Sex differences may play a critical role in modulating how chronic or heavy alcohol use impacts the brain to cause the development of alcohol use disorder (AUD). AUD is a multifaceted and complex disorder driven by changes in key neurobiological structures that regulate executive function, memory, and stress. A three-stage framework of addiction  has been useful for conceptualizing the complexities of AUD and other addictions. Initially, alcohol drinking causes short-term effects that involve signaling mediated by several neurotransmitter systems such as dopamine, corticotropin releasing factor, and glutamate. With continued intoxication, alcohol leads to dysfunctional behaviors that are thought to be due in part to alterations of these and other neurotransmitter systems, along with alterations in neural pathways connecting prefrontal and limbic structures. Using the three-stage framework, this review highlights examples of research examining sex differences in drinking and differential modulation of neural systems contributing to the development of AUD. New insights addressing the role of sex differences in AUD are advancing the field forward by uncovering the complex interactions that mediate vulnerability. Diagnosticand Statistical Manual of Mental Disorders (DSM-5), published by the American Psychiatric Association, describes AUD by mild, moderate, and severe subclassifications depending on the number of criteria met for the diagnosis.Addiction is a chronic relapsing disorder characterized by continued substance misuse despite harmful consequences. Alcohol use disorder (AUD) is specific to the maladaptive consumption of alcohol.The goal of this review is to discuss AUD using the three-stage framework of addiction—binge/intoxication, withdrawal/negative affect, and preoccupation/anticipation7This review focuses primarily on preclinical animal studies using self-administration procedures to elicit alcohol exposure and/or to measure drinking behaviors to allow for more direct comparison to key findings about drinking behaviors in humans. Preclinical drinking models are summarized in other reviews.In this phase, individuals consume enough alcohol to induce intoxication and cause impairment of physical and mental abilities. An example of this is binge drinking—the excessive consumption of alcohol that results in blood alcohol levels of 0.08 gram percent (g/dL) or higher—typically reached by consumption of five or more drinks in men and four or more drinks in women within a 2-hour period.The lifetime prevalence of AUD is 29% in the United States, with a higher prevalence in men than women.17Higher risk-taking tendencies can lead to early-onset use and subsequent alcohol misuse—especially in males.21Another reason that individuals may drink alcohol is for its acute anxiolytic, or anxiety-reducing, properties. Experimenter-administered alcohol intoxication can temporarily reduce anxiety-like behavior in rodents.Overall, these studies suggest that sex plays a distinct role in the motivating factors leading to drinking initiation. Risk-taking behaviors are more likely to influence adolescent boys to consume alcohol, whereas adolescent girls are more likely to consume alcohol due to its anxiety-reducing properties. Understanding the factors underlying early alcohol drinking onset may produce better strategies to prevent and dissuade alcohol consumption in adolescence and may help create specialized alternatives to alleviate the need for this coping mechanism.Drinking during adolescence has been shown to lead to higher levels of drinking in adulthood in both male and female mice.32Imaging studies in humans show other prefrontal regions are also altered with alcohol drinking in adolescence and early adulthood. The dorsolateral prefrontal cortex is thinner in younger adults who frequently engage in heavy drinking (≥5 drinks) compared to controls, and the magnitude of this effect is more robust in young adult men compared to young adult women.Reward comprises learning (cue associations), hedonic (“liking”), and motivational (“wanting”) components.42The mesocorticolimbic dopamine pathway may contribute to sex differences in appetitive and consummatory behaviors, given its essential role in conditioning and associative learning of environmental and physiological cues that predict alcohol reward availability.The interaction between gonadal hormones and dopamine may provide insight into the molecular mechanisms underlying sex differences in the rewarding properties of alcohol.Lastly, in vitro treatment with ICI 182,780—an antagonist of estrogen receptor subtypes alpha and beta —attenuated alcohol-induced excitation of VTA dopamine neurons in mice with moderate levels of estradiol (diestrus II); this suggests that estradiol’s modulation of dopamine sensitivity to alcohol may be occurring through its acute interaction with ERα and/or ERβ subtype in the VTA slice. The acute interaction between estradiol and its receptors appears to depend on moderate or high estradiol levels, as the ERα/ERβ antagonist did not measurably attenuate alcohol-induced increases in dopamine firing under conditions of low estradiol (estrus).Through its effects on mesocorticolimbic dopamine, estradiol appears to mediate association-based learning and the rewarding properties of alcohol in context, which could ultimately promote drinking. Indeed, estradiol-treated ovariectomized mice show both increased dopamine signaling in the VTA in response to alcohol and increased preference of an alcohol context compared to vehicle-treated ovariectomized mice.53Progesterone and its metabolites also have been implicated in the modulation of mesocorticolimbic dopamine neurons in response to alcohol.54Binge drinking can cause injuries and other adverse outcomes, with high-intensity (extreme binge) drinking  resulting in more severe consequences such as blackouts, alcohol overdose, and even death.57After repeated episodes of binge drinking, individuals can begin to experience a negative affective state when alcohol is withdrawn voluntarily or involuntarily. This includes dysregulated stress hormone levels, dysphoria, anxiety, depression, and irritability—a symptomology thought to be due in part to adaptations in stress-related neural pathways.Chronic heavy alcohol consumption eventually can lead to severe AUD. A hallmark feature of AUD is the negative emotional and physiological state that arises when alcohol wears off.65Individuals with AUD report having negative and unpleasant feelings during withdrawal, such as low self-concept, neuroticism, depression, and hostility—all of which predict alcohol craving.Male rats and mice show a more pronounced display of negative affective-like behaviors and neuroactivity after withdrawal from chronic alcohol exposure compared to female rats and mice.77Withdrawal from alcohol is associated with a dysregulation of stress hormones. The hypothalamic pituitary adrenal (HPA) axis governs the neuroendocrine response to stress by releasing corticotropin-releasing factor (CRF) from the hypothalamus, which activates the release of the adrenocorticotropic hormone (ACTH) from the anterior pituitary, resulting in the release of the glucocorticoids from the adrenal glands (cortisol in primates and corticosterone in rodents).Studies in humans show that, compared to men, women had lower ACTH and cortisol levels under baseline (resting) conditions in the morning, but were more sensitive to peripheral stimulation of the HPA axis as indexed by the dexamethasone/CRF test.83In male rats, dampened HPA responsivity has been observed after withdrawal from chronic intermittent alcohol vapor exposure, and to a lesser extent following chronic alcohol drinking alone.87Dysregulation of the HPA axis is thought to result from alcohol-induced neuroadaptive changes within this neuroendocrine axis itself.91Stress can increase alcohol drinking, but this depends on sex, age, and the type of stress exposure.Another study of mice used the “Drinking in the Dark” (DID) binge drinking procedure for 2 weeks followed by 11 days of unpredictable, chronic, mild stress.93The studies discussed above and others96The neural systems implicated in the interactive effects of stress and alcohol include not only structures of extended amygdala, but also brain regions thought to be involved in the third stage of AUD (preoccupation/anticipation).67Prolonged heavy alcohol use leads to a state of a constant preoccupation with alcohol and compulsive drinking despite negative consequences.103After long bouts of abstinence, alcohol-related cues can trigger incentive salience, which heightens cravings and precipitates relapse.Bdnf gene expression in the VTA and low alcohol drinking behavior; this effect was not observed in female offspring.Bdnf +/− mice, with males showing higher TrkB phosphorylation than females in the prefrontal cortex and striatum.In mice, male offspring of alcohol-exposed fathers have high As discussed earlier, multiple cycles of binge intoxication followed by withdrawal can transition individuals from light to moderate drinking to severe AUD.112One strategy used to measure inflexible drinking is the assessment of a persistent motivation to drink despite increasing the response requirement to obtain alcohol. In animal studies, this can be tested by training subjects to press a lever or nose poke for alcohol in operant boxes.Alcohol solutions also can be manipulated to devalue reward and to test for signs of inflexible drinking. One approach to devaluing alcohol is the addition of an unpleasant substance to change the flavor of alcohol by adding the bitter taste of quinine hydrochloride dihydrate or lithium chloride.118Another approach used to test for inflexible drinking is to measure shock-resistant alcohol intake.122Altogether, these studies suggest that inflexible drinking promotes heavy and continued use of alcohol and, consequently, may lead to further neuroadaptations in the brain. However, some of the devaluation strategies show limited evidence of sex differences. The inclusion of female subjects in these studies to directly compare the effects is vital to evaluate the role of sex in compulsive-like drinking under these different paradigms.Deficits in executive function can result from early-onset drinking or chronic heavy use, and this may lead to a higher chance of relapse following abstinence.125With long-term abstinence in both men and women, there is increased resting-state connectivity to brain regions that control executive function and decreased connectivity within reward processing regions.127Animal studies have advanced our understanding of neural connectivity at the axonal and microstructural level, giving insight into the mechanisms by which prefrontal function improves across development and can be impaired after alcohol exposure. During adolescent development in rats, prefrontal axons undergo robust increases in myelin ensheathment, which corresponds with a twofold increase in neuronal transmission speed.130Longitudinal studies show that white matter growth is attenuated in the frontal lobes in humans who started drinking during adolescence—an effect that was comparable in both sexes.135Despite more robust effects in males, examination of myelinated axons at the microstructural level shows that alcohol alters the nodal domain in both male and female rats.Administration of extreme binge-like doses of alcohol damages the hippocampus and prefrontal cortex, and impairs memory in rats.The preclinical and clinical studies outlined in the current review show sex differences in behavioral risk factors and neural systems implicated in AUD, as summarized in Adolescent drinking in the context of stress, negative affect, and increased cue-reactivity is greater in females. Males show vulnerability with regard to higher levels of impulsivity and, compared to females, they are less sensitive to the aversive effects of intoxication, making males less likely to stop drinking. Sex also was found to be a predictor of the negative impact that chronic alcohol use has on the brain  self-report questionnaire. A metabiological study recently reported that resting state connectivity functional magnetic imaging can be useful for assessing AUD.


These efforts can help improve existing therapeutic approaches by taking into consideration the contribution of cellular plasticity/heterogeneity in enabling drug resistance.Intratumoral heterogeneity is a major ongoing challenge in the effective therapeutic targeting of cancer. Accumulating evidence suggests that a fraction of cells within a tumor termed Cancer Stem Cells (CSCs) are primarily responsible for this diversity resulting in therapeutic resistance and metastasis. Adding to this complexity, recent studies have shown that there can be different subpopulations of CSCs with varying biochemical and biophysical traits resulting in varied dissemination and drug-resistance potential. Moreover, cancer cells can exhibit a high level of plasticity or the ability to dynamically switch between CSC and non-CSC states or among different subsets of CSCs. In addition, CSCs also display extensive metabolic plasticity. The molecular mechanisms underlying these different interconnected axes of plasticity has been under extensive investigation and the trans-differentiation process of Epithelial to Mesenchymal transition (EMT) has been identified as a major contributing factor. Besides genetic and epigenetic factors, CSC plasticity is also shaped by non-cell-autonomous effects such as the tumor microenvironment (TME). In this review, we discuss the latest developments in decoding mechanisms and implications of CSC plasticity in tumor progression at biochemical and biophysical levels, and the latest  Heterogeneity in cancer biology has long been recognized and exploited in the clinical management of the disease . Intertua priori defined; rather stemness can be thought of as a cell state can that be reversibly gained or lost. In other words, cellular plasticity can allow CSCs and non-CSCs to switch among one another  were shown to be capable of inter converting among themselves as well as give rise to non-CSCs  – a hallmark process of cancer cell plasticity in which cancer cells transdifferentiate and acquire endothelial cell like characteristics . In tripructures . In renaructures . Thus, CCSC plasticity is controlled by both cell-intrinsic and cell-extrinsic factors . SeveralMany studies have pointed out various mechanisms of epigenetic regulation such as bivalent chromatin state, DNA methylation, histone modifications in mediating CSC plasticity . For exain vitro and enhanced tumorigenesis in vivo (EMT is a reversible, dynamic process which is critical during embryonic development and also aberrantly activated during various pathological processes like wound healing, fibrosis and cancer progression . EMT is  in vivo . Recent  in vivo . A recen in vivo . Another in vivo . It woulBesides juxtacrine crosstalk among cancer cells and stromal cells, there are factors secreted by the different cell types that form complex interacting networks in a TME . Accumulin vivo (in vivo and not in tumor cells treated by hypoxia in vitro alone (The physical and chemical composition of the microenvironment such as acidic pH, low oxygen and nutrient availability, rigidity and porosity of the ECM can also play an important role in regulating the cancer stem cell behavior . A classin vivo . Using fro alone .These studies underscore the importance of the microenvironment in sculpting intra-tumoral heterogeneity and CSC plasticity and highlight the need to better understand the tumor-microenvironment crosstalk for the development of effective therapeutic strategies . HoweverCSC plasticity can be instigated by various components in the microenvironment such as the secretion of cytokines and chemokines, communication with different stromal cell types and extracellular matrix and hypoxia . Consequin vivo as a consequence of plasticity and adaptation to the microenvironment (in vitro clonogenic and in vivo limiting dilution xenotransplantation assays are of paramount importance to validate the stemness trait of the cells (One of the serious and longstanding challenges in studying CSCs is the determination of appropriate methodology for the isolation and characterization of CSCs . One of ironment . These ohe cells ; Table 2The properties of stem cells such as self-renewal and multipotency can be governed by intra-cellular and extracellular components constituting the stem cell niche . SimilarCancer cells display variations in response to extrinsic biomechanical stimuli, leading to heterogeneity in their biophysical properties, which influences the overall nature of cells such as stemness and differentiation. Recent studies have shown that mechano-transduction cues greatly influences the generation and maintenance of CSCs and eventually metastasis . In addiin vitro. The more deformable flexible phenotype was associated with expression of many genes involved in motility, metastasis and greater mammosphere formation efficiency (in vivo, deformability has been shown to be crucial for efficient extravasation of tumor-repopulating cells during metastasis as seen in zebrafish models (For instance, CSCs were enriched based on their adhesive traits using a microfluid chip having micro-channels coated with basement membrane extract. The cells entered into the chip driven by hydrodynamic forces. While highly adhesive cells were captured in micro-channels, less adhesive cells were collected from the outlet, which were shown to be enriched in CSCs, had greater motility and were resistant to chemotherapeutic drugs . This stficiency . Consisth models .in vitro and greater tumor development in vivo. In addition, the authors isolated IBC cells based on their adhesive property using a microfluidic device and showed that the less adhesive cell population was ALDH– enriched, displayed enhanced in vitro invasion and migration as well as increased in vivo tumor development. Further, exogenous alteration of cell stiffness also resulted in changes in metastatic potential of these cells with less stiff cells showing greater invasion and migration (Similar to biochemical heterogeneity observed within CSCs , CSCs caigration . The resigration .Therefore, with increasingly detailed characterization of biomechanical properties of various subpopulations of cancer cells and ECM, a “mechanosome” or “matrisome” signature may be helpful in identifying and isolating the most aggressive cancer cell subpopulations .Much of our current understanding about CSCs comes from studies performed on bulk cancer cell populations. However, bulk analysis masks the underlying intra-tumor heterogeneity and does not inform much about rare cell subpopulations within the tumor . Therefoin vivo lineage tracing method in a genetic mouse model of breast cancer, A recent study integrated FACS analysis of CSC markers with functional assays such as proliferation, self-renewal and multipotency tests, and observed that glioblastoma stem cell heterogeneity results from tumor plasticity which is determined by the microenvironment cues . LineageThe advent of single-cell omics approaches has revolutionized our knowledge on CSC biology. Single cell genomic and transcriptomic analyses have provided invaluable insights regarding intra-tumor heterogeneity and clonal evolution in different cancers . In addiin situ hybridization or sequencing will enable the decoding of the spatial regulation of cellular heterogeneity at single-cell resolution (Nonetheless, most of the single-cell omics analyses does not preserve spatial information as it requires the cells to be isolated from their microenvironment . Anothersolution . Single-solution . Using asolution . Integrasolution . A recensolution . Live sisolution . The numsolution .With emerging evidence about the potential of single-cell analysis in understanding the biology of CSCs, development of newer tools and analysis methods and integrative approach are required for better comprehending the cell state transitions and improved therapeutic strategies.in vitro and in vivo (in silico the coupling between core regulatory circuits of EMT, CSCs and other connected signaling pathways such as Notch signaling; this model predicted that altering the coupling strength between EMT and CSC networks and/or modulating Notch signaling can change the position of “stemness window” on the “EMT axis,” thus generating various subsets of CSCs in terms of EMT phenotypes (With the deluge of preclinical and clinical data being generated at a high-dimensional level, computational approaches to extract meaningful information and generate testable hypotheses are becoming more common . Various in vivo  and has  in vivo . How areenotypes . Such CSenotypes . A commoenotypes . Phenotyenotypes ; thus, benotypes .“Data-based” models have also been valuable in decoding CSC signatures. A stemness index was derived using one-class logistic regression and observed to be higher in the metastatic breast cancer cells compared with primary tumors , suggestde novo generation of CSCs (Another set of questions where mathematical models have been useful is estimating the fraction of CSCs in a tumor. Many studies have used population-level models to understand the difference in growth kinetics of CSCs and non-CSCs, and used that to offer a potential mechanistic underpinning of “tumor growth paradox,” i.e., accelerated tumor growth with increased cell death . Typical of CSCs  and thei of CSCs .Spatial heterogeneity is a fundamental feature of TME that contains diverse cell types . One can–CD44+ mesenchymal breast CSCs were found in the invasive edge of the tumor, while the more epithelial or hybrid E/M CSCs, identified by ALDH1+, were localized in the interior regions of the tumor (In vitro knock down of JAG1 in SUM149 human breast cancer cells significantly reduced their tumor organoid formation, confirming the role of Notch-Jagged signaling in tumor progression (Varying levels of nuclear β-catenin expression was observed in different sub-populations of well-differentiated colorectal cancer, suggested to be regulated by TME . Cells ihe tumor . Put toghe tumor . In vitrgression . Future Spatial heterogeneity of tumors can be used as a predictor of cancer prognosis and treatment response across different cancer types . For exaEver since Otto Warburg’s observation that cancer cells, unlike normal cells preferentially rely on glycolysis for energy production even under aerobic conditions, referred to as aerobic glycolysis or Warburg effect , metabolIn addition to switching between glycolytic and OXPHOS phenotype, CSCs have also been shown to coopt these two pathways and exist in a hybrid metabolic state as suggested by recent studies . So, it Although most studies have focused on the glucose metabolism, the involvement of other metabolic pathways like lipid and amino acid metabolism still needs to be investigated further. A better understanding of the metabolic phenotypes and plasticity of CSC is required for the effective elimination of these cells.The issue of phenotypic plasticity presents a clear and present danger in the treatment of cancer patients. Accumulating evidence suggests that CSCs consist of different sub-populations that can interconvert among different states through intracellular and intercellular regulatory networks. Over expression of one or more transcription factors or activating trans-differentiation processes such as EMT and metabolic alterations can drive the switch among CSCs and non-CSCs as well as between different subsets of CSCs. These adaptive strategies adopted by cells must be taken into account while devising new therapeutic strategies in the clinic in order to target all populations effectively.The challenges to addressing this issue are multifold. The ability to identify the plastic CSC population using markers has its inherent problems which are further confounded by each individual patient’s unique biochemical and biomechanical signatures. The general consensus is that drug resistance is achieved through a transition to a slow cycling state which is reversible once the stress is removed . UnderstUsing latest developments in computational and experimental methods will allow us to map the different states of tumor cells within multiple locations in a tumor, thus enabling a more comprehensive view of the genetic and non-genetic heterogeneity that exists within a cancer. Correlating such intratumoral heterogeneity with cellular phenotypes will be key to devising better therapeutic options in patients to ablate the tumor cells stably within a patient.RN and MJ designed the review. AT, KS, RM, MJ and RN wrote, read, and approved of the manuscript.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.


This editorial review of the Research Topic “Integrating predation risk across scales: from neurons to ecosystems and milliseconds to generations” explores prey responses to predation risk from an array of different perspectives. It includes work from neurobiologists, ecologists, and other disciplines interested in predator-prey interactions at varying spatial and temporal scales. Taken together, the 15 papers in this Research Topic represent an attempt to synthesize work across disciplines in search of intellectual synergies and new avenues of research collaboration.Madin et al. and Atwood et al. report on research addressing the phenomena of coral reef “halos,” zones of unvegetated seafloor surrounding coral reef patches. These patches have long been thought to result from herbivorous fish seeking shelter from predators within reef cracks and crevices; they only feed on vegetation growing near the coral because foraging too far from shelter might expose them to predators. Madin et al. report that coral reef halos found in the Great Barrier Reef result from both prey fear and the action of bioturbators, sediment-dwelling organisms that churn up the seafloor and affect algal settlement. Atwood et al. conducted complementary research confirming the importance of predation risk in changing algal grazing rates and demonstrating that these changes decrease sedimentary carbon storage within the reef halos.Papers by Chizzola et al. and Bleicher et al. describe work exploring the “landscape of fear” in terrestrial systems. Fear of lions has long been thought to shape the habitat choices and foraging decisions of many herbivorous mammals; Chizzola et al. combine behavioral, physiological, and isotopic information to reveal the highly dynamic and scale-dependent responses of wildebeest and impala to this fearsome threat. At a smaller spatial scale, Bleicher et al. explore how desert rodents on two continents respond to the common threat of owls and snakes, two dangerous but very different predators. They found that the two rodent communities differ in their assessment of which predator is the most threatening and discuss how local environments can profoundly shape prey responses.Both Trakimas et al. discuss the connections between development rate and physiological stress in the cricket Gryllus integer. They found that slow-developing crickets had higher resting metabolic rates than fast-developing ones, presumably reflecting the energetic costs associated with a higher resting metabolism. They also resumed activity faster following a stressor than did fast developers, a relationship that may translate into higher foraging rates. Second, Baranowski and Preisser report the results of work testing the impact of predation risk on the survival of luna moth (Actias luna) larvae. They found that caterpillars exposed to a threatening but non-lethal predator (a wasp with its jaws and stinger immobilized) would “freeze” in place, stop feeding, and die at a higher rate than larvae exposed to a similarly-sized but harmless fly. Finally, Eason et al. describe fascinating work addressing how threat type and refuge availability affect the trajectory of fleeing Eastern Gray Squirrels (Sciurus carolinensis). Briefly, they found that squirrels preferred not to run to refuges (trees) that were directly behind them and would actually run toward threats to reach a tree in front of them.The special feature also contains several articles exploring the impact of risk on individuals. First, Weiss summarizes our current understanding of how Daphnia, a crustacean that is a model system for both ecology and evolutionary biology, detect and avoid predators. In addition to defending themselves against predators, Daphnia exposed to predator cues can produce offspring with spines and “helmets” that make them more difficult for predators to subdue and consume. The review traces response from the initial cues to the underlying neurophysiological mechanisms that structure prey responses, providing a concise overview of the molecular mechanisms underlying both short- and long-term phenotypic adaptation. In the second, Draper and Weissburg provide a review and synthesis of the impact of global warming and increased CO2 levels on the sensory ecology of predator-prey interactions. Both factors affect multiple parts of the predator-prey encounter sequence, including the ability of predators to detect prey and the ability of prey to flee approaching predators. Although cue production, transmission, and reception will be globally affected by such large-scale environmental changes, they highlight the fact that aquatic predator-prey interactions may be disproportionately sensitive to these disruptions. Altered CO2 levels are likely to have especially strong effects in marine systems due to ocean acidification, which can change Ph levels sufficiently to make the calcium-based shells of prey difficult or impossible to produce.The Research Topic includes two large-scale reviews of different ecological processes that affect predator-prey interactions. In the first,  Cinel and Taylor explored how chronic exposure to bat calls affected transcription in fall armyworms (Spodoptera exigua) that were either continuously exposed to foraging and attack calls or held in silence. The 290 transcripts they found either up- or down-regulated were involved in a broad range of cellular functions, information that lays the foundation for research addressing how these changes affect insect physiology, behavior, and demography. Working in a very different system, Tong et al. report on how testosterone affects both mouse fear and physiology. Exposing male mice to this chemical reduced their innate fear of predator cues (cat urine) when allowed to explore potentially risky habitats. In a complementary experiment, they also found that testosterone produced hypomethylation of the promoter region of the arginine vasopressin gene. The latter compound plays a key role in socio-sexual behavior; they suggest that testosterone-induced reductions in fear occur via manipulations of the trade-off between fear (reduced activity) and social/sexual interactions (increased activity).Moths and other night-flying prey are often targeted by bats that use ultrasonic calls to detect and help capture them. As a result, many moths possess tympanic organs capable of detecting bat sonar. Franklin provides a cogent assessment of our current understanding. Work in rodent model systems demonstrates the role of the rostrolateral PAG in hunting behaviors and how the dorsal-ventral and rostral-caudal axes of the PAG differ in their impact on defensive behavior. The fact that brain imagining work in humans shows threat-induced PAG activity suggests that large portions of PAG function are likely conserved across an array of mammalian species.Recent technical advances in neurobiology have revolutionized our understanding of how predators and prey detect and respond to each other. This progress has heightened our appreciation for the role of the periaqueductal gray (PAG) in modulating such interactions; Mitra reviews the role of these neurons and how they change in response to predator odors. While neurons in the basolateral amygdala respond quickly to risk cues, continued exposure to stress-related hormones can change them in ways that also facilitate rapid endocrine responses to future stressors. Current evidence suggests that the basolateral amygdala plays an important role in risk-related activities across multiple taxa, and that it may be generally responsible for anticipatory defensive behaviors in threatening situations.The amygdala, and the basolateral neurons within it, also play a critical role in detecting and responding to predator cues; Asok et al. explore the behavioral, neural, genetic, and biochemical mechanisms involved in this process. As fear generalization is a hallmark of many anxiety and stress-related disorders , Asok et al. highlights the importance of sex differences and remote timescales in rodent models to improve the dialogue between human and animal studies. Factors that contribute to PTSD were also considered in the review by Bhattacharya et al. Recent research in both humans and animals suggests that experience of the parent may influence its offspring. Bhattacharya et al. review studies in both humans with PTSD and laboratory predator stress models of PTSD that suggest changes to DNA methylation may underlie the generational effects of trauma transmission. Both Asok et al., and Bhattacharya et al., argue that a deeper understanding of the mechanisms that promote stress-induced psychopathology will accelerate the development of effective therapeutics.Fear generalization is an adaptive process that promotes survival in complex and dynamic environments. In their review, 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.


From fertilization to onset of gastrulation, a mammalian embryo goes through several rounds of cellular morphogenesis resembling phenomena of epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET), collectively referred to as EMTs. How these EMT events play a role in shaping the three-dimensional (3-D) architecture of the developing embryo is not well-understood. In this review, we present a model in which cellular morphogenesis, represented primarily by dynamic changes in its epithelialization status, is the driving force of embryonic 3-D organization. This is achieved through the integration of three key components of mammalian early development, the pluripotency regulation, morphogenetic signaling, and biomechanical force anisotropy. Although cells in an early embryo do not exhibit full mesenchymal characteristics, our model underscores the importance of investigating molecular regulation of epithelial cell polarity and partial EMT/MET in understanding mammalian early development. After fertilization, a mammalian embryo undergoes several rounds of early cleavages before acquisition of apicobasal polarity through compaction and segregation of inner and outer cells through asymmetric divisions  and/or mesenchymal (M) status, pluripotency, biomechanical forces and morphogen signaling.in vivo or in vitro, varying from totipotency, corresponding to the status of blastomeres before lineage segregation between the TE and ICM, to unipotency which corresponds to cells that can differentiate only into a single cell type (Cellular potency is defined by its ability to differentiate into various cell types either ell type . Status in vitro or in vivo) of cells isolated in vivo and maintained in vitro. Recent efforts have been made to associate pluripotency with features of either the original developmental stage during pluripotent cell derivation or the putative developmental stage after those pluripotent cells have achieved a steady state under a given culture condition. Four pluripotency states have so far been characterized using cell morphological and molecular criteria. The first one is termed “naïve pluripotency,” which corresponds to the ICM cell after TE specification and segregation. Naive pluripotent cells do not have apicobasal polarity. They express a particular set of associated genes   and the amniotic epiblast (contributing to the ectoderm portion of the amnion), and the appearance of the proamniotic cavity. The molecular mechanisms that link changes in cellular morphology and cellular potency are still not well-explored. GRHL2, an epithelial state inducer, has been reported to control the expression of a subset of the pluripotent network genes upon transition from naive to primed pluripotency state  or its causal relationship with human primitive streak initiation is unclear.Non-polarized naïve epiblast precursors at this stage do not respond to mechanical stimuli  involve oriented cellular intercalation and morphology changes that can be understood from the perspective of polarized cytoskeletal organization and directional biomechanical tension (Ferro et al., in vitro models of early epiblast-like PSC organization (Deglincerti et al., in vitro platform, with high reproducibility and programmability, to investigate how mechanical signals are integrated into the biochemical network that regulates cellular specification and tissue patterning and whether cell morphological changes might be the missing link reconciling how mechanical and chemical signals influence cell identity during development.During early embryonic development, it is unclear why cells need to undergo successive morphological transformation in order to achieve the complexity in cellular differentiation. Is it a necessity to ensure proper control over the morphogenetic process or just a direct consequence of morphology changes? The former would imply that it is necessary to restrict cellular potency as embryonic morphogenesis progresses in order to reduce potential differentiation errors. However, this paradigm seems to be contradicted by a recent study on totipotency loss and trophectoderm differentiation in which the authors showed that ICM-derived naïve PSCs retain the ability to differentiate into the trophectoderm lineage (Guo et al., The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.GS conceived the manuscript topic. GI, SH, and GS discussed about the writing and wrote the manuscript. All authors agreed on the content of manuscript.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.


Non-invasive ventilation (NIV) and continuous positive airway pressure (CPAP) are effective treatments for children with severe sleep disordered breathing (SBD). However, some patients may present too severe SDB that do not respond to NIV/CPAP or insufficient compliance to treatment. A careful revaluation of the interface and of ventilator settings should be performed before considering alternative treatments. In patients with obstructive sleep apnea (OSA), alternatives to CPAP/NIV rely on the underlying disease. Ear-nose-throat (ENT) surgery such as adeno-tonsillectomy (AT), turbinectomy or supraglottoplasty represent an effective treatment in selected patients before starting CPAP/NIV and should be reconsidered in case of CPAP failure. Rapid maxillary expansion (RME) is restricted to children with OSA and a narrow palate who have little adenotonsillar tissue, or for those with residual OSA after AT. Weight loss is the first line therapy for obese children with OSA before starting CPAP and should remain a priority in the long-term. Selected patients may benefit from maxillo-facial surgery such as mandibular distraction osteogenesis (MDO) or from neurosurgery procedures like fronto-facial monobloc advancement. Nasopharyngeal airway (NPA) or high flow nasal cannula (HFNC) may constitute efficient alternatives to CPAP in selected patients. Hypoglossal nerve stimulation has been proposed in children with Down syndrome not tolerant to CPAP. Ultimately, tracheostomy represents the unique alternative in case of failure of all the above-mentioned treatments. All these treatments require a multidisciplinary approach with a personalized treatment tailored on the different diseases and sites of obstruction. In patients with neuromuscular, neurological or lung disorders, non-invasive management in case of NIV failure is more challenging. Diaphragmatic pacing has been proposed for some patients with central congenital hypoventilation syndrome (CCHS) or neurological disorders, however its experience in children is limited. Finally, invasive ventilation via tracheotomy represents again the ultimate alternative for children with severe disease and little or no ventilatory autonomy. However, ethical considerations weighting the efficacy against the burden of this treatment should be discussed before choosing this last option. Long term non-invasive ventilation (NIV) and continuous positive pressure (CPAP) are increasingly used in children with sleep disordered breathing (SDB) –4. The cAlternatives to a non-invasive ventilatory support vary according to the underlying disease severity, the patient's behavioral and cognitive status, the family support and the expected benefit of NIV/CPAP treatment. Treatment options change with child's age and the need for NIV/CPAP requires a constant evaluation. From a practical point of view, treatment failure and therapeutic options must be evaluated in the light of the underlying pathological mechanism and the expected benefit. This review analyzes the current alternative therapeutic options to CPAP and NIV in children with OSA and in children with nocturnal hypoventilation due to neuromuscular, neurological, thoracic, and lung disorders.OSA is defined as the “recurrent partial or complete upper airway obstruction  with disruption of normal oxygenation, ventilation and sleep pattern .” The prObjective assessment of CPAP/NIV utilization is mandatory. First of all, in case low adherence, the first step is to check the equipment. The combined analysis of in-built software data and nocturnal pulse oximetry or a sleep study with CPAP/NIV may identify residual respiratory events that require settings changes , 37, 38.The patient's and caregiver's motivation and support should be assessed if the compliance remains low after checking of all technical issues. Most studies reported a suboptimal compliance with CPAP uses ranging from 4.7 to 5.3 h/night despite a behavioral program and a close follow-up , 41. SupOSA may change with child's growth, with some children improving spontaneously while other deteriorate, justifying a continuous multidisciplinary assessment . DespiteRapid maxillary expansion (RME) is an orthodontic treatment that widens the palate and nasal passage, thereby increasing airway caliber and reducing nocturnal upper airway obstruction. The technique can only be used prior to midline fusion of the maxilla, which generally occurs shortly prior to puberty. RME is proposed after the 4th year of life, when the second deciduous molars of the upper jaw have erupted, and the device is usually removed after a period of 12 months . RME is MDO consists in the application of internal or external distraction devices that are placed on each mandibular branch. MDO has been used with success to treat severe OSA due to mandibular hypoplasia, particularly in infants with congenital mandibular hypoplasia such as Pierre Robin sequence or Treacher Collins –69. SuccTracheotomy is an invasive technique that represents the ultimate rescue therapy for children with severe OSA for whom no other therapeutic options are available. On the other hand, in many cases tracheostomy represents a safe and easy to manage therapy. Moreover, practice often depends on local training habits and on the expertise of the different centers, with some team preferring tracheostomy for failure of conservative methods . AlthougHypoglossal nerve stimulation is a technique that consists in an implantable device that delivers electrical impulses to tongue protrusor muscles during inspiration aiming at reducing the collapsibility of the upper airway. Hypoglossal nerve stimulation has been shown to be effective in selected adults with OSA and no obesity or circumferential velopharynx collapse , 83. ThiNasopharyngeal airway (NPA) refers to the placement of a modified endotracheal tube in the hypopharynx. In infants with Pierre Robin syndrome or other congenital abnormalities of the upper airway, NPA has shown to be effective and well-tolerated, even if 10–20% of children still require tracheostomy –88. The 2) that may be set from 21% to nearly 100%. The nasal cannulas used with HFNC are less invasive and more comfortable than nasal masks or nasal prongs used for CPAP therapy. A few case series have reported the use of HFNC in children with OSA  is a non-invasive ventilatory device that is increasingly used for the treatment of acute and chronic respiratory failure in adults and children , 91. HFNwith OSA . Our growith OSA . Also, twith OSA .Some adjuvant therapies may be indicated for selected patients. Weight loss should be the first line therapy for obese children with OSA , 15. HowIntranasal corticosteroids or leukotriene modifier therapy have been shown to improve children with mild surgery-naïve OSA or mild residual OSA after AT. This treatment may decrease nasal inflammation and improve nasal obstruction especially in those children with chronic nasal obstruction due to chronic allergic rhinitis or who had difficulties in tolerating CPAP/NIV nasal interfaces.Positional therapy may be considered when a school-aged child, adolescent, or older teenager presents obstructive respiratory events, occurring exclusively or predominantly in the supine position as compared to the other positions. Several tools such as pillows and belts are commercially available. Positional therapy has not been well-evaluated in children, and a sleep study should be performed to verify its efficacy case per case.Hypoventilation, defined by hypercapnia and hypoxemia, is the consequence of an imbalance between respiratory load and the ability of the respiratory muscles to generate an adequate alveolar ventilation . Some paPatients with congenital central hypoventilation syndrome (CCHS) and those with spinal cord injury and elective diaphragm palsy may be eligible for diaphragm pacing (DP). DP consists on an implantable device that sends electrical impulses to the diaphragm via the phrenic nerve. Successful decannulation and management of SDB in pediatric CCHS patients have been reported, even though treatment efficacy should be carefully assessed because this approach may not be sufficient or cause obstructive respiratory events , 104.Finally, when respiratory insufficiency progress, especially in children with advanced neuromuscular disease, NIV may fail to guarantee efficient correction of nocturnal and daytime gas exchange. Some groups reported successful experience with NIV in totally ventilator-dependent children with neuromuscular disease , while oCPAP and NIV are widely used to treat SDB in infants and children. However, their efficacy depends on treatment adherence and underlying diseases severity. Checking of equipment and patient's and caregiver's education are of paramount importance to achieve an optimal CPAP/NIV use. For patients with OSA and CPAP failure, a multidisciplinary approach is recommended in order to choose the most appropriate therapeutic option. In patients with nocturnal hypoventilation due to neuromuscular or neurological conditions, the range of alternative therapies is limited. Tracheostomy with or without invasive ventilation represents an option that need to be discussed with the patients and families, and ethical considerations weighting the efficacy against the burden of this treatment should be discussed.AA is the main author of the manuscript. BF, SK, LG, TT, and AL contributed to the conception of the manuscript. All authors approved the final version of the manuscript.SK is employed by the company ASV Sante but does not have any commercial of financial conflict of interest with the content of this review. The remaining 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.


Both acute brain injuries and chronic neurological disorders affect millions of patients worldwide every year, such as traumatic brain injuries, ischemic stroke, Alzheimer's disease (AD), and Parkinson's disease (PD). Due to the high mortality or catastrophic neurological and cognitive dysfunction of these brain injuries and neurological disorders, enormous efforts have been made to unravel the underlying pathophysiologic mechanisms in the past decades. Despite the respective progression in different cases, there are some notable commonalities among these different conditions, such as the cell‐to‐cell network.In this special issue, we collected nine research articles and four review articles, aiming to compile the recent research concerning the pivotal role of NVU and BBB in different neurological disorders. In three review articles, the topics mainly covered the mechanisms of neuroinflammation and BBB disruption in neurovascular diseases, especially in ischemic stroke. Wang LY et al.Huang XW et al.Zhao FF et al.In this special issue, we also assembled nine research articles, generally comprised topics over different neurovascular diseases, including both experimental and clinical research. Two research articles focus on ischemic stroke in animal models. Venkat et al.Consistent with the review article by Wang LY et al, Akihiro et al.Intracerebral hemorrhage (ICH) could induce profound pathological changes in the NVU and leads to catastrophic consequences to patients’ neurological outcomes. It is important to identify the high‐risk patients. In a clinical research on spontaneous ICH, Zhu FP et al.Collectively, this special issue gathers reviews and recent researches in the field of NVU protection under different pathological conditions. We anticipate that the novel findings in the current issue will give rise to a better understanding of the roles of NVU not only in the above‐mentioned neurological diseases, but also in those previously undervalued conditions, such as post‐operative cognitive decline or delirium. Meanwhile, future works are important to figure out the clinical relevance and translational perspectives in the mechanisms that underlying the NVU dysfunction after acute brain injury or neurological disorders, with a better use of the emerging high‐end and rapidly evolving technologies.


Epilepsy comprises more than 40 clinical syndromes affecting millions of patients and families worldwide. To decode the molecular and pathological framework of epilepsy researchers, need reliable human epilepsy and control brain samples. Brain bank organizations collecting and supplying well-documented clinically and pathophysiologically tissue specimens are important for high-quality neurophysiology and neuropharmacology studies for epilepsy and other neurological diseases. New development in molecular mechanism and new treatment methods for neurological disorders have evoked increased demands for human brain tissue. An epilepsy brain bank is a storage source for both the frozen samples as well as the formaldehyde fixed paraffin embedded (FFPE) tissue from epilepsy surgery resections. In 2014, the University of Saskatchewan have started collecting human epilepsy brain tissues for the first time in Canada. This review highlights the necessity and importance of Epilepsy Brain bank that provides unique access for research to valuable source of brain tissue and blood samples from epilepsy patients. The pioneer of brain banking in Canada was Dr. Ali Rajput at the University of Saskatchewan. In 1968, Dr. Ali Rajput created the first brain bank in Canada dedicated only to patients with Parkinson's disease (PD) and other movement disorders. Development of the Bank led to numerous international and local contributions and critical breakthroughs in the modern treatment of PD, only possible because every case has the full documentation of In 1980, McGill University created the next brain bank in Canada. The bank is based at the Douglas Mental Health University Institute and has become one of the largest brain banks in the world. Currently, it houses more than 3,000 brains as well as a large relational database containing demographic, clinical and developmental histories from the donorsIn 1993, Dalhousie Medical School established the Maritime Brain Tissue BankIn 2014, the University of Saskatchewan created the first epilepsy brain bank in CanadaEpilepsy comprises more than 40 clinical syndromes affecting 50 million people worldwide. People with epilepsy experience unprovoked recurring seizures. This is a disease that poses significant challenges for modern medicine, as ~36% of patients receiving anti-convulsant medication have inadequate seizure control . In addiPartial seizures account for up to 50–60% of epilepsy cases in adults  and temporal lobe epilepsy (TLE) is the most common type of partial epilepsy . ApproxiAn epilepsy brain bank is a storage source for pieces of tissues from epilepsy surgery resections. The tissues are donated by living patients undergoing brain resection for the treatment of their medically refractory epilepsy. Brains are properly extracted, dissected and orderly stored either in formalin-fixed-paraffin-embedded (FFPE) blocks or cryopreserved in −80°C freezers. Control tissues can be obtained from brain surgeries in the same center from patients with other neurological conditions such as brain tumors, vascular malformations and other focal lesions. These patients may have associated seizures but also, they could be seizure-free being an adequate control for some studies. In addition to accepting donated control brain tissues we will seek other brain banks to find and receive suitable control brain tissues as well, for example normal tissue. Alternatively, the aspirated tissue from resection margins, if available, could also serve as the healthy control tissue for each patient's seizure focus.The discovery of the molecular underpinnings and potential treatments of human epilepsy based on new molecular targets is demonstrably lagging behind progress made in neurodegenerative diseases such as AD and PD , partly Many fundamental questions in epilepsy remain unanswered: why do some epilepsy patients fail to respond to antiepileptic drugs(AEDs)? Why, despite newly developed epilepsy drugs, has their efficacy not improved? Why do 30 to 40% of epilepsy patients never respond to AEDs despite new drugs appearing in the market every year? Are there any better targets for developing new AEDs? Is it possible to develop antiepileptogenic drugs as opposed to antiepileptic drugs? What are the mechanisms of neurodegeneration in the epileptic brain? Are there targets for drug development with ability to prevent neurodegeneration and memory impairment in patients with epilepsy?As per current standard of care in most epilepsy centers, the brain tissue obtained from temporal lobectomies or other types of temporal resections is fixed in formaldehyde to undergo routine pathological analysis. No further research studies are usually performed on the specimen. In addition, formaldehyde-fixed tissue is not suitable for most advanced research methods such as molecular biology, proteomics, and genomics. It is extremely crucial to perform further studies on brain samples using advanced methods, such as bulk or single cell RNA sequencing (RNAseq), mass spectrometry and transcriptomic analysis, coupled with traditional techniques of molecular biology, biochemistry, and immunohistochemistry to answer important questions. For example, how does prolonged seizure damage hippocampal neurons, and what is the role of glutamate receptor expression, and specifically do anti-seizure medications that attenuate glutamate receptors overexcitation protect hippocampal neurons and preserve memory?The initial step is saving fresh brain specimens for molecular biology/ biochemical studies with storage at −80°C in a freezer. Concurrently obtained blood samples from patients will also be banked for future epilepsy-related genetic studies. Our center has started saving the brain from epilepsy surgeries and we encourage other Canadian epilepsy centers to begin this process, as expanding the tissue source will provide a richer resource for sharing epileptic and control tissue that could be used to address the following: (1) To detect the chronic differences in regions of the brain prone to seizures using proteomics, immunohistochemistry, and molecular biology; (2) To identify biomarkers of refractory epileptic activity, the common abnormal pathways shared by many patients with temporal epilepsy will be used as biomarkers of refractory epileptic activity; (3) These biomarkers will be translated into therapeutic targets, which will be tested in animal models to determine efficacy prior to clinical trials; (4) Target binding of these compounds in humans at therapeutic doses will be assessed using Positron Emission Tomography (PET) scanning with specific ligands .There are several advantages:No large storage space is needed. In neurodegenerative diseases, brain donation is post mortem and therefore the whole brain tissue is stored , however, in epilepsy brain bank a small piece of surgically dissected tissue is stored.In brain banks for neurodegenerative diseases, the tissue is harvested post-mortem and therefore, hypoxic-ischemic changes in the brain are unavoidable. However, in an epilepsy brain bank, the tissue can be processed and stored quite quickly after surgical excision with minimal chance of hypoxic-ischemic changes. In addition, in samples from an epilepsy brain bank the entire tissue is preserved evenly since the small pieces of tissue are rapidly frozen, resulting in the inner depth of the tissue being frozen almost instantaneously as the tissue surface, whereas the freezing procedure required for a brain hemisphere with degenerative conditions could take longer thereby adding increased risk of hypoxic/ischemic brain damage.The samples are a better representation of the population as they are usually harvested during ongoing, active disease in living patients as opposed to neurodegenerative samples that are usually harvested post mortem and much later in the course of the disease.After applying for and obtaining human biomedical ethics approval (often the most time-consuming step), a reliable storage location for both the frozen samples as well as the formaldehyde fixed paraffin embedded (FFPE) tissue specimens has been established with funding support from institutional and provincial funding agencies . All samples were coded and kept in locked locations, with the decoding data and patient's clinical information stored on a password protected computer using reliable software. The freezer must be equipped with emergency power as well as an alarm system to indicate any temperature change. Tissue collection, processing and storage was a collaborative process involving an epilepsy surgeon, two epileptologists, research coordinator, and two neuropathologists. After specimen excision, the tissues are placed in saline solution in sterile plastic containers, labeled based on anatomical location, for transport to the neuropathology lab. Neuropathologists then confirm the anatomical locations and cut the samples into smaller pieces, selecting the required amount of tissue for clinical pathological staining and diagnosis, with the rest left for banking. Any portion of formalin fixed samples saved for clinical pathology but not ultimately used will also be later transferred to the brain bank for storage. Fresh pieces of tissue are slow frozen with isopropyl alcohol in cryostat Eppendorf tubes and then stored in the locked −80°C freezer. The location of tissue in the freezer , patient coding information and clinical data are then entered into secure software developed by the University of Saskatchewan information technology department, on a password protected computer located in the same locked room where the freezer is located. Only the two epileptologists who are the biomedical ethics applicants have the access keys to the tissue storage room. A sample of patient's blood is also stored in the same freezer for possible future analysis. The neurosurgical suctioning and/or ultrasonic aspiration supernatant if available can also be centrifuged and saved for use in proteomics experiments.Functional magnetic resonance imaging (fMRI) is a non-invasive tool increasingly used for mapping eloquent cortex in patients prior to brain surgery. The most common clinical application of fMRI is for functional mapping of basic language , 16 and Patient's scans are taken during interictal periods allowing for a neurocognitive assessment of the state prior to surgery. Our pre-surgical planning includes fMRI localization of eloquent cortex comprising spoken language, motor, attention, memory, emotional face and place processing and visual cortical excitability. Our fMRI battery not only provides a neurocognitive assessment useful for pre-surgical planning but also for basic research. Images are collected using a 3 Tesla Siemens Skyra MRI and have been summarized elsewhere . During Epilepsy surgery has become an accepted treatment option for children with medically refractory or treatment-resistant epilepsy . PediatrEn bloc resection techniques are used whenever possible to maximize preservation of brain tissue. Once the epileptic focus is identified as a target for resection, the margins of resection are planned along anatomic borders. If the target gyrus is surrounded by non-eloquent brain, trans gyral resection through neighboring gyri allows for en bloc removal of the seizure focus along with a margin of normal brain. The trans gyral approach has the additional advantage of tending to result in less bleeding compared to trans sulcal and intralesional approaches. If neighboring gyri are eloquent and need to be preserved, the target gyrus can be isolated and removed trans sulcally. Sub-pial dissection along gyral borders helps preserve the integrity of neighboring gyral cortex, as well as the any vasculature within the sulci ; a completed phase 3 trial with patients with TSC who have refractory partial-onset seizures . mTOR hyperactivation signaling in epilepsy makes it an efficient target for therapeutic intervention and have driven the significant effort to pharmacologically target this pathway  signaling Everolimus  and focal cortical dysplasia (FCD) , ethosuximide (a T-type Ca+ channel blocker), benzodiazepines , and felbamate/topiramate  are current therapeutic approaches for epilepsy , the most common form of epilepsy, do not response to current antiepileptic drugs (AEDs). The AEDs used for non-epileptic disorders, such as rapamycin, everolimus, celecoxib, bumetanide, amiloride, resveratrol, and losartan have been studied in animal models of epilepsy with good outcomes . Losartain vivo samples and autopsy cases have produced new attitudes toward neurological disorders. Well-documented autopsy material has been applied to study the human brain proteome in neurological diseases and in normal aging , therefore, it is crucial to have a readily available ante-mortem brain tissue bank to generate protein-protein interaction network map that most resembles the native interactions occurring in the healthy or diseased brains –61.In the brains of affected individuals, peptide and protein patterns are complex, and new molecular tools, such as MALDI-M, are needed to characterize these patterns and to identify specific proteins involved in neurological diseases such as epilepsy. MALDI-MS can be used to generate peptide and protein profiles of brain-tissue sections, and also enables the localization of neuropeptides and proteins in much smaller areas. These proteins at a high spatial resolution with markers of cellular components can document the localization patterns of molecular entities implicated in epileptic brain at the subcellular level –54. The In recent years, some studies have yielded substantial progress in the classification of genetic mutations involved in different forms of epilepsies –64. SomeThe development of novel neuroprotective therapies for patients suffering from pharmaco-resistant Temporal Lobe Epilepsy (TLE) is urgently needed; therefore, the human epilepsy brain bank will be utilized to confirm pre-clinical data gathered using animal models for TLE. Adenosine, an endogenous nucleoside that acts as a neuromodulator in the brain, plays an integral role in the pathophysiological etiology of TLE, where adenosine-based treatment strategies have proven to be efficacious. Increasing hippocampal adenosine signaling has been found to have anti-epileptiform actions through adenosine A1 receptors (A1Rs), inhibitory G protein coupled receptors that can induce profound synaptic depression . A1R actPrevious and ongoing investigations have demonstrated the involvement of adenosine A1 and A2A (A2AR) receptors in the pathophysiology of neurodegeneration in animal stroke models, which display post-stroke depression, cognitive dysfunction and increased post-stroke seizures . An impoIn preliminary studies, elucidation of the signal transduction pathways activated by A1R stimulation in the hippocampus showed evidence of modulation of the expression, localization, and function of ionic channels, including the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels  and the HERG potassium channels (Kv11.1). Importantly, key alterations in these channels have been shown to underlie some forms of epilepsy . These mMore than 50 million people worldwide suffer from epilepsy and about one third of these patients are considered resistant to pharmacotherapy, despite the availability of more than 20 anti-epileptic drug choices. In addition to incurring a higher drug cost to treat epilepsy, the consequence of drug resistant epilepsy in these patients is much more severe as judged by a four to seven-fold increase in mortality rates in this group compared to drug-responsive epileptic patients , 74, 75.In recent years, studies on epilepsy animal models and post-surgical TLE specimens resulted in the discovery of several novel therapeutic targets  and signaling pathways including the potential involvement of interleukin 1β, mammalian target of rapamycin (mTOR), nuclear erythroid-2-related factor 2 (Nrf2), sphingosine 1-phosphate receptors, and peroxisome proliferator-activated receptors (PPARs). Although these discoveries are promising and considered a great step forward in combating epilepsy, they are still far away from patients' medicine cabinets. More research and studies are needed to further confirm and validate previous observations as well as revealing as yet unidentified epilepsy-related signaling pathways and entities. Indeed, the epilepsy brain bank will be a pivotal establishment for this purpose.The main aim of this article is to help to stablish and spread the concept of an epilepsy brain bank. We believe that more institutions around the world should be interested in having epilepsy brain banks, especially in centers with active neurosurgical and epilepsy surgery programs. The epilepsy brain bank is also an opportunity for national and international collaborations in order to share tissue but also to share research ideas and technology to analyze the samples. Our article also explores the benefits of correlating clinical information of patients with the observations obtained from the human tissue. Once we have reached a large number of samples in our bank, we will work in the internationalization of our bank to collaborate with other centers.LH-R, JT-Z, and FM-A develop the idea of the epilepsy brain bank. JT-Z and LH-R are the holders of the bank in the University of Saskatchewan. HM collaborated to write the whole document. AW wrote the section related with surgical techniques. RA and VZ wrote the section on neuropathology. RB and MM wrote the section of imaging. RH wrote the section on pediatric neurology. MV wrote the section neuropsychology. FC and CT wrote the sections on physiology and pharmachology. AC reviews the whole document. All authors contributed to the article and approved the submitted version.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.


Brain Injury Awareness Month, recognized each March, provides an important opportunity to bring attention to the prevention of traumatic brain injury (TBI) and to promote strategies to improve the quality of life for persons living with TBI and their families.MMWR found a nationwide 17% increase in the rate of fall-related TBI deaths during 2008–2017, with increases in most states  initiative includes resources and tools for health care providers to improve identification of patients at risk for a fall, as well as effective strategies to reduce the risk for fall-related injuries, including TBI.Evidence-based prevention efforts to decrease falls are important to reducing the incidence and prevalence of TBI among older adults. CDC’s STEADI (Stopping Elderly Accidents, Deaths & Injuries;


This bias can occur when individuals identified as a result of the treatment group assignment are included in analyses.Pragmatic trials provide the opportunity to study the effectiveness of health interventions to improve care in real-world settings. However, use of open-cohort designs with patients becoming eligible after randomization and reliance on electronic health records (EHRs) to identify participants may lead to a form of selection bias referred to as To demonstrate the importance of identification bias and how it can be addressed, we consider a motivating case study, the PRimary care Opioid Use Disorders treatment (PROUD) Trial. PROUD is an ongoing pragmatic, cluster-randomized implementation trial in six health systems to evaluate a program for increasing medication treatment of opioid use disorders (OUDs). A main study objective is to evaluate whether the PROUD intervention decreases acute care utilization among patients with OUD (effectiveness aim). Identification bias is a particular concern, because OUD is underdiagnosed in the EHR at baseline, and because the intervention is expected to increase OUD diagnosis among current patients and attract new patients with OUD to the intervention site. We propose a framework for addressing this source of bias in the statistical design and analysis.The statistical design sought to balance the competing goals of fully capturing intervention effects and mitigating identification bias, while maximizing power. For the primary analysis of the effectiveness aim, identification bias was avoided by defining the study sample using pre-randomization data . To expand generalizability of study findings, secondary analyses were planned that also included patients newly diagnosed post-randomization, with analytic methods to account for identification bias.As more studies seek to leverage existing data sources, such as EHRs, to make clinical trials more affordable and generalizable and to apply novel open-cohort study designs, the potential for identification bias is likely to become increasingly common. This case study highlights how this bias can be addressed in the statistical study design and analysis.ClinicalTrials.gov, NCT03407638. Registered on 23 January 2018. Pragmatic clinical trials provide the opportunity to study the effectiveness of interventions in real-world medical settings , 2. Unliidentification bias [Yet pragmatic trials also introduce several methodological challenges –13. One ion bias –20 and rThis potential for bias is well-recognized in trial settings in which individuals are formally recruited to participate (i.e. recruitment bias ). HoweveIn this paper, we illustrate how identification bias poses a threat to valid inferences and propose methods for mitigating this source of bias in the statistical design of the PRimary care Opioid Use Disorders treatment (PROUD) Trial, an ongoing cluster-randomized, open-cohort, pragmatic implementation trial  to evaluAddiction to opioids has reached epidemic proportions in the United States, resulting in increases in drug overdose and death , 23. AltPROUD is a pragmatic, cluster-randomized implementation trial to evaluate a program of office-based addiction treatment of OUD in primary care settings. The study funds the health system to hire a nurse care manager (“the nurse”) who coordinates care with the site’s primary care team to provide evidence-based OUD medication treatment , 31. TheThe trial aims to evaluate whether the PROUD intervention successfully (1) increases evidence-based medication treatment for OUD  and (2) improves health outcomes among patients with OUD . To illustrate methods for addressing identification bias, the current case study focuses on this latter effectiveness aim, where the optimal approach to addressing identification bias was not clear. The effectiveness outcome is a patient-level measure of the number of days of acute care utilization , an adverse health outcome among patients with OUD.The study includes an open-cohort sample of patients with a primary care visit to one of the randomized sites during the period from three years before randomization (“baseline period”) until two years after randomization (“follow-up period”). However, as we will discuss below, the analytic sample for our effectiveness objective was restricted to a subset of this cohort.To model the patient-level number of days of acute care utilization over the follow-up period, the planned analysis is to apply a Poisson mixed-effect model with a random intercept for site that adjusts for pre-specified covariates known (or hypothesized) to be associated with acute care utilization, such as the baseline value of the outcome , 31.Because there is no patient contact for research purposes, eligibility criteria for identifying the population of patients to be included in trial analyses must be defined solely using automated data sources as discussed above. However, identifying the target population of interest (patients with OUD) is difficult, because OUD is underrecognized within general primary care populations and, in particular, is frequently not documented in the EHR see Fig. . Based oAdditionally, the PROUD intervention is expected to increase identification of OUD, both by increasing diagnosis of OUD among individuals who are already receiving primary care in the site due to nurse assessments, as well as by attracting individuals with OUD who are seeking OUD treatment into primary care who have not been seen previously at the intervention site Fig. . These lThe under-documentation of OUD in the EHR, together with the likelihood that the intervention increases documentation of OUD and attracts new patients with OUD to the site, makes defining eligibility criteria for inclusion in statistical analyses of PROUD trial outcomes challenging. Restricting analyses to individuals identified pre-randomization avoids the potential for identification bias at the cost of missing benefits to patients (i.e. decreased acute care utilization) that result from the intervention. In particular, such an approach would miss the benefits to new patients who are attracted by the nurse care manager program because of a lack of treatment options elsewhere, or patients who only disclosed their OUD symptoms to clinicians (who then documented this newly disclosed OUD in the EHR) after treatment was available. On the other hand, allowing individuals to be identified for inclusion in the sample post-randomization, as is often done in open-cohort designs, introduces the potential for estimates of intervention effects to be biased. Table An initial step in the statistical design for a particular study objective is to first determine whether the objective is susceptible to identification bias (Step 0). In order for identification bias to occur in a randomized trial, the randomly assigned intervention must affect who is identified for inclusion in the statistical analysis of the study outcome . For theStep 1. Identification of candidate analytic samples. Consider different options for the choice of analytic sample to be used for the primary analysis. Determine which options include patients identified using post-randomization data  and which rely solely on pre-randomization data (and therefore avoid identification bias).Step 2. Choice of unbiased analytic sample for the primary analysis. Conduct a power analysis to identify which of the unbiased, pre-randomization samples identified in Step 1 achieves the maximal power. Use this sample for the primary analysis. Details on the methods used in the PROUD case study are below.Step 3. Determine whether secondary analyses are needed to assess generalizability. If generalizability of results from using the analytic sample identified in Step 2 is of concern (e.g. for the reasons described above), alternate samples that may be affected by identification bias may be considered as secondary analyses.Step 4. Statistical analysis plan for secondary analyses in alternate analytic samples. Specify a statistical analysis plan for secondary analyses that include individuals identified after randomization that accounts for the potential for identification bias.To address the competing goals of fully capturing intervention effects with mitigating identification bias for the effectiveness objective, in the statistical design stage we considered several options for defining the study population to be used in the analysis. Hereafter, we will use the term “analytic sample” to refer to the population that would be included in an intent-to-treat statistical analysis for the effectiveness outcome. Our framework to address identification bias in the statistical study design then comprised the following steps.Here we describe the statistical methods used in our case study to select a primary analytic sample for the effectiveness objective that avoids identification bias by using pre-randomization data (Step 2). Because the goal of the power evaluation was to provide a general comparison of the operating characteristics of the different choices of analytic sample, we used a closed-form sample size formula based on Poisson regression  that didFor the formula-based power evaluation, we considered different scenarios that varied the true prevalence of OUD in the sites (parameterized by π) and we calculated power across a range of effect sizes for five different options for the analytic sample. Based on prior literature on the prevalence of OUD, which suggested that the true prevalence is approximately 1% in general US populations, with higher rates in certain subpopulations, study investigators hypothesized a likely range of values of 1%–4% to be used in the power evaluation . The five different options for the analytic sample corresponded to using the population of patients with a prior OUD diagnosis , weThe present case study highlights the potential impact of identification bias in pragmatic, cluster-randomized trials with open-cohort designs and how this source of bias is being addressed in a study using EHR data to evaluate the impact of a primary care OUD treatment program on reducing acute care utilization among patients with OUD. We addressed identification bias in the statistical design by selecting a primary analytic sample that used only pre-randomization data and we proposed secondary analyses that would allow individuals identified with OUD after randomization to be included, thereby capturing more of the potential effect of the intervention. Because such secondary analyses may be affected by identification bias, the proposed analytic approach uses regression-based methods to adjust for measured factors that could differ between patients newly identified in the intervention and control arms, along with a sensitivity analysis to investigate the potential impact of unmeasured confounders.In determining the analytic sample for the effectiveness aim using pre-randomization data, it was initially thought that identifying individuals at “increased risk” of OUD would have higher power relative to using the entire population of primary care patients or restricting to those patients with a prior diagnosis. However, the results of our evaluation suggested that this was not always the case. In particular, using the population of individuals with documented OUD at baseline had higher power under scenarios of low (1%) or moderate (2%) prevalence of true OUD compared to the other options considered. This finding demonstrates the importance of conducting preliminary analyses to evaluate different choices for the analytic sample. Prior information on the properties of algorithms used to identify the analytic sample , along While the framework considered in this paper prioritized selecting a population using pre-randomization data for the primary analysis, other studies have taken alternate approaches . One exaThis work has several limitations. First, because the PROUD Trial is still in the data collection phase, we are not yet able to evaluate how the choice of analytic sample would affect final inferences in the effectiveness analysis. This case study was therefore intended to highlight issues relating to identification bias for others designing pragmatic trials. Second, although the framework proposed here illustrates how to address identification bias within a given case study, there remain several gaps in knowledge on the optimal statistical design to address identification bias in open-cohort pragmatic trials more generally. Future work could evaluate the operating characteristics of these choices by conducting comprehensive simulation studies across a wide range of scenarios. Third, our proposed secondary analysis that includes patients newly identified with OUD after randomization uses just one of many possible analytic approaches to account for identification bias, regression adjustment. Principal stratification is an alternate approach that could be adopted in this setting . For exaIn the present paper, we proposed a framework for addressing identification bias in pragmatic cluster-randomized trials. In the study design, identification bias can be avoided by specifying the analytic sample using baseline (pre-randomization) data. In the analysis plan, methods can be applied to adjust for identification bias and alternate analytic samples that include participants who enter the study after randomization can be considered in sensitivity analyses. With more studies seeking to leverage existing data sources such as EHRs to make clinical trials more affordable and generalizable, the potential for identification bias is likely to become increasingly common in the future. The framework developed in this case study can serve as a model for any pragmatic trial seeking to mitigate this source of selection bias due to the intervention affecting identification of the population included in trial analyses.Additional file 1. Details of the power evaluation for selecting an unbiased analytic sample for the PROUD effectiveness analysis.


Significance: Speech processing tasks can be used to assess the integrity and health of many functional and structural aspects of the brain. Despite the potential merits of such behavioral tests as clinical assessment tools, however, the underlying neural substrates remain relatively unclear.Aim: We aimed to obtain a more in-depth portrait of hemispheric asymmetry during dichotic listening tasks at the level of the prefrontal cortex, where prior studies have reported inconsistent results.Approach: To avoid central confounds that limited previous studies, we used diffuse correlation spectroscopy to optically monitor cerebral blood flow (CBF) in the dorsolateral prefrontal cortex during dichotic listening tasks in human subjects.Results: We found that dichotic listening tasks elicited hemispheric asymmetries in both amplitude as well as kinetics. When listening task blocks were repeated, there was an accommodative reduction in the response amplitude of the left, but not the right hemisphere.Conclusions: These heretofore unobserved trends depict a more nuanced portrait of the functional asymmetry that has been observed previously. To our knowledge, these results additionally represent the first direct measurements of CBF during a speech processing task recommended by the American Speech-Language-Hearing Association for diagnosing auditory processing disorders. The auditory pathway alone includes both ascending and descending pathways that begin with sensory hair cells in the cochlea and ultimately project to prefrontal cortical regions. Thus, damage to or dysfunction of any aspect of the central nervous system often interferes with aspects of speech perception, such as comprehension in noisy environments.,,,,,One robust empirical characteristic of dichotic listening tests is that right-handed subjects consistently retain and repeat information presented to the right ear more accurately than the left. This phenomenon is termed a “right ear advantage” (REA).–At the level of prefrontal cortex, however, the left hemisphere dominance during dichotic listening tasks is not as apparent. In fact, imaging studies have found that dichotic listening tests can evoke comparatively larger activation in the right prefrontal cortex.,One approach for assessing the relevance of right prefrontal cortex to more general, modality-neutral performance optimization would be to monitor the time-course of regional functional activation during repeated behavioral tasks. Repetition on any behavioral task alters performance, an outcome that involves dynamic plasticity in myriad cognitive processes such as working memory management, attention, and information processing. Even at the level of concise sensory-evoked responses, repeated stimuli elicit progressively smaller responses only in regions closely involved with execution of that task or handling of that information.Dynamic aspects of functional activation during dichotic listening, however, have been challenging to acquire at high temporal resolution using magnetic resonance imaging (MRI)-based modalities because of technical limitations related to scanner noise. For example, in their functional MRI (fMRI) study on dichotic listening, Jancke and Shah found that the background level of noise was 70 to 80 dB sound pressure level (SPL),To avoid these systematic confounds, we monitored regional cerebral blood flow (CBF) during a repeated, free-recall dichotic digits listening task in healthy subjects using an optical technique, diffuse correlation spectroscopy (DCS). DCS takes advantage of the dynamic scattering of light from moving red blood cells to directly measure CBF and is particularly sensitive to flow in the cortical microvasculature due to the high absorption (and thus low probability of photon escape) in larger blood vessels.22.1Thirteen healthy, right-handed subjects  participated in this study, which was approved by the Institutional Review Board at New York Medical College and Westchester Medical Center. All methods were carried out in accordance with the relevant ethical guidelines and regulations of both institutions. Informed consent was obtained from all participants and/or their legal guardian/s prior to the measurement session.2.2Subjects wore insert earphones  and performed diotic and dichotic digits listening tasks. During dichotic digits task blocks , each eaDuring diotic digits task blocks, both ears were simultaneously presented with the same numbers in unison from a single randomly selected set of three numbers so that in total only three unique numbers were heard. Speaking can introduce mechanical artifacts in both CBF and EEG measurements. To ensure that there was not an uneven influence of mechanical artifact on diotic versus dichotic listening tests, subjects were instructed during the diotic tests to twice recite the three numbers they heard, e.g., “five one two, five one two.”Spoken numbers were selected and stimulus waveforms assembled using custom LabVIEW code  and output as analog waveforms through a USB-based DAQ unit . The voices used for constructing the stimulus were computer-generated male voices. Analog waveforms were amplified by a stereo power amplifier  and input to earphone amplifiers  that were coupled to insert earphones through plastic tubing. The average output at the earphones was slightly adjusted between subjects but was within 2.3Experimental sessions lasted The experiment followed a block paradigm and consisted of the following phases: (1) initial rest (2 min), diotic digits . Potential errors in the BFI calculation (semi-infinite homogenous model) due to a mismatch between the assumed and actual optical properties are minimized by utilizing the ratio between the BFI at each time point and that from the baseline period. We performed the fit to the normalized intensity autocorrelation function, 2.5EEG was recorded wirelessly using the 10-10 international electrode placement system . The headcap was fitted with wet electrodes  which were then filled with Sigma Gel . Channels with an impedance 2.6Subjects’ verbal responses were recorded with a monomicrophone . Acoustic artifacts and experimental pauses such as the silent periods between trials and rest periods were removed from the waveform using Audacity software (Audacity Team). The data were subsequently analyzed using custom Matlab functions. We quantified the duration of responses as the time elapsed between the first and last reported numbers. Specifically, vocal response duration was measured from the first to the last point in time of each trial where the absolute magnitude of the recorded waveform exceeded that of the ambient room noise.33.1,Supplemental Materials. Among subjects who performed a second dichotic listening block . We found that within this range of values, the absolute BFI could vary by as As with other diffuse optical techniques, hemodynamic calculations reported by DCS are dependent on the estimated tissue optical properties used in the physical modeling. The simplicity of the apparatus and measurement configuration in this study makes it attractive from the standpoint of clinical implementation; however, because a multispectral characterization of tissue optics was not integrated, errors in estimated absorption and scattering properties can potentially have an impact on the results.,,in vivo for profound physiological perturbation  are An additional consideration when characterizing task-related BFI changes is the potential influence of functional changes in tissue absorption and scattering properties. In terms of absorption, major physiological perturbations such as breath-holding or hyperventilation have been reported to elicit, at maximum, a ,–In terms of the clinical interpretation of the optical measurements reported here, as with other clinical measurement modalities such as fMRI, systemic responses during imaging experiments involving behavioral tasks are a ubiquitous confound. Relevant to this study, listening tasks that involve digit retention, such as the dichotic listening task, have been shown to elicit increases in heart rate of 5% to 10%.,Because the optical measurement technique we employed is relatively silent, inexpensive, and tolerant of head motions, a natural clinical implementation that our approach is relevant for is speech-language pathological assessment. The present study demonstrates the feasibility of using noninvasive optical measurements to monitor brain activity during listening task drawn from a battery of tests recommended by the American Speech-Language Hearing Association for diagnosing auditory processing disorders.On a broader, societal level, the absence of quantitative metrics for speech-language pathological disorders serves to preserve current inequalities in the assessment and treatment of children. Speech-language pathological assessment and treatment procedures are generally not reimbursed by health insurance policies because it is not quantitatively demonstrable that therapy influences specific neurological functions or deficits. This leads to a significant socioeconomic bias in the referral and intervention process. For example, children in private schools are twice as likely to be diagnosed, and the vast majority of those who receive therapy are Caucasian.5Assessing auditory processing disorders involves reducing the results of complex listening tasks into simple readouts such as accuracy. In efforts to establish quantitative diagnostic metrics that are more intimately related to specific neural structures, prior investigation has revealed hemispheric amplitude differences as one possible distinguishing parameter. Our findings identify additional parameters that can be used to classify normative responses, namely kinetics and asymmetric adaptation during repeated tasks. The variability of the laterality findings in the context of other studies still suggests that further investigation is needed before prefrontal cortical activity can be used to infer anatomical mechanisms underlying performance on clinical speech-language tests. Future measurements with the same apparatus and different sensory modalities may help elucidate the degree to which the asymmetries that we observed are behavioral task- or sensory modality-neutral.Click here for additional data file.


Daboia russelii), a priapism (painful erection) developed rapidly in a 16-year-old male and only subsided after administration of antivenom 3 hours later. Potential mechanisms for this snakebite-induced priapism are unclear but likely due to venom toxins causing nitric oxide (NO) release and subsequent vasodilation of endothelium in the corpus cavernosum, although the possible involvement of other mechanisms cannot be ruled out. We strongly believe that this unusual case report may lead to further scientific research in order to improve the clinical understanding of the pathophysiology of envenomation due to Russell’s viper bites. Although it is too early to speculate, further research may also discover the possibilities of developing venom-based candidate molecules to treat sexual dysfunction in males and females.Following a bite from a juvenile Russell’s viper ( Daboia russelii). The history of priapism following venomous bites is primarily limited to spiders, particularly, the Brazilian wandering spider (Phoneutria nigriventer), where injection of the venom in male mice caused a sustained priapism, frequently before death  are highly commercially viable with ongoing research for better alternatives. When NO is released from endothelial cells, as a result of sexual stimulation, it activates soluble guanylate cyclase and consequently causes an increase in cyclic guanosine monophosphate (cGMP), which relaxes smooth muscle, leading to vasodilation and increased blood flow: an erection. These treatments for ED protect cGMP from degradation by inhibiting PDE5 . Therefoin edema . Moreoveon rates  could pre venoms . Interes and KCa , hence t and KCa . The preUnusual cases, such as this, are starting points for unique research to determine the unprecedented mechanisms of venom toxins which may lead to better understanding of the envenomation pathology. At this stage, it is unclear what the causative agent is from Russell’s viper venom to induce priapism and what the evolutionary benefit of such a toxin or if it is a rare response from the patient’s own physiology. Indeed, priapism is known to be induced by various factors such as sickle cell disease in men , the lonThe data collection, consent form, and information sheets were approved by the Institutional Ethics Committee at Toxiven Biotech, Tamil Nadu, India (Reference number: ICMR-Toxiven Ethics 2019–001/002). A written consent was obtained from the victim’s parents (as the victim was a minor) to collect and publish the data presented in this article.


Promoting bone repair and regeneration in bone fractures by local delivery of growth factors possessing osteoinductive activity has been extensively investigated with significant advancements. Methods such as a combination of biomaterial-based scaffold and local bone active molecule delivery has been used . Thus, the targeted accretion in locally implanted particulate HAP is achieved by systemic drug administration, loading the biomaterial which is biologically activated  particles as a recruiting moiety for different drug classes administered systemically, showing that their affinity to HAP binding sites can activate the particulate material to exert a biological effect (Raina et al. 186Re for metastatic bone disease (de Klerk et al. Importantly, several issues need to be controlled to achieve successful results. First, the targeted delivery of drugs should have a specific and high affinity to HAP binding sites, where micro- to nanoparticles of HAP at the site acting as both a carrier and a recruiting moiety for systemically administered drugs. Examples are bone-seeking drugs like bisphosphonates for bone regeneration, HAP binding antibiotics like tetracycline for infection Perrin , and bonStaphylococcus aureus for example is a causative agent of osteomyelitis and has a high affinity for bone, and can induce osteonecrosis and resorption of bone matrix (Lucke et al. The current promising results using HAP as a recruiting and reloadable particulate apatite moiety to which systemically administered drugs circulating in the bloodstream could bind due to a high chemical affinity provides a novel method for treatment of various bone diseases. Apart from its promise for bone regeneration, it is also potentially applicable in the treatment of bone infections, tumors, and osteoporosis. Since HAP is already abundant in bone, implantation of HAP would not necessarily be required. Several bone mineral seekers are known apart from bisphosphonates, tetracycline, and fluorine—certain peptides for example (Rotman et al.