Artificial intelligence in computer vision has been increasingly adapted in clinical application since the implementation of neural networks, potentially providing incremental information beyond the mere detection of pathology. As its algorithmic approach propagates input variation, neural networks could be used to identify and evaluate relevant image features. In this study, we introduce a basic dataset structure and demonstrate a pertaining use case.n = 1493) rating a variety of features including fracture certainty was used to confirm its usability for separating input variations. We trained a customized neural network on the task of fracture detection using a state-of-the-art preprocessing and training protocol. By grouping the radiographs into subsets according to their image features, the influence of selected features on model performance was evaluated via selective training.A multidimensional classification of ankle x-rays (The models trained on our dataset outperformed most comparable models of current literature with an ROC AUC of 0.943. Excluding ankle x-rays with signs of surgery improved fracture classification performance (AUC 0.955), while limiting the training set to only healthy ankles with and without fracture had no consistent effect.Using multiclass datasets and comparing model performance, we were able to demonstrate signs of surgery as a confounding factor, which, following elimination, improved our model. Also eliminating pathologies other than fracture in contrast had no effect on model performance, suggesting a beneficial influence of feature variability for robust model training. Thus, multiclass datasets allow for evaluation of distinct image features, deepening our understanding of pathology imaging.The online version contains supplementary material available at 10.1007/s11548-023-02839-9. Artificial intelligence (AI) via neural networks is becoming an increasingly important tool in diagnostic imaging. Its accuracy in detecting pathologies has been increased to and even beyond the level of human radiologists –5. BecauApplication of neural networks in computer vision is widely established on two-dimensional real world images , allowinIn this study, we present a multiclass image dataset of ankle x-ray examinations as well as improvements in image preprocessing and augmentation on the performance of a modified Inceptionv3-network in a clinical image classification task. The dataset consists of ankle x-ray images taken over 6 years at a single medical center, its intended primary purpose is to be used as a training set for a variety of deep learning algorithms in clinical application with a focus on trauma diagnostics. Its multiclass design enables an analysis of influencing factors as well as performance optimization, as we demonstrate on a fracture prediction task.The dataset included x-ray examinations of the ankle region from adult patients at the trauma center of our institution between 2017 and 2019. In order to balance the training datasets regarding the proportion of fracture-containing examinations , fracture-positive ankle examinations from the years 2013 to 2016 were selectively included. The image data were imported from PACS into the NORA application for further processing. NORA is a software designed to improve the connection between research and clinics in the field of medical imaging and offers customized integrated solutions to visualize and process medical imaging data [Images were then classified into anteroposterior (ap) and lateral projections of the ankle, disregarding mortise projections for their lack of prevalence in routine ankle trauma workup at our institution. Exclusion due to clinical reasons was not performed, neither did we exclude examinations with osteosynthetic materials, prostheses, obstructed images or those displaying pathologies other than ankle fractures, but rather classified them into respective selective and partially overlapping training subsets.Aside from image classification according to projection, data was also classified at examination level using an NORA-embedded custom rating tool to rate relevant aspects. These included image quality, existence and graded certainty of fracture, plaster casts, internal foreign bodies, joint configuration, previous surgery, other pathologies of the bones as well as soft tissue lesions and swelling . Selective training was performed by defining training subsets according to the assigned labels of their images.In particular, we intended to investigate the effect of visible signs of therapy such as osteosynthetic materials and plaster casts as indirect indicators of fracture, such that models trained on an image set consisting of all ankles (unrestricted set) were compared to those trained on a set defined by excluding images displaying therapeutic appliances (pretherapeutic set). Further excluding images displaying non-traumatic osseous lesions such as tumors or excessive degeneration (filtered set) enabled training of a “baseline” model on only normal and fractured ankles without other pathologies Fig. . The anaAn imagenet-pretrained Inceptionv3-network architecture was modified by removing the top layers and adding one two-dimensional global average pooling, dropout and dense convolutional layer each onto the network as provided by the Keras API , 12. TheNetwork architecture, training runs and dataset augmentation were implemented using the Tensorflow 2.6 library . PerformTraining runs were uniformly set to 200 epochs of 100 steps using a batch size of 15 images. In order to achieve equally-mixed training batches, label-based balancing was performed on batch generation, as well as data set augmentation. Augmentation consisted of random vertical image flipping, rotation of up to 20°, contrast adjustment and image cropping. Afterward, images were resized to an input size of 340 × 512 pixels, which was determined by taking into account the oblong shape of ankle radiographs as well as GPU memory capacity. All hyperparameters were set empirically and kept constant for all training runs. Validation results were calculated using integrated Tensorflow prediction functions and Scikit-learn 0.24.2. For training, we used NVidia GTX 1080 TI-GPUs on dedicated server machines.As our total training dataset was biased in favor of its fracture prevalence, no epidemiologically valid conclusions can be drawn from it as is. For the unbiased subselection from 2017–2019, 1,255 patients were included. The male predominance of 689 vs 566 female patients suggests a predisposition of male gender to relevant trauma exposure and subsequent imaging diagnostics in the setting of radiographic imaging numbers. While in agreement with , 15 the Unrestricted set: The initial image inclusion criteria yielded 1,493 images .Pretherapeutic set: Reduction to pretherapeutic x-rays yielded 1,313 images .Filtered set: Limiting the pretherapeutic images to those devoid of other bony pathology and foreign bodies lead to 1,225 studies .Filtering images by non-fracture features as explained above produced the following subsets:The results were compared between the described fracture certainty classes 1 to 3 and the fracture-negative class 0 cases  containing about 12% fractures, which achieved an AUC of 0.983 for the ankle region without artificial data augmentation .Given the obvious difference in training set size between this large-scale study with the number of images one order of magnitude higher than ours, we attribute the largest part of the difference in performance to quantity and quality of the training set, and consider the choice and rigorous training of the network the second largest factor. Additional factors include augmentation, pretraining status of the network and the duration of training. Comparing the performance difference of our study to those of comparable set sizes and network structure especially in light of this very different study, the effects of improved preprocessing, optimized network layers as well as refined image data augmentation methods become evident.Utilizing the remaining two color channels by copying especially the edge-enhanced filtered image might have provided additional benefit, although the exact extent of this needs to be addressed in dedicated investigations. In general, isolating and emphasizing imaging features for easier identification by a human investigator is thought to improve network performance at least in terms of convergence speed as well, but to our knowledge, no relevant evidence has been published at the time of writing.In this regard, our aim to achieve a high precision using several improvements over common practice on average-sized image datasets could contribute to this question.In order to gauge the influence of distinct image features on fracture classification performance, we performed training runs with and without images containing the features in question.When training on the pretherapeutic image set without therapeutic appliances, models consistently performed better than on the unrestricted data set. The reason for this behavior might be the follow-ups with and without visible fracture features leading to lower correlation of fracture with therapeutic materials. Limiting the set to pretherapeutic images removed the palpable, confounding feature set of implants and bandages, effectively reducing the variability of possible image features, presumably simplifying the classification task.On the other hand, reducing the image feature set even more by excluding other pathologies in the filtered set might have decreased the variability of training image features to a degree that overall prediction performance was reduced again to the initial level of the model trained on the unrestricted set.Limiting training to ap-projections of the ankle while having rated fracture certainty as maximum visibility on both ap- and lateral projections invariably leads to discrepancy, which we chose not to address for the results presented here.On the one hand, to some degree, misclassifications in a sufficiently large and diverse data set serve to improve network robustness, as in principle, fracture and non-fracture status exist on a spectrum of possible pathologies with overlapping imaging and clinical features, so that imaging findings conceptually need to be differentiated from objective diagnosis. A desirable network prediction thus would recreate that degree of overlap so that the variety of possible morphologies can rather be accounted for during interpretation of said prediction. To what level additional findings like secondary fracture signs could serve to aid that interpretation on a network level needs yet to be examined.On the other hand, we would argue that rating an objective property apparently not visualized on the rated image conceptually extends the prediction process over those features recognizable to the human reader, whereas implementing a decision logic for separately trained, self-contained network predictions on different views equals a potential limitation to the level of optimal human performance. To make full use of a machine’s potential to permit complementary and comprehensive interpretation, it would therefore be necessary to expose it to a more comprehensive set of information than that contained in a single view.In conclusion, the results presented here demonstrate the multidimensional nature of influence on neural network performance and the advantage of training on a dataset containing ratings of additional features instead of only those the detection of which it is intended to train. Systematic analysis of various subsets offers insight into the relations of distinct image features and ultimately may help training classification networks by optimizing their training protocol. To this end, we defined a multiclass dataset of ankle x-ray images and compared the performance of a neural network trained on it to contemporary models, where additional beneficial factors for classification performance on medical images could be identified in preprocessing and customization of network top layers.We plan to expand our multidimensional dataset in size and maximize its use in creating a robust supportive diagnostic tool for ankle x-rays as well as explore the relations of distinct image and clinical features to further our own understanding of pathology detection in conventional musculoskeletal imaging.Supplementary file1 (DOCX 13 KB)Below is the link to the electronic supplementary material.


Glaucomatous optic neuropathy (GON) is the major cause of irreversible visual loss worldwide and can result from a range of disease etiologies. The defining features of GON are retinal ganglion cell (RGC) degeneration and characteristic cupping of the optic nerve head (ONH) due to tissue remodeling, while intraocular pressure remains the only modifiable GON risk factor currently targeted by approved clinical treatment strategies. Efforts to understand the mechanisms that allow species such as the zebrafish to regenerate their retinal cells have greatly increased our understanding of regenerative signaling pathways. However, proper integration within the retina and projection to the brain by the newly regenerated neuronal cells remain major hurdles. Meanwhile, a range of methods for in vitro differentiation have been developed to derive retinal cells from a variety of cell sources, including embryonic and induced pluripotent stem cells. More recently, there has been growing interest in the implantation of glial cells as well as cell-derived products, including neurotrophins, microRNA, and extracellular vesicles, to provide functional support to vulnerable structures such as RGC axons and the ONH. These approaches offer the advantage of not relying upon the replacement of degenerated cells and potentially targeting earlier stages of disease pathogenesis. In order to translate these techniques into clinical practice, appropriate cell sourcing, robust differentiation protocols, and accurate implantation methods are crucial to the success of cell-based therapy in glaucoma.Translational Relevance: Cell-based therapies for glaucoma currently under active development include the induction of endogenous regeneration, implantation of exogenously derived retinal cells, and utilization of cell-derived products to provide functional support. Glaucoma is the major cause of irreversible visual loss worldwide, second only to the reversible causes of uncorrected refractive error and cataract.,,Cross-sectional studies have demonstrated IOP within the general population to follow an approximately Gaussian distribution with an exaggerated central peak and modest skew toward higher pressures.,Among the pathogenic features of glaucoma, characteristic cupping of the ONH as a result of tissue remodeling appears to be the most specific. In a healthy retina, over 1 million RGC axons converge at the ONH before turning sharply to exit the globe via the optic nerve. Prior to passing through the lamina cribrosa, RGC axons have very high energy requirements, as evidenced by the elevated concentration of cytochrome C oxidase and mitochondria seen in the prelaminar and lamina cribrosa regions.The ONH is constantly subject to static and dynamic biomechanical forces, in part determined by IOP in its anterior aspect and intracranial pressure (ICP) in its posterior aspect.,,,The lamina cribrosa is a specialized porous collagenous plate, which provides structural integrity to the ONH. Situated between RGC axons and the lamina cribrosa are astrocytes, the major glial cell type found in this region, which provide structural and metabolic support to the RGCs.At times of excess pressure-induced stress, studies have demonstrated ONH astrocytes to become reactive and decrease their expression of glial fibrillary acidic protein, a finding that consistently coincides with morphologic changes, axonal transport delay, and astrocyte swelling.Another important aspect of the ONH structure is the blood supply to this region. Indeed, impaired blood flow is currently thought to be an important pathogenic factor for normal-tension glaucoma (NTG).,Maintenance of healthy RGCs requires the availability of essential metabolic substrates, specific trophic factors, and prompt removal of environment stressors.In summary, potential sources of axonal injury are counterbalanced by a range of protective mechanisms within the healthy individual. However, eyes of individuals who are particularly susceptible to GON can become overwhelmed in the presence of significant detrimental factors . While tThe retina is particularly well suited for the development of cell-based therapies for several reasons. First, most cell types within the retina are derived from a common neuronal lineage; second, the retina is arranged into well-defined layers that contain distinct cell typesRegeneration of the retina is a phenomenon that has been well described in certain species such as the zebrafish, where cell lineage tracing attributed the source of the regenerated cells to MGCs present in the inner nuclear layer of the central retina.,stat3, ascl1, and its downstream target lin-28 in allowing the reentry of MGCs back into the cell cycle.A host of differentially expressed factors by zebrafish MGCs following injury has been found by both genetic and proteomic methods.The detailed interactions of these pathways during the regenerative process have been extensively reviewed elsewhere.–An alternative approach to treat degenerative retinal conditions is to prepare functional cells in vitro, followed by implantation into the damaged tissue. Although current technologies can derive therapeutic cells to closely match damaged cells, designing a method of delivery that promotes integration within the retinal circuit has proven to be exceptionally difficult.,,Embryonic stem cells (ESCs) are derived from the inner cell mass of the blastocyst and have the ability to differentiate to any cell type from the three germ layers, mesoderm, endoderm, and ectoderm.–The use of human ESCs (hESCs) has been explored for use in a wide range of retinal degenerative conditions. Established protocols include the differentiation of RPE  for AMD (age-related macular degeneration)-based therapies–,–Both hESCs and human iPSCs (hiPSCs) have been used to generate three-dimensional retinal organoids that are shown to closely mimic in vivo development.,–Differentiation of photoreceptor precursors has also been increased in retinal organoids by the addition of COCO, an antagonist of the Wnt, Notch, TGF-β, and BMP pathways.In some cases, methods to isolate cells from stem cell–derived retinal organoids may be more advantageous than trying to develop protocols to promote direct differentiation. RGCs are especially well suited for this, as they can be produced more rapidly due to being one of the first cell types to develop in retinal organoids.–––,Mesenchymal stem cells (MSCs) are adult stem cells that can be derived from the bone marrow or umbilical cord. They are considered multipotent and have the capacity to differentiate toward cells of mesodermal lineage such as osteocytes, adipocytes, and chondrocytes.MSCs have been extensively explored for their therapeutic use in the treatment of glaucoma. Intravitreal injections of MSCs in glaucoma-induced rodent eyes align along the ILM  and can survive for several weeks, resulting in increased RGC survival.,,MGCs are the main glial cell type in the retina, as they span radially across all neuroretinal layers to form contacts with all cells of the neurovascular unit. The main functions of MGCs are to provide structural and metabolic support to retinal neurons similar to astrocytes within the ONH. MGCs become reactive in response to retinal injury, which can lead to the regeneration of lost neural cells in some species. Although this regenerative ability is not replicated in mammalian retina, MGCs with stem cell characteristics have been identified in the adult human eye. These cells can be isolated from postmortem tissues and spontaneously immortalize in vitro.Extensive research has been conducted on the potential for MGCs in the treatment of glaucoma. In a rodent model of NMDA-induced retinal ganglion cell depletion, transplantation of human MGC-derived RGC precursors into the vitreous space resulted in a partial recovery of RGC function as judged by the negative scotopic threshold response of the electroretinogram.Isolation of MGCs has been performed successfully from hiPSC-derived retinal organoids in vitro. Subsequent intravitreal transplantation of these MGCs into a rat model of RGC depletion has been demonstrated to induce a partial recovery of visual function.As retinal regeneration has proven to be an immensely difficult goal, attention in recent years has shifted somewhat into strategies of neuroprotection. As we gain deeper understanding of the pathogenesis of glaucoma, various experimental approaches have emerged that aim to inhibit detrimental pathways or enhance endogenous protective mechanism to prevent GON.,The dependence of RGCs on neurotrophic factors is evident during optic nerve development,,Despite these promising data, there remain concerns over the duration of any therapeutic effects from direct delivery of these neurotrophins, whereas a whole-cell transplant-based strategy may offer a longer-lasting solution. In support for this hypothesis, rat and human bone marrow–derived MSCs induced to secrete high levels of BDNF, GDNF , and VEGF  were found to exert a marked neuroprotective effect in a rodent optic nerve transection model following intravitreal injection.In order to realize the potential of neurotrophic factors in the future management of glaucoma, a safe, stable, and continuous system for their delivery remains a major challenge. Furthermore, despite deprivation of neurotrophic factors being characteristic of the glaucomatous retina, prolonged delivery of BDNF and TrkB receptor to axotomized optic nerve was unable to maintain long-term RGC survival.,MicroRNAs (miRNAs) are endogenous, small, noncoding RNAs. Their primary function is the posttranscriptional regulation of protein-coding gene expression by binding to the targeted messenger RNA (mRNA), which leads to the inhibition of translation or mRNA degradation. Through this system of interference, miRNAs have been demonstrated to play pivotal roles in cell proliferation, differentiation, and apoptosis.In recent years, significant research interest in neuroprotective strategies has been focused upon the phosphatase and tensin homolog (PTEN) gene, the master inhibitor of the proregenerative mTOR/PI3K/Akt pathway.–Over the past decade, secreted EVs comprising cytosol enclosed in a lipid bilayer membrane have been identified as a major mode of intercellular communication,Since EVs are capable of delivering their cargo directly into target cells, EVs derived from healthy cells may potentially have a comparable therapeutic potential as the cells themselves. Cell-derived EVs also contain a range of miRNAs to potentially activate multiple antiapoptotic and prosurvival pathways at once. As evidence accumulates for their neuroprotective efficacy, low immunogenicity, and high stability, EVs appear to be promising candidates as an adjunctive therapy to IOP-lowering medications and thus a potential future treatment for glaucoma.In recent years, there has been tremendous progress in the development of cell-based therapies for glaucoma. In addition to established strategies for endogenous regeneration and cell replacement, there is growing interest in more novel approaches using cell-derived products such as EVs. Appropriate cell sourcing, robust differentiation protocols, and accurate implantation techniques are all crucial to the development of successful cell-based therapy in glaucoma. In this rapidly progressing field, the increasing diversity of approaches and incremental refinements of existing techniques are bringing the prospect of clinical application ever closer to reality.


Case Presentation. The ureteropelvic junction obstruction imaging with significant renal function deterioration in an adult patient is described in this report. Laparoscopic exploration revealed aberrant vessels that compress the ureteropelvic junction against the lower pole of the kidney.  The ureteropelvic junction obstruction is more common in children; however, it can also affect adults. The management of this condition has shifted toward a nonoperative approach with serial ultrasonography and renography.  It is important to consider that some of the ureteropelvic junction obstruction cases can get worse even in a short period of time. Hydronephrosis related to ureteropelvic junction obstruction (UPJO) is more commonly diagnosed in children but can also occur in adults . A junctA 32-year-old woman with a previous history of left mild hydronephrosis, nonspecific intermittent left flank pain, and equivocal DRG result was on close observation Figures .After 7 years of surveillance and ruling out the vesicoureteral reflux, her symptoms got worsen during 6 months with remarkable changes in her imaging findings Figures . ThroughHaving been referred to our center, she was scheduled for transperitoneal laparoscopic evaluation due to persistent pain and obvious deterioration in renal function. There was an aberrant vessel during laparoscopy, and after releasing and transpositioning this vessel, the left renal pelvis showed an ever-increasing dilation, which meant an intrinsic obstruction at UPJ may be responsible for the situation . So, we UPJO is increasingly being diagnosed prenatally through ultrasonographic screening. The condition can be due to intrinsic or extrinsic factors and can be congenital or acquired. As a result of UPJO, there are several causes, including ureteral kinking, an aperistaltic segment, or a high insertion of the ureter, respectively . HoweverAn active surveillance study conducted by Gulur et al. found that during follow-up, adults diagnosed with UPJO had significantly reduced renal function without developing any symptoms . In the There is a wide spectrum of UPJO, ranging from a reversible radiological finding, which occurs without producing renal damage, to well-defined histological alterations of the UPJ, which are associated with clinically significant renal parenchyma injury. It remains unclear whether the parenchymal damage in congenital UPJO is caused by chronic obstruction or genetically histological alterations in the renal parenchyma independent of obstruction degree . In addiThere is a lack of consensus on the frequency of diuretic renography during follow-up, which should be into consideration to ensure appropriate and timely intervention in the event of renal function deterioration. While taking a conservative approach to asymptomatic UPJO in adult patients seems reasonable, some cases may experience worsening even in a short period of time. More studies are required to determine the best management and follow-up protocols for UPJO in adult patients.


With the emergence of soft robotics, there is a growing need to develop actuator systems that are lightweight, mechanically compliant, stimuli-responsive, and readily programmable for precise and intelligent operation. Therefore, “smart” polymeric materials that can precisely change their physicomechanical properties in response to various external stimuli  are increasingly investigated. Many different types of polymers demonstrating stimuli-responsiveness and shape memory effect have been developed over the years, but their focus has been mostly placed on controlling their mechanical properties. In order to impart complexity in actuation systems, there is a concerted effort to implement additional desired functionalities. For this purpose, elastin-like polypeptide (ELP), a class of genetically-engineered thermoresponsive polypeptides that have been mostly utilized for biomedical applications, is being increasingly investigated for stimuli-responsive actuation. Herein, unique characteristics and biomedical applications of ELP, and recent progress on utilizing ELP for programmable actuation are introduced. Soft robotics as a scientific field has grown tremendously over the last two decades. Compared to the traditional robotics that involves the design and fabrication of controllable electromechanical parts with hard materials such as metals and hard plastics, soft robotics rely more on flexible and mechanically compliant materials that allow much greater complexity in movement and device fabrication More recently, stimuli-responsive and compliant materials are actively investigated for biomedical applications, such as drug delivery, biosensor, and tissue engineering, as they can impart complex control over their functionalities, which are not generally possible for conventional materials. For example, drug release from soft materials, such as hydrogels and nanovesicles as drug delivery systems, is governed by the laws of diffusion. Even though the release rates can be controlled to a certain degree by controlling the mechanics of the materials, the release is continuous and not amenable for “switching on and off” under desired conditions. The stimuli-responsive polymers that undergo reversible phase transition between swollen and collapsed states under aqueous environment in response to changes in external stimuli, such as pH, temperature and ionic strength, have been widely adopted to develop the materials that allow more precisely block or facilitate the drug release at specific conditions A crucial factor for developing soft robotic systems is the proper actuation mechanism, which is also directly related to the type of material. For the actuator to undergo highly specific changes, the material must be designed so that there is a precise correlation between the amount of stimulation and the degree of shape deformation. In addition, this correlation must be completely reversible for repeated operations. This means it should be possible to “program” their stimuli-responsive physical change, while maintaining their structural integrity and processability.EAPs are defined broadly as the materials that can change their physical dimensions in response to externally applied electrical field Ionic EAPs rely on the mobility of ions embedded in the polymeric material under the electric field Tg), and the “hard” segment that holds its original position to which the deformed polymer can revert. Therefore, thermoplastic polymers engineered to form physical or chemical crosslinking are generally explored as SMPs. This actuation mechanism of SMP is entropically driven to the original disordered state. The most notable class of SMPs is polyurethane (PU) In addition to electrical field, temperature has been another potent stimulus for inducing physical changes of soft actuators. Thermoresponsive polymers that can undergo reversible phase transition at a particular temperature, called lower critical solution temperature (LCST), have received significant research interest  Zhang etThe most important attribute of thermoresponsive polymers is the reversibility of actuation, which is a clear advantage over SMPs that need repeated thermomechanical reprogramming. In addition, because of this innate reversible thermoresponsiveness, these polymers can be easily hybridized with other polymers and nanomaterials to impart the thermoresponsive actuation capability. However, because of the low mechanical strength, it is difficult to fabricate actuators by themselves, and as a consequence, they generally need to be integrated with the existing platform for mechanical reinforcement. Furthermore, the actuation mechanism for the thermoresponsive polymers is mostly based on hydrophobic interaction and hydrogen bonding which are mostly realized under aqueous environment. Therefore, thermoresponsive polymers are ideally suited for materials used for biomedical applications, such as hydrogels, nanofibers, and nanovesicles With the continued maturation of the field of bioengineering, there is a growing trend to develop and adopt new materials having unprecedented functionality. In this regard, materials that can function as soft actuators are extensively investigated to impart biomaterials with controllable properties. In addition to known polymers such as PNIPAm, there is a new class of biopolymers, called elastin-like polypeptide (ELP), which are steadily emerging as a biocompatible, stimuli-responsive material system that possesses similar thermoresponsive properties, while allowing more sophisticated control of their properties via genetic engineering. In the following, the principles of genetic engineering technology to produce ELP and its basic properties are introduced. In addition, the notable examples of implementing ELP as a stimuli-responsive soft actuators for biomedical application are provided with a historical perspective.As thermoresponsive polymers relied on the hydrophobic interaction at elevated temperature, there are polymers that can undergo phase transition in response to the changes in pH. For example, polyanions, the polymers containing numerous acidic functional groups such as carboxylic acid and sulfonic acid, can form hydrogen bonds at lower pH leading to chain collapse, while the deprotonation at higher pH leads to ionization and the loss of hydrogen bonding. Polycations, on the other hand, possess amino groups that acquire positive charge upon protonation at lower pH. In addition, these polymers can acquire charge at different pH’s, making them ionic EAP’s that can be actuated via external electrical potential. Olvera Bernal et al. develop a nanofiber-based soft actuator consisting of chitosan, a natural cationic polysaccharide, and poly . There are photoactive polymers that can change their conformations in response to light at a specific range of wavelength. The most notable example is the polymer containing azobenzene moieties, which undergoes reversible trans-cis transition via UV light, called photoisomerization It is truly fascinating to know that soft actuators based on EAP and thermoresponsive polymers are largely inspired based on human physiology. The movement of muscle tissue, contraction and relaxation of myofibers, is governed by the electrical signals relayed via neural network connected by neuromuscular junction, which is essentially the basis for EAPs Elastin is found in many soft connective tissues, such as skin, blood vessel, tendon and ligament, as well as those in larger internal organs such as lung and heart Because of this thermoresponsive properties as well as biocompatibility, there have been significant research efforts to utilize elastin for biomedical applications in addition to basic biochemical research  Lima et Despite the recent success in the synthesis and application or rTE, there are several limitations that prevent more efficient production and application of rTE. The biosynthesis of rTE via recombinant technology is often technically challenging, as rTE often becomes degraded by the bacterial host or bacterial membrane, often requiring fusion with other protein using lysogenic host The advancement in the recombinant technology for ELP was remarkably boosted by a key experimental techniques, called recursive directional ligation (RDL), developed by Chilkoti and co-workers Another advantage of recombinant technology for ELP production is the simple purification process. Because of the thermoresponsiveness, ELP undergoes aggregation at higher temperature above LCST. Therefore, after cell lysis to dissolve the expressed ELP in the aqueous buffer, the temperature is increased above LCST to induce the ELP aggregation which is then collected via centrifugation. This process can be repeated to maximize the yield. This process, called inverse transition cycling, is by far more efficient and less arduous than a typical polyhistidine tagging and liquid chromatography. The most important and distinguishable biological function of elastin is imparting elasticity and mechanical toughness to biological tissues. Naturally, ELP has been often adopted to generate a variety of biomaterials with improved mechanical and thermoresponsive properties for biomedical applications Since ELP is synthesized by recombinant DNA technology, it is feasible to develop fusion protein with other polypeptides and proteins to acquire synergistic properties. 8, where X was tyrosine, phenylalanine or tryptophan. The size of the nanofibers as well as their LCST’s could be effectively controlled by the type of X residue. These ELP-based nanofibers have been utilized as scaffolds for tissue engineering applications. Natsume et al. developed the self-assembled ELP nanofibers presenting cell-adhesive REDV peptide as a scaffold for vascular graft generation Like other biopolymers, ELP can be processed into nanofibers via electrospinning Another advantage of thermoresponsive self-assembly of ELP nanofibers is the ability to precisely control their length and morphology, which is not generally feasible for electrospun nanofibers. These self-assembled nanofibers have been used to develop drug delivery vehicles and tissue engineering scaffolds. It is also possible to conjugate other polymers or lipids to tune nanofiber dimensions and morphologies. Aluri et al. demonstrated that the size of self-assembled nanofibers could be controlled by conjugating 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), a phospholipid, to ELP . Aluri e2VPGEG (VPGVG)2]n above a critical concentration of 6% (w/v) -rich hydrophobic endblocks and glutamic acid (E)-rich midblock, [(VPGVG)6% (w/v)  Lao et a6% (w/v)  Dai et aNaturally, being a polypeptide, most of the research on ELP has been focused on designing and implementing ELP for biomedical applications. The characteristic thermoresponsive properties are like those of widely explored PNIPAm, while the recombinant technology allows other peptide moieties to be integrated with ELP for added functionalities. This is a clear advantage over synthetic polymers like PNIPAm, which need to be hybridized with other functional materials. As PNIPAm over the recent years has been widely explored as thermoresponsive soft actuators, there has been a rising research trend in adopting ELP for this purpose as well.Urry and co-workers were one of the first to introduce the possibility of the mechanical actuation of chemically-crosslinked ELP hydrogels While the study by Trabbic-Carlson et al. focused more on the basic mechanical characterization of ELP hydrogels, Valiaev et al. demonstrated the practical potential of ELP to modulate the actuation of microcantilever  Valiaev One common method of producing a soft actuator is to attach two layers consisting of stimuli-responsive “active” layer and a non-responsive “passive” layer, so the actuator can bend in one direction upon stimulation in a controlled manner based on the difference in mechanics. Kamada et al. developed a bilayer actuator consisting of polyacrylamide (PAAm)-ELP hydrogel layer and non-crosslinked ELP hydrogel The ELP hydrogels that are structurally robust enough can also function as thermoresponsive soft actuators. Increasing the temperature above LCST to induce the shape deformation can be easily accomplished with a variety of heat sources. However, it is technically challenging to precisely control the thermoresponsive actuation in a localized manner using conventional heat sources. One innovative strategy to accomplish this task is by incorporating reduced graphene oxide (rGO) into the ELP hydrogel. rGO has been shown to display photothermal effect, in which thermal energy is generated upon photoexcitation using near IR (NIR) range. Wang et al. successfully demonstrated that the irradiation of NIR laser upon rGO-ELP nanocomposite hydrogel generated enough heat at the area of the irradiation, causing the hydrogel to display highly coordinated motions  Wang et More recently, Chiang et al. presented a more expansive exploration of rGO-laden ELP hydrogel as a soft actuator mediated by the photothermal effect In addition to creating a hybridized network of silk fibroin and ELP by copolymerization, it is also possible to create a fusion protein of silk fibroin and ELP by recombinant DNA technology. While hybridization leads to randomized distribution of silk fibroin and ELP, it can be inferred that the silk fibroin unit directly attached to ELP would have more intimate effect on the thermoresponsive properties of ELP. Wang et al. developed a fusion protein consisting of the characteristic domain (GAGAGS) of silk fibroin and ELP (“SELP”), in which the silk domain provided greater hydrophobicity and mechanical strength  Wang et Tg, necessitating significant thermomechanical energy for shape deformation, the transition temperature of ELP is much lower and the thermoresponsive deformation occurs naturally at higher temperature, requiring much less external energy for shape programming. It should be noted that it differs from the traditional SMPs in that the deformed hydrogel at higher temperature could not maintain its structure and reverted to its original shape at lower temperature.ELP has also been used to generate SMPs, in which the ability of ELP to undergo phase transition above LCST and crosslinking could serve as the soft segment. For example, Zhang et al. developed a semi-interpenetrating network hydrogel consisting of chemically crosslinked PAAm and physically-crosslinked ELP  Zhang etThe biocompatible nature of ELP, being a natural biopolymer, also allows the ELP-based actuators to be used for biomedical applications. For example, the thermoresponsive shape change of ELP could be used as a “switch” to induce conformational changes of nanostructures for drug delivery and biosensing applications. Kim et al. developed a fusion protein consisting of calmodulin (CaM) and ELP via recombinant DNA technology to fuse two genes  Kim and Photocrosslinking is one of the most popular methods to create hydrogels. Like other biopolymers, ELP has been modified with photolabile groups to generate hydrogel via photocrosslinking. Compared to physical crosslinking of ELP copolymers, mechanical properties of photocrosslinked ELP hydrogels can be more efficiently and broadly tuned. Guo et al. developed ELP hydrogel by photocrosslinking methacrylic functionalized ELP (“ELP-MA”) Elastin has long fascinated biomedical researchers for its unique role in tissue physiology; controlling mechanical properties of ECM as well as providing biophysical cues to influence cellular behavior. Especially, elastin is largely responsible for the high mechanical strength and elasticity of biological tissue, which is originated from the coiled secondary structure of tropoelastin polypeptides that are enzymatically crosslinked. The tropoelastin consists of repeated pentapeptide units, rich in valine, glycine and proline that mediates hydrophobic interaction-driven coiled morphology above a certain transition temperature. Due to the difficulty of obtaining pure elastin from natural sources and the advantages of imparting desired functionalities, elastin-like polypeptide (ELP), which contains characteristic pentapeptide repeats and whose specific sequences can be precisely designed and produced by recombinant DNA technology, have been more widely utilized. The thermoresponsive phase transition behavior of ELP has proven to be quite useful for developing biomaterials, such as hydrogels, nanofibers and self-assembled nanostructures for biomedical applications including drug delivery and tissue engineering.The ability of thermoresponsive polymeric materials to undergo shape deformation, such as widely popular poly(N-isopropylacrylamide) (PNIPAm), is being widely adopted to create actuators for soft robotics and multifunctional biomedical devices. Just as ELP has followed the footsteps of PNIPAm in the field of biomedical engineering, the recent research trend regarding ELP is suggesting that it is also being seriously considered as thermoresponsive soft actuators. The number of published reports is not significant at this point to imply that it is becoming a broad trend, but it is not surprising given that ELP itself has not reached the status of more popular biopolymers, such as alginate, chitosan, and gelatin for biomedical applications, largely due to the fundamental limitation of mass producibility. However, with its unique thermoresponsive properties and precise control of physicochemical properties that other biopolymers do not possess, ELP has found its niche in the field of biomedicine as a stimuli-responsive biomaterial. In the same manner, ELP has the potential to provide the similar attributes to developing soft actuators by providing new functionalities afforded by the genetic engineering.Even though ELP can be produced in relatively large quantities via recombinant technology, it is still not economically viable enough to develop large-scale actuators solely using ELP. Furthermore, the mechanical strength of ELP is not high enough to develop robust structures required for actuators. Therefore, ELP is more ideally suited as a functional moiety to impart thermoresponsiveness and bioactivity to a known material. As the direction of the research in soft actuators is shifting towards developing multifunctional actuators with different modes of actuation and a diverse array of properties, it is also expected that more hybridization strategies would be utilized to generate ELP-based composite materials. As a burgeoning material system for soft actuators, the ELP-based actuators developed thus far have been mostly geared toward biomedical applications. However, with further maturation, it can be envisioned that the ELP-based soft actuators could also find their place in other areas, such as electronics and environmental engineering.


The purpose of this review is threefold: (i) to give an overview of well-established invasive methods for assessing patients with ischemia with no obstructive coronary arteries (INOCA) in the cardiac catheterization laboratory; (ii) to describe the prognostic and treatment implications based on these findings, and (iii) to discuss current knowledge gaps and future perspectives.Recent studies have demonstrated that invasive coronary function testing not only allows for risk stratification of patients with INOCA but also guides medical therapy with improvement in symptoms and quality of life. Based on these findings, invasive coronary function assessment is now a class 2a recommendation in the 2021 ACC/AHA chest pain guideline to improve the diagnosis of coronary microvascular dysfunction and to enhance risk stratification.Invasive functional testing for patients with INOCA is well established and easily performed in the catheterization laboratory. Comprehensive invasive assessment is a key to differentiating INOCA endotypes and optimizing both medical therapy and preventive strategies including lifestyle modification. Patients with angina and/or signs of ischemic heart disease are often found to have no obstructive coronary artery disease (CAD) . This coDue to a growing recognition of INOCA and technical advances in diagnostic testing, multiple studies have proposed and validated various invasive approaches for the differentiation of INOCA endotypes, prognostication, and individualization of therapy in recent years. The current review gives an overview of each well-established invasive assessment and discusses prognostic value and treatment implications in INOCA.The coronary arterial system consists of the epicardial coronary artery (>400 μm), pre-arterioles 100–400 μm), and arterioles (40–100 μm) each of which is regulated by different mechanisms  are the two vasoactive agents most used in the cardiac catheterization laboratory and act on the coronary arterial system differently. Adenosine primarily induces endothelium-independent relaxation of vascular smooth muscle cells , 15, wheTraditional cardiovascular testing, such as exercise treadmill testing, stress echocardiography, and SPECT, have limited diagnostic accuracy for detecting coronary vasomotor dysfunction  , 19. NonInvasive coronary angiography and coronary computed tomography angiography are the mainstay for the anatomic assessment of CAD and are required to confirm the absence of no hemodynamically significant obstructive stenosis. Moreover, coronary computed tomography angiography is the gold standard for the identification of myocardial bridging . HoweverDirect measurement of coronary flow velocity using a Doppler-tipped guidewire is one of the established techniques for the invasive assessment of coronary flow reserve (CFR) Fig. . Peak flThe use of Doppler-derived CFR in INOCA was first validated in a cohort from the WISE  study . DopplerCFR can also be estimated using the thermodilution technique, which uses a pressure-temperature sensor-tipped guidewire to monitor temperature changes in response to intracoronary saline administration Fig.  37]. Ro. Ro37]. To date, a number of studies have assessed the correlation between CFRs derived from bolus thermodilution, Doppler, and non-invasive techniques , 42–44. Aside from the diagnosis of CMD based on CFR, it is important to recognize that CFR is a continuous predictor of adverse outcomes rather than a step-like threshold . In the The index of microcirculatory resistance (IMR) is calculated as hyperemic mean distal intracoronary pressure multiplied by hyperemic mean transit time both of which can be measured using a pressure-temperature sensor-tipped guidewire in a similar manner to bolus thermodilution-derived CFR Fig.  47]. Ho. Ho47]. Based on prior studies in healthy subjects –53, the Alternatively, microvascular resistance can be assessed with hyperemic microvascular resistance (hMR), defined as the ratio of hyperemic mean distal pressure to hyperemic average peak velocity, using a pressure/Doppler sensor–tipped guidewire . hMR >2.Evidence suggests that high IMR/hMR accompanied by low CFR or vasospastic angina, but not high IMR/hMR alone, is associated with long-term cardiovascular events in INOCA , 62. ThiACh-mediated increase in coronary blood flow volume measured using a Doppler-tipped guidewire is a surrogate of coronary endothelial microvascular function . CoronarImpaired endothelial function in INOCA as assessed with coronary blood flow response was first reported in the early 1990s and is known to be highly prevalent , 66•, 67Although the current ACC/AHA guideline does not specifically recommend invasive coronary endothelial function testing ••, the aImpaired coronary vascular response to ACh is independently associated with an increased risk of cardiovascular events as well as decreased time free of adverse events in patients with INOCA , 69. EndACh is also used for the assessment of epicardial and microvascular vasospastic angina (VSA) . InvasivThe reported prevalence of VSA is approximately 40% in INOCA . IdentifMicrovascular vasospasm (MVS) is defined as symptom reproduction with ischemic ECG changes but no epicardial vasospasm during provocative spasm testing . AlthougRecent, long-term follow-up data in a total of 736 INOCA patients who underwent ACh testing demonstrated that the risk of all-cause and cardiac deaths was low irrespective of spasm provocation results . HoweverThe coronary slow flow phenomenon is an angiographic finding first reported in 1972 and characterized by the delayed flow of contrast medium in the absence of obstructive CAD . The TIMThe prognostic value of the TIMI frame count was reported in a pilot study from the WISE study . AlthougA variety of agents such as dipyridamole and simvastatin were studied as a potential pharmacologic intervention for the coronary slow flow phenomenon and were found to improve coronary flow as assessed with the TIMI flow count , 82. HowThe risk of invasive coronary assessment is reported to be low. In the WISE study, serious adverse events related to comprehensive invasive testing occurred in 2 out of 293 women with INOCA (0.7%) and included 1 iatrogenic coronary artery dissection and 1 ST-segment elevation myocardial infarction due to coronary artery spasm . A recenIt is currently believed that the additional time and cost related to invasive coronary assessment is offset by preventing unnecessary testing/treatment and future adverse clinical events as a consequence of optimal management guided by invasive testing . HoweverAlthough obstructive CAD is only one of the many causes of myocardial ischemia, false reassurance after being found no obstructive CAD is often given to patients who might have other structural and/or functional abnormalities in the coronary arterial system. Consequently, these patients continue to have angina with an increased risk of adverse clinical events. INOCA should be recognized as a clinically important diagnosis for which further invasive coronary assessment is indicated to differentiate its endotypes, risk stratify patients, and individualize treatment. This guideline-directed approach needs to be implemented in our clinical practice so that patients with INOCA will receive symptomatic and prognostic benefits.


The challenge of adapting clinical trials to decentralized care models is increasingly pressing as virtual care becomes more common. In the United States, the patchwork of state and federal policies and regulations governing virtual care, such as limited interstate physician licensure, was a major barrier to the development of hybrid care models, blending in-person and telehealth, and the implementation of decentralized clinical trials (DCTs) . During in situ measurements using sensors [Multiple existing frameworks are frequently employed to guide the process of identifying and addressing barriers to DCTs; however, none integrate perspectives from stakeholders across the translational spectrum . As DCTs sensors . No publWe propose a novel conceptual framework to identify barriers to DCTs using stakeholder engagement that incorporates the broad perspectives of patients, their local providers, and state-based policymakers. Through the discovery of barriers experienced by these groups, this framework integrates the dynamic experiences of key stakeholders across the translational spectrum to identify and address policies that hinder the conduct of DCTs. Unlike previous frameworks, this framework addresses the hurdle of nonuniform policy landscapes that modulate the scope and scale of DCTs on a state-by-state basis in the United States. CARE-P ’s framew3 framework, denoting: Clinical Adaptive Research Engagement – Patients, Providers, and Policymakers. CARE-P3 uses stakeholder-engaged perspectives to identify barriers to DCTs and output actionable recommendations for state policymakers. This interdisciplinary framework engages patients and providers (Fig. Limited communication between scientists and policymakers has long been established as a barrier to integrating new technologies in clinical practice . Additioers Fig. , using mers Fig. .In the U.S., state laws frequently require healthcare entities and agencies to submit reports to the legislature ; yet, no3 framework, DCT case studies characterized by a range of interventions and representative of various socioeconomic and diagnostic conditions are critical. While the pilot implementation of this framework has been trialed in both rural and urban settings, refining this approach will require multiple rounds of analysis and feedback to identify major gaps and arrive at a robust framework that can be used by researchers before, during, and after a DCT [To implement, evaluate, and refine the CARE-Per a DCT –24. A chDepending on which temporary flexibilities introduced during the PHE are made permanent, this work may be more urgently needed for specific states or patient populations. Another challenge associated with this work is disparate terminology and communication styles that are stakeholder specific. For instance, language used by patients is not identical to terminology employed by providers. Similarly, policymakers use different terms than health care professionals and assess research findings with domain-specific methodologies .Understanding the challenges faced by patients of different socioeconomic groups who elect or decline to participate in DCTs is crucial to diversifying clinical trials in the digital age. To develop safe and robust DCTs, it is essential that provider concerns and hurdles are addressed. Future research should also consider how to engage federal policymakers in the development of national DCT networks. Effectively communicating these findings and identifying actionable recommendations for policymakers are integral to developing equitable DCTs that yield high-quality data to fully realize the promise of decentralized clinical research.


Studies have consistently demonstrated that cognitively stimulating activities can protect against the negative effects of brain pathology on cognition in aging  as a form of intervention. Their results suggest that attenuated self-referential attention in specific emotion-inducing social situations may be a neurocognitive mechanism through which tDCS reduces emotional reactivity.Yet, the effect on some specific CR proxies have been investigated to a lesser extent. Some of these proxies include emotion regulation (Huang et al., Jansen et al. that focuses on the effect of cognitive reserve on cognition in older adults with different levels of cognitive decline: from subjective cognitive complaints to Alzheimer's dementia. Their results demonstrate how with more advanced levels of cognitive impairment, the positive effects of cognitive reserve proxies on cognition become less pronounced. This suggests that our ability to adapt, presumably mediated via cognitive reserve, may be functioning best in case of relatively intact cognition or only mild levels of cognitive impairment. That being said, this relationship has been proven not to be linear, but to be most likely moderated by many other factors, like, for example, the time given to perform divergent thinking tasks (Fusi et al., Jansen et al. seeing how different interventions might affect brain coping mechanisms would be important.Another open question is how differently our brains cope with damage when pathology is present if compared to normal aging. A first answer can be found in the study by Stalpaert et al. provides a first answer with a specific reference to patients with Primary Progressive Aphasia. Their results highlight how patients who received speech-language therapy showed specific neurophysiological changes, suggesting that this therapy might be more effective for patients with a non-fluent variant of aphasia.The study by In summary, the Research Topic of studies published in this Frontiers Research Topic shed light on a range of understudied aspects of CR including the relationship between CR and other cognitive, behavioral, and neural components, the modulation of CR proxies through non-invasive brain stimulation, and the role of CR in therapeutic outcomes and in protective effects during pathological aging. While the relationship between CR and cognition is complex and moderated by various factors, promising results warrant further investigation of interventions targeting CR to enhance cognitive abilities.BC: Writing—original draft, Writing—review and editing. A-KB: Writing—original draft, Writing—review and editing. JO: Writing—original draft, Writing—review and editing.


Synaptic plasticity such as Long-term potentiation (LTP) is a key mechanism for learning in central synapses including the cortex. There are two least two major forms of LTPs: presynaptic LTP and postsynaptic LTP. For postsynaptic LTP, the potentiation of AMPA receptor-mediated responses through protein phosphorylation is thought to be a key mechanism. Silent synapses have been reported in the hippocampus, but it is thought to be mainly present in the cortex during early development, and may contribute to maturation of the cortical circuit. However, recent several lines of evidence demonstrate that silent synapses may exist in mature synapses of adult cortex, and they can be recruited by LTP-inducing protocols, as well as chemical-induced LTP. In pain-related cortical regions, silent synapses may not only contribute to cortical excitation after peripheral injury, but also the recruitment of new cortical circuits as well. Thus, it is proposed that silent synapses and modification of functional AMPA receptors and NMDA receptors may play important roles in chronic pain, including phantom pain. The study of synaptic plasticity therefore is essential for our understanding of almost most of brain key functions.Long-term potentiation (LTP) is a key cellular mechanism for our understanding of physiological and pathological mechanisms of learning and memory, chronic pain, emotional disorders, drug addiction and neuronal development.10 Among several possible mechanisms that contribute to the formation of LTP, silent receptors have caused much of attention. It serves as one of the most direct evidence for postsynaptic mechanism for the expression of LTP. Our understanding of basic mechanisms of memory has also greatly improved our understanding of synaptic mechanisms in many chronic diseases such as chronic pain.4 In this review, I will summarize recent work of silent synapses in pain-related cortex; and propose that it may exist as a key synaptic mechanism for cortical circuit LTP and spreading in chronic pain.The study of synaptic mechanism in the hippocampus, especially CA1 region, lead major progress of our understanding of synaptic transmission and plasticity.10 Silent synapses are the synapses that lack functional receptors to detect the release of presynaptic transmitter such as glutamate. At silent glutamatergic synapses, there are lack of functional AMPA receptors to respond to the release of glutamate. The existence of silent synapses and their contribution to synaptic plasticity are well demonstrated by LTP induction protocols that can quickly recruit postsynaptic AMPA receptors, thus turn silent synapses to functional synapses.12 In the CA1 region of the hippocampus, activation of NMDA receptors is required for this recruitment. For intracellular signaling pathways, it is shown that postsynaptic CaMKII plays critical roles in triggering the recruitment of silent synapses.14 Therefore, this is the most direct evidence for postsynaptic expression of hippocampal CA1 LTP. Due to the limit of electrophysiological experiments, most of these experiments were performed in young hippocampal neurons, and few report of silent synapses in adult hippocampus.Silent synapses were first reported in young hippocampal neurons in the CA1 region.17 After peripheral injury, such silent afferent fibers became active. It has also been reported that spinal dorsal horn neurons became active after peripheral injury. However, extracellular recordings of dorsal horn sensory neurons did not allow further investigation of possible synaptic mechanisms. It is also quite possible that such recruitment of neuronal spikes can be due to disinhibition in the spinal dorsal horn.19 While there are several possible mechanisms contribute to recruitment of silent neurons in spinal cord, silent synapses may be one of strong candidates. Li and Zhuo (1998) reported that many spinal dorsal horn synapses are silent. Postsynaptic protein-protein interaction between AMPA receptors and postsynaptic traffic proteins is required for the recruitment of silent synapses. There are several subsequent reports, however, suggest that silent synapses may be mainly limited to young neurons in the spinal cord. One limitation of electrophysiological studies in adult spinal cord is that it is too difficult to clamp sensory neurons using classic whole-cell patch clamp, since many of these dorsal horn neurons send ascending projection to the thalamus  cells). It is likely that while the soma potential is at −70 mV, the membrane potentials at distal dendrites may be near positive potentials. In fact, in several early electrophysiological reports, slow EPSPs that are sensitive to the inhibition of NMDA receptors are also detected at the soma resting membrane potential (−65 to −70 mV). Furthermore, co-application of adenylyl cyclase (AC) activator forskolin and serotonin can recruit new AMPA receptors mediated responses into these pure NMDA synapses. Thus, the leaking current will be tremendous. Using intracellular recording, Wang et al. (1992) reported that some synapses are completely mediated by pure NMDA receptors. Thus, it is likely that silent synapses may still exist in adult sensory synapses (or filopodia); and such silent ones are difficult to be detected by whole-cell patching recording.In early studies of chronic pain after peripheral injury, it has been reported that some of sensory afferent fibers are inactive or ‘silent’ before the injury.  may contribute to spinal injury-related chronic pain.A recent study in the cortex suggests that filopodia or thin spines are likely silent synapses or synapses containing NMDA receptors see . In adul Issac et al. (1997) reported that the loss of ability to undergo LTP is accompanied by the disappearance of silent synapses in adult cortex. These studies strongly suggest that silent synapses may mainly play roles in central plasticity during neurodevelopment. Interestingly, these silent synapses seem to have less or no role in adult LTP. In the adult cortex where LTP can be detected, it is thought that LTP is mainly achieved by postsynaptic AMPA receptor phosphorylation and/or AMPA receptor insertion; at least for the postsynaptic form of LTP. These consistent results discourage others to search for possible silent synapses in adult cortex, and it is generally believed that silent synapses are more easily to be found in young synapses in the brain.Several previous studies have nicely demonstrated that silent synapses exist in developing somatosensory cortex, and it contributes to development-related synaptic potentiation (LTP).10 When postsynaptic membrane is clamped at resting potentials, voltage-sensitive NMDA receptors are not available to respond to released glutamate. Thus, the minimal stimulation used can be used to mimic one or a few synaptic events. These silent synapses turn not silent when the postsynaptic membrane is depolarized, and NMDA receptor-mediated responses can be observed. However, if we put these silent synapses to the distal dendrites of adult pyramidal cells in the cortex where it is impossible to be inactivated by clamping potentials at the soma, it is possible that these ‘silent’ synapses cannot be detected. In fact, several previous studies using field or intracellular recording method found that some synapses responses at physiological potentials (−65 to 70 mV at the soma) are sensitive to the inhibition of NMDA receptor antagonists. Furthermore, in freely moving adult mice, it has been reported that there are evoked responses recorded in the ACC by stimulating the other side of ACC that are mainly mediated by NMDA receptors, since AP-5 blocks most of synaptic responses. In adult ACC neurons, NMDA receptor-mediated fEPSPs and spikes have been reported. Additional evidence for pure NMDA receptor-mediated responses in the cortex is so-called NMDA spikes. Cortical basal dendrites are a major target for synaptic inputs to pyramidal cells, and there are reports of abundant NMDA receptor-mediated spikes in pyramidal neurons including layer 5 neurons.29Previous characterization of silent synapses is mostly by using whole-cell patch-clamp recording technique. These experiments provide strong evidence that filopodia can serve as a marker for silent synapses in the adult brain. Are there filopodia or filopodia-like structures reported in the adult cortex? Future experiments are clearly needed to investigate these possibilities in other areas of the cortex.A recent study by Vardalaki et al. (2022) using different experimental approaches demonstrates that indeed silent synapses exist in deep layer V pyramidal cells of adult visual cortex. They found that there are substantial amount of filopodia (protrusions lacking distinct heads) in adult pyramidal cells. More interestingly, such filopodia are not just limited to layer V pyramidal cells; they can be also found in layer II/III pyramidal cells. Using selective AMPA and NMDA receptor makers, filopodia are found to be exclusively NMDA receptor-positive, and AMPA receptors negative; indicating a possible pure NMDA receptor expressed synapses. Electrophysiological experiments further confirm that these filopodia are indeed silent, pure NMDA synapses. They further found that an LTP induction protocol (spike-timing protocol) is sufficient to unsilence silent synapses. These experiments meet all previously established silent synapses in the hippocampus of young animals and spinal cord, strongly suggest that silent synapses that are sensitive to LTP recruitment exist in adult cortical neurons, and filopodia are a structural substrate for such silent synapses.31 Neurons in the ACC receive peripheral sensory inputs through thalamic projections.32 In adult ACC of human and animals, ACC neurons respond to peripheral noxious stimuli, and the neuronal responses are related to the intensity of noxious stimuli. In conscious human brain imaging studies, it has been reported that the intensity of ACC activation is correlated with the intensity of pain unpleasantness. Recent studies have further expanded our understanding of the ACC. Glutamatergic synapses in the ACC, just like those found in the hippocampus, are highly plastic.3 LTP and long-term depression (LTD) can be found in layer II/II and V pyramidal cells of ACC of adult animals.3 Both presynaptic and postsynaptic forms of LTP (pre-LTP and post-LTP) have been identified, and post-LTP is activated by NMDA receptors, and the expression is mediated by GluA1 containing AMPA receptors.34 Biochemical and genetic studies suggest that post-LTP is likely mediated by the phosphorylation of AMPA receptor at Ser 845 through cAMP-dependent protein kinase A (PKA).ACC and IC are two key cortical regions for pain perception. Due to the complexity of cortical pyramidal neurons in adult mice, it is difficult to have a good space clamp. In our preliminary studies, we have also found filopodia-like structures in pyramidal cells of ACC of adult mice and tree shrews (unpublished observation). Interestingly, recent studies using MED 64 recording channels found that some silent responses can be detected in the ACC.38 Although field recording with such microelectrodes is difficult to assure the exact origin of electronic source of responses, LTP inducing protocol, however, can clearly recruit active responses from the same sites . Here I propose a hypothesis that such silent responses are actually the results of recruited responses in filopodia or filopodia-like silent synapses. Future studies are clearly needed to examine such possible. Results from filopodia and silent responses in MED studies both consistently suggest that silent synapses do exist in the adult cortex.In a recent study of young mice , it has been reported that silent synapses in the ACC pyramidal neurons that receive thalamic projections.me sites . A recen36 Chen et al. (2014) reported that TBS-induced late-phase LTP (L-LTP) in the ACC of Fmr1 WT mice were able to recruit silent responses that were inactive during baseline recordings  further confirmed the recruitment of silent responses by TBS-induced L-LTP in the ACC and found that the recruitment of silent responses was blocked in GluA1 845 knock-in mice, indicating that GluA1 is required for such recruitment. This result is in parallel with blocked LTP of active channels in the same slices. These results consistently suggest that there are at least two populations of individual synapses that undergo LTP in the ACC. The first one is active synapses, the potentiation in these synapses is likely due to modification of existing AMPA receptors by intracellular signaling pathways. The second one is silent synapses where there is no functional AMPA receptor before LTP induction. The recruitment of GluA1-containing receptors is likely to occur after LTP. Once it is induced, these new synapses are stable, and mediated increased synaptic responses in the ACC.Furthermore, genetic and pharmacological evidence strongly demonstrated that those recruited synaptic responses in silent units are mediated by AMPA receptors, or more precisely, GluA1-containing receptors.ings see . These n40 Li et al. (2019) reported that bath application of calcitonin gene-related peptide (CGRP) can recruit silent responses in the ACC of adult mice, and this effect is likely mediated through CGRP receptors  further suggest that the reduced level of BDNF may be one of causes, since BDNF or a selective TrkB receptor agonist can rescue this effect in middle-aged mice.In addition to LTP, these silent responses can be also recruited chemically by different signaling peptides.eceptors . FurtherIn summary, it is likely that glutamatergic synapses in adult ACC are heterogenous . In addi37 Postsynaptic increases in Ca2+ triggered by activation of NMDA receptors or other types of signaling sources. Among several types of ACs, AC1 is the most sensitive form to Ca2+. Increases in 2nd messenger cAMP then leads to activation of PKA, and the phosphorylation of AMPA receptors by PKA play important roles in the potentiation of AMPA receptor-mediated responses as well as the recruitment of silent responses.36 In addition, PKMzeta (PKMζ) activity maybe also required. It is likely that these signaling pathways are shared by silent synapses and the potentiation of AMPA receptors in the ACC. It is important for the future to investigate if other signaling molecules may also contribute to the recruitment of silent synapses as well as the potentiation of AMPA receptor-mediated responses. Fragile X mental retardation protein (FMRP), a key intracellular signaling molecule is found to be important as well.Previous studies of intracellular mechanism of ACC LTP provide a possible explanation for the basic mechanisms of the recruitment of silent synapses. It is clear that the recruitment of silent synapses serves as a key mechanism for a postsynaptic form of LTP in the ACC, in addition to the potentiation of active, existed AMPA receptor-mediated responses. It is likely that calcium-stimulated ACs are important for the recruitment of silent synapses in pyramidal cells . GeneticCortical excitation or potentiation has been proposed to contribute to chronic pain. More specially, two major forms of LTPs have been found in the ACC and IC. Pre-LTP is thought to contribute to injury-related emotional changes, post-LTP plays important roles in chronic pain and injury-related behavioral hyperalgesia and allodynia. Silent synapses (or filopodia) add new mechanisms for critical potentiation/excitation. Furthermore, the recruitment of silent synapses may lay the basis for spreading the excitation within the ACC circuit. Such spreading of excitation is likely limited.43 Postsynaptic potentiation of AMPA receptor-mediated responses has been reported after trace fear training. Postsynaptic increased level of AMPA receptors on the membrane has been reported in the ACC. The recruitment of silent synapses may serve as one of key mechanisms to fear-induced LTP in the ACC. Functionally, these silent synapses may contribute to the induction and expression of fear memory.In addition to chronic pain, ACC neurons have also been indicated in fear memory.45 Facilitated responses of dorsal horn neurons may contribute to behavioral hyperalgesia and allodynia in case of chronic pain. Increased postsynaptic responses of ACC pyramidal cells after LTP may enhance such top-down modulation of spinal sensory transmission. Silent synapses in the deep pyramidal cells may thus contribute to top-down descending facilitation.ACC pyramidal cells in deep layers have been reported to project to the spinal cord dorsal horn and exert facilitatory modulation of spinal nociceptive transmission. In case of chronic itch, it is likely that the recruitment of silent synapses in the ACC may contribute to long-lasting itch.In addition to pain, fear and sensory modulation, ACC has also been indicated in itch.47At the spinal cord level, the recruitment of silent synapses may serve as a key synaptic mechanism for endogenous descending facilitatory modulation from supraspinal structures such as the brain stem and cortex.Recent evidence more and more suggest that silent synapses are unlikely limited to the developmental stage. Previous studies that missed these synapses are likely due to the technical limitation, and distal dendrite location. Silent synapses (0 to 1) together with postsynaptic receptor modification (1 to 1x) offer unique distinct mechanisms for synaptic potentiation in the ACC. Such potentiation not only happens at existing connected circuits, but also can recruit new connections or ‘silent’ connections through silent synapses. Future studies are clearly needed to investigate if these two mechanisms may share exact intracellular signaling pathways or if any distinct signaling molecules may be involved. For physiological and pathological significance of silent synapses, it is important to understand possible different mechanisms or molecules that may be involved, and this new information may help us to find better treatment for patients with chronic pain, depression and other mental diseases.


Over the last decade, utilization of clinical genetics services has grown rapidly, putting increasing pressure on the workforce available to deliver genetic healthcare. To highlight the policy challenges facing Canadian health systems, a needs-based workforce requirements model was developed to determine the number of Canadian patients in 2030 for whom an assessment of hereditary cancer risk would be indicated according to current standards and the numbers of genetic counsellors, clinical geneticists and other physicians with expertise in genetics needed to provide care under a diverse set of scenarios. Our model projects that by 2030, a total of 90 specialist physicians and 326 genetic counsellors  will be required to provide Canadians with indicated hereditary cancer services if current growth trends and care models remain unchanged. However, if the expansion in eligibility for hereditary cancer assessment accelerates, the need for healthcare providers with expertise in genetics would increase dramatically unless alternative care models are widely adopted. Increasing capacity through service delivery innovation, as well as mainstreaming of cancer genetics care, will be critical to Canadian health systems’ ability to meet this challenge. Hereditary cancer syndromes are caused by heritable genetic variants in tumor suppressor genes or proto-oncogenes and account for 5–10% of all cases of cancer in North America ,3 by infThe Canadian clinical genetics workforce as a whole, which includes GCs as well as clinical geneticists and other specialist physicians with expertise in genetics , is facing significant pressure as a result of this rapid growth in utilization. While it currently includes approximately 110 clinical geneticists and 485 GCs ,7,8, aboTo explore this nation-wide challenge and inform strategic planning for cancer genetics healthcare in Canada, we estimated the number of GCs and GMDs that will be required to meet the projected need for hereditary cancer services in 2030 under a range of scenarios.We constructed a workforce requirements model using the needs-based approach to health human resources planning  to characterize and explore uncertainty in the model’s projections. The DSA varied each parameter by ± 25% while holding other variables constant , while tThe base case model estimated that in 2020 there was a need for 53 physicians with expertise in genetics  and 199 genetic counsellors  to provide hereditary cancer services in Canada . There wIn our deterministic sensitivity analysis in which a set of 20 inputs were varied by +/−25%, six factors had the largest impact on workforce requirements for both GMDs and GCs . Three oIn contrast, Our model suggests that under existing trends, referrals to hereditary cancer services in Canada will likely increase by more than 50% from 2020 to 2030. Concomitantly, a total of 91 GMDs  and 329 GCs  will be required to meet the projected need in 2030 . The senThe Canadian clinical genetics workforce in 2020 for all clinical areas was estimated to include 111 clinical geneticists , a smallIn 2018, Cancer Care Ontario also performed a needs-based analysis that considered both projected change in the incidence of new cancers by primary site and the estimated proportion of patients for whom a genetics assessment would be clinically indicated , though In addition to the expected growth in eligibility for hereditary cancer services, it is also worth considering the implications of a potential acceleration in the move towards integrating hereditary cancer genetics into oncology clinics. On the one hand, commercial next generation sequencing (NGS)-based tumour testing panels often include numerous genes linked to hereditary cancer  and receOn the other hand, the mainstreaming of hereditary cancer-specific testing is being tested in a wide range of clinical settings in oncology . While tIn ongoing work, we are expanding our workforce requirements model to incorporate all major clinical areas of genetic medicine  and deveOne limitation of modeling workforce requirements on a Canada-wide basis is that our model does not consider inter-provincial differences in funded testing eligibility, private pay and self-pay options, mainstreaming practices, or service delivery models. Along with the use of input parameters drawn from non-Canadian sources, and the fact that our model projects need rather than actual referrals or appointments , our model should not be used alone to project future utilization. However, in previous work we have found that trends in clinical genetics utilization are consistent across high-income countries . MoreoveOverall, our results suggest that, under standard care provision models, growth in the need for hereditary cancer services in Canada by 2030 is likely to, at best, severely strain the capacity of the clinical genetics workforce to meet that need, and, at worst, completely outstrip it. However, our scenario analyses also point to innovations in service delivery that could help GMDs and GCs serve a greater number of patients . Indeed,


Multiple potential hurdles will be examined, including: a) toxicity of agents, b) the likely range of subtypes of cancers affected by a given treatment , c) the availability of practical endpoints besides the blocking of cancer formation or pharmacokinetics related to the agents administered in a primary prevention study, and d) the interpretation of the regression or blockage of new preinvasive lesions in the anti-progression study. Such an anti-progression approach may help address some of the factors commented on regarding primary prevention  but still leaves major questions regarding interpretation of modulation of preinvasive endpoints when it may not be clear how frequently they progress to clinical cancer. Additionally, we address whether certain recent preclinical findings might be able to reduce the toxicities associated with these agents and perhaps even increase their potential efficacy. Antibodies and TKIs other than the EGFR inhibitors are not discussed because few if any had been tested as monotherapies in humans, making their efficacy harder to predict, and because a number have relatively rare but quite striking toxicities. Furthermore, most of the practical hurdles raised regarding the EGFR inhibitors are relevant to the other TKIs. Finally, we briefly discuss whether early detection employing blood or serum samples may allow identification of high-risk groups more amenable to agents with greater toxicity.There are major hurdles to the use of tyrosine kinase inhibitors (TKIs) and any other agents with significant toxicities  in the context of prevention/anti-progression (interception) studies. We will discuss epidermal growth factor receptor (EGFR) inhibitors as examples, both in a primary prevention setting, where agent(s) are administered to individuals with no cancer but who might be considered at higher risk due to a variety of factors, and in anti-progression/interception studies, where agent(s) are administered to persons with known preinvasive lesions (e.g., colon adenomas, lung nodules, ductal carcinoma  The primary focus of this review is on some of the first generation EGFR inhibitors, e.g., gefitinib, erlotinib, lapatinib. This has been in contrast to certain of the third generation inhibitors, e.g., osmertinib, which preferentially interact with mutated forms of EGFR. This is predicated on the consideration that, as proposed for use in bladder, colon and pancreas, it appears that one is dealing with inhibition of wild type EGFR. Therefore, osmertinib which minimally effects wild type EGFR is unlikely to be highly effective. However, one could probably run a test for CT-scan identified lesions in an Asian population which has a very high incidence of EGFR mutant tumors with osmertinib since a high percentage of those lesions even without sequencing would be expected to have EGFR mutations. A systematic discussion of the widest range of small molecule EGFR inhibitors examining their specificity, target kinases, etc., has recently been published , with inPrimary prevention involves administering an agent(s) to a group of individuals with no cancer but who might be considered at higher risk for that type of cancer based on other known factors  .A primary obstacle to long-term clinical primary chemoprevention studies and acceptance is toxicity. For example, tamoxifen and the aromatase inhibitors have proven highly effective in the prevention of estrogen receptor-positive (ER+) breast cancer in large-scale clinical trials . In the In the case of tamoxifen and the aromatase inhibitors, one had clear data from large adjuvant trials that these agents used as monotherapies would prevent the development of the preponderance of ER + PR + HER2-negative breast cancers  . This waThese specific examples of EGFR inhibitors or HER2 inhibitors exemplify the problem for TKIs, or probably most targeted therapies in a primary prevention setting. EGFR mutant lung adenocarcinomas in a Caucasian population represent <15% of all adenocarcinomas. Since adenocarcinomas represent at most 50% of all lung cancers worldwide, EGFR mutant lung adenocarcinomas will represent <7% of all lung cancers. Similarly, an HER2 inhibitor by itself is likely to affect only the roughly 20% of breast cancers that overexpress HER2 and are either ER positive or ER negative. Thus, it would not be the basis for a primary prevention trial, although it might significantly improve a trial in conjunction with an anti-hormonal agent .In the metastatic setting, one routinely expects to continue treatment until the tumor progresses or until you have a striking pathologic response. In the adjuvant setting, as in the case of tamoxifen and the aromatase inhibitors, it was demonstrated that 5 years of treatment is roughly as good as 10 years. However, the prevention trial with the aromatase inhibitor anastrozole raises the question of how long to treat . This reThe cost of a large-scale clinical prevention trial is quite substantial. However, if the result obtained is relatively striking then it may be more than worth the cost. However, if the agent employed is still on patent, then the cost is likely to prove prohibitive for general use in a population. Quite obviously, if an agent has significant toxicity and may require observation by medical practitioners due to toxicity, this likely makes it untenable for primary prevention.in situ (DCIS) in the breast or transitional cell carcinomas (TCCs) in the bladder are routinely treated with surgery and some adjuvant treatment.Although primary prevention is often what people think of when they discuss prevention as listed above, the use of such an approach is complex in general and potentially fraught for a class of agents such as the TKI inhibitors (using as an example the EGFR inhibitors) . The exi CT scannDue to germline mutations in APC (adenomatous polyposis coli), individuals with FAP have literally scores to hundreds of adenomatous polyps. Because of the presence of a great number of lesions and the expectation that one or more of these lesions will progress to invasive colon cancer, some clinical intervention appears necessary. There was a positive FAP trial combining the EGFR inhibitor erlotinib together with a relatively low dose of the NSAID sulindac which resulted in a fairly striking clinical effect . This smDCIS are precursor pre-invasive lesions that are frequently identified at screening mammography or during breast cancer surgery. These are commonly treated with surgery, often followed by adjuvant radiation and hormonal therapy. While this is considered therapy by most oncologists, the objective is to prevent the development of invasive cancer and could serve as an interception (anti-progression) model. For DCIS lesions overexpressing HER2, trastuzumab has demonstrated activity in clinical trials . A trialPanIN is a histologically well-defined precursor to invasive ductal adenocarcinoma of the pancreas. PanINs are relatively common lesions, particularly in an elderly population. These lesions typically have a KRAS mutation and often alterations in P16 as well. These are the two most common genomic alterations observed in invasive ductal adenocarcinomas of the pancreas. However, it is still not clear what percentage of PanIN lesions progress to pancreatic adenocarcinoma .TCC is the most frequently observed tumor in the bladder. TCC arises from the cells lining the bladder and is routinely diagnosed during the pre-invasive stage. At the molecular level, there are 5 major subtypes of bladder cancer, including luminal  , HER2-exCarcinogenesis of the head and neck area provides a classical example of the multistep process leading to invasive malignancy. With easily observable oral premalignant lesions that could undergo serial biopsies, several trials assessed the activity of EGFR TKIs alone or in combinations with other agents for prevention of invasive cancer, or evaluating surrogate biomarkers in patients with oral premalignant lesions. The combination of erlotinib and celecoxib led to 63% histologic response rate that correlated with EGFR pathway inhibition . Green tPreclinical prevention studies have raised the possibility of reducing toxicity by employing intermittent dosing, weekly dosing, lower dosing, or a combination of agents  Table2)Table2). A problem with targeted therapies developed for cancer treatment, such as the EGFR inhibitors erlotinib, gefitinib, and lapatinib, is that they all can cause acneiform rash and diarrhea when given daily. Studies in humans treating brain metastases from either EGFR mutant lung cancers or HER2-overexpressing breast cancers demonstrated that pulsatile weekly dosing with erlotinib or lapatinib, at up to 7-fold the standard daily dose, was effective and paradoxically caused less toxicity than daily dosing. This high dose pulsatile dosing was initiated to deal with brain metastases from EGFR mutant lung cancers and HER2-overexpressing breast cancers. Daily dosing of these agents did not reach effective levels in the brain following standard daily dosing, presumably due to the blood brain barrier. Therefore, weekly pulsatile dosing was employed on the rationale that one would achieve higher levels in the brain. It appears that the approach has been relatively effective in numerous human trials . In prec1. Weekly dosing with an EGFR inhibitor in an Asian population (that has a high incidence of EGFR mutant lung cancers) with pulmonary nodules determined by CAT scans 2. Weekly dosing of an EGFR inhibitor together with an NSAID (see below) in individuals with a) FAP, b) pancreatic precursor lesions (PanINs), and c) basal TCC of the bladder 3. Potential trial of lapatinib in DCIS. HER2 overexpression is often observed in DCIS in the breast and appears to be associated with a higher incidence of recurrence and progression to invasive breast cancer. This is routinely treated with surgery plus radiation. Trastuzumab can be employed in this setting with at least hints of efficacy, but it requires intravenous administration.Potential trials employing weekly EGFR inhibitor administration :1. WeeklThere are both preclinical and some clinical data that combinations of certain agents may yield strong efficacy. However, any potential toxicity problems that may arise may have to be determined clinically. Common toxicities, such as acneiform rash or diarrhea for EGFR inhibitors or gastric bleeding for NSAIDs, could be determined in a limited Phase I trial.Two of the more successful Phase II clinical prevention trials that involved combinations of agents were performed in colon and were based on preclinical data. In the first, the combination of an NSAID (piroxicam) and DFMO was shown to be highly effective preclinically in a colon model almost 30 years ago . This le1. Repeat of FAP Study: The prior FAP study examining erlotinib plus an NSAID can be performed using a weekly dose of erlotinib. This is likely to be both highly effective and plausibly less toxic than the standard dosing. The combination of pulsatile EGFR inhibitor and intermittent NSAID was found to be profoundly effective in a rat model of FAP .2. Interception (anti-progression study) on pre-invasive pancreatic lesions : The combination of an EGFR inhibitor and a Cox 1/2 inhibitor is highly effective preclinically .3. Interception (anti-progression) study in basal subtype of bladder cancer: Erlotinib plus an NSAID can be administered using a weekly dose of erlotinib. This is likely to be both more effective and plausibly less toxic. The combination of pulsatile EGFR inhibitor and intermittent NSAID was found to be profoundly effective in a rat model of basal bladder cancer . PerhapsPotential trials employing EGFR inhibitor plus an NSAID:In science in general and oncology in particular, significant advances, at least conceptually, often come out of some technical advance. The potential technical advance that is starting to come into clearer focus is the possibility of early detection of incipient tumors in blood or some other body fluid . By defiA. As outlined above, there are multiple hurdles to any potential use of EGFR inhibitors in particular or any other TKIs generally in a primary prevention trial. These include the fact that a potential subtype of sensitive tumors (EGFR mutant lung cancer or HER2-amplified breast cancer) to target in such a blind primary prevention trial is unclear. Furthermore, there are too many toxicities associated with daily dosing to make it viable unless some of the newer TKIs have more limited toxicity.B. In an interception trial, it would seem that one can determine subtypes of cancers of particular organs (EGFR mutant lung adenocarcinomas) or precursor lesions (HER2-overexpressing DCIS) that might be susceptible to an EGFR receptor inhibitor. There are still questions about potential endpoints to employ. However, the presence of lesions raises the possibility of examining alterations in lesions by various -omic methodologies. Furthermore, if someone has existing lesions, some increase in toxicity will be more acceptable.C. There are alterations in dosing and potential combinations of agents which might make the use of TKIs more acceptable. These were discussed above. It appears that pulsatile dosing of TKI inhibitors may prove useful in certain subtypes of cancer. Furthermore, the combination of an EGFR inhibitor plus an NSAID (a known preventive agent) appears particularly promising in anti-progression studies such as FAP.