Due to the Internet of Things (IoT) requirements for a high-density network with low-cost and low-power physical (PHY) layer design, the low-power budget transceiver systems have drawn momentous attention lately owing to their superior performance enhancement in both energy efficiency and hardware complexity reduction. As the power budget of the classical transceivers is envisioned by using inefficient linear power amplifiers (PAs) at the transmitter (TX) side and by applying high-resolution analog to digital converters (ADCs) at the receiver (RX) side, the transceiver architectures with low-cost PHY layer design  are mandated to cope with the vast IoT applications. Therefore, in this paper, we propose the orthogonal shaping pulses minimum shift keying (OSP-MSK) as a multiple-input multiple-output (MIMO) modulation/demodulation scheme in order to design the low-cost transceiver architectures associated with the IoT devices. The OSP-MSK fulfills a low-power budget by using constant envelope modulation (CEM) techniques at the TX side, and by applying a low-resolution one-bit ADC at the RX side. Furthermore, the OSP-MSK provides a higher spectral efficiency compared to the recently introduced MIMO-CEM with the one-bit ADC. In this context, the orthogonality between the in-phase and quadrature-phase components of the OSP are exploited to increase the number of transmitted bits per symbol (bps) without the need for extra bandwidth. The performance of the proposed scheme is investigated analytically and via Monte Carlo simulations. For the mathematical analysis, we derive closed-form expressions for assessing the average bit error rate (ABER) performance of the OSP-MSK modulation in conjunction with Rayleigh and Nakagami-m fading channels. Moreover, a closed-form expression for evaluating the power spectral density (PSD) of the proposed scheme is obtained as well. The simulation results corroborate the potency of the conducted analysis by revealing a high consistency with the obtained analytical formulas. Recently, the dawn of the Internet of Things (IoT) has intensified the research efforts to fulfill the escalating demand for low-power budget wireless transceiver systems. Overnight, the communication industry experienced an explosion in the number of sensors and terminals that connect mobile users to the internet ,2. HowevThe IoT technology is protruded today as one of the main use cases in the upcoming generations of the wireless communication networks such as the fifth generation (5G) and beyond . As suchIn the massive MIMO, equipping the BSs with an enormous number of antennas provides a worthwhile enhancement in the system capacity, however, it results in a considerable increase in both the power consumption and the hardware complexity, since, each antenna unit requires a dedicated radio frequency (RF) chain circuit . TherefoReferring to the existing literature, the concept of using a one-bit ADC along with the massive MIMO systems was touched upon in , where aThe use of the one-bit ADC with massive spatial MIMO (SM-MIMO) was first tackled in , in whicThe multiple-input multiple-output constant envelope modulation (MIMO-CEM) with the one-bit ADC ,23,24,25The problem of the low spectral efficiency of the MIMO-CEM with the one-bit ADC was implicitly introduced in . This prThe OSP-MSK Modulation/Demodulation Scheme: The OSP-MSK is proposed as a low-power and low-cost modulation scheme. The OSP-MSK relaxes both the power budget and the hardware complexity by using nonlinear PAs at the TX side and by deploying a low-resolution one-bit ADC at the RX side. Furthermore, the OSP-MSK achieves a higher spectral efficiency than the recent MIMO-CEM system by increasing the number of bits per symbol without additional bandwidth.OSP-MSK Performance Analysis: Firstly, a generic closed form expression for assessing the average bit error rate (ABER) performance of the proposed OSP-MSK modulation over an additive white Gaussian noise (AWGN) is presented thoroughly. Afterwards, this expression is used to derive closed-form expressions for the ABER performance of the OSP-MSK over the generalized Rayleigh and Nakagami-m fading channels.OSP-MSK Bandwidth Utilization: The bandwidth utilization of the OSP-MSK is evaluated by deriving a closed form expression of its power spectral density (PSD). This PSD is used to assess the ability of the OSP-MSK to boost the spectral efficiency compared to the MIMO-CEM system.Ensuing the above concepts, the major contributions of this paper can be summarized as follows: The rest of this paper is structured as follows. µ and Q-function, the complementary error function, and the gamma function, respectively. Finally, we use a, b, and c are Gaussian hypergeometric function arbitrary parameters, and Notation: Throughout this paper, vectors are represented using bold lowercase letters and matrices are represented using bold capital letters. Complex Gaussian distributed random variables (RVs) are expressed by As such, the constant envelope signal of each dataset is given by:Therefore, the transmitted signal of the proposed OSP-MSK is expressed as follows:Confusion may be arisen here that both gnal see , becauseplifying . Hence, On its path, the OSP-MSK transmitted signal undergoes a fading channel, and it is corrupted by AWGN. At the RX side, a coherent demodulation is applied to the received signal, as shown in Finally, the MSK in-phase components are separated by exploiting the orthogonality between the shaping pulses In the same way, the MSK quadrature-phase components The quadrature-phase component of each dataset is then obtained by the same orthogonality principle used above. The extracted MSK in-phase and quadrature-phase components are then parallelly to serially converted. Then, a convolutional decoder and deinterleaver are applied in pursuit of estimating the input binary data.It should be noted here that the use of low-resolution one-bit ADCs to detect the in-phase and the quadrature-phase components at the RX side of the OSP-MSK removes most of the analog stages , analog filters, etc.). Moreover, the use of the coherent demodulation along with the one-bit ADCs at the RX side of the OSP-MSK eliminates the need for the complex maximum-likelihood sequence estimation (MLSE) equalizer mandated at the RX side of the MIMO-CEM system ,23,24,25In this section, the analytical average bit error rate (ABER) performance of the proposed OSP-MSK modulation is derived. Furthermore, to evaluate the spectral efficiency of the OSP-MSK, its analytical power spectral density (PSD) is obtained in detail.The ABER performance of the OSP-MSK modulation relies mainly on the correlation receiver , as shown in Accordingly, the output of the sampler can be expressed as follows:However, in Equation (11), Hence, to correctly recover Therefore, the noise Accordingly, the probability of error  in Equation (20), the probability of error can be expressed as follows: The probability of error in Equation (24) is the probability of error of the first arm correlation receiver of the OSP-MSK demodulator. Likewise, the probability of error of the second arm correlation receiver can be acquired, and it is equal to the probability of error of the first arm. However, it should be mentioned here that the second arm correlation receiver constitutes a quadrature multiplexed version of the first arm. Therefore, the probability of correctness due to the two arms can be expressed as follows:Hence, the probability of error due to the first two arms can be expressed as follows:Likewise, the probability of error due to the other two arms Lastly, the OSP-MSK demodulator can be interpreted as two independent correlation receivers, each with a probability of error of ε, and the instantaneous signal power will be attenuated by ε is a random variable (RV) with a mean square value β = To obtain the ABER of the proposed OSP-MSK modulation over different fading channels, the previous analysis can be expanded by averaging the ABER of AWGN provided by Equation (27) over the PDF of fading channels . More spTherefore, the ABER performance of the OSP-MSK over any fading channel can be determined by integrating the channel fading effect in Equation (28) in the formula of the ABER over AWGN in Equation (27) to acquire the conditional ABER . Then, the ABER due to the presence of fading can be expressed as follows:Following the aforementioned procedure, the conditional Substituting Equation (31) into Equation (30), the ABER of the OSP-MSK over Rayleigh fading channel  and (38) into Equation (33), the ABER over the Rayleigh fading channel can be expressed as follows:Using the same procedure, the ABER in the case of the Nakagami-m fading channel can be expressed as follows: Integral Integral Substituting Equations (41) and (43) into Equation (40), the ABER over the Nakagami-m fading channel can be expressed as follows:In the following, a closed form expression for the PSD of the OSP-MSK is analytically derived to evaluate the effectiveness of the proposed OSP-MSK in boosting the spectral efficiency compared to the MIMO-CEM. As depicted in Therefore, the spectrum of tion (4) . As suchHowever, Since the in-phase and quadrature-phase components of the OSP-MSK signal are independent and the PSDs of Substituting Equation (53) into Equation (52), the baseband PSD of Using some mathematical manipulations, the baseband PSD of Firstly, the ABER performances of the proposed OSP-MSK over AWGN, Rayleigh fading channel, and Nakagami-m fading channel are evaluated analytically and via comprehensive Monte Carlo simulations. To compare meaningfully, the analytical formulas and the simulation results are assessed in both uncoded and coded scenarios. It should be mentioned here that the relation between the code symbol energy per  follows :(56)EcNoSecondly, the ABER performance of the proposed MIMO OSP-MSK modulation is evaluated and compared to the ABER performance of the MIMO-CEM system using the traditional GMSK modulation, and by using the quadrature multiplexed Gaussian minimum shift keying (QMGMSK) modulation ,33. The Finally, the PSD of the proposed OSP-MSK modulation is evaluated analytically and via numerical simulation and compared with the PSDs of the above-mentioned schemes in order to evaluate the spectral capabilities of the proposed OSP-MSK modulation.m = two and m = four) compared to the numerical simulation results for the uncoded scenario. Furthermore, The high consistency between the analytical formulas and the simulation results dates primarily to the absence of any mathematical assumptions in deriving the ABER of the OSP-MSK modulation. This, without a doubt, evinces the potency of the mathematical analysis conducted throughout this paper.In As shown in As shown in In this paper, the multiple-input multiple-output orthogonal shaping pulses minimum shift keying (MIMO-OSP-MSK) has been presented as a low-cost modulation/demodulation scheme. The OSP-MSK fulfills a low-power budget by using a nonlinear PA at the TX side and by applying a low resolution one-bit ADC at the RX side. Moreover, the OSP-MSK harnesses in a new fashion the orthogonality between the shaping pulses to boost up the spectral efficiency. By means of this orthogonality, the OSP-MSK increases the number of transmitted bits per symbol of the MSK/GMSK without additional cost for extra bandwidth. Mathematical closed-form expressions for assessing the ABER performance of the OSP-MSK modulation over the AWGN channel, Rayleigh fading channel, and Nakagami-m fading channel were derived and analyzed thoroughly. In addition, a mathematical closed-form expression for the power spectral density of the proposed OSP-MSK was also derived and studied in detail. The simulation results corroborate the effectiveness of the conducted analysis by revealing a close match to the obtained analytical formulas. Furthermore, the simulation results evinced that the proposed OSP-MSK outweighs the MIMO-CEM with the one-bit ADC, in sense of the spectral efficiency and without any deterioration in the ABER performance.


The stability behaviors and interaction mechanisms between molten Ti-47Al-2Cr-2Nb alloy and Y2O3-Al2O3 composite crucibles were investigated at high temperature. Results showed that the surface morphology of crucibles and the degree of interfacial reactions between the composite crucibles and the metal melts varied with the change of Al2O3 content in the crucible matrix. The pure Y2O3 crucible was the densest and its chemical stability was the highest. With the increase in Al2O3 content, the number of pores on the crucibles surface gradually increased and the interfacial reactions between the composite crucibles and the molten alloys became weaker. When the content of Al2O3 in composite crucibles increased from 3.5 wt % to 10.5 wt %, the thickness of the interface layer of melt-crucible decreased from 150 µm to 50 µm, and the equilibrium contact angles between metal and crucibles gradually decreased from 69.3° to 64.2° at 1873 K.In this work, Y Intermetallic TiAl-based alloys with the advantages of low density, high specific strength and elastic modulus, excellent creep properties and high resistance to oxidation, have extensive application in aerospace, automotive and energy industries ,2,3,4. TThe melting process of an alloy is one of the most important factors affecting the properties of the alloy . Many me2O3, CaO, ZrO2 and Y2O3 are the most commonly used  (AM)  was used as a monomer, N,N′-methylenebisacrylamide [(C2H3CONH)2CH2] (MBAM)  as a coupling agent, N,N,N′,N′-tetramethylethylenediamine (TEMED) as a catalyst, ammonium persulphate  as an initiator and ammonium polyacrylate  as a dispersant. The crucible’s sintering temperature is 1873 K and the holding time is 4 h.In this study, four different types of composite crucible with various components were designed and characterized. The compositions of the crucibles are listed in −3 Pa and backfilled with pure argon  up to 0.05 MPa. The superheating temperature was 1873 K, measured and controlled by a thermocouple thermometer. The alloy ingots collected in composite crucibles were superheated for 10 min. Afterwards, the molten alloy was allowed to solidify in the composite crucible down to 300 K.The master alloy, with nominal composition of Ti-47Al-2Cr-2Nb (at %), was prepared by an arc-melting method using Nb sheet (99.87%), commercial Ti sponge (99.76%), Al ingot (99.99%) and Cr granule (99.98%) as raw materials. The melting procedure was performed in a modified Bridgeman vacuum directional solidification furnace equipped with a removable composite crucible. Before each heating cycle, the chamber was evacuated down to ~102O3 and Al2O3 mixture powders were ground. Polyvinyl alcohol (PVA)  with a content of 5 wt % was added as a binder. The wetting experiment was carried out by the improved sessile drop equipment. Before heating, the furnace chamber was vacuumed to 9.9 × 10−4 Pa and further backfilled with a deeply purified Ar (99.999%) atmosphere to prevent active elements from evaporation and oxidation. When the temperature reached 1873 K, the alloy with the size of 3 × 3 × 3 mm3 in the top of the equipment was dropped. The spreading process was recorded by a CCD camera . The contact angles were obtained by the ADSA-SESDROP and FTA32 software  [In order to further clarify the mechanism of interaction between molten metal and crucible, high temperature wetting experiments between TiAl-based alloys and crucibles were conducted by using composite ceramic substrates which were made under the same conditions of making composite crucibles. The oxide ceramic substrates with cylindrical shape were prepared by the dry pressing method using a uni-axial pressure machine . Then, the YAfter the melting experiment and wetting experiment, the microstructures of the alloys were examined by an electron probe micro-analyzer , and the microstructures of crucibles were observed via scanning electron microscopy . Energy-dispersive spectrometry  was used to analyse the chemical composition and impurity elements at specified positions from the surface to the inside of the ingots to establish the homogeneity. The phase identification of the compounds on the alloy matrix and contact interface was performed by X-ray diffraction  with Cu Kα radiation by scanning on the designated area.2O3 crucible  system. The Al2O3 powder with size of 5 µm was added to the Al2O3-A, Al2O3-B and Al2O3-C crucibles, and an alternative part of the 325 mesh Y2O3 powder formed the mixed fine powder. Because the particle size of Al2O3 powder was much smaller than that of the 325 mesh Y2O3 powder, and the 325 mesh Y2O3 of 3.5 wt % in the Al2O3-A crucible was replaced by Al2O3 powder of 5 µm, and the surface grain of the small crucible formed by sintering was finer than that on the surface of the pure Y2O3 crucible. The proportion of Al2O3 powder in Al2O3-B and Al2O3-C crucibles continued to increase. The particles were in close contact with each other, and the sintering degree was good. Therefore, compared to the Al2O3-A crucible, the porosity of the Al2O3-B and Al2O3-C crucible was only slightly increased, and the surface micromorphology of the two was similar.The surface microstructure and the porosity of the crucibles showed that the addition of AlR. Zhang , Y2O3 an the XRD  detected product . Kolitscperature . Howeverreported . The rea2O3 crucible was the smallest. However, in the composite crucibles, the amount of oxide inclusions decreased with the addition of Al2O3. When the amount of Al2O3 added was 3.5 wt %, the proportion of inclusions in the metal was largest.The interfacial reactions between melt metals and ceramic crucibles are very complicated physical-chemical process. The experimental results in this study showed that the alloy melting experiments were carried out in the crucibles with different compositions under the same experimental conditions, and the amount of oxide inclusions in the metal was different after the experiment. The amount of oxide inclusions in the alloy melted with pure Y2O3 crucible without Al2O3 was more thermodynamically stable than composite crucibles with Al2O3. The Al2O3 and TiO2 detected in the interfacial layer  is stronger than that between the pure Y2O3 crucible and the melt, and weaker than that between the Al2O3-C crucible and the melt. The crucible structure was relatively loose, and the wettability between metal and interface was relatively poor. Therefore, the corrosion of metal in the crucible was considerable, and the content of oxide inclusions in molten alloys was higher than that in pure Y2O3 and Al2O3-C.From the chemical point of view, the pure Yal layer  were pre surface  showed t2O3-based composite crucibles with an Al2O3 content of 0 wt %, 3.5 wt %, 7.0 wt % and 10.5 wt %, based on which the interfacial reactions and associated mechanisms between TiAl-based alloy and composite crucibles were discussed. The primary conclusions of this work were as follows.The TiAl-based alloy was melted in Y2O3 crucible was the best. After the melting experiment, the number of inclusions in the interior of the metal particles and the reactants at the interface layer with the pure yttrium crucible were the lowest. With the increase in the amount of Al2O3, the interfacial reaction between the composite crucible and the molten alloy became weaker. When the content of Al2O3 in the composite crucible increased from 3.5 wt % to 10.5 wt %, the thickness of the interface layer of the melt-crucible decreased from 150 µm to 50 µm. The addition of Al2O3 increased the wettability of the composite crucible with the metal melt. The equilibrium contact angles between metal and crucible under 1873 K decreased from 69.3° to 64.2°.The chemical stability of the pure Y


Secondly, the aim was to establish a large animal model to enable future translational studies and further graft development.® Propaten heparinized graft or a Jotec® Flowline Bipore heparinized graft was used, both 5 mm in diameter.; End-to-side bypass of the common carotid artery was performed bilaterally in sheep. Either a GoreFollowing graft implantation, the sheep were kept on pasture for 6 months, with monthly duplex scans to determine patency. At termination, the grafts were duplex scanned a final time, with the animals sedated, and the grafts were removed for heparin activity analysis.® grafts, and 6 Jotec® remained. Mean patency time was 106.7 ± 21.9(SD) days and 96.2 ± 25.9(SD) days for Gore® and Jotec®, respectively. Log-rank test showed no significant difference at final follow-up after 6 months. Post mortem heparin analysis showed no significant difference in mean activity.; 14 sheep were operated, 11 survived total follow-up time. At final follow-up, 4 patent Gore; Based on patency data alone, no significant difference between these grafts were found. In accordance, heparin activity analysis showed no difference between the grafts. The model itself, proved easily implementable, and provides many possibilities for future studies, though some adjustments should be made to improve survival. •First direct comparison of heparinized graft.•No significant difference in patency or heparin activity concerning the two grafts.•Animal model is easily implementable, and expandable to include numerous innovations. However, recent studies show, that ePTFE grafts bonded with heparin using the CARMEDAy to ASV . Below ty to ASV .A Swedish study from 1988  showed hOngoing development have improved the general patency of prosthetic grafts, although patency is still lacking compared to that of autologous vein grafts ,7. The mAdditionally, risks such as acute hemorrhaging or heparin induced thrombocytopenia ,9, assoc®. The first commercially available graft with CBAS bonded heparin, was released in 2004, around the same time a animal study showed a reduction in NH as well as improved patency when compared to crude grafts [A Swedish company, Carmeda, achieved this and consequently patented their method, which was later purchased by Goree grafts . This wae grafts .® warrants end-point bound heparin [® graft is also marketed as coated with heparin, however Jotec® does not specify how the heparin molecules are bound, therefore it could bound at random. This may reduce heparins efficiency as an anticoagulant, because the bio-availability may be reduced when compared to the Gore® grafts. To our knowledge a study concerning this specific comparison was performed by Vermassen et al., in 2009, but so far, no results have been published [Currently two name brands promote their grafts as being bonded with heparin. Gore heparin , anchori heparin ,13,14. Tublished .This pilot study's objective was to investigate, the primary patency rates of these differently heparin bonded grafts, during a 6 months' follow-up period. Additionally, remaining heparin activity after 6 months would also be assessed, as well as the feasibility of the animal model itself.22.1This pilot study's design and experimental animal model was based on a previous study published by Pedersen et al.  using a The different grafts were alternately implanted on the right and left side by a fixed randomization key.The study protocol was approved by the Danish animal experiments inspectorate.2.2® grafts branded as Propaten® grafts were 5 mm in diameter, and removable ring inforcing. They were made from expanded polytetrafluoroethylene, and had their rings removed to better match the Jotec® grafts. Heparin is bound covalently to the graft surface, by a proprietary method allowing Gore® to guarantee end-point bonding of the heparin molecules.The Gore® grafts branded as FlowLine Bipore heparin® were also 5 mm in diameter and had no ring reinforcement. The grafts were also made from expanded polytetrafluoroethylene. Jotec® markets these grafts with innovative heparin coating, bound covalently, however makes no statement as to any specific binding of the heparin molecules.The Jotec2.3Sheep were selected, because of their relatively stable body weight throughout a long follow-up period. Moreover, they usually recover rapidly following surgery with minimal care from animal technicians needed. This allows fast transfer from the stables to stay in pastures, which reduces costs and increases animal welfare.We used female sheep (n = 14) wild type crossbreeds, but predominantly of the Texel race, which are usually more docile, thus easier to handle during duplex-scans without the need for sedation. Mean weight was 52 kg (range: 36–62 kg), mean age 4.2 years (range: 1–7 years).2.4All operations were done by the same two trained vascular surgeons, and a medical student assisting them, and during surgery an animal technician observed the vital parameters of the animal.® graft which was ring-enforced had the rings removed to make it more comparable to the Jotec® graft.The common carotid artery was exposed for this bilaterally, using standard open surgical technique, exposing approximately 5–10 cm of the artery, depending on the muscular configuration of the sheep, to avoid damaging muscles, and the vagus nerve. Both gr®, B Braun, Melsungen, Germany) leaving the grafts approx. 6 cm in length. After confirming the patency of the anastomosis and the grafts themselves, with palpable pulse, the native artery was ligated using 2–0 prolene, approx. 1 cm distally from the proximal anastomosis.Before clamping the common carotid artery, 5000 IU Heparin was administered, to prevent clotting, while suturing the anastomosis. Graft-ends were cut at a 45-degree angle and anastomosis were done in end-to-side fashion using 5–0 Prolene  were used for all analyses and illustrations.t-test.The heparin activity was expressed as quantitative results, which represented the amount of active heparin at removal at the end of follow up. The differences between the individual implanted grafts were compared by paired 33.114 Sheep were operated, one had to be put down 3 days following the operation, due to post-operative complications. Another was euthanized after 9 days due to suspected aspiration pneumonia. Lastly one sheep had a wound defect, resulting in a prosthesis infection, which had to be removed. The same animal was put down at the first monthly follow-up, possibly due to paralytic bowels syndrome. The remaining animals did not suffer any complications and stayed on pasture for the entire follow-up period.3.2® and n = 6 (54.5%) Jotec® grafts were still patent  Gorel patent . It shou3.3® grafts were 116.4 ± 21.6 (SD) days compared to Jotec® 104.9 ± 26.7(SD) days. Paired t–test analysis of the difference between the pairs of grafts showed no significant difference (p = 0.74).Mean patency for the Gore2 for each graft. Mean heparin activity in Gore® grafts was 16.6 pmol/cm2 ± 2.3 (SD), and 15.6 pmol/cm2 ± 4.3 (SD) for the Jotec®. Paired t-test showed no difference between grafts, with a mean difference of 1.00 , p = 0.554.Heparin activity was recorded as picomol/cm® grafts did not occlude as rapidly as the Jotec® grafts with 6 patent grafts of each make after three months. However, after this initial period an increasing number of Gore® graft failures were observed, and at six months’ final follow up, the initially observed difference between the grafts had diminished (p = 0.88 by log-rank test) with four remaing patent Gore® grafts and six Jotec® grafts.The Kaplan-Meier plot , shows t® grafts, had significantly better patency than the Jotec® grafts until the 3rd month, were most of the occlusions occurred in both groups (p = 0.027 by log-rank test).Interestingly, post-hoc analysis, showed that the Gore3.4Concerning ultrasonic visibility of the implanted grafts, it is notable that duplex scans were carried out with the animals un-sedated. This was done to reduce the stress on the animals, sometimes requiring operator patience. Depending on the muscular configuration of the sheep, and how the grafts themselves behaved in-vivo, visibility was remarkably good, with clear duplex signal , and in 4Heparin bonded grafts have proven to improve patency, at least transiently .Differences in heparin bonding to the grafts may influence the outcome.This pilot study demonstrates, as the first comparative study, no immediate significant difference, in primary patency between the tested grafts at 6 months’ follow-up. However a trend showing a potential short-term effect is found. Due to the high numbers and long observation time needed in clinical trials, examining different grafts patency is often done retrospectively in registry studies ,7, whichConcerning our findings regarding patency, the Kaplan-Meier plot  suggest ® grafts, with n = 2 (18.2%) failures at the time of first duplex, compared to n = 4 (36.4%) Jotec®. As previously shown the crucial point remains at 3 months [® grafts, which might protect the grafts from acute occlusion within the first three months. Additionally, if our hypothesis is right, it also explains why the Jotec® grafts occlude earlier on. However, this remains speculative as it was not planned initially and would require additional heparin analysis, but it does show an interesting trend.Nevertheless, there seems to be a trend indicating that initial patency is better in the Gore3 months , where mIn comparison with other animal studies investigating heparin bonded grafts, the occlusion rate of this series is higher than expected ,17,18. TLooking at the removed grafts reported heparin activity, no difference was found. This corresponds with our survival analysis. Retrospectively it could have been interesting to perform the heparin activity analysis both prior to implantation and after explanation.4.1, [Our intension was to challenge the bonded heparin and patency of grafts as much as possible by choosing 5 mm grafts and furthermore, creating turbulent flow by ligation of the native common carotid artery approximately 1 cm distally from the proximal anastomosis. These modifications of the model may have overly encouraged thrombogenicity, compared to previous reports , [13,17,, . This mi, ,14,19and, [Additionally, patency studies done in baboons, canines, and sheep all show better patency in grafts with CBAS immobilized heparin , [13,17,, , than whIf a larger diameter graft had been used, there might have been a clearer difference between the grafts, than in this current setup. The strain on the grafts in our series, might censor the actual difference between the grafts.It could be argued that antiplatelet therapy, which ought always to be administered to patients with known atherosclerotic disease, would improve the patency of these grafts as shown by an arteriovenous-graft model in porcine . However4.2In our series, there were some early censoring of the grafts, following the complications mentioned above. These could have been due to surgery, although all surgical procedures were performed proficient, and up to clinical standards. One graft however, did get infected most likely because of a wound defect, and not because surgical aseptic failure.Considering that three out of all 14 (21.4%) sheep did not live through the length of the study, this is of course a disadvantage to the series, but on the other hand, the required sample size was achieved.Another limitation of this series, and potentially in any series using sheep, is that their atherosclerotic status, is unknown, if there is any. This could have potential implications when using data derived from sheep studies, when treating patients.It is still too early to reach a conclusion concerning the differences in primary patency between these two types of heparinized grafts. Mainly because our model might have challenged the grafts to a clinically irrelevant level. But this could probably be avoided using a larger diameter graft in a future series. It could also be argued that the grafts could be interpositioned with end-to-end anastomosis instead of end-to-side, to reduce the strain on the grafts, when testing the antithrombotic capabilities. But this would not reproduce real-world conditions for most below knee bypasses.4.3This study, has continued the foundation of earlier animal studies, to create a large model that allows a direct comparison of grafts.This large animal model makes a direct comparison a real possibility. We are now able to compare the effects of these modern grafts in-vivo, which provides us with an extended evidence base to choose the best treatment available for patients.Regarding the patency results, more research is still required. This could be done using a graft size which is more commonly used in revascularization surgery, i.e. a 6 mm graft. This might reduce the strain on the grafts, and therefore provide a more accurate result. A future study could include a 3-month end-point, were grafts would be removed for heparin analysis, since it seems that most occlusions occur around this point in time.This study model shows promise to enable future studies to be conducted investigating the future development of artificial vascular grafts. It could even be adapted to feature an infection model, that could test grafts for their ability to fight of infections, or timed drug release.® responded and chose to provide the utilized grafts. The Jotec® grafts were bought through the vascular surgery department at Odense University Hospital. Additionally, the Carmeda® labs performed the heparin analysis of the removed graft material.We reached out to both companies, however only GoreThe study protocol was approved by the Danish animal experiments inspectorate.University Hospital Odense Grant ID 15-A915 and through Aase & Ejnar Danielsens fond Grant ID 10-001454.Funding was obtained at Mads Liisberg/Jes Lindholt – Conceptualisation, planning and funding.Mads Liisberg/Michael Stenger/Carsten Behr-Rasmussen – Surgery, analysis and follow-up.Mads Liisberg – Writing.Jane Stubbe/Jes Lindholt/Michael Stenger/Carsten Behr-Rasmussen – editing.® provided grafts for the study – we reached out to Jotec but they did not reply after several advances.GoreHeparin analyses was conducted at Carmeda labs.Not relevant.Mads Liisberg, M.D.Not commissioned, externally peer reviewed.


The success of prosthetic vascular grafts in the management of peripheral arterial disease is frequently limited by the development of anastomotic neointimal hyperplasia (ANIH), with the host response to prosthetic grafts beginning soon after implantation. To address this, we combine a platform of polyethylene terephthalate (PET) fabric with an applied cryogel layer containing biologic agents to create a bioactive prosthetic graft system, with the ability to deliver therapeutics targeting modulators of the ANIH-associated transcriptome response, along with antithrombotic agents.Hybrid graft materials were synthesized by cryopolymerization of methacrylated alginate and heparin onto electrospun (ePET), knitted PET (kPET), or woven PET (wPET). Arg-Gly-Asp (RGD) peptides were added to increase cell adhesion. Scanning electron microscopy (SEM) was used to study the microstructure at 1 day, and 2, 4, and 8 weeks. Physical properties such as swelling ratio, pore connectivity, shape recovery, and stiffness were evaluated. Human aortic endothelial cell (HAoEC) adherence was visualized using confocal microscopy after 24 hours and proliferation was evaluated with a resazurin-based assay for 7 days. Confocal microscopy was used to assess delivery of adeno-associated virus (AAV-GFP) after incubation of hybrid grafts with HAoECs. Heparin activity of the materials was measured using an anti-Xa assay.SEM demonstrated large interconnected pores throughout the entire structure for all graft types, with minimal degradation of the cryogel after 8 weeks. Hybrid materials showed a trend towards increased shape recovery, increased stiffness, decreased swelling ratio, and no difference in pore connectivity. HAoECs incorporated, adhered, and proliferated over 7 days on all materials. HAoECs were successfully transduced with AAV-GFP from the hybrid graft materials. Anti-Xa assay confirmed continued activity of heparin from all materials for over 7 days.We have developed a bioactive prosthetic graft system with a cryogel coating capable of delivering biologic agents with antithrombotic activity. By applying the cryogel and selected agents onto PET prior to graft implantation, this study sets the stage for the system to be individualized and tailored to the patient, with bioengineering and targeted gene therapy strategies dovetailing to create an improved prosthetic graft adaptable to emerging knowledge and technologies. Peripheral arterial disease (PAD) affects more than eight million Americans and, despite advances in endovascular technologies, open surgical revascularization using vein or prosthetic graft is a key component in management of these patients . AnastomDecades of research elucidating the biology and pathophysiology leading to neointimal hyperplasia and graft failure have established several fundamental factors critical in predicting the success of a prosthetic vascular graft, such as adequate runoff, technical aspects in constructing the anastomoses, contact activation, and the thrombogenic potential of the graft material. To improve the patency of prosthetic grafts, it is essential to focus both on improvement of the graft as a conduit, and also on creating a bioactive material capable of modulating the acute inflammatory and wound healing response that begins following any vascular intervention . Our preOur goal is to create a bioactive prosthetic graft system (BPGS) incorporating a clinically used prosthetic graft material made of polyethylene terephthalate (PET) with a clinically approved delivery vehicle such as alginate cryogel to deliver biologics combined with an antithrombotic agent such as heparin. This cryogel-coated prosthetic graft material can be premade before complete assembly of the BPGS, allowing for customized selection of agents for gene delivery near the time of graft implantation.Hybrid graft materials were constructed by coating electrospun PET , knitted PET , or woven PET  with an alginate cryogel to form hybrid ePET, hybrid kPET, and hybrid wPET grafts, respectively.Cryogel components methacrylated alginate , methacrylated heparin (MA-heparin), and Arg-Gly-Asp (RGD) containing peptide (ACRL-PEG-RGD), to enhance cell adhesion through integrin binding, were synthesized as previously described . MethacrMethacrylated alginate was synthesized with sodium alginate  dissolved 1 g in 0.6% w/v 100 mM 2-morpholinoethanesulfonic acid  buffer (pH 6.5), and 2.8 g 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride  was added to activate carboxylic acid groups, followed by 1.3 g N-hydroxysuccinimide  and 2.24 g aminoethyl methacrylate . This was precipitated in excess acetone after 24 h.Methacrylated heparin was synthesized with heparin sodium  dissolved 1 g to 50 mL in a 2% w/v 50 mM MES and 0.5 M sodium chloride buffer (pH 6.5). Activation of carboxylic acid groups was achieved by addition of 1.62 g EDC followed by 0.5 g NHS and 1 g N-(3-Aminopropyl) methacrylamide hydrochloride . After 24 h, this was precipitated in excess acetone/ethanol in a 1:1 ratio.1H NMR, with MA-alginate macromonomer found to have approximately a degree of methacrylation of 50% using this method  dissolved in anhydrous N,N-dimethylformamide  containing 4 M excess of triethylamine , and ACRL-PEG-NHS  dissolved in anhydrous DMF and mixed with 1.1 M excess peptide for 3 h before precipitation twice in cold anhydrous ether and vacuum drying overnight. The peptide coupling reaction was monitored with nuclear magnetic resonance spectroscopy.MA-alginate , MA-heparin , and ACRL-PEG-RGD  were dissolved in 470 µl deionized water . CryopolHybrid graft samples were incubated in phosphate-buffered saline (PBS) or in human aortic endothelial cell media  at 37 °C on a shaker, and collected at 1 day, and 2, 4, and 8 weeks. Samples were immersed in increasing concentrations of ethanol, followed by hexamethyldisilazane , vacuum dried, and sputter-coated with gold. Ultra55 Field Emission Scanning Electron Microscope  was used to obtain overview and cross-sectional SEM images to assess the microstructure of the hybrid grafts. Images at each time point represent separate samples due to technique used for SEM imaging. Average cryogel pore size was calculated by averaging the diameters of the pores observed by SEM.To assess the impact of combining prosthetic material to the cryogel, properties of the cryogel-coating, such as swelling ratio, pore connectivity, and shape recovery, were measured as previously described, and compared to cryogel alone . Hybrid The swelling ratio was defined as the fully hydrated gel weight divided by dried weight. Pore connectivity, which allows for migration and proliferation of cells within an increased area of the polymer, was calculated by subtracting the dehydrated weight from the hydrated weight, then dividing by the hydrated weight . Shape rTo quantify the stiffness of the hybrid grafts, samples were hydrated for one day prior to mechanical testing, then compressed at a rate of 1 mm/min on an Instron 3342 mechanical tester  equipped with a 50 N load cell. Young’s modulus was calculated from the linear region of the resulting stress–strain curve between 0–10% strain, and all measurements were carried out in triplicate for each type of material.Hybrid grafts were labeled with fluorescein isothiocyanate isomer I  as described before . SteriliSterilized hybrid ePET, hybrid kPET, and hybrid wPET graft materials were incubated with 40,000 HAoECs for 4 h. Resazurin-based assay  was used to evaluate cell proliferation and repeated at 3, 5, and 7 days, with the ratio normalized to the respective initial absorbance value. This data was compared to control absorbance values obtained for cells only samples and empty resazurin only samples, and measured at 570 nm and 600 nm (spectrophotometer), completed in triplicate samples. Data are presented as mean ± SEM.Sterilized hybrid grafts were incubated with adeno-associated virus tagged with green fluorescent protein AAV-GFP  at multiplicity of infection (MOI) of 1000 or 10,000. Successful transduction of AAV-GFP was demonstrated by cellular expression of GFP, resulting in a green fluorescent signal. Hybrid graft materials were incubated with AAV-GFP for 3 h, and 40,000 HAoECs added and incubated for 7, 14, and 21 days prior to fixation, permeation, and staining with DAPI and Alexa Fluor 647 for confocal microscopy. Data are presented as representative images.Heparin activity of the hybrid graft materials was evaluated by incubating samples in PBS and HAoEC media, which was replaced daily. Heparin concentration was calculated by measuring absorbance values using a chromogenic anti-Xa assay  per manufacturer’s instructions. The activity of heparin was measured using a chromogenic peptide substrate, a chromophore (405 nm), which is inversely proportional to the initial amount of heparin in the tested sample.First, 25 µL of sample is added to 200 µL of antithrombin-III (AT-III) provided in the kit, and incubated at 37 °C for 2 min, allowing an AT-III/heparin complex to form. Following this, 200 µL of Factor Xa is added and incubated at 37 °C for 1 min, resulting in Factor Xa/AT-III/heparin as well as residual Factor Xa in solution. Finally, 200 µL of Xa Substrate is added and incubated at 37 °C for 5 min, which reacts with the residual Factor Xa in solution to form the chromophore, and after 5 min 200 µL of acetic acid is added to end the measurement.Absorbance at 405 nm is measured using a spectrophotometer and standard curve for heparin concentrations constructed by plotting the mean absorbance at a wavelength of 405 nm value for each heparin standard versus its corresponding concentration and drawing the best-fit calibration curve. Data are presented as mean ± SEM of the cumulative total of units of heparin released by hybrid graft materials over time.t-test was performed. Differences were considered to be statistically significant at p-values less than 0.05.Data from testing physical properties and heparin activity from the materials were obtained in triplicate, with statistical analysis performed using Prism software . For comparison of data across multiple groups over time, 2-way ANOVA was used. For direct comparison of data for 2 groups, Scanning electron microscopy demonstrated the interconnected porous structure of the cryogel coating for all hybrid graft types, with porous network visible in the cryogel material after 8 weeks in PBS and in cell culture media . AverageCompared to the cryogel material alone, all hybrid graft materials demonstrated statistically significant increase in shape recovery, statistically significant decrease in swelling ratio, and no statistically significant difference in stiffness (Young’s modulus) or pore connectivity .Cell incorporation into all the hybrid materials was seen on confocal microscopy after incubation for 24 h, confirming that HAoECs were able to adhere and infiltrate on the cryogel coating  and 4.Resazurin-based cell viability assay for all samples demonstrated that HAoECs were able to adhere and proliferate on cryogel, hybrid ePET, hybrid kPET, and hybrid wPET over 7 days , albeit At day 7, cellular expression of GFP from transduction of AAV-GFP into HAoECs was seen in the hybrid ePET graft at MOI of 1000, and on the cryogel, hybrid kPET, and hybrid wPET at MOI of 10,000. The green fluorescence signal continued to days 14 and 21 for the cryogel, hybrid kPET and hybrid wPET, suggesting continued expression of GFP by HAoECs, and continued proliferation of cells on the hybrid grafts at 21 days .Anti-Xa assay confirmed that heparin-containing hybrid grafts demonstrated continuous release of heparin for over 7 days . A two-wWe have created a bioactive prosthetic graft system composed of clinically used prosthetic graft material, PET, and clinically approved alginate in a cryogel polymer delivery vehicle that can successfully deliver viral vectors to human aortic endothelial cells. These findings suggest that our BPGS can be used as a delivery vehicle for modulators of the injury response at the time of graft implantation to reduce the development of intimal hyperplasia (IH).in vitro for up to 21 days as demonstrated with the transduction with AAV-GFP. Although limited, these results suggest that the hybrid grafts have the capacity to adsorb and release viral vectors that are capable of transducing the cells. Further studies are required to examine the kinetics of release over longer periods of time.We demonstrated that the interconnected porous microstructure of the cryogel is maintained when combined with PET material, and remains intact with minimal degradation after 8 weeks. Our BPGS is non-toxic and biocompatible with HAoECs, showing cell adherence and proliferation With autologous saphenous vein unavailable in up to 30% of patients due to previous surgeries or size mismatch, creating an improved prosthetic graft material is crucial in the continued management of patients with PAD . A numbeOur experiments demonstrated that, compared to the cryogel material alone, all hybrid materials demonstrated statistically significant increase in shape recovery with addition of the prosthetic graft material and statistically significant decrease in the swelling ratio, with no difference in stiffness or in pore connectivity. Shape recovery represents the ability of the material to regain shape after dehydration and hydration. All types of PET combined with the polymer gel coating had increased shape recovery, potentially due to the PET material providing a structured backbone for the polymer gel, which acts like a coating on the fabric. We found that the swelling ratio was significantly decreased in all hybrid materials compared to cryogel alone. The swelling ratio is calculated as the fully hydrated weight divided by the dried weight. Since the cryogel is used as a coating on the PET surface, potentially it would swell less when combined with the fabric backbone. All the hybrid materials would also likely have less total difference in weight at fully hydrated weight and the dried weight, compared to the cryogel polymer alone. With the addition of the PET in creating the hybrid material, this could lead to a decrease in the calculated swelling ratio, since a proportion of the weights would be due to the presence of PET. Hybrid wPET and kPET were noted to have a greater swelling ratio decrease but this was not statistically significant, and potentially due to variations in measurement and differences in weight between similar sizes of the graft materials, as increased weight of prosthetic graft leads to decrease in swelling ratio.Our data did not demonstrate a difference in pore connectivity between materials, which would suggest that the cryogel polymer, when combined with PET, is able to maintain a porous network. During the cryogelation process, ice crystals form within the polymer structure and leave pores upon thawing of the cryogel, and the measurement of pore connectivity is calculated by subtracting the dehydrated weight from the hydrated weight and dividing it by the hydrated weight. This technique assumes that the volume of fluid removed equals the volume of the interconnected pores. Although the weight of the PET material affects the calculation of the pore connectivity, this alone would be expected to result in decreased pore connectivity compared to cryogel polymer. However, our data demonstrated no change in pore connectivity, which could be due to the PET material promoting increased removal of fluid from the cryogel when combined together during the water wicking technique.Besides these conventionally used grafts, new materials made with 3D printing technology and stem cells are also currently under investigation . HoweverTo create practical and effective drug delivery from a prosthetic graft material, we have created a modular platform of PET with an applied cryogel polymer containing biologic agents such as adeno-associated virus. Our BPGS is assembled in separate components, and thus we have the unique capability to customize the selection of biologic agent. This eliminates the need to obtain a pre-made specialized graft loaded with the biologic of choice for each target. In our BPGS, biologics can be added directly onto the graft system just prior to use.in vivo experiments are also needed to determine the effect of high-pressure forces of the arterial system on these grafts.Cryogels are networks of polymers with high water content that undergo a freezing cycle during free radical polymerization, leading to the formation of ice crystals throughout the gel, which become pores once the cryogel is thawed. The interconnected porous structure acts like a scaffold similar to the extracellular matrix (ECM) and facilitates the uptake of molecules, with enhanced cell attachment due to the addition of the amino acid sequence arginine-glycine-aspartic acid (RGD) into the polymer solution . A variein vivo studies using our BPGS in a rabbit carotid interposition bypass model to assess the antithrombotic properties in vivo and determine whether the release of heparin will affect rates of thrombosis.The cryogel layer of our BPGS demonstrated continuous heparin activity over 7 days through potential surface interactions, which could be further controlled in the future by modifying the dissolution and breakdown of the polymer. In our data, the measured hybrid kPET heparin activity in serum was greater than the other materials, however this was not statistically significant. It is possible that a difference in the surface structure of knitted versus woven or electrospun PET may contribute to increased heparin assay activity, or that variations in measurement, assay activity, or polymer layer could contribute, but the difference in hybrid kPET was not statistically significant compared to the cryogel polymer or the other hybrid materials. The proportion of heparin added to the cryogel may also require optimization to maximize its antithrombotic activity. However, at this time it is difficult to predict whether this will have a significant clinical effect. We plan to conduct With the majority of gene signaling events occurring soon after graft implantation, the ideal length of time for drug delivery remains to be determined but may range from days to weeks . Our preWe have developed a BPGS with an applied cryogel polymer coating that allows for cell adherence and growth, with antithrombotic activity and the ability to deliver therapeutics. This system allows application of the gel and biologic agents to PET prior to implantation. Hybrid materials using cryogel-coated ePET, kPET, and wPET were able to demonstrate cell adhesion and successfully deliver viral vectors to human aortic endothelial cells. We did not find a statistically significant difference between types of PET used in our preliminary experiments. Further studies are needed in order to define and assess which, if any, of the prosthetic graft materials may provide a more advantageous fabric backbone for our hybrid cryogel-coated material. The present study sets the stage for future investigation to test personalization of combined bioengineering and targeted gene therapy approaches to create an improved prosthetic graft adaptable to evolving treatment strategies.10.7717/peerj.7377/supp-1Supplemental Information 1Included is the raw data for measurements of porosity of the cryogel and all hybrid materials.Click here for additional data file.10.7717/peerj.7377/supp-2Supplemental Information 2This includes the raw data for the human aortic endothelial cell viability assay, including data points for each group over time.Click here for additional data file.10.7717/peerj.7377/supp-3Supplemental Information 3Raw data for all materials tested for release of heparin in vitro, over time.Click here for additional data file.10.7717/peerj.7377/supp-4Supplemental Information 4Combined raw data points for measurements of stiffness for cryogel and hybrid materials.Click here for additional data file.10.7717/peerj.7377/supp-5Supplemental Information 5Instron measurements and calculations of Young’s modulus for cryogel material.Click here for additional data file.10.7717/peerj.7377/supp-6Supplemental Information 6Instron measurements and calculations of Young’s modulus for cryogel material.Click here for additional data file.10.7717/peerj.7377/supp-7Supplemental Information 7Instron measurements and calculations of Young’s modulus for cryogel material.Click here for additional data file.10.7717/peerj.7377/supp-8Supplemental Information 8Instron measurements and calculations of Young’s modulus for hybrid electrospun PET.Click here for additional data file.10.7717/peerj.7377/supp-9Supplemental Information 9Instron measurements and calculations of Young’s modulus for hybrid electrospun PET.Click here for additional data file.10.7717/peerj.7377/supp-10Supplemental Information 10Instron measurements and calculations of Young’s modulus for hybrid electrospun PET.Click here for additional data file.10.7717/peerj.7377/supp-11Supplemental Information 11Instron measurements and calculations of Young’s modulus for hybrid knitted PET.Click here for additional data file.10.7717/peerj.7377/supp-12Supplemental Information 12Instron measurements and calculations of Young’s modulus for hybrid knitted PET.Click here for additional data file.10.7717/peerj.7377/supp-13Supplemental Information 13Instron measurements and calculations of Young’s modulus for hybrid knitted PET.Click here for additional data file.10.7717/peerj.7377/supp-14Supplemental Information 14Instron measurements and calculations of Young’s modulus for hybrid woven PET.Click here for additional data file.10.7717/peerj.7377/supp-15Supplemental Information 15Instron measurements and calculations of Young’s modulus for hybrid woven PET.Click here for additional data file.10.7717/peerj.7377/supp-16Supplemental Information 16Instron measurements and calculations of Young’s modulus for hybrid woven PET.Click here for additional data file.


Correction to: International Journal of Obesity42, 1823–1833;10.1038/s41366-018-0230-y;published online 09 October 2018This paper was originally published under a standard licence. This has now been amended to a CC BY licence in the PDF and HTML.


Rhinovirus A, predicting four epitopes recognized by antibodies present in 87% of sera (n = 250). An analysis of 2,908 proteins from 400 viral taxa that infect humans predicted seven enterovirus epitopes and five Epstein-Barr virus epitopes recognized by >30% of specimens. Analysis of Staphylococcus and Streptococcus proteomes similarly predicted 22 epitopes recognized by >30% of specimens. Twelve of these common viral and bacterial epitopes agreed with previously mapped epitopes with p-values < 0.05. Additionally, we predicted 30 HSV2-specific epitopes that were 100% specific against HSV1 in novel and previously reported antigens. Experimentally validating these candidate epitopes could help identify diagnostic biomarkers, vaccine components, and therapeutic targets. The K-TOPE approach thus provides a powerful new tool to elucidate the organisms, antigens, and epitopes targeted by human antibody repertoires.Antibodies are essential to functional immunity, yet the epitopes targeted by antibody repertoires remain largely uncharacterized. To aid in characterization, we developed a generalizable strategy to predict antibody-binding epitopes within individual proteins and entire proteomes. Specifically, we selected antibody-binding peptides for 273 distinct sera out of a random library and identified the peptides using next-generation sequencing. To predict antibody-binding epitopes and the antigens from which these epitopes were derived, we tiled the sequences of candidate antigens into short overlapping subsequences of length k (k-mers). We used the enrichment over background of these k-mers in the antibody-binding peptide dataset to predict antibody-binding epitopes. As a positive control, we used this approach, termed K-mer Tiling of Protein Epitopes (K-TOPE), to predict epitopes targeted by monoclonal and polyclonal antibodies of well-characterized specificity, accurately recovering their known epitopes. K-TOPE characterized a commonly targeted antigen from  Immunological memory allows for rapid antibody responses towards diverse antigens long after initial exposure. For example, the adaptive immune response to many vaccinations is often sustained throughout an individual’s lifetime . This imA disease with prominent antibody responses is the common viral infection HSV, which causes human infections in the orofacial region (“cold sores”) and the genital region  . In 2012a priori. Furthermore, these methods require large specimen volumes (>10 mL) to obtain a sufficient quantity of cells —conformation-specific (ab201059) (Abcam), and rabbit V8137 Anti-V5 polyclonal antibody (Sigma). Antibodies were spiked into healthy donor serum at a concentration of 25 nM. All sera (n = 273) were obtained as deidentified specimens from biobanks according to institutional guidelines  AND identity:0.9” and yielded 2,908 proteins. The Staphylococcus proteome search used a Uniref search of “uniprot:(taxonomy:"Staphylococcus [1279]" fragment:no reviewed:yes) AND identity:0.9” and yielded 3,071 proteins. The Streptococcus proteome search used a Uniref search of “uniprot:(taxonomy:"Streptococcus [1301]" fragment:no reviewed:yes) AND identity:0.9” and yielded 2,976 proteins. HSV analysis used a UniProt search of “reviewed:yes AND organism:"Human herpesvirus 1 (strain 17) (HHV-1) (Human herpes simplex virus 1) [10299]" AND proteome:up000009294” for HSV1, yielding 73 proteins and a Uniprot search of “reviewed:yes AND organism:"Human herpesvirus 2 (strain HG52) (HHV-2) (Human herpes simplex virus 2) [10315]" AND proteome:up000001874” for HSV2, yielding 72 proteins.Protein sequences were obtained from UniProt or by using the Biopython module . AccessiStaphylococcus aureus using protein database searches [Poliovirus 1, the epitope PALTAVETGATNPL was found to be a cross-reactive epitope in many enteroviruses [For EBNA1, it was noted that RRPFF antibodies were found in the serum of healthy individuals . KRPSCIGsearches . For Poloviruses . The remhttps://github.com/mlpaull/KTOPE) and all 278 antibody-binding peptide files are available from the Dryad Digital Repository (https://doi.org/10.5061/dryad.v7d0350). The imune-processor.jar file is available for research, non-profit, and non-commercial use, but requires a license for commercial use. All other software is available under the MIT license. The algorithms for generating nonredundant sequence lists from FASTQ files, outputting enrichment values for subsequences, and exhaustively calculating k-mer statistics were adapted from IMUNE  [® Core™ i7-4790 CPU @ 3.60 GHz, 64-bit operating system, and 32.0 GB of RAM. Processing FASTQ files into subsequences from 12 specimens, each containing approximately 1.5 million unique sequences, required 2.3 hours and calculating k-mer enrichment required 7.7 minutes. The duration of these calculations scales approximately linearly with the number of specimens and sequences.All software files are posted on GitHub (rns.jar) . We adde2 2 + log2 4 + log2 8 = 6. The frequency counters are compiled into a histogram which is smoothed using a moving window. For this analysis, to provide adequate smoothing, the window had width 7 and used linear weighting with 1 in the center and 0.1 at the edges. Minima and maxima are identified in the smoothed histogram. All intervals between 2 minima that contain a maximum are used to define epitopes. Epitopes were limited to a minimum length of 6 and a maximum length of 15 to roughly approximate the size of actual linear epitopes [The K-TOPE algorithm  is writtepitopes . EpitopeRhinovirus A genome polyprotein (P07210) for 250 specimens required 24.4 seconds. The proteome searches for viruses with human tropism, Staphylococcus, and Streptococcus for 250 specimens required 3.1, 2.3, and 1.9 hours, respectively.After determining epitopes for individual specimens, K-TOPE can determine consensus epitopes for a population. Each epitope is characterized by a “centroid” which is the weighted central position of the epitope, indexed as a position in the protein sequence. Centroids for all epitopes that meet the epitope percentile threshold are compiled. They are then clustered using k-means to associate close centroids with the KMeans function from scitkit-learn . A repreWe calculated expected membership of epitope groups by multiplying the proportions of the population that bound each epitope. For example, if epitope 1 was bound by 32% of the population and epitope 2 was bound by 67%, then the expected membership of epitope group ‘1+2’ would be 21%. We ranked the overlaps between K-TOPE derived epitopes and literature epitopes in a list of 10,000 randomly generated epitope overlaps to determine a p-value. To remove redundant epitopes found in the proteome searches, we used the PAM30 similarity matrix to align two epitopes and compare each position to calculate a similarity score. Epitopes that had similarity scores >10, were in the same protein, and were from different organisms were considered redundant. We removed the less prevalent of the two redundant epitopes.The HSV analysis used “disease” group specimens to predict epitopes and “control” group specimens to subtract epitopes. Epitopes were predicted in the disease group that met the epitope percentile threshold (95%) and the minimum prevalence (30%). Then, all disease epitopes were evaluated in the control group. For an epitope to be considered disease-specific, its score had to be below the epitope percentile threshold (80%) in all control specimens. To predict HSV2-specific epitopes that were also in the HSV1 proteome, we identified epitopes that exactly matched a subsequence in an HSV1 protein.S1 FigHistograms were generated for antibodies against cMyc (P01106), V5 (P11207), and amyloid beta (P05067). The most prominent peaks were present regardless of whether antibodies were added to serum or buffer. This suggests that the binding signature of a single antibody was not obscured by the many other antibody specificities present in serum.(TIF)Click here for additional data file.S2 FigRhinovirus A genome polyprotein (P07210) with the prevalence fixed at 30%. The base 10 logarithm of the number of epitopes appeared to decrease linearly with increasing epitope percentile threshold. The value 95% was chosen for analysis because it corresponds to a p-value of 0.05 and ensures that the total number of epitopes predicted was of order one. By predicting a total number of epitopes of order one, fewer false positives should to be included in this analysis.Epitopes were generated for 250 specimens using the (TIF)Click here for additional data file.S3 FigRhinovirus A genome polyprotein (P07210) with the epitope percentile threshold fixed at 95%. The base 10 logarithm of the number of epitopes appeared to decrease exponentially with increasing prevalence. There were 123 epitopes bound by at least one member of the group. The value 30% was chosen arbitrarily from the prevalence values that predicted a total number of epitopes of order one. By predicting a total number of epitopes of order one, fewer false positives should to be included in this analysis.Epitopes were generated for 250 specimens using the (TIF)Click here for additional data file.S4 FigEnterovirus revealed that the epitopes found for the Rhinovirus A strain analyzed in Enterovirus strains, as demonstrated by the bands at approximately positions 212–221, 569–578, 577–590, and 602–613 . Epitopes 1, 2, and 4 were only found in Rhinovirus, whereas epitope 3 was found in many Enterovirus strains. The heat map was restricted to positions 0–700 to show relevant epitopes. A binary decision was made for each position in each protein to determine whether it was in an epitope.Analyzing multiple strains of (TIF)Click here for additional data file.S1 Tableonly bound the epitopes in the groups e.g. specimens in group ‘1’ did not bind ‘2’ or ‘3’. Most of the actual and expected membership values agreed except for the ‘1+2+3’, ‘3+4’, ‘1+2+4’, ‘1+2+3+4’, and the ‘None’ groups which had higher membership than expected and the ‘1+3’ group which had lower membership than expected. Additionally, the group targeting only epitope 4 was 40% smaller than expected suggesting that it was generally bound along with other epitopes. All groups that had percent differences equal to or greater than 50% are in bold.The expected membership of epitope groups was calculated by multiplying the proportions of the population that bound each epitope. For example, if epitope 1 was bound by 32% of the population and epitope 2 was bound by 67%, then the expected membership of epitope group ‘1+2’ would be 21%. Note that specimens in groups (DOCX)Click here for additional data file.S2 TableRhinovirus A epitopes. The average age is given with the standard deviation.The average age for the 138 specimens for which there was age data was 35. The ‘None’ group had an average age of 52 which was approximately 50% higher than the average age of 35 (in bold). Additionally, specimens targeting 3 or more epitopes had an average age of 17 (in bold), which was approximately 50% lower than the average age of 35. This discrepancy suggests that older people targeted fewer (DOCX)Click here for additional data file.S3 TableEnterovirus revealed that the epitopes found for the Rhinovirus A strain analyzed in Rhinovirus-specific, but not Enterovirus-specific. Similarity comparisons used the PAM30 similarity matrix with similarity defined as a similarity score > 10.Analyzing multiple strains of (DOCX)Click here for additional data file.S4 TableTwelve of the epitopes (in bold) were similar to epitopes found in prior studies with p-values of < 0.05. However, 30 of the epitopes were in proteins with no reported epitopes, and 3 epitopes were in organisms with no reported epitopes. Only 6 of the epitopes were in well-characterized proteins but were not found in the literature, suggesting that these epitopes were false positives or novel epitopes. Additionally, only two bacterial epitopes were in previously described proteins, suggesting that the remainder of the bacterial proteins were false positives or novel antigens. Epitopes 2, 3, and 4 differ slightly from those in (DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 TableNotably, epitope 1 (FVLPHWYM) contains the 3-mer LPH which is also in the first epitope of the HSV1 specific epitopes, RIRLPHI . Additio(DOCX)Click here for additional data file.S1 Code(TXT)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.


To the Editor:We thank Massart et al for their commentsCCDC67) and rs7976329 (gene annotation PTPRO), were well imputed in our study (INFO>0.95) and neither reached genome‐wide significance in our reported analyses.The 2 recipient genetic loci highlighted in their paper,Table The lack of replication signal in our study, despite greater numbers of cases, may be due to a number of factors. We agree with Massart et al that one reason may be the differences in phenotype definition. Our study was primarily designed and powered to detect genetic variation in donor and recipient genomes associated with long‐term graft survival, as this is the key unmet medical need in clinical renal transplantation outcomes, with currently no effective therapeutic options. Our acute rejection phenotype was established from reported national registry‐based outcomes and was not specific to acute T cell–mediated rejection, and thus signal attenuation may be responsible for the difference. However, we note that in our study the recipient minor allele frequency differences between cases and controls are less than 1%, indicating almost complete attenuation. Alternatively, it is possible that the signals found by Ghisdal et alWe agree that genetic variation outside the HLA region is an important consideration in seeking to understand the pathogenesis of long‐term graft survival and potentially identifying novel therapeutic targets to reduce cumulative allograft loss over time. We look forward to working with already established international collaborationsAmerican Journal of Transplantation.The authors of this manuscript have no conflicts of interest to disclose as described by the


Treatment of chronic Rockwood’s type V Acromioclavicular (AC) joint dislocation remains controversial. We describe a surgical technique to reduce and maintain AC joint using a combination of gracilis autograft with GraftMax™ button . Graft was prepared using running whip stitch technique with No. 5 Hi-Fi high strength suture . Our technique reduces intraoperative clavicular and coracoid tunnel fracture and restores anatomical coracoclavicular ligament. At sixth week and third month postoperatively, the patient demonstrated good clinical and radiographic outcome. TM button.To date there is no consensus on gold standard treatment for chronic Rockwood’s type V acromioclavicular (AC) joint dislocation. We report an open surgical technique, early clinical and radiographical outcomes of AC joint reconstruction using autologous gracilis tendon grafts and GraftmaxA 63-year old motorcyclist presented to our center one day after a motor vehicle accident. He complained of left shoulder pain and inability to abduct his left shoulder. His left shoulder contour was asymmetrical with left acromioclavicular joint tenderness. No neurological deficit was noted. Plain radiograph revealed left acromioclavicular joint dislocation, Rockwood’s type V .Following the diagnosis, left acromioclavicular joint reconstruction using autologous gracilis graft was planned to reduce the acromioclavicular joint and improve his shoulder function.At week five, on the next available operating date, the procedure was performed under general anaesthesia. Ipsilateral gracilis tendon was harvested and prepared by running whip stitch technique using No.5 Hi-Fi high strength sutures  . Left acThe base of the coracoid process was drilled using Schanz pin size 4mm in superior to inferior direction, followed by another two clavicular tunnels, around 1 cm apart, drilled 2.5cm lateral to the acromioclavicular joint . The graSatisfactory clinical and radiological outcome were achieved at six weeks and three months postoperative . Functio1.High grade acromioclavicular joint dislocation (Grade IV and above) requires surgical intervention to prevent painful shoulder joint movement. The available surgical options include acromioclavicular joint fixation (hook plates), coracoclavicular fixation , and ligament reconstruction 1. Meanwhile, tightrope system  and mersilene tape  serve as a non-rigid fixation that secure superoinferior translation but not anteroposterior translation. AC joint reconstruction was historically used to treat chronic AC joint instability. The earlier technique such as Weaver and Dunn procedure exploits coracoacromial ligament for AC joint reconstructions but demonstrated only 25% of biomechanical strength as compared to intact coracoclavicular ligament, which resulted in up to 30% of failure rate2.Bosworth screw and hook plate fixation provide rigid AC joint fixation but are often related to hardware failure requiring hardware removal3. However, Cook et al reported up to 80 % loss of reduction in patients treated with GraftRope at the mean of seven weeks4. In addition, several studies have also shown significant increased risk of clavicular fracture in clavicular tunnels of 5mm or greater in diameter5. Interference screw used to secure graft end may increase risk of tunnel fracture. Our population has smaller clavicle size and is at greater risk of clavicular fracture. Therefore, we developed a modified reconstruction technique to prevent clavicular tunnel fracture. Both clavicular and coracoid tunnel size 4mm were prepared as compared to previous 5mm size. We introduced the concept of hybrid graft by preparing the graft using running whip stitch technique as compared to previous technique that only sutured both tail end of graft. Looping our graft through coracoid tunnels will reduce the motion of graft bone interface and thus tunnel widening as reported in previous case series4.Anatomical coracoclavicular joint reconstruction using GraftRope  has shown equivalent biomechanical strength as the native intact ligamentThe limitations of our surgical technique are graft length estimation, large surgical wound, and short-term follow-up. The graft is elastic and tends to crumple during passage through the tunnel. We successfully passed the graft through only after multiple attempts. The large surgical wound in our case gave us better access to the anatomy . With better understanding of anatomy and the use of image intensifier, this procedure can be performed by mini-open or arthroscopy assisted. Long- term follow-up is also needed to look at the clinical and radiological outcome.In conclusion, positive outcome is achieved using the modified technique of AC joint reconstruction in treating Rockwood’s type V chronic AC joint dislocation. Patient outcome may also improve if the procedure is conducted using mini open or arthroscopy assisted technique.


A 35-year-old left-handed man who works as a mechanic and firefighter presented to our institution with hand pain and cold intolerance for 3 months. He was referred by a local vascular surgeon who had made the diagnosis of hypothenar hammer syndrome (HHS) following an angiogram of the right upper extremity showing complete occlusion of the ulnar artery . On initIntraoperatively, Doppler ultrasound revealed a segmental thrombotic defect extending from the superficial palmar arch, across the Guyon canal, extending 10 cm proximally in the forearm. The ulnar artery was resected back to healthy bleeding pulsatile flow, resulting in a segmental defect of 12 cm. Final pathology of the submitted ulnar artery segment revealed organizing thromboembolus in the lumen, which at the time of resection was adherent to the lumen.The DLCFA was harvested, with care taken to prevent injury to the lateral femoral cutaneous nerve. A 12-cm segment of the artery was circumferentially dissected, as well as approximately 2 cm of 2 branching vessels at the distal end of the segment that would be used to reconstruct the superficial palmar arch and common digital arteries with one graft. With the arterial graft completely dissected, it was ligated both distally and proximally.The arterial graft and recipient vessels were examined under the operative microscope. The proximal anastomosis was preformed first. The two branches at the distal end of the arterial graft were anastomosed to the superficial palmar arch stump, and common digital artery to the ring and small finger, respectively. A bolus of 5000 units of heparin was given intravenously before release of the microvascular clamps. Adequate pulsatile flow was achieved though the graft and confirmed with Doppler ultrasound to each finger , Video 1The patient remained in the hospital overnight for monitoring and was discharged the following morning on oral aspirin as the only anticoagulant. On follow-up examination 2 days later, strong Doppler signals were elicited at the ulnar artery, palmar arch, and radial and ulnar aspect of each digit. At 1 month, Doppler signals remained strong and the ulcer previously present on the right long fingertip had completely resolved , Video 2What is hypothenar hammer syndrome?Who gets hypothenar hammer syndrome?How is hypothenar hammer syndrome typically treated?What are the benefits of the lateral circumflex femoral artery as an interposition graft?Hypothenar hammer syndrome is a condition characterized by ulnar arterial insufficiency secondary to thrombosis or aneurysm.,The overall incidence of HHS in the general population is unknown, although large prospective cohort studies have estimated it to be close to 1.6%.2,2,5,Operative versus nonoperative management in HHS is often based on the acuity and severity of symptoms, as no established guidelines currently exist in the literature. Studies suggest that asymptomatic HHS may be treated conservatively with oral antiplatelet agents and vasodilators.2Reconstruction of the ulnar artery in HHS may involve direct end-to-end arterial repair or an interpositional graft. Typically, a local venous graft harvested from the flexor aspect of the ipsilateral forearm has been the preferred option. In the HHS population—which typically consists of active young to middle aged males—durability and long-term patency is a primary concern.Hypothenar hammer syndrome is a rare cause of ulnar arterial insufficiency that, if left untreated, may lead to severe digit ischemia and potential amputation. While end-to-end anastomosis and interposition venous grafts have been the mainstay of surgical management, arterial grafts utilizing the DLCFA may be a viable reconstructive option.


The relationships between the vertical stiffness (in the countermovement and take-off phases) and the stiffness estimated using the Myotest allow us to conclude that despite the significantly overestimated stiffness value, the Myotest accelerometer can still be used for determination of the stiffness trends, e.g., following training. The overestimated stiffness values can result both from inaccuracy in the determination of ground contact time and flight time by the Myotest accelerometer and from the use of an equation that assumes that the movement of the center of mass has a harmonic profile.Currently, there are several computational methods for stiffness during a hopping task, but they do not necessarily yield the same values. Therefore, it is essential that the simplicity of the equipment used does not affect the measurement validity. The aim of this study is to compare the stiffness values during a hopping task recorded in a laboratory environment and those acquired using the Myotest accelerometer. The measurements were performed on a group of 30 untrained female students . According to the manual for the Myotest accelerometric system, each study participant performed three sets of 5 hops. Vertical stiffness was determined based on two measurement methods, one using the Myotest accelerometer and the other using Kistler force plates. The mean value (±SD) of vertical stiffness was 19.0 ± 9.3 kN/m in the countermovement phase and 15.1 ± 5.9 kN/m in the take-off phase. Furthermore, the stiffness determined using the Myotest was 30.7 ± 13.3 kN/m. However, significant relationships between the vertical stiffness in the countermovement phase and the Myotest stiffness ( Evaluation and monitoring of biomechanical variables have become an important element in the quantitative analysis of athletic performance. Sports coaches will obtain valuable information from measurements carried out under conditions as close as possible to those during competitions. Therefore, they are often skeptical of analyses performed under isolated laboratory conditions. However, recent technological innovations related to the miniaturization of wearable sensors that do not influence the technical movements of athletes allow movement analysis to be performed during sporting activities. An example of a tool that allows the measurement of acceleration during motion and under training conditions is the Myotest performance measuring system .muscular rigidity, which is usually called stiffness, is an interesting indicator enabling you to find the ideal muscular tension for bouncing in running events or team sports, for instance.” However, the problem of the “stiffness” estimated by the Myotest seems more complex than this definition.The Myotest accelerometric system is a wireless handheld device weighing just a few ounces (59 g) and is attached to a specially designed belt at the pelvic level. This 3-D accelerometer allows the estimation of variables such as jump height, time of contact, reactivity, and stiffness during a hopping task. Time of contact refers to time when the feet (at least one) are in contact with the ground between the flight phases. Reactivity should be understood as the reactive strength index (RSI), i.e., as the ratio of jump height to contact time . We can bouncing gait” has been used to describe the human body during hopping tasks where lower limbs perform the function of “springs” responsible for center of mass (COM) movements [leg spring” and a point that represents the total body mass [spring length” representing both lower limbs) and vertical stiffness  are commonly used to describe the mechanical properties of a “spring” representing the lower limbs during a hopping task [Stiffness is a quantitative measure of the elastic properties of the body and is expressed as a ratio of the deforming force to the deformation length  . Therefoovements , 5, 7. Tody mass . Leg stiing task .spring length.” Moreover, estimations of stiffness using an accelerometer also do not provide insights about the ratios of stiffness in individual joints. Therefore, it should be assumed that the stiffness value estimated by the Myotest is vertical stiffness. The Myotest accelerometric system is recognized as a reliable and valid tool for the estimation (despite significant overestimation) of jump height based on the flight time method [The Myotest guide does not give an unambiguous answer as to which of the above types of stiffness  is the value provided by the Myotest accelerometric system during the hopping test. Some authors equate the stiffness value estimated by the Myotest with leg stiffness –12. Howee method –17. Howee method , 10 haveIn the process of sports training control, it is necessary to quantify the effects of the exercises and loads applied. Therefore, the use of a portable measuring device is a compromise between measurements under laboratory conditions and those under training conditions. However, there are currently several computational methods for  stiffness, but they do not necessarily yield the same values , 18–20. The measurements were performed on a group of 30 untrained female students from the University School of Physical Education. They were persons with no competitive-level sports training (within a period of at least 5 years before the experiment) and with no injuries to the musculoskeletal (motion) system. The study group was characterized by the following mean parameters (±SD): body height: 1.72 ± 0.07 m, body mass: 64.8 ± 10.0 kg, and age: 23.0 ± 1.7 years. The tests were carried out in the Biomechanical Analysis Laboratory (with PN-EN ISO 9001: 2009 certification). Each subject completed all trials in the same time period of test days (in the morning) to eliminate any influence of circadian variation. Subjects refrained from physical activity for 24 hours before testing, to avoid any interference in the experiment. Prior to the measurements, the participants were familiarized with the purpose of the study and gave written consent for participation in the experiment. Before the test, the subjects were informed of the activities they were supposed to perform and were motivated to properly perform the task. The research project was approved by the Senate's Research Bioethics Commission, and the procedure complied with the Declaration of Helsinki regarding human experimentation. We followed the methods of Struzik and Pietraszewski .Each study participant performed three sets of 5 hops (hopping test). The measurement procedure was conducted in accordance with the Myotest performance measuring system: quick start guide (jump–plyometry test). The trials were simultaneously recorded by the Myotest accelerometric system  and by two force plates . The sampling frequencies of the signal from the force plates and the accelerometric system were set at 500 Hz. This sampling frequency is the maximum common value for both systems. The use of force plates is usually considered the gold standard , 15.at the short beep from accelerometer, perform a countermovement jump, then bounce back up five times as high as possible and with a ground contact time that is as short as possible, while keeping your hands on your waist .” After 5 hops, the participant reassumed a vertical standing posture, and the double beep from the accelerometer signals the end of the test. During the experiment, the participant was asked to rest her palms on her hips to exclude the effect of arm swing on hopping performance. Landings were performed on the same plates as take-offs. According to the Myotest guide, a one-minute rest took place between test repetitions. Errors in hopping task execution are signaled by a deep beep from the accelerometer. The Myotest accelerometric system tolerates two errors before automatically stopping the test. An error message is generated if the following points are not observed (according to Myotest guide): “(1) execute the movements energetically so that the Myotest can clearly detect them, (2) stand still before the starting beep, (3) ground contact time must be short and clearly below the time of flight, and (4) perform a total of 5 bounces.” During performance of the hopping test, the participant should take-off with the knees and ankles extended and land in a similarly extended position. The test was repeated if the lower limbs were flexed at the knee and/or hip joints during the flight phase (incorrectly performed hopping task).Prior to the measurements, a 10-minute-long warm-up, which included jogging , a series of hops, and a familiarization test task, was administered. Each study participant started performing a trial series after becoming familiar with the test. After the trial series, the proper research procedure began. Next, the participant was asked to perform a series of 5 bilateral hops (3 sets) from the standing position to the maximum height (performed as a bounce action on the fore foot) and with minimal time of contact with the ground. The whole part of the hopping test took place on a rigid surface (force plates). As indicated by the guide, the participant wore a belt with the Myotest accelerometer attached vertically on the left side of the body at the pelvic level . Before each trial, the subjects were asked to stand over the force plates (each foot on a separate plate) while assuming a vertical posture with arms akimbo, looking straight ahead and standing still . The hopF) recorded by the force plates (the ground reaction forces registered by both force plates were added up), it was possible to determine the flight time (tf) and ground contact time (tc) during the hopping task. The instantaneous pattern of changes in the height of the COM (y) was calculated by double integration of the COM vertical acceleration, as calculated from the vertical ground reaction force [Kv = ΔF/Δy) of the human body during the hopping task was determined as the ratio of the change in the ground reaction force (ΔF) to the corresponding change in the height of the COM (Δy) separately for the countermovement and take-off phases, similar to the method described by Struzik and Zawadzki [F(Δy) shown in F(Δy) equals the numerical value of stiffness in this range. Vertical stiffness was calculated for the parts of the countermovement and take-off phases where the slope of the F curve with respect to the Δy axis was relatively constant and the F(Δy) profile was nearly linear. For the countermovement phase (marked green in ymax). The boundaries of the part for the take-off phase (marked blue in Fmax from which ground reaction forces decreased only) and the moment of take-off from the plates [R2 that expresses the quality of adjustment of the trend line to the relevant part of the F(Δy) curve is sufficiently high (over 0.6) [ymax and Fmax occur at exactly the same time, then the whole F(Δy) curve is analyzed. If not, then the part of the F(Δy) curve between the Δymax and Fmax points (marked in black in F(Δy) curve intersects [Fmax point to appear before the Δymax point. Then, the profile of the F(Δy) curve should be considered individually, and the boundary of the analyzed parts of the countermovement and take-off phases should be modified. For example, the analyzed countermovement phase part will end at the point Fmax, and the analyzed part of the take-off phase will begin at the point Δymax.Further analysis focused on the attempt with the highest mean height of hops obtained by each participant. From the hopping task, 5 hops were analyzed without taking into account the starting countermovement jump. The values of all presented variables were averaged for the five analyzed hops to obtain results analogous to those obtained from the Myotest. The hopping test was used with some simplifications that resulted from the use of the spring-mass model, which characterizes both running and hopping. The model assumes that the human body consists of a material point representing the total mass of the body; a massless “spring” representing both lower limbs, which performs the supporting function; and a parallel source of force resulting from the active action of the muscles involved in the take-off . Based oon force . The verZawadzki . To relie plates . This obver 0.6) . If the tersects , for exatersects , which chMyo), ground contact time (tc‐Myo), and stiffness (KMyo). In the Myotest guide, the manufacturer did not explain how the values of individual variables are estimated. However, based on the accelerometer capabilities, one can guess that the values of jumping height (hMyo) and ground contact time (tc‐Myo) were determined based on the duration of the flight and ground contact phases [hMyo) recorded by the Myotest accelerometer, the flight time (tf‐Myo) could be determined using the following formula:g is the acceleration due to gravity [Kv‐Myo) can be evaluated using the equation described by Dalleau et al. [Kv‐Myo is the vertical stiffness, m is the body mass, tf‐Myo is the flight time, and tc‐Myo is the ground contact time. Therefore, it may be accepted that the stiffness value estimated by the Myotest is the vertical (quasi-) stiffness.The Myotest accelerometer was used to record the following variables during the hopping test: jump height  is close or equal 1. The Shapiro-Wilk (W) and Lilliefors tests were used to examine the distribution of individual variables. All the studied variables had a distribution close to normal. Therefore, parametric tests were used for further analyses. Pearson's r correlation coefficient was used to evaluate the concurrent validity of the Myotest accelerometric system and force plate. The significance of the correlation coefficient value was verified with the t-test. To demonstrate possible differences between the values of the variables obtained from different measuring devices, Student's t-test of significance of differences for dependent variables was used. In all tests performed, the level of significance was set at α = 0.05. Statistical calculations were made by means of the Statistica 13.3 software package . Furthermore, the remaining calculations were made using a Microsoft Excel 2016 spreadsheet . Additionally, concurrent validity was analyzed through a Hopkins [r correlation coefficient. The criterion was the dependent variable, and the practical was the predictor in a consecutive pairwise manner. The typical error of the estimate was standardized (SEE) by dividing by the SD of the criterion. SEE was evaluated using half the thresholds of the modified Cohen scale: <0.1, trivial; 0.1–0.3, small; 0.3–0.6, moderate; 0.6-1.0, large; 1.0-2.0, very large; and >2.0, extremely large. Uncertainty in the estimates was expressed as 90% confidence limits. To complement the correlation analysis, Bland-Altman plots were used to visualize the mean of the difference (bias) and the limits of agreement .The sample size was determined based on the power analysis. For  Hopkins  spreadshr = 0.79, SEE = 0.77, r = 0.89, SEE = 0.50, p < 0.001) was also found.The mean value (±SD) of vertical stiffness was 19.0 ± 9.3 kN/m in the countermovement phase and 15.1 ± 5.9 kN/m in the take-off phase during the hopping test. Furthermore, the stiffness determined using the Myotest accelerometric system was 30.7 ± 13.3 kN/m. Therefore, the stiffness values determined using the Myotest were significantly higher than the stiffness values determined using the force plate in both the countermovement (Δ = 11.7 ± 8.2 kN/m) and take-off phases (Δ = 15.6 ± 8.5 kN/m). However, significant relationships between the vertical stiffness in the countermovement phase and the Myotest stiffness (r = −0.51) and take-off phases (r = −0.90) also increases.Bland-Altman plots are presented in Figures tc and tf) and the equation described by Dalleau et al. [KD = 15.6 ± 5.7 kN/m. KD was significantly lower than the vertical stiffness in the countermovement phase  and the Myotest stiffness (Δ = 15.1 ± 8.4 kN/m) and was at a similar level as the vertical stiffness in the take-off phase.Moreover, the value of stiffness determined using the force plate time measurements , modern measurement equipment provides more accurate information about a particular ability or variable. The development of technology also allows a more objective and precise evaluation. It is also becoming easier to collect more data than was previously possible using conventional methods and tools. Therefore, it is fundamental that coaches should utilize available methods for applying scientific output in sports training. Utilizing these methods is likely to provide them with feedback on the current skill level of an athlete and the efficiency of the particular practice stimuli used and will help them plan future training programs. Modern measurement tools also offer possibilities for the detection of irregularities in an athlete's body that might lead to injuries.Reliability can be defined as the consistency of measurements (test-retest) . Choukoutc and tf values, it can be concluded that the hopping frequency chosen by the participants in this study was low.The stiffness determined using the Myotest accelerometric system during the hopping test was significantly higher than the vertical stiffness determined using the force plate measurements in the countermovement and take-off phases. Therefore, the Myotest overestimated measurements of stiffness, as in other studies , 10. Chotc and tf) and the equation described by Dalleau et al. [On the other hand, Hobara et al.  reportedu et al.  were alsu et al. . In thisu et al. , 13. Chou et al.  stated tu et al. . The Myou et al. , 13, 24.y values are scattered widely above and below the regression line  curve as accurately as possible. The increase in the ground reaction force with respect to the COM displacement should be linear or close to linear over the whole duration of the contact with the ground phase. If the moment of occurrence of Fmax divided tc into two halves (harmonic movement), it would theoretically mean the same values of vertical stiffness during the countermovement and take-off phases. Meeting the above conditions would justify using one value as vertical stiffness for a specific movement while neglecting the calculations for the take-off phase [Fmax and Δymax do not necessarily occur at the same time. It is assumed that for a hopping frequency lower than 2 Hz, lower limbs stop behaving as linear springs, thereby distorting the F(Δl) profile [F(Δy) relationship, especially during transient states [Determination of the vertical stiffness during the hopping task requires several assumptions that sometimes seem to have been omitted, whereas measurement validity would require verification of these assumptions. The simplest case is when ff phase . Ferris ff phase  emphasiz profile , 37. In  profile  estimateDespite the clearly established procedures for hopping test performance and Myotest accelerometer fixation, between-subject differences in hopping technique  and the stiffness estimated using the Myotest accelerometric system allow us to conclude that, despite the significantly overestimated value of stiffness, the Myotest accelerometer can be used for determination of the stiffness trend. Therefore, this measurement device offers only an approximate stiffness value that can provide information about changes, e.g., following training. Therefore, the Myotest stiffness is not interchangeable with respect to the values obtained from other measurement devices and methods because of systematic overestimation. The overestimated stiffness value can result both from inaccuracy in the determination of ground contact time and flight time by the Myotest accelerometer and from the use of an equation that assumes that the movement of the center of mass has a harmonic profile.