Trigeminal neuralgia (TN) is a disabling pain condition that has classically been treated using either surgical or medical techniques. Several researchers have shown that stereotactically delivered radiation can be an effective tool in the amelioration of this condition. For these studies, the Gamma Knife was used to deliver the radiation treatment. The target location was designated as the proximal nerve at the root entry zone, and doses greater than 70 Gy to the maximum point in a single fraction were found to be effective in controlling pain in 80% of the patients treated. LINAC‐based stereotactic radiosurgery has been notably absent from the treatment of TN, even though it has many similarities to Gamma Knife‐based stereotactic radiosurgery.The aim of this paper is to describe our LINAC‐based stereotactic technique for treatment of TN. We also compare treatment of TN using our technique to that using the Gamma Knife. We found that a LINAC‐based treatment of TN can be accomplished with accuracy comparable to treatments delivered using the Gamma Knife. The dose distributions are essentially equivalent for the two treatment approaches. The LINAC‐based system is easy to plan and offers the ability to reduce the involvement of sensitive structures from the treatment fields as well as the Gamma Knife system does. A disadvantage of the LINAC‐based system is the time involved for treatment.PACS number: 87.53.Tf 87.56.Da The 4‐mm aperture was used for these treatments, and the target location was designated as the proximal nerve at the root entry zone. Doses greater than 70 Gy to the maximum point (100%) in a single fraction were found to be effective in controlling pain in 80% of the patients treated. Patients with no prior surgery had complete or near complete pain relief, while complications due to treatment were nearly nonexistent in the published studies. What makes this particular treatment unique from other stereotactic treatments is the extreme accuracy that is required for all steps for successful treatment. This is primarily due to the small size of the target, the difficulty in identifying the target region, the small cone sizes used for treatment, and the extremely high single‐shot doses >70 Gy that could result in neural injury if they were misguided (or misdirected).Trigeminal neuralgia (TN) is a disabling pain condition that has classically been treated using either surgical or medical techniques. Recently, research in several publications has shown that stereotactic radiosurgery can be an effective tool in the amelioration of this condition.–8 In(–) even though many of the steps in the process of this stereotactic treatment are similar, if not identical, to those undertaken for a Gamma Knife treatment. Due to the effectiveness of this treatment for pain relief, we initiated a program of LINAC‐based stereotactic radiosurgery at our institution. We evaluated our technique to determine if our LINAC‐based system had the required accuracy to properly treat such small target volumes. Positioning accuracy and dose distributions are compared against what is expected for Gamma Knife treatments, and application of LINAC‐based stereotactic radiosurgery for this clinical problem is discussed.The use of LINAC‐based stereotactic radiosurgery for the treatment of TN has been limited in the historical literature,–8 evWe compared the mechanical accuracy of the two systems, the imaging accuracy of the techniques, and the dosimetric aspects of the treatment including coverage of the target, and the dose to uninvolved normal structures.5×5 cm2 field.Our LINAC‐based system uses the Radionics, Inc. MRI‐compatible head ring for patient fixation; the larger diameter Brown‐Roberts‐Wells (BRW) head ring is attached to the MRI head ring for CT scanning and to secure the patient to the treatment stand. Depth helmet measurements are taken after the head ring is fixed initially to the patient and immediately before treatment to ensure that ring placement has remained constant throughout the process. The high‐precision beam delivery is provided by a Philips (Elekta) SRS 200 independent support stand and subgantry assembly installed on a 6‐MV X‐ray Varian 6/100 LINAC. A 5‐mm circular cone is used for the treatments, with the LINAC collimators set for a  It is the extreme accuracy of this system that allows for the treatment of TN using a LINAC; couch‐based stereotactic systems are completely dependent on the rotational accuracy of the couch and gantry of the LINAC itself. This Philips SRS 200 system, no longer marketed, is the precursor to the LINAC Scalpel™ System currently available from Zmed, Inc. .The Philips SRS 200 mechanical system, which is the key source of the mechanical accuracy required by this technique, consists of an independent subgantry assembly, which has a head support system that is independent of the treatment couch, and a secondary gantry, which holds the cones for patient treatment. This independent subgantry is mated to the LINAC through a gimbal assembly. The force to rotate the subgantry assembly is supplied by the LINAC and transmitted to the subgantry via the gimbal assembly. Any small misalignment between the LINAC rotation and the subgantry rotation is handled by the play available in the gimbal assembly. This integrated subgantry is the source of the extreme accuracy required for this particular radiosurgery technique. The independent head support is much more accurate for head positioning than the LINAC couch, while the subgantry is much more accurate than the gantry rotation of a standard LINAC. The capabilities of the system are well documented,11 IIn our procedure, we first obtain MRI scans of the head with the MR head ring and universal localizer (UCLF) on the patient using a 3D data acquisition sequence (MP‐RAGE) on a Siemens Magneton scanner. Author Sagittal, axial, and coronal scans of 1 mm thickness at 1 mm spacing are reconstructed from this data acquisition sequence. CT scans are then acquired with the BRW head ring attached to the MR head ring with the CT fiducial localizer in place. CT scans are obtained at 1 mm spacing through the target region with a Siemens Somatom Plus scanner with 2‐mm scans taken outside the treatment region. Additional checks are performed to ensure the accurate transfer of the MR and CT scan data from the scanners to the treatment‐planning system.The target is determined using the sagittal and axial MR scans. The target volumes are drawn using the MR image sets obtained with the Radionics universal localizer (UCLF) in place during the MR scans and using the Siemens MR head coil. The MR image sets are transformed into the BRW coordinate system using the fiducials on the MR images. The CT data set is required for planning by the Radionics software, and we use the CT image set to delineate the external contour, the eyes, optic nerves, and brain stem. The treatment field arrangement for our LINAC‐based treatment contains 13 arcs spaced at 15° intervals for approximately 1300 total degrees of arc rotation . TreatmeTo evaluate the dosimetric component of our treatment technique, we compared our planning results to a Gamma Knife B unit that we simulated in our Xknife planning computer. The Gamma Knife unit consisted of 201 individual cobalt‐60 sources arranged radially in five rings of 35, 39, 39, 44, and 44 sources . We obtaThe accuracy of the beam modeling for the Gamma Knife data in Xknife was verified before doing the test comparisons of dose distribution for the LINAC and Gamma Knife trigeminal treatments. We first tested the accuracy of the representation of a single 4‐mm Gamma Knife Co‐60 beam in the Xknife planning system. This Gamma Knife beam was simulated in the Xknife software as a single beam having a one‐degree arc. At the isocenter, the profile obtained from this test matched the data of the input dose profile. The treatment plan of the trigeminal nerve using the Gamma Knife data was simulated in Xknife using 201 single arc beams with the same isocenter location  as our LOur treatment technique using 13 arcs totaling approximately 1300 arc degrees of rotation was designed to deliver approximately the same amount of dose along the arc path as that given along a Gamma Knife beam. Our rationale for choosing this beam arrangement was based on the assessment that each of the individual 201 fields of the Gamma knife could be closely approximated by a 5° arc, and that our 1300 degrees of rotation would equal about 260 five‐degree arc segments. Each Gamma Knife cobalt field would essentially deliver 7000 cGy/201 beams, or approximately 35 cGy to the isocenter per beam. 7000 cGy delivered using 260 five‐degree segments would deliver about 27 cGy per 5° arc, close to the 35 cGy per beam for the Gamma Knife unit. Thus the dose through normal brain tissue for each of these 5° segments would be close to the dose delivered along the track of each Gamma Knife beam.<0.3 mm deviation average with a maximum deviation of 0.5 mm.The mechanical isocenter accuracy of the SRS 200 system comes from the design, which minimizes the couch inaccuracy effect by supporting the patient's head independent from the LINAC treatment table. In addition, the subgantry system minimizes the inaccuracies of gantry rotation.The mechanical isocenter accuracy of the Gamma Knife system is approximately 0.25 mm, as reported in the literature. This represents the total clinically relevant uncertainty and includes image reconstruction accuracy, coordinate system transformation, and mechanical errors. The application accuracy of the Leksell frame used with the Gamma Knife was reported to be 1.7 mm ± 1.0 mm (SD), while the BRW frame was 1.9 mm ± 1.0 mm (SD).The application accuracy,14 Tx, y, and z  coordinates of these structures in the transformed computer stereotactic coordinate system.Imaging accuracy depends on the scanning system employed, the coordinate transformation accuracy of the planning system, and the accuracy of the transfer of imaging data from the scanner to the planning computer. Imaging accuracy for our system was determined using the Radionics™ head test phantom as the standard of comparison. The Radionics™ test phantom consists of four internal structures—a cube, cone, cylinder, and sphere—whose location in BRW space is very accurately known . For tesCT resolution for the Gamma Knife system has been reported to be approximately 1.7 mm. our tests of the MR accuracy of the UCLF localizer have consistently yielded the indicated results. For each patient treatment, the reconstructed locations of the localizer frame are checked, and they routinely match the theoretical location of the localizer rods.Calculated dose comparisons were done between the 5‐mm, 6‐MV X‐ray LINAC‐based beam and the 4‐mm Co‐60 fields from the Gamma Knife unit.  shows thA test case on which to make the comparison was a patient treated for TN using the LINAC‐based system. Dose distributions were done for the 13‐arc LINAC‐based treatment approach and compared to the Gamma Knife plan. The locations of the 201 Co‐60 sources were from a B model unit, and the beams were represented as 1° arcs in the treatment‐planning system. As stated above, actual OAR and TMR data from an existing clinical Gamma Knife B unit were input into the system to provide a realistic comparison of delivered dose.By comparison of imaging accuracy, mechanical accuracy, dosimetric comparisons, and clinical results, we feel that LINAC‐based stereotactic radiosurgery using this subgantry assembly is an effective means of treating TN and can be comparable to Gamma Knife treatments.


The exact positions of critical points in the charge density in enzyme active sites reflects electrostatic preorganization. Electrostatic preorganization is thought to be a principle factor responsible for the impressive catalytic capabilities of enzymes. The full protein structure is believed to facilitate catalysis by exerting a highly specific electrostatic field on the active site. Computationally determining the extent of electrostatic preorganization is a challenging process. We propose using the topology and geometry of the electron charge density in the enzyme's active site to asses the effects of electrostatic preorganization. In support of this approach we study the convergence of features of the charge density as the size of the active site model increases in Histone Deacetylase 8. The magnitude of charge density at critical points and most Bader atomic charges are found to converge quickly as more of the protein is included in the simulation. The exact position of critical points however, is found to converge more slowly and be strongly influenced by the protein residues that are further away from the active site. We conjecture that the positions of critical points are affected through perturbations to the wavefunctions in the active site caused by dipole moments from amino acid residues throughout the protein. We further hypothesize that electrostatic preorganization, from the point of view of charge density, can not be easily understood through the charges on atoms or the nature of the bonding interactions, but through the relative positions of critical points that are known to correlate with reactivity and reaction barriers. While only a handful of amino acids in the active site participate in the reaction, the rest of the protein macromolecule is proposed to have an important function too, and that is the creation of a highly specific electric field. That field would influence the active site and bound substrate, and interact most beneficially with the transition state of the catalyzed reaction.3Understanding enzymatic function, including the exact mechanism, and the factors making these catalysts efficient and incredibly selective, is central to chemistry, biology, and medicine. It is also important in industrial catalysis if we wish to design equally efficient artificial enzymes for unnatural functions of interest to humankind.4 The full enzyme, particularly the polar groups, are thought to perturb the electron density at the active site to create an electrostatic field that lowers the activation barrier to the catalytic reaction. Electrostatic interactions are long-ranged, and this gives insight into the need for the large size of enzymes. When a reaction proceeds in a polar solution such as water, the solute molecules see a fluctuating dipole environment caused by the motion of the solvent molecules. For a great many catalytic reactions, the transition state energy is highly sensitive to this environment, with the lowest transition state energy characterized by a distinct organization of the solvent dipoles. The free energy difference between the reactant and transition states is what determines the rate constant in enzyme catalysis, making the transition state free energy a key component to optimize for reaction speed. It is suggested that the charged and polar residues – even at some distance from the active site – are positioned in enzymes so as to permanently preorganize the electric field and thereby minimize the free energy of activation.This phenomenon is known as electrostatic preorganization, and has been argued to be the origin of the enormous catalytic power of enzymes.6 assessing its extent remains a challenge. Some computational and experimental methods have been used to determine the role of electrostatic preorganization and quantify its influence on activation barriers. Computationally, empirical valence bond (EVB) methods have been used to determine the importance of electrostatic interactions based on contributions from ionic resonance forms along the reaction coordinate. The stabilization of the ionic resonance form at the transition state has been found to be linearly related to the difference in activation free energies for an enzymatic reaction occurring in a general solvent compared to the enzyme.7 A similar relationship is not found for the covalent resonance forms, suggesting that electrostatic stabilization of ionic structures is a key feature for enzymatic reactions. Experimentally, vibrational Stark spectroscopy has been used to measure local dipole moments in the active site that are influenced by an external field. Direct relationships between dipole moments in carbonyl and nitrile functional groups to activation energies for enzyme catalyzed reactions have been found.11While electrostatic preorganization is gaining acceptance as the largest factor in catalytic ability of enzymes,ρ, due to successive perturbations to the environment around an enzyme active site. The electrostatic potential is determined by the electron charge density via the Poisson equation, making ρ a logical platform for studying electrostatics in enzymes. Furthermore, the active site is electronically polarizable, so certain structural features in the electron density will change when a polar environment, i.e. the full protein, is added around the active site. We use Histone Deacetylase 8 (HDAC8), which catalyzes a deacetylation reaction, as our model. HDAC8 is a well-studied enzyme for which a variety of mechanisms have been proposed,15 but we have found the proton shuttle mechanism presented in 16 Through this mechanism, H143 acts first as a base, abstracting a proton from the water molecule, and second as an acid, transferring the proton to the substrate.We take a novel approach to investigate electrostatic preorganization by following the evolution of the electron charge density, 2+ ion  was found to be linearly correlated to metal binding affinity for HDAC8.16 In this study, we thus focus on the charge density around these five atoms, which are highlighted in Key bonding interactions for this reaction were found to involve the substrate carbonyl (C1 and O1), the oxygen on the water molecule (Ow) and the Zn ion see . These f2+, C1, O1, Ow, and NHis atoms , and those that do not. The latter region is an extended environment whose effect on the deacetylation reaction is to perturb the electronic structure of the reactive region. The magnitude of the perturbation can be assessed by the induced changes to the charge density in the central region which are mirrored by changes to the corresponding CPs in this region.Our approach is as follows: for three different nuclear configurations obtained from quantum mechanics/discrete molecular dynamics (QM/DMD) simulations we approximate the enzymatic structure around the divalent zinc ion with five essentially spherical clusters of increasing radii. We then use density functional methods to compute the one electron wave functions and charge density for each of the 15 resulting model systems. By analyzing the changes to the density through a series of increasingly larger clusters, we are able to separate the enzyme's atoms into those that are part of a local environment that directly contributes charge density to the region under study, consisting of the ZnOur paper is organized by first determining the boundary of the local environment in the active site of HDAC8. We find this boundary by measuring where Bader atomic charges and the amount of charge density at CPs in the central region converge due to direct overlap of one-electron wave functions. Next we discuss the implications of perturbation theory on the CP structure. We study the perturbative effect of the extended environment on the central region by measuring the exact positions of CPs in the central region, which we propose can be used as an indicator of electrostatic preorganization. Then we discuss the importance of the potassium ion near the HDAC8 active site on the motion of CPs. Finally, a discussion of results and their implications for enzyme design is presented.2ρ in the central region has converged. First we measure the Bader atomic charges of the Zn2+, C1, O1, Ow, and NHis atoms. Next we find the size of the active site cluster model for which the magnitude of charge density at bond and ring CPs in the central region converges. For details on how Bader charges and CPs are defined and calculated, see ESI.In order to analyze the effects of the protein's extended environment on electrostatic preorganization we must first determine the position of the boundary surrounding the local environment, outside of which all other atoms will act as a perturbation on the central region charge density. The boundary is found by moving radially out from the zinc ion and determining when additional atomic wavefunctions no longer contribute significant charge density to the central region. We use two methods based on the topology of the electron charge density to measure when the magnitude of et al. were used to create our active site models for these calculations  were created by removing any amino acid residues from the 7 Å models that did not include atoms within the cut-off distance and again optimizing the positions of capping hydrogens. Single point calculations were performed on systems of all five sizes for the three configurations using all combinations of double-zeta polarized (DZP) and triple-zeta polarized (TZP) Slater-type orbital basis sets with four functionals: LDA, PBE, M06L, and TPSS, within the Amsterdam Density Functional (ADF) package version 2016.21 This resulted in a total of 120 single point calculations. The topology and geometry of the electron charge density was analyzed using the standard Bader package within ADF23 to calculate Bader atomic charges and magnitude of charge density at CPs.Nuclear coordinates from the three lowest energy configurations obtained from QM/DMD simulations performed by Nechay ε = 4 and 20 to represent the full protein, which are common values used in enzyme calculations for buried and surface active sites, respectively. The second approach was to add point charges from the surrounding protein environment to the single point calculations. Point charges were obtained using NWChem24 and were calculated using a Co2+ HDAC8 system from 2+ and Zn2+ systems was nearly zero. The electrostatic embedding calculation performed in ADF included 3000 point charges calculated from atoms within a 25 Å radius of the Zn2+ ion.Calculations larger than 7 Å were not computationally feasible due to system sizes exceeding 600 atoms. We instead approximated the effects of the protein beyond our largest cluster model using two methods for the 7 Å LDA TZP configuration 1 active site model. The first approximation method was to add a dielectric constant to the calculation using COSMO solvation with 33.12+, C1, O1, Ow, and NHis from the single point LDA TZP calculations are shown in Bader atomic charges for ZnMost Bader atomic charges for HDAC8 converge once a full residue sphere is included around each atom in the central region. The O1 charge initially appears to converge at 4 Å, once Tyr306 is included (which hydrogen bonds to O1), with the most pronounced change occurring for configuration 1. However, from 6–7 Å the O1 charge decreases slightly, most notably for nuclear configuration 1. The Ow charge does not converge until the model sizes reaches 5 Å, which includes the His142 and 143 that hydrogen bond to the water molecule , which is also the atom with the least converged charge for the 7 Å cluster model. The C1 charge increases when adding a dielectric constant or point charges, but still remains in the same range of values from the 5–6 Å model calculations for configuration 1 without the full protein effect added. Since the C1 charge changes in the opposite direction of O1, this indicates that the overall substrate carbonyl charge is remaining constant, but the group gets polarized and activated. In general, inverse behavior was found for atomic volumes, i.e. an increase in atomic charge (decrease in number of electrons) correlates to a decrease in atomic basin volumes , as hasρ, but also on the locations of their boundaries that are influenced by the exact position of CPs. As will be discussed in Section 3.3, Bader charges may also be affected by perturbations from the extended environment and their convergence rate may not be the best metric to determine the boundary of the local environment. Interestingly, previous work showed that Bader atomic charges in the active site of 2-cysteine peroxiredoxin thioredoxin peroxidase B converge rapidly for neutral atoms as the model size increases, but not for negatively charged atoms.26 Here we have tested one positively charged atom, Zn2+, and found its charge to rapidly converge.Atomic charges are generally converged when one residue sphere is included around the atom of interest, but have slight variations for some values at larger sized cluster models. Bader atomic charges are based not only on the magnitude of 3.2ρ at CPs with the exceptions of the C1–O1 and Zn2+–Ow configuration 1 values. These critical points are one bond path away from the incipient C1–Ow bond CP, a result of the close proximity to the ring CP. These two CPs can annihilate each other as a result of slight nuclear motions, as occurs in nuclear configurations 2 and 3 . In conρ are the C1–O1 and Zn2+–Ow bond CPs. The magnitude of ρ at the Zn2+–Ow bond CP is converged for the 5 Å system once His142 and His143 are included. The C1–O1 bond CP reaches a converged value of ρ for the 4 Å model size, once Tyr306 is included. Configurations 2 and 3 have a slight depression in the amount of ρ at the C1–O1 bond CP from 3–4 Å but there is a larger change in configuration 1. The differences in the change in ρ are likely due to the Tyr306 hydrogen bonding occurring with an oxygen atom in configuration 1, and a carbon atom on Tyr306 in configurations 2 and 3.Once all direct bonding interactions to the central region are included in the model size, the CP charge density converges. The only critical points where a system larger than the 3 Å base model is required for converged values of ρ at CPs. The converged values that are reached by the 5 Å model remain constant when approximating electrostatic effects from the full protein, emphasizing that the magnitude of ρ at CPs is due to the local environment around the central region.Adding either point charges or a dielectric constant to the active site cluster model calculation does not affect the magnitude of ρ is most affected by direct changes in ρ due to an overlap of wavefunctions in the base system with new atomic wavefunctions that are introduced from directly neighboring amino acids. Since the charge density drops off exponentially, only nearby molecular orbitals have large effects on the magnitude of ρ. Once a full residue sphere is included around a critical point in the central region, the magnitude of ρ at the point is converged. Results from other functionals and basis sets give similar results and are shown in Fig. S4 j, are given byH(1) is the first order correction to the Hamiltonian. ψ(1)i is the perturbed wavefunction and can be viewed as a superposition of the unperturbed states of ψ(0)i where the state j will make no contribution if H(1)ij = 0 and can only make a significant contribution when E(0)i – E(0)j is small.Since the changes to the charge density in the central region are subtle, we use a perturbative approach to examine the effect of the extended environment of the protein. We conjecture that the perturbation from the full protein can be observed through the position of CPs that are defined by points where the gradient of the charge density, ∇M is a monopole term, μ̂ is the dipole operator, and Q is the quadrupole operator, which are all due to the addition of the amino acid residues beyond the base model.The form of an electric field represented as a multipole expansion from the extended protein environment goes asψ(0)j may couple, those of similar nodal character will couple most strongly. The dipole operator on the other hand is antisymmetric with respect to a nodal plane and most strongly couples orbitals that have different nodal character. For the example of a multipole expansion centered around Zn2+, the dipole operator will preferentially couple one-electron orbitals that are of the same phase on one side of Zn2+ and of different phase on the other side of the ion. The resulting perturbed wavefunctions will have different nodal character around the zinc atom which will in turn change where ∇ρ = 0, moving the positions of critical points.The result of eqn (2) for each term in eqn (3) must contain the totally symmetric irreducible representation of the group in order to be non-zero. For a high symmetry system, the monopole operator is fully symmetric and thus most strongly couples MOs that are of the same symmetry and hence have the same nodal character. In a system lacking symmetry, such as our system, while all 2+–O1, Zn2+–Ow, and Zn2+–NHis bond CPs for all three nuclear configurations as the system size increases for LDA TZP calculations. The motion of the C1–Ow bond CP and ring CP for configuration 1 is shown in a separate plot in N = 4–7. Data from all calculation methods is included in Fig. S5–S7  can have a significant effect on atomic energies.3.42+ that is in the central region. The second is a K+ ion, which is included in the 7 Å models for configurations 1 and 2, but is not included in any configuration 3 models. The CPs in the central region move less when the model size changes from 6–7 Å for configuration 3 compared to the other two models. The effect of K+ on the electrostatics in the active site is undeniable, and in a way represents the most graphical model for what the environment can do to the features of the charge density. We therefore use this K+ to particularly illustrate the point of this work. We want to know which features of the charge density are affected by K+. It is enchanting that Nature placed such an electrostatic bomb right next to the active site. What exactly is the reason?The orientation and magnitude of an electric field in a protein will be most affected by polar and charged molecules and ions. In HDAC8, there are two cations near the active site. The first is the Zn+ ion included in the simulation. + is included in the simulation, indicating that the ion does indeed have a large influence on the electrostatic environment in the active site. For configuration 2, all of the CPs except for the C1–O1 bond CP move a greater distance when the K+ is included. However, in configuration 1, only the ring CP and Zn2+–O1 bond CP move a larger distance with K+. The C1–Ow, Zn2+–N, Zn2+–Ow CP move less when K+ is included and the C1–O1 has no difference in its motion with or without K+.We investigated the effect the potassium ion has on CP motion in the central region by calculating the charge density for all three of the 7 Å systems, both with and without the K2+–O1 bond CP has greater motion when K+ is included in the model for all nuclear configurations tested. This bonding interaction is critical for the substrate to bind to the active site of HDAC8. When performing QM/DMD simulations for 2+ if the potassium ion was not included in the QM region. These observations suggest that the potassium ion is vital to the proper electrostatic environment in the active site for substrate binding to occur.The Zn4ρ in the central region of HDAC8 is directly influenced by the local environment, which we find here to be represented by a cluster model that includes a full residue sphere around the point of interest. Rapid convergence of the magnitude of ρ was expected since the charge density is known to be near-sighted and decay exponentially.32 This has also been observed for organic molecules with varying functional groups.33The magnitude of The sensitivity of the position of active site CPs due to the addition of atoms far away indicates that CP position is due to perturbations to the wavefunction in the active site caused by the protein's extended environment.35 Perturbations to wavefunctions that move CPs are due primarily to the orientations and positions of dipole moments from each atom in the surrounding protein, and will be most prominent for charged and polar residues, including the K+ present in the 7 Å model for configurations 1 and 2.Studies have shown similar magnitudes of changes in position of bond CPs on small organic molecules due to the addition of electron withdrawing and donating groups.et al.38 In the rate-determining step of the deacetylation reaction, ts2, the ring CP must move towards one of the Zn2+–O bond CPs to annihilate both CPs, opening the tetrahedral ring structure pictured in 39 The directions and magnitudes of curvature of ρ at CPs involved in the reaction step can be used to determine how a reaction will proceed.38 A flat charge density between CPs can occur through two methods, either by a change in the magnitude of ρ at the CPs, or by a change in the distance between the two points. Here we find the magnitude of ρ at CPs to be directly affected by the local environment and not the full protein. The positions of CPs, however, are influenced by the extended environment of the protein. Electrostatic preorganization may be causing the CPs in the active site to move into the most favorable positions in order to create a flat charge density along the reaction coordinate. We propose that the exact positions of CPs can be used as a measure of electrostatic preorganization, which is also influenced by polar and charged species.The distances between CPs, as well as the curvature of the charge density at these points, can also be related to reaction coordinates through the electron preceding picture presented by Ayers + has been shown to have a large influence on the electrostatic environment in the central region. In an electrostatically preorganized active site, it should not only be easy for the critical points directly participating in a reaction to move, but the CPs surrounding the active site should show the opposite behavior and be difficult to move. When an enzyme is electrostatically preorganized, the dipoles around the active site should not change throughout the reaction coordinate as has been demonstrated by Boxer et al.8 CPs surrounding the active site with high curvatures (non-flat regions of ρ) should be more difficult to move and could be used to predict the preorganization of an active site, especially for enzyme design.To test this theory, a non-metal enzyme that catalyzes a simple one-step reaction would be preferred. In this case, the direction of the motion of CPs necessary for the reaction to occur would be clear. For HDAC8, the rate determining step is the second step, so it is difficult to determine the preferred direction for the CPs to move in the enzyme–reactant complex to facilitate catalysis and increase the reaction rate. Furthermore, the fact that HDAC8 has two metal cations near the active site may make the effects of electrostatic preorganization from the rest of the protein less important, as K5ρ at CPs.Electrostatic preorganization is considered to be the dominant factor in the high catalytic efficiencies of enzymes. Quantifying the effect of the protein's extended environment on the active site electrostatics is a challenging problem, but is vital to improving enzyme design and the general understanding of enzyme structure and kinetics. We have tested the convergence of the geometry of the electron charge density in the active site of Histone Deacetylase 8 as increasing amounts of the surrounding protein are included in a cluster model. The magnitude of the charge density at critical points is found to be dependent on the local environment and converges once one residue sphere is added around the point of interest. The Bader atomic charges of atoms usually converge at this same limit, with slight variations in charge values for some atoms at larger size models. Adding a dielectric constant or point charges to the simulation has a slight effect on atomic charges, but does not alter the converged values for the magnitude of The positions of critical points, however, are generally not converged up to the limit of active site model sizes tested here and are shown to be affected by the extended environment of the protein. This finding elucidates the effect of electrostatic preorganization on the charge density. It is not the charges on atoms, or the nature of the bonding interactions, but the relative positions of CPs that reveals the signature of electrostatic preorganization. The locations of critical points are influenced by dipole moments from residues in the full protein, which manifest as perturbations to the wavefunction in the active site. Critical point positions are known to correlate with reactivity and reaction barriers. The positions and curvatures of the charge density at CPs could then be used to predict the extent of electrostatic preorganization in computationally designed enzymes.


The pathogenesis of venous thromboembolism (VTE) is still not completely understood. Experimental animals in which human deep vein thrombosis can be modeled are useful tools to investigate the pathogenesis of VTE. Besides the availability of transgenic and genetically modified mice, the use of high frequency ultrasound and intravital microscopy plays an important role in identifying thrombotic processes in mouse models. In this article, an overview about the application of various new technologies and existing mouse models is provided, and the impact of venous side branches on deep vein thrombosis in the mouse model is discussed. Approximately 40,000 people die annually in Germany from venous thromboembolism, which is usually the result of deep vein thrombosis . Due to be able to ideally model the human disease,contribute to animal protection aspects by keeping the number of experimental animals to be examined to a minimum, andoptimize existing models – also in terms of their comparability.Deep vein thrombosis and resulting pulmonary embolism in the sense of venous thromboembolism (VTE) are among the most common diseases worldwide and are associated with a high mortality rate . Along wThrombotic processes can be observed in a murine small vein model, for example, in mesenteric veins, or in large veins of the animal model. To induce venous thrombosis, the inferior vena cava is mainly used. Studies in small vessels, such as the mesenteric veins, are usually designed to analyze acute thrombogenetic processes, while the inferior vena cava thrombosis model can be used to analyze both acute and chronic stages . The sizThe use of various mouse knockout strains in experimental animal models has still further advantages with respect to comparability with the human organism. Thus, some parallels arise from the species comparison, including the initial infiltration of inflammatory neutrophils and monocytes, fibrin and collagen production in thrombus formation, and clot retraction and new infiltrating vascular channel formation in thrombus tissue during thrombus resolution .Currently, there are various approaches to induce thrombus formation in the inferior vena cava. To access the surgical site, a midline laparotomy is performed initially under deep anesthesia. After exteriorization of the bowel to the side, the inferior vena cava can be visualized. The section to be used is located caudal to the renal vein and extends to the bifurcation of the two iliolumbar veins Fig. . The 5 mA small piece of filter paper saturated with a 3.5% solution of ferric chloride is directly applied to the exposed vein and removed after 2–3 min. The oxidative damage induced to the vessel wall results in the formation of an occlusive thrombus within a matter of minutes. The rate of thrombus initiation and the size of the resulting thrombus can be influenced by varying concentration (10%/20% ferric chloride) and exposure time.Another method is the injection of photoreactive substances , which accumulate in the lipid bilayer of endothelial cells. Irradiation with light initiates a photochemical reaction that leads to the formation of oxygen radicals, thus, resulting in damage to the endothelium .These models are mainly used to study acute thrombogenesis. Because of the toxic or redox chemical stimulus, these models do not ideally represent the pathophysiology of the majority of clinically occurring deep vein thromboses .Stasis in a vessel is achieved by complete ligation of the inferior vena cava in the region caudal to the renal vein. The side branches opening laterally into the vena cava are additionally ligated and partially posterior branches cauterized. This model reflects complete occlusion of the vessel in the clinical scenario. The thrombus size is relatively constant; however, the endothelial damage resulting from total ligation is not negligible. This model can also be used to study both acute and chronic processes; the focus is more on observation of chronic stages with the analysis of recanalization and thrombus resolution.A silver-coated copper wire attached to a 25-gauge needle is inserted into the subcutaneous tissue and into the caudal inferior vena cava. Application of direct current (250 μA) over about 15 min promotes the formation of free radicals, which results in endothelial cell activation . LateralAs in the stasis model, ligation caudal to the renal veins is performed in the inferior vena cava (IVC) stenosis model; however, only an object  is used to create the stenosis and is removed again during the course of the procedure, thus, a decrease in blood flow of approximately 80% is guaranteed Fig. . Brühl eIn the literature, various modifications of this model are described, including the use of vascular clips which result in not only a reduction in flow Fig.  but alsoIn summary, vena cava ligature with induction of stenosis without any modifications represents the best model for deep vein thrombosis in human disease. There is only a decrease of the flow rate, the endothelium remains intact, and the thrombus forms under existing circulation.The technique of intravital video fluorescence microscopy allows the real time visualization of cellularprocesses in vivo. By using fluorescent dyes in vivo or ex vivo that are well-tolerated by the animal, different cellsand cellular components can be visualized in the living animal by fluorescence microscopy. In most cases, theresulting image data are recorded using a fluorescence light microscope with a water-immersion objective incombination with a digital camera and sent to a computer where the entire data analysis is performed withsoftware. The microscope used is equipped with specific filters that are compatible with the fluorescentdyes. Nucleated blood cells are often labeled in vivo with various DNA markers ,while the platelet and microparticle fractions are isolated first from the blood of a donor animal and are transfusedinto the recipient animal after ex vivo staining with dichlorofluorescein or rhodamine B . Cell-spIntravital microscopic examination of the various venous thrombosis models is performed immediately afterinduction of thrombosis and depending on the model usually not longer than 6 h on deeply anesthetized animals understrict control of anesthesia. Thus, especially in the IVC stenosis model, the initial phase of venous thrombusformation can be examined in the area between the sinister renal vein and the common iliac veins, as well as cell–celland cell–vessel wall interactions  and cell velocities (frame-to-frame method) can be detected and analyzed . Using dUsing the IVC stenosis model in combination with intravital video fluorescence microscopy, the kinetics of leukocyte subpopulations of monocytes and neutrophils, and the role of NETs (neutrophil extracellular traps) in the early phase of venous thrombus formation could be detected . FurtherUsing high-frequency ultrasound, e. g., with the VisualSonics Vevo 770® High-Resolution Imaging System , the2). In addition, a fixation unit which can be heated and can record an electrocardiogram is needed. The area to be imaged must be completely free from fur prior to the study. To prevent excessive cooling of the animal, the ultrasound gel should be warmed to 37 °C prior to use. It is imperative that the bodytemperature be measured using a rectal probe during the period of investigation. First, the long-axis view provides a view of the ligated section , each mouse was tested 48 h after successful surgery. Thrombus size and the distance between the opening of the side branches from the long axis were measured. Furthermore, flow velocity in the side branch was determined using pw-Doppler.The analysis of our results showed no association between ligation of the lateral side branches and a more constant thrombus length or thrombus formation in general. The variation in size and formation process remained unchanged, which is probably due to the dorsal side branches that were not ligated.Although we identified side branches as being determinants in thrombus formation (at least in the mouse model), the role of venous side branches and their influence on the events of deep vein thrombosis remains insufficiently understood. In humans, collateral vessels appear to represent natural ways around obstructions . AnotherHigh-frequency ultrasound is a noninvasive method that is particularly useful to study venous thrombosis. Thus, the formation and the course of venous thrombosis can be followed in various IVC thrombosis models.The influence of venous side branches, particularly the dorsal branches, on thrombus formation should always be considered when using the vena cava stenosis model in order to interpret the results correctly.In addition to our results, it must be taken into consideration that some factors such as the sex and age of the animals, which are not modifiable in the experiments , have an


This drastic evolution of the carriers and phonons dynamics might be due an important electron-phonon coupling evolution due to the quantum confinement. These properties have to be taken into account for future TIs-based spintronic devices.Heralded as one of the key elements for next generation spintronics devices, topological insulators (TIs) are now step by step envisioned as nanodevices like charge-to-spin current conversion or as Dirac fermions based nanometer Schottky diode for example. However, reduced to few nanometers, TIs layers exhibit a profound modification of the electronic structure and the consequence of this quantum size effect on the fundamental carriers and phonons ultrafast dynamics has been poorly investigated so far. Here, thanks to a complete study of a set of high quality molecular beam epitaxy grown nanolayers, we report the existence of a critical thickness of around ~6 nm, below which a spectacular reduction of the carrier relaxation time by a factor of ten is found in comparison to bulk Bi After an intense effort to develop a comprehensive understanding of the bulk properties2, the age of the investigation of integration of TIs at nanoscale is heralded by now3. Among exciting perspectives for nano-spintronics, TIs can be used for charge-to-spin current conversion3 or Dirac fermions based nanometer Schottky diode4. However, as known in quantum wells, dots or nanocrystals6 or in metallic nanostructures9, the downscaling usually leads to apparition of size dependent properties and an enhancement of the quantum confinement effect. In the case of TIs films, it has been reported that the electronic properties are deeply affected indeed under a reduction of the layer thickness where the electronic structure deviates from the bulk one with the lost of surface Dirac states properties11. A gap opening in the surface Dirac states appears as well as a profound modification of the bulk electronic levels . This phenomenon revealed by Angle-Resolved Photoemission Spectroscopy (ARPES), appears around below 4 nm in Bi2Se3 (BS) compound11 and 3–4 nm in Bi2Te312. A sketch of the thickness dependence electronic structure is shown in Fig. 12. Different mechanisms have been discussed to elucidate this phenomenon, including substrate-mediated Rashba effect10 and the existence of a crossover from a 3D to a 2D topological insulator13. The band gap opening has been reproduced theoretically and it has been reported that additional quantum size effects should appear leading to topological quantum phase transitions that depend on the film thickness16. While this drastic evolution of the electronic structure submitted to a strong confinement has been well described in TIs at the thermodynamic equilibrium with photoemission spectroscopy12 and with transport properties measurements18, the consequence of this confinement on the ultrafast dynamics of carriers and phonons has been debated only recently in BS20. The ultrafast dynamics of carriers and phonons are only well characterized in bulk crystals of TIs with either time-resolved ARPES or optical methods26. Moreover, while a theoretical prediction of the enhancement of the coupling between the surface electrons and the acoustic phonons has been reported for a confined structure27, no direct experimental reports confirm these predictions so far while ultrafast optics can provide new insights.Topological Insulators (TIs) provide new perspectives for next generation spintronics devices thanks to a natural spin polarized surface current that is topologically protected11 and BT12 compounds. Secondly, thanks to femtosecond optical pump-probe methods, we reveal several anomalies on the carriers and coherent phonons ultrafast dynamics in Bi2Te3 (BT) when the layers thickness is typically smaller than the critical value of ~6 nm. We report indeed a dramatic modification of the hot carriers relaxation time with a reduction from around 2.2 ps (bulk) down to around 200 fs for layer as thin as 4 nm. This transition appears somehow universal since it has been observed for two different samples exhibiting both thickness gradient but having been grown according either to a step or wedge geometry. In parallel to the electron-phonon relaxation time determination, we also report a modification in the spectrum of the photoinduced phonons in the confined nanostructures. While for so-called thick BT layers, the optical phonon mode A1g is clearly generated and detected as expected from bulk behavior, a clear softening and a decrease of its lifetime are observed for ultrathin layers. Surprisingly, while the A1g phonon signal vanishes for ultrathin layers, an increasing contribution of coherent acoustic phonons signal appears with a clear resonant effect. These carriers and phonons dynamics size dependence suggests the existence of a transition in the mechanism of the electron-phonon coupling when reducing the TIs layer which has to be taken into account in the perspectives of the development of TIs nanodevices.In this article, we study the confinement effect on the electronic structure as well as on the ultrafast dynamics of carriers and phonons. We first confirm the appearance of the evolution of the electronic structure for very thin layers with X-ray Photoemission Spectroscopy (XPS) in accordance with what was observed in BS2Te3 (BT) ultrathin films having thickness varying from around 2 nm up to 10 nm and grown with molecular beam epitaxy with PREVAC system28. Different samples were grown with a step or a wedge geometry as shown in Fig. In-situ XPS measurements  for the thick layer (10 nm)25. However, a clear difference is visible at the valence band offset where the lowering of the top of valence band by about 0.2 eV with respect to the thicker films is detected for the 4 nm thick film. At the same time the intensity in the vicinity of the Fermi level is unchanged. This is related with the position of the Fermi level which is crossing the bottom of the bulk conduction band. This indicates a n-type doping for our BT films. Regarding the core-levels XPS data . In Fig. 5/2 line for the as grown film and after the oxidation in air 29. It is important to underline that under surface passivation by the oxides, the core levels (Bi and Te lines) characteristic to BT TIs remain unchanged confirming the stability of the bulk properties of the layer over several months. We are aware that the passivation may affect the surface states31 since they are very sensitive to a small amount of extrinsic surface defects and are submitted to a rapid aging (few minutes) even in high vacuum. However, we did not observe any changes in the in-situ obtained XPS spectra within several minutes and hours after deposition. Consistently with our XPS analysis, we will see in Figs The investigations were carried out with single crystalline Bints Fig.  performeelectron + phonons, where the electronic contribution is composed of a fast rising signal due to the carriers excitation induced by the pump beam followed by a decay described by two exponential functions with a fast decay time ranging from 200 fs to 2 ps (depending on the layer thickness) and a slower component with a time ranging from 30 to 60 ps associated to long living carriers and thermal relaxation process  indicates that the softening does not come from hot electrons-driven potential softening as known for bismuth crystal41. A detailed description of our pump power dependence of the A1g mode is shown in Supplementary Figure −1. However a resonant-like effect is observed in the thickness dependence of the coherent acoustic phonons amplitude as depicted in Fig. Some typical time-resolved and thickness dependent optical reflectivity signals are given in Figs. entclass1pt{minima fs Fig. . This in 36 Fig. . An addi 36 Fig.  as well  36 Fig. , confirmess Fig.  without L) dependence of the electronic levels  when the nanocrystal diameter is reduced from 4 nm to 2 nm. Similar decrease of the carrier lifetime 8. In our case, a numerical adjustment leads to the phenomenological thickness dependent relaxation time as:43) and on the final density of states according to the Golden Fermi rule, but the quantitative relationship between this experimental law and the size dependent electronic structure is not an easy task since a complete electronic and phonon dispersion curves are required for bulk and surface states. No complete theoretical formulation of the size dependence relaxation time of the photoexcited population in CdSe and CdS was reported so far due to the same complexity5. Different possible contributions can be proposed. When the size of the system is reduced, the carriers surface trapping is known to be more efficient since the probability per unit of time for an electron to reach the surface increase as soon as the mean free path of electrons is comparable with the size of the object. This has been already discussed in the case of semiconductor nanostructures5. This surface recombination was one channel of relaxation in semiconductor nanocrystals but quantum size effect on the electron-phonon coupling was also evoked5. In the case of metallic nanoparticles, the proposed interpretation was based on the reduction of electron-ion screening interaction due to a decrease of the carrier density close to the surface which contains a non-negligible number of atoms compared to volume ones for few nm nanoparticles radius R8. A phenomenological scattering rate equation was proposed with α and 7. In the case of BS thin films, a decrease of the relaxation time driven by a size dependent Fröhlich interaction was proposed19 but many other mechanisms can contribute. In the case of BT nanolayers, we can of course think about possible trapping at the oxide cap layer/BT layer interface which could increase the hot carrier damping, even if it is difficult at that stage to evaluate this contribution. Furthermore, it has been also shown that the range of the bulk-surface interband scattering channel has been estimated to occur up to 5 nm in BT compounds24, which matches well the critical thickness we estimate and could indicate that the anomaly we observe is a crossover from the 3D to the 2D system. This is supported by the fact that it is known that the coupling between surfaces Dirac fermions wavefunctions of two opposite sides of a thin layer has been reported to occur over few nm for BS, and even detectable in film of 10 nm of thickness18. Moreover, additional effect could be possible such as band bending. It was previously observed at the free surface of a bulk crystal4 which means that such situation can be enhanced in ultrathin layers of BT with the closeness of the two opposite surfaces. As a consequence, the hybridization of two opposite surfaces states, the surface band bending, the modification of the bulk electronic levels . The question is how much this deformation potential varies as a function of the thickness. If we consider that the hot electrons (holes) rapidly thermalize down (up) to the conduction  bands, the relevant deformation parameter is the one close to the band gap (Eg) with:47The electron-hole acoustic phonon deformation potential is expected to be thickness dependence in topological insulators ultrathin films since the energy bands evolve quite a lot as we observed Fig.  and acco47. As mentioned above in the description of our results, the coherent acoustic phonons under discussion are those corresponding to the thin film eigenmodes, i.e. those inducing an out-of-plane strain L so that the relevant strain associated to the deformation potential is dL/L /. Consequently the relevant deformation potential becomes:This approximation is justified as soon as the frequency of the detected coherent acoustic phonons is smaller than the inverse of the time of thermalization in the conduction and valence bands respectively with12. As a first element of discussion, we have estimated the experimental parameter 10 . The thickness dependence is shown (blue dashed curve) in Fig. 50. We have performed this calculation by considering the first acoustic eigenmode  in the integral term. We remind that z = 0 corresponds to the free surface of BT layer (we have neglected the contribution of the passivation oxide layer at this level). k0 and Eprobe and 32). From the literature32 one can extract both the real and the imaginary part of the derivative of the refractive index at the probe energy (2.2 eV) and we found that n as n2 = 1.6. The result of the calculation gives a time-dependent sinusoidal function for transient optical reflectivity signal with the corresponding frequency of the eigenmode. We focus on the thickness dependence of the magnitude of this signal. This maximum amplitude is not the absolute one, since A as well as L dependence at least. Considering that the sound velocity of the BT layers does not change  and the photoexcited carriers concentration N only as:47We have access to the thickness dependence of the band gap in the literature with different precisions for BS and BT compoundssee Fig. , we can N varies versus the layer thickness L and show that N is roughly multiplied by two in a layer of 4 nm compared to a 10 nm thick one. We have taken this into consideration in the calculation.As shown in Fig. L variations; as matter of fact we have normalized the curve to their maximum for discussing the tendency. It appears that the detection mechanism reproduce an enhancement of the detection of the coherent acoustic phonons as well as a decrease of the detected signal for ultrathin layers. If now we include in this calculation the thickness dependence of the deformation potential, we obtain the green curve (curve labelled as model). Some similitude in the thickness dependence appears but there is a clear shift between theory and experiments that might comes from the approximations we did. Among them, we have used the The calculated thickness dependence of the maximum of amplitude of the detected eigenmode is shown in Fig. et al27. have shown that the quantum size effect leads also to an enhancement of the Dirac surface electron-acoustic phonon coupling which shows that both approaches go in the same direction suggesting an enhancement of coherent acoustic phonon generation in quantum-confined BT layer. This size dependence of the electron-hole acoustic phonon deformation potential parameter needs to be confirmed with ab-initio calculations.At this level we do not claim that we have definitive conclusion but we have to admit that the comparison between experiments and the theoretical calculation, supports a quantum size effect on the electron-hole acoustic phonon deformation potential as never reported before. As an intermediate important conclusion, this model could establish a possible partial correlation between the size dependent electron-hole acoustic phonon deformation potential and the observation of the increase of the carrier relaxation rate, in the case of predominant scattering of carriers by the acoustic phonons. As discussed above, surface trapping also play probably a role. Moreover, it is worth to underline that our model Eq.  is relatL can be explained at least partially by a enhancement of the electron-hole acoustic deformation potential parameter according to our theoretical proposition. The experimental optical measurement integrates bulk and surface electrons, but non negligible surface carriers contribution could exist since our film thickness scales with the required characteristic distance for surface Dirac fermions states to hybridize18, but it is difficult to give a quantitative estimate by now of separated contributions. Apart this quantum-confinement effect, one cannot exclude the contribution of the carrier surface recombination that could enhance the carrier relaxation as well. All these new physical insights show that downscaling the topological insulators properties is not straightforward and these new experimental reports have to be taken into account for potential TIs based spintronic nanodevices.As a conclusion, this work reveals a clear modification of the out-of-equilibrium carriers and phonons dynamics when the BT layer is reduced to few nanometers. This time-domain investigation provides a new insight in the size-dependent physical properties of topological insulators while size-dependent electronic properties were probed in the past only at the thermodynamic equilibrium. While known for semiconductor and metallic nanostructures up to now, we report a similar drastic decrease of the electron-phonon relaxation time with an increase of the confinement. We can assign this effect to a size-dependent electron-hole phonon deformation potential parameter which need to be confirmed by ab-initio calculation. In particular, the increase of the coherent acoustic phonons signals with decreasing in-situ with the use of XPS  measurements were done on the wedge sample to evidence the gradient of the thickness  some deviations are observed probably due to the electronic properties changes as revealed by XPS  of around 1.5 cm (very smooth gradient for the wedge sample) and a width of 1 cm, we were able to investigate many positions along and perpendicular to the gradient which permitted to get a very good statistic of transient reflectivity signals, crucial to reveal subtle properties changes.The pump-probe technique used here is based on a 80 MHz repetition rate Ti:sapphire femtosecond laser 120 fs). The pump wavelength is fixed at the harmonic of the Ti:sa laser  while the probe beam is introduced in a synchronously pumped OPO to tune the wavelength to 582 nm (2.13 eV). The experiments were conducted with the front-front configuration with incident pump and probe beams perpendicular to the surface as shown in Fig. 20 fs. ThThe authors declare that the data supporting the findings of this study are available within the article and its Supplementary Information.Supplementary Information


Cohort studies must collect data from their participants as economically as possible, while maintaining response rates. This randomized controlled trial investigated whether offering a choice of online or paper questionnaires resulted in improved response rates compared with offering online first.Eligible participants were young people in the Avon Longitudinal Study of Parents and Children (ALSPAC) study . After exclusions, 8,795 participants were randomized. The “online first” group were invited to complete the questionnaire online. The “choice” group were also sent a paper questionnaire and offered a choice of completion method. The trial was embedded within routine data collection. The main outcome measure was the number of questionnaires returned. Data on costs were also collected.Those in the “online first” arm of the trial were less likely to return a questionnaire . The “choice” arm was more expensive . It cost an extra £47 to have one extra person to complete the questionnaire in the “choice” arm.Offering a choice of completion methods (paper or online) for questionnaires in ALSPAC increased response rates but was more expensive than offering online first. The main drivers are presumed improved response rates and reduced costs. The online approach is also expected to speed up the process of questionnaire administration and data entry, improve data accuracy, and reduce environmental costs. The anticipated improvements from the participants' point of view include choice about how and when to complete the questionnaire  and instant and easy submission of data (reducing unwanted reminders and the need to find a postbox).The Avon Longitudinal Study of Parents and Children (ALSPAC) is a birth cohort study which is following up children born in a 21-month period in 1991–1992. Questionnaire data in ALSPAC have traditionally been collected by postal questionnaires. For large cohort studies, particularly in times of austerity, online data collection is a financially attractive option However, there are concerns about using an online-only approach for data collection. Evidence from both market research 22.1ALSPAC recruited 14,541 pregnant women resident in Avon, UK, with expected dates of delivery April 1, 1991, to December 31, 1992; 14,541 is the initial number of pregnancies for which the mother enrolled in the ALSPAC study and had either returned at least one questionnaire or attended a “Children in Focus” clinic by July 19, 1999. Of these initial pregnancies, there was a total of 14,676 fetuses, resulting in 14,062 live births and 13,988 children who were alive at 1 year of age.When the oldest children were approximately 7 years of age, an attempt was made to bolster the initial sample with eligible cases who had failed to join the study originally. As a result, when considering variables collected from the age of 7 years onward , there are data available for more than the 14,541 pregnancies mentioned above. Further phases of enrollment are described in more detail in the cohort profile http://www.bristol.ac.uk/alspac/researchers/data-access/data-dictionary/).The total sample size for analyses using any data collected after the age of 7 years is therefore 15,247 pregnancies, resulting in 15,458 fetuses. Of this total sample of 15,458 fetuses, 14,775 were live births and 14,701 were alive at 1 year of age. These children, now young people (YP) of around 24 years of age, and their parents (or step parents) have been followed in detail until the present day. Please note that the study website contains details of all the data that is available through a fully searchable data dictionary ”]. The study participants included in this data collection exercise are all YPs eligible to receive the questionnaire planned for 2012. Exclusions were due to death, withdrawal from the study, a noncontact status flag on the study database , because returned mail has previously indicated that ALSPAC does not have the correct address, or because they have opted out of questionnaires. The “online first” arm of the trial received a letter with a link to the online questionnaire. The “choice” arm of the trial also received a letter with a link to the online questionnaire, in addition to a paper copy of the questionnaire (with prepaid return envelope) so that a choice of method was offered. Participants were not aware of the trial, as it was carried out as part of routine data collection.Assuming an expected response rate of 50% (based on previous YP questionnaires), it was calculated that 1,605 participants in each arm of the trial would have 80% power to detect a difference in response rates of 5% between the two arms.2.3The paper version of the questionnaire was an A5 booklet of 44 pages. Excluding the three administrative questions at the end, there were five sections: “Children of the 90s”; “Gambling”; “Deliberate Self-Harm”; “Employment, Education and Training”; and “Tobacco and Alcohol.” The number of questions in each section ranged from 7 to 43 (median 17), but many of these questions had several parts. Skip statements were used to divert participants around questions that were not relevant to them. The number of questions in each section that were followed by a skip statement ranged from 1 to 9 (median 5).2.4The online questionnaire was designed to be as similar as possible to the paper questionnaire, acknowledging that certain functions, such as skip statements, would be different because participants would be automatically directed to the next relevant question. This also affected the numbering of questions, which would have been nonconsecutive if not allowed to be dynamic in the online version.Generally, the number of questions per page was less in the online version than in the paper version. The ability to have a variable page length was one of the key differences between paper and online as it allowed more logical grouping of questions. Finally, for online completion, an approximate progress indicator bar was given for each section.2.5The reminder schedule was the same for both arms of the trial . The firA reminder was sent only if a paper questionnaire had not been received from the participant and the online submission was not complete . The exception to this was the reminder at 8 weeks enclosing a paper questionnaire—this was only sent if a paper questionnaire had not been received and an online submission had not been initiated . If a participant contacted ALSPAC to request a paper questionnaire at any stage in the process, then this was recorded and one was sent.A £10 Amazon voucher was offered to compensate participants for their time and to encourage response.2.6To test that both arms of the trial and the reminder process, which was more intensive than any used previously in ALSPAC, were acceptable to participants, and to identify any logistical problems, the RCT was piloted on 200 participants. To maximize the efficiency of the pilot study, we chose participants with a high probability of responding. They were randomly chosen from among those who had responded to the YP questionnaire administered around age 18 years. These YPs were then assigned to each arm of the trial using simple randomization. Because it was important to complete the pilot quickly, to administer the main questionnaire on schedule, the reminders were issued at 2 weekly intervals rather than the timeframe set out in 2.7The remaining YPs were randomly assigned with equal probability to either the “online first” arm or the “choice” arm. Randomization was stratified on important confounders—national tertiles of Index of Multiple Deprivation (IMD) score 2010, based on postcode Randomization of participants was carried out using the runiform function in Stata with anonymous identifiers. This was performed by a researcher who was not involved with the ALSPAC administration process. The results of the randomization were passed to the questionnaire administration team, who implemented the mailings and reminders for both arms of the trial.2.8The primary outcome measure was the number of questionnaires returned (with at least one question answered) in each arm of the trial. For the purposes of this analysis, return rates were calculated 30 weeks after the initial mailing.A secondary outcome was completeness of questionnaires. For returned questionnaires, the number of questions answered was compared, to see if either approach (online first or choice of online/paper questionnaire) encouraged more complete responses. ALSPAC staff compiling the completion statistics were blinded to group assignment. Of the 314 possible questions included in the full questionnaire, 109 of these were not dependent on skip statements, and the analysis of completeness was repeated for this core subset of questions.Other secondary outcomes included: mode of response (online or paper); time taken to respond; the number of reminders issued; and requests for paper questionnaires.Resources used in the administration of each arm of the trial were identified. These included: printing, packing and posting of letters and questionnaires (including address labels and envelopes); printing and posting of reminder postcards; and text and phone call reminders. The amount of administrative time spent on individual calls and texts was based on an average for a sample of these communications spread over time.Costs were applied to these resources. Administration time was valued using the wage rate per minute for casual staff. Consumables  were valued on a per item basis. Resources were only compared where there was a difference in costs between the two arms. Setup costs, for example, creation of database, design of questionnaire, and design of the data entry form, were not included as both the online version and the paper-based version had to be available for both arms in this trial. Some of the higher level administration costs (which included the time to send emails) for each stage of the reminder processes were also excluded as they were the same across both arms. The actual costs of telephone calls were also excluded because business telephone packages are based on a monthly fee.2.9Analysis was carried out on an intention-to-treat basis. Logistic and robust multiple linear regression was used to compare responses, and other secondary outcomes, between the two arms of the trial, adjusting for stratification variables .The mean cost per participant for each item of resource use for the two arms of the trial was calculated as the mean resource use per participant multiplied by the unit cost for that resource. The total mean cost per participant for the two arms of the trial was then calculated by summing up the individual cost items and dividing by the number of participants in each arm. An estimate of the extra cost to have one extra person to complete a questionnaire was calculated as the difference in total costs between the two arms divided by the difference in the number of people who completed a questionnaire, defined as completed at least one question.2.10Ethical approval was obtained from the ALSPAC Ethics and Law Committee (ref: E201215).33.1P = 0.6), suggesting that the planned RCT would not have a detrimental effect on response rates. These percentages are based on 100 participants in each arm and a cutoff of approximately 24 weeks after the initial mailing. As expected, the number of responses that were submitted online was greater in the “online first” arm (69) than in the “choice” arm (37). ALSPAC monitored feedback from participants and concluded that both arms of the trial and the reminder process were acceptable to participants.Overall, response rates were very similar in both arms of the pilot trial—84% in the “online first” arm and 81% in the “choice” arm—with no evidence of a difference , more reminders were sent in the “online first” arm (P < 0.05).A flow diagram of participant numbers in the RCT is shown in 3.3P = 0.04].P = 0.003). Other secondary outcomes are shown in www.jclinepi.com.The mean time to respond  was 3.5 days shorter in the “online first” arm than in the “choice” arm of the trial  in the “choice” arm and £2.43  in the “online first” arm. Adjusting for the stratification variables, this led to a mean difference per participant of £0.71 . The total cost was £13,792 in the “choice” arm and £10,690 in the “online first” arm. Hence, it cost an extra £47 to have one extra person to complete the questionnaire in the “choice” arm.A detailed economic breakdown for each arm of the trial is given in 44.1In an RCT of two approaches to questionnaire completion in a population cohort study, we found that response rates were higher in the group offered a choice of online or paper questionnaire from the outset compared with those initially offered only online completion and that offering a choice of method cost an average of £0.71 more per participant than offering only online completion. The additional cost per completed questionnaire in the group offered a choice was £47.4.2The effects of offering alternative methods of data collection have not been sufficiently evaluated in cohort studies The effectiveness of electronic questionnaires for data collection in cohort studies may differ from that in cross-sectional studies, and the use of online questionnaires as a primary data collection method in this setting has been relatively limited It seems likely that a mixed-mode approach  will be important in the gradual shift from paper to online data collection in cohort studies. Although there has been much debate in the survey literature on whether a concurrent or sequential mixed-mode approach is better In this trial, we found that response rates were 10% lower among participants in the “online first” arm, compared with those offered a choice of online or paper completion.4.3Due to attrition throughout the 21 years of the study, the cohort included in this study is not representative of the initial ALSPAC cohort or of the general population. The effects of attrition bias are documented elsewhere There were pragmatic differences between the two arms which reduced their comparability. For example, the questionnaire layout was not identical in the two methods (paper/online), and reminders and the compensatory gift voucher were not triggered in exactly the same way.No complaints about the questionnaire or reminder process were received. However, a small number of participants (thirteen) reported having difficulties logging on to complete the questionnaire online. Considerable numbers contacted the study to report that they had lost their login details (256) or that never received them (115). Although login details were provided when such cases were reported, it is conceivable that these problems might have resulted in reducing the number of participants who chose to complete the questionnaire online, thereby possibly reducing the efficacy of the “online first” arm of the trial.Another important limitation is that the invitation to complete the online questionnaire was by post rather than by email (which would make it easier to access). This was because, at the time of this questionnaire, email addresses were not routinely held for participants. As such contact details are updated, it will be possible to send out invitations by email, which may increase the number of people accessing the online questionnaire and will certainly reduce costs.Finally, we note that this is a rapidly evolving field and that a limitation of any research in this field is the speed with which technology evolves. For example, the effectiveness of online data collection may be quite dependent on not only the proportion of the cohort who own a Smartphone but also on the functionality of online questionnaires when completed on Smartphones. It is a challenge for methodological research to keep up with new and emerging data collection tools 4.4Future work should attempt to replicate research into optimal approaches to mixed-mode data collection published in the survey methodology literature in the context of cohort studies, where a more cautious approach to experimentation has been noted Further studies should assess the effectiveness of different types of reminders. In this study, the reminder method was dependent on the contact details available, but ideally, participants would be randomized to different reminder methods to assess their relative impact on response rates  compared with offering online-only first  to maximize response rates to questionnaires. The results are likely to be generalizable to other cohorts of similar age and will help cohort studies weigh up the extra cost against anticipated improvements in response rates.


An essential component of quality assurance in radiation therapy is verifying the accuracy of monitor unit calculations. Differences between sophisticated algorithms using 2.5D or 3D calculations and simpler monitor unit check algorithms or hand calculations assuming a flat water phantom must be expected. For many anatomical sites, such differences are small and of little or no consequence in the context of expected clinical impact. However, for tangential breast fields the discrepancies are considerably larger than those that would generally be considered acceptable. A simple model to reconcile the differences between sophisticated and simple algorithms is presented, based on replacing the breast contour with a triangular or elliptical contour and using this to estimate an equivalent rectangular prism providing equivalent scatter to the prescription point. The elliptical approximation reconciles the observed differences in calculated monitor units. The analysis we present can assist the treatment planning physicist in selecting a method and tolerance window for verifying monitor unit calculations for tangential breast fields.PACS: 87.53.Kn, 87.53.Tf, 87.53.Xd External beam therapy quality assurance (QA) requires that plans be validated,Missing tissue, field “flash,” and an oblique irradiation of the surface combine to make the dosimetry of breast irradiation using tangent fields complex. Modern treatment‐planning systems (such as Pinnacle™) meet this challenge by accounting for the external body surface (hence field “flash” and missing tissue) and oblique incidence inherently in their algorithm.Validation of the monitor unit calculation is performed either using hand calculations or one of many software packages, both “home‐grown”Due to the approximations in monitor check routines, strategies for validating breast plans have been developed. Many of these involve some adjustment of the field size to an “equivalent square” field and are frequently based on experience. For example, Ayyangar et al.We have investigated alternative schemes for correcting monitor units calculated for tangential breast fields under simple geometrical conditions. The schemes are based on the breast profile and the location of the prescription point. The notion of equivalent square is extended, replacing the breast contour with an “equivalent rectangular parallelepiped,” which would provide similar scatter to the prescription point. Performance of this method appears similar to that of Ayyangar et al.Currently, our clinic plans in 2.5D using a single digitized breast profile (in Pinnacle™) and checked either with software  or a hand calculation, resulting in the disagreements described here. We anticipate that full 3D planning will make reconciliation of the monitor unit calculations more difficult. This initial study showed that an equivalent scattering area approximation works well in 2.5D, and extension to an equivalent scattering volume in 3D is a promising strategy for using current second check tools and reconciling the differences observed.Eighty‐six tangential breast plans, each with a medial and a lateral field, and each treated at 6 MV, were culled at random from our clinical database. These 2.5D plans were prepared by acquiring a 2D contour of the breast and using ADAC Pinnacle™ to calculate both the dose distribution and the monitor units for each field. According to our planning protocol, 2 cm of flash is added to each field, and the dose is prescribed to a point approximately midway between the beam entry points and one‐third of the distance from the posterior field border to the anterior breast surface . The fie−4.4% and a standard deviation of 1.4%. The resulting plans were exported to RadCalc™ for a second check. Comparing monitor units from Pinnacle™ and RadCalc™, we observe a difference of −0.7%, with a standard deviation of 1.6%, consistent with the work of Leszczynski and Dunscombe.(~8%) occurred for fields treating the arm, axilla, or sinus, where contour, flash, and inhomogeneity are expected to produce discrepancies. It should be noted that these other fields are almost all full 3D conformal plans and usually included corrections for tissue heterogeneity, whereas our breast tangent fields are usually calculated assuming homogeneous tissue density. Yet for these other 405 fields, agreement is much better than for the breast fields, indicating that it is this particular geometry that is pushing RadCalc™ beyond the valid limits of its assumptions.As a point of comparison, 405 fields treating sites other than breast/chest wall were examined. The mean difference between the monitor units calculated by RadCalc™ and Pinnacle™ is R2 is in excess of 0.999 for both the breast and nonbreast data. The slope of the regression line is about 1.04 for the breast data versus 1.007 for the nonbreast site. More revealing are the histograms of the percentage disagreement of RadCalc™'s monitor units from that of Pinnacle™'s  is to reduce the field size for calculating phantom scatter by the amount of flash that we will denote by d, to the prescription point as half of one dimension of the rectangle as shown in We next need to account for an overestimation of tissue providing scatter to the prescription point. Consider an audacious assumption, that the amount of scatter to the prescription point would remain constant if the breast tissue were rearranged into a rectangular prism. The corrections we investigated are based on either a triangular or an elliptical approximation of the breast contour as shown in w’ d is equal to the area of the triangle or the half ellipse in d and the depth to the isocenter is ignored. This “equivalent rectangle” would be similar to the field illuminated by a beam of width w' on a flat water phantom, ignoring the divergence of the field's upper border. For our two approximations:The rectangle width w’ is chosen such that the area L and width w+f had an equivalent square field size F=2L(w+f)/(L+w+f). The equivalent rectangular scatterer has a width w' as given above. The equivalent square field size F is then replaced by F', calculated using the original field length L, but the revised field width w' so that F'=2Lw'/(L+w'); henceThe original rectangular field of length (w+f) by L field of equivalent square size F, with a new field w' by L and equivalent square size F'. This change of field size should account for most of the missing scatter, by changing the values of Sp and tissue phantom ratio (TPR) used in the calculation. This can be carried out with a simple multiplicative correction factor:F' related to F' as given above. This factor is greater than 1, and dividing the number of monitor units calculated for a flat water phantom by this factor should approximate the correct number of monitor units (as given by Pinnacle™ or other modern planning systems) within the geometrical assumptions of the triangle and ellipse.When calculating our second check , we replace the Monitor units were recalculated in RadCalc™ for our selected 86 pairs of tangential breast fields, reducing the field width by 2 cm to correct for field flash. This leaves the effect of surface contour uncorrected. The results are summarized in the histogram in The proposed correction factor in 9.1±1.6cm and field widths of 10.2±3.1cm. The sample data have a range of depths to the prescription point of 19.0±2.2cm). This alters the data only slightly to that shown in As a further simplification, consider approximating the superior/inferior length of the treatment field to be a constant nominal length of 20 cm  in the original uncorrected data is 0.0% to ±2%. The data in −1.2% difference, 68% of the plans would pass this test. As a point of comparison, for the 405 fields delivered to sites other than breast 75% would meet the 2% tolerance level, and 25% would require checking by a physicist.TG‐40−6.4% to −2.4% to pass the checking procedure, encompassing 80% of the breast plans. Implementation of such a policy provides the same assurance of accuracy as for other anatomical sites and results in a site‐independent workload for physicists performing plan checks.However, the question does remain as to whether or not one needs to apply a correction factor to check monitor units for tangential breast fields. If the analysis above, together with the experimental validation of Pinnacle™ by Ayyangar et al.,1.7/1.4×2%=2.4% or 1.4 standard deviations. This would pass about 84% of plans, and 16% would still be checked. Or one could select a range that conforms to one's comfort level based on an estimation of the frequency of a serious error. The approach presented here allows each center to make an informed decision.If one is confident that the increase of standard deviation from 1.4% to 1.7% in We have considered the problem of disagreement in monitor unit calculations between a primary and a second check for 2.5D planning of tangent breast fields. The disagreement arises when the second check is performed with a simplified algorithm that does not account for the absence of full scatter conditions.We have described a simplified method of correcting for the absence of full scatter conditions in the case of tangential breast fields based on a geometrical estimate of an equivalent scattering rectangular parallelepiped. The assumptions are justified on physical grounds and supported by the statistics demonstrating the efficacy of the method. The results presented give the clinical physicist the option of correcting monitor units for better agreement with more accurate 2.5D calculations or simply shifting the tolerance window to accommodate the approximations made during checking. The analysis can, and should, be performed by any user by a retrospective examination of plans to ensure that they get acceptable performance before implementing it as a solution in their clinic.Extension of this method to the case of full 3D planning is obvious in principle; an estimate of the treated volume of breast tissue could be used to calculate an equivalent rectangular parallelepiped. Extension of our ellipse to a half ellipsoid is tempting, but may prove to be a poor approximation to real anatomy. As our clinic moves to 3D breast planning, we hope to collect the data that would allow us to test extensions to this simple model.Thanks to Mr. S. Morgan for his assistance collating and analyzing this data.


Amblyomma americanum, since fluralaner was not re-administered 56 days after the initial treatment. The Authors disagree with this assessment and confirm that the design was appropriate, and therefore the results and conclusions for the entire study period are valid.In a recent Letter to the Editor, Armstrong raises concern that the design of the study reported by Six et al. was not consistent with the product label for treatment of  A. americanum ticks within 24 h than a single dose of fluralaner from 6 weeks after initial treatment [Six et al. reported the results of a study demonstrating that monthly administration of sarolaner consistently killed significantly more reatment . In his (i.)requires that fluralaner be re-administered at 56 days for the treatment A. americanum when in fact the label [may be re-administered every 8 weeks in case of potential exposure to A. americanum . This language is very confusing and may be interpreted by a pet owner or veterinarian to mean that fluralaner may be effective against A. americanum for up to 12 weeks. This supports the study design which evaluated efficacy for 12 weeks and therefore we believe the results and conclusions for the entire study period are valid.The author states that the approved Bravecto® label he label , 4 state(ii.)A. americanum as reported in the Bravecto® Freedom of Information Summary [The author states that the manuscript reports in the Discussion section that the Bravecto® label directions were not followed when in fact the Discussion section simply references the efficacy of fluralaner against  Summary ; the des(iii.)P ≤ 0.0006) [The author accurately summarizes the manuscript which reported that at the 8 and 12 h counts on Day 42 the efficacy of both products was lower than expected for unknown reason; however, at the 24 h count on Day 42 and for the duration of the study through Day 90, sarolaner provided significantly lower live tick counts compared to fluralaner  period and from Day 42 onwards this efficacy was significantly better than that provided by fluralaner.The design, conduct and reporting of this study are therefore accurate and appropriate. Under the conditions of this study, monthly administration of Simparica® provided consistently high (> 70%) efficacy against A. americanum.Veterinarians and pet owners cannot predict at the time of treatment the genera and species of tick to which a dog will potentially be exposed. This consideration is of even greater importance when a long-acting product (where the tick genera and species must be predicted months in advance) is used. With Bravecto® this is further complicated by the fact that the duration of efficacy and therefore the ideal treatment interval varies depending upon the tick species. The design of the study is consistent with the fluralaner re-treatment interval recommended on the product label (anywhere between 8 and 12 weeks), and demonstrates the efficacy observed under controlled conditions from a re-treatment interval that will possibly be used on dogs exposed to A. americanum provided by monthly dosing with Simparica® will reduce the direct deleterious effects of tick feeding and should reduce the risk of tick-borne disease transmission better, and more reliably than a single dose of fluralaner over a 90-day period.This study therefore clearly demonstrates that the rapid and consistent kill of