IMPT) takes uncertainties into account during spot weight optimization and leads to dose distributions that are resilient to uncertainties. Previous studies demonstrated benefits of linear programming (LP) for IMPT in terms of delivery efficiency by considerably reducing the number of spots required for the same quality of plans. However, a reduction in the number of spots may lead to loss of robustness. The purpose of this study was to evaluate and compare the performance in terms of plan quality and robustness of two robust optimization approaches using LP and nonlinear programming (NLP) models. The so‐called “worst case dose” and “minmax” robust optimization approaches and conventional planning target volume (PTV)‐based optimization approach were applied to designing IMPT plans for five patients: two with prostate cancer, one with skull‐based cancer, and two with head and neck cancer. For each approach, both LP and NLP models were used. Thus, for each case, six sets of IMPT plans were generated and assessed: LP‐PTV‐based, NLP‐PTV‐based, LP‐worst case dose, NLP‐worst case dose, LP‐minmax, and NLP‐minmax. The four robust optimization methods behaved differently from patient to patient, and no method emerged as superior to the others in terms of nominal plan quality and robustness against uncertainties. The plans generated using LP‐based robust optimization were more robust regarding patient setup and range uncertainties than were those generated using NLP‐based robust optimization for the prostate cancer patients. However, the robustness of plans generated using NLP‐based methods was superior for the skull‐based and head and neck cancer patients. Overall, LP‐based methods were suitable for the less challenging cancer cases in which all uncertainty scenarios were able to satisfy tight dose constraints, while NLP performed better in more difficult cases in which most uncertainty scenarios were hard to meet tight dose limits. For robust optimization, the worst case dose approach was less sensitive to uncertainties than was the minmax approach for the prostate and skull‐based cancer patients, whereas the minmax approach was superior for the head and neck cancer patients. The robustness of the IMPT plans was remarkably better after robust optimization than after PTV‐based optimization, and the NLP‐PTV‐based optimization outperformed the LP‐PTV‐based optimization regarding robustness of clinical target volume coverage. In addition, plans generated using LP‐based methods had notably fewer scanning spots than did those generated using NLP‐based methods.Robust optimization of intensity‐modulated proton therapy ( The two most important sources of uncertainty in IMPT are the beam range and patient setup uncertainties. These uncertainties can result in deviation of the delivered IMPT dose distribution from the planned distribution, which may lead to suboptimal treatment decisions and unforeseen outcomes. Therefore, these uncertainties must be considered during IMPT plan optimization.In photon therapy, the conventional approach to handling patient setup uncertainties and organ motion is to expand the clinical target volume (CTV) by an empirically determined margin to form a planning target volume (PTV). The underlying assumption in the determination of the CTV‐to‐PTV margin is that the CTV will be sufficiently covered with high probability e.g., 95%) in the face of uncertainties. This approach works well for photon therapy because the variations in photon dose distributions when patient anatomy changes are relatively small.5% in theOne such approach is robust optimization, which aims to produce optimal, resilient IMPT plans in the face of uncertainties. Researchers have conducted several probabilistic and scenario‐based studies to incorporate uncertainties into IMPT plan optimization.Fredriksson et al. first proposed minmax robust optimization. This nonlinear constrained model does not assume a probability distribution for uncertainties.To date, only a handful of studies have compared different IMPT robust optimization approaches. Fredriksson generalized a class of robust optimization methods, including expected value and minmax optimization.In all of these studies, researchers investigated robust optimization techniques for IMPT using either an LP or NLP model. However, a comparison of the performance of LP and NLP models with one or more robust optimization approaches has yet to be reported. Therefore, we performed this study to identify and outline differences in the behavior of various LP‐ and NLP‐based approaches and models for IMPT robust optimization. To that end, we developed and evaluated worst case dose (voxel‐wise) and minmax methods for both LP and NLP formalism models. To better understand the influence of LP‐ and NLP‐based models on robust optimization results, we also performed PTV‐based conventional optimization with both LP and NLP objective functions. Thus, we compared six optimization methods: LP‐PTV‐based, NLP‐PTV‐based, LP‐worst case dose, NLP‐worst case dose, LP‐minmax, and NLP‐minmax. Specifically, we compared the plan optimality and robustness and number of scanning spots (surrogate of plan efficiency) for the plans created with each of the six methods. Although Fredriksson and Bokrantz previously compared quadratic worst case dose and minmax approaches to account for setup uncertainties for two patients with prostate cancer, the performance of different methods may depend on the treatment site.22.AThe relative performance of various robust optimization approaches was evaluated by regenerating treatment plans for two patients with prostate cancer, one with skull base cancer, and two with head and neck cancer, all of whom had undergone proton therapy at our institution. Two lateral fields were used for prostate cancer cases, whereas three fields were used for the other three cases. For each patient, eight uncertainty scenarios were assumed: two setup uncertainty scenarios (±5 mm for prostate cancer and ±3 mm for the other cancers) in the x, y, and z directions and two range uncertainty scenarios . For each patient, the six optimization methods described above and below were used to account for range and setup uncertainties. The PTV was chosen as the target for the conventional optimization approach and, appropriately, the CTV was used as the target for the robust optimization approaches.2.BFor each patient, three optimization approaches were evaluated: conventional PTV‐based, worst case dose robust, and minmax robust optimization. For each approach, LP‐ and NLP‐based optimization models were developed.2.B.1et al. described details of the NLP‐based optimization model.In conventional optimization, uncertainties were accounted for by expanding the CTV to a PTV via margins and treating the PTV as the target. The PTV was formed via isotropic expansion of 5 mm for prostate cancer and 3 mm for the other types of cancer to account for setup uncertainties. Cao et al. described details of the LP‐based optimization model, and Liu 2.B.2dijr is the influence matrix, which denotes the dose contributed by the Jth beamlet per unit weight, and is received by voxel i under scenario r.For a voxel inside the target, the minimum dose of the voxel of all dose distributions corresponding to different uncertainty scenarios was selected. For any voxel outside the target, the maximum dose of the voxel was selected. This formed the worst case dose distribution as follows:xj was the intensity of beamlet j. The optimization models applied to both CTV and OARs were as follows:λi is the penalty weight, and Dipresis the prescription dose for each voxel. In linear programming, hard constraints are imposed on dose to each voxel, that is, ensuring that all constraints have to be satisfied otherwise no solution exists. LBi and UBi are lower and upper reference bounds on dose to each voxel. However, in nonlinear programming, only soft constraints are imposed. This means that the model penalizes deviations between delivered and prescribed doses, but does not require each voxel to meet certain dose limits.Robust optimization was performed by substituting the worst case dose distribution for the nominal dose distribution in each iteration. The decision variable 2.B.3Dir=∑jdijr·xj,∀i.An alternative approach to worst case dose robust optimization is the minmax method described by Fredriksson et al.Both CTV and OARs are incorporated in the optimization model. Relative strengths of the minmax and voxel‐wise robust optimization methods are described in the literature.2.CSix IMPT plans were generated using the six optimization methods and compared in terms of plan quality and robustness and delivery efficiency. Only dose constraints (hard constraints for LP‐based models and soft constraints for NLP‐based models) were used in plan optimization. Both dose and dose‐volume constraints of nominal and worst case dose distributions were reviewed after optimization. If an optimized plan failed to meet such constraints, the plan could be re‐optimized by adjusting objective weights or dose constraints until all constraints were satisfied. Some of the key criteria for plan quality we used are listed below.Rectum: volume receiving a dose of 70 Gy  no greater than 20%.Bladder: volumes receiving doses of 65 Gy  and 40 Gy  no greater than 25% and 50%, respectively.Prostate cancer:Brainstem: maximum dose no greater than 60 Gy (RBE).Spinal cord: maximum dose no greater than 45 Gy (RBE).Oral cavity: mean dose no greater than 35 Gy (RBE).Temporal lobe: volume receiving a dose of 60 Gy  no greater than 1%.Skull base and head and neck cancer:v and Vd) were used to evaluate the quality of the plans. Dv denotes the amount of the dose received by more than v percent of the organ, and Vd denotes the percent volume of the organ receiving more than d Gy (RBE). Dv is proportional to the target coverage, whereas Vd is inversely proportional to OAR sparing. To illustrate differences in nominal plan quality and robustness among the six optimization methods, we selected several critical DVH indices for comparison for each cases. Those comparisons are discussed in the Results section.For the sake of comparison, all plans were renormalized to cover at least 99% of the CTV by the prescribed dose in the nominal dose distribution. Dose‐volume histogram (DVH) indices (Dv) denotes the width of the DVH band at volume v, and Δ(Vd) denotes the width of the DVH band at dose d. This robustness evaluation technique effectively determined the robustness of the IMPT plans in the face of setup and range uncertainties. Next, to estimate the delivery efficiency of the plans, the resulting number of scanning spots with positive intensity generated using each method was assessed. Note that we used the same base scanning spot placement  for all optimization methods.To compare the robustness of the IMPT plans generated using the different methods, families of DVHs corresponding to different uncertainty scenarios were plotted along with the nominal DVHs. The resulting envelopes were used to assess the sensitivity of the plans under the uncertainty scenarios.3We compared the plan quality and robustness of the six optimization strategies in terms of DVH indices for nominal doses and DVH band widths for uncertain scenario doses for five cancer patients. In addition, we compared the numbers of scanning spots after performing the six methods. Figure To further evaluate plan robustness, we compared the DVH‐family band widths at key dose‐volume indices for the six methods for patient 1 Fig. . For robDVH indices for nominal doses and DVH‐family band widths for patient 2 (the other prostate cancer patient) are shown in Fig. The robustness of CTV coverage for the PTV‐based methods was inferior to that for the robust optimization methods for patient 3 (the skull base cancer patient) Fig. . In thisFor patient 4 (with head and neck cancer), the NLP‐based optimization methods were more robust in covering the CTV than were the LP‐based methods as shown in Fig. Figure In addition, we compared the PTV coverage for the six optimization methods. Table Next, we determined the resulting number of spots  for each optimization method. Figure 4The inclusion of plan robustness (resilience in the face of uncertainties) in optimization of dose distributions is widely recognized as essential for IMPT. Researchers have developed different planning strategies to account for uncertainties in IMPT planning. In this study, we assessed and compared two mathematical models (LP‐ and NLP‐based) and two robust optimization approaches (worst case dose and minmax) as well as the conventional PTV‐based optimization approach. Our results demonstrated that the robust optimization methods created more robust IMPT plans in terms of target coverage and OAR sparing than did the PTV‐based method. However, the robust optimization methods behaved differently from site to site, and no method emerged as superior to the others under any circumstance.We performed IMPT planning studies to identify circumstances under which the LP‐ and NLP‐based robust optimization methods behaved differently. It is important to note that the LP‐based models used in this study have hard constraints on dose limits, whereas the NLP‐based models do not. The constrained LP models require model‐specific parameters such as lower and upper dose reference bounds for various organs. These parameters are critical to controlling the outcome of optimization.Moreover, an optimal dose distribution from a constrained model such as an LP‐based one (see section 2.2) must satisfy all dose‐limit constraints under all possible scenarios. This can be a burden under some scenarios and may result in wider DVH‐family bands than those for an unconstrained model. If a cancer case is not challenging and all scenarios are favorable for finding a feasible solution using constrained LP, the resulting DVH will exhibit better control of plan robustness than that from an unconstrained model, especially for target coverage, as we observed for the two typical prostate cancer cases , the worst case dose of each voxel was calculated independently among all uncertainty scenarios considered. Thus, if one voxel's dose were the worst in one uncertainty scenario, another voxel's dose might be the worst in a different scenario. In other words, all scenarios were used in calculating the objective function value in optimization. However, the minmax methods select one scenario that is the worst case among all scenarios in terms of its objective value; the remaining “easy” scenarios are discarded in the optimization model.We observed a clear difference between the LP‐ and NLP‐based models in terms of the number of spots included in the optimized plans Table . One of We also observed that LP‐PTV‐based optimization was outperformed by NLP‐PTV‐based optimization in terms of robustness of CTV coverage in all five cases. The robustness of sparing critical structures for NLP‐PTV‐based optimization was either superior to or comparable with that for LP‐PTV‐based optimization. This may have resulted from the fact that the plans generated using the LP‐based methods were sparser than the plans created using the NLP‐based methods. A sparse solution, in which few spots are selected or have positive intensities, is naturally sensitive to uncertainty if plan robustness is not considered in optimization. However, we did not observe a correlation between the reduction in the number of spots caused by LP‐based robust optimization and loss of robustness. LP‐based robust optimization created considerably more robust plans than did PTV‐based optimization (either LP or NLP) in all cases. It also outperformed NLP‐based robust optimization in plan robustness for the two prostate cancer cases. Overall, plan robustness is primarily determined by whether uncertainty is taken into account in optimization, not by the choice of LP or NLP. Furthermore, whether LP‐ or NLP‐based robust optimization outperforms each other in terms of resulting plan robustness can vary case by case.A limitation of the robust optimization approaches evaluated in this study was the limited set of predefined uncertainty scenarios. These scenarios accounted only for setup uncertainties along the x, y, and z axes and range uncertainties. We did not consider movements in other directions, nonrigid patient movements, changes in patient anatomy, or other sources of uncertainties. However, incorporating more error scenarios requires more time to solve problems, which may not be practical. Some studies have used a larger number of scenarios.5The findings of this study reiterate the importance of implementing robust optimization for IMPT. Our results also demonstrate that the robust optimization method ideally should be chosen on a site‐by‐site basis. None of the robust optimization methods (consisting of worst case dose and minmax methods with both LP‐ and NLP‐based models) consistently outperformed the others in terms of either nominal plan quality or plan robustness against uncertainties. The LP‐based methods provided more robust target coverage than did the NLP‐based methods where all uncertainty scenarios were able to meet tight dose constraints, as shown for prostate cancer cases. However, the robustness of the plans created using the LP‐based methods was inferior in more challenging cases, such as head and neck cancer cases, in which some scenarios prevented LP with relatively tighter constraints from finding a feasible solution. In addition, the LP‐based methods resulted in significantly fewer scanning spots than did the NLP‐based methods for the same quality and robustness. With conventional PTV‐based optimization, NLP‐based method outperformed LP‐based method regarding robustness of CTV coverage.The authors declare no conflict of interest.


GSK) for ex vivo hematopoietic stem cell (HSC) gene therapy for the treatment of adenosine deaminase (ADA)‐deficient severe combined immunodeficiency (SCID), a very rare congenital disorder of the immune system. The new medicine, named Strimvelis™, is an advanced therapy medicinal product (ATMP) , a joint venture between Telethon Foundation and San Raffaele Scientific Institute. This ATMP is the first ex vivo stem cell gene therapy to receive regulatory approval anywhere in the world. Strimvelis™ consists of a single infusion of autologous gene‐corrected HSC and is prepared from the patient's own bone marrow (BM) HSCs, which are genetically modified using a gamma‐retroviral vector to insert a functional copy of the ADA gene.Gene and cell therapy research recently reached a fundamental milestone toward the goal to deliver new medicines for orphan diseases. In 2016, the European Commission granted market approval to GlaxoSmithKline ( Importantly, gene‐modified cells were detectable in multiple hematopoietic lineages and stable engraftment persisted throughout long‐term follow‐up in the majority of patients, indicating that, although at low levels, correction of multipotent stem cells was achieved.Market approval of Strimvelis™ in the European Union (EU) was based on data collected from a total of 18 ADA‐SCID children treated from 2000 to 2011, with a median follow‐up of about 7 years , applied its expertise in product development to optimize and standardize the manufacturing process achieving robustness and suitability for commercial supply.The interactions with local ethics committees, regulatory authorities at the national  and Istituto Superiore di Sanità) and European levels (EMA committees and working parties) have been constructive throughout the process. Protocol assistance and pre‐submission meetings with these agencies provided effective advice on manufacturing and preclinical and clinical development. Despite the complexity and the many challenges faced by such a highly innovative product, the EMA Committee for Human Medicinal Products Use (CHMP)'s positive opinion was received within 10 months from the validated submission, and EU marketing was approved by the EU Commission within 12 months. AIFA approved Strimvelis™ pricing and reimbursement in Italy in < 2 months through an accelerated procedure. Similar agreements are under discussion in other EU countries.ex vivo HSC gene therapy must comply with high standards in product management and supply chain, from the procurement of the autologous BM HSC to the medicine infusion. A robust quality system certified by the Joint Accreditation Committee (JACIE) has been established at the San Raffaele Stem Cell Program.Strimvelis™ is available for ADA‐SCID patients without a suitable HLA‐matched related donor and administered at present only at the San Raffaele Hospital in Milan due to the short current product shelf life and requirements for expertise in gene therapy and HSC transplantation. Indeed, Patients treated with the experimental and licensed product will continue to be monitored since the long‐term effects of gene therapy (> 10–15 years) are not known. A long‐term prospective observational study is being implemented by GSK in accord with EMA recommendations to monitor the long‐term risks of insertional mutagenesis, oncogenesis, immunogenicity, and hepatic toxicity as well as the persistence of efficacy of ADA‐SCID gene therapy.As of end of 2015, only six ATMPs were authorized in the EU since the implementation of ATMP regulations (1394/2007). Of these, only one has been reported to be actually administered as a commercial product, while the others have been discontinued or are still struggling to secure reimbursement. The approval of two new ATMPs in 2016 represents a turning point for the field, along with the extraordinary number of agreements between industrial biotech and academia that promise to reshape the field and increase our expectations on its potential impact on human health. The innovative legislative and regulatory pathways recently implemented in the EU  initiative, Adaptive Pathway, and Accelerated Review), in the USA (Breakthrough Therapy Designation and Priority Review), and in Japan are expected to accelerate the development of ATMPs and help to bring safe and effective products to the market.Although Strimvelis™ represents the first generation of gene therapy vectors for stem cell gene therapy, its safety and efficacy track record charts a clear path for ATMP development from early clinical experimentation to drug registration and marketing. The valuable outcomes of this process reach well beyond the benefit of ADA‐SCID patients and include the strategies developed to support the quality assessment, specifications and manufacturing of the ATMP, as well as its administration at the hospital bed, and finally, but not less importantly, the policies adopted for establishing its cost and reimbursement policy. These advances pave the way to further clinical development of newer generations of vectors with higher efficiency and safety of gene transfer, such as lentiviral vectors. Based on the current safety and efficacy data from clinical studies using the lentiviral vector platform, we expect a rapid growth of the applications of stem cell gene therapy in several inherited diseases and in cancer. Marketing authorizations for new ATMPs have already been requested in the EU or are likely to be filed within the next 2–3 years. Once approved, the next challenge will be to make these therapies accessible to patients worldwide. Pharmaceutical industries will be confronted with the necessity to standardize process development across different countries, guarantee adequate manufacturing capacity for the growing needs of expanded applications, establish a network of qualified treatment centers for administration of the new ATMPs, ensure long‐term monitoring and pharmacovigilance, and maintain sustainability and cost‐effectiveness of these personalized therapies. The successful collaboration between academia and industry established for ADA‐SCID gene therapy could represent a model to follow and to further expand upon.AA is the principal investigator of the ADA‐SCID gene therapy trial sponsored by GSK. GSK acquired the license from Telethon and Ospedale San Raffaele, which are entitled to receive milestone payments and royalties upon commercialization of this and other gene therapies for genetic diseases.


Hum Brain Mapp 38:3163–3174, 2017. © 2017 The Authors Human Brain Mapping Published by Wiley Periodicals, Inc.Functional MRI is valuable in presurgical planning due to its non‐invasive nature, repeatability, and broad availability. Using ultra‐high field MRI increases the specificity and sensitivity, increasing the localization reliability and reducing scan time. Ideally, fMRI analysis for this application should identify unreliable runs and work even if the patient deviates from the prescribed task timing or if there are changes to the hemodynamic response due to pathology. In this study, a model‐free analysis method—UNBIASED—based on the consistency of fMRI responses over runs was applied, to ultra‐high field fMRI localizations of the hand area. Ten patients with brain tumors and epilepsy underwent 7 Tesla fMRI with multiple runs of a hand motor task in a block design. FMRI data were analyzed with the proposed approach (UNBIASED) and the conventional General Linear Model (GLM) approach. UNBIASED correctly identified and excluded fMRI runs that contained little or no activation. Generally, less motion artifact contamination was present in UNBIASED than in GLM results. Some cortical regions were identified as activated in UNBIASED but not GLM results. These were confirmed to show reproducible delayed or transient activation, which was time‐locked to the task. UNBIASED is a robust approach to generating activation maps without the need for assumptions about response timing or shape. In presurgical planning, UNBIASED can complement model‐based methods to aid surgeons in making prudent choices about optimal surgical access and resection margins for each patient, even if the hemodynamic response is modified by pathology.  Presurgical mapping aims to identify essential functional brain regions which are close to the lesioned tissue to be excised. The process allows the best surgical approach to be determined and allows the neurosurgeon to plan the resection extent and identify post‐operatory risks to the patient. The “gold standard” cortical mapping technique is Direct Cortical Stimulation (DCS), which is performed during awake craniotomy. As well as being invasive, this approach presents an additional risk of cortical damage and seizures to the patient. In contrast, functional MRI (fMRI) provides a repeatable, non‐invasive means of localizing essential cortical regions. Used in a presurgical planning context, this technique may allow the extent of the resection to be increased, surgery time to be reduced and may improve preservation of function after surgery  and regions such as the edge of the brain, white matter regions, and high contrast interfaces  where false positive activation elicited by task correlated motion generally manifests.The effectiveness of the identification of compromised runs in UNBIASED was assessed by reference to the GLM results for individual runs. Additionally, subject‐level UNBIASED results generated with and without the exclusion of the runs identified as “bad” were compared by calculating the percentage change in reliability values in a generous ROI (drawn by hand) containing activation in the primary motor area.rUG [as defined in Cardoso et al., It has been shown that UNBIASED provides improved detection of consistent non‐model‐conform responses [Cardoso et al., The effect of the inclusion of compromised runs and of habituation of the response on the sensitivity of the GLM, UNBIASED, and Finite Impulse Response (FIR) methods is reported in Supporting Information.t‐values are shown in Figure t‐value activations present in the GLM maps (not shown).Unthresholded UNBIASED results and positive GLM UNBIASED identified compromised runs in four out of ten patients' results: P2 (Run 4), P6 (Run 1), P8 (Run 1 and 4), and P10 (Run 5). GLM analysis of individual runs in these patients Fig.  shows noPrimary motor regions with reproducibly modified response shape were identified in three patients . In addition to the sustained smooth response present in a large extent of the dorsal region of the motor cortex see Fig. , temporaIn this study, we applied a model‐free analysis method, UNBIASED, to presurgical planning fMRI at ultra‐high field. UNBIASED integrates information from a number of runs with identical timing, using the signal change in each voxel of each run as a predictor for the signal change in the same voxel in other runs. This allows innate quality assurance—identifying low quality runs—and provides a measure of how consistently each voxel is activated by the task performed. Since it is not based on assumptions about the response shape and/or duration, UNBIASED is sensitive to activation‐related signal changes even if there are deviations from task timing and non‐model‐conform BOLD responses.Multi‐run fMRI acquisitions allow patients short breaks between runs, which facilitates cooperation and reduces the likelihood of motion during acquisition. Generally, the responsible clinician determines the number of fMRI runs to be acquired on the basis of how challenging the detection of activation is in the affected cortical area and patient compliance. However, even if the number of runs is as low as two (the minimum required in UNBIASED), the method was shown to accurately depict activation [Cardoso et al., UNBIASED offers automated data quality assurance via identification of runs that compromise the quality of the subject‐level analysis either because of technical artifacts  or poor performance. This is particularly desirable in clinical fMRI, as the activation maps need to be available shortly after the examination for therapeutic decision‐making purposes. This study confirms that such runs, which inevitable occur in a clinical context, are correctly identified.Gonzalez‐Castillo et al.  reportedOther model‐free methods have been applied to ultra‐high field presurgical planning data. ICA has been used to identify non‐model‐conform activation foci and separate activation from artifacts, for instance [Robinson et al., Localization uncertainties are critical, particularly in presurgical planning. A measure based on reliability is of utmost importance. Using a model‐based analysis, Beisteiner et al.  showed tThe age range of the participating patients in this study varies from 13 to 55 years old . Precise and nearly artifact‐free identification of the motor cortex was attained in this case.The main limitations of UNBIASED are that the fMRI experiment must be constructed as a number of runs with identical timing, and that it is incapable of distinguishing activation from different tasks or contrasting conditions [Cardoso et al., High reliability values could, in principle, arise from physiological fluctuations if the acquisition of the fMRI runs were temporally synchronous with some physiological cycle . No coherent relationship exists, in general, between physiological cycles across runs [Cardoso et al., Application of the model‐free analysis method UNBIASED to ultra‐high field presurgical fMRI resulted in reliable activation maps of the primary representation of the motor cortex. Unreliable runs were identified automatically and UNBIASED functional maps suffered from less artifact contamination than those from a GLM analysis. Additionally, cortical regions exhibiting consistently modified response shape and timing could be identified with UNBIASED but not with the GLM.The ability to identify consistent but atypical BOLD responses is a valuable feature when compromised task performance or modified HRF are expected. This makes it particularly attractive as a complementary approach for presurgical planning, aiding clinicians in decisions concerning surgical approach, and necessity and extent of therapeutic resection of impaired brain tissue.The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this article.Ethics Committee of the Medical University of Vienna, Austria.Supporting InformationClick here for additional data file.


Due to a growing demand of food production worldwide, new strategies are suggested to allow for sustainable production of food with minimal effects on natural resources. A promising alternative to the application of chemical pesticides is the implementation of crops resistant to insect pests. Plants produce compounds that are harmful to a wide range of attackers, including insect pests; thus, exploitation of their natural defense system can be the key for the development of pest‐resistant crops. Interestingly, some plants possess a unique first line of defense that eliminates the enemy before it becomes destructive: egg‐killing. Insect eggs can trigger (1) direct defenses, mostly including plant cell tissue growth or cell death that lead to eggs desiccating, being crushed or falling off the plant or (2) indirect defenses, plant chemical cues recruiting natural enemies that kill the egg or hatching larvae (parasitoids). The consequences of plant responses to eggs are that insect larvae do not hatch or that they are impeded in development, and damage to the plant is reduced. Here, we provide an overview on the ubiquity and evolutionary history of egg‐killing traits within the plant kingdom including crops. Up to now, little is known on the mechanisms and on the genetic basis of egg‐killing traits. Making use of egg‐killing defense traits in crops is a promising new way to sustainably reduce losses of crop yield. We provide suggestions for new breeding strategies to grow egg‐killing crops and improve biological control. A growing demand of an increasing world population, estimated to reach 9 billion people in 2050, requires a drastic increase of food production  were observed to desiccate and/or drop off the plants   , which is defined as a rapid cell death usually activated by pathogens resulting in necrosis restricting the pathogens to the inoculated regions  . In Viburnum shrubs (Adoxaceae), twigs produce wound tissue in response to eggs of the Viburnum leaf beetle  laid into cavities, leading to beetle eggs being crushed inside the cavity  (Mazanec Prunus serotina) (Karban Persea americana)  Karban , and avovia manipulation of egg‐induced plant infochemicals that enhance egg parasitoid foraging abilities, but this promising strategy has not yet been implemented. Egg‐induced plant defenses guide egg parasitoids toward the plant infested with herbivore eggs either by volatile attractants from a distance or by contact chemical cues at short range  and black mustard (B. nigra) showed that egg deposition by lepidopteran pests resulted in the emission of OIPVs that attract polyphagous Trichogramma egg parasitoids as well as larval parasitoids that eventually kill the caterpillars  and by pine sawflies on pine (Pinus sylvestris) also induces OIPVs attracting specialist egg parasitoids  . OIPVs often consist of complex mixtures of volatiles including green leaf volatiles, terpenoids, and isothiocyanates  attraction toward N. viridula egg‐induced volatiles. Interestingly, regardless if nonhost beetle chewing damage was inflicted by larvae feeding on roots, or by adults feeding on leaves, the composition of the OIPV blend was significantly altered resulting in a decrease in attraction of the wasps  is also feeding on the plant. A growing body of literature suggests that, under multiple herbivore attack, the emission of OIPVs can be altered depending on several aspects of the nonhost herbivore attack such as insect feeding guild , leaf chewing by caterpillars of native (P. brassicae) and invasive alien herbivores (Spodoptera exigua) disrupt Trichogramma species attraction toward P. brassicae egg‐induced volatiles , which inhibits the growth of Pseudomonas syringae strains  . The dated phylogeny and associated defense traits, according to Table So far, little is known on the evolutionary history of the various plant defense traits against herbivore eggs. To explore this issue, we draw a dated phylogeny of the 32 plants listed in Table The most parsimonious reconstruction proposes that the defense trait at the most ancestral node of this evolutionary tree was an indirect one, and more particularly the emission of volatile chemical cues (OIPVs). The taxa with the most outgroup positions in this analysis  displayed this indirect defense trait; thus, it is logical that the inferred most ancestral trait was attraction of egg parasitoids by “volatile chemical cues”. Including more gymnosperms in a similar analysis would allow confirming whether this trait is really the most likely ancestral one among seed plants.According to the distribution of defense traits, it appears as most parsimonious that the last common ancestor of eudicots had the direct defense trait “wound tissue growth”. In Brassicales, most of the species display an HR‐like necrosis (Shapiro and De Vay Brassica oleracea or Z. mays), whereas their wild relatives or landraces emit OIPVs. The loss of OIPV emission could thus be a result of domestication. Indeed, in Z. mays, egg‐induced volatile emission is very rare in commercial hybrids but common in landraces  that triggers egg‐killing responses in the plant of great potential for pest control  genes involved in resistance against viruses, bacteria, fungi, oomycetes, nematodes, and sucking insects are characterized and efficiently used in crop improvement programs. So far, no R genes are known to be involved in the recognition of herbivore‐associated molecular pattern (HAMPs) from leaf‐chewing insects including caterpillars of generalist moths Spodoptera spp. or Plutella xylostella, which are destructive pests that also show increasing resistances to pesticides  varieties against egg deposition of a serious pest, the whitebacked planthopper (Sogatella furcifera). When eggs are laid into air spaces of leaf sheaths, they cause necrotic discolorations, or “watery lesions”, which contain an ovicidal substance, benzyl benzoate  oviposition attracting egg and larval parasitoids. In particular, in landraces, stemborer eggs induce increased emission of some terpenoids, including (E)‐4,8‐dimethyl‐1,3,7‐nonatriene (DMNT), a key compound for parasitoid attraction (Tamiru et al. Tamiru et al.  studied Besides these two recent approaches, more studies are needed and we hope that future efforts will focus on the genetic aspects underlying lethal egg‐killing traits as such information could be the basis to develop a novel strategy for sustainable pest control.via OIPV emission seems lost in cultivated plants compared with wild relatives or landraces. However, our database is restricted to only a small subset of plant species, and expanding the knowledge on the evolutionary history of egg‐killing traits is necessary to fully understand the role played by artificial selection for high‐yield traits on plant defenses. Considering the advantages and the ubiquity of egg‐induced plant defenses, especially in wild species, we believe that egg‐killing traits have a strong potential to be implemented in pest control programs.In this review, we have highlighted the strategies adopted by plants to kill insect eggs, thus minimizing the damage inflicted as the herbivore is killed before the crop feeding stage. Such egg‐killing traits have been documented so far in about 30 plant species belonging to different plant orders, and this number is likely to increase rapidly as research in this area is still in its infancy. Our phylogenetic analysis supports the hypothesis that plant domestication negatively affected oviposition‐induced defense traits in brassicaceous and graminaceous crops, particularly the capacity of attracting parasitoids It is recommended to breed for inducible defenses rather than select for continuous expression of defenses in order to avoid costs when herbivores are not attacking the plant. Direct egg‐killing defenses are likely to be more attractive for plant breeders, who have traditionally focused on bitrophic interactions. Furthermore, as pest suppression is not dependent on the third trophic level, the results of implementing direct egg‐killing traits into crops are likely to be less variable and less context dependant. Breeding specifically for parasitoid‐attracting traits is more challenging, because the extra level of complexity represented by the actions of egg or larval parasitoids can increase the failure risks in controlling the pest population. Furthermore, as crops protected by these traits do not achieve the complete elimination of pests, farmers may be more interested in other alternatives for pest control. When possible, using an integrated approach in which crops are protected with both direct and indirect egg‐induced defenses is encouraged, as in the case of brassicaceous plants. Evidence accumulates that priming by egg deposition can enhance defenses against subsequent attack due to oviposition‐mediated cross‐resistance effects. Thus, crops with high resistances to eggs might also become better protected against feeding stages of pests or even pathogens. In a scenario in which multiple sustainable strategies are used, such crops equipped with egg‐killing traits can be supplemented with floral resources to maximize the pest control service provided by parasitoids using an attract‐and‐reward approach.None declared.


BAV) is recommended before transcatheter aortic valve implantation (TAVI), despite limited data around the requirement of this preprocedural step and the potential risks of embolization. This study aimed to investigate the trends in practice and associations of BAV on short‐term outcomes in the UK TAVI registry.Aortic valve predilation with balloon aortic valvuloplasty  of whom had no BAV before TAVI valve deployment. We observed heterogeneity in the use of BAV nationally, both temporally and by center experience; rates of BAV in pre‐TAVI workup varied between 30% and 97% across TAVI centers. All endpoints were similar between treatment groups in SAPIEN  valve patients. After correction for multiple testing, none of the endpoints in CoreValve  patients were significantly different between patients with or without predilation.Eleven clinical endpoints were investigated, including 30‐day mortality, myocardial infarction, aortic regurgitation, valve dysfunction, and composite early safety. All endpoints were defined as per the TAVI without predilation was not associated with adverse short‐term outcomes post procedure, especially when using a balloon‐expandable prosthesis. Randomized trials including different valve types are required to provide conclusive evidence regarding the utility of predilation before‐TAVI.Performing  Transcatheter aortic valve implantation (TAVI) is an effective treatment option for multimorbid patients with severe symptomatic aortic stenosis who are either not suitable for conventional surgical aortic valve replacement or who are deemed high‐risk surgical candidates.During the TAVI procedure, recommendations have included the use of balloon aortic valvuloplasty (BAV) to predilate the aortic valve before deployment of the transcatheter valve. Such predilation is intended to aid delivery of the prosthesis across the valve, enhance prosthesis expansion in the aortic annulus, provide information about the aortic annulus size, and, potentially, improve hemodynamic performance during the TAVI procedure.Therefore, this analysis was undertaken in the UK TAVI registry to investigate patterns of BAV use across the UK and its association with short‐term clinical outcomes following TAVI.The UK TAVI registry uses a Web‐based interface provided by the National Institute of Cardiovascular Outcomes Research to collect data prospectively on every TAVI procedure conducted in the UK.This study analyzed data from January 2007 to December 2014. The Edwards SAPIEN  and the Medtronic CoreValve  prostheses were available to all centers throughout the study period.Endpoints in this study were 30‐day mortality and the following events occurring up to hospital discharge: myocardial infarction (MI); stroke; paravalvular leak (PVL)/moderate‐severe aortic regurgitation (AR); coronary artery obstruction (CAO); valve dysfunction requiring repeat procedure; permanent pacemaker implantation requirement; device migration; kidney injury; major vascular complications (MVC); and composite early safety. All endpoints were defined as given in the VARC‐2 definitions.This analysis defined BAV procedures based on the timing of any such procedure relative to the time of TAVI. Specifically, we distinguished the following timings: (1) BAVs completed before the date of TAVI (Before‐TAVI BAV); (2) BAVs completed as part of the TAVI procedure, but before valve deployment (During‐TAVI BAV); and (3) no BAV before or during the TAVI procedure (Direct TAVI). Because the aim of this study was to investigate the effect of predilation on TAVI outcomes, the main analysis excluded any patient who had a BAV before the date of TAVI; hence, the main analysis compared endpoints across patients with a During‐TAVI BAV (but none before) and Direct TAVI, with the latter group taken as the reference. All patients with missing treatment group identifiers were excluded.Additionally, to investigate whether the timing of the BAV relative to the TAVI procedure was associated with outcomes, we conducted a sensitivity analysis that did not exclude those patients who had a BAV before the date of TAVI. Hence, the sensitivity analysis compared outcomes across all 4 possible treatment groups: (1) Before‐TAVI BAV and During‐TAVI BAV; (2) Before‐TAVI BAV and No During‐TAVI BAV; (3) No Before‐TAVI BAV and During‐TAVI BAV; and (4) No Before‐TAVI BAV and No During‐TAVI BAV (Direct TAVI). Here, groups 3 and 4 comprised exactly those patients as in the main analysis.Given that the effects of BAV on outcomes post‐TAVI were potentially dependent on the expansion method of the valve type , all analyses were completed in device‐specific subgroups . Patients were excluded only from the valve‐subgroup analyses if they were not treated with a SAPIEN or CoreValve prosthesis or if the valve type was unknown.Continuous data were presented as means and SDs, with group comparisons done with ANOVA. Categorical data were presented as counts and percentages with group comparisons done using the chi‐squared test.Every variable with missing data was imputed using multiple imputation.To investigate clinical outcomes across treatment groups, propensity scores (PSs) for being in each treatment group were calculated for all patients to control for potential confounders and baseline differences.Patient characteristics that resulted in a higher probability to perform predilation were identified by deriving a logistic regression model with During‐TAVI BAV (no before BAV) as the dependent variable. Predictors associated with the use of predilation were investigated by backward selection using Akaike information criterion in each imputed data set, resulting in 10  sets of selected predictors. Predictors that were selected in more than 50% of the 10 imputed data sets were identified as independent predictors of During‐TAVI BAV, following the so‐called majority method of selecting variables in multiple imputed data.R From January 2007 to December 2014, 7431 patients underwent a TAVI procedure in the UK. The flow of patients through the steps of exclusion criteria is illustrated in Figure P<0.001), although the proportion of patients with impaired left ventricular function at the time of the TAVI procedure was similar (P=0.47). The LES and STS score models were calculated in each multiply imputed data set using the variables and coefficients previously published.Summary statistics of baseline characteristics for the main analysis are given in Table P<0.001) and by access route (P=0.001) , who represent a specific complex group of patients. All outcomes were similar between those who had a BAV before the date of TAVI (with or without subsequent BAV during TAVI) and those undergoing Direct TAVI. However, although we were able to distinguish the patients who had a BAV as a bridge to TAVI, the UK registry does not capture the reasons a BAV was conducted. Hence, this study could not investigate the full impact of BAVs conducted before TAVI. Before‐TAVI dilation is often conducted when a patient has presented with severe AS or when there are questions regarding the clinical benefit of a TAVI procedure. Therefore, one could argue that TAVI might not be feasible in such cases, without the period of convalescence after the preparatory BAV. Further work in such patients is recommended, given that there are a paucity of data for this specific cohort of patients.One limitation of the current work is that outcomes associated with the decision to use BAV were studied in this retrospective study. Such a design may introduce significant selection biases given that the UK TAVI registry does not capture the reasons why or how each BAV was conducted. As such, any reported relationships cannot be interpreted as causal and they may relate to unmeasured confounders or selection bias. The inclusion of most patient demographic, procedural information, and TAVI center experience in the PS models should mitigate the effects of this as much as possible. Likewise, patients who undergo a BAV are generally more‐severe cases with complex anatomy and would hence be expected to have poorer outcomes over those who do not undergo BAV; the use of PS in the correct work aims to correct for such confounding by indication. Finally, the absence of information regarding hemodynamic performance, valve failure rates, and echocardiographic outcomes means that such outcomes were unable to be analyzed. Similarly, we were unable to investigate technical difficulties, which have previously been indicated in Direct TAVI patients.This large‐scale study highlights that a no‐BAV (Direct TAVI) approach has similar clinical outcomes to the current practice of using BAV to predilate the diseased valve, especially when using a balloon‐expandable prosthesis. Although this analysis provides evidence that omitting the BAV step is feasible, this warrants prospective, randomized studies to define further the utility of BAV.This research was funded by the Medical Research Council, through the Health e‐Research Centre, University of Manchester (MR/K006665/1), and the North Staffordshire Heart Committee.None.


We recently described postzygotic activating mutations in GNA11 or GNAQ as causes of phakomatosis pigmentovascularis (PPV), and GNAQ mosaicism as a cause of extensive dermal melanocytosis (EDM).GNAQ mosaicism has previously been found to cause Sturge–Weber syndrome (SWS) and isolated nonsyndromic port‐wine stain‐type capillary malformations.q and Gα11.What is not yet understood is how the same mutations can lead to such differing skin phenotypes, either vascular alone (SWS), pigmentary alone (EDM) or a combination of both (PPV). The existence of a common precursor cell leading to both the vascular and pigmentary birthmarks is likely, as the same mutation has been identified in both types of lesion in patients with PPV. In mosaic disorders we would usually therefore invoke the issue of the timing of the mutation as the cause of differing phenotypes, or in other words that the mutation leading to PPV would be expected to occur earlier in embryogenesis than that for SWS or EDM. If this were the case we would expect the multiorgan phenotype in PPV to be more severe than in SWS, owing to an earlier embryological mutation. This was suggested in one case series of PPV;Looking at our patient cohorts we hypothesized that ethnicity may be associated with a phenotype‐modifying effect in this spectrum of diseases. Although ethnicity is a loosely defined classification, it is already known to be clearly associated with self‐resolving dermal melanocytosis (Mongolian blue spots), which is far more common in Afro‐Caribbean than white populations.Ethnicity data are collected routinely upon attendance at our hospital, where patients and families choose their own ethnicity from a standard list. We have assumed that there should be no inherent bias in this choice relating to the type of birthmark with which the child presents. Ethnicity data were extracted for all patients seen in our department over a 2‐year period, between March 2011 and March 2013, with a diagnosis of SWS, isolated facial port‐wine stain, congenital melanocytic naevi, congenital epidermal naevi and infantile haemangioma. For the rarer conditions PPV and EDM, where patient numbers were small, we obtained information relating to all patients with the condition rather than restricting our observations to this time period, to maximize the size of this cohort.A review of the clinical records was used to complete missing data where possible. To ensure that the common occurrence of self‐resolving Mongolian blue spots did not bias our data collection, dermal melanocytosis was considered to be relevant only where it was ‘atypical’. On the basis of observational studies of normal Mongolian blue spotsP < 0·001, Fig. GNAQ or GNA11 mosaicism) the same pattern is in evidence; logistic regression modelling of having a pigmentary component to the cutaneous phenotype produced an odds ratio for white ethnicity of 0·017 . There was no effect of sex.The percentages of different ethnicities in our whole referral cohort revealed a substantial preponderance of white ethnicity in all birthmark groups, as would be expected for our geographical referral population, except for PPV and EDM Fig. a. In theMC1R, which encodes a G‐protein‐coupled receptor. Variants in MC1R are already known to modify the phenotype of congenital pigmentary disorders such as oculocutaneous albinism and congenital melanocytic naevi.q and Gα11.The mechanism of this association between cutaneous phenotype within this diagnostic spectrum and ethnicity is not yet clear. However, we hypothesize that germline ethnicity‐associated variants in pigment genes could be involved, perhaps including the melanocortin‐1 receptor gene GNA11 and GNAQ mutations causes an overlapping phenotypic spectrum of vascular and melanocytic birthmarks, with associated ophthalmological, neurological, overgrowth and malignant complications. Ethnicity appears to be associated with congenital phenotypic variation in the cutaneous component of this spectrum of mosaic disease.In conclusion, postzygotic mosaicism for


Microelectrode arrays (MEAs) have proved to be useful tools for characterizing electrically active cells such as cardiomyocytes and neurons. While there exist a number of integrated electronic chips for recording from small populations or even single cells, they rely primarily on the interface between the cells and 2D flat electrodes. Here, an approach that utilizes residual stress‐based self‐folding to create individually addressable multielectrode interfaces that wrap around the cell in 3D and function as an electrical shell‐like recording device is described. These devices are optically transparent, allowing for simultaneous fluorescence imaging. Cell viability is maintained during and after electrode wrapping around the cel and chemicals can diffuse into and out of the self‐folding devices. It is further shown that 3D spatiotemporal recordings are possible and that the action potentials recorded from cultured neonatal rat ventricular cardiomyocytes display significantly higher signal‐to‐noise ratios in comparison with signals recorded with planar extracellular electrodes. It is anticipated that this device can provide the foundation for the development of new‐generation MEAs where dynamic electrode–cell interfacing and recording substitutes the traditional method using static electrodes. While the standard design of MEAs feature flat or pillar‐shaped electrodes, Spira and co‐workers developed gold spine electrodes in which cells engulf mushroom‐shaped 3D protrusions of 1–2 µm diameter.In this study, we exploit stress‐based roll‐up and differential residual stress in nanoscale bilayer constructs22.1Figure2 bilayer is released and the panels with the embedded electrodes self‐actuate and fold toward the middle of the structure. The electrodes in the shell are individually addressable via photolithographically patterned interconnects. The integration of such multielectrode structures with living, electrogenic cells allows for a new approach for cell recording with 3D spatiotemporal control, physically gripping the cells and providing a robust cell‐electrode interface.The concept of the self‐folding, individually addressable multielectrode shell is illustrated in 2. Passivated Au interconnect lines are first patterned using photolithography and subsequently covered with a dissolvable sacrificial layer. Next, a bilayer of SiO/SiO2 is e‐beam evaporated on top and a subsequent wet oxide etch defines the general cross‐shape of the multielectrode shell. Passivated Au interconnect lines are extended to each of the four cardinal points of the structure and rigid panels of SiO2 are then patterned on top. Finally, the Au interconnects are extended upward again through the panels and the exposed Au surface area serves as the electrode that contacts the cell and transfers any recorded membrane potential back to the bond pads .We fabricated the devices on a (100) Si wafer coated with 500 nm of thermal chemical vapor deposited (TCVD) SiO2.2Figure2. The majority of shells had electrodes patterned in each of their four cardinal points, but electrodes could alternatively be incorporated in the center at the bottom of the shell, or elsewhere as needed. Additionally, each panel electrode can be individually addressed, as used in our studies, or could be connected to a single bond pad if needed. After the full fabrication flow, the wafer was diced into individual chips that were glued and wire bonded on top of a printed circuit board (PCB). Depending on their thickness, the panels can either fold in a rigid way or display some curvature. For example, by utilizing an ultrathin film bilayer, the stress can also cause each panel to be flexible and curve during folding which accentuates the shell shape. For optimal enclosure of the shell around the cell, and thus optimal cell–electrode contact, we chose to work with such panels that we hypothesize can more readily conform to the cell  water, the chips were sterilized using 70% EtOH, coated with fibronectin and seeded with primary cardiomyocytes. Over time, the cells settle and attach to the substrate while the cell culture medium continues to dissolve the remaining sliver of Cu under the panels. Once the actuation force from the release of the differential stress of the hinges exceeds the adhesive force, the panels are released from the substrate  and any cell laying on top will be captured by the multielectrode shell . Simply by changing the focal plane of the microscope, cardiomyocytes contained within the arms of the multielectrode shell could clearly be distinguished from the rest of the monolayer. Contained cells conformed to the shape of the shell while still exhibiting their typical cytosolic Ca2+ transients, indicating viable and functional cells with a maintained bioelectrical activity . Moreover, with each contractile event the electrodes were observed to move in conjunction with the cell, highlighting tight cell–electrode contacts . Subsequent characterization using scanning electron microscopy (SEM) shows how the structures intimately integrate in the cellular monolayer, with cardiomyocytes growing in between cell‐harboring shells . Spontaneous and uniform Ca2.32 bilayer thickness on the folding angle. Details are discussed in the Supporting Information. Overall, the folding angle increases as the mismatch strain increases  is in close agreement with the FEA modeling results.Enabling multipoint 3D spatiotemporal electrical sensing, the incorporation of individually addressable embedded electrodes increases the complexity of fabrication and impacts the final folding angles. An optimal device should ensure good contact between the electrodes and the cells, as well as provide enough electrical conductivity and necessary insulation. During design and fabrication, it is important to determine the parameters and materials for optimal function. Finite element analysis (FEA) provides a powerful approach to predict deformations of structures with complex shape. Using FEA, we modeled the effect of key variables, including mismatch strain, interconnect dimensions, and SiO/SiOes Figure3a; thi2.4Figure2 bilayer that pushes the electrodes against the membrane of the captured cell. The gap between cell and electrode will therefore be smaller compared to the conventional situation where the cell is laying on top of the electrode. Hence, the resistance between this gap and the surrounding solution  is increased, causing less leakage current and consequently a higher measured signal amplitude.FigureVM, the measured signal amplitude or output voltage Vo, the membrane capacitance CM, the membrane resistance RM, the electrode capacitance CS, and the electrode resistance RS. In order to demonstrate the dependence of the recorded amplitude on the gap between the electrode and cellular membrane, we performed an analytical simulation based on the area‐contact model described by Joye et al. (see the Supporting Information),Vs approximately doubles when the cell is twice as close to the electrode. Based on our measured signal amplitudes, we therefore assume that the force exerted by the stressed bilayer decreased the cell–electrode distance approximately by half. It would be interesting for future investigation to determine the relationship between the intrinsic stress of the bilayer and measured amplitude, together with other relevant electrogenic cell types such as neurons.Primary cardiac cell signals were recorded after three DIV using a MEA‐headstage with preamplifier. A significant finding was that the signal picked up by electrodes that wrapped around the cell was consistently and approximately twofold higher compared to electrodes that remained in the planar, open configuration Figure4. The Figure−1; these values match conduction velocities reported for cardiac tissue, and more specifically for ventricular cells.The overall design of the individually addressable multielectrode shell allowed simultaneous recordings of every folded electrode located on each of the four cardinal points Figure6a. Sup3In summary, a novel and first‐of‐its‐kind multielectrode shell chip has been developed with self‐folding electrodes that bridges the gap from 2D planar recordings to more complex 3D interfacing, allowing parallel readout of all cardinal points of electrogenic cells with higher signal‐to‐noise ratios. The fabrication is done using conventional cleanroom technology allowing cost‐effective, wafer‐level‐based batch processing and future integration with CMOS modules for switching and signal processing. As demonstrated, this device can provide the foundation for the development of new‐generation MEAs where dynamic electrode–cell interfacing and recording substitutes the traditional method using static electrodes. Accordingly, future research effort will have to focus on the next major step of combining these cell‐sized multielectrode shells with microelectromechanical systems (MEMS) in order to have precise control over their position and force, ultimately acting as impactive or even ingressive end effectors. Also the integration of microfluidic layers can be of great interest, which is currently in the scope of our future research and development activities. Finally, by scaling electrode density, highly parallel 3D spatiotemporal electrical maps could be recorded from few or potentially individual cells with micrometer‐scale resolution which would advance our understanding of cellular function, heterogeneity, and response times.4Cell Culture: Neonatal rat ventricular cardiomyocytes were harvested from 2 d old Wistar rats. Animals were handled in accordance with international (EU Directive 86/609/EEC) and national laws governing the protection of animals used for experimental purposes, minimizing distress during procedures. The use of animals and procedures was approved by the Ethical Committee for Animal Welfare  of KULeuven and Imec. The extracted ventricles were washed in Hank's balanced salt solution (HBSS), followed by overnight incubation at 4 °C in 0.05% trypsin. Next, the tissue was dissociated by adding collagenase for 15 min at 37 °C. Cells were separated through trituration and centrifugation and added to primary cardiomyocyte medium, after which they were pre‐plated to allow for selective attachment of remaining fibroblasts. After counting and a final centrifugation step, a desired concentration of cardiomyocytes was added to cell culture medium and seeded on the substrate.Electrophysiological Experiments: Extracellular action potentials were recorded using a preamplifier with a blanking circuit  with a gain of 1100, a sampling rate of 50 kHz, and band‐pass filter from 1 Hz to 3 kHz. Each recording session lasted for at least 2 min.Fluorescent Imaging and Scanning Electron Microscopy: Relative fluorescent changes in the intracellular calcium concentration were visualized using the fluorescent marker Fluo‐4 AM , loaded in the myocytes using the cell‐permeant AM ester form. Fluo‐4 was excited at 488 nm and emission signals over 516 nm were collected using an upright Examiner microscope . For the actin staining, the cardiomyocytes were fixed in prewarmed 4% paraformaldehyde fixation buffer for 10 min, washed three times with PBS and permeabilized in 0.1% Triton for 5 min. After another PBS wash, an Alexa Fluor 633 Phalloidin solution  was added for 1 h and finally washed one last time in PBS prior to imaging using a Carl Zeiss LSM 780 confocal laser scanning microscope. For SEM imaging of folded shells without cells, the entire Cu sacrificial layer of the sample was etched using APS‐100, subsequently transferred to a 100% ethanol solution and washed at least three times. If cells were cultured on top, samples were first fixed for 10 min using a 4% paraformaldehyde fixation buffer. Next, cells underwent postfixation in a 2% osmium tetroxide (OsO4) solution for 2 h, followed by dehydration using a series of increasing concentrations of ethanol . Finally, all samples (with and without cells) were dried in a liquid CO2 critical point dryer , sputter‐coated with 2 nm of Pt to improve conductance and subsequently imaged using SEM .Statistical Analysis: Statistical analysis was performed using the GraphPad Prism 5 software. Normality was examined by means of a D'Agostino & Pearson omnibus normality test, and numerical data were compared directly using an unpaired t‐test.The authors declare no conflict of interest.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.


SCI) possesses a significant health and economic burden worldwide. Traumatic SCI is a devastating condition that evolves through two successive stages. Throughout each of these stages, disturbances in ionic homeostasis, local oedema, ischaemia, focal haemorrhage, free radicals stress and inflammatory response were observed. Although there are no fully restorative cures available for SCI patients, various molecular, cellular and rehabilitative therapies, such as limiting local inflammation, preventing secondary cell death and enhancing the plasticity of local circuits in the spinal cord, were described. Current preclinical studies have showed that fibroblast growth factors (FGFs) alone or combination therapies utilizing cell transplantation and biomaterial scaffolds are proven effective for treating SCI in animal models. More importantly, some studies further demonstrated a paucity of clinical transfer usage to promote functional recovery of numerous patients with SCI. In this review, we focus on the therapeutic capacity and pitfalls of the FGF family and its clinical application for treating SCI, including the signalling component of the FGF pathway and the role in the central nervous system, the pathophysiology of SCI and the targets for FGF treatment. We also discuss the challenges and potential for the clinical translation of FGF‐based approaches into treatments for SCI.Spinal cord injury ( Acute spinal cord injury (SCI) is physically and psychologically grievous condition that dramatically affects most people in their most productive life Fibroblast growth factor (FGF) is present in the central and peripheral nervous system during development and throughout whole life. FGFs stimulate neuronal cell fate determination, migration and differentiation via a classical secretory pathway  subfamily) In mammals, the FGF system is include 18 ligands, of which at least 10 are located in brain. According to numerous researches, four previous members, now called FGF homologous factors (FHF1–4), have been removed from the original list of 22 ligands, because unlike the others, it is comprised of intracellular proteins acting on intracellular targets without activating a cell surface, transmembrane receptor tyrosine kinase There are four receptors bounding with membrane‐bound and the fifth one truncated (soluble) receptor with lower affinity for most ligands. FGF receptors consist of one transmembrane domain and two intracellular kinase domains 2+ pathway, has been demonstrated in the stimulation on neurite extension by FGF2. The PI3 kinase/Akt pathway mediates some of the activities of FGFs, although there is little evidence for its role in neural development in vivo downstream of FGFRs, our recent data indicated bFGF shows a neuronal protective effect in stroke rat model via the activation of both the PI3K/Akt and ERK1/2 signals The receptors signal primarily through four main pathways, phospholipase Cγ (PLCγ), mitogen‐activated protein kinase (MAPK), FRS2 α and AKT to influence gene transcription. Briefly, the MAPK/Erk signalling cascade is particularly important and the pathway most commonly employed by FGFRs and results in stimulation of the expression and/or activation of various transcription factors that act as effectors of the pathway, including Ets proteins, AP1, GATA proteins, c‐myc and CREB in vivo improves rat sensorimotor functions, reduces focal ischaemia‐induced infarct size, stimulates functional recovery of rats subjected to motor cortex lesion and promotes rat hippocampal cell survival upon kainic acid‐induced seizure et al. demonstrated that FGF20 almost completely enhanced survival these tyrosine hydroxylase (TH)‐positive neurons against 6‐hydroxydopamine (6‐OHDA)‐induced toxicity. It indicated that important role for FGF20 in protecting dopamine neuron integrity and preserving of some motor function Accumulated evidence shows that multiple FGFs are expressed in the CNS and are crucial regulators in the development of brain or spinal cord and the functions from adult CNS. Evidence of FGF1 and FGF2 mitogenic and neurotrophic activities includes their ability to improve the viability and outgrowth of various neuronal cell types, including neocortical, hippocampal, cerebellar, dopaminergic, spinal cord and isolated sensory neurons et al. tested one of the FGF‐8 isoforms, FGF‐8b, and compared its activity with bFGF for neuroprotective in hippocampal neurons from oxidative insult. They report that FGF‐8b affords dose‐dependent protection against an oxidative insult The knowledge of the roles of other FGFs in the CNS injury is currently limited, especially in SCI. Mark et al. demonstrated that SUN111602 also ameliorate memory and learning defects in the hippocampally lesioned model rats by preventing neuronal death and/or promoting neurite outgrowth, suggesting SUN11602 is a potential candidate for treating Alzheimer's disease (AD) 2+ homeostasis as observed in the case of bFGF via phosphorylation of the cytosolic tyrosine kinase domain of FGFR‐1, but does not have promoting effect on cell proliferation unlike bFGF Recently, some synthesized small molecules, such as SUN13837 and SUN11602, are reported to mimic the neuroprotective effect of bFGF via receptors, activating various differentiating molecules, performing the axon chemotactic function, guiding and quickening the axon regeneration, performing the chemotactic function of inflammatory cells and stimulating the blood vessel formation, stimulate axon sprout; (2) to protect the injured neurons, lower the death of injured neurons and mediate the gene expression of injured neurons; (3) to attenuate the major pathological process of SCI secondary injury including inflammation, astrocyte activation and scar formation.The pathological course of SCI is a ‘two‐staged’ process involving primary and secondary injurious events et al. et al. found that aFGF‐intercostal nerves graft combination resulted in functional recovery in a complete spinal cord transection model. The Basso, Beattie and Bresnahan (BBB) score was remarkably improved. Moreover, detection of somatosensory (SSEP) and motor evoked potentials (MEPS) showed both sensory and motor signal across the lesion site aFGF, also known as FGF‐1, was first extracted and purified in 1975 from the brain and pituitary based on their capability to stimulation on the proliferation of mouse fibroblasts et al. SCI elicits a robust inflammatory response that recruits macrophages to the injured site of spinal cord et al. has demonstrated that aFGF is able to protect PC12 cells from glutamate‐induced toxicity via the activation of the phosphatidylinositol 3‐kinase (PI3K)/Akt kinase signalling pathway in vitroet al. also revealed that aFGF induced an alternative possible protective pathway in SCI. Stimulation of aFGF may induce production of neurotransmitters (Rab‐GDI) and alleviate the production of an inhibitor (RKIP) of the Raf/MEK/ERK pathway. As a result, this should create a favourable conditions for the regeneration process and the repair of injured tissues in SCI et al. aFGF has been demonstrated to ameliorate the dieback procedure of the corticospinal tract and to enhance sprouting and regeneration of Schwann cell guidance channels or peripheral nerve grafts across the injured site in adult rats with complete thoracic transection et al. reported that their combined treatment using peripheral nerve graft and aFGF led to reduced GFAP immunoreactivity, attenuated macrophage activities and decreased KSPG deposition 14 days post‐spinal cord transection injury Scar formation is believed as a major physical and chemical obstacle to axonal regeneration after SCI, and these areas contain reactive astrocytes as a major component et al. found that restriction of acute ischaemic spinal cord damage leads a specific temporal and spatial induction of both aFGF and bFGF protein generation and their previous results have also showed a dramatic increase in the levels of mRNA for bFGF in the spinal cord after injury immediately et al. found that focal injection of bFGF into the lesion centre 5 min. after SCI at T8 ameliorate spinal ventral horn (VH) and intermediolateral (IML) motor neurons adjacent to the lesion site at 1 day after injury and preserved their cholinergic phenotype bFGF, also known as FGF2, was the first FGF to be cloned in the rat. bFGF is highly expressed in the nervous system. Previous studies have shown that bFGF supports neuronal survival and growth, as well as neural stem cell proliferation and differentiation. Treatment of bFGF significantly rescues neuronal and improves functional recovery following experimental brain trauma and stroke. In 1996, Mocchetti in vitro. Furthermore, bFGF is also a mitogen factor and chemoattractant for oligodendrocyte precursor et al. study, using the injectable drug delivery system, co‐delivery of epidermal growth factor (EGF) and bFGF was performed in the subarachnoid space of Sprague Dawley rats bFGF stimulates glial progenitor proliferation and oligodendrocyte maturation In an early study, bFGF has been shown to prevent EAA‐mediated neuronal cell death in several neuronal populations, possibly by down‐regulating NMDA receptor function. bFGF can prevent oligodendrocyte apoptosis after injury and induce mature oligodendrocytes to dedifferentiate and proliferate. It has been proven that endoplasmic reticulum (ER) stress is involved in cell apoptosis after SCI, especially in neurons and oligodendrocytes but not in astrocytes via induction of progenitor cells progenitor and differentiation after SCI in vitro and in vivoet al. et al. via the activation of the JNK and ERK or FAK signalling pathways. bFGF has also exerted an effect of disrupting myelin production in mature oligodendrocytes, causing reactive changes in oligodendrocyte progenitor cells, and increased the density of microglia at high concentrations in vivo. Microglia also strongly responds to bFGF and transform to an amoeboid phagocytic cell type from a ramified phenotype in vitroin vitro.Astrocytes are the most abundant cell type and enriched in the mammalian CNS system. Protoplasmic astrocytes performed various functions, including maintaining the BBB integrity, neurotransmitter turnover, ion homeostasis and synapse formation. However, another major effect of these astrocytes involves their activation in response to injury Functional recovery of SCI in studies aiming a single component, nevertheless, is often modest. Combination of therapies may help overcome multiple obstacles to regeneration and provide synergistic effects on functional recovery. Because growth factors are prone to burst in the body with a short half‐life, it often is difficult to reach ideal drug concentrations et al. Combining cell transplantation with growth factors delivery may reinforce restoration following SCI. Compared to unmodified OPCs, oligodendrocyte precursor cells (OPCs) modified to express ciliary neurotrophic factor survived to a greater extent after transplantation into the injured spinal cord et al. reported that transplantation with bFGF/HEMA‐MOETACL provided a scaffold for the intrinsic growth of regenerating tissue 8 weeks after implantation. Combination of bFGF and HEMA‐MOETACL implantation enhanced both axonal regeneration and functional recovery after SCI Combining growth factors delivery with biomaterial scaffolds exhibit a useful effect on treatment for SCI due to its capacity to act both as a scaffold and medium supporting the delivery of therapeutic agents in vivo researches relative to therapy of SCI are performed in rodents. Although preliminary studies of FGFs in animals are obbligato and fundamental, research on SCI should focus on trials in humans. In 2004, Cheng et al. et al. from Taiwan reported a clinical trial and involved nine patients who had cervical SCI for longer than 6 months received direct spinal cord implantation of fibrin glue containing aFGF. Six‐month postoperative follow‐up showed a significant improvement in their patients’ ASIA motor and sensory scale scores. Interestingly, modest nerve regeneration occurred in all nine patients after this procedure without any observed adverse effects bFGF and FGF7  are currently approved by the Pharmaceuticals and Medical Devices Agency and Food and Drug Administration in Japan and United States, respectively. FGFs have been used in clinical settings for several years for treating skin ulcers mucositis Designing an effective administration of FGF is an elusive but urgent task. Several obstacles prevent the application of FGF are as follows: (1) How to use pharmaceutical technology and biomaterial to assist FGFs with its best bioactivity. (2) It is reported that bFGF had therapeutic effects in Strok, and it is worth for considering that intravenous drip of bFGF might be useful for SCI in clinic. (3) Many FGFs with neuronal protection, except aFGF and bFGF, whose effect and mechanism in SCI are significantly worth digging, such as FGF20 or FGF13.i) minimalization of the deleterious effects of secondary events, such as inflammation and scar formation; (ii) optimization of the survival and function of spared CNS fibres in partial lesions of the spinal cord; (iii) replacement of lost neural tissue at the impact site by nervous transplant or various stem cells; (iv) addressing the multiple components of CNS inhibition to promote and guide axonal regrowth; and (v) establishment of appropriate connections by regenerating axons to restore damaged neural circuits As presented in this review, it appears that treatment of FGFs could efficiently promote functional recovery after SCI based on findings from pre‐clinical research and clinical trials. Despite their widespread application in the clinic, major challenges remain with the use of FGFs, such as short half‐life, susceptibility to inactivation, and rapid dilution and metabolism if used topically. Notable progress has been made in the research of modifications to cell transplantation and biomaterial scaffolds materials for sustained release of growth factors in SCI. Ideally, future research direction will comprise a multifactorial approach or combination therapies addressing the following points: (i) the spinal cord is site sensitive and dangerous; (ii) combination therapy involving stem cells and FGFs is a new treatment and entails a number of complex issues, such as risk for tumorigenicity, and interactions of cells with drugs; and (iii) clinical deficit could be worsened by inappropriate stem cell differentiation and tissue hyperplasia. A great deal of additional work is needed to ensure the safety and efficacy of FGF before large, multicentre and controlled clinical trials are undertaken.Although extensive studies have demonstrated that FGFs promote functional recovery after SCI, there still lack of clinical translational evidence of benefit among patients with SCI. Moving from preclinical to clinical SCI trials is very complex, and randomized controlled trials (RCTs) will definitely offer more scientific evidence on one hand, but on the other hand, RCTs are more difficult to be conducted, however. The benefit of FGFs in human SCI, however, can only be established by further clinical phase II/III trials. Clinical trials basically also showed the safety of the transplantation procedures, especially the combining FGFs delivery with cell transplantation or biomaterial scaffolds, in SCI. Companies, researchers and patients should be aware of the regulatory issues that must be addressed before starting human experimental trials because of some special concerns: (Although a great deal of additional work is needed to ensure the safety and efficacy of these therapies before large, multicentre and controlled clinical trials are undertaken, the very preliminary clinical trials reviewed in this study offer novel data supporting their potential efficacy of FGFs in SCI.The authors declare no conflict of interest.


Vaccinium myrtillus) by nine different treatments using clipping  with or without chemical treatment by methyljasmonate (MeJA). We subsequently measured consequences of induced defenses through vegetative growth and insect herbivory during one growing season. Our results showed that only MeJA‐treated plants showed consistent defense responses through suppressed vegetative growth and reduced herbivory by leaf‐chewing insects, suggesting an allocation of resources from growth to defense. Leaf tissue removal reduced insect herbivory equal to the effect of the MeJa treatments, but had no negative impact on growth. Branch removal did not reduce insect herbivory or vegetative growth. MeJa treatment and clipping combined did not give an additional defense response. In this study, we investigated how to induce defense responses in wild plant populations under natural field conditions. Our results show that using the chemical elicitor MeJA, with or without biomass clipping, may be a better method to induce defense response in field experiments than clipping of leaves or branches that often has been used in ecological field studies.Inducible plant defense is a beneficial strategy for plants, which imply that plants should allocate resources from growth and reproduction to defense when herbivores attack. Plant ecologist has often studied defense responses in wild populations by biomass clipping experiments, whereas laboratory and greenhouse experiments in addition apply chemical elicitors to induce defense responses. To investigate whether field ecologists could benefit from methods used in laboratory and greenhouse studies, we established a randomized block‐design in a pine‐bilberry forest in Western Norway. We tested whether we could activate defense responses in bilberry ( Plants Induced plant defense systems rely on a complex signaling and regulatory network of plant hormones where jasmonic acid and its derivative methyl jasmonate (MeJa) are important elicitors of plant defense systems against leaf‐chewing insects and necrotrophic pathogens  is a dominant evergreen dwarf shrub and an important food source for insects and vertebrate herbivores, pollinators, and fruit‐eating birds and mammals , graminoids and bryophytes, and is located in the southern boreal zone with an annual precipitation of 700–900 mm and a mean summer temperature of 12–16°C . Within each block, nine bilberry ramets, ranging from 10 to 25 cm in height, were randomly selected and individually marked and exposed to the nine different treatments , or a control solution of water and ethanol until the point of runoff  and annual shoots  at the selected ramets three times in a 2‐week period. Insect herbivory was simulated by removing two circles, 2 mm in diameter, from each of the five lower leaves once a week over the treatment period Figure ; Table 1Geometridae caterpillars . Quantifying leaf tissue consumption by the leaf area removed by leaf‐chewing insects was not considered as an appropriate field method as it would have been too time‐consuming and also could stress the plants considerably. Therefore, we counted the number of leaves per plant that had chewing marks as a proxy for the abundance of leaf‐chewing insects and as an indicator of bilberry defense. This is a simple and cost‐efficient method, often applied in field studies where the objective is to study possible changes in leaf palatability by leaf‐chewing insects in response to experimental treatments  of the ramets from in situ morphological measurements at each census based on the biomass formula developed for bilberry by Hegland et al.  and caused changes in resource allocation from plant growth to defense . Therefore, we tested how the increase in the proportion of insect‐grazed leaves and plant biomass changed from June to August in response to the treatments applied in early June. As there were few flowers on the plants in the study area, probably due to shading effects of trees and annual variation , treatment effects on flowering frequency and berry production could not be tested. Twenty‐three individual ramets with <2/5 of their leaf or shoot tissue untreated  were removed from the dataset prior to the statistical analysis to avoid potential bias from severe experimental (clipping) damage. As also excessive loss of leaves between registrations constitutes a potential bias (as we don't know whether or not these leaves were insect grazed and why they were lost), we removed 11 individuals which lost more than 1/5 of their leaves during the experiment. Six more individuals were entirely lost during the experiment, most likely due to ungulate grazing, resulting in a final sample size of 94 independent individual ramets for analysis.p‐values , MeJa concentration , and clipping  as well as their interactions were used as fixed effects predictors. The nested spatial structure of ramets within block were used as random effects to account for both dependencies within blocks and ramets and variability in size and grazing pressure at the start of the experiment. We allowed for both random intercepts and slopes  for ramet and block. Random slopes and fixed effects elements were removed from the models when not contributing significantly to model performance in a backwards selection procedure using likelihood ratio tests. All statistical analyses were performed in R version 2.15.0  on Norway spruce (Picea abies)  reduced the growth rates of sawflies, whereas Rodriguez‐Saona, Polashock et al. (Lymantria dispar) herbivory, mechanical tissue damage, and exogenously applied MeJa activated plant defense genes and decreased caterpillar attack on American cranberry (Vaccinium macrocarpon). Our study adds to this growing body of evidence by showing that boreal plants under natural conditions may have their defense system triggered by MeJa which reduces the herbivory on bilberry leaves by leaf‐chewing caterpillars.In this study, we showed that exogenously applied MeJa elicits a defense response in a wild bilberry population at the expense of plant growth. This study was specifically designed to test various treatment methods, both physically and chemically, in order to induce defense responses under natural field conditions. In contrast to laboratory and greenhouse studies or studies in agricultural systems, ecological field studies have to deal with considerable variation in growth conditions, plant age, genetical composition, pathogenic infestation, and previous herbivory. This variation is also of ecological importance as variation is symptomatic of how nature works. Moreover, in field studies of plant defense responses, precipitation, evaporation, and wind can dilute the concentration of airborne plant defense volatiles and thereby reduce their repellent role in plant–herbivore interactions. Nevertheless, our study showed that leaf‐chewing insects reduced their attack on MeJa‐treated plants, which supported our prediction I. These findings are consistent with a field experiment testing winter browsing effects on bilberry only applying chemical treatment by MeJa  significantly reduced their berry production over two succeeding years, probably caused by an allocation of resources from growth and reproduction to defense. In contrast to MeJa‐treated plants, the clipping treatments  alone did not suppress plant growth, suggesting that there was no allocation of resources from growth to defense following such clipping treatments. The leaf tissue removal, however, did reduce attack by leaf‐chewing insects similar to the reduction in insect herbivory observed for MeJa‐treated plants (Table The result that only MeJa‐treated plants appeared to divert resources from growth to defense during the growing season was consistent with our prediction II. Shifts in resource allocation from growth to defense in MeJa‐treated plants has been reported for wild tobacco (Nabity et al., ts Table . Insect ts Table , it is lThe activation of plant defense systems by both exogenously applied MeJa and mechanical tissue removal will clearly differ from the activation caused by herbivores (Koo & Howe, None declared.