The suggested safe zone demonstrates that inserting the mini-plate with plate-to-lateral mass inferior pole distances of 4–5 mm and 5–6 mm at the C3–C5 and C6–C7 levels, respectively, would avoid instrument-related complications. The risk of plate impingement was higher at the proximal level, whereas the risk of screw facet violation was higher at the distal level in open-door cervical laminoplasty. These risks coincide with anatomical differences at each level. Despite inappropriate positioning of the mini-plate, clinical outcomes were not adversely affected.This study aimed to describe a safe zone for mini-plate positioning that can avoid instrument-related complications in laminoplasty. Fifty-one patients who underwent laminoplasty and were followed up for at least 1 year were retrospectively reviewed. The posterior surface length and inferior pole angle of the lateral mass were measured at each level using computed tomography. The safe zone was defined based on these measurements. Incidences of screw facet violation and plate impingement were recorded. Patient-reported outcome measures were compared between the appropriate position (AP) and inappropriate position (IP) groups. Among 40 patients included, 15 (37.5%) had inappropriate plate positioning, causing screw facet violation or plate impingement, which more commonly occurred at distal  and proximal  levels, respectively. Lateral mass posterior surface length was shorter at the proximal levels, and the inferior pole angle of the lateral mass was smaller at the distal levels, signifying that the lateral mass became thin and long at the distal levels. Patient-reported outcome measures were not significantly different between the two groups. However, cervical range of motion at the final follow-up was significantly less in the IP group ( Various instruments have been used to maintain the opening of the lamina, including bone block, stay suture, wire, and mini-plate3. Mini-plate fixation has been reported to reduce the risk of lamina reclosure while enhancing bone healing at the trough side5. Fixation of the mini-plate is generally a safe procedure, and critical complications related to its instrumentation are rare since it is fixed with short screws6.Open-door laminoplasty is a safe and widely applied surgical technique for the treatment of cervical myelopathy9. Considering the anatomy of the lateral mass, mini-plates located too caudally would result in screw facet violation. However, when the plate is located excessively cranially, the inserted plate would impinge on the lateral mass at the proximal level. Previous studies have demonstrated that screw facet violation is not rare in mini-plate fixation for laminoplasty, where incidence ranges from 34.1 to 37.4%9. Screw facet joint violation or plate impingement caused by the mini-plate raises concern for possible facet degeneration and postoperative neck pain9. Aggravation of neck pain and kyphosis after laminoplasty are common, possibly caused by instrument positioning12. It has also been reported that facet joint violation by mini-screws can decrease cervical range of motion (ROM)9.Although mini-plate fixation with screws is free from adverse complications such as vertebral artery injury or nerve root irritation, which could occur with lateral mass screw or pedicle screw fixation, instrument-related complications can still occur14. Therefore, this study was conducted to (1) describe the incidence of screw facet joint violation and plate impingement in open-door laminoplasty using mini-plate fixation, (2) define a safe zone to avoid instrument-related complications, and (3) identify whether inappropriately positioned mini-plates would adversely affect clinical outcomes.While previous studies have described optimal insertion points and trajectories for lateral mass screws or pedicle screws, not many have demonstrated an optimal method for mini-plate fixation for laminoplastyThis was a retrospective cohort study approved by the institutional review board of our institute . All methods were carried out in accordance with relevant guidelines and regulations. Informed consent was waived owing to the study’s retrospective nature. The study was conducted in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement for cohort studies.The medical records of 51 patients who underwent laminoplasty for cervical myelopathy caused by spondylosis or ossification of the posterior longitudinal ligament between September 2012 and March 2019 were retrospectively reviewed. Patients (1) who underwent surgery due to trauma, infection, or tumor; (2) who lacked radiographic or clinical data; and (3) who had a follow-up period of less than 1 year were excluded.Patients with screw facet joint violation or possible plate impingement with cranial lateral mass observed on postoperative computed tomography (CT) were classified into the inappropriate position patient group (IP group). Patients with no identifiable mini-plate-related complications were defined as the appropriate position patient group (AP group).15.Patients were placed in a prone position with their heads located on the Mayfield headrest. A midline posterior approach was used to expose the spinous process and lamina of the indicated levels. Dissection was performed until the lamina-lateral mass junction was exposed. The spinous processes were resected at the base. Open-side and hinge-side troughs were made at the lamina-lateral mass junction. After the lamina was carefully opened to avoid complete fracture of the hinge side, a mini-plate  was used to maintain the lamina opening. We attempted to position the mini-plate at the center of the posterior surface of the lateral mass. Two 5-mm screws were used to fix the plate at the lamina, and two 5-mm screws were inserted at the lateral mass to anchor the plate. Screws into the lateral mass were inserted perpendicular to the posterior surface of the lateral mass. For C7, partial laminectomy rather than open-door laminoplasty was performed to preserve muscle insertion in the spinous process16.The neck pain visual analog scale (VAS), arm pain VAS, and neck disability index (NDI) were recorded preoperatively and at each postoperative follow-up. Three-dimensional CT scans were taken preoperatively for surgical planning and at 2 days postoperatively to evaluate adequate decompression and instrument positionPossible plate impingement was diagnosed when the cranial edge of the mini-plate reached the caudal edge of the lateral mass of adjacent proximal level Fig. A. Screw The posterior surface length of the lateral mass was measured as the distance between the cranial and caudal edges of the lateral mass on sagittal CT images. A sagittal image showing a pedicle-lateral mass junction was selected because the mini-plate is usually fixed to the lateral mass at this location. Within the same sagittal image, the inferior pole angle of the lateral mass was measured as the angle between the line drawn through the posterior surface of the lateral mass and the line drawn through the ventral-inferior border of the lateral mass that composed the facet joint. Measurements were performed bilaterally, and the mean value was used for evaluation Fig. .Figure 2Cervical lordosis was measured in the lateral view in the neutral position based on the angle between the lines passing through the lower margin of C2 and C6 or C7. The sagittal vertical axis (SVA) of C2-C7 was defined as the horizontal distance between the vertical line from the center of C2 and the posterior-superior aspect of C7. Cervical ROM was measured as the change in the angle between the lower margin of C2 and the lower margin of C7 on dynamic (flexion and extension) radiographs.A safe zone that can avoid both screw facet violation and plate impingement at the proximal adjacent level was defined. To define the safe zone, we first measured the size of the mini-plate  used for all patients included in the study. The distances between the edge of the plate and the center of the screw fixation hole, between the center of each screw fixation hole, and between the cranial and caudal edges of the plate were 2 mm, 4 mm, and 8 mm, respectively Fig. A.Figure Second, the minimum distance from the inferior pole of the lateral mass needed to avoid screw facet violation was calculated. The minimum distance for screw placement (x) was calculated using the inferior pole angle (a) and the length of the screw using the following equation: [x = length of screw ÷ tangent (a)] where tangent (a) would be the same as [length of screw ÷ x] Fig. .Finally, a safe zone that could avoid both screw facet violation and plate impingement was defined as the distance between the caudal edge of the mini-plate and the inferior pole of the lateral mass. We assumed that the screw was inserted perpendicular to the posterior surface of the lateral mass. The minimum distance of the safe zone would be [distance needed to avoid screw facet violation (x) – 2] where the plate caudal edge-to-plate caudal screw hole center distance is 2 mm. The maximum distance of the safe zone would be [lateral mass posterior surface length – 8] since the plate cranial edge-to-plate caudal edge distance is 8 mm Fig. B.p < 0.05.Student’s t-test was performed to compare the patient-reported outcome measure results between the IP and AP groups. Paired t-test was performed to compare preoperative and postoperative values. The Mann–Whitney U test and Wilcoxon signed rank test were used to analyze cervical sagittal alignment and ROM, as these parameters did not demonstrate a normal distribution according to the Shapiro–Wilk test. All data management and statistical analyses were performed using SPSS version 21.0 software . Statistical significance was set at Forty patients with 120 levels met the inclusion criteria and were included in the study. Screw facet violation was observed in 8 patients (20.0%) and in 8 levels (6.7%). Furthermore, plate impingement was detected in 9 patients (22.5%) and in 11 levels (9.2%). In total, 15 patients (37.5%) had inappropriate plate positioning and were classified as the IP group . The remaining 25 patients (62.5%) were classified into the AP group . One patient (4.0%) in the AP group demonstrated adjacent segment degeneration during follow-up, which required anterior operation revision. Baseline patient characteristics were not significantly different between the AP and IP groups The lateral mass posterior surface length was longer at the distal levels. Furthermore, the inferior pole angle of the lateral mass tended to decrease at these levels. Because of the smaller inferior pole angle at the distal levels, the calculated minimum distance from the inferior pole to insert screws by [x = length of screw ÷ tangent (a)] was higher at the distal levels Table  Fig. 4)4).Table p < 0.01), while it did not change significantly in the AP group (p = 0.91). Cervical ROM at the final follow-up was significantly smaller in the IP group than in the AP group (p < 0.01)  Table .Table 4CAmong the 40 patients included, 25 (62.5%) underwent a 1-year follow-up CT, which enabled evaluation of hinge site union status. Among the 76 segments evaluated, three (3.9%) demonstrated hinge site non-union. One segment was associated with screw facet joint violation, while the other two segments were not associated with screw violation or plate impingement.P < 0.01). No significant difference in patient-reported outcome measures between the AP and IP groups was observed at each follow-up period  and maximum distances (to avoid plate impingement) from the inferior pole of the lateral mass.Two previous studies have suggested safe mini-screw insertion points for laminoplastyIn this study, screw facet violation was more common at the distal levels, including C5 and C6, whereas plate impingement was more common at the proximal levels, such as C3 and C4. The results of radiological measurements demonstrate that this trend is consistent with the anatomical differences between each level. The possibility of plate impingement would be higher at the proximal level because the posterior surface length is shorter at these levels. However, a smaller inferior pole angle at the distal level signifies a thin lateral mass at these levels, which increases the possibility of screw facet joint violation. Considering such anatomical differences at each level, locating the mini-plate more caudally at the proximal levels and more cranially at the distal levels would help avoid instrument-related complications.3. It is known that the lateral mass is generally thin at C7, which makes pedicle screw a more preferred choice than lateral mass screw21. The results of the present study also demonstrated that inserting a 7-mm screw for mini-plate fixation in C6–C7 would be unsafe owing to the thin lateral mass at these levels as demonstrated by the small inferior pole angle. This finding supports performing partial laminectomy rather than laminoplasty at C7 due to a higher chance of instrument-related complications at this level22.The safe zone was described based on the distance between the inferior pole of the lateral mass and the caudal edge of the mini-plate. Although previous reports have used the screw insertion area as the reference point, we used the caudal edge of the mini-plate because it is easier to identify intraoperatively. The suggested safe zone demonstrates that inserting the mini-plate with a plate-to-lateral mass inferior pole distance of 4–5 mm for the C3–C5 levels and 5–6 mm for the C6–C7 levels would avoid instrument-related complications. Min et al. also demonstrated that more distance from the inferior border of the lateral mass is needed at distal levels to avoid screw facet joint violation7. Although the suggested safe zone in the present study could be used as a reference while placing the mini-plate, such distortion of anatomic landmarks would make it difficult to identify appropriate insertion points. Therefore, individual assessment and preoperative planning with radiographic measurements used in this study would further enhance the safety of laminoplasty.Laminoplasty is often performed in patients with cervical spondylosis, which distorts the anatomical landmarks due to bony spurs and spondylolisthesis. Lee et al. demonstrated that screw facet joint violation is more common in severely degenerative cervical spine than in mildly degenerative spine5. Park et al. recommended using two screws with relatively longer screw length for lateral masses in order to increase resistance to output force17. Therefore, fixation with a longer screw at the optimal area is needed, rather than a decrease in the screw length.Although shorter screws can prevent facet joint violation, weak fixation strength caused by decreased screw length can lead to screw pull-out. While this phenomenon was not observed in the present study, previous reports have demonstrated that screw pull-out can occur even with 5-mm screw fixation9. However, both studies included a small number of patients with instrument-related complications, which warrants further evaluation.The results of the present study demonstrate that clinical results, such as neck pasin VAS or NDI, were not adversely by affected screw facet joint violation or plate impingement. However, cervical ROM was adversely affected by inappropriate positioning of the instrument. This corresponds to the findings of Chen et al., which suggested that screw facet joint violation is related to decreased ROM8. Second, as previously discussed, the study has limited capacity to demonstrate the clinical impact of inappropriate instrument positioning due to the small sample size. Finally, the study is not free from the possibility of selection bias because it was a retrospective, single-center study.This study had several limitations. First, the lateral mass posterior surface is not a plane surface, but rather has a round curvature, and the measuring method of the present study would have limitations reflecting such curved surfaces. However, within the confinement of using two-dimensional images, the round curvature of the lateral mass cannot be completely measured. Furthermore, the minimal safety distance to avoid screw facet violation demonstrated in this study corresponds to that reported in other previous reportsIn conclusion, the risk of plate impingement was higher at the proximal level, whereas the risk of screw facet violation was higher at the distal level in open-door cervical laminoplasty. These risks coincide with the anatomical differences at each level. The demonstrated safe zone can be used as a reference for plate positioning. Despite inappropriate positioning of the mini-plate, the clinical outcomes were not adversely affected.


The interference screw is the most popular device that fixes the graft for anterior cruciate ligament reconstruction, reducing the incidence of windshield effect and bungee effect. For the screw, either metallic, “bioresorbable,” or polyetheretherketone (PEEK) material is available. PEEK is popular and extensively used due to its stability, biocompatibility, radiolucency, and elastic modulus. Rare relevant complications were reported, but here, we report two cases of extra-articular migrations of PEEK interference screw from the tibial tunnel after anterior cruciate reconstruction.An 18-year-old boy and a 56-year-old woman underwent anterior cruciate ligament reconstruction using a PEEK interference screw to fix the graft in the tibial tunnel. They suffered from screw extrusion from the tibial tunnel after 40 days and six months, respectively, with an incision rupture or palpable subcutaneous mass. They underwent a second operation and recovered well.The exact incidence of extra-articular migrations of PEEK interference screws is unknown, but it seems to be quite low; despite this and its uncertain cause, the negative effects caused by the PEEK material need to be considered. A secure fixation of the graft in the femoral and tibial tunnels is the prerequisite of a successful anterior cruciate ligament (ACL) reconstruction. Interference screws can be used either for soft-tissue grafts or bone–patellar tendon–bone grafts to reduce the incidence of windshield effect and bungee effect, and it has been shown to be a highly successful and reproducible technique with excellent long-term outcomes . RegardiHere, we present two cases of extra-articular migration of PEEK interference screws from the tibial tunnel, with or without wound rupture, at 40 days and 12 months after ACL reconstruction, respectively. To our knowledge, this is the first case report of this complication of commonly used PEEK screws.9/L. The knee joint fluid evaluation revealed no signs of infection, with a nucleated cell count of 900 × 106/L. The joint fluid smear of bacterium and fungus was negative. The MRI scan revealed the intact screw extrusion from the tibial tunnel to subcutaneous tissue  at the femoral tunnel. An 8 mm × 25 mm PEEK interference screw  was used to fix the graft in the 7-mm tibial tunnel. The patient recovered well and was discharged two days post-operation. His wound healed well after two weeks, so the sutures were removed in an outpatient department, and knee flexion was encouraged. Forty days after the operation, the patient came back and complained of tibial wound rupture for one day after a 1-week history of swelling and pain over the anterior aspect of the proximal tibia. Knee examination showed a 1-cm rupture in the tibial wound without obvious redness and effusion, but with focal soft-tissue swelling and moderate tenderness around the wound. The knee joint also had no obvious swelling. Laboratory evaluation revealed no systemic signs of infection, with an ESR of 35 mm/h, CRP of 2.9 mg/L, and WBC of 6.9 × 10Surgery was performed to remove the screw. When the wound rupture was incised and enlarged, the screw was found in the subcutaneous tissue surrounded by a small amount of clear fluid. The whole screw was removed, and its surrounding soft tissue was fetched for germiculture. After the debridement of the adjacent soft-tissue and tibial tunnel, sufficient irrigation was performed, and a vacuum sealing drainage dressing (VSD) covered the wound with a continuous negative pressure suction to promote healing.VSD was removed after three days, and wound dressing was performed every three days. The cultures of preoperative joint fluid and intraoperative soft tissue were both negative. Therefore, he was discharged from the hospital with a smoothly healed wound two weeks post-reoperation. After two years, he recovered uneventfully, with a knee joint range of motion of 120° of flexion to 0° of extension. The Lachman test was positive, but the pivot-shift test negative. He was able to return to simple sport activities, such as jogging, without continued symptoms. The MRI scan showed a continuously regenerating ACL  at the femoral tunnel. A 9 mm × 25 mm PEEK interference screw  was used to fix the graft in the 9-mm tibial tunnel. The patient recovered well and was discharged two days post-operation. Her wound healed well, and the sutures were removed in an outpatient department. Knee flexion was encouraged two weeks post-operation. The patient regained the normal range of knee motion, but she complained of discomfort in the pretibial wound six months post-operation. Physical examination revealed a bulge under the wound scar, with a firm and immobile palpable mass. The MRI scan showed a part of screw extrusion from the tibial tunnel to the subcutaneous tissue Fig. .Fig. 3THowever, she refused to undergo a second operation due to economic pressure and less obvious symptoms. After six months, the patient complained of increased subcutaneous bulge with tenderness Fig. . The X-rInterference screw migration is uncommon. Several cases of metallic interference screw migration have been reported in literatures, with femoral screws migrating into the joint cavity. All patients underwent ACL reconstruction with bone–patellar tendon–bone graft and received the reoperation after pulling out the femoral interference screw –18. The To our knowledge, this is the first case report of PEEK interference screw migration after ACL reconstruction. Because a non-routine MRI scan was performed after the operation, a postoperative radiology of the interference screw with normal position was absent. However, we confirmed that the screw migrated after operation, because the well-trained surgeons (Dr Zhang and Dr Li) recruited the interference screws of proper size and made sure they had been inserted into the tibial tunnel completely with palpation during operation. However, unlike the metallic screw, which was pulled out of the femoral tunnel, the PEEK screw was extruded out of the tibial tunnel in these two cases. This is more similar with the cases of “bioresorbable” interference screw, which manifests as a palpable mass with or without wound dehiscence. The reason cannot be excluded as screw divergence, bone resorption, and tunnel enlargement resulting from windshield effect and bungee effect. The biological reactions of PEEK cannot also be neglected, with several cases recently reported in literatures. A rare case of allergic reaction seven days following PEEK cranioplasty was reported, with light yellow and transparent epidural effusion at the bilateral PEEK implant site. This reaction was successfully treated with dexamethasone and drainage . One casThe exact incidence of extra-articular migrations of PEEK interference screws is unknown, but it seems to be quite low; despite this and its uncertain cause, the negative effects caused by the PEEK material need to be considered.


N-oxide (NO) formation, the major circulating metabolite, by CYP2C19, CYP2C9 and CYP3A4, was determined in incubations of human recombinant CYP enzymes and liver and intestine microsomes. The contribution of the individual enzymes to NO formation was 63.1% CYP2C19, 13.4% CYP2C9 and 29.5% CYP3A4 as determined by specific CYP inhibition in microsomes and intersystem extrapolation factors. The type of inhibition and inhibitory potential of VRC, NO and hydroxyvoriconazole (OH–VRC), emerging to be formed independently of CYP enzymes, were evaluated by their effects on CYP marker reactions. Time-independent inhibition by VRC, NO and OH–VRC was observed on all three enzymes with NO being the weakest and VRC and OH–VRC being comparably strong inhibitors of CYP2C9 and CYP3A4. CYP2C19 was significantly inhibited by VRC only. Overall, the quantitative in vitro evaluations of the metabolism contributed to the elucidation of the pharmacokinetics of VRC and provided a basis for physiologically-based pharmacokinetic modeling and thus VRC treatment optimization.The small-molecule drug voriconazole (VRC) shows a complex and not yet fully understood metabolism. Consequently, its in vivo pharmacokinetics are challenging to predict, leading to therapy failures or adverse events. Thus, a quantitative in vitro characterization of the metabolism and inhibition properties of VRC for human CYP enzymes was aimed for. The Michaelis–Menten kinetics of voriconazole  Scedosporium sp. And Fusarium sp., as well as prophylaxis in patients with haematopoietic stem-cell transplantation ) and inhibitor ([I]) concentration to determine KM, Vmax and Ki of the reaction. The obtained data were used to fit the three main models of reversible inhibition, i.e., competitive (Equation (10)), noncompetitive (Equation (11)) and uncompetitive (Equation (12)) using the “nls” function in R. The type of inhibition was determined according to the best model fit assessed with Akaike’s information criteria (AIC) [The type of reversible inhibition as well as the inhibitory constant (Kia (AIC) . A modelN-oxidation in all enzymatic systems were characterized by Michaelis-Menten kinetics . HIM also relevantly metabolized VRC to NO. The reaction kinetics were characterized by a CLint of 1.52 µL/min·mg (1.27–1.86 µL/min·mg) and were thus 5.76-fold lower than CLint observed in HLM.In HLM, this resulted in a CLint of 1.25 µL/min·pmol (0.857–2.05 µL/min·pmol). Kinetics for rhCYP2C9 were determined to have a CLint of 0.00173 µL/min·pmol (0.00138–0.00224 µL/min·pmol), which was 723-fold lower than that in rhCYP2C19. Lastly, in rhCYP3A4, Michaelis–Menten kinetics of VRC N-oxidation were described by a CLint of 0.00742 µL/min·pmol (0.00523–0.0115 µL/min·pmol), which was 168-fold lower and 4.29-fold higher than the CLint observed in rhCYP2C19 and rhCYP2C9, respectively.In recombinant enzymes, rhCYP2C19 showed the highest VRC turnover indicated by a CLDifferent methods can be applied to determine the contribution of CYP enzymes to the overall formation of a certain metabolite. The two approaches implemented for VRC comprised: (i) the individual inhibition of CYP enzymes in HLM and (ii) the extrapolation from recombinant enzymes using ISEF.N-oxidation were evaluated using four VRC concentrations ≤ KM (2.98 µM). CYP2C19 had the largest share in VRC N-oxidation with 62%, followed by CYP3A4 with 48% and CYP2C9 with 36%  The effect of the inhibitors loratadine (CYP2C19), ketoconazole (CYP3A4) and sulfaphenazole (CYP2C9) on the reaction velocities of VRC with 36% . Evidentwith 36% . In briewith 36% . OverallN-oxidation. To compare their clearance to the overall observed CLint in HLM, the respective CYP abundance in HLM was considered, as well as an ISEF to account for activity differences in the two metabolic systems. Michaelis-Menten kinetics of marker reactions for CYP2C19, CYP2C9 and CYP3A4 were evaluated under laboratory individual conditions in HLM and rhCYP , 1.37% (CYP2C9) and 12.5% (CYP3A4) were obtained.(ii) Incubations of rhCYP2C19, rhCYP2C9 and rhCYP3A4 all revealed distinct VRC nd rhCYP . When VmN-oxidation of VRC were determined.Considering a mean of both ISEF approaches and the inhibition investigation in HLM, contributions of 63.1% CYP2C19, 13.4% CYP2C9 and 29.5% CYP3A4 to the hepatic int,hepatic,invivo of 540 mL/min (32.4 L/h), and applying the well-stirred liver model resulted in a CLhepatic of 227 mL/min (13.6 L/h). When individual CLint values of rhCYP were summed up, CLint,hepatic,invivo values of 652 and 278 mL/min (39.1 and 16.7 L/h) were obtained, depending on whether ISEFVmax or ISEFCLint was applied, respectively. Consequently, CLhepatic values of 266 and 127 mL/min (16.0 and 7.62 L/h) were determined using ISEFVmax or ISEFCLint, respectively.In vitro in vivo extrapolation (IVIVE) from HLM resulted in a CLNO and OH–VRC showed no depletion over time when incubated directly with HLM. After 60 min at the low NO concentration, 110% and 105% NO compared to a control incubation without HLM were present in the two replicates. At the high NO concentration, 96.9% and 109% NO compared to the control were determined. The same was observed for OH–VRC, which yielded a recovery of 92.5% and 108% at the low, and 94.7% at the high, OH–VRC concentration after 60 min. Additionally, no trend, i.e., decreasing concentration over time, was observable, neither for the HLM containing incubation nor for the control .50, differed for the three enzymes. The metabolism of S-mephenytoin by CYP2C19 was most affected by VRC, with an IC50 of 3.72 µM . NO and OH–VRC showed minor inhibition of CYP2C19, resulting in IC50 values of 288 µM (65.0–31623 µM) and 41.7 µM (26.9–89.1 µM), respectively. Thus, estimated values exceeded the experimentally applied inhibitor concentrations and hence precluded a precise evaluation of IC50. On CYP2C9, OH–VRC had the largest influence on the formation of 4-hydroxydiclofenac, with an IC50 of 3.67 µM (3.16–4.26 µM); followed by VRC, with an IC50 of 4.17 µM (2.54–6.51 µM); and NO, with 13.4 µM (9.90–19.1 µM). The strongest inhibition by all substances was caused on the 1-hydroxylation of midazolam by CYP3A4, which resulted in IC50 of 1.02 µM (0.796–1.27 µM) for OH–VRC, 1.76 µM (1.26–2.36 µM) for VRC and 4.48 µM (3.78–5.29 µM) for NO  for NO .50. In two cases an IC50 shift of >1.5-fold was observed: for the effect of OH–VRC on CYP3A4 in the absence of NADPH and for NO on CYP3A4 in the presence of NADPH  and 11.6 µM (9.65–14.0 µM), respectively. Inhibition by NO was noncompetitive, with a Ki of 58.6 µM (46.8–75.2 µM), and was low overall, with the estimation exceeding the experimentally applied concentrations. Therefore, the assessment of NO has to be interpreted with caution. All three substances inhibited CYP2C9 in a competitive manner and yielded Ki values of 2.57 µM (2.16–3.14 µM) for VRC, 5.47 µM (4.32–7.00 µM) for NO and 2.80 µM (2.20–3.61 µM) for OH–VRC. The inhibition of VRC, NO and OH–VRC on CYP3A4 was noncompetitive and resulted in Ki values of 2.75 µM (2.35–3.22 µM), 5.24 µM (4.68–5.86 µM) and 2.53 µM (2.24–2.87 µM), respectively  and 9.3 pmol/min·mg (±11.1 pmol/min·mg) and were based on HLM derived from three individuals [M 2.98 µM, Vmax 26.1 pmol/min·mg). In recombinant enzymes for CYP2C19, KM and Vmax values of 14 ± 6 µM and 0.22 ± 0.02 nmol/min·nmol [M and a 7- and 4-fold lower Vmax value compared to our determinations. For rhCYP3A4, KM and Vmax of 16 ± 10 µM and 0.05 ± 0.01 nmol/min·nmol [M of 1.20 µM and a Vmax of 0.00893 pmol/min·pmol were 13- and 196-fold lower with regard to KM and 6- and 16-fold lower with regard to Vmax. For rhCYP2C9, one study reported a KM of 20 µM and a Vmax of 0.056 pmol/min·pmol [The recision ,33. In Hividuals . Consequmin·nmol  have beemin·nmol  were obsmin·pmol , which wv/v) as low as possible, with a rule-of-thumb of <1% methanol in final incubations [N-oxidation of VRC had a maximum of 0.5% methanol. As VRC is a lipophilic drug, it is difficult to reproduce how the solubility as well as the organic solvent limit was maintained at VRC concentrations of 2500 or 5000 µM [In particular, the different magnitudes of reported parameters demonstrate the necessity of carefully planned and executed in vitro experiments to generate reliable results. However, previously reported studies carried some limitations, potentially resulting in discrepancies between in vitro predicted and in vivo observed PK properties. First, kinetic investigations were performed using HLM prepared from only a few individuals. Yet, the individual predisposition for CYP enzymes is very heterogenic, as shown by Achour et al.’s meta-analysis of hepatic CYP expression , with vaubations ,71. All  5000 µM ,33. Four 5000 µM ,33, pote 5000 µM . Therefo 5000 µM . However 5000 µM —and thus 5000 µM . ReactioN-oxidation; (ii) the contribution of CYP2C9 was negligible; and (iii) besides CYP3A4, no further enzymes were contributing significantly to NO formation. While conclusion (ii) is in accordance with the reported safety of VRC in a poor metabolizer of CYP2C9 in a clinical trial [With regard to the contributions of the respective enzymes to the overall formation of NO, different approaches for the determination have been explored, yielding results with mean contributions of 63.1% for CYP2C19, 13.4% for CYP2C9 and 29.5% for CYP3A4. The determination by the application of specific CYP inhibitors in HLM was interfered by cross-inhibition of the inhibitors loratadine and sulfaphenazole on the enzymes of the same family, CYP2C19 and CYP2C9. This problem is not unknown, and even the U.S. Food and Drug Administration states that for CYP2C19 no specific inhibitor is yet available ,67. Addial trial , conclusal trial . Furtheral trial . OverallN-oxidation, was taken into account. The extrapolation from HIM and the determination of the fraction of the intestinal (intrinsic) clearance of the total VRC metabolic clearance is a promising next step from the current work by the application of PBPK modeling.The performed IVIVE additionally confirmed the validity of the generated data. In clinical studies, VRC clearances of 244 mL/min   and 272 circulating metabolite, the formation of (di-)hydroxy-VRC has been claimed to be the major pathway, and low plasma concentrations have been explained by a high clearance [The role of other pathways, e.g., (di-)hydroxylation of VRC at the fluoropyrimidine moiety, has been controversially discussed in literature. Although NO is the major learance ,23. Howeint was approximately 6-fold lower than in HLM, which was probably due to the lower CYP enzyme abundance per gram of microsomal protein in the small intestine compared to the liver. For CYP2C19, CYP2C9 and CYP3A4 in the small intestine abundances of 2.1, 11 and 58 pmol/mg have been described, which were respectively 5.2-, 5.5- and 1.6-fold lower than those in the liver . On CYP2C19 the authors also described a competitive inhibition of CYP2C19; however, their determined Ki of 5.07 µM was 2.7-fold higher than our result. Lastly, on CYP3A4 Jeong et al. found a mixed competitive and noncompetitive inhibition by VRC with Ki values of 0.66 and 2.97 µM, respectively [i 2.75 µM). Deviations might arise from the different evaluation methods; Jeong et al. used a graphical tool, Dixon plots, which are based on a linear transformation of the data, while our results were based on the best nonlinear model fit and thus less error prone. Furthermore, in this case also the choice of marker reactions might influence the determined parameters [In adults, VRC exhibits nonlinear PK, an observation assumed to originate in saturation or an auto-inhibition of its metabolism ,79. The <10.5 µM ,44,81; hectively , while wrameters ,57.50 have been published, both resulting in different conclusions. We demonstrated a relevant inhibitory effect of NO on CYP3A4 and, although to a lesser extent, CYP2C9, but only a minor one on CYP2C19, which is in line with Giri et al.  [50 40.2 µM) than CYP3A4 (IC50 146 µM) [i for CYP3A4). The clinical relevance of the investigated inhibition is challenging to predict. Although plasma concentrations of OH–VRC are up to 30-fold lower than those of NO [Time-dependent inhibition of CYP2C19, CYP2C9 and CYP3A34 was not observed in our experiments, which is in line with two previously reported findings ,44, but ctively) . Hohmann 146 µM) . OH–VRC Indeed, for prospective predictions of VRC exposure in humans, developing a PBPK model is suggested to provide a mechanistic framework to integrate VRC’s physicochemical properties, the presented in vitro hepatic metabolism data, and other physiological processes and parameters ,85. The Furthermore, IVIVE–PBPK linked models enable the extrapolation of the PK of VRC to vulnerable patient populations, e.g., pediatrics , to suppUltimately, the combination of a VRC PBPK model with a pharmacodynamic (PD) model could be used to estimate the time course of the drug response for various dosing regimens in different populations or disease states ,90. SimuIn vitro investigations in HLM, rhCYP and other enzyme sources are a powerful tool for the assessment and evaluation of human metabolism. However, there is a substantial difference in the identification and basic kinetic determinations of enzymes involved in the metabolism of a (new) drug and a profound quantitative characterization suitable for PBPK modeling. Thus, we established a coherent framework of VRC’s metabolism assessing relevant aspects of its properties as a substrate and inhibitor to contribute to the elucidation of the complex PK of VRC.


Background: Acid phosphatase and its regulation are important objects of biological and clinical research and play an important role in the development and treatment of prostate and bone diseases. The newly patented aminoalkanol (4-[2-hydroxy-3-(propan-2-ylamino)propyl]-1,7-dimethyl-8,9-diphenyl-4-azatricyclodec-8-ene-3,5,10-trione hydrochloride) (I) and (4-[3-(dimethylamino)-2-hydroxypropyl]-1,7-dimethyl-8,9-diphenyl-4-azatricyclodec-8-ene-3,5,10-trione hydrochloride) (II) derivatives have potential anticancer activity, and their influence on enzymatic activity can significantly impact the therapeutic effects of acid phosphatase against many diseases. Therefore, in this study, we investigated the action of compounds (I) and (II) on acid phosphatase. Methods: Capillary electrophoresis was used to evaluate the inhibition of acid phosphatase. Lineweaver–Burk plots were constructed to compare the Km of this enzyme in the presence of inhibitors (I) or (II) with the Km in solutions without these inhibitors. Results: Compound (I) showed a stronger competitive inhibition against acid phosphatase, whereas derivative (II) showed a weaker competitive type of inhibition. The detailed kinetic studies of these compounds showed that their type and strength of inhibition as well as affinity depend on the kind of substituent occurring in the main chemical molecule. Conclusions: This study is of great importance because the disclosed inhibition of acid phosphatase by compounds (I) and (II) raises the question of whether these compounds could have any effect on the treatment possibilities of prostate diseases. Depending on its origin, acid phosphatase can have different forms. This enzyme can be found in the cellular components of the bone, spleen, kidney, liver, and intestine, as well as in the blood. It also occurs in postpubertal prostatic epithelial cells in exceptionally high concentrations ,3,4,5,6.The serum levels of prostatic acid phosphatase (PAP) are determined to evaluate the success of the surgical treatment of prostate cancer . In the Thus far, many works have focused on acid phosphatase ,8. Some An important aim of many works is to analyze the possibility of using acid phosphatase activity as a parameter to assess the degree of treatment of prostate cancer-related diseases. Activity of prostatic acid phosphatase increases with the proliferation of malignant tissues .Acid phosphatase is also associated with bone formation processes and may have an important role in the treatment of osteoporosis or osteomalacia. The increasing interest of researchers in the biochemistry of this enzyme is related to its use as a marker of osteoclasts, which are the bone-resorbing cells. Bone demineralization may contribute to an increase in the activity and concentration of acid phosphatase in the serum of affected individuals ,23.A pathological increase in bone resorption arises when osteoclasts are stimulated to carry out resorption at an increased rate ,29,30,31The activity of acid phosphatase can be regulated under the influence of many substances ,33. SomeConsidering the versatility of acid phosphatase and its influence on many biochemical processes in the human body as well as its diagnostic properties, this study aimed to investigate the characteristics and inhibition type of the new derivatives of 1,7-dimethyl-8,9-diphenyl-4-azatricyclodec-8-ene-3,5,10-trione  with a pThe present work is a continuation of our studies on aminoalkanol derivatives of dicarboximides ,39,40,41Compounds (I) and (II) were tested to assess their inhibitory activity toward PAP. The structure–activity relationships observed with the enzyme inhibition were consistent with those already observed for the inhibitory activity of parent compounds (I) and (II) toward TNAP .Moreover, the propan-2-ylamino-substituted compound (I) and the derivative (II), which bears the dimethylamino group, exhibited excellent stronger competitive (I) and weaker competitive (II) inhibitory activity toward PAP, respectively.CE enabled us to carry out selective monitoring of p-nitrophenol (PN-OL), p-nitrophenyl phosphate (PN-TE), and compound (I) and compound (II), as well as to eliminate any interference with the endogenous components originating from the serum samples . In this work, we used, to a large extent, the method developed earlier by us ; howeverLinearity of the reaction for each substrate -product concentrationhas been shown in The statistical power for each dataset and each value of the kinetic parameter was achieved by measuring six times each value obtained for the nine concentrations of substrates and reaction products in the system without inhibitors and for four systems in the presence of 0.01 mM, 0.03 mM, 0.04 mM and 0.05 mM inhibitor (I) or (II). After exceeding the value of 0.05 mM, there was no further increase in inhibition, therefore the value of 0.05 mM was considered the cutoff value for both inhibitors.The inhibitory effects of compounds (I) and (II) on PAP are shown in the form of electrophoregrams in Lineweaver–Burk curves intersecting at one common point on the 1/Vmax axis indicate the stronger competitive type of inhibition (compound (I)), whereas curves having one common intersection on the 1/S axis indicate the weaker competitive type of inhibition (compound (II)) . The Km 50/([S]/Km + 1), Ki was calculated at 0.786 mM and 0.924 mM for the competitive (I) and competitive (II) inhibitor, respectively.Inhibition constants were also calculated for the two enzymatic systems. Using equation Ki = ICTo determine the type and strength of PAP inhibition by compounds (I) and (II) and the strength of affinity of inhibitors (I) and (II) to the enzyme, we estimated the differences in the slope angles of straight lines, values of Michaelis–Menten constant (Km), and inhibition constants (Ki) between the control systems (without an inhibitor) and the systems containing inhibitors (I) and (II) at 0.05 mM concentration .50) of PAP in the presence of a competitive (I) and competitive (II) inhibitor were also determined  and 39 μM for compound (II) (In order to accurately determine the effect of inhibitors (I) and (II) on prostatic acid phosphatase, apart from the parameters defining the type of inhibition, the affinity of the inhibitor to the enzyme and the maximum speed, the parameters of the inhibition constant (Ki), turnover number (kcat), catalysis rate (kcat/Km) and half-maximal inhibitory concentration (ICtermined . Both thund (II) .Taking into account the structure of the acid phosphatase and the structures of a competing inhibitor molecules, we also analyze the possibility of their interaction  with theIn order to estimate the potential bioactive conformation of the compounds and possible interactions stabilizing the protein–ligand interaction, molecular docking methods were used. Both compounds (I and II) as well as ligand  crystalized in active site of PAP in 1nd5 PDB structure (classified as strong inhibitor) were docked in the crystallographic structure of the human prostate acid phosphatase active site using the Autodock Vina program. The obtained values of estimated binding free energy for compound (I) were slightly higher and amounted to −8.0 kcal/mol and for compound (II) −7.8 kcal/mol, what was in agreement with the experimental data. Both compounds scored higher in comparison to the crystallized ligand for which binding interaction was estimated as −7.5 kcal/mol.Analysis of bioactive conformations showed that both compounds adopt nearly identical conformations at the binding site. In the case of both compounds’ non-polar part this was stabilized in the active pocket by interactions with, TYR123, LEU124, ARG127, PHE171 and polar fragments form hydrogen bonds with ARG79, THR259 HIS257.Predicted differences was due to structural diversity within the amino group and resulting from the occurring hydrogen interactions with polar amino acids at the binding pocket. In both cases, the protonated amino group formed an intramolecular hydrogen bond with the carbonyl group. The differences were due to the presence of an intermolecular hydrogen bond between ASP258 and the second hydrogen atom bonded to the amine group which was present in compound (I) but not possible for compound (II) due to the methylation. Additionally unfavorable donor–donor interaction with HIS12 occurred for compound II. The receptor–ligand interaction pattern is depicted in The simultaneous formation of two bonds between the inhibitor (I) and (II) and the active site proves the high binding strength and affinity of this inhibitor to PAP, as shown by the experimental results presented in Using the electrophoresis method developed, we investigated the necessary kinetic parameters of the PAP-catalyzed enzymatic reaction in the presence and absence of compounds (I) and (II) in a very short time. The method was found to be characterized by very good validation parameters, which enabled its use in studying enzymatic kinetics. From the results obtained, we determined the type of inhibition and the inhibitory strength of compounds (I) and (II) as well as their affinity to the enzyme (PAP).50 and scoring function parameters (The results indicated compound (I) to be a stronger competitive inhibitor and compound (II) as a weaker competitive inhibitor of PAP. All data and the calculations of the slope angles of straight lines in the course of enzymatic reactions and the Km, Ki, kcat, kcat/Km, ICrameters  clearly rameters .The comparison of the slope angles of straight lines for compound (I) and (II) showed that compound (I) was a 1.02 times more potent competitive inhibitor of PAP than compound (II). The slope of the line for inhibitor (I) was 1.312 degrees greater than for inhibitor (II), which also indicated a greater degree of inhibition of compound (I) compared to compound (II).Comparing of the Km values for the system without inhibitor (1.124 mM) with systems containing inhibitor (I) (2.429 mM) and inhibitor (II) (2.216 mM) and Vmax values , it is clearly seen that in both cases the Vmax values remain practically unchanged, while the Km values clearly increase relative to the reference system (without the inhibitor). This clearly indicates the competitive type of inhibition of compounds (I) and (II), and comparison of the Km value for inhibitor (I) with the Km value for inhibitor (II) shows that compound (I) is a stronger competitive inhibitor of PAP than compound (II).Differences in the inhibitory potency of PAP can also be seen when comparing the values of inhibition constants (Ki) characterizing the strength of the inhibitor’s affinity for the enzyme. Low Ki values in all cases indicate a high affinity of inhibitors (I) and (II) to PAP.Comparing the values of the Ki constants for the competitive (I) (0.786 mM) and competitive (II) (0.924 mM) inhibitor, it can be seen that affinity of the inhibitor (I) to the enzyme was 1.176 times stronger than competitive inhibitor (II). The difference between the Ki value for inhibitor (I) and (II) was 0.138 mM which also indicated a stronger affinity of compound (I) to the PAP compared to compound (II).−2 s−1 for the enzyme inhibited by a competitive inhibitor (I) and 1.304 × 10−2 s−1 for non-inhibited enzyme. It follows from the above data that the turnover number of PAP and thus the number of substrate molecules converted into the reaction product in the presence of a competitive inhibitor (I) was 1.004 times less than in the presence of the non-inhibited enzyme, and the difference between kcat values of the inhibited and uninhibited enzyme was 0.005 × 10−2 s−1. In the case of a competitive inhibitor (II) the values of the catalytic constants (kcat) were 1.300 × 10−2 s−1 for the enzyme inhibited by competitive inhibitor (II) and 1.304 × 10−2 s−1 for the non-inhibited enzyme; therefore, turnover number in the presence of competitive inhibitor (II) was 1.003 times smaller than in the presence of the non-inhibited enzyme, and the difference between kcat for inhibited and non-inhibited enzyme was 0.004 × 10−2 s−1.The values of the catalytic constants (kcat), characterizing the turn over number of the enzyme, were 1.299 × 10Thus, when comparing the kcat values for inhibitor (I) and (II) and the differences between them, it is clearly seen that compounds (I) and (II) have no major influence on the turnover number of the enzyme and confirm the inhibition competency of both compounds.−2 M−1 s−1) and the non-inhibited enzyme (1.160 × 10−2 M−1 s−1) showed that in the presence of a competitive inhibitor (I) the catalytic efficiency of the enzyme was 2.11 times lower than the non-inhibited enzyme. Comparison of the specificity constants (kcat/Km) for the enzyme in the presence of compound (II) (0.595 × 10−2 M−1 s−1) and the non-inhibited enzyme (1.160 × 10−2 M−1 s−1) showed that in the presence of a competitive inhibitor (II) the catalytic efficiency of the enzyme was 1.95 times lower than the not inhibited enzyme.Comparison of the specificity constants (kcat/Km) for the enzyme in the presence of compound (I)  and thus the enzyme’s overall ability to convert substrate to product is lower for inhibitor (I) than for inhibitor (II), which is further evidence that compound (I) is a stronger competitive inhibitor of PAP than compound (II).The differences in kcat/Km values between the inhibitor (I)-non-inhibited enzyme system and the inhibitor (II)-non-inhibited enzyme system were 0.611x1050 values for inhibitors (I) (37 µM) and (II) (39 µM). The comparison of these values shows that compound (I) is 1.05 times more potent PAP inhibitor than compound (II). Such a small difference is due to the small, but well registered by our method, difference between the substituents present in the main structure of compounds (I) and (II).The influence of the type of substituent is also clearly visible when comparing the ICThe obtained values of the binding free energy for compound (I) were slightly higher and amounted to −8.0 kcal/mol and for compound (II) −7.8 kcal/mol, which also confirmed the stronger bond of compound (I) to PAP than compound (II).There are many papers describing the influence of various compounds on prostatic acid phosphatase. The most popular are works describing the effects of inhibitors such as benzylphosphonic acids ,43 And L50 range from >500,000 nM do 4 nM  dec-8-ene-3,5,10-trione, which have potential anticancer activity, on the activity of prostatic acid phosphatase.We used the CE method developed earlier by us . This meAs aminoalkanol derivatives were not recognized as PAP inhibitors and not reported in the literature so far, we attempted to analyze their effect on the activity of PAP. Our study showed that PAP inhibition can be assessed simply with an in vitro drug metabolic system using a CE analysis. The results showed that the studied derivatives exerted different effects on PAP. One of the analyzed compounds (I) was identified as a stronger competitive inhibitor, and the other (II) as a weaker competitive inhibitor of the PAP. Furthermore, the findings indicated that the type of substituent present in the main chemical molecule determines the inhibition type, and that the competitive inhibition of compound (I) is stronger than the competitive inhibition of compound (II). The investigations performed also showed differences in the inhibitory potency and affinity of compounds (I) and (II) to prostatic acid phosphatase (PAP).The described properties of compounds (I) and (II) and the knowledge of their effect on the activity of prostatic acid phosphatase can be of use in designing therapy for many diseases. In addition, such an analysis of properties can indicate the different possibilities of influencing the regulation of specific biochemical processes, which can be considered important information on substances to be used in medicine.


For slowly compressed hexagonal ice pressure-induced amorphisation to high-density amorphous ice (HDA) takes place below and at 130 K, but polymorphic transformation to ice IX takes place at 140–170 K. Stable ice II only forms above 170 K. Ice IX impurities trigger ice IX growth even at 120 K. HDA and ice IX are equally long-lived, where both can be regarded as metastable phases. Slow compression of ice at 100 K usually results in pressure-amorphisation, unless there are ice IX seeds and temperature gradients. This necessitates that activation barriers against crystallisation are sufficiently high, i.e., thermal fluctuations at 110–160 K cause them to turn into ultraviscous liquids, without crystallisation. In their recent work Tulk et al. negate this possibility by claiming that crystallisation to ice IX rather than formation of HDA takes place for slow compression experiments of hexagonal ice Ih at 100 K.9 Specifically, they compress ice Ih at 100 K at two different rates. At the higher rate (∼50 MPa min−1) they observe the well-known pressure-induced amorphisation (PIA) Ih → HDA near 1 GPa,1,10 followed by crystallisation of HDA to ice VII near 3 GPa. By using a slower, stepwise compression they obtain a different transition sequence: Ih → IX at 0.3 GPa, followed by IX → XV above 1 GPa, and at 3 GPa ice VIII forms. They interpret HDA formation as a “kinetically inhibited result of interrupted crystallisation of Ih”, i.e., HDA seems to be a transition state between ice Ih and ice XV that is not thermodynamically connected with high-density liquid water.Liquid water is anomalous, especially in its supercooled state. One of the key properties distinguishing it from most other liquids is “polyamorphism”.et al.1,10 that were later confirmed by many groups, including Klotz et al.,11 Strässle et al.,12 Gromnitskaya et al.13 and our own group.14 While failing to reproduce the results in these literature studies, the results of Tulk et al. are quite similar to results in our own group obtained at much higher compression temperature.15,16 Specifically, we have shown in M. Bauer et al. that compression rate as well as compression temperature are of crucial importance to determine the outcome of compression experiments of ice Ih.15,16 In that work we have shown that ice III/IX forms as the major product quite easily at 170 K, but ice II predominates at higher temperature. Faster compression favours the metastable form ice III/IX, and slower compression favours the thermodynamically stable form ice II. In order to obtain thermodynamically stable, pure ice II upon compression of ice Ih, the compression needs to be slower than 2 MPa min−1 at 170 K, but can be as fast as 100 MPa min−1 at 220 K  forms at higher temperature. The compression rate dependence and the phases emerging found by Tulk et al.9 are exactly the same as that reported by us in ref. We here take a stand on the controversy emerging from these findings, where they challenge the idea that HDA is a glassy form thermodynamically related to the liquid. Their results contradict the results obtained in the pioneering work by Mishima et al.18 repeated the experiment by Tulk et al.9 at a different high-pressure neutron diffraction setup (J-PARC MLF), where they find that HDA indeed does form at 100 K in slow compression experiments, not ice III/IX. In spite of the contradicting results, R. Bauer et al. come up with an interpretation similar to the one of Tulk et al., i.e., formation of HDA is the result of an incomplete transition between two crystalline forms of ice, that is, ice Ih and ice VI/XV.18 In order to clarify what the experimental findings actually are and how to understand the nature of HDA we conducted slow compression experiments following their protocol, but with direct measurement of pressure and temperature in our piston-cylinder setup and using powder X-ray diffraction for sample characterisation. This extends our earlier work done at 170–230 K15,16 to lower temperature. Furthermore, we have purposefully contaminated ice Ih samples with ice III/IX to study the impact of these impurities on the outcome of the slow compression experiment.R. Bauer 19,20 400 μL of ultrapure liquid H2O were pipetted into a precooled container made of ∼0.7 g indium foil. After crystallisation of water to hexagonal ice, the sample container was inserted into the 8 mm bore of our custom-made high-pressure piston cylinder setup that is pressurised using a commercial “universal material testing machine” . The high-pressure cell was cooled by liquid nitrogen flowing through a copper loop around the cylinder. Heating elements as well as a Pt-100 temperature sensor were inserted in additional bores in the cell, 1 cm from the sample position  at temperatures <80 K and subambient pressure.On the methodological side, the slow compression experiments were done following the procedures established over the last 20 years in our group.h at 100 K at ∼4 MPa min−1, which corresponds to the linear equivalent of the stepwise compression profile used by Tulk et al.9 These curves exhibit linear densification up to 0.94 GPa when a steplike densification of about 20% occurs. X-Ray diffractograms  become apparent , and at 1.4 GPa only the halo peak is observed, with no signs of any Bragg peaks. That is, pressure-induced amorphisation is complete and HDA easily survives for hours at p > 1 GPa at 100 K, whereas ice IX does not appear at all.rams see  reveal tet al. even more closely, we have also used a stepwise compression protocol at 100 K. For this purpose, pressure was increased using a step-profile, using 50 MPa min−1 for each step, and maintaining the plateaus for 1 hour  line in ESI,et al. (2 MPa min−1) we do not find any signs of crystalline ice IX at 100 K. The respective (a) ΔV(p) and (b) p(t) lines (red) are compared with the ones of an experiment conducted at 4 MPa min−1 (black) in ESI,h). Even though this experiment takes about 10 hours, HDA survives. There are no signs of any crystalline high-pressure ice phases after such a long experiment. This demonstrates the longevity of HDA and implies that at 100 K the activation barrier is high enough and the prefactor small enough so that none of the attempts to cross the barrier for HDA crystallisation is successful. There might be an even slower compression experiment that allows formation of metastable ice III/IX (or even stable ice II) at 100 K from ice Ih. However, such an experiment would certainly exceed the limit of available time resources for a PhD thesis. We surmise that the ice Ih sample would need to be kept for years at 100 K to actually reach conversion to an ice that is thermodynamically more stable than HDA. Put in other words, HDA is an ice form of longevity at 100 K, maybe even an infinitely stable phase.In order to mimic the experiment by Tulk V(p) line in h and ice IX, indicating that the transition is in progress, but incomplete at 0.7 GPa (see red middle diffractogram in V(p) line in h → IX at 4 MPa min−1. Yet, the onset pressure of the transformation (0.65 GPa) is significantly higher compared to the one reported by Tulk et al. (0.3 GPa). Finally, at 170 K . In such situations the temperature fluctuations increase, and a bit larger deviations from the set temperature appear for a few minutes, after which the set temperature is reached again  is at the origin of the difference between the results obtained by Tulk et al. and ourselves, we conducted a control experiment compressing ∼400 μL D2O ice Ih at 100 K at a rate of 4 MPa min−1  curves reveal only a single Ih → HDA densification step at ∼1 GPa  as well as the volume change incurred through HDA formation (ΔVIh→HDA). h → IX now. When comparing the onset pressure of ice IX growth at 140 K between compression of pure ice Ih  as a result of shock-wave heating could explain the incompatibility with the experiments in other groups.For this reason, we studied the influence of ice IX contamination by slowly compressing mixtures of I40 K see . Dependicase see . There ie ice Ih  and compIh ,27 while only the much more stable expanded HDA (eHDA) was found to be directly related to ultraviscous water.25 This leads to the conclusion that the activation barriers for HDA crystallisation are significantly higher than claimed by Tulk et al.9 as well as R. Bauer et al.18 In fact, they are high enough to keep HDA stable above its glass-to-liquid transition temperature, so that high-density liquid water (HDL) may equilibrate instead of crystallisation to ice IV and ice XII. This has been demonstrated in the recent work of our group by Stern & Loerting30 as well as Handle & Loerting.31This view is inconsistent with all experimental findings on PIA of ice Iet al.18 are consistent with the results presented in h and ice VI/XV, contrasting our claim above of HDA being a metastable phase related to the high-pressure liquid state. Similarly Shephard et al. claim HDA to be a derailed state along the ice Ih to ice IV conversion.32 The resistance of HDA against crystallisation for many hours at 120 K speaks against both ideas of HDA being a transient state in a polymorphic conversion. In slow heating experiments HDA even resists crystallisation up to 161 K.30 Also the conjecture by R. Bauer et al.18 that amorphisation represents the onset from a single H-bond network to an interpenetrating H-bond network defies experimental evidence. In reality, HDA always crystallises first to the single H-bond network ice IV and/or ice XII,33,34 but not directly to ice VI/XV with interpenetrating H-bond networks. In fact, the transition to interpenetrating networks does not require an amorphous intermediate, but directly takes place in the polymorphic ice IV → ice VI or XII → ice VI transition.While the recent experimental findings presented by R. Bauer 35 temperature-induced amorphisation of ice,36 decompression-induced melting of high-pressure ice,37 or compression of hyperquenched water,38 to name just a few. This is only possible if HDA/HDL is describable as a deep basin (as sketched in Taken together this experimental evidence allows regarding HDA/HDL as a “metastable phase” in the thermodynamic sense rather than a “transient state”, in spite of its glassy nature. Unlike “simple amorphous states”, metastable HDA/HDL can be reached through many different thermodynamic paths, where pressure-induced amorphisation studied in the present work is only one of them. Other ways include cooling of the pressurised liquid,C. M. T. and T. L. conceived and designed the experiment, analysed the data and wrote the manuscript. C. M. T. and M. B. conducted the experiments.There are no conflicts to declare.CP-024-D1CP03922A-s001


Non-IgE-mediated gastrointestinal food allergy (non-IgE-GI-FA) is the name given to a series of pathologies whose main entities are food protein-induced allergic proctocolitis (FPIAP), food protein-induced enteropathy (FPE), and food protein-induced enterocolitis syndrome (FPIES). These are more uncommon than IgE-mediated food allergies, their mechanisms remain largely unknown, and their diagnosis is mainly done by clinical history, due to the lack of specific biomarkers. In this review, we present the latest advances found in the literature about clinical aspects, the current diagnosis, and treatment options of non-IgE-GI-FAs. We discuss the use of animal models, the analysis of gut microbiota, omics techniques, and fecal proteins with a focus on understanding the pathophysiological mechanisms of these pathologies and obtaining possible diagnostic and/or prognostic biomarkers. Finally, we discuss the unmet needs that researchers should tackle to advance in the knowledge of these barely explored pathologies. Food allergy (FA) is a specific immunological response that occurs reproducibly on exposure to a given food. This immune reaction can be due to an IgE-mediated mechanism or a non-IgE-mediated mechanism. IgE-mediated FA is much more common than non-IgE-mediated FA, the symptoms are easily recognized, and the underlying mechanisms are better established .Although the relationship between food allergy and gastrointestinal dysfunction has been known for several years, the exact mechanisms involved have not yet been clearly defined. The diagnosis of non-IgE-mediated gastrointestinal food allergy (non-IgE-GI-FA) is clinical and—due to a lack of a specific biomarker—not always easy. The diagnosis is based on compatible symptoms, the exclusion of other diseases, resolution of the symptoms once the offending food is removed from the diet, and the reappearance of the symptoms when it is reintroduced.Non-IgE-mediated gastrointestinal food allergy typically comprises three entities: (i) food protein-induced allergic proctocolitis (FPIAP); (ii) food protein-induced enteropathy (FPE), and (iii) food protein-induced enterocolitis syndrome (FPIES).Food protein-induced allergic proctocolitis (FPIAP) is a non-IgE-mediated food allergy characterized by bloody stools in a healthy infant. The symptoms begin within the first months of life; usually in the first two months. It affects both exclusively breastfed infants and formula-fed infants; although it appears to be slightly more common in exclusively breastfed infants (60%) .The most common offending triggers are cow’s milk (CM) proteins (76%), followed by hen’s egg (16%), soy (6%), and corn (2%). In 8% of the cases, the etiology could be multiple . Any foo+ lymphocytes, Th-2 type lymphocytes, and eosinophil infiltrate in all layers of the colonic mucosa [The pathophysiological mechanism of FPIAP is not well established. It seems that it involves CD8c mucosa . Additioc mucosa .The typical clinical manifestations are mucus bloody stools in a healthy appearing infant. The bleeding is usually visible although it can appear as occult blood in the stools. Some patients have iron-deficiency anaemia and less frequent hypoalbuminemia and peripheral eosinophilia . SymptomFPIAP is a very common cause of rectal bleeding in infants although the real prevalence is unknown. The population prevalence in infants is approximately 1% to 2%. The prevalence among infants with rectal bleeding ranges from 18% to 64% . Other sThe diagnosis is clinical and based on the following criteria: (a) small amount of rectal bleeding in an otherwise healthy infant; (b) disappearance of symptoms after all causal food is removed from diet; and (c) exclusion of other causes of rectal bleeding . AlthougThe differential diagnosis includes anal fissure, which is the most common cause of rectal bleeding in children under 12 months; swallowed maternal blood, or other diseases, such as enteric infection, malrotation with volvulus or Hirschsprung disease—but these usually affect the general condition .FPIAP can be an over-diagnosed entity, which is why some authors advocate the performance of an early oral food challenge (OFC) to avoid unnecessary elimination diets .The prognosis is excellent and nearly all infants outgrow the disease by 1 to 3 years of age .Food protein-induced enteropathy (FPE) is also a non-IgE-GI-FA. It is probably the most forgotten disorder of the three, and in recent years there are hardly reports of FPE in the literature. The symptoms begin in the first year of life and manifest as chronic diarrhea and intermittent vomiting. If the symptoms persist, they will lead to poor weight gain, failure to thrive, and villous atrophy. Some children develop an iron-deficiency anemia associated with the presence of occult blood in the stools . The patThe most common offending triggers are CM proteins and soy. The diagnosis is based on a compatible clinical history, the anatomopathological findings, and the disappearance of the symptoms once the food is removed from the diet. Intestinal lesions are patchy with villus atrophy, crypt hyperplasia, increased number of intraepithelial lymphocytes, and eosinophilic infiltration .The main differential diagnosis is with celiac disease. Clinically and pathologically, they are indistinguishable, but the typical celiac disease antibodies are absent in FPE .FPIES has been considered a rare disease, whose cumulative incidence rates are between 0.15–0.7% . HoweverFPIES is a non-IgE-GI-FA and is considered a heterogenous disorder with a wide spectrum of clinical phenotypes. These are based on: (i) time and duration of the symptoms, (ii) age of presentation, and (iii) specific IgE-positive of the causal food. Thus, we can classify FPIES into acute, chronic, later onset, and atypical .Acute FPIES usually presents in infancy, with repetitive protracted vomiting that begins 1 to 4 h after food ingestion. Vomiting is often accompanied by lethargy and pallor and can be followed by diarrhea. Some patients have an isolated fever peak and severe cases can progress to hypothermia, methemoglobinemia, acidemia, and hypotension, mimicking sepsis . ChildreChronic FPIES is less well characterized. In this presentation, the symptoms occur over several days or weeks and do not have a clear relationship with the offending food. Children have intermittent vomiting and/or diarrhea, failure to thrive, or poor weight gain. The severe form can develop dehydration and hypovolemic shock. In chronic FPIES, once the offending food is removed from the diet, the symptoms subside within two weeks, but re-exposure to it triggers the typical symptoms of acute FPIES. When the disease onsets in adolescence or adulthood, the symptoms appear after years of tolerance to the food and the predominant symptoms are, in most cases, colic abdominal pain and diarrhea ,20,21,22Although FPIES is considered a non-IgE-mediated FA, in some patients, it is possible to detect specific IgE against the causal food, either at the beginning or during the course of the disease. This form of presentation is known as atypical FPIES and it seems to have a protracted course, with a further risk of developing IgE-mediated symptoms. In adults, it has been also described that severe FPIES phenotypes developed IgE and this is linked to the worst prognosis of the disease ,24,25,26Any food can trigger FPIES; however, there are geographical differences, and some foods are more prevalent than others. In the USA, the most causative foods are CM proteins and soy. Children with either CM- or soy-induced FPIES can also react to both foods and have an increased likelihood of reacting to a solid food, most commonly oats and rice. In Asia, CM, soy-based formulas, and rice are the most common causal foods. Soy is especially frequent in Korea, but it has a better prognosis and earlier resolution than in other countries. Rice has been observed to be the main trigger of FPIES among the Australian pediatric population, and in European countries, CM and fish are the most frequent triggers of FPIES ,28,29.The development of tolerance varies by type of trigger and geographic location. CM- and soy-induced FPIES are reported to occur at an earlier age than other foods. The average age of tolerance of grains-FPIES is 35 months and fish-FPIES usually resolves within the first 5 years ,28.Nowadays, there is not a specific biomarker for FPIES that allows for confirmation of the diagnosis or to predict when the tolerance to the offending food has been achieved. For this reason, all patients with FPIES should undergo an oral food challenge (OFC) at least once in life. The OFC is a risky procedure and patients can develop moderate or severe symptoms after it, so it should be always performed under medical supervision ,30.The diagram shown in The most important diagnostic tool in the evaluation of non-IgE-GI-FA is a careful clinical history. However, when the clinical history is not sufficiently thorough, the OFC should be used as the gold standard to confirm the diagnosis of this disease, as has been mentioned above ,31,32.There are no other specific diagnostic procedures for these diseases, mainly because their pathophysiological mechanisms are poorly understood. However, there are a variety of laboratory tests to support the diagnosis and, more importantly, to rule out other conditions such as lactose intolerance, sepsis, necrotizing enterocolitis, anaphylaxis, gastrointestinal reflux disease, and celiac disease, among others . In thisFor the in-depth study of non-IgE-GI-FA, feces are the sample of choice. This is due to the fact that the main system affected is the gastrointestinal tract and to their non-invasive collection . There aAnimal models are a useful tool to study allergic diseases in vivo. Several mouse models have been developed to study the gastrointestinal reactions related to food allergy ,41,42,43In another model, OVA23-3 mice, which express an OVA-specific T-cell receptor, were used to demonstrate that Peyer’s patches (PPs) and mesenteric lymph nodes (MLNs) promote intestinal inflammation in AE . This isClosely related to non-IgE-GI-FA is eosinophilic esophagitis (EoE), which is a chronic allergic disease of the esophagus. A mix of immediate, IgE-mediated and delayed, non-IgE-mediated immunological reactions to foods is thought to play a role in EoE . EoE murThe use of animal models has been crucial to the understanding not only of the pathology associated with the disease but also to the pre-clinical evaluation of new therapeutics. That makes it of high importance in the development of new and more accurate models that allow a better understanding of non-IgE-GI-FAs.The human microbiota and its genetic composition—known as the human microbiome—began to be studied on a large scale at the beginning of the 21st century within the framework of major projects such as the Human Microbiome Project . Among tApproximately 70% of the primary colonization of the gut microbiota of newborns is vertically transmitted, occurring directly from mother to offspring. However, the composition of the gut microbiota is dynamic and changes throughout life due to factors such as delivery mode, age, diet, antibiotic consumption, ethnicity, lifestyle, environmental factors, and diseases and disorders, among others ,55,56,57The development of the gut microbiota during the first years of life correlates with the development and maturation of the immune system. Moreover, the gut microbiota and the production of gut microbiota-derived short-chain fatty acids  have been shown to be crucial for the proper expression and function of regulatory T cells (Treg), which are important in the induction and maintenance of oral tolerance to food antigens in humans ,63,64,65All the omics are based on the analysis of a large volume of data, and they have allowed a paradigmatic change in the development of research strategies ,69,70,71Bacteroides and Alistipes, and a single Firmicutes, Sarcina, at diagnosis, in children with non-IgE-mediated CMA when compared to healthy controls. However, at the phylum level, only Bacteroidetes was significantly enriched in children with non-IgE-mediated CMA. In addition, the authors observed that these children had a significantly lower fecal concentration of butyrate compared to healthy controls.Gut microbiota composition and fecal butyrate levels in children affected by non-IgE-mediated CMA were evaluated by Berni Canani et al. . The autBacteroides compared to infants with non-IgE-mediated CMA. In contrast, some members of the Clostridiales order, such as Lachnospiraceae and Coriobacteriaceae families, were significantly higher in allergic infants. In addition, children fed with hydrolyzed rice formulas were the only ones which did not develop tolerance to CMA and the microbiota colonization pattern was characterized by a low abundance of sequences of Actinobacteria, in particular the genus Bifidobacteria. Regarding metabolomics, two branched-chain fatty acids  presented significantly higher levels in allergic infants, but SCFAs were not statistically significant.Furthermore, Díaz et al. analyzed the composition of gut microbial communities and fecal associated parameters in 17 infants with non-IgE-mediated CMA (under a milk elimination diet) compared to 10 healthy infants  to establish potential links between the type of formula substitutes, gut microbiota, and desensitization . In thisBifidobacterium breve M-16V) amino acid-based formula (AAF) in patients with non-IgE-mediated CMA [Bifidobacteria and lower Eubacterium rectale/Clostridium coccoides percentages compared with those in the control group—thus, resembling healthy breastfed infants. Differences in gut microbiota were maintained in these patients over a 26-week follow-up period [Ruminococcus and Alistipes genera [Bifidobacterium spp. and Lachnospiraceae spp. Finally, the authors observed an increase of L-lactate and a decrease of valeric and isobutyric acids in the test versus control.Looking further into feeding, Candy et al. investigated the effect of a synbiotic-containing (prebiotic blend of fructo-oligosaccharides and the probiotic strain ated CMA . Infantsp period . Furtherp period . Wopereis genera . The heaLactobacillus) increased. Microbial metabolites such as BCFAs and p-cresol were lower. However, skatole, a microbial degradation product from tryptophan, was significantly increased.In this regard, Guadamuro et al. analyzed changes in gut microbiota composition, before and after milk challenges, from infants with non-IgE-mediated CMA consuming hypoallergenic hydrolyzed formulas . One monn = 12), non-IgE mediated-CMA (n = 5), proctocolitis (n = 5), or included as healthy controls (n = 8), were recruited. The concentrations of Eggerthellaceae, Lachnospiraceae, and Peptostreptococcaceae (at family level) and of Rothia (at genus level) was higher in fecal CMA samples than in those from other groups. In contrast, the abundance of Bifidobacterium genus decreased in the CMA group. Erysipelatoclostridium abundance was higher in controls and Intestinibacter was higher in the proctocolitis group followed by the CMA one. Regarding the bacterial composition of breast milk, the relative abundance of the family Eggerthellaceae was significantly higher in CMA than in the control and colic groups and Prolixibacteraceae was higher in the proctocolitis group than in the CMA group.The recently published study by Aparicio et al. aimed to evaluate the fecal microbiome and different immunological parameters in the feces of infants with gastrointestinal disorders and their maternal milk . A cohorThe trend in publications of studies analyzing the intestinal microbiota through omics techniques in patients with non-IgE-GI-FA is growing. For this reason, this field is likely to evolve very rapidly in the coming years. The attempts to evaluate the use of fecal biomarkers for the diagnosis of non-IgE FAs have been reviewed recently . The maiCalprotectin is an immunomodulatory, antimicrobial, and antiproliferative protein that is present in the cytoplasm of neutrophils, in the membranes of macrophages, in activated monocytes, and in mucosal epithelial cells . The meaOn the other hand, α-1 antitrypsin is an inhibitor of serine proteases present in serum which is synthesized in the liver . It can A promising group of proteins as potential biomarkers for CMA are the eosinophilic proteins EDN and ECP. They are proteins released by the degranulation of eosinophils  which coAn interesting example is the work of Kalach et al., who studied the most complete panel of possible biomarkers , as well as intestinal permeability and fecal microbiota, in a pilot study of 11 children with CMA  compared to 14 controls. From the panel, they found EDN to be the most promising, highlighting as well the obvious need for studies with a higher population to confirm the results . Other sIn most cases, FPIAP, FPE, and FPIES have a good prognosis, resolving during childhood. This leads to efforts in the knowledge of pathology to be directed to the search for diagnostic and prognostic biomarkers, relegating the treatment to the withdrawal of the causal food and to the treatment of the acute reaction, in the case of acute FPIES.For symptomatic infants who are exclusively breastfed, the food should be withdrawn from the maternal diet. For exclusively formula-fed infants, an EHF or AAF should be started. In infants who have already been introduced to other foods and we suspect that one is the cause of the symptoms, the food should be removed from their diet, and tolerance to other foods from the same group should be checked to avoid unnecessarily restricted diets. In the specific case of proctocolitis, if the symptoms are mild, an expectant attitude can be maintained for a few days, since in some cases, a spontaneous resolution occurs without it being necessary to remove the food from the diet ,95,96.In the case of an acute reaction in FPIES, the treatment will be staggered according to the severity of the symptoms. Thus, a mild reaction—one or two vomits without lethargy or other symptoms—can be controlled with oral hydration and, optionally, ondansetron. When the reaction is moderate—if the number of vomits is ≥three and is also accompanied by mild lethargy—intravenous fluid therapy should be administered early along with ondansetron. Finally, if the reaction is severe—severe lethargy appears, with hypotonia or cyanosis—in addition to intravenous fluid therapy and ondansetron, a corticosteroid bolus should be administered ,95,97,98There is an ongoing increase in the prevalence of food allergy worldwide, but the cause of this increment remains uncertain . FactorsThe development of non-invasive methods to study immune cells activated by food exposure in the gastrointestinal tract is needed to understand how symptoms are triggered . In thisIn this context, the identification of a metabolic fingerprint of non-IgE-GI-FA by the application of the abovementioned omics techniques can lead to the development of better diagnostic tools and/or new treatment options. Indeed, it is expected that with a multiomic approach, a combination of different biomarkers will provide the opportunity to monitor allergic conditions and predict whether a patient will respond to a specific therapeutic approach or not. The study of predictive metabolic biomarkers of the disease could be also reached by these techniques in a near future.Lactobacillus rhamnosus GG, and the synbiotic composed of a prebiotic blend of fructo-oligosaccharides and the probiotic strain Bifidobacterium breve M-16V to ameliorate non-IgE-mediated CMA symptoms [One rising strategy for both prevention and intervention in non-IgE-GI-FA is the use of probiotics, prebiotics, and synbiotics to modulate gut microbiota. In this regard, some authors have described beneficial alterations of the intestinal microbiota by adding to certain diets compounds such as the probiotic symptoms ,72. Wopesymptoms .To sum up, future efforts in non-IgE-GI-FA research should be aimed at elucidating the underlying pathological mechanisms of the diseases in order to understand the etiology of the symptoms and develop diagnostic and prognostic tools as well as innovative treatment targets.


Horses are used in a variety of equestrian disciplines predisposing them to musculoskeletal injury or disease including osteoarthritis and tendinopathy. As a result, a number of preventative measures are used within equine medicine and husbandry, ranging from therapeutic shoeing to the use of nutraceuticals. Despite their popularity and routine use evidence base and clinical outcomes are variable, bringing into question the efficacy of these prophylactic measures. In recent years a small number of studies have been performed examining the effect of specific strategies in order to quantify the preventative and protective claims such modalities have on joint and forelimb health. Few have robustly demonstrated a capacity to protect the limb by reducing inflammation, or promoting regenerative pathways. This review focusses on performance horses specifically, and the resounding theme that emerges in current research is the need for longitudinal studies to inform scientific conclusions surrounding single and multi-modal use. Furthermore, there is a requirement to prioritise evidence-based medicine to inform optimal clinical practice. The equine musculoskeletal system has significant demands placed on it , specifiEquine hoof trimming aims to maintain a healthy, functional hoof and prevent lameness, supporting articular structures and subsequent soft tissues within the limb . There aA study by Maeder et al.  describein vitro effect of hoof wall thinning on hoof structure and functionality, leading to increased deformability and resulting in damage to laminar tissue. This was undertaken using paired cadaver forelimbs of 12 horses. Each pair had the hoof wall from a single limb thinned by 25%. The contralateral hoof was used as a control within the study. Trimming the hoof was found to result in deformability of the entire hoof capsule, postulated to reduce damage to suspensory apparatus in the distal limb. In addition, there was significant disruption to suspensory apparatus of the distal phalanx in untrimmed hooves. This suggested a thinning of the hoof wall can decrease disruption to laminar tissue, supportive of hoof trimming as a prophylactic measure. However, it should be noted that this study was conducted in vitro and not in vivo. Therefore, the effect of the sole and frog were not been included. These structures may also be overloaded, and have a significant effect on the health and functionality of the distal limb and its joints . AT is funded by Horserace Betting Levy Board Equine Post-Doctoral Fellowship (VET/2020−2 EPDF 8). Our work is also supported by the Medical Research Council (MRC) and Versus Arthritis as part of the MRC Versus Arthritis Centre for Integrated research into Musculoskeletal Ageing (CIMA).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.