Vocal learning is a complex acquired social behavior that has been found only in very few animals. The process of animal vocal learning requires the participation of sensorimotor function. By accepting external auditory input and cooperating with repeated vocal imitation practice, a stable pattern of vocal information output is eventually formed. In parallel evolutionary branches, humans and songbirds share striking similarities in vocal learning behavior. For example, their vocal learning processes involve auditory feedback, complex syntactic structures, and sensitive periods. At the same time, they have evolved the hierarchical structure of special forebrain regions related to vocal motor control and vocal learning, which are organized and closely associated to the auditory cortex. By comparing the location, function, genome, and transcriptome of vocal learning-related brain regions, it was confirmed that songbird singing and human language-related neural control pathways have certain analogy. These common characteristics make songbirds an ideal animal model for studying the neural mechanisms of vocal learning behavior. The neural process of human language learning may be explained through similar neural mechanisms, and it can provide important insights for the treatment of language disorders. Vocal learning is a rare animal behavior that learns to replicate conspecific or heterologous sounds or even artificial sounds through a process of repetitive neural activity from auditory to vocal motor . The rolAlthough mammals and birds evolved from different sources, there is growing evidence that the vocal learning processes of the two species are highly similar . A compavia neural projections to regulate brainstem vocal motor nuclei. Two parallel neural pathways are responsible for forebrain regulation of vocalization: the limbic vocal control pathway controls innate non-verbal and emotional vocalization; the laryngeal motor cortical pathway regulates fine motor control of voluntary vocalization, such as speaking and singing, as well as the spontaneous production of innate vocalization  without the involvement of auditory feedback. All vocal vertebrates, from fish, amphibians, reptiles, birds to mammals, including humans, share similar brainstem vocal control pathways . The othlization .Heterocephalus glaber) from different regions can produce sounds with unique group information, similar to dialects , juveniles learn the songs of their relatives during the sensitive period and gradually develop their own personalized and lifelong repertoire (Lonchura striata domestica), has the ability to control the ordering of syllables, which is similar to human control of the syntactic structure of language (Biziura lobata), a member of the Anseriformes family, also have vocal learning behavior, which provides more diversified information for deciphering the evolution of human language  plays a central role in this pathway, as evidenced by the fact that damage to PAG results in complete vocal inability in cats, monkeys and humans . PAG recIn order to combine individual articulation into sentences, human speech requires the involvement of the laryngeal motor cortical pathway. The human laryngeal motor cortex (LMC), located ventral to the primary motor cortex, is responsible for fine motor control of voluntary vocalization such as speech and singing, as well as regulating the spontaneous production of innate vocalization . NaturalThe laryngeal motor cortical pathway is directed from dLMC/vLMC to Am, which coordinates laryngeal muscle movements and respiratory rhythm to precisely control vocalization . dLMC/vLIn primates, another connection between LMC and the limbic vocal control pathway exists at the brainstem reticular formation (RF) , particuIn addition to the vocal motor pathway (VMP), human language learning, including the memory of vocabulary and grammar, relies on an additional forebrain pathway, the cortex-striatum-thalamus loop, consisting mainly of motor language center Broca’s area , the anterior striatum (ASt) and the anterior thalamus speech area (aT) , and thiVoluntary production of words and sentences through the motor cortex requires a large amount of memory and involves the activity of a large number of neurons, many of which are located in the Broca’s area . Broca’sMeanwhile, as a core component of human motor skill learning, ASt receives signal input from Broca’s area and remains activated for learning new words during the process of learning mother tongue in early childhood and second language in adulthood, indicating that ASt plays a key role in the process of language learning and memory . And theIt is traditionally believed that the superior temporal regional cortex (the sensory language center Wernicke’s area and its surrounding areas) is involved in the perception and memory of speech . AlthougThe differences between human and other primates in the language system are not only in the function of brain regions, but also in the location of the vocal organ, i.e., the larynx. In early childhood, the position of the larynx in humans is not much different from that of chimpanzees, but the human larynx rapidly descends to neck in early juvenile life. The descending position of the larynx contributes to the development of the human respiratory and digestive tracts and the formation of language function . HoweverThe instinctive vocalization of all birds relies on a mammalian-like brainstem vocal control pathway, from the dorsomedial nucleus of the intercollicular complex (DM) in the midbrain to the tracheosyringeal part of hypoglossal nucleus (nXIIts) in the brainstem , similarZonotrichia leucophrys) was first described by Serinus canaria).Comparative studies of gene expression pattern of adult animals and molecular embryology indicate that avian and mammalian brains share an analogous cortex-basal ganglia-thalamus-cortex circuit associated with vocal learning behavior . It is sThe accurate song of songbirds depends on the regulation of VMP, which consists of the song premotor nucleus HVC (used as a proper name) and the robust nucleus of the arcopallium (RA) in the telencephalon and nXIIts in the brainstem . HVC is + current inactivation kinetics, and large resurgent Na+ currents , the avian basal ganglia area X and the medial portion of the dorsolateral nucleus of the anterior thalamus (DLM) to form the cortex-basal ganglia-thalamus circuit . AFP is Area X is a unique region in the basal ganglia of songbirds that is critical to song learning, which receives afferents from both LMAN of AFP and HVC of VMP, and is analogous to the mammalian striatum . All majCortical nucleus LMAN receives afferents from DLM and is the output nucleus of AFP that projects to RA of VMP , which iDLM receives afferents from area X, and is analogous to the intralaminar nuclei of the mammalian thalamus , which iIn summary, the brainstem vocal control pathways that control innate vocalizations exist in almost every species of mammals and birds, but only a few species, including humans and some birds, possess vocal learning abilities and related neural pathways. Accidental discoveries of individual cases of animals imitating human speech, such as elephants, seals, and parrots, suggest that there may be other species’ vocal learning behaviors that have not yet been deciphered. Meanwhile, the differences of complex vocal behaviors and their neural control between mammals and birds predict that the formation of vocal learning behaviors in different species may have multiple independent origins . In receThe ethology of songbird song learning has been studied for decades from its inception to its interdisciplinary study with neurobiology. The anatomical structure of songbird song control system and its role in regulating song behavior have been comprehensively understood . The effMore challengingly, how vocal learning changes with time and experience in different ages, the patterns of activity and association of relevant brain regions, and how auditory feedback plays a role are key issues that both human language and bird song research fields share and need to address . HoweverYZ: investigation and writing—original draft. LZ: investigation. JZ: visualization. SW: funding acquisition. WM: conceptualization, validation, supervision, funding acquisition, and writing—review and editing. All authors contributed to the article and approved the submitted version.


The development of bio-based materials is of great importance in the present environmental circumstances; hence, research has greatly advanced in the valorization of lignin from lignocellulosic wastes. Lignin is a natural polymer with a crosslinked structure, valuable antiradical activity, unique thermal- and UV-absorption properties, and biodegradability, which justify its use in several prospective and useful application sectors. The active functionalities of lignin promote its use as a valuable material to be adopted in the composite and nanocomposites arenas, being useful and suitable for consideration both for the synthesis of matrices and as a nanofiller. The aim of this review is to summarize, after a brief introduction on the need for alternative green solutions to petroleum-based plastics, the synthesis methods for bio-based and/or biodegradable thermoplastic and thermosetting nanocomposites, along with the application of lignin nanoparticles in all green polymeric matrices, thus generating responsiveness towards the sustainable use of this valuable product in the environment. The rising global demand for fossil resources for nonenergy purposes, as in the case of plastics production, has intensely motivated research to find alternative solutions to petrochemical plastics; however, progress has still not reached a commercially viable scale. The demand for cost-effective, ecofriendly materials has also increased to reduce waste management and pollution issues, thus academic/industry interest in sustainable bio-based materials has accordingly exploded in recent years. Even if numerous synthetic biopolymers have been used to this purpose, the need to suit different applications has allowed for progress even in the field of natural biopolymers. According to this, significant progress in lignin valorization and the use of sustainable and natural resources has been accomplished in the last years, in particular with regard to bio-based and biodegradable natural polymers based on this source, which are facing increasing consideration as they are environmentally green and economically reasonable. Replacing petroleum-based-derived materials with sustainable and environmentally friendly materials has been also considered as a crucial activity in the current period, so consideration has been given to the progress of lignin bio-based and/or biodegradable materials with thermomechanical performance that can compete or even surpass the petroleum-based products presently used. Lignin is considered an excellent substitute feedstock for the preparation of chemical products and polymers, even if one of the main difficulties still remaining is the lack of a well-defined structure and the partial flexibility linked to its origin, including extraction fragmentation procedures . AlthougFollowing its fast development, efforts have incessantly been made to advance its compatibility with other additives in multicomponent systems, as in the case of its mixing with thermoplastics and thermosetting green matrices . On the Lignin is characterized by a complicated three-dimensional structure, obtained by polymerizing three phenylpropane units that arise from three aromatic alcohols: p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol a 5]..5].Its complexity can be controlled by accurate deconstruction, so the synthesis of polymers starting from aromatic compounds ideally obtained from lignin has attracted a growing curiosity. A large quantity of polymers obtained from lignin derivatives originate from different products b, gainedDifferent approaches can be considered to realize high-performance polymers from lignin. The different adopted depolymerization methods have great prospective to get several monomer precursors, e.g., as alcohols, aldehydes, and acids, from lignin a. VanillThe use of valorized lignin increases the potential to realize biodegradable biopolymers, such as polyhydroxyalkanoates, polyhydroxybutyrates, polylactic acid, or nonbiodegradable matrices, as in the case of polyurethane, polyolefins, polyamides, etc.  Figure .Oceanimonas doudoroffii (PHA from sinapinic acid and syringic acid), Cupriavidus basilensis B-8, Pseudomonas putida A514, and Pseudomonas putida KT2440. The produced PHA can be adapted to convert into varied chemical precursors, such as alkenoic acids and hydrocarbons, which indicate that lignin can be adapted to become fuel-range hydrocarbons, chemical precursors, and biomaterials [The first case of polymerized vanillic acid to make polyesters was defined in 1955 . Carboxyaterials .Lignin has been also explored as a natural resource for the synthesis of polyurethanes because it retains hydroxyl groups on its surface . It fitsMeng et al. studied a different method that utilizes lignin extracted from the cosolvent lignocellulosic fractionation (CELF) of poplar wood to realize bio-NIPUs . In thisAccording to this, the mechanical behavior of NIPUs based on lignin can be definitely varied from rigid to elastic by basically changing the lignin constituents of the polymeric material. The thermal characteristics of NIPUs were enhanced thanks to the addition of aminated lignin, and NIPU containing 55 and 23 wt% of lignin exhibited a high elongation at break (~140%) and tensile strength (~1.2 MPa), respectively. The obtained experimental results reveal that the reaction of cyclic carbonate with aminated lignin can be considered as a significant strategy for the synthesis of lignin-based NIPU with a relatively high lignin amount . CopolymGraft copolymerization, or in general, derivatization reactions, can generate new lignin containing thermoplastics by incorporating technical lignin and synthetic material. By appropriately choosing low-glass-transition temperature chemistries, lignin derivatives with variable thermomechanical properties can be obtained. On the other hand, the new progress on controlled/living polymerization and specific and effective synthetic techniques  propose new pathways for the design and realization of high-performance thermoplastic materials based on lignin derivatives having functional properties . Even wiThermosetting polymers can be produced from many bio-based resources, as in the case of vegetable oils, lactic acid, and citric acid, giving polymeric materials with more than 90% of a renewable amount . VariousTwo approaches are currently considered to obtain lignin-based thermosets. The first method utilizes lignin itself or incorporates lignin with other components to synthesize copolymers, with the key restriction that the reactivity of bulk lignin is lower than that of monomers. To overcome these restrictions, another method was developed that uses aromatic molecules obtained directly from lignin depolymerization. This approach enables molecular design and structural modification and can increase the performance of the resulting polymeric materials, as in the case, for example, of vanillin and other derivatives . The maiCompatibility and reactivity of lignin with compounds can be enhanced by means of chemical changes . Nevertheless, steric-hindrance influence and the limited compatibility of lignin-based epoxy resin weakened the crosslinking density, deteriorating the thermomechanical behavior of the thermosetting polymeric materials. Consequently, limitations such as the complex process, limited effect, low-lignin loading, and waste liquid recovery are unavoidable and still need to be solved. In lignin-modified phenolic resins, lignin is considered as the phenol able to react with formaldehyde in basic conditions or as an aldehyde to react with phenol in acidic conditions. Nevertheless, the replaced amount of phenol is partial due to scarce reactive sites and steric hindrance in lignin. To overcome these restrictions, lignin-derived phenols have been exploited to produce phenolic resins  Figure b. The prLignin nanoparticles have received much attention in the last years concerning the effort to utilize and apply lignin into more valued sectors . In ordeXanthomonas axonopodis pv. vesicatoria and Xanthomonas arboricola pv. pruni Gram-negative bacteria over time, to positively influence the innovation, and to induce a positive effect against hazardous bacterial plant pathogens. The disintegration test under composting conditions revealed that the tested formulations reach a value up to 90% after 15 days; however, the presence of LNPs did not affect the disintegrability of different films, as shown in Xanthomonas axonopodis pv. vesicatoria and Xanthomonas arboricola pv. pruni Gram-negative bacteria over time, to positively influence the innovation, and to induce a positive effect against dangerous bacterial plant pathogens. The disintegration test under composting conditions revealed that the tested formulations reach a value up to 90% after 15 days; however, the presence of LNPs did not affect the disintegrability of different films, as shown in The use of nanolignin as a reinforcing phase in macromolecules  is a key methodology to advance in the realization of sustainable polymeric composite systems. Recently, lignin was utilized as a nanoscaled reinforcement to improve the structural characteristics of polysaccharides, proteins, natural rubber, and synthetic polymeric matrices ,45. In tChollet and coauthors considered the nanolignin as a new additive for flame-retardancy of poly (lactic acid) . Lignin 3, Ag2O, Fe2O3, TiO2, and ZnFe2O4 [2O4 particles exhibited the best antioxidant behavior. Radical scavenging activity was also observed in ternary-based nanocomposites, where lignin and metal oxide nanofillers operated together synergically to boost the functional properties . Lizund ZnFe2O4   copolymer. The toughness also rose 1.5 times above the PLA/PCL. Similar results were observed for the crystallization values and UV protection. Nanolignin–PLA/PCL-based systems can be utilized in the food packaging industry as an impact-resistant and UV-protectant material.The central role of lignin nanoparticles as UV barrier filler was also investigated by Yang and coauthors . They coMicrococcus luteus and Gram-negative Escherichia coli bacteria) behavior to the PLA films, and a higher consequence was indeed found when increasing the filler content. Acetylation (aLNP) of the fillers moderately limited the antioxidant characteristics and the UV protection of the obtained composite systems, but it affected positively the nanoparticles distribution and aggregation, improving ductility and aesthetic quality of the films by decreasing at the same time the characteristic dark color of the lignin. Migration tests and disintegration test realized in simulated composting conditions of the nanocomposites showed that, irrespectively of their system, the realized active nanocomposites behaved likewise to neat PLA.Cavallo et al. suggested the use of polylactic acid (PLA) films containing 1 wt% and 3 wt% of lignin nanoparticles (pristine (LNP) chemically modified with citric acid (caLNP) and acetylated (aLNP)). The different polymeric films were produced by extrusion and filming, and after that, the formulations were analyzed by determining the overall performance needed for the food packaging sector . The obtThe use of LNPs was proposed as a valid possibility to develop promising wound dressing. Pahlevanneshan and coauthors  proposedLignin can be blended, considered as a filler in a composite/nanocomposite formulation, both in its native form or chemically modified, combined in the presence of particular additives: in all cases, it has been proved that lignin can beneficially improve the overall performance of the resulting polymers. While the literature reports numerous examples of LNPs in thermosets ,54,55, lA new approach was adopted to realize lignin phenol formaldehyde (LPF) resin: in the paper of Chen et al. , the preIn a quite recent paper , a simplAnother example of lignin nanoparticle exploitation in a novel manner is represented by the study reported by , where tResearchers from Washington State University, part of the NSF-supported Industry–University Cooperative Research Center for Bioplastics and Biocomposites (CB2), considered the use of a deep eutectic solvent to extract oligomeric lignin (nanoDESL) from plant biomass at a high yield and also nanosized . NanoDESAll these studies provide awareness on potentialities of lignin nanosized fractions and allows for the design of reproducible and foreseeable material characteristics. It is essential to know these characteristics if we would exploit lignin as a raw material for a sustainable and innovative design. These preliminary and updated works confirm that nanolignin, if combined with green thermosetting matrices, can give fully green nanocomposites and, if effective, multifunctionality is often achieved in the presence of this nanoscaled filler.This review, divided into two main sections, firstly provided an overview of preparation and applications of green thermoplastic and thermosetting nanocomposites based on lignin, and thereby a glance to the use of nanolignin in biopolymeric nanocomposites was also considered. Even if various structures and different properties of lignin at the nanoscale effectively can be challenging and interesting from the research point of view, the preparation and application of nanolignin-based green composites in high-value sectors are still in their infancy. Limiting factors include the achievement of uniform dispersion, and, additionally, the morphology, size, and chemistry of lignin nanoparticles, which need to be the prerequisites for the high-value-added and multifield applications of lignin. The structural and functional properties of lignin are the key points for its conversion into aromatics, polymers, and high-performance materials. Regarding this issue, we should emphasize that the production of thermoplastics or thermosetting polymers from the depolymerized lignin still involves the use of several chemicals, often causing a high environmental impact for the synthesis of bio-based and/or biodegradable polymers. Consequently, methods to chemically functionalize lignin to useful products without the use of expensive reagents or complicated synthetic routes must still be identified, with the main aim of competing with commercial commodity polymeric materials. The success of synthesizing thermoplastics and thermosetting from lignin opens up, on the other hand, new avenues to incorporate lignin as a component of value-added polymers while utilizing renewable resources. To balance the negative impact of chemically treated lignin and lignin derivatives to produce bio-based matrices, the use of lignin nanoparticles (obtained applying green process) in lignin-derived polymeric nanocomposites can be considered as a valid strategy to guarantee multifunctionality in sustainable biopolymeric matrices.


Brain metastases are amongst the most invasive and lethal cancers. Yet, effective methods for their diagnosis and therapy have remained elusive. While several factors are responsible for this, the primary hurdle has been the absence of an appropriate delivery strategy for the diagnostic/therapeutic agents to the brain. Fortunately, the advent of nanobiotechnology and the advances in bioengineering techniques have provided some hope to circumvent this hurdle. The aim of this review is to evaluate the suitability of novel smart nanobiotechnological approaches in brain cancer therapy. We discuss several state-of-the-art strategies for specific delivery of drugs to brain cancer sites with minimal side effects. We further delineate several classes of nanoplatforms that have generated tremendous interest among contemporary scientists as potential agents for brain cancer therapy. In doing so, the authors hope that the review will serve as a platform for further studies for the discovery of brain cancer strategies.Despite their low prevalence, brain tumors are among the most lethal cancers. They are extremely difficult to diagnose, monitor and treat. Conventional anti-cancer strategies such as radio- and chemotherapy have largely failed, and to date, the development of even a single effective therapeutic strategy against central nervous system (CNS) tumors has remained elusive. There are several factors responsible for this. Brain cancers are a heterogeneous group of diseases with variable origins, biochemical properties and degrees of invasiveness. High-grade gliomas are amongst the most metastatic and invasive cancers, which is another reason for therapeutic failure in their case. Moreover, crossing the blood brain and the blood brain tumor barriers has been a significant hindrance in the development of efficient CNS therapeutics. Cancer nanomedicine, which encompasses the application of nanotechnology for diagnosis, monitoring and therapy of cancers, is a rapidly evolving field of translational medicine. Nanoformulations, because of their extreme versatility and manipulative potential, are emerging candidates for tumor targeting, penetration and treatment in the brain. Moreover, suitable nanocarriers can be commissioned for theranostics, a combinatorial personalized approach for simultaneous imaging and therapy. This review first details the recent advances in novel bioengineering techniques that provide promising avenues for circumventing the hurdles of delivering the diagnostic/therapeutic agent to the CNS. The authors then describe in detail the tremendous potential of utilizing nanotechnology, particularly nano-theranostics for brain cancer imaging and therapy, and outline the different categories of recently developed next-generation smart nanoformulations that have exceptional potential for making a breakthrough in clinical neuro-oncology therapeutics. Brain disorders are among the most prevalent of disorders, yet strategies for their diagnosis and treatment remain mediocre at best. The global surge in drug development for neuropathologies has grown rapidly with the increase in the elderly population and the number of confirmed clinical cases. Intriguingly, drug development for diseases of the central nervous system (CNS) has a very poor success rate. The failures of clinical trials of the potential drugs/agents are largely attributed to the complexity of the brain and the multifactorial nature of its disorders, undesirable side effects and the highly selective nature of the blood brain barrier (BBB). The latter has in particular been a major hindrance, since only low molecular weight lipophilic molecules can effectively cross the BBB, rendering the vast majority of potentially neuroprotective molecules inefficient .Among the diverse cancer types, brain cancers have one of the lowest incidences; however, they are amongst the most invasive and with the highest mortality rates . In addiThe standard clinical therapy in malignant brain tumors is notoriously complicated and involves maximal surgical resection in combination with high-dose radiotherapy and chemotherapy; however, the median survival time and recurrence time is still very small ,10. In aThe ever-evolving field of nanotechnology has opened new avenues for the study and regulation of biological systems at the nanoscale level by mimicking or manipulating endogenous cellular mechanisms. In particular, because of their versatilities and unique potential for manipulation/functionalization, smart nanomedicines hold tremendous promise for theranostic diagnosis and management of brain tumors ,17. The Nevertheless, while NPs have been in clinical use for other cancer types, their applications in neuro-oncology have been somewhat delayed. The blood brain barrier is the most important physiological component maintaining brain homeostasis. The BBB is a highly selective barrier lining the blood vessels in the CNS, acting as the first physical barrier against the potential toxic effects of both endogenous and exogenous chemicals, as well as in the regulation of the movement of essential nutrients and metabolites between the systemic circulatory system and the brain cells. Its protective role against the toxicity of molecules to the CNS and its formidable physico-chemical and electrostatic barriers in therapeutic medicine research are well-known. Indeed, BBB-mediated regulation of the transport of both endogenous and exogenous chemicals between the blood and the brain poses enormous challenges to neuro-therapeutic and diagnostic agents ,25.The BBB is comprised of highly specialized endothelial cells present in the brain capillaries, brain microvascular endothelial cells (BMECs) and other perivascular cells  that surround the BMECs. Each cellular component of the BBB has specialized functions ,27. WhilApart from small molecular compounds, such as water, and gaseous molecules, such as carbon dioxide, which passively diffuse across the endothelial cells of the BBB, other metabolites have to employ specialized transporters to gain access through the BBB ,30. PassSeveral studies have indicated that neuropathologies can alter the structure, organization and function of the BBB ,28,32. TThe BBTB is itself disrupted with the progression of the brain tumor leading to a leaky vasculature and resulting in the pathophysiological phenomenon of EPR, which is characterized by the formation of gaps in the BMECs . This leOf particular note, other endogenous mechanisms associated with cancer pathology may also be targeted for delivery of NPs to the tumor environment. For example, tumor-associated macrophages have been recently proposed as an alternate and effective strategy for drug delivery to cancerous cells . HoweverSpecific targeting of the drug to cancerous cells is a hurdle that is common to all types of cancers. The challenge for any systemic or localized treatment here is to limit the damage to noncancerous healthy tissue. In this regard, NPs have prominent advantages as they can be composed in a versatile manner with inclusion of biochemical moieties that enable specific recognition of cancerous cells  . One or As stated, when it comes to brain cancers, conventional therapeutic options offer modest therapeutic benefits, poor tendencies to delay progression/recurrence and enhanced side-effects. In this regard, nanotechnology is increasing being perceived as useful alternatives for both the imaging and therapy of brain cancers. Recent advances in nanotechnology and bioengineering have accelerated the use of NPs in the treatment of cancers and other brain pathologies, such as neurodegeneration ,19,51,52The superiority of nanomedicines for oncological applications relies on the ability to finely calibrate the physico-chemical properties of the NPs: size, shape, surface charge and brain tumor targeting . In partOne of the major theranostic advantages of nanomedicine in cancer therapy is that it is not limited to chemotherapy. Indeed, with the advent of newer tools and techniques and advancements in biological science research, some novel therapeutic strategies have arisen, including immunotherapy, gene therapy, oncolytic virotherapy and protein therapy (reviewed in ), which Lastly, because of the versatility of the inherent physico-chemical properties as well as the unlimited prospects for manipulative bioengineering, nanomedicinal formulations have tremendous potential in drug-free therapeutic approaches . In partIn conclusion, while it is clear that recent advances in smart bioengineering of nanoplatforms have provided huge promises for therapeutic, diagnostic and theranostic successes in clinical neuro-oncology, further research is warranted both for optimization of the strategies and for their comprehensive evaluation in clinical settings.As discussed, tumor targeting to CNS sites bypassing the BBB/BBTB barriers at the required therapeutic concentrations, while reducing non-specific effects on the peripheral and non-tumor cells, is the major challenge in neuro-oncology. There are several potential techniques for bypassing the BBB for delivery of NPs at the tumor site in the CNS .The intracranial pathway involves the direct and localized delivery of the drug/imaging agent to the tumor site . SevInfusion of drugs in the intrathecal space  either in the Ommaya reservoir or directly into the CSF in the spinal column has been a known alternative for bypassing the BBB . While tThe intranasal mode of drug delivery is made possible by the unique and direct nose-to-brain route through the olfactory area of the nasal sub-mucosa to the CSF, thus bypassing the BBB . Brain dIt should be noted, however, that this technique of delivery is still relatively nascent, and as such more studies are warrantied for further evaluation of its suitability for neuro-oncological purposes. For example, the intranasal route of drug delivery may be limited by the dosing volume through the nasal cavity and spillage (and hence the amount of drug entering the CNS). Moreover, the accumulation and curative activity of drugs delivered through the intranasal route are critically dependent on the location of the tumor tissue in the CNS. In this regard, several aspects of intranasal delivery can be optimized by nanobiotechnological strategies, including minimizing clearance/degradation by mucosal enzymes, increasing target-specificity , and finer control of the spatiotemporal aspects of release, for example, by endogenous (pH and reactive oxygen species) or external (magnetic field and ultrasound) stimuli.Since the brain is one of the most highly perfused organs with a dense vascular network, minimally invasive systemic delivery of therapeutics can be a realistic option if appropriate strategies are employed to bypass the BBB/BBTB barriers and to reduce non-specific peripheral distribution. Because of their versatility and manipulative potential, NPs are tremendous prospective candidates in this respect. In fact, the systemic delivery of NPs remains the most researched route for brain cancer therapies, and several possible strategies have been proposed to increase their efficiency for brain tumor targeting.Systemic intravenous administration of NP functionalization with suitable targeting ligands can aid in their BBB/BBTB penetration, exploiting both the transcellular and the paracellular pathways ,93. A loIn addition to the transferrin receptor ligands/antibodies, ligands against LDL receptors on endothelial membranes can also be harnessed to facilitate the CNS uptake of bioengineered functionalized NPs. Indeed, examples of the ligands for LDL receptors coated on NPs abound in the literature . Accordingly, NPs functionalized with the cyclic RGD peptides offer promising alternative targeting strategies for therapeutic agents in glioma [Integrins, extracellular matrix (ECM) transmembrane receptors associated with tumor progression, invasion and neovasculation, constitute an interesting target of receptor-mediated transcytosis utilizing the pathological EPR effect. In particular, ligand-targeting strategies for αications . It is in glioma ,107.Lastly, examples of adsorptive transcytosis-mediated drug entry utilizing conjugation with cationic proteins or cell-penetrating peptides are also known in the literature . The adsTo address the non-uniformity of the tumor vasculature  and hencCa) channels [An interesting smart strategy is the use of focused ultrasound (FUS) for temporary disruption of the BBB and hence convenient delivery of NPs to gliomas ,116. Thichannels  and the channels  have alschannels ,121,122.channels . Intereschannels . In factchannels .Another novel technique for transient BBB/BBTB disruption and facilitation of drug delivery in the brain is the transcranial magnetic stimulation and consequent NMDAR-mediated neuronal activation ; howeverThe ability of some cell-types to migrate towards malignant tumor cells in the brain without attracting immunological responses can provide an interesting option for drug delivery by NPs. The various cells that can be employed for this purpose include neural stem cells (NSCs) , bone maIt is clear that there has been an exponential increase in the development of potentially successful strategies to allow penetration through the BBB/BBTB, raising hopes for effective brain drug delivery pathways in clinical settings. In this regard, nanocarriers have emerged as the most suitable candidates for mediating the delivery of diagnostic, therapeutic, theranostic agents . RegrettIn recent decades, several kinds of NPs have been studied in in vivo and in vitro models of cancers. For example, the veteran liposomal nanoformulations have been widely used in non-CNS clinical practice since their inception in 1995. These nanoformulations have excellent credibility as safe and versatile agents for the encapsulation of various drugs (both lipophilic and hydrophilic), and of a wide range of molecular weights. Moreover, the drugs are not required to be chemically modified for encapsulation ,143. UsePolymeric NPs are a class of nanocarriers composed of natural or synthetic polymers. Their main advantage lies in their inherent biocompatible, non-immunogenic and biodegradable characteristics and the ability to entrap hydrophobic drugs in the hydrophobic core, protecting them against endogenous degradative and clearance mechanisms. Though they have been long thought to harbor the potential for therapy of brain cancers, significant challenges must be identified and addressed .In the neuro-oncological perspective, carbohydrate-based polymeric NPs have been proposed as platforms with significantly improved brain delivery agents. In addition to the ease and cost-efficiency of their preparation and their inherently biodegradable and biocompatible nature, the ability to functionalize carbohydrate polymeric NPs with diverse groups of ligands offers multiple pathways of therapies and imaging strategies for brain cancers . In partDendrimers  are a novel class of synthetic polymeric smart nanoformulations that have recently garnered immense interest for neuro-oncology applications. These highly branched polymers with modifiable surface functionalities and available internal cavities with a high loading capacity are emerging candidates as nanocarriers with immense potential for a directed theranostic approach to brain cancer imaging and therapy ,158. OwiSimilar to natural carbohydrates, protein-based NPs offer a convenient-to-formulate, low-cost, biocompatible and biodegradable pathway of drug delivery to the brain. Moreover, they harbor immense potential for functionalization by a wide range of ligands and therapeutic/imaging agents for a targeted approach in brain cancer treatment. An appropriate example is the transferrin-based NPs that have elicited tremendous abilities to cross the BBB/BBTB by targeting the transferrin receptor-mediated transcytosis see also  for braiOne of the most widely used protein-based nanoformulations in the therapeutic studies of brain cancers involves the use of serum albumin, possibly due to the high energy/nutrient requirement-induced overexpression of albumin binding proteins  on cancerous cells ,171. CatGold-based nanoconjugates synthesized on Au cores come in different shapes and have some unique properties that can be harnessed for brain cancer treatment. In addition to their optimal size, Au NPs are biocompatible and can be conjugated with appropriate cell-targeting ligands ,177 and 2O3 core were found to be effectively delivered to the brain of a non-human primate with little toxicity [The magnetic and electric properties of the MENPs (such as those employing gadolinium and iron oxide cores) aid in specific targeting to the tumor site and can also be utilized for magnetic resonance imaging (MRI) traceability . Furthertoxicity . In additoxicity ,196.3O4–Au composite magnetic NPs were used for a combinatorial magneto-photothermal (magnetic fluid hyperthermia and near infrared hyperthermia) therapeutic approach against glioma cells [In particular, the unique ability of MENPs for localized hyperthermia therapy harbors huge implications for brain cancer theranostics . In ma cells . Nanoengma cells . Aminosima cells ,199.Another interesting class of smart magnetic NPs that could potentially prove to be beneficial in neuro-oncology research are what are known as the magnetosomes, which are membranous structures derived from magnetotactic bacteria containing iron-rich magnetic NPs. They are a particularly relevant alternative to traditionally synthesized iron oxide magnetic NPs with regards to their increased biocompatibility while maintaining the hyperthermia and tracing properties ,201. MorQuantum dots are luminescent/fluorescent semi-conducting nanovectors having unique optical properties and immense potential for surface chemistry alterations, making them exceptional candidates for traceable and targeted delivery of diagnostic, therapeutic and theranostic agents ,207,208.Lanthanide upconversion nanoparticles are an interesting class of nanocarriers that convert deep tissue penetrating near-infrared light into visible/ultraviolet emissions . As suchDevelopments in the understanding of (and the techniques related to) stem cell biology have resulted in an exponential surge in research studies aimed at exploiting their biomedical applications . In partIn recent years, the combination of nanotechnology and stem cell therapy has been put forward as an interesting option for brain cancer therapy with tremendous potential benefits. Nano-engineering of stem cells allows for optimization of the cellular microenvironment, brain penetration and in vivo survival ,246. ForThe unique and versatile morphological, biological and surface properties of viruses combined with their efficiency in escaping endogenous immune clearances and targeting of specific cells can potentially serve as a platform for the design of suitable NPs ,252. NotWith the advances in the basic knowledge of the molecular mechanisms of brain tumor pathology, several genetic targets have emerged, which in turn has increased the prospects of developing suitable gene therapy strategies in clinical settings of neuro-oncology. Conventionally, DNA delivery has relied on viral vectors; however, smart nanoplatforms offer several potential advantages, such as enhanced brain penetration, biocompatibility and flexibility of cargo capacity. Indeed, smart nanoengineering can potentially allow the delivery of DNA/RNA for a multi-targeted gene therapeutic approach against brain cancer. Various aspects of cancer cell biology can be targeted simultaneously; induction of the expression of tumor suppressor genes, reduction of the expression of oncogenes, stimulation of cell death programming, sensitization of the tumor cells for subsequent cytotoxic treatment, induction of differentiation of tumor-initiating cells and regulation of the proliferation and migration of cancer cells . InteresBroadly, nucleic acid-NPs can be divided into DNA- and RNA-NPs. Ideally, the DNA-based NPs are expected to drive expression of genes that can induce cell death or make the cancer cells susceptible to other cytotoxic agents. RNA-NPs are more versatile in the payloads and can incorporate almost all of the known agents of RNA-based therapeutics: mRNAs, miRNAs, siRNAs and ribozymes. While miRNAs and siRNAs are gene therapeutic candidates for controlling the expression of particular endogenous mRNAs, mRNA-based NPs are increasingly been perceived as a tool for vaccine-based immunotherapeutic strategies against brain cancer ,264. ForExosomes are extracellular vesicles secreted by endomembrane-containing cells  and are implicated in intercellular communication, both under normal and pathological conditions ,268. TheIn conclusion, while it is clear that exosomes could potentially be a very important tool for the delivery of various kinds of therapeutic agents to the CNS across the BBB, more research studies are needed for a thorough understanding of the choice of exosome donor cell, the optimization of encapsulation procedures, the evaluation of loading efficiencies and toxicity and pharmacokinetic properties. It is relevant to stress the interesting potential utility of endogenous exosomes, particularly isolated from body fluids such as blood and CSF, in serving as targets for biomarker identification for neuropathological conditions, including brain tumors .This review revisits some of the challenges in translational neuro-oncology research and discusses the recently developed smart nanotechnological strategies with the potential to overcome them. In spite of the tremendous potential of nanomedicine for the treatment of brain cancers as evidenced by preclinical animal experiments, the clinical human trial data has remained less than satisfactory. The reasons for this discrepancy are not quite understood, but it is speculated that the less-than-pronounced EPR effect and the heterogeneity of human brain tumors are the major contributors. There is clearly still hope and much room for improvement and optimization. For example, the variable ERP effect in humans can be circumvented by nanomedicines with better pharmacokinetics and tumor deposition. Combinatorial approaches  and personalized medicine (theranostics) are the future lines of research that may prove beneficial for addressing the tumor heterogeneity issue (and drug resistance) in human brain cancers. In particular, the incorporation of imaging and therapeutic agents into single nanoplatforms may lead to significant clinical successes in dealing with brain cancers in a clinical setting. Stimulus-responsive strategies, especially focused ultrasound and magnetic field activation, may elicit the highest clinical potential, as they offer no tissue- or BBB-penetrating limitations. These and other smart bioengineered options for superior diagnosis, monitoring and treatment of brain cancers are already being rigorously perused by researchers globally . More st


Drosophila melanogaster, decision making on the cellular and organism level is achieved with temporally controlled cell polarization events. The initial polarization of Par proteins occurs as early as in the germline cyst, when one of the 16 cells becomes the oocyte. Another marked event occurs when the anterior–posterior axis of the future organism is defined by Par redistribution in the oocyte, requiring external signaling from somatic cells. Here, we review the current literature on cell polarity events that constitute the oogenesis from the stem cell to the mature egg.Cell polarity is a pre-requirement for many fundamental processes in animal cells, such as asymmetric cell division, axon specification, morphogenesis and epithelial tissue formation. For all these different processes, polarization is established by the same set of proteins, called partitioning defective (Par) proteins. During development in  Cell polarization is the canonical process of distributing cellular components—from molecules to organelles—unevenly, leading to intracellular and morphological asymmetry. The acquisition and definition of cell functions are often linked to this asymmetry. In broader terms, cell polarization can be viewed as cellular decision-making process generating spatial information. A well-studied example is apicobasal axis formation in epithelial cells. The apical membrane faces the outside of the cell, while the basal membrane contacts the inside. This polarization is the foundation for compartmentalization, organ formation and physical separation of the vertebrate body from the environment . AnotherA subclass of metazoans, called bilaterians, define two body axes during embryonic development, the anterior-posterior (AP) axis being the first and the dorsal-ventral (DV) axis following as second symmetry break . For mosDrosophila melanogaster oogenesis occurs inside ovarioles, structures composed of the germarium at the anterior tip, and sequentially more mature egg chambers towards the posterior  hosts a number of germline stem cells (GSC). These cells divide asymmetrically to produce another stem cell and a differentiating daughter cell called cystoblast. This cystoblast undergoes four rounds of incomplete cell divisions giving rise to a cyst of 16 cells, which are called cystocytes. Due to these cell divisions being incomplete, the cystocytes remain connected through cytoplasmic bridges called ring canals. The cystoblast divides into two cystocytes, which go through three more rounds of division and, thus, have four ring canals. Due to the history of divisions, two of the sixteen cystocytes have three, four have two, and eight have only one ring canal , bottom.The identity of the oocyte is established following two distinct events. In the nucleus, the oocyte arrests in the prophase of meiosis I, while the other cytocytes exit meiosis I and start the endoreplication cycle. In the cytoplasm, the microtubule network is organised such that, while passing the ring canals, the microtubule minus-ends predominantly accumulate in one of the two pro-oocytes and form a non-centrosomal microtubule organizing centre (ncMTOC) . This leIt has long been assumed that both pro-oocytes are equally competent to become the oocyte. This assumption was based on the naïve observation that the early transitions of the cell cycle in the two pro-oocytes are indistinguishable. Both pro-oocytes, as well as several cells with three ring canals, enter meiosis I and form synaptonemal complexes between homologous chromosomes . Only asDrosophila homologue of Spectraplakin, Short Stop (Shot), a component of the fusome, is necessary for oocyte specification, and appears to stabilize microtubules and link them to the fusome  and aPKC localize to adherens junctions that form around the ring canals   . This sudominant . This moDrosophila, the development from stem cell to mature oocyte is critically determined by a series of spatial decision-making processes. These processes go back to canonical cell polarity events governed by the Par protein network. Despite recent advances in our understanding of cell polarity and the downstream processes leading to cell selection and differentiation, intracellular reorganisation of the oocyte, and body axes formation, several major gaps of knowledge persist. In our view the most critical open questions relate to the interplay between follicle cells and the oocyte. Unlike in other species, for example in C. elegans, where polarization of the zygote and first body axis determination occurs cell autonomously, an intimate mechanistic relationship between soma and germline has been retained in Drosophila. Most importantly, the identification of the follicle cell signal that determines the second polarity event and body axis formation is long overdue, but all the past studies so far suggest that this would require analyses beyond genetic screens and knockout studies. Biophysical approaches that test the cell-cell interface between oocyte and posterior follicle cells could give new insight.In


As the incidence of cancer continues to increase, so too will the use of various forms of cancer therapeutics and their associated oral and dental complications. Although many of the acute and chronic oral toxicities of cancer therapy are largely unavoidable, appropriate and timely management of these complications has the potential to alleviate morbidity and improve outcomes. Undoubtedly, the substantial short- and long-term impacts of cancer therapy on the health of the oral cavity requires increased awareness, prevention, and treatment by multidisciplinary healthcare teams consisting of medical oncologists, dentists, and other oral healthcare specialists. This mini review provides a brief purview of the current state of clinical oncology and its impact on oral health. The topics introduced here will be further investigated throughout the remainder of the “Oral Complications in Cancer Patients” mini-review series. Cancer accounted for roughly 10 million deaths in 2020, serving as a leading cause of mortality globally –5.Many cancer treatment modalities, such as surgery, radiotherapy, chemotherapy , and hematopoietic stem cell transplantation, as well as supportive care measures  have the potential to cause various oral complications  6]. Mor. Mor6]. This mini review provides a brief purview of the current state of clinical oncology and its impact on oral health. Clinical oncology consists of three primary disciplines: surgical oncology, radiation oncology, and medical oncology. Basic principles of clinical oncology, recent advancements in cancer therapeutics, and various oral health complications associated with cancer treatment will be discussed. Finally, the authors will consider various approaches to promoting oral health before, during, and after cancer treatment. The topics introduced here will be further investigated throughout the remainder of the “Oral Complications in Cancer Patients” mini-review series.According to the Global Burden of Disease (GBD) study, cancer imposes the largest burden of any disease in the world, exceeding that of ischemic heart disease and stroke . In 2018Cancers of the head and neck (HNC) are a heterogeneous group of malignancies that comprise the ninth and seventh most common cancer in the US and world, respectively , 4. EachSurgical management remains a mainstay of modern cancer treatment, including for HNSCC. While removal of simple, early stage tumors may result in minimal side effects, surgical treatment of more advanced stage lesions can produce numerous esthetic, functional, and psychological sequelae. Potential impacts of surgery on oral function include difficulty tasting, speaking, chewing, and swallowing, whereas the excision of mucosal surfaces, loss in soft tissue volume, and removal of bone may result in substantial esthetic deformities . MaxilloThe primary goals of maxillofacial prosthodontics are to restore oral function, improve facial esthetics, and enhance quality of life . For exaRadiotherapy remains one of the primary treatment modalities for both localized and late-stage cancers. Further, various randomized control trials have demonstrated a superior tumor response to radiotherapy and concurrent chemotherapy, rather than radiotherapy alone, for numerous advanced tumors including HNCs –25. The Oral mucositis (OM) is fairly ubiquitous among patients receiving radiation therapy to the head and neck . SymptomRadiotherapy to the head and neck region is a common cause of salivary gland dysfunction, with doses of 50 Gy or higher imparting the highest risk for this complication , 33. PatWhile various pharmacologic agents have been suggested as possible interventions for preventing radiation-induced salivary gland dysfunction, such as parasympathomimetic drugs, parasympatholytic drugs, and cytoprotective agents, the evidence to support their efficacy is of admittedly poor quality , 39–42. Osteoradionecrosis (ORN) of the jaw is a potentially severe iatrogenic disease of devitalized bone caused by radiation therapy of the head and neck that fails to heal or remodel –48. EarlChemotherapeutic agents comprise a vast group of chemicals designed to halt the growth of cancer cells, either through inducing apoptosis or preventing their replication. These agents produce their toxic effects by targeting rapidly proliferating cells, such as the basal cells of the mucosal layer as well as the acinar and ductal cells of the salivary glands . The oraHematopoietic stem cell transplantation (HSCT), on the other hand, involves the transplantation of healthy hematopoietic stem cells to patients with dysfunctional or depleted bone marrow for the treatment of various cancers, immune-deficiency syndromes, and hemoglobinopathies . HSCT caGraft-vs.-host disease (GVHD) is a major cause of morbidity and non-relapse mortality in patients undergoing allogeneic HSCT, with over 50% of patients developing chronic GVHD , 59. ChrMedication-related osteonecrosis of the jaw (MRONJ) is a potentially debilitating condition characterized by non-healing exposed bone in patients who have used either antiresorptive or antiangiogenic agents , 65. HigDentists and other oral healthcare specialists play a vital role in preventing or minimizing a patient's risk of developing MRONJ. Dental assessments and the provision of prophylactic dental care prior to initiation of antiangiogenic or antiresorptive therapy have been shown to decrease a patient's chances of developing MRONJ . In patiTargeted therapies, which target specific genes and proteins involved in the growth and survival of cancer cells, and immunotherapies, which stimulate a patient's own immune system to combat cancer, are quickly becoming central pillars of cancer treatment. Although the introduction of targeted therapies and immunotherapies have revolutionized treatment for numerous types of malignancies, they have also produced novel side effects known as immune-related adverse events (irAE). While these adverse events have been observed in nearly all parts of the body, their impact on the oral cavity is notable .Although immunotherapy has made an indelible mark on the field of cancer therapeutics, irAEs associated with their use are unfortunately commonplace . These aObtaining dental clearance is an essential step prior to the initiation of cancer treatment, particularly for patients to be exposed to radiation to the head and neck. The rationale for dental screening prior to cancer therapy derives from numerous studies linking an increased incidence of intra-therapy complications, such as acute dental infections, with poor oral health –81.A complete oral evaluation prior to the initiation of cancer treatment should elucidate the following information: presence of a dental home, date of last dental visit, recognition of past and current dental problems, an oral/dental evidence-based risk assessment such as CAMBRA (caries management by risk assessment), and an evaluation of past and current medication use.Additionally, the patient should receive oral prophylaxis including professional hygiene therapy as well as eliminate any existing low-grade infections and possible sources of trauma  . The NatThe increased risk of acute oral complications resulting from radiation and/or chemotherapy highlights the importance of continued proper oral hygiene practices and maintenance. Patients should continue to use the modified Bass brushing method with fluoride toothpaste at least two times per day , floss o3; abnormal clotting factors present; and/or absolute neutrophil count of <1,000/mm3 [It is crucial that dentists and other oral healthcare specialists maintain communication with the oncologist throughout the duration of cancer therapy and obtain proper consultation with the oncologist prior to any dental procedures, including prophylaxis. A blood sample should be obtained from patients undergoing chemotherapy roughly 24 h prior to any invasive oral surgical procedures and postponed when the following are present: platelet counts <75,000/mm,000/mm3 .Following the completion of cancer therapy, patients should remain on a regular recall schedule as recommended by their dentist and continue practicing proper oral hygiene including the use of fluoride toothpaste and varnish. In patients at higher risk for dental caries  as well as those with a history of oral malignancy (such as OSCC), a more intensive and frequent recall schedule may be necessary. Further, a patient's hematologic status, such as the resolution of immunosuppression and/or thrombocytopenia following the cessation of cancer therapy, should be assessed prior to dental treatment. Furthermore, the dentist should update the patient's medication list and assess for any anti-resorptive and/or anti-angiogenic medications used throughout the duration of cancer treatment.Finally, childhood cancer therapy has the potential to result in numerous dental, craniofacial, and soft tissue complications. Children are particularly susceptible to the long-term effects of cancer therapy as treatment typically occurs during the most active stage of growth and organ development . For exaInnovations and improvements in cancer therapy have substantially increased survivorship in recent years. As a result, there is a growing need for continuing management of the oral health needs of this population. Although many of the acute and chronic oral toxicities of cancer therapy are largely unavoidable, appropriate and timely management of these complications has the potential to alleviate a considerable amount of morbidity. Further, with advances in computational modeling and “Deep Learning” protocols, individuals at risk for developing drug toxicities may be identified and providers may be better equipped to predict which patients and drugs are most likely to induce oral side effects. Finally, the successful management of this complex patient population requires interprofessional collaboration and the utilization of a comprehensive, patient-centered approach with an emphasis on oral health.JH and GO wrote the first draft of the manuscript. GH guided and revised the manuscript. NT conceptualized and guided the project. All authors contributed to the article and approved the submitted version.GO was supported in part by the Piano di Sostegno alla Ricerca (PSR) 2020, Linea 2: Dotazione Annuale per attività istituzionali, Department of Biomedical, Surgical and Dental Sciences, Università degli Studi di Milano, Milan, Italy.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Inflammatory bowel diseases, including Crohn’s disease and ulcerative colitis, are incurable autoimmune diseases characterized by chronic inflammation of the gastrointestinal tract. There is increasing evidence that inappropriate interaction between the enteric nervous system and central nervous system and/or low activity of the vagus nerve, which connects the enteric and central nervous systems, could play a crucial role in their pathogenesis. Therefore, it has been suggested that appropriate neuroprosthetic stimulation of the vagus nerve could lead to the modulation of the inflammation of the gastrointestinal tract and consequent long-term control of these autoimmune diseases. In the present paper, we provide a comprehensive overview of (1) the cellular and molecular bases of the immune system, (2) the way central and enteric nervous systems interact and contribute to the immune responses, (3) the pathogenesis of the inflammatory bowel disease, and (4) the therapeutic use of vagus nerve stimulation, and in particular, the transcutaneous stimulation of the auricular branch of the vagus nerve. Then, we expose the working hypotheses for the modulation of the molecular processes that are responsible for intestinal inflammation in autoimmune diseases and the way we could develop personalized neuroprosthetic therapeutic devices and procedures in favor of the patients. Autoimmune diseases occur when the immune system (ImS) does not discriminate correctly between molecules that are self and molecules that are non-self to the organism. When this happens, ImS attacks its own cells as if they were non-self. Most of these diseases are accompanied by chronic inflammation, such as systemic lupus erythematous, type I diabetes mellitus, or inflammatory bowel disease (IBD) . IBD is A key role in IBD pathogenesis is deserved to the central nervous system (CNS) and its interactions with the enteric nervous system (ENS). ENS is embedded within the gut wall and is widely interconnected with the CNS, the enteroendocrine and gastrointestinal ImS, the gut microbiota, and the peripheral nervous systems (PNS). ENS regulates gastrointestinal motility, nutrient absorption, secretion, immunity, and defense . A failuTherefore, the restoration of CNS-ENS-ImS impairments by modulating the output of the VN could lead to a therapeutic approach for IBD ,7,8. ThiIn the present paper, we provide a comprehensive overview of the cellular and molecular bases of CNS-ENS interactions and the way these interactions could affect the ImS that underlies IBD. Furthermore, we discuss the therapeutic application of electrical stimulation of the vagus nerve (VNS), with an emphasis on the electrical stimulation of the auricular branch of the vagus nerve, highlighting the action mechanisms that modulate the molecular processes responsible for intestinal inflammation in IBD patients ,12,13.ImS protects the organism against harmful and potentially detrimental agents that could trigger disease onset and progression through two different defense strategies: the innate and the acquired response  14]. . 14]. These responses are cell-mediated  or molecule-mediated  . ForeignThe innate immune response represents the first line of defense of the ImS. It is an antigen-independent defense mechanism that acts non-specifically and immediately  to eliminate microbes and prevent infection . Innate The acquired immune response is mediated by cells (lymphocytes T or B) and antibodies. T cells express surface receptors that allow them to detect and generate responses to various agents whilst B lymphocytes are responsible for the antibody-mediated responses. Antigen-presenting cells (APCs) allow B cells to produce specific antibodies against the identified antigen; in addition, like APCs, B cells can also present the antigen to T cells initiating an adaptive immune response . In addiInflammation is a consequence of ImS activation and represents a defensive reaction: after antigen identification, it attracts defense cells of the ImS and grants access to the site of damage . HoweverThe bowel is innerved by an intrinsic and an extrinsic system, provided by ENS and CNS, respectively ,29. ENS Below the intestinal mucosa, a network of neurons and glia, called the enteric nervous system (ENS), is responsible for the regulation of all sensory and motor functions within the gastrointestinal tract and independently of the brain ,35 Figu.Enteric neurons synthesize and release a wide range of neurotransmitters and neuropeptides, allowing them to communicate with smooth muscles, epithelium, other neurons, and immune cells . ImportaENS regulates intestinal motility ,37 throu+2 levels by communicating junctions and with neurons, especially by purines [While enteric neurons lie below intestinal crypts within the submucosa as well as between longitudinal and circular smooth muscle layers, enteric glial cells are located within the intestinal layers, around crypts, and throughout intestinal villi and are involved in the synaptic transmission into the myenteric plexus , in the  purines . ProjectThe most abundant subclass are enterochromaffin (EC) cells that release 5-HT with a controversial effect between pro and anti-inflammatory, being in the case of enteric neurons an anti-inflammatory neurotransmitter that exerts neuroprotective and neurogenic effects for the CNE to survive inflammation .Evidence suggests extensive bidirectional crosstalk between enteric neurons and enteric glial cells and between the epithelium and ENS, playing a role in the maintenance of intestinal epithelial barrier integrity  and contTherefore, these interactions may also excerpt an effect on inflammatory processes within the intestine. This close interaction between ENS cells and ImS has led to a novel concept of neuroimmune cell units at the core of gut homeostasis and defense . However14), which not only reside in the interior of the digestive tube but also interact with the host organism, participating in important functions like nutrition, immune responses, brain activation, and contributing to the homeostasis between systems [The gut represents a perfect environment for the life of a very large number of microorganisms , enabling the translocation of microbiota, food particles, or toxins and leading to an immune response characterized by cytokine production, mast cell, and macrophage activation . These iVN is the tenth cranial nerve and has the longest pathway with most territories outside the head . VN origFrom the jugular ganglion and within the jugular foramen, VN also gives off the auricular branch, also termed Arnold’s nerve or Alderman’s nerve, that provides somatosensory innervation to the external ear . The aurCaudally from the nodose ganglion, the vagus nerve can be anatomically divided into three segments, the cervical, the thoracic, and the abdominal.In the neck, the VN enters the carotid sheath that contains the common carotid artery and the internal jugular vein and gives off the pharyngeal branch for constrictors and most of the palatine muscles and the superior laryngeal nerve, providing sensory innervation of the laryngeal mucosa  and motor innervation of the inferior constrictor of the pharynx  and cricothyroid muscles . At the lower neck, VN gives off to the superior and middle cervical cardiac branches and the aortic branch , whose axons innervate the aortic arch on the left side and bifurcation of the brachiocephalic trunk on the right side.In the thoracic region, VN enters the superior mediastinum, passing anteriorly to the aortic arch on the left and the subclavian artery on the right side and then behind the lung’s roots. Here VN forms two plexuses around the esophagus  that pass through the diaphragm muscle and constitute the abdominal VN. During its pathway along the mediastinum, VN gives off the inferior cervical cardiac branch, the thoracic cardiac branches, the inferior laryngeal nerve (recurrent nerve), and the pulmonary plexus.VN enters the abdominal cavity with the esophagus through its hiatus and with an anterior/posterior disposition, giving off branches that innervate the entire gastrointestinal tract until the splenic flexure of the colon. The posterior abdominal VN gives rise to the criminal nerve of Grassi, along the lesser curvature, and the dorsal coeliac branch that converges with the ventral coeliac branch of the anterior abdominal VN into the coeliac ganglion, from which vagal fibers depart to the intestines, pancreas, and potentially the spleen. The anterior trunk of the VN, moreover, gives off the common hepatic branch for the liver and bile ducts and completes the innervation of the stomach along the lesser and greater curvatures, the pyloric sphincter, the pancreas, and the proximal duodenum.According to the nerve fiber composition, VN is a mixed nerve, with both sensory (80–90%) and motor (including postganglionic parasympathetic) (10–20%) fibers, distinguished in afferent general somatic, afferent general and special visceral, general and special visceral efferent axons . Most afEfferent fibers of the VN originate from both the ventral motor nucleus (nucleus ambiguous) and the dorsal motor nucleus and provide the innervation of the pharyngo-laryngeal muscles and parasympathetic innervation of the neck, thoracic, and abdominal organs up to the splenic flexure of the colon, respectively .The different anatomical and molecular profiles of the VN fibers innervating the gastrointestinal tract constitute the substrate for the regulation of its main functions, including feeding behavior, digestion, and metabolism. Vagal afferent axons form different kinds of sensory endings along the gastrointestinal tube that allow the detection of chemical and mechanical stimuli. VN A-type sensory fibers present intraganglionic laminar endings (IGLE), tension slowly/rapidly adapting receptors for control of muscle distension, or mucosal and muscular-mucosal receptors, sensitive to muscle distension and mucosal deformation. Mucosal endings are largely C-fibers terminating in the lamina propria of the gastric and intestinal mucosa and allow the detection of chemical stimuli from the lumen  but also mechanical stimulation. The distribution of mucosal endings changes from the stomach to the intestine, being more expressed in the proximal small intestine. In the small intestine, mucosal endings do not make contact with the intestinal lumen and communicate with second-order chemosensory cells, the enterochromaffin cells, the principal source of serotonin in the body, forming neuropod-mediated synapses.Enterochromaffin cells of the gastrointestinal tract include the enteroendocrine cells of the gut epithelium . These cInnervation of the muscular layers of the gastroenteric tube is provided by intramuscular arrays, consisting of branching fibers in the circular and longitudinal muscular sheath acting as stretch receptors. These fibers are mainly unmyelinated and respond to gastrointestinal distension and chemical stimuli (gut hormones). Muscular-mucosal receptors of vagal endings lie in the lamina propria layer, creating a subepithelial plexus that responds to both light mucosal stroking and distension. These receptors are sensitive to harmless mechanical stimuli in inflammatory conditions that sensitize and trigger mechano-sensitivity in muscular-mucosal and vascular afferents.Finally, the strict link between vagal afferent fibers and ImS modulation has been recently investigated, showing that cytokine-specific information is present in sensory neural signals within the vagus nerve .Therefore, VN and the dorsal vagal complex, through the organization of vagovagal reflexes, establish interconnections between the entire neuroaxis of the CNS and the gut and is responsible for a bidirectional communication pathway between the intestine and brain .Sympathetic innervation of the GI tract drives noradrenergic (NA) fibers from the prevertebral ganglia and splanchnic nerves to the enteric tube smooth muscle wall, intramucosal/submucosal plexus ganglia, and arteries ,99. In pIn the gastrointestinal tract, sympathetic postganglionic neurons provide vasoconstrictor input to arterioles while enteric submucosal vasomotor neurons and afferent neurons innervate the vessels and can lead to their vasodilatation ,101. In In experimental setups, NA fibers of the enteric nervous system are marked using antibodies against tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of dopamine and NA. Evidence has shown a direct and inverse correlation between age and the TH staining in the circular smooth muscle .Sympathetic nerves also provide an important role in intestinal inflammatory response through their connections with the cholinergic anti-inflammatory pathway (CAP). The relevance of the sympathetic system to intestinal inflammatory mechanisms derives from patients with IBD, showing an enhanced sympathetic tone. Accordingly, experimental evidence has demonstrated that NA depletion using the reserpine, an irreversible reuptake-inhibitor of VMAT-2 (vesicular monoamine transporter-2), prevented the anti-inflammatory action of vagal stimulation in rats , thus reThe effects of the sympathetic system on the inflammatory mechanism and their cross-interaction with the counterpart parasympathetic need to be further investigated. Contrasting results of stimulation protocols need to be interpreted in light of the different experimental setups for their ability to enhance or reduce the inflammatory cascade.Crohn’s disease (CD) is an autoimmune disorder subsequent to an inappropriate innate and acquired immune response to harmless antigens that can normally pass through the gastrointestinal tract. This disease is characterized by inflammation of the entire gastrointestinal wall and can be located anywhere from the mouth to the anus. Although the origin is unidentified, it seems to be the consequence of a combination of genetic and environmental factors that alter the immune response in the gastrointestinal tract . The risAt the beginning of the disease, the intestinal epithelial barrier is damaged, whereby the antigens existing in the intestinal lumen penetrate the epithelium. Specific ImS cells  can recognize these antigens and trigger an immune response, producing pro-inflammatory mediators that chemo-attract other immune cells to the site of inflammation and induce differentiation of undifferentiated T cells. T cells can differentiate into Th1 or Th17 cells, producing more inflammatory mediators and sustaining inflammation and damage to the intestinal epithelium. When these processes are maintained, inflammation becomes chronic, and it can generate local and systemic complications ,112.Current treatment of CD is based on the use of corticosteroids, aminosalicylates, thiopurine, and immunomodulatory and biological drugs . SulfalaThis wide pharmacological approach, however, not always provides positive effects, hypothesizing that other factors are involved and reduce the drug efficacy. Among others, the diet is able to change intestinal barrier function, affect the structure and function of intestinal flora, and promote immune disorder, thus promoting inflammation . TherefoBifidobacteria, Lactobacilli, Actinomycetes) and inhibiting pathogenic bacteria , exerting effects similar to prebiotics [A non-pharmacological therapy that is increasing in importance is treatment with polyphenols. Phenolic compounds or polyphenols are secondary metabolites with a wide presence in the human diet, such as fruits, vegetables, and products derived from commonly consumed plants such as cocoa, tea, or wine. These compounds have demonstrated through in vitro, ex vivo, and animal tests, different beneficial effects on health due to their antioxidant, anti-inflammatory, estrogenic, cardioprotective, cancer chemopreventive, and neuroprotective properties, acting either directly when absorbed or indirectly, after being processed by the intestinal microbiota ,120,121.ebiotics ,122,123.The human body identifies ingested polyphenols as xenobiotics making them less available than micro- and macro-nutrients. The majority of these polyphenols, those with oligomeric and polymeric forms of high molecular weight, reach the colon, where they are broken down by intestinal microbiota and transformed into absorbable metabolites with biological activity; a small portion of these polyphenols, those with monomeric and dimeric structures, are absorbed in the small intestine ,118,124.The low final concentration of polyphenols in plasma suggests that direct antioxidant effects would be expected only in the tissues that are directly exposed to these molecules , although they could also accumulate in some compartments interacting with other membranes or that they could deconjugate to reach different tissues . The degPolyphenols affect the composition of the intestinal microbiota with their prebiotic activity while providing precursors for microbes-produced metabolites that protect the intestinal immunomodulation barrier . In vitrOf particular interest are the microbiota–gut–brain axis and the modulatory activity of the vagus nerve. Brain behavior and mental health are indirectly modulated by polyphenols through this axis in a bi-directional direction in which, in addition to the vagus nerve, also participate the immune system, neuroendocrine pathways, and bacteria-derived metabolites  Figure . Microbiota dysregulation is linked to neuroinflammatory and neuroimmune diseases , so polyphenols’ action on the microbiota (either directly or through the byproducts of the metabolism of the bacteria) could modulate oxidative stress, inflammation, and neurotransmitter imbalance through the activation of the hypothalamus–pituitary–adrenal axis (HPA axis).In vitro and in vivo results suggest that both food polyphenols and their microbial metabolites contribute to the effect of these molecules on host health ,121,124.The intestinal epithelium normally blocks the paracellular passage of antigens present in the intestinal lumen through junctions between intestinal epithelial cells . In CrohPaneth cells are highly specialized intestinal epithelial cells that excrete granules with antimicrobial peptides, such as α-defensins, and control the commensal microbiota . SeveralIn the absence of disease, ImS does not trigger immune responses to many antigens (tolerance). In Crohn’s disease, the immunological tolerance of dendritic cells and/or macrophages could be diminished, and once they recognize antigens, they polarize undifferentiated T lymphocytes (naive T lymphocytes) to differentiate into Th1 or Th17 by means of specific inflammatory mediators: IL-12 or IL-23, respectively . This caThe innate immune response acts as the first line of defense in the gastrointestinal tract. In Crohn’s disease, the innate immune response in the gastrointestinal tract is triggered by (1) the increased permeability of the intestinal epithelium, (2) the dysfunction of Paneth cells, and/or (3) the decreased tolerance of dendritic cells and macrophages to intestinal lumen antigens ,129 Fig.The intFinally, another factor to consider is dysbiosis, understood as an unfavorable alteration of the composition and function of the intestinal microbiota. The increase in pathogenic bacteria with the ability to adhere to the intestinal epithelium can affect the permeability of the intestine, also altering the diversity and composition of the microbiota and inducing inflammatory responses. Although a direct causal relationship has not been established, many studies have reported that the composition of the microbiota in IBD patients is altered compared to healthy subjects ,60.The acquired immune response is also dysregulated in Chron’s disease and characterized by (1) an imbalance in the number of lymphocytes, (2) an increase of Th1 cytokines, and (3) an increase of lymphocytes in the mucosal lamina propria  Figure . As mentioned above, macrophages and/or dendritic cells participate in the differentiation of naive T lymphocytes into Th1 or Th17 lymphocytes. If this process becomes chronic, an imbalance between effector T cells (Th1 and/or Th17 lymphocytes) and regulatory T cells can appear . IL-12 cAfter the polarization of Th1 lymphocytes from naive T lymphocytes, Th1 produces a great amount of cytokines, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and IL-2 that increase even more when these cells are stimulated by the inflammatory mediator IL-12 . The cytAlterations of innate and adaptive immune responses are currently correlated to persistent bowel inflammation and also dysfunction of the ENS and gut–brain axis ,142. TheTherefore, non-pharmacological therapies seem to be of interest in IBD treatment and are popular among patients: neuromodulation, based on the bidirectional interaction between CNS and ImS, VNS being the most likely therapeutic procedure ,147.To date, electrical left cervical VNS has been approved and used for the treatment of refractory epilepsy since 1997, treatment-resistant depression since 2005, and cluster headaches since 2016 ,148. TraThe stimulation parameters, which are typically in the range of 10–20 Hz, 0.25–2 mA intensity, and 0.1–0.5 ms pulse width, are optimized to activate mainly vagal afferent fibers . ActivatThis extensive neural network, also involving the sympathetic nervous system and neuroendocrine systems, together with local microglia and astrocytes, underlies appropriate behavioral, autonomic, neuroendocrine, and inflammatory responses.The CAP: STN, activated by general visceral efferent fibers of VN, project to the dorsal motor nucleus of the vagus (DMx), i.e., the parasympathetic nucleus of the VN, that synapses in celiac ganglia and sends sympathetic fibers to the spleen through the splenic nerve  due to splenectomy ,163. MorThe hypothalamic–pituitary–adrenal axis: the cholinergic anti-inflammatory pathway also involves a response of the hypothalamic–pituitary–adrenal axis  was reported in an animal model with behavioral deficits induced by ischemic/reperfusion cerebral deficits and treated with VNS . The decTargeting the inflammatory process of IBD with artificial stimulation of certain branches of VN could be achieved through neuroprostheses to restore intestinal homeostasis. The main disadvantages of stimulating the cervical VN are the highly invasive approach and the likely stimulation of nerve fibers that are not in the cervical trunk of the VN, which can cause adverse side effects . For thiNeuroprostheses are a novel therapeutical tool that can be used to restore specific neural functions . Recent Finally, a matter still to be clarified is the effect of VNS on the intestinal microbiota, either beneficial or harmful, which are the changes induced to the resident microbiota and, consequently, its contribution in triggering other chronic diseases or in modulating the effect of treatment. Inversely, VNS’s direct and indirect beneficial role should also be elucidated. Moreover, since the employment of VNS as a therapeutic strategy, either “per se” or as a complementary treatment for a multitude of disorders, it is essential to understand the secondary effects of this treatment on gut microbiota and the possibility of a consequent triggering (or facilitation) of the development of IBD-like diseases.


Idiopathic generalized epilepsy (IGE) was characterized by 3–6 Hz generalized spike-wave discharges (GSWDs), and extensive altered interactions in subcortical-cortical circuit. However, the dynamics and the causal relationship among these interactions were less studied. Using resting-state functional magnetic resonance imaging (fMRI) data, the abnormal connections in the subcortical-cortical pathway in IGE were examined. Then, we proposed a novel method of granger causal analysis based on the dynamic functional connectivity, and the predictive effects among these abnormal connections were calculated. The results showed that the thalamus, and precuneus were key regions representing abnormal functional network connectivity (FNC) in the subcortical-cortical circuit. Moreover, the connectivity between precuneus and adjacent regions had a causal effect on the widespread dysfunction of the thalamocortical circuit. In addition, the connection between the striatum and thalamus indicated the modulation role on the cortical connection in epilepsy. These results described the causality of the widespread abnormality of the subcortical-cortical circuit in IGE in terms of the dynamics of functional connections, which provided additional evidence for understanding the potential modulation pattern of the abnormal epileptic pathway. Idiopathic generalized epilepsy (IGE) was typically characterized by 3–6 Hz generalized spike-wave discharges (GSWDs). The mainstream concept believed that the burst of GSWD was related to the inter-regional interactions in the subcortical-cortical circuit , 2005, iRegarding the origin of generalized epileptic discharge, previous studies have proposed some different discharge model hypotheses, with two representative concepts of the cortical focus theory and the thalamic pacemaker theory. The hypothesis of cortical focus theory assumed that epileptic discharges originated in a certain local area of the cortex and then spread through the interaction of subcortical structures with the cortex, thus recruiting specific neuronal networks into typical oscillatory behavior . Under tOther structures under the cortex, such as the striatum and cerebellum, also played an important role in regulating epilepsy. Some animal studies have shown that the striatum, whose fibers connected to the substantia nigra reticulum, was involved in the regulation of epileptic discharges . PreviouIn this study, we investigated the altered subcortical-cortical pathway of IGE, and the causality relationship among these abnormal connections was examined using the dynamic functional of the resting-state network. The dynamic interactions within the abnormal epileptic networks may provide evidence for the origin and modulation of epileptic activity.In total, seventy-eight patients with IGE  including 32 patients with juvenile myoclonic epilepsy (JME) and 46 patients with generalized tonic–clonic seizures (GTCSs) were recruited in this study. Diagnosis and classification was made by neurologists in accordance with the International League Against Epilepsy (ILAE) guidelines . RoutineIn this study, the MRI data of all the participants were recorded from the 3T MRI scanner  with an eight channel-phased array head coil in UESTC. Resting-state fMRI data were acquired using a gradient-echo echo planar imaging sequences . A total of 255 volumes were collected in the resting-state scan for each participant. During fMRI scanning, all the participants were instructed to keep still and close their eyes without sleeping. In addition, high-resolution T1-weighted images were also acquired using a 3D fast spoiled gradient echo (T1-3D FSPGR) sequence .1 and NIT toolboxes2 , and also cerebellum network, each of network consisting of a few components, that is the ROIs. The MNI coordinates for the selected ROIs were shown in Spatial ICA was performed to the group fMRI, and the functional networks identified by the ICs were obtained. Here, the number of ICs was 100 and determined according to the previous studies. In ICASSO,min = 0.01 Hz). Then, the variation of the weights for the dynamic FNC in the time course was calculated, representing the dynamics of the connections in the subcortical–cortical circuit.The static FNC and dynamic FNC was constructed among ROIs. Static FNC was computed by the Pearson’s correlation of the time courses between any two selected components. Based on the static FNC, we calculated the degree of FNC nodes by summarizing the weights of the connections of each node. Dynamic FNC was also computed using correlations between windowed time-courses of different ROIs. Here, the length of the slide window was 50TR, with the sliding step width 1TR. The selection of the window length was constrained by the minimum frequency of the BOLD signal .Finally, According to the previous studies, we selected the components in DMN, SMN, BGN, SN, and cerebellum from 100 ICs obtained in ICA analysis. Thus, each network was constituted of a few components, i.e., the ROIs. Here, DMN included the frontal_sup_medial area, the angular, cingulum_post (PCC), and two precuneus components. SMN included bilateral post-central areas, pre-central, paracentral_lobule, and supp_motor_area (SMA). BGN was comprised of the thalamus, putamen, and caudate. SN involved the left and right frontal_mid area, the cingulum_ant (ACC), cingulum_mid (MCC), and insula. The cerebellum network included the cerebellum posterior lobe and the declive. The maps of these five networks were illustrated in t-test was conducted to the static FNC, and the difference of FNC between IGE and HC was shown in P < 0.001). In between-group comparisons, age and gender were regressed out as the covariates. The results showed that thalamus, precuneus were the key nodes representing significant alteration in the subcortical–cortical circuit in epilepsy. Increased connectivity between thalamus and striatum, as well as the insula, and increased connectivity between precuneus and motor area was found in IGE comparing to HC. Decreased connectivity between the thalamus and frontal area, between precuneus and the frontal area, and also the thalamus/striatum was found in IGE. In addition, striatum showed increased connectivity with insula and cerebellum in epilepsy brain. Moreover, we compared the degree of FNC nodes between IGE and HC, and the thalamus, precuneus, ACC and post-central areas had decreased static FNC in IGE (P < 0.05)  .Besides, comparison of static FNC was also performed between two epilepsy groups, i.e., JME and GTCS. Comparing to GTCS, JME showed decreased connectivity in thalamus-precuneus, and thalamus-frontal connections. The results may be related to the longer epilepsy duration of these patients with JME in this study.P < 0.05) (P < 0.01) were shown in After the abnormal connections of static FNC in the epilepsy group were labeled, the dynamic changes of these connections were extracted according to the weights of the dynamic FNC matrices. The variation of the dynamic FNC showed that the connections of thalamus-frontal_mid, thalamus-precuneus, precuneus-frontal_mid, and frontal_mid-post_central had increased dynamics in IGE ( < 0.05) . Then, tTo sum up, patients with epilepsy had increased cerebellum-striatal-thalamic connections and widespread decrease in thalamocortical circuits. The causal relationship between these abnormal connections was summarized in This study investigated the subcortical–cortical pathway in IGE, and showed that thalamus, precuneus were key nodes representing abnormal functional connectivity in subcortical–cortical circuit. Furthermore, the causality relationship among these abnormal connections in IGE was examined. It was shown that the increased connections between precuneus and the adjacent regions had the causal effect on the widespread decreased connections in thalamocortical circuit. In addition, the two-way causal effect of the connection between the thalamus and the striatum on the cortical connection indicated the modulation role of subcortical circuit in epilepsy.Numerous studies have shown that there were abnormalities in a wide range of functional connections in IGE . AlteredAccording to the hypothesis of generalized epileptic discharges , the epiIn one of our previous study, we detected the synchronous network during the stage of epileptic discharges, and found the connectivity within frontal areas and between frontal–parietal areas was the main network pattern after the discharge onset . Also, eTo re-test the stability of the causal analysis, we also conducted the multivariate GCA to describe the directed causal effect from this seed connection to the other connections, as well as the reverse impact intensities from other connections to this seed connection. The results showed similar directed patterns with the bivariate GCA, including the predictive effect from precuneus-related connections to the thalamocortical connections, and then to the cortical–cortical connections. Moreover, much extensive effects were found from the striatum-thalamus to both the thalamocortical and the cortical–cortical connections. The results were shown in To sum up the results, we concluded the causal relationship among the abnormal functional connectivity in epileptic brain, which described the trigger role of precuneus with its extensive hyperconnectivity, and the widely distributed dysconnectivity of thalamocortical circuit, and also the following alteration of frontal and motor-related connectivity. This causality in epileptic circuit provided additional evidence for the theory of epileptic origin and propagation on the level of macro functional connectivity. However, the main limitation of this study is that the causal relationship of the abnormal connectivity was based on the resting-state data rather than the discharging state. Therefore, the result provided the potential modulation pattern in terms of the epileptic activity, and the inference of the epileptic propagation circuit in this study was urgently needed to test by the physical stimulation and clinical evaluation.This study investigated the subcortical–cortical pathway in IGE, and showed that thalamus, precuneus were key regions of the abnormal FNC in epileptic brain. Furthermore, the causality analysis among these abnormal connections demonstrated the predictive effect of precuneus on the widely distributed thalamocortical dysconnectivity. In addition, the increased connectivity between thalamus and the striatum indicated the modulation role of subcortical circuit on the cortical connection in epilepsy. These results provided additional evidence for the widespread abnormality in epilepsy brain based on the dynamics of functional connections, and the predictive relationship in the subcortical–cortical circuit enlightened the theory of epileptic origin and propagation.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of the University of Electronic Science and Technology of China. The patients/participants provided their written informed consent to participate in this study.YQ, DY, and CL conceived and designed the work. YQ and SL acquired the data. YQ analyzed the data. YQ and CL wrote the manuscript. All authors revised the work for important intellectual content and read, and approved 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.