The Substantia Nigra pars reticulata (SNpr) is the major information output site of the basal ganglia network and instrumental for the activation and adjustment of movement, regulation of the behavioral state and response to reward. Due to both overlapping and unique input and output connections, the SNpr might also have signal integration capacity and contribute to action selection. How the SNpr regulates these multiple functions remains incompletely understood. The SNpr is located in the ventral midbrain and is composed primarily of inhibitory GABAergic projection neurons that are heterogeneous in their properties. In addition, the SNpr contains smaller populations of other neurons, including glutamatergic neurons. Here, we discuss regionalization of the SNpr, in particular the division of the SNpr neurons to anterior (aSNpr) and posterior (pSNpr) subtypes, which display differences in many of their features. We hypothesize that unique developmental and molecular characteristics of the SNpr neuron subtypes correlate with both region-specific connections and notable functional specializations of the SNpr. Variation in both the genetic control of the SNpr neuron development as well as signals regulating cell migration and axon guidance may contribute to the functional diversity of the SNpr neurons. Therefore, insights into the various aspects of differentiation of the SNpr neurons can increase our understanding of fundamental brain functions and their defects in neurological and psychiatric disorders, including movement and mood disorders, as well as epilepsy. Basal ganglia, a network of brain structures and nuclei in the forebrain and anterior brainstem, evaluate external and internal signals and use this information to select, activate and invigorate appropriate behavior, often including voluntary movement . These bThe basal ganglia include separate structures for information input, modulation and output. The main input areas sending afferent projections to the basal ganglia are the striatum and the subthalamic nucleus (STN), which in turn receive projections from the cortex and thalamic nuclei . The modHow the basal ganglia output is controlled by specialized SNpr neurons, responsible for distinct behavioral outcomes, remains incompletely understood. A prerequisite for understanding the cell type-specific output is to have information on the properties and diversity of the output neurons at multiple levels . In thisThe SNpr is an anatomically easily recognizable nucleus in the most ventral brainstem, next to the dopaminergic neurons in the Substantia Nigra pars compacta (SNpc) and Ventral Tegmental Area (VTA) . In the Gbx2CreERT2) labeled the posterior part of the SNpr (pSNpr), but not its anterior part (aSNpr). Studies of the development of GABAergic neuron subtypes in the rhombomere 1 (r1) of the anterior hindbrain have further refined the origin of the pSNpr neurons in the ventrolateral r1 neuroepithelium expressing the homeodomain transcription factor (TF) NKX6-1, and in particular the anterior part of this neuroepithelial region close to the midbrain-hindbrain border   . In contecursors . Further TF TAL2 .Thus, the early precursors of the pSNpr and aSNpr neurons are dependent on different combinations of GATA and TAL TFs that function as GABAergic fate selectors.The SNpr GABAergic neurons are heterogeneous in their cytoarchitecture and expression of neurochemical markers, including Parvalbumin (PVALB), Calretinin, and Nitric oxide synthase, with the subtypes differing in their spatial distribution along the medio-lateral and antero-posterior axes of the SNpr .Gad1), and markers of the GABAergic neurons in the posterior diencephalon-midbrain-ventral hindbrain region, such as Gata3 , is preferentially, although not exclusively, expressed in the ral SNpr . Also GAral SNpr .In summary, the SNpr GABAergic neurons are molecularly heterogeneous. The subtypes of mature SNpr neurons that differ in their developmental origins and regulation can also be distinguished by differential gene expression . Recent + aSNpr neurons to their location ventral to SNpc dopaminergic neurons , and periaqueductal gray (PAG) , and glutamatergic input from the STN and a subset of neurons in the midbrain locomotor region (MLR) (the RBP4 expressing glutamatergic neurons in the midbrain reticular formation) . The stray (PAG) .The striatal input to the SNpr is also topographically organized in primates. The dorsal striatum in primates is composed of two nuclei, the Caudate and the Putamen. Of these, the Putamen projects primarily to the GPi, whereas the main target of the Caudate nucleus is the SNpr . The Cauvia striatonigral GABAergic projection neurons, which also express D1R.In addition to these inhibitory and excitatory projections, the SNpr receives dopamine released by the dendrites of adjacent SNpc dopaminergic neurons . SNpr GAThe SNpr sends GABAergic output to the dorsal midbrain , brainstem nuclei and the thalamus . This ouPvalb-expressing neurons are connected to the SC and MLR, brain regions related to locomotion, while medial Gad2-expressing SNpr neurons project more widely to brainstem regions involved in brain-state regulation  in the dorsal midbrain are located in the anterolateral SNpr, whereas the neurons sending projections to the brainstem nuclei in the hindbrain, including the dorsal raphe and pontomedullary reticular formation, are located in the posteromedial SNpr . This isgulation . It is pgulation .In addition to SNpr neurons having region-specific output targets, there is regional specification also within the shared targets. In particular, most SNpr neurons are characterized by a projection to the thalamus. The target areas in the thalamus, however, differ between the SNpr neurons connected to the dorsal midbrain and the brainstem: the SNpr neurons projecting to the dorsal midbrain send collaterals to the more lateral thalamus, whereas the SNpr neurons projecting to brainstem send collaterals to the more medial regions of the motor and intralaminar thalamic nuclei . These cAs mentioned above, the SNpr is thought to contain only sparse local interneurons. Rather than local interneurons, the axon-collaterals of the SNpr projection neurons provide feedback and mutual inhibitory interactions within the SNpr, as well as project to the adjacent dopaminergic SNpc . The SNpIn summary, the SNpr receives region-specific input and is a source of region-specific output. The regional organization of the input and output show similarities, suggesting separate information channels , but theSimilar to the SNpr GABAergic neurons, the SNpr glutamatergic neurons also receive inhibitory input from the striatum and project to the thalamus . HoweverPvalb-expressing SNpr neurons, were also shown to fire at higher frequencies and have higher baseline activity than the Gad2- and Vgat (Slc32a1) expressing SNpr neurons  and CD(t) (see above). In primates, the activity of the CD(t)-pSNpr circuit was specifically observed during the saccade toward previously learned high-value objects, while the activity of the CD(h)-aSNpr circuit was associated with the selection of objects with short-term, changing value .Pvalb- and Gad2-expressing SNpr neurons  as well as more focused (for example activating a specific movement) functions. The functional repertoire of SNpr appears to be based on distinct SNpr neuron subtypes and their cell-type specific connectivity that form parallel, segregated pathways regulating specific behaviors . We haveAs a center for behavioral gating, understanding the SNpr has broad implications for brain disease . A defecJP and KA wrote the article and compiled the figure. JP conceptualized the study. Both authors contributed to the article and approved the submitted version.


The beginning decades of the 21st century have been marked by multiple emergence and re-emergence phenomena of viral diseases. Climate change and changes in ecosystems due to biodiversity loss and modifications in land use pose environmental threats to human and animal health [Flavivirus is a genus of positive-strand RNA viruses in the family Flaviviridae that are mainly transmitted by the bite of an infected arthropod (such as a mosquito or tick) and hence are classified as arboviruses. Amongst the instances which deserve special attention are the recent expansion of emerging neurotropic arthropod-borne viruses, such as West Nile virus (WNV), Usutu virus (USUV), and tick-borne encephalitis virus (TBEV), beyond their natural range of distribution, especially in Europe.Therefore, in the last decade, the number of emerging flaviviruses identified worldwide has considerably increased, causing a rise in human outbreaks which represents an emerging threat to human health . FlaviviUSUV and WNV are characterised by overlapping geographic distribution, host and vector species, and similar clinical manifestations. They are maintained in the environment through an enzootic cycle involving mosquitoes and birds, but they also occasionally infect humans and other mammals. Disease symptoms in vertebrates can range from mild febrile illness to severe neurological disorders . USUV and WNV co-circulate in parts of southern Europe, but the distribution of USUV extends into central and northwestern Europe. Few European countries implement routine surveillance programmes, and the majority of USUV and WNV infections in humans are asymptomatic, making it likely that the circulation of these viruses is underestimated. Despite this, it is now documented that USUV is rapidly spreading in several European countries and that WNV has widely spread in central and southern European countries, causing larger epidemics ,5. A larFor its part, TBEV is one of the most frequently reported tick-borne diseases in Europe. It is endemic in many European countries, including the Baltic and Scandinavian countries, Slovenia, and the Czech Republic. The rise in the number of reported cases in recent years is likely driven by several factors, including improved diagnostic tests, increased disease awareness, an increase in time spent outdoors, as well as rising temperatures. TBEV is maintained in an enzootic cycle involving hard ticks and rodents as the main reservoir . It is sThe control of arboviruses expansion relies mainly on surveillance and the adoption of preventive measures where cases are detected. The spatial and temporal overlap of flaviviruses poses significant challenges for surveillance and control, highlighting the need for reinforced measures of monitoring and surveillance programmes. Indeed, the current situation calls for a rigorous analysis of the epidemiological situation in Europe. This information would be essential to the design of future surveillance strategies. One of the main public health issues related to the expansion of flaviviruses is the associated neurological damage. Numerous flaviviruses, such as WNV, USUV, and TBEV, can reach the CNS and cause neuronal defects through pathogenic mechanisms which remain largely unknown. Understanding the mechanisms behind neuroinvasion and its associated neuroinflammation during arbovirus infection is urgent, as symptoms can prove severe, trigger long-term impairment, and sometimes even be fatal . TherapeThis Special Issue entitled “Usutu, West Nile, and tick-borne encephalitis viruses” assembles a collection of original research articles and scientific reviews highlighting the critical advances in our understanding of all aspects of viral infection related to these arboviruses (or other neurotropic flaviviruses), including their cellular, molecular, and immunological aspects, alongside epidemiological, vector competence, and diagnostic studies.


To evaluate the application of hydrogel scaffold materials and triple antibiotic paste in endodontic regeneration through literature review. Results and Conclusion. Hydrogels regulate stem cell fates, modulate growth factor release, and encapsulate antibacterial and anti-inflammatory drugs. The triple antibiotic paste is composed of metronidazole, ciprofloxacin, and minocycline, which exhibits promising antibacterial effects and duration at appropriate concentrations, with low cytotoxicity, and effectively promotes the preservation and regeneration of pulp tissues and the formation of dental hard tissues. However, issues such as tooth discoloration and bacterial drug resistance also exist. The present article reviews the progress of research on the application of hydrogel scaffold materials and triple antibiotic paste in endodontic revascularization. An electronic search of the literature published on PubMed, Wangfang database, and CNKI database using the search terms “endodontic regeneration,” “pulp blood flow reconstruction,” “recanalization,” “triple antibiotic paste,” and “scaffold material” was conducted. The searched literature was used for analysis.  Pulpal periapical disease is a common disease in dentistry . EndodonPulpal periapical disease is conventionally treated by apical induction molding followed by filling with nonbiologically active material . HoweverThis study focuses on the investigation and analysis of hydrogel scaffold materials and triple antibiotic paste in endodontic regeneration. The results are as follows.Collagen-based hydrogels are one of the earliest and most widely used hydrogel scaffold materials of biological origin . CollageHyaluronic acid-based hydrogels: hyaluronic acid is a proteoglycan that forms the extracellular matrix, and due to its richness in hydrophilic groups, it can be chemically modified and chemically cross-linked to form hydrogel materials under mild conditions with excellent biocompatibility . A caseiChitosan-based hydrogels are deacetylated products of the natural polysaccharide chitin, a polysaccharide composed of randomly distributed N-acetylglucosamine and glucosamine units, which forms chitosan-based hydrogels by physical association or covalent cross-linking and obtains the desired pore size, degree of cross-linking, and solubility precisely. Studies have shown that chitosan-based hydrogels constitute a dexamethasone release system that can modulate the drug release rate and can also be used as a slow/controlled release carrier for a variety of drugs due to their good biocompatibility, biodegradability, and natural antibacterial effects , 19, andSelf-assembled peptide hydrogels (SAP): in tissue engineering applications, SAP hydrogels feature good biocompatibility and degradability and can build nanoscale scaffolds and slow-release systems , which aDecellularized matrix hydrogels: decellularized matrix hydrogels are scaffold materials that have been well studied in the last decade and are more bionic in composition and structure, offering unparalleled advantages and potential . A decelThe triple antibiotic paste is a paste made of metronidazole, ciprofloxacin, minocycline, and wood slip oil .Metronidazole is a highly effective and less expensive drug of the nitroimidazole class, which is strongly potent against various G+, G-, and anaerobic bacteria and mainly targets anaerobic infections to kill specialized anaerobic bacteria. Metronidazole is selectively toxic to anaerobic microorganisms. Upon entry into the bacteria, the nitro group in the drug molecule is reduced by certain redox proteins to highly reactive nitro radicals, which are capable of disrupting the helix structure of deoxyribonucleic acid (DNA), leading to rapid bacterial death. An analytical study showed that the use of metronidazole as an adjunct to nonsurgical treatment of aggressive periodontitis resulted in better clinical outcomes.Ciprofloxacin is a broad-spectrum antibacterial drug that belongs to the 3rd generation of quinolone drugs, with strong antibacterial activity, is less liable to produce resistance, and can be used in combination with metronidazole and other drugs for root canal disinfection. Ciprofloxacin inhibits DNA-associated substances such as DNA procyclase in the nucleus of bacteria, causing degradation of DNA and thus producing a bactericidal effect. Ciprofloxacin has very strong antibacterial activity against Gram-negative bacteria, but Gram-positive bacteria and most anaerobic bacteria are resistant to ciprofloxacin. Therefore, ciprofloxacin is often used in combination with metronidazole to treat mixed infections to compensate for the limited antibacterial spectrum. In addition, although ciprofloxacin can cause side effects, the drug can be used safely in clinics at low dose effects.Minocycline, also known as semisynthetic tetracycline and dimethylaminotetracycline, is a broad-spectrum antimicrobial drug, with fewer resistant bacteria, and only a small amount of use can produce a high local drug concentration, with high efficiency and long-lasting effect. Minocycline accompanies the passive diffusion of cell membranes across the outer membrane of bacteria and invades the inner membrane through active transport to reach the surface of ribosomes within the bacterium and inhibit protein synthesis, ultimately leading to bacterial death. Because minocycline tends to cause discolouration of the tooth structure, it has also been studied as a replacement for other antibiotics to form modified TAP.Triple antibiotic paste possesses strong antimicrobial activity, high penetration, low irritation, less cytotoxicity while exerting antimicrobial disinfection, and good biocompatibility to promote pulp regeneration . CurrentIn tissue engineering, how to achieve a uniform distribution of cells in the scaffold after implantation into the scaffold and how to place the scaffold into the site of action are the key issues that remain to be addressed , which aRegulation of stem cell fate: biomaterials in direct contact with stem cells largely influence the behaviour and differentiation fate of stem cells, including the physical and chemical properties of the material. During endodontic regeneration, hydrogel scaffold materials are modified to promote cell adhesion and migration, increase the rate of material degradation, and maximize the induction of stem cell differentiation for endodontic regeneration. Modulation of growth factor release: it has been reported that inadequate blood supply constitutes one of the challenges for pulp regeneration, and that in spite of sufficient dentin-derived growth factors to promote dentinogenic differentiation of stem cells, insufficiency exists to provide vasculogenic differentiation of stem cells. The direct use of growth factors that promote angiogenesis is associated with failure to maintain long-lasting effects due to their short half-life and rapid metabolism in vivo. In a study using a hydrogel scaffold with cell adhesion and matrix degradation, adding a series of growth factors to the hydrogel via heparin binding and encapsulating pulp stem cells in the scaffold material showed better pulp stem cell proliferation and spreading, superior intercellular cluster formation, and collagen secretion.Loading of anti-inflammatory drugs: hydrogels can absorb large amounts of water to encapsulate therapeutic drugs to prevent their rapid degradation and control the release rate of encapsulated drugs. Numerous studies have reported that self-assembled peptides can mimic the extracellular matrix structure, promote the growth and differentiation of pulp stem cells, and serve as a satisfactory vehicle for the attachment of growth factors. Colombo et al. predicted that self-assembled peptide hydrogels could be a reliable vehicle for the attachment of abscisins, and by adding bioactive scaffold materials, they could achieve the slow release of abscisins, modulate the inflammatory state of the pulp cavity, and regenerate the pulp-dentin complex.Loading of antimicrobial drugs: after total pulp necrosis, the bacterial biofilm colonizing the root canal wall is difficult to be completely removed due to the complexity of the root canal system. A foreign study reported for the first time that injectable nanofiber hydrogel slow-release antimicrobial drugs (triple antibiotic paste) are available to avoid direct contact of high local concentrations of drugs with stem cells around the apical foramen, and its sustained release can provide a relatively sterile microenvironment in the root canal, resulting in more stable pulp regeneration.The advantages of the triple antibiotic paste are high antibacterial activity, high penetration, long duration of action, and low cytotoxicity, which facilitate the formation of hard tooth tissues and the continued development of the root. Most of the bacteria in the dentin tubules are anaerobic and are the target bacteria for root canal disinfection. The metronidazole in the triple antibiotic paste kills specialized anaerobic bacteria, and is unassociated with dysbiosis and drug-resistant strains, with no contraindications to the combined application of other antibiotics. Its ineffectiveness against parthenogenic anaerobic bacteria can be compensated by minocycline. Minocycline is effective against both parthenogenic anaerobic bacteria and anaerobic bacteria, with stable efficacy and little damage to adjacent tissues, and promotes regeneration of adjacent tissues. Ciprofloxacin is available in combination with metronidazole and other drugs thanks to its broad-spectrum antimicrobial properties and a good bactericidal effect. Based on the pharmacological properties of the three antibiotics, the combination of the three drugs achieved a good root canal disinfection effect .Tooth discoloration: during pulpal vascular regeneration treatment, minocycline chelates with Ca2+ in dentin to form insoluble complexes are deposited in the hard tissues of the teeth resulting in tooth discoloration. Porter et al. found that triple antibiotic paste containing doxycycline resulted in lighter dentin staining than triple antibiotic paste containing minocycline, which can be prevented by presealing the dentin tubules through acid etching bonding to inhibit chelation and avoid tooth staining. Alternatively, the use of an injectable nanofiber hydrogel slow-release triple antibiotic paste to avoid direct contact between the high local concentrations of the drug and the stem cells around the apical foramen is also available for the prevention of tooth discoloration. Moreover, Porter et al. also proposed the replacement of minocycline with double antibiotic paste or with other generic antibiotics such as amoxicillin and cefaclor to reduce dentin staining. Results of previous studies have confirmed that the substitution of minocycline with cefaclor and its application in the teeth of animals obviated tooth discoloration.Destruction of the dentin surface: the results of several studies have shown that triple antibiotic pastes can significantly reduce the hardness of the dentin surface and induce demineralisation of the dentin surface. Yasen et al. found that triple antibiotic paste had a significantly stronger effect on the root canal wall roughness than sodium hypochlorite solution and calcium hydroxide. The countermeasure may involve the selection of an appropriate concentration of triple antibiotic paste or the use of a drug-laden scaffold to slow-release drugs to protect the root canal and reduce erosion.Bacterial resistance and drug allergic reactions: given the complexity of microorganisms in the root canal, prolonged use or high concentrations of the triple antibiotic paste may produce resistant strains and lead to reinfection and treatment failure. Allergic reactions to drugs such as urticaria or pruritus have been reported with metronidazole, oral mucosal oedema with ciprofloxacin, and pneumonia or eosinophilia with minocycline. Therefore, bacterial culture, drug sensitivity test, exploration for appropriate triple antibiotic paste concentration, and application length are essential. Drug allergic reactions are mostly associated with systemic medications and uncommon with topical medications, for which dentists should be vigilant to protect the physical and mental health of patients.Research on hydrogel scaffold materials has focused on inflammation control, promotion of blood supply, and mimicking the microenvironment of healthy pulp tissues. In recent years, although hydrogel scaffold materials have made milestones in the study of pulp regeneration, the efficient and stable generation of functional pulp-dentin complexes still needs to be explored. The ideal hydrogel scaffold material should have excellent biological properties in endodontic regeneration, mimic ECM, participate in the release of signaling molecules, modulate the behavior of stem cells, and possess excellent physicochemical properties . In termIn summary, it is currently feasible to obtain stable endodontic regeneration by using injectable nanofiber hydrogel slow-release triple antibiotic paste. A multidisciplinary combination of biomaterials, biomedicine, and tissue engineering is necessary to achieve functional endodontic regeneration in the future to realize the shift from laboratory to clinical practice.


Depression and anxiety disorders are the two most prevalent psychiatric diseases that affect hundreds of millions of individuals worldwide. Understanding the etiology and related mechanisms is of great importance and might yield new therapeutic strategies to treat these diseases effectively. During the past decades, a growing number of studies have pointed out the importance of the stress-induced inflammatory response in the amygdala, a kernel region for processing emotional stimuli, as a potentially critical contributor to the pathophysiology of depression and anxiety disorders. In this review, we first summarized the recent progress from both animal and human studies toward understanding the causal link between stress-induced inflammation and depression and anxiety disorders, with particular emphasis on findings showing the effect of inflammation on the functional changes in neurons in the amygdala, at levels ranging from molecular signaling, cellular function, synaptic plasticity, and the neural circuit to behavior, as well as their contributions to the pathology of inflammation-related depression and anxiety disorders. Finally, we concluded by discussing some of the difficulties surrounding the current research and propose some issues worth future study in this field. Depression and anxiety disorder are the most common mental diseases affecting hundreds of millions of people worldwide . The comIn recent years, many studies have consistently shown that stress-induced neuroinflammation and subsequent changes in the brain regions involved in emotion regulation play an essential role in the pathological development of neuropsychiatric diseases including depression and anxiety disorders ,9,10,11.Accumulating evidence from the past decades has shown that inflammation induces psychological and neuropathological disorders by influencing neuronal excitability, neurotransmitter release, receptor, and transporter expression through peripheral hormones and autonomic responses ,23. A nuIn this review, we first summarized the mechanisms of stress-induced inflammation  and the crosstalk between peripheral inflammation and neuroinflammation in the brain. Then, the effects of inflammation on the functional changes in the amygdala and the relationship between inflammation and stress-related depression and anxiety disorders were systematically discussed from the perspectives of molecular signaling, cellular function and neural plasticity, circuit and behavioral output. Finally, we addressed some of the current issues and thoughts surrounding the amygdala research on the mechanism of inflammation-induced depression and anxiety disorders and proposed some research questions worthy of future research in this field.All living organisms strive to reach and maintain a dynamic equilibrium state called homeostasis, which is threatened by real or perceived physical, emotional, or psychological events, known as “stressors” ,32. FromIn recent years, numerous studies have indicated that the stress-induced activation of macrophages and microglia, which in turn leads to aberrant peripheral and central inflammation, plays a pivotal in the pathogenesis of mental diseases. Most noteworthy, inflammation can occur throughout the lifespan, making it a high risk for depression and anxiety disorders. For example, a recent study showed that exposing mice to LPS during early developmental stages increases the capacity of microglial engulfment, which induces long-lasting neuronal maladaptation, thus promoting the development of depression-like symptoms during adolescence .During injury, disease, or infection by bacteria and viruses, the immune system is activated, and plenty of pro-inflammatory cytokines are produced and released by immune cells such as macrophages and T and B lymphocytes, which act as mediators of the innate immune response. Notably, a growing body of studies have shown that psychological or emotional stress can also trigger the activation of the peripheral immune system, leading to the release of multiple pro-inflammatory cytokines  Figure . PsycholIt should be noted that stress is common throughout life. The level of stress-induced inflammation and its influence on emotional behavior are closely related to stress intensity. In the face of acute stress, the body’s immune system will respond quickly in a short time by releasing inflammatory factors in response to stress. However, pro-inflammatory cytokines usually disappear after completing their task; when the stress persists (chronic stress), these cytokines continue to increase. Then, the persistently existing cytokines begin to have harmful effects on the body and induce changes in the brain regions related to emotion regulation, which subsequently cause mental illnesses such as depression and anxiety disorders.Unlike peripheral inflammation responses mediated by macrophages, central inflammation is mainly caused by microglia activation. Microglia are resident macrophages of the central nervous system (CNS), which constitute 5–10% of total brain cells and are the brain’s first line of defense against trauma, pathogens, infection, or brain diseases, a function similar to peripheral macrophages ,45. MicrAccumulating evidence from animal studies has revealed that restraint stress, unavoidable footshock stress, and chronic unpredictable stress can lead to the aggregation and activation of microglia in emotion-related brain regions such as the amygdala, prefrontal cortex, and hippocampus in rodents ,53,54. ONumerous animal studies have shown that peripheral inflammatory stimuli can activate microglia cells in the brain . Due to Notably, cytokines cannot be passively diffused across the BBB because of their high molecular weight and hydrophilic properties, but they can enter the brain parenchyma through the humoral route . These pIn addition to the humoral routes, cytokines can also target the autonomic nerve afferents that innervate the infection site and transmit signals into the brain through the vagus nerve  Figure . The vagClinical and animal studies have consistently shown that peripheral inflammation can disrupt the normal function of the BBB through multiple pathways, leading to secondary inflammatory responses in the CNS and the developing of neurological diseases . The BBBStress-induced inflammatory processes and microglia-activation-related neuroinflammation have numerous effects on the structure and function of neurons in the brain and are associated with most pathophysiological diseases in the CNS ,45. IncrThe amygdala was first identified by Burdach in the early 19th century. Burdach originally described a group of cells that are now known as the basolateral complex . SubsequThe LA is traditionally known as the primary target of multimodal sensory information from the thalamus and cortex ,84. UponThe amygdala plays an essential role in regulating the activation of the HPA axis and SAM axis, which are hallmarks of the stress response . It shouEmerging evidence suggests that the hyperactivity of amygdala neurons is a fundamental cause of chronic stress-induced anxiety disorder and depression. Neuroimaging studies have shown that amygdala activation in patients with anxiety disorders is significantly higher than that in controls in response to the same stimulus , which iThe refined synaptic communication between excitatory and inhibitory synapses is essential for regulating neural activity and normal brain function . An exciIncreasing evidence indicates that elevated pro-inflammatory cytokine levels caused by the recruitment of peripheral cytokines to the brain and microglia activation play an essential role in the development of stress-induced anxiety disorder and depression. The possible mechanisms may involve the regulation of neurotransmitter metabolism, the influence of neuroendocrine function, synaptic plasticity, and the neural circuitry of emotion ,98. For A α2 and GABAA γ2 was significantly decreased [A α2 and GABAA γ2 were dramatically increased in the BLA of CFA-treated mice [Numerous studies have implicated that inflammation-related anxiety disorders are associated with the aberrant activation of amygdala neurons. For example, studies have shown that in an inflammatory pain mouse model induced by complete Freund’s adjuvant (CFA) injection, the anxiety-like behavior in mice was markedly increased, which is associated with the upregulation of the excitatory postsynaptic receptors NMDA receptor and AMPA receptor in the BLA, as well as the postsynaptic dense protein PSD95, while the expression of inhibitory receptors GABAecreased . Howeverted mice . In termted mice . While cted mice . These fted mice . Howeverted mice . The AMPted mice . The exated mice ,94. GiveARα2 subunit expression and GABA-induced current density, and ultimately caused the disruption of the E/I balance and the shift toward excitation, resulting in anxiety disorder [The specific mechanisms of how inflammation affects the BLA neuronal function and the occurrence of anxiety disorders are still vague. Such an effect may likely be related to the effect of cytokines on monoamine and glutamate. For example, studies showed that neonatal LPS treatment reduced TGF-β1 expression in the BLA during adulthood, which lead to the down-regulation of GABAdisorder . Therefodisorder . In addidisorder , while tdisorder . In vitrdisorder . Furtherdisorder . These fThe CeA also plays a vital role in regulating the immune stress response. The CeA was activated in male and female rats after LPS injection 14 days after birth . CeA lesAlthough the vast majority of studies revealing the role of the amygdala in modulating emotional function have been focused on fear and anxiety, recent studies have shown that amygdala inflammation has an undoubtful function in depression. For example, intravenous or subcutaneous IL-1β injection increased depression-like behavior in mice and enhanced the expression of pro-inflammatory cytokines TNF-α and IL-6 in the amygdala, which is regulated by the CORT/GR system. Furthermore, serum corticosterone levels were markedly increased with IL-1β injection, while GR inhibitor RU486 reduced IL-1β-induced depression-like behavior and TNF-α and IL-6 expression . In addiIn mammals, the downregulation of serotonin (5-HT) levels in the CNS has been implicated in the pathophysiology of depression. With the development of the cytokine theory of depression, it has been well documented that pro-inflammatory cytokines, including IL-1β, IFN, and TNF can reduce the bioavailability of neurotransmitters such as serotonin (5-HT) . The synA large amount of evidence has shown that inflammatory response is accompanied by IDO dysfunction in depression ,144. EarThe structural and functional connectivity between the amygdala and other emotion-related brain regions, such as the mPFC, hippocampus, and anterior cingulate cortex, play crucial roles in stress-induced anxiety and depressive behaviors ,148,149.It is worth noting that many studies have shown that the abnormal activation of the mPFC-BLA-vHPC circuit contributes to chronic stress-induced anxiety ,96,153, There is also evidence showing that changes within CeA circuit are implicated in inflammatory-related mental disease. For example, a recent study showed that in a mouse model of sepsis induced by intraperitoneal infection, the mice exhibited anxiety-like behavior and exaggerated fear memories, which are similar to anxiety and PTSD-like symptoms seen in human sepsis survivors . It was A growing body of evidence shows that stress-mediated immune response and neuroinflammation contribute to anxiety disorder and depression upon stress exposure, which is closely related to the structural and functional remodeling of the amygdala. Nevertheless, there are still a series of unsolved questions that need further investigation. Foremost, a critical question that needs to be addressed is how amygdala inflammation is incorporated into the development of depression and anxiety disorders. Although numerous studies have indicated that inflammation-induced anxiety and depression are accompanied by microglia activation and inflammatory gene expression in the amygdala, the causal relationship between amygdala inflammation and the development of psychiatric disorders is still vague. Second, there is still conflicting evidence of stress in the inflammatory response and neuronal function in the amygdala. For example, footshock stress has been shown to change the activity of BLA neurons in the amygdala to mediate the occurrence of anxiety disorder, while a recent study found that footshock did not induce inflammation in the amygdala, thus raising an intriguing question that the inflammation-induced functional remodeling and behavioral changes in the amygdala caused by psychogenic stress are associated with the stress type. More comprehensive studies are needed in the future to help better understand the immune-neuro-psychological role of the amygdala. Third, since it has been widely documented that communication between neurons and microglia is highly coordinated, the possible interaction between these cells in the amygdala and the potential mechanism in the pathology of depression and anxiety disorder is a venue meriting further exploration. Fourth, the distinct neural circuits in the amygdala are implicated in different emotional behavior outputs; whether those circuits are differentially affected by inflammation remains an open question, which adds another layer of complexity toward understanding the exact role that amygdala inflammation plays in stress-induced depression and anxiety disorder. Further exploration of these questions will improve our understanding of the pathogenesis of mental diseases and help to develop better treatments to improve the quality of life of individuals suffering from anxiety disorders and depression.


Extracellular vesicles (EVs) are tiny biological nanovesicles ranging from approximately 30–1000 nm in diameter that are released into the extracellular matrix of most cell types and in biofluids. The classification of EVs includes exosomes, microvesicles, and apoptotic bodies, dependent on various factors such as size, markers, and biogenesis pathways. The transition of EV relevance from that of being assumed as a trash bag to be a key player in critical physiological and pathological conditions has been revolutionary in many ways. EVs have been recently revealed to play a crucial role in stem cell biology and cancer progression via intercellular communication, contributing to organ development and the progression of cancer. This review focuses on the significant research progress made so far in the role of the crosstalk between EVs and stem cells and their niche, and cellular communication among different germ layers in developmental biology. In addition, it discusses the role of EVs in cancer progression and their application as therapeutic agents or drug delivery vehicles. All such discoveries have been facilitated by tremendous technological advancements in EV-associated research, especially the microfluidics systems. Their pros and cons in the context of characterization of EVs are also extensively discussed in this review. This review also deliberates the role of EVs in normal cell processes and disease conditions, and their application as a diagnostic and therapeutic tool. Finally, we propose future perspectives for EV-related research in stem cell and cancer biology. Diverse communication systems mediate intercellular communication, both in physiological and pathological conditions, such as cellular junctions , integrins, and selectins  with the plasma membrane during the differentiation of reticulocyte for the first time , Nanog, Rex-1, stem cell leukaemia (SCL), and GATA binding protein 2 (GATA-2)) and proteins (such as Wnt family member 3 (WNT3) and OCT4) induce the overexpression of genes responsible for pluripotency. The mESCEVs increase survival and expansion of HPCs by inducing phosphorylation of mitogen-activated protein kinases (MAPK) p42/44 and serine-threonine kinase AKT. Further, the self-renewal phenotype of HPCs diminishes after inhibition of the function of mESCEVs by heat inactivation or pre-treatment with RNase, suggesting the critical effect of the protein or mRNA cargo within the mESCEVs on maintaining stemness of HPCs  facilitate the sustenance of self-renewal and expansion of adult stem cells; hematopoietic progenitor cells (HPCs) via delivery of specific mRNAs  and Drosophila . Cuticle development is blocked upon depletion of V-ATPase, suggesting that EV-mediated release of Hh-related proteins in C. elegans provides a unique way to transport lipid-modified or transmembrane proteins during development -EVs release  and Hh signalling , multicellular organisms , and mammals  of in vivo condition), can be isolated and cultured as ESCs with the capacity of complete development. The ICM plays a crucial role by receiving various autocrine- or paracrine- signals to facilitate the processes that lead to embryogenesis , EGF, FGF, Wnt, and bone morphogenetic proteins  of the blastocyst, which produces the entire embryo (The core components of the hedgehog (Hh) signalling pathway are Hh ligand, transmembrane receptor Patched 1, intracellular G-protein Smoothened and transcription factors GLI1-3. Tanaka et al. showed that FGF signalling promotes secretion of EVs containing SHh and retinoic acid at the node of the gastrula’s primitive streak. They also revealed that fluid flow at the node promoted EV movement towards the left in embryos. The asymmetric accumulation of EVs specifically activated the non-canonical Hh signalling pathway in the left of the embryo, resulting in differential changes to intracellular calcium levels and gene expression, leading to a left-right gradient of morphogens  belong to the superfamily of transforming growth factor-beta (TGF-β), responsible for morphogen-regulating organization of the body axis, tissue patterning, and development and preservation of stem cell niches at the time of embryonic development in diverse species. On the surface of the cell, BMPs bind to the receptors; bone morphogenetic protein receptors (BMRPs), by promoting the phosphorylation of members of the SMAD family that are translocated into the nucleus and regulate the expression of downstream genes , including the brain. The intercellular communication via neuronal EVs in neurons is vital in supporting various functions of the nervous system including myelination and maintenance of axonal integrity. The release of EVs is promoted by glutamate neurotransmitter via stimulation of ionotropic glutamate receptors, followed by the uptake of these EVs that transport their constituents to neuronal cells. Further, the released EVs from oligodendrocytes can protect neurons by elevating their tolerance to stress , cardiac muscle cells release EVs, especially under hypoxic conditions  effector lymphocytes in human leukocyte antigen A2 (HLA-A2) transgenic mice (HHD2). Therefore, EVs could be associated with the transfer mechanism of functional MHC class I/peptide complexes to DCs for effective activation of cytotoxic T lymphocytes (CTL) in vivo . In the main, DC-derived EVs express major histocompatibility complex (MHC)-I, MHC-II, and co-stimulating factor  dramatically reduces release of EVs in a mouse model of colon carcinoma, because these two channels control intracellular calcium levels , favorably localized on the membrane of EVs via its affinity to heparin. The interaction between VEGF189 and EVs significantly augmented the half-life of the ligand and decreased its recognition by bevacizumab (a VEGF antibody). Therefore, tumor angiogenesis stimulated by EV-associated VEGF (VEGF-EVs) could not be neutralized by bevacizumab. Moreover, the augmented VEGF-EVs were linked to lethality of disease when patients were treated with bevacizumab, suggesting that resistance to bevacizumab was potentially due to raised amounts of VEGF-EVs , and Total Exosome Isolation reagents (Thermo Scientific). The essential process requires mixing samples with reagents followed by simple centrifugation steps. The precipitation method is useful for isolating exosomes from low volume samples. Nonetheless the reagents for exosome isolation become expensive if the process is scaled-up for a large sample size , and micro-RNA  has recently been applied in EV research  non-invasively, and continuous time-dependent monitoring of a patient’s response is possible with EV-based therapeutics. Because of their small size, EVs can cross physiological barriers, including the BBB, making them useful in drug delivery. The benefits of EVs in therapy of various diseases including cancer have also been studied. Other critical aspects of the application of EVs involve their self-renewal capacity, ability to arbitrate communication between stem cells and niche, and their functions in stem cell differentiation or cell fate determination. The role of EVs in normal physiological processes and disease conditions has also been extensively studied, including malignant progression of cancer. The exploration of various multidimensional roles of EVs have become possible with technological advancements in methods for isolation and characterization of EVs, and the development of advanced microfluid based systems for studies associated with EVs.The role of EVs has yet to be thoroughly explored. In the study of disease progression via intercellular communication of EVs, the potential of EVs to carry various cargoes to recipient cells warrants attention. In the context of cancer progression, there are several drugs such as paclitaxel, which causes neuropathic pain, or drugs against which drug resistance has developed such as temozolomide for glioma, that could be a cargo for EVs. Furthermore, the exploration of potential changes in the molecular constituents of stem cells, after uptake of EVs released from a different subset of cancer cells, may help future research into the mechanisms that underlie the development of drug resistance.The use of EVs as a carrier for either genetic materials such as lncRNA, miRNA, or small molecules such as anti-cancer drugs, holds great promise for the development of novel delivery methods. One of the important advantages of EVs as a delivery carrier is their ability to escape the reticuloendothelial system (RES). Nonetheless the synthetic nano-delivery systems are often prone to be captured by RES and therefore very likely to prompt an immune response. In the prevailing scenario, the use of EVs, particularly autologous EVs, has strong potential for targeted delivery after surface modifications with specific ligands against the target receptor, for example, epidermal growth factor receptor (EGFR) in lung cancer and EGFRvIII in glioma.The application of EVs to track disease progression, including metastatic progression of cancer, is an exciting prospect. The conventional approaches to diagnosis of cancer, such as investigation of molecular changes in tissues via biopsy, imaging techniques like magnetic resonance imaging or computed tomography in glioma, and digital rectal examination in prostate cancer, are limited by their resolution in detecting the development of tumor. In the prevailing scenario, the development of biosensors is required to detect EVs and their constituents with high sensitivity and specificity for tracking disease diagnosis. Additionally, mapping of various proteins on the surface of EVs could be used to potentially demarcate the disease state.Although there is significant ongoing research associated with EVs, there remains a need to develop a universal protocol for their isolation and characterization, as well as for their clinical application. When available, it could be certified and applicable for good clinical practice (GMP). A specific road map could be developed for the translation of EVs from bench to bedside.


Among the different air pollutants, particulate matter (PM) is of great concern due to its abundant presence in the atmosphere, which results in adverse effects on the environment and human health. The different components of PM can be classified based on their physicochemical properties. Carbonaceous particles (CPs) constitute a major fraction of ultrafine PM and have the most harmful effects. Herein, we present a detailed overview of the main components of CPs, e.g., carbon black (CB), black carbon (BC), and brown carbon (BrC), from natural and anthropogenic sources. The emission sources and the adverse effects of CPs on the environment and human health are discussed. Particularly, we provide a detailed overview of the reported toxic effects of CPs in the human body, such as respiratory effects, cardiovascular effects, neurodegenerative effects, carcinogenic effects, etc. In addition, we also discuss the challenges faced by and limitations of the available analytical techniques for the qualitative and quantitative detection of CPs in atmospheric and biological samples. Considering the heterogeneous nature of CPs and biological samples, a detailed overview of different analytical techniques for the detection of CPs in  biological samples is also provided. This review provides useful insights into the classification, toxicity, and detection of CPs in biological samples.  Th10 . . 249]. TCPs, being a major fraction of ultrafine PM, are abundant in the atmosphere and originate from different anthropogenic and natural sources. Their abundant presence in the atmosphere not only contributes to environmental impacts such as global warming (by their absorption of solar radiation) but also presents a likely pathway for their inhalation by humans. The inhalation of CPs by humans results in a wide range of adverse effects, ranging from respiratory to carcinogenic effects, as ultrafine CPs can translocate through the bloodstream to different organs. The qualitative and quantitative detection of CPs in atmospheric and biological samples is very important to understand their adverse effects. Different analytical techniques such as laser-induced incandescence and optical absorption are used for CPs’ detection in gaseous samples. However, CPs’ detection in biological samples is very challenging due to their heterogeneous nature and complexity associated with biological samples. The different analytical techniques used for CPs’ detection in model studies in biological samples include radiolabeling detection, electron microscopy, mass spectrometry, etc. Whereas Raman spectroscopy and FPLM are employed for CP detection in models and real-exposure samples.Different techniques such as FPLM or Raman spectroscopy are very sensitive for the qualitative and quantitative detection of CPs in real-exposure biological samples; however, these microspectroscopy techniques are very labor-intensive and time-consuming due to the related sample preparation, microscopy measurements, and data analysis. Therefore, to detect CPs in biological samples for clinical applications, there is still a need for the development of robust, sensitive, and time-effective analytical techniques. There is also growing evidence that the harmful effects of PM pollutants are size-dependent, in wherein ultrafine particles are the most harmful . Hence, Although BC and CB constitute a major fraction of CPs, recently, BrC has also received significant attention from scientists. The complexity and heterogeneity of CPs, the variability of their molecular properties, and the nature of their emission sources pose great challenges to their detection and discrimination in biological samples. Hence, there is a need for the development of advanced and sensitive analytical tools for the qualitative and quantitative detection and discrimination of different components of CPs to understand their adverse effects on human health. The currently available approaches will require optimization to not only detect very small particles (<100 nm) but to also discriminate between different CPs for diagnostic purposes. In addition to understanding the adverse effects of CPs on human health, their atmospheric concentrations from different anthropogenic sources need to be controlled by better policy making (and implementation) at the regional and national levels. Recently, the WHO released new Global Air Quality Guidelines intending to save millions of lives from the harmful effects of air pollution. Among the different constituents of air pollution, particulate matter (PM) is on the top of the agenda of the WHO guidelines. In addition, the EU key directive (2008/50/EC) on ambient air quality and cleaner air for Europe also stressed reducing air pollution levels to minimize the adverse effects on human health.


Circular RNAs (circRNAs) are covalently closed single-stranded RNA molecules, which have many biological functions. Previous experiments have shown that circRNAs are involved in numerous biological processes, especially regulatory functions. It has also been found that circRNAs are associated with complex diseases of human beings. Therefore, predicting the associations of circRNA with disease  is useful for disease prevention, diagnosis and treatment. In this work, we propose a novel computational approach called GGCDA based on the Graph Attention Network (GAT) and Graph Convolutional Network (GCN) to predict circRNA-disease associations. Firstly, GGCDA combines circRNA sequence similarity, disease semantic similarity and corresponding Gaussian interaction profile kernel similarity, and then a random walk with restart algorithm (RWR) is used to obtain the preliminary features of circRNA and disease. Secondly, a heterogeneous graph is constructed from the known circRNA-disease association network and the calculated similarity of circRNAs and diseases. Thirdly, the multi-head Graph Attention Network (GAT) is adopted to obtain different weights of circRNA and disease features, and then GCN is employed to aggregate the features of adjacent nodes in the network and the features of the nodes themselves, so as to obtain multi-view circRNA and disease features. Finally, we combined a multi-layer fully connected neural network to predict the associations of circRNAs with diseases. In comparison with state-of-the-art methods, GGCDA can achieve AUC values of 0.9625 and 0.9485 under the results of fivefold cross-validation on two datasets, and AUC of 0.8227 on the independent test set. Case studies further demonstrate that our approach is promising for discovering potential circRNA-disease associations. Circular RNAs (circRNAs) produced by reverse splicing of pre-mRNAs are single-stranded, covalently closed RNA molecules, which are lacking a 5′ cap and a 3′ polyadenylated tail . HoweverIn recent years, through various biological experiments and studies, more and more manually curated databases have been designed for studying circRNAs and the associations between circRNAs and diseases. These databases fall into two broad categories. The first category is databases of recording circRNA annotation resources, such as circBase , CircNetGGCDA constructs a novel deep learning model based on multi-head GAT and GCN. Specifically, the multi-head Graph Attention Network is used to obtain different weights of circRNA and disease features, and GCN is used to aggregate the features of adjacent nodes in the network and the features of the nodes themselves. So, it can learn not only the node feature information, but also the information contained in the network structure.Multiple similarities of circRNAs and diseases are integrated as the original features of nodes in the GGCDA, including circRNA sequence similarity, disease semantic similarity and corresponding Gaussian interaction profile kernel similarity.RWR is used to capture the multi-faceted relationship between two nodes, capturing the overall structural information of the graph.An independent dataset that contains more data is used to verify the generalization of GGCDA, besides common comparisons and case studies.In this paper, we propose a novel approach based on the Multi-Head Graph Attention Network and Graph Convolutional Network to predict circRNA and disease associations, called GGCDA. Our model offers the following contributions:https://github.com/hhhhcccc22/GGCDA (accessed on 23 May 2022).Comparisons with several state-of-the-art methods and case studies were applied to verify the performance of the proposed method; the experimental results demonstrate the capability of GGCDA for predicting circRNA-disease associations. The source code and run environment of our model are available at http://bioinfo.snnu.edu.cn/CircR2Disease/ (accessed on 23 May 2022). Based on the original CircR2Disease database, we removed duplicate associations, non-human associations and some circRNA-disease associations of circRNAs with no sequence information in the circBase database or diseases with no disease ontology identity information in the Disease Ontology (DO) database [To measure model performance, we used CircR2Disease, an experimentally validated circRNA-disease association database. The CircR2Disease database includes 739 manually collected circRNA-disease associations from published literature, covering 661 circRNAs and 100 diseases, which can be downloaded from database . ConsequTo validate the stability of the model, we constructed a larger dataset called DATA containing more data information from three databases: CircR2Disease, Circ2Disease and CircRNADisease. After de-duplication and the same operation as above, we integrated the data from these three databases, and finally, the DATA dataset containing 944 associations of 809 circRNAs and 119 diseases was obtained.To investigate the generalization of the model, we tested the models on an independent dataset that was used in the reference . We collIn order to facilitate the expression of the calculation formula, we used To obtain the sequence similarity of circRNAs, we downloaded all the human circRNAs in the circBase database, which contains 140,790 pieces of circRNA sequence information. Then, we extracted the circRNAs sequence information corresponding to the circRNA-disease associations. The similarity between any two circRNAs sequence is calculated based on Levenshtein distance . LevenshTo calculate the semantic similarity of diseases, we downloaded disease terms including DOID, name and associations with parents from the DO database. Given a disease  disease , we useds method .Due to the sparse sequence similarity of circRNAs and the semantic similarity of diseases, we introduced the Gaussian interaction profile kernel  (GIP) siIn order to solve the problem that single similarity of circRNAs and diseases is too sparse and causes the instability of the prediction results, we combined the GIP similarity of circRNAs with sequence similarity, and the GIP similarity of diseases with semantic similarity, respectively. The combination formula is as follows:As mentioned above, t to 0.9 . S^ reprBased on the above data processing, we constructed a heterogeneous network  is aimed at learning high-level feature representation vectors from a graph structure . In applBased on the heterogeneous graph GAT introduced the self-attention mechanism, which represents the weight of adjacent nodes by the attention coefficient, so as to learn the hidden representation of nodes on the graph. To be specific, to learn the importance of the first-order neighbor nodes to a particular node, GAT calculates the attention coefficient for each node through a forward linear transformation, where the attention coefficient The output result of the final node was shown as follows: However, different from GAT, GCN can learn the representation of nodes on the graph level. Its basic idea is to conduct convolution operations on the graph to obtain node embedding. After obtaining the attention features through the GAT, GCN is used to fully aggregate node information from its neighbors and its own node information on circRNA and disease feature, so as to update node features and generate embedding. The specific embedding representation is shown as follows: The above GAT layer and GCN layer can be performed multiple times. Through the hybrid GNN of GAT and GCN, we obtain the potential feature representation of diseases and circRNA. To predict potential circRNA-disease associations, we use fully connected neural networks as classifiers for prediction, as they are widely used in classification. In the previous According to the previous study , we set In the process of model training, we optimize the loss function by cross entropy loss and L2 regularization; here, we randomly select negative sample data with an equal number of positive samples from the association matrix, which may be potential candidates but are labeled as 0, where the loss function defined as follows:In this study, fivefold cross-validation (FFCV) was used to evaluate the performance of the GGCDA model. Specifically, we randomly divided all samples into five subsets. In each fold, four subsets are used as training sets and one subset is used as the test set. Here, the known circRNA-disease pairs are regarded as positive samples, whereas the randomly selected unknown circRNA-disease pairs with the same number of positive samples are negative samples.We adopted evaluation indexes that are frequently used to evaluate the performance of the machine learning model, including Accuracy (ACC), Precision (PRE), Recall (REC) and F1-Score. The larger the above evaluation index value is, the more outstanding the performance of the model is. In order to further explore the model effect, we describe the receiver operating characteristic curve (ROC) and precision-recall curve (PR). From these, the area under the receiver operating characteristic curve (AUC)  and the Since the random allocation of negative samples may lead to slight errors in the experimental results, we repeated the experiment 10 times. From the experimental results listed in In this part, we discuss the impact of model parameters on the performance of GGCDA based on the CircR2Disease database, including the number of attention heads, the number of GAT layers, the number of GCN layers, embedding numbers and the penalty factors.Specifically, the attention model using multiple heads in the GAT layer will be more robust. As shown in Since our model is composed of several sections, in this part, ablation studies were conducted based on the CircR2Disease database. In order to judge the influence of each component on the overall performance of the model, FFCV was performed, and ablation studies were mainly conducted on the following variants. GGCDA without RWR: only circRNA similarity matrix and disease similarity matrix are used instead of features calculated after RWR.GGCDA without GAT: only the convolution features extracted by GCN are used.GGCDA without GCN: only features with attention mechanisms extracted by GAT are used.GGCDA without FC: use the inner product instead of FC to predict.So far, a growing number of researchers have designed numerous models to predict circRNA-disease associations. To demonstrate the superiority of GGCDA, we calculated the AUC values against recently published models on the CircR2Disease dataset, including DMCCDA , NCPCDA In this section, we also used the value of AUC as a comparison indicator. It can be seen from In order to further verify the robustness of the model and explore the performance on a large dataset, we performed FFCV on the DATA dataset.In this experiment, the composition of the model we adopted is the same as the previous one, and there are only differences in the data. The detailed results can be seen in To investigate the generalization of the model, we tested the model on an independent test set, the dataset MNDR, which is described in detail in To further validate the superiority of GGCDA, case studies on multiple diseases were implemented. Specifically, we set all circRNA-disease samples to be validated as unknown and then used GGCDA to generate all association scores for three common diseases: hepatocellular carcinoma, breast cancer and colorectal cancer. Then, we sorted them in descending order according to the scoring of the model, and finally screened out the top 10 circRNAs corresponding to these diseases.The results of case studies on hepatocellular carcinoma , breast Table 6Identifying associations between circRNAs and diseases has important implications for disease diagnosis, treatment and identification of biomarkers. In this work, we propose an effective method based on multi-head GAT and GCN for predicting potential circRNA-disease association, and use three datasets to evaluate the performance of GGCDA. Moreover, we conducted case studies on hepatocellular carcinoma, breast cancer and colorectal cancer. The experimental results demonstrate that our method has suitable performance and can effectively detect more circRNA-disease associations.The superior performance of the GGCDA model can be attributed to several factors. Firstly, GGCDA makes full use of circRNA sequence information and integrates various information to construct a heterogeneous network. Secondly, the graph-based neural network method is used to learn the topology structure and internal information in the network of circRNAs and disease, GAT learns features on bipartite networks and GCN is embedded on weighted similarity networks, so as to obtain the representation of depth features of nodes. Moreover, the random walk with restart algorithm is used to capture the overall structure of the graph information to enhance the feature representation of the similarity network.However, there are some limitations of our model. Firstly, GGCDA only uses circRNA sequence similarity, disease semantic similarity and Gaussian kernel similarity. In the future, we will combine more biomarkers, such as miRNA and protein, to construct more complex heterogeneous networks. With the development of the graph neural network, more and more advanced models have been proposed to learn the low-dimensional representation of graph structure, such as heterogeneous graph attention network, heterogeneous graph transformer and so on. In future research, we will continue to explore the positive role of these graph-embedding methods to identify circRNA-disease associations.