Ganoderma microsporum (GMI) in an in vitro rodent model of primary cultures. Using primary neuron/glia cultures consisting of neurons, astrocytes, and microglia, a GMI showed an alleviating effect on lipopolysaccharide (LPS)/interferon-γ (IFN-γ)-induced inflammatory mediator production and neuronal cell death. The events of neuroprotection caused by GMI were accompanied by the suppression of Nitric Oxide (NO), Tumor Necrosis Factor-α (TNF-α), Interleukin-1β (IL-1β), and Prostaglandin E2 (PGE2) production, along with the inhibition of microglia activation. Mechanistic studies showed that the suppression of microglia pro-inflammatory polarization by GMI was accompanied by the resolution of oxidative stress, the preservation of protein tyrosine phosphatase and serine/threonine phosphatase activity, and the reduction of NF-κB, AP-1, cyclic AMP response element-binding protein (CREB), along with signal transducers and activators of transcription (Stat1) transcriptional activities and associated upstream activators. These findings suggest that GMI may have considerable potential towards the treatment of neuroinflammation-mediated neurodegenerative diseases.Microglia polarization of classical activation state is crucial to the induction of neuroinflammation, and has been implicated in the pathogenesis of numerous neurodegenerative diseases. Fungal immunomodulatory proteins are emerging health-promoting natural substances with multiple pharmacological activities, including immunomodulation. Herein, we investigated the anti-inflammatory and neuroprotective potential of fungal immunomodulatory protein extracted from  Neuronal dysfunction and/or death are common events contributing to both acute and chronic neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease, and stroke. Among the etiology and pathogenesis responsible for neuronal degeneration, neuroinflammation is an emerging mechanism for the initiation and progression of degeneration. Activation of resident glial cells, particularly the microglia, along with the recruitment of peripheral leukocytes, and the overproduction of inflammatory mediators within the nervous system, form the basis of neuroinflammation. These neuroinflammatory responses are present in the vicinity of degenerating neurons, cerebrospinal fluids, and even systemic blood circulation ,2,3. TheOn the basis of cell levels, neurons, astrocytes, microglia, oligodendrocytes, pericytes, and other minor cell types orchestrate the structural and functional units of the nervous system. Among those component cells, microglia in particular show remarkable immune competency and are intimately associated with neuronal function and fate. Adequate microglial activation modulates both synaptic function and plasticity, while maintaining tissue homeostasis. Once overactivated, microglia release a wide range of inflammatory and bioactive molecules which impose detrimental impacts on neurons ,2,3,6,7.Ganoderma lucidum has been widely used as both an alternative medicinal remedy and dietary supplement in order to promote health and longevity. Ganoderma lucidum-derived spores, extracts, triterpenoids, and polysaccharides have a wide range of biological activities, including neuroprotection. Those extracts and ingredients alleviate microglial activation both in vitro and in vivo, as well as protect the brain against lipopolysaccharide-, streptozotocin-, Aβ-, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-, and cerebral ischemia-induced neuronal degeneration , ERK inhibitor (U0126), JNK inhibitor (SP600125), p38 inhibitor (SB203580), Akt inhibitor (LY294002), Jak inhibitor (AG490), Src inhibitor (PP2), COX-2 inhibitor (NS398), and antioxidant  all showed alleviating effects on LPS/IFN-γ-induced NO, TNF-α, IL-1β, and PGE2 production , IL-4, and IL-10, with the consequences of suppressing inflammation and restoring homeostasis. Moreover, IRF5, IRF8, P2X4R, P2X7R, and P2Y12R promote microglia polarization towards the classical activation state. By contrast, CD206 (a mannose receptor) and arginase 1 polarize microglia towards the alternative activation state ,30,34,35LPS/IFN-γ is commonly used to induce the microglia classical activation state in vitro. Upon exposure to LPS, the engagement with the transmembrane Toll-like Receptor 4 (TLR4) turns on intracellular phosphorylatory cascades to spread diverse signals, and eventually impact on the nuclear influx activity, DNA binding activity, and transcriptional activity of transcription factors, including NF-κB, AP-1, CREB, and Stat1. The linking adaptors and signaling molecules include MyD88, TAK1, TBK1, mitogen-activated protein kinases , IKK-α/β, p65, c-Jun, c-Fos, CREB, Src, as well as Akt. Likewise, the binding of IFN-γ with receptors activates Stat1 to drive IRF1 expression, by which Stat1 and IRF1 work in concert to boost another wave of transcriptional program. The activation of the Stat family latent transcriptional factors is promoted by upstream stimulatory tyrosine kinases such as Jak and Src family kinases, and inhibited by Src Homology 2 domain containing Phosphatase (SHP), Suppressor of Cytokine Signaling (SOCS), and Protein Inhibitors of Activated Stats (PIAS) ,10,25,31Free radicals are commonly multiplied during the course of neuroinflammation. Apart from being a primary neurotoxic mediator, free radicals indirectly cause neuronal cell death through their action on redox-sensitive intracellular signaling molecules and transcription factors to augment neuroinflammation. Moreover, oxidative stress represents an alternative mechanism to cause inactivation of protein phosphatase ,7. GMI-mGanoderma lucidum offer another view on the health-promoting effects of natural substances [Biological products are emerging as therapeutic drugs in human health care. In traditional Oriental medicine, plants are a good source of small molecular compounds. The identification and translational application of LZ-8 immunomodulatory proteins of bstances ,14. Dailbstances ,18,19,20bstances . Althougbstances . FindingOur results clearly demonstrate that the anti-inflammatory and neuroprotective effects of GMI are mediated through the inhibition of pro-inflammatory phenotype of microglia in the model of LPS/IFN-γ-stimulated neuron/glia cultures. The suppression of microglia pro-inflammatory polarization and a switch towards the alternative activation state by GMI was accompanied by the resolution of oxidative stress, preservation of protein tyrosine phosphatase and serine/threonine phosphatase activity, and reduction of NF-κB, AP-1, CREB, and Stat1 transcriptional activities and associated upstream activators. These findings suggest that GMI may have considerable potential for the treatment of neuroinflammation-mediated neurodegenerative diseases.d-lysine-coated dishes. For the preparation of neuron/glia, the cells were maintained in a minimum essential medium, supplemented with 10% Fetal Bovine Serum (FBS) and 10% horse serum for 10–12 days in vitro. Mixed glia were maintained in Dulbecco’s Modified Eagle Medium (DMEM)/F12 containing 10% FBS for 14 days in vitro. Microglia were isolated from confluent mixed glia through shaking at a speed of 200 rpm for 24 h, and maintained in DMEM/F12 containing 10% FBS. The retained astrocytes were replated and maintained in DMEM/F12 containing 10% FBS. Neuron/glia consisted of 30–40% neurons, 40–50% astrocytes, and 10–15% microglia. Mixed glia were composed of ~85% astrocytes and ~15% microglia. The purity of microglia and astrocytes was more than 95%. Cell composition was identified and estimated by immunocytochemical staining with antibodies recognizing neuron (Microtubule-associated Protein 2 (MAP-2), Transduction Laboratories, Lexington, KY, USA), astrocyte , and microglia , respectively.All experimental protocols for animal study were reviewed and approved by the Animal Experimental Committee of Taichung Veterans General Hospital  and subsequently conducted in strict accordance with the guidelines. Neuron/glia, mixed glia, and microglia cultures were prepared from cerebral cortices of postnatal day 1 Sprague-Dawley rats in accordance with previously reported methods ,27,33. ARecombinant GMI proteins were manufactured by Mycomagic Biotechnology Co., Ltd. . The detailed procedures for GMI cloning, expression, purification, and verification were described as in previously reported methods ,23.™ LDH Cytotoxicity Assay Kit  was used to assess cell damage in accordance with the manufacturer’s instructions. The cytotoxicity index was indicated by the ratio of released LDH and total LDH.A quantitative measurement of Lactate Dehydrogenase (LDH) released into the culture media by the PierceFor the conduction of immunocytochemical staining, neuron/glia (24-well plates) were washed with Phosphate-buffered Saline (PBS), fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100, in accordance with our previously reported methods ,27,33. ANeuron/glia, mixed glia, and microglia were seeded onto 24-well plates. For the conduction of ELISA, supernatants were collected and subjected to measurement using commercial ELISA kits, including Tumor Necrosis Factor-α (TNF-α), Interleukin-1β (IL-1β), and Prostaglandin E2 (PGE2), according to the manufacturer’s instructions .For NO (nitrite/nitrate) determination, neuron/glia, mixed glia, and microglia were seeded onto 24-well plates. The supernatants were collected and subjected to measurement using a Griess Reagent Kit, according to the manufacturer’s instructions .x 485 nm and Em 510 nm).The levels of intracellular free radicals were assayed by measuring intracellular oxidation of dichlorofluorescein, as in our previously described methods . Neuron/To conduct Western blot analysis, neuron/glia were seeded onto 6-well plates. Cultured cells were homogenized with a Laemmli SDS sample buffer, and the resultant cell lysates were subjected to SDS-PAGE separation and PVDF electrophoretical transfer . The memNeuron/glia were seeded onto 6-well plates. A commercially available nuclear extraction kit and EMSA kit were used to prepare nuclear extracts and conduct EMSA, respectively, according to the manufacturer’s instructions . As with our previous studies ,33, nuclp value was less than 0.05.All statistical results are presented as the mean ± standard deviation. A one-way analysis of variance was performed to evaluate experimental values between groups with a consequent Dunnett’s test or Tukey post-hoc test performed for the purpose of comparison. It was considered statistically significant when the


Vascular remodeling is a characteristic feature of cardiovascular diseases. Altered cellular processes of vascular smooth muscle cells (VSMCs) is a crucial component in vascular remodeling. Histone deacetylase inhibitor (HDACI), butyrate, arrests VSMC proliferation and promotes cell growth. The objective of the study is to determine the mechanism of butyrate-induced VSMC growth. Using proliferating VSMCs exposed to 5 mM butyrate, immunoblotting studies are performed to determine whether PI3K/Akt pathway that regulates different cellular effects is a target of butyrate-induced VSMC growth. Butyrate inhibits phosphorylation-dependent activation of PI3K, PDK1, and Akt, eliciting differential effects on downstream targets of Akt. Along with previously reported Ser9 phosphorylation-mediated GSK3 inactivation leading to stability, increased expression and accumulation of cyclin D1, and epigenetic histone modifications, inactivation of Akt by butyrate results in: transcriptional activation of FOXO1 and FOXO3 promoting G1 arrest through p21Cip1/Waf1 and p15INK4B upregulation; inactivation of mTOR inhibiting activation of its targets p70S6K and 4E-BP1 impeding protein synthesis; inhibition of caspase 3 cleavage and downregulation of PARP preventing apoptosis. Our findings imply butyrate abrogates Akt activation, causing differential effects on Akt targets promoting convergence of cross-talk between their complimentary actions leading to VSMC growth by arresting proliferation and inhibiting apoptosis through its effect on dual targets, HDAC activity and PI3K/Akt pathway network. Vascular remodeling is a characteristic pathological trait of many of the cardiovascular diseases and disorders, including atherosclerosis and restenosis through the changes in cell proliferation, cell growth, apoptosis, and differentiation of vascular cells in concert with vascular homeostasis. Cardiovascular disease continues to be a major health concern in spite of great strides that have been made in understanding the pathogenesis and management of the disease ,2,3. ThiHistones are targets of several different transient and reversible site-specific covalent post-translational modifications (PTMs) such as acetylation, methylation, phosphorylation, sumoylation and ADP-ribosylation. Reversible modifications of histones by acetylation alter chromatin structure, dynamics, promoter accessibility and recruitment of histone modifiers to promote changes in gene functions by affecting gene expressions ,20,21,22Butyrate, a fermentation product of dietary fiber and a long known natural HDACI, also causes a variety of cellular effects such as cell-cycle arrest in G1-phase, change in cellular morphology and size, stimulate cell differentiation and/or induce apoptosis in different non-cancerous and cancerous cells ,35,36,37The PI3K/Akt pathway is central to cellular processes such as cell proliferation, growth, differentiation and cell death. Several studies have shown that inhibition of Akt activation is involved in butyrate-inhibited cell proliferation leading to cell differentiation or cell death. However, not much is known regarding the role of butyrate-inhibited Akt activation in cell growth. Therefore, the focus of our present study is to determine whether butyrate-inhibited proliferation and stimulation of growth  of VSMCs is linked to its role as a dual inhibitor of PI3K/Akt pathway and HDAC activity. Results of our study demonstrate that butyrate inhibits activation of PI3K/Akt signaling and differently alters downstream targets of Akt that appear to support cell-cycle arrest and promote cell growth by changing morphology and size of VSMCs. This action apparently affects dual targets: histone deacetylase ,36, actiTreatment of VSMCs with butyrate for 72 h alters cellular morphology of VSMC from an elongated bipolar morphology to a round spread-out phenotype with increase in cellular size A. The exIn response to various factors and conditions, cells are stimulated to produce second messenger phosphatidylinositol-3,4,5-triphosphate (PIP3) by activating PI3K. The important role of PIP3 is to trigger activation of signaling components that regulate proliferation, growth and survival, particularly Akt. A number of proteins having pleckstrin homology (PH) domains have strong affinity to PIP3. Since PIP3 is restricted to plasma membrane, PIP3 formation by the activation of PI3K plays an important role in the recruitment of PH domain-containing signaling proteins. These proteins include phosphoinositide–dependent protein kinase 1 (PDK1) and Akt that translocate from the cytoplasm to the plasma membrane to mediate proliferation, growth and survival signals ,45,46,47PDK1 is a serine/threonine kinase and is an essential upstream activator of Akt. Activation of PI3K is necessary for the activation of PDK1, a member of the AGC superfamily of protein kinases, at the membrane to activate Akt, where it binds to PIP3 on plasma membrane formed by PI3K catalytic activity through its PH domain ,46,47,48Akt, a serine/threonine kinase that is also referred to as protein kinase B (PKB), regulates several cellular effects including, cell proliferation, cell growth, survival, cell death and migration by regulating several downstream signaling effector proteins ,45,46,47Akt controls prosurvival and antiapoptotic activities. Upon activation Akt triggers several cellular effects including, cell proliferation, survival, cell growth, cell death, and migration by activating or inactivating several downstream target effector proteins. These proteins include FOXO transcription factors, mTOR, GSK3, and pro and antiapoptotic proteins in response to various cellular stimuli . To deteActive Akt supports survival of cells by blocking apoptosis through inhibition of function or expression of proapoptotic proteins and processes. FOXO proteins, a subgroup of Forkhead transcription factors, are direct targets of Akt, which regulate transcription of distinctly different groups of genes, depending on the cellular context to produce context-specific phenotypic effects. Primarily, Akt-mediated phosphorylation of FOXO proteins suppresses activation of FOXO proteins blocking transcription of genes linked to cell-cycle arrest or apoptosis ,49,50. PAkt also participates in both promoting and inhibiting apoptosis by modulating expression of pro- and antiapoptotic proteins, including BAD, BAX, BAK, Bcl-2, CREB, MDM2, as well as modulating intrinsic mitochondrial pathway of apoptosis through its direct targets FOXO proteins and GSK3 ,50,51,52The serine/threonine kinase mTOR is the catalytic component of two distinct multiprotein signaling complexes, rapamycin-sensitive mTORC1 and rapamycin-insensitive mTORC2. As a part of the PI3K/Akt/mTOR pathway, mTOR exhibits a dual role as both an upstream activator of Akt and the downstream effector of the PI3K/Akt/mTOR pathway activity influencing cell growth, differentiation and metabolism by regulating protein synthesis and mRNA translation ,54,55,56Upon phosphorylation-dependent activation, mTOR upregulates protein synthesis by exerting its kinase activity on its downstream target proteins p70S6 kinase and eukaryotic translation initiation factor-4E (eIF4E)-binding protein (4E-BP1), the key regulators of ribosome synthesis and mRNA translation ,54. ActiVSMCs play critical roles in atherosclerosis and restenosis-associated vascular remodeling leading to neointimal hyperplasia ,57,58,59Butyrate is a natural HDACI and a fermentation product of dietary fiber that potentially mediates the beneficial effects of dietary fiber. The physiological concentration of butyrate is about 10 to 20 mM based on the type of dietary fiber consumed. For most cell culture and animal studies, mM concentrations of butyrate are used ranging from 0.5 mM to 10 mM. In many studies 5 mM concentration appears to be effective concentration ,33,37, iPI3Ks are a family of dual protein and lipid kinases, which upon activation phosphorylate the hydroxyl group at position 3 of the inositol ring of inositol phospholipids present in the plasma membrane. Class I PI3Ks are heterodimers of 85kDa regulatory subunit p85 and 110 kDa catalytic subunit p110, which are linked to an array of different functions. These functions include cell growth, proliferation, differentiation, survival and migration ,45,46,47Our present study reveals increased phosphorylation of Thr308 and Ser473 residues of Akt in 6 h and 24 h untreated VSMCs, compared to corresponding butyrate-treated VSMCs. However, compared to 6 h untreated VSMCs, 24 h and 30 h untreated VSMCs exhibited significant reduction in phosphorylation of both residues of Akt. This inhibition of Akt activation in 24 h and 30 h untreated VSMCs is not unusual. Studies have shown that stimulation of cells with serum or growth factors, induces Akt activation ,63,64,65Inhibition of cell proliferation by butyrate promotes different cellular effects including cell survival, cell growth, cell differentiation or cell death depending on the cell type and cellular context ,37,40,41Activated Akt elicits many cellular effects including cell survival, growth, proliferation, migration, metabolism and apoptosis that are important for cardiovascular functions and structural integrity through the mediation of serine and/or threonine phosphorylation of an assortment of downstream targets. Phosphorylation by Akt can be inhibitory or stimulatory, thus, negatively or positively suppressing or enhancing the activity of target proteins. Moreover, some crucial Akt targets including FOXO transcription factors, mTOR and GSK3, control more than one cellular function and their role may vary in a cell and signal-context dependent manner. As such, FOXO transcription factors, mTOR and GSK3 control several cellular processes that include cell survival, growth, proliferation, metabolism, and apoptosis. The role of these Akt targets in butyrate-induced cellular effects are not well understood, particularly in butyrate-inhibited VSMC proliferation and altered cellular morphology. Our present study reveals abrogation of Akt activation by butyrate differentially affects FOXO1 and FOXO3 transcription factors, mTOR, caspase 3 and PARP, and GSK3 . ConvergFOXO1 and FOXO3 proteins, members of a subgroup of Forkhead transcription factors, play pivotal roles both in cell-cycle arrest, and/or apoptosis signaling by coordinating a cast of gene expression that regulate cell survival and cell death ,49,50. AA range of unrelated factors and conditions, including chemotherapeutic agents, loss of contact with the extracellular matrix, DNA damage, and growth factor withdrawal can initiate the intrinsic mitochondrial pathway of apoptosis, resulting in the activation of caspase cascade. Although several studies reveal that butyrate arrests cell proliferation by inducing apoptosis ,28,29, o+ and ATP, causing caspase-independent apoptosis or necrotic cell death. Unwarranted hyperactivity of PARP has been alluded to the pathogenesis of several diseases and disorders, including stroke, myocardial infarction, inflammation, diabetes, cancer and neurodegenerative disorders [Interestingly, in butyrate-treated VSMCs, intact uncleaved PARP level was not only significantly downregulated, but there was also no cleavage of PARP, compared to untreated VSMCs B,C. Furtisorders ,70. In tBesides arresting proliferation and causing no cell death, butyrate alters cellular morphology of VSMCs from their elongated bipolar morphology to a round and spread-out phenotype with increase in sizes, implicating inhibition of cell proliferation by butyrate promotes cell growth ,24,36,53Our previous studies also reveal that increase in cyclin D1 levels is linked to increased synthesis and increased stability, based on cyclin D1 transcription and phosphorylation state of GSK3, respectively . GSK3 isInduction of protein synthesis in G1-phase is required for cell growth, and it is acknowledged that mTOR signaling pathway and its downstream targets, p70 ribosomal S6 kinase (p70S6K) and eukaryotic translation initiation factor 4E binding protein (4EBP1) dependent signals are key to regulating growth by increasing protein synthesis ,72,73,74Rat aortic VSMCs obtained from commercial source  were used for studies. They were cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM) augmented with 10% fetal bovine serum, 100 Units of penicillin and 100 µg/ml streptomycin , used as complete medium, in a humidified atmosphere of 5% CO2 at 37 °C. Confluent cultures were subcultured by seeding the cells at a ratio of 1:4. One day after seeding, proliferating cells were untreated or treated with 5 mM butyrate  for indicated periods of time as described previously ,36. For To determine the effect of butyrate on VSMC growth, one day after seeding, proliferating VSMCs were treated with complete medium containing butyrate (BA) or no butyrate (Con) for 72 h. After 72 h of treatment, cultures were washed with PBS and fixed in cold methanol for 5 minutes as described previously, to assess butyrate-induced altered cellular morphology and increase in size that promotes VSMC growth ,36,41. ATo demonstrate butyrate-induced VSMC growth is linked to proliferation arrest, VSMC cultures exposed to butyrate for 72 h, as described above. After 72 h of treatment, cultures were washed three times with sterile PBS and trypsinized with trypsin-EDTA. Cells were collected in DMEM and centrifuged briefly at 3000 rpm at room temperature. The cell pellets were suspended in PBS and cell numbers were counted by trypan blue exclusion method, using TC20 Automated cell counter from Bio-Rad, as described by the manufacturer .For Western blot analysis, one day after seeding the cells, cultures were treated with complete medium containing (BA) or no butyrate (Con) for indicated periods without replacing with fresh culture medium. At the end of treatment period, cultures were washed with ice cold PBS. Total cell lysates from experimental cultures were prepared and quantitated for protein concentration as described previously . BrieflyWestern blotting was performed first blocking the blots with Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% non-fat milk powder  for 1 h at room temperature. Then the blots were incubated overnight at 4 °C with appropriate primary antibodies to detect activation and/or levels of target proteins with gentle agitation ,41. The Following primary antibody incubation, the blots were incubated with species appropriate horse radish peroxidase (HRP) conjugated anti-rabbit-HRP-linked secondary antibody  from Cell Signaling Technology  or IRDye-conjugated goat anti-mouse IRDye 680LT  and goat anti-rabbit IRDye 800CW  secondary antibodies from LI-COR, Inc. . Blots probed with HRP-conjugated secondary antibodies were processed for chemiluminescence immunodetection using luminol reagent from Santa Cruz Biotechnology . Band intensities were measured and analyzed by using Molecular Imager Pro Plus MultiImager system and Quantity One software from Bio-Rad . Blots that were probed with IRDye-conjugated secondary antibodies were imaged on Odyssey CLx imager and band intensities were measured and quantitative analysis was performed using Image Studio software from LI-COR, Inc. . For normalizing protein loading, blots were probed with anti-ERK1/ERK2 antibodies, which is unaffected in response to butyrate treatment ,36,41 unp < 0.01 and p < 0.001.Statistical analysis was performed using GraphPad Prism version 5 software from GraphPad Software Inc . Statistical differences between controls and treatment groups were analyzed by one-way analysis of variance (ANOVA) with Bonferroni multiple comparison test. Statistically significant differences between data sets were calculated at In conclusion, butyrate’s cellular effects on VSMCs are mediated through disparate effects on dual targets, HDAC activity and PI3k/Akt signaling network. Butyrate arrests VSMC proliferation and inhibits cell death, promoting cell growth by increasing cellular size. Through its HDAC inhibitory functionality, butyrate not only alters expression of epigenetically regulated genes associated with cell-cycle regulation and PI3K/Akt signaling network, but also interferes with PI3K/Akt signal transduction pathway through PTMs of Akt and Akt targets. Increased expression of cyclin DI and D3, p15INK4, p21Cip1/Waf1 ,36 and c


Background: Allogeneic hematopoietic stem cell transplantation  following chemotherapy is part of standard treatment protocol for patients with acute myeloid leukemia (AML). FUS-ERG+ AML is rare but has an extremely poor prognosis even with allo-HSCT in remission, possibly due to its a leukemia stem cell (LSC)-driven disease resulting in chemotherapy resistance and a novel therapy is urgently required. It has been reported that FUS-ERG-positive AML expresses CD123, a marker of LSC, in some cases. CD123-targeted CAR T cell (CART123) is promising immunotherapy, but how to improve the complete remission (CR) rate and rescue potential hematopoietic toxicity still need to explore.Case Presentation: We used donor-derived CART123 as part of conditioning regimen for haploidentical HSCT (haplo-HSCT) in a patient with FUS-ERG+ AML who relapsed after allogeneic transplantation within 3 months, resists to multi-agent chemotherapy and donor lymphocyte infusion (DLI) and remained non-remission, aiming to reduce these chemotherapy-resistant blasts and rescue potential hematopoietic toxicity. The blasts in BM were reduced within 2 weeks and coincided with CAR copies expansion after CART123 infusion. The patient achieved full donor chimerism, CR with incomplete blood count recovery, and myeloid implantation.Conclusion: Our results hints that CART123 reduces the chemotherapy-resistant AML blasts for FUS-ERG+ AML without affecting the full donor chimerism and myeloid implantation. FUS-ERG+ AML patients are one of them  , which is a rare reciprocal chromosomal change. This translocation has been most frequently reported in AML, with an incidence of 1%  compared to that of MRD-positive, possibly due to its a leukemia stem cell (LSC)-driven disease and cannot be successfully eradicated with current treatment protocol   is part of standard treatment protocol for patients with high-risk acute myeloid leukemia (AML). In some cases, however, even allo-HSCT in remission still could not overcome the poor prognosis,  of them –3. FUS-Ece of 1% . To datechildren . In chiltion t16;1  T cell targeting CD19 have demonstrated remarkable potential in B cell malignancies –17. At pFUS-ERG+ AML relapse after allo-HSCT.Although a high objective response rate was achieved after CART19, the high relapse rate remains the major problem –25. It iA 25-year-old male was diagnosed with AML-M2 1 year ago, according to the French-American-British classification. Bone marrow (BM) morphology revealed 62.2% blasts, and peripheral blood (PB) was manually sorted by 39% blasts. The morphology examination exhibited megakaryocyte dysplasia, erythrophagocytosis, vacuolation in both cytoplasm and nucleus in leukemia cells in BM .Immunophenotyping by flow cytometry (FCM) analysis revealed positive results for CD34, CD38, HLA-DR, CD13, CD33, CD15, CD64, CD11b, CD56, CD117, CD123, MPO, and CyCD3 in BM. His karyotype showed 46, XX, t    /46, XY Then, he successively received DCAG , DMA , and CLAG  combined with donor lymphocyte infusion (DLI) and achieved transient CR with MRD positive. He developed an anal fissure and perianal abscess, and the infection was controlled by anti-infective therapy. He subsequently relapsed 1 month later with central nervous system leukemia (CNSL) and was administered four cycles of Ara-c, MTX, and DXM by intrathecal injection and CLAG + DLI. CNSL was controlled, but the disease progressed .8 cells, and 9 × 107 cells (CAR+ 80.2%) were CAR+ cells (1 × 106/kg). This second-generation CAR consisted of anti-CD123 single chain fragment variable (scFv), CD8a hinge region, CD8 transmembrane domain, 41BB costimulatory domain, and CD3ζ cytoplasmic region. Truncated human Epidermal Growth Factor Receptor (EGFR) polypeptide (tEGFR) was integrated with CAR gene through a P2A peptide  and CD4+ subset (54.7%), and 8.43% and 90% of CAR+ cells were characterized with the central memory phenotype (CD45RO+/CD62L+) and effect memory phenotype (CD45RO+/CD62L−), respectively  6 days after CART123 infusion. Considering the potential hematopoietic toxicity of CART123, the second infusion of PBMC will be performed to promote implantation if the hematopoietic system remains unrecovered within about 14 days (The patient received reduced-intensity conditioning (RIC) regimen of TVFB  and CART123 1 day after preconditioning. The total infused CART123 was 1.1 × 10 peptide . The viaectively . Both th 14 days .8/Kg, CD34+ 4.74 × 106/Kg, CD3+ 4.44 × 108/Kg). On day 32, blasts in BM were 0.5, 0.05, 0.042, and 0.02% by morphology, FCM, Wilms tumor-1 (WT1) and FUS-ERG detection, respectively. Compared to the first allo-HSCT, the second allo-HSCT was conducted in non-remission status using a RIC regimen, indicating the anti-leukemic activity of CART123. The patient achieved myeloid implantation on day 42 but was not weaned from platelet (PLT) and red blood cell (RBC) transfusion  was administered as conditioning regimen and G-PBSC was infused again on day 18 and day 19  in PBMC was significantly increased after CART123 infusion. Then, T cells were sharply reduced after the administration of medications, such as methylprednisolone, ATG, and basiliximab (The proportion of T lymphocytes (CD3iliximab . Direct iliximab .The patient developed a fever (>39°C), hypotension (92/58 mmHg) and pneumonia within 24 h after infusion, and these effects were evaluated as grade 3 CRS. He was immediately administered tocilizumab, a pressor agent and empirical anti-infective therapy. Assessment of cytokines in serum revealed an increasing trend for IL-6 and IFN-γ, and the effects in IL-6 was most obvious. Four days later, dyspnea, progressive pneumonia, and fever persisted (up to 41°C), and these features were evaluated as grade 4 CRS. The changing trend of C-reactive protein (CRP), lactate dehydrogenase (LDH), and body temperature was consistent with the level of cytokines and the clinical symptoms of the patient. Considering that tocilizumab on days −5 (240 mg) and −3 (400 mg) was invalid, methylprednisolone was administered from days −2 to 8  and the dose was gradually decreased. CRS was rapidly controlled after the infusion of methylprednisolone and ATG, with the decline of CRP, LDH, body temperature, and IFN-γ .The patient has an anal fissure before transplantation, and then it progressed to anal fistula with perianal infection after transplantation. However, the perianal infection caused repeated sepsis and pneumonia. Intermittent fevers occurred and were accompanied by sharp elevations in CRP and LDH after allo-HSCT. Repeated anti-infective, symptomatic and supportive treatment was administered to the patient and exhibited effective results. On day 28, he developed disseminated intravascular coagulation (DIC) due to infection and was controlled by the symptomatic treatment .7 umbilical cord blood mesenchymal stem cells (UCB-MSC) were administered for the treatment of aGVHD. Finally, he was diagnosed with grade IV aGVHD involving liver and gut. In the final stage, creatinine increased progressively, reflecting the deterioration of renal function. Unfortunately, the patient died of aGVHD, severe pneumonia, intestinal obstruction, and multiple organ failure on day 56  progressively increased, mainly direct bilirubin (DBiL). He was diagnosed with aGVHD and administered by CsA, glucocorticoids (GC), MMF, basiliximab, tacrolimus, and maraviroc were successively for the treatment of aGVHD. On day 48, a total number of 7 × 10n day 56 .Still, relapse after allo-HSCT remained a ticklish question . In addiFUS-ERG+ patient with AML relapsed within 3 months after the first allo-HSCT and maintained non-remission before the second allo-HSCT. The patient had progressive disease after a total of nine prior lines of therapy including ineffective DLI. Overall, this patient had an extremely dismal prognosis. He received a donor-derived CART123 as part of conditioning regimen for haplo-HSCT and achieved CRi status, full donor chimerism, and myeloid implantation. This is the first report of allogeneic CART123 as part of conditioning regimen for haploidentical HSCT in the treatment of relapsed AML after transplantation. This regimen could provide possible options for AML patients with an abysmal prognosis, including who is FUS-ERG positive.Numerous reports suggested that second allo-HSCT may produce a better prognosis for patients with favorable performance status, remission at the time of the second transplant and most importantly, a long interval between initial transplant and relapse , 38. Howin vivo CAR T cell expansion, such as disease burden, intensity of precondition regimen, the CAR T cell dose, and design of CAR , and GVHD, which closely related to his disease status. Park et al.  showed tPGF, which can be a life-threatening complication, occurs in 5–27% of patients after allo-HSCT –63. The In this study, the patient developed a fatal aGVHD after achieved CRi. It is most likely related to recurrent infections after transplantation. Infectious diseases can theoretically promote the elevation of inflammatory cytokines after allogeneic HSCT and the activation of various immune effector cells, which might lead to aggravation of acute GVHD . UnfortuFUS-ERG-positive AML without affecting the full donor chimerism and myeloid implantation. However, the long-term anti-leukemic effect, the interval between infusions of CART123 and G-PBSC, and the prophylaxis of GVHD still require further study.Taken together, our results hint that haploidentical CART123 reduces the chemotherapy-resistant AML blasts for See the Materials and Methods section in the All datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by Institutional Review Board at the Affiliated Hospital of Academy of Military Medical Sciences . The patients/participants provided their written informed consent to participate in this study.CH and ZB designed the trial and experiments and analyzed the data. HL and CJ performed experiments, analyzed the data, and revised the manuscript. SY performed experiments, analyzed the data, and wrote the paper. LY, LB, WQ, NH, FH, ZL, and WP performed experiments and analyzed the data.LY, FH, ZL, and WP was employed by the company HRAIN Biotechnology. The remaining 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.


Candida albicans has been one of the most prevalent human fungal pathogens, and recently, the prevalence of Candida tropicalis as a causative agent of candidiasis has increased. In C. albicans, the ability to switch between yeast and hyphal forms is thought to be a key virulence factor and is regulated by multiple signaling cascades—including the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA), calcineurin, high-osmolarity glycerol (HOG), and mitogen-activated protein kinases (MAPK) signaling pathways—upon receiving environmental cues. The cAMP/PKA signaling pathway also triggers white-opaque switching in C. albicans. However, studies on C. tropicalis morphogenesis are limited. In this minireview, we discuss the regulation of the yeast-hypha transition, virulence, and white-opaque switching through the cAMP/PKA pathway in the closely related species C. albicans and C. tropicalis.Fungal species undergo many morphological transitions to adapt to changing environments, an important quality especially in fungal pathogens. For decades,  Candida species are common causative agents of superficial and invasive fungal infections worldwide [Candida species, C. albicans is the most prevalent and has been responsible for >60% of candidemia cases worldwide for decades [albicans Candida species (NACs)—such as C. tropicalis, C. glabrata, and C. parapsilosis—in patients with candidiasis or candidemia has increased [C. tropicalis is the first or second most commonly isolated species from patients with candidiasis, especially in tropical regions such as Brazil and Asia [C. tropicalis is closely related to C. albicans based on genomic comparisons, but the natural habitats of these pathogens differ: C. albicans is a human commensal organism and is rarely found in the environment, whereas C. tropicalis is not only a commensal of the human oral cavity, but is also present in soil, compost, plants, beaches, and seawater in tropical or subtropical areas [orldwide ,2. Of th decades . Recentlncreased ,5. Amongand Asia ,8,9,10. al areas ,12,13,14C. albicans, the transition between yeast and filamentous (pseudohyphae and hyphae) forms as well as white-opaque switching have been observed and characterized. Cells that are locked in either yeast or hyphal forms have altered virulence, and white-opaque switching is associated with biofilm formation [2, and high temperature, govern the morphological switch between different cell types in C. albicans [C. tropicalis in various environmental conditions such as CO2, pH, and N-acetylglucosamine (GlcNAc) [C. albicans and C. tropicalis.In order to propagate and cause disease, pathogens have developed several strategies to deal with the harsh conditions associated with survival within a host. In particular, morphological plasticity is an important tool for fungi to adapt to changing environments within a host. In ormation ,23,24,25albicans ,31,32,33albicans ,35,36,37(GlcNAc) ,41,42,43C. albicans, the PKA holoenzyme contains two “inactivate” catalytic subunits (Tpk1 and Tpk2), each bound to a homodimer regulatory subunit (Bcy1)  . Environcircuits ,55,56,57S. cerevisiae, the cAMP/PKA signaling pathway is involved in the regulation of cell growth and cellular differentiation [S. cerevisiae PKA catalytic subunits  show distinct roles in pseudohyphal growth. ScTpk2 positively regulates pseudohyphal growth upon nitrogen starvation, while the other two catalytic subunits, (ScTpk1 and ScTpk3) negatively influence pseudohyphal growth [C. albicans, the cAMP/PKA pathway plays critical roles in growth, morphogenesis, white-opaque switching, and virulence in murine models of systemic infection [bcy1/bcy1 mutant is only viable in a tpk2/tpk2 mutant background, which has lower PKA activity compared with the wild type. Meanwhile, the conditional tpk1/tpk1 tpk2/tpk2 mutant can be acquired, but has severe growth defects [bcy1/bcy1 mutant that exhibited severe growth defects, Ding et al. showed that Bcy1 controls filamentation in C. albicans [tpk1/tpk1 tpk2/tpk2 double deletion mutant that exhibited severe growth defects [Cryptococcus neoformans and Aspergillus fumigatus—showing that PKA is not essential for, but does regulate, fungal growth [In ntiation ,59,60,61l growth ,63. In Cnfection ,18,64,65nfection . In cont defects ,67. Rece defects ,69. By oalbicans . Soon afl growth ,70.C. tropicalis is an emerging pathogen, studies related to its cAMP/PKA signaling pathway are limited. A pioneer study in C. tropicalis showed that Efg1, a transcription factor that is a potential downstream target of cAMP/PKA, is involved in the yeast-hypha transition, biofilm formation, and white-opaque switching [C. albicans [C. tropicalis and C. albicans. The initial study of C. tropicalis PKA revealed that Tpk1 and Tpk2 have redundant functions in growth and filamentation, but the PKA catalytic null mutant was not viable. However, a few years later, two separate laboratories obtained tpk1/tpk1 tpk2/tpk2 mutants that exhibited severe defects in cell growth, filamentation, and white-opaque switching [CYR1 in C. tropicalis results in severe growth defects and reduced hyphal development [C. tropicalis adenylyl cyclase and PKA catalytic subunits have been identified and characterized, knowledge is limited on the functions of the regulatory subunit Bcy1 in growth and morphological transitions. Study of these PKA null mutants in C. albicans and C. tropicalis has facilitated the reevaluation of the global regulatory roles of the cAMP/PKA pathway.Although witching . Similaralbicans ,57,71,72witching ,73. In aelopment . Althoug2. Intracellular cAMP levels are tightly regulated by adenylyl cyclase (Cyr1) and phosphodiesterase (Pde2), which produce and degrade cAMP, respectively  regulates hyphal growth induced by serum, CO2, or bacterial peptidoglycan [PDE2 results in reduced hyphal growth and attenuated virulence in a murine model of systemic infection [CaCYR1 and CaPDE2 homologs are also involved in filamentation in C. tropicalis: The loss of CtCYR1 and CtPDE2 results in attenuated and enhanced filamentation, respectively [cAMP is required to trigger the activation of the PKA pathway in response to various environmental stimuli, including GlcNAc, serum, amino acids, and COectively . In C. adoglycan ,31,74, wnfection ,76. The ectively . Interesectively .C. albicans, Tpk1 and Tpk2 have redundant roles in growth and dimorphic switching, but each has distinct roles in hyphal development in various filamentation-inducing conditions [tpk2/tpk2 mutant, but not the tpk1/tpk1 mutant, exhibited reduced virulence in murine model of systemic infection [C. albicans tpk1/tpk1 tpk2/ptk2 double deletion mutant showed no hyphal growth in the various tested conditions, including solid and liquid Spider medium and Lee’s medium supplemented with glucose or GlcNAc as well as YPD with serum in liquid [tpk1/tpk1 tpk2/tpk2 mutant has not been characterized, but it is predicted to have attenuated or abolished virulence due to its severe defects in cell growth, hyphal growth, and white-opaque switching [In nditions ,72,77,78nditions ,72,78,79n liquid . The virwitching .C. albicans tpk1/tpk1 tpk2/tpk2 double deletion mutant, the bcy1/bcy1 mutant is not viable due to its high PKA activity in early studies. However, the bcy1/bcy1 mutant was obtained in a tpk2/tpk2 mutant background, which has reduced PKA activity, and showed defects in hyphal growth [bcy1/bcy1 null mutant in a C. albicans SN152 background that had two distinct phenotypes: smooth and filamentous [bcy1/bcy1 cells had defects in hyphal growth, while the filamentous bcy1/bcy1 mutant exhibited hyphal growth indistinguishable from that of the wild type. Interestingly, the byc1/bcy1 mutant was able to switch between these two phenotypes on YPD plates and in liquid SD medium, but had a preference for the filamentous phenotype under both conditions [Similar to the l growth . Recentlamentous . Smooth nditions .C. tropicalis was published in 2016 and suggested that Tpk1 and Tpk2 have redundant roles in filamentation and cell growth [tpk1/tpk1 tpk2/tpk2 double mutant, but did not characterize the impact on filamentation [C. tropicalis tpk1/tpk1 tpk2/tpk2 mutant despite severe defects in cell growth, filamentation, and virulence in a murine model of systemic infection [C. tropicalis tpk1/tpk1 and tpk2/tpk2 mutants showed virulence similar to the wild type in murine model of systemic infection. In addition to the redundant roles in cell growth and virulence, this group also found that Tpk1 and Tpk2 have distinct roles. For example, Tpk1 contributes to stress responses and antifungal drug tolerance, while Tpk2 plays a key role in filamentation, flocculation, and biofilm formation in C. tropicalis [C. albicans and C. tropicalis  whereas C. albicans opaque cells are not [2 concentrations (5~20%) induce white-opaque switching and stabilize opaque cells at 37 °C in C. albicans, but inhibit the white-opaque transition in C. tropicalis [In addition to transitioning between yeast and hyphal growth, l change ,81,82,83id media ,84,85,86 surface ,82. Howeopicalis ,86,87.C. albicans cAMP/PKA pathway is highly involved in white-opaque switching [tpk1/tpk1 or tpk2/tpk2 mutant in C. albicans has a similar rate of white-opaque switching compared to the wild type in various culture conditions [CYR1 results in low intracellular cAMP concentration, and a tpk1/tpk1 tpk2/tpk2 mutant without obvious PKA activity showed severe defects in white-opaque switching [pde2/pde2 knockout, bcy1/bcy1 knockout, or TPK2 overexpression results in cAMP/PKA pathway activation, thus promoting the white-opaque transition  in acidic or high CO2 conditions, whereas C. tropicalis cAMP levels are higher in alkaline and normal air conditions [C. albicans is a commensal that lives in different niches within humans with relatively high CO2 concentrations, while the environmental microorganism C. tropicalis is well adapted to normal air.Although the cAMP/PKA pathway is involved in white-opaque switching in nditions ,69. ThisC. albicans and C. tropicalis are close relatives, their different natural habitats have shaped their regulatory systems via conserved signaling pathways (such as the cAMP/PKA pathway), which can facilitate the establishment of infection. In C. albicans and C. tropicalis, the cAMP/PKA singling pathway regulates the yeast-hypha transition, virulence, and white-opaque switching. In general, the components of the cAMP/PKA signaling pathway in the two species have similar roles in vegetative growth, hyphal development, and white-opaque switching. Interestingly, the catalytic subunit Tpk2 shows distinct roles in virulence between the species: CaTpk2, but not CtTpk2, is required for virulence in a murine model of systemic infection. Furthermore, different conditions induce or suppress the yeast-hypha transition and white-opaque switching in these two fungal pathogens, suggesting that cAMP/PKA might respond to different environmental cues. In addition to morphogenesis, the C. albicans cAMP/PKA pathway has been shown to regulate glycogen synthesis, energy metabolism, and mitochondrial activity [C. albicans [C. albicans and C. tropicalis, this pathway might be a promising target for antifungal drug development. A similar approach in the calcineurin pathway [C. albicans and C. neoformans [C. albicans, knowledge of this important pathway in C. tropicalis is limited. Comparative studies on the roles of Hsp90 and small molecules in morphogenesis and virulence and the roles of cAMP/PKA in metabolic adaptation in C. tropicalis will contribute to the understanding of how these fungal pathogens utilize the cAMP/PKA pathway to cause human infections.Although activity ,89. Inhialbicans ,90, and albicans ,92,93. F pathway  has showoformans ,96. Alth


Taxus chinensis is a precious woody species with significant economic value. Anthocyanin as flavonoid derivatives plays a crucial role in plant biology and human health. However, the genes involved in anthocyanin biosynthesis have not been identified in T. chinensis. In this study, twenty-five genes involved in anthocyanin biosynthesis were identified, including chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, anthocyanidin synthase, flavonoid 3’-hydroxylase, flavonoid 3’,5’-hydroxylase, dihydroflavonol 4-reductase, anthocyanidin reductase, and leucoanthocyanidin reductase. The conserved domains and phylogenetic relationships of these genes were characterized. The expression levels of these genes in different tissues and different ages of xylem were investigated. Additionally, the anthocyanin accumulation in xylem of different ages of T. chinensis was measured. The results showed the anthocyanin accumulation was correlated with the expression levels of dihydroflavonol 4-reductase, anthocyanidin synthase, flavonoid 3’-hydroxylase, and flavonoid 3’,5’-hydroxylase. Our results provide a basis for studying the regulation of the biosynthetic pathway for anthocyanins and wood color formation in T. chinensis. Secondary metabolites are the products of plant adaptation to the environment during their evolution. Anthocyanins as a kind of secondary metabolite play a vital role in various processes of plant development, such as providing color to organs to attract pollinators, and of antioxidant activity to protect plants from injury by biotic and abiotic stress ,2,3,4. IArabidopsis thaliana, Medicago sativa, Petunia hybrida, and Salvia miltiorrhiza .,35.T. chPinus radiata, Picea sitchiensis, and Picea glauca, whereas TcF3H3, TcF3H4, and TcF3H5 were clustered with F3H from Ginkgo biloba   , which bam00067) . The proPohlia nutans (lower plant), whereas TcF3’H3-TcF3’H4 clustered together with F3’Hs of Plectranthus scutellarioides (dicot). TcF3’5’H clustered together with F3’5’Hs of Glycine max and Salvia miltiorrhiza. TcF3’H1 showed higher expression level in roots than other tissues, while TcF3’H2 showed the lowest expression level in roots  have neither Asn nor Asp in the specific site, indicating that there are different substrate specific recognition mechanisms of DFR. Multiple amino acid sequence alignment of TcANR and ANRs from other species showed they were highly conserved. TcANR had active sites  and the NAD/NADH combination domain (G-G-X-G-X-X-A).DFR catalyze dihydroflavonols into leucoanthocyanidins, which are the substrates of ANS. The products of ANS have two possible outcomes: one is conversion into anthocyanins by chemical modification, and the other is the production of flavane-3-alcohol catalyzed by ANR. DFR and ANR belong to the NAD/NADH-dependent epimerase family, which use nucleotide sugar as substrate for a variety of chemical reactions ,42,43. AGinkgo biloba), which is consistent with the results of a previous study [Gossypium arboreum), GmDFR (Glycine max), and NtDFR (Nicotiana tabacum) especially for TcDFR1, TcDFR2, and TcDFR5. TcANR clustered with PsANR (Picea sitchensis) and GbANR (Ginkgo biloba), which are gymnosperms. The analysis of expression profiles revealed that TcDFR5 exhibited similar expression in the tissues analyzed; TcDFR2 and TcDFR6 exhibited prominently high expression level in leaves, TcDFR3 and TcDFR7 exhibited mainly high expression level in roots and TcDFR1, and TcDFR4 exhibited high expression levels in phloem and xylem -flavane-3-alcohol, which is the precursor of procyanidin . LAR hasanalyzed ,48,49,50TcLAR1 and TcLAR2 are different in different tissues , clade 2 (gymnosperms), and clade 3 (dicots) a. TcLARs tissues b. The exT. chinensis was measured by HPLC; 1% methanol hydrochloric acid was used as solvent in combination with ultrasonic extractor-assisted extraction at low temperature and dark conditions to obtain the best extraction. The results showed that one-year-old xylem had relatively higher anthocyanin content, which increased gradually from two-year-old to four-year-old xylem (In order to investigate the relationship between the expression levels of genes involved in anthocyanin biosynthesis and anthocyanin accumulation, the content of anthocyanin and the expression level of genes involved in anthocyanin biosynthesis of xylem ranging in age between one to four years in ld xylem a. The chT. chinensis were analyzed using qRT-PCR (TcCHS exhibited higher expression levels in one-year-old xylem than any other sample. TcCHI1 and TcCHI2 both exhibit higher expression levels in one-year-old xylem than others, especially for TcCHI2. This may suggest that the enhanced effect on anthocyanin biosynthesis in one-year-old xylem is also higher than that in older xylem. TcF3H1, TcF3H2, and TcF3H3 exhibited higher expression levels in one-year-old xylem than older xylem; TcF3H4 and TcF3H5 exhibited similar expression level in the different ages of xylem, except for relatively high level of TcF3H4 in four-year-old xylem. TcF3’H2 and TcF3’H4 exhibited higher expression levels in one-year-old xylem and three-year-old xylem, respectively, while TcF3’H1 and TcF3’H3 exhibited higher expression levels in four-year-old xylem. TcF3’5’H exhibited higher expression level in one-year-old xylem than that in older xylem. TcDFRs exhibited similar expression patterns except for relatively high levels for TcDFR1, TcDFR4, and TcDFR8 in one-year-old xylem. TcANR exhibited similar expression level in each tissue and higher levels in one-year-old xylem than other ages. TcLAR1 showed similar expression, while TcLAR2 showed the highest expression in one-year-old xylem. Taken together, the anthocyanin accumulation was correlated with the expression levels of TcDFR1, TcANS, TcF3’H1, and TcF3’5’H, suggesting these genes may play a vital role in the anthocyanin biosynthesis of xylem.Expression levels of genes involved in anthocyanin biosynthesis in  qRT-PCR b,c. The T. chinensis. In this study, up to 25 genes putatively related to anthocyanin biosynthesis were identified in T. chinensis, including TcCHS, TcCHI1-TcCHI2, TcF3H1-TcF3H5, TcF3’H1-TcF3’H4, TcF3’5’H, TcDFR1-TcDFR8, TcANS, TcANR, and TcLAR1-TcLAR2. Only three of these have been previously reported. The sequence features and tissue-specific expression patterns of these genes in T. chinensis were analyzed. The conserved domains of deduced amino acid sequences of these genes and their phylogenetic relationships were performed and found to have high identity and close phylogenetic relationships with the corresponding genes from gymnosperms. Anthocyanins are an important type of secondary metabolites which play significant roles in colored plant tissues, such as flowers, fruits, and wood. Although the genes related to biosynthesis of anthocyanin have been identified in many plant species, they have not been systematically identified in T. chinensis is thought to have evolved in the Paleozoic era approximately 300 million years ago, surviving the harsh environment of the Quaternary glacier period. It belongs to relict plants of the Tertiary period and has a longer evolutionary period than angiosperms. Analysis of phylogenetic trees conducted in this study showed that most of the identified T. chinensis genes are located at the root of the phylogenetic tree, which is consistent with the evolutionary relationship between T. chinensis and other angiosperms [TcF3’H1 and TcLAR2 of which deduced proteins were closely related to angiosperms in phylogenetic trees may have more variation in their selection process. The gene family originated by gene duplication and divergence from a common ancestor. Interestingly, TcCHI1 possesses two Chalcone_3 domains in a tandem array in T. chinensis, whereas one CHI gene contains only one conserved domain similar to that found in other species. The sequence analysis of Lotus japonicus showed that there were four CHI genes in a tandem array in its genome [T. chinensis precedes Lotus japonicus in the evolutionary relationship, we speculate that the ancestor gene of CHI family existed with several tandemly conserved domains and became a cluster of tandem genes through the long period of evolution. The number of genes identified in anthocyanin biosynthesis-related gene families in T. chinensis were different from other plant species such as Arabidopsis thaliana, salvia miltiorrhiza, and Oryza sativa, indicating that genes encoding the same enzyme may have different evolution patterns in different species (T. chinensis DFR genes is significantly more than the number in Arabidopsis thaliana, Salvia miltiorrhiza, and Oryza sativa, suggesting that more gene duplication events occurred for DFR genes in T. chinensis (iosperms ,52. Howeiosperms . Genes ss genome . Since T species  [12,28,5hinensis .DFR, ANS, and F3H [DFR and ANS are considered as very important enzymes in anthocyanin biosynthesis, as they could direct the flavonoid flux into the anthocyanin branch [DFR, ANS, and F3H and some transcription factors were positively correlated with anthocyanin accumulation. The expression levels of TcDFR1, TcDFR4, and TcDFR5 showed higher expression levels in phloem than in other tissues. Similarly, the expression levels of TcDFR3and TcDFR8 showed high expression in roots, possibility resulting in high anthocyanins accumulation in phloem and root. This is consistent with the red bark and red root of T. chinensis.The anthocyanin biosynthesis pathway has been extensively studied in plants. A number of studies about transcriptomics and metabonomics in anthocyanin biosynthesis revealed that the expression levels of structural genes play an important role in anthocyanin accumulation, especially  and F3H ,56,57,58n branch ,56,57,58T. chinensis has high commercial value because of its aesthetic appearance, straight texture, high density, mechanical strength, rich elasticity, corrosion resistance, and purple red brown colored wood. The color of the wood is an important trait reflecting its quality and economic value. It has been shown that the formation of wood color is due to the existence of secondary metabolites in heartwood, including different kinds of phenolic compounds [T. chinensis was conducted. Analysis of the expression levels of these genes in different tissues showed different expression patterns, suggesting potential different functions. The expression levels of TcCHS, TcANS, and TcDFR1 showed relatively higher in xylem than in other tissues. The expression levels of TcDFR1, TcANS, TcF3’H1, and TcF3’5’H were observed to increase gradually from two-year-old to four-year-old xylem, especially for TcDFR1 and TcF3’5’H that showed the highest expression level in one-year-old xylem, which is consistent with the trend of anthocyanin content accumulation (T. chinensis has a close association with TcDFR1, TcANS, TcF3’H1, and TcF3’5’H their expression levels not only are consistent with the trend of anthocyanin content but also showed relatively high expression levels in xylem (T. chinensis.The wood of ompounds . In ordemulation a,b. In tin xylem b,c, impl


ATP and nucleic acids, the universal cellular actors, were shown to modulate LLPS of FUS in a unique manner: enhancement and then dissolution. Currently, the driving force for LLPS of FUS is still under debate, while the mechanism for the modulation remains completely undefined. Here, by NMR and differential interference contrast (DIC) imaging, we characterized conformations, dynamics, and LLPS of FUS and its domains and subsequently their molecular interactions with oligonucleic acids, including one RNA and two single-stranded DNA (ssDNA) molecules, as well as ATP, Adenosine monophosphate (AMP), and adenosine. The results reveal 1) both a prion-like domain (PLD) rich in Tyr but absent of Arg/Lys and a C-terminal domain (CTD) abundant in Arg/Lys fail to phase separate. By contrast, the entire N-terminal domain (NTD) containing the PLD and an Arg-Gly (RG)-rich region efficiently phase separate, indicating that the π-cation interaction is the major driving force; 2) despite manifesting distinctive NMR observations, ATP has been characterized to modulate LLPS by specific binding as oligonucleic acids but with much lower affinity. Our results together establish a unified mechanism in which the π-cation interaction acts as the major driving force for LLPS of FUS and also serves as the target for modulation by ATP and oligonucleic acids through specific binding. This mechanism predicts that a myriad of proteins unrelated to RNA-binding proteins (RBPs) but with Arg/Lys-rich disordered regions could be modulated by ATP and nucleic acids, thus rationalizing the pathological association of Amyotrophic lateral sclerosis (ALS)-causing C9ORF72 dipeptides with any nucleic acids to manifest cytotoxicity. FUS is an RNA-binding protein that is mutated and/or aggregated in several neurodegenerative diseases; this study shows that both ATP and nucleic acids modulate liquid-liquid phase separation of FUS in the same manner by specific binding. Fused in sarcoma (FUS)/Translocated in Sarcoma (TLS) was first identified as a fusion oncogene in human liposarcomas, which is composed of the FUS residues 1–266 fused with a transcription factor C/EBP Homologous protein (CHOP) . On the FUS is a 526-residue protein that is composed of three major domains : the N-tRecently, it was recognized that the formation and dissolution of LLPS in cells are under tight regulation by various cellular factors/events, and their misregulation will result in a large spectrum of human diseases –17,21–254 types of proteins and nucleic acid molecules -15N steady-state heteronuclear Nuclear Overhauser Effect (hNOE)  , as wellt (hNOE) , the PLD15N backbone relaxation data  of Cα, Cβ, and Hα protons—the difference between the experimental and random coil chemical shifts—is a sensitive indicator of secondary structure population for both folded and unfolded proteins ,47,50,53A close examination of the CSI  and SSP A detailed examination of their sequences revealed that the FUS ZnF contains no large hydrophobic residues Val, Ile, or Leu, while other two have both large hydrophobic residues and more aromatic residues. This implies that the folding of the FUS ZnF might be predominantly driven by Zn-coordination. To confirm this, we denatured the FUS CTD by 8 M urea followed by purification with reverse-phase HPLC to remove zinc ion. The zinc-depleted FUS CTD had no well-dispersed HSQC peaks . On the Recently we found that in addition to dissolving LLPS of FUS as a hydrotropic molecule at high concentrations , ATP cou2, which was previously used for characterizing the mediation of FUS LLPS by ATP [4, because both of them have been previously shown to have the functionally relevant binding to the FUS domains by various methods including NMR [Here, we further conducted a systematic NMR and DIC characterization of LLPS of FUS and its three domains , namely S by ATP . In ordeding NMR ,59. Howeding NMR ,59,60, wWe first assessed the conformations of two ssDNA molecules in different buffer conditions by far-UV CD spectroscopy . Indeed,We next assessed the effect of two ssDNA molecules under exactly the same solution conditions. For TssDNA, at a molar ratio of 1:1 (FUS:TssDNA), LLPS of FUS appeared to be significantly enhanced by forming a large number of droplets with a maximal diameter up to 5.4 μm IV of . FurtherHere, we showed that one RNA and two ssDNA molecules with distinctive structures are all able to modulate LLPS of FUS in the same two-stage manner. The effect appears to be dependent on the binding affinities of nucleic acids with FUS. Indeed, previously, FUS has been shown to bind various nucleic acids, including RNA and ssDNA, with highly degenerative specificity to types and sequences of nucleic acids ,59,61,62Recently, we found that ATP could monotonically dissolve LLPS of the FUS NTD, as characterized by the intensity reduction of all HSQC peaks . FurtherSubsequently, we assessed the effects of three oligonucleic acids on LLPS of the entire NTD at different molar ratios, including 1:0.1, 1:0.5, 1:1, 1:2, 1:5, 1:10, :1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, and 1:50, by DIC imaging . InteresTo gain a residue-specific view of the interaction between the FUS NTD and ssDNA in mediating LLPS, we characterized dissolution of the liquid droplets of the FUS NTD induced by two ssDNA molecules with NMR HSQC titrations under exactly the same solution condition and at the same molar ratios as used for the above DIC imaging. Interestingly, at a molar ratio of 1:1, only a small set of HSQC peaks either became shifted or disappeared . AdditioWe then analyzed the intensity change of HSQC peaks of the NTD  in the pThe present NMR results of the NTD titrated with two ssDNA molecules are unexpected in consideration of the fact that ATP is one building block of nucleic acids, as well as both ATP and ssDNA inducing the same monotonic dissolution of LLPS of NTD. So why are the patterns of the intensity changes of the NTD HSQC peaks induced by ATP and oligonucleic acids so different? While ssDNA induced the differential intensity changes of HSQC peaks for PLD and RGG1, ATP triggered the uniform intensity reduction of HSQC peaks of all NTD residues. To understand the underlying mechanisms for this observation, we titrated the PLD at 20 μM in the same buffer with ATP and two ssDNA molecules as monitored by both DIC imaging and NMR. The results showed that at 20 μM, no LLPS was induced for the PLD by adding ATP up to 10 mM as well as two ssDNA molecules, even at a ratio up to 1:50 (1 mM). Consistent with DIC results, for ATP, only very minor shifts of several HSQC peaks were observed, but no significant change of the peak intensity was found for PLD even in the presence of ATP at 10 mM . FurtherThe results also imply that ATP and two ssDNA molecules dissolved LLPS of the FUS NTD by binding to the same or at least highly overlapped set of the residues over RRG1. To test this hypothesis, we added ATP into the FUS NTD sample to reach a final concentration of 3 mM to mimic the ATP concentration in neuron cells of approximately 2.7 mM , and conSo, an interesting question arises: if ATP and two ssDNA both bind to the same set of residues within RRG1 to dissolve LLPS of the FUS NTD, why does the binding of ATP induce uniform intensity reduction of HSQC peaks of all NTD residues? Here, we propose that this unique NMR observation for ATP is most likely due to the hydrotropic nature of ATP as previously reported ,64–68. ABoth the FUS F-CTD and U-CTD lack the intrinsic capacity to phase separate. However, we recently found that ATP could induce the FUS F-CTD to phase separate at 1–2 mM, followed by subsequent dissolution at high ATP concentrations >4 mM in vitro . DetaileSubsequently, here we titrated the U-CTD with ATP and found that ATP was also able to induce and then dissolve LLPS with the same pattern as we previously observed on the F-CTD . StrikinOn the other hand, we also characterized the effects of one RNA and two ssDNA molecules on LLPS of F-CTD at different molar ratios, including 1:0.1, 1:0.5, 1:1, 1:2, 1:5, 1:10, :1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, and 1:50. Upon adding RNA at a molar ratio of 1:2, many small droplets formed with a maximal diameter of approximately 1.1 μm I of . HoweverWe then monitored the two-stage effect of oligonucleic acids by HSQC titrations at the same molar ratios used for the above DIC imaging. For the F-CTD, upon adding TssDNA at 1:0.1, no significant shift of HSQC peaks was observed, but the peak intensity appeared to slightly reduce. At 1:0.5, there was still no shift of peaks, but the peak intensity reduced significantly . At 1:1,On the other hand, for the FUS U-CTD, addition of TssDNA at 0.1 and 0.5 led to no significant shift of HSQC peaks but only the reduction of the peak intensity, while at 1:1, all HSQC peaks also disappeared, which is similar to what was observed on the F-CTD. Further addition to 1:5, at which LLPS was completely dissolved, resulted in the reappearance of some HSQC peaks, and the reappearance became saturated at 1:10 . T24 alsWe subsequently calculated the CSD between the FUS U-CTD in the free state and in the presence of TssDNA or T24 at a molar ratio of 1:10 . Most inTherefore, a speculative model was proposed for the induction and dissolution of LLPS of both the F-CTD  and U-CTRecently, we found that ATP enhanced LLPS of full-length FUS at low ATP concentrations, followed by dissolution of LLPS at high concentrations and characterized by gradual intensity reduction of all HSQC peaks . FurtherIn parallel to DIC imaging , we alsoWe subsequently titrated full-length FUS with both TssDNA and T24 at different molar ratios, including 1:0.1, 1:0.5, 1:1, 1:2, 1:5, 1:10, :1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, and 1:50. Briefly, two ssDNA molecules triggered highly similar changes of HSQC spectra of FUS. At low molar ratios, addition of TssDNA led to no significant shift of HSQC peaks but changes in peak intensity . HoweverTo test whether ATP and oligonucleic acids also share the same binding sites in the context of full-length FUS, we added ATP into an FUS sample to 3 mM, and consequently, the HSQC peak intensity was significantly reduced . SubsequTherefore, by binding to almost the same residues of the FUS molecule, ATP and oligonucleic acids achieve modulation of LLPS of the full-length FUS in the same manner: enhancement at low concentrations but dissolution at high concentrations . BrieflyPreviously, LLPS of well-folded proteins such as lysozyme has been extensively characterized to occur only at very high concentrations (>mM) because they were mostly driven by nonspecific molecular interactions ,39,70–72Here, we first assessed LLPS of FUS and its six differentially dissected domains. The results indicate that the isolated PLD containing 24 Tyr residues but lacking Arg/Lys failed to efficiently phase separate, thus revealing that the π–π interaction alone is relatively weak in driving LLPS. This is further supported by the results that both ATP and oligonucleic acids also failed to induce LLPS of the PLD. On the other hand, despite containing 5 Tyr and 5 Phe as well as 35 Arg and 4 Lys residues, the FUS CTD was also unable to phase separate regardless of being folded or unfolded for ZnF, thus implying that without other interactions, most proteins rich in RGG motifs are not able to phase separate by themselves. Indeed, the intrinsically disordered PGL-3 protein responsible for forming the P-granule, which has an Arg-rich C-terminus, only phase separates in the presence of mRNA . StrikinSubsequently, we successfully defined the mechanism for ATP and nucleic acids to modulate LLPS of FUS. We found that ATP and oligonucleic acids not only modulate LLPS of full-length FUS but also its dissected domains in the same manner. Interestingly, although ATP and oligonucleic acids manifested different NMR observations upon interacting with FUS, both of them were revealed to modulate LLPS by primarily binding to Arg/Lys residues, as evidenced by the residue-specific NMR results. However, oligonucleic acids have a much higher capacity than ATP in binding with FUS, which most likely results from their ability to establish dynamic but multiple-site binding to the Arg/Lys residues within the disordered regions through both π-cation and electrostatic interactions ,51,52,83Very importantly, we found that regardless of being folded or unfolded for ZnF, FUS CTD could be induced to phase separate at low concentrations followed by dissolution at high concentrations by both ATP and oligonucleic acids through specific binding to the Arg/Lys residues, which even include those Arg/Lys residues located within the unfolded ZnF domain without “RG/RGG” sequence motifs. Furthermore, in the context of full-length FUS, oligonucleic acids bind all domains except for the PLD and are also able to displace ATP from binding to FUS. This strong ability for nucleic acids to displace ATP may bear significant implications in understanding the interplay between ATP and nucleic acids in modulating the physiological and pathological roles of FUS in cells. In the cellular environments where nucleic acids are available at high concentrations, ATP is expected to be largely displaced from binding with the disordered regions, and consequently, FUS is predominantly bound with nucleic acids with LLPS dissolved. As a result, its PLD will be available for functioning as a transcriptional activation domain by binding to various partners, which include the CTD of RNA polymerase II and hormone and retinoid receptors –20,84–86Our results together reveal that unlike binding to the well-folded domains, which requires the specific pockets constituted by different residues ,88, out Recently, the ALS-causing C9orf72 dipeptide repeats extremely rich in Arg have been shown to impair the assembly, dynamics, and function of SGs –91. Our In consideration of the general involvement of nucleic acids in forming various membraneless organelles and very high concentrations of ATP in all living cells, in the future, it is of fundamental and physiological significance to understand the interplay of ATP and nucleic acids in binding and/or modulation of LLPS of a myriad of proteins with intrinsically disordered regions rich in Arg/Lys residues. In fact, previously, it has been already revealed that ATP determined the viscosity of the droplets formed by RNA and proteins in the oocyte nucleolus ,95. Our In conclusion, our study indicates that LLPS of FUS appears to be primarily driven by the π-cation interaction between aromatic and Arg/Lys residues, which also serve as the target for modulation by ATP and nucleic acids through specific binding. Specific and multiple-site interactions among intrinsically disordered regions in driving LLPS provide a mechanism for the observation that unlike the well-folded proteins, proteins such as FUS with intrinsically disordered regions can phase separate at very low concentrations. Furthermore, one possible reason for the fact that only a small number of organelle types are observed in eukaryotic cells might result from the specificity of the interactions driving LLPS as well as the specific and direct modulation of LLPS in cells by ATP and nucleic acids. Noticeably, many pathological factors appear to manifest their cytotoxicity by hacking into this mechanism, and consequently, to safeguard this mechanism might represent a promising avenue to develop therapeutic strategies/molecules.Previously, we have cloned DNA fragments encoding FUS and its differentially dissected fragments into a modified vector pET28a with a C-terminal His-tag . Althoug15NH4)2SO4 for 15N-labeling and (15NH4)2SO4/13C-glucose for 15N-/13C-double labeling [To generate isotope-labeled proteins for NMR studies, the same procedures were used, except that the bacteria were grown in M9 medium with addition of  [2, and the final solution pH was adjusted to 6.0 with aliquots of very diluted NaOH or HCl.ATP, AMP, and adenosine were purchased from Sigma-Aldrich  with the same catalog numbers as previously reported ,27. MgCl complex ,27. The ngapore) . The proCD characterization of TssDNA and T24 was performed on a Jasco J-1500 spectropolarimeter  equipped with a thermal controller as previously described . The speAlthough in cells, it was estimated that FUS has a concentration of approximately 12 μM , and we We have characterized the effect of three oligonucleic acids (one RNA and two ssDNA molecules) on LLPS of FUS and its domains by measuring the turbidity at 600 nm in parallel with monitoring formation of droplets by DIC microscopy. We found that turbidity value is much more dependent on the size than the number of the droplets: while the solution containing a large number of droplets with diameters <1 μm had negligible absorption values at 600 nm, the solution containing only a few droplets but with diameters >5 μm could have very high absorption values. This is likely due to the fact that the ability for droplets to reflect and scatter light is highly dependent on their diameters. Therefore, we further attempted to obtain a quantitative measurement of the droplet formation and dissolution by picking up all droplets and measured their diameters by the software built in the microscopy. However, as for the small droplets with diameters <1 μm, there are more than one layer of droplets, and consequently, it is extremely challenging to precisely pick up all droplets in the view field. To the best of our knowledge, currently there is no other better method to quantify LLPS without need of labeling with external reagents. As for our current study, we just need a qualitative estimation of enhancement and dissolution of LLPS at different ratios of nucleic acids. Therefore, to avoid misleading, we only presented semiquantitative descriptions of DIC imaging and also provided the key video files in Supporting Information.2 in the absence and in the presence of ATP-Mg complex at 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 mM, as previously conducted [2 was also added to the sample of full-length FUS and the CTD with the folded ZnF to reach a final concentration of 4 mM.The formation of liquid droplets was imaged at 25 °C on 50 μl of different FUS samples at a protein concentration of 20 μM in 5 mM sodium phosphate buffer at pH 6.0 containing 1 mM MgClonducted ; in the onducted ,31. ZnCl2 was also added to the sample of full-length FUS and the CTD with the folded ZnF to reach a final concentration of 4 mM.All NMR experiments were acquired at 25 °C on an 800 MHz Bruker Avance spectrometer  equipped with pulse field gradient units and a shielded cryoprobe as described previously ,41,46,9715N-/13C-double labeled sample, while cross-space 15N-edited HSQC-TOCSY and HSQC-NOESY were collected on 15N-labeled samples of the FUS CTD with ZnF either folded or unfolded at a protein concentration of 500 μM in 5 mM sodium phosphate buffer at pH 6.0 [1H chemical shifts were referenced to external DSS at 0.0 ppm. NH, N, Hα, Cα, and Cβ chemical shifts of FUS CTDs were further analyzed by SSP program [For achieving sequential assignments of the FUS CTD with ZnF folded or unfolded, NMR three-dimensional experiments including cross-bond triple-resonance experiments HN(CO)CACB, CBCA(CO)NH, CCC(CO)NH, and HNN were acquired on a t pH 6.0 . NMR 1H  program  to gain  program  and anal program .1H-15N NMR HSQC spectra were collected on 15N-labeled samples at a protein concentration of 20 μM in 5 mM sodium phosphate buffer (pH 6.0) containing 1 mM MgCl2 at 25 °C in the absence and in the presence of ATP, AMP, and adenosine, as well as two ssDNA molecules at concentrations or molar ratios exactly as those used for the above DIC imaging.For NMR titration studies of the interactions between FUS/domains and ATP/ssDNA, one-dimensional proton and two-dimensional To probe the interactions of FUS and its domains with ATP and two ssDNA molecules by NMR line broadening, the peak intensity of HSQC spectra was respectively normalized by dividing the intensity with that of a reference HSQC spectrum of FUS/domains in the free state at the same protein concentration (20 μM) and buffer conditions (5 mM sodium phosphate at pH 6.0) as previously reported . To calc15N backbone T1 and T1ρ relaxation times and {1H}-15N steady-state NOE intensities were collected on a 15N-labeled FUS (1–267) at 25 °C at protein concentration of 50 μM in 5 mM sodium phosphate buffer (pH 6.0) on an Avance 800 MHz Bruker spectrometer with both an actively shielded cryoprobe and pulse field gradient units [1H}-15N steady-state NOEs were obtained by recording spectra with and without 1H presaturation, a duration of 3 s, and a relaxation delay of 6 s at 800 MHz.nt units ,44,46,4715N backbone relaxation data of FUS (1–267) were analyzed by direct mapping of the reduced spectral density with simplified approximations [0hγN γH/8π2)(r -3) and c = ωNΔδ/31/2.The imations ,47. Brie15N transverse relaxation dispersion experiments were acquired at 25 °C on the 15N-labeled FUS (1–267) on a Bruker Avance 800 spectrometer [TCP = 50 ms) was used with a series of CPMG frequencies, ranging from 40 Hz, 80 Hz, 120 Hz (×2), 160 Hz, 200 Hz, 240 Hz, 320 Hz, 400 Hz, 480 Hz, 560 Hz, 640 Hz, 720 Hz, 800 Hz, and 960 Hz (×2 indicates repetition). A reference spectrum without the CPMG block was acquired to calculate the effective transverse relaxation rate by the following equation:CPMG) is the peak intensity on the difference CPMG frequency and I0 is the peak intensity in the reference spectra.trometer ,45,46. A2O at protein concentration of 50 μM in 1 mM sodium phosphate buffer (pD 6.0). The experiments were performed using the Bruker pulse sequence and the Bruker macro diffusion ordered spectroscopy (DOSY) [Ds) were calculated using the Bruker DOSY analysis program. Each sample was run in triplicate, and Ds values were averaged over the three experiments. The resulting decay curves were fitted, and Ds values were calculated with the equation below:3 Hz/G, δ is 4.000 ms, and Δ is 150 ms.PGF-NMR experiments were run on an 800 MHz Bruker Avance spectrometer at 25 °C. The different protein samples were prepared in Dy (DOSY) ,50. TypiS1 DataCTD, C-terminal domain; FUS, Fused in sarcoma; ZnF, zinc finger.(XLSX)Click here for additional data file.S1 Fig15N-labeled NTD (1–267) (blue) and PLD (1–165) (red) at 20 μM in 5 mM sodium phosphate buffer at pH 4.0 and 6.8. CTD, C-terminal domain; DIC, differential interference contrast; FUS, Fused in sarcoma; HSQC, Heteronuclear single quantum coherence spectroscopy; LLPS, liquid–liquid phase separation; NTD, N-terminal domain; PLD, prion-like domain; RGG1, RG/RGG-rich region 1.(A) Amino-acid compositions of the FUS PLD (1–165), RGG1 (166–271), and CTD (371–526). DIC microscopy images of liquid droplets formed by FUS (B) and NTD (C). (D) HSQC spectra of the (TIF)Click here for additional data file.S2 Fig15N NMR backbone relaxation data of the FUS NTD as measured at 800 MHz. (A) {1H}-15N steady-state NOE intensities. (B) R1. (C) R2. FUS, Fused in sarcoma; NOE, Nuclear Overhauser Effect Spectroscopy; NTD, N-terminal domain.(TIF)Click here for additional data file.S3 Fig15N-labeled FUS CTD with the assignment of the folded ZnF residues labeled. Residue-specific values of the FUS CTD with the unfolded (blue) or folded (red) ZnF for (ΔCα–ΔCβ) (C), (ΔHα) (D), and SSP (E). CTD, C-terminal domain; FUS, Fused in sarcoma; F-CTD, ZnF folded CTD; HSQC, Heteronuclear single quantum coherence spectroscopy; SSP, Secondary Structure Propensity; U-CTD, ZnF unfolded CTD; ZnF, zinc finger.(A) Amino-acid sequence of the FUS CTD with the ZnF residues underlined. Arg residues are colored in blue and Lys in green. (B) HSQC spectra of the (TIF)Click here for additional data file.S4 FigCD spectra over 200–340 nm collected in different buffers at pH 6.0 for T24 (A); and TssDNA (B). CD, circular dichroism; ssDNA, single-stranded DNA; TssDNA, telomeric ssDNA.(TIF)Click here for additional data file.S5 Fig15N-labeled FUS NTD in the absence (blue) and in the presence of AMP (A) and adenosine (B) at 10 mM, respectively. HSQC spectra of the 15N-labeled FUS PLD in the absence (blue) and in the presence of ATP at 10 mM (C) and TssDNA at a ratio of 1:50 (D). AMP, Adenosine monophosphate; FUS, Fused in sarcoma; HSQC, Heteronuclear single quantum coherence spectroscopy; NTD, N-terminal domain; PLD, prion-like domain; ssDNA, single-stranded DNA; TssDNA, telomeric ssDNA.HSQC spectra of the (TIF)Click here for additional data file.S6 FigDIC images of liquid droplets formed by the FUS NTD in the absence (A) and in the presence of RNA (B); TssDNA (C) and T24 (D) at different molar ratios. DIC, differential interference contrast; FUS, Fused in sarcoma; LLPS, liquid–liquid phase separation; NTD, N-terminal domain; ssDNA, single-stranded DNA; TssDNA, telomeric ssDNA.(TIF)Click here for additional data file.S7 Fig15N-labeled FUS F-CTD in the absence and in the presence of AMP (A), adenosine (B), and TssDNA (C) at different molar ratios. (D) Normalized HSQC peak intensity of the 15N-labeled FUS F-CTD in the presence of TssDNA at molar ratios of 1: 0.1 (blue) and 1:0.5 (red) as divided by that in the free state. (E) A speculative model to rationalize the specific binding of ATP and ssDNA to Arg/Lys residues within the FUS F-CTD to induce LLPS at low concentrations but to dissolve at high concentrations. AMP, Adenosine monophosphate; CTD, C-terminal domain; FUS, Fused in sarcoma; F-CTD, ZnF folded CTD; HSQC, Heteronuclear single quantum coherence spectroscopy; LLPS, liquid–liquid phase separation; ssDNA, single-stranded DNA; TssDNA, telomeric ssDNA; ZnF, zinc finger.HSQC spectra of the (TIF)Click here for additional data file.S8 Fig(A) DIC microscopy images of liquid droplets formed by the FUS F-CTD in the presence of RNA and two ssDNA molecules at different molar ratios. (B) DIC microscopy images of liquid droplets formed by FUS U-CTD in the presence of RNA and two ssDNA molecules at different molar ratios. The videos for outputting these images are provided in Supporting Information. CTD, C-terminal domain; DIC, differential interference contrast; FUS, Fused in sarcoma; F-CTD, ZnF folded CTD; LLPS, liquid–liquid phase separation; ssDNA, single-stranded DNA; U-CTD, ZnF unfolded CTD; ZnF, zinc finger.(TIF)Click here for additional data file.S9 Fig15N-labeled FUS in the absence and in the presence of AMP (A) and adenosine (B) at different concentrations. AMP, Adenosine monophosphate; FUS, Fused in sarcoma; HSQC, Heteronuclear single quantum coherence spectroscopy.HSQC spectra of the (TIF)Click here for additional data file.S10 FigSuperimposition of HSQC spectrum of the full-length FUS (blue) with that of RRM (red) (A), that of the F-CTD (red) (B), and that of the NTD (red) (C). CTD, C-terminal domain; FUS, Fused in sarcoma; F-CTD, ZnF folded CTD; HSQC, Heteronuclear single quantum coherence spectroscopy; NTD, N-terminal domain; RRM, RNA-recognition motif; ZnF, zinc finger.(TIF)Click here for additional data file.S11 Fig15N-labeled FUS in the presence of TssDNA at 1:10 (red) and the FUS NTD (1–267) in the presence of TssDNA at 1:5 (blue). FUS, Fused in sarcoma; HSQC, Heteronuclear single quantum coherence spectroscopy; LLPS, liquid–liquid phase separation; NTD, N-terminal domain; ssDNA, single-stranded DNA; TssDNA, telomeric ssDNA.HSQC spectra of FUS in the absence (blue) and in the presence of TssDNA (red) at 1:0.5 (A); and at 1:10 (B). (C) HSQC spectra of the (TIF)Click here for additional data file.S12 Fig15N-labeled FUS in the presence of ATP at 3 mM and TssDNA at a ratio of 1:15 (red) and the FUS NTD (1–267) in the presence of TssDNA at 1:5 (blue). FUS, Fused in sarcoma; HSQC, Heteronuclear single quantum coherence spectroscopy; NTD, N-terminal domain; ssDNA, single-stranded DNA; TssDNA, telomeric ssDNA.HSQC spectra of FUS in the absence (blue) and in the presence of ATP at 3 mM (red). (B) HSQC spectra of the full-length FUS in the absence (blue) and in the presence of ATP at 3 mM with an extra addition of TssDNA at a ratio of 1:15 (red). (C) HSQC spectra of the (TIF)Click here for additional data file.S1 VideoDIC, differential interference contrast; FUS, Fused in sarcoma.(WMV)Click here for additional data file.S2 VideoDIC, differential interference contrast; FUS, Fused in sarcoma.(WMV)Click here for additional data file.S3 VideoDIC, differential interference contrast; FUS, Fused in sarcoma.(WMV)Click here for additional data file.S4 VideoDIC, differential interference contrast; FUS, Fused in sarcoma; ssDNA, single-stranded DNA; TssDNA, telomeric ssDNA.(WMV)Click here for additional data file.S5 VideoDIC, differential interference contrast; FUS, Fused in sarcoma; ssDNA, single-stranded DNA; TssDNA, telomeric ssDNA.(WMV)Click here for additional data file.S6 VideoDIC, differential interference contrast; FUS, Fused in sarcoma.(WMV)Click here for additional data file.S7 VideoDIC, differential interference contrast; FUS, Fused in sarcoma.(WMV)Click here for additional data file.S8 VideoCTD, C-terminal domain; DIC, differential interference contrast; FUS, Fused in sarcoma; ZnF, zinc finger.(WMV)Click here for additional data file.S9 VideoCTD, C-terminal domain; DIC, differential interference contrast; FUS, Fused in sarcoma; ssDNA, single-stranded DNA; TssDNA, telomeric ssDNA; ZnF, zinc finger.(WMV)Click here for additional data file.S10 VideoCTD, C-terminal domain; DIC, differential interference contrast; FUS, Fused in sarcoma; ZnF, zinc finger.(WMV)Click here for additional data file.S11 VideoCTD, C-terminal domain; DIC, differential interference contrast; FUS, Fused in sarcoma; ZnF, zinc finger.(WMV)Click here for additional data file.S12 VideoCTD, C-terminal domain; DIC, differential interference contrast; FUS, Fused in sarcoma; ssDNA, single-stranded DNA; TssDNA, telomeric ssDNA; ZnF, zinc finger.(WMV)Click here for additional data file.S13 VideoCTD, C-terminal domain; DIC, differential interference contrast; FUS, Fused in sarcoma; ZnF, zinc finger.(WMV)Click here for additional data file.


In the past two decades, mangrove-derived microorganisms, along with mangrove plants, proved to be rich sources of bioactive secondary metabolites as exemplified by the constant rise in the number of publications, which suggests the great potential of this important ecological niche. The present review summarizes selected examples of bioactive compounds either from mangrove endophytes or from soil-derived mangrove fungi and bacteria, covering the literature from 2014 to March 2018. Accordingly, 163 natural products are described in this review, possessing a wide range of potent bioactivities, such as cytotoxic, antibacterial, antifungal,  In our research group, this intriguing metabolite was isolated from the endophyte Phomopsis longicolla that was derived from the mangrove plant Sonneratia caesolaris, collected on Hainan Island, South China (19 showed pronounced growth inhibition of cisplatin-sensitive and -resistant cell lines (IC50 in the range from 0.7 to 5.2 μM) in our bioassays, confirming the literature data . Co. CoL. thAspergillus sp. 16-5B, obtained from leaves of Sonneratia apetala , was investigated, and this resulted in the characterization of polyketides with in vitro α-glucosidase inhibitory activity (43–45) (43) showed significant inhibition of enzyme activity, with an IC50 value of 9 μM, which is approximately 60-fold more potent than the clinically used α-glucosidase inhibitor acarbose (IC50 = 554 μM). Moreover, a new isocoumarin derivative (44), which was isolated as a racemate, along with the known metabolite pestaphthalide A (45), displayed considerable inhibitory activities against α-glucosidase with IC50 values of 90 and 70 μM, respectively. Subsequent kinetic analysis of the most active compound (43) revealed that it exhibits noncompetitive inhibition characteristics. A later study from the same research group led to the isolation of fifteen isocoumarins (46–60), including six new natural products, which were derived from Talaromyces amestolkiae YX1, an endophyte of Kandelia obovata  .The endophytic fungus  (43–45)  36]. In. InAsper, China)  37]. Re. ReAsperDiaporthe sp. SYSU-HQ3, isolated from the mangrove plant Excoecaria agallocha, afforded three isoprenylisoindole alkaloids with a rare 1,4-benzodioxan moiety, namely diaporisoindoles A–C, in addition to the known derivative tenellone C (62) hexane, in addition to nafuredins A and B (114) , with IC50 values in the range from 1.2 to 9.8 μM, whereas penitalarins A–C and nafuredin A proved to be inactive.Addition of the DNA methyltransferase inhibitor 5-azacytidine to the fungus  A (113)  52]. In. InPenic B (114) , which wAspergillus ustus 094102  afforded a series of cytotoxic drimane sesqui- and meroterpenoids . Co. CoStrepStreptomyces sp. strain CHQ-64, obtained from rhizosphere soil collected from the mangrove conservation area of Guangdong province, China, was reported to be a source of intriguing natural products . Th. Th146),ynthesis . Moreoveynthesis . Interesma cells . TherefoFurther bioactive metabolites derived from mangrove soil/sediment microorganisms that could serve as potential lead structures are summarized in 100–102) [19) [143) [40 and 41) [Mangrove-associated microorganisms have gained considerable attention as a rich source of structurally diverse secondary metabolites with pronounced biological activities, which could be utilized in the discovery of new drug leads . In this100–102) , phomoxa02) [19) , and pie9) [143) , as well and 41) . Neverth and 41) . This is and 41) ,82. Taki and 41) .25) and lasiodiplactone A (42) from the endophytic fungi P. brocae MA-231 [L. theobromae ZJ-HQ1 [The biosynthetic potential of mangrove-associated microorganisms has been associated with activation of silent genes by unique environmental stimuli imparted on this special ecological niche . Thus, pe MA-231  and L. te ZJ-HQ1 , respect68) and B (69) from co-culture of the mangrove endophyte P. citrinum MA-197 with the bryozoan-derived B. felina EN-135 [75) and lasiodiplodin derivatives (76–79) with α-glucosidase inhibitory activity from co-culture of the endophytic fungus Trichoderma sp. 307 with A. johnsonii B2 [Co-cultivation of microorganisms has likewise been exploited to enhance the accumulation of constitutively present natural products and/or to trigger the production of cryptic metabolites , which ca EN-135 , as wellsonii B2 .P. variabile HXQ-H-1 afforded the cryptic metabolite varitatin A (113) with potent cytotoxic and protein kinase inhibitory activities [Aspergillus nidulans was triggered by co-cultivation with the bacterium Streptomyces rapamycinicus due to targeted histone modification, thus shedding light on the connection between epigenetic modification and microbial crosstalk [In the last few years, epigenetic manipulation has attracted great interest as a strategy for activation of silent biosynthetic pathways. Several regulatory proteins, such as chromatin-modulating agents and transcription factors, are known to control the secondary metabolome of microbes . The epitivities . Notablyrosstalk . Streptomyces and Penicillium. Even though these microorganisms still hold enormous biosynthetic potential, as demonstrated by recent genome sequence studies, a vast amount of novel fungal and/or bacterial taxonomic groups still lies unexplored [144) from Streptomyces sp. MSB090213SC12 [Saccharomyces cerevisiae, E. coli, or Streptomyces lividans for large-scale production of the desired lead compounds or derivatives thereof [The majority of compounds described in this review have been derived from common genera, such as explored . To a laexplored . To overexplored ,88, and 0213SC12 ,89,90. T thereof ,91,92.Overall, mangrove-associated microorganisms have gained considerable attention due to their unique ecological characteristics, diversity, and wealth of novel bioactive secondary metabolites. Nevertheless, pharmaceutical development of these metabolites is still in its infancy, with only the proteasome inhibitor, salinosporamide A, being hitherto in phase I clinical trials for the treatment of multiple myeloma ,93. In o