Glioma, a kind of central nervous system (CNS) tumor, is hard to cure and accounts for 32% of all CNS tumors. Establishing a stable glioma model is critically important to investigate the underlying molecular mechanisms involved in tumorigenesis and tumor progression. Various core signaling pathways have been identified in gliomagenesis, such as RTK/RAS/PI3K, TP53, and RB1. Traditional methods of establishing glioma animal models have included chemical induction, xenotransplantation, and genetic modifications . Recently, CRISPR/Cas9 has emerged as an efficient gene editing tool with high germline transmission and has extended the scope of stable and efficient glioma models that can be generated. Therefore, this review will highlight the documented evidence about the molecular characteristics, critical genetic markers, and signaling pathways responsible for gliomagenesis and progression. Moreover, methods of establishing glioma models using gene editing techniques and therapeutic aspects will be discussed. Finally, the prospect of applying gene editing in glioma by using CRISPR/Cas9 strategy and future research directions to establish a stable glioma model are also included in this review. In-depth knowledge of glioma signaling pathways and use of CRISPR/Cas9 can greatly assist in the development of a stable, efficient, and spontaneous glioma model, which can ultimately improve the effectiveness of therapeutic responses and cure glioma patients. Glioma is a common human brain tumor that accounts for approximately 50% of intracranial tumors. Although the current incidence of center nervous system (CNS) tumors is 14.3% of all tumors, 49.1% of nervous system tumors are malignant gliomas In recent decades, the survival rate of glioma patients has remained low due to poor prognosis, with less than 5% five-year survival rate, and the prognosis is even worse in elderly patients with glioma Cancer cells have the fundamental features of growth and survival beyond a nominal homeostatic or favorable environment, and their growth, survival, oncogenic proliferation, and apoptosis are governed by signaling pathways Here, we presented a systematic view of the molecular characteristics of gliomagenesis in the context of genetic markers and core signaling pathways. Furthermore, comparisons are drawn among traditional and modern methods for establishing glioma models. Moreover, the advantages and limitations of traditional and modern gene editing techniques are critically discussed. The potential applications of CRISPR/Cas9 system for generating glioma models are specifically highlighted, and the clinical trials associated with gene therapy are briefly discussed. Finally, clinical trials and available glioma therapies are also briefly discussed in the current study. A summary highlighting the pros and cons of traditional and modern glioma models, gene editing techniques, key signaling pathways and their regulatory function is presented in the form of a schematic diagram Fig. .Previous studies indicated that the new molecular classification system for gliomas might be valuable for predicting the prognostic of the glioma patients TP53 mutation is one of the most frequently event in GBM patients. However, compared with the highly frequency of EGFR mutation, TP53 mutation is rarely observed in classical subtype CDKN2A, was a frequent and significantly associated event in classical subtype, and accompanied with EGFR amplification in most of classical subtype samples. Furthermore, previous report showed that the neural precursor and stem cell marker NES, and the regulators of Notch and Sonic hedgehog signaling pathways, including NOTCH3, JAG1, LFNG, SMO, GAS1 and GLI2, were frequently amplified in classical subtype Chromosome 10 (Chr 10) loss combined with chromosome 7 (Chr 7) amplification was detected in all classical subtype, and frequently occurred in GBM patients. Although the Chr 7 amplification was also observed in other subtypes of glioma, the highly expression of EGFR was infrequently determined in other subtypes. It is known that The neural subtype was identified by the amplification of several neuron markers, including SYT1, GABRA1, NEFL and SLC12A5, which were involved in the processes of neuron projection and axon and synaptic transmission PDGFRA and IDH1 were two major features in proneural subtype. Although PDGFRA was amplified in almost all subtypes of GBM, it was amplified with a much higher rate in proneural subtype PDGFRA gene PIK3CA/PIK3R1 mutations were observed in proneural subtype, most of which did not have PDGFRA abnormalities, whereas eleven out of twelve mutations of IDH1 gene were determined in this subtype with no PDGFRA abnormality TP53 were also frequently detected in this subtype. In contrast, the classical GBM event, the amplification of Chr 7 paired with the loss of Chr 10, were distinctly less prevalent and only occurred in 54% proneural subtype samples. The highly expressed oligodendrocyte development genes, including PDGFRA, NKX2-2 and OLIG2, were detected in proneural subtype OLIG2 could promote proliferation and induce tumorigenesis through downregulating CDKN1A expression The mutations of CHI3L1 and METNF1 mutated samples. In addition, most of mesenchymal subtype samples were detected as NF1 and PTEN dual mutations, which play an important role in the RTK/PI3K pathway The mesenchymal subtype mainly exhibited the amplification of the mesenchymal markers, including Pten, Tp53 and Rb1) in mice that induced high-degree malignant tumors in astrocytes With the continuous development of sequencing technologies and high-throughput gene editing, it is possible to analyze the genetic and epigenetic changes in tumors Ink4a mutation and TP53 mutation in the EGFR signaling pathway in primary GBMs In RTK/Ras/PI3K pathway, receptor tyrosine kinases (RTKs) can mediate many growth signals through diffusive growth factors  or heterozygous (Tp53+/-) mice and zebrafish fail to induce gliomagenesis Tp53 mutation might be insufficient to initiate gliomagenesis.In gliomas, another commonly identified pathway is TP53 signaling pathway  Transgenic models involve molecular-level manipulation of the animal genome in the form of gene editing strategy to induce tumorigenesis Arf, Ink4a-Arf and Tp53, has also been determined during tumorigenesis Genome editing nucleases, including ZFNs, TALENS and CRISPR/Cas9, are also emerging tools to detect abnormal gene function in cancer. Gene editing technology has been successfully utilized in mice to study abnormal gene expression during tumorigenesis in liver and lung cancers In contrast to ZFN and TALEN systems, CRISPR/Cas9 system could efficiently identify any target sequences with convenient design nf1, tp53, or rb1, in gliocytes of brain tissue Tumorigenesis is often caused by factors such as chromosome aberrations, base deletions or other mutations that ultimately lead to tumor-related genetic modifications. With this novel genomic technology, interactions among molecular mechanisms can be identified in different stages of glioma development. Coupled with the exploitation of relevant signaling pathways, various glioma subtypes can be classified via specific gene expression profiles. CRISPR/Cas9 system is therefore specific, fast, and easy, can lead to stable glioma tumor outcomes and can overcome the limitations of traditional gene editing methods. The CRISPR system can also provide a method to establish tumor models that will be used to study various novel drug target genes and resistance genes for drug treatments In conclusion, this review highlights the molecular aspects of glioma induction and provides a theoretical basis for establishing a glioma model. Furthermore, the expression and regulation of relevant genes in terms of core pathways have been discussed. Moreover, the method of establishing glioma models using the novel gene editing technology CRISPR/Cas9 and the significance of targeted gene therapy were explained. We believe that the CRISPR/Cas9 technology has a significant potential to enhance the understanding of the mechanisms of gliomagenesis, and enable the development of glioma models and novel and efficient treatment methods.


Fibroblast growth factor (FGF) 21 is one of the FGF members with special endocrine properties. In the last twenty years, it has attracted intense research and development for its physiological functions that respond to dietary manipulation, pharmacological benefits of improving the macronutrient metabolism, and clinical values as a biomarker of various human diseases. Generally, FGF21 can be produced by major metabolic organs, but only the subgroup from the liver shows canonical endocrine properties, which emphasizes the special value of delineating the unique secretory and functional characteristics of hepatic FGF21. There has been a growth in literature to address the extra-hepatic activities of FGF21, and many striking findings have therefore been published. Yet, they are fragmented and scattered, and controversies are raised from divergent findings. For this reason, there is a need for a systematic and critical evaluation of current research in this aspect. In this review, we focus on the current knowledge about the molecular biology of endocrine FGF21, especially present details on the regulation of circulating levels of FGF21. We also emphasize its emerging roles in inter-organ crosstalk and cancer development. Figure 1). With the expansion of knowledge of FGF21, our understanding of its biology is constantly undergoing modification, especially for the liver-secreted FGF21, which is considered the main source of circulating FGF21 and has been implicated in various diseases (Table 3). As a dominant metabolic organ, the liver can sense the stress from both external and internal environments, and accordingly activate its intra- and extra-organ metabolic activities The mammalian fibroblast growth factor (FGF) superfamily consists of 23 members, which are grouped into 7 subfamilies based on their sequence homology and function. FGF19 (FGF15 for mice) is the only endocrine-acting subfamily that can secrete into the circulation and function as hormones Table 1. Generally, FGF21 is mainly under the control of peroxisome proliferator activated receptor α (PPARα) as an adaptive response to stress Mechanically, the expression and secretion of hepatic FGF21 are coordinately regulated by a set of proteins which are partly listed in Figure 2) For both humans and mice, serum FGF21 shows circadian rhythm during fasting with the nocturnal rise and diurnal fall, but this pattern is largely diminished in obese individuals and under standardized meals or cold exposure For both rodents and humans, exercise resulted in a significant increase in serum FGF21 levels, which were in line with the elevated lipolysis and decreased glucose levels The sensitivity and tendency of FGF21 to dietary intervention are different between mice and humans. Both hepatic and circulating levels of FGF21 are robustly induced by fasting, high-fat diet, low-carbohydrate, and high protein ketogenic diets in mice at el. at el. While increased FGF21 levels, decreased half-life, and retained diurnal rhythm during cold exposure have been consistently observed in mice On the other hand, FGF21 expanded overall thermogenic capacity by stimulating fat browning activity The regulation of FGF21 is complex and still not being fully understood. In this regard, establishing strict eligibility criteria for participant selection, measuring FGF21 levels at multiple time points, and setting paired samples are indispensable for studies to decipher the precise response of FGF21 under certain conditions. in vivo administration of FGF21 stimulated the response of white adipose tissue (WAT) and breast tissue but not the liver Table 3) show its pleiotropic effects. Up to now, the role of FGF21 has been unearthed in several organs/tissues, and the existence of FGF21-based inter-organ crosstalk was implied .FGF21-FGF receptors (FGFR) axis could activate a multitude of signaling cascades, including the RAS-MAPK pathway, the JAK-STAT pathway, the PI3K-AKT pathway, the PLCγ pathway, and JNK pathways Though undetectable in the nervous system, FGF21 could cross the blood-brain barrier and act in the CNS There has been a focus on neuroprotection activities of FGF21 in brain disorders in recent years. Decreased serum FGF21 levels were detected in patients with Parkinson's disease and Alzheimer's disease Clostridioides difficile infection patients receiving fecal microbiota transplantation therapy B. adolescentis, the dominant species in the human intestine, showed reduced hepatic FGF21 levels, which were believed as an improvement of the non-alcoholic fatty liver disease (NAFLD)-related FGF21 resistance state Gut microbiota (GM) represents a mysterious group of microorganisms living in the digestive tract, and host-GM crosstalk has been discussed in various human diseases Matla knockdown prevented and reversed obesity, insulin resistance, and hepatosteatosis via stimulating the expression and secretion of hepatic FGF21, which subsequently activated the liver-adipose tissue axis resulting in increased BAT thermogenesis, WAT lipolysis, and secretion of adiponectin While the liver is the major contributor to circulating levels of FGF21, WAT is the target contributing greatly to the metabolic effects of FGF21. The expression of KLB in conjunction with FGFR1c in adipose tissue confers FGF21 activities there The interactions between FFA and FGF21 create the loop between WAT and the liver. The FFA level in the serum of healthy individuals showed a similar oscillatory pattern with FGF21 under both fasting and feeding conditions, though the pick time was preceded. On the one hand, hepatic p38 stimulated WAT lipolysis and FFA release via upregulating FGF21 secretion from the liver to WAT Adiponectin is the most abundant adipokine secreted by adipocytes Table 3). Interestingly, one recent study detected that the cardiac FGF21 gene expression levels in heart failure patients were as low as healthy subjects, but the protein levels of FGF21 in both the serum and cardiac section were aberrantly increased and accompanied by elevated cardiac FGFR3 expression, which indicated the existence of a FGF21-dependent cardiohepatic signaling circuit in vivo FGF21 treatment failed to activate ERK signaling in the heart The evaluated levels of FGF21 were predictive of poor outcomes in cardiac diseases Retinal and choroidal ocular pathologic neovascularization is aggravated in FGF21 knockout mice and suppressed via the FGF21 analog-activated adiponectin pathway The data discussed above indicate that the aberrant expressions of FGF21 observed in circulation and organs in different contexts are likely to be an ex officio action of the liver to realize the pleiotropic effects of FGF21, especially regarding the adaptive starvation response in the CNS system, nutrition metabolism in GM, and energy homeostasis in adipose tissue. A crosstalk network is therefore delineated between the liver and other organs/tissues, which is no doubt striking and should be an area of fruitful future study.Figure 2), suggesting the biomarker value of FGF21 in cancer diagnosis and treatment. Theoretically, the well-studied findings of the oncogenic FGF-FGFR axis attest to this hypothesis. FGF21-related FGF receptors FGFR1c, 2c, 3c, and 4 are widely expressed in tumor cells and aberrantly activated in cancers As a stress-responsive hepatokine, the implications of FGF21 in cancers have been focused on in recent years. Overall, overexpression of FGF21 in both circulating and tissue levels has been found in different types and clinical stages of cancers  and contributed to tumor development Atg7 deletion induced hepatic tumor process, implicating the suppressive effect of FGF21 on autophagy-insufficient hepatoma On the other hand, some studies have discussed the tumor suppressor effects of FGF21 on HCC under both physiological and pharmacological levels. FGF21 knockout mice were more sensitive to a high fat, high sucrose diet-induced HCC In vitro FGF21 treatment promoted tumor cell migration and invasion by upregulating FGFR-EMT signaling. It is worth noting that FGF21 expression was detected in neither normal thyroid tissues nor tumor tissues, which indicated that FGF21 might induce thyroid tumor progression in an endocrine way Serum FGF21 levels were significantly higher in the papillary thyroid carcinoma patients than in controls and were positively associated with pathological stage, recurrence, and mortality. FGF21 expression was decreased in clinic prostate cancer tissues, and overexpression of FGF21 inhibited prostate cancer cell viability In vitro study showed that FGF21 was elevated in tumor cells compared with normal lung cell lines, which facilitated tumor progression by promoting cell growth, migration, and defending oxidative stress via Sirtuin 1/PI3K/AKT signaling FGF21 was upregulated in non-small cell lung cancer tissues, and the higher expression level was associated with shorter overall survival time and advanced histological type. Overexpression of FGF21 in mouse melanoma cell line enhanced cell aggressiveness and tumorigenicity under hypoxia and low-nutrition double deprivation stress The circulating levels of FGF21 in pancreatic cancer patients are still unclear, but both pancreatic cancer specimens and cell lines had lower levels of FGF21 than normal human pancreatic tissues and cells, respectively. The pharmacological dosage of FGF21 supplementation effectively inhibited pancreatic intraepithelial neoplasia lesions, inhibited liver metastasis, and prolonged the overall survival of KRAS-mediated pancreatic cancer under the high-fat-diet challenge. Though serum FGF21 level was not influenced by pancreatic KRAS mutation, the effect of pathological levels of circulating FGF21 under a high-fat diet, in turn, on pancreatic tumor development was not addressed As mentioned, adipose tissue is not only a source of autocrine FGF21 but also a direct and predominant target of hepatic FGF21, which may result in a substantial enrichment of FGF21 in the microenvironment of the tumors growing in the anatomical vicinity of adipose tissue, such as breast, renal and colon cancers. Even though the activities of FGF21 in these types of cancers have not been explored yet, elevated serum levels of FGF21 were reported in the early stages of breast cancer Increased FGF21 levels were observed in patients with different stages of gastric cancer, suggesting it is a suitable biomarker for early-stage gastric cancer So far, there is limited information available to explain the role of FGF21 in tumor initiation and progression. Though the results listed above are promising, they need more detailed and in-depth investigations as it is hard to discern whether the increased level of FGF21 contributes to cancer development or is just a result of carcinogenesis or a stress response for maintaining body system homeostasis. As a potential biomarker, the aberrant expression of FGF21 has been generally observed in different types and stages of cancers, which indicates its low specificity. Instead, FGF21 may be an ideal non-invasive biomarker applied in regular health tests for discovering and preventing cancers at an early stage, though the sensitivity and cancer-specificity for clinical use need to be evaluated critically.Since 2000, the scientific appreciation of FGF21 in various human diseases has been rapidly expanding. As an unfolded area, the increasing roles of FGF21 generate a series of inspiring ideas, such as whether exclusive FGF21 secretion ability confers liver extra-hepatic functions, whether endocrine FGF21 is the holistic hub point for interorgan crosstalk, whether there are concomitant modulators that collaborate with FGF21 or whether FGF21 is an actionable biomarker with therapeutic values for cancers. However, studies in this area are still in their infancy. To boost the research process of FGF21, several factors need to be considered.in vitro concentrations to in vivo doses (and vice versa) is even thornier since it is hard to set a clear line between pathological and pharmacological dosages for in vitro study. In this case, in vivo study is more reliable for observing the biological effects of FGF21 under different conditions.The first factor is about distinguishing between pathological and pharmacological doses of FGF21. The physiological actions of FGF21 occur at much lower concentrations and in more restricted organ systems and tissues than its pharmacological actions Secondly, the elimination half-life of FGF21 is only 0.5~2 hours Besides, the similarities and differences between mice and humans need to be considered. While it may sound hypercritical, the aforementioned distribution and activity difference between human and mouse FGF21 warn us of the existence of species specificity in various contexts. Therefore, the clinical translation of FGF21 activities demonstrated in mice to humans needs to be further investigated. For example, mouse adipose tissues express detectable levels of FGF21, which has been implicated in the magnificent effects of FGF21 on energy metabolism. However, human adipose tissues do not express FGF21, which questions the clinical value of autocrine activities of adipose FGF21 observed in rodent-based studies Last but not least, it is plausible that the physiology and function of FGF21 may be sexual dimorphism considering the substantial difference between genders in sex hormone levels and macronutrient metabolism patterns. Besides the different sensitivity to circadian rhythms Focusing on FGF21, the findings discussed in this review indicated its high sensitivity to adaptive stress response, its important role in modulating liver-participated inter-organ crosstalk, and its potential value in cancer diagnosis and treatment. In-depth studies to establish a comprehensive framework of the biology and activities of endocrine FGF21 are called upon.Supplementary information.Click here for additional data file.


Our understanding of eosinophil biology, development, and regulation has dramatically increased in the past decade, leading to new paradigms for the role of eosinophils in human health and disease and, perhaps more importantly, providing insights toward novel treatment strategies in the fight against eosinophil-mediated inflammation. In this review, we discuss recent advances regarding the role of eosinophils in host-viral defense, eosinophil heterogeneity, and eosinophil-targeted therapies. Over the last decade, however, the role of eosinophils in a wide variety of homeostatic processes, including antitumor responses, tissue remodeling and fibrosis, metabolism, and immunoregulation, has become evident. This, combined with the development of novel therapeutic agents that target eosinophils3, has led to an increasing appreciation of the multifunctional nature of this enigmatic cell. In 2021, a total of 4,764 publications were identified in PubMed using the search term “eosinophil OR eosinophilia,” a 72% increase from 2,770 in 2011. In comparison, the number of publications retrieved using “mast cells OR mastocytosis” increased only 28.4%  during the same time frame. Given the breadth of recent information pertaining to eosinophil biology and function, a comprehensive summary is beyond the scope of this review. Instead, we will focus our discussion on three topics of considerable recent interest: (1) the antiviral response, including findings related to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), (2) eosinophil development and heterogeneity, and (3) lessons learned from the rapidly evolving landscape of anti-eosinophil therapy.Conserved throughout evolutionary history, the eosinophil has long been recognized for its role in innate parasite immunity and type 2 inflammation4. Despite this early observation, the role of eosinophils in the immune response to viral infection was virtually unstudied until the 1969 description of pulmonary eosinophils in two children who died from a hypersensitivity reaction to the respiratory syncytial virus (RSV) following immunization with a formalin-inactivated RSV vaccine5. These initial findings and the association between early RSV infection and wheezing in childhood6 paved the way for studies exploring the role of eosinophils in viral infection  particles in a process that leads to eosinophil activation and release of interleukin 6 (IL-6) and IL-8. Eosinophils indirectly modulate viral immune responses through their function as antigen-presenting cells, as evidenced by in vitro studies demonstrating antigen-specific activation of virus-specific T cells by eosinophils exposed to rhinovirus or influenza virus and by adoptive transfer experiments of lung eosinophils from fungal allergen-challenged mice into the tracheas of mice with influenza infection16. More recently, the same group showed that lung eosinophils rapidly upregulate the adhesion molecules, VLA-4 and ICAM-1, in murine influenza infection, migrate to the T-cell zones of the mediastinal lymph nodes, and protect the airway barrier from cytotoxic damage, possibly through downregulation of pro-apoptotic genes in infected epithelial cells17.Recruitment of eosinophils to the lung has been demonstrated in mice infected with a wide variety of respiratory viruses, including RSV and influenza virus. Moreover, several lines of evidence support the direct and indirect roles of eosinophils in the immune response to viral pathogens. Human and mouse eosinophils express a variety of surface receptors, including Toll-like receptors and retinoic acid-inducible gene I (RIG-I), that recognize viral nucleic acid motifs18. Whereas most data suggest impairment of innate immune mechanisms in asthmatics22, this appears to be counterbalanced by increased allergen-activated eosinophils in the lung, raising concern that therapeutic depletion of eosinophils might lead to increased frequency or severity of viral infection. This was explored directly in a placebo-controlled study of patients with mild asthma treated with the anti-IL-5 monoclonal antibody (mAb), mepolizumab, and challenged with rhinovirus23. Although mepolizumab treatment affected both the cellular and humoral responses to rhinovirus challenge and resulted in increased viral loads, eosinophil activation by rhinovirus was preserved and there was no effect of eosinophil depletion on clinical symptoms of viral infection. This lack of adverse outcomes is supported by the data from multiple clinical trials of eosinophil-depleting therapy in asthmatic patients, which have shown no increase in frequency or severity of viral infections but rather a reduction in acute asthma exacerbations26. Ultimately, continued investigation is needed to better understand eosinophil-viral interactions in the context of atopic disease.Viral respiratory infections remain a leading cause of asthma exacerbations and hospitalizations28. The pathogen would quickly be identified as the novel beta-coronavirus, SARS-CoV-2, and the infection as coronavirus disease 2019 (COVID-19)29. The rapid spread of the virus has led to an unprecedented health crisis and a global pandemic with over 167 million infections and more than 3.4 million deaths worldwide in the year following the initial report30. Although the association between eosinopenia and disease severity in patients with COVID-1933 has led some to suggest that eosinopenia might distinguish SARS-CoV-2 infection from other viral respiratory illnesses35, it is important to recognize that febrile eosinopenia was first recognized by Zappert36 in 1893, nearly 10 years prior to the description of the first human viral pathogen. Moreover, eosinopenia is a well-established diagnostic sign and prognostic indicator in critically ill patients with sepsis42 and was a marker of severe disease during the 2009 H1N1 influenza pandemic, affecting 50% of patients at the time of hospital admission43. The mechanism underlying febrile eosinopenia remains incompletely understood, although recent data implicate CD49d-mediated homing of eosinophils to tissues44 and exuberant type 1 immunity or type I interferon-mediated eosinophil apoptosis46.In December 2019, reports of a mysterious viral pneumonia emerged from Wuhan, China48, and histopathological studies demonstrated a proinflammatory infiltrate comprised predominately of neutrophils, monocytes, macrophages50, and lymphocytes53. More recent studies have documented enhanced eosinophilic inflammation in the lung in patients with fatal COVID-19 infection55. By using flow cytometric analysis of whole blood samples obtained longitudinally during the acute and recovery phases of COVID-19 infection, Rodriguez et al. demonstrated transient expansion of activated CD62L+ eosinophils mediated by interferon-gamma (IFN-γ) and suggest that this immunomodulation may promote eosinophil trafficking to the lung following acute infection, providing a potential mechanism for COVID-19-associated eosinophilic lung pathology56. Albeit uncommon, case reports describing the temporal association of COVID-19 immunization and the development of eosinophilic disorders, including acute eosinophilic pneumonia and eosinophilic granulomatosis with polyangiitis59, are intriguing in this regard and merit further investigation.The role of eosinophils in COVID-19-associated pathology continues to be controversial. Early studies suggested that eosinophilic pulmonary inflammation was uncommon in COVID-1961. In fact, some studies have suggested that type 2 inflammation may exert a protective effect by limiting viral entry through modulation of angiotensin-converting enzyme 2 (ACE2) expression at respiratory and gastrointestinal (GI) epithelial surfaces66. Consistent with this hypothesis, analysis of a large database of hospitalized patients with COVID-19 infection demonstrated improved survival in patients with a history of eosinophilic gastrointestinal disorders (EGIDs) compared with non-EGID patients matched for demographic and clinical factors67, and pre-existing eosinophilia was associated with a lower hospitalization rate and lower mortality in a retrospective analysis of 951 asthma patients presenting with COVID-1968. These findings have led to the hypothesis that eosinophilia or Th2-high inflammation (or both) may be protective in COVID-1969 and conversely that the use of eosinophil-depleting therapies may be harmful. Although definitive clinical studies addressing these concerns are lacking, case reports of mild disease in high-risk patients with asthma on eosinophil-depleting biologic therapy71 and data from published studies examining the association between biologic therapy and the incidence and severity of COVID-19 infections in asthmatic patients  characterizing the phenotype of rEos compared with that of eosinophils involved in the inflammatory response (iEos) and (2) functional differences between rEos and iEos.The dual roles of the eosinophil in producing and downregulating inflammation suggest that, like other immune cells, functional subtypes of eosinophils may exist. This hypothesis is supported by the fact that eosinophils can be detected in healthy tissues in the absence of inflammation. Although these “resident eosinophils” (rEos) are most prominent in the GI tractintCD62L+CD101lo surface expression. These cells have a ring-shaped nucleus and a regulatory gene expression profile95. Functionally, they inhibit dendritic cell maturation and Th2 sensitization to an allergen. In contrast, iEos in the same model were localized in peribronchial areas, had segmented nuclei, were characterized by a Siglec-FhiCD62L–CD101hi surface phenotype, and exhibited a proinflammatory gene signature and Th2 effector functions95. Eosinophil surface expression of Gr1 and Ly6G in the murine lung in the setting of allergen challenge or viral infection (or both) also identifies two populations of eosinophils characterized by distinct granule-derived cytokine repertoires suggestive of inflammatory  versus regulatory (CXCL10 and IL-10) functions99.Phenotypic differences between eosinophils, often with functional consequences, have been demonstrated by multiple groups . In the 101. The functionality of these receptors was confirmed in vivo using a ligated intestinal loop model of luminal antigen uptake in allergen-sensitized mice. It should be noted that upregulation of MHC class II and CD80 and antigen presentation by eosinophils has also been described on bronchoalveolar lavage eosinophils from aerosol allergen-challenged mice106 and on human blood eosinophils exposed to various allergens and bacterial products in vitro107, suggesting that the functional distinctions between resident eosinophils (rEos) and those recruited from the blood (iEos) may not be rigid.Varied eosinophil surface phenotypes have also been described in the murine GI tract , but their presence enhanced local inflammatory responses to an unrelated inhaled allergen (house dust mite). This site-dependent effect was also seen using a combined asthma/arthritis model, where increases in synovial eosinophils were associated with attenuation of joint inflammation in mice sensitized and challenged with ovalbumin89. In this model, the joint eosinophils had phenotypic, genotypic, and functional characteristics consistent with rEos (in contrast to iEos in the lung) and were induced to proliferate in situ by increased systemic IL-5 produced primarily by ILC2 and CD4+ cells in the lung. Sensitization and challenge with house dust mite  caused a dominant Th17 response and exacerbation of arthritis89, suggesting that both the site of sensitization and the antigen are important drivers of the type of eosinophilic response. A recent study demonstrating that exposure of murine eosinophils to type 1 (IFN-γ or Escherichia coli or both) or type 2 (IL-4) conditions in vitro were sufficient to produce distinct transcriptional signatures further supports the hypothesis that the environment plays an important role in driving eosinophil heterogeneity109.Interesting recent work has begun to explore the role of eosinophils in crosstalk between different sites of allergic sensitization. Olbrich hi human eosinophils that is increased in allergic donors and after allergen provocation110, phenotypic differences between eosinophils in normal lung tissue and those from the sputum of individuals with allergic asthma95, and an increased number of eosinophils with a regulatory phenotype in the peripheral blood and joint synovium of patients with rheumatoid arthritis in remission compared with patients with active disease89. However, the functional heterogeneity of human eosinophils has been examined directly in only a few studies to date. In one such study, Lingblom et al. demonstrated greater suppression of T-cell proliferation by CD16hi eosinophils from healthy donors compared with CD16lo eosinophils from the same donors105. This suppression was at least partly dependent on galectin-10  released in extracellular DNA traps when eosinophils come into contact with activated T cells111. An area of ongoing controversy, especially in humans, is whether there are developmentally distinct populations of eosinophils that maintain their phenotype and function irrespective of their environment or whether the observed heterogeneity is entirely due to plasticity in response to external cues. This has important implications in the context of the ever-increasing number of targeted therapeutic agents that deplete eosinophils or alter their cytokine milieu (or do both).Although regulatory functions and phenotypic heterogeneity of eosinophils have clearly been demonstrated in humans , the rel114, a general discussion of the differences between eosinophil-targeted agents will be provided, and specific examples will be utilized to illustrate the concepts outlined above.The advent of eosinophil-targeted therapies not only has revolutionized the landscape of treatment for eosinophilic disorders by providing less toxic and more effective therapeutic options for patients with common and rare eosinophil-associated disorders but has provided a unique opportunity to enhance our understanding of the basic biology of eosinophils in humans and their role in homeostasis and disease. Whereas a comprehensive discussion of the many novel therapies currently available and in clinical development for eosinophil-associated diseases is beyond the scope of this article and can be found in numerous reviewsEosinophil-targeted therapies can be broadly classified by their mechanisms of eosinophil reduction: (1) depletion of IL-5 (mepolizumab and reslizumab), (2) antibody-dependent cytotoxicity , (3) interference with eosinophil trafficking to tissues , and (4) other or unknown . The var116, leading to concern that eosinophils were unimportant in asthma pathogenesis. Instead, these initial stumbling blocks led to the recognition of asthma endotypes that continue to help define the pathogenesis and treatment of severe asthma117. More recently, it has been suggested that variability exists even within the endotype of eosinophilic asthma, with implications for the selection of targeted therapies118. In a recent retrospective analysis of 3,531 patients with severe asthma, 384 (11%) switched biologics once and 45 switched twice, consistent with this hypothesis119. Similar findings have been reported in a multicenter retrospective study of patients with PDGFRA-negative hypereosinophilic syndrome (HES), which described successful switching in the subset of patients who did not respond to the initial biologic tried120.The earliest example of the utility of eosinophil-targeted therapy in understanding disease pathogenesis in humans comes from the initial trials of mepolizumab and reslizumab in asthma, which failed to show significant improvement in clinical outcome measures122, reslizumab123, and benralizumab  have not met clinical endpoints despite substantial or, in the case of benralizumab, near complete reduction of blood and tissue eosinophilia. An analysis of GI pathology specimens from a phase 2 clinical trial of benralizumab in HES demonstrated persistent epithelial changes in the stomach and esophagus despite eosinophil depletion, consistent with a role for factors other than eosinophils in disease pathogenesis in EGIDs124. The promising clinical outcome results from phase 2 trials of lirentelimab, a mAb to Siglec-8 that both depletes eosinophils and inhibits mast cell degranulation125, and dupilumab, a mAb that targets IL-4 receptor alpha reducing tissue eosinophilia and blocking IL-4/IL-13 signaling126, also suggest that targeting multiple pathways may be more effective. Unfortunately, these findings have not been replicated in phase 3 studies to date . More studies using targeted therapies are needed to disentangle the role of eosinophils in the pathogenesis of EGIDs and other eosinophil-associated diseases. These should include head-to-head studies, such as the ongoing trial comparing mepolizumab with benralizumab for the treatment of eosinophilic granulomatosis with polyangiitis , to directly assess the effects of differing degrees and methods of eosinophil depletion in the different disorders and patient populations.The apparent disconnect between eosinophil reduction and clinical outcomes also reminds us that multiple cell types and pathways may play a role in eosinophil-associated disorders. This has been particularly evident in EGIDs, where clinical trials with mepolizumab132. That said, the relatively few human studies that have looked directly at more subtle changes in immune responses have not replicated the data from murine models  and should provide a definitive answer to the question of whether eosinophil depletion is safe in pregnancy. Other potential consequences of prolonged eosinopenia, including effects on susceptibility or severity of helminth infection, have not been formally studied in humans.Despite convincing evidence supporting a homeostatic role for eosinophils, long-term safety studies using eosinophil-depleting agents have failed to demonstrate major negative consequences after 3 to 11 years of therapy109.Even if (as current data suggest) eosinophil depletion is safe in humans, this may reflect the fact that essential eosinophil functions are only part of a network of responses. In this regard, the potential for biologics that target multiple cells or pathways and/or the concomitant use of multiple biologics to unmask adverse consequences remains a theoretical concern. Although studies using lirentelimab and case reports describing dual biologic therapy have not supported this hypothesis to date, long-term data are lacking. Similarly, there is little evidence that dupilumab, which decreases tissue eosinophilia and blocks IL-4 and IL-13 signaling, adversely affects tissue repair, despite a growing body of evidence pointing to the importance of eosinophil secretion of IL-4 in this processet al. demonstrating that Gal-10 protein, the major component of Charcot-Leyden crystals, promotes goblet cell metaplasia and bronchial hyperreactivity in a mouse model of asthma. Importantly, antibodies against Gal-10 were found to dissolve Charcot-Leyden crystals and ameliorate type 2 airway inflammation140 . Although the mechanism of action is unknown, bone marrow sampling during the study in HES was consistent with induction of maturational arrest of eosinophils at the promyelocyte stage.Novel targets are of particular interest in the management of eosinophilic disorders and may provide alternatives for individuals or subpopulations who do not respond to existing therapeutics and may help define the roles of novel molecules and pathways in eosinophil biology. This is illustrated by recent work from Persson ation140 , providiRecent advances in our understanding of eosinophil biology have continued to highlight the multifunctional and complex nature of this mysterious cell. Currently available drugs that target eosinophils, as well as new additions to the therapeutic arsenal, provide not only hope for patients with eosinophilic disorders but a unique opportunity to illuminate the role of eosinophils in different contexts. Careful attention to trial design and associated mechanistic studies is essential in this regard.


This Research Topic is aimed at collecting contributions that help to shed more light on pathogenesis, diagnostics, and prospective therapeutic tools in critical clinical conditions resulting from impaired oxygen availability, such as cardiac arrest, hypoxia, ischemia, and sepsis. Emphasizing translational research, we invited the scientific community to contribute studies conducted with experimental animal models, as these allow the detailed analysis of the complex pathophysiological processes for developing new therapeutic strategies. Here we present nine contributions. Seven studies describe experimental results, mainly animal experiments, and two are reviews that expand our knowledge of the physiological and pathophysiological events associated with impaired oxygen delivery.Hof, Marcus et al. offers a “tool-box,” comprising a combination of analyses to determine structural and functional changes of the microcirculation and of mitochondria that are applicable in studies using experimental animals for modeling septic complications and hemorrhagic shock. In order to accurately assess the consequences of impaired oxygen delivery on organ function, cell metabolism, and inflammatory processes, the authors further suggest magnetic resonance imaging as a non-invasive method.The review of Bjertnæs et al. gives us a well-written overview about the physiological consequences of accidental hypothermia, a medical condition, which still has a high mortality rate. The authors describe guidelines for the rewarming procedure, which are of relevance not only for the treatment of victims of accidental hypothermia, but may also interest those considering hypothermia as a therapeutic approach.The review of in-vitro study presented by Woyke et al., describing the combined effect of lowering temperature and increasing CO2 levels on the oxygen dissociation curve in unbuffered human whole blood. The authors found that decreasing temperature leads to an increased oxygen affinity to hemoglobin. However, this temperature effect is outweighed by the CO2-effect, inducing a right shift of the oxygen dissociation curve. Interestingly, the relative CO2-effect was higher with decreasing temperature, indicating a significant interaction of temperature and pCO2. Additionally, the CO2-effect was diminished at higher CO2 levels suggesting saturation effect. The study further suggests that levels of pCO2 exceeding 40 mmHg may result in a too poor oxygen binding and an insufficient oxygen delivery within the system.An additional insight in the association of hypothermia and hypoxia is given in the Hof, Truse et al. compared the effect of locally applied CO2 and O2 on gastric and oral microcirculation in dogs subjected to hemorrhagic shock. The idea behind it was, that when applied locally, CO2 at high concentrations increases the oxygen release from hemoglobin locally, which can be used to intentionally improve the oxygenation of hypoxic tissue. Indeed, local hypercapnia improved microvascular oxygenation and was associated with a continuous blood flow. Thus, local CO2-application is able to improve oxygen saturation of hypoxic tissues better than application of oxygen and represents an interesting new minimally invasive approach to improve gastric microcirculation during hemorrhagic shock.This study presented by Graf et al. They used succinyl phosphonate as an inhibitor of 2-oxoglutarate dehydrogenase complex (OGDHC) in cerebellar tissue of pregnant and non-pregnant rats. Both, OGDHC inhibition by limiting NADH, and hypobaric hypoxia by limiting oxygen supply, perturb the respiratory chain function. OGDHC inhibition produced similar changes in the cerebellar amino acid pools, which were different between pregnant and non-pregnant rats. The authors suggest that chemical OGDHC inhibition is suitable for mimicking the metabolic changes induced by insufficient oxygen supply.A deeper insight into the cellular metabolism associated with insufficient oxygen supply is given by the contribution of Rutai et al. used a clinically relevant porcine model for mimicking human sepsis and septic shock and developed a standardized research protocol that allowed characterizing the progression of sepsis-related events. Their approach is to cluster the host responses into sepsis and septic shock groups using a specific porcine SOFA score including quantitative and qualitative assessment of blood CFUs along with the measurement of macro- and microcirculatory variables.The study by Denoix et al. used a porcine long-term model of hemorrhagic shock and resuscitation with pre-existing atherosclerosis to investigate whether sodium thiosulfate, an H2S releasing compound, exerts neuroprotective effects. However, despite its lung-protective efficacy in this model, neuro-histopathological analysis revealed no differences between groups, possibly because the blood brain barrier remained intact and neuronal tissue appeared relatively unaffected by the induced hemorrhagic shock. This research group additionally examined H2S as a potential therapeutic in mice with deleted H2S-producing cystathionine-γ-lyase (CSE). In a previous study, they could demonstrate sodium thiosulfate-mediated protection against traumatic/hemorrhagic shock induced injury . Although ECMO can save lives in respiratory distress, it has serious side-effects as it frequently induces acute kidney injury. Therefore, the group developed a clinically relevant model using Vietnamese minipigs for a prolonged ECMO protocol (30 h). The described protocol induced acute renal impairment, which was demonstrated by a significantly decreased renal function with signs of structural damage and impaired mitochondrial function. It is therefore a good tool to further study therapeutic interventions for decreasing acute kidney injury introduced by ECMO.Finally, the contribution of JCD and WW planned the Editorial. WW wrote the first draft. JCD and AVK performed major revisions of the text. All authors contributed and approved the submitted version of the article.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.


To cope with biotic and abiotic stress conditions, land plants have evolved several levels of protection, including delicate defense mechanisms to respond to changes in the environment. The benefits of inducible defense responses can be further augmented by defense priming, which allows plants to respond to a mild stimulus faster and more robustly than plants in the naïve (non-primed) state. Priming provides a low-cost protection of agriculturally important plants in a relatively safe and effective manner. Many different organic and inorganic compounds have been successfully tested to induce resistance in plants. Among the plethora of commonly used physicochemical techniques, priming by plant growth regulators (phytohormones and their derivatives) appears to be a viable approach with a wide range of applications. While several classes of plant hormones have been exploited in agriculture with promising results, much less attention has been paid to cytokinin, a major plant hormone involved in many biological processes including the regulation of photosynthesis. Cytokinins have been long known to be involved in the regulation of chlorophyll metabolism, among other functions, and are responsible for delaying the onset of senescence. A comprehensive overview of the possible mechanisms of the cytokinin-primed defense or stress-related responses, especially those related to photosynthesis, should provide better insight into some of the less understood aspects of this important group of plant growth regulators. The crisis of feeding the world’s rapidly growing population with annual growth of around 80 million  is compoSimilar to immunization in animals and humans, where an infectious agent or vaccine helps the immune system develop immunity to a disease, plants can also be immunized. This type of “acquired physiological immunity” in plants is now referred to as priming and can generally be described as the ability to elicit a faster and/or stronger defense response after a prior exposure to a biotic or an abiotic stress condition . Both biWhile defense priming is typically associated with bacterial and fungal pathogens or other biotic stressors, an altered response to abiotic stressors  is no less important. Previous exposure to a mild abiotic stress including heat, cold, osmotic or water stress can also improve resistance of various environmental stresses . PrimingIt has been recently reported that priming of wheat seeds with benzylaminopurine (BAP) and kinetin (KIN) applied by spraying the wheat plants during the grain filling stage resulted in up to 14% higher productivity, higher fresh and dry weight, and chlorophyll content index of flag leaves . In addiTriticum aestivum L.) seeds were pre-soaked in BAP and KIN solutions, and the primed and non-primed seeds of salt-intolerant MH-97, as well as salt-tolerant Inqlab-91, were compared. KIN was effective in increasing the germination rate in the salt-intolerant cultivar and early seedling growth when compared with hydropriming under salt stress. Furthermore, the KIN-primed seeds showed a consistent promoting effect in the field and improved growth and grain yield in both cultivars under salt stress  with zeatin resulted in significant changes of endogenous CK pools in both shoots and roots of 7-day-old seedlings . Also, rt stress . The autt stress .Triticum aestivum cv Hereward and spring barley (Hordeum vulgare), malting variety Bojos or winter oilseed rape treated by picking with a 50 µM solution of urea-based CK derivatives  or increase in CO2 assimilation rate, stomatal conductance and transpiration was recorded under salt stress in eggplant after seedlings’ exposition to BAP , WUE, carboxylation efficiency and leaf yield  was observed in Anthurium plants sprayed with KIN along with its possible effects on gas exchange and antioxidant enzyme activities (N-2-(chloro-4-pyridyl)-N-phenyl urea) reversed the drought mediated suppression of RAF1 and the RuBisCO activase proteins, implied in the assembly and activation of RuBisCO complex  adapted state, better transfers of electrons beyond QA, enhanced electron transport rate and lower relative variable fluorescence intensity at the J-step were detected in the BAP primed wheat , ΦPSII and increase of pq  at the expense of non-photochemical quenching (NPQ) leading to a lower dissipation of excitation energy in the PSII antennae  was detected in wheat plants sprayed with BAP, which simultaneously increased their endogenous zeatin levels. A higher effective and maximal quantum yields of PSII photochemistry in the light (ΦPSII) and dark , jasmonic acid (JA) and ethylene (ET) are interlinked in a complex network regulating plant immunity . These cPseudomonas syringae in Arabidopsis via interaction with SA response factor TGA3 and binding to the PR1 promoter. In addition to the direct activation of defense responses by ARR2, CK pretreatment led to a hyperactivation of PR1 transcription after pathogen inoculation  in poplar leaves after herbivory . Consequently, the mortality dropped to 30% in the case of OsCKX1 knock-out plants upon an infestation by brown planthopper, compared to the 90% wild-type mortality upon infestation. A simple CK treatment positively regulated JA biosynthetic and an expression of JA-responsive genes. In addition, the CK-mediated resistance against the brown planthopper infestation was diminished in JA-deficient mutant og1  appears to play a major role in this process  responses and underlying epigenetic modifications cannot be ruled out. Experiments with these aromatic CK derivatives confirmed the synergistic relationship between CK and JA in defense priming, as the levels of JA and its metabolites were significantly elevated (In addition, our aromatic CK derivatives that have been synthesized and tested retain certain properties of CK responses, such as delayed senescence and defense against pathogens, while the classical CK signaling pathway is only negligibly affected ; therefoelevated . Howeverelevated . An ET pelevated . Togetheelevated . Intereselevated .ahk2 and ahk3, show significant tolerance to dehydration. CK crosstalk with ABA may be significantly involved in dehydration tolerance, as ahk mutants have an increased level of expression of ABA-upregulated genes (ahk single mutant lines after subsequent dehydration stress (While the effects of CK priming under biotic stresses are relatively well documented, mechanistic studies on CK priming in relation to abiotic stresses are just beginning to emerge. Previous works are mainly devoted to CK’s role in priming against heat stress and drought . Kang eted genes . CK pretn stress . This phIn the last decade, many studies have pointed to the practical use of plant growth regulators that can mitigate the negative effects of biotic and abiotic stress conditions and improve overall crop condition and yield. Hormopriming is widely accepted as an efficient way to create memory imprints in seeds or young developing plants that can later, under limiting or stressful conditions, help shift the endogenous hormonal balance towards defense responses. Given the persistent social barriers to genetically modified crops, targeted modification of stress-related pathways through treatment with low amounts of highly bioactive substances offers an interesting alternative for agricultural use. The plant hormone CK is emerging as a promising new type of priming agents, the use of which appears to be closely linked to the control of the photosynthetic apparatus Figure 1MH and VN wrote the original draft of the manuscript. VN designed the figure with help of OP. OP and VN conceived the idea and made the final adjustments. All authors contributed to the article and approved the submitted version.


If food and feed production are to keep up with world demand in the face of climate change, continued progress in understanding and utilizing both genetic and epigenetic sources of crop variation is necessary. Progress in plant breeding has traditionally been thought to be due to selection for spontaneous DNA sequence mutations that impart desirable phenotypes. These spontaneous mutations can expand phenotypic diversity, from which breeders can select agronomically useful traits. However, it has become clear that phenotypic diversity can be generated even when the genome sequence is unaltered. Epigenetic gene regulation is a mechanism by which genome expression is regulated without altering the DNA sequence. With the development of high throughput DNA sequencers, it has become possible to analyze the epigenetic state of the whole genome, which is termed the epigenome. These techniques enable us to identify spontaneous epigenetic mutations (epimutations) with high throughput and identify the epimutations that lead to increased phenotypic diversity. These epimutations can create new phenotypes and the causative epimutations can be inherited over generations. There is evidence of selected agronomic traits being conditioned by heritable epimutations, and breeders may have historically selected for epiallele-conditioned agronomic traits. These results imply that not only DNA sequence diversity, but the diversity of epigenetic states can contribute to increased phenotypic diversity. However, since the modes of induction and transmission of epialleles and their stability differ from that of genetic alleles, the importance of inheritance as classically defined also differs. For example, there may be a difference between the types of epigenetic inheritance important to crop breeding and crop production. The former may depend more on longer-term inheritance whereas the latter may simply take advantage of shorter-term phenomena. With the advances in our understanding of epigenetics, epigenetics may bring new perspectives for crop improvement, such as the use of epigenetic variation or epigenome editing in breeding. In this review, we will introduce the role of epigenetic variation in plant breeding, largely focusing on DNA methylation, and conclude by asking to what extent new knowledge of epigenetics in crop breeding has led to documented cases of its successful use. FLOWERING WAGENINGEN (FWA) between related species, and DNA methylated regions are enlarged by the generation of tandem repeats. Therefore, this falls under “obligatory” epigenetic variation . DNA methylation states are stably inherited and play a role in transcriptional regulation not only of protein coding genes but also of transposable elements (TEs) . Intraspariation Figure 1ariation . In Arablowering . The latlowering , and thilowering .A. thaliana, developmental abnormalities and transposition of transposons were observed. Some developmental abnormalities in A. thaliana were due to the loss of function by insertion of endogenous transposons in genes or their regulatory regions  is an epimutation, and a heritable phenotypic change caused by epimutations/epialleles is called an epimutant. Inheritance of epigenetic alterations in natural populations or breeding populations needs to be evaluated to understand how epimutants contribute to evolution or crop domestication . The impA. thaliana. Its relatively small genome size, relatively simple genome structure, and ease of use for research purposes makes it an excellent model species and the knowledge gained in studies of A. thaliana is largely translatable to crops. While the genomes of most crops are much larger and more complex than that of A. thaliana, the genic complements of all higher plant species are relatively similar and the molecular biology of epigenetics is largely conserved across A. thaliana and cultivated crops. However, there are important differences  and other crop species, and studies of inter- and transgenerational inheritance that is often important for short-term response or adaptation to environmental changes and stress in crop plants.A great deal of progress has been made in studying the molecular mechanisms underlying the induction, maintenance, and transmission of epigenetic variation in the model plant ferences . These lde novo DNA methylation. In maintenance of DNA methylation, during replication, methylated DNA strands are separated, and a new unmethylated daughter strand is synthesized. Hemi-methylated DNA is recognized by maintenance DNA methyltransferases that methylate the unmethylated cytosine (De novo DNA methylation is mediated by RNA-directed DNA methylation (RdDM) coordinating with 24 nucleotide small interfering RNA (24nt-siRNA). DNA sequences complementary to the 24nt-siRNA that have been generated are methylated , CHROMOMETHYLASE 3 (CMT3), and CMT2 have been shown to be predominantly responsible for maintaining DNA methylation of CG, CHG, and CHH contexts, respectively, and DOMAINS REARRANGED METHYLASE 2 (DRM2) in RdDM  is associated with transcriptional activation, while removal of the acetyl group by histone deacetylase (HDAC) is associated with transcriptional repression. In the case of histone methylation, lysine residues are methylated and the number of methyl groups  is typically associated with transcriptional activation or repression. These changes are catalyzed by the histone lysine methyltransferase (HKMTase) and histone demethylases (HDMases). For example, trimethylation of lysine 4 of histone H3 (H3K4me3) and H3K36me3 are often associated with transcriptional activation, while H3K9me2 and H3K27me3 are associated with transcriptional repression .Linaria vulgaris is due to transcriptional repression of the Lcyc gene by DNA methylation in its genic region (colorless non-ripening (cnr) mutant in tomato (Solanum lycopersicum L.). Non-ripening of tomato fruit is due to the silencing of the LeSPL-CNR gene caused by high levels of DNA methylation in its promoter region  yield, is solely based on epigenetic components, as a genetically identical canola population was used for selection. The epigenetic EUE component was stably inherited, which allowed for selection (Oryza sativa L.); recurrent selection for EUE resulted in enhanced yield in field trials which again was due to heritable epigenetic change , which is an important factor in determining canola (election . This inc change .MutS HOMOLOG1 (MSH1) via RNA interference (RNAi) ] (Sorghum bicolor L. Moench) (A second example of an approach to breeding for enhanced yield that takes advantage of epigenetics involves silencing of e (RNAi) . Silencin tomato , soybean (Merr)] , and sor Moench) . These rA. thaliana accessions determined 78% of methylated cytosines were differentially methylated across accessions. DNA methylation is correlated with place of origin and its climate, suggesting it plays a role in plant adaptation (-4 methylation polymorphisms per CG site per generation) is four to five orders of magnitude higher than the rate of spontaneous genetic mutations (7x10-9 base substitutions per site per generation) (The development of the WGBS method that enables the detection of DNA methylation at single base pair resolution has increased the accuracy of epimutation detection . Compariaptation . Differeaptation . A compaeration) .A. thaliana accessions, five generations of selection produced variations in flowering time and plant architecture within a population, and changed phenotypes were stably inherited for two to three generations. These variations resulted in altered DNA methylation because of the presence of a small number of single nucleotide polymorphisms (SNPs) between selected individuals and their original plants  that is due to an increase in DNA methylation at a locus with a previously low methylation allele gaining methylation to resemble the more heavily methylated allele, and trans-chromosomal demethylation (TCdM) that is due to loss of DNA methylation at one of the methylated genomic segments . Epi-RILs were generated by crossing between wild type and hypomethylated mutants, followed by repeated self-pollination of individual plants creating homozygosity of epigenetic states of each allele . Epi-RILe growth . DNA metthaliana , and a sthaliana . These fA. thaliana, hypermethylation in the CHH methylation pattern is observed in columella relative to other root cell types, perhaps reinforcing silencing in neighboring stem cells  genes, which encode chromatin remodelers  genes (rice seed b-Zipper (RISBZ1) and rice prolamin box binding factor (RPBF) genes, repress aleurone formation  genes . These g3) genes . Similar3) genes . In cont3) genes . In riceormation . Rice DNe grains .Cucumis melo L.), the expression of the 1-aminocyclopropane-1-carboxylic synthase (CmACS-7) gene encodes an ethylene biosynthesis enzyme that represses stamen development in female flowers. In male flowers, wound inducible protein 1 (CmWIP1), a zinc-finger transcription factor, indirectly represses the expression of CmACS-7 and, consequently, aborts carpel development and results in the development of unisexual male flowers. In addition, a DNA methylation change in the DNA transposon inserted into the CmWIP1 promotor causes CmWIP1 silencing, and the conversion from male to female flowers (Diospyros lotus L.), an individual plant has either male or female flowers, and a pair of genes encoding the homeodomain transcription factors, Male Growth Inhibitor (MeGI) and the Y-chromosome encoded pseudogene Oppressor of MeGI (OGI), governs sex determination . Although the characteristics of PRC2 complexes have not been fully understood in crops, some rice studies reported the roles of PRC2 . Although DNA methylation levels often differ between lines within a species, H3K27me3 patterns are mostly conserved between different lines or varieties in the same species . Howevernsitions ; EMBRYON of PRC2 . The ric of PRC2 . Two recass PRC2 , althougass PRC2 . PhenotyVTE3 gene in tomato, mediated by differential methylation of a SINE retrotransposon inserted in the promoter, determine nutritionally important vitamin E levels in tomato fruit  gene encoding the GTP-binding protein, Epi-d1, shows a metastable dwarf phenotype  gene encoding a component of the rice PRC2. This change causes ectopic expression of FIE1, decreasing H3K27me3 and activating the PRC2-mediated genes. Likewise, change in DNA methylation levels on other rice epialleles, Epi-rav6 and Epi-ak1, alters leaf angle by modulating brassinosteroid homeostasis, and photosynthetic capacity, respectively , is highly methylated in wild cotton; however, COL2D in domesticated cottons is hypomethylated and activated; perhaps resulting in photoperiod dependent flowering in domesticated cotton. Similarly, a large number of DMRs contribute phenotypic variation between maize and teosinte and show signals of selection during domestication  pigmentation in the whole plant body, while the Booster-Intense (B-I) allele is highly transcribed and provides dark pigmentation. When the B-I allele is exposed to B’ alleles, B-I is converted to B’ and pigmentation changes from dark to light in all F1 plants  and doesn’t involve tandem repeats  paramutant alleles, and also in supporting both normal somatic development and gametophyte function, via the maintenance of hypermethylated “off” states of given alleles. Furthermore, since they both function to maintain “off” states, CHDs and the RNA polymerase II catalytic subunit RPD1, which plays a role in generating the small RNAs important to paramutation, are mechanistically linked. sulfurea paramutation in the tomato is dependent on the function of the tomato ortholog of A. thaliana’s KYP and CMT3. Furthermore, this result indicates that models of paramutation should include CMT3/KYP and its role in changing the chromatin structure, in addition to mechanisms that involve small RNAs and the RdDM pathways, and that these models should accommodate both locus-specific and species-specific mechanisms.Advances in the study of the molecular biology of paramutation in maize was facilitated by the isolation of stable, genetic mutations of genes that are functionally critical to this phenomenon, yet the genetics of paramutation is not fully elucidated. While the first case of paramutation, the rouge phenotypes in pea, was documented over 100 years ago, no such genetic studies had been reported until the recent work of A. thaliana  in maize found that 145 genes displayed patterns of non-Mendelian e-QTL inheritance that was paramutation-like: where all the segregating progeny from a cross had expression levels for a given gene similar to either one or the other of the two parents . Genome-thaliana  and in tthaliana . ClearlyR locus in maize , has been documented to impact paramutation . In a stin maize , testcroA. thaliana, preferential expression of imprinted genes is explained by strict epigenetic reprogramming during gametogenesis. In the central cell of a female gamete, the DNA methylation level is widely reduced by the action of DNA demethylase  or from the paternal allele, a paternally expressed gene (PEG). Based on studies mostly in ethylase . Howeverive cell . The difive cell , resultiive cell . These aMeg1 is a maternally imprinted gene and involves establishing and differentiating endosperm nutrient transfer tissue . The explant is then placed on media to induce shoot and root formation. The phytohormone concentration required for each developmental stage can depend on the plant species and regeneration of plants from callus can take weeks or even months depending on the plant species. Regenerated plants from the same tissue are clones harboring the same genome, but has phenotypic variation, named somaclonal variation. Somaclonal variation is known to be caused by both genetic and epigenetic changes of the genome that occur during the tissue culture. DNA methylation levels are reported to increase with the 2,4-dichlorophenoxyacetic acid  concentration that is used for callus induction and increases with kinetin concentration that induces shoot formation . Tissue thaliana . EstabliTriticum aestium L.) created a line with a heritable increase of spikelet number possibly caused by epigenetic modification  and zebularine are cytosine analogs that are randomly incorporated into the genome during DNA replication, replacing cytosine. In the case of 5-azadC, the azacytosine-guanidine dinucleotides are recognized and bound by DNA methyltransferase but cannot accept the transfer of the methyl group and become trapped as an adduct ultimately leading to the inhibition of DNA methylation . Use of fication . Howeverfication . As a rafication  because fication . Seedlinfication . The efffication .DNA hypomethylation can result in the activation of mobile TEs that can translocate within the genome by a cut-paste (DNA transposons) or copy-paste (retrotransposons). The transposing TEs may land in a position that will disrupt gene function, leading to heritable genetic changes. These mutations can induce useful agronomical traits. Epigenetic breeding can result in genetic changes and is important to understand the underlying mechanism of the phenotypic change in order to consider the appropriate breeding strategy for crop improvement. For this, the combination of efficient phenotypic, genetic, and epigenetic screening systems may be required to identify useful traits from a large-scale random epigenome modification.via epigenetic modifications. RNAi can be induced by the expression of transgenes designed to form an inverted repeat separated by a spacer that folds into a hairpin RNA (hpRNA) structure (RNAi was developed in the late 90’s as a means to regulate specific gene expression tructure . This hptructure . These mtructure . The exatructure . The hervia the use of genome editing enzymes. Transcription activator-like effectors (TALEs) and Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) are enzymes that have been used for targeted epigenome editing. TALEs are proteins that are naturally occurring transcriptional activators secreted by the plant pathogen Xanthomonas spp. Fusing a DNA methylation or demethylating enzyme such as DNA methyltransferase 3 alpha (DNMT3A) or ten-eleven translocation 1 (TET1) to TALE has been used for DNA methylation modification in mammalian cells . sgRNA consists of a 20 nucleotide sgRNA recognition sequence containing a protospacer adjacent motif (PAM) that has 5’-NGG-3’ sequence and a scaffold sequence for Cas-binding. Cas9/sgRNA complex is directed to bind to the recognition sequence on the genomic DNA. For epigenome editing, a dead Cas9 (dCas9) is used so the Cas9 will still bind to the target DNA but will not cleave the site. Similar to TALEs, an epigenomic modifying enzyme can be fused to dCas9 and direct the epigenome modification in the target region , and thiOne of the important considerations for using artificially induced epigenome modification is the legal or political regulation concerning the safety of the consumers and environment. Genetically modified organisms (GMOs) have been regulated in many countries to limit their use to within a closed environment to prevent the release of any transgenic plant material (including pollen) to an open environment. For releasing transgenic plants for commercial use, a strict and often expensive risk assessment is often required, which may outweigh the benefit of transgenic plants.Conventional mutagenesis using ionizing/non-ionizing irradiation and chemicals have created useful traits for various crops. These techniques can induce structural changes of the genome including deletions, insertions, and chromosomal rearrangements . These mOn the other hand, RNAi, TALE, and CRISPR/Cas9 technologies all require the introduction of transgenes to induce targeted epigenome modification. Therefore, any of these plants would be under GMO regulation, and would be difficult to use for any breeding that requires the release of the plant to an open environment. Recently some countries such as United States, Argentina, Australia, and Brazil have indicated that if no foreign DNA is present in the variety, it would not be regulated as a GMO and would be regulated equivalently to conventional crops . Taking via endocytosis  scion was grafted to a potato (Solanum tuberosum) rootstock and TGS induced via the RdDM pathway; the TGS was maintained in the progeny lacking the siRNAs. Rootstock-to-scion transfer of small RNAs conferring virus resistance in the scion of cherry tree was also demonstrated by grafting of a virus resistant rootstock variety. Grafting of lines that express RNAi or CRISPR/Cas9 transgenes for epigenome modification to an elite variety may allow more efficient epigenetic breeding without the need for finding a graft compatible line with the desired epiallele  by the cabbage butterfly (Pieris rapae L.) resulted in transgenerational priming of the adaptive response of increased trichome density; a greater-in-magnitude response to herbivory was observed in progeny whose parent was stressed than in those whose parent was not stressed , although it was referred to as a transgenerational effect by the authors  in response to a combination of drought and heat stress and in the soybean in response to drought stress  locus which encodes a long-noncoding RNA that is processed into 21-nt phasiRNAs that accumulate in pollen (outer cell layer 4 (ocl4) locus in maize. They found that in one derivative line male fertility restoration by warm temperatures was enhanced as compared with other lines of similar genotype, that this was mediated by the elevated production (as compared with sterile counterparts) and pollen accumulation of a 21-nt phasiRNA, and that this conditioned fertility was perpetuated over several generations but apparently only when pollen maturation occurred under warm temperatures. Thus, it is debatable whether or not this is a case of epigenetically-mediated transgenerational inheritance, according to the strict rules discussed above. Looking forward, one attractive hypothesis concerning pollen-accumulated phasiRNAs is that they could possibly play a role in transmitting epigenetic code to the next generation following pollination.Transgenerational adaptation may be conditioned by a unique type of siRNAs, the 21- and 24-nt “phased siRNAs” (phasiRNAs) that accumulate during male gametogenesis in pollen . Pollen n pollen . Yadava via the MSH1-RNAi approach of the Mackenzie group , seed obtained from parents grown with varying levels of the macronutrients nitrogen, phosphorus, and potassium, produced plants which provided substantially more yield when grown with fertilizer than did seed obtained from plants not grown with fertilizer -containing solution that results in RNAi suppression of selected target genes, such as MSH1, that results in transgenerational enhancement of crop performance. Over the last decade this type of approach has been developed and documented to work intragenerationally for the management of plant viral and fungal pathogens (Enhanced Green Fluorescent Protein (EGFP) and Neomycin phosphotransferase II (NPTII) reporters in A. thaliana resulted in DNA methylation of target gene sequences and regions. Key advantages of this approach over the use of stress treatments is its ease of use and that it would avoid negative impacts of stress treatments on the parent plants, insuring optimal seed yield and seed quality. What if simply spraying plants in a seed production field, or male parents in a hybrid seed production field, with a solution containing various dsRNAs, can result in a transgenic enhancement of productivity by 10% to 25%?Another alternative to the athogens . This tySimple use of parental treatment or stress in seed production to produce crops with enhanced performance in optimal or non-stressful environments would represent an “appropriate technology” . It woulKarma and the mantling trait in the oil palm  and genomic selection, which exploit genetic variation, have made a significant contribution to the efficiency of breeding. In order to apply the same methods to epimutation, it is necessary to develop a method for selecting epialleles that is high-throughput. Inducing epimutation by chemical treatment or epigenomic editing may be able to produce new traits. Since DNA methylation is associated with gene expression, change of DNA methylation of regions that regulate gene expression such as cis-elements may induce new gene expression associated with new phenotype.The stable inheritance of traits is important for the use of epialleles in breeding perhaps more so than for applications in crop production It is clear that taking practical advantage of the potential of epigenetics in crop breeding is in its infancy. While a great deal of interest and enthusiasm has been generated, as evidenced by the numerous review articles addressing the potential of this new science and technology  of Japan Society for the Promotion of Science (JSPS) granted to KT, and Grant-in-Aid for Challenging Research (Exploratory) (20K21313) granted to RF and KO, and Kobe University Strategic International Collaborative Research Grant (Type B Fostering Joint Research), Fund for the Promotion of Joint Research (16KK0171), Grant-in-Aid for Scientific Research (B)  of Japan Society for the Promotion of Science (JSPS) granted to RF.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.


As a newly emerging field, connectomics has greatly advanced our understanding of the wiring diagram and organizational features of the human brain. Generative modeling-based connectome analysis, in particular, plays a vital role in deciphering the neural mechanisms of cognitive functions in health and dysfunction in diseases. Here we review the foundation and development of major generative modeling approaches for functional magnetic resonance imaging (fMRI) and survey their applications to cognitive or clinical neuroscience problems. We argue that conventional structural and functional connectivity (FC) analysis alone is not sufficient to reveal the complex circuit interactions underlying observed neuroimaging data and should be supplemented with generative modeling-based effective connectivity and simulation, a fruitful practice that we term “mechanistic connectome.” The transformation from descriptive connectome to mechanistic connectome will open up promising avenues to gain mechanistic insights into the delicate operating principles of the human brain and their potential impairments in diseases, which facilitates the development of effective personalized treatments to curb neurological and psychiatric disorders. The human brain is a fascinating machine in which the interactions of vast numbers of distributed circuits and networks give rise to complex cognitive functions such as perception, attention, decision making and memory. Delineating the brain’s anatomical wiring diagram and its functional operation principles constitutes an important first step to decipher the underlying mechanisms of cognition. Connectomics is a newly developed field dedicated to providing a complete map of neuronal connections in the brain . Fueled As the essential components of human connectomics, structural and functional connectomes are driven by two major imaging modalities, diffusion MRI and functional MRI, respectively. Diffusion MRI is a method based on measuring the random Brownian motion of water molecules within the white matter , which cFirst of all, such macroscopic connectivity analysis is largely descriptive and superficial  than long-range  connections , which is defined as the directed causal influence among neuronal populations . DiffereThis review paper attempts to give a concise overview of existing generative modeling approaches in fMRI analysis and how they contribute to the emerging and rapidly growing field of human connectomics. We first define generative modeling in a general sense, discuss their important roles in neuroscience, and introduce three major generative modeling frameworks in fMRI connectome analysis. Next we review the state-of-the-art of each of the three major generative modeling approaches and sample their applications to cognitive or clinical neuroscience problems. Lastly, we summarize the developments and achievements of generative models in fMRI connectome analysis and discuss future research directions for this dynamic and promising field in neuroscience.Generative models are computational models that are built to simulate the response profile of a complex system based on the physical mechanisms of interactive components . Such moFirst, they focus on the estimation of effective connectivity or coupling strengths among neural populations. Second, they are driven and constrained by neuroimaging data such as diffusion MRI, fMRI and MEG, and seek to reveal the neuronal mechanisms underlying the observed data. Third, they generally apply population-level  models to describe network dynamics as such dynamic models relate more closely to macroscopic neuroimaging data by describing the collective activity of neuronal populations  a neuronal model that generates population-level neural activity; (2) an observation model that transforms the neural activity to simulated neuroimaging data; and (3) an optimization scheme to estimate connection parameters.The generative models for human connectomics have several distinct features compared with detailed microcircuit models for non-human data. ulations . Lastly,Depending on the differences in neuronal models, the scope of parameter estimation and the optimization scheme, existing generative models for connectomics can be broadly classified into three major types: (1) Dynamic Causal Model (DCM); (2) Biophysical Network Model (BNM); and (3) Dynamic Neural Model (DNM) with direct parameterization. Below we review the foundation and development of these three types of generative models and survey their applications to cognitive or clinical neuroscience.Dynamic causal modeling (DCM) is a predominant analysis framework to infer effectivity connectivity of individual connections at single subject level using standard Variational Laplace procedures , 2007. AThe original deterministic DCM applies to task-fMRI only and is restricted to relatively small networks (< 10 brain regions) due to computational burden . To accoThe original DCM is limited to modeling one neural population for each brain region, which is later extended to two-state DCM to account for the intra-regional interactions between excitatory and inhibitory neural populations . DespiteDespite the significant technical advance of DCM in both model scope  and model complexity , computational efficiency remains a major limitation. Even for the most efficient spectral DCM, inversion of a medium size network with 36 nodes takes about 20–40 h , limitinThe original DCM uses a bilinear state space model :x(t) denotes the hidden neuronal states for multiple brain regions, u(t) represents exogenous experimental inputs and the matrix C models the influence of external inputs on neuronal activity. A is the baseline effective connectivity and Bk represents the modulation on effective connectivity due to the input uk(t). DCM estimates the parameters A, Bk and C based on fMRI data. For the generative neural models of other variants of DCM, refer to related publications  to observed BOLD response yi(t)  leads to a vasodilatory signal si(t) which is subject to self-regulation. The vasodilatory signal induces change in the blood flow fi(t) resulting in subsequent change in blood volume vi(t) and deoxyhemoglobin content qi(t). The hemodynamic model equations are given as follows  . Specifi follows :where κ is the rate of decay, γ is the rate of flow-dependent elimination, τ is the hemodynamic transit time, α is the Grubb’s exponent and ρ is the resting oxygen extraction fraction. The predicted BOLD response is calculated as a static non-linear function of blood volume and deoxyhemoglobin content that depends on the relative contribution of intravascular and extravascular components:v0 is the resting blood volume fraction, and k1, k2 and k3 are the intravascular, concentration and extravascular coefficients, respectively.where (1) experimental design and hypothesis formulation; (2) selection of regions and extraction of fMRI-BOLD timeseries; (3) selection of DCM model depending on the fMRI modality (task or resting-state), network size and the problem of interest ; (4) specification of the neural model including network connectivity and experimental inputs; (5) model estimation at subject-level; and (6) group-level analysis with Parametric Empirical Bayes  approach, In summary, BNM and DCM share similarities but also bear significant differences. At the common side, they both have a generative model for neuronal dynamics and a measurement model to convert the neuronal activity to BOLD signal, and usually integrate an optimization scheme to estimate EC. At the different note, DCM focuses on estimating individual EC at single subject level, while BNM focuses on simulating fMRI data. Based on such distinct design objectives, DCM utilizes a relatively simple bilinear neural model while BNM employs more realistic spiking or neural mass models. Also, BNM usually utilizes SC as a proxy for synaptic efficacy or a backbone for EC, while DCM does not necessarily require structural information, though it can be used as a prior constraint . BesidesVm) is governed by the following equation:For the “direct simulation” approach in BNM, individual neurons are commonly described by the classical integrate-and-fire (IF) spiking neurons  connecteCm is the membrane capacitance, gm the leak conductance, VL the resting potential, and IExt the external input. When Vm crosses a threshold Vth in the upward direction, a spike is generated and the membrane potential is reset to a value Vres for a refractory time period τres. The total synaptic current Isyn is a summation of recurrent excitatory/inhibitory inputs from local region and excitatory inputs from other brain areas; Excitatory inputs are mediated by both AMPA and NMDA receptors, while inhibitory inputs are mediated by GABAA receptors. Synaptic currents follow the following biophysical model  = 1/(1 + exp(−0.062Vm)/3.57) for NMDA currents and B(Vm) = 1 for AMPA and GABAA currents. The gating variable s follows the following dynamics:where A current:For AMPA or GABAFor NMDA current:x models the neurotransmitter concentration with the rise time constant τx. τAMPA, τGABA and τNMDA are the decay time constants for AMPA, GABAa and NMDA synapses, respectively, ti is the presynaptic spike times, and αs controls the saturation properties of NMDA.where The dynamic mean field model (MFM) has been frequently used in BNMs , which iSi, xi and H(xi) represent the average synaptic gating variable, the total input current, and the population firing rate at brain region i, respectively. G is a global scaling factor, J is the value of synaptic coupling, w is the recurrent connection strength, and Cij denotes the SC between region i and region j. τs and r are kinetic parameters, and a, b and d are parameters of the function H(xi). vi represents uncorrelated Gaussian noise with standard deviation σ. The simulated neural (synaptic) activity Si is fed to the same hemodynamic model as DCM (Eqn. 2–6) to generate simulated BOLD timeseries.where (1) parcellating the brain into discrete regions; (2) extracting whole-brain fMRI-BOLD timeseries and calculating FC; (3) computing SC based on diffusion MRI data; (4) representing each network node with populations of spiking neurons or neural-field/NMM of local neuronal populations; (5) linking individual network nodes with long-range connections based on SC; (6) transforming the network activities to simulated BOLD signals via a hemodynamic model; and (7) fitting model parameters to FC via an optimization scheme.Development of BNMs includes the following major steps: There are a number of computational software packages to facilitate the development and implementation of BNMs including NEURON , BRIAN , NEST G, and Thein silico stimulation predicted by the BNM can accurately force transitions between different brain states, and in particular, from deep sleep to wakefulness and vice versa. These findings provide valuable insights how and where to induce a brain state transition using whole-brain BNM, which may potentially apply to restore the pathological brain state to normal state using external stimulation. The new brain state definition and modeling framework were recently applied to study the effect of external stimulation on functional networks in the aging healthy human brain (N = 310 for each group). A whole-brain BNM was then developed and fit to the PMS and in silico stimulation with region-specific intensity was applied to induce transitions from the brain states of the older group to those of the middle-age group. The authors discovered that the precuneus, a brain region belonging to DMN and involved in a variety of complex functions such as episodic memory and visuospatial processing, is the best stimulation target for brain state transition. This elegant study suggests that generative models for neuroimaging data could potentially serve as gateway for the design of novel brain perturbation techniques to reverse the adverse effects of aging on cognitive functions.One fundamental question in cognitive neuroscience is how different cognitive states such as attention, sleep and wakefulness are defined mechanistically and switch from one state to the other. Existing definitions focus on resting networks and statistical description of functional activity patterns , which dan brain . The autAnother important contribution of BNM to cognitive neuroscience is the manifestation of the dynamical origin of slowness of thought during task-based cognition . This diSince its introduction, the MOU-EC model  has beenIn addition to DCM and BNM, the two major types of generative models for fMRI, there exist other modeling frameworks that estimate EC and study connectome in a generative fashion . One fraOne recent development of DNM is termed Mesoscale Individualized Neurodynamic (MINDy) modeling that fits non-linear dynamical neural models directly to fMRI data . To achivia long-range fibers whose baseline connection strengths are determined by SC from diffusion MRI; weak inter-regional connections are removed to construct sparse networks and avoid the problem of over-parameterization. The regional neural activity is converted to corresponding BOLD signal via a hemodynamic model  is random additive noise following a normal distribution. The long-range connectivity strength from region k to region j is represented by Wkj scaled by empirical SC (Ckj). The non-linear response function S is modeled as a sigmoid function Wkj, u based on individual SC and FC.where (1) parcellating the brain into discrete regions; (2) selecting brain regions to model and extracting fMRI-BOLD timeseries; (3) calculating FC and/or SC if necessary; (4) representing each network node with a NMM of local neuronal populations; (5) linking individual network nodes with long-range connections with or without SC constraint; (6) deconvolving the empirical BOLD signals with a hemodynamic response function  fitting model parameters to deconvolved BOLD signals or FC via an optimization scheme. Due to the flexibility in neuronal model, SC utilization, choice of objective function and optimization algorithm, DNMs are generally implemented using customized scripts with MATLAB or other computing languages .The development steps of DNMs are generally similar to BNMs: ion HRF,  or transic model ; and (7)DNM has been applied to study the circuit mechanisms of major depressive disorder (MDD). MDD is a leading cause of chronic disability worldwide with a lifetime prevalence of up to 17% , but theThe MNMI framework is well suited to address these questions due to its biological realism utilizing a physiologically informed NMM and its multiscale nature incorporating both intra-regional and inter-regional neural interactions. By applying the MNMI framework to a large sample-size resting-state fMRI dataset consisting of 100 MDDs and 100 normal control (NC) healthy subjects, Driven by the rapid advances in non-invasive neuroimaging techniques, the young emerging field of human connectomics has made significant accomplishments in characterizing the large-scale organizational features of both structural and functional brain networks. Notwithstanding, the potential of connectomics to answer fundamental neuroscience and clinical questions has yet to bring into full play. To achieve such important goal, computational connectomics has moved beyond the anatomical and statistical description of connectivity to more mechanistic formulation of the neural processes underlying neuroimaging data . Mechanistic connectome based on generative modeling of fMRI offers a natural and principled tool to link microscopic or mesoscopic neural process with macroscopic BOLD dynamics, which enables mechanistic understanding of brain cognitive functions in heath and dysfunction in diseases. It is important to note that, unlike static structural connectome, mechanistic connectome based on effective connectivity is parameterized by the state of the brain. That is, one would obtain a very different mechanistic connectome when the task demands change, or when the stimuli challenge, or when endogenous activity switches to a different state  . Thus, tRecent years have seen tremendous development and expansion of generative model-based connectome analysis toolsets. Two well-established and widely used modeling frameworks include DCM and BNM, which represent two approaches at the opposite end of biological realism and estimation tractability. Specifically, while DCM allows estimation of full connection parameters at individual subject level, the physiological interpretability for model parameters is limited due to the abstract bilinear state model. On the other hand, though BNM incorporates more physiologically grounded neuronal models for fMRI generation, its identifiability is limited to one or a small subset of parameters often at the group-average level. Effort to combine the advantages of DCM with BNM has led to the development of a different type of modeling framework that can be categorized as Dynamic Neural Model (DNM) with direct parameterization. DNM seeks to model population-level neuronal dynamics accurately with biophysically plausible NMMs and estimate physiologically meaningful parameters for individual connections at single subject level. It should be noted that the boundary among these three types of generative models is diminishing with the latest developments of DCM and BNM which utilize more biophysically informed models and are equipped with the capability for efficient EC estimation of large-scale networks at both individual connection and individual subject levels. One should expect the convergence of DCM, BNM and DNM continues in the future.via Bayesian model selection, a process where different candidates of models are iteratively generated and compared to reach the models that have the optimal level of complexity , while not being more complex than the data can accommodate (given the limited resolution of fMRI). This principle has been well implemented in DCM mplexity . Specifimplexity ,b. The tmplexity . AddressGL and P-TY contributed to the conception of the work, revised the manuscript, and approved the final version of the manuscript. GL drafted the manuscript. Both authors contributed to the article and approved the submitted version.