Conifer (Pinaceae) needles are the most frost‐hardy leaves. During needle freezing, the exceptional leaf anatomy, where an endodermis separates the mesophyll from the vascular tissue, could have consequences for ice management and photosynthesis. The eco‐physiological importance of needle freezing behaviour was evaluated based on the measured natural freezing strain at the alpine treeline. Ice localisation and cellular responses to ice were investigated in mountain pine needles by cryo‐microscopic techniques. Their consequences for photosynthetic activity were assessed by gas exchange measurements. The freezing response was related to the microchemistry of cell walls investigated by Raman microscopy. In frozen needles, ice was confined to the central vascular cylinder bordered by the endodermis. The endodermal cell walls were lignified. In the ice‐free mesophyll, cells showed no freeze‐dehydration and were found photosynthetically active. Mesophyll cells had lignified tangential cell walls, which adds rigidity. Ice barriers in mountain pine needles seem to be realised by a specific lignification patterning of cell walls. This, additionally, impedes freeze‐dehydration of mesophyll cells and enables gas exchange of frozen needles. At the treeline, where freezing is a dominant environmental factor, the elaborate needle freezing pattern appears of ecological importance. The frost‐hardiest conifer species are in the northern hemisphere, where the coldest climatic zones with long‐lasting winter periods are found . The mesophyll is separated from the central vascular cylinder by a distinctly differentiated, thick‐walled endodermis. Within the endodermis, there are one or two vascular bundles embedded in the transfusion tissue. Transfusion tracheids transport water radially. Strasburger cells transport assimilates from the mesophyll to the phloem. Transfusion parenchyma cells scavenge solutes from transpiration water  in these experiments may, however, not allow drawing conclusions about freezing under natural conditions. In a cryo‐SEM study, freezing of cold‐acclimated Pinus radiata did not change the shape of the needles or the relative areas occupied by the central vascular cylinder and mesophyll and the endodermis was no ice barrier  naturally grows on Mt. Patscherkofel within the area of the Alpine Garden of the University of Innsbruck . The Alpine Garden is situated within the treeline ecotone in the alpine zone. The experiments were conducted during fall, winter and spring; consequently, the plants were in a cold hardened state. Twigs (approx. 20 cm) with attached needles were cut, stored in a cooler bag and transported to the respective laboratories. For gas exchange measurements, mountain pine branches were collected from potted plants.Mountain pine (2.2n ≥ 5). Additionally, air temperatures in close vicinity to the leaves were measured as references. While a measurement frequency of one measurement every 5 min was used in the above +5°C range, data were monitored at a high frequency (0.5 s−1) at lower temperatures to detect needle‐freezing events. The climate station data was uploaded automatically every 6 h to a server, which allowed online data monitoring.At the investigation site, micrometeorological data was recorded from 5 October 2018 till 27 June 2019 with a climate station . The soil temperature was recorded in a depth of 5 cm. Photosynthetic active photon flux density was measured with a quantum sensor . Needle temperature records were done with copper constantan thermocouples . To ensure good contact, the thermocouple tips were glued to the needles with cyanoacrylate glue . The harsh weather conditions required high maintenance and resulted in an unequal and changing number of temperature replications   equipped with a 532 nm laser line, a spectrometer , a CCD camera  and a 100x oil immersion objective . Microsections were scanned with a laser power of 20–30 mW and one full spectrum (100–3780 cm−1) was acquired at every pixel with a step size of 333 nm and an integration time of 0.1–0.4 s. Microsections from three different needles were scanned, whereby long overview runs were included as well as zooms into selected regions. The best representative overview scans from one cross‐section and one EtOH‐washed longitudinal‐section are exemplarily shown. From all hyperspectral datasets, cosmic rays were removed and the baseline corrected before generating chemical images based on True Component analysis using WITec project PLUS 5.2 software. This software locates the most different spectra within the mapped area and calculates every pixel as a linear combination of the established component spectra with a basis analysis algorithm  or by external ice crystal application  and attached leaves was monitored with a digital infrared camera . Subtracting the infrared image prior to the freezing event from the following images enabled to spatiotemporally visualise freezing. This procedure is called infrared differential thermal analysis (IDTA)  with the infrared camera.Freezing of mountain pine branches (2.6n = 8) were placed in this environment and gradually exposed to freezing temperatures. Sectioning was done either transversally or longitudinally with a microtome  and the cut surface was immediately inspected.To visualise in which tissues ice crystals are accommodated during sub‐lethal freezing, cryo‐microscopy in reflected‐polarised‐light was applied  were continuously cooled to sub‐lethal temperatures and fixed before ice nucleation at −2°C and in the frozen state at −6°C/−15°C. For fixation, the samples (n = 3) were transferred to an automatic freezing unit according to a previously published protocol, which prevents samples from thawing during the transfer  photochemistry (Fv/Fm) was determined . To prevent measuring errors due to the constant temperature change, the internal infrared gas analyser of the GFS 3000 system was calibrated in 6 min intervals. Needle temperature monitoring allowed the detection of needle freezing by exotherm detection. Furthermore, the set parameters were: Flow 750 μmol s−1, CO2 400 ppm, H2O 4000 ppm, O 4000 ppm.Gas exchange of mountain pine needles was measured before, during, and after ice formation with a GFS3000 gas exchange measurement system . Three terminal shoots were detached and put into vials filled with tap water and placed inside a temperature‐controlled freezer. There, 6–8 needles were sealed into the standard gas exchange measurement cuvette  that, as a whole, was inside the freezer. It was possible to cool the needles inside the cuvette until the freezing took place and beyond. Needle temperature and the net photosynthesis rate were measured simultaneously throughout cooling. To allow photosynthesis and to keep the stomata open, the needles were irradiated with a moderate photosynthetic active photon flux density of 100 μmol photons m2.9Numerical data processing and analysis were performed with R . Results are given as mean ± standard deviation.33.1n = 40). All detected freezing exotherms were single‐peak shaped.Needle freezing temperatures were detected for about seven months between 21 October 2018 (day of year 294) and 16 May 2019 (day of year 136)  or frozen (−8°C) Figure . Ice cry3.5Semi‐thin sections of frozen‐fixed mountain pine needles were investigated for cellular changes induced by freezing. Most significant changes were observed in the endodermis and the transfusion tissues when unfrozen controls Figure  and −15°3.6Hedera helix L. and Helleborus niger L., ice formed in the intercellular spaces of the spongy tissue (data not shown) and rapidly inhibited gas diffusion. For the frozen mountain pine needles, even 1 h after ice nucleation and a further decrease in freezing temperature, a positive carbon assimilation rate was still recorded.Gas exchange of mountain pine needles that were successively cooled did not decline abruptly after ice formation (arrow), as exemplarily shown by the carbon assimilation rate in Figure 44.1Pinus bungeana revealed higher solute permeability for Casparian strips in needles compared to roots, explained by primary pit fields and no suberin in the needle endodermis . In our experiments, we could not verify such a two‐phase freezing event. However, in our freezing experiments, cooling rates were as in nature; this suggests that the high‐speed freezing protocol employed by Kaku ; consequently, the shape of needles did not change during freezing, which was similarly observed by Roden et al. . In P. r4.2P. radiata needles, despite ice in intercellular spaces, distinct gas spaces remained in the mesophyll. Contrarily, in frozen Eucalyptus pauciflora leaves, ice filled the intercellular spaces of the spongy tissue, blocking gas exchange immediately  than in our study , after ice nucleation, the assimilation rate was lowered to zero within 17 min and stomatal conductance decreased to a minimum after 45 min. Thereafter, stomatal conductance increased again, leading to the conclusion that the photosynthetic depression was not caused by stomatal closure (Lindfors et al., 2 concentration in frozen needles was attributed to a missing photosynthetic activity (Gaumont‐Guay et al., Already before ice nucleation, the photosynthetic rate decreased. This was as well observed for s et al.  reported4.3R. glacialis leaves: in the ice‐free palisade tissue, water is shifted via the symplast to the spongy cells and from there released into intercellular spaces (Stegner et al., The invaginations of arm palisade parenchyma cells are air‐filled and thus extensions of the leaf air space system (Wiebe & Al‐Saadi, Why the mesophyll cells of frozen mountain pine needles succeed in maintaining photosynthesis and their shape might be found in microchemistry and structure (Stegner et al., 5In nature, needles of mountain pine froze around −2°C, could stay permanently frozen for longer than a month and can be subjected to freeze–thaw cycles throughout the whole year. After ice nucleation, ice propagation was uninterrupted from stems into needles due to the lack of ice barriers. Within the needles, the ice masses were strictly confined to the central vascular cylinder. This suggests an important putative function of the endodermis. It is hypothesised that the particular lignification pattern blocks the penetration of ice from the frozen transfusion tissue into the mesophyll. This, together with the structurally rigid mesophyll cell walls, prevented mesophyll freeze‐dehydration. As a result, the intercellular gas spaces of the mesophyll remained free of ice, which may allow needle gas exchange during prolonged periods of frost at the alpine treeline. Similarities in needle anatomy suggest that the results may also apply to other frost‐hardy species of the Pinaceae.Gilbert Neuner designed the research. All authors performed experiments. Matthias Stegner, Othmar Buchner, Jasmin Lindner, Alexander Flörl, Nannan Xiao, Andreas Holzinger, Notburga Gierlinger, and Gilbert Neuner analysed the data. Matthias Stegner collected and merged the data. Matthias Stegner, Othmar Buchner, Nannan Xiao, Andreas Holzinger, Notburga Gierlinger, and Gilbert Neuner wrote the manuscript. All authors carefully revised the manuscript.The authors declare no conflicts of interest.Appendix S1. Supporting Information.Click here for additional data file.


Furthermore, the effects of different inhibitors, specifically monovalent ions, were tested on DAT inhibition by vanoxerine. Interestingly, these ions also reduced vanoxerine potency in a similar manner. The homology models of DAT suggested a potential secondary inhibitor binding site that affects inhibition in an allosteric manner. These findings imply that the use of combination therapy with monoamine reuptake inhibitors should be approached cautiously, as antagonistic effects may occur.Monoamine transporters, including dopamine, norepinephrine, and serotonin transporters , are important therapeutic targets due to their essential roles in the brain. To overcome the slow action of selective monoamine reuptake inhibitors, dual- or triple-acting inhibitors have been developed. Here, to examine whether combination treatments of selective reuptake inhibitors have synergistic effects, the pharmacological properties of DAT, NET, and SERT were investigated using the selective inhibitors of each transporter, which are vanoxerine, nisoxetine, and fluoxetine, respectively. Potencies were determined via fluorescence-based substrate uptake assays in the absence and presence of other inhibitors to test the multi-drug effects on individual transporters, resulting in antagonistic effects on DAT. In detail, fluoxetine resulted in a 1.6-fold increased IC O-methyl transferase in the cleft, and some are reuptaken by specific transporters on the presynaptic cell. DAT, NET, and SERT are members of the solute carrier 6 (SLC6) transporter family. While SERT is highly selective to serotonin, DAT and NET are less selective; their endogenous substrates are dopamine, norepinephrine, and epinephrine. Importantly, the force driving monoamine transporters to uptake neurotransmitter is the concentration gradients of Na+ and Cl− [+ and one Cl− with one dopamine, and NET transports one Na+ and one Cl− with one norepinephrine. SERT transports one Na+ and one Cl− with one serotonin and anti-transports one K+. The crystal structures of a bacterial leucine transporter (LeuT) in the SLC family have been determined, revealing that a substrate (leucine) binding site is located between the first and sixth transmembrane domains [Monoamine transporters, including the dopamine (DAT), norepinephrine (NET), and serotonin transporters (SERT), are localized to presynaptic sites, where they terminate monoamine transmission signaling via reducing the synaptic concentrations of neurotransmitters and maintain presynaptic monoamine storage . Neurotr and Cl− . DAT tra domains .Monoamine transporters are important therapeutic targets . DAT is v/v) fetal bovine serum , penicillin , and streptomycin  as well as selection antibiotics, geneticin G418  in a humidified 5% CO2 incubator at 37 °C. Cells were passaged every three days. Eighteen to twenty hours prior to the cell-based uptake assay, cells were seeded onto the poly-L-lysine-coated  96-well black wall/clear-bottom plates  at a density of 5 × 104 cells per well.HEK293 cells stably expressing human DAT (HEK-hDAT), human NET (HEK-hNET), or human SERT (HEK-hSERT) were kindly provided by Professor Bryan Roth from the University of North Carolina at Chapel Hill. Cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% . Fluorescent neurotransmitter substrates were added to cells and incubated for 10 min at room temperature. The changes in intracellular substrate concentrations were monitored for 30 min as a ratio of the measured fluorescent intensity to the initial fluorescent intensity (Ft/F0). In detail, cells were selectively exposed to 440 nm light for excitation, and the emitter fluorescence light through a 515 nm long-pass filter was passed with a freezing digital CCD camera mounted on the device. Data were collected every 90 s at 520 nm using a digital fluorescent analyzer. For the uptake inhibition assay, cells were pretreated with test inhibitors for 15 min in a humidified 5% CO2 incubator at 37 °C prior to the substrate addition. Vanoxerine (GBR12909), nisoxetine, and fluoxetine  were used as selective drugs for DAT, NET, and SERT, respectively. To obtain the dose–response curves, the percent transporter activity (% of control activity) was calculated using the fluorescence ratios (Ft/F0) at 30 min for the inhibitor-treated and untreated cells. All data were acquired and analyzed using an FDSS6000 system and its software.Dopamine, norepinephrine, and serotonin uptake activities were measured using a Neurotransmitter Transporter Uptake Assay Kit   with an https://alphafold.ebi.ac.uk/ (accessed on 13 May 2022)). AutoDock Vina (https://vina.scripps.edu/ (accessed on 13 May 2022)) was employed to predict the complex structure between hDAT and the inhibitors. Protein dynamics was simulated using the GROMACS MD simulation package 2022.1 (https://www.gromacs.org/ (accessed on 13 May 2022)).The three-dimensional structure of human DAT was obtained from the AlphaFold Protein Structure Database , where %Ap value of <0.05 was considered statistically significant, where * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NS is not significant.All results are presented as the mean ± SEM. The significance of observed differences was evaluated using Kruskal–Wallis and post hoc Dunn’s tests. A t/F0) in To investigate the effects of multiple drugs on monoamine transporter inhibition, selective drugs for each monoamine transporter were tested using HEK293 cells expressing DAT, NET, or SERT. The time-dependent uptake of neurotransmitters was observed in all of the three transporters, which is presented as a ratio of the measured fluorescent intensity to the initial fluorescent intensity  was measured in the presence of other drugs at their ICuoxetine . Quantit potency .min) values of DAT by vanoxerine seem to also be affected by co-treatment with other drugs . Thus, choline does not appear to have any significant effects on DAT in vivo. It is also worth noting that NMDG produces a stiff sigmoidal curve with an apparent Hill coefficient (h) of 11, while other ions have h values of 1. This suggests that DAT has multiple binding sites for NMDG.To further investigate the antagonistic effects of multiple inhibitors, we examined ion dependency in DAT inhibition by vanoxerine. First, various ions, different in size and charge, were tested for DAT inhibition a. Both md charge . Overall50 values of each monovalent ion were used, i.e., 3.7 mM NMDG, 36.6 mM choline, 1.4 mM gluconate, or 3.3 mM isethionate. Like the multiple drug effects described above, monovalent ions reduced vanoxerine potency by two to three folds. In particular, isethionate appeared to alter DAT inhibition by vanoxerine the most, increasing the IC50 value by three folds. This might be because isethionate contains a chemical moiety with a sulfur atom, as observed in some atypical DAT inhibitors. This result is consistent with that of a previous report that isethionate reduced the binding of a DAT inhibitor, mazindol, by more than 50% [Then, DAT inhibition by vanoxerine was examined in the presence of monovalent cations, NMDG or choline, or in the presence of monovalent anions, gluconate or isethionate . For thethan 50% . Cholinethan 50% ,14,15,16Molecular docking studies with the X-ray crystal structures of LeuT have revealed that inhibitors bind either to the central binding site (S1 site) that overlaps the substrate (leucine) or to the secondary binding site (S2 site) in the extracellular vestibule ,18,19. DIn order to describe this antagonistic effect at atomic scale using the three-dimensional structure of hDAT, a molecular modeling study was conducted with the hDAT model. The S1 site is located at the center of the transport pathway of hDAT, and the S2 site is reported to be at the entrance of the transport pathway. Docking simulations were performed on sites S1 and S2, respectively, to predict the binding sites of nisoxetine and fluoxetine in the presence of vanoxerine. Based on the docking results, it was predicted that vanoxerine preferentially binds to the S1 site of hDAT, but nisoxetine and fluoxetine are expected to bind to the S2 site in the presence of vanoxerine a–c.As a result of calculating the average binding energies of vanoxerine by analyzing the simulation trajectories, the binding energy of vanoxerine to hDAT is weakened when nisoxetine or fluoxetine is bound to the S2 site d. This eIn the present work, we observed the antagonistic effects of multiple drugs on DAT inhibition by monoamine transporter selective inhibitors. It was also observed in a different class of inhibitors, monovalent ions. Also, molecular modeling studies were performed to understand the antagonistic effects of vanoxerine with nisoxetine or fluoxetine. This persistent antagonistic effect on DAT inhibition observed here suggests that a combinatorial therapeutic strategy using monoamine reuptake inhibitors should be carefully considered. Since this study has a limitation in that multiple drugs were only tested on the cells stably overexpressing monoamine transporters, it is necessary to conduct additional verification on primary neuronal cells in the future. Moreover, if the arguments proposed in this study can be verified through in vivo animal models mimicking DAT-related diseases such as depression and schizophrenia, they can be extended to the individual level.


Meta-analysis and systematic review.Robot-assisted pedicle screw placement technique offers greater accuracy than the traditional freehand screw placement technique. However, it is controversial whether there is a difference between the two procedures in terms of improved clinical outcomes.We systematically searched PubMed, EMBASE, Cochrane, and Web of Science to identify potentially eligible articles. Indispensable data such as the year of publication, study type, age, number of patients, sex distribution, and outcomes were extracted. The outcome indicators of interest included Oswestry disability index (ODI), visual analog scale (VAS) score, operative time, intraoperative blood loss, and post-operative length of stay. RevMan 5.4.1 was used for the meta-analysis.P = 0.0003) and ΔODI , robot-assisted pedicle screw placement technique scored higher than traditional freehand technique. Additionally, the intraoperative blood loss  and the length of hospitalization  for patients who underwent robotic-assisted pedicle screw placement were less than that of those who underwent the conventional freehand screw placement. No significant difference was found between robot-assisted techniques and conventional freehand techniques in pedicle screw placement in surgical time .A total of eight studies with 508 participants were included. Eight were related to ΔVAS, six were related to ΔODI, seven were related to operative time, five were related to intraoperative blood loss, and seven were related to the length of hospitalization. The results showed that, in terms of ΔVAS (95% CI, −1.20 to −0.36, Robot-assisted technique helps improve short-term clinical outcomes, reduce intraoperative blood loss and patient suffering, and shorten recovery time compared to the freehand technique. Pedicle screw fixation is an effective treatment option for all spinal disorders. Pedicle screw placement provides an excellent three-column fixation and is widely used in spinal surgery. However, the traditional freehand screw placement method has limitations because the operator's field of vision and body posture are limited by space, which affects the accuracy of screw placement and can lead to pedicle violations. The misplacement rate of conventional freehand pedicle screws ranges from 5 to 41% in the lumbar spine and 3% to 55% in the thoracic spine . Feng etHowever, whether robot-assisted techniques are superior to freehand techniques in terms of post-operative clinical outcomes remains unclear. Only a few reviews and meta-analyses have focused on this issue. Karamian et al.  concludeThis meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis procedures. We systematically searched the PubMed, EMBASE, Cochrane, and Web of Science databases to identify potentially eligible articles. All the databases were updated on November 10, 2022. Medical subject headings (MeSHs) and free-text words were used to search for potential literature. The PubMed, EMBASE, Cochrane, and Web of Science databases were searched using the following keywords: ‘‘robotics’’ (or ‘‘robot’’ or ‘‘robotic’’ or ‘‘robotics’’) (MeSH) and ‘‘pedicle screws’’  (MeSH) . The exclusion criteria were as follows: duplicate publications; articles without traditional freehand pedicle screw placement in the control group; and articles that did not include patient visual analog scores, Oswestry disability index, and post-operative length of stay. Both RCTs and retrospective cohort studies (RCSs) were eligible for inclusion. Only human studies were considered. The inclusion of studies was not limited by sample size or publication type. Review articles and commentaries were excluded from the analysis.The two reviewers independently assessed all included studies according to the Cochrane risk-of-bias criteria and the Newcastle–Ottawa Quality Assessment Scale using the risk-of-bias tool. The Cochrane risk-of-bias criteria were used to assess the quality of RCTs in terms of selection, performance, detection, attrition, reporting, and other biases. We defined other biases as differences in baseline characteristics between the experimental and control groups. Retrospective cohort studies were evaluated using the Newcastle–Ottawa Quality Assessment Scale, rated from 0 to 9 stars. Six stars or higher indicated sufficiently high quality.The two reviewers independently performed data extraction. Disagreements were resolved through discussion or consultation with the third reviewer. Indispensable data such as the year of publication, study type, age, number of patients, sex distribution, and outcomes were extracted. The outcome indicators of interest included ODI, VAS score, operative time, intraoperative blood loss, and post-operative length of stay.The process of inclusion of these studies is illustrated in Fig. P = 0.0003; Fig. P = 0.004; Fig. Eight studies provided data on pre- and post-operative VAS scores; seven studies provided data on pre- and post-operative ODIs. After obtaining the data from each study, we calculated the ΔVAS and ΔODI for each study, using statistical methods. The results showed that in terms of ΔVAS (SMD = −0.78, 95% CI, −1.20 to −0.36, P = 0.10; Fig. Seven studies provided data on operative time. No significant difference was found between robot-assisted techniques and conventional freehand techniques in pedicle screw placement in surgical time (SMD = 12.04, 95% CI, −2.24 to 26.32, P = 0.02; Fig. Five studies provided data on intraoperative blood loss. The results showed that the intraoperative blood loss for patients who underwent robotic-assisted pedicle screw placement (SMD = −75.64, 95% CI, −140.34 to −10.94, P = 0.01; Fig. Seven studies provided data on the length of hospitalization. The results showed that the post-operative length of hospitalization for patients who underwent robotic-assisted pedicle screw placement (SMD = −1.45, 95% CI, −2.59 to −0.31, Robot-assisted pedicle screw placement has been designed to improve the accuracy and safety of pedicle screw placement. Theoretically, robot-assisted screw placement technology has the advantages of high precision, repeatability, and fatigue resistance compared with traditional freehand screw placement technology. A meta-analysis  was condIn this meta-analysis, eight studies with a total of 2408 screws were included to compare the differences between robot-assisted and traditional freehand pedicle screw placement methods in terms of five clinical indicators: ΔVAS, ΔODI, operative time, intraoperative blood loss, and length of hospitalization. The results for four metrics were significantly different between the two methods. The robot-assisted technique was superior to the traditional freehand screw placement technique in four metrics. Hence, the results recommend the use of the robot-assisted technique for screw placement.Notably, we compared the difference between the pre- and post-operative VAS scores and ODIs of patients who underwent the two procedures. Because the VAS scores and ODIs gradually decrease with time, as reported in previous studies, there is no significant difference in long-term clinical outcomes between the two surgical techniques . It is eThe original data of each clinical study were not available; therefore, we used statistical methods to calculate the mean and standard deviation of ΔVAS and ΔODI in each study. The calculation of VAS score is presented as an example of a specific calculation method.The pre-operative visual analog score was set as In previous studies, patients were followed up over time to investigate the advantages of robot-assisted screw placement technique over the traditional freehand screw placement technique in terms of clinical outcomes. The follow-up results were compared directly. Su et al.  and Lee We believe that the ultimate goal of developing orthopedic surgical robots is to reduce patient suffering and that patients' subjective perceptions of post-operative recovery outcomes are an important measure of the success of robot-assisted technology. This study identified significant differences between the robot-assisted and freehand screw placement techniques as indicators of short-term post-operative clinical outcomes. The results showed that the robot-assisted screw placement technique was more effective in reducing the patient's VAS score, ODI, and intraoperative blood loss than the freehand screw placement technique and thus reduced the patient's post-operative hospital stay. Furthermore, the robot-assisted screw placement technique did not significantly prolong the operative time.The present study has some limitations. First, four of the included studies were retrospective cohort studies, and the level of evidence was not as high as that of RCTs. Second, only two robot models were included in this study, and six of the eight included studies used the same robot model, which may have rendered the conclusions of this study inapplicable to other robot models. Despite these limitations, all RCSs included in this meta-analysis had scores ≥ 7 stars and were of high quality.The robot-assisted pedicle screw placement technique showed a significant reduction in the patients’ VAS scores, ODIs, intraoperative blood loss, and post-operative length of hospitalization compared with the traditional unassisted technique. And the robot-assisted screw placement technique did not significantly prolong the operative time. These results suggest that robot-assisted techniques help improve short-term clinical outcomes, reduce patient suffering, and shorten recovery time compared with the freehand technique.


This study aimed to compare whether Calcium phosphate cement (CPC) promotes the stability of osteoporotic lumbar pedicle screw by enhancer-injecters with different number of holes.Through a self-designed bone cement injection device, the pedicle screw canal was strengthened with calcium phosphate bone cement, and divided into 4-hole group, 6-hole group, 8-hole group, straight pore group and the control group. The screw was inserted into the mechanical test module, the Maximum insertion torque and Maximum axial pull-out strength were recorded, and the distribution of calcium phosphate bone cement was analyzed by CT and X-ray. The data results were analyzed using SPSS19.0 statistical software package.The distribution of bone cement in different reinforcement groups was different and showed regularity. The bone cement in the 4-hole group was roughly located in the head 1/3 of the screw, the 6-hole group was located in the middle 1/3 of the screw, and the 8-hole group was located in the caudal 1/3 of the screw. Compared with the control group, the maximum axial pull-out force of screws in the lateral hole and full screw tunnel reinforcement group was significantly increased. There was no significant difference between the 4-hole, 6-hole and straight pore groups. There was no difference in the screw-in torque between the reinforcement groups, and they all increased significantly compared with the control group, and the difference was statistically significant. After the screw was pulled out, the interface between the bone cement and the polyurethane material was fractured, and a tight package was formed with the screw.Enhancer syringes with different hole numbers combined with CPC bone cement injection can significantly increase the maximum screw pull-out force. The 8-hole group has a smaller pull-out force and is relatively prone to leakage of reinforcing material, which lacks safety in use. The local reinforcement of 4-hole and 6-hole sheath can play a similar role to that of total nail tunnel reinforcement. Vertebral compression fractures (VCF) are one of the most common fragile fractures and often require internal fixation . PedicleSince the chemical composition of calcium phosphate cement is the same as that of bone mineral , it is oIn our study, previously designed enhancer-injecters with different number of holes were used to verify the effect of CPC injection on the augmentation of pedicle screw in the Chinese osteoporotic lumbar pedicle channel through biomechanical analysis .In this study, we used the biomechanical test module  synthesized by polyurethane material, which had the characteristics of isotropy and homogeneity. Furthermore, this module contains more than 95% open pore structure with an average pore size of 1.5 to 2.5 mm, simulating the cellular structure of cancellous bone under osteoporosis and suitable for penetration of injectable bone cement materials.In this study, we performed biomechanical experiments based on the three groups, including a local augmentation group, a full-length augmentation group, and a control group. We designed the hole of the local augmentation group to be located on the side wall of the sheath, which including 4-hole group, 6-hole group and 8-hole group. Then We designed a full-length augmentation group consisting of injectors with straight pore but no side holes. The design of enhancer injecter, bone cement injector, straight pore design can be seen in our previous study [According to the difference of whether bone cement was injected or not and the number of holes in the syringe with fortification agent, they were divided into 5 groups, namely, 4-hole group, 6-hole group, 8-hole group, straight pore group and the control group. The sample size of each group was 6.The center point was selected according to the plane of PU module 13 cm×4 cm, and the pedicle screw path was prepared conventionally. Use a 3.5 mm diameter hand drill to carefully place holes on the module. Ensure that the drill bit is always perpendicular to the module. The entry depth is 45 mm. During the process, try to avoid the shaking of the hand drill and the expansion of the nail path. Place holes in all nail paths in turn.We made the calcium phosphate bone cement (CPC) into a paste according to the protocols. The CPC was produced by Shanghai rebone Biological, a mixture of 3.8 g powder and 1.85ml water. For the perfusion of CPC, the lateral foramen sheath inserted into the nail canal was inserted at the same depth to 45 mm, and the CPC was pushed into the lateral foramen sheath at a constant speed. The sheath is then left still, unattached to the syringe, and the pushrod is quickly inserted into the sheath. The remaining CPC in the sheath was completely perfused into the material surrounding the nail path.We used a torque wrench to make a stable connection to the pedicle screw entrer. Screw the screw into the mechanical test module at 3 RPM according to ASTM F 543-02. And record the maximum torque value.The pedicle screw extraction force test was performed at a speed of 5 mm/min according to ASTM F 543-02. When the screw is pulled out after the module is damaged, it is defined as the maximum axial pulling force of the screw.SPSS19.0 statistical software was used for statistical analysis. The maximum screw torque and maximum axial pulling force were expressed by measurement data (mean ± standard deviation) and one-way analysis of variance was used. SNK-q test was used for comparison between groups, and P < 0.05 meant the difference was statistically significant.The SNK-q test showed that the maximum torque of 4-holes, 6-holes, 8-holes and straight pore groups was significantly different from that of control group P < 0.01). There was no significant difference in the torque between the groups 4-holes, 6-holes, 8-holes and straight pore group (P > 0.05). The comparison of screw extraction forces among different groups was conducted by one-way analysis of variance and SNK-q test. The results showed that the 4-holes, 6-holes groups and straight pore group were higher than the 8 well group, and the differences were statistically significant (P < 0.01). However, there was no significant difference in pairwise comparison among all groups in 4-holes, 6-holes and straight pore groups (P > 0.05). There were significant differences between all groups and control group (P < 0.01)  bone cement can provide effective axial extraction force as a common method . HoweverOur study found that the maximum torque value increased significantly after material perfusion, from 0.06 N·m to 0.12 N·m. This is closely related to the surrounding porous cancellous bone-like void structure filled by bone cement injection. However, because the amount of bone cement injected into the lateral pore sheath with different number of holes is similar, the distribution position is different, and the shape and scale of the inserted screws are the same, which are cylindrical screws rather than conical screws, so the influence on the maximum torque may not be very great. The insertion of the pedicle screw on the strengthened cancellous bone module is equivalent to the insertion of the screw into the bone with increased bone density, thus enhancing the maximum torque of the insertion .The biomechanical test module used in this study is composed of polyurethane material. It has the characteristics of uniform material and uniform density, which can reduce the influence of bone density factors on the extraction force of pedicle screw and reduce the bias of experimental results, which has been recognized as a commercial mechanical test material in the world , 12. It We found that the distribution of calcium phosphate cement around the screw showed a certain regularity after the juxtaposition of different lateral pore sheathes. The distribution of hardened bone cement blocks in the 4-holes, 6-holes and 8-holes groups was almost helical. The four holes were distributed in about 1/3 of the distal end of the screw, the six holes were distributed in about 1/3 of the middle part of the screw, and the eight holes were distributed in about 1/3 of the proximal end of the screw. According to the shape of the wrap around the pedicle screw after CPC injection, the wrap formed in the 8-holes group was roughly located at the screw head. In clinical application, bone cement overflow was likely to occur due to its proximity to the spinal canal. The main factors affecting the maximum axial pull-out strength are the shear strength between the material and the screw, the strength of the surrounding medium, the resistance formed by the proximal “bone” of the CPC along the axis of the screw after the wrapping of the screw and the material, and the shear force formed during the pulling out of the CPC bone cement and the surrounding medium , 15. TheEnhancer syringes with different hole numbers combined with CPC bone cement injection can significantly increase the maximum screw pull-out force. The 8-hole group has a smaller pull-out force and is relatively prone to leakage of reinforcing material, which lacks safety in use. The local reinforcement of 4-hole and 6-hole sheath can play a similar role to that of total nail tunnel reinforcement.


In the present study, the concentrations of flumatinib and its major metabolite M1 were rapidly determined using an stable ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method. The half-maximal inhibitory concentration (IC50) was 6.66 μM in human liver microsomes (HLM), while 0.62 μM in rat liver microsomes (RLM) and 2.90 μM in recombinant human CYP3A4 (rCYP3A4). Furthermore, the mechanisms of inhibition of flumatinib in human liver microsomes, rat liver microsomes and rCYP3A4 by isavuconazole were mixed. Moreover, ketoconazole, posaconazole, and isavuconazole showed more potent inhibitory effects than itraconazole, fluconazole, and voriconazole on HLM-mediated flumatinib metabolism. In pharmacokinetic experiments of rats, it was observed that isavuconazole could greatly change the pharmacokinetic parameters of flumatinib, including AUC(0−t), AUC(0−∞), Cmax and CLz/F, but had no effect on the metabolism of M1. According to the results of in vitro and in vivo studies, the metabolism of flumatinib was inhibited by isavuconazole, suggesting that isavuconazole may raise the plasma concentration of flumatinib. Thus, it is important to take special care of the interactions between flumatinib and isavuconazole in clinical applications.As the validated agent for the treatment of chronic myelogenous leukemia (CML), flumatinib is a novel oral tyrosine kinase inhibitor (TKI) with higher potency and selectivity for BCR-ABL1 kinase compared to imatinib. Many patients experience aspergillosis infection and they may start using isavuconazole, which is an inhibitor of CYP3A4. However, there is no study on their interaction  Chronic myelogenous leukemia (CML), also known as chronic granulocytic leukemia, usually occurs in or after middle age which is a slowly progressive disease of the blood and bone marrow . A recipFlumatinib is a new oral TKI that is more selective and potent for BCR-ABL1 kinase compared to imatinib . It was The important cytochrome P450 (CYP450) enzyme participating in the metabolism of flumatinib is CYP3A4. There are two main metabolites after treatment with CYP3A4, namely, M1 (N-desmethyl flumatinib) and M3 (amide hydrolysis product) . M1 has Various microbial-induced infections are the most common complications of leukemia and may have a serious impact on the survival rate of patients, where fungal secondary infections are one of the most devastating infections . In 2015in vivo. The findings are anticipated to provide basic data to promote the clinical application of flumatinib favorably.Isavuconazole is a substrate and moderate inhibitor of CYP3A4 , and fluFlumatinib , and M1 m/z 563.1 → 463.0, m/z 549.3 → 462.9, and m/z 488.0 → 401.1 for flumatinib, M1 and dasatinib, respectively.Ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) equipped with a Waters Acquity UPLC BEH C18 column  was used to detect the concentrations of flumatinib and M1. Quantification of the analytes was made by using a Waters XEVO TQS triple quadruple mass spectrometer with a multiple reaction monitoring (MRM) in positive ion mode. The mobile phase was consisted of 0.1% formic acid aqueous solution (A) and acetonitrile (B) at a flow rate of 0.4 mL/min. An elution program of 90% A (0–0.5 min), 90%–10% A (0.5–1.0 min), 10% A (1–1.5 min), 10%–90% A (1.5–1.6 min) and 90% A (1.6–2 min) was performed. The whole elution took 2 min. During the analysis, the temperature of the column and autosampler rack was controlled at 40°C and 10°C, respectively. In addition, optimized parameters for the mass spectra of flumatinib, M1 and dasatinib included: cone voltage were all 10 V, and collision energy was 20 eV, 25 eV, 20 eV, respectively. The monitoring transitions were in vitro liver microsome incubation system was set up, then the drug was added to the system and incubated for 45 min as to determine the metabolism of the drug by the microsomes. With a total volume of 200 μL, the system contained 1 M PBS, flumatinib , HLM (0.3 mg/mL), and NADPH (1 mM) in the HLM, while the RLM system contained 1 M PBS, flumatinib , RLM (0.4 mg/mL) and NADPH (1 mM). In addition, the rCYP3A4 system contained 1 M PBS, flumatinib , rCYP3A4 (0.5 pM) and NADPH (1 mM). The three systems were used to detect the Michaelis-Menten constant (Km) values of flumatinib in HLM, RLM and rCYP3A4, respectively. The NADPH-free mixture was preincubated at 37°C for 5 min, followed by the addition of 1 mM NADPH to activate the reaction. The mixture was incubated at 37 °C for 40 min until 20 μL of IS solution (200 ng/mL) and 400 μL of cool acetonitrile were added. It was then centrifuged at 13,000 rpm for 5 min. After that, 100 μL of the supernatant was taken to conduct the analysis by UPLC-MS/MS.An m values. Then, we determined the half-maximum inhibitory concentration (IC50) of isavuconazole, and the concentration of isavuconazole was set to 0, 0.01, 0.1, 1, 10, 25, 50 and 100 μM in HLM, RLM and rCYP3A4. Ketoconazole, a strong inhibitor for CYP3A4, was used as a positive control. In addition, several additional azoles  in RLM and HLM were also evaluated for their IC50 on flumatinib metabolism.For the detection of possible DDIs between flumatinib and isavuconazole, the concentrations of flumatinib were 2.17 µM, 29.28 µM, and 3.06 µM in RLM, HLM and rCYP3A4, respectively, according to their K50 curves for flumatinib were obtained from the incubation system described above. In vitro inhibition mechanisms were investigated in microsomes. In HLM, the concentrations of isavuconazole and ketoconazole were set to 0, 2.66, 6.66, 13.31 μM and 0, 0.0049, 0.049, 0.098 μM, respectively, and the concentrations of flumatinib were established as 1.46, 5.86, 11.71 and 29.28 μM. In RLM, the concentrations of isavuconazole and ketoconazole were set to 0, 0.15, 0.62, and 1.23 μM and 0, 0.08, 0.16, 0.32 μM, respectively, and the levels of flumatinib were established as 0.55, 1.1, 2.2, and 4.4 μM. In rCYP3A4, the concentrations of isavuconazole were set to 0, 1.16, 2.90, and 5.80 μM, and the levels of flumatinib were established as 0.33, 1.32, 2.64, and 6.60 μM.The ICn = 5): 60 mg/kg single oral dose of flumatinib (Group A); orally taken 20 mg/kg isavuconazole and 60 mg/kg flumatinib (Group B). The rats in both groups started fasting for 12 h ahead of the experiment, but were free to water. During the experimental day, group B was orally administered with isavuconazole dissolved in 0.5% carboxymethylcellulose sodium salt (CMC-Na) solution, while group A was orally administered with the same dose of CMC-Na solution. After administration of isavuconazole or CMC-Na for 30 min, all groups were given 60 mg/kg flumatinib. Tail vein blood samples were collected from rats in both groups at multiple time points . Subsequently, an additional 300 μL acetonitrile and 20 μL dasatinib (200 ng/mL) were added to 100 μL plasma. Then the mixture was vortexed to mix thoroughly for 2 min and centrifuged at 13,000 rpm for 10 min to obtain the final supernatant for UPLC-MS/MS analysis.Sprague–Dawley male rats  were purchased from the Animal Experimental Center of The First Affiliated Hospital of Wenzhou Medical University . Ten SD rats were then divided randomly and equally into two groups . Non-compartmental analysis was performed by using Drugs and Statistics (DAS) software  to obtain pharmacokinetic profiles. Mean plasma concentration-time curves were generated using Origin 8.0, and the parameters were statistically investigated between groups using independent samples t-test by SPSS 24.0 software . All data were expressed as mean ± SD and p < 0.05 was considered statistically significant.ICA successful method for the quantification of flumatinib and its metabolite M1 in plasma was developed. Flumatinib, M1 and dasatinib were all effectively separated without interfering with each other, with retention times of 1.13, 1.13 and 1.19 min, respectively. In the range of 1–500 ng/mL for flumatinib and 1–100 ng/mL for M1, the standard calibration curves for both flumatinib and M1 had correlation coefficients greater than 0.99. The lower limit of quantitation was 1 ng/mL for both compounds.m values of flumatinib in HLM, RLM and rCYP3A4 were 29.28 μM, 2.20 μM and 3.06 µM, respectively. The obtained IC50 by calculation were 6.66 μM, 0.62 μM, and 2.90 μM for HLM, RLM and rCYP3A4, respectively. These results could be found in The K50 values of ketoconazole in HLM and RLM were 0.05 μM and 0.16 µM, respectively, with the Ki value of 0.53 μM and 0.33 μM. And, the inhibitory mechanisms of ketoconazole on flumatinib metabolism were mixed inhibitory mechanisms in both HLM and RLM. These results were presented in 50 values of other azoles  in RLM and HLM, could be found in In addition, the ICin vivo were presented in (0–t), AUC(0–∞) and Cmax were 1.72, 1.88 and 1.70-fold, while CLz/F was 48% lower than those of group A, respectively . However, it was not found the pharmacokinetic parameters of M1 were significantly different between the two groups of rats.The pharmacokinetic parameters of flumatinib and M1 were shown in CML is a myeloproliferative neoplasm that constitutes roughly 15% of newly diagnosed leukemia cases in adults . As a noIsavuconazole, an azole antifungal drug, is suitable for the first-line option to treat invasive aspergillosis and the second-line choice for treating mucormycosis . Like otin vitro and in vivo. Compared to the control group, it was demonstrated that isavuconazole inhibited flumatinib to 7.75% in RLM. In vitro, the IC50 value of flumatinib by isavuconazole in HLM, RLM and rCYP3A4 were 6.66 μM, 0.62 μM and 2.90 μM, respectively. It could be presumed that the metabolism of flumatinib in vitro was inhibited by isavuconazole. As a further study, the mechanisms of inhibition between these two drugs were investigated, which was revealed that the inhibition of flumatinib metabolism in three systems by isavuconazole was a mixed type. In addition, the constant inhibition Ki of isavuconazole on flumatinib in HLM, RLM, and rCYP3A4, were 3.59, 1.44, and 5.48 μM, respectively. The higher inhibition potential of isavuconazole in RLM with a lower Ki value pointed out that it is more likely to display DDIs in RLM than in HLM and rCYP3A4.Although increased systemic exposure to flumatinib has been reported with voriconazole, the co-administration of flumatinib with isavuconazole has never been investigated . In the in vitro inhibitory effects were found at distinct degrees. Of note, ketoconazole showed the more potent inhibitory effects than isavuconazole on flumatinib metabolism. Also, based on the IC50 values of HLM-mediated metabolism by all azoles, ketoconazole, posaconazole, and isavuconazole, had stronger inhibitory capacities than itraconazole, fluconazole, and voriconazole on the metabolism of flumatinib.In addition, the inhibitory effects of five additional azoles on flumatinib metabolism were investigated in HLM and RLM, in which ketoconazole was used as a positive control for it was found to be the most potent inhibitor of flumatinib metabolism in RLM. The results clearly showed that (0−t), AUC(0→∞) and Cmax of flumatinib were increased by isavuconazole in vivo, and these parameters were 1.72, 1.88 and 1.70 times higher than those in the control group. Other pharmacokinetic important parameter, such as CLz/F, was decreased significantly in the combined dose group. No statistical significances were found between the combination and control groups in investigating the pharmacokinetic parameters of the metabolite M1. With these outcomes, it was clear that isavuconazole increased the exposure of flumatinib in rats.The AUCClinically, many patients may use flumatinib and triazole antifungal agents concurrently because secondary fungal infections are prone to occur during the use of flumatinib. Hence, there are profound clinical importance in studying their interactions. The results of the current study could also provide an accurate application basis for the combination of the two drugs in the future. Further research should be illustrated the impact of isavuconazole on the flumatinib in humans.in vivo and in vitro. And, mechanisms of in vitro inhibition in HLM, RLM and rCYP3A4 were a mixture of competitive and non-competitive inhibition. In HLM-mediated metabolism by all azoles, ketoconazole, posaconazole, and isavuconazole, had stronger inhibitory capacities than itraconazole, fluconazole and voriconazole on the metabolism of flumatinib. In addition, it was demonstrated that isavuconazole remarkably changed the main pharmacokinetic profiles of flumatinib, increasing the systemic exposure in rats. The current study found that the interaction between isavuconazole and flumatinib, which may be helpful for therapeutic drug monitoring, clinical dose reference and provide a valuable tool for drug-drug interactions. The rat in vivo study does not predict accurately the in vivo situation in humans, since the contribution of rat CYP3A enzymes to metabolism of CYP3A4 substrates is different from that in humans. Further research should be done to confirm this result in clinical studies.To summarize, isavuconazole had an inhibitory effect on flumatinib metabolism


HIV and gender-based violence (GBV) are syndemic in sub-Saharan Africa and provision of support for participants who disclose GBV constitutes part of comprehensive care. Consequently, a process was undertaken to develop, implement, and evaluate standard operating procedures (SOPs) in MTN-025/HOPE, a study of the dapivirine vaginal ring for HIV prevention. The SOP was developed using needs assessment surveys in addition to World Health Organization (WHO) guidelines and other literature. Sites tailored and implemented the SOP through HOPE implementation. At study end, staff reported increased training 32/35 (91.43%); improved confidence ; and improved vicarious trauma prevention onsite . Leadership reported increased staff competence in GBV response. Obstacles included limited referral organizations and time for follow-up, continued training needs, and cultural norms. Development and implementation of an SOP is a feasible strategy to build a GBV response to improve health systems and support sustained effective use of HIV prevention products.The online version contains supplementary material available at 10.1007/s10461-022-03864-6. Research into antiretroviral-based HIV pre-exposure prophylaxis (PrEP) methods has expanded prevention possibilities, introducing a variety of user-initiated methods—and efforts to support effective use of these methods—that are key to reducing HIV incidence among women, who have been disproportionately impacted by the pandemic . In MarcDue to the syndemic relationship between HIV and GBV, multiple international donors have recently recognized that expanded and improved GBV response and prevention is a key component of comprehensive HIV services . SimilarMulti-disciplinary groups, such as sexual assault response teams, have long been used to enhance the quality, accessibility, and predictability of services for violence survivors , 19. TheIn MTN-025/HOPE, an open-label extension trial of the ring among women in sub-Saharan Africa, it was thought that open conversation and disclosure fostered by study-specific counseling, coupled with routine questions examining experiences of violence in a context of high rates of GBV within communities where the study took place and among likely participants, would increase the likelihood of GBV disclosure during study participation . To bettThe HIV Open-label Prevention Extension (HOPE) study NCT02858037) was a phase 3B trial of the ring for HIV prevention, which enrolled 1456 former participants of the MTN-020/ASPIRE (NCT01617096) phase 3 trial at 14 clinical research sites in Malawi, South Africa, Uganda, and Zimbabwe . HOPE pa58037 wasTo ensure that site teams were prepared to identify and respond to participants who reported GBV, the study management team conducted two needs assessment surveys—one among site staff and one among site leadership—and developed and implemented a template GBV Response SOP based on identified needs, best practices in multidisciplinary GBV response, and international guidelines regarding provision of services for individuals experiencing GBV , 22. BecTo understand existing resources and needs at HOPE sites, and inform SOP development, site team members were requested via email to complete two needs assessment surveys in Qualtrics. Surveys were anonymous and contained 10–15 multiple choice, LIKERT scale, and open-ended questions, depending on respondent experience and resources to report. Sites were requested to assign 3–4 individuals to complete the survey: 1 leadership representative  and 2–3 staff per site including counselors, nurses, interviewers, and community educators who most commonly interacted directly with participants . This sampling was predicted to be broadly representative as each site had 1–3 individuals in study leadership and, while sites likely had more than 15 staff assigned to the study, only about 5–10 of these staff had regular in-depth interactions with HOPE participants. The survey addressed: GBV training, referral resources and staff requirements for understanding these resources, confidence in responding to GBV, standardized procedures for GBV response, and feelings of support within their teams. The leadership survey, completed by site investigators of record (IoRs), study coordinators, sub-investigators, and site leaders, focused on GBV training (including trainers and follow-up requirements), referral partners, standardized GBV response procedures, and mechanisms for receiving participant feedback on referral organizations.The HOPE GBV Response SOP template provided a framework for sites to increase basic staff training on the dynamics of GBV and the provision of first-line, trauma-informed care, expand and strengthen referral networks through investigation and relationship development, improve staff support to prevent vicarious trauma by establishing regular debrief sessions and psychological support options, encourage participant sensitization on GBV, and build a shared understanding of staff roles and site approaches to identifying and responding to GBV among participants .Key resources, such as the WHO Clinical and Policy Guidelines for Responding to Intimate Partner Violence and Sexual Violence Against Women and United Nations Population Fund Multi-Sectoral Response to GBV Guide, were cross-referenced with results of the needs assessment surveys to identify key components for the SOP template , 36. ThiStudy management supported sites to adapt the SOP template to reflect local resources, site structure, and staff roles. Site teams reviewed and revised lists of referral organizations based on existing needs and template SOP recommendations and conducted outreach when needed. Site leadership engaged local organizations to deliver staff trainings and support participant GBV sensitization via on-site visits and attendance at participant engagement activities (PEAs), which also provided an opportunity for participants to become familiar with referral organizations . Where lAt study end, a second set of anonymous surveys was conducted—one each for staff and leadership—comprising 19–25 multiple choice, LIKERT scale, and open-ended questions, depending on referral resources and experience with management team support, to assess SOP adaptation and implementation. Although these surveys were administered to the same categories of staff members and leaders as the first surveys, with the same instructions for participant selection, respondents differed due to staff turnover and availability. Because the surveys were anonymous and respondents were not identified, it is not known how many respondents completed both surveys or the overlap in responses. Topics addressed included trainings, on-site support for staff, confidence in responding to GBV, and standardized site processes.All four surveys were summarized from Qualtrics data using descriptive statistics and synthesis of qualitative responses to open-ended questions. Response proportions were calculated with number of respondents selecting a given response representing the numerator, while total respondents to the question served as the denominator. Comparisons were made between responses on the needs and process evaluation surveys and proportion of respondents selecting a given response to capture changes reported by site staff and leadership. Illustrative quotes highlighting key themes are presented in the Results section. Site PEA reports were reviewed for mention of referral partner engagement and inclusion of GBV-related topics with instances of referral partner engagement summarized.A total of 34 site staff (1–5 per site from 13 of 14 sites) and 17 site leadership representatives (1–3 respondents each from 11 of 14 sites) completed the baseline needs assessment.Of staff, 38% (13/34) had received some previous training in GBV-related topics as part of their work at the research site. Among 26 staff with prior experience supporting participants reporting violence, 21 (80%) had reached out to site leadership for support, 13 (50%) reached out to fellow staff and 6 (23%) contacted outside organizations. Staff were split on standardized responses to GBV: 12 of 26 (46%) reported a standard site-wide process, an additional 12 of 26 (46%) reported following study-specific processes, and 2 of 26 (8%) reported that there was no standard process for GBV response. Nearly all respondents  reported that there was at least one organization in their area that offered services to survivors of GBV via written referral  or walk-in . Only 35% (8/23) of respondents reported using site staff accompaniment during referrals. When asked what they did well when responding to GBV, most respondents mentioned listening  or supportive counseling .Eight (47%) leadership respondents indicated that staff received prior training on GBV prevention, support, and response, with 71% (10/14) indicating that there were no SOPs or other standard resources on-site for responding to participants who disclosed GBV. While nearly all respondents  indicated that their site provided referrals for GBV services, referrals reportedly occurred yearly  or less . Of sites with known referral organizations, 54% (7/13) collected follow-up information on participant contacts with at least some of these organizations. Like staff, site leadership identified counseling as a strength in responding to GBV . Although site leadership indicated few referrals, 58% (7/12) also cited access to referral options as a strength.Finally, respondents were asked what was needed to improve GBV response at their site. Responses to this open-ended question are outlined thematically in Table Because the SOP process was conducted to improve study services, all participants at the 13 sites received care per the SOP. Per standard study practice, all staff cadres were trained on their site-specific SOP. Sites then employed multiple strategies to ensure sufficient GBV training as required by the SOP, including inviting community advisory board members, consultants, and external service organizations to provide trainings, or utilizing online trainings provided by groups such as MOSAIC Training, Service, and Healing Centre and the United Nations High Commissioner for Refugees. Trainings were recommended for all cadres, from outreach workers to clinicians, to foster a supportive research environment. Per SOP recommendations, some sites also initiated multi-disciplinary GBV response teams including staff cadres who received GBV training. Training organizations were added to site referral directories, with staff providing accompanied referrals when accepted by participants.In addition, study management provided two mandatory vicarious trauma trainings for the full protocol team. Of seven sites not already offering no-cost professional counseling to staff responding to GBV, five began doing so as a result of SOP implementation. Sites also shared experiences with providing GBV support, including lessons learned from referral provision and follow-up, and preventing vicarious trauma, via presentations during regular multisite HOPE team meetings. Debriefing and problem solving regarding individual and systemic challenges were also provided directly to counselors and site leadership by study management during site assessment visits.Anonymous process evaluation surveys were completed by 17 site leaders (1–3 respondents at each of the 14 sites) and 32 staff (1–5 staff at 13 of 14 sites) after SOP implementation.“I am now spontaneous in my responses as I am sure of the information I am providing,” mentioned one counselor, continuing that “due to debriefing, I am able to give off my best at every intimate partner violence counseling session.” Improved support for staff was frequently mentioned, with counselors reporting that “I feel more supported by leadership and do not have grey areas,” and “debriefing with other counselors always helps with lifting the burden of being the carrier.” Site leadership also mentioned the importance of a supportive atmosphere, stating “the environment has to be welcoming from the onset… participants need to be clearly aware of the assistance they might get from the study team.”Post SOP implementation, site leadership respondents also reported improvements as illustrated in Fig. As illustrated in Fig. Improvements related to referral networks as reported by both site staff and leadership were minimal, however Fig. . ImproveSite leadership made multiple recommendations for SOP implementation in future trials across all study populations, including integration of GBV-related topics into all participant engagement activities and monitoring and documentation of all instances of GBV and resulting follow-up.Although the SOP encouraged sites to invite organizations to PEAs, only two sites reported that a GBV service organization attended an event—both sites were in Zimbabwe and engaged the same organization. Overall, four sites, two each in South Africa and Zimbabwe, reported carrying out discussions related to GBV with participants during these events—two as a result of SOP implementation, with three of these sites addressing the topic of GBV multiple times. One counselor emphasized the importance of this collaboration: “the participants have managed to acquire more knowledge on how to take care of themselves as we now interact more with the support system since we invite them to address participants [in] retention meetings.”Our experience in HOPE illustrates that the use of tailored SOPs to identify and respond to GBV is a feasible strategy to build standardized GBV services within research sites and potentially within other clinics providing HIV prevention services in a syndemic setting. SOP development can be informed by the use of survey tools and accepted best practices to create a tailored, systematic approach through which services for GBV survivors can be improved, and support systems and clinic atmospheres around GBV can be strengthened to benefit both staff and the populations they serve .HOPE teams were able to utilize their SOPs and reported increased training, improved confidence, and more on-site support to prevent and address vicarious trauma. Leadership at research sites also reported improved response skills among their teams. Enhanced atmospheres of support for participants and staff were noted by site team members when asked to reflect on the SOP process.The combination of needs and process evaluation surveys also allowed the HOPE team to identify ongoing gaps, such as limited time for providing counseling and follow-up to participants. Leadership reports of insufficient referral networks in the process evaluation, and limited engagement of referral organizations at PEA events, highlight the importance of adequate community-based referral networks and ongoing efforts to address societal norms that lead to GBV and represent challenges to the implementation of a GBV Response SOP. While clinic teams can be equipped to provide first-line support and offer a comprehensive network of warm referrals, survivor access to crucial services such as emergency housing and financial assistance, legal and protection services, and ongoing counseling is limited by what is available nearby . EffortsOur assessment of the SOP process aimed to determine what impacts this process had on site GBV response. However, recall bias and social desirability bias due to the timing of the process evaluation and respondent knowledge that study management would have access to survey results may have impacted findings. Because leadership from one site did not respond to the initial needs assessment, it is possible that not all site needs were accounted for during the process. In addition, because the SOP development and implementation process was carried out to improve services for all participants rather than as part of the trial, participants were not randomized to SOP implementation or standard of care. Despite the importance of understanding participant experience, data were not collected regarding the SOP and its use or impact on participant wellbeing to avoid creating an undue data collection burden on participants while allowing for collection of data related to the primary study objectives. Therefore, indicators of improved counseling abilities and increasingly supportive site atmospheres are based on site team report. Collecting data on participant experiences, needs, and recommendations before and after the process, and devoting resources to understanding and applying learning from this data, would enhance SOP development and implementation in future similar efforts. Finally, there were few identified instances of GBV during HOPE, limiting our ability to explore how GBV response guided by SOP implementation related to HIV prevention and adherence to study product.Our experience also suggests that supportive research staff and clinic environments for GBV survivors can be fostered as a complement to biomedical HIV prevention research services, even if extensive resources are not available. Specifically, systematically assessing the needs of research and clinic staff and working to address identified needs through standardized procedures is a low-cost process that can be applied in a variety of settings. Although more research is needed, studies suggest that delivering survivor-centered support to users of biomedical HIV prevention products may support them to continue using PrEP as long as they feel it is necessary, rather than discontinuing use or struggling with consistent use due to partner objection or trauma related to experiences of violence , 7, 43.The HOPE GBV Response SOP Template  implementation experience contributes to ongoing efforts to expand GBV identification and support in research and service provision related to PrEP . The proSupplementary file1 (PDF 188 KB)—MTN-025/HOPE GBV Response SOP Template. Word document distributed to sites for adaptation and implementation during the MTN-025/HOPE study.Supplementary file2 (PDF 436 KB)—CHARISMA-CHOICE SOP for Addressing Partner Relationships and IPV in PrEP Services. Word document version of the SOP developed by the CHOICE Collaboration, finalized in October 2020.Below is the link to the electronic supplementary material.


Outcomes for half of patients with melanoma remain poor despite standard-of-care checkpoint inhibitor therapies. The prevalence of the melanoma-associated antigen chondroitin sulfate proteoglycan 4 (CSPG4) expression is ~70%, therefore effective immunotherapies directed at CSPG4 could benefit many patients. Since IgE exerts potent immune-activating functions in tissues, we engineer a monoclonal IgE antibody with human constant domains recognizing CSPG4 to target melanoma. CSPG4 IgE binds to human melanomas including metastases, mediates tumoricidal antibody-dependent cellular cytotoxicity and stimulates human IgE Fc-receptor-expressing monocytes towards pro-inflammatory phenotypes. IgE demonstrates anti-tumor activity in human melanoma xenograft models engrafted with human effector cells and is associated with enhanced macrophage infiltration, enriched monocyte and macrophage gene signatures and pro-inflammatory signaling pathways in the tumor microenvironment. IgE prolongs the survival of patient-derived xenograft-bearing mice reconstituted with autologous immune cells. No ex vivo activation of basophils in patient blood is measured in the presence of CSPG4 IgE. Our findings support a promising IgE-based immunotherapy for melanoma. IgE antibodies targeting cancer antigens can be used for immunotherapy. Here the authors present an IgE antibody targeting the melanoma-associated antigen, chondroitin sulphate proteoglycan 4, that recognises human melanoma, stimulates tumour cell cytotoxicity, and restricts tumour growth in humanised mouse models. Although CSPG4 expression has been reported in some normal tissues, including in the vascular system, skeletal and cardiac myoblasts, and chondroblasts2, CSPG4 is overexpressed in several solid tumors, including malignant melanoma, subsets of breast cancer, mesothelioma, and neuroblastoma. Therefore, CSPG4 is considered a promising target for cancer-targeting immunotherapies1. Several CSPG4 targeting therapeutics have been evaluated in pre-clinical and clinical studies; showing preliminary efficacy and favorable safety profiles8.The tumor-associated antigen chondroitin sulfate proteoglycan 4 (CSPG4) is a highly glycosylated transmembrane proteoglycan9. Many patients’ tumors do not respond to existing immune and targeted therapies or acquired resistance develops quickly10. Checkpoint inhibitor antibody immunotherapies are designed to activate immune cells irrespective of their antigen reactivity. Therefore, immunotherapies targeting a melanoma-associated antigen, such as CSPG4, may effectively direct immune cells against cancer and address a significant unmet need.Despite substantial progress with the approval of checkpoint inhibitor antibody immunotherapies, the 5-year survival rates remain poor (<55%) for patients with late stage disease18. Strong adaptive immune responses triggered by IgE, may protect from cancer growth23. IgE has a high affinity for its cognate high-affinity Fcε receptor (FcεRI) and slower dissociation compared to IgG for its respective Fcγ receptors. The high affinity of IgE for its Fcε receptors may translate to long retention on immune effector cells in tissues, such as within a tumor, for long periods (up to 14 days)25. Unlike IgG, IgE has no known inhibitory Fc receptors. Studies to-date have also demonstrated that localized immune cell activation and release of mediators can uniquely activate immune cells to perpetuate anti-tumoral activities26. Pre-clinical studies of the first anti-cancer IgE, MOv18 IgE  have now been translated to a first-in-human, first-in-class Phase I clinical trial (NCT02546921)27. However, an IgE class antibody targeting a melanoma-associated antigen has not yet been evaluated.Immunotherapeutic antibodies used to treat cancer belong to the immunoglobulin G (IgG) antibody class (most often IgG1). However, tumor antigen-specific immunoglobulin E (IgE) antibodies may offer significant potential advantages and have shown favorable results in both in vitro and in vivo modelsHere we confirm the expression of CSPG4 in human melanomas compared with normal tissues and demonstrate that a recombinant anti-CSPG4 antibody (clone 225.28), generated with human IgE class constant domains, binds human CSPG4-expressing cancer cells and melanoma tissues including lymph, and distant metastases. We assess the anti-tumor effects of CSPG4 IgE in vitro and in vivo in human melanoma xenograft models, and a melanoma patient-derived xenograft (PDX) model. Cellular immunity in these animal models is reconstituted using human immune effector cells from healthy volunteers and from patients with melanoma. We study the effects of IgE Fc-mediated immune effector cell functions, activation and signaling pathways ex vivo and in vivo. Furthermore, we evaluate CSPG4 IgE in an ex vivo basophil activation assay in whole patient blood to consider the potential of type I hypersensitivity.n = 8–127 per cancer type) and human cancer tissues (n = 102–1075 per cancer type)29  compared to normal skin (n = 558) tissues , and across all stages of melanoma  indicated detectable CSPG4 protein expression in 63% of malignant melanomas 33. SDS polyacrylamide gel electrophoresis (SDS-PAGE), under reduced and non-reduced conditions, and size-exclusion high performance liquid chromatography (SEC-HPLC) confirmed a monodisperse product with subunit composition and molecular mass consistent with that of equivalent IgEs33, and purity typically above 95% one Fig. 32. Affin95% Fig. .Fig. 2Ge34. As expected, we did not detect binding to IGROV1 ovarian or SKBR3 breast cancer cells , respectively, confirmed by binding of target antigen-specific IgEs) , and when divided into cutaneous lesions, lymph node and distant metastases (n = 302). Benign nevi (n = 18) showed low/intermediate CSPG4 expression detected with CSPG4 IgE  was either negative or low, other than the cerebrum, digestive, endocrine, eye, and female reproductive tissues where low or intermediate CSPG4 expression was observed in a proportion of tissues  Fig. . FurtherEs) Fig. , right. IgE Fig. , left, aIgE Fig. , right. ues Fig. .Thus, CSGP4 expression was detected in malignant melanomas and showed low and restricted distribution in normal tissues. The chimeric CSPG4 IgE and a mouse anti-human CSPG4 clone showed comparable binding to human tissues by IHC, and recognized CSPG4-expressing melanoma cells.We next evaluated the potential anti-tumoral functions of CSPG4 IgE. We analyzed the relative expression of CSPG4 in human melanoma cell lines  in comparison to cells not known to express CSPG4  Fig. . We sele35. Since the Fab-mediated and Fc-mediated effector functions of anti-tumor IgE antibodies directed against tumor cells may restrict cancer cell growth38, we investigated whether CSPG4 IgE could impair cancer cell function in vitro. Firstly, we studied the Fab-mediated direct effects of our antibody on cancer cell functions, in the absence of cross-linking or effector cells. A375 cell adhesion, migration, and invasion were partly inhibited by CSPG4 IgE, compared a non-specific isotype control  and the proportion of monocytes overall within PBMCs  and of monocytes expressing the high-affinity IgE Fc receptor FcɛRI . We found no significant differences in any of these parameters  mice. Since human IgE does not react with mouse FcεR-expressing immune cells, mice were engrafted with healthy volunteer peripheral blood immune cells and challenged subcutaneously with A375 melanoma cells Fig. . Tumors 13, immunohistochemical analysis of excised tumors showed infiltration of human CD45+ leukocytes, and significantly higher CD68+ macrophage infiltration in tumors from mice treated with CSPG4 IgE, compared to tumors from mice given isotype control IgE or CSPG4 IgG  and patients with melanoma (n = 15) triggered no degranulation above background    by its specific multimeric antigen (NIP-BSA) triggered significant degranulation, incubation with CSPG4 IgE with sera from healthy volunteers  Fig. . Exogeno15) Fig. . Basophi15) Fig. . Stimula15) Fig. .Fig. 8CSCSPG4 IgE did not induce RBL-SX38 cell degranulation in the presence of patient sera and did not activate basophils in whole blood of patients with melanoma. These findings provide preliminary indications for the absence of early signals of ex vivo basophil activation with CSPG4 IgE in patient sera and blood.42. CSPG4-specific antibodies, and engineered chimeric antigen receptor (CAR) lymphocyte therapies, have previously been shown to significantly reduce lung metastases and tumor recurrence in mouse models of melanoma48. Anti-CSPG4 antibodies designed with immune-activating features and able to restrict tumor growth are still required. Based on emerging pre-clinical efficacy, immune activatory functions and promising clinical studies of the first IgE class antibody specific for tumor-associated antigens37, here we designed CSPG4 IgE by combining the CSPG4-specific variable domains of a mouse clone (225.28), with a human IgE backbone.Malignant melanoma is the most aggressive skin cancer. Despite the emergence of targeted and immune therapies, many patients do not benefit sufficiently due several resistance mechanisms. Cancer-associated antigen targets, such as chondroitin sulfate proteoglycan 4 (CSPG4), may be promising for cancer treatment45, together supporting the potential of CSPG4 as a specific tumor-associated therapeutic target. Previous studies also showed that the anti-CSPG4 225.28 antibody with high affinity (1 × 10−9) for CSPG449, binds to a unique epitope on CSPG4 compared to other clones, and not to ubiquitously expressed carbohydrates52. Furthermore, clone 225.28 has been safely introduced to melanoma patients, suggesting a low risk of off-target-related toxicities53 for a 225.28-based treatment for melanoma.Transcriptomic analyses confirmed higher CSPG4 gene expression in melanoma cells and tissues compared to other cancer types and normal skin tissues. Using commercially available and our own engineered CSPG4-specific antibodies, we demonstrated CSPG4 expression in malignant melanoma, including in lymph and distant metastases, and absent/low expression in normal tissues Figs. , 21,42,454. CSPG4 IgE conferred Fc-mediated functions in vitro in the presence of high and intermediate CSPG4-expressing melanoma cells: ADCC mediated by human effector cells, including monocytes from healthy individuals and patients with melanoma; and RBL-SX38 cell degranulation. No effector functions were observed in the presence of low or non-CSPG4-expressing cells 59. Furthermore, the BAT has been incorporated as an eligibility criterion, and monitoring companion alongside other clinical parameters, for the phase I clinical trial of this therapeutic candidate (NCT02546921)27. Therefore using the BAT, here we evaluated the potential of CSPG4 IgE to trigger basophil activation. Ex vivo stimulation of whole unfractionated blood samples from melanoma patients with CSPG4 IgE did not induce basophil activation, despite clear activation by well-described IgE and non-IgE specific immune stimuli in the same samples. Taken together, our data suggest that any molecules present in the melanoma patient circulation, or intermediate-low CSPG4-expressing cells, did not have the capacity to activate basophils. However, the ex vivo BAT remains to be evaluated alongside a clinical trial.The main perceived risk of IgE immunotherapeutic agents is that of type I hypersensitivity and anaphylaxis which may be induced by circulating multivalent tumor antigen, shed from the tumor, and in the presence of autoantibodies. These may have the propensity to form immune complexes with CSPG4 IgE in the patient circulation, potentially resulting in cross-linking and activation of IgE Fc receptor-bearing immune cells such as basophilsays Fig. . CSPG4 I4. In this model, repeated administration of a surrogate rat CSPG4 IgE to immunocompetent rats did not trigger overt toxicities or anaphylaxis. Although previous findings of CSPG4 IgE administration in a fully immunocompetent rat model may indicate lack of on-target off-tumor toxicity against minimally CSPG4-expressing normal tissues, the safety of CSPG4 IgE requires further investigation.In this study, we show that our clone does not cross-react with mouse CSPG4 antigen . Both male and female human participants were included. Written informed consent was obtained from all participants.63.Cells lines (human and rat effector cells and human cancer cell lines) were sourced and cultured as described in Supplementary Methods. Primary melanocytes (ATCC PCS-2000-012), human peripheral blood mononuclear cells (PBMCs), human peripheral blood lymphocytes (PBL) and purified monocytes were derived from healthy volunteer and melanoma patient blood by standard Ficoll separation  and as described in detail in Supplementary MethodsDetection and visualization of CSPG4 expression in normal and melanoma tissues, and analyses of human xenograft samples following in vivo tumor growth, are described in Supplementary Methods.64. Antibody purity was assessed by SDS-PAGE and SEC-HPLC as we previously described32. The binding of the antibody to cell-surface CSPG4 (on human tumor cell lines) and human FcεRI (on rat basophilic leukemia RBL-SX38 cells) were assessed by flow cytometry as in prior studies13. IgE antibodies of different specificities were used as isotype controls: NIP IgE specific for the hapten 5-iodo-4-hydroxy-3-nitrophenyl described earlier65. Further analyses of CSPG4 IgE specificity and cross-reactivity are described in Supplementary Methods.CSPG4 human/mouse chimeric IgE antibody, derived from clone 225.28, was engineered and produced in stable cell lines expressing anti-CSPG4 IgE24, a flow cytometric antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) tumor cell killing assay39, monocyte stimulation31, and the basophil activation test (BAT)62 were performed as described in Supplementary Methods.Direct anti-tumor effects of the antibody on A375 melanoma cells, the RBL-SX38 cell degranulation assay63 or injected intravenously leading to the formation of lung lesions as described in detail in Supplementary Methods. Antibody treatments were dosed at 10 mg/kg in concordance with previous in vivo studies of MOv18 IgE and SF25 IgE66. At experimental endpoints, mice were humanely euthanized using CO2 asphyxiation followed by cervical dislocation in accordance with The Animals (Scientific Procedures) Act 1986 (ASPA) regulated by the Parliament of the United Kingdom.Male and female NOD/scid/IL-2Rγ−/− mice , aged 6–10 weeks, were maintained in accordance with Institutional Committees on Animal Welfare of the UK Home Office and the Biological Services Animal Welfare & Ethical Review Body (AWERB), Guy’s Campus, King’s College London. The mice were kept on a 12 hour light/dark cycle (light of 350–400 lux). Housing conditions were maintained at 20 °C, and at a relative humidity of 40 to 60%. Malignant melanomas were established as subcutaneous tumorst test or One-way ANOVA for normally distributed data; Mann–Whitney or Kruskal-Wallis tests for non-parametric data; and where appropriate, Two-way ANOVA). Comparisons of Kaplan-Meier survival curves were performed using a Log-rank Mantel–Cox test. Details of the statistical tests applied are included in the figure legends. Statistical analyses were performed throughout, and statistically significant differences are shown in the graphs. p values: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.Data are presented as mean ± Standard Error of the Mean (SEM). Statistical analyses were performed on GraphPad Prism (version 9.0). Evaluation of normal distribution of data was performed using a Shapiro–Wilk normality test. The most appropriate statistical analysis to compare data between experimental conditions was then selected (two-tailed unpaired Student’s Further information on research design is available in the Supplementary InformationReporting Summary