Historically piezoceramics have been the most

Historically, piezoceramics have been the most commonly used materials in ultrasound transducer arrays but, recently, the improved performance offered by relaxor-based piezocrystals in the Pb(Mg1/3Nb2/3)O3–PbTiO3 (PMN-PT) family has been recognized. Piezocrystals are now used widely in biomedical ultrasound imaging and investigation of their possibilities is under way in nondestructive evaluation, particle manipulation and underwater sonar [7–10] based on piezoelectric properties including ≈90% and >1500pC/N [11]. Bulk, monolithic piezocrystal has little advantage over piezoceramic, with the values of being similar, but improved performance can be realized in configurations such as the planks used in biomedical imaging arrays and the pillars used in piezocomposites.
PMN-PT itself, termed a Generation I material, is susceptible to changes in functional properties at elevated temperature and pressure [12], but Generations II and III, respectively exemplified by ternary PIN-PMN-PT and doped ternary Mn:PIN-PMN-PT, are under development to reduce these effects whilst maintaining much of the performance advantage over piezoceramic, e.g. in terms of and that determine piezocomposite performance. Piezocrystal composites thus have potential to improve the effectiveness of focused ultrasound transducers and the work reported here contributes to this by describing the fabrication and testing of a 2D concave phased array made with Generation I piezocrystal.
A widely-reported method to fabricate concave transducer arrays is to place individual single-element transducers in a prefabricated frame at predetermined positions [6,13]. This requires manufacture of each individual array PF-3758309 separately and the presence of the frame can significantly reduce the array fill-factor, i.e. the percentage surface coverage of the active piezoelectric material, and thus the focusing gain and acoustic energy output of the transducer. As an alternative, Raju et al. [14] proposed an array design using a spherically-focused ceramic bowl with printed electrodes defining the elements. However, fabrication of curved sections from piezocrystal boules would be wasteful of material which costs $0.5−3/mm3, and more importantly, performance would be decreased by the lack of alignment between the piezocrystal axes and the surface of the array. Mechanical forming to focus piezocrystal transducers has been achieved successfully using mechanical hard-press [15] and dimpling techniques [16], but reports are limited to single-element transducers. Other possibilities are to thermally form a 1–3 connectivity piezoelectric-polymer composite into the desired shape [17] or to use flexible polymers for flexible composites [18], but these may be difficult at the frequencies of interest here, below 1.0MHz, and the values of volume fraction, e.g. VF>50%, used in focused ultrasound applications.

Geodesic structure
A section of an icosahedron dome structure with dome frequency 6V, referring to a structure with six subdivisions of the original triangles in one icosahedron [19], was chosen for the proposed array. In total, the section requires 24 triangular flat plates positioned as shown in Fig. 1. To match a commercial piezoelectric bowl used as a reference, the target operating frequency of the array was 1.0MHz and its aperture was 62mm across its largest diagonal, with a natural focal distance of 75mm, equal to the dome radius, giving f#≈ 1.2. Table 1 lists the four triangular geometries in this design, with their side lengths and corresponding angles. Each triangle is named for its sides taken in a clockwise direction. Five side lengths in total are needed, denoted D, F, G, H and I. Triangles HGG and HII are isosceles and triangles DFG and DGF are scalene; six of each type are required. These triangles were machined by a precision dicing saw (MicroAce 66, Loadpoint Ltd., Wiltshire, UK) with dicing position accuracy of 0.001mm and theta axis resolution of 0.0005°.

As it can be seen in Fig the values

As it can be seen in Fig. 2, the values of the effective indentation modulus determined for the ULK-350nm and ULK-200nm films differ significantly, despite the same nominal porosity. The lack of a pronounced dependence of M on the applied static load indicates that the results are not influenced by the elastic properties of the substrate. The initial increase in the values of the effective indentation modulus and presence of the hysteresis in the load–unload curve is caused most probably by a small plastic deformation of the film\’s upper layer. Similar behavior was observed for porous organosilicate glasses with a porosity of 30% and a thickness of about 700nm [42]. The mean values of M calculated for the 350nm and 200nm thin films from all the measurement points acquired during the measurements were 6.3GPa±0.2GPa and 7.2GPa±0.2GPa, respectively.
The values of the effective indentation modulus determined for the ULK-46nm sample are presented in Figs. 3 and 4. In the first attempt, we used the rounded tip and relatively large values of the static load ranging from 270nN to 3350nN. The results obtained in this series of measurements are presented in Fig. 3a. The load–unload curve represents the mean values of M calculated as a function of the static load from 20 single point measurements. The error bars represent the corresponding values of the standard deviation. In p2y inhibitor to those presented in Fig. 2, the curve presented in Fig. 3a shows a very strong dependence on the applied static load. The mean values of M increase from 14GPa to 18GPa during load and decrease to about 15GPa during unload. The difference between the load and unload values of M has similar character to that visible in Fig. 2, namely it is smaller than the values of the standard deviation of the corresponding mean value and it decreases with the increasing value of the static load.
The strong increase in the values of the effective indentation modulus suggests a substrate influence. To minimize it, we had to reduce the size of the stress field expanding into the sample. This can be done by applying an AFM tip with a smaller tip radius and/or lower values of the static load. To ensure that radical change in the tip geometry will not influence the results obtained for the indentation modulus of the samples, we performed an additional test. The test sample was also a film of porous organosilicate glass but with a nominal porosity of 40% and thickness of 350nm. Due to the higher porosity content, the modulus of the test sample was lower than that of the ULK-350 sample. Thus, the test sample would deform more under the same load and the influence of the tip geometry would be even more pronounced. The measurement and data evaluation procedures were the same as for the thin-film samples. The mean value of the indentation modulus determined for the test sample with the blunt tip (cantilever #1) in the force range from 650nN to 4750nN was 3.6GPa±0.2GPa. Then we used a cantilever equipped with a standard sensor tip having radius in the range from 30nm to 50nm (cantilever#2). The force ranged from 160nN to 1500nN. The mean value of the indentation modulus obtained with the sharp tip was 3.9±0.1GPa. The result obtained for the indentation modulus with the sharp tip was in a good agreement with that obtained with the blunt tip. The small difference of 0.3GPa could be caused by differences in the actual and the assumed tip geometries, and the corresponding tip–sample contact conditions. We will use this difference in further analysis of the experimental data obtained for ULK-thin film samples sample. Namely, any difference in the data obtained with blunt and sharp tips smaller than 0.3GPa will be treated as negligible.
We repeated the measurements on sample ULK-46nm, but this time we used the cantilever #2 with a smaller tip radius. The applied static load ranged from 200nN to 1400nN. We performed 24 single point measurements. The curve representing the mean values of the M as a function of the applied static load is presented in Fig. 3b. The error bars represent the values of the corresponding standard deviation. As it can be seen in Fig. 3b, the mean values of M obtained for the ULK-46nm sample with standard tip still show dependence on the applied static load. The values of M increase from 8.6GPa to 10.2GPa during load and decrease to 9.2 during unload. The maximal value of M determined during this series of measurements is less than the minimal value of M determined for the same sample with help of the rounded tip.

By good fortune I had

By good fortune, I had hired as my first postdoc Johann Tafto, from the powerful group of Jon Gjonnes in Oslo. Johann arrived full of his Ph.D. work on the theory of membrane transporter channeling, which I\’d been simulating in the hope of reproducing on EELS spectra some of the standing-wave effects on X-ray fluorescence which Batterman had observed using X-rays at Cornell. But the EELS case involved double-channeling, difficult to interpret, and instead Johann conducted a brilliant series of experiments on channeling effects on EDX spectra from polyatomic crystals containing dopants. (Since multiple scattering affects host and dopant atoms equally, the effect cancels). This lead to our Alchemi technique for locating foreign atoms in crystals, used recently in the study of turbine-blade alloys [20] and ceramics [10]. But in a brilliant collaboration with Ondrej and his new spectrometer, they were able to combine the standing-wave effect with the sensitivity of near-edge structure (ELNES) to chemical valence for site-specific valence determination in iron oxide [47]. Johann went on to use the channeling effect to study localization in EELS by a clever experimental arrangement [43], solved the double-channelling problem (through reciprocity and a large beam divergence) and established the usefulness of ELNES with a famous series comparing spectra from octahedral and tetrahedrally coordinated Mg and K in oxides, showing how these could be distinguished [46]. All this provided an important market for future applications of Ondrej\’s wonderful spectrometers.

EELS
My own education in energy-loss spectroscopy (EELS) began with my Ph.D. in Melbourne, and was continued by Ray Egerton at Oxford when Ondrej was working in Cambridge on his Ph.D. My Ph.D. was devoted to detection of a second-order \”double-plasmon\” process [40], and, with David Johnson, to developing the logarithmic deconvolution method of removing multiple-scattering effects from EELS spectra [18], now described in detail in Ray Egerton\’s book [9]. Cowley, at that time in the Physics department in Melbourne, had purchased one of the first DEC PDP8 computers in order to automate EELS data collection from our JEOL JEM 7 TEM [3]. These early \”mini\” computers were based mainly on discrete components, with, for example, a complete removable printed circuit board for one binary half-adder, and an associated paperback book for the manual of each board. The retarding-field electrostatic energy filter had a bias voltage applied to it (across the high voltage) using a set of nylon strings running down into the oil tank from relays (driven by the PDP8) which operated binary switches floating at high voltage. Writing instrument-control machine code and constantly rebuilding this system with others, occasionally getting results membrane transporter (mainly plasmon spectra and elastically filtered Bragg scattering for quantification), constituted the four years of my Ph.D., all of us working very long hours against the background of the Vietnam war protests and the music of the Beatles. We were surprised to see the paper [34,35] reporting energy gain in the beam from interaction with previously beam-excited surface plasmons, and immediately reversed the polarity of the bias batteries in the high-voltage tank to seek this effect, without success. Now, 43 years later, the sad recent death of Ahmed Zewail reminds of the energy-gain possibility in his fascinating PINEM observations, discussed elsewhere in this volume. Later at ASU, starting around 1978, with my first student Mark Disko\’s Ph.D. we developed a tight-binding theory of near-edge structure [7] and spent fruitless hours in a search for optical pumping effects from a laser on both high-resolution lattice images and inner-shell EELS spectra during Ondrej\’s time at ASU. In the first case, Naoki Yamamoto and I had built a cathodoluminescence apparatus for STEM [48] for study of luminescence from individual dislocation in diamond at 25K (this part was very successful), and this could be run backwards to illuminate the sample through a spectrometer tuned to the optical absorption edge of strongly absorbing dopants in a crystal. We expected to see the dopant atoms change appearance in the HREM lattice image when they were optically illuminated (because the low-angle electron scattering, well within the resolution of our TEM, is so sensitive to changes in valence due to ionization) but didn\’t. Later calculations showed we\’d have needed to melt the sample to get an effect, and a superlattice of dopants would be needed. Similar attempts to observe optical pumping effects on ELS spectra with this apparatus failed, as did our attempts to detect co-incidence spectra between EDX or CL and EELS (to reduce background in EELS), where it finally dawned on us that we could only expect the statistics of the channel with the poorest counting statistics in such experiments. All this work was done on the superb Philips EM400 TEM/STEM which we shared with Ondrej\’s development of his serial EELS system amid intense discussion at morning teas.

br Gradient based DIC for

Gradient-based DIC for simulation-based HR-EBSD
As commented above, Bragg (kinematic) simulations allow the construction of EBSPs in a computationally efficient way. However, the overall intensities, especially in the neighborhood of a zone axis, are far from the actual intensities of real EBSPs. Dynamical simulations, albeit more accurate, require long computational times (each pattern may take about an hour to be generated on a modern desktop computer [16]).
The algorithm proposed here is similar to the approach of Villert et al. [11]. It is based on the Bragg condition used to determine band locations, together with a modified band profile to emulate excess and deficiency lines. Thus, the intensity of the simulated EBSP is given by the additive contribution of each diffracting plane according to its structure factor, weighted in a very simplistic way by the following criterion: the intensity of the pixel is weighted by 0.8 if it lies within sin  < sin θ < 1.2sin  and by 1.2 if it lies within 0.8sin  < sin θ < sin  (where θ is the angle to the corresponding crystal plane and is the Bragg angle). This modified Bragg model is of course still insufficient to simulate with high accuracy the intensities of an EBSP. In order to improve the fitting quality, a new GB-DIC procedure is proposed, different from the conventional DIC procedure. GB-DIC algorithm is based on the approach suggested by Tzimiropoulos et al. [17]. From an EBSP image, a new image (gradient image) is created based on the intensity gradients in the following form: where I(x, y) is the intensity of the EBSP image (either experimental or simulated) and i is the imaginary unit (see Fig. 1). The cross-correlation image can be obtained from the Fourier transform of the gradient image of a real () and a simulated () EBSP as follows: where is the inverse Fourier transform and is the complex conjugate of . The peak position in corresponds to the image shift between the two images. According to Tzimiropoulos et al., this kind of antimalaria medication brings a more robust and accurate estimation of image translations, rotations and scale changes between images. Moreover, this image transformation leads to a pair of complex images where the Kikuchi lines are visibly highlighted and the overall background intensities are flattened. As a consequence, the quality of the fitting (peak height to background ratio in the FFT correlation image) is largely increased as shown in Fig. 2. A more robust fitting of kinematically simulated EBSPs is then possible.
Image filtering has been shown to be critical for an accurate HR EBSD procedure. Wilkinson et al.[4] studied different filtering parameters and finally applied a Hamming-like window in order to prevent aliasing problems and a low-pass filter in the frequency domain to remove noise. In this case, with the aim of highlighting the benefits of the gradient-based procedure, no windowing was considered and only a low-pass filter was applied (equivalent to the one proposed by Wilkinson et al.[4]) with a double objective: first, remove the residual noise from the EBSP image; and second, to smooth down the edges of the Kikuchi bands in the simulated pattern.

New procedure of calibration of the pattern center
Traditionally, PC location has been calibrated by different methods including feature mirror symmetry methods, screen movement methods or shadow casting methods [18,19]. However, the typical resolution of these methods was of the order of 0.5% of the camera width. With the advent of HR-EBSD this resolution has been shown to be inadequate [15,20] (as it will be demonstrated below) and new PC calibration procedures have been developed. Maurice et al [21], for example, tried to improve the resolution of screen movement methods by cross-correlation techniques but the mechanical insertion/retraction and geometric uncertainties limit its application. Mingard et al. [22] also extended the use of shadow casting techniques with relevant results. Finally, Basinger et al. [23] using an EBSP spherical remapping technique and simulated patterns and reached a resolution of 0.045% in a Ni polycrystal limited by the uncertainty in the exact crystal orientation. In this paper we present a novel method (based on the method proposed by Kacher et al. [13]) with improved resolution that adapts the simulation-based HR-EBSD using GB-DIC procedures to the problem of PC calibration with minimum assumptions on the stress state of the crystal.

A further set of experiments was designed to

A further set of experiments was designed to test the limits of this proportional shift. This was done by varying the size of the test disk in one eye, while presenting the other eye with a large homogeneous test field. The smaller the test field, the closer its area is to its outer contour. Varying the size of the monocular test field from 7 to 5, 3 and 1 degree of visual angle, the contribution of that test field to the binocular brightness impression increased (over 8 observers) from w=.851 to .857 to .887 to .932. In other words, a half-image’s contribution to binocular brightness approaches w=1 when its average distance to a contour approaches zero. This was coined the “contour mechanism”. The perceived brightness remains quite stable for all sizes of the test field, i.e. it is subject to a “constancy rule”. A summary classroom demonstration of all these brightness averaging effects can be experienced by fusing the stereo pair in Fig. 1C.
In additional experiments it was then shown that it takes time for the eye weighting coefficient to settle after bepridil onset. In other words, the contour mechanism shows some degree of inertia. It takes about 100–200ms for an appearing contour to induce w=1 in its immediately adjacent visual field. This delay predicts that a pair of orthogonal gratings, the classic demonstration stimulus for binocular rivalry, should look like a grid (commonly referred to as a plaid) if briefly flashed on and off. That indeed turned out to be the case, indicating that temporarily ==1/2. This settling mechanism is the same as what can be observed and precisely measured in metacontrast (Alpern, 1952; Stigler, 1910) and in “false fusion” (Wolfe, 1983).
Together, these experimental findings led to the following account of the conflict in binocular rivalry. The law of complementary shares and the contour mechanism necessarily come into conflict for any dichoptic bepridil area A where adjacent but non-corresponding contours are presented to the two eyes. The contour mechanism for the left eye induces, with some degree of inertia, →1 in area ; the contour mechanism for the right eye similarly induces →1 in area . This, however, violates the law of complementary shares (+1). The system apparently resolves this conflict by abrogation of the constancy rule, while preserving the law of complementary shares: both →1 and →1, but they do so in turn, steadily alternating. In addition, there was no pressing reason to assume that this process would be different for the normal fused state in binocular vision. The still dominant “permanent rivalry hypothesis” was not challenged, but rather supported by this account for the conflict in binocular rivalry.
The contour mechanism, a contour inducing w=1 for its immediately adjacent visual field, also formed the starting point for the analysis of the alternation process in rivalry. If a contour is presented to one eye only, the resulting percept will be stable, whatever the strength (formally denoted by λ) of that stimulus may be (not considering “Troxler’s fading”, which was shown to be irrelevant for the rivalry mechanism). The only way to make such a stimulus perceptually disappear is by presenting a strong enough stimulus to the other eye. This notion led to the hypothesis that the duration of a monocular contour’s dominance period in rivalry is determined by the strength of a contour in the corresponding area of the other eye. This had never been studied before. Since Breese (1909), there existed a literature on the alternation rate in rivalry that showed that increasing luminance, contrast or amount of contours in one or both eyes increased alternation rate. There also existed an even larger literature on factors affecting the predominance of monocular patterns in rivalry (following Breese, 1909 and Roelofs & Zeeman, 1919), which was affected by the same or similar factors as alternation rate, in particular by the contrast or sharpness of contours. However, at the time no explicit model had been proposed to relate the two, alternation rate and predominance, to each other. Four propositions were intended to fill this gap (Levelt, 1965). In their formal description, they involved a number of symbols: and , denote the mean durations of the left and right eye dominance periods in a two-choice rivalry situation, where the mean duration of the complete cycle is . Predominance () of left-eye stimulus () is calculated as and similarly so for the right eye stimulus (). Stimulus strengths of and are given by and respectively. Operationally, stimulus strength was defined (up to order relations) by two variables: (i) the amount of contour per area, and (ii) the contrast of contour (taking into account the local difference threshold). The above hypothesis can now be stated as and , where f is a monotonic decreasing function of . These two equations together yielded Levelt’s (1965) four propositions in their original form:

br Introduction br Experiment br Experiment br General

Introduction

Experiment 1

Experiment 2

General discussion
The results obtained in SEM are in line with previous studies showing that the global effect diminishes with time (Coeffe & Oregan, 1987; Findlay, GW5074 Supplier 1982; Ottes, Vangisbergen, & Eggermont, 1985; Van der Stigchel & Theeuwes, 2005). Whereas saccades were aimed at an intermediate location in the immediate-response condition, saccades were aimed at the target in the deferred-response condition. Importantly, the results obtained in the no-delay condition of TEM demonstrate that the decline in the global effect even occurs in the presence of an intermediate saccade. That is, the distributions of landing positions of second eye movements in the no-delay conditions of TEM were, similarly to those obtained in the deferred-response conditions of SEM, bimodal rather than unimodal.
Our results, in particular those obtained in Experiment 2, are difficult to reconcile with the center-of-gravity account (Coren & Hoenig, 1972; Ottes, Vangisbergen, & Eggermont, 1984). This account implies that the global effect is primarily caused by poorly resolved spatial signals. Since the spatial GW5074 Supplier improves over time, the global effect becomes less pronounced and should eventually disappear. Crucially, even in the case that the identity of objects is not yet determined, observers should gradually become more capable of aiming their eyes at single objects. However, in the current study, a global effect was observed in the identity-delay condition of TEM, a condition in which the locations of the objects were given from the start of the trial. Accordingly, the global effect appears to arise from a deficiency of identity information, rather than a deficiency of location information.
The results are consistent with the weighted-average account (Godijn & Theeuwes, 2002; Marino et al., 2012; Meeter, Van der Stigchel, & Theeuwes, 2010; Trappenberg et al., 2001).This account explains that visual stimuli induce peaks of activity in a saccade map, possibly situated in the intermediate layers of the superior colliculus (Schall, 1991). If these signals originate from locations relatively close together, they overlap and conjoin, such that in the absence of target information, eye movements tend to be directed towards the weighted average of the signals. When target information is obtained over time, the peak associated with the target strengthens relative to the peak associated with the distractor. As a result, eye movements become more target-directed and thus the global effect less pronounced. Indeed, the current results confirm that target information is required to prevent the eyes from being directed towards the middle of the object pair. It was only in the no-delay condition of TEM, the condition in which the identity of the target was revealed prior to the first saccade, that the global effect was diminished. Importantly, this finding also suggests that identity information is preserved over eye movements and continuously modulates the activity in the retinotopic saccade map.
The present results are different from those found by Zelinsky et al. (1997). Zelinsky et al. observed sequences of eye movements that were aimed in between objects, even though objects were presented without any delay. Importantly, in contrast to the current stimuli, Zelinsky et al. employed real-world objects. Therefore, whereas the identity information obtained prior to first eye movements sufficed to discriminate the target from the distractor in the present experiments, this was not the case in Zelinsky et al. In this respect, the difference in findings between ours and those obtained by Zelinsky et al. leads to the same conclusion: the global effect primarily arises from uncertainty about a target’s identity rather than insufficiently resolved location information.

Acknowledgments
This research was partly financed by NWO (ORA grant 464-11-015 to M. Donk).

Despite evidence from a variety of attenuated

Despite evidence from a variety of attenuated alphaherpesviruses suggesting that mutations within ICP4 may be a causative factor driving attenuation of the virus, the two recombinant viruses tested containing high frequency candidate SNVs within ICP4 did not cause any reduction in virulence of MDV, although the Mut ICP4-2 recombinant virus did fail to transmit Lomustine horizontally to contact birds. Recombinant MDV viruses that are virulent and induced MD in challenged Lomustine but failed to transmit horizontally to cause MD in contact birds have been previously reported for mutations in UL44 (gC) and UL13 (protein kinase) (Jarosinski et al., 2007; Jarosinski and Osterrieder, 2010), as well as with a recombinant virus with a point mutation within LORF2 (Hildebrandt et al., 2014). One possible explanation for the failure of Mut ICP4-2 to infect contact birds is due to lower levels of in vivo replication at days 7 and 14, during the times traditionally classified as early cytolytic and latent phases of infection (Baigent and Davison, 2004). The lowest virus load among the three time points was seen during the transition between the early cytolytic phase to latency (7dpi) for Mut ICP4-2. This could affect transmission of the recombinant virus because as chickens mature they develop an age-related resistance towards MD. Therefore, young chicks are most susceptible to MDV infection and due to a decrease or delay in shedding of virulent MDV by Mut ICP4-2, the potential for transmission and infection to older contact birds would decrease (Jarosinski et al., 2007). Alternatively, these mutations within ICP4 may impair replication within the feather follicles, leading to an inability to spread through shed feather dander. Despite the indication that mutations within ICP4 appear to be correlated with attenuation in a multitude of alphaherpesviruses, recombinant viruses containing point mutations within ICP4 show that mutations within ICP4 alone are not sufficient for attenuation in MDV. This conclusion is supported by experiments using the Oka vaccine strain of VZV in which transactivation of downstream promoters regulated by ICP4 was compared between mutated versions of ICP4 from the vaccine strain versus the parental virus. Cohrs et al. (2006) established that regulation and transcription of downstream promoters between the mutated and wild-type ICP4 were comparable, leading to their conclusion that mutations within ICP4 alone are not sufficient to cause attenuation.
Mutations within the VZV Oka vaccine strain did not affect transactivation of viral promoters, but it is known that ICP4 contains different regions with specific functions in addition to transactivation, such as DNA binding, nuclear localization, and regulation of late genes (Wyrwicz and Rychlewski, 2007). Therefore, we sought to computationally determine what function observed mutations within MDV ICP4 may impact, particularly those within amino acids 60–63, which contained mutations in three completely attenuated MDV replicates via sequence comparisons to other alphaherpesviruses. Alignment of the complete amino acid sequence of ICP4 revealed significant variation among alphaherpesviruses, which has previously been described. It is known that certain regions of ICP4, such as regions 2 and 4, are highly conserved among herpesviruses, while other regions are not particularly well conserved among species, such as regions 1, 3 and 5 (Wyrwicz and Rychlewski, 2007; McGeoch et al., 1986). Specifically, comparisons among genera show that Mardiviruses have a significantly longer ICP4 of approximately 2173 amino acids in length compared to the average length of 1500 amino acids for non-Mardiviruses. Furthermore, in our attenuated MDV replicates containing several high frequency mutations around amino acids 60–63, these mutations occur within a region found uniquely in MDV yet absent in other closely related Mardiviruses. Considering that MDV is not only virulent, but is also an oncogenic virus, unlike all other alphaherpesviruses which do not cause tumors, it is difficult to predict what role these unique regions in MDV ICP4 provide during the life cycle of this oncogenic herpesvirus, particularly in light that closely related apathogenic Mardiviruses lack this commonly mutated region of ICP4.

Potential correlations between in vitro findings in apoptosis assays and

Potential correlations between in vitro findings in apoptosis assays and the biological role of apoptosis during in vivo infections must be made cautiously. Nevertheless, it is possible to speculate that the presence of specific Ab at the time of MAP infection could locally promote apoptosis in the macrophages of the intestinal mucosa. Taking into account that intracellular viability of mycobacteria has been demonstrated to decrease in apoptotic cells (Keane et al., 2000) and that apoptosis favors cross-presentation of mycobacterial antigens potentiating T cells response (Winau et al., 2006), the increase in apoptosis induced by Ab might be beneficial in the in vivo response to infection. Indeed, the idea of developing a pro-apoptotic vaccine has been proposed as an alternative for improving tuberculosis control (Behar et al., 2011). In this sense, evidences consistent with a beneficial role of apoptosis in JD infection have been described in deers. BMDM from JD-resistant red deers showed significantly higher apoptosis rates than cells from susceptible animals, when infected ex vivo with MAP (Dobson et al., 2013).
In summary, in this work we provide evidence of a pro-apoptotic effect of buy ambroxol hydrochloride in an in vitro infection model of bovine macrophages with MAP. Collectively, the findings reported herein and in our previous works (Mundo et al., 2008; Jolly et al., 2011) suggest an active role of specific antibodies that might be proposed as beneficial for the host in the MAP–macrophage interaction. Whether the induction of an early potent specific antibody response could serve for JD protection needs to be further elucidated.

Conflict of interest statement

Acknowledgements
We gratefully thank PhD Daniela Ureta and Vet. Bárbara Fernández for their helpful technical assistance in flow cytometry and serum ELISA assays, respectively. This work was financially supported by Agencia Nacional de Promoción Científica y Técnica (BID PICT 2010-2672) and Universidad de Buenos Aires (UBASeCyT 20020100100912, 20020130100607BA).

Introduction
Contagious bovine pleuropneumonia (CBPP) is a respiratory disease of cattle caused by Mycoplasma mycoides subsp. mycoides (Mmm) and listed by the World Organization for Animal Health (OIE, 2014) as one of the most economically important livestock diseases in Africa.
Currently, a live attenuated culture of the causative organism strain T1/44 is used as a vaccine of choice. Although it confers some level of immunity, the T1/44 vaccine has certain drawbacks that include low efficacy (Thiaucourt et al., 2000) and a short duration of immunity. Further, the vaccine causes adverse post-vaccinal reactions at the site of inoculation leading to poor acceptance by farmers (Kusiluka and Sudi, 2003; Sori, 2005). Finally, the vaccine has poor stability (short shelf life), hence the requirement for a cold chain during delivery (Rweyemamu et al., 1995) and there is the possibility of reversion to virulence (Mbulu et al., 2004). For this reason, an efficient inactivated vaccine would be a useful addition to the existing prophylactic measures.
Vaccinations using inactivated vaccines have been successful in a number of mycoplasma diseases including contagious agalactia (Buonavoglia et al., 2008) and contagious caprine pleuropneumonia (Rurangirwa et al., 1987). Similar trials for CBPP with a saponin-inactivated vaccine (Nicholas et al., 2003) and with an Immunostimulating Complex (ISCOM) formulation (Hübschle et al., 2003) have not yielded success.
The inactivation method and quantity of mycoplasma administered may play an important role. Inactivation by heat or by sodium hypochlorite can substantially alter the antigens from Mycoplasma agalactiae and hence reduce the immunogenicity (Tola et al., 1999). However, two doses of 20ml at a protein concentration of 14.5mg/ml of heat inactivated mycoplasma formulated with a suitable adjuvant induced immunity against CBPP (Gray et al., 1986). This suggests that mycoplasma may have to be present in large numbers, either alive or dead, to induce a sufficient protective response and confirms that an inactivated vaccine can confer immunity. Protection by the live T1 strain of Mmm has also been shown to be dose dependent, with a low dose of 105 mycoplasma conferring low protection, while there was no significant difference between doses of 107 and 109 (Gilbert and Windsor, 1971; Masiga et al., 1978; Thiaucourt et al., 2000).

In patients with BC the group from Lund Sweden performed

In patients with BC, the group from Lund, Sweden, performed a study aiming to detect the sentinel LNs in 75 patients who underwent RC and PLND for BC. In 10 patients (13%), no sentinel LN could be detected by collimator. A median of 40 LNs per patient were removed. Thirty-two patients showed LN metastases at histopathology. In 6 of these 32 patients, LN metastases were only detected at histopathology and not by collimator, resulting in a false-negative rate of 19%.
In patients with PC, the group from Augsburg, Germany, combined preoperative planar films with intraoperative gamma probe identification of sentinel LNs after transrectal injection of 99mtechnetium into the prostate gland. In 228 men who underwent RP and PLND for high-risk PC (prostate-specific antigen [PSA] > 20 ng/mL or biopsy Gleason score 8-10), a median of 18 LNs were resected per patient. LN metastases were found in 96 men at histopathology. In 26 patients (27%), LN metastases were found only at histopathology and not by collimator. If the sentinel (radioactive) LNs had been the only nodes resected, then approximately one-third of LN metastases would not have been removed. Therefore, the high rate of metastases in nonradioactive LNs makes this group of patients not suitable for a limited radio-guided LN dissection or focused poly ic therapy.
Lymphoscintigraphy in BC and PC has further drawbacks: the shining-through effect (sentinel LN visualization is impeded by radioactivity from the primary injection site and 99mtechnetium- containing urine in the bladder); the procedure has to be performed in a shielded room; the preoperative SPECT has a limited resolution; the bulky collimator for intraoperative nodal search limits access to nodes and therefore smaller LNs may be missed; and last but not least, it entails significantly higher costs. These drawbacks and especially the high false-negative rate for the detection of metastatic nodes are the major reasons why the SN technique has not gained wide acceptance.

Fluorescent Dyes
The data on the use of fluorescent dyes for LN imaging in BC are limited. In 12 patients who underwent open RC and PLND (median of 17 LNs removed per patient) for BC, ICG lymphangiography was performed. Five patients had LN metastases in histopathology but none had metastases in the sentinel LN.
In another study on 10 patients who underwent robotically assisted RC and PLND (a median of 16 LNs removed per patient) for BC, the authors report a sensitivity of 75% with ICG in detecting metastatic nodes. One quarter of metastatic LNs were therefore missed when relying on ICG node fluorescence alone.
Recently a group showed that not only where the fluorescent dye is applied (serosally or mucosally bycystoscopic injection) but also the filling status of the bladder may play a crucial role, underlining possible pitfalls associated with the injection technique of fluorescent dyes into the bladder wall.
The experience is greater with fluorescent dyes for LN imaging in PC. In 50 patients who underwent robotically assisted RP and PLND for PC, the mean number of LNs resected was 14. In a quarter of patients, no sentinel LNs could be detected. LN metastases were found in 4 patients. In a recently published series of 38 patients who underwent laparoscopic RP and PLND (median number of resected LNs was 18) for intermediate- or high-risk PC, the detection rate of sentinel LNs and metastases with the help of ICG was 97% and 98%, respectively. Without performing an extended backup PLND, however, the number of missed LNs also containing metastases cannot be known.

Pelvic Nodal Imaging Using MRI or CT
CT and conventional MRI are widely used for preoperative detection of LN metastases in patients with BC or PC. Their diagnostic accuracy, however, is less than optimal. In BC and PC, CT may miss 30% to 40% of LN metastases. Thus, upstaging of clinical N0 to pathological N+ is frequently found despite negative preoperative imaging. These imaging techniques rely mainly on morphologic criteria (LN size, shape, and internal architecture), although the major shortcomings of these criteria are readily apparent. The size of nonmetastatic LNs varies widely and may overlap with the size of LN metastases. There is a consequent lack of consensus regarding the normal limit for size in the diagnosis of pelvic LN metastases. Further, the normal range for LN size varies even within different anatomical regions as well as for different cancers. Using the criterion of a short-axis diameter larger than 6 mm on CT images of patients with PC, the sensitivity and specificity for the detection of a malignant node were found to be 78% and 97%, respectively. If the threshold is changed to a short-axis diameter larger than 5 mm for metastases (in pelvic malignancies), the sensitivity may improve slightly to 86% at the price of a lower specificity of 78%. So small metastases often remain undetected and enlarged LNs due to reactive hyperplasia may be misinterpreted as metastatic LNs. It is obvious that novel imaging techniques are needed to identify the correct nodal status.

Materials and Methods Statistical analysis was

Materials and Methods
Statistical analysis was conducted by IBM SPSS Statistics Version 22 (Armonk, NY). An independent t test for continuous variables and a chi-square test for qualitative variables were performed to confirm homogeneity within the group variables as well as to correlate infection and explantation rates with various risk factors. A multivariate logistic regression analysis was conducted to predict explantation and infection of the implant using both groups separately and various risk factors (diabetic disease, history of pelvic irradiation, prior surgery for urethral stenosis, lengths of administered antibiotic, postoperative bleeding, impaired wound healing, mechanical failure of device, urethral erosion) as predictor. Infection-free survival in comparison to the non-IZ and IZ groups was estimated by Kaplan-Meier method and analyzed by the log-rank test.

Comment
Device infection is a greatly feared complication in prosthetic surgery that commonly leads toward device infection, and has a major impact on affected patients. Regarding the AUS, the infection rate varies between 0.5% and 10.6% and occurs either immediately after order ALW-II-41-27 with a mean of 3.7 months or delayed mostly within the first 2 years. The most frequent organism of deviceinfection in genitourinary prosthesis is the Staphylococcus species.
The pathophysiology of device-associated infections is well understood. It depends on device, host, and bacterial characteristics. Because of various pathophysiological processes, the existence of a biofilm is the basic survival mechanism for bacteria in order to resist external and internal environmental factors. The bacterial colonization of the device itself does not only precede clinical infection but also can adversely affect the function of the device. In these cases, frequently no clinical evidence of infection is present. Therefore, infection rates of medical devices are most probably underestimated due to the lack of clinical signs of infection, inadequate sample acquirement for antimicrobial cultures, or the failure of current microbiological techniques to consistently cultivate organisms in a biofilm.
Infection of AUS is not always properly defined in literature. Frequently, infection of the device is referred only to primary implant surgery and therefore is limited to the first months after surgery. Later infections are referred to as secondary infections due to urethral erosion or urethral manipulation and are often excluded. However, in a recent long-term follow-up study of AUS, 2 infections are described after 3.5 and 13 years. Thus, there is currently no consensus whether or not infection is causing erosion or vice versa. Furthermore, the time frame of postoperative device infection and the relationship with urethral erosions are not clearly defined in the current literature. Lai et al retrospectively investigated 218 patients and included wound infection as well as infection of the device without erosion as infection. Kim et al reported perioperative infection and infection separately but did not define the criteria to distinguish these findings clearly. Because of the fact that device erosion and infection are often coherent, many publications report combined erosion and infection rates.
In consideration of the pathophysiology of device infection, resistance mechanisms of bacteria due to biofilm formation, and clinical data, we gathered all documented device infections irrespective of time of infection or existence of concomitant urethral erosion. Furthermore, chronic device infection without clinical signs of infection might also be the cause of, or aggravate the incidence of urethral erosion as well. This raises the question whether or not antibiotic coating of the device can diminish infection and erosion rates. Thus, urethral erosion was taken into consideration in the current study as well. Additionally, combined infection and erosion rates were investigated and an early onset infection within the first 6 months was taken into consideration as well. Furthermore, infection of the device normally leads to temporary or permanent explantation. As chronic infection without clinical signs could lead not only to urethral erosion but also to dysfunctions of the device, we could expect a difference in explantation rates. In test cross study population, no statistically significant differences in infection, early onset infection, infection and erosion, or explantation rates could be identified. These results support the evidence that the antibiotic coating does not significantly reduce infection rates. The overall infection and explantation rates correlated well with current rates in other publications ranging from 0.5%-10.6% to 9.5%-64%, respectively.