Analysis of major upstream regulators suggested that melatonin may activate PKB/mTOR signaling pathways to program the metabolism of KGN cells to support slower growth and differentiation and to prevent follicular atresia. Similarly, PKA activation through stimulation of cAMP synthesis with FSK seemed to exert the same effects as melatonin in reducing follicular growth and regulating differentiation. This study suggests that melatonin may act through PKA and PKB simultaneously in human granulosa cells to prevent follicular atresia and early luteinization at the antral stage.Female reproduction depends on the metabolic status, especially during the period of folliculogenesis. Even though it is believed that melatonin can improve oocyte competence, there is still limited knowledge of how it can modulate metabolic processes during folliculogenesis and which signaling pathways are involved in regulating gene expression. To investigate the effects of melatonin on metabolic signals during the antral stage of follicular development, human granulosa-like tumor cells (KGN) were treated with melatonin or forskolin, and gene expression was analyzed with RNA-seq technology. Following appropriate normalization and the application of a fold change cut-off of 1.5 (FC 1.5,  Female reproduction is sensitive to the metabolic status, particularly nutrition intake during gestation, but also in the period of gametogenesis . In monoThe oocyte acquires its maturation competence during folliculogenesis, when the bidirectional communication between somatic cells and the oocyte creates an intrafollicular microenvironment that maintains the growth of primordial follicles into antral follicles, from which one is selected to ovulate a high-quality oocyte. This microenvironment depends on the maternal metabolic environment, which can affect the oocyte epigenetic and metabolic programming throughout follicular growth .Over the last few years, studies have shown melatonin (N-acetyl-5-methoxytryptamine), a neurohormone secreted by the pineal gland, controls the daily coordination of physiological, neuroendocrine, and behavioral processes. Most tissues, including ovaries, can synthesize melatonin in their mitochondria. Melatonin is expressed in oocytes, cumulus, and granulosa cells, where it acts through several molecular cascades via its specific receptors ,7. It isThere is very little knowledge of how melatonin regulates and controls metabolic processes during follicular growth and development and of which potential signaling pathways are used by melatonin to trigger specific gene expression events. A few studies examined the effects of melatonin on oocyte metabolic programming and showed that supplementation of porcine oocyte culture medium can affect lipid metabolism and modulate mitochondrial metabolic functions ,13, but The overall objective of this study was to investigate the effects of melatonin on metabolic signals during the antral stage of follicular development as assessed by transcriptomic analysis. The human granulosa-like tumor cell line (KGN), which originated from a diagnosed stage-3 granulosa cell tumor from an antral or pre-antral stage follicle of a Japanese woman in 1984, was selected as a model for this study . By usinGene Expression Analysisp-value of 0.05. A full list of DEGs for each treatment is available in supplemental data  software was used to identify the principal canonical pathways and upstream regulators associated with the observed DEGs in each treatment , were also identified in the FSK treatment. Vascular endothelial growth factor A (VEGFa), a major upstream regulator, was inhibited (z-score = −2.090) following PKA stimulation. Studies showed that inhibition of VEGF stimulates fatty acid oxidation and decreases glucose uptake and anaerobic glycolysis in endothelial cells ; howeverTumor proteins TP53 and TP73 were scored as activated in the FSK treatment. The TP53 regulates glucose metabolism by repressing the expression of glucose transporters and downregulating rate-limiting enzymes of glycolysis. It activates the pyruvate dehydrogenase complex (PDH) to reduce the conversion of pyruvate to lactate and to increase the conversion of pyruvate to acetyl-CoA to maintain oxidative phosphorylation. Besides, TP73 is a stimulator of glutamine uptake to replenish the tricarboxylic acid (TCA) cycle by decreasing the expression of the transcription factor MYC, which was inhibited in our treatment (z-score = −2.976). In turn, this gene is responsible for suppressing the mRNA expression of the glutamine uptake receptor SLC1A5 . HGF wasTo obtain a biological interpretation of the combination of differentially expressed genes, we used IPA to highlight the functions and pathways affected in each treatment. A list of canonical pathways and the principal putative regulators that were more significant to the input data set was identified by IPA .Granulosa cells are specialized cells that act as a barrier to protect the oocyte against the extra ovarian microenvironment by metabolic and signaling interplay. An increased understanding of their roles in metabolism and reproduction continues to be instrumental in the improvement of infertility prevention methods and treatments to ameliorate IVF outcomes. During the final stages of maturation, granulosa cells supply the growing follicle with energy substrates to produce the necessary ATP for the oocyte to reach the preovulatory stage. This process is highly regulated by the metabolic and signaling coupling between cumulus and granulosa cells and between cumulus cells and the oocyte.The use of the KGN granulosa cell line as opposed to in vivo granulosa cells or primary cultures gave us the possibility to repeatedly establish very specific and controlled conditions that allowed us to explore the molecular pathways involved in the regulation of granulosa cell metabolism by melatonin. The metabolic profile of granulosa cells obtained from growing follicles seemed to resemble the profile of highly proliferating cells, such as cancer cells and pre-implementation embryonic cells . While nIt is well known that reproduction and metabolism are interconnected and reciprocally regulated. Several studies showed that female fertility depends on the maternal metabolic environment, which affects the intrafollicular microenvironment and consequently oocyte quality ,29. The This study revealed key target genes implicated in the downstream signaling network used by melatonin to exert its function of “metabolic programmer” in human granulosa-like cells (KNG). Some reports claimed that the effects of melatonin on the modulation of many reproductive functions are transduced by two G-protein-coupled receptors, MTNR1A and MTNR1B, which were detected in human granulosa cells  The signIn line with these findings, our data confirmed that melatonin is a regulator of the granulosa cell metabolism-related factors DAP3, MT-TE, LONP1, MALSU1, and MRPL14, which are mitoribosomal proteins involved in the regulation of mitochondrial translation, mitochondrial ribosome biogenesis, mtDNA metabolism, and cell-stress response. Moreover, the most significant upstream regulators in this treatment, DAP3, LONP1, and STOX1, are PKB signaling downstream factors. DAP3, a substrate of Akt kinase, has a dual function in both mitochondrial physiology and cell death regulation , but itsOur results confirmed that melatonin plays an essential role in controlling human granulosa cell metabolism homeostasis and apoptosis under hypoxia conditions. The actions of melatonin are mediated via HIF1α and HIF2α (EPAS1) expression, transcription factors expressed in response to low cellular oxygen levels in granulosa cells of numerous species ,45. In tThe expression of aging-related sirtuin genes in mural granulosa cells and KGN cells is well documented ,58. The Folliculin (FLCN) is a tumor suppressor causing the Birt–Hogg–Dubé syndrome and is involved in energy homeostasis regulation by controlling several metabolic pathways. Its roles in several organ cells are well studied; however, to the best of our knowledge, FLCN expression in human granulosa cells was reported in our data for the first time. It is known that the transcriptional activity of TFE3, a member of the MiTF/TFE transcription factor family, is negatively regulated by FLCN, which was confirmed in our data. The knockdown of FLCN in salivary glands and cystic kidneys triggered metabolic reprogramming pathways by modulating PGC1α and TFE3 transcription and the AMPK-mTOR pathway to support cell proliferation ,72. LossMelatonin seems to be implicated in regulating steroid receptors in human granulosa cells The G protein-coupled estrogen receptor 1  is expressed in KGN cells  and had The effects of melatonin are mediated through specific receptors, which activate numerous signaling pathways, such as the PKA pathway, in several tissues ,78,79,80The cell cycle inhibitors TP53 and CDKN1A, which are related to granulosa cell differentiation, were among the upstream regulators identified by IPA and had a strong tendency to be activated through PKA activation in the FSK treatment. Characterized by its ability to induce cell cycle arrest, apoptosis, and senescence, TP53 was also reported to play an important role in metabolic regulation  through Hepatocyte growth factor (HGF) was highlighted as an inhibited upstream gene by IPA in response to PKA activation by FSK in agreement with previous reports from our group . This grAdenylate cyclase activation regulates the expression of several cell proliferation and cell cycle genes, such as FOXM1, CCDN1, CDKN2A, E2F4, RABL6, and CDK4. According to IPA, most of these upstream regulators were inhibited in our FSK treatment. These results revealed that PKA activation may inhibit granulosa cell proliferation, which was previously highlighted by our team . Both cAERBB2 and AREG, two ovulation-related factors, were among the most significant upstream regulators and were inhibited by PKA stimulation. It is known that ERBB2 and AREG stimulate glucose uptake and could regulate mitochondrial functions through switching cancer cell metabolism from OXPHOS to glycolysis to enhance cell growth ,99,100. Unless otherwise stated, all chemicals, such as 4-androstene-3,17-dione (product A9630), melatonin (MLT) (product M5250), and forskolin (FSK) (product F3917), were purchased from Sigma-Aldrich . Each was dissolved in 95% ethanol to make stock solutions.The granulosa cell line KGN was purchased from the RIKEN Bioresource Centre . This cell line presents some physiological characteristics of normal human granulosa cells, such as steroidogenic activities and functional FSH receptors .2/95% O2 at 37 °C. The cells were thawed on day 1, placed in a cell culture flask , and sub-cultured on day 3. Confluence was never over 80%. On day 6, cells were placed in a 6-well dish at a density of 1.5105 cells per well in 1.5 mL of medium and grown for 72 h, at which point they were reaching almost full confluency. For all experimental conditions, the medium was replaced with charcoal-stripped (SVFA) DMEM/F12 containing antibiotics and 100 nmol L−1 of 4-androstene-3,17-dione. The 24 h treatments consisted of adding either ethanol as a control  or additional stimulators in ethanol to obtain either 1 nmol of melatonin L−1 or 1 μmol of FSK L−1 as final concentrations. The experiment was repeated four times (four different weeks) to generate four biological replicates for RNAseq analysis.The KGN cells were cultured as previously described . BrieflyTotal RNA was isolated with the AllPrep DNA/RNA Mini Kit  according to the manufacturer’s instructions. Total RNA integrity and concentrations were evaluated on a 2100-Bioanalyzer . The RNA Integrity Number of all samples was 10. The total RNA input used for sequencing was 360 ng for each treatment, which consisted of 90 ng from each biological replicate. The mRNA was isolated from total RNA using the NEBNext Poly (A) mRNA Magnetic Isolation Module .PolyA-selected mRNA was fragmented to a mean size of 200 nt, reverse transcribed to generate double-stranded cDNA, and converted to a paired-end library using the NEBNext Ultra RNA Library Prep kit for Illumina  according to the manufacturer’s instructions, with Agencourt AMPure XP beads and NEBNext Mutliplex Oligos for Illumina . Libraries were sent to the Génome Québec Innovation Centre at McGill University for quality control tests. Libraries were then pooled at equimolar concentrations and sequenced on an Illumina HiSeq4000 in paired-end mode with 100 base pair reads (PE100) to a depth between 55 and 67 million reads.Ensembl (release 91) was used as the source of annotated genes and transcript isoforms. Trimming of adapters was performed using Trimmomatic; sequencing adapters were removed, and base calls with a quality score below 30 were removed from the end of the reads . TrimminFunctional analysis was applied using the ingenuity pathway analysis software (IPA), a web-based software that enables the interpretation of data derived from omics experiments, to create a global picture of the principal upstream regulators and genomic mechanisms implicated in the treatments. This software can compare our dataset to the ingenuity knowledge Base to provide biological interactions and functional annotations and attribute a probability of association between genes in our data and biological functions. We uploaded, separately, the lists of differential expressed genes from RNA-seq analysis of each treatment into the IPA software to be analyzed. Then, we extracted the major biological functions as well as the most significant upstream regulators to our dataset.The results of this study suggest that melatonin supplementation in a dominant-like plateau phase follicular context may activate PKB/mTOR signaling to program granulosa cell metabolism during the switch in the hormone-dependent state and following the FSH decline. These metabolic effects include balancing between glycolysis and oxidative phosphorylation and between mitochondrial fission and fusion to support slower growth and proliferation of granulosa cells as well as balancing between follicular atresia and cell survival and regulating granulosa cell early differentiation. Protein kinaseA activation by FSK stimulation of cAMP synthesis exerted similar effects as melatonin. Additionally, the PKA and cAMP-dependent activation of PKB suggests that melatonin may act through PKA and PKB simultaneously in human granulosa cells via its membrane receptors and its free radical scavenging activity to prevent follicular atresia or luteinization at the antral stage. These findings confirmed previous reports suggesting that melatonin, acting through the PKB/mTOR signaling pathway, plays an essential role in follicular somatic cell metabolic programming to improve oocyte developmental competence. To the best of our knowledge, this is the first study to investigate the signaling pathways involved in the “metabolic programmer” role of melatonin in human granulosa cells. Given the crosstalk between PKA and PKB signaling in ovarian granulosa cells, this study supports these findings and suggests that melatonin may act through both PKB/mTOR and PKA pathways to prevent follicular atresia and delay ovarian aging. We also showed that the KGN cell line is a unique and useful in vitro model to study the transcriptomics and metabolomics of follicular dominance/growth and oocyte competence acquisition processes. Future studies focused on the effects of specific activators and inhibitors of the melatonin signaling pathways and metabolic activity are required to demystify further the intricate PKB signaling involved in granulosa cell metabolism and antral follicle growth.


Plasmodesmata (PD) are membrane-lined pores that connect adjacent cells to mediate symplastic communication in plants. These intercellular channels enable cell-to-cell trafficking of various molecules essential for plant development and stress responses, but they can also be utilized by pathogens to facilitate their infection of hosts. Some pathogens or their effectors are able to spread through the PD by modifying their permeability. Yet plants have developed various corresponding defense mechanisms, including the regulation of PD to impede the spread of invading pathogens. In this review, we aim to illuminate the various roles of PD in the interactions between pathogens and plants during the infection process. We summarize the pathogenic infections involving PD and how the PD could be modified by pathogens or hosts. Furthermore, we propose several hypothesized and promising strategies for enhancing the disease resistance of host plants by the appropriate modulation of callose deposition and plasmodesmal permeability based on current knowledge. Throughout their life span, plants are constantly challenged by pathogens, namely fungi, bacteria, and viruses  and sugar canemosaic virus MPs was found necessary and sufficient for PD localization  by the double-gene block module  assembles five MPs to facilitate its intercellular movement, including four viral components-an Hsp70h, a 64kDa protein, and two capsid proteins-and a none-structural 6-kDa hydrophobic protein  to target to and dilate PD  2 encodes a chloroplast DEAH RNA helicase, whose mutation increases the branched PD formation and intercellular trafficking  can move rapidly from the initially injected mesophyll cells which are interconnected by PD into neighboring cells, whereas it was retained in mature guard cells lacking PD connections. The PSTVd consists of 27 RNA loop motifs flanked by short helices, of which 11 loops were identified as critical for its intercellular movement  and BIOTROPHY-ASSOCIATED SECRETED (BAS1) produced by Phytophthora brassicae can interact with and inhibit CalS1, CalS2, and CalS3, to reduce the callose deposition around PD, so as to promote symplastic trafficking  is a phloem-inhabiting bacterium that causes a destructive disease of citrus trees called Huanglongbing (HLB), which is achieved by its spread via sap flow in the phloem throughout the host plants  DC3000, a putative mono-ADP-ribosyltransferase. The amino acids in position 41 to 283  of HopO1-1 are required for its localization to PD  is increased by 10 to 50% (Vatén et al., CALS3 can foster the increased production of callose and reduced aperture of PD that together impair cell-to-cell trafficking activity. This raises an intriguing hypothesis: the introduction of same-site mutations of cals3m into other native CalS genes that are quickly and dramatically induced by pathogen attacks, may function similarly as cals3m, precluding the introduction of an exogenous gene resource. CRISPR technology is a suitable choice for gene editing (Zaynab et al., CalS1 and CalS12 were highly induced in response to biotic stresses (Dong et al., cals1m and cals12m similarly. However, a pre-test for screening those modifications that do not interfere with the normal functioning of plants in the absence of pathogens is still necessary. When pathogens attack, these improved CalS proteins are then functioning at high efficiency.Vatén et al.  identifiOver the last few decades, findings have increasingly emerged which are helpful for addressing how pathogens modify the PD structure and permeability to facilitate their intercellular movement and how plants manipulate PD to impede pathogenic infections. It is known that various PD-localized components are involved in the interactions between pathogens and plants, but many questions about mechanistic differences in how PD are regulated remain unanswered. For example, is there any conserved molecular mechanism conferring symplastic mobility to various pathogens? How do some pathogens or effectors overcome the blockage of PD by callose, and why do others fail to? How do plants manage themselves to gain control over the modification of PD when competing for this with pathogens during an infection? Previously, high-resolution electron microscopy and genetic approaches have greatly advanced our understanding of PD structure and function. Methodological improvements in the isolation and purification of PD may be helpful for identifying new PD components and examining their modifications that occur during interactions between pathogens and plants. PD regulation by pathogens and plants could provide us with a new perspective for the genetic improvement of plant disease resistance.JL, LZ, and DY wrote the manuscript. DY and JL designed the figures. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Androgens are synthesized in the brain, gonads, and adrenal glands, in both sexes, exerting physiologically important effects on the structure and function of the central nervous system. These effects may contribute to the incidence and progression of neurological disorders such as autism spectrum disorder, schizophrenia, and Alzheimer's disease, which occur at different rates in males and females. This review briefly summarizes the current state of knowledge with respect to the neuroplastic effects of androgens, with particular emphasis on the hippocampus, which has been the focus of much of the research in this field. For example, in comparison to their male counterparts, women are more likely to develop AD and experience a more rapid decline in memory.4 The severity of cognitive symptoms associated with many of these disorders also appears to differ between the sexes.2 That these sex differences might be attributable, at least in part, to the actions of sex steroid hormones has prompted increased interest in the mechanisms mediating hormonal effects on the brain.The incidence and progression of several neurological and psychiatric disorders, including Alzheimer's disease (AD),5–7 In addition to wide-ranging effects on neurotransmitter and neuropeptide systems9 and cerebral energy metabolism,5 estradiol has also been demonstrated to induce dramatic, reversible changes in cellular morphology in many regions of the brain. Estrogen-induced changes in neuroplasticity include alterations in dendritic morphology, spine synapse density, and neuron number.11Much of the work in this field has focused on the effects of the principal ovarian steroid, estradiol, because of data indicating that circulating and locally synthesized estradiol may play a fundamental homeostatic role in the brain.12 Androgen-induced changes in cognitive functions and behavior have been reported in humans13–15 as well as experimental animals.17 In men, the gradual decline in testosterone with age has been correlated with cognitive performance, with lower levels of free testosterone in older men being associated with reduced performance on verbal and spatial memory tasks.18–20Relatively less is known about the effects of androgens on the brain, although there are similarities between their effects and those of estrogens, particularly in females, at least in part because testosterone is a substrate for estradiol biosynthesis in the brain.16 Associated with these effects, morphological analysis has demonstrated that androgens induce marked changes in hippocampal and cerebral cortical dendritic morphology, spine and spine synapse density, and hippocampal neurogenesis.21–26In male rodents, gonadectomy and testosterone supplementation has been found to influence spatial and working memory, although the responses appear to be mixed, possibly due to methodological differences across studies, including the type of cognitive test used and age of the animals.Although the catalogue of reported neuroplastic effects of androgens on the brain continues to expand, how these effects are mediated remains only partially understood. In this review, we provide a brief summary of the current research on androgens and their influence on various aspects of neuroplasticity, with a particular focus on the effects of these hormones in the hippocampus and the possible mechanisms mediating these effects. We also highlight areas of uncertainty and suggest possible directions for future research.Androgens, similar to other steroid hormones, are derived from the metabolism of cholesterol . The iniThese steroids are precursors for the synthesis of dehydroepiandrosterone (DHEA), androstenediol, and androstenedione, weak androgens that are themselves substrates for conversion to testosterone, the predominant androgen circulating in the bloodstream. In androgen target tissues, testosterone is further metabolized to a range of biologically active metabolites, including 5α dihydrotestosterone (DHT), via the actions of 5α-reductase, as well as estradiol via cytochrome P450-aromatase.A receptor.27 Both 3β-diol and, to a lesser extent 3α-diol, are ligands for estrogen receptor β (ERβ), possibly mediating some of the analgesic, anxiolytic, and cognitive-enhancing effects of circulating testosterone.28In addition to being a more potent androgen than testosterone, DHT is also the precursor for two other biologically active metabolites: 5α-androstane-3α,17β-diol (3α-diol) and 5α-androstane-3β,17β-diol (3β-diol). 3α-diol is an allosteric modulator of the GABA30This pattern of local tissue metabolism complicates elucidation of the mechanisms involved in mediating the effects of testosterone on the brain, since not only may multiple different neuronal and glial cell types be involved, but also there may be contributions from multiple receptor systems, responding to locally produced metabolites with a range of bioactivities. More detail regarding the enzymes and pathways involved in steroidogenesis is provided elsewhere.31 The majority of circulating testosterone is synthesized by the Leydig cells of the testes in males and in the ovarian stroma in females,32 although the amounts produced by the testes are ∼7 to 8 times higher than those produced by the ovaries.31 Biosynthesis of testosterone also occurs in the cortex of the adrenal gland in both sexes.34Although testosterone is often considered to be primarily a male sex hormone, it is present in females as well as males and exerts important physiological effects in both sexes.35 However, extensive evidence indicates that the brain itself is capable of local steroid synthesis.36 This can occur by either local metabolism of circulating steroid intermediates or via de novo synthesis from cholesterol .Since androgens are nonpolar and are capable of easily diffusing across the blood–brain barrier, it was long assumed that androgens were synthesized in the periphery and passively distributed throughout the brain to exert their effects.38The idea that androgens could be synthesized in the brain was first developed by Baulieu and colleagues in the 1980s. These authors found that androgens  and their sulfoconjugate were present in higher concentrations in the nervous system than in the plasma, and these high levels remained long after gonadectomy and adrenalectomy of male rats.38 Subsequently, the presence of the necessary steroidogenic enzymes in the brain was established in several species, including rodents and humans (reviewed in39–42). Along with the enzymes required for conversion of the primary sex steroids into biologically active metabolites.43–45 the discovery of de novo neurosteroid synthesis has greatly expanded our appreciation of the diverse roles of gonadal steroids in the regulation of neuronal structure, function, and activity.46These findings led to the introduction of the term “neurosteroids” to refer to steroids synthesized locally in the brain.48 and more rapid effects initiated via membrane receptors linked to kinase signaling cascades.49 Nuclear steroid receptors  may in some instances subserve both roles, with a portion of the receptors in the cell being translocated to the plasma membrane.50Androgen action on the brain involves effects mediated via both steroid receptors of the nuclear receptor family, which bind to hormone response elements on DNA to regulate the expression of target genes,52 as well as on oligodendrocytes and astroglia.53 Estradiol, whether synthesized locally or delivered via the circulation, acts on both nuclear estrogen receptors (ERα and ERβ) and the G-protein coupled membrane estrogen receptor 1,54 which are present in several subregions of the hippocampus and dentate gyrus (DG).56The AR have been found in both nuclear and extranuclear sites in hippocampal neurons,57–59 Gonadal hormones such as androgens have a profound influence on the induction of LTP and LTD in the hippocampus.60Electrophysiological studies of long-term potentiation (LTP) in hippocampal slice preparations provided the first indication that androgens might induce functional plasticity in the hippocampus. LTP and long-term depression (LTD) are lasting changes in synaptic response following an initial stimulus that are believed to reflect key molecular mechanisms underlying learning and memory.61 Orchidectomy also altered paired-pulse facilitation of fEPSP within the hippocampus,61 suggesting that the removal of circulating androgens has a significant effect on presynaptic transmission, as well as on synaptic maturity.61 These effects are multifaceted, including both positive and negative effects on hippocampal neural activity.After orchidectomy, male mice and rats displayed a reduction in LTP and a decrease in the amplitude of field excitatory postsynaptic potentials (fEPSP).62 found that in adult male rats, orchidectomy resulted in increased synaptic transmission and LTP in the mossy fiber pathway, associated with a long-lasting upregulation of mossy fibers brain-derived neurotrophic factor (BDNF) immunoreactivity, compared with sham-operated rats.62 Thus, the overall effects of androgen on hippocampal function may be mixed, enhancing some aspects of hippocampal neurotransmission while simultaneously limiting hyperexcitability and some forms of plasticity in the mossy fiber–CA3 pathway.62Thus, Skucas et al.63 The AR deletion in an AR mutant mouse model demonstrated a reduction in N-Methyl-D-aspartate (NMDA) receptor-mediated EPSP and high-frequency stimulation LTP of CA1 pyramidal neurons, indicating impairments in glutamate receptor activation.64In addition to orchidectomy-based studies, pharmacological and molecular targeting of the AR has also been used to investigate the effects of androgens on hippocampal LTP and LTD. Although administration of the AR antagonist flutamide has minimal effects on hippocampal LTP, it impairs the induction of LTD of CA1 pyramidal neurons, indicating a disruption in signaling processes.64Although NMDA receptor levels did not differ between AR mutant and wild-type mice, this suggests that eliminating AR action leads to an overall decrease in the activation and function of NMDA receptor.Ex vivo administration of testosterone has been reported to facilitate LTP, increase overall excitability of CA1 pyramidal neurons in both gonadectomized male and female rats, and normalize CA3 mossy fiber fEPSP of orchiectomized males back to levels comparable to those observed in intact rats.62 DHT has effects on LTP similar to those of testosterone, suggesting that local conversion to estradiol is not required for the response.The administration of androgens such as testosterone and its major 5α-reduced metabolites DHT and 3α-diol has also been shown to directly affect hippocampal synaptic transmission. 65 This response was reversed with the application of DHT, but not testosterone.65 DHT has been shown to reverse increases in mossy fiber transmission of orchiectomized rats back to the levels observed in sham-operated animals.62 However, 3α-diol had no effect on CA3 fEPSP,62 consistent with the view that the effects were mediated via AR-dependent mechanisms.Treatment with the 5α-reductase inhibitor finasteride to block the metabolism of testosterone to DHT and 3α-diol in gonadally intact males resulted in a reduction in LTD and decreased fEPSP of CA1 pyramidal neurons.66 and the DG of the hippocampus.67 Newly proliferated neurons in the DG subgranular zone migrate to the inner granule cell layer, where they extend long axons along the mossy fiber pathway to the CA3 subregion.68–70The electrophysiological evidence for functional alterations in hippocampal neuroplasticity after androgen exposure led to studies aimed at defining the effects of these hormones on the hippocampal circuitry. Studies over the past 40 years have demonstrated that neurogenesis continues in regions of the adult vertebrate brain that are involved in learning and memory, notably in the vocal control centers of songbirds72 A growing body of research indicates that, in the hippocampus of adult rodents, gonadal steroids affect multiple steps in this pathway. This subject is covered in detail in the article by Blankers and Galea in this special issue,21 so we will only provide a brief summary here.Neurogenesis is a complex multistep process that involves the proliferation, differentiation, migration, and survival of new neurons.73 whereas the reverse is true for the non-aromatizable androgen, DHT.74 By contrast, in adult male voles, testosterone appears to enhance the survival of newly generated neurons in the DG, with no significant difference in cell proliferation.75The effects of gonadal steroids on neurogenesis appear to be both sex- and hormone-specific. Thus, estradiol modulates new neuron survival and cell proliferation in the DG of adult female, but not male, rats,24 However, in castrate adult male rats, 30 days of hormone replacement via injections or implants of testosterone propionate have been shown to increase neurogenesis in the DG via cell survival, relative to controls.76 The dose and duration of testosterone administration appears to be an important determinant of the effects of testosterone on neurogenesis.77Similarly, castration of adult male rats led to a decrease in cell survival of new neurons within the DG, with no effect on cell proliferation.76 Consistent with this hypothesis, Okamoto et al. found that in male rats, mild exercise increased local intrahippocampal DHT synthesis, resulting in significantly increased neurogenesis, a response blocked by systemic administration of the AR antagonist, flutamide.78Studies using metabolites of testosterone, as well as receptor-specific antagonists, suggest that the effects of testosterone on neurogenesis in the male hippocampus are probably mediated via AR, as opposed to local estrogen biosynthesis.79 suggesting that androgens may influence cell survival directly via AR in newly generated neurons. However, whether the effects are actually mediated directly within the DG remains uncertain because of evidence indicating that AR are expressed in the CA1 and CA3 subregions of the hippocampus, but not the DG.80–83The molecular mechanisms underlying these effects are not yet fully understood. Some studies in male rodents have detected AR protein and mRNA expression in the DG,84 In particular, androgens may bind to AR on CA3 pyramidal cells, initiating the release and retrograde transport of a survival factor that may act on newborn neurons in the DG.85 This hypothesis is consistent with observations indicating that newly formed neurons in the DG send projections to CA3, whereas damage to the CA3 has significant effects on neurogenesis in the DG.87Because AR are not expressed by immature neurons within the DG of young male mice and rats, it is possible that their effects may be mediated indirectly via target cells located elsewhere in the brain.88It also provides a striking parallel to work in the developing spinal cord, in which motoneurons in the spinal nucleus of the bulbocavernosus (SNB) are dependent for survival on androgen-dependent trophic support mechanisms via the target muscles that they innervate, even though the SNB neurons themselves do not express AR.89 These rapid synaptic modulations may be mediated by sex steroids such as androgens because of their local and rapid synthesis within the brain.91 Local synthesis results in elevated DHT, testosterone, and estradiol levels within the hippocampus, when compared with plasma levels.92A large body of evidence has demonstrated the effects of androgens on hippocampal plasticity, which may play important roles in normal physiological responses to changes in the environment. For example, in response to a stressful stimulus, males exhibit higher hippocampal CA1 dendritic spine density in comparison to their female counterparts.93 After orchidectomy, studies in both rats and non-human primate St. Kitts vervet monkeys demonstrated a significant reduction in dendritic spine synapse density in the CA1 region of the hippocampus.23The majority of studies, however, have not been based on examination of the effects of physiological variations in steroid levels, but rather on experiments in which gonadal hormone levels are controlled by surgical gonadectomy and hormone replacement. Morphological studies often correlate changes in dendritic spine density with spine structure and the numbers and structure of spine synaptic contacts.23 Subsequently, it was demonstrated that androgen replacement restores the loss of spine synapse density in the male hippocampus that occurs after surgical gonadectomy94  male rats expressing defective AR, results with both androgen and antiandrogen treatment were indistinguishable from those observed in wild-type male controls.96 This suggests that at least in the CA1 region of the hippocampus, the synaptic effects of testosterone are retained even under conditions in which nuclear AR function is impaired.Flutamide did not block the synaptoplastic effects of either DHT or DHEA: Indeed, the responses to flutamide and the two androgens appeared to be additive, rather than inhibitory.22 whereas flutamide also exerts additive effects on CA1 synaptogenesis in combination with the non-aromatizable androgen, DHT.95This cannot be ascribed to local conversion of the androgen to estradiol, since estradiol does not affect hippocampal synapse density in males95 flutamide is not a completely specific antagonist of AR-mediated responses and may itself exert some agonist effects. Under in vitro cell culture conditions, the main bioactive metabolite of flutamide, hydroxyflutamide, has been reported to activate the mitogen activated protein kinase (MAPK)/extracellular regulated protein kinase (ERK) pathway, even in AR-negative cell lines.97A definitive explanation for these observations remains lacking. One possibility is that the effects of flutamide may involve mechanisms other than those activated by the cell nuclear AR. As previously noted,98 Membrane-associated AR may be involved. Sato et al.99 have reported that the self-reinforcing behavioral effects of androgen appear to involve membrane AR. These effects are preserved in Tfm rats, suggesting that membrane receptor-mediated effects of androgens are retained in these animals despite the deficit in nuclear AR-activated responses.99 Similar considerations may apply to androgen-induced synaptogenesis, which could explain the continued expression of normal androgen-induced increases in hippocampal synaptogenesis in Tfm males.It also exerts weak benzodiazepine-like effects in the mouse brain.in vivo.The downstream signaling pathways mediating the neuroplastic effects of testosterone and its metabolites also remain only partially resolved. There are two main possibilities: first, that androgens may act directly on neurons to regulate intracellular neuronal signaling pathways contributing to growth and differentiation; and second, that androgen action on other cells, including both neurons and glia, may affect neuronal structure and function indirectly, via neurotrophic and metabolic response mechanisms. These two possibilities are, of course, not mutually exclusive and both may well contribute to testosterone action in vitro studies, using neurons in tissue culture or hippocampal tissue slices. Androgen treatment increases spine formation in hippocampal neurons in culture.101 Using hippocampal slices from male adult rats, 2-hour treatment with DHT resulted in a significant increase in the densities of middle and large-head spine whereas 2-hour treatment with testosterone resulted in a significant increase in the density of small head spines.93That androgens can act directly on neurons to modulate neuroplasticity has been established by a number of 93 Using primary cultures of rat hippocampal neurons, Guo et al. demonstrated that in vitro testosterone and bovine serum albumin-conjugated testosterone treatment similarly resulted in a rapid increase in spine density, consistent with the hypothesis that these effects may be mediated via membrane-associated AR.101Incubation with letrozole did not affect testosterone's induction of spine density, suggesting that these effects may be attributed to its androgenic effects, as opposed to local conversion to estradiol.102–105 By modulating intracellular signaling cascades, sex steroids may continuously regulate neuronal structure, dendritic spine density, and the functional plasticity of the hippocampal circuitry106  pathways and the phosphoinositide 3-kinase/PI3K/Akt/mammalian target of rapamycin (PI3/Akt/mTOR) pathway.uitry106 .107–110 Androgen-mediated increases in spine density have been correlated with increased expression of structural proteins such as synaptophysin and activation of ERK and cAMP-response element binding protein (CREB).101MAPK cascades are well known for their roles in synaptic modulation, and activation of PKC has been shown to be necessary for LTP in hippocampal slices.111–114 In cultured hippocampal neurons, PKC activation has been shown to rapidly upregulate NMDA receptor-mediated signaling115 whereas multiple PKC isoforms are expressed within dendritic spines themselves.116 Activation of PKC has been shown to induce activation of extracellular receptor kinase and rapid formation of new dendritic spines in cultured hippocampal neurons.109For many years, it has been known that androgens stimulate rapid activation of PKC and ERK MAPK phosphorylation in the hippocampus and other tissues.111 New spine formation has been associated with increased functional synaptic connectivity and network activity in cultured neurons.109 Within CA1 specifically, it has been shown that ERK MAPK mediates synaptic modulation of LTP through activation of both PKA and PKC mechanisms.110 Although AR-dependent PKC and ERK activation has been well established in a variety of tissues,112–114 findings from Hatanaka et al.93 suggest that AR activation by DHT and testosterone results in activation of both PKA- and PKC-mediated ERK signaling.ERK-1 and ERK-2 have been shown to be transiently phosphorylated after AR activation, and this has been implicated in mediating the neuroprotective effects of androgens such as testosterone and DHT.51 Androgen-mediated activation of ERK signaling is further supported by the data of Guo et al.,101 who showed that in the presence of U0126 (a selective inhibitor of MAPK/ERK kinases 1 and 2), rapid testosterone-mediated increases in spine density were reversed and subsequent CREB phosphorylation was inhibited, suggesting that this rapid effect is mediated through AR-dependent ERK signaling.101 Similarly, testosterone and DHT-induced dendritic spine formation is reduced by inhibition of ERK MAPK, PKA, PKC, or LIMK, whereas inhibition of CAMKII or PI3K has been shown to have no effect on total spine density.93Within dendritic spines, concurrent expression of these kinase cascades and ARs suggests that they may associate post-synaptically.118 CREB activity has been shown to be regulated by many other cell signaling mechanisms, including PI3K/Akt,119 PKA,121 CaMKIV,123 and PKC.110 Coupling of the PKA and PKC to CREB phosphorylation is mediated by the MAPK cascade within hippocampal CA1.110The CREB is an important downstream transcriptional effector of MAPK/ERK activation.124 Intriguingly, subsequent studies demonstrated that androgen-induced formation of dendritic thorns within the CA3 stratum lucidum occurs via MAPK and PKC but independently of PKA signaling.100Although CREB has been shown to be rapidly activated by DHT in hippocampal neurons, Nguyen et al. investigated the upstream signaling mechanisms responsible for this and determined that DHT mediates CREB phosphorylation through AR-dependent activation of PKC.in vitro conditions, tissue culture studies suggest that AR-mediated neuroplastic responses can be initiated directly in neurons; however, under in vivo conditions, lesion studies have demonstrated that androgen effects on neurogenesis and synaptic plasticity are at least partially dependent on afferent input from other areas of the brain.126 Indirect androgen mediated modulation of cellular responses could theoretically involve synaptic/neurotransmitter input, and/or trophic factors released into the extracellular milieu.Under 128 as well as hippocampal LTP.129 Several studies have investigated androgen regulation of BDNF in the context of AR activation and synaptogenesis. For example, Jia et al. determined that testosterone treatment significant increased CA1 spine density, BDNF, PSD-95, and CREB levels, whereas these endpoints were reduced in animals treated with flutamide.130A likely mediator of at least some androgen effects is the neurotrophin BDNF. BDNF and its downstream targets such as post-synaptic density protein 95 (PSD-95) play important roles in regulating spine formation. BDNF modulates functional synaptic properties in the mature and developing nervous system,131 In other regions of the hippocampus, interactions between androgens and BDNF may have different consequences. Thus, after orchidectomy in rats, Skucas et al. reported increased synaptic transmission and LTP of the mossy fiber pathway along with increased BDNF levels, suggesting that androgens may play an inhibitory role in CA3 to prevent hyperexcitability and aberrant mossy fiber synaptic transmission.132Conversely, orchidectomy has been reported to reduce protein levels of PSD-95 and BDNF in the CA1 region of the hippocampus in male mice, an effect reversed by testosterone replacement.133 For example, cortactin redistribution has been shown to be the result of a balance between BDNF and NMDA receptor activation during the development of the postsynaptic actin cytoskeleton.133 Cortactin is an important structural protein that is known to be a target of ERK activation.Within the cell, BDNF appears to be involved in regulating the distribution of key synaptic proteins that mediate actin cytoskeletal development.133 Cortactin contains multiple phosphorylation sites that may be phosphorylated by kinases such as PKA or PKC, which have been shown to be activated after DHT or testosterone treatment.134 However, multiple cortactin phosphorylation sites may need to be activated to mediate androgen-induced spine formation.93Once phosphorylated, cortactin may play an important role in spine reorganization and remodeling by grouping actin cytoskeletal matrices.135 The potential importance of these interactions is illustrated by data indicating that defective neuronal–glial interactions may play a key role in the etiology of common neurological disorders such as depression,136 schizophrenia,137 and AD.138Astroglia are critical regulators of synaptic plasticity and neurotransmission.139 but they may also contribute to androgen-mediated regulation of neural plasticity.140 Androgen and estrogen receptors are expressed in astrocytes,141–143 whereas oligodendrocytes express ERβ, which is involved in estrogen-mediated enhancement of myelination.144 In rats, at least after intrahippocampal injury, microglia also express immunoreactive AR.142Not only are astroglia capable of androgen and estrogen biosynthesis,23 demonstrated a significantly increased density of immunoreactive astrocytes in the cerebral cortex after orchidectomy (145 used an orchidectomized rat model to investigate and compare the effects of testosterone, estradiol, and non-aromatizable DHT treatment on reactive astroglia and microglial staining after a stab wound injury to the brain.Although the study of androgen modulation of glial–neuronal interactions remains relatively limited, a few studies have reported the effects of testosterone on astroglial morphology. In male rats, Leranth et al.idectomy . AndrogeEarly and delayed subcutaneous treatment with either testosterone or estradiol significantly reduced immunoreactive astrocyte and microglial staining within the hippocampus. Intriguingly, DHT had no effect on astrocyte staining but significantly reduced the volume fraction of immunoreactive microglial staining after early subcutaneous treatment. This suggests that early and late androgenic regulation of astroglia and microglia reactivity to neural injury is, at least in part, mediated through aromatization to estradiol whereas the early effects of androgens on microglia reactivity may also involve AR-specific mechanisms.Androgen-regulated neural plasticity may play a role in both the normal development and function of the brain and in pathological states, leading to the development of sexually differentiated neurological disease states. Although progress in understanding the mechanisms involved in these effects is less well developed than is the case for estrogen-induced neuroplasticity, in part because of the range of bioactive metabolites involved in testosterone's actions, there are hints that the effects of androgens may involve unique elements that are distinct from the effects of androgens on non-neural target tissues.Understanding these mechanisms may help to clarify the effects of androgens on neurological disorders that are expressed more commonly in males than females, potentially helping to provide improved therapeutic modalities for these disorders. In concluding this review, we will briefly consider two elements of the neurotrophic actions of androgens in which significant gaps in our understanding remain, hopefully to stimulate additional research in these remaining areas of uncertainty.in vivo are mediated. These effects are clearly not mediated simply via conversion to estradiol.22 As discussed earlier, although testosterone induction of hippocampal spine formation is mimicked by DHT, suggesting AR involvement, it is also reproduced by the much weaker androgen DHEA—at doses that do not induce a significant peripheral androgenic effect.95A major unresolved question is: how the effects of testosterone on hippocampal synaptogenesis 96 Taken together, these observations suggest that the neurotrophic effects of androgens on hippocampal spine synapse density may be mediated via mechanisms distinct from those mediated through the “classical” intranuclear AR—despite the extensive evidence indicating the involvement of AR-linked kinase networks in the actions of DHT on spine plasticity in hippocampal neurons in culture.101Administration of flutamide, at a dose sufficient to block the trophic effects of DHT on the prostate, does not antagonize, but rather potentiates the effects of this androgen on hippocampal spine synapse density . In malein vivo remains unclear. We have previously speculated that the effects of testosterone in vivo may involve contributions from other mechanisms, such as potentiation of GABA-ergic transmission via the actions of 3α-diol.94 Contributions from afferent input originating from other parts of the brain may also be involved.126 Membrane AR-mediated responses may be involved,101 since this response pathway appears to be preserved in Tfm rats.99Exactly how androgens exert their effects 146 How are new neurons integrated within existing neural networks? What processes ensure that androgen-induced increases in synapse density enhance function, rather than simply increasing “noise” in preexisting circuits?A second area of uncertainty concerns the question of how the effects of gonadal steroids on neural plasticity are integrated within the preexisting neural circuitry. For steroid induction of neurogenesis and new synaptic connections to be beneficial, it needs to be integrated with the existing neural circuitry. Otherwise, it may have detrimental consequences—disrupting rather than enhancing function.148 may direct functional connectivity in androgen-sensitive circuitry in the brain. In addition to growth factor-dependent regulation, however, it seems likely that mechanisms exist to regulate connectivity at the local level. What processes determine how newly formed synapses connect? Or are the mechanisms similar to those operating normally in development, with initial synaptic over-production being subsequently refined and focused by selective pruning of under-utilized connections?149An obvious possibility is that interactions between new neurons and potential synaptic targets, as has so elegantly been described for the SNB in the spinal cord,These questions cannot be answered definitively at the present time. One possibility that may merit further investigation, however, is that androgens may regulate cell–cell communication via the Wnt-β-catenin-cadherin signaling pathways. Wnts are critically involved in the organization of different cell types during development, regulating interactions between neighboring cells. A key component of the Wnt signaling pathway, β-catenin, also functions as a component of the cadherin complex, which controls cell–cell adhesion and influences cell migration.150 are also of critical importance during embryonic development for morphogenesis of the central nervous system. This pathway is of particular interest, in the context of androgen action on the brain, for two reasons. First, the Wnt-β-catenin pathway is androgen-sensitive in the male reproductive tract,151 as well as in prostate cancer in which androgen regulation of the Wnt signaling contributes to the progress of the disease.152Cell adhesion molecules, including the cadherins and protocadherins,153–155 This raises the possibility that the relatively greater sensitivity of the male to these disorders might involve a genetic-hormonal interaction, with the effects of the mutations being enhanced by testosterone-induced responses.Second, several recent studies have demonstrated that developmental neurological disorders such as autism spectrum disorder and schizophrenia, which occur more often in males than in females, are frequently associated with mutations in the cadherin signaling pathway.156 and the hippocampus.157 In the hippocampus, this effect was also induced by estradiol, but not by DHT,157 suggesting possible estrogen receptor involvement. Further studies of androgen regulation of the Wnt-β-catenin-cadherin signaling pathway may shed additional light on the mechanisms involved in androgen-induced neuroplasticity.Consistent with this hypothesis, Monks et al. have reported in rats that testosterone treatment increases expression of N-cadherin in both spinal motoneuronsIn summary, extensive evidence supports a role for androgens, locally synthesized in the brain as well as derived from adrenal and gonadal androgens delivered via the circulation, in the regulation of cellular plasticity in the central nervous system. This plasticity involves both androgen-induced enhancement of synapse formation and increases in the formation and survival of new neurons.These effects may play a role in the normal development and function of the brain as well as in pathological states leading to the development of sexually differentiated neurological disorders. The underlying cellular mechanisms responsible for these processes, however, still remain incompletely defined. A better understanding of these mechanisms may allow the development of improved therapeutic interventions to correct the problems associated with androgen-sensitive neurological disease.


In equivocal or suspected cases of Brugada syndrome (BrS), ajmaline testing is frequently used in the diagnostic approach. However, the administration of sodium channel blockers can not only elicit the coved ST-segment elevation characteristic of type 1 Brugada pattern but also induce right bundle branch block (RBBB), which can preclude the electrocardiographic manifestations of BrS. We describe a case report wherein RBBB posed a diagnostic challenge during the ajmaline test for suspected BrS. His physical examination was unremarkable. There was no family history of syncope or sudden cardiac death.A 28-year-old Caucasian man was referred to our center because of a possible right bundle branch block (RBBB) electrocardiographic (ECG) pattern recorded during a medical evaluation for suspected coronavirus disease 2019 infection 1 However, the presence of it in subjects affected by cardiomyopathies or channelopathies may conceal the underlying ECG changes, thus making the diagnosis of such conditions more challenging. Its atypical appearance in the current case led us to consider that a type 1 Brugada ECG pattern would be hidden or “masked” by the underlying RBBB2; then, we ordered a higher intercostal space ECG, which showed the resolution of RBBB . However, although the changes related to the RBBB also resolved on the frontal leads, we could not rule out a history of intermittent RBBB. The diagnostic drug challenge performed by intravenous administration of ajmaline (1 mg/kg over five minutes) by specifically designed electrograms covering the right ventricular outflow tract3 unmasked a diffuse abnormal response compatible with the type 1 Brugada syndrome (BrS)-like ECG pattern superimposed on the pre-existing atypical RBBB during 30 seconds of infusion , and the infusion was immediately stopped at that stage. After complete resolution of the electrogram , we readministered ajmaline, which also showed ST-segment elevation in the precordial leads as a reproducible finding .At first glance, the patient had sinus rhythms with an atypical appearance of RBBB. Indeed, RBBB is generally considered a benign finding that does not imply an increased risk when found in asymptomatic healthy individuals.3 Thereafter, it was shown that, although the presence of the RBBB pattern is not necessary for reaching a diagnosis of BrS, it is supportive. In RBBB in healthy subjects, the ST segment is usually not elevated in the right precordial leads. In RBBB patients, activation of the entire right ventricle was delayed, while, in contrast, delayed activation was confined to the right ventricular outflow tract in BrS patients.4 Therefore, BrS patients do not have the wide final S-waves in the left leads  necessary for RBBB. However, the presence of a wide and/or large S-wave in lead I has been found to be a powerful predictor of life-threatening ventricular arrhythmias in patients with BrS.5 Furthermore, the characteristic ECG pattern of BrS can be masked by complete RBBB and exposed by the resolution of the block or pharmacological or pacing maneuvers.7 In the presence of complete RBBB, the premature activation of the right ventricle by apical pacing caused fusion beats, with narrower QRS complexes disclosing the distinctive coved-type ST-segment elevation in the right precordial leads.8 Thus, BrS should be ruled out whenever a slight ST-segment elevation is observed in the right precordial leads in the presence of RBBB.8Indeed, Brugada and Brugada initially described an RBBB pattern among their eight cases of BrS.12 However, some authors have reported a potential 5% false-positive rate, thus making the universal interpretation of provocative drug testing challenging.14 Therefore, not only diagnostic decisions but also therapeutic choices with long-term implications were adopted worldwide before the potential for false-positive results of the ajmaline test was fully appreciated.13 Clinicians should consider all alternative causes in unexplained cardiac arrest and avoid ajmaline doses of greater than 1 mg/kg.15 Furthermore, it should be kept in mind that the administration of sodium channel blockers could not only elicit the coved ST-segment elevation characteristic of type 1 Brugada pattern but also induce an RBBB that can preclude the ECG manifestations of BrS.16 As placing the right precordial ECG leads at an upper position  increases the sensitivity of ECG to detect all types of BrS,3 it would be nice to take an ECG at upper intercostal spaces and perform an ajmaline test to unmask the true RBBB effect.In equivocal or suspected cases of BrS, ajmaline testing is frequently used in the diagnostic approach.


Proteases play a central role in regulating renal pathophysiology and are increasingly evaluated as actionable drug targets. Here, we review the role of proteolytic systems in inflammatory kidney disease. Inflammatory kidney diseases are associated with broad dysregulations of extracellular and intracellular proteolysis. As an example of a proteolytic system, the complement system plays a significant role in glomerular inflammatory kidney disease and is currently under clinical investigation. Based on two glomerular kidney diseases, lupus nephritis, and membranous nephropathy, we portrait two proteolytic pathomechanisms and the role of the complement system. We discuss how profiling proteolytic activity in patient samples could be used to stratify patients for more targeted interventions in inflammatory kidney diseases. We also describe novel comprehensive, quantitative tools to investigate the entirety of proteolytic processes in a tissue sample. Emphasis is placed on mass spectrometric approaches that enable the comprehensive analysis of the complement system, as well as protease activities and regulation in general. Enzymatic proteolysis controls myriad physiological and pathophysiological processes, such as differentiation  control glomerular function and phenotype  of end-stage kidney disease is a result of glomerular diseases USRDS . AnatomiThe aims of this article are to (1) describe our latest understanding of pathomechanisms of proteolytic systems in inflammatory glomerular kidney disease, particularly focusing on the role of the complement system in lupus nephritis and membranous nephropathy, and (2) highlight novel proteomics strategies using state-of-the-art mass spectrometry for the study of protease function in the context of glomerular function in inflammatory kidney disease.The complement system is a well-studied and relevant proteolytic system whose activation is widely accepted to be triggered during glomerular kidney disease. The complement system is an essential part of the innate immune system and is vital for maintaining tissue homeostasis , OMS721 targeting MASP2 (NCT03608033), or C1INH targeting C1r and C1s (NCT02547220) are currently under investigation and may potentially be integrated as new treatments of selected diseases. Currently, 28 clinical trials, including six phase III trials, all with relevance to glomerular kidney disease, are ongoing and the C5 inhibitor Eculizumab is already available on the market  and this upregulation is a hallmark of the disease. The so-called “full-house” immunofluorescent staining pattern carried out as the gold-standard clinical testing in lupus nephritis biopsies with colocalization of IgG, IgM, IgA, C1q, and C3 (C4) is almost solely seen in lupus nephritis   is an antibody-mediated proteinuric kidney disease. Glomerular complement deposition can be readily detected by immunofluorescence- and mass spectrometry-based approaches in patient biopsies  and thrombospondin type-1 domain-containing 7A (THSD7A)  can be used to detect and quantify the entirety of proteases within the glomerulus to gain insight of the intricate protease-networks, making this an indispensable tool for understanding kidney disease and defining novel markers of kidney inflammation. Modern proteomics has matured to provide deep maps of protein compositions  , a heterotrimeric sodium channel which is often targeted by aldosterone antagonists and channel inhibitors . It has been suggested that ENaC cleavage leads to increased sodium retention and that the proteolytic cleavages are mainly carried out by serine proteases . These proteins may also contain active proteases. From studies chiefly in nephrotic syndrome, it has been suggested that these proteases contribute to uncontrolled cleavages of ion transporters and channels, with functional consequences (Artunc et al. A preclinical model was used to analyze proteolytic processing in a model of cisplatin-induced acute kidney injury (Späth et al. Proteases are key modulators of glomerular function, and the complement is an important proteolytic system that communicates between the epithelia and the innate immune system. While several inflammatory kidney diseases show that proteolysis is active and can be targeted genetically, it remains under investigation—both clinically and preclinically—if protease inhibition can emerge as therapeutic strategy in glomerular inflammatory kidney disease.Within this context, novel aspects of complement system characterization can be useful. Traditionally regarded as a simple proteolytic cascade, the complement system exhibits increasingly recognized complex interactions with other proteolytic enzymes and inhibitors (auf dem Keller et al.


Research in ultrahigh magnetic field strength combined with ultrahigh and ultrafast gradient technology has provided enormous gains in sensitivity, resolution, and contrast for neuroimaging. This article provides an overview of the technical advantages and challenges of performing clinical neuroimaging studies at ultrahigh magnetic field strength combined with ultrahigh and ultrafast gradient technology. Emerging clinical applications of 7-T MRI and state-of-the-art gradient systems equipped with up to 300 mT/m gradient strength are reviewed, and the impact and benefits of such advances to anatomical, structural and functional MRI are discussed in a variety of neurological conditions. Finally, an outlook and future directions for ultrahigh field MRI combined with ultrahigh and ultrafast gradient technology in neuroimaging are examined. Increased signal-to-noise ratio and high spatial resolution conferred by ultrahigh field magnetic resonance imaging (MRI) can be used to improve the delineation of small anatomical structures and subtle pathology.The challenges associated with ultrahigh field MRI include artifacts associated with increased susceptibility effect, inhomogeneity of the radiofrequency transmit field, and increased radiofrequency energy deposition in tissues.High-performance gradient systems equipped with high maximum gradient amplitudes and slew rates enable increased spatial resolution and faster acquisitions.Technical limitations of high-performance gradient systems include the biological effects of rapidly switching, large magnetic fields on the human body, which can induce peripheral nerve stimulation.The last 20 years have seen a push in the development of magnetic resonance imaging (MRI) scanner technology with remarkable advances in field strength and progress in gradient coil design and performance. Clinical MRI scanners with magnetic field strengths up to 7 T and gradient strengths up to 80 mT/m with a peak slew rate of 200 T/m/s have now become the state-of-the-art imaging technology for clinical and research studies of the brain. For neuroimaging, research in ultrahigh magnetic field strength and ultrahigh, ultra-fast gradient technology has led to a multitude of benefits for high-resolution anatomic, structural, and functional imaging, opening an array of opportunities for changing the way that patients are imaged and evaluated, and providing new insights into the pathophysiology of neurological diseases.Since the first ultrahigh field human MRI images were obtained at 8 T in 1998 .The benefits and challenges of imaging at higher static magnetic field strength directly follow from the dependence on magnetic field strength of the physical properties of tissues and other magnetic fields involved in image formation. The main advantage of increasing the magnetic field strength for MRI is increased sensitivity. The signal scales greater than linearly with magnetic field strength . The resTissue relaxation properties also change with magnetic field strength, with an expected prolongation of the longitudinal relaxation time T1 and shortening of the transverse relaxation times T2 and T2* at higher field. These changes in relaxation properties along with increased SNR benefit certain applications; for example, longer T1 relaxation times offer better signal-to-noise ratio for applications such time-of-flight (TOF) magnetic resonance (MR) angiography  and artee.g., fast spin-echo and inversion-recovery) is limited at higher field strengths. Spatial variations of the RF transmit field due to standing waves can also produce localized heating. Metallic implants may also lead to heating due to RF absorption, although many studies have shown that heating may be less of a problem than initially anticipated, with only minor temperature changes incurred by small, passive implants  .–42.39–42The contribution of 7-T MRI for detecting MS white matter lesions is less robust than that for the cortex  with ongversus 3 T demonstrated better visualization of spinal cord anatomy and 50% improvement in detection of MS spinal cord lesions at 7 T compared to 3 T [The increased spatial resolution and high SNR afforded by 7-T MRI could also be beneficial in imaging demyelinating lesions of the spinal cord, which remains a challenge at lower field strengths. For example, a small study that compared spinal cord imaging of MS patients at 7 T d to 3 T . Ouelletd to 3 T  used 7-Td to 3 T , 43.In summary, even though the use of ultrahigh field MRI is not yet part of standard-of-care imaging protocols in MS, it has allowed for improved imaging characterization and identification of the different aspects of MS pathophysiology, thereby offering new insights into inter-individual variability and disease evolution.Several studies suggest that 7-T MRI can provide superior information regarding microvascularity and necrosis than 1.5-T and 3-T MRI and could facilitate early stratification of patients with gliomas Fig. , in accoTOF MR angiography at 7 T has recently been used to characterize intratumoral vessels successfully in a small sample of 12 patients with gliomas . The adv23Na) MRI at 7 T correlated with isocitrate dehydrogenase (IDH) mutation status in glioma patients with potential application in image-guided biopsy, surgery, and radiotherapy [23Na concentration from regions of central necrosis to normal appearing white matter suggesting a correlation with tumor infiltration.High-resolution FLAIR images at 7 T were compared with clinical FLAIR sequences at 3 T in patients with glioblastoma prior to radiotherapy planning and yielded significantly higher SNR in white matter and better contrast between gray and white matter compared to clinical FLAIR sequences . The autotherapy . The autversus normal appearing brain and successfully discriminate between short-echo metabolites, particularly glutamate and glutamine, although with low spatial resolutio n[Magnetic resonance spectroscopy (MRS) at higher field strengths improves SNR and spectral dispersion, leading to improved sensitivity and specificity, respectively, to different chemical species and metabolites. MRS has the potential to improve the detection of metabolites for tumor characterization; however, the technical challenges of increased SAR, magnetic field inhomogeneities, and fast T2*-relaxation can impose constraints on the acquisition of MRS data at 7 T –62. Despsolutio n; this disolutio n. The lassolutio n, 64–67. solutio n used 7-Tsolutio n successfsolutio n, 69. ForChemical exchange saturation transfer imaging (CEST), a novel contrast technique that can detect low concentration metabolites with higher sensitivity than MRS, has been used both at clinical and ultrahigh field strengths to differentiate gliomas –74. PaecIn summary, ultrahigh field MRI improves the detection of small metastases, delineation of tumor extent, characterization of the intratumoral microenvironment, and detection of post-treatment effects when compared to conventional strength MRI. However, technical challenges related to MRS and novel CEST techniques remain and warrant additional research.Studies at 7 T within the field of neurodegenerative disorders have focused mainly on (1) dementia including Alzheimer’s disease (AD) and vascular dementia and (2) Parkinson’s disease (PD). Although hyperphosphorylated tau and amyloid beta are considered key mediators in the pathogenesis of AD, an increasing number of studies suggest that altered iron metabolism also plays an important role –80. T2*-A series of studies have used 7-T MRI to delineate the pattern of atrophy of different substructures of the hippocampus in AD with implications for understanding the neuropathogenesis involved in memory loss in these patients –84. BouvDeep brain stimulation (DBS) of target nuclei such as the subthalamic nucleus, globus pallidus pars interna, and ventralis intermedius nucleus of the thalamus is a widely performed surgical treatment for patients with PD in which an electrode is placed to stimulate the motor components while avoiding the associative and limbic components . ImproveDifferences in anatomical patterns of the substantia nigra between PD patients and healthy volunteers using 7-T MRI have also been described , which cIn summary, increased image resolution at 7 T MRI enables the visualization of small structures of interest in neurodegenerative pathophysiology including cortical layers, hippocampal subfields, brainstem nuclei, plaques, and microbleeds. 7-T MRI has been successfully employed to detect biomarkers of disease in both AD and PD and may offer a powerful complement to other noninvasive imaging modalities in the evaluation of dementia.MRI remains the most sensitive imaging technique to detect acute infarction. Clinically feasible stroke imaging protocols have been designed to image subacute and chronic infarcts at 7 T . The excMR angiography at 7 T has been used successfully to image occlusive changes in small vessels such as the lenticulostriate arteries in acute and chronic strokes; these vessels are difficult to image with conventional modalities , 100. Fuin vitro intravascular sonography and 7-T MRI findings with histology concluded that 7-T MRI is a reliable method for detecting atherosclerotic burden within the intracranial arteries [Intracranial atherosclerosis is a major risk factor of ischemic stroke . Beyond arteries . A recenarteries . Lindenharteries  investigarteries .The increased SNR, higher spatial resolution, and better fluid suppression seen at 7 T have been leveraged to evaluate patients with aneurysms that are at high risk for rupture, predict potential for atherosclerotic plaque rupture, and characterize infarct morphology . For exaIn summary, ultrahigh field vessel wall imaging in the evaluation of cerebrovascular disease may enable earlier diagnosis, better differentiation of the underlying disease processes, and provide valuable information in the management intracranial vascular disease. However, the exclusion of DWI limits use of 7-T MRI in the evaluation of acute stroke patients at this time.As a major component of the scanner, the gradient system plays a key role in MR image generation by enabling spatial selection and image encoding. The performance characteristics of the gradient system are parameterized by the maximum gradient amplitude, which is measured in mT/m, and the slew rate, which describes how fast a gradient can attain a desired amplitude within a given amount of time (T/m/s). The magnetic field gradients have tremendous influence on the overall quality of the acquired image, a point that has gained increasing focus from the major scanner manufacturers in the quest for improved image quality and ever-faster image encoding speed. Considerable engineering efforts have been made in the last 30 years to maximize the performance of gradient systems for clinical use. The most recent generation of commercial scanners such as the Siemens Prisma, GE Premier, and Philips Achieva 3T X-series  feature integrated whole-body gradient systems with a maximum gradient amplitude of 80 mT/m and maximum slew rates of 200, 200, and 100 T/m/s, respectively.For applications such as diffusion MRI, gradient coils capable of producing gradient fields with high amplitudes significantly reduce the time required for diffusion encoding and improve the SNR of such measurements by reducing signal decay from T2 relaxation. Diffusion MRI has been the driving motivation behind recent efforts to engineer dedicated gradient coils with large maximum gradient amplitudes. For example, customized gradient coil inserts such as the Siemens SC72 gradient insert that was designed for the Washington University-Minnesota Consortium Human Connectome Project scanner are capable of generating maximum gradient strengths of 100 mT/m . The ConHigher performance gradient systems equipped with higher maximum gradient amplitudes and slew rates enable increased spatial resolution and faster acquisitions. To achieve higher spatial resolution, larger gradient areas are required, noting that the maximum area under a gradient encoding pulse determines the highest achievable spatial resolution. This can be achieved using lower gradient strengths by lengthening the gradient encoding pulses without increasing their amplitudes; unfortunately, this approach leads to a loss of SNR due to T2-related signal decay during the image readout period. To shorten the image readout time without sacrificing resolution, the gradient amplitude must increase, and the time to achieve the desired gradient amplitude must be minimized. High-performance gradient systems are thus designed to maximize gradient amplitude and slew rate within technical and biological limits. Shortening the gradient pulses during image readout must be carefully balanced against decreasing the SNR of the image beyond an acceptable limit, as the SNR is inversely related to the square root of the bandwidth, which is set by the gradient amplitude.Technical limitations include the engineering challenges of achieving a spatially linear magnetic gradient field, removal of heat from the gradient coil, and efficiency and power requirements of the coil. While these technical challenges have largely been addressed in the modern era, including advances in gradient and shim technology –115, theStronger and faster gradients benefit anatomical imaging by enabling more efficient image readout, with a concomitant reduction in echo time and repetition time, depending on the image contrast of interest. The shortened acquisition time can then be used to increase spatial resolution and/or SNR through signal averaging within a similar overall scan time. For example, using the Connectome whole-body gradient system with a maximum gradient strength of 300 mT/m and slew rate of 200 T/m/s, and comparing against a whole-body gradient system with 60 mT/m and 200 T/m/s maximum gradient strength and slew rate, improvements in anatomical imaging include ~ 25% reduction in the repetition time and ~ 50% reduction in echo time for the 3D T1-weighted MPRAGE sequence due to shorter echo spacing, which translates into a roughly ~ 25% reduction in acquisition time.The constant push for shorter scan times has led to the development of more efficient k-space sampling schemes for 2D and 3D imaging, which benefit from better gradient performance. Spiral k-space encoding makes efficient use of the gradient system hardware  and has High-performance gradient systems equipped with large maximum gradient amplitudes are particularly beneficial for diffusion MRI as they boost the efficiency of diffusion encoding. The use of stronger gradients enables a given diffusion-encoding gradient area to be achieved in less time and effectively shortens the entire diffusion-encoding period and echo time, resulting in less signal loss from T2 relaxation. Beyond the increase in SNR, larger gradient amplitudes enable stronger diffusion encoding to be achieved with shorter effective diffusion times, which improves the sensitivity of the diffusion MRI measurement to microscopic structures on the micron scale  and imprAmong the tissue microstructural properties that can be measured using diffusion MRI, axon diameter estimation requires high gradient amplitudes to sensitize the diffusion MRI signal to intra-axonal water diffusion , 137–139The availability of ultrastrong gradients for diffusion MRI has enabled the translation of novel diffusion-encoding paradigms to probe brain tissue microstructure in patients, including oscillating-gradient waveforms , double-The results of clinical research studies demonstrate the potential of high-gradient diffusion MRI to uncover changes in axonal and cellular microstructure and motivate the continued development, application, and dissemination of high-gradient technology for use in commercially available human MRI scanners.e.g., cardiac and respiratory motion around the brainstem). Given that the pathophysiological hallmark of Parkinson’s disease is a degeneration of several neurotransmitter systems in the brain stem, these new techniques may make it possible to test for altered activity and functional connectivity of these nuclei in PD and other neurodegenerative disorders.Higher gradient strengths and faster slew rates facilitate the development and application of accelerated fMRI acquisitions at high spatial resolution. Higher gradient strengths and faster slew rates translate directly into better image quality for echo-planar imaging, the workhorse of functional MR image readout, by boosting the SNR, reducing distortions, and enabling higher in-plane resolution and thinner slices. Beyond the benefits to image quality, the fast slew rates available on high-performance gradient systems promise to enhance the acquisition efficiency and dense sampling of T2 and T2* signal evolution for fast quantitative mapping of the BOLD signal arising from the micro- and microvasculature. Acquiring fMRI data at higher temporal and/or spatial resolution promises to provide new information about neuronal activation, particularly in the early phases of the BOLD response that are thought to reflect the fast response of smaller blood vessels in the brain to local neuronal activity. Traditionally, fMRI has been thought to be too slow to measure brain oscillations due to the dependence of the fMRI signal on slow changes in blood flow. However, recent studies using ultrafast fMRI at 3 and 7 T have revealed fast neural oscillations at up to 0.75 Hz in the visual cortex following high-frequency visual stimulation , 153. AlMRI technology development over the last 20 years has focused on pushing the limits of static magnetic field strength and gradient amplitude and speed. Advances in imaging at ultrahigh field, particularly at 7 T, have effectively ushered in next-generation image quality for clinical neuroimaging, with better SNR and higher resolution scans that provide better visualization of small and subtle lesions such as cortical lesions in MS and cortical malformations in epilepsy. At the same time, advances in gradient technology have enabled high-fidelity image acquisition within shorter scan times, and have also enabled better resolution of tissue microstructure through diffusion MRI, allowing for the first time the characterization of microscopic tissue features in the living human brain paralleling what can be seen directly on histopathology. Building on the success of these advances, the next few decades will likely see a continued push toward even higher field strengths and higher gradient strength systems, while encouraging the more widespread adoption of ultrahigh field and ultrahigh, ultrafast gradient technology for clinical use.


There are only 30 reported cases of primary malignant melanoma of the bladder in the literature so far. Of those, 17 cases were reported as deceased within three years of presentation. Our case reported here is that of a 78-year-old female who presented with a new-onset incontinence and intermittent hematuria. She had no evidence of primary melanoma anywhere else in her body. The patient was treated with cystectomy and ileal conduit with plans for adjuvant chemotherapy. Unfortunately, the patient succumbed to her disease with diffuse metastatic involvement within 16 months of presentation. Melanoma of the skin had an incidence of roughly 23% of all new US cancer diagnoses in 2017 , making Extracutaneous melanomas are extraordinarily rare, aggressive, and extremely lethal . PMM of At this time, the PMM of the bladder has an unknown etiology and risk factors . Both meA 78-year-old female presented to the Emergency Department for new onset of incontinence and gross hematuria. Upon initial workup, computed tomography (CT) urogram Figure  demonstrEight months since her symptoms of hematuria and incontinence began, and approximately seven months following her initial diagnosis of PMM, the patient underwent radical cystectomy with ileal conduit and bilateral pelvic lymph node dissection, lysis adhesions, removal of the left ureteral stent, bilateral ureteral stent placement, hysterectomy and bilateral salpingo-oophorectomy, and excision of the anterior vaginal wall. The specimen was sent for analysis, and pathological analysis showed pT3N0M0 PMM of the bladder.Six months later , the patient presented with abdominal pain. A CT scan of the abdomen and pelvis showed new retroperitoneal lymphadenopathy and infrarenal occlusion of the aorta. A month later, FDG-PET CT Figure  showed cThe patient had planned to proceed with adjuvant chemotherapy following cystectomy. However, the patient presented with recurrent abdominal pain and had a complicated hospital course. During that hospitalization the patient eventually succumbed to her metastatic disease.Extracutaneous lesions of melanoma as discussed are very rare. Most cutaneous melanomas typically arise from genetic alterations of the melanocyte precursors due to direct ultraviolet (UV) radiation exposure leading to mutations and development of malignancy. However, these melanocyte precursors are also found in areas of squamous-covered mucous membranes such as the respiratory and urogenital tracts . MucosalTo differentiate metastatic melanoma of the bladder from primary melanoma of the bladder, the following criteria have been used: (1) detailed history ruling out cutaneous, regressed, or visceral melanoma; and 2) recurrence pattern that is consistent with the primary origin of melanoma  recurren. HistoloPMM of the bladder has a poor prognosis because it presents at an advanced stage, has ambiguous pathological features making it difficult to diagnose, and is very aggressive, with two-thirds of patients succumbing to metastatic disease by three years ,5. In adDiagnosis and treatment of PMM of the bladder is difficult due to its similar histological presentation similar to that of metastatic melanoma, its advanced disease at presentation, aggressive nature and mortality, and rarity of cases with survival after treatment, as described above. At this time, there is no universally accepted method of treatment, but its poor prognosis is consistent throughout.In conclusion, due to the rarity of PMM of the bladder, evidence regarding etiology, risk factors, histopathological diagnosis, and treatment options remains in question. Further analysis of all reported cases and head-to-head clinical trials regarding treatment options is warranted to adequately identify and describe these unknowns.


Metastasis is the end stage of cancer progression and the direct cause of most cancer-related deaths. The spreading of cancer cells from the primary site to distant organs is a multistep process known as the metastatic cascade, including local invasion, intravasation, survival in the circulation, extravasation, and colonization. Each of these steps is driven by the acquisition of genetic and/or epigenetic alterations within cancer cells, leading to subsequent transformation of metastatic cells. Epithelial–mesenchymal transition (EMT), a cellular process mediating the conversion of cell from epithelial to mesenchymal phenotype, and its reverse transformation, termed mesenchymal–epithelial transition (MET), together endow metastatic cells with traits needed to generate overt metastases in different scenarios. The dynamic shift between these two phenotypes and their transitional state, termed partial EMT, emphasizes the plasticity of EMT. Recent advances attributed this plasticity to epigenetic regulation, which has implications for the therapeutic targeting of cancer metastasis. In this review, we will discuss the association between epigenetic events and the multifaceted nature of EMT, which may provide insights into the steps of the cancer metastatic cascade. Metastasis is a complex process by which tumor cells spread to distant sites and is the leading cause of mortality among patients with cancer . To succCancer cells with the epithelial phenotype display apical–basal polarity, specialized intercellular junctions, and contact with the basement membrane . These din vivo is difficult owing to the transient and reversible nature of this process and tumor cells that have undergone EMT are indiscernible from stromal cells in the tumor microenvironment  program to drive disseminated cells to revert to the epithelial state , 16, 17.In this review, we will discuss the epigenetic changes involved in the regulation of EMT in metastatic cascade and emphasize herein the difficulties and controversies of epigenetic therapies that target EMT for cancer treatment, with the aim to provide a perspective for future research.For local migration, epithelial cells must acquire traits that favor detachment from the primary sites and penetration into the adjacent parenchyma Figure 1ZEB2 promoter , may form cell clusters by associating with each other or other cells such as platelets to survive under the immune system and shear stress Figure 1However, it remains controversial which state  plays a more crucial role in metastasis. Previously, it was revealed in human breast cancer specimens that elevated levels of mesenchymal CTCs during metastasis are correlated with acquired drug-resistance and poor prognosis . This wain vivo imaging visually demonstrated that overexpressing Snail in tumor cells triggered the breakdown and remodeling of the basal lamina to facilitate transendothelial migration  inhibitors, histone methyltransferases (HMTs) inhibitors and HDACs inhibitors.Owing to the reversible nature of epigenetic modification, it will be of prime interest to target epigenetic mechanisms to manipulate different cancer cell states Figure 4SPR–Cas9 , 80. As SPR–Cas9 . Accordiin vitro. As the DNMT1 inhibitors 5-aza-2’-deoxycytidine (decitabine) and 5-azacytdine have already been approved by FDA for treating acute myeloid leukemia and myelodysplastic syndromes, a clinical trial is underway to evaluate Aza-TdC for patients with advanced solid tumors (NCT03366116). A series of HDMs inhibitors and HMT inhibitors involving LSD1 inhibitor SP-2577 (Seclidemstat) (NCT03895684), EZH2 inhibitor CPI-1205 (NCT03525795), Tazemetostat (NCT01897571) are in trials against multiple cancer types. Moreover, many HDAC inhibitors such as Vorinostat, Tefinostat mocetinostat, and Panobinostat are also being extensively tested. Furthermore, as miRNAs have displayed critical roles in the regulation of EMT, the development of miRNA-based approaches has a promising potential. Progress has been made employing miRNAs in combination with multiple cytotoxic therapies, efficiently sensitize resistant cancer cells to chemotherapy in prostate cancer, pancreatic cancer and breast cancer  to reverse back to the epithelial state , 93. HenPreviously, a mathematical model integrating multiple feedback loops was proposed, in which the reversible transition from epithelial to hybrid phenotype was attributable to the miR-34-Snail feedback loop, followed by the conversion to the mostly irreversible mesenchymal state that was driven by the miR-200-Zeb1 feedback loop, moreover, the autocrine TGF-β-miR-200 feedback loop further stabilized the mesenchymal state to reinforce the irreversibility of EMT , 97. Thiin vivo using lineage-tracing strategies  National Natural Science Foundation of China ; (2) China Postdoctoral Science Foundation ; (3) Sichuan Science and Technology Program (2019YFS0263); (4) Post-Doctor Research Project, West China Hospital, Sichuan University ; and (5) 1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University (2016105).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.