Cartilage matrix remodelling homeostasis is a crucial factor in maintaining cartilage integrity. Loss of cartilage integrity is a typical characteristic of osteoarthritis (OA). Strategies aimed at maintaining cartilage integrity have attracted considerable attention in the OA research field. Recently, a series of studies have suggested dual functions of microRNA-140 (miR-140) in cartilage matrix remodelling. Here, we discuss the significance of miR-140 in promoting cartilage formation and inhibiting degeneration. Additionally, we focused on the role of miR-140 in the chondrogenesis of mesenchymal stem cells (MSCs). Of note, we carefully reviewed recent advances in MSC exosomes for miRNA delivery in OA treatment. Osteoarthritis (OA) is a prevalent joint disease, and the main characteristic of OA is cartilage degeneration. As the absolute number and percentage of the ageing population grows, OA will become a leading cause of disability . ArticulCartilage is a specialized connective tissue without blood or lymphatic vessels that are mainly composed of collagen type II (COL2) and proteoglycan. Chondrocytes are stable, postmitotic, differentiated cells and are the only cell type found in the cartilage matrix . These cHealthy adult articular cartilage contains approximately 5% of its volume as chondrocytes and 95% as extracellular matrix (ECM) , 10. TheOn the other hand, factors produced by chondrocytes affect the synthesis of ECM . HyalinePromoting cartilage matrix anabolism is a feasible strategy to treat OA by maintaining cartilage integrity. Clinical studies have suggested that autologous chondrocyte implantation (ACI) is an effective way to repair cartilage defects, and it is the only cell-based therapy approved for OA treatment by the Food and Drug Administration (FDA) . Due to Cartilage matrix catabolism is a crucial factor in maintaining the dynamic balance of cartilage matrix metabolism. The increased degradation of cartilage collagen and the proteoglycan aggrecan (ACAN) principally leads to the degeneration of articular cartilage in arthritic joint diseases. Of the proteases that degrade cartilage collagens and proteoglycans, MMPs and aggrecanases have received the greatest attention . CollagemiRNAs are 20- to 23-nucleotide-long single-stranded noncoding RNA molecules that act as transcriptional repressors by binding to the untranslated region (UTR) of the target messenger RNA (mRNA) . The priBoth miR-140-3p and miR-140-5p are expressed in the cartilage, and previous research has revealed that miR-140-3p is more abundant than miR-140-5p in human cartilage tissue . The tarIn the chondrogenic differentiation of MSCs and uncultured articular chondrocytes, miR-140-3p and miR-140-5p are both highly expressed. However, the expression of miR-140-3p and miR-140-5p is markedly and significantly downregulated in both chondrocytes and synovial fluid (SF) from knee OA . The abuThe transcription factors L-SOX5/SOX6/SOX9, C/EBP, NFAT3, and ERα upregulate miR-140 expression while TGF-ß/SMAD3 and IL-1ß downregulate miR-140 expression. miR-140 inhibits OA progression by (1) inhibiting chondrocyte senescence by targeting the Notch pathway  and PI3K-AKT , (2) inhibiting chondrocyte hypertrophy by targeting HDAC4 and SMAD1, (3) inhibiting chondrocyte apoptosis and autophagy by targeting FUT1, (4) inhibiting inflammation via targeting TLR4 and CXCR4, and (5) inhibiting cartilage matrix degradation by directly targeting MMP-13, ADAMTS-5, and IGFBP-5. For subchondral bone metabolism, miR-140 targets BMP-2 and DNPEP, thus inhibiting subchondral bone development.Although WWP2 is the host gene of miR-140, different expression levels of WWP2 and miR-140 in OA chondrocytes have been detected, suggesting that other regulatory elements affect miR-140 expression independently of WWP2. Studies have identified the regulatory elements in the sequence upstream of pre-miR-140  domain-containing transcription factor, activates transcription by binding to a specific heptameric DNA sequence ((A/T)(A/T)CAA(A/T)G). As a pivotal transcription factor in chondrocytes, SOX9 regulates cartilage-specific genes, including COL2A1, COL9A1, COL11A1, ACAN, and others . ExistinOA is a progressive disease of the joints that is characterized by degradation of articular cartilage and is mainly mediated by MMPs and ADAMTSs. To date, no effective treatment to terminate the gradual degeneration of the cartilage matrix has been developed. Thus, there is an urgent need to develop new strategies to target proteases. The synovial membrane shows signs of inflammation in OA patients, and synovial fibroblasts contribute to OA development by secreting inflammatory cytokines . TherefoUnmodified MSCs or MSCs that have been modified using miRNA transduction are under active exploration as therapeutic agents , 60. In In the context of joint diseases such as OA, intra-articular administration of MSCs could be more beneficial than the intravenous/intraperitoneal route by applying the cells directly to the affected tissues. Preclinical models have detected the effect of MSCs in reducing cartilage degradation and joint inflammation. Ninety-nine clinical trials involving MSCs for OA treatment have been registered on ClinicalTrials.gov to date. Some results from clinical trials have provided initial evidence of the safety and efficacy of MSCs for OA treatment. However, the results of MSCs in OA therapy are quite inconsistent , 65. TheSome approaches have been implemented, such as overexpressing specific miRNAs, to improve the therapeutic effect of MSCs against OA. Building on the observation that miR-140 expression increased in parallel with SOX9 and COL2A1 expression during chondrogenic culture of MSCs , our groExosomes are of endosomal origin and range in size from 40~160 nm in diameter. Almost all cell types secrete exosomes into the extracellular environment under both physiological and pathological conditions . ExosomeRecently, exosomes have gained increasing attention in the study and research of OA. Purified exosomes isolated from inflamed SF are functionally active in their ability to stimulate the release of the pro-inflammatory cytokines IL-6 and tumour necrosis factor-alpha (TNF-α) from M1 macrophages , which mPrevious studies have demonstrated a protective role of miR-140 in OA development. However, unprotected miR-140 is easily degraded by endogenous nucleases, especially in the context of an inflammatory disease such as OA. The lack of a practical or safe method to deliver miR-140 is an obstacle for real progress towards its intended use as a clinically usable therapeutic. To protect miR-140 from degradation and maintain its integrity and activity within the OA joint and synovium, considerable efforts have been made to develop strategies to improve miRNA delivery efficiency, such as chitosan nanoparticles , virusesViruses are very efficient at infecting human cells, and thus viral vectors are widely used for ex vivo delivery of genes. However, there are several disadvantages of using viral vectors such as oncogenicity from insertional mutations . These dExosomes derived from miR-140-overexpressing cells or miR-140-encapsulated exosomes could be used to promote MSC chondrogenesis or inhibit cartilage matrix degradation for OA therapy.Articular cartilage is composed of chondrocytes and cartilage matrix. The dense matrix is synthesized by chondrocytes. miR-140 plays an important role in the stability and maintenance of the cartilage matrix in chondrocytes. Recent advances in epigenetic studies have shown the enormous potential of miR-140 as a therapeutic target for OA. miR-140 can act on different targets to regulate chondrocyte differentiation and OA development at different stages. The effect of miR-140 on chondrocytes and cartilage matrix as well as OA requires deeper investigation and additional experimental research. Concerning miR-140 delivery, exosomes are a promising cargo carrier in OA treatment. In particular, exosomes from autologous SF-MSCs are a safe miR-140 delivery tool without any unwanted immune responses.However, before consideration as clinical therapeutics, several unanswered aspects of exosomes should be explored: (i) Large-scale amounts of exosomes should be produced to meet clinical requirements. Exosome isolation requires a considerable amount of labour and starting cells, and the preparation should be carefully examined to ensure that its content is consistent over time and devoid of contaminants. (ii) The content of exosomes is cell type- and stimulation-dependent, and given the low amount of available primary cells, the production of exosomes for therapeutic purposes will be limited to exosomes from cell lines, which might not contain the same beneficial exosomal content. (iii) Exosomes contain not only multiple miRNAs but also other biological effectors , which might affect the phenotype of uptaking cells. In addition, multiple cell types might uptake these exosomes with unpredictable effects on other tissues. (iv) The methods by which miR-140 is packaged into exosomes should be optimized to maintain high loading efficiency. (v) Cellular metabolism of synthetic miR-140 in vivo should be highly considered. Further studies should be performed to monitor miR-140 function and systematic response after injection into the joint tissue. (vi) Exosomes should be endowed with precise targeting abilities. To accomplish this goal, more precise targeting strategies should be developed. If these challenges are adequately addressed, delivery of miR-140 by exosomes from autologous SF-MSCs will be a reliable and promising method to counteract OA.


Signal transduction at the neuromuscular junction (NMJ) is affected in many human diseases, including congenital myasthenic syndromes (CMS), myasthenia gravis, Lambert–Eaton myasthenic syndrome, Isaacs’ syndrome, Schwartz–Jampel syndrome, Fukuyama-type congenital muscular dystrophy, amyotrophic lateral sclerosis, and sarcopenia. The NMJ is a prototypic cholinergic synapse between the motor neuron and the skeletal muscle. Synaptogenesis of the NMJ has been extensively studied, which has also been extrapolated to further understand synapse formation in the central nervous system. Studies of genetically engineered mice have disclosed crucial roles of secreted molecules in the development and maintenance of the NMJ. In this review, we focus on the secreted signaling molecules which regulate the clustering of acetylcholine receptors (AChRs) at the NMJ. We first discuss the signaling pathway comprised of neural agrin and its receptors, low-density lipoprotein receptor-related protein 4 (Lrp4) and muscle-specific receptor tyrosine kinase (MuSK). This pathway drives the clustering of acetylcholine receptors (AChRs) to ensure efficient signal transduction at the NMJ. We also discuss three secreted molecules ) that we recently identified in the Wnt/β-catenin and fibroblast growth factors (FGF) signaling pathways. The three secreted molecules facilitate the clustering of AChRs by enhancing the agrin-Lrp4-MuSK signaling pathway. The neuromuscular junction (NMJ) is a prototypic synapse between an ectoderm-derived element  and a mesoderm-derived element . The NMJ uses acetylcholine (ACh) as a neurotransmitter, which is recognized by the nicotinic acetylcholine receptor (AChR) embedded in the postsynaptic membrane. Genetic defects in molecules expressed at the NMJ result in a group of neuromuscular diseases called congenital myasthenic syndromes (CMS), which are characterized by muscle weakness, accelerated muscle fatigue, muscle hypotrophy, and sometimes minor facial anomalies. In addition, signal transduction at the NMJ is compromised in a variety of diseases including myasthenia gravis, Lambert–Eaton myasthenic syndrome, Isaacs’ syndrome, Schwartz–Jampel syndrome, Fukuyama-type congenital muscular dystrophy, amyotrophic lateral sclerosis, and sarcopenia ,2.The NMJ in vertebrates has a typical structure. The axon terminal of a motor neuron sinks into the muscle membrane, creating a characteristic depression referred to as the primary gutter . Each guCHRNA1), β1 subunit (CHRNB1), δ subunit (CHRND), and ε subunit (CHRNE). Other causes include mutations in genes encoding (i) structural proteins for scaffolding AChR clusters, (ii) presynaptic molecules involved in ACh release and resynthesis, (iii) intrasynaptic molecules to degrade ACh and to facilitate juxtaposition of the nerve terminal and the motor endplate, (iv) enzymes that glycosylate extracellular domains of transmembrane proteins and extracellular matrix (ECM) proteins at the NMJ, and (v) signaling molecules that drive AChR clustering [AGRN, LRP4, MUSK, and DOK7 have been identified in CMS patients. The best-characterized synaptic signaling molecule to drive AChR clustering is the neural isoform of agrin (encoded by AGRN). Neural agrin is released from the nerve terminal. Two postsynaptic receptors for agrin at the motor endplate are the low-density lipoprotein receptor-related protein 4  and muscle-specific receptor tyrosine kinase . Dimeric Lrp4 and dimeric MuSK form a hetero-tetrameric receptor for agrin, and the receptor is associated with ECM molecules including laminin α5 (encoded by LAMA5), laminin β2 (encoded by LAMB2), collagen type XIII α1 (encoded by COL13A), and collagen-like tail subunit of asymmetric acetylcholinesterase , all of which belong to category (iii) above.The identification of CMS-associated mutations in genes specifically expressed at the NMJ contributed to the discovery of molecules involved in the NMJ signal transduction. Mutations in more than 30 genes have been identified in CMS patients. The major causes of CMS are mutations in genes encoding the AChR subunits, namely, the α1 subunit , and newly identified ones  .The synaptic structure of the NMJ depends on several molecules. Transmembrane molecules at the NMJ form a large protein complex together with ECM molecules, intracellular molecules , and downstream signaling molecules to stabilize AChR clusters at the NMJ. For example, the extracellular domain of α/β-dystroglycans anchors an ECM protein, perlecan. Perlecan binds to another ECM protein, ColQ, and ColQ binds to transmembrane protein, MuSK. The sarcoglycan complex comprises the transmembrane proteins and the α, β, δ, and γ subunits; the α subunit binds to another transmembrane protein, Lrp4 ,8,9. α/βLAMA2, LAMA5, and LAMB2, respectively), and proteoglycans . Six collagens  are enriched at the NMJ, and two of them (XIII and Q) are defective in CMS [ACHE) at the NMJs [Representative synaptic ECM molecules are collagen fibrils, nidogen 2, laminins  . FurtherAgrn in mouse spinal motor neurons, as well as of Lrp4 and Musk in the mouse skeletal muscle, has been reported. Homozygous agrin-deficient mice develop normally until the last embryonic day (E18.5) but die around birth due to respiratory failure [Lrp4 [Musk [Targeted disruption of  failure ,29. Furtre [Lrp4  and Muskp4 [Musk  show no nic day E8.5 but dp4 [Musk .The N-terminus of agrin binds to the ECM network via laminins α2/α5/β2. Agrin is also anchored to α/β-dystroglycans at the NMJ. By binding to these ECM molecules, agrin contributes to stabilize the synapse and maintain its structural integrity via connection to the ECM network ,34,35. AGRN variants have been reported in severe forms of CMS and some of them have been partially characterized to determine their effect on the NMJ [Domains: More than 10  the NMJ ,41,42,43 the NMJ ,36. The The sperm protein, enterokinase, and agrin (SEA) domain is important for the glycosylation and secretion of agrin, and also protects agrin from degradation by proteases such as neurotrypsin . Indeed,The first and second laminin G-like domains (LG1 and LG2) are essential for anchoring agrin to the NMJ using α-dystroglycan or other muscle-specific cell surface molecule(s) ,47. Two The LG2 domain includes codons encoded by the neuron-specific alternative exon A/y ,48 and bThe third LG (LG3) domain contains codons encoded by two neuron-specific alternative exons named B/z, and is essential for inducing AChR clusters by binding to Lrp4 and thus activating MuSK . p.G1871In addition to agrin and MuSK, Lrp4 binds to App ,52, App-LRP4 cause not only CMS but also (i) Cenani–Lenz syndactyly syndrome (CLSS), which is characterized by syndactyly; (ii) sclerosteosis 2 (SOST2), which is characterized by sclerosing bone dysplasia; (iii) Richter syndrome (RS), which is characterized by the transformation of chronic lymphocytic leukemia to aggressive lymphoma; and (iv) a predisposition to low-trauma fracture (LTF) [Domains: Lrp4 is a transmembrane molecule that contains extracellular and intracellular domains . The intre (LTF) . CLSS isre (LTF) . In the re (LTF) , while pre (LTF) ,63. We sre (LTF) ,63. Thusre (LTF) ,65,66. Tre (LTF) , and there (LTF) ,67.In addition to Lrp4, the extracellular domain of MuSK directly binds to ColQ and biglycan . A triplMUSK mutations are clustered in the intracellular tyrosine kinase domain, whereas three missense mutations have been reported in CMS patients without functional characterizations: p.N103S [MUSK mutations that cause CMS have been reviewed by others [Domains: The extracellular domain of MuSK has four immunoglobulin-like domains (Ig1–4) . Ig1 and p.N103S ,72 and p p.N103S  in the I p.N103S  in the Fy others . Additioy others , and wilChrng), as well as AChR α1 subunit (Chrna1) and Musk, in the extrajunctional region. The changes in gene expressions cause decreased stability of AChR in the plasma membrane. Muscle contractions suppress the expression of extrajunctional AChR subunits by increasing calcium flux [Although ACh is a neurotransmitter for the NMJ synapse, it also modulates postsynaptic AChR clustering via regulation of extrajunctional gene expression, as has been recently reviewed elsewhere . When anium flux ,78, whicium flux ,81,82,832+ pathway [Wnt ligands can activate three pathways: (i) the canonical Wnt/β-catenin signaling pathway, (ii) the non-canonical planar cell polarity pathway, and (iii) the non-canonical Wnt/Ca pathway . The Wnt pathway ,86. The  pathway ,88. In m pathway . β-caten pathway ,90. NMJ  pathway .LRP4 cause not only CMS but also CLSS, SOST2, RS, and low-trauma fracture (LTF) [does require the frizzled-like domain of MuSK but does not require Lrp4. The difference between mice and zebrafish is likely due to an additional Kringle domain in the MuSK extracellular region in zebrafish. In addition to the cytoplasmic crosstalk between the agrin and Wnt signaling pathways stated above, these pathways also engage in crosstalk at the extracellular level of the NMJ. A representative example at the extracellular level is observed in Lrp4 . Lrp4 inre (LTF) . Interesre (LTF) . In contEach Wnt ligand has a unique role in AChR clustering . Wnt4, WXenopus preferentially extend filopodia toward co-cultured muscle cells [Fgf18-/-) mice showed an abnormal aggregation of multiple nerve terminals in the diaphragm. In particular, the nerve terminals made a giant pre-synapse, but had few synaptic vesicles within each terminal [Fgf18-/- mice showed simplified motor endplates and reduced gene expression of NMJ-specific Chrne and Colq in the diaphragm [Muscle-derived factors regulate synaptogenesis by promoting differentiation of the nerve terminal at the NMJ. The assembly of the NMJ is initiated when the nerve and the muscle first contact each other via filopodial processes of the nerve terminal. These filopodial processes enable a close interaction between the synaptic partners, which facilitates synaptogenesis. Embryonic spinal neurons of le cells . This file cells . While mle cells . Presynale cells . We repoterminal . Thus, Fiaphragm .Chrne [Nrg1-knockout mice showed a 50% reduction in the density of postsynaptic AChR; however, neither homozygous Nrg1-, ErbB2-, nor ErbB4-knockout mice showed embryonic or perinatal lethality [Four types of neuregulins and their receptors, ErbB receptors (ErbB1-4), are expressed in motor neurons, muscle cells, and Schwann cells . NeureguChrne , in the Chrne . Homozygethality ,103,104.ethality . The intethality . Yap1 in mice exhibits remarkable presynaptic deficits, as well as abnormal AChR clustering. Yap1-deficient mice show reduced protein levels of β-catenin in skeletal muscle, suggesting that Yap1 promotes synaptogenesis through β-catenin [Hippo signaling pathway: Yes-associated protein 1 (Yap1) is a major intracellular effector of the Hippo pathway that regulates cell proliferation and differentiation during development. Activation of the Hippo/Yap1 signaling pathway restricts tissue growth in adult animals. Skeletal muscle-specific knockout of -catenin . HoweverCRTC1), show fragmented AChR clustering at 12 months of age. The roles of TOR in the development and maintenance of the NMJ were recently reviewed in detail [Target of rapamycin (TOR) signaling pathway: TOR proteins mediate a key signaling pathway that is responsible for muscle metabolism. Namely, TOR activity is essential for muscle development and growth; however, overactive TOR signaling is also implicated in aging and sarcopenia . Mice den detail . Still, NT-4/TrkB signaling pathway: TrkB kinase activity maintains synaptic function and structural integrity of adult NMJs. Inhibition of TrkB kinase activity or a lack of NT-4 results in reduced complexity of AChR clustering in mice ,111.Ache expression [ATP/P2X2 signaling pathway: Extracellular ATP can affect the development of NMJs with its purinoreceptors P2X2 and P2Y ,113. In pression . EphA/Ephexin1 signaling pathway: Ephexin1 is an intracellular protein linking Eph receptors and RhoA signaling in muscle cells . EphexinDrosophila [Dok7, Wnt11, and Musk in this cell type. However, the effects of BMP ligands on AChR clustering at the NMJ remain to be studied.BMP signaling pathway: The growth, assembly, and maintenance of the NMJ are regulated by retrograde signaling of Decapentaplegic, a BMP homolog, in osophila . In mammosophila  and bindThe NMJ is a prototypic synapse, and the molecules and mechanisms that drive the formation and maintenance of the NMJ are likely to be similar to those of synapses in the central nervous system (CNS). An understanding of the mechanisms of formation and stabilization of the NMJ will thereby propagate our understanding of the less accessible synapses in the CNS.Colq-deficient mice to the levels of wild-type mice [Therapeutic intervention at the level of protein-anchoring is possible in human neuromuscular diseases and during aging . An ECM ype mice . We nameype mice . We alsoype mice . The proFurther insights into the mechanisms of extracellular signaling molecules at the NMJ are expected to lead to the development of therapeutic approaches for alleviating human neuromuscular diseases.


Brassinosteroids (BRs) are important plant growth hormones that regulate a wide range of plant growth and developmental processes. The BR signals are perceived by two cell surface-localized receptor kinases, Brassinosteroid-Insensitive1 (BRI1) and BRI1-Associated receptor Kinase (BAK1), and reach the nucleus through two master transcription factors, bri1-EMS suppressor1 (BES1) and Brassinazole-resistant1 (BZR1). The intracellular transmission of the BR signals from BRI1/BAK1 to BES1/BZR1 is inhibited by a constitutively active kinase Brassinosteroid-Insensitive2 (BIN2) that phosphorylates and negatively regulates BES1/BZR1. Since their initial discoveries, further studies have revealed a plethora of biochemical and cellular mechanisms that regulate their protein abundance, subcellular localizations, and signaling activities. In this review, we provide a critical analysis of the current literature concerning activation, inactivation, and other regulatory mechanisms of three key kinases of the BR signaling cascade, BRI1, BAK1, and BIN2, and discuss some unresolved controversies and outstanding questions that require further investigation. Brassinosteroids (BRs) are plant-specific steroid hormones and play essential roles in a broad range of plant growth and developmental processes, including cell elongation, cell division, and differentiation, seed germination, stomata formation, root development, vascular differentiation, plant architecture, flowering, male fertility, and senescence ,2,3,4. Bbzr1-1D1), an F-box E3 ubiquitin ligase, leading to BIN2 ubiquitination and proteasome-mediated degradation , and Arg1032 (at the beginning of the β9-strand), thus stabilizing the activation loop and establishing an active kinase conformation (1044 (pSer1044) and Arg922 of the αC-helix is likely critical for creating the so-called regulatory-spine (R-spine) consisting of 5 residues   in the “P+1 loop”  revealeignaling . It willIn contrast to these four proteins that enhance BR signaling by preventing nuclear localization of BIN2/GSK3s, at least two members of the Arabidopsis HSP90 family whose mammalian homologs were known to assist the folding and activation of GSK3β , were shbin2-1 (Glu264-Lys) mutation greatly stabilized the mutant kinase, which was partially responsible for its increased BES1/BZR1 phosphorylation activity [Proteasome-mediated BIN2 degradation is another important mechanism to keep this negative regulator of BR signaling at low levels. An earlier study suggested that the activity . A recenactivity . In addiactivity . It remaactivity . It is iactivity .The basic scheme of the BR signaling pathway is quite simple, involving a PM-localized BR receptor complex, a cytosolic/nuclear-localized inhibitor BIN2, and two key transcription factors, BES1 and BZR1. In the absence of BR, BIN2 is constitutively active to phosphorylate and inhibit BES1/BZR1. BR binding to BRI1 triggers its conformational changes to allow stable heterodimerization, transphosphorylation, and activation of BRI1 and BAK1, leading to inhibition of BIN2 and nuclear accumulation of non/de-phosphorylated BES1/BZR1. These BES1/BZR1 proteins bind to their target promoters to control expression of thousands of genes important for plant growth, development, and stress tolerance. Studies in the last twenty years have provided molecular understanding of key BR signaling events and discovered a wide range of biochemical and cellular mechanisms that regulate the abundance, subcellular locations, and biochemical activities of these key signaling components, which have dramatically enhanced our understanding of BR signaling processes. Despite these great achievements, there are still outstanding questions and unresolved controversies (discussed throughout this review) that require further investigation. Emerging technologies and novel experimental approaches will not only help to answer questions and resolve controversies, but will also lead to new discoveries that will provide a high-resolution atomic portrait of the BR signaling events to further our understanding of the BR signaling pathway and its interactions with other plant signaling processes.


Pospiviroidae viroids must move into the plant nucleus. However, the mechanisms of viroid nuclear import are not entirely understood. To study the nuclear import of viroids, we established a nuclear import assay system using onion cell strips and observed the import of Alexa Fluor-594-labeled citrus exocortis viroid (CEVd). To identify the plant factors involved in the nuclear import of viroids, we cloned the Viroid RNA-binding Protein 1 (VIRP1) gene from a tomato cultivar, Seokwang, and heterologously expressed and purified the VIRP1 protein. The newly prepared VIRP1 protein had alterations of amino acid residues at two points  compared with a reference VIRP1 protein (AJ249595). VIRP1 specifically bound to CEVd and promoted its nuclear import. However, it is still uncertain whether VIRP1 is the only factor required for the nuclear import of CEVd because CEVd entered the plant nuclei without VIRP1 in our assay system. The cause of the observed nuclear accumulation of CEVd in the absence of VIRP1 needs to be further clarified.Before replicating,  Pospiviroidae and Avsunviroidae. Avocado sunblotch viroid, an Avsunviroidae viroid, has been detected in chloroplasts of infected cells [Pospiviroidae, are found in the nuclei.Viroids are plant pathogens consisting of a single-stranded non-coding circular RNA molecule with 246–401 nucleotides ,4. The ped cells , while Ped cells  and citred cells , membersPospiviroidae viroids enter plant cells, they move into nuclei, replicate there, exit the nuclei, and move to other parts of the host plants [Pospiviroidae viroids are associated with plant RNA polymerase II (RNA Pol II) [When t plants . In studt plants ,17 and Ct plants ,18 were t plants , coconutt plants  was imag Pol II) , which g Pol II) , viroid  Pol II) , is an a Pol II) . There a Pol II) . Althoug Pol II) . Viroids Pol II) .The nuclear import of PSTVd was found to be specifically competitive, and insensitive to GTP-γ-S and cytoskeleton-disturbing chemicals . StudiesSilencing of the gene coding the plant-specific 4/1 protein changed the accumulation and transport of PSTVd . Plant-sViroid RNA-binding Protein 1 (VIRP1) from tomato plants has a bromodomain  and is lEcoRI  to provide a proper termination point. To prepare the Alexa-Fluor-594-labeled CEVd (red fluorescence), the CEVd-containing plasmid vector (1 μg) was transcribed in vitro with 100 units of T7 polymerase  in the presence of 200 μM of ChromaTideTM Alexa-Fluor-594-5-dUTP  in a 30-µL reaction mixture at 37 °C for 3 h, following the manufacturer’s instructions. The reaction was stopped by treating the mixture with 2.5 units of RNase-free recombinant DNase I  at 37 °C for 20 min. Finally, the Alexa-Fluor-594-labeled CEVd was purified with NucAway Spin Columns .The DNA fragment corresponding to the linear monomeric CEVd (Access No. M34917)  was consSolanum lycopersicum cv. Seokwang) leaves and reverse transcribed with M-MLV . The cDNA was used to amplify the full-length coding sequence of the VIRP1 gene (AJ249595) [EcoRI site and a reverse primer (AAA CTC GAG AGA GTG TGC ATC ATC AGC ATC AG) containing a XhoI site. The DNA fragment amplified by PCR using a high fidelity Taq polymerase  was cloned into a pET28a expression vector containing a C-terminal 6xHis-tag , and the cloned gene was confirmed by sequencing. Escherichia coli strain BL21 was transformed with the completed expression vector, and the expression of VIRP1 was induced with 0.1 mM  at 30 °C for 3 h. The induced cells in 1 L of Luria-Bertani (LB) medium were harvested by centrifugation at 5400× g for 5 min at 4 °C . The pellet was resuspended in 9 mL of ice-cold 1 × STE buffer and treated with 10 mg/mL of lysozyme  for 30 min in the presence of 3% phenylmethylsulfonyl fluoride (PMSF)  in 10 mL of the final mixture. The sample volume was increased with 10 mL of 1 × STE buffer, and the sample was then sonicated  for 10 s on ice, and this was repeated 60 times with cooling intervals. The sonicated preparation was then treated with 1% Triton X-100  for 20 min on ice and cleared by centrifugation . The supernatant was collected and subjected to the following purification steps. VIRP1 protein was purified from the crude extract with a Ni-NTA column  following the manufacturer’s instructions. The final fraction containing 65.6 kDa protein was obtained by a native electrophoretic separation method . The Ni-NTA-purified and size-fractionated VIRP1 protein was concentrated with Amicon Ultra-15 Centrifugal Filter Units . The final sample was resolved by SDS-PAGE to confirm the purity and the size of the protein.Total RNA was isolated from 20-d-old tomato (J249595)  by PCR, Allium cepa) was cut with a razor blade and thin cell strips were isolated. Pieces of onion cell strips (3 × 4 mm) were floated on and permeabilized with 0.2% Triton X-100 in import buffer  following Merkle et al. [https://imagej.nih.gov/ij/).A fresh bulb of onion  in an ultraviolet cross-linker . The Dig signal on the membrane was detected by following the manufacturer’s instructions . Briefly, the fixed membrane was washed with 1 × maleic acid buffer  and blocked with 1 × blocking buffer  containing 0.5% TWEEN 20  for 30 min at 27 °C in a hybridization oven with a rotating wheel . Then, the membrane was incubated with anti-Dig polyclonal antibodies (0.1 μg/μL) conjugated with horseradish peroxidase (HRP)  for another 30 min at 27 °C. The membrane was washed twice with maleic acid buffer (pH 7.5) containing 0.5% TWEEN 20. It was then equilibrated with the alkaline (pH 9.5) detection buffer  containing 0.5% TWEEN 20. HRP activity was detected with West-Zol plus  and recorded with an image analyzing system .CEVd was labeled with digoxigenin (Dig) by in vitro transcription in the presence of Digoxigenin-11-UTP  following the protocols supplied by the manufacturer. The Dig-labeled CEVd and purified VIRP1 protein were mixed and incubated in the binding buffer  in the presence of 1 μg/μL of yeast tRNA  for 1 h at 22 °C. The mixed sample was resolved on a non-denaturing 6% polyacrylamide gel (29:1) in 0.5 × TBE at 70 V. The resolved materials were transferred to a sheet of nylon membrane  in the flow of 10 × SSC buffer. The transferred materials were cross-linked to the nylon membrane by UV illumination , the cloned sequences revealed changes in two amino acid residues . However, the characteristics of the amino acid residues were the same as those of the amino acid residues of AJ249595. Therefore, it was vital to examine the binding activity of the newly prepared VIRP1 before investigating its effect on the CEVd nuclear import. We purified the recombinant VIRP1 protein using a Ni-NTA column  from the total RNA obtained from tomato leaves and cloned the cDNA fragment in a modified pET28a vector with C-terminal 6xHis-Tag. Although the PCR primers were designed based on the nucleotide sequences of A column A and furA column C. The exTo evaluate the ability of VIRP1 to bind CEVd, CEVd–VIRP1 interactions were analyzed by EMSA with the purified VIRP1 and Dig-labeled CEVd. A band shift, indicating the binding of VIRP1 to CEVd, was observed following the reactions with Ni-NTA-purified VIRP1 B and theWe used competition experiments to confirm the specificity of the CEVd–VIRP1 interaction. The addition of a 30 to 200 molar excess of unlabeled CEVd as a competitor to the binding mixture reduced the signals corresponding to the labeled CEVd–VIRP1 complex (data not shown). This showed that the newly prepared VIRP1 specifically bound to CEVd, even with the amino acid residue changes at two points. The EMSA patterns observed with Ni-NTA-purified VIRP1 B and theThe red fluorescent Alexa Fluor 594-labeled CEVd (AF594-CEVd) passed through the plasma membrane and concentrated in the nucleus  during tIt has been assumed that viroids use or are helped by host proteins to enter the plant nucleus . We postP < 0.05)  B. Becaus < 0.05) . FurtherVIRP1 protein originating from a tomato cultivar Seokwang was found to have amino acid residue changes at two sites  compared with the published one (AJ249595). However, the properties of the two amino acid residues were unchanged, and VIRP1 specifically bound to CEVd as expected. The nuclear import of fluorescently labeled CEVd was observed in an assay system based on onion cell strips. When the purified VIRP1 was added to the nuclear import assay system, the nuclear import of CEVd was significantly increased. However, it is unclear whether VIRP1 is a unique factor involved in the nuclear import of CEVd, as the nuclear accumulation of CEVd was observed in the absence of VIRP1.


This review focuses on the current knowledge on the involvement of metal ions in signaling processes within the cell, in both physiological and pathological conditions. The first section is devoted to the recent discoveries on magnesium and calcium-dependent signal transduction—the most recognized signaling agents among metals. The following sections then describe signaling pathways where zinc, copper, and iron play a key role. There are many systems in which changes in intra- and extra-cellular zinc and copper concentrations have been linked to important downstream events, especially in nervous signal transduction. Iron signaling is mostly related with its homeostasis. However, it is also involved in a recently discovered type of programmed cell death, ferroptosis. The important differences in metal ion signaling, and its disease-leading alterations, are also discussed. Signal transduction and spreading is a key cellular process in maintaining life and its development. Typical chemical signaling comprises the release of a transmitter from one cell and its interaction with selected detectors on the surface of another. If the transmitter is taken up into the cell, it may bind to receptors on the inner part of cell membrane and, thus, stimulate it to react in a required manner to this signal. The molecular mechanisms of reaction to the signal are regulated by a strict spatiotemporal dynamic . The sigThe major groups of chemical neurotransmitters in excitable cells are amino acids, amines, or neuropeptides. Recent studies also indicate that metal ions, such as zinc and copper, may be released to the synaptic cleft ,8,9. Thi2+ can act as an antagonist to reduce Ca2+ signaling in endothelium . Montezano and coworkers have shown that magnesium prevents vascular calcification and osteogenic differentiation by restoring TRPM7 activity, counteracting calcium actions, and increasing expression of anticalcification proteins 2+ increase in immature neurons independently of calcium signals. Moreover, cytosolic magnesium regulates the signaling activities of extracellular signal-regulated kinase (ERK) negatively, cyclic AMP response element binding protein (CREB) positively, and mammalian target of rapamycin (mTOR) sigmoidally  is maintained at approximately 100 nM. More calcium is stored in some organelles, such as the Golgi apparatus and endoplasmic reticulum (ER), amounting to hundreds of microM [2+ after its release [2+]i which coexist in cells: the release from intracellular stores, mainly ER, or the influx from the extracellular medium through the opening of calcium-permeable channels and Ca2+ transporters located in plasma membrane i acts as a signal that influences a broad range of dendritic cell (DC) functions ..182].HFE interacts with TFRC, at the binding site for holo-Tf . TherefoTfr2 knockout mice exhibited decreased BMP-SMAD signaling and hepcidin expression. The effect is even more visible in double-knockout Hfe/Tfr2 mice  is maintained at approximately 100 nM; much greater levels are stored in the ER and mitochondria. In electrically non-excitable cells, the Ca2+ ions are released from internal stores mainly in a process known as the store-operated calcium entry (SOCE). This mechanism has two important functions in the cell: cellular signaling and store refilling. Ca2+ flux through the excitable cell membrane is strictly limited in time due to biophysical properties of VGCCs and ligand-activated receptors. During calcium-mediated signal transduction these receptors and channels associate temporally and form the transient signaling complexes.Many controversies still exist regarding the roles of magnesium in cell signaling. These controversies are caused mainly by the versatility and complexity of this metal ion. Studies show that it can be trophic or toxic, an activator or an inhibitor, and a cause of disease progression or regression. Magnesium has a crucial role in numerous cellular processes including enzymatic reactions, ion channel functions, metabolic cycles, and DNA/RNA stabilities. Disorders of MgZinc and transition metal ions, such as copper and iron, have been traditionally thought to be kinetically inert cofactors that are bound and buried within proteins and other molecules. However, recent data reveal that beyond their traditional functions as metabolic cofactors, they have also an important role in cell signaling. Copper, iron, and zinc are the three most abundant non-alkali metal ions in the brain, for this reason both deficiency and excess of these metal ions results in central nervous system disease.2+ ions facilitate the transduction of a variety of signaling cascades in response to extracellular stimuli. Zinc pathways interact with calcium, redox, and magnesium signaling. The influx of Zn2+ into the cell triggers the Ca2+ related processes, what was observed not only in neurons but also in other cells. In physiological conditions, the majority of Zn2+ is bound by proteins and small chelators in both excitable and non-excitable cells. Zinc has an impact on the activity of many enzymes, transcriptional processes, cell growth and differentiation, and the immunological response. Very interesting events caused by zinc ions are called “zinc sparks”; functionally, the zinc sparks mediate a decrease in intracellular zinc content that is necessary for continued egg cell cycle progression. Recent studies reveal that this mechanism is dependent on intracellular calcium transients, which are tightly associated with embryonic development.Zinc might regulate the plasticity of synapses, however, how much zinc is released during synaptic activity remains highly elusive. Some research confirms that Zn+ and Cu2+. Because of their potential toxicity to cause oxidative stress and free-radical damage, copper ions must be strictly controlled, e.g., through binding to specific proteins. The exchange of copper between a variety of target-specific cytosolic chaperones and their targets is driven presumably by an increase in the copper binding affinity. Copper containing enzymes and transcription factors are essential for cellular integrity, energy production, and proliferation. The intracellular level of copper is managed by a set of different proteins such as Cu-transporters and copper chaperones. Copper signaling may influence synaptic transmission indirectly by modulating the synthesis of neurotransmitters, or directly by being released into the synaptic cleft from the nerve terminals of glutamatergic neurons.Copper ions may exist in two different forms in biological systems—Cu3+ and Fe2+. There is no regulatory mechanism for the removal of excess iron from the body. Therefore, the perfect balance between iron absorption, distribution, accumulation, and excretion should be maintained. Regulation of the proteins involved in iron metabolism is a form of iron-sensing mechanism during post-transcriptional modification. Elevated iron levels were found in tumors, as they exhibit increased metabolism and rapid proliferation of the cells. Iron ions, like copper ions, take part in the redox signaling process. The distinction between signaling and toxic redox processes is not always obvious. There is growing evidence that iron excess is a major risk for carcinogenesis, suggesting the importance of ferroptosis-resistance. Ferroptosis is a relatively recently described type of non-apoptotic cell death, induced by iron-dependent lipid peroxidation, that remains to be fully characterized.Iron is present in the living cells in the form of ferric and ferrous ion, i.e., FeThere are suggestions that copper and iron redox mechanisms are interesting therapeutic targets for treating some diseases such as cancer and chronic lung inflammation.This review shows the role of chosen metal ions in signaling processes. As we can see, variable thermodynamics and kinetics of metal actions blur the lines between metabolism and signaling, placing metals in a unique chemical and biological space.Nonetheless, deficiency, as well as excess, of any essential metal ion, can lead to many disorders. Not only redox-inactive alkali metals, but also some transition metal ions, have important roles in signaling and other biological processes. Recent studies focus on the disruption of transition and non-transition metal ions homeostasis by such factors as the disturbances of their bioavailability, and changes in the chemical status of essential cations. This knowledge can help to prevent many disorders, and to design proper medication, such as inhibitors of the destructive signaling pathways, where these metal ions play an important role.Many conclusions on the distribution of metal ions in excitable cells are based on conventional histochemical studies of nerve tissues. This technique is used primarily to show sites of only relatively high metal accumulation. Hence, the main far-reaching goal of novel research seems to be the improvement and development of techniques able to detect the chosen chemical form, concentration, and location of metal ions, especially zinc, iron, and copper, e.g., in the living brain. As described in this review, the proper cellular management of the copper and zinc levels is relevant for signal transduction and viability of neuronal cells. Additionally, the potent regulation of copper and zinc signals in excitable cells strongly influences calcium concentration.


Frontonasal dysplasia (FND) is a rare congenital craniofacial cleft syndrome associated with a spectrum of midline facial bone and soft-tissue malformations. When present, the physical features of FND are often obvious and classified at birth. The resultant facial deformities have the potential to negatively influence psychosocial health and quality of life. Reconstructive surgical intervention in early childhood can serve to restore facial contour and alleviate psychological stress. In this report, a case of a 14-year-old female with previously undiagnosed mild form of FND presented for reconstructive surgery evaluation and underwent several procedures including sliding advanced genioplasty, submucosal resection of the nasal turbinates, open rhinoplasty, and bilateral transnasal medial canthopexies. The patient had subsequent nasal tip recontouring for persistent supratip fullness. The patient achieved an acceptable esthetic outcome and was satisfied with her physical appearance. This case emphasizes the subtle presentation and reconstructive surgical options of a mild case of FND that was diagnosed at a later age, unlike the more severe phenotypes of the syndrome and other common craniofacial anomalies that are usually diagnosed and treated in early childhood. Multidisciplinary craniofacial care teams should be able to correctly diagnose and implement the appropriate surgical interventions in patients with milder forms of FND. Frontonasal dysplasia (FND) is a rare congenital orofacial cleft syndrome of early embryogenic development resulting in a broad phenotypic presentation of midline head and face malformations ,2. PatieAs the classification of craniofacial clefts changed over the years, FND adopted several names including Tessier cleft no. 0, frontonasal dysostosis, median cleft face syndrome, median facial dysplasia, and most recently median craniofacial hyperplasia ,9. ThereAlthough the mode of inheritance of FND is not definitive, most cases are sporadic, with familial and multifactorial patterns reported in the literature . FND is Literature describing the reconstructive surgical management of FND is limited and encompasses various multistaged bony and soft-tissue interventions tailored to the craniofacial features expressed in each individual ,7,12. ThThese operations, among other surgical options for associated defects, are generally performed in prepubescent children beginning in infancy or early childhood to restore normal function and anatomy of the aforementioned disfigurements, as well as mitigate social stigmatization during critical childhood development ,12,19. MA 14-year-old female presented to the outpatient pediatric reconstructive surgery clinic for evaluation and correction of a previously undiagnosed syndromic congenital facial malformation. The patient did not have a history of developmental delay or family history of syndromic diagnoses. On physical examination the patient had characteristic features classified by the senior author as FND including a low forehead with a central hair pattern resembling a widow’s peak, mild telecanthus, substantial retrogenia, and profound nasal deformity. The patient showed a very large upper dorsal kyphosis extending well out in front of her forehead, shortened columella, and a very broad almost bifid nasal tip with extremely narrow nostrils causing airflow obstruction Figure -1C. The The senior author began with a subperiosteal dissection of the mandibular symphysis through a lower buccal incision in preparation for a sliding genioplasty. Horizontal osteotomies were performed above the lower border of the mandible and below the mental nerves to free the advanced segment, which was advanced anteriorly 6.5 mm to increase the projection of the chin. Next, a coronal incision was made and dissection was carried out to the supraorbital ridges and down to the medial canthal tendons where the dorsal kyphosis was visualized coming off of the nose at a higher level than the forehead. Due to the small nostril size, access to the nasal elements for an open rhinoplasty was made via a columellar base incision. The nasal kyphosis was removed en bloc, leaving a large flattened area on the dorsum of the nose otherwise called an open roof deformity which was closed with infractures. The previously harvested nasal cartilage was used to recontour the columellar strut in order to increase the structural integrity of the tip and provide a scaffold on which the nasal tip can be corrected. The boxy nasal tip was reshaped by trimming the lower lateral cartilages and the alar lining was raised with two percutaneous sutures taken through the alar crease. To increase the size of the nasal passages and improve airflow, the surgeon subsequently performed a submucosal resection of the nasal turbinates. Next, the intercanthal distance was measured at 42 mm, and bilateral medial canthopexies were performed with transnasal wires, acting to stabilize the nasal bones as well as tighten and approximate the canthi. There were no intraoperative complications, and the patient was taken to the recovery room in good condition. Postoperative follow-up at one month showed significant improvement of her facial, nose, and chin contour. At one year postoperatively, the patient returned to the clinic for concerns of a persistent wide nasal tip. Thus, a nose revision was performed to thin out the nasal tip with no complications. Following the revision, the nasal tip continued to show mild supratip fullness, but the patient was satisfied with her current appearance and did not wish to undergo further surgical intervention Figure -1F.  Patients with FND may present with a broad constellation of features and of varying severity. However, the pattern of features seen in FND should be appropriately recognized, leading to a correct diagnosis, understanding the underlying pathogenesis, and formulating a sound surgical plan. This case is unique due to the mild form of the syndrome with subtle features, and the late presentation in adolescence without a previous diagnosis. Although this patient did not receive genetic testing, it is hypothesized that she may have had type 1 FND due to this type’s association with milder forms, the presence of characteristic facial features, and absence of a family member with a syndromic diagnosis hinting at a sporadic or an autosomal recessive inheritance pattern . The recThe patient’s mild physical deformities were treated with surgical repair using several standard osteoplastic operations, resulting in significant improvement. She was able to achieve a satisfactory result without unnecessary invasive procedures such as facial bipartition for more severe forms of FND exhibiting pronounced orbital hypertelorism . InsteadAs implied by this case, patients with mild presentations of the syndrome may potentially go undiagnosed at an early age until the facial malformations become socially apparent, driving the patient to seek reconstructive evaluation for their facial esthetics. Multidisciplinary craniofacial care teams should be aware of milder forms of FND as the manifestations can be more subtle and present at a later age, but also lead to psychological morbidity due to social stigmatization and self-perception in affected individuals ,20. FND is a rare orofacial cleft syndrome of varying severity. The resultant facial deformities may lead to psychological morbidity in affected individuals and may present as subtle abnormalities at older ages. This case discusses the clinical indications and surgical management of a rarely encountered mild case of frontonasal dysplasia in an adolescent female.


Cerebrovascular diseases are one of the leading causes of death worldwide, however, little progress has been made in preventing or treating these diseases to date. The transforming growth factor-β (TGF-β) signaling pathway plays crucial and highly complicated roles in cerebrovascular development and homeostasis, and dysregulated TGF-β signaling contributes to cerebrovascular diseases. In this review, we provide an updated overview of the functional role of TGF-β signaling in the cerebrovascular system under physiological and pathological conditions. We discuss the current understanding of TGF-β signaling in cerebral angiogenesis and the maintenance of brain vessel homeostasis. We also review the mechanisms by which disruption of TGF-β signaling triggers or promotes the progression of cerebrovascular diseases. Finally, we briefly discuss the potential of targeting TGF-β signaling to treat cerebrovascular diseases. According to the latest Global Burden of Disease Study, cerebrovascular disease is the second leading cause of mortality worldwide . EmerginBlood vessels of the brain form a highly specialized vascular network, which have complex interactions with the central nervous system, and has important physiological functions in the development and maintenance of the central nervous system . The devCerebrovascular development is a highly conserved and complex process involving multiple signaling pathways. Using various model organisms, researchers have successively identified many genes and signaling pathways that regulate the formation and homeostasis of blood vessels of the brain, including vascular endothelial growth factor (VEGF), sonic hedgehog/Patched (Shh/PTC-1), platelet-derived growth factor B/platelet-derived growth factor receptor β (PDGFB/PDGFRβ), Wnt/β-catenin, orphan G protein coupled receptor 124 (GPR124), as well as transforming growth factor β/SMAD (TGF-β/SMAD) signaling . As one The TGF-β signaling pathway is highly conserved in evolution, and plays multiple and complex physiological functions in the regulation of embryonic development and tissue homeostasis in a highly context-dependent manner .The TGF-β signaling pathway comprises of more than 30 kinds of ligands, mainly divided into subfamilies such as TGF-βs, bone morphogenetic proteins (BMPs), activins, inhibin, Nodal, anti-Müllerian hormone, and growth and differentiation factors (GDFs) . Most TGActivated TGF-β ligands, which are usually disulfide-linked homodimers, directly bind to the serine/threonine protein kinase type II receptors on the cell membrane surface, sometimes with the assistance of co-receptors such as endoglin and β-glycan . VariousThe type II receptors phosphorylate the type I receptors to form a receptor complex, which then phosphorylates the receptor regulated SMADs (R-SMADs) intracellularly. Type I receptors for the TGFβ subfamily  mainly phosphorylate SMAD2 and SMAD3, whereas type I receptors for the BMP subfamily  mainly phosphorylate SMAD1, SMAD5, and SMAD8. The activated R-SMADs form a complex with the central mediator SMAD4 and translocate into the nucleus, where it binds to specific gene loci under the guidance of signal-driven transcription factors (SDTFs) and lineage-determined transcription factors (LDTFs) as well as tripartite motif 33 (TRIM33) to regulate chromatin accessibility and gene transcription . A negatIt is intriguing that this seemingly simple “two-step” signal transduction of the TGF-β pathway has various and even opposite biological effects on a wide range of physiological processes, thereby reflecting the high spatiotemporal specificity of TGF-β signaling. The complexity of TGF-β signaling is manifested in the abundance and different combinations of its ligands, receptors and intracellular co-factors collaborating with SMADs. A single ligand can trigger multiple receptors [one of the 5 type II receptors  in combination with one of the 7 type I receptors (ALK1-7)], and single receptor can interact with different ligands as well. The diversity of TGF-β ligand-receptor combinations leads to superimposed, synergistic or antagonistic effects on cells harboring different transcription factors and co-factors interacting with SMADs, resulting in complex biological effects .The cerebrovascular network is developed via sprouting angiogenesis. The primary vessels of PNVP penetrate the CNS parenchyma and undergo remodeling to form a hierarchical vascular system composing of branched arteries and veins as well as capillaries, which is regulated by various signaling pathways including TGF-β signaling .Endothelial TGF-β signaling has been shown to be essential for cerebral angiogenesis, since Tgfbr2 or Alk5 gene knockout blood vessels fail to invade into the neuroepithelial layers and exhibit intracerebral hemorrhage . GeneticA previous study has revealed the anti-angiogenic effect of TGF-β signaling in CNS vascular development . ActivatTransforming growth factor-β signaling has also been shown to promote angiogenesis. TGF-β1 derived from radial glial cells promotes murine microcapillary brain EC migration and tube formation in vitro and stimulates cerebrovascular branching angiogenesis in the cerebral cortex, and this effect may be mediated by the balanced expression of pro-angiogenic gene GPR124 or anti-angiogenic gene, brain-specific angiogenesis inhibitor-1 (BAI-1) .Bone morphogenetic protein-ALK1 signaling has been shown to limit EC number and maintain the quiescence of nascent vessels. BMP9 and BMP10 are physiological ligands of ALK1 during vascular development . In zebrThe mechanisms by which BMP-ALK1 regulates cerebrovascular development are quite limited; therefore, certain studies on the developmental mechanisms of mouse retinal vasculature can help us understand the related processes. The study using heterozygous Acvrl1+/- mice revealed that BMP9-ALK1 signaling inhibits EC proliferation and migration by activating PTEN to inhibit PI3K/Akt and MEK/ERK cascades, thereby maintaining retinal vascular quiescence . ConsistTransforming growth factor-β signaling has been implicated in BBB formation and permeability by regulating tight and adherens junctions. The BBB is mainly composed of ECs which are characterized by the presence of tight and adherens junctions, and pericytes play an important role in the formation and maintenance of the BBB . EndotheBone morphogenetic protein signaling has also been demonstrated to participate in the maintenance of BBB function. In zebrafish, BMP3 has been shown to regulate BBB integrity by promoting pericyte coverage . In rat Cerebral angiogenesis is not only programmed in ECs, but also orchestrated by dynamic TGF-β signaling in other cell types within or outside the NVU, including pericytes, astrocytes, oligodendrocyte precursor cells, neural progenitors, preosteoblasts and periosteal dura cells.Brain pericytes have been shown to induce and upregulate the functions of BBB through continuous TGF-β production . PericytAstrocytes, whose endfeet interact with ECs of the neural capillaries, play a critical role in cerebral angiogenesis and BBB formation though BMP signaling. Targeted disruption of BMP type IA receptor (BMPR1A) in telencephalic neural stem cells leads to upregulated expression of VEGF in mutant astrocytes, impaired EC-astrocyte interaction, and cerebrovascular malformation, demonstrating that BMP signaling in astrocytes is essential for a functional BBB . A very Oligodendrocyte precursor cells have also been shown to maintain BBB integrity through TGF-β signaling. TGF-β1 derived from oligodendrocyte progenitor cells can activate the MEK/ERK signaling pathway in ECs to promote tight junction protein expression and improve BBB integrity, and knockout of Tgfbr1 in oligodendrocyte progenitor cells leads to cerebral hemorrhage and disruptive BBB in mice .Several neural progenitors have also been shown to play important roles in brain region-specific angiogenesis via TGF-β signaling. Tgfbr2 silencing in forebrain-derived neural progenitors and neural cells impedes EC migration and sprouting, decreases vessel density and branching via altered secretion of pro- and anti-angiogenic factors, thereby leading to intracerebral hemorrhage in the telencephalon . Neural In addition, BMP2 and BMP4 derived from preosteoblasts and periosteal dura are essential for dural cerebral vein formation. Loss of Twist1 or BMP2/4 signaling in skull progenitor cells and dura leads to cerebral vein malformations, similar to that in humans with craniosynostosis .Endothelial-to-Mesenchymal Transition is a complex biological process and mainly refers to the trans-differentiation of ECs into mesenchymal stem cells, fibroblasts, SMCs or pericytes . During Activation of the TGF-β signaling pathway is the most important onset of EndMT . All thrEmerging studies have revealed that BMP signaling serves as a gatekeeper by antagonizing TGF-β-induced EndMT in ECs. BMP7 has been shown to inhibit hypoxia-induced EndMT and gremlin-1-mediated EndMT , 2020. LPhysiologically, EndMT plays essential roles during cardiovascular development, such as angiogenic sprouting and cardiac valve formation . DysreguEmerging evidence indicates that dysregulated EndMT contributes to certain cerebrovascular diseases . The firRecent studies have shown that defects in TGF-β signaling are associated with human cerebrovascular diseases. Pathogenic mutations in TGF-β signaling, such as ENG, ALK1 gene mutations, are associated with type 1 and type 2 hereditary hemorrhagic telangiectasia (HHT), as well as Loeys-dietz syndrome with cerebrovascular events . Some geCCM1/KRIT1, CCM2/malcavernin, or CCM3/PDCD10  and KLF2/4 in CCM pathogenesis, which is independent of TGF-β/SMAD signaling . EndotheGDF2 (HHT5 or HHT like), type I receptor ALK1 encoding gene ACVRL1 (HHT2), co-receptor endoglin encoding gene ENG (HHT1) and intracellular mediator SMAD4 encoding gene MADH4 (JP-HHT)  .BMP9/10-ALK1 signaling suppresses HHTs through SMAD-dependent or SMAD-independent pathways. Endothelial-specific knockout of Alk1 triggers cerebral AVMs mimicking the pathologic characteristics of HHT . In adulMice with homozygous or heterozygous deletion of Eng with VEGF treatment exhibit brain AVMs , 2014, aThe essential role of endothelial SMAD4 in the maintenance of cerebrovascular integrity has been demonstrated by the study using a brain endothelial specific Smad4 knockout mouse, which develops phenotypes partially simulating HHT patients, such as dilated vessels, increased EC proliferation, intracranial hemorrhage and BBB breakdown . PostnatThese studies based on mouse models that mimic human HHT patients have provided the causal link between dysregulated TGF-β signaling and the pathogenesis of HHT. Blood flow stimulates BMP9-ALK1-ENG-SMAD4 signaling to maintain EC quiescence by suppressing EC proliferation and inducing pericyte recruitment , which iHTRA1) gene have been identified to be associated with CARASIL (Cerebral Autosomal Recessive Arteriopathy with Subcortical Infarcts and Leukoencephalopathy is a rare autosomal recessive cerebrovascular disease that mainly occurs in cerebral white matter and basal ganglia, causing early adult-onset dementia, gait disturbance, alopecia, and low back pain . Histolo CARASIL . In pati CARASIL .HTRA1 mutation carriers exhibit no significant change in pSMAD2/3 expression (HTRA1 mutations leading to different outcomes (The mechanistic role of TGF-β signaling in the pathogenesis of CARASIL, is a debatable topic. HRTA1 is a serine protease which is strongly expressed in ECs, vessel SMCs and pericytes . HtrA1 kpression . The posoutcomes . Future Current therapies for CCM, HHT, and CARASIL patients mainly rely on surgery or relieving complications . Recent Activated TGF-β/BMP signaling has been demonstrated to contribute to the onset and progression of CCMs in patients and mouse models . TherefoThe majority of HHT patients have pathogenic loss of function mutations in TGF-β signaling. Although many studies have uncovered the molecular mechanisms underlying HHTs caused by dysfunctional TGF-β signaling, there is currently no efficient drug for HHT treatment. Current drug therapy regimens mainly focus on interfering with the downstream core signaling pathway such as activated VEGF and PI3K/Akt signaling . Since hGenome editing may serve as the final solution. CRISPR-based genome editing has been demonstrated as a powerful tool for treating genetic diseases . The CRIPrevious studies have demonstrated the crucial function of TGF-β signaling in cerebral vasculature development and integrity, and uncovered the causal link between the dysfunctional TGF-β signaling and the onset or progression of several cerebrovascular diseases such as CCM, HHT and CARASIL. However, the related mechanisms underlying the dysregulation of TGF-β signaling resulting in cerebrovascular diseases remains to be further elucidated. In recent years, using the rapidly developed single cell sequencing technology and advanced graphics algorithm, researchers have revealed the unappreciated heterogeneity and plasticity of human and mouse cerebral blood vessels, discovering not only new markers for different subtypes of ECs but also a new cell type adjacent to the blood vessel . FurtherThere are not many animal models that can accurately mimic the genetic and pathological characteristics of human cerebrovascular diseases. Rapid advances in genome editing technologies based on CRISPR-Cas systems provide powerful tools for generating animal models carrying genomic mutations precisely mimicking the ones in human patients . StudiesAlthough recent advances have indicated that targeting TGF-β signaling will be a potential strategy for the treatment of cerebrovascular diseases, clinical transformation is still challenging. Considering the cell context-dependent pleiotropic roles of the TGF-β signaling pathway, the selectivity and dosage of targeted drugs may be crucial for the desired therapeutic effects. Previous research has identified various TGF-β inhibitory drugs involving almost every level in the TGF-β signaling cascade, some of which have been proved safe and effective for treating systemic sclerosis, cancers or idiopathic pulmonary fibrosis in clinical trials . These eIncreased clinical research data shows that there is a close correlation between cerebrovascular and central nervous system diseases. Abnormal cerebrovascular structure and function are closely related to brain atrophy, dementia and various neurodegenerative disorders and cognitive impairment . DysreguYZ and XY wrote the review. All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Melanoacanthoma is a benign, deeply pigmented condition of the oral mucosa characterized by the proliferation of melanocytes and keratinocytes. A 60-year-old male, with no history of systemic diseases or medical treatment, presented with an asymptomatic diffuse pigmentation involving the maxillary and mandibular gingiva. The histopathology of the anomalous pigmented area was analyzed and the dendritic melanocytes expressed positively for Masson-Fontana, S-100, and HMB-45. The clinical and microscopic findings were indicative of melanoacanthoma. Depigmentation with a diode laser was performed in the areas of esthetic concern. A regular periodic screening was done to rule out any alteration in color, size, and shape. A one-year follow-up disclosed no new lesions. The observations noted in this case are rare, and our literature review identified only a single previously documented case of gingival melanoacanthoma in the Indian subpopulation. Melanoacanthoma is an uncommon, reactive, pigmented mucocutaneous lesion characterized by the presence of keratinocytes admixed with dendritic melanocytes. An alarming feature of oral melanoacanthoma is its rapid growth rate, potentially masquerading as a melanoma. The first evidence of cutaneous melanoacanthoma was described by Bloch in 1927 [The buccal mucosa is the most commonly reported intraoral site of melanoacanthoma (51.4%), with fewer lesions originating on the palate 22.2%), lips (15.2%), and gingiva (5.6%) 2.2%, lip. Herein,A sixty-year-old male patient reported to the department of periodontics Maulana Azad Institute of Dental Sciences (MAIDS) with a chief complaint of pigmentation of the gums in the lower arch. The patient first noted the pigmentation due to the dislodgement of his prosthesis in the 46 region one month ago. He was unaware of the onset of the pigmentation, however, the extent and appearance made the patient anxious. Subsequently, the patient reported to the hospital to negate any grievous condition. Extraoral examination revealed no relevant findings. Intraoral examination revealed multiple, widespread gingival macular bluish-black pigmentations on the buccal surface of 42-47 and lingually from 37-47. The edentulous region also showed a diffuse area of pigmentation Figure .The pigmented area was smooth, non-tender with irregular margins involving free and attached gingiva. The patient denied any cutaneous pigmentary changes, foods, or dental hygiene agents that had caused any oral irritation, usage of tobacco products, and awareness of oral habits of compulsion. The widespread nature and clinical appearance of the lesion were perplexing for the authors, thereby necessitating an incisional biopsy. The area to be biopsied was the edentulous region (46). The gross specimen comprised an irregular, blackish-brown soft tissue measuring 1.5 x 0.6 x 0.2 cm in size  under local anesthesia (1:80000) , the case presented here is that of an Indian male. Based on the cumulative data, the majority of patients present with a solitary lesion (13 out of 16) whereas three cases noted multiple lesions. To the best of our knowledge, this is the first reported case of multifocal gingival melanoacanthoma in an adult Indian male. The prognosis of oral melanoacanthoma is excellent, however, in three of the reviewed cases, the authors reported recurrence 5%, the c. No evidThe definitive pathogenesis of melanoacanthoma has not been identified, although these lesions are generally considered to be reactive in origin . The etiMelanoma, post-inflammatory lichen planus, oral melanotic macule, acquired melanocytic nevus, hemochromatosis, and so on have a similar clinical appearance to melanoacanthoma and are crucial for the differential diagnosis. Other conditions associated with increased melanin deposition include physiologic racial pigmentation, Addison disease, and syndromes such as McCune-Albright and Peutz-Jeghers . ConsumpAll of the biopsied specimens seen with our case were consistent with the histopathologic features of melanoacanthoma. Immunostaining for HMB-45 and S-100 highlighted the presence of benign melanocytes and dendritic melanocytes and was in agreement with previous investigations. Dendritic melanocytes are not pathognomonic for melanoacanthoma, however, they may occur with various neoplasms, most notably melanoma, squamous cell carcinoma, mucoepidermoid carcinoma, blue nevus, and Spitz nevus .Based on the features described in the available literature, oral melanoacanthoma (OMA) is a rare, reactive lesion affecting the oral mucous membrane with no malignant potential. Its treatment should be directed towards the elimination of all local potentially irritating factors and excluding any other cause of oral pigmentation.Taking everything into account, gingival melanoacanthoma is an uncommonly encountered pigmented lesion and should undergo a histopathologic examination for timely identification to rule out malignancy. This case report underlines the relevance for clinicians to consider oral melanoacanthoma in the differential diagnosis of multifocal gingival pigmentations.


Here, by combining sophisticated long-term live fluorescent imaging techniques and CRISPR technology in simian- and human-derived cell cultures, the authors show that RV-infected cells signal to surrounding uninfected cells through a purinergic signaling pathway. The data show that RV infection induces intercellular calcium (Ca2+) waves (ICWs) that propagate between infected and neighboring uninfected intestinal epithelial cells. This process is driven by adenosine diphosphate (ADP) release from RV-infected cells and its subsequent interaction with P2Y1 receptors expressed by neighboring cells. This mechanism appears to be independent of traditional RV-induced signaling pathways that involve paracrine signaling molecules such as prostaglandin E-2 (PGE2), nitric oxide (NO), or RV enterotoxin nonstructural protein 4 (NSP4). Ca2+ responses in surrounding non-infected cells require P2Y1 receptors, and disrupting intercellular purinergic through P2Y1 receptors decreases the rate of RV replication and expression levels of interleukin-1α (IL-1α), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) mRNA following RV infection. Purinergic signaling through P2Y1 receptors also contributes to diarrhea by stimulating serotonin (5-HT) secretion from RV-infected cells, and small molecule inhibitors of P2Y1 receptors reduced diarrhea severity in infected animals. Thus, P2Y1-ADP signaling is a promising target for future RV infection therapies.Rotavirus (RV) infection causes life-threatening diarrhea, despite only infecting a limited number of intestinal cells. A long-held concept of how this occurs is that RV infection induces the release of potent signaling molecules that can dysregulate neighboring, uninfected cells. However, the mechanisms of RV-induced signaling changes are multifactorial and incompletely understood. Ghang-Graham et al. [RV is among the most severe, life-threating diarrhea-inducing viruses, and it affects millions of children worldwide. RV causes these effects by infecting enterocytes and enteroendocrine cells in the small intestine in which RV replicates by disrupting host cell calcium signaling. However, not all susceptible cells become infected, and how they are involved in RV-disease progression has remained an open question. RV induces major alterations to host cell homeostasis which result in the release of potent paracrine signaling molecules including enterotoxin NSP4, PGE2, and NO (see Chang-Graham et al.  for refe. . Thus, a. addressed this issue by studying intercellular signaling between RV-infected and bystander African green monkey epithelial kidney cells expressing the genetically encoded calcium indicator GCaMP5G or GCaMP6s. The authors observed large increases in cytosolic calcium in host cells following RV infection which were followed by increases in cytosolic Ca2+ in uninfected neighboring cells. ICWs such as these are considered important for the spatial coordination of cellular functions [Functional cell-to-cell signaling had not been directly observed during RV infection prior to the study by Chang-Graham et al., and the signaling pathways involved in this process were incompletely understood. Chang-Graham et alunctions . Antibod2+ in infected cells. However, anti-NSP4 antibodies were not able to reduce ICWs, suggesting that enterotoxin NSP4 was not required for ICW propagation to neighboring cells. Instead, the authors found that the extracellular messenger and receptor complex responsible for mediating RV-induced ICWs were ADP acting on P2Y1 receptors. This conclusion was supported by data showing that apyrase, an enzyme that degrades extracellular ADP and adenosine triphosphate, decreased Ca2+ responses in uninfected bystander cells. Further, by reducing P2Y1 receptor signaling with the antagonist BPTU or by CRISPR-Cas9-mediated knockdown of P2RY1 prevented the formation of ICWs but did not prevent infection. Similar results were observed when the authors tested a different RV variant strain, which suggests that purinergic signaling is a common mechanism induced by multiple strains of RV. These effects were independent of previously identified RV-induced signaling molecules, and the addition of PGE2 and NO did not elicit Ca2+ fluctuations or directly transmit ICWs. The authors replicated these findings in human jejunal-derived intestinal enteroids expressing GCaMP and determined that human enterocytes utilize ADP-P2Y1 Ca2+-induced signaling under basal conditions, and that this is later exploited during RV infection. These findings are in accordance with the pivotal role of purinergic signaling in regulating gut homeostasis and pathology [Virally encoded NSP4 is responsible for inducing ICWs in RV-infected cells and is, therefore, the usual suspect for disease onset and progression . Chang-Gathology .1 purinergic signaling significantly decreased the expression of IL-1α, COX-2, and iNOS mRNA, which function to produce PGE2 and NO and subsequently activate fluid secretion process leading to diarrhea [1 receptors with BPTU reduced diarrhea severity in neonatal mice and shortened recovery times.In addition to inducing ICWs, purinergic signaling was also necessary for the replication rate of RV and induced the production of cellular factors canonically known to play a role in the development of RV-associated pathology. Inhibiting ADP-P2Y1 receptors decreases RV-induced diarrhea severity in mice. Several drugs targeting P2Y receptors have emerged as candidate therapies for cardiovascular and inflammatory diseases and neurodegeneration [This study provides a detailed mechanism that explains how RV induces pathology through intercellular purinergic signaling. Perhaps, the most important observation by Chang-Graham et al. is that blocking the formation of ICWs in bystander cells through the inhibition of P2Yneration . The res.’s work suggests that expanding research in this area to include noninfected neighboring cell populations would improve the mechanistic understanding of disease progression and ultimately, drug development. This is certainly true when it comes to defining the molecular mechanism of enteric pathogen-induced pathologies. For example, norovirus is known to require very few viral particles to induce severe pathology [Another exciting development from this paper was the identification of the integral role that uninfected cells play in disease progression. Historically, infectious disease research has primarily focused on infected cells. However, Chang-Graham et alathology , and it athology  highligh