The enhanced permeability and retention (EPR) effect has underlain the predominant nanomedicine design philosophy for the past three decades. However, growing evidence suggests that it is over-represented in preclinical models, and agents designed solely using its principle of passive accumulation can only be applied to a narrow subset of clinical tumors. For this reason, strategies that can improve upon the EPR effect to facilitate nanomedicine delivery to otherwise non-responsive tumors are required for broad clinical translation. EPR-adaptive nanomedicine delivery comprises a class of chemical and physical techniques that modify tumor accessibility in an effort to increase agent delivery and therapeutic effect. In the present review, we overview the primary benefits and limitations of radiation, ultrasound, hyperthermia, and photodynamic therapy as physical strategies for EPR-adaptive delivery to EPR-insensitive tumor phenotypes, and we reflect upon changes in the preclinical research pathway that should be implemented in order to optimally validate and develop these delivery strategies. One of nanomedicine's primary contributions to cancer research is its illustration of the power of intentional agent design as a tool for enhancing drug delivery. This philosophy has spurred decades of research into nanoparticle formulations and functionalization of existing drugs, pushing forward the concept of personalizing medical treatment by tailoring drug design for specific applications. The coming years hold the potential to revolutionize treatment options for a variety of ailments, chief among them being solid tumor cancers.et al. in their discovery that elevated interstitial fluid pressure (IFP) and heterogeneous blood supply limited macromolecular delivery to tumors In the wake of the massive intellectual and financial investment in nanomedicine design, scrutiny of the founding principles and assumptions upon which the discipline has been built is warranted. Few would argue that that the most prevalent nanomedicine designs principle in cancer treatment is the idea of the Enhanced Permeability and Retention (EPR) effect. First introduced by Matsumura and Maeda in the 1980's The observation of the EPR effect provided a clear design philosophy for cancer therapy development - increase drug concentration at the site of interest relative to healthy tissue as a means of alleviating treatment side effects. This was explored mainly through optimizing formulations for increased blood circulation time, such as encapsulation of agents within liposomes and surface chemistry modification using PEG chains. These changes led to longer exposure of the tumor site to circulating nanoparticles, increasing relative accumulation. Time-dependent optimization was coupled with careful size selection of agents, which were chosen to act optimally for slow renal excretion, low off-target liver uptake, and maximal tumor uptake. Many of the first and arguably most successful clinically approved nanoformulations, including Doxil® and Abraxane®, capitalize on these principles. While these drugs provide clinical benefits by means of reduced toxicity, the high level of specificity and sensitivity seen by Maeda and Matsumura's early work has not be recapitulated to the same degree in a clinical setting et al. showed accumulation of radiolabelled PEGylated liposomes ranging from as low as 2.7% ID/g in ductal breast cancer to as high as 53.0% ID/g in head and neck cancers, with other spontaneous lung, brain, and cervical cancers spanning this range et al. showed increased accumulation in carcinomas relative to sarcomas in spontaneous canine solid tumors This unrealized potential has led to a questioning of the ubiquity of the EPR effect, including critiques by its founders To overcome these limitations in achieving consistent delivery to a varied clinical target, many researchers are exploring a more inclusive nanomedicine design philosophy - delivery strategies that do not rely on the fixed, passive accumulation capacity inherent to a given tumor. Such strategies complement the EPR effect by modifying tumor accessibility and susceptibility in order to increase nanomedicine delivery across many solid tumor phenotypes, thereby maximizing their clinical applicability. This review hopes to provide a timely overview of the current strategies that fall under this classification of EPR-adaptive delivery, and it seeks to critically compare the advantages and challenges inherent to the associated design processes. This includes a review of the translational potential of these design strategies - both in a clinical and interdisciplinary sense - as well as an identification of the necessary pre-clinical tools required to effectively evaluate EPR-adaptive delivery strategies.The passive nature of the EPR effect means that it has the capacity to affect the biodistribution of all nanosized agents for cancer targets. In this manner, it is true that all nanomedicine delivery strategies benefit in some form from the EPR effect, albeit to varying degrees across different tumor types. EPR-based delivery can be conservatively thought of as a delivery strategy that does not attempt to modify inherent tumor accessibility in an effort to increase agent accumulation and improve therapeutic potential.in situThis design philosophy places focus upon modification to nanomedicine formulation to improve passive accumulation of the agent, primarily by increasing circulation time and optimizing nano-bio interactions, for example through size selection. Beyond static designs, formulations can also be chemically functionalized to actively target biochemical signatures of a tumor et al. illustrated that three different prostate cancer cells lines from vastly different tumor phenotypes all displayed similar longitudinal accumulation of radiolabeled albumin when xenografted into mice, all reaching 5% ID/g What has been identified as one of the primary detractors of the clinical success of the traditional EPR-based delivery strategy is the over-representation of the EPR effect in preclinical models of cancer, most notably subcutaneous xenografts. Heneweer It is clear that there exists a phenotypic spectrum for tumors with regards to how treatable they are under an EPR-based delivery strategy, with preclinical models exhibiting more sensitivity and clinical models showing more insensitivity Figure . The speEPR-adaptive delivery, is characterized by its ability to modify tumor accessibility in an effort to increase agent delivery and therapeutic effect. While the EPR effect itself may still be partially responsible for the overall delivery efficacy of certain agents developed under this principle, these EPR-adaptive strategies are generalizable to a broader range of clinical targets, including those historically inaccessible to traditional EPR-based strategies. In alleviating the dependence of these strategies on the EPR effect as a primary determinant of delivery success, these approaches overcome the pitfalls commonly attributed to conventional nanomedicine delivery.While the traditional EPR-based design philosophy has led to the clinical success of some formulations, it reneges on the promise for nanomedicine to be Ehrlich's “magic bullet”, capable of selectively targeting tumors across a wide range of cancer phenotypes. Alternative drug delivery strategies that forego the requirement of an EPR-sensitive tumor phenotype do exist, and they have been receiving increased attention in recent years as disenchantment with the EPR effect grows. This class of techniques, herein referred to as EPR-adaptive delivery encompasses both chemical and physical techniques used to improve nanomedicine biodistribution. Chemical mediators seek to improve tumor access through direct modification of the tumor vessels, stroma, and cancer cells. Vascular agents, such as bradykinin, prostaglandins, and nitrous oxide, induce an inflammation-like state to restore tumor blood flow and improve tumor influx, thus boosting permeability to circulating drugs Physical techniques represent the other arm of the EPR-adaptive delivery strategy. These involve the use of external stimuli to modify a delivery site either prior to or concurrent with nanomedicine delivery. This includes radiation, ultrasound, hyperthermia, and photodynamic therapy, and these strategies will be the focus of discussion in this review. Each of these capitalize on unique physiological responses to improve agent uptake and augment retention in a manner that is applicable to a majority of solid tumor phenotypes Table .Given the common usage of radiotherapy with adjunctive chemotherapy in cancer treatment, it is only natural that one of the first physical modalities used to enhance nanomedicine delivery was radiation. Traditional radiotherapy delivers ionizing radiation to the tumor site under image guidance, leading to damage at tumor areas and minimal dosing of neighbouring healthy tissue. This has a variety of effects on cancer, endothelial, and stromal cells, including increased hypoxia, decreased interstitial fluid pressure, and lowered solid tumor pressure et al. conducted some of the original studies in this area combining nanoformulations of chemotherapy and radiation pre-treatment. They showed that ionizing radiation delivered 24 hours before injection of PG-TXL had a 4.4-fold enhancement of tumor growth delay compared to PG-TXL treatment alone in ovarian OCa-1 intramuscular tumors, extending survival time by 46 days  et al. conducted comparable studies using liposomal doxorubicin as a model agent and radiation delivered to osteosarcoma tumors 24 hours post-treatment. They showed a 2-4-fold improvement in nanoparticle uptake in both xenograft and orthotopic tumors when either single or fractionated-dose ionizing radiation was administered Li et al.'s seminal paper, as they showed that liposomal doxorubicin penetrated more deeply in both xenograft and orthotopic tumors when combined with radiation therapy. Tumor histology revealed that, while accumulation in untreated tumors was largely perivascular, irradiated tumors showcased a more uniform intratumoral drug distribution The ability of radiotherapy to decrease intratumoral pressure is an asset to its ability to enhance nanomedicine delivery. Reductions in pressure are due to a combination of factors, including vascular regression, death of tumor cells which compress microvasculature, and decreased resistance to blood flow However, the efficacy of radiotherapy to improve drug delivery has strong temporal and spatial dependence. The remodelling that occurs in tumors in response to fractionated radiation treatment can lead to upregulation of the ECM, and new vessel growth may exacerbate tumor hypoxia and lead to insufficient perfusion for delivery Ultrasound is an incredibly versatile clinical modality, seeing universal applicability as a diagnostic imaging technique due to its high tissue penetration depth (~10 cm at 1 MHz), non-invasiveness, and ease of use. To improve contrast of vessels during imaging, gas-filled agents called microbubbles have long been used, as they produce non-linear oscillations called cavitations that dramatically increase contrast between vascularized structures. Beyond imaging, these oscillations can be exploited in two primary ways - at low intensities, microbubbles undergo stable cavitation, leading to transient disruption of endothelium and an increase in vascular permeability. At high intensities, microbubbles undergo inertial cavitation, creating shockwaves that can perforate cell walls and lead to elevations in temperature and reactive oxygen species. While the latter can be exploited for therapeutic delivery with high-intensity focused ultrasound, the former is ideal for invoking biomechanical effects that transiently disrupt vascular endothelium to increase local drug extravasation Figure A 60,61.in vitro cell lines and in vivo animal models, suggesting that the mechanism involves both paracellular  and transcellular  transport et al. pioneered this application for notoriously inaccessible brain tumors. Their application of MRI-guided focused ultrasound improved liposomal doxorubicin accumulation in healthy brains in a manner linearly dependent on microbubble dosage, showing a 3.5-fold increase in delivery with no surrounding tissue damage The efficacy of pre-treatment with ultrasound and microbubbles in improving nanoparticle delivery is observed across both et al. showcased that two EPR-insensitive phenotypes  showed increased accumulation of liposomes after ultrasound irradiation compared to untreated controls et al., where uptake of lipid-coated CdSE quantum dots with sizes ranging from 30 nm to 180 nm in CT-26 colorectal adenocarcinoma xenografts was measured after pre-treatment with ultrasound and Sonovue® microbubbles. This pre-treatment strategy showed that the greatest increases in extravasation were achieved with the smaller, 30 nm nanoparticles et al. have shown that low-intensity ultrasound enabled with Sonovue® microbubbles and pre-administered gemcitabine nearly doubled median survival of patients with inoperable pancreatic ductal adenocarcinoma compared to gemcitabine alone The most common delivery strategies employed with ultrasound are co-injection and conjugation. Co-injection involves injection of free nanoparticles either preceding, following, or concurrent with ultrasound treatment, the latter of which would involve mixing of the individual agents immediately prior to injection Figure B 77. Thiet al. were one of the first groups to fabricate microbubbles with liposomal doxorubicin and showed a 12-fold increase in drug accumulation in DSL6A murine pancreatic xenografts compared to non-irradiated tumors in a bilateral tumor model  to mild hyperthermia (42ºC) for improving extravasation of fluorescent liposomes of various sizes in SKOV-3 ovarian carcinoma xenografts using a window chamber model. They showed that the size range of extravasating agents extended from 7-100 nm to 7-400 nm, as well as observing overall increases in accumulation for all sizes tested. The magnitude of the effect was greatest for smaller nanoparticles and was limited to tumor vasculature Seminal work by Kong et al. saw a doubling of agent permeability in murine melanoma and lung models compared to murine sarcoma and human melanoma models et al. compared the effects of hyperthermia and radiation to improve accumulation of HPMA co-polymers in both aggressive and slow-growing variants of Dunning R-3327 prostate cancer. They observed that hyperthermia improved uptake of 65kdA poly(HPMA) in the slow-growing model to a level that matched uptake in the aggressive model. However, while radiation treatment was found to increase uptake across all sizes and tumor types, the hyperthermic effect was not recapitulated when tested with smaller, doxorubicin-loaded polymers, suggesting that the result is limited to nanoparticles larger than a certain size threshold While the extent of improvement appears to be dependent on tumor model, hyperthermia has been shown to work in both EPR-sensitive and insensitive phenotypes. Li et al. showed that the increased nanoparticle accumulation seen after hyperthermia treatment was partially regulated by the heat shock response. Their results showed that the increase in accumulation of fluorescent PEGylated iron oxide nanoparticles was hampered after progressive hyperthermia treatments, with no improvement for injections delivered after the third successive hyperthermia treatment. However, when tested in mice with genetically disabled HSF1, a heat shock protein, consistent accumulation dynamics were observed for progressive treatments More recently, Bagley in vivoTemperature-sensitive liposomes are one class of nanotherapeutics that capitalizes on these principles. The delivery benefits of the physiological effects of hyperthermia are augmented using liposomes designed to release their contents upon a non-ablative increase in temperature. While it was first explored in the 1970's Photodynamic therapy (PDT) has been largely studied in the context of cancer therapy as a means of non-invasively and selectively treating superficial tumor targets. It operates on the principle of stimulating a light-activatable agent (photosensitizer) with NIR light such that, either through direct electron transfer to a biological substrate (type I photosensitization) or to molecular oxygen (type II photosensitization), cytotoxic molecular species are generated. Because the photosensitizer is generally non-toxic in the absence of light and light doses are chosen to be low enough to spare surrounding tissues, the required co-registration of light and sensitizer ensures high localization of cellular and tissue damage to the area of the field of illumination that contains the photosensitizer. Because the penetration depth of NIR light is on the order of millimetres in tissue, this limits application to superficial sites or those that can be probed with cylindrically diffusing optical fibres, such as the prostate et al. was one of the first to explore whether or not this mechanism could be utilized to improve agent delivery. In 2003, they illustrated that the application of low fluence phototherapy with a porphyrin-based photosensitizer led to 5-12-fold increase in uptake of fluorescent microspheres ranging in size from 0.1-2 µm but impermeability to microspheres at a smaller size of 0.02 µm. This was complemented with therapeutic studies using liposomal doxorubicin, which showed enhanced accumulation at all doses and improved survival for doses of 3 mg/kg or greater et al., who saw increased uptake of 100 nm FITC-dextran in both subcutaneous and orthotopic murine models of prostate cancer when given 15 minutes after pre-treatment with verteporfin-based PDT et al. with liporubicin delivery to Visudyne-based PDT-treated sarcomas xenografted into rodent lungs, showing both a two-fold increase in accumulation and no change in adjacently treated healthy lung tissue The ability of PDT to cause transient vascular leakiness followed by vessel shutdown has been known since the 1980's, and it was broadly considered to be a detriment to its usage as an adjunctive therapy et al. saw that photodynamic therapy of A431 EGFR+ xenografts 24 hours after injection of IR700 conjugated to panitumumab, a monoclonal antibody specific to EGFR, led to tumor shrinkage via damage to selectively-bound pericytes at the tumor site et al. also showed the delivery benefits of using active targeting, observing a 20.08-fold increase in accumulation of quantum dots in PDT-treated tumors in a bilateral 4T1 breast tumor xenograft model. They also highlighted a pronounced effect for larger nanoparticles, hypothesizing that the improvement is magnified for agents with size-limited extravasation rates. A lower but apparent effect was still observed for smaller nanoparticles, evidenced by albumin showing increased uptake in breast , prostate , melanoma , and glioblastoma  xenografts Active targeting strategies have also been employed to improve specificity of PDT-based tactics for improving drug delivery. Mitsunaga Overall, the physical EPR-adaptive delivery strategies described here have all been shown to improve nanomedicine uptake and treatment outcomes in preclinical models. While they still require optimization and validation in more clinically relevant models, their basis on mechanisms that can alter tumor structures regardless of the target tumor's level of EPR-sensitivity gives them a greater chance of achieving broad clinical translatability.Whether intentional or through sheer convenience, there is no denying that the current testing paradigm for new innovations in nanomedicine-characterization of a new nanoparticle, assessment of its gross tumor uptake in a xenograft model, and assessment of reduction in tumor growth across a relatively short timeline - is biased towards traditional EPR-based delivery. From the methods used to gauge efficacy to the disease models in which they are validated, there exist numerous implicit advantages which increase the likelihood of observing benefit in any agent that capitalizes on a highly expressed EPR effect. While this has generated excitement with regards to the volume of agents that have shown preclinical promise, its effect on stunting clinical translation has ultimately weakened the field as a whole, giving the impression to many that nanomedicine translation stops at the mouse The primary shortcoming of EPR-based nanomedicine delivery is its heterogeneous response in clinical tumors, an outcome which is thought to be the result of inconsistent traits in clinical tumors compared to the common EPR-sensitive phenotype of most preclinical models. In order to both better determine the applicability of a formulation and the eligibility of patients for various treatment options, a reliable means to assess the EPR-sensitivity of tumors is of critical importance.The classification of tumor phenotypes on the basis of EPR-sensitivity necessitates specific criteria that can be easily assessed, the choice of which are still in early stages of research. This is in part due to a lack of knowledge as to what exactly enables the EPR effect on a mechanistic level. Even if this was discovered, it would still be required that these qualities were valid and measurable at the clinical level. The current techniques characterizing the EPR effect rely mainly on histology and imaging, the latter of which is most attractive due to its non-invasiveness. et al. combined ceUS, micro-CT, and MRI as tumor imaging modalities to assess vascular characteristics that enable increased uptake of 100 nm fluorospheres in five different animal tumor models. They observed that blood vessel density (via CT) and wash-in time (via ceUS) correlated with improved uptake, while Ktrans, an MRI parameter often used to represent degree of extravasation and vessel leakiness, showed no correlation et al. in a variety of xenograft and orthotopic tumor types using MRI and fluorescence imaging to observe lipo-nanocapsule delivery was not able to find any significant relationship between tumor permeability, vessel size, and blood volume fraction that functioned predictively in their model systems Indirect imaging of tumor vasculature is an attractive prospective method for evaluating EPR-sensitivity, as it would allow for a non-invasive, versatile implementation that fits cleanly into established clinical workflows. It is predicated upon first connecting characteristics of tumor vessels or perfusion with agent uptake and assuming that this correlates with improved treatment response, hence its notation as indirect. The Lammers group has made considerable strides in this area using CT While these works individually suggest some relationship between vascular features quantifiable on imaging and tumor characteristics representative of EPR-sensitivity, no group has yet found an individual measure that has been shown to be predictive. A unified metric combining multiple features seen on imaging and weighted by degree of correlation may eventually be a useful tool for the prediction of EPR-sensitive phenotypes, but considerable work would be required to validate such a tool across a variety of preclinical and clinical tumor types.et al. showed that FMX, a magnetic nanoparticle used as an MRI contrast agent, could effectively predict nanoparticle accumulation in subcutaneous HT1080 human fibrosarcoma xenografts using PLGA-PEG NPs and therapeutic response using liposomal paclitaxel in subcutaneous 4T1 breast cancer xenografts, respectively. Subjects stratified by high, medium, or low uptake based upon an increase in T2-weighted MRI signal between 1 and 24 hours showed strong correlations with differences in DNA damage, apoptotic fraction, drug uptake, and percent change volume in tumor size et al. showed similar capabilities with PET imaging, using a liposomal 89Zr-nanoreporter and liposomal doxorubicin in mice bearing subcutaneous 4T1 breast cancer xenografts. They illustrated very strong correlations between intensity of PET images at the tumor site and agent accumulation (r=0.97) and were able to provide predictions for therapeutic outcome by stratifying mice into high and low accumulation subgroups (threshold of 25 mg/kg interpolated uptake at the tumor site), displaying categorical differences in relative tumor size increase and median survival A direct measure of EPR-sensitivity would seek to correlate increased agent uptake with improved efficacy. While this may lead to a more powerful predictive tool for determining tumor phenotype, it also requires additional work to develop trackable components for each new therapeutic to be tested, likely in the form of a companion nanodiagnostic or an incorporable chelator for radiolabeling 64Cu-labelled HER2-targeted PEGylated liposomal doxorubicin and transtuzumab, monitoring biodistribution using PET/CT scans and biopsies for comparison with therapeutic response. Lesion volume was not found to correlate with agent uptake, and lesion deposition was found to vary between 0.5 and 14% ID/g. When stratified by lowest uptake lesion, agent accumulation was found to significantly correlate with therapeutic response, giving clinical evidence that EPR-sensitivity is a determinant of nanomedicine efficacy The clinical viability of companion nanodiagnostics has been trialed by Merrimack Pharmaceuticals in two studies in human patients. The first study related therapeutic response from liposomal irontecan with accumulation of FMX in 28 patients with advanced, metastatic cancer. While they observed only marginal correlation between irontecan uptake and FMX signal, they did see a relationship between reduction in lesion size and FMX signal after stratifying patients by median degree of FMX accumulation The combined use of indirect and direct methods for evaluating EPR-sensitivity, while still early in development, poses significant benefit to the developmental paradigm for nanomedicine. For existing formulations, it would provide a means by which patients can be pre-selected on the basis of their EPR-sensitivity and thereby potential to see a positive therapeutic response. For new platforms, it would be an invaluable preclinical validator for studies that want to investigate if agent accumulation and treatment response in EPR-insensitive phenotypes can be made comparable to EPR-sensitive phenotypes through the use of EPR-adaptive delivery strategies.There is no doubt that the rapid rate at which new nanomedicines have been developed is partially due to the incredibly convenient and accessible preclinical models available for testing. Primary among these are subcutaneous and orthotopic xenografts, a system almost synonymously used for early proof-of-efficacy studies. While these models possess highly desirable features including rapid growth and relative homogeny between subjects, they exhibit a radically different microenvironment than clinical tumors, whereby the majority of these distinctions make them more amenable to traditional EPR-based nanomedicine delivery. High rates of angiogenesis in xenografts lead to uniformly low pericyte coverage and large endothelial fenestrations In addition to exaggerating efficacy of most nanomedicines, these models are poorly suited for testing EPR-adaptive delivery strategies. Given that EPR-adaptive techniques attempt to elevate delivery to cancers with microenvironmental features that prohibit conventional delivery, a more representative test of efficacy would use a comparison between an EPR-sensitive tumor type and a more clinically-relevant EPR-insensitive tumor type. The latter would provide the largest margin for improvement compared to conventional delivery strategies, while the comparison would illustrate generalizable efficacy across large changes in the tumor microenvironment.et al. in an orthotopic pancreatic PDX showed comparable collagen and fibroblast-activated protein levels to the native tumor and, when treated with either IGF1R-targeted IONP-doxorubicin or free doxorubicin, showed increased tumor growth regression and apoptotic cell death in the nanoformulation treatment arm Alternative models that may be more clinically representative include patient-derived xenografts (PDX), organoids, and spontaneous cancers, including genetically-engineered models. PDX allow for the transplantation of tumor cells directly from a patient into an animal model, often preserving stromal structures and allowing for testing against specific, low-incidence cancer phenotypes in vivo models.Organoids, 3D cancer cell cultures that better recapitulate the ECM and stroma when compared to 2D systems, may be useful as a supplementary tool to assess the impact of EPR-adaptive delivery strategies on various characteristics of the tumor microenvironment, including stromal density et al. illustrating that treatment using DACHPt-loaded micelles showed significant benefits for survival, reduced tumor burden, and decreased metastasis when compared to free oxaliplatin in a spontaneous pancreatic cancer model et al. compared liposomal and free prednisolone in the treatment of spontaneous breast carcinomas, and they observed accumulation of their liposomal formulation at comparable levels to their studies in B16F10 melanoma and C26 colon xenografts Finally, spontaneous cancers are claimed to be one of the tumor models most representative of slow-growing human cancers. They have shown some success, with Cabral Each new model systems brings with it its own set of advantages and disadvantages, with no single choice superior to others when assessed cumulatively based on clinical relevance, throughput potential, technical difficulty, and resource investment. Thus, rather than being seen as an indictment of the xenograft, this discussion should be viewed as an argument for a more informed choice of experimental model that best suits the research question being put forward. Diversifying the variety of models in nanomedicine research will not only allow for better options in testing EPR-adaptive therapies, but it will also better reflect the clinical reality of a heterogeneous tumor population, yielding a more fruitful environment for nanomedicine development.The early decades of nanomedicine research equivocated improvements in overall accumulation of nanoparticles within tumors with improvements in delivery, as this was often found to be correlated with therapeutic benefits in the form of decreased tumor burden and prolonged survival. In the wake of our growing appreciation of the complexity of the tumor microenvironment, it is surprising that this relationship is still accepted with little question - a large number of preclinical works claim improved delivery using only gross measurements such as UPLC, fluorescence spectroscopy, mass spectroscopy, and gamma counting of excised and digested tumors. High-resolution imaging has the capacity to provide spatial localization of agents within a tumor, but many studies that quantify the delivery of functionalized or multimodal nanodiagnostics do so by averaging fluorescence, PET, or MRI signal across the entire tumor area. While this is in some cases a technological limitation, there is often little discussion of how gross measurements and averaged signals are not directly representative of improved delivery to the intended, cellular target. Since several of the key advantages conferred by an EPR-adaptive delivery approach are spatial and temporal in nature, the use of appropriate measurements is key in proving their mechanism and illuminating their benefits for delivery.in vivo tool for evaluating extravasation distance over time, although it often necessitates the use a window-chamber model. Immunohistochemistry of tumor sections can also help to correlate protein expression and intratumoral distribution, although it becomes less suitable if extravasation measurements at multiple timepoints are desired. Cellular localization of delivered nanomedicine is another measure that is more informative of therapeutic outcome than gross tumor accumulation. From in vitro studies, ultrasound-assisted delivery is known to induce cellular localization through several mechanisms in vitro work, but few researchers have made the needed transition to re-validating these findings for in vivo studies. This is especially pertinent to experiments performed in immunocompetent models, as immune cells like tumor-associated macrophages are known to uptake a significant proportion of accumulated nanoparticles As spatial measures, penetration depth and cellular localization are two areas where EPR-adaptive delivery may provide significant improvements compared to passive accumulation. It has long been accepted that nanoparticles uptaken by virtue of the EPR effect remain close to blood vessels, leading to the phenomenon of the “rim effect” wherein the hypervascularized tumor periphery exhibits greater accumulation than the less vascularized, more necrotic tumor core et al. utilizes an in vivo conversion of microbubbles to stable nanoparticles for enhancing agent loading fraction. They showcased peak intact nanoparticle accumulation as early as 15 minutes after ultrasound exposure, exhibiting an atypical mechanism of uptake (Figure Temporal resolution of nanoparticle uptake is another aspect of the delivery paradigm that is rarely questioned. Many studies that rely upon end-point metrics of nanoparticle accumulation, such as histology, UPLC, and spectroscopy, choose a single timepoint for measuring accumulation. This timepoint is often chosen to be 24 hours or later, given the time dependence of the EPR effect for progressive deposition up to 72 hours after injection et al. used a variant of the Hill concentration-effect equation alongside data from PDT-treated and untreated tumors to better evaluate whether or not their observed therapeutic benefits were due to additive or synergistic effects of their combination treatment et al. produced a data-driven mathematical model to simulate the effects of focused ultrasound treatment on diffusive and convective transport out of tumor vasculature and through the tumor microenvironment, helping them interpret their observations. Similarly, Miller et al.'s modelling of uptake of nanoparticles and companion nanodiagnostics allowed them to hypothesize tumor characteristics that primarily drive uptake for different particle types Finally, computational modeling may also be useful as a companion tool for testing EPR-adaptive delivery. Models that incorporate research data to help better describe an observed effect can be useful for both hypothesis generation and elucidation of mechanism. For instance, Snyder While the EPR effect has been an incredible motivating force behind decades of nanomedicine development, the era of embracing it as an infallible solution has passed. Three decades of nanomedicine research has produced only a handful of useful clinical formulations - a far cry away from the “magic bullet” as it had been marketed. While this does not compromise its validity, it defines the scope of its function; namely, its primary efficacy against EPR-sensitive cancer phenotypes rather than the vast heterogeneity of all clinical cancers for which it has been purported to benefit. To broaden the applicability of past decades of research, EPR-adaptive delivery strategies are necessary to deliver agents in a manner suitable for a larger selection of tumor types. Generalizability is one of the strongest aspects of this emerging delivery paradigm, and it seeks to bridge the long-lasting issue of clinical translatability in the field of nanomedicine.In order to aid in the development of these techniques, the field needs to critically reflect on the biases inherent to the current methodologies we use in preclinical research, and a conscious decision must be made to use more representative models and systems. To achieve this larger gain for the community, it may be necessary for journals to use firmer criteria with regards to evaluation of preclinical studies, requiring stipulations on how researchers frame their results based on the limitations of the models, measurements, and techniques that they have used.Because EPR-adaptive delivery techniques do not rely upon the tumor-specific EPR effect, it also enables their translation to other clinical targets. As nanoparticles can be extensively functionalized to provide contrast for almost all clinically available imaging modalities, a tool that improves their access to cells and structures beyond the vasculature would be invaluable to the diagnosis of disease across almost all organ systems. There is potential for circulation-privileged areas such as the brain and spinal cord to be accessed and treated non-invasively. Immunotherapy applications would benefit from functionalized T-cell delivery to specific organ systems, a task that sees natural benefit from these localized delivery platforms. A shift towards a non-invasive delivery archetype that is highly localized benefits the whole genre of design-based clinical research - it is time we start giving it the attention that it deserves.


Many complex real-world decisions, such as deciding which house to buy or whether to switch jobs, involve trying to maximize reward across a sequence of choices. Optimal Foraging Theory is well suited to study these kinds of choices because it provides formal models for reward-maximization in sequential situations. In this article, we review recent insights from foraging neuroscience, behavioral ecology, and computational modelling. We find that a commonly used approach in foraging neuroscience, in which choice items are encountered at random, does not reflect the way animals direct their foraging efforts in certain real-world settings, nor does it reflect efficient reward-maximizing behavior. Based on this, we propose that task designs allowing subjects to encounter choice items strategically will further improve the ecological validity of foraging approaches used in neuroscience, as well as give rise to new behavioral and neural predictions that deepen our understanding of sequential, value-based choice. For many decades, neural processes governing reward maximization have been a central interest in decision neuroscience. One framework that has contributed to our understanding of these processes is reinforcement learning (RL), in which an animal learns to maximize long-run future reward by trial-and-error  dilemmas, in which an individual experiences an option with diminishing returns and must decide when it is best to leave that option. The other deals with accept-reject (or diet-selection) dilemmas, in which an individual chooses whether to engage with an option or ignore it in search of a better one . These firing rates scale with increasing travel time to the next patch, suggesting that dACC neurons may set a threshold for switching and that this threshold is sensitive to the time cost of switch decisions   control over the successive items they encounter  in a directed encounter design. Because low value options can be avoided, this local threshold would also be met more quickly by the current option. Whether subjects use a global or local leaving threshold in directed designs is an empirical question. For our purposes, what is most important is that both perspectives predict lower leaving thresholds, compared with a random encounter design in which MVT provides the optimal solution.With these changes in mind, the empirical work reviewed above implies several behavioral and neural predictions. Behaviorally, one prediction is that patch selection will become less random over time. From an RL perspective, this can be viewed as a declining policy parameter that reduces the amount of exploration relative to exploitation (Sutton & Barto, From a neural perspective, if the firing rate of dACC neurons reflects a threshold for switching (Hayden et al., These suggestions represent a first step towards the development of new foraging approaches in neuroscience, which allow directed encounters with choice items. More complex designs could consider different relationships between replenish rate and travel time. The importance of these factors has been highlighted by Possingham and Houston , who shoIn addition to these considerations, dACC has many competing theories about its role in decision-making. As a starting point, we have framed our neural predictions under the view that dACC may encode a threshold for switching away from current options (Hayden et al., In this respect, one limitation of the stay-switch designs we have covered is that there may be an insufficient range of foreground and background values to fully decouple flexibility (Kolling et al., Neuroscience has made great strides in using foraging as a tool to understand the neural computations that underlie sequential, value-based choice. In reviewing these insights, alongside those from behavioral ecology and computational modelling, we have presented evidence demonstrating that, while useful, current task designs involving random encounters with choice items do not reflect situations in which animals can make use of their knowledge in the environment to encounter items strategically (Fagan et al.,


Osteoporosis represents the most common bone disease worldwide and results in a significantly increased fracture risk. Extrinsic and intrinsic factors implicated in the development of osteoporosis are also associated with delayed fracture healing and impaired bone regeneration. Based on a steadily increasing life expectancy in modern societies, the global implications of osteoporosis and impaired bone healing are substantial. Research in the last decades has revealed several molecular pathways that stimulate bone formation and could be targeted to treat both osteoporosis and impaired fracture healing. The identification and development of therapeutic approaches modulating bone formation, rather than bone resorption, fulfils an essential clinical need, as treatment options for reversing bone loss and promoting bone regeneration are limited. This review focuses on currently available and future approaches that may have the potential to achieve these aims. Osteoporosis represents a polygenetic, environmentally modifiable bone disease, which often results in fragility fractures and poses a high risk of fractures in low impact trauma. Furthermore, the molecular perturbations leading to osteoporosis are also associated with delayed fracture healing and impaired bone regeneration. Based on a steadily increasing life expectancy in modern societies, the worldwide implications of osteoporosis and impaired bone healing are tremendous. Therefore, the clinical need to reverse bone loss, to stimulate bone formation and to boost bone regeneration is increasing and has become a crucial challenge for professional health care providers. A range of drugs approved by the United States Federal Drug Administration (FDA), which work by inhibiting bone resorption, are available for the prevention and treatment of osteoporosis. These substances including bisphosphonates, the monoclonal antibody denosumab and selective oestrogen receptor modulators, only inhibit the breakdown of bone but do not stimulate the formation of new bone. Research in the last decades has revealed several pathways that stimulate bone formation and could be applied to treat both osteoporosis and impaired fracture healing. This review focuses on currently available and future approaches that may be employed to target bone formation and bone regeneration in every day clinical practice.Skeletal tissue represents a highly dynamic tissue that continues to change throughout a lifespan. This process of skeletal turnover is called bone remodelling and is required to protect the structural integrity of bone tissue and to contribute metabolically to the body’s balance of calcium and phosphate. Remodelling includes the resorption of old or damaged bone (bone resorption), which is followed by the formation of new bone (bone formation). In bone tissue, three different and highly specialized cell types are thought to be responsible for the resorption and formation phases of bone remodelling.First, osteoclasts, originating from the hematopoietic/monocyte-macrophage lineage, are the only cells within the organism capable of bone resorption. Under the influence of specific cytokines, including receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor, osteoclast progenitors fuse to form multinucleated osteoclasts, which attach to the bone surface and commence resorption . A combiThe activity of bone cells is influenced directly or indirectly by a large variety of different factors. Local factors including cytokines, chemokines and growth factors among others, are expressed and secreted by cells within the bone microenvironment and exert auto- and/or paracrine effects governing bone turnover. A large array of different systemic factors including hormonal signals have been demonstrated to regulate bone metabolism, for example parathyroid hormone and oestrogen which play a crucial role in the balance between bone formation and bone resorption . In a healthy organism, the processes of bone resorption and formation are tightly regulated, resulting in the maintenance of sufficient bone mass with adequate structure and mechanical quality. If this balance is disturbed, osteoporosis may develop, which represents the most prevalent bone disease worldwide . In mostPatients with osteoporosis have a disrupted bone architecture, a lower quality of bone tissue and, as a result, compromised bone strength and increased risk of fracture ,12. OsteAs stated above, a major complication of osteoporosis is an increase in fracture risk. Every fifth man and every other woman over the age of 50 will sustain a fracture due to increased bone fragility in their lifetime . FracturIn the light of these facts it is apparent that osteoporosis requires effective treatment. The foundation of treatment and prevention of osteoporosis has been reviewed elsewhere and includes weight-bearing exercises, fall avoidance and adequate nutrition to ensure sufficient calcium, vitamin D and protein intake . These gIt appears evident that the inhibition of bone resorption prevents loss of bone mass and architecture, explaining the fact that antiresorptive drugs represent a widely used substance class. Antiresorptive agents including bisphosphonates and the monoclonal antibody to RANKL (denosumab) target the generation, function and survival of osteoclasts and thus reduce the rate of bone resorption. As bone formation is coupled to bone resorption, inhibition of bone resorption is followed by a decrease in osteoblast activity. While this is initially associated with an increase in bone mineral density and some improvement of structural and material properties of bone tissue, increasing evidence points towards an association of long-term suppression of osteoclast activity with increased microdamage accumulation and an alteration in both bone mineralization and collagen formation . AlthougIn principle, stimulating bone formation by pharmacologic means (anabolic therapy) can increase bone mass to a greater extent than antiresorptive drugs. While there is a variety of different antiresorptive agents employed in every day clinical practice , the only currently available treatment regimen to stimulate bone formation is represented by daily injections of parathyroid hormone (PTH) or one of its analogues such as teriparatide and abaloparatide.In a healthy organism, PTH functions as an essential endocrine regulator of calcium and phosphate concentrations in the extracellular space, which is crucial for maintaining serum and urinary calcium levels within the physiological range. Chronically elevated PTH levels, as observed in primary and secondary hyperparathyroidism, cause a high bone-turnover state with bone resorption exceeding bone formation, ultimately resulting in osteoporosis . HoweverIn skeletal tissue, PTH primarily binds to and exerts its biologic effects through the parathyroid hormone 1 receptor (PTH1R). Among other cell types, this G protein-coupled receptor is expressed in mesenchymal stem cells, osteoblasts and osteocytes but not in osteoclasts . It is nAnother significant effect of PTH is the activation of the Wnt signalling pathway in cells of the osteoblast lineage, including osteoblasts and their precursors, as well as osteocytes . Wnt ligPTH may not only stimulate bone formation through a direct effect on Wnt signalling in osteoblasts but also indirectly through reducing sclerostin production by osteocytes ,34. ScleAlthough iPTH or teriparatide primarily stimulate bone formation through its high affinity for the R0 conformation of the PTH1R, a gradual increase in bone resorption can be observed during prolonged usage . TherefoIn order to possibly prolong the anabolic window, abaloparatide, a structurally related agent has been developed and recently approved by the FDA for the treatment of postmenopausal osteoporosis. Abaloparatide is a synthetic analogue of parathyroid hormone-related protein (PTHrP) which binds transiently to the RG conformation of PTH1R and also requires daily subcutaneous injections. Experimental studies demonstrated that abaloparatide increases trabecular thickness and improves trabecular microstructure . In a phSearching for novel targets to increase bone formation, researchers soon became interested in a rare, autosomal-recessive form of a high bone mass disorder, which resulted in the identification of sclerostin as a key regulator of osteoblast activity. Patients with sclerosteosis—a loss of function mutation—or Van Buchem disease—a genetic mutation affecting sclerostin expression—display high bone mass with excellent biomechanical stability due to an excessive osteoblast activity . SimilarAs a rational consequence of these observations, the therapeutic effect of inhibiting sclerostin with neutralizing antibodies in various animal models was subsequently tested. Data from these experimental studies showed a consistent effect of sclerostin immunoneutralization to increase bone formation, bone mass and biomechanical stability at various skeletal sites ,52. ThesPhase III clinical trials (FRAME and STRUCTURE) in female patients suffering from postmenopausal osteoporosis have shown that romosozumab increases bone mineral density at the lumbar spine and hip and reduces the risk of vertebral and clinical fractures in comparison with placebo ,54. RomoApart from the agents discussed above, various cytokines, chemokines, growth-factors and other signalling molecules have been identified to be of crucial importance in regulating bone formation ,57,58 anBone tissue is not only continually remodelled by the combined and tightly regulated activity of bone cells but also has the remarkable capacity for scar-free repair following fracture. The processes governing bone turnover in health and disease are also effective during bone regeneration, as fracture healing can be regarded to represent a juxtaposition of tissue formation (anabolism) and tissue resorption (catabolism or remodelling). These concepts are useful for understanding bone repair and have led to the evaluation of osteoporosis drugs for the treatment of impaired fracture healing. Fracture healing or bone regeneration, results from a complex interplay of cellular and molecular signalling events that reiterate embryonic skeletal development. Traditionally, fracture healing is subdivided into four main phases that show a significant degree of overlap: (1) inflammatory phase, (2) soft callus phase, (3) hard callus phase and (4) remodelling phase . Bone reFracture healing is an evolutionary highly conserved process which functions effectively and efficiently without significant complications in the majority of affected patients. However, in up to 10–20% of patients with fractures, impaired bone regeneration including fracture non-union can be observed, despite the considerable progress in the advance and optimization of surgical fracture care . Non-uniThe use of bisphosphonates in osteoporosis for the prevention of fragility fractures is well established, their value in promoting fracture healing and in preventing and treating non-union much less so ,73,74. IThe monoclonal antibody denosumab binds to RANKL, prevents it from binding to its receptor RANK on the cell surface and therefore inhibits osteoclast recruitment and differentiation. Similar to bisphosphonates, denusomab has been shown to increase callus formation and delay remodelling in animal studies, however the formed callus seems to have better biomechanical properties compared to bisphosphonate treatment . In the As osteoclast function is required to remove necrotic bone fragments and the cartilaginous tissue intermediate during bone regeneration, it is assumed that the stimulation of bone formation is more favourable to improve bone regeneration than the inhibition of bone resorption. Based on the anabolic effect of iPTH, teriparatide and abaloparatide in intact bone, this has led researchers to investigate their use for the prevention and treatment of impaired fracture healing. Both iPTH and teriparatide have been shown to promote fracture healing in animal studies employing various species . Callus Because most animal studies used PTH or its analogues in supraphysiological doses, associated with the potential risk of osteosarcoma development following long-term application, there were significant concerns that clinical studies using only physiological doses would not show the desired results for treatment efficacy . AvailabOne family of peptides, on which more profound information regarding pharmacologic application and clinical value to boost bone regeneration is available, is represented by bone morphogenetic proteins (BMP). BMPs are a family of cytokines pertaining to the TGF-β superfamily and function as key regulators of tissue development in embryonic and adult animals. BMPs were first discovered in 1965 for their capacity to induce ectopic bone formation . To dateIn bone defects, BMPs promoted healing when used in combination with a variety of scaffolds and autologous or allogenic grafts. This was shown in both small and large animal models with cranial and maxillary defects, as well as with segmental bone defects otherwise resulting in non-unions ,94,95. HIn conjunction with rhBMP-2 anti-sclerostin antibodies were reported to improve bone regeneration in a rat femoral defect model when compared to rhBMP-2 alone . HoweverDue to the potential side effects associated with the systemic application of a number of substances with high potential for bone regeneration, some research groups have focused on establishing local delivery methods to defect sites. Both non-genetic methods, such as conjugating oligoaspartic acid, which has a high affinity for hydroxyapatite in fracture sites and promotes elevated concentrations of the chosen agent within the fracture site, and methods using gene therapy for enhancing local transcription of growth factors have been described . For exaBased on the current demographic development, the number of patients with diseases of the musculoskeletal system including osteoporosis and impaired fracture healing is expected to rise steadily. The understanding of the complex cellular and molecular interactions that govern bone metabolism and bone regeneration in health and disease has given rise to novel compounds with high therapeutic potency and a potential low risk for adverse effects. The nature of osteoporosis and impaired bone regeneration, as well as the presence of different comorbidities in affected patients, may require individualized treatment regimens employing more than just one bone drug to achieve the best possible outcomes. The further development and study of therapeutic approaches targeting bone formation, rather than bone resorption, fulfills an essential clinical need, as treatment options for reversing bone loss and promoting bone regeneration are currently limited.


The unique case of a rare 3-level extensor mechanism failure in a 28-year-old male, involving a tibial tubercle avulsion fracture, a patellar tendon avulsion off the tibial tubercle fragment, and a severely comminuted patella fracture, and the surgical technique required to repair such an injury is presented. Focus is spent on the unique repair of a tendon injury when both proximal and distal bony attachments are damaged. Trifocal knee extensor mechanism is a rare clinical entity with minimal literature available—to date, this injury has only been reported in a retrospective review of combat-related injuries in military personnel. It is important to maintain an understanding of knee extensor mechanism anatomy and perform thorough investigation of high-energy knee injuries to ensure adequate treatment of all injuries. The outcome presented in this case shows that positive results after complex extensor mechanism injuries may be achieved, but limited data exists to elucidate optimum treatment. It is essential for surgeons to have firm grasp of techniques used to treat each segment of the extensor mechanism so that they may be combined when a patient presents with complex, multifocal injury. Traumatic extensor mechanism injuries are common, especially in those under 40 years of age. Most commonly, when the extensor mechanism fails, the failure occurs at a single location along the chain, with patella fractures being more common than other locations.  This papA 28-year-old male presented to the emergency department after a motor vehicle accident (MVC). The patient had a body mass index (BMI) of 36, no significant past medical history, and no history of smoking. There was a possible history of remote, pediatric “knee surgery,” but this history could not be confirmed by the patient. He was driving an 18-wheeler truck in the accident, which had the front end of the cab collapse into the cabin. At time of presentation to the trauma bay, he was initially assessed by the trauma service via Advanced Trauma Life Support (ATLS) standards and was determined to be hemodynamically stable with an isolated orthopaedic injury to his left lower extremity. The left lower extremity had two visible approximately 5 cm wounds over the anterior knee . InitialThere were two large wounds, one overlying the anterior tibia  as well as one overlying the patella . The disAttention was then turned to the patellar fracture. Patellar retinaculum was investigated and determined to be intact, with a comminuted patella fracture underlying with minimal displacement of fragments. A small retinacular window was created to observe the largest fragment, and a point-to-point bone clamp was placed on the medial and lateral aspect of the patella to compress the fracture fragments. Following compression, a #2 fibre wire was placed in a cerclage, purse-string fashion around the patellar retinacular soft tissue and hand tied under tension . Clamp wFinally, the patellar tendon insertion avulsion was addressed. Due to the associated tibial tubercle fracture, this presented a unique challenge in acquiring adequate fixation. Two Arthrex 5.5 mm corkscrew anchor sutures  were placed at the tibial insertion just medial to and through the previously fixed fracture fragment. The anchors were advanced from anterior to posterior through the fracture fragment and into a stable tibial bone . The ancFollowing surgery, the patient was hospitalized for 24 hours of antibiotic treatment, compartment monitoring, and pain control. He was discharged with partial weight bearing in a hinged knee brace locked in extension on postoperative day 1. First follow-up visit occurred 10 days after surgery and showed well-healing wounds without evidence of skin necrosis . Final fWe have described a case of trifocal knee extensor mechanism failure including a tibial tubercle avulsion fracture, a patellar tendon avulsion off the tibial tubercle fragment, and a severely comminuted patella fracture. Failure of the knee extensor mechanism in three locations is exceedingly rare with the only identified report in a retrospective review of combat-related injuries in military personnel . In thatIn adults, patellar tendon avulsions/ruptures are more common in patients with elevated body mass index (BMI), underlying systemic illness, or steroid use . The patIn limited case series, patella fractures were open in 7-13% of cases and were treated with antibiotics, incision, and debridement and various forms of fracture fixation with or without internal fixation . In the Although examples of trifocal extensor mechanism failure are lacking in nonmilitary series, there are a number of reports of bifocal failure. Kang et al. summarized these reports and presented a case of bifocal knee extensor mechanism disruption in an 84-year-old male after a motorcycle accident resulting in an open avulsion fracture of the inferior patellar pole and avulsion fracture of the tibial tubercle . In thisThough reports of such injuries are limited in adult literature, several repair techniques have been described in pediatric cases of combined tibial tubercle and patella tendon avulsion injuries. Common to most of these reported techniques is open reduction and internal fixation of the tubercle fragment with screw fixation, but K-wire and staple fixation have also been described . MultiplAlthough the injury described in this case does appear to be extremely rare, it is important to maintain a complete understanding of knee extensor mechanism anatomy and perform thorough investigation of high-energy knee injuries to ensure adequate treatment and improved outcomes in injuries that could be otherwise devastating to future function. The case and technique reviewed in this case demonstrate that acceptable outcomes after complex extensor mechanism injuries are possible, but limited data exists to elucidate optimum treatment. It is essential for surgeons who may encounter similar injuries to have firm grasp of techniques used to treat injury to each segment of the extensor mechanism so that they may be combined accordingly when a patient presents with complex, multifocal injury.


Nephrotic syndrome is classically categorized by the histopathology with examples including focal segmental glomerulosclerosis (FSGS) and minimal change disease. Pediatric patients are also classified by whether their nephrotic syndrome is sensitive to, dependent on, or resistant to steroids. However, this traditional classification system overlooks the frequent clinical conundrum when, for example, one patient with FSGS responds briskly to steroids, and another quickly progresses to end stage kidney disease despite therapy. Two patients may have similar histopathologic appearances on kidney biopsy but entirely different clinical characteristics, rates of progression, and treatment responses. Transcriptional regulation of gene activation and posttranscriptional processing of mRNA may drive the unique and heterogeneous phenotypes which are incompletely understood in kidney disease and are a recent focus of research. Gene expression profiles provide insight on active transcriptional programs in tissues, are being used to understand biologic mechanisms of progressive chronic kidney disease, and may help to identify patients with shared mechanisms of kidney damage. This mini-review discusses clinically relevant techniques of bulk tissue and single cell transcriptomics, as well as strengths and limitations of each methodology. Further, we summarize recent examples in kidney research achieved through transcriptomics. This review offers an outlook on the role of transcriptomics in an integrative systems biology model with the goal of defining unique disease subgroups, finding targets for drug development, and aligning the right drug with the right patient. Nephrotic syndromes are kidney diseases which result in proteinuria, hypoalbuminemia, edema, and hyperlipidemia. Traditionally nephrotic syndrome is categorized into diagnoses by their histopathology , membranous nephropathy) or their response to steroids, however pathology and clinical phenotype do not predict or uniquely correlate with rate of progression or response to therapy. The transcriptome refers to all of the RNA expressed from genes of a particular organism, tissue, or cell type. Understanding the transcriptome can help elucidate the crucial link between genotype and phenotype, as gene expression and control of gene expression can vary under different states of health and disease. In addition, progressive kidney diseases may upregulate particular common transcriptional pathways—allowing scientists to identify prognostic biomarkers and develop interventions targeted at these shared pathways in chronic kidney disease (CKD). By defining kidney diseases more granularly than their histopathology and clinical presentation and incorporating data from transcriptomics as well as genomics, proteomics, and metabolomics—scientists work toward advancing the field of nephrology toward targeted treatments for kidney disease.In this mini-review, we briefly review techniques used to analyze the transcriptome, specifically comparing and contrasting techniques that use “bulk” methods profiling the mRNA expression from whole tissue (in our case kidney biopsy tissue) vs. “single cell” methods profiling the mRNA expression within individual cells extracted from tissue or biofluids, with the goal of describing how these techniques are being used to improve our understanding and treatment of kidney disease.a priori understanding of the model organism's genome and transcriptome and must be regularly updated with known sequence data. When using microarray technology, whole tissues can be analyzed en masse or can be divided into compartments. In human kidney research, the glomerular compartment and the tubulointerstitial compartment are commonly separated through microdissection, and are then analyzed separately , eGFR slope, and “progressors”—those who advanced to a higher CKD stage within the study follow up—were more likely to have low uEGF/Cr. Most recently, in a cohort of 623 children, low uEGF/Cr was associated with increased risk of CKD progression  as an independent predictor of CKD progression . EGF is ved eGFR . In a coved eGFR . Again igression .In summary, low uEGF/Cr level is a robust predictor of eGFR in cross-section, and it has also been shown to be a promising early biomarker to predict progression of kidney disease across several cohorts and disease types, including children. In many kidney diseases, EGF may be a general marker for tubular health, not captured by serum creatinine and urine protein. In this example, transcriptomic data was used to identify a novel biologic process and then applied to develop a non-invasive marker which reliably captures intra-renal transcriptional activity, and predicts kidney disease progression.Transcriptomics has also found a role in elucidating mechanisms of disease which escape traditional animal models. While mouse models of diabetic nephropathy mimic early stage disease in humans, they fail to replicate the progressive histopathological, and biochemical changes that are classic for advanced diabetic nephopathy . Using mIn this example, transcriptomic data from kidney biopsy was used to identify a targetable disease pathway in diabetic nephropathy that led to a successful early stage clinical trial. Insights gained from this foundational work have been applied to other kidney diseases such as FSGS, as well as models of polycystic kidney disease  and unilBulk tissue transcriptomics has been used to identify biomarkers, novel mechanisms of disease, and potential targets for treatment. However, using tissue in bulk, such as tissue from a renal biopsy, to evaluate gene expression poses challenges because of the admixture of many cell types. While bulk methods advance our understanding of global transcriptional changes in the whole tissue, elucidating transcriptional changes in particular cell types remains a challenge. Bioinformatic methods have been developed to infer cell composition from bulk expression data based on unique cell markers, but characterizing single cell expression relies on inference based on marker gene expression levels, rather than direct measurement . MethodsWhile bulk sequencing is useful for identifying gene expression patterns across a whole tissue or tissue compartment, single cell sequencing techniques allow researchers to identify gene expression patterns of a single cell type within a whole tissue.Microdissection and sorting by flow cytometry to process bulk tissue into single cells for RNA analysis are limited by the inability to reliably purify a single cell type and interrogate a single cell's transcriptome. In other words, the resulting transcriptomic data is an average of RNA transcripts from a mixed group of cells . Newer sSingle cell RNA sequencing involves five basic steps: single-cell isolation, cell lysis, reverse transcription of mRNA, amplification, and library generation , 35–37. One of the most common graphical representations of single cell data is a T-Distributed Stochastic Neighbor Embedding (tSNE) plot, which is useful for visualizing cell populations . In a tSThe advantages of single cell techniques are the ability to identify rare and new cell types and trace developmental relationships between heterogeneous cells  and cellular states  . Methodoin vitro transcription to combine barcoding and amplication steps into a single reaction  project is working to create reference maps of all human cells.In this example, single cell data was used to comprehensively identify cell types within mouse kidney tissue. It allowed direct comparison of mouse and human kidney cell types to assess similarities and differences, critical for using mouse models to study human disease. It also identified novel cell types with potential implications for identifying biological processes, previously not appreciated in humans. The human cell atlas  to study human disease. For example, Harder et al has shown through single cell transcriptional profiling of podocytes from kidney organoids, that organoid podocyte lineage cells have two distinct transcriptional states . These p cohorts . These dOrganoids have also been used in combination with single cell transcriptomics to better understand specific pathology, such as polycystic kidney disease. Cyst size and total kidney volume in polycystic kidney disease are associated with disease progression and mortality. However, phenotypes in polycystic kidney disease differ by family and mutation, which is an area of continued research. To interrogate different phenotypes of polycystic kidney disease, Czerniecki et al. edited genes of kidney organoids to produce cysts. DropSeq analysis of these gene-edited models showed that inhibition of non-muscle myosin II increased cyst formation, and this process is potentially regulated though polycystin-1, a pathway for future investigation .These examples show that analysis of cell specific transcriptional changes in cell development, health, and disease, can lead to identification of novel genes and pathways that may be relevant to human disease.Bulk and single cell transcriptomic profiling techniques each have unique contributions to advancing our understanding of kidney disease. These techniques can help us identify cell types and mechanisms relevant to disease processes that thus far have largely been defined by histopathology, which does not well-capture the underlying biologic heterogeneity of CKD and nephrotic syndromes. As demonstrated in the examples provided in this mini-review, interrogations of the kidney transcriptome have helped researchers understand mechanisms and biomarkers of disease. Exciting applications now and in the near future include further interrogation of these discoveries in animal models, targeting these disease pathways with medications with the goal of delaying progression of CKD, and helping researchers identify subgroups of patients who might be most likely to respond to specific pharmacologic therapies. These approaches may benefit all patients with kidney disease, but in particular pediatric patients with a higher likelihood of a germline genomic mutations have much to potentially gain. Translation of this technology to clinical practice in nephrology is not yet available, however application of similar techniques in this bench to bedside paradigm has revolutionized the field of oncology. Ultimately, the goal of applying transcriptomic analyses to blood, urine, and kidney biopsy specimens of individual patients to better provide targeted treatments for their nephrotic syndrome and CKD is part of the future of nephrology.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The challenges of effective vaccination against influenza are gaining more mainstream attention, as recent influenza seasons have reported low efficacy in annual vaccination programs worldwide. Combined with the potential emergence of novel influenza viruses resulting in a pandemic, the need for effective alternatives to egg-produced conventional vaccines has been made increasingly clear. DNA vaccines against influenza have been in development since the 1990s, but the initial excitement over success in murine model trials has been tempered by comparatively poor performance in larger animal models. In the intervening years, much progress has been made to refine the DNA vaccine platform—the rational design of antigens and expression vectors, the development of novel vaccine adjuvants, and the employment of innovative gene delivery methods. This review discusses how these advances have been applied in recent efforts to develop an effective influenza DNA vaccine. Seasonal influenza epidemics continue to challenge public health systems worldwide, causing 3–5 million cases of severe respiratory disease and 290–650 thousand deaths annually . DespiteDNA vaccines possess numerous properties ideal for influenza control and have been trialled for a range of diseases, including viral and bacterial infections, and some cancers –7. WhilsInactivated influenza vaccines and live attenuated influenza vaccines (LAIV) are the most widely used forms of influenza vaccine, and are generated by harvesting viruses grown in embryonated hen’s eggs . The delvia the major histocompatibility complex (MHC) pathways. CD4+ T helper cell activation following MHC class II presentation of secreted DNA vaccine protein is critical for the production of antigen-specific antibodies  . This me2 to transport a liquid inoculum to the intradermal or intramuscular layer (Alternative delivery devices have been developed to improve upon traditional needle and syringe inoculation for parenteral administration. In small animal models, the gene gun induces immune responses successfully with low doses of DNA by delivering gas-propelled plasmid-coated gold microparticles directly into epidermal cells , 61. Humar layer –64. Recear layer –67. HA Dar layer .Parenteral gene delivery has been further enhanced by electroporation, which temporarily increases the permeability of local cell membranes with electrical pulses . Its earThe mucosa is an appealing site of inoculation for influenza vaccines as it is easily accessible and is the clinical site of entry for influenza viruses . Existinin vivo. The mechanisms of action for licensed conventional adjuvants include the formation of antigen depot at the inoculation site, the activation of inflammatory pathways, and the recruitment of APCs (The co-administration of adjuvants with influenza DNA vaccines is a common strategy to elicit adequate levels of protection  of APCs .The goal of adjuvant design is to increase the immune response to vaccine antigens, a critical hurdle in the DNA vaccine field. Mineral salts such as alum are widely used in human vaccines and have resulted in up to fivefold increase in HA DNA vaccine-induced antibody titres . CytokinAdjuvant compounds developed as delivery reagents aim to improve the transfection efficiency of DNA vaccine plasmids. The efficiency of the cellular uptake of DNA vaccines is determined by cell membrane permeability and the susceptibility of foreign DNA to host enzymes. It is estimated that only 1% of a naked plasmid inoculation is able to reach the nuclei of target cells for protein expression—most plasmids remain in the extracellular space to be cleared by host processes . SynthetThe administration of novel vaccine types including adenovirus vectors , 100, suDespite this, studies have indicated that DNA vaccines may have a clinical application in pandemic settings. Chang et al.  demonstrAfter two decades of research, DNA vaccine technology is gaining maturity—several veterinary DNA vaccines are currently licensed for West Nile virus and melanoma , and sigAll authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that they have no commercial or financial relationships that could be construed as a potential conflict of interest.


In the early nineties of the twentieth century Jaak Panksepp coined the term “Affective Neuroscience” (AN) today being accepted as a unique research area in cross-species brain science. By means of (i) electrical stimulation, (ii) pharmacological challenges, and (iii) brain lesions of vertebrate brains , Panksepp carved out seven primary emotional systems called SEEKING, CARE, PLAY, and LUST on the positive side, whereas FEAR, SADNESS, and ANGER belong to the negative affects. Abundant research into human clinical applications has supported the hypothesis that imbalances in these ancient primary emotional systems are strongly linked to psychiatric disorders such as depression. The present paper gives a concise overview of Panksepp's main ideas. It gives an historical overview of the development of Panksepp's AN thinking. It touches not only areas of neuroscience, but also shows how AN has been applied to other research fields such as personality psychology. Finally, the present work gives a brief overview of the main ideas of AN. The ancestral voices (to use Ross Buck's phrase) guide their choices as they navigate life. Each of the primal emotional affects is evaluative, that is, has a valence that is either pleasant or aversive and signals objects or situations to approach in the case of the pleasant ones  or to avoid in the case of the aversive ones .Yet, experiencing a primary affect does not necessarily mean all mammals can self-reflect on their emotional experiences. That capacity may be reserved for the more cortically endowed mammals. However, “raw” primary affects are experienced as pleasant or aversive qualia, which alter behavior and provide for secondary-level learning (conditioning principles). The emotional minds of most mammals may be limited to displaying “intentions-in-action” rather than a more reflective “intentions to act.” In Endel Tulving's terminology, the capacities of most mammals likely combine anoetic (without knowing) and noetic (knowing) consciousness without necessarily attaining autonoetic consciousness—being able to sufficiently hold experiences in memory to review the past and anticipate the future Tulving, . We knowApart from experiencing primary-process emotional affects, what is much more difficult to study in the non-human mammalian world is Tulving's autonoetic consciousness characterized by the capacity of humans to experience affective nuances often reflecting human higher-order cognitions and language including having thoughts about thoughts. This represents a limitation of cross-species affective neuroscience. However, subtler models of affective concepts such as pessimism in dogs  the details of the hornet nest's appearance and location. This event will be forever embedded in your memory, and you will have learned to avoid repeating this experience by remaining more vigilant when outdoors walking through unfamiliar terrain.Each primary-process emotional command system likely encompasses a separate reward or punishment system. These learning systems can be thought of as secondary-processes integrating new experiences into the primary framework allowing for previously neutral environmental stimuli to elicit the emotion and for novel reactions to become associated with such stimuli. For an example of novel reactions to an emotional arousal, over the ages, humans when threatened have learned to reach for their swords, and more recently their pistols instead of clenching their fists. However, more evidence is needed regarding the extent to which these different emotional systems encompass different learning and memory parameters.While deep brain stimulation (DBS) allows researchers to demonstrate that brain stimulation at specific sites evokes distinct emotional behaviors that are accompanied by corresponding affects, there remains the question of whether the emotional affects elicited at these sites are similarly distinct. We do know that rats can learn to discriminate between DBS in the hypothalamus and septal regions of the brain  and the knowledge that the symptoms of frontal lobe damage generally resemble ADHD, it was hypothesized that right frontal lobe damage might be a useful rodent model of ADHD. They found that rat right frontal lobe lesions (performed at three or four days of age) significantly increased playfulness  as well as increasing activity levels confirming the hypothesis  significantly reduced the number of pins and overall activity, “ameliorating” the elevated play urges and activity levels. Indeed, the play therapy even significantly reduced overall activity in the sham lesion control subjects.Additional tests in new subjects that had no lesions indicated that play experiences also increased behavioral inhibition not specific to increased fear. Combining both experiments, the authors suggested “the possibility that one of the long-term functions of social play is to promote maturation of various higher brain areas, including frontal cortical ones” . The initial chick predisposition and the acquired preference were hypothesized to represent independent brain systems. That is, selective cortical lesions impaired the acquired preferences but not the predisposition  relationship requires the ongoing involvement of our affective states influencing our perceptions, thoughts, and urges as well as the acquired cortical capacity to modulate our emotional evolutionary foundations in order to achieve an appropriate seesaw positioning for ongoing life events. Yet, much research remains to be done to illuminate the mechanisms involved in these evolutionarily integrated processes.At least since the ancient Greeks first speculated about the four humors and Ptolemy proposed the Sun, Moon, and planets set our behaviors in motion, humans have speculated about the basis of personality and psychopathology. More modern theories have ranged from Freud's theory of sex and aggression on personality development Freud,  to the tCattell pioneered using the lexical hypothesis—the assumption that any important human characteristic was embedded in language—and factor analysis and first reported four personality factors Cattell, , but as However, the lexical Big Five personality bastion now seems to have splintered into multiple competing theories proposing six and seven personality dimensions Saucier,  and evenPanksepp took a different approach to carving nature at its joints by using electrical stimulation to directly probe the brain for its secrets. He followed in the tradition of Hess  who in tThese ESB sites are concentrated in subcortical regions of the brain, and correspond with Panksepp's primary-process emotional-action command systems. Cases of such dramatic and unambiguous emotional behaviors or affects have not been elicited from the neocortex. However, more muted displays of laughter have been reported from evolutionarily more ancient cortical areas such as the anterior cingulate cortex  were developed  on the low end of Emotional Stability making Emotional Stability a confusing scale to interpret but also conflating the distinctions between these problematic emotions that are so closely linked to the etiology and treatment of psychopathology. These associations are stable across cultures, and the same patterns between individual differences in primary emotional systems as assessed with the ANPS and the Big Five of Personality have been observed in many countries including studies in the USA, Germany and China, potentially hinting at “a global ancestral neuro-biological effect”  examined the use of low doses of buprenorphine (a “safe” opioid that has low respiratory depression effects and becomes a mu-opioid blocker at high doses) to counteract suicide in a population of subjects with chronic suicidal thoughts, many of which had already attempted suicide. A small pilot study provided encouraging results  could alleviate treatment-refractory depression. A German group conducted an early small trial study. The site selected for DBS was the nucleus accumbens, which proved somewhat successful but largely limited to acute effectiveness  the interactions of early experience and brain development, (2) gene expression linked to primary-process experience, (3) the relationship between primary emotional experience and memory, (4) how many distinct subcortical emotional circuits exist, (5) whether the neocortex can generate new emotions independent of the subcortical brain, and (6) the capacity of bottom-up subcortical primary-process brain activity to guide cortical development and eventual top-down regulation of primary emotional expression . It wilKD wrote the first draft of the manuscript, which was critically revised by CM.KD was employed by company Pegasus International. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Medications can be used to treat alcoholism by decreasing craving for alcohol or by blocking the development of processes that lead to addiction. Medications also can enable alcoholic patients to participate more effectively in treatment by decreasing psychiatric symptoms associated with alcoholism or by improving alcohol-related intellectual impairment. Many promising medications for treating alcoholism appear to play a role in normalizing communication among nerve cells. Such medications include naltrexone and acamprosate, which help prevent relapse in patients undergoing psychotherapy for alcoholism. Medications that affect the neurotransmitter serotonin, however, may alleviate symptoms of anxiety and depression in alcoholics without affecting the alcoholism itself. Recent advances in neuroscience have stimulated the search for medications to treat alcoholism. Medications development relies on both preclinical and clinical investigations. Preclinical research studies the effects of experimental chemicals on the physiology and behavior of laboratory animals or on the function of cells grown in culture. Results of such research provide the basis for clinical studies  of specific medications in humans.Preclinical research has established that alcohol affects brain function by interfering with communication among nerve cells . Researchers have identified specific neural communication systems that contribute to the rewarding effects of alcohol consumption, thereby promoting alcoholism. Preexisting defects in such systems also may underlie vulnerability to excessive alcohol consumption. Neurons communicate by synthesizing and releasing chemicals called neurotransmitters, which bind to specific receptorClinically, medications can be used to treat alcoholism or its effects by (1) decreasing symptoms associated with the initial stages of recovery, such as anxiety or depression; (2) decreasing a person’s desire to consume alcohol; (3) blocking a component of the addictive process ; (4) treating psychiatric disorders that might coexist with alcoholism; and (5) improving alcohol-related intellectual impairment . Althoug®), selective serotonin reuptake inhibitors (SSRI’s), and acamprosate.This article discusses medications whose effects fall into one or more of the first four categories, including naltrexone . Behavioral and pharmacological data in animals and humans suggest a link between the endogenous opioid system and alcohol consumption  affects multiple actions in the brain, including the regulation of mood states, appetite, and sleep. This diversity of function has been linked to the existence of several specific serotonin receptor subtypes as well as variability in the mechanisms whereby receptor activation is translated into neuronal function.Preclinical studies suggest a relationship between serotonin function and alcohol consumption. For example, serotonin administration causes a decrease in alcohol consumption in animals selectively bred for alcohol preference as well as in animals that are genetically heterogeneous  prescribed to treat depressive disorders. SSRI’s increase the activity of serotonin by preventing its reabsorption into the neuron that released it. In several clinical trials, Results of preclinical and clinical laboratory studies may help explain these disappointing results. First, if altered levels of serotonin underlie alcoholism, serotonin agonists might be more effective in reducing alcohol consumption than would reuptake inhibitors, which rely on adequate pre-existing levels of serotonin for their action. Second, different aspects of alcohol consumption and alcoholism may involve different serotonin receptor subtypes. Medications targeted at specific receptor subtypes may affect alcohol consumption more effectively than a medication like fluoxetine, which increases serotonin activity nonspecifically.®) is an antianxiety medication that affects the serotonin system. Although buspirone decreases alcohol consumption in animals, its clinical application in human alcoholics is unclear. Several small clinical trials found that buspirone decreased symptoms of anxiety in alcoholics but not alcohol consumption. In a 12-week clinical trial evaluating the use of buspirone in combination with coping-skills treatment in anxious alcoholics, patients treated with buspirone showed a trend toward a lower number of drinking days and a lower number of drinks per drinking day  through the NMDA receptor. Studies in humans provide evidence that NMDA antagonists produce alcohol-like properties in humans and can modulate the effects of alcohol intoxication, including euphoria, a “high” feeling, and sedation. In addition, evidence suggests that alcohol’s reinforcing properties may be related in part to its effects on the NMDA receptor  to help compensate for alcohol’s continued antagonistic effect. At the end of a drinking bout, the now hyperactive glutamate system is no longer balanced by the presence of alcohol, leading to tremor, high blood pressure, sweating, and seizures . Administration of NMDA antagonists is one form of treatment that can suppress withdrawal-induced seizures in animals. Persistent alteration in NMDA receptor function in alcoholics may increase the risk of seizures and contribute to the development of alcohol-related organic brain disease. Thus, abnormalities of glutamate function may be involved in effects of alcoholism ranging from acute intoxication and addiction to long-term nervous system effects. This possibility suggests the recent hypothesis that alcoholism may be understood as a glutamate-related neuropsychiatric disorder (reviewed in These preclinical and clinical studies have suggested that glutamatergic medications may reduce alcohol consumption. The leading candidate in this area is acamprosate. Preclinical research suggests that acamprosate may block glutamate reuptake . A serieBecause alcoholism is a complex disorder, its treatment should be multifaceted. Effective treatment must target the extensive social, psychological, and medical complications of alcoholism. Medications can play various, complementary roles in alcoholism treatment. For example, acamprosate may decrease symptoms associated with early recovery, naltrexone may decrease the desire to drink or may block the reinforcing aspects of alcohol, and medications such as fluoxetine or buspirone may help in treating co-occurring psychiatric disorders. Clinicians can use medications to ease patients into psychosocial treatments or to enhance the effect of psychosocial treatments in decreasing alcohol consumption. The optimal clinical applicability for these medications has yet to be fully examined. Clinical and neuroscience research will continue to increase our understanding of the applications and limitations of proposed treatments while stimulating the development of new medications.