However, the immediate and short-term effects of cSiO2 in this widely used model of autoimmune disease are not well-understood. In the present study, we tested the hypothesis that a single acute cSiO2 dose triggers early presentation of cellular, histopathological, transcriptomic, and protein biomarkers of inflammation and autoimmunity in lupus-prone mice. Eight-week old female NZBWF1 mice were intranasally instilled once with 2.5 mg cSiO2 or saline vehicle and necropsied at 1, 7, 14, 21, and 28 d post-instillation (PI). Analyses of bronchoalveolar lavage fluid (BALF) and lung tissue revealed that by 7 d PI, acute cSiO2 exposure persistently provoked: (i) robust recruitment of macrophages, neutrophils, and lymphocytes into the alveoli, (ii) cell death as reflected by increased protein, double-stranded DNA, and lactate dehydrogenase activity, (iii) elevated secretion of the cytokines IL-1α, IL-1β, IL-18, TNF-α, IL-6, MCP-1, and B cell activation factor (BAFF), and (iv) upregulation of genes associated with chemokines, proinflammatory cytokines, lymphocyte activation, and type I interferon signaling. The appearance of these endpoints was subsequently followed by the emergence in the lung of organized CD3+ T cells (14 d PI) and CD45R+ B cells (21 d PI) that were indicative of ectopic lymphoid structure (ELS) development. Taken together, acute cSiO2 exposure triggered a rapid onset of autoimmune disease pathogenesis that was heralded in the lung by unresolved inflammation and cell death, proinflammatory cytokine production, chemokine-driven recruitment of leukocytes, an interferon response signature, B and T cell activation, and ELS neogenesis. This short-term murine model provides valuable new insight into potential early mechanisms of cSiO2-induced lupus flaring and, furthermore, offers a rapid venue for evaluating interventions against respirable particle-triggered inflammation and autoimmunity.Occupational exposure to crystalline silica (cSiO Systemic lupus erythematosus (lupus) is a devastating autoimmune disease affecting diverse tissues and characterized by intermittent flaring and remission that, over time, can inflict irreversible organ damage , 2. Lupu2)  –11. Whenilicosis , alveoliilicosis , pulmonailicosis , chronicilicosis , and lunilicosis .2 exposure mimics lupus flaring by triggering onset and progression of autoimmunity (2 elicits development of ectopic lymphoid structures (ELS) in the lung, systemic autoimmunity, and early onset of lupus nephritis over the following 3 months post-instillation (PI)  activity, complement activation, and adhesion molecules were evident in subsequent weeks. Expression of these genes intensified as disease progressed. Pulmonary transcriptomic signatures were consistent with observed elevations of neutrophils, macrophages, dendritic cells, B cells, and T cells, and collectively, these were consonant with progression of autoimmunity. Taken together, airway exposure to cSiO2 in lupus-prone mice evoked aberrant pulmonary transcriptomic signatures for both innate and adaptive immune responses, suggesting that the lung functioned as the nexus for initiating systemic autoimmunity and, ultimately, glomerulonephritis.In lupus-prone mice, short-term repeated cSiOimmunity , 18. Usiion (PI) , 20. In ion (PI) . Transcr2-triggered human autoimmune flaring can be recapitulated chronically in lupus-prone mice, the short-term responses to this particle in these models are still not well-understood. In this study, we hypothesized that a single bolus cSiO2 exposure triggers early presentation of cellular, histopathological, transcriptomic, and protein markers of inflammation and autoimmunity in lupus-prone mice. The specific objective of this study was to determine the immediate and short-term effects  of a single acute intranasal instillation of 2.5 mg cSiO2 on triggering inflammatory markers and autoimmunity in the lungs of female NZBWF1 mice. The results suggested that within 1 week, cSiO2 triggered persistent cell death, recruitment of leukocytes, increased proinflammatory cytokine and chemokine expression, and a prominent IFN gene signature in the lung. Remarkably, appearance of these biomarkers was followed within 3 week by the onset of ectopic lymphoid neogenesis.While cSiO2 that was not seen in normal C57BL/6 mice  in accordance with the guidelines of the National Institute of Health. Six-week-old female NZBWF1 mice were purchased from Jackson Laboratories  and housed 4 animals per cage. This mouse strain was chosen because it has been previously used to recapitulate robust autoimmune triggering by cSiOL/6 mice –24. MiceL/6 mice , 20. Exp2 as previously described  were acid-washed, oven-dried, and suspended in sterile phosphate buffered saline . Fresh stock suspensions were prepared prior to use, sonicated, and vortexed for 1 min before intranasal instillation. Mice were anesthetized by inhalation with 4% isoflurane in O2, held in the supine position, then instilled once with 1 or 2.5 mg cSiO2 in 25 μl PBS or 25 μl of PBS vehicle (Veh). Mice were held in the same position for a short period after instillation to ensure proper distribution inside the respiratory tract. The processing time of the intranasal instillation was <10 s per mouse, and no injury or death occurred during the procedure. In a preliminary study, mice were intranasally instilled with 1 mg cSiO2 (n = 12) or Veh (n = 12). Cohorts of cSiO2- and Veh-treated mice  were terminated at days 1, 3, or 7 d post-instillation (PI). For the main study, mice were instilled with either 2.5 mg cSiO2 (n = 40) or Veh (n = 40). Cohorts from cSiO2- and Veh-treated mice  in the complete study were terminated at days 1, 7, 14, 21, or 28 d PI. The 2.5 mg cSiO2 per mouse dose has been widely used to study silicosis  of 50 μg/m3/d established by the U.S. Occupational Safety and Health Administration  was instilled through the cannulated trachea to recover BALF. For each mouse, this procedure was repeated twice and BALF fractions combined for subsequent cell and protein analyses. The caudal lung lobe was then removed and stored in RNAlater  at −80°C for RNA analysis. Finally, the left lung lobe was intratracheally fixed with 10% (v/v) neutral-buffered formalin  at a constant pressure (30 cm H2O) for 2 h then immersed and stored in a large volume of formalin fixative for 24 h. After fixation, the left lung lobe was processed in 30% ethanol for histological preparation and further microscopic analysis.Mice were anesthetized by intraperitoneal injection with 56 mg/kg body weight sodium pentobarbital then euthanized by exsanguination via the abdominal aorta. Bronchoalveolar lavage fluid (BALF) was collected from the lungs as described previously . BrieflyTotal cell counts in BALF were determined by standard counting on a hemocytometer. Cytospin slides were prepared by centrifuging 150 μL of BALF from each mouse at 400 × g for 10 min. Slides were allowed to dry overnight at 25°C and were then stained with Diff-Quick (ThermoFisher). Differential cell counts for macrophages/monocytes, neutrophils, and lymphocytes in BALF were performed based on morphological criteria from a total of 200 cells present on the slides.Cell damage and death were assessed by measuring release of protein, dsDNA, and lactate dehydrogenase (LDH) in BALF. Total protein concentration, an indicator of epithelial and cell membrane integrity, was quantified using a Pierce™ BCA Protein Assay Kit . Double-stranded DNA, a measure of nuclear damage, was assessed using Quant-iT™ PicoGreen™ dsDNA reagent  according to the manufacturer's protocol. LDH activity was used to evaluate cytotoxicity and performed as described . BrieflyELISA DuoSets  were used to measure IL-1β (cat. #DY401), IL-1α (cat. #DY400), IL-6 (cat. #DY406), TNF-α (cat. #MTA00B), IL-18 (cat. #DY122-05), MCP-1 (cat. #MJE00B), and BAFF (cat. #MBLYS0) in the BALF according to the manufacturer's instructions.Formalin-fixed left lung lobes were routinely processed and embedded in paraffin. Serial sections of tissue (5 μm) were deparaffinized and stained with hematoxylin and eosin (H&E). H&E stained sections were used for brightfield microscopic imaging and semi-quantitative scoring for the following lung lesions: (a) presence of centriacinar inflammation, (b) presence of centriacinar epithelial hyperplasia, (c) presence of alveolar proteinosis, and (d) presence of perivascular lymphoid cells. Individual lungs were semi-quantitatively graded for these lesions as percent of total pulmonary tissue examined based on the following criteria: (0) no changes compared to control mice; (1) minimal (<10%); (2) slight (10–25%); (3) moderate (26–50%); (4) severe (51–75%); or (5) very severe (>75%) of total area affected. Microscopic and semiquantitative scoring of lung lesions were performed by a certified veterinary pathologist without knowledge of individual animal exposure history.+ and CD3+ lung tissue were calculated by projecting a point grid over randomly sampled images with the STEPanizer v1.8 stereology tool and totaling the number of points falling onto positive staining or reference tissue.Lung sections were stained with mouse-specific monoclonal antibody to CD45R  or mouse-specific polyclonal antibody to CD3  as described previously to identify B and T lymphocytes, respectively, which are indicative of ELS. Tissue morphometry was carried out as previously reported . Slides RNA was extracted from lungs of 7, 14, 21, and 28 d PI mouse groups using TissueLyser II (Qiagen) and Tri Reagent . Extracted RNA was purified using a Zymo RNA Clean and Concentrator Kit with DNase . RNA was dissolved in nuclease-free water and quantified using the Qubit (Thermo Fisher Scientific). RNA integrity was also assessed using the Bioanalyzer (Agilent Technologies) at the MSU Genomics Core. Equal amounts of RNA from individual samples within each group were pooled to yield 8 samples for autoimmune profiling across all time points . Samples (RNA integrity >8) were analyzed using Nano String Autoimmune Gene Expression assay (XT-CSO-MAIP1-12) at the MSU Genomics Core.2 transformed normalized counts were used for Heatmap and PCA plot generation using ClustVis . Prciations . InteracActb, Gapdh, and Hprt) at the MSU Genomics Core. Gene expression levels were normalized to the endogenous housekeeping genes and reported as fold change relative to the experimental control group using the 2−ΔΔCT method . TaqMan assays (Thermo Fisher Scientific) were then performed on a Smart Chip Real-Time PCR System with technical triplicates for selected genes of interest and housekeeping genes (T method .www.graphpad.com). Data were inspected using the Grubb's outlier test (with Q = 1%) to identify potential outliers. Data that did not meet the normality assumption as determined by the Shapiro-Wilk test (p < 0.01) were analyzed using the Kruskal-Wallis non-parametric test with Dunn's post-hoc test. Data meeting both normality and variance assumptions were analyzed using a two-way analysis of variance (ANOVA) with Sidak's post-hoc test (comparisons of treatments at each time point) and Tukey's post-hoc test (comparisons across timepoints within treatment groups) as appropriate. Data are presented as mean ± standard error of the mean (SEM). A p ≤ 0.05 was considered statistically significant.All statistical analyses except nCounter data were performed using GraphPad Prism version 8 for Windows . A total of 7 clusters were identified. Based on the number of contained genes, the top five clusters were associated with proinflammatory cytokines, chemokines, lymphocyte activation, interferon signaling, and complement  and B cells (21 d PI) in the lung, indicative of ELS development. Collectively, these observations suggest that acute exposure to cSiO2 contributed to fast-developing unresolved inflammation, cell death, and innate immune responses in the lung that were, in turn, followed by adaptive immune responses heralding autoimmunity onset.This investigation is the first to explore how a single bolus exposure to cSiO2 caused slight induction of total cells in BALF that quickly returned to basal level, the 2.5 mg dose elicited elevated leukocyte counts throughout the 28-d experimental period. These findings for the lower dose of cSiO2 corresponded with a lack of histopathologic effects in the lungs of mice treated, whereas the higher dose stimulated rapid and prolonged inflammatory cell recruitment reflected by persistent infiltration of neutrophils, macrophages, and later, lymphocytes. Consistent with our observations, when pulmonary inflammation in C3H/He mice was compared 4 week after intratracheal instillation with 0, 0.375, 0.75, 1.5, 3, 5, and 6 mg cSiO2, protein concentration and cell numbers in the BALF increased in linear dose-response fashion and reached a maximum effect at the 3 mg dose  participate in phagocytosis, (ii) undergo degranulation leading to discharge of enzymes, proinflammatory mediators, and reactive oxygen species, (iii) secrete extracellular traps; and finally die by apoptosis or necrosis—all of which contribute to robust inflammation and tissue injury  was observed only at 28 d PI, is consistent with the possibility that most IL-1α is constitutively produced. Rider et al. , CXCL5 (neutrophil chemoattraction and activation), and CXCL9,10 (activated T cell chemoattraction). Accordingly, prolonged and increased expression of these chemokine genes likely leads to increased migration of neutrophils, macrophages, and lymphocytes that were observed here in the BALF and lungs. Consistent with this premise, we observed early appearance of the MCP-1  in the BALF of cSiO2-treated mice which continued to rise throughout the remainder of the experiment. MCP-1 is released predominately by monocytes/macrophages and mediates recruitment of monocytes from systemic circulation to local sites of inflammation after tissue injury . BAFF is produced as a membrane-bound protein that can be cleaved to a soluble form by furin and other proteases of the pro-protein convertase family .PC: study design, data analyses/interpretation, figure preparation, manuscript preparation, investigation, and manuscript editing. JW: study design, necropsy, and lab analyses. AB: study design, data analyses, figure preparation, and manuscript writing. RL: necropsy and lab analyses. OF and KW: necropsy, lab analyses, and manuscript writing. KG and ER: animal handling and lab analyses. JH: study design, lung/kidney histopathology, morphometry, data analyses, manuscript preparation, and project funding. JP: planning, coordination, oversight, manuscript preparation/submission, and project funding. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fusarium fungal pathogens. Fumonisins are structural analogs of sphingosine and potent inhibitors of ceramide synthases (CerSs); they disrupt sphingolipid metabolism and cause disease in plants and animals. Over the past three decades, researchers have used fumonisin B1 (FB1), the most common fumonisin, as a probe to investigate sphingolipid metabolism in yeast and animals. Although the physiological effects of FB1 in plants have yet to be investigated in detail, forward and reverse genetic approaches have revealed many genes involved in these processes. In this review, we discuss the intricate network of signaling pathways affected by FB1, including changes in sphingolipid metabolism and the effects of these changes, with a focus on our current understanding of the multiple effects of FB1 on plant cell death and plant growth. We analyze the major findings that highlight the connections between sphingolipid metabolism and FB1-induced signaling, and we point out where additional research is needed to fill the gaps in our understanding of FB1-induced signaling pathways in plants.Fumonisin toxins are produced by  Fusarium molds such as Fusarium verticillioides and Fusarium moniliforme, which infect many cereal crops such as maize (Zea mays), wheat (Triticum aestivum), and barley (Hordeum vulgare)  . Fumonisvulgare) . In the seedling . Contamiseedling .F. verticillioides (F. moniliforme) MRC 826 cultures as novel mycotoxins with cancer-promoting activity in rat livers  and FB2 were isolated from t livers . FB1 andt livers . Howevert livers . Three yynthesis . Accumulde novo biosynthesis of LCBs resulting from the condensation of serine and palmitoyl-CoA moieties  are encoded by a multigenic family named after the yeast protein Lag One Homolog (LOH) and are responsible for the formation of the amide bond that links long-chain (<C20) fatty acids (LCFs) or very-long-chain (≥C20) fatty acids (VLCFAs) to LCB, leading to Cer formation . Cers caSphingolipid compounds have a multitude of functions. In addition to functioning as structural components of the plasma membrane and other endomembrane systems, sphingolipids serve as bioactive molecules and second messengers for an array of cellular signaling activities in plant cells, such development, biotic and abiotic stress responses, and programmed cell death (PCD). However, compared with animals, much less is known about sphingolipid metabolism and signaling in plants. For instance, the receptors, targets, and mediators involved in these processes are almost totally unclear , limitinΔ4, sphingosine; d18:0, sphinganine; DPL1, dihydrosphingosine phosphate lyase; FAH1/2, fatty acid hydroxylase; GCS, glucosylceramide synthase; GINT1, glucosamine inositolphosphorylceramide transferase 1; GMT1, GIPC mannosyl-transferase 1; GONST1, Golgi localized nucleotide sugar transporter 1; IPC, inositol phosphorylceramide; IPUT1, inositol phosphorylceramide glucuronosyltransferase 1; LCB, long-chain-base; LCB1/2, subunit of serine palmitoyltransferase 1/2; LCBK1/2, long-chain base kinase 1/2; LCB-P, long-chain-base phosphate; LOH1/2/3, LAG1 Homolog 1/2/3, ceramide synthase; NCER, neutral ceramidase; ORM1/2, orosomucoid-like protein 1/2; SBH1/2, sphingoid base hydroxylase 1/2; DPL1, dihydrosphingosine phosphate lyase; SL, sphingolipid; SPHK1/2, sphingosine kinase 1/2; SPP1, sphingoid phosphate phosphatase; SPT, serine palmitoyl transferase; ssSPT, small subunit of serine palmitoyl transferase; t18:0, phytosphingosine; TOD1, turgor regulation defect 1.Schematic representation of the sphingolipid biosynthetic pathway in plants. 3-KSR, 3-ketosphinganine reductase; ACD11, accelerated cell death 11, Cer-1P transporter; ACD5, accelerated cell death 5, ceramide kinase; ACER, alkaline ceramidase; CoA, coenzyme A; d18:1Solanum lycopersicum) Alternaria stem canker resistance-1 (Asc-1) was the first CerS identified in plants  from yeast and Asc-1 as queries identified three Asc-1/LAG1 Homologs, LOH1–3  (Z. mays) likely contains at least two ceramide synthase isoforms with different substrate specificities, which may result in susceptibility/resistance to F. verticillioides  , which wpersici) , pointinllioides .Pseudomonas syringae pv. tomato DC3000 also provoked a rapid increase in t18:0 (phytosphinganine) levels, implying that LCBs function in pathogen resistance  subunit of SPT. SPT mediates FB1-initiated PCD by catalyzing the first rate-limiting step of de novo sphingolipid biosynthesis (ssSPTa) resulted in the accumulation of LCBs and reduced tolerance to FB1, whereas ssSPTa suppression lines displayed lower levels of LCBs but enhanced tolerance to FB1  negatively regulate SPT activity to maintain sphingolipid levels in humans and yeast, and are required for sphingolipid homeostasis in plants, as well as FB1 resistance . The tolterparts . By contild type .AtLCBK1  increased plant resistance to FB1 treatment, whereas AtLCBK1 knockdown plants showed increased sensitivity to FB1 treatment , contains lower levels of trihydroxy-LCBs and higher levels of total LCBs than wild-type plants  exhibited increased sensitivity to FB1, whereas these phenotypes were rescued by the overexpression of AtBI1  mutants, possibly due to different growth conditions such as photoperiod  plants, which are insensitive to JA, exhibited much lower susceptibility to cell death induced by FB1 than FB1-treated wild-type protoplasts (OPR3), which is involved in JA biosynthesis, enhanced the resistance of protoplasts to FB1 upon a dark/light shift (opr3 mutants are often unclear (The role of the plant hormone jasmonate (JA) in plant resistance to fungi is well known . The roltoplasts . Similarht shift . However unclear . FB1 hij unclear . Further unclear .In addition to ethylene and JA, plant susceptibility to FB1 appears to be associated with salicylic acid (SA) signaling, whereas FB1-induced PCD does not require the SA signal transmitter non-expresser of PR genes 1 . Indeed,Taken together, these findings indicate that FB1-elicited cell death requires ethylene, JA, and SA. Nevertheless, the molecular mechanisms underlying the roles of these hormone-signaling pathways in FB1-induced cell death remain to be investigated.Mitogen-activated protein kinases (MAPKs) were activated in FB1-treated monkey kidney samples, but the functional role of MAPKs in FB1-induced toxicity has not been addressed in this system . In Arab2+ levels increase via FB1-induced phytosphingosine (PHS)  . Meanwhine (PHS) .2+ levels and the accumulation of ROS are critical for inducing PCD. The disruption of Arabidopsis super sensitive to aba and drought 2 (SAD2) (an importin beta-like gene) enhanced H2O2 accumulation and cell death in the sad2-5 mutant under FB1 treatment  have key roles in PCD; in fact, cell death is considered to be triggered by ROS  and ROS reatment . The FB1ignaling . SAD2-me fbr11-1 . In fact fbr11-1 , attenua fbr11-1 .Fumonisin B1 triggers the rapid depletion of extracellular ATP in Arabidopsis, which initiates cell death; this process was rescued by exogenous ATP treatment . ADP, AMAutophagy, a mechanism used to degrade unwanted constituents in eukaryotic cells, is essential for maintaining cell homeostasis and nutrient recycling. We recently demonstrated that FB1 and LCBs induce autophagy in Arabidopsis . The losautophagy-related (atg) mutants  of autophagy in regulating cell death is determined remains elusive . In plan mutants , implyin mutants . Cer andt manner . This obt manner , 2013, cacd11-associated cell death -domain putative transcription factor, conferred FB1 resistance in fbr6 plants , and transgenic plants constitutively overexpressing FBR12 exhibited phenotypes consistent with precocious cell death   . These f2+, ATP signaling, and so on. LCBs regulate the activities of cell death-associated proteins such as MPK6, CPK6, and 14-3-3 proteins. LCBs also induce autophagosome formation, which maintains cellular homeostasis, protecting the cell from unnecessary death. ER stress appears to be involved in this process. LCBs induce the expression of sphingolipid biosynthesis, PCD, and senescence-related genes through transcription factors such as EIN2, CTR1, SPL14, and ANAC032. 14-3-3, 14-3-3 family protein; ANAC032, NAC domain containing protein 32; ATG8, autophagy 8; AUX1, auxin resistant 1; BI1, bax inhibitor 1; Cers, ceramides; CPK6, calcium dependent protein kinase 6; CTK, cytokinin; CTR1, constitutive triple response 1; EIN2, ethylene insensitive 2; ET, ethylene, ER stress, endoplasmic reticulum stress; FB1, fumonisin B1; FBR12, fumonisin B1 resistant 12; GluCers, glucosylceramides; GIPCs, glycosyl-inositolphosphoryl-ceramides; hCers, hydroxyceramides; IAA, indole-3-acetic acid; IGS, indole glucosinolate; JA, jasmonate; LCBs, long-chain bases; LOH1, LAG1 Homolog 1; MPK6, MAP kinase 6; MPK6-P, MPK6 phosphorylated form; PE, phosphatidylethanolamine; PHS, phytosphingosine; PIN1, PIN-FORMED 1; RbohD/F, respiratory burst oxidase homolog; ROS, reactive oxygen species; SA, salicylic acid; SPL14, squamosa promoter binding protein-like 14; TFs, transcription factors; VLC, very-long-chain; VPE, vacuolar processing enzyme.The solid arrows indicate established links and the dashed arrows indicate putative links. Reduced VLC sphingolipid levels lead to disordered trafficking, membrane incorporation, and secretion. LCBs produced in response to FB1 to trigger cell death signaling, such as ET, JA, SA, ROS, CaLOH1 and LOH3 showed increased growth and cell division, with enhanced production of Cers with VLCFAs and trihydroxy LCBs  BY2 cells showed severe effects on cell growth and cell shape and a delay in cell division  and PIN1 in the cytosol . Defecti2+, ROS, and ATP signaling, which ultimately contribute to cell death. However, this process is sufficiently complex such that interplay with other factors in each route and even across routes determines the final outcomes. Black, targeted by FB1 (purple), orange, inhibited or reduced by FB1; blue, promoted or elevated by FB1. CerS, ceramide synthase; ET, ethylene, FB1, fumonisin B1; IGS, indole glucosinolate; JA, jasmonate; LCBs, long-chain bases; ROS, reactive oxygen species; SA, salicylic acid; SLs, sphingolipids; VLCFA, very-long-chain fatty acid.Route 1 (Orange): FB1-induced decreases in VLCF levels strongly affect membrane properties, which are responsible for normal plant growth. Route 2 (Blue): FB1-induced increases in LCB and C16 Cer levels primarily affect hormone, CaFUM1, which encodes a polyketide synthase (PKS)  .F. verticillioides and maize, investigation of the role of fumonisin production on disease development has yielded controversial results. Only strains of F. verticillioides that produce fumonisins cause foliar disease symptoms on seedlings of the sweet maize hybrid “Silver Queen”  encodes a genetic repressor of fumonisin production, is directly adjacent to the FUM cluster, and was induced in response to pyrrocidines. This suggests that other microbes may manipulate the fumonisin biosynthetic program of F. verticillioides to control F. verticillioides growth in the host  signaling are activated in response to F. verticillioides infection (F. verticillioides (Lipoxygenase (LOX) genes encode non-heme iron-containing dioxygenases that catalyze the oxygenation of polyunsaturated fatty acids , flavones, and 2-amino-3H-phenoxazin-3-one , ROS, NO, ATP, and CaIn addition to being used as a ceramide inhibitor or a fungal toxicant, FB1 represents a highly useful tool for revealing details about sphingolipid-controlled cell death and growth inhibition. These details would benefit agriculture by helping us overcome the subtle strategy of FB1-producing pathogens and others foes sharing similar mechanisms in plant–pathogen interactions.Based on the complicated signaling network induced by FB1, we propose a model to classify the global effects of this compound into basic two routes . HoweverSphingolipids remain an underexplored, large group of metabolites . The stuRecent studies using molecular genetics and biochemical approaches have enhanced our understanding of this issue in plants and provided a deeper understanding of the roles of sphingolipids in plant life and death. Despite these important findings, our understanding of the importance of sphingolipids for the regulation of plant cell death and growth, and consequently crop yields remains limited and sphingolipids represent an exciting area for further study. Many efforts have been made to functionally characterize individual genes downstream of FB1. However, little is known about the FB1 signal transduction network. Therefore, apart from screening more participants involved in the FB1 response, studies should be designed to elaborate how the known participants operate simultaneously in a coordinated signaling network under FB1 treatment. In particular, it is crucial to investigate the connection between the regulators of FB1 resistance and sphingolipid metabolism. Specifically, sphingolipid profile analysis of FB1-resistant or -sensitive mutants with and without FB1 treatment should be performed to determine whether these proteins engage in sphingolipid metabolism. Furthermore, our lack of knowledge about the molecular targets of individual sphingolipid components, as well as their biological significance to plants, is still a major impediment to understanding FB1 signaling. Thus, there is a need for interactome association analysis between proteins downstream of FB1 and LCB, as well as sphingolipid enzymes, to untangle the interplay between these signals and sphingolipid metabolites or metabolism pathways. Recent studies have demonstrated the great potential of using the photoactivatable and clickable analog of sphingosine (pacSph) and azide-tagged sphingolipids to identify sphingolipid targets . In addiBeside Arabidopsis, the FB1 signaling network should be elaborated in crop plants. Relatively little is known about the physiological effects of fumonisins from fungal pathogens in the natural environment on the development of plant diseases. The introduction and manipulation of parts of the FB1 signaling pathway in crop plants will have broad and overarching impacts on agriculture to help overcome the effects of FB1-producing pathogens. In view of the broad host range of FB1-producing pathogens, the eradication of plant diseases  caused by FB1 appears to be elusive. However, better insight into the global effects of FB1 on plants will undoubtedly contribute to the development of a rational, effective approach to reducing the hazards of FB1 contamination.H-YZ and NY designed the manuscript and wrote the manuscript. C-YL and NY contributed to the reagents and materials. All authors have discussed and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Circulating renin-angiotensin system (RAS) and local paracrin-autocrin-intracrin tissue-based RAS participate in numerous pathobiological events. Pro-inflammatory, pro-fibrotic, and pro-thrombotic consequences associated with local RAS activation have been detected at cellular and molecular level. Regenerative progenitor cell therapy in response to RAS modulating pharmacotherapy has emerged as an adjunct in the context of endothelial cell injury and regeneration to improve regeneration of the vascular endothelium. Local hematopoietic bone marrow (BM) RAS symbolizes the place of cross-interaction between vascular biology and cellular events from embryogenesis to definitive hematopoiesis underlying vascular atherosclerosis. The BM microenvironment also contains Mas receptors, which control the proliferative role of Ang 1-7 on hematopoietic stem cells. Ang 1-7 is produced from Ang-II or Ang-I with the help of ACE2. Various tissues and organs also have an effect on the RAS system. The leukocytes contain and synthesize immunoreactive angiotensinogen species capable of producing angiotensin in the basal state or after incubation with renin. The significance of RAS employment in atherosclerosis and hypertension was indicated by novel bidirectional Central Nervous System (CNS) RAS–BM RAS communications. Myeloid cells generated within the context of hematopoietic BM RAS are considered as the initiators and decision shapers in atherosclerosis. Macrophages in the atherosclerotic lesions contain angiotensin peptides by which RAS blockers inhibit monocyte activation and adherence. Furthermore, vascular biology in relation to inflammation and neoplasia is also affected by local tissue RAS. The purpose of this article is to outline interactions of circulating and local angiotensin systems, especially local bone marrow RAS, in the vascular pathobiological microenvironment of CNS. As previously known, renin-angiotensin system (RAS) was described as an endocrine system that regulates blood pressure and body electrolyte balance . RecentlRAS molecules, particularly ACE, ACE2, AGT, AGTR1, AGTR2, AKR1C4, AKR1D1, ANPEP, ATP6AP2, CMA1, CPA3, CTSA, CTSD, CTSG, CYP11A1, CYP11B1, CYP11B2, CYP17A1, CYP21A2, DPP3, EGFR, ENPEP, GPER, HSD11B1, HSD11B2, IGF2R, KLK1, LNPEP, MAS1, MME, NR3C1, NR3C2, PREP, REN, RNPEP, THOP1 are available in the BM . These mMajor RAS molecules, including renin, angiotensinogen, angiotensin receptors and ACE, are all found in the BM microenvironment . HaznedaBM stromal microenvironment includes AT1R and AT2R  and inhibitory tetrapeptide AcSDKP (N-acetyl-Ser-Asp-Lys-Pro) , 12. ThePhase I/II clinical trials of a pharmaceutical agent of peptide angiotensin 1-7 (Ang-1-7) have been directed to assess the role of local BM RAS in different diseases , 24, 25.The supposition that local autocrine BM RAS may be effective in neoplastic hematopoiesis supports the prominent functions of local RAS in primitive embryonic hematopoiesis –33. LikeLocal BM RAS has significant effects on hematopoietic systems, particularly on myeloid, and erythroid cells , 12, 19.+, CD38− cells, and lymphocytes  and BM cells. This relationship between BM stromal cells, HSCs and nerve terminals has been defined as the ‘neuro-reticular complex’ , 65. A dAll components of RAS are found in the brain , 71. TheHypertension is a very significant risk factor for cardiovascular diseases. It remains a global public health problem . VariousBM has an essential function in hematopoiesis regulation . BM HSPCAng II related HT lead to a recommendation that activation of microglia in the pre-sympathetic cardio-regulatory brain fields may precede both the activation of the sympathetic drive and the increase in blood pressure , 75. ProOne study demonstrated that a dysfunctional BM ANS is correlated with imbalanced EPCs and inflammatory cells in hypertension . They deBM has an important role in neurogenic hypertension. Memory T cells are present in the BM. T-cell activation has a significant function in hypertension . The BM There are lots of described important therapeutic usages for Angiotensin (1-7) and analogs on treating cardiovascular diseases and atherosclerosis. Some studies showed new pharmacological approaches. One of them investigated a BM-specific adrenergic beta 1 and beta 2 knock out mouse chimera (AdrB1.B2 KO) to research how sympathetic impulse to the bone influences transcripts and miRNAs in the hypothalamic paraventricular nucleus . The resIn light of all these data, it is thought that the autonomous control of the BM has an important function in hypertension and there is bidirectional communication between the brain and the BM in this procedure . It is tRC was responsible for the writing of the article. IH served as scientific adviser, drafted the article, and revised it critically for important intellectual content. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Magnaporthe oryzae strains YC81-2, including one virus in the family Tombusviridae, one virus in the family Narnaviridae and four viruses in the family Botourmiaviridae. Since the RNA-dependent RNA polymerase (RdRp) of one botourmiavirus show the highest identity (79%) with Magnaporthe oryzae ourmia-like virus 1 (MOLV1), the virus that was grouped into the genus Magoulivirus was designated as Magnaporthe oryzae botourmiavirus 2 (MOBV2). The three other novel botourmiaviruses were selected for further study. The complete nucleotide sequences of the three botourmiaviruses were determined. Sequence analysis showed that virus 1, virus 2, and virus 3 were 2598, 2385, and 2326 nts in length, respectively. The variable 3′ untranslated region (3′-UTR) and 5′-UTR of each virus could be folded into a stable stem-loop secondary structure. Each virus consisted of a unique ORF encoding a putative RdRp. The putative proteins with a conserved GDD motif of RdRp showed the highest sequence similarity to RdRps of viruses in the family Botourmiaviridae. Phylogenetic analysis demonstrated that these viruses were three distinct novel botourmiaviruses, clustered into the Botourmiaviridae family but not belonging to any known genera of this family. Thus, virus 1, virus 2, and virus 3 were designated as Magnaporthe oryzae botourmiavirus 5, 6, and 7 , respectively. Our results suggest that four distinct botourmiaviruses, MOBV2, MOBV5, MOBV6, and MOBV7, co-infect a single strain of Magnaporthe oryzae, and MOBV5, MOBV6, and MOBV7 are members of three unclassified genera in the family Botourmiaviridae.Via virome sequencing, six viruses were detected from  Mycoviruses, viruses of fungi, are ubiquitous and diverse in all major phyla of fungi . They caAlphaflexiviridae, Barnaviridae, Gammaflexiviridae, Deltaflexiviridae, Narnaviridae, and Botourmiaviridae [Botourmiaviridae, a new family established by the International Committee on the Taxonomy of Viruses (ICTV) [Ourmiavirus, including plant ourmiaviruses, and three mycovirus genera, Scleroulivirus, Botoulivirus, and Magoulivirus, that include botourmiaviruses (previously named ourmia-like mycoviruses). However, several botourmiaviruses do not belong to the above three mycovirus genera, and have been clustered into an undefined clade.The genomes of most mycoviruses are double-stranded RNA (dsRNA) or linear positive-sense single-stranded RNA (+) ssRNA . Only a aviridae ,11. AmonMagnaporthe oryzae, the pathogen of rice blast, was discovered in 1971 [M. oryzae. Of these mycoviruses, dsRNA viruses are clustered into three families: Magnaporthe oryzae virus 1, 2, and 3  are classified into the family Totiviridae [Chrysoviridae [Partitiviridae. In addition, several (+) ssRNA mycoviruses have also been found to be associated with M. oryzae. Among them, Magnaporthe oryzae virus A is closely related to plant viruses of the family Tombusviridae. Magnaporthe oryzae ourmia-like virus 1 (MOLV1) and Magnaporthe oryzae ourmia-like virus 4 (MOLV4) are in the family Botourmiaviridae [P. oryzae isolate infecting wheat [M. oryzae, in the family Narnaviridae, Magnaporthe oryzae narnavirus virus 1 (MoNV1), was identified [The first mycovirus in  in 1971 . Since tiviridae ,14,15. Moviridae ,17,18. Moviridae , and 3 baviridae ,22,23. Ang wheat . Recentlentified .Chrysoviridae and Partitiviridae, have been reported to impact the the biological characteristics of their fungal host, M. oryzae. Among them, three chrysoviruses, MoCV1-A, MoCV1-B, and MoCV1-D, confer hypovirulence to M. oryzae. The infection of these hypovirulent viruses diminishes the pathogenicity of their fungal hosts by changing the biological characteristics of M. oryzae, such as by impairing hyphal growth, causing abnormal colony morphology, and reducing pigmentation [M. oryzae [M. oryzae sporulation and virulence to barley substantially decreased. However, the effects of the (+) ssRNA mycovirses from three families, Tombusviridae, Narnaviridae, and Botourmiaviridae, on M. oryzae have not been previously mentioned.To the best of our knowledge, only four dsRNA mycoviruses in two families, entation . In addi. oryzae . After iA single fungal strain can generally be infected by multiple mycoviruses. This kind of co-infection can be facilitated by chemical compound- or virus-mediated suppression of heterogenic recognition and RNA silencing ,27,28,29M. oryzae, we isolated and characterized four novel (+) ssRNA botourmiaviruses, designated as Magnaporthe oryzae botourmiavirus 2 (MOBV2), MOBV5, MOBV6, and MOBV7, from a single M. oryzae strain, YC81-2, that was isolated from a field in China. Sequence evaluations and phylogenetic analysis of their RdRps showed that MOBV2 belongs to the genus Magoulivirus, while MOBV5, MOBV6, and MOBV7 are clustered into three undefined clades.In this study, to expand the knowledge of mycoviral diversity in M. oryzae strain, P131, was used as a control [Diseased rice leaf samples with the typical symptoms of rice blast were collected from a rice field in Yichang, Hubei province, P. R. China. Single-spore  isolation was performed from lesions of the diseased rice leaf samples. Then, a single-spore isolate, YC81-2, was selected for further study. A virus-free  control . The colFor RNA extraction, a fresh mycelial sample of strain YC81-2 was ground into fine powder in liquid nitrogen. To detect RNA viruses in strain YC81-2, dsRNA extraction was performed as previously described . The dsRM. oryzae were filtered out from the raw data. Then, de novo assembly was performed with CLC Genomics Workbench (version: 6.0.4). Primary UniGenes were spliced to generate final UniGenes using CAP3 EST. The final UniGenes were then subjected to BLAST, using BLASTx to search for homology with viral sequences against non-redundant (NR). The contigs corresponding to the same viruses were assembled to generate viral fragments by DNAMAN.Virome sequencing was carried out on the Illumina HiSeq2000/2500 platform by GENEWIZ Technology Services . In brief, one microgram of total RNA was used to obtain ribosomal-depleted RNA and construct a cDNA library using a VAHTSTM Total RNA-seq (H/M/R) Library Prep Kit for Illumina . After sequencing, adapter-polluted, contaminated, paired-end reads shorter than 100 bp, low quality, high content of unknown base (N) reads, and the RNA and DNA sequences of 4 RNA ligase . The PC3-T7 loop-ligated ssRNA was purified, denatured in dimethyl sulfoxide , and then reversely transcribed into cDNA. The terminal sequences of the viral cDNA were amplified with the primer PC2 (5′-p-CCGAATTCCCGGGATCC-3′), a complementary primer to the PC3-T7 loop, and sequence-specific primer F1 or R1, corresponding to the 5′- or 3′-terminal sequences of all viral fragments. Then, using the first round PCR products as templates, primer pairs PC2/F2 and PC2/R2 were applied to conduct Nested PCR amplification of the 5′- and 3′-ends of the viruses, respectively. After purification with a gel extraction kit , the purified PCR amplicons were cloned into the pMD18-T vector  and sequenced. The experiment was repeated three times independently. Primers used in this study are listed in The ligase-mediated terminal amplification was performed to determine the terminal sequences of the viruses . The adaThe ORFs of the viruses were predicted based on standard genetic code, and their homologous amino acid sequences were searched in the NCBI database, using ORF finder and the BLASTp programs, respectively. Sequence alignment was performed using the CLUSTALX 1.8 program . AnnotatThe morphological characteristics of the strains YC81-2 and P131 on PDA were observed. The colony of strain YC81-2 was black and flat, with little mycelia. The colony of strain P131 was grayish white and flat, with dense mycelia A. There Tombusviridae, Magnaporthe oryzae virus A (MoVA), one novel virus in the family Narnaviridae, named as Magnaporthe oryzae narnavirus virus 2 (MoNV2), and four novel viruses in the family Botourmiaviridae. Based on the alignment with non-redundant protein sequence collection in the NCBI database using BLASTp, the RdRp of one novel botourmiavirus displayed the highest identity with the RdRp of MOLV1. Thus, this virus was classified in the genus Magoulivirus and designated as Magnaporthe oryzae botourmiavirus 2 (MOBV2). The other three novel botourmiaviruses were selected for further study. Since five botourmiaviruses (previously named ourmia-like mycoviruses), MOLV1, MOLV4, PoOLV1, PoOLV2, and PoOLV3, had been found in M. oryzae, the viruses found in this study were designated as MOBV5, MOBV6, and MOBV7, respectively. The 5′- and 3′-ends of each viral cDNA were determined using ligase-mediated terminal amplification , as shown in MOBV6 was also found to be a (+) ssRNA virus B. It was−42; query cover: 64%) in the genus Scleroulivirus  and the 3′-terminal sequence (nt positions 2576–2598) of MOBV5 could be folded into terminal stable stem-loop structures with ΔG values of −14.00 and −9.40 kcal/mol, respectively A. The MOBased on multiple alignments and a conserved domain database search with the previously identified Magnaporthe botourmiaviruses, typical conserved motifs of the RdRps of (+) ssRNA mycoviruses , includiBotourmiaviridae and Narnaviridae (Naranaviridae (mitovirus and narnavirus), while the third clade embraced viruses of the new proposed and accepted family Botourmiaviridae. Botourmiaviruses were clustered into four classified genera and two unclassified groups in the phylogenetic tree. The phylogenetic tree showed that MOBV5, MOBV6, and MOBV7 indeed belonged to the family Botourmiaviridae, but were not clustered into any classified genera. They were relatively distant from MOLV1, which belongs to the genus Magoulivirus. MOBV2 was grouped into the genus Magoulivirus. The results suggest that new genera should be supposed in the family Botourmiaviridae to embrace the new members.To define the evolutionary relationship of MOBV2, MOBV5, MOBV6, and MOBV7, a phylogenetic tree was constructed using the maximum likelihood (ML) method with 1000 bootstrap interactions for the complete RdRp amino acid sequences of viruses from the families aviridae . The ML M. oryzae were identified via virome sequencing. Of them, the RdRp of MOBV2 displayed 79% identity with that of MOLV1 and belongs to the genus Magoulivirus. Using ligase-mediated terminal amplification, the full-length nucleotide sequences of MOBV5, MOBV6, and MOBV7 cDNAs were determined as 2598, 2385, and 2326 nts, respectively. The genome of each virus consists of a single unique ORF. The proteins encoded by MOBV5, MOBV6, and MOBV7 show 52%, 34%, and 34% amino acid sequence identity with RdRps of three botourmiaviruses in the family Botourmiaviridae; PoOLV1, PoOLV2, and SLOV2, respectively. Multiple alignments with the RdRps of certain botourmiaviruses showed that these proteins contain conserved domains of the RdRp in (+) ssRNA viruses (motifs I-VIII) [Botourmiaviridae, but are significantly distant from known Botourmiaviruses, and are not clustered into any classified genera in this family.In this study, four novel (+) ssRNA mycoviruses  that co-infect an isolate of the plant pathogenic fungus  I-VIII) ,51, inclBotourmiaviridae was established by ICTV in 2018, and includes viruses that infect plants and filamentous fungi containing a (+) ssRNA genome that could be mono- or multi-segmented. It consists of four genera: Ourmiavirus (plant viruses), Botoulivirus, Magoulivirus, and Scleroulivirus . Members of the genera Botoulivirus, Magoulivirus, and Scleroulivirus that infect fungi have a genome with a single segment of 2000–3200 nts encoding an RdRp. The demarcation criteria for these three mycovirus genera are based on differences in the RdRp amino acid sequence. Members of different genera in the family Botourmiaviridae should be >70% different in their complete RdRp amino acid sequence. However, in the last two years, several ourmia-like viruses that could not be classified into any of these three established genera have also been reported, such as SsOLV4, PoOLV1, and MOLV4. The identities of amino acid sequences of these are all lower than 30% compared with representative mycoviruses  in three established genera [Botourmiaviridae. Our results also demonstrate that compared with BOLV, MOLV1, or SsOLV1, the identities of the amino acid sequences of MOBV5, MOLV6, and MOLV7 were all close to or lower than 30%. We also compared the identities of amino acid sequences between MOBV5 and MOBV6 (28%), MOBV5 and MOBV7 (27%), and MOBV6 and MOBV7 (31%). Taken together, these results indicate that MOVB5, MOBV6, and MOBV7 cannot be classified into any of the three established genera, and each of them may represent a new genus.The family d genera ,24, whicBotourmiaviridae might evolve quickly and have diverse functions. Their terminal sequences exhibit several different structural features. For example, MOLV1 and MOLV4 have 3′- and 5′-UTRs with variable lengths and sequences, which can be folded into a stable stem-loop secondary structure. BOLV contains stretches of 28 and 50 nt sequences at the 5′- and 3′- non coding regions (NCRs) that can form a slightly less stable stem-loop structure [Saccharomyces [Magoulivirus, has a poly (A) tail at its 3′-end. In mycoviruses, this structure was first reported in SsMV2/KL-1, a mitovirus infecting Sclerotinia sclerotiorum [Botourmiaviridae associated with M. oryzae.Due to the simple single strand structure, the botourmiaviruses in the family tructure . Furtheraromyces . The typrotiorum , and wasrotiorum ,56. Morerotiorum . Our preBotourmiaviridae have been found to be associated with fungal hypovirulence. However, mitoviruses that belong to the genus Narnaviridae and are closely related to botourmiavirus have been found to be relevant to the variable virulence of their fungal hosts. For example, Cryphonectria cubensis mitovirus 1b and Cryphonectria cubensis mitovirus 2a have no significant impact on their hosts [Botourmiaviridae is still unknown. It has been reported that certain mycoviruses infecting M. oryzae, such as MoCV1-C, MoV3, and MOPV2, have been successfully removed from their fungal hosts through single-spore isolation. However, in the present study, we failed to generate any virus-free strains of YC81-2 using the same strategy. This result indicates that MOBV2, MOBV5, MOBV6, and MOBV7 might vertically transfect via spores. In follow-up research, we will try to obtain virus-free strains using other methods, such as protoplast regeneration technology, to determine the impact of the four botourmiaviruses investigated here on host pathogenicity and the interactions among themselves.So far, none of the mycoviruses in the family ir hosts . On the ir hosts . The co-ir hosts . Whether


Background: Although there are multiple ways to manage immunoglobulin G4–related disease (IgG4-RD), including treatment with glucocorticoids, “steroid-sparing” immunosuppressive drugs, or biologic agents, few clinical trials on IgG4-RD have been conducted. This study aimed to compare the efficacy and safety of glucocorticoids (GCs) combined with cyclophosphamide (CYC) or mycophenolate mofetil (MMF) in IgG4-RD patients. This cohort study was registered at ClinicalTrials.gov (ID: NCT01670695).Methods: This retrospective study included 155 IgG4-RD patients who received GCs with CYC or MMF at the Department of Rheumatology at Peking Union Medical College Hospital between January 2012 and July 2018. Propensity score matching (PSM) was conducted to match two groups of patients based on their baseline clinical characteristics. Treatment response, relapse rate, and drug safety were analyzed. The treatment response was evaluated based on complete response (CR), partial response (PR), and no change (NC), and the cumulative relapse rate and adverse events in each treatment group were compared using Kaplan–Meier curves and log-rank test, respectively.Results: Of the 155 IgG4-RD patients, 90 were treated with GCs plus CYC (group I) and 65 with GCs plus MMF (group II). After propensity score–matched (PSM) analysis, 108 patients were selected (54 in each group), 49 of whom had “definite” IgG4-RD, 8 “probable” IgG4-RD, and 51 “possible” IgG4-RD. At the last follow-up, the total response in groups I and II was 98.15 and 96.3%, respectively, and within 12 months, the cumulative relapse rate in group II was significantly higher than that in group I . Recurrence occurred at the paranasal sinus, lacrimal glands, skin, lung, pancreas, and bile ducts, and the relapsed patients achieved remission after switching immunosuppressants or/and increasing the GC dose.Conclusions: In IgG4-RD patients with internal organ involvement, GCs plus CYC or MMF are both effective with similar effects in disease response, while GCs plus CYC reduced the relapse rate better than GCs plus MMF. Immunoglobulin G4–related disease (IgG4-RD) is a systemic fibroinflammatory condition that affects multiple organs including the pancreas, bile duct, lacrimal gland, salivary gland, thyroid, lung, liver, gastrointestinal tract, kidney, and retroperitoneum . It is cGlucocorticoids (GCs) are the first-line therapy for achieving clinical remission in active or untreated IgG4-RD patients . RelapseMMF, an immunomodulatory agent with antifibrotic effects, can be used for the treatment of IgG4-RD because of its ability to inhibit the transforming growth factor beta pathway . CYC is This prospective observational cohort study to investigate the disease course and treatment response of IgG4-RD patients was registered on ClinicalTrials.gov (ID: NCT01670695). From January 2012 to July 2018, consecutive patients treated with GCs plus CYC or MMF were retrospectively identified for this study. The study protocol was approved by the ethics board of Peking Union Medical College Hospital, and all study subjects provided written informed consent for study participation.The inclusion criteria were as follows:18 to 75 years of age.Agreement with the 2011 comprehensive diagnostic criteria for definite, probable, or possible IgG4-RD as follows: (1) characteristic diffuse/localized swelling, sclerosis, or inflammation affecting single or multiple organs; (2) elevated serum IgG4 concentrations (135 mg/dL); (3) increased number of lymphocytes, infiltrated IgG4+ plasma cells (IgG4+ cells/IgG-positive cells >40% and >10 IgG4+ plasma), and fibrosis. Patients who met criteria (1), (2), and (3) were diagnosed with definite IgG4-RD; those who met (1) and (3) were diagnosed with probable IgG4-RD; and those who met (1) and (2) were diagnosed with possible IgG4-RD .Presence of internal organ involvement.Use of GC and CYC or MMF for initial treatment and maintenance until the 12-month follow-up.Regularly followed up at months 1, 3, 6, and 12.The exclusion criteria were as follows:Presenting only with IgG4-related dacryoadenitis and sialadenitis or lymphadenopathy of IgG4-RD.Serious infection.Presence of other rheumatic diseases.Presence of malignant diseases.Women with childbearing potential or currently planning a pregnancy.All patients underwent laboratory tests for complete blood count, erythrocyte sedimentation rate (ESR), hypersensitivity C-reactive protein (hs-CRP) levels, serum immunoglobulin, and IgG subclass concentrations, urinalysis, liver, and renal function tests; and at least one imaging examination, including ultrasonography, digital radiography (DR), computed tomography (CT), magnetic resonance imaging (MRI), or positron emission tomography/computed tomography (PET-CT). In total, 78 patients (72.22%) underwent CT scan, 24 (22.22%) underwent PET scan, 33 (30.56%) underwent MRI, and 49 (45.37%) underwent other imaging examinations, including ultrasound and DR. When necessary, tissue biopsies were analyzed and reviewed by pathologists using previously described methods , and labClinical features, laboratory results, imaging data, adverse events, and details of treatment protocol and disease relapse were evaluated at baseline and at 1, 3, 6, and 12 months after treatment or until relapse.Disease activity per visit was assessed by the IgG4-RD responder index (RI), with a system score of 0–4 for each site/organ system . An IgG4Three categories of clinical response were defined: (1) complete response (CR), in which IgG4-RD RI <3 and declined ≥2 points after treatment; (2) partial response (PR), in which IgG4-RD RI declined ≥2 points after treatment but still remained ≥3, and if IgG4-RD RI was 3 at initial treatment a partial response was defined as a 1-point decline after treatment; and (3) no change (NC), in which there was no significant improvement in affected organs and clinical symptoms and change of IgG4-RD RI <2 points , 9, 15.Relapse was defined as the recurrence of worsened/new disease manifestations or abnormality of organ-specific imaging findings despite treatment, with or without elevated serum IgG4 .t-test, whereas categorical variables were compared by the chi-square test, Fisher Exact test, or Mann–Whitney test. The Kaplan–Meier curves and log-rank test were used to calculate the cumulative relapse rate. Statistical analyses were performed using IBM SPSS statistics .Continuous variables are presented as mean ± standard deviation (SD), median, or interquartile range (IQR), and compared by p < 0.05), pancreas , and bile duct  involvement.From January 2012 to July 2018, 155 IgG4-RD patients who conformed to the inclusion criteria were selected. Among them, 90 were treated with GCs plus CYC (group I) and 65 were treated with GCs plus MMF (group II). Compared to group I, patients in group II had more paranasal sinus . In group I, patients received CYC at a mean initial dose of 54.75 mg/day . They maintained the initial dose for 3 months, after which it was reduced to 50 mg per day or every other day, consistent with previous treatment regimens  analysis to match group I patients with group II patients based on gender, age, involvement organs , and other involvement and constitutional symptoms not attributable to the involvement of a particular organ . At the end of the analysis, 108 patients were selected (54 in each group), of whom 49 were diagnosed with definite IgG4-RD , 8 with probable IgG4-RD , and 51 with possible IgG4-RD . In the post-match model, the differences of baseline characteristic between group I and group II was not statistically significance. All patients were treated with prednisone, which was gradually tapered down according to the individual's clinical response. The analyses indicated that the initial prednisone dosages were similar between the two groups  , and 2 had recurrence in the 12th month . In group II, recurrence at the paranasal sinus, lacrimal glands, skin, lung, bile ducts, and pancreas accounted for 25, 50, 12.5, 12.5, 12.5, and 25%, respectively, with the lacrimal glands being the most vulnerable to recurrence. The cumulative recurrence rate in groups I and II was 3.7 and 14.8%, respectively, and group II had a higher relapse rate than group I (= 0.046) .The follow-up data after retreatment in these 10 patients showed that they all achieved remission after the immunosuppressants were changes and/or the dose of GCs was increased according to their condition. In group I, for one patient, we increased the GCs dosage and replaced CYC with MMF, whereas for the second patient, the GCs dosage was increased. Of the 8 relapsed patients in group II, the treatment was converted to GCs and CYC for 3, iguratimod was added for 1, GC dose was increased and MMF was replaced with MTX for 1, and previous regimens with increased dosages were maintained in 3 patients. The details of relapsed patients in group I and group II are shown in Based on comprehensive assessment of disease response, relapse rate, and adverse reactions, it was suggested that GCs plus CYC had a better therapeutic effect than GCs plus MMF .Several immunosuppressants have been used as a steroid-sparing treatment for IgG4-RD, but there has been no head-to-head study comparing the efficacies between steroid-sparing drugs. In this retrospective cohort study, we aimed to explore the efficacies of GCs plus MMF therapy and GCs plus CTX and compare them in order to determine a better therapeutic strategy for IgG4-RD patients and prevent disease recurrence.In this study, CYC and MMF were used because their efficacies in IgG4-RD had been proven in previous studies. In a recent study, CYC plus GCs yielded a lower relapse rate and higher response rate relative to GC monotherapy. Similar to the results of combined CYC regimens, a study from India showed clinical improvement in patients who were treated with GCs plus MMF, with no case of relapse within a median duration of 8 months. Furthermore, an RCT study from China reported that GCs plus MMF was more effective than GCs monotherapy.In our study, IgG4-RD patients responded well to GCs plus CYC or MMF, and a majority of them experienced marked improvements within a month of treatment, which were manifested as improvement in the affected organs; reductions in the concentrations of serum ESR, CRP, IgG, and IgG4; as well as successful GC tapering. Both combined treatment regimens were efficient for IgG4-RD patients, having almost the same response rate during a year follow-up period. Comparing the ability of the two immunosuppressants to prevent flare by relapse rate, our results demonstrated that 10 patients (2 with CYC and 8 with MMF) relapsed, and relapse occurred earlier and more frequently in those receiving MMF than CYC (14.8 vs. 3.7%). Additionally, patients receiving CYC had less cumulative GCs than those receiving MMF, indicating that the steroid-sparing effect of CYC is superior to that of MMF. Therefore, the combined treatment with CYC decreased organ recurrence and maintained disease remission much better than combined treatment with MMF. Therefore, it was suggested that a low-dose CYC combination treatment regimen might be a better choice than low-dose MMF therapy in IgG4-RD patients with multiple organ involvement.In a recent RCT study that enrolled IgG4-RD patients with multiple organ involvement, it was pointed out that lacrimal glands and paranasal sinus were most susceptible to recurrence in patients receiving low-dose MMF plus GCs . HoweverRegarding safety issues, since low doses of CYC and MMF were applied in this study, both drugs were well tolerated, and this is consistent with previous IgG4-RD studies. The main adverse reactions in the CYC or MMF groups were infections and gastrointestinal reactions, and they did not differ between both groups . PreviouSo far, this is the first study to compare the effects of different immunosuppressive agents in IgG4-RD, although there are some limitations. Since the study is a retrospective cohort study, unknown variables might affect the results, leaving room for potential bias such as adverse effects that were as retrieved from the patients' hospital records. However, the results are quite reliable because there were no statistically significant differences in the baseline characteristics between the two groups.In conclusion, most IgG4-RD patients had a good response to both treatment regimens, but GCs plus CYC had a lower rate of relapse within a year than GCs plus MMF. This suggests that GCs plus CYC might be a more effective treatment option for IgG4-RD patients with internal organ involvement. However, larger prospective and RCT studies are required to understand the role of CYC and MMF in IgG4-RD.All datasets generated for this study are included in the article/supplementary material.XL and YP designed the research, analyzed data, and wrote the manuscript. PZ, ZL, JL, and HL collected and analyzed data and revised the manuscript. XZh, FZ, and XZe optimized the research and proofread the paper. YF and WZ designed the study and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Owing to a sessile lifestyle in nature, plants are routinely faced with diverse hostile environments such as various abiotic and biotic stresses, which lead to accumulation of free radicals in cells, cell damage, protein denaturation, etc., causing adverse effects to cells. During the evolution process, plants formed defense systems composed of numerous complex gene regulatory networks and signal transduction pathways to regulate and maintain the cell homeostasis. Among them, ubiquitin-proteasome pathway (UPP) is the most versatile cellular signal system as well as a powerful mechanism for regulating many aspects of the cell physiology because it removes most of the abnormal and short-lived peptides and proteins. In this system, the ubiquitin-conjugating enzyme (E2) plays a critical role in transporting ubiquitin from the ubiquitin-activating enzyme (E1) to the ubiquitin-ligase enzyme (E3) and substrate. Nevertheless, the comprehensive study regarding the role of E2 enzymes in plants remains unexplored. In this review, the ubiquitination process and the regulatory role that E2 enzymes play in plants are primarily discussed, with the focus particularly put on E2′s regulation of biological functions of the cell. Arabidopsis thaliana genome) which are core components of the UPS (Ubiquitin-proteasome system) are still unclear, especially E3s  and E2s  [Researchers have successfully extracted a small molecular protein  from reticulocytes, and this protein is a key factor in ATP-dependent substrate proteolysis and is universally present in many organisms ,3,4. Bas genome) ,21. Ther10 helix against a five-strand mixed β-sheet and seven reverse turns [A. thaliana [N-terminal Met1 residues in ubiquitin is likely to be involved in ubiquitin chain formation in vivo, and different ubiquitin linkages lead to the formation of different types of ubiquitin chains and induce diverse molecular signals in the cell [Ubiquitin, a small protein of 76 amino acids acting as a tag in post-translational modification of proteins, is absolutely conserved in vertebrates and higher plants, and only two or three amino acid residues are different even among animals, plants, and fungi . The higse turns . Ubiquitse turns , and these turns ,27,28. Use turns ,30. Six thaliana ,32. Any the cell ,34,35,36the cell ,37,38. Sthe cell . Besidesthe cell , but alsthe cell . Lys-33-the cell . HoweverIn comparison, the small ubiquitin-related modifier (SUMO) which contains approximately 100–115 amino acids is similar to ubiquitin. The SUMO emerges as an influential class of conjugation of protein tags or a modifier to target proteins, and evidence proves that it plays a regulatory role in transcriptional control, subcellular trafficking, and cell cycle ,44,45. SThe ubiquitin-proteasome system/ubiquitin-proteasome pathway is a prevalent protein turnover system that can degrade or modify proteins in eukaryotic cells. The system can modulate numerous cellular processes, and is almost involved in all aspects of growth and development, so the modification of ubiquitination may be more important than modification of phosphorylation. The UPS consists of ubiquitin (Ub), ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), ubiquitin-ligase enzymes (E3), 26S proteasome, DUBs and target proteins ,50,51, aC-terminal Gly carboxyl group of ubiquitin and the active site Cys of the E1 enzyme by an ATP-dependent reaction. Then, the E1 transfers the activated ubiquitin to the Cys residue of the E2 enzyme to form an E2-ubiquitin thioester-linked intermediate by transesterification. Eventually the E2 transfers the ubiquitin to the substrate protein by E3 [C-terminal glycine (Gly76) and the ε-amino group of a lysine residue [The covalent attachment of ubiquitin to specific target proteins is mainly accomplished by stepwise enzymatic cascade reactions, and ubiquitin is attached to the substrates via the concerted action of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3) ,53,54. Tin by E3 . Ubiquit residue . There a residue ,54,57,58 residue ,60. Subs residue . In most residue  , HECT (Homologous to the E6AP carboxyl terminus) and Cullin–RING ligases (CRLs) [The family of E3 enzymes is large and diverse. Over 1400 E3s have been found in s (CRLs) ,64. CRLss (CRLs) . An E3 is (CRLs) ,66. The The E2 enzyme is a key transfer site of ubiquitination, which interacts with E1 and E3 respectively. There are two puzzling questions about the ubiquitin-proteasome system. One is why E2 enzymes are present in all eukaryotes and the other is why we do not directly transfer ubiquitin from E1s to E3s. Some researchers believed that an E2 enzyme could combine with different types of E3s that recognized and selected distinct targets, and thereby played a decisive role in determining whether the labeled protein would be degraded or involved in nonproteolytic processes. In other words, E3s mainly select the substrate, while E2s determine the fate of the substrate . Another10 helix [N-terminal and UBC domain; class III is present in both C-terminal and UBC domain; and class IV is present in N-terminal, C-terminal and UBC domain [Caenorhabditis elegans [A. thaliana [Saccharomyces cerevisiae [Vitis vinifera [Carica papaya [Dimocarpus longan Lour. [Each E2 contains a core UBC domain composed of about 150–200 amino acid residues, in which the cysteine is an active site for the formation of thioester linkages ,70. The 10 helix ,73,74. A10 helix . Asparti10 helix ,76. AccoC domain . E2 enzy elegans , 40 in Athaliana , 14 in Srevisiae , 37 in hrevisiae , 48 in rrevisiae , 75 in mrevisiae , 72 in brevisiae , 43 in Vvinifera , 34 in Ca papaya , 59 in ta papaya , 40 in Dan Lour. , and 57 an Lour. .OsUEV1s is also capable of functionally rescuing the yeast mms2 mutant from death caused by DNA-damaging agents [AtUev1s, 4 OsUev1s, and 3 BdUev1s have been found in A. thaliana, rice and Brachypodium distachyon, respectively [The ubiquitin E2 variant (UEV) is similar to the E2 enzyme in both sequence and structure, but it cannot form a thioester linkage with ubiquitin because of the lack of the catalytic cysteine residue ,88. The g agents . Much mog agents ,93,95. 4ectively ,96,97. Cectively . Besidesectively . It remaThe E2 enzymes are one of the key parts of the ubiquitin-proteasome system, having special significance and functions .VrUBC1 is a ubiquitin-conjugating enzyme gene from mung bean, and responds to dehydration, high salinity and abscisic acid (ABA) treatment. VrUBC1-overexpressed plants are more sensitive to abscisic acid-mediated stomatal closure and have more tolerance to drought stress, indicating that VrUBC1 is a positive regulator under osmotic stress in A. thaliana [OgUBC1 is a ubiquitin-conjugating enzyme gene from wild rice, triggered by salicylic acid in leaves [NtUBC1 exhibits increased resistance to cadmium (Cd) compared to WT [LeUBC1 under the conditions of heat shock and cadmium chloride (CdCl2) indicates that LeUBC1 can respond to these two stresses [AhUBC2 is a ubiquitin-conjugating enzyme gene in peanuts, which can respond to PEG6000, high salinity, abscisic acid (ABA) and low temperature. Higher levels of P5CS1, RD29A, and KIN1 under a normal condition, and a higher level of proline under both soil-drought stress and control conditions are found in 35S:AhUBC2 transgenic plants, suggesting that the constitutive expression of AhUBC2 improves water-stress tolerance by activating an ABA-independent signaling pathway [GmUBC2 from soybean is the yeast homolog Rad6, capable of improving salt and drought tolerance, and it is involved in the regulation of ion homeostasis, osmolyte synthesis, and oxidative stress responses in A. thaliana [CmUBC in transcript level in melon plants under drought and salinity stresses indicates that CmUBC can respond to physiological water stress [Hevea brasiliensis shares a similarity of over 96% with OsRad6 in rice and AtUBC2 in A. thaliana, and its expression can be markedly induced by the latex stimulator ethephon (ET) and methyl jasmonate (MeJA) [RCE1 from A. thaliana is homologous with the human UBC12 gene, and its conjugation with the ubiquitin-like protein RUB1 that combines with AtCUL1 affects the functions of SCF E3s and thus influences the auxin response [OsUBC13 under different biotic and abiotic stresses, suggesting OsUBC13 as a housekeeping gene [A. thaliana response to low temperature [ERF1 between UBC18 mutants and UBC18 overexpression lines are caused by ubiquitination. Down-regulation of UBC18 leads to increased ERF1 and proline accumulation. Besides, UBC18 negatively regulates responses to drought and salt stresses [AtUBC32, AtUBC33, and AtUBC34 expression in both single and triple mutant lines can promote abscisic acid-mediated stomatal closure and improve tolerance to drought stress, indicating that AtUBC32, AtUBC33, and AtUBC34 play a negative regulatory role in drought stress through mediation of the abscisic acid [AtUBC32, AtUBC33, and AtUBC34 belong to group XIV UBCs, and UBC32-GFP is located in ER membranes [UBC32 mutants are extremely sensitive to paraquat treatment compared with overexpression plants, suggesting that UBC32 is an essential ERAD component and plays an important part in the response to oxidative stress and salt stress [A. thaliana and rice are not induced by cold stress treatment [UBC24 (pho2) is regulated by microRNA399 (miR399) to control inorganic phosphate (Pi) homeostasis and Pi translocation and remobilization [thaliana . OgUBC1 n leaves . Tobaccoed to WT . A signistresses . AhUBC2  pathway . The GmUthaliana . Similarr stress . The Rade (MeJA) . RCE1 frresponse ,108. A sing gene . Recent perature   proteins and their cognate effectors (Fni3/Sl-Ubc13-2 and Suv positively regulate plant immunity [Nicotiana benthamiana via VIGS (gene silencing), it is found that these genes are necessary for plant development, immunity-associated ROS production, and suppression of immunity-associated PCD (programmed cell death) by AvrPtoB [TaU4 gene which encodes a ubiquitin-conjugating enzyme 4 in Triticum aestivum by VIGS can slow down the development of disease symptoms and reduce Septoria sporulation , indicating that TaU4 is a negative regulator for defense against the phytopathogen Septoria in wheat [OgUBC1 in A. thaliana defends plants from UV-B-mediated cell damage and enables them to resist Botrytis cinerea [Fni3, Lys-63-linked ubiquitination, is a homolog of the UBC13-type ubiquitin-conjugating enzyme. A decreased expression ffectors B. This simmunity . By knoc AvrPtoB ,135. Besin wheat . The groin wheat . Heterol cinerea .A. thaliana carrying UBC13 mutants can display strong phenotypes and is unable to grow root hairs in case of iron deficiency. Especially, A. thaliana containing UBC13A and UBC13B mutants have a significantly reduced root-hair density. Therefore, it is concluded that UBC13A/B is involved in epidermal cell differentiation and iron deficiency responses [A. thaliana can influence primary and lateral roots [A. thaliana, indicating that UBC22 is required for the development of female gametophytes [A. thaliana [A. thaliana [esponses ,137. UBCal roots  , which is proven to play a role in DNA repair, damage-induced mutagenesis, and sporulation [To date, UBC13 is the only E2 known to be able to catalyze K63-linked polyubiquitin chain assembly in eukaryotes ,145,146.rulation ,158. In rulation ,160. Besrulation ,161. Therulation ,163,164.E2 enzymes are the intersection between E1 and E3 enzymes and determine the ubiquitination of specific target proteins by tagging different types of E3 enzymes. This review elaborates on some of the reported functions of E2 enzymes. Since the direct interaction between E3 enzymes and substrates results in the substrate ubiquitination, previous studies mainly focused on the mechanisms and functions of E3 enzymes. E3-mediated substrate degradation has always been the concern of research, but E2 enzymes that play a crucial role in this mechanism have not gotten much attention. The present model of ubiquitination only expounds the process of Ub transferring. The findings on the driving mechanism of E2 enzymes recognizing diverse E3 and specific substrates are very limited. Moreover, it is wondered whether E2 enzymes have other biological functions independent of the ubiquitin-proteasome system, such as regulating other signaling pathways to regulate the expression of downstream genes through signaling pathways. Much more importantly, only a few studies have investigated the role of E2 enzymes, especially in plants. Therefore, future research should focus on clarifying above important issues and explore new concerned research directions.


Furthermore, the MR relaxometry-based detection of accumulated biogenic iron in the brain tissue is useful in disease evaluation. The early description and understanding of the developing pathological process might be critical for establishing clinically effective MS-modifying therapies.Multiple sclerosis (MS) is an autoimmune disease with expanding axonal and neuronal degeneration in the central nervous system leading to motoric dysfunctions, psychical disability, and cognitive impairment during MS progression. The exact cascade of pathological processes  causing MS onset is still not fully understood, although several accompanying biomarkers are particularly suitable for the detection of early subclinical changes. Magnetic resonance (MR) methods are generally considered to be the most sensitive diagnostic tools. Their advantages include their noninvasive nature and their ability to image tissue in vivo. In particular, MR spectroscopy (proton  Multiple sclerosis (MS) is a multifactorial neurodegenerative disease that affects over 2.5 million individuals worldwide ,2. It isMS is predominately characterized as an inflammatory disease of the CNS involving the initiation of myelin loss and of axonal and neuronal damage leading to progressive neurological dysfunction ,14. Howe2 relaxation times  in the CNS provides a unique possibility for assessing Mg2+ homeostasis and its implementation in brain pathophysiological processes.The indirect om α-ATP , taking -ATP  [2+ [2+] has been suggested to be particularly important in neurological diseases in which energetic mitochondrial dysfunction is probably the primary causative or putative pathogenic factor  [2+ decline in pathological conditions and the availability of this ion in the neural tissue after its administration are still unclear [2+], correlating with motor deficits [Magnesium is involved in a myriad of biochemical processes including the activation of more than 300 enzymes ,166,167 entified ,170), thmembrane . Further–1.5 mM) ,164. The mM) [2+ ,109. On e, etc.) . However unclear . Neverth unclear , anxiety unclear , sleep d unclear , pain [1 unclear , and con unclear . In expedeficits  that shodeficits .2+ [+ and Ca2+ accumulation [2+ mechanisms might include the inhibition of the release and uptake of glutamate through the NMDA receptor and the blockade of Ca2+ channels [2+ deficiency might also induce the apoptotic process, mainly through increased oxidative stress  and eventually corrected, as this may at least moderate MS manifestations [Magnesium has been established to reduce brain edema and cerebral vasospasms, to restore blood–brain barrier effectiveness ,180, and2+ . These rmulation ,181. Furchannels ,183, cauchannels ,184. ExtA damage ,185), acentation . From thentation ,183. Thientation . Alterna content ). Conseq content . A declideficits . In MS p lesions . This is plaques . In prinstations .31P MRS is a modern noninvasive MR technique providing useful in vivo assessment of pathophysiological brain conditions, it is currently not accepted as a diagnostic tool in clinical practice. One reason is the gyromagnetic ratio  of 31P nuclei, which is approximately 2.5 times lower than for 1H, resulting in lower resonance frequency and much lower detection sensitivity [31P spectrum, it is necessary to apply special MR equipment  able to work at the resonance frequency of the 31P nuclei [31P MRS has become more easily implemented [31P metabolites, it is not advisable to use common in-built scanner sequences for 1H MRS such as stimulated echo acquisition mode (STEAM) and point-resolved spectroscopy (PRESS), but it is recommended to apply rarely implemented techniques such as image-selected in vivo spectroscopy (ISIS) or pulse acquire techniques [31P MRS metabolites are in a spectral curve presented as double, triple, or multiple peaks because of J-coupling, which needs to be correctly fitted or, even better, reduced by decoupling techniques [31P MRS examinations under higher magnetic fields  provide a better spectral resolution, higher repeatability, and signal improvements that enable the measurement of an extended range of biomarkers  exploiting the phase coherence of nuclear spins after a 90° radiofrequency pulse followed by a series of 180° refocusing pulses [2 relaxation measurement, single/multi-slice images that are repeatedly collected at several echo times are usually suitable, enabling the exponential loss of the signal to be fitted at each image pixel [2 is the time required for the transverse magnetization to fall to approximately 37% of its initial value [Tuding MS ,29,33,41on times  that refg pulses ,189. Thig pulses ,188,190.2 relaxation times are exhibited by small, free, and fast-moving molecules, unlike the more efficient T2 relaxation of larger macromolecules with a stable molecular structure supporting the existence of strong local magnetic fields [2 relaxation time is a potential biological parameter enabling the noninvasive in vivo evaluation of changes in tissue composition, chemical environment, or interactions [2 relaxation times in healthy brain tissue  that are characterized by prolonged T2 relaxation times [2 relaxation in MS lesions is thought to be caused by increased extracellular space and by larger cellular water intake in the less hydrophobic environment triggered by demyelination in WM [2 relaxation times have been found in several CNS regions, including the NAWM of MS patients [2 relaxation times are correlated with the tNAA concentration in RRMS patients [Substantially longer Tc fields ,192. Sevractions ,193,194.n tissue  have beeon times ,198. Theon in WM . Althougpatients ,200, othpatients , suggest2 relaxation is also affected  by iron accumulation [Several studies have shown that Tmulation ,9,202. Imulation . In the mulation ,205) or mulation ,18,206) mulation ,207.2-weighted MRI is thought to come from the ferric oxyhydroxide particles formed only in ferritin and hemosiderin [Cerebral iron ions released from oligodendrocytes and myelin fibers during demyelination is thus physiologically amplified in the healthy aging human brain ,208,209.osiderin .2 relaxation times of CNS structures can be considered as a biomarker of MS progression. Moreover, T2 shortening throughout the T2-hypointensities in GM has been shown to be more closely associated with neurologic status and cognitive impairment [2-hypointensities in GM is the best predictor of whole-brain atrophy [However, iron is essential for physiological neuronal metabolism, including brain oxygen transport, electron transfer, neurotransmitter synthesis, and myelin production and maturation; its excessive accumulation, especially in pathologically affected brain tissue, is however harmful ,208. Poopairment ,220 thanpairment ,206. Thi atrophy ,28. Thes atrophy ,209,210; atrophy ,221. As  atrophy ,221. Fur atrophy . Therefo2, T2*, T2’, T2 rho, R2 = 1/T2, R2* = 1/T2*, R2’= R2* − R2 [2 relaxation measurements is in general multiexponential, consisting of a very long relaxation associated with the free water in cerebrospinal fluid (T2 relaxation time >2000 ms), intermediate relaxation attributable to intra- and extracellular water (T2 relaxation time ~80 ms), and short relaxation typical for the water trapped between the myelin bilayers (T2 relaxation time ~30 ms) [Even though several assessment MR parameters (TR2* − R2 ) have beR2* − R2 ,223,224.R2* − R2 ), which R2* − R2 . Further ~30 ms) ,225. For ~30 ms) ,199. Mor ~30 ms) ,208,226. ~30 ms) , gradien ~30 ms) , asymmet ~30 ms) ,205, mag ~30 ms) ), no con ~30 ms) . In prin ~30 ms) ,208. The ~30 ms) ,228, whi


The MiT/TFE family of transcription factors , which control transcriptional programs for autophagy and lysosome biogenesis have emerged as regulators of energy metabolism in cancer. Thus, their activation increases lysosomal catabolic function to sustain cancer cell growth and survival in stress conditions. Here, we found that TFEB depletion dramatically reduces basal expression levels of the cyclin-dependent kinase (CDK) inhibitor p21/WAF1 in various cell types. Conversely, TFEB overexpression increases p21 in a p53-dependent manner. Furthermore, induction of DNA damage using doxorubicin induces TFEB-mediated activation of p21, delays G2/M phase arrest, and promotes cell survival. Pharmacological inhibition of p21, instead, abrogates TFEB-mediated protection during the DNA damage response. Together, our findings uncover a novel and direct role of TFEB in the regulation of p21 expression in both steady-state conditions and during the induction of DNA-damage response (DDR). Our observations might open novel therapeutic strategies to promote cancer cell death by targeting the TFEB-p21 pathway in the presence of genotoxic agents. However, accumulating evidence also indicates that it can play a role in a variety of cellular functions such as the regulation of transcription, DNA repair, and the modulation of apoptosis . p21 proWhile studying the effects of TFEB modulation in cell cycle proteins, we found that the depletion of TFEB severely impairs the basal expression levels of p21, while its overexpression transcriptionally upregulates p21 through direct binding to its promoter in a p53-dependent manner. Most interestingly, pharmacological induction of DNA damage using doxorubicin can induce TFEB/p21 pathway, uncovering a physiological role of TFEB in regulating the cell cycle during stress responses. Thus, TFEB activation can promote G1 arrest through p21 upregulation, delaying G2/M phase arrest, and increasing cell survival upon chemotherapeutic treatment of cancer cells. These observations envisage novel therapeutic strategies in cancer therapy by targeting the TFEB-p21 pathway in combination with genotoxic drugs.HeLa and ARPE-19 cells were purchased from ATCC. ARPE-19 is a spontaneously immortalized cell line of human retinal pigment epithelium. These cells are diploid and non-transformed. HeLa TFEB and TFEB/TFE3 knockout and p53-null human osteosarcoma (SAOS-2 p53-null) cells were kindly provided by Richard J. Youle  and Dr. Caterina Missero , respectively. HeLa cells stably expressing TFEB-GFP or TFEB 3xflag were obtained as previously described ,28. CellCells were transfected using Trans-IT reagent  according to the manufacturer’s protocol with plasmids encoding TFEBS211A x3flag , CMVp53 -ΔΔCt method. HPRT was used as the reference gene for the normalization of target gene expression levels. Total RNA was purified from cells by using the RNeasy Mini Kit . Reverse transcription was performed using the QuantiTect Reverse Transcription Kit . qPCR was performed with a LightCycler® System 2.0 , using SYBR Green PCR master mix  and the following primers: p21 , p53 , TFEB , HPRT . Relative gene expression was calculated by the 2Cells were rinsed twice with cold PBS, resuspended in lysis buffer  containing protease  and phosphatase  inhibitors, and sonicated. Protein concentration was determined by the Bradford method. Then protein samples were separated by SDS–PAGE, transferred to nitrocellulose membranes, and immunoblotted with specific primary antibodies followed by peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies . Chemiluminescence was detected by a chemiluminescence imager . Densitometric quantification of bands was performed using ImageJ (NIH). Primary antibodies used were: TFEB , p53 , p21 , pWee1(Ser642) , Myt1 , cyclin B1 , aurora B , pHH3(Ser10) , cyclin E2 , pCDC2(Tyr15) , cyclin A , p18 , and β-actin , Caspase-3 , pULK757 , ULK1 , pP70 , 4EPB , p4EPB , TFE3 . Chromatin immunoprecipitation assay in HeLa cells transfected with either 3xflagCMV14 empty or TFEB S211A 3xflag plasmids and in HeLa cells stably expressing TFEB 3xflag was performed as previously described . Anti-flHeLa cells were harvested and resuspended in PBS. For the fixation, 9-fold volume of ethanol 70% was added and incubated a 4 °C for at least 1 h. Next, cells were centrifuged, washed in PBS and resuspended in PBS containing RNase A 0.1 mg/ml. After incubation for 1 h at 37 °C, propidium iodide was added to a final concentration of 10 µg/ml and samples were analyzed in a flow cytometer Accuri C6 or with BD FACSAriaIII.Cell cycle analysis by high content imaging was performed by using the Click-iT Plus EdU Alexa Fluor 488 Imaging Kit  according to manufacturer instructions. In addition, immunofluorescence detection of histone phosphorylation to identify cells in mitosis was performed by using an anti-pHH3 (Ser28) antibody , in combination with an Alexa Fluor 647 anti-rat antibody . Images were acquired with an automated confocal microscopy  and analyzed through Columbus Image Data Storage and Analysis System , by applying an elaborated script. In essence, cells in S-phase, which have incorporated EdU , are visualized in green; cells with low Hoechst 33342 sum intensity (low DNA) and EdU/pHH3 negative are G1-phase cells; cells with high Hoechst 33342 sum intensity (high DNA) and EdU/pHH3 negative are G2-phase cells; finally, cells in mitosis are visualized in red (pHH3 positive). Low/High Hoechst 33342 sum intensity threshold is calculated by the quantification of Hoechst 33342 sum intensity mean in EdU positive nuclei. Immunofluorescence detection of endogenous TFEB was performed by using the antibody anti-TFEB . Alexa Fluor 488 anti-rabbit antibody  was used to detect the anti-TFEB antibody. Images were acquired with an automated confocal microscopy  and analyzed through Columbus Image Data Storage and Analysis System . A dedicated script was applied as previously reported  to evaluRecent evidence suggests that depletion of TFEB/TFE3 transcription factors can dysregulate the expression of genes involved in the DNA-damage response induced by etoposide treatment . Thus, wp21 has been originally identified as a downstream effector of the tumor suppressor transcription factor p53 . InteresIntrigued by the TFEB-mediated modulation of p21, we tested whether genotoxic induction using the chemotherapeutic agent doxorubicin could activate the TFEB-p21 pathway. Doxorubicin causes severe DNA double-strand breaks, promoting p53-dependent induction of p21 and leading to a block of the cell in the G2-phase of the cell cycle . As expeThen, we investigated whether doxorubicin could activate TFEB by looking at the subcellular localization of endogenous TFEB . We founThen, we asked whether the modulation of TFEB protein levels could be relevant modulating the cell cycle of cancer cells in untreated conditions or after the treatment with doxorubicin, which causes a block of G2-phase of the cell cycle  Figure A. We fouTo evaluate the role of the TFEB-p21 pathway in cancer cell survival, we performed colony formation assays using cells with different levels of TFEB and p21 , in presence or absence of doxorubicin. While the number of colonies formed was similar in all cell lines treated with DMSO, the average size of these colonies was dramatically reduced in cells depleted of TFEB A,B. MoreIn normal cells, the induction of DNA damage or other stress signals activate the tumor suppressor p53, leading to transient expression of the cyclin-dependent kinase inhibitor (CKI) p21. In these conditions, p21 plays a role as an antiproliferative effector and mediator of cellular senescence. However, recent evidence suggests that p21 can acquire either tumor suppressor or oncogenic properties depending on cell type, cellular localization, p53 status, and the type and level of genotoxic stress. In cancer cells, the overexpression of p21 may promote the generation of cancer cell clones that escape from senescence, harbor extreme genome instability, and present a drug-resistant phenotype . Thus, aThen, we found that one of the major regulators of p21, the tumor suppressor p53 ,42,43,44Several genotoxic stressors inducing DDR such as etoposide, cisplatin, UVC, and doxorubicin can induce TFEB nuclear translocation ,45. IndeGiven the implications of p21 in chemotherapeutic drug resistance ,43, we e