe that this finding represents an important new insight into the role played by contacts in mitigating the impact of the applied bias on the embedded molecule (possibly via interface states), which is an essential point to consider for improving the performance of molecular rectifiers.Contrary to what one expects intuitively based on the potentiometer rule, CnT junctions exhibit very modest rectification ratios (RR ≈ 1.5). By corroborating the experimental results on nanojunctions with state-of-the art 1-Heptanethiol (C7T) 98%, 1-octanethiol (C8T) 98.5%, 1-nonanethiol (C9T) 99%,1-decanethiol (C10T) 99%, and 1-dodecanethiol (C12T) 98% were purchased from Sigma Aldrich. Gold nuggets (99.999% pure) were purchased from Mowrey, Inc. . Silver pellets (99.99% pure) were purchased from Kurt J. Lesker Company. Evaporation boats and chromium evaporation rods were purchased from R. D. Mathis . Platinum and titanium for e-beam evaporation were purchased from Kamis, Inc. . Silicon (100) wafers were obtained from WaferNet . Contact mode AFM tips (DNP-10 silicon nitride probes) were purchased from Bruker AFM Probes.2.Contact mode AFM tips were coated with Ag, Au and Pt. Template-stripped flat metal substrates were used to grow high quality self-assembled monolayers (SAMs) for sample characterization and reproducible electrical measurements. The preparation of AFM tips and template stripped flat substrates with Ag, Au and Pt has been described previously.I–V characteristics were recorded; V > 0 means a positive tip  resistance. The low bias resistance was measured between ±0.1 V except the data of C12T, which were collected between ±0.5 V due its low conductivity, and ±1.5–2 V was applied to the tip to get the transition voltage Vt.The electrical measurements were completed by mounting the substrates in the AFM and bringing the metal coated tip into contact with the SAM under ∼1 nN of applied compressive load, –4 a.u. = 0.05142 V nm–1) along the negative y axis, we set “FIELD = Y-8 NoSymm EPT = OVGF TRAN = FULL IOP(9/11 = 100)” in the GAUSSIAN*.com input file. Within the OVGF framework the HOMO energies (shown in The quantum chemical calculations were based on the OVGF method.shown in  are estiThere are no conflicts to declare.Supplementary informationClick here for additional data file.


Pathology archives with linked clinical data are an invaluable resource for translational research, with the limitation that most cancer samples are formalin-fixed paraffin-embedded (FFPE) tissues. Therefore, FFPE tissues are an important resource for genomic profiling studies but are under-utilised due to the low amount and quality of extracted nucleic acids. We profiled the copy number landscape of 356 breast cancer patients using DNA extracted FFPE tissues by shallow whole genome sequencing. We generated a total of 491 sequencing libraries from 2 kits and obtained data from 98.4% of libraries with 86.4% being of good quality. We generated libraries from as low as 3.8 ng of input DNA and found that the success was independent of input DNA amount and quality, processing site and age of the fixed tissues. Since copy number alterations (CNA) play a major role in breast cancer, it is imperative that we are able to use FFPE archives and we have shown in this study that sWGS is a robust method to do such profiling. The samples used in this analysis were derived from the METABRIC cohort, which encompassed a large biobank of fresh frozen tumor samples collected across five major teaching hospitals in the UK and Canada (Comparative Genomic Hybridisation (CGH)  has had d Canada .Formalin-fixed paraffin-embedded (FFPE) tissue samples are more routinely collected and hence more representative of cancer in the general population. These FFPE archives are a valuable resource for molecular profiling in cancer research. Whilst the fixation process is essential to protect cellular morphology and protein expression, it is detrimental to nucleic acids and results in their chemical modification and degradation. As a result, extraction of DNA from FFPE tissues results in lower yields when compared to extraction from fresh frozen tissues. DNA extracted from FFPE works well for downstream applications using polymerase chain reaction (PCR), particularly for small size amplicons (<300 base pairs), but for other applications, including microarray based CGH, where efficient labelling of the DNA is dependent on its integrity, its use is more challenging. There have been several studies describing different methods for DNA extraction , qualityNext generation sequencing has revolutionised cancer genomics. It is now relatively easy and inexpensive to sequence an entire genome. However, as with microarrays, the robustness of the results obtained are dependent on the quality of the input DNA. Two recent studies have demonstrated the feasibility of doing shallow whole-genome sequencing (sWGS) for CNA profiling using DNA extracted from FFPE tissue material . The firHere we present extensive sWGS data generated from DNA extracted from FFPE breast cancer samples to describe steps to ensure successful libraries.22.1NCT00432172 & NCT00841828). Detailed information on the sample cohort is collated in FFPE tissue samples from invasive breast cancer patients diagnosed between 1997 and 2014 were obtained from several tumor repositories: Addenbrooke's Hospital in Cambridge (n = 62), a consortium of hospitals participating in clinical trials (GEICAM) in Spain (n = 172), and Samsung Medical Center in South Korea (n = 122). In some cases, we extracted DNA from adjacent normal (n = 15) and DCIS (n = 115) samples. Some of the clinical trials samples were biopsies taken at diagnosis (n = 107) and/or surgery (n = 106) where 41 are paired. All tumor samples were collected with informed patient consent and their use for genomics profiling had ethics approval from the institutional review board for each of the biobanks , or 4-6 × 10 μm sections (Spain) from FFPE blocks, using Qiagen QIAmp DNeasy Kits  according to the manufacturer's instructions. All DNA samples were quantified fluorometrically using the Qubit dsDNA High Sensitivity Quantification Reagent . The DNA quality was assessed using Illumina's FFPE QC kit, a quantitative PCR (q-PCR) assay. All test DNAs and the template control provided in the kit (ACD1) were diluted to 0.25 ng/μl and PCR reactions set up in triplicate as per manufacturer's instructions. DNA quality was quantified as the difference between the Ct (cycle threshold) value of the test FFPE-extracted DNA against the Ct value of the control DNA template.2.3DNA samples of different concentrations (4-500 ng) were diluted in water to a final volume of 15 μl in Covaris microTUBE-15 8 strip tubes  and fragmented to an average size distribution of 150-180 bp with Covaris LE220 Focused Ultrasonicator with Adaptive Focused Acoustics technology. The following parameters were used for shearing: Peak Incident Power: 180 W; Duty Factor: 30%; Cycles per Burst: 50; with the fragmentation time: 250 s for DNA with ΔCt <10, and 200 s for DNA with ΔCt ≥10.2.4Sequencing libraries were generated using either the beta testing version of the Illumina FFPE TruSEQ kit  or the Rubicon Genomics Thruplex DNASeq , as per manufacturer's instructions. For four samples, we generated sequencing libraries using both kits to compare their performance a–b. The 8 reads per lane.The ILMN libraries were generated manually whilst RGT libraries were generated either on the Agilent Bravo (n = 228) or manually . Final libraries were purified using magnetic beads  and eluted libraries were quantified using Kapa Library Quantification kit . Fragment size distributions were analysed utilising a 2100 Bioanalyzer with a DNA High Sensitivity kit . Two nanomoles (nM) of each library were prepared and 48 samples were pooled in one lane for sequencing on a HiSeq4000 . The pools were re-quantified and normalised to 10 nM. Single end sequencing was conducted for 50 cycles, generating on average 4.3 × 102.5https://broadinstitute.github.io/picard) or Novosort . Circular binary segmentation on the aligned files was performed in 100 kb windows using the QDNAseq R package available on Bioconductor, which corrects for mappability and GC content  yielding low amounts of DNA . Therefore, to successfully generate libraries for CNA profiling using limited input DNA, we needed to understand how different variables could influence the quality of libraries and steps that can be taken to ensure good sequencing results .Fig. 1Ov3.1We examined the copy number plots by manual inspection and categorised them based on the variance in the CN data for each case into categories: “Very Good”, “Good”, “Intermediate” and “Poor” a. We als3.2We tested two kits (Illumina FFPE TruSEQ kit and Rubicon Genomics Thruplex DNASeq) using four FFPE samples to generate sequencing libraries and found comparable results a–b. The 6 reads) for six libraries with high read counts (up to 24 × 106 reads). We found a significant improvement in the quality of copy number plots with increasing number of reads  and found improvement in the data quality in 20 out of 23 libraries  libraries failed and 12 (2.7%) generated poor quality libraries out of 446 libraries. To recover some of these failed/poor samples, we prepared fresh libraries from samples with sufficient DNA (n = 6) or repeated the sequencing using three-fold more library material for samples with insufficient DNA to generate new libraries (n = 8). Thirteen of these new/re-sequenced libraries generated good quality data. The one repeat sample that failed was from the re-sequencing group. Consequently, only two out of 446 RGT libraries (taking into consideration the repeated libraries and re-sequencing) failed, resulting in a 99.5% success rate. Good sWGS data produced from 379/446 (84.9%) samples.3.5The FFPE samples were collected from three different tissue banks, spanning 20 years . The eff3.6t-test, p = 5.24e−06; 500 ng: 23.0 nM, Welch Two Sample t-test, p = 0.0121). There was no difference in library yield when using either 200 or 500 ng of DNA . This is probably due to the quality of the input DNA as libraries produced from 200 ng of DNA had lower ΔCt values  than those using 500 ng  value of the test FFPE-extracted DNA against the Ct value of the control DNA template provided in the kit. Increasing ΔCt values indicate decreasing DNA quality with Illumina quality thresholds set at: ΔCt < 1.5 denotes high quality (HQ), ΔCt < 3.0 denotes medium quality (MQ), and ΔCt > 3 denotes low quality (LQ) DNA. The Illumina DNA-input recommendations for sWGS are 50 ng DNA with HQ DNA, 200 ng with MQ DNA, and exclusion of LQ DNA. Using the ILMN kit, we could generate good quality sWGS using 50 ng HQ and MQ DNA, and 200-500 ng of LQ DNA. Unsurprisingly, for eight samples with paired libraries generated from 50 ng and 200 or 500 ng of input DNA using the ILMN kit, we found that the sequencing library yields generated with more DNA was significantly higher than when using only 50 ng . This is probably due to the fewer library-washing steps using the RGT kit (six washing steps in the ILMN protocol versus one in RGT).Using the RGT kit, we found no correlation between amount of input DNA and sequencing library yield  groups a, fragmeUsing our copy number output categorisation scoring, we examined if quality of the libraries  can be attributed to the different features of the input DNA and library yields , Fig. 5.4In this study, we have looked at the effect that quantity and quality of DNA from FFPE tissues has on successful sWGS library preparation for CN profiling of human breast cancers. Both the quantity and quality of DNA have always been an important consideration for sample selection and in deciding which genomic application to use. For example, microarrays require 100 ng–2.5 μg of DNA depending on the resolution of the arrays whereas PCR based methods require only 10 ng of DNA. In our hands, we have not had much success in obtaining CN data with DNA extracted from FFPE DNA using microarrays, especially when the extracted DNAs are more fragmented and of lower quality .Here we have robustly shown that we can generate CN data from virtually all archived FFPE samples using sWGS. We show good CN profiling data irrespective of the quality of input DNA, as inferred by whether it can be amplified with Q-PCR (ΔCt). Previous work has extensively tested the utility of FFPE DNA for mutation analysis  but to dAnother advantage of generating sWGS libraries is the ability to use the same library for targeted sequence enrichment to identify mutations. There have been other methods reported for CN profiling using DNA extracted from FFPE samples but these methods do not generate sequencing libraries that can then be used for target enrichment and sequencing  or if th5We have shown that sWGS is a robust and cost-effective method for obtaining good quality CN data from FFPE cancer samples, irrespective of the DNA quality and quantity used. In the case of breast cancer, CN profiles can be used to stratify breast cancers into one of the 10 Integrative Clusters , reiteraa–b Four separate libraries made with different kits - QDNASEQ copy number plots from four samples made with Rubicon Genomics Thruplex  or Illumina TruSEQ (C–F). We downsampled the Illumina TruSEQ  to achieve a more comparable results to the Rubicon Genomics Thruplex . The actual sequencing depth for Illumina TruSEQ is presented in plot E-F.Supplementary Fig. 1Increasing sequencing depth improves the resolution of the copy number plots - QDNASEQ copy number plots from two samples made with Rubicon Genomics Thruplex with increasing read depth. Sample on the left has a ΔCt of 4.79 and on the right has a ΔCt of 4.31.Supplementary Fig. 2a6 reads and plotted against the measured standard deviation (from QDNASEQ plots).Increasing sequencing depth improves the resolution of the copy number plots – For six libraries, the number of reads were down-sampled stepwise at 1 × 10Supplementary Fig. 2bComparing the input DNA and libraries generated from different sites.Dot plots represent the range (minimum-maximum) observed values for each category and the red dot (•) represents the median. Lines represent comparison between sites with associated statistical p-value.A. The quality of input DNA inferred by ΔCt.B. Fragment sizes of the libraries in base pair. C. The library yield in nanomoles.Image 1Library yield and its association with amount of input DNA using the Illumina TruSEQ kit.A. For eight samples, libraries were generated with 50 ng and either 200 or 500 ng of input DNA. Each coloured line represents paired libraries drawn connecting the lower and higher input DNA for each sample. B. Dot plots represent the range of library yield when generated with different amount of input DNA and the red dot (•) represent median. Red line represents statistical test between the groups spanning the line with p-value. C. Dot plot represent range of input DNA quality inferred by ΔCt for the samples from the different groups of input DNA and the red dot (•) represent median. Red line represents statistical test between the groups spanning the line with p-value.Image 2Features of libraries generated using Illumina TruSEQ or Rubicon Genomics Thruplex kits. ΔCt = difference between the cycle threshold of test to the control template ACD1 provided in the kit. ng = nanogram, PCR = polymerase chain reaction, bp = base pairs, nM = nanomoles. Supplementary Table 2 Comparing the DNA input requirement and cost for different commercial platforms available for copy number profiling. sWGS – shallow whole genome sequencing. Supplementary Table 3 Description of the libraries generated using the Rubicon Genomics Thruplex kit. Data given in range and median in brackets. ΔCt = difference between the cycle threshold of test to the control template ACD1 provided in the kit. ng = nanogram, bp = base pairs, nM = nanomoles.Supplementary Table 1The following are the supplementary data related to this article.We disclose no conflicts of interest.


The dependence of the resistivity with changing diameter of heavily-doped self-seeded germanium nanowires was studied for the diameter range 40 to 11 nm. The experimental data reveal an initial strong reduction of the resistivity with diameter decrease. At about 20 nm a region of slowly varying resistivity emerges with a peak feature around 14 nm. For diameters above 20 nm, nanowires were found to be describable by classical means. For smaller diameters a quantum-based approach was required where we employed the 1D Kubo–Greenwood framework and also revealed the dominant charge carriers to be heavy holes. For both regimes the theoretical results and experimental data agree qualitatively well assuming a spatial spreading of the free holes towards the nanowire centre upon diameter reduction. Semiconducting nanowires are in the focus of research due to their potential applications in electronics and optics –9. GermaAmong different synthetic routes for obtaining Ge NWs –13, the 2 substrates and contacted lithographically in four-point probe configuration [For our study we chose heavily doped self-seeded Ge NWs predominantly having the same crystallographic direction ,14–15. Iguration . Electriguration ,16. FromUpon NW diameter reduction, first the resistivity revealed a rapid drop by two orders of magnitude, followed by a region of weakly varying resistivity below 20 nm diameter. Prior to this region, an increased scattering of the resistivity values was observed coinciding with the NW diameter reaching the excitonic Bohr radius (≈24 nm ), indicad near the interface [d can be calculated by solving the Poisson equation in cylindrical coordinates [nh. One finds the expression [To describe our findings, we first recall that in self-seeded Ge NWs the majority charge carriers are free holes whose concentration depends on the number of charge traps at the NW core/shell interface ,18. In pnterface –21 whichrdinates –21 and fpression 0 is the electrostatic potential at the core/shell interface, ε0 is the vacuum permittivity, εr the dielectric constant of germanium, and e the elementary charge.where Φd will become comparable to the NW radius R which we find in our calculation to occur for a diameter of about 18 nm −1 with ηct being the charge trap density per cm2. The NW resistivity can be therefore written as (We first address the data in the region with the largest resistivity variation (diameters ≥ 20 nm). Here we presume itten as 0 = 0.3 eV [r = 16 [ct = 1013 cm−2 [−3 cm2 V−1 s−1. Interestingly, the mobility increases with reducing diameter, in contrast to lightly doped semiconductor NWs [where µ is the free hole mobility. Taking Φ= 0.3 eV , εr = 16 [r = 16  and ηct 013 cm−2  we can cctor NWs .d << R for nanowires ≥ 18 nm diameter; d = R). We consider the simple model of a NW being represented by a cylindrical well with infinite potential walls. It is shown below that despite this simplification we find qualitative correspondence with our experimental findings.Fitting the mobility data by a linear function and inserting it into Within this approach using the parameters of bulk germanium we determined the positions of the heavy and light hole sub-band minima along with the corresponding density of states . The denAs dominating resistivity contributions, we considered phonon and (remote) Coulomb scattering from the charge traps at the core/shell interface . SurfaceFor diameters between 11 and 22 nm, the NW resistivity and mobility values were calculated . A peak −3 Ω cm for a 19 nm diameter NW, the estimated 3.4 × 10−3 Ω cm differs only by a factor of three. However, for the top of the peak feature, the calculated 4.2 × 10−3 Ω cm is underestimated by more than two orders of magnitude. We attribute this discrepancy to the simplifications used. The infinite potential well approximation (rather than a self-consistent solution to the Schrödinger and Poisson equations) requires the carrier wavefunctions to be strictly zero at the cylinder surface which in turn results in an underestimate of the charge carrier density within the NW surface region. This influences the actual Coulomb-scattering contribution to the resistivity as the Coulomb-scattering amplitude between any two sub-bands is strongly dependent on the NW diameter (Although the position of the peak-like feature at 14 nm is well qualitatively reproduced, there is less quantitative agreement: For the lowest value of 9 × 10diameter . While fdiameter .File 1Details on theoretical calculations.


We observed both concentration- and duration-dependent inhibitory effects of metformin. Concentrations as little as 20 μM (nearing circulating therapeutic levels) were sufficient to reduce insulin secretion following 3-day treatment. Concentrations of 200 μM and 1 mM produced more pronounced effects more rapidly. With 1 mM metformin, islets showed severe impairments in calcium handling, inhibition of insulin secretion, and increased cell death. No stimulatory effects of metformin were observed for any experimental endpoint. We conclude that the direct effects of metformin on islets are inhibitory at near-physiological concentrations. Beneficial effects of metformin observed on islets under various stressors may occur by “resting” fatigued cellular processes. However, metformin may have unintended consequences on normally functioning islets within the circulating range that require further evaluation.Metformin is considered the first-line treatment for type 2 diabetes. While metformin primarily increases insulin sensitivity, evidence also suggests that metformin affects the activity of insulin-secreting pancreatic islets. This study was designed to systematically examine the direct effects of metformin by measuring insulin secretion and the kinetics of the calcium response to glucose stimulation in isolated mouse islets using varying concentrations (20  The first line of pharmacological treatment for patients with type 2 diabetes (T2D) is metformin . MetformAlthough the mechanism of action for metformin is fairly well understood in hepatocytes, the direct effects metformin has on pancreatic islets and beta cells are less clear. In conditions of stress, metformin treatment appears to ameliorate the effects of diabetes on islets by reducing or inhibiting damaging levels of activity related to hyperglycemia and deranged lipids , nitric While there is an apparent restorative and protective effect of metformin on beta cells in a stressed environment, there is relatively little evidence of stimulatory effects of metformin on insulin secretion . Biguani2+]i responses, we demonstrate a clear inhibitory effect of metformin that increases with increasing concentration and/or duration. Importantly, these in vitro effects of metformin are observed at concentrations surprisingly close to the therapeutic range of circulating levels found in blood i levels as previously described [2+]i ratio from emitted light at 510 nm. Data were analyzed using cellSens Dimension 1.13 imaging software .Fura-2 AM fluorescence imaging was utilized to measure [Caescribed . Briefly2+]i imaging, all islets were loaded with 1 μM fura-2 AM and CellTracker Red was also included during fura-2 loading for one of the two treatment groups in order to pair together and simultaneously image islets from untreated and metformin-pretreated groups as published previously [2+]i responses to glucose stimulation i  at 5-sec intervals in response to acute glucose stimulation. Islets were treated with metformin at 20 μM, 200 μM, and 1 mM concentrations for 1, 2, or 3 days. Fresh media and metformin were given each day. In To acquire more detail on the kinetics of metformin's effects on the coupling between glucose stimulation and insulin release, we measured changes in intracellular [CaμM metformin had no significant effect on [Ca2+]i  and virtually no discernible [Ca2+]i response to glucose stimulation . Because intrinsic oscillations in glycolysis and [Ca2+]i are easily disrupted by changes in glucose, islets should be maintained in a steady-state 11 mM glucose condition for proper recording and analysis i data for 3 mM glucose and 28 mM glucose are summarized in 2+]i is tightly regulated and kept low in conditions of low/basal glucose. Issues with basal [Ca2+]i are often described as indicators of excessive calcium influx and/or calcium release from the endoplasmic reticulum [2+]i relative to control were not found for 20 μM or 200 μM metformin exposure, however, a time-dependent increase in basal [Ca2+]i relative to untreated controls was observed for 1 mM metformin. Shifts in fura-2 ratio as little as 0.02 to 0.05 have been associated with islet dysfunction [2+]i responses suggest severe dysfunction similar to the stress-induced effects observed in cytokine-induced cell death i response to 28 mM glucose stimulation were also concentration- and duration-dependent. Peak [Ca2+]i in 28 mM glucose was only slightly reduced for 20 μM, even after 3 days of exposure (and mildly increased at 2 days). This is somewhat incongruous with the insulin secretion data showing ~50% reduction at 3 days but not entirely unexpected since there are well-established dissociations between [Ca2+]i and insulin release [2+]i for 200 μM metformin appears to reflect a concentration-dependent reduction in insulin secretion , as opposed to an improvement in function between 2 days and 3 days of exposure to 1 mM metformin. This is also consistent with the substantial increases in cell death observed for 1 mM metformin.Declines in peak i changes in response to glucose stimulation. Our findings support other previous studies of metformin's effects on normal islets from mice, rats, and humans [Our work shows that metformin inhibits islet function as measured by insulin release and [Cad humans . NotablyMetformin's mechanism of action is still under debate. A large amount of evidence points to AMP-activated protein kinase (AMPK) activation as a primary mechanism of action (reviewed in ). In betIt is possible that the beneficial effect of metformin on islets under various stressors occurs by inhibiting or “resting” fatigued cellular processes. There is evidence showing protective effects of therapies to reduce beta cell workload including intensive insulin therapy at diagnosis with or without metformin, or increasing KATP-channel activity with diazoxide to reduce beta cell excitability , 47. We Overall, our findings indicate that the direct effects of metformin on insulin secretion from islets in isolation are inhibitory. If also true in vivo, our data suggest metformin may benefit individuals with islets that are fatigued by elevated metabolic demands by reducing islet metabolic activity. However, individuals with normally functioning islets may see reduced insulin secretion from metformin treatment. These islet effects are likely secondary to the insulin-sensitizing effects of metformin since the overwhelming clinical evidence indicates that metformin has beneficial metabolic effects overall.


The study of infinite matrices is important in the theory of summability and in approximation. In particular, Toeplitz matrices or regular matrices and almost regular matrices have been very useful in this context. In this paper, we propose to use a more general matrix method to obtain necessary and sufficient conditions to sum the conjugate derived Fourier series. A-summability and almost convergence.The idea of mentclass2pt{minimL if L is denoted x.Let x. For x (see Lorentz ).for allif and only iffor allnandWe need the following well-known Dirichlet-Jordan criterion for Fourier series see , 9)..9]).The trigonometric Fourier series of a 2π-periodic functionfof bounded variation converges tofor everyx, and this convergence is uniform on every closed interval on whichfis continuous.We writeLetbe a periodic function with period 2πand Lebesgue-integrable overand letdenote the partial sums of series (Let the methodbe regular. Then, for everyfor whichis continuous and of bounded variation onuniformly iniif and only iff series . Let theK is a positive constant.We havehow that  holds if0,π] and  holds, wSince condition  is satisThis completes the proof of the theorem. □Letbe a periodic function with period 2πand Lebesgue-integrable overThen, for everyfor whichis continuous and of bounded variation onissummable tocosecif and only ifissummable to 0 for everyIt follows easily by choosing Letbe a periodic function with period 2πand Lebesgue-integrable overThen, for everyfor whichis continuous and of bounded variation onissummable toif and only ifissummable to 0 for everyIt follows easily by choosing In this paper, we have characterized the


Structure-function relationships in tendons are directly influenced by the arrangement of collagen fibres. However, the details of such arrangements in functionally distinct tendons remain obscure. This study demonstrates the use of quantitative polarised light microscopy (qPLM) to identify structural differences in two major tendon compartments at the mesoscale: fascicles and interfascicular matrix (IFM). It contrasts functionally distinct positional and energy storing tendons, and considers changes with age. Of particular note, the technique facilitates the analysis of crimp parameters, in which cutting direction artefact can be accounted for and eliminated, enabling the first detailed analysis of crimp parameters across functionally distinct tendons.IFM shows lower birefringence (0.0013 ± 0.0001 [−]), as compared to fascicles (0.0044 ± 0.0005 [−]), indicating that the volume fraction of fibres must be substantially lower in the IFM. Interestingly, no evidence of distinct fibre directional dispersions between equine energy storing superficial digital flexor tendons (SDFTs) and positional common digital extensor tendons (CDETs) were noted, suggesting either more subtle structural differences between tendon types or changes focused in the non-collagenous components.By contrast, collagen crimp characteristics are strongly tendon type specific, indicating crimp specialisation is crucial in the respective mechanical function. SDFTs showed much finer crimp (21.1 ± 5.5 µm) than positional CDETs (135.4 ± 20.1 µm). Further, tendon crimp was finer in injured tendon, as compared to its healthy equivalents. Crimp angle differed strongly between tendon types as well, with average of 6.5 ± 1.4° in SDFTs and 13.1 ± 2.0° in CDETs, highlighting a substantially tighter crimp in the SDFT, likely contributing to its effective recoil capacity.This is the first study to quantify birefringence in fascicles and interfascicular matrix of functionally distinct energy storing and positional tendons. It adopts a novel method – quantitative polarised light microscopy (qPLM) to measure collagen crimp angle, avoiding artefacts related to the direction of histological sectioning, and provides the first direct comparison of crimp characteristics of functionally distinct tendons of various ages.A comparison of matched picrosirius red stained and unstained tendons sections identified non-homogenous staining effects, and leads us to recommend that only unstained sections are analysed in the quantitative manner.qPLM is successfully used to assess birefringence in soft tissue sections, offering a promising tool for investigating the structural arrangements of fibres in (soft) tissues and other composite materials. Collagenous tissues are no exception, therefore there is considerable interest in understanding the mechanical performance of tissues, based on studying the details of their composition and structural arrangement. Tendon is a highly ordered unidirectional fibrous composite, consisting mainly of hierarchically organised collagen type I molecules. Collagen is the most abundant protein and main load-bearing constituent of tendons and many other tissues. It attains its mechanical properties through hierarchical assembly. In tendon this spans from the tropocollagen triple helix at the molecular level, to collagen fibrils, fibres, fascicles, fascicle bundles and whole tendons Because of its simple structure relative to other connective tissues, tendon tissue is often treated as a calibration material for a variety of techniques A series of recent studies have focused on understanding the composition and function of fascicles and their connecting interfascicular matrix In addition to the composition and structural arrangement of fibres, other features, like collagen fibre crimp morphology, may differ between functionally distinct tendons, and therefore may have a crucial impact on tendon mechanical behaviour Multiple observations of biological samples with polarized light have been used There are two types of birefringence that contribute to the total birefringence observed in a sample: intrinsic and form birefringence. Birefringence resulting from the molecular bonds in a material is called the intrinsic birefringence. Collagen, the major component of most tissues, is highly intrinsically birefringent, which allows for direct observation of collagenous tissues with polarized light. The amount of intrinsic collagen birefringence can be related to the amount (or volume fraction) of collagen fibres in the imaged section. Form birefringence relates to the spatial arrangement of a fibrillar structure, and can also be observed in collagenous tissues under polarized light. Form birefringence of collagenous structures is related to the spatial dispersion of collagen fibres (tissue anisotropy). The tissue total birefringence measured with polarized light is a combination of the intrinsic birefringence of its birefringent components and their arrangement in space. Adopting polarised light analysis tools, fibrillar arrangement has successfully been evaluated in hard A common problem when analysing collagen arrangement with polarised light is the issue of fibres altering their appearance when observed with the polariser at different angles In this study, we used quantitative polarised light microscopy to investigate morphological features of collagen fibres in functionally distinct equine tendons: the energy storing superficial digital flexor tendon (SDFT) and positional common digital extensor tendon (CDET) in order to identify potential differences in fibre arrangement and collagen crimp across fascicles and the IFM of these functionally distinct tendons, and the optical anisotropy in fascicles and interfascicular matrix, reflecting the distinct composition and collagen fibre arrangements within these regions. We investigated if aging would result in altered fibre arrangements and altered collagen crimp patterns in both tendon types. Finally, we tested if birefringence patterns and relative signal magnitude are preserved with staining in site-matched picrosirius red stained and unstained tendon sections.22.1The superficial digital flexor tendon (SDFT) and common digital extensor tendon (CDET) were dissected from a single forelimb of 6 horses directly post mortem, all acquired as waste material from a licensed abattoir. None of these twelve tendons exhibited macroscopic evidence of injury. Horses were divided into two age groups: three young horses aged 3–7 years and three old horses aged 17–20 years. Additionally, a visibly injured SDFT of a 17 years old horse was also dissected and considered independently. A sample (approximately 10 mm × 5 mm × 5 mm) was cut from the core of the mid-metacarpal region of each of the 13 tendons, ensuring a matching region of each tendon was assessed and that this encompassed the region of damage in the injured tendon.Samples were prepared following standard histological preparation methods, first fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 48 h at 4 °C, then stored in 70% ethanol for 24 h, followed by processing to paraffin wax blocks. Six 5 µm-thin longitudinal sections were cut from each paraffin block and mounted on glass slides, after which three sections from each block were stained with picrosirius red using standard protocols 2.2, Irvine, CA, USA). Both polarisers were manufactured by Werner Kaminsky, University of Washington. It should be noted that a conventional microscope can be easily converted into a (quantitative) polarised light microscope by placing these specific filters in the light path . A schematic showing the arrangement of the components and details of the subsequent light path through the system, alongside typical output images collected from an unstained 5 µm-thick tendon section are shown in Samples were imaged using a circularly polarised light technique, also referred to as quantitative polarised light microscopy (qPLM) 0, see φ  images obtained from the Rotopol software, and subsequently transformed to grey scale with values ranging from 0 to 255 2.4The second birefringence related imaging channel of qPLM is the slow axis angle measurement, which denotes the refraction angle of the slow ray (or extraordinary ray direction) through the material, and reflects the in-plane ordering within a section, see e and no are the refractive indices in the direction of the extraordinary (slow) ray and the ordinary (fast) ray.The total birefringence is related to the orientation angle of the slow axis of a material (φ) in the following manner:crimp and crimp length L Slow axis images were utilised for quantitative evaluation of collagen crimp angle θIn order to accurately calculate crimp parameters, it was first necessary to restrict analysis to those samples for which the crimp was in plane with the cutting direction, so no information was lost in out of plane crimp orientation, and the maximal crimp angle was observed in plane. Sections were rarely cut exactly with crimp propagation plane, so most sections showed a small amount of crimp in both the out-of-plane and in-plane directions (birefringence and slow axis images). However, images with clearly out-of-plane crimp that would cause major underestimation of crimp angle, were excluded from the crimp angle analysis. To achieve consistency in the selection of in-plane images, grey-scale thresholding was adopted, whereby out-of-plane images were excluded if crimp pattern  were still visible in the fascicular region after thresholding to grey scale level of 54 , which showed approximately in-plane crimp, and were used for investigating crimp angle.to-peak, from-peak, crimp) was calculated from the two mean in-plane orientations based on simple trigonometry:Remaining slow axis images were directly transformed from the grey scale range of 0–255 to 0–180° as the potential range of the slow axis orientation. The Rotopol system was calibrated to align the 0˚ direction perpendicular to the long axis of an image see a. This eTo assess the extent of error associated with simply estimating crimp angle in regular histological sections, we compared qPLM crimp angle measures with those acquired from a classic manual crimp angle measurement Average crimp length L, independent of sectioning plane, was measured between two adjacent crimp peaks in at least 10 regions of interest in each slow axis image 2.5A Lilliefors (Kolmogorov-Smirnov) normality test and quantile-quantile (qq) plots were performed on all data sets, in order to check if data were normally distributed. For data sets that were normally distributed, arithmetic mean values were calculated for the birefringence of thresholded IFM and fascicular regions in each image. Similarly, arithmetic mean of slow axis angles (thresholded: to a peak/away from a peak) were calculated in each image. Mean crimp length was calculated in each image as an arithmetic mean of the length between 10 crimp peaks. Further, means for tendon type , age group  and staining condition  were calculated and compared using unpaired two-tailed t-tests for samples with equal or unequal variances. Comparisons of data sets for which normality could not be ensured were performed using the Kruskal-Wallis non-parametric tests. Significance level of α < 0.05 was used.33.1Total birefringence in tendon tissue is related to the presence of birefringent collagen fibres (intrinsic birefringence), as well as to the extent of alignment of these fibres (form birefringence). When viewing longitudinal tendon sections, the presence of collagen will ensure some birefringence is always present, but increased levels of birefringence denote increased dispersion in the out-of-plane orientation of collagen within the sample. Total birefringence was quantitatively compared between fascicular and interfascicular regions of energy storing SDFTs and positional CDETs of various ages.3.1.1Tendon sections of 5 µm thickness showed sufficient birefringence to be directly investigated under circularly polarised light without staining. Birefringence levels were 0.0040 ± 0.0005 and 0.0042 ± 0.0005 in fascicles of young and old SDFTs, and 0.0058 ± 0.0007 and 0.0042 ± 0.0005 in young and old CDET fascicles, respectively. No significant differences between the age groups were noted in birefringence values for fascicles of either tendon type (p > 0.05), so samples of all ages were combined for further birefringence analysis. The differences between birefringence in fascicles of CDETs c and SDFAverage birefringence levels in the IFM of young and old SDFTs were 0.0013 ± 0.0001 and 0.0012 ± 0.0001, respectively and 0.0013 ± 0.0001 in the IFM of both young and old CDETs, highlighting no significant differences with age. Similarly, there were no significant differences in birefringence in the IFM regions of different tendon types, indicating that variations in the dispersion of fibre directions were not tendon specific in either the fascicles or IFM.In contrast, a comparison of total birefringence levels between fascicles and IFM showed highly statistically significant differences between regions in both tendon types, with significantly lower total birefringence in the IFM than fascicles . As qPLM3.1.2The comparison of birefringence in picrosirius red stained and unstained sections was performed exclusively in the fascicular regions of tendons. The variability in IFM area fraction between images meant average birefringence levels across a complete section were heavily affected by the presence of the IFM, and in some stained sections, birefringence in the IFM region was removed by the automatic threshold in the image capturing software (as explained in methods section). Choosing fascicular regions only for a comparison of staining effects ensured appropriate analysis of the picrosirius red stain.As reported in unstained sections, differences in birefringence with age in stained samples were insignificant, so samples were combined for further analysis, and average birefringence data is provided in p < 0.00001 for both SDFTs and CDETs). Whilst an initial visual comparison of the matched stained and unstained sections seemed to suggest that birefringence increased homogenously across stained sections . Directly comparing the mean increase in birefringence with staining highlighted an increase of a factor of 2.3 (± 0.8) × for CDETs as compared to 1.8 (± 0.4) × for SDFTs, which may indicate that some components of fascicular matrix in CDETs attract the picrosirius red stain more intensively.Higher birefringence was measured in fascicular regions of picrosirius red stained sections relative to matched unstained sections in both tendon types, and these differences were highly significant , crimp pattern  was conserved across IFM boundaries, suggesting an order to fascicle structure across multiple fascicles see a, c. How3.3Six unstained sections from a visibly injured SDFT tendon were additionally investigated for birefringence and collagen crimp characteristics.p = 0.0003) than the birefringence levels observed in SDFTs without visible damage (0.0043 ± 0.0005 [−]). This reduction is likely to be partially related to the significantly flatter crimp in the injured SDFT (4.2 ± 0.6°) than the healthy SDFTs . However, it also indicates either more uniaxially arranged collagen fibres, more sparse packing of fibres and/or a change in composition of the injured tendon. The crimp length in the injured tendon (17.6 ± 7.6 µm) was somewhat shorter than that in non-injured SDFT tendons (21.1 ± 5.5 µm), but no significant differences were seen (p > 0.05).Mean birefringence measured in the fascicles of the unstained sections of the injured SDFT was 0.0037 ± 0.0003 [−], significantly lower  collagenous tissues in a quantitative manner, resulting in values ranging from approximately 0.002 [−] in 10 µm unstained sections of mammalian cochlear tissue Differences in birefringence within fascicles between tendon types were weak, and no significant differences with ageing were evident in either tendon type. However, it was notable that differences in fascicular birefringence between SDFT and CDET fascicles approached significance, and with increased sample numbers, significant differences between fibre arrangements in the two tendon types may become apparent. Nevertheless, the data collected suggests that there is little specialisation in collagen fibre packing or spatial dispersion within the fascicles of functionally distinct tendons and no notable alterations with ageing. As such, specialisation and age changes may be more pronounced in terms of the composition of non-collagenous (and non-birefringent) constituents of tendon (like proteoglycans or elastin) and/or structural changes at other hierarchical levels of the tissue, like the fascicle bundles and/or collagen fibres. For example, it has been suggested that there may be considerable changes in the helical organisation of tendon fascicles, which may not be evident in histological sections By contrast, this study provides the first quantification of clear differences in birefringence levels between fascicular and interfascicular matrix in tendons. Across all samples, mean levels of birefringence were substantially lower in the IFM (0.0013 ± 0.0001 [−]), as compared to fascicular regions (0.0044 ± 0.0005 [−]), confirming considerable variation in birefringence between the two regions. These data match histological and proteomics findings, suggesting a far more sparse fibrillar structure in the IFM and relatively higher amounts of collagen type I in fascicles Of note, this study also shows no significant differences in IFM birefringence between energy storing and positional tendons, indicating that the arrangement and amounts of birefringent collagen fibres in the IFM do not differ largely between functionally distinct tendons. Recent studies have indicated variations in the amounts of a range of non-birefringent components in the IFM of functionally distinct tendons, such as elastin and proteoglycans  p < 0.05), but no significant differences between age groups. Further, direct comparisons of crimp parameters and mechanical properties can be carried out in the eight matched samples, demonstrating clear trends of higher stiffness in SDFTs (638 ± 172 MPa) associated with smaller crimp angles (6.5 ± 1.4˚) and crimp lengths (21.1 ± 5.5 µm), as compared to CDFTs with a lower stiffness (461 ± 157 MPa) associated with a larger crimp angle (13.1 ± 1.8˚) and crimp length (135.4 ± 20.1 µm), see In agreement with previous studies CDET fascicles, that showed a larger crimp angle and crimp length, were seen to fail at higher strains (15.0 ± 2.1%) than SDFT fascicles (11.3 ± 2.6%) Interestingly, in this study we have found no significant differences in crimp length between skeletally mature young and old horses, which suggests there may be few changes in collagen crimp length after animal skeletal maturation. While this contrasts with previous studies, which have indicated crimp length changes with aging, it is notable that there is little consistence between studies: as both shortening Indeed, it has been hypothesised that crimp length in tendon may not be directly associated with animal age, but more specifically with the tissue age, with a finer crimp in newly laid fibrils Whilst crimp patterns were often preserved across the IFM boundary between fascicles, some samples in all tendon groups demonstrated altered crimp patterns across two adjacent fascicles. It is possible that these inhomogeneities occur naturally, but they may also be a result of damage and repair within the tendon. Indeed, they were observed more frequently in the injured tendon samples but also in young SDFT and old CDET samples. The more frequent discontinuities across fascicle boundaries seen in young relative to old SDFT samples may be indicative of a higher remodelling rate in the younger group, whilst more discontinuities in old than young CDET samples may be indicative of more damage accumulation with age in CDETs. Further investigation of these changes would be of interest for future studies. Inhomogeneous regions of crimp may pose certain mechanical consequences, creating inhomogeneous damping across the tendon, and increasing the degree of shear within the IFM during normal tendon use. As such, it may be hypothesised that inhomogeneous crimp patterns increase the risk of further tendon damage with use. Further, no gradual transition zone between fascicles and interfascicular matrix was observed, but rather a sharp transition between the zones was seen. This certainly has consequences for the mechanical behaviour of tendons and is important for prescribing proper boundary conditions for modelling tendons mechanical behaviour While measures of collagen crimp length in 2D longitudinal histological sections are independent of the cutting plane, the crimp angles seen in these sections are strongly dependent on the relative orientation of the sectioning plane to the crimp propagation plane, and subsequently subjected to underestimation errors Crimp angle was lower in the energy storing SDFT (6.5 ± 1.4°), as compared to the positional CDET (13.1 ± 1.8°) and this dependence was not significantly affected by age. This finding was surprising, and does not support previous results showing crimp flattening with age In the literature, crimp angles measured from traditional histology images of equine SDFT tendons have ranged from 4.6 ± 0.1° to 14.3 ± 0.4° Little previous data depicting the crimp characteristics of equine CDETs are available, and that available is limited to young, skeletally immature horses (1.5 years old). However, the average crimp angle of these young CDETs was 7.0 ± 1.0° Indeed, the observed combination of collagen crimp angle and length data from the energy storing and positional tendons highlight how much greater crimp height is in the CDET. This is surprising, as a previous study of functionally different rat tendons  reported a similar crimp height for the two tendon types  As the qPLM technique enables crimp orientation to be assessed relative to section cutting plane, it also provides additional insight into the question of crimp organisation as either a planar or helical structure. Within the field of view of current images (at 20× magnification), no clear change in crimp orientation relative to sectioning plane is evident, suggesting that either crimp is a planar wave Previous studies have used picrosirius stained soft tissue sections to investigate composition and arrangement of fibres in collagenous structures This study demonstrates the use of quantitative polarised light microscopy (qPLM) to assess birefringence and the arrangement of collagen fibrillar structures in soft tissues. We report quantitative information on the structural differences between the functionally distinct tendons. Namely we demonstrate significant crimp length and crimp angle differences between the energy storing SDFT and positional CDET. Importantly the qPLM technique enables assessment of collagen crimp avoiding artefacts related to regular histology measurements.


The integrity of the human genome is constantly threatened by a wide variety of challenges  . SuboptiRecent data suggested that the DDR also impacts alternative splicing (AS), a highly regulated process evolved in eukaryotes to diversify their transcriptomes and proteomes , 3. For We were surprised to discover that IR rapidly inhibits the activity of the SMG1 kinase, an enzyme previously implicated in the regulation of nonsense- mediated RNA decay . This fip53β is generated by retention of a cryptic exon inside intron 9 of the p53 gene via alternative splicing . The retIn exploring the mechanisms by which p53β might be affecting specific cellular outcomes after damage, we found that cells lacking p53β expression fail to transcriptionally repress negative regulators of aging such as BCL6 and SIRT1. Interestingly, both of these two genes epigenetically regulate downstream target gene expression. Therefore, transient induction of p53β after IR may be able to alter cellular phenotypes after damage by regulating epigenetic modifications that drive cells toward a senescence pathway.Transcriptional profiling revealed over 600 distinct AS events in cells after IR treatment and we speculate that some of these events, like p53β splicing, are regulated by the “SMG1-RPL26-SRSF7” pathway and contribute to senescent phenotypes in damaged cells. In addition to intron retentions (p53β), we also identified some AS events that are generated by alternative exon usages (e.g. the CDYL gene), suggesting a potentially broad range of targets for this pathway. We are in the process of validating some of these events in the hope of identifying novel determinants of cellular aging and potential therapeutic targets for aging-associated diseases and some of the toxic side-effects of irradiation and chemotherapy.