Ascomycota. Additionally, we demonstrated that biochemical warfare of Fusarium verticillioides factory against animal cells is not only due to mycotoxins such as fumonisins, but acute cytotoxic firing is based on different excreted secondary metabolite series, potentially leading to animal and human diseases. In this study, fumonisins, which can be followed by in situ localization, quantification, or expression of the key gene implicated in their synthesis, are used to understand secondary metabolite production by this fungus. It is known that F. verticillioides produces mycotoxins such as fumonisins on cereals, but until now, there is no evidence demonstrating a method to totally block fumonisin production on feed and food. In this paper, we explained, what was never clearly established before, that fumonisin production depends on two bottlenecks. The fumonisin synthesis and secretion in fungal articles of the mycelium are medium-independent and follow the fungal cell cycle developmental program (ontogenesis). Conversely, the fumonisin excretion into the medium depends on its composition, which also impacts fumonisin biosynthesis level. Using a high-pressure freezing method, we showed that, in non-permissive fumonisin excretion (NPFE) medium, FB1 is sequestered inside extra-vesicles and in the first third of the cell wall next to the plasmalemma, leading to the hypothesis that the fungus develops mechanisms to protect its cytosolic homeostasis against this cytotoxic. In permissive fumonisin excretion (PFE) medium, leading to very high quantities of excreted fumonisins, FB1 localized inside extra-vesicles, crosses the entire cell wall thickness, and then releases into the medium. Our results demonstrated a delayed and lower expression of Fvpks gene in mycelium developed on NPFE medium as compared to PFE medium. Conversely, higher amounts of fumonisins were accumulated in NPFE-grown mycelium than in PFE-grown mycelium. Thus, our results demonstrated for the first time that we have to take into account that the synthesis and secretion inside the article of secondary metabolites depend on the occurrence of cryptic biochemical specialized articles, differentiated in the mycelium. However, those are not morphologically different from other colonial hyphae.This study presents fungi infrequently viewed as fungal factories for secondary metabolite production resources such as mycotoxins in  Mycotoxin is a human and animal health concern due to the daily meal consumption. The common accepted idea is that the biosynthesis of secondary metabolite (SM) in fungi is quantitatively variable and mysterious as to their appearance or onset, and is dependent mainly on metabolism adaptation in response to the environment . HoweverBasidiomycota and Ascomycota  Nirenberg  is a fungus, naturally present in the fields, that causes stalk rot and ear rot in cereals such as maize and rice, in which it has been discovered as Gibberella fujikuroi species complex  are first synthesized and sequestered inside the mycelium, or alternatively excreted into the culture medium. Thus, we developed permissive fumonisin excretion (PFE) medium to obtain high excreted fumonisin quantities, and non-permissive (= retention) fumonisin excretion (NPFE) medium. Then, we proceeded for FB1 localization in fungal articles, which were developed in these permissive or non-permissive media, to give evidence for in vitro fumonisin production. In addition, by coupling fungal extracts from chemical extractions or fungal culture supernatants with a cellular cytotoxic assay [effect-directed assay (EDA)], we attempted to determine the potential existence of other cytotoxic compounds (SM), different from FB1, which are also excreted in the medium or sequestered in the mycelium.In this paper, we demonstrated for the first time Strain 7600 (FRC M3125 = NRRL 20956) F. verticillioides from ARS Culture Collection  is maintained on PDA Petri dishes until experimentation at 4°C during 6 months of storage at the maximum. Agar covered with growing mycelium 7 days after inoculation (d.a.i.) was spotted (75 mm3). Ten spots were shown in 50 ml of CMC  medium. Five d.a.i., “spores” were separated from mycelium and agar was removed by filtration on the cheesecloth. Using Neubauer cell, 105 “spores” were inoculated per milliliter of NPFE  or PFE  liquid media (50 ml) or on corresponding solid media [1.4% (w/v) agar covered with PVDF membrane]. Solid cultures were maintained static whereas liquid cultures were continuously stirred at 220 rpm. Fungal structures were recovered by two centrifugations . After microscopic control, supernatants were stored at −20°C until fumonisin quantification. To eliminate the residues adsorbed on the cell wall, inoculum (5 × 106 “spores”), fungal pellets, and mycelia from PVDF membranes were washed three times in 10% (w/v) glucose in water and then recovered by centrifugation . After lyophilization of fungal material, dried mycelium was aliquoted precisely in batches of 10 mg before being stored in liquid nitrogen until use.F. verticillioides growth kinetic by estimating mycelium dry weight (MDW)/ml PFE and NPFE liquid  and solid (10 d.a.i.) media, relative Fvpks/a-Fvtef subunit expression from dried mycelium obtained from liquid  or from solid (10 d.a.i) PFE and NPFE media, and fumonisin quantification in the media  or in the dried weight mycelia  after culture in liquid  or on solid  PFE and NPFE media.The following parameters were evaluated: 6 “spores”) and from 7 ml of 5-day-old CMC-inoculated medium.Fumonisins were quantified from 7 ml of supernatant liquid  or 7 ml of lyophilized solid  PFE and NPFE media. FB1, FB2, FB3, and HFB1 were also quantified from inoculum (5 × 10n = 9).Triplicates, for all time points of each condition, were carried out using three independent experiments /the housekeeping gene α-Fvtef subunit (FVEG_02381) was determined using real-time PCR conditions and the 2−ΔCT method  using Trizol reagent  as described in the manufacturer’s instructions, then treated with DNAse I (Sigma Aldrich). Using oligo-dT columns , mRNAs were purified from total RNA. The quality and integrity of mRNA were confirmed by denaturing agarose gel electrophoresis (data not shown), monitored using Nanodrop technology, and also confirmed by Agilent technology . One graThe following primers were used:Fvpks gene expression, a fragment of 628 bp was amplified at 60°C using Fvpks-forw (5'-GATGCTCTTGGAAGTGGCCTACG-3') and Fvpks-rev (5'-CCTGGACCCCAAGGACACTTGT-3') primers  and α-Fvtef subunit-rev (5'-AACATGATRACTGGTACSTCC-3') primers  using 2x SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad), 100 ng cDNA from each sample, and 250 pM from each primer (Forw and Rev) in each 96-well plate. The reactions were cycled 35 times according to the following parameters: 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min, with an initial cycle of 95°C for 10 min. At each cycle, an accumulation of PCR products was detected by monitoring the increase in fluorescence of SYBR Green binding to dsDNA. A non-template control and a no-primer control (negative controls) were run with each assay, and all determinations were performed at least in triplicate to achieve reproducibility.T), and thus the number of templates (mRNAs) present in the reactions. The results were expressed as the relative expression of Fvpks normalized to the α-Fvtef subunit, which is the housekeeping gene, always expressed constitutively whatever the stage of development of the mycelium considered.Analysis of PCR data: the software determines the cycle number, the “cycle threshold” (Cn = 9 × 3 (27).For each fungal culture, three independent RNA extractions and a real-time PCR triplicate were performed. Means ± standard deviation (SD) and CV% were calculated from g.min), and supernatants were kept for fumonisin purification. Briefly, filtrate was adjusted to pH 7.0 and enriched fumonisin fraction was eluted by using Bond Elut Strong Anion Exchange (SAX) column system (Agilent) and 1% (v/v) acetic acid in methanol as described by the manufacturer. After evaporation to dryness under a nitrogen stream, dry pellets were stored at −20°C until fumonisin quantification (Fumonisins were extracted from filtrates by using an adaptation of fication .β-mercaptoethanol). One minute later, 20 μl of this mixture was injected on the chromatographic system composed of an M2200 pump  connected to a Prontosil C18 column  with a porosity of 5 μm and a size 250 × 4.6 mm. The latter was connected to a fluorescence detector RF 10A XL  and a PIC3 acquisition system . The mobile phase was composed of methanol/0.1 M phosphate buffer pH 3.35 . The flow rate was 1 ml/min. The excitation and emission wavelengths were respectively 335 nm and 440 nm. The quantities of FB1, FB2, FB3, and HFB1 were determined by linear regression measuring the peak area and comparing it to a standard calibration curve obtained with ranges of standards  of known concentrations. For each culture condition, three independent cultures and triplicates of fumonisin-enriched methanol fraction were obtained. Results  were calculated for three independent HPLC injections for each sample (n = 9 × 3).Dried samples were dissolved in 200 μl of methanol before high-performance liquid chromatography analysis with fluorescence detection (HPLC-FLD) according to Main results obtained demonstrated that the quantification procedure is linear, repeatable (CV = 4%), and reproducible (CV = 4.7%). The mean of HPLC-FLD recovery was 98%. These results are in agreement with the AFNOR EN13585 standard.F. verticillioides articles that produced SM was carried out with mycelium developed on solid media in order to focus samples on differentiated articles, which was too difficult using mycelium developed in liquid media. Moreover, using solid media, excreted FB1 could be immunodetected because of accumulation due to PVDF membrane. This immunolabeling would not have been possible using liquid cultures due to the different washings in procedures.Structure analysis of 3 each were high-pressure frozen with a Leica EMPACT system.4), 0.1% (w/v) uranyl acetate, and 0.5% (w/v) glutaraldehyde at −90°C for 84 h. Temperature was then raised up to −50°C at a rate of 3°C/h and then samples were kept at −50°C for 38 h. Samples were subsequently washed three times for 20 min in 100% acetone and then three times for 20 min in 100% ethanol. Samples were embedded by two impregnations for 2 h in 20% (v/v) Lowicryl HM20 in ethanol, then 2 h in 50% (v/v) Lowicryl HM20 in ethanol, and overnight in 75% (v/v) Lowicryl HM20 in ethanol. After three baths of 2 h each in 100% Lowicryl HM20, polymerization was run out under UV for 48 h at −50°C and then 48 h at 20°C under UV. After ultrathin microtone sectioning, sections of 80 nm deep were deposited onto gold-coating grids and samples were observed using a TEM . Experiences were conducted in triplicate.Fungal material was produced on solid PFE or NPFE media (PVDF membrane) in Petri dishes. The high-pressure freezing method was adapted from Gold-coating grid-deposited fungal sections were treated for 10 min with 20 μl of 0.02 μm filtered 0.1% (w/v) glycine in water at 20°C. Grids were then saturated using 50 μl of 1 mg/ml BSA in TBS  containing 2% (v/v) tween 20 for 15 min at 20°C. Immunoreactions were carried out at 20°C for 45 min using 20 μl of anti-FB1 rabbit polyclonal antibodies  diluted at different concentrations  in 0.05% (v/v) Tween 20 in TBS (TBST). Slides quickly washed five times using TBST were then incubated with 20 μl of 5 nm of gold-labeled goat anti-IgG rabbit polyclonal antibodies  at different concentrations  in TBST for 40 min at 20°C. After five quick washings with TBST, slides were rinsed with 20 μl of 0.02-μm-filtered water and dried at 20°C before TEM observations. Negative control was carried out omitting anti-FB1 rabbit polyclonal antibodies. Experiences were triplicated.g.min), supernatants were kept at −80°C, whereas pellets, containing fungal material, were re-extracted in the same conditions. The supernatants coming from the same mycelium were pooled and then lyophilized. The water-extracted mycelia were extracted twice in 50% (w/v) ethyl acetate (ETAC). ETAC supernatants were lyophilized and then stored at −20°C for further experimentations. Lyophilized fungal water extracts (PFE-MycW and NPFE-MycW) were resuspended in water whereas lyophilized ETAC fungal extracts (PFE-MycETAC and NPFE-MycETAC) were resuspended in 10% (v/v) methanol/water.Frozen supernatants  from liquid PFE or NPFE culture media were lyophilized and then resuspended in 7.5 ml of sterile water and directly used for effect-guided cellular testing (PFE-SN and NPFE-SN). Lyophilized fungal materials were water (PFE-MycW and NPFE-MycW) or ethyl acetate (PFE-MycETAC and NPFE-MycETAC) extracted as follows: lyophilized fungal materials were resuspended at 50% (w/v) in water before being extracted overnight at 4°C under agitation. After centrifugation .Extracts  were diluted at different concentrations in Neuro-2A cell culture medium for acute cytotoxic assays. Each extraction was triplicated from three independent experiments (F. verticillioides molecules were carried out using EDAs (The cytotoxicity test is based on the OECD guidance document n°129 . Acute cing EDAs  against 2 humidified incubator at 37°C.Neuro-2A cells were cultured in RPMI medium containing 10% (v/v) FCS, 2% (w/v) L-glutamine, 2% (w/v) glucose, and 1% (w/v) tobramycin in a 5% (v/v) CO5 cells per milliliter. Neuro-2A cells (100 μl of 105/ml) were deposited in each 96-well plate, incubated 24 h at 37°C in a 5% (v/v) CO2 humidified incubator, washed with 100 μl of PBS, and then exposed to different extract concentrations , Neuro-2A cell culture medium (100% viability positive control), or vehicle diluted in Neuro-2A cell culture medium (negative controls) that consisted of addition of 5% (v/v) methanol for Myc-ETAC or 5% (v/v) water for MycW fractions (corresponding to the highest concentration used in cytotoxicity assay: dilution 1/2), respectively, or 50% pre-inoculated PFE or NPFE liquid media (highest concentration used in cytotoxicity assay: dilution 1/2). About 0% viability control (negative control for MTT assay) was obtained by omitting cells in wells. After 24 h of incubation, cell viability was estimated by MTT assay  of each extract (n = 8 × 3) using statistic estimated by GraphPad Prism (V 6.05).After supernatant removal, cells were washed with 10 ml of PBS, individualized by 3 ml of 0.05% trypsin . After addition of 7 ml of Neuro-2A cell culture medium, living cells were counted using Neubauer cell. Culture medium was adjusted to obtain 10TT assay . Experien = 9 to n = 27, depending on experiences. We used variance analysis and Tukey honestly significant difference tests. Statistical analysis was carried out by using the R software  and values of p < 0.05 were considered to be significant.Data were expressed as mean ± SD and CV% was calculated from n = 9.Using liquid media, the growth rate, evaluated by the mycelium dry weight (MDW) during 10 days, is 1.65 times higher in the PFE medium (6.13 ± 0.02 mg/ml) than in the NPFE medium (3.71 ± 0.07 mg/ml). Using GraphPad Prism (V 6.05) software, the optimized curve shows that fungal growth reaches a stationary state 5 d.a.i. in PFE medium, whereas only a growth inflection is obtained 10 d.a.i. in NPFE medium . CV% wasn = 9. Statistically, the MDW depending on each solid medium used is not significantly different and CV% reaches 21.59 and 21.61, for PFE and NPFE media, respectively. These CV% are under 30% which is the maximum generally used in biological experiments; nevertheless, we generally use maximum CV% to 20 to keep significant in all our experiments. Under 10 d.a.i., results on MDW could not be included in this study due to CV% >36, which could not be acceptable.Using solid media, MDW could only be evaluated 10 d.a.i. when the mycelia covered the PVDF membrane. MDW is 35.2 ± 7.6 mg on PFE medium and 27.3 ± 5.9 mg on NPFE medium using Fvpks/α-Fvtef subunit expression in mycelia developed in liquid or on solid PFE and NPFE media. Relative Fvpks/α-Fvtef subunit expression increases during fungal culture in mycelia developed in liquid PFE medium, reaching 3 d.a.i. 368.2 ± 6.8, then 2,867 ± 15 and 4,791 ± 30 7 and 10 d.a.i., respectively.Data show relative Fvpks mRNA is detected 3 d.a.i., relative Fvpks mRNA accumulation reaches 406.5 ± 3 7 d.a.i. and 1727 ± 68 10 d.a.i. .Any Fumonisins were extracted from culture media 1.5, 3, 5, 7, and 10 d.a.i. and then quantified.n = 27.FB1, FB2, and FB3 concentrations were nearly similar (about 54 ± 0.6 mg/L) 1.5 d.a.i. and HFB1 quantity represents about half of them (25.7 ± 0.5 mg/L). Except for this time point, FB1 was the major excreted fumonisin in PFE medium whereas FB2, FB3, and HFB1 were also detected at the lower quantity at each time point . From thAt day 10, very few amounts of FB1 > FB3 > FB2 are detected in mycelia developed in PFE medium, reaching 38.5 ± 1.76 > 28.8 ± 2.74 > 22.4 ± 1.6 mg/g MDW, respectively, with FB3 quantity significantly slightly higher than the FB2 one.Conversely to PFE medium, higher fumonisin amounts were unexpectedly detected in mycelia grown in NPFE medium. In that event, FB1 >>> FB3 > FB2, reaching 796 ± 25.45 >> 65.4 ± 4.18 > 61.4 ± 4.84 mg/g MDW, respectively, with no significant differences between FB2 and FB3 quantities .n = 27.Fumonisin quantities are enhanced in mycelia during culture time but to a lesser extent in PFE liquid medium than in the NPFE one . In myceAny fumonisin could be detected in the inoculum (not illustrated).Amounts of FB1 >> FB3 > FB2 were quantified (887.78 ± 76.93 >> 168.22 ± 14.25 > 84.22 ± 6.14 mg/L) in solid PFE medium 10 d.a.i. , but theFumonisins were quantified in mycelia developed on solid PFE medium: FB1 > FB3 > FB2 .Fungal articles were analyzed using an adaptive embedding and high-pressure freezing protocols .F. verticillioides is an Ascomycota. Vesicles are small enough to go through the septal pore (dolipore) and may use this way to be transported from article to article .After immunolabeling optimization (as described in M&M), anti-FB1 rabbit polyclonal antibodies were used at 1/200 and 5-nm gold-labeled secondary antibodies at 1/30 in TBST. No labeling could be detected omitting primary antibody, demonstrating anti-FB1 antibody specificity: negative control [On PFE medium, FB1 diffuses throughout the cell wall and is excreted outside fungal articles. FB1 is detected inside small vesicles and MVB in the cytosol [Unexpectedly, on the NPFE medium, FB1 is localized inside fungal articles, confined in ectosomes  with some organized as MVB in the cytosol [Fusarium verticillioides acute cytotoxicity was determined against Neuro-2A cells by in vitro EDA. Fungal culture media cytotoxicity (PFE-SN and NPFE-SN) was higher than those obtained with the corresponding mycelial extractions (compare  compare –F.50(PFE-SN) = 2 × 10−3 (50(NPFE-SN) = 3.5 × 10−4 (−3 (PFE-SN) and 10−4 (NPFE-SN), viable cells represented 100%.NPFE-SN without fumonisin was unforeseen to be more than 10 times more acute cytotoxic than PFE-SN containing FB1: EC2 × 10−3  whereas 5 × 10−4 . Until d50(PFE-MycW) = 8.7 × 10−3, 50(PFE-MycETAC) = 9 × 10−3, 50(NPFE-MycW) = 1.9 × 10−2, 50(NPFE-MycETAC) = 3.8 × 10−2, n = 24.All fungal extracts from mycelium developed in PFE medium were highly toxic compared to this grown in NPFE medium: [ECNeither 5% methanol, 5% water, nor 50% pre-inoculated PFE and NPFE liquid media diluted in Neuro-2A cell culture medium affected Neuro-2A viability, which reached 100% (data not shown), suggesting that both pre-inoculated liquid PFE and NPFE media were not toxic against Neuro-2A cells.In our studies, all CV% were under 16.33%, except for MDW on solid medium where CV% reaches 21.59 and 21.61. CV% until 30% is usually accepted for biological experimentations , but we G. fujikuroi with Gfpks multicopy gene. F. moniliforme, leading to confusion whether FB1 is in the mycelium or excreted in the medium. Moreover, 280 mg of FB1 + FB2/L malt extract medium was obtained 10 d.a.i. in a jar using F. moniliforme  to sow media. PFE medium was optimized to achieve a high amount of excreted FB1 into medium and, to a lesser extent, FB3, FB2, and HFB1 excretions. Differences in fumonisin excretion could not be explained by differences in mycelium amount obtained according to media. In NPFE medium, any fumonisin could be detected neither in liquid nor in solid media. Before us, previous works underlined that fumonisin production depends on the fungal strain and media composition . Howeveriliforme . Solid-sr on PDA . Conversmonisins . Thus, t factory .in vitro by several authors in their medium. Fumonisin quantities in PFE medium found in our experiments represent about 43 times higher amounts of FB1 + FB2 obtained in medium by 3 of fumonisin-contaminated maize grains, to obtain this same quantity, if we consider that grain contamination in the field reaches 2000 μg of fumonisins/maize grain kg , we hypothesize a common phenomenon for production of all SMs, and all fungi are concerned whether they belong to gulation . Hence, gulation . Implemegulation ,b.Using NPFE medium, FB1 is detected inside vesicles and MVB, in ectosomes (ve) or confined in the inner 1/3 of cell wall next to the plasmalemma. Thus, growing on NPFE medium allows one to hypothesize that toxic molecules are trapped in vesicles and MVB in order to certainly protect fungal cytosol homeostasis against potential deleterious/toxic FB1 effects. FB1 trapped in the inner 1/3 of the cell wall shows also that this fumonisin is not authorized to diffuse freely through the entire cell wall, indicating that broadcast blocking is provided by the cell wall physicochemical composition, which may itself depend on cultural environment. Because high quantities of fumonisins are detected inside mycelium, we hypothesize that when fumonisins are detected in cereals without the presence of the fungus, this fact could be due to disappearance of dead mycelia, leaving fumonisins in grains as remaining traces of their dislocated corpses.Phanerochaete chrysosporium peroxidases  way to be transported from articles to subapical articles to the apex of hyphae. In PFE medium, FB1 diffuses from mycelium to medium. However, we cannot specify whether FB1 scatters from each fungal cell article or whether a specific apex diffusion mechanism of sequestering vesicles is used as for oxidases , A. nigeamylases , or Picn laccase .via regulation of critical nutrients  generate at the plasmalemma (ectosomes = microparticles/microvesicles) or inside vesicles (= exosomes) that could be organized as MVB . It is wutrients . F. vertF. verticillioides cannot be viewed as a strict plant pathogen because it can easily be in vitro cultured and SM can relatively be well expressed. Moreover, fumonisins are undoubtedly produced by in vitro culture isolates, suggesting that plant–fungus interaction is not required for fungal SM productions. Our results match with previous works, emphasizing that F. verticillioides develops a non-close invasive relationship with the plant  affects Neuro-2A cells. We hypothesize that F. verticillioides synthesizes and excretes other cytotoxic SM against neuronal cells. Thus, it appears that (i) biochemical armaments of this fungal factory against animal cells seem multiple, (ii) this Ascomycota develops different biochemical warfare processes to ensure its survival in different environmental conditions, and (iii) that fungal cytotoxic firing is based on different excreted SM series. Our results match with the existence of 39 Fvpks clusters in F. verticillioides genome  The synthesis and secretion of fumonisins or SM inside the articles are under control and closely related to the natural fungal development program, the understanding of which is still in infancy, whereas (ii) fumonisin or SM excretion into the medium is under environment dependence. Discrete changes in cellular biochemical differentiation in articles certainly trigger the high expression of the Fvpks gene of the fum cluster and the production of exogenous fumonisins, without drastic morphological modification of these specialized articles. Nevertheless, depending on the medium composition, mycotoxins can remain endogenous inside articles where cell wall structure seems to be not permissive to its diffusion. We thus hypothesize that, like mycotoxins, SM productions depend on fungal ontogenesis for each article, leading to SM excretion as an armament to fight for life depending on environmental conditions. Moreover, this phenomenon should be ubiquitous to all fungus belonging to different families, and it could be extrapolated to animal- or vegetal-specific organs to preserve the survival of the species during life constraints. In contrast, all of this opens up the field of SM potentialities in public and animal health, especially for developing prevention strategies or therapeutic methods that medicine could use.In conclusion, our theory is that productions of The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ralstonia solanacearum population. Artificial disruption of Gram-positive bacteria in HRS using 500-μg/mL vancomycin increased bacterial wilt occurrence in tomato. To identify HRS-specific and plant-protective Gram-positive bacteria species, Brevibacterium frigoritolerans HRS1, Bacillus niacini HRS2, Solibacillus silvestris HRS3, and Bacillus luciferensis HRS4 were selected from among 326 heat-stable culturable bacteria isolates. These four strains did not directly antagonize R. solanacearum but activated plant immunity. A synthetic community comprising these four strains displayed greater immune activation against R. solanacearum and extended plant protection by 4 more days in comparison with each individual strain. Overall, our results demonstrate for the first time that dysbiosis of the protective Gram-positive bacterial community in DRS promotes the incidence of disease.Enrichment of protective microbiota in the rhizosphere facilitates disease suppression. However, how the disruption of protective rhizobacteria affects disease suppression is largely unknown. Here, we analyzed the rhizosphere microbial community of a healthy and diseased tomato plant grown <30-cm apart in a greenhouse at three different locations in South Korea. The abundance of Gram-positive Actinobacteria and Firmicutes phyla was lower in diseased rhizosphere soil (DRS) than in healthy rhizosphere soil (HRS) without changes in the causative  Rhizoctonia solani, Pythium ultimum, Gaeumannomyces graminis var. tritici, Plasmodiophora brassicae, and Fusarium oxysporum, and a bacterial pathogen Ralstonia solanacearum [Rhizosphere microbiota play an important role in plant fitness, development, and immunity –4. The nnacearum –15. In cnacearum .G. graminis var. tritici by producing the antibiotic 2,4-diacetylphloroglucinol [Arabidopsis and tomato [Rhizosphere microbes play a complex role in the establishment of disease-suppressive soils. Early studies on diseasesuppressive soils focused mainly on the direct mechanism of microbes against a target pathogen. The best example of an antagonistic microbe in disease-suppressive soil is the fluorescent pseudomonads, which directly inhibited the growth of glucinol , 17. On glucinol –20. ISR glucinol , 20. ISRd tomato , 22. Cerd tomato , 24.Pseudomonas [Bacillus [Paenibacillus [Streptomyces [Most studies on disease-suppressive soils have focused on the effect of a selected microbial strain, rather than that of a microbial consortium, on the target pathogen , 25, 26.udomonas , 17, 25,Bacillus , 28, Paebacillus , and Strptomyces , 26. Oveptomyces . Recentlptomyces , 31. Howptomyces . Thus, tptomyces –34.Arabidopsis leaves [The eubiosis of host-associated microbial communities can potentially alter disease occurrence –39. In as leaves . ReductiRalstonia solanacearum, in their rhizosphere. Analysis of the rhizosphere samples by 16S rRNA amplicon sequencing and cultivation-based approaches revealed a decline of Firmicutes and Actinobacteria in the diseased rhizosphere soil (DRS). Strikingly, the disruption of Firmicutes and Actinobacteria in the healthy rhizosphere soil (HRS) using the antibiotic vancomycin led to the DRS-mimicking phenomenon. Furthermore, a SynCom of HRS-specific strains activated JA signaling-dependent ISR against R. solanacearum in tomato. Based on our results, we suggest that dysbiosis of the protective microbial community in the soil disrupts disease suppression. Our results will help to broaden the agricultural applications of synthetic microbial communities as biological control agents against plant pathogens.Here, we examined the rhizosphere of two adjacent paired tomato plants with and without disease symptoms grown within a 30-cm distance in a greenhouse in three different geographic locations in South Korea. Both plants showed drastic differences in the incidence of bacterial wilt, despite the presence of a similar amount of the causal pathogen, HRS and DRS samples were collected from three tomato plastic greenhouses, one each in Damyang (35°15′53.4″N and 126°55′11.8″E), Yongin (37°06′20.6″N and 127°08′14.3″E), and Gwangju (37°29′24.1″N and 127°18′22.6″E), in South Korea. Each greenhouse was 6.6 × 100 m in size and contained 950–1200 tomato plants. The temperature in each greenhouse was maintained at 30 ± 5 °C. Tomato plants in the selected DRS samples showed severe bacterial wilt symptoms including stem blight; wilting of petioles, main stem, branch tips, and leaves; and chlorosis and necrosis of foliage. Tomato plants in HRS samples did not show bacterial wilt symptoms and were adjacent to DRS samples (within a distance of 30 cm). The HRS and DRS were filtered through a 2-mm mesh to remove large soil particles and collected only rhizosphere soil without plant root tissue and debris. Then, the HRS and DRS were suspended in sterile distilled water for 30 min to remove tightly attached soil particles. The soil solution was centrifuged at 8000 rpm for 10 min, and the soil pellet containing the microbiome was stored at −80 °C until needed for microbial community analysis.R. solanacearum, the causal organism of bacterial wilt. At 10 days post inoculation (dpi), the severity of bacterial wilt was recorded on a 0–5 scale [Soil microbiome was extracted from HRS and DRS samples as described previously . Briefly–5 scale , with 0 R. solanacearum was grown in casamino acid-peptone-glucose (CPG) broth  at 30 °C for 24 h, and 10-mL pathogen suspension (OD600 = 1) was applied to the soil by drench application. All experiments were performed in triplicate, with 12 plants per treatment.Seedlings of the bacterial wilt susceptible tomato variety, Juiken, were cultivated in sterile soil for 14 days after germination. Roots of tomato seedlings were treated with soil microbial fractions using the root-dipping method. On day 14, the treated roots were washed with distilled water to remove the attached soil particles and were immersed in 20-mL soil fraction for 30 min. Then, tomato seedlings were transplanted in sterilized soil and grown at 28 °C for 5 days. To inoculate tomato seedlings with the pathogen, Microbial genomic DNA was extracted from HRS and DRS samples using the FastDNA Spin Kit  and quantified using Epoch Spectrometer . PCR amplification was performed using primers targeting V3 and V4 regions of 16S rRNA genes. The first round of amplification was carried out using primers 341F and 805R Table  under thhttps://docs.qiime2.org/2019.1/data-resources/). Relative proportions were calculated because changes in proportions at the Phlyum level are related to soil conditions. For this analysis, samples were normalized within the group using DESeq2.The quality of the native sequence was evaluated by FastQC, and low-quality cutoffs for forward and reverse readings were determined. Then I brought forward and reverse readings to QIIME2 (v 2020.2)  for qualP value < 0.05) [We also used linear discriminant analysis effect size   to identThe ratio of viable Firmicutes and Actinobacteria bacteria to the total number of Gram-negative bacteria in HRS and DRS samples was measured using two methods. A total of 180 bacterial colonies were first tested using the 3% KOH string test , and theTo optimize the vancomycin treatment, HRS and DRS microbial fractions (1 g/mL each) were treated with three different concentrations of vancomycin  for three different durations . The HRS and DRS fractions treated with 500-μg/mL vancomycin were centrifuged, and pellets were washed twice with 2.5-mM MES buffer to remove residual vancomycin. Soil fractions treated with or without vancomycin were inoculated on TSA medium containing 0- or 5-μg/mL vancomycin. To induce the dysbiosis of Firmicutes and Actinobacteria, each soil fraction was added to 500-μg/mL vancomycin and incubated at 30 °C for 3 h.600 = 1.0) of R. solanacearum was plated on a CPG medium containing 2,3,5-triphenyl tetrazolium chloride (TZC), and that of each of the four selected protective bacterial strains HRS1, HRS2, HRS3, and HRS4 was plated on TSA medium. Then, 10-μL vancomycin  or kanamycin  was dropped on each inoculated plate and incubated at 30 °C for 2 days. The plates were examined after 2 days to examine the development of an inhibition zone. All experiments were performed in triplicate.To examine the vancomycin sensitivity of the bacterial wilt pathogen and protective bacteria, a suspension (ODhttp://blast.ncbi.nlm.nih.gov/Blastcgi), and the closest match was identified.To isolate spore-forming bacteria, HRS and DRS fractions were incubated at 80 °C for 30 min , plated 600 = 20) were prepared at 0, 1, and 2 weeks post inoculation (wpi). The rhizosphere soil suspension was incubated on TSA medium containing 100-µg/mL rifampicin for 2–3 days at 30 °C, and then the bacterial population was measured. All experiments were performed in triplicate, with five plants per treatment.To assess the colonization of tomato roots by the four selected bacterial isolates, CFU values of the four spontaneous rifampicin-resistant strains of HRS1, HRS2, HRS3, and HRS4 were measured as described previously , 53. Bri600 = 1.0). Tomato seedling roots were washed with sterile water and immersed in each bacterial suspension for 30 min. Then, the tomato seedlings were transplanted in sterilized soil and grown at 28 °C for 5 days. Subsequently, tomato roots were inoculated with R. solanacearum via drench application, as described above. This experiment was performed in triplicate, with 12 plants per treatment.The selected bacterial species were cultured on TSA medium for 24 h and then suspended in sterile distilled water . After 7 days, 50-μL R. solanacearum were examined using the co-culture method [R. solanacearum (OD600 = 1.0) was plated on TSA medium. Then, 10 μL each of HRS1, HRS2, HRS3, and HRS4 (OD600 = 1.0) or of gentamycin  was dropped on the pathogen-inoculated plate. The plates were then incubated at 30 °C, and the development of an inhibition zone was examined after 2 days. This experiment was performed in triplicate.Antagonistic effects of each of the four selected isolates against e method . A suspeUbiquitin3 mRNA. All qRT-PCR experiments were performed in triplicate.Total RNA was extracted from tomato leaves harvested at 0 and 12 h after pathogen inoculation, and first-strand cDNA was synthesized as described previously . Then, qt test were performed in the R program [P < 0.05.Two-way analysis of variance and two-tailed Student’s  program  to analyR. solanacearum, was 9.3 × 105 and 1.1 × 106-CFU/g soil in HRS and DRS, respectively, and this difference in population size was not statistically significant (P = 0.05)  and did not spread throughout the greenhouse at the end of season in any of the three locations, where the physicochemical properties of soil differed Fig. . A patch05) Fig. . The inoial wilt . TherefoR. solanacearum without antagonism [Because the composition of rhizosphere microbiome can be used to determine the suppression of bacterial wilt in tomato, as demonstrated previously , 58, we tagonism , reducedTo detect differences in microbial composition between healthy and diseased rhizosphere samples, seven HRS and eight DRS collected from Damyang, Yongin, and Gwangju were subjected to 16S rDNA amplicon sequencing Fig. . RelativTo explore the most discriminating amplicon sequence variant (ASV) in Firmicutes and Actinobacteria in HRS, we selected the top three Firmicute and Actinobacteria ASVs showing a significant difference in relative abundance between HRS and DRS fractions using the Lefse method Fig. . Among FR. solanacearum  at 5 wpi Fig. ; by contpi Figs.  and S5. Brevibacterium frigoritolerans) and 12 Bacillales  were isolated from the Damyang HRS sample  and nine Bacillales  were isolated from the Yongin HRS sample  and ten Bacillales  were isolated from the Gwangju HRS sample , B. niacini HRS2 (HRS2), S. silvestris HRS3 (HRS3), and B. luciferensis HRS4 (HRS4) were selected as keystone taxa isolates conferring HRS, with culturable bacterial abundance >5%   Fig. . HRS1 waly) Fig. . These fly) Fig. . The relant Fig. .Fig. 4IdB. frigoritolerans (HRS1) and B. niacini (HRS2) play an important role in bacterial wilt suppression in tomato.Compared with the control, strains HRS1 and HRS2 significantly reduced disease severity at 13 dpi  and 14 dpi  Fig. . By cont7, 1.6 × 107, 2.4 × 107, and 1.7 × 106-CFU/g soil, respectively, at 0 wpi; 3.5 × 106, 5.2 × 104, 7.6 × 104, and 1.3 × 105-CFU/g soil, respectively, at 1 wpi; and 6.1 × 105, 3.1 × 102, 7.3 × 103, and 3.6 × 103-CFU/g soil, respectively, at 2 wpi  signaling in systemic leaves at 0 and 12-h post inoculation (hpi) Figs.  and S6. nes Fig. . In addihpi Fig. , whereashpi Fig. . HoweverPlants manipulate rhizosphere microbiota to establish disease suppression in the soil –4, 63. RTo confirm the role of rhizosphere microbiota disruption in disease suppression, we used vancomycin to disrupt populations of bacteria belonging to Firmicutes and Actinobacteria phyla in tomato rhizosphere. Previous studies employed a pasteurization method, involving the use of moist heat, methyl bromide, or chloropicrin, which kills a wide range of microbes in the soil ; therefoR. solanacearum reduced the diversity and abundance of non-pathogenic rhizobacteria [R. solanacearum was similar in HRS and DRS fractions  than in the Damyang and Yongin HRS samples  and B. subtilis (Firmicutes) increased the production of undecylprodigiosin, which suppressed the fungal pathogen, Verticillium dahliae [A previous study showed that Firmicutes taxa, c effect ; howeverdent ISR , 77–80. ns Figs.  and 6a. les Fig. . Recent les Fig.  and 6. Tgers ISR , 88. A mresponse , 90. In  dahliae , 92. HowR. solanacearum in soil and the limitation of the control method, it is difficult to control the incidence and spread of bacterial wilt [R. solanacearum.In this study, we first demonstrated that the specific disruption of the protective Firmicutes and Actinobacteria community in tomato rhizosphere enhanced the incidence of bacterial wilt disease. Although sustained monoculture can lead to a considerable decline in rhizosphere microbiota diversity and consequently local disruption of disease suppression, this diversity can be recovered by amending the soil with a minimal SynCom. Further investigations are needed to (1) identify the factors responsible for the local collapse of disease suppression, (2) identify an early diagnostic marker of microbiota disruption by microbiome analysis before disease occurrence, and (3) understand individual microbial determinants that activate plant immunity. Because of the high stress tolerance of ial wilt . Our resSupplementary TableSupplementary Figures


V1.3 knock-out (KO) and otoferlin KO mice, where auditory brainstem nuclei are deprived from spontaneous cochlea-driven neuronal activity, neuronal circuitry is disturbed. So far it was unknown if this disturbance is also accompanied by an impaired topography of LSO astrocyte networks. To answer this question, we immunohistochemically analyzed the expression of astrocytic connexin (Cx) 43 and Cx30 in auditory brainstem nuclei. Furthermore, we loaded LSO astrocytes with the gap junction-permeable tracer neurobiotin and assessed the network shape and orientation. We found a strong elevation of Cx30 immunoreactivity in the LSO of CaV1.3 KO mice, while Cx43 levels were only slightly increased. In otoferlin KO mice, LSO showed a slight increase in Cx43 as well, whereas Cx30 levels were unchanged. The total number of tracer-coupled cells was unaltered and most networks were anisotropic in both KO strains. In contrast to the WTs, however, LSO networks were predominantly oriented parallel to the tonotopic axis and not orthogonal to it. Taken together, our data demonstrate that spontaneous cochlea-driven neuronal activity is not required per se for the formation of anisotropic LSO astrocyte networks. However, neuronal activity is required to establish the proper orientation of networks. Proper formation of LSO astrocyte networks thus necessitates neuronal input from the periphery, indicating a critical role of neuron-glia interaction during early postnatal development in the auditory brainstem.Anisotropic gap junctional coupling is a distinct feature of astrocytes in many brain regions. In the lateral superior olive (LSO), astrocytic networks are anisotropic and oriented orthogonally to the tonotopic axis. In Ca In many brain regions, astrocytes and oligodendrocytes form large panglial gap junction (GJ)-mediated networks ,2,3,4. IV) 1.3 or the calcium sensor otoferlin in inner hair cells of the cochlea, cause hereditary deafness [V1.3 knock-out (KO) mice [Before hearing onset, which takes place at around postnatal day 12 in mice, circuits undergo substantial refinement ,18,19. Ideafness ,30,31,32KO) mice  and otofKO) mice . In thesKO) mice ,35,36. IV1.3 KO and otoferlin KO mice. The expression of connexin (Cx) 43 and Cx30 was increased, but the degree of GJ coupling was unaltered. However, network topography was strongly altered in CaV1.3 KO and otoferlin KO mice. Most networks were anisotropic, but in contrast to the WT, networks were now predominantly oriented parallel  to the tonotopic axis. Thus, our data show that spontaneous cochlea-driven neuronal activity is not only mandatory for proper formation of neuronal circuitry, but in addition is required for proper orientation of astrocyte networks in the LSO.Our results show that LSO astrocytes assessed at postnatal days 10–12 maintain an electrophysiologically earlier developmental phenotype in CaV1.3 KO and otoferlin KO mice, neuronal circuitry and nuclei topography are altered in the auditory brainstem [V1.3 KO: p = 0.020; otoferlin KO: p = 0.010) and in the LSO from otoferlin KO  and LSO (p < 0.001) from CaV1.3 KO, but not from the otoferlin KO , however, the coronal MNTB area was not altered  and a very low membrane resistance , which is typical for LSO astrocytes [V1.3 KO: −83.2 ± 4.8 mV, n = 21, p = 0.814; otoferlin KO: −79.0 ± 8.1 mV, n = 17, p = 0.413) or membrane resistance .Neuronal circuitry in both KO models shows impaired development, but it was unknown, if the loss of spontaneous cochlea-driven neuronal activity interferes with astrocyte development. We patch-clamped individual LSO astrocytes and characterized their basic electrophysiological properties. Astrocytes from the WT exhibited a highly negative membrane potential  relationship were designated as nPAs. In turn, astrocytes that primarily expressed ohmic currents and hence displayed a preferentially linear I/V relationship, represented PAs. Both astrocytes subtypes are present in the WT and both KO models  and orientation (α) relative to the dorsoventral axis  and otoferlin KO mice (4/8) were oriented parallel to the tonotopic axis , the WTsotropic Bb,Cb. Hopic axis Bb,Cb,Db.Taken together, our results show that deprivation of spontaneous cochlea-driven neuronal activity does not per se distort astrocyte coupling in the LSO. However, LSO astrocyte network orientation is largely altered.V1.3 KO and an otoferlin KO. As we have previously demonstrated a strong anatomo-functional correlation between neuronal circuitry and glial GJ network topography, we hypothesized that the altered neuronal circuitry in these two mouse models is also reflected in altered astrocytic networks. Our data show that the expression of astrocytic Cx is partially increased in the LSO, but the extent of tracer coupling is not altered. Most GJ networks are still anisotropic, but are oriented along the tonotopic axis in the KOs, thus correlating with the disturbed neuronal circuitry.In the present study, we have investigated the influence of absent spontaneous cochlea-driven neuronal activity on gap junctional tracer coupling in LSO astrocytes. To do so, we used two mouse models of hereditary deafness—a CaV1.3 KO and otoferlin KO mice  times the samples plus 5 μL of the PCR product.At 3–5 days after birth and directly after preparation of brain tissue, a tail biopsy was taken. First, biopsies were digested in 200 μL 25 mM NaOH and 0.2 mM ethylenediaminetetraacetic acid (EDTA) for 1 h at 95° Celcius (C) at 300 rpm in a twitter  to isolate the DNA. Afterwards, 200 μL 40 mM tris(hydroxymethyl)aminomethane (Tris)—HCl, pH 5, was added to neutralize the solution and products were centrifuged for 9–10 min at 15–20 °C at 13,000 rpm . For the following polymerase chain reaction (PCR), 200 μL of the supernatant was decanted, since this contained the DNA. The PCR solution contained the master mix  as well Next, visualization of the DNA bands in the gel was achieved by adding 4 μL sample buffer . Then, 5 μL DNA ladder  was loaded into the first lane of each 1.5% agarose gel (1.5% agarose and 0.001% EtBr diluted in tris-acetate-EDTA buffer). The other lanes were filled with 14 μL of each probe and were run for 30–35 min at 90–95 V. To develop the gel and visualize the bands, gels were put into a chamber .Animal perfusion and tissue preparations were performed as described earlier . The tisn = 3–5/3–8; CaV1.3 KO: n = 19–23/11–12; otoferlin KO: n = 5/6–8. The number of slices used for the analysis of nucleus area is the cumulated number of slices used for Cx43 and Cx30 for each nucleus and genotype. Selection and documentation of slices was done blind. For background correction of signal intensities, negative controls were performed and resulting mean background levels for each nucleus were subtracted.Overview images were documented using a motorized upright widefield microscope  equipped with a DS-Q1Mc camera  and a FITC filter set . All settings were kept constant when comparing immunolabeled areas and stainings. High-resolution images showing the center of auditory brainstem nuclei were taken on a motorized confocal laser scanning microscope . Fluorophores were detected with an Argon laser  in combination with EZ-C1 3.91 Silver Version software . A minimum of 3 slices were analyzed per nucleus and genotype: WT (Cx43/30): 3, 1.25 NaH2PO4, 2.5 KCl, 1 MgCl2, 2 CaCl2, 260 d-glucose, 2 Na-pyruvate, and 3 myo-inositol, pH 7.4, bubbled with carbogen . Thereafter, slices were transferred to artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCl, 25 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 1 MgCl2, 2 CaCl2, 10 d-glucose, 2 Na-pyruvate, 3 myo-inositol, and 0.44 ascorbic acid, pH 7.4, bubbled with carbogen. 270-μm-thick slices were cut using a vibratome . For a priori identification of astrocytes, slices were incubated for 30 min at 37 °C in 0.5–1 μM SR101 dissolved in ACSF and washed for another 30 min at 37 °C in SR101-free ACSF. Afterwards, slices were kept at RT until experiments were performed.Acute coronal brainstem slices were prepared as described earlier . In brieN-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid), 1 MgCl2, 2 Na2ATP, and 0.3 Na2GTP, pH 7.3. The pipette solution additionally contained a cocktail of the GJ-impermeable dye AF568  and the GJ-permeable tracer neurobiotin  to mark the patched cell and label the coupling network, respectively [Whole-cell patch-clamp experiments were performed at RT with an upright microscope equipped with infrared differential interference contrast  and an infrared video camera  using a patch-clamp EPC10 amplifier and “PatchMaster” software . The pipette solution contained (in mM): 140 K-gluconate, 5 EGTA (glycol-bis(2-aminoethylether)-N,N′,N′,N′-tetraacetic acid), 10 HEPES (RIn) consists of membrane resistance (RM) and series resistance (RS) that are arranged in series [U = 5 mV). RIn is given by (Equation (1)):U1 is −85 mV, U2 is −90 mV. I1 and I2 are the recorded steady-state currents at U1 and U2, respectively. RS was calculated by (Equation (2)):U1, U2, and I2 are the same parameters as given in Equation (1) and Ipeak is the maximal current at the initial phase when clamping from U1 to U2. Finally, RM was calculated by (Equation (3); [Astrocytes were patched in the central part of the LSO, where the mediolateral and dorsoventral axes are roughly tangential and orthogonal to the tonotopic axis, respectively. Astrocytes were recorded in voltage-clamp mode and held at −85 mV, which is close to their resting membrane potential ,37. The n series . They weion (3); ):(3)RM=RRS exceeded 15 MΩ to ensure sufficient electrical and diffusional access to the patched cell [I/V relationship. The resulting current traces were sampled at 50 kHz and online filtered at 2.9 kHz. Data were analyzed using “IGOR Pro” software .Measurements were rejected if the hed cell . The liq2HPO4*2H2O, NaH2PO4*H2O; pH 7.4). Membrane permeabilization was achieved by incubation in 0.25% triton X-100 for 30 min. Thereafter, slices were washed again in PBS. Neurobiotin was identified by incubating slices for 3 h with avidin AF488  and slices were washed again. Since tracer coupling differs within and dorsal to the LSO [GJ networks and nucleus boundaries were visualized as described earlier ,9. FixedSR101-labeling, network tracing and immunohistochemical stainings were documented at a confocal microscope  in combination with ZEN software , respectively. Fluorophores were detected as follows (excitation wavelength/filtered emission wavelength): AF488 (488 nm/505–530 nm), AF568 (543 nm/>560 nm), AF647 (639 nm/>640 nm), and SR101 (561 nm/580–620 nm). To improve the quality of confocal micrographs and reduce background fluorescence, a Kalman filter was used . Images were processed using “FIJI” software [R is the quotient of the normalized y value and the normalized x value (Equation (4)):1An, 1Bn, 2An and 2Bn are the number of cells in respective sectors. Then, the coordinate system was rotated and the ratio was recalculated in 15° steps. A sinusoidal function (max = A0 + AR) of the fit reveals the angle of maximal anisotropy of a single network. Therefore, networks were classified into three groups depending on maxR and their preferential orientation α  maxR ≤ 1.1, round and (3) maxR > 1.1 and 0° ≤ α < 45° or 135° < α ≤ 180°, anisotropic and parallel to the tonotopic axis.To avoid unconscious experimenter-based corruption of data, coupled cells were identified using an intensity-based detection method . Only cetation α Da: (1) Rt-tests. Otherwise, results were assessed by a Mann–Whitney U-test. Differences in distribution of classes were analyzed between the WT and the two mouse models using a Χ2 test. p represents the error probability, * p < 0.05, ** p < 0.01, *** p < 0.001. n represents the number of recorded cells or analyzed networks (/slices). In case of multiple comparisons data were statistically analyzed by the tests described above under post hoc Šidák correction of critical values [p < 0.025, ** p < 0.005, *** p < 0.0005.Results are provided as mean ± SD. Data were statistically analyzed using WinSTAT  and tested for normal distribution with the Kolmogorov–Smirnov test. In case of normal distribution, results were assessed by two-tailed, unpaired Student’s l values : two comhttp://creativecommons.org/licenses/by/4.0/).The WT data, as well as


Artemisia species, has inhibitory effects on neuroinflammation and tau phosphorylation. We treated mouse macrophages and microglia cells with lipopolysaccharides (LPSs) to activate inflammatory signals, and we treated neuronal cells with a protein phosphatase 2A inhibitor, okadaic acid (OA), or transfection with pRK5-EGFP-Tau P301L plasmid to induce tau phosphorylation. The results indicated that eupatin significantly reduced the LPS-induced protein expression and phosphorylation of p65 and inducible nitric oxide synthase as well as downstream products interleukin 6 and nitrite, respectively. Furthermore, eupatin markedly inhibited the expression of phospho-tau in response to OA treatment and plasmid transfection. We discovered that this inhibition was achieved through the inhibition of glycogen synthase kinase 3β (GSK3β), and molecular docking results suggested that eupatin can sufficiently bind to the GSK3β active site. Our results demonstrate that eupatin has neuroprotective effects, making it suitable for AD treatment.Alzheimer’s disease (AD), which is among the most prevalent neurodegenerative diseases, manifests as increasing memory loss and cognitive decline. Tau phosphorylation and aggregation are strongly linked to neurodegeneration, as well as associated with chronic neuroinflammatory processes. The anti-inflammation effects of natural products have led to wide recognition of their potential for use in treating and preventing AD. This study investigated whether eupatin, a polymethoxyflavonoid found in  N-methyl-d-aspartate receptor antagonist; however, these agents only provide symptomatic relief, and their benefits remain limited [Alzheimer’s disease (AD) is a common progressive neurodegenerative disease and constitutes a global health concern. Approximately 50 million people have dementia worldwide, and this number is expected to nearly triple by 2050 . AD caus limited ,4. ThereStudies have indicated that extracellular amyloid β (Aβ) plaques and intracellular tau neurofibrillary tangles (NFTs) are two neuropathological hallmarks of AD. Evidence suggests that a crosslink exists between these two markers, although the detailed mechanisms remain unclear . Aβ formArtemisia annua L., commonly known as Qinghao, has traditionally been used for treating chills and fever in China. A. annua and other Artemisia species are traditional components of treatment for febrile conditions, inflammation, and pain [Artemisia species, has been recognized to possess nitric oxide–reducing and acetylcholinesterase-inhibitory effects [For many years, flavonoids have been used in a variety of disease treatments. Their great diversity and distribution class them as a good choice among the available therapeutic agents. As one of the main components used for synthesizing various drugs that also have potential for use with natural products  , flavonoand pain . Eupatin effects . HoweverThe structure of eupatin is displayed in The results indicated that LPS treatment significantly increased the phosphorylation of p65 , therebyOkadaic acid (OA) potently inhibits protein phosphatase 2A (PP2A) and is capable of inducing in vitro and in vivo AD-like tau hyperphosphorylation ,16. We oIn addition, Neuro-2a cells transfected with pRK5-EGFP-Tau P301L plasmid, which encodes human mutant P301L-tau, caused tau phosphorylation at multiple sites, including Ser262, Ser202/Thr205 and Ser396 . This moGSK3β is a major kinase involved in aberrant tau hyperphosphorylation . GSK3β a0–t of casticin, a methoxy-derivative in chromone ring of eupatin, the oral administration 400 mg/kg and intravenous injection 50 mg/kg in rat are 18,652.72 ± 4030.88 ng h/mL and 41,225.92 ± 1403.37 ng h/mL, respectively [Neuroinflammation is known to contribute to the neuronal damage that underpins neurodegenerative disorders such as AD. Several flavonoid compounds have undergone comprehensive investigation and been recognized for their potential for preventing and treating neurodegenerative diseases . The mosectively . The meaTau is known to play a key role in microtubule assembly and stabilization. One study indicated that phosphorylation is central to the regulation of tau’s physiological functions, such as microtubule binding, and is thus responsible for regulating microtubule stabilization and assembly . ResearcEupatin was obtained from the National Cancer Institute (NCI No. 122412). We purchased primary antibodies against p-GSK3β (Tye216) and p-p65 (Ser536) from Cell Signaling Technology . We obtained antibodies against p-tau (Ser396) from Abcam  and those against p-tau (Ser202/Thr205), p-tau (Ser262), and tau w from Thermo Fisher Scientific . Antibodies against GSK3β and α-tubulin were obtained from GeneTex Inc. , and antibody against iNOS was purchased from Santa Cruz Biotechnology . Jackson ImmunoResearch Inc.  was our source for horseradish peroxidase (HRP)-conjugated antimouse and antirabbit immunoglobulin G secondary antibodies, and OA was purchased from Cayman Chemical Company . Unless otherwise stated, we obtained all chemicals not mentioned here from Sigma-Aldrich .2 air atmosphere.Murine RAW264.7 cells, a mouse macrophage cell line, were obtained from the Bioresource Collection and Research Center . Mouse BV-2 microglia were kindly provided by Prof. Shiow-Lin Pan . Dulbecco’s modified Eagle’s medium (DMEM)  with supplementation of 100 μg/mL streptomycin , 10% (v/v) fetal bovine serum , and 100 U/mL penicillin was used for cell culturing. We obtained the murine neuroblastoma cell line (Neuro-2a) from the Bioresource Collection and Research Center. After culturing the cells in minimum essential media consisting of streptomycin (100 μg/mL), penicillin (100 units/mL), and 10% fetal bovine serum, we incubated them at a temperature of 37 °C in a humidified 5% CO4 cells were incubated in 96-well plates in 1 mL of medium with the control vehicle or vehicle containing the experimental compound. The next step after the application of different treatments was the addition of MTT (1 mg/mL) and incubation of the plates at 37 °C for another 2 h. We then pelleted and lysed the cells in 10% sodium dodecyl sulfate (SDS) with 0.01 M HCl and used a microplate reader to measure the absorbance at 570 nm.Colorimetric MTT assay was employed for measuring cell cytotoxicity. For 48 h, 1 × 106 cells in 6-well plates and stimulating them using 100 ng/mL LPSs for 24 h, we mixed a Griess reagent with the cell supernatant (100 μL each) and measured the optical density at 550 nm. A standard curve based on obtained concentrations of sodium nitrite dissolved in DMEM was generated for calculating the nitrite concentration.Nitrite production was measured in RAW264.7 macrophage and BV-2 microglia cell supernatants. In brief, after culturing 1 × 10Thirty-minute incubation of the cells with the experimental compounds was applied prior to and during 24-h incubation with LPSs (100 ng/mL). After collection of the medium, an enzyme-linked immunosorbent assay kit  was used to perform an assay for IL-6.2 atmosphere.Cells were seeded 1 day before transfection. After 20 min of mixing 1 μL of TurboFect transfection reagent and pRK5-EGFP-Tau P301L plasmids (1 μg) at room temperature and addition of the mixture to the cells, the suspensions underwent 24-h incubation at 37 °C in a humidified 5% CO6 cells were incubated in lysis buffer . Next, the cells were removed, placed on ice for 10 min, and subjected to 30-min centrifugation  at 4 °C. Next, we electrophoresed 20-μg protein samples on SDS polyacrylamide gels before transferring them onto a nitrocellulose membrane. The nitrocellulose membrane was subsequently blocked through 30-min incubation with 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20 (TBST) at room temperature. Immunoblots were obtained through incubation overnight at 4 °C with primary antibodies in TBST and subsequent 1-h incubation at room temperature with secondary antibodies conjugated with HRP. Measurement of antibody binding was performed through photographic film exposure and application of an enhanced chemiluminescence reagent .For 10 min at 4 °C, 1 × 10Molecular docking analysis was performed using the Maestro docking module (Schrödinger). The GSK3β crystal structure was obtained from the Protein Data Bank (PDB ID: 1UV5) . The cocp < 0.05.One-way analysis of variance was used for analyses, and data are presented as mean ± standard error of the mean. When significant differences were detected between groups, Tukey’s post hoc test was applied for identifying the significantly different group pairs. The significance level for parameters was set at Eupatin treatment significantly inhibited LPS-triggered p65 activation and inflammatory factors production, and markedly reduced tau phosphorylation on Ser262, Ser202/Thr205, and Ser396. Our novel findings indicate that eupatin has neuroprotective potential against AD.


EMD) cause Emery–Dreifuss muscular dystrophy (EDMD1), an inherited disorder characterized by progressive skeletal muscle wasting, irregular heart rhythms and contractures of major tendons. The skeletal muscle defects seen in EDMD are caused by failure of muscle stem cells to differentiate and regenerate the damaged muscle. However, the underlying mechanisms remain poorly understood. Most EDMD1 patients harbor nonsense mutations and have no detectable emerin protein. There are three EDMD-causing emerin mutants  that localize correctly to the nuclear envelope and are expressed at wildtype levels. We hypothesized these emerin mutants would share in the disruption of key molecular pathways involved in myogenic differentiation. We generated myogenic progenitors expressing wildtype emerin and each EDMD1-causing emerin mutation  in an emerin-null (EMD−/y) background. S54F, Q133H, and Δ95–99 failed to rescue EMD−/y myogenic differentiation, while wildtype emerin efficiently rescued differentiation. RNA sequencing was done to identify pathways and networks important for emerin regulation of myogenic differentiation. This analysis significantly reduced the number of pathways implicated in EDMD1 muscle pathogenesis. Mutations in the gene encoding emerin ( X-linked Emery–Dreifuss muscular dystrophy EDMD1) is an inherited disorder caused by mutations in EMD, which encodes emerin. EDMD1 is characterized by progressive skeletal muscle wasting, irregular heart rhythms, and contractures of major tendons  is an in,3,4. IneEmerin residues 46–221 is intrinsically disordered ,14 and c−/y mice revealed compensatory upregulation of skeletal muscle regeneration genes [Rb) and MyoD genes were altered. Rb protein was found to be inappropriately hyperphosphorylated at key developmental stages, including when myoblasts exit the cell cycle to commit towards differentiation [−/y myogenic progenitors display impaired myogenic differentiation and fail to fuse into myotubes [MyoD, Myf5, Pax3, and Pax7, is disrupted in EMD−/y myogenic progenitors [Emerin knockout mice display delayed skeletal muscle regeneration and repair, mild atrioventricular alterations, and motor coordination defects ,28. Biopon genes ,29. In pntiation ,29. Emermyotubes ,31,32, dmyotubes  and pertmyotubes ,35. The genitors  due to tgenitors ,29,35.Pax3, Pax7, MyoD, and Myf5 genes, and rescued myotube formation in EMD−/y myogenic progenitors [−/y myogenic progenitors [−/y myogenic progenitors [Emerin interacts directly with the core components of the nuclear corepressor (NCoR) complex, which is involved in repressing genes by stably binding chromatin . HDAC3 igenitors ,36. Inhigenitors . Recent genitors . These s−/y myogenic progenitors exhibited impaired differentiation [−/y progenitors failed to exit the cell cycle appropriately, resulting in delayed myoblast commitment and inhibition of myoblast formation. RNA sequencing (RNAseq) showed that EMD−/y myogenic progenitors failed to completely transcriptionally reprogram upon differentiation induction, which signals the progenitors to exit the cell cycle and commit to myotube formation. More than 1600 genes were differentially expressed in EMD−/y myogenic progenitors at this important differentiation transition [−/y progenitors. Studying differentiation in myogenic progenitors containing EDMD1-causing emerin mutants was predicted to narrow down the potential genes and pathways responsible for EDMD pathogenesis. Here we show, for the first time, that EDMD1-causing emerin mutant myogenic progenitors exhibit impaired differentiation. Transcriptional profiling of these EDMD1-causing myogenic progenitors during differentiation significantly narrowed the pathways implicated in the muscle regeneration pathology of EDMD1.We previously showed that EMDntiation ,34,35. E−/y mice were obtained from Tatiana Cohen and Terence Partridge  [l-glutamine  and 20 units/mL γ-interferon . Proliferating cells were plated on gelatin at a density of approximately 650 cells/cm2 and grown at 33 °C and 10% CO2. Differentiating cells were plated on gelatin at a density of 25,000 cells/cm2 in proliferative conditions for 24 h, then switched to differentiation media consisting of DMEM supplemented with 5% horse serum  and 2% l-glutamine, and grown at 37 °C and 5% CO2. Cells between passage six and twelve were used for all analyses.Myogenic progenitors from H2K Wildtype and EMDWA, USA) . ProlifeWA, USA) . Prolife−/y mouse myogenic progenitors (EMD−/y) were seeded at a density of 1000 cells/well in 96-well plates coated with 0.01% gelatin. Cells were incubated at 33 °C and 10% CO2 overnight in proliferation media and replaced with infection medium containing lentiviral particles  at a multiplicity of infection of 350 and 8 µg/mL polybrene . Polybrene is a cationic polymer known to increase lentiviral transduction efficiency [−/y cells transduced with control vector, S54F and Δ95–99 were selected using 15 µg/mL puromycin. EMD−/y cells transduced with Q133H and M179 vectors were selected using 10 µg/mL puromycin. EMD−/y cells transduced with wildtype emerin (+EMD) was selected using 6 µg/mL. Once the cells were 40–60% confluent, cells were subsequently moved to six-well dishes, followed by transfer to 10 cm dishes, and finally expanded to multiple 15 cm dishes. Cells were kept sparse to ensure cells don’t undergo spontaneous differentiation or senescence. Media containing puromycin was replaced every 2–3 days.H2K myogenic progenitors expressing wildtype emerin (+EMD) and EDMD causing emerin mutations , an emerin mutation that does not cause the disease (M179), and a vector only control were generated using the following protocol. EMDficiency  by neutrficiency ,41. The –/y cells were seeded at a density of 7800 cells per well into 96-well plates or 92,800 cells per well into 12-well plates coated with gelatin. For experiments in 12-well plates, +EMD, Q133H and Δ95–99 cells were plated at a density of 118,800 cells per well coated with gelatin. For 96-well plate experiments, +EMD, vector control, S54F and M179 were plated at a density of 10,000 cells per well coated with gelatin. Cells were incubated overnight in proliferation media. Differentiation was induced by replacing proliferation media with differentiation media  and incubating cells at 37 °C and 5% CO2.Wildtype and EMDEdU  was used as a proliferation marker. EdU is a thymidine analog which gets incorporated into newly synthesized DNA during S phase of the cell cycle. Ten micromolar EdU was incubated with cells for 2 h prior to fixing. Cells were fixed at 0, 24, 48, and 72 h post differentiation induction. After fixing, cells were washed three times and were processed according to the manufacturer’s instructions . Cells were treated with 0.5% Triton-X100 in phosphate-buffered saline (PBS) for 20 min, washed twice with 3% bovine serum albumin in PBS, and treated with a Click-IT EdU reaction cocktail for 30 min. Cells were washed with PBS and then blocked for an hour with 3% BSA in PBS containing 0.1% Triton-X100.Cells were then treated with emerin antibodies  for an hour, washed three times with PBS, and incubated with monoclonal antibodies against MyHC  for an hour. Cells were washed three times with PBS, incubated with secondary antibodies Alexa Fluor 488 goat anti-rabbit  for one hour followed by Alexa Fluor 594 goat anti-mouse antibodies  for one hour. Cells were washed twice with PBS and incubated with 0.2 µg/mL DAPI to label nuclei. Cells were washed once with PBS and imaged using the EVOS-FL Auto microscope . Multiple images were acquired at random (at least three) for each well using a 40× objective. A total of three wells were analyzed for each treatment for a given biological replicate. At least two biological replicates were done for each treatment. Each field was imaged in four different channels: green fluorescent protein channel for emerin positive nuclei, blue fluorescent channel for DAPI stained nuclei, Cy5 channel for Edu positive nuclei, and Texas Red channel for MyHC positive nuclei. Image analysis was done using the cell counter plugin in ImageJ. Cell cycle withdrawal was determined by dividing the total number of cells expressing EdU by the total number of nuclei in an image. Differentiation index (DI) was defined as the percentage of nuclei expressing the terminal differentiation MyHC. Myotube formation was determined by measuring the percentage of myotubes containing at least three nuclei and expressing MyHC.Proliferating H2K myogenic progenitors in 15 cm dishes were harvested using SDS-PAGE sample buffer. A total of 50,000 cell equivalents for all cell lines except for +EMD and M179 expressing cells  were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. Blocking of the membrane was done using 5% dried nonfat milk in PBS containing 0.1% Tween 20 (PBST) at room temperature for an hour. The membrane was then incubated with emerin antibodies  for an hour followed by three washes in PBST for five minutes. The membrane was then incubated for an hour at room temperature with goat anti-rabbit HRP antibodies , followed by three PBST washes for five minutes each. The blot was incubated with Amersham ECL western blotting detection reagents  and imaged on the Bio-Rad Chemidoc equipment . The HRP enzyme was then inactivated with 0.1% sodium azide in PBST for 15 min followed by five washes (five minutes each) with PBS. The blot was incubated with mouse gamma-tubulin antibodies  for an hour at room temperature followed by incubation with donkey anti-mouse HRP antibodies  for an hour at room temperature. The blots were then incubated with Amersham ECL western blotting detection reagents  and processed according to the manufacturer’s instructions. ImageLab software (Bio-Rad Laboratories) was used to perform volume analysis.n = 2). Cells were trypsizined, washed three times with PBS, and total RNA was isolated using the mRNeasy Plus Kit  per manufacturer’s instructions.For isolation of total RNA during proliferation, each H2K myogenic progenitor cell line was seeded at a density of 158,000 cells per 15 cm dish coated with 0.01% gelatin and incubated in proliferative media for two days. Two 15 cm dishes were combined and represent one technical replicate. Three technical replicates per sample were performed for each biological replicate , 24 h, 48 h, and 72 h of differentiation. RNA was isolated using the mRNeasy Plus Kit  per manufacturer’s instructions. Three technical replicates per sample were performed for each biological replicate (n = 2).Wildtype and EMDq-value <0.05. The q-value is a p-value corrected for the False Discovery Rate (FDR) and represents the standard in the field for determining significance. All differentially expressed transcripts were also ≥2-fold increased or decreased. KEGG pathway enrichment analysis was done using phyper, a function of R.Purity of RNA was assessed using Nanodrop (ND-2000), and quantification was done using the Quant-IT RNA assay kit . BGI Genomics performed library preparation and sequencing using BGISEQ-500 platform. BGI Genomics filtered reads using SOAPnuke and genome mapping was done to the mouse genome (mm10) using hierarchical indexing for spliced alignment of Transcripts-2 (HISAT2). Differentially expressed genes was detected using DEseq2. Transcripts were considered to be significantly differentially expressed if the RNA sequencing data is available through the NCBI Gene Expression Omnibus (Accession number GSE152226).−/y myogenic progenitor lines expressed wildtype emerin , Δ95–99 (0.99-fold), S54F , and M179 (0.76-fold) at levels similar to wildtype myogenic progenitors. EMD−/y myogenic progenitor lines expressing Q133H had 2.38-fold more protein. EMD−/y and vector alone failed to express any emerin, as expected , +EMD, M179, and S54F myogenic progenitor lines were induced to differentiate by serum withdrawal. Cell cycle withdrawal was analyzed after 24 h. 9.1% of wildtype myogenic progenitors incorporated EdU (−/y and 17.1% of control cells incorporated EdU (p < 0.02). In total, 14.4% of +EMD cells incorporated EdU. In total, 26.4% of M179 and 20.8% of S54F progenitors incorporated EdU  and 40.2% of vector-alone cells (p < 0.02) expressed MyHC after 48 h of differentiation. In total, 61.3% of wildtype cells expressed MyHC. In total, 55.7% of +EMD cells expressed MyHC, demonstrating successful rescue of myoblast commitment. Expression of M179 and S54F in EMD−/y progenitors failed to rescue MyHC expression and the differentiation index, with 32.4% and 37.9% expressing MyHC, respectively (p < 0.02).In total, 44.6% of EMD−/y and control cells, as only 19.2% of EMD−/y cells and 19.4% of control cells (p < 0.02) formed mature myotubes compared to 35.4% of wildtype cells. Wildtype emerin expression rescued myotube formation, as 35.8% of +EMD cells formed myotubes. Both M179 and S54F cells were significantly impaired in myotube formation, as only 19.5% of M179 cells and 21.5% of S54F cells fused to form mature myotubes (p < 0.02).We next analyzed myotube formation, which was defined as the percentage of myotubes containing at least three nuclei and expressing MyHC. Myotube formation was impaired in EMD−/y cells maintained a significantly higher percentage of cells in the cell cycle  compared to wildtype cells  was significantly lower than wildtype progenitors (58.0%) after 48 h of differentiation (p < 0.02). In total, 58.1% of +EMD progenitors expressed MyHC after 48 h of differentiation. Both Q133H and Δ95–99 progenitors failed to commit to differentiation, as 20.6% of Q133H cells and 21.2% of Δ95–99 cells expressed MyHC (p < 0.02).As expected, the differentiation index of EMD−/y cells failed to form myotubes (16.4%), as compared to wildtype cells . In total, 39.9% of +EMD progenitors formed myotubes after 72 h. Only 3.2% of Q133H progenitors and 6.7% of Δ95–99 progenitors fused to form mature myotubes (p < 0.02), demonstrating a massive failure in myotube formation and maturation in these EDMD-causing emerin mutants.After 72 h of differentiation, EMD−/y, and each emerin mutant myogenic progenitor line throughout differentiation using Beijing Genomic Institute (BGI) Americas. Differentially expressed genes were identified by BGI by comparing all datasets to each other. The first comparison compared the changes in transcript expression at each day of differentiation for each EDMD-causing emerin mutant or EMD−/y myogenic progenitors to wildtype progenitors. Transcripts were considered to be significantly differentially expressed if the q-value <0.05. It was expected that the expression of a large number of differentially expressed transcripts would be seen during differentiation of each EDMD-causing mutant based on previous studies [RNA sequencing (RNAseq) was done on wildtype, EMD studies .−/y progenitors plated at high-density for differentiation  were identified in EMDn day 0, A. S54F, n day 0, A. There n day 0, A. In totn day 1, A. S54F, n day 1, A. There s day 2, A. S54F, s day 2, A. There −/y, S54F, Δ95–99, Q133H, and M179 differentially expressed 795 genes , 1111 genes , 1464 genes , 1287 genes , and 1647 genes  when actively proliferating, respectively , 1279 genes , 2441 genes , 3792 genes , and 2526 genes  in mature myotubes, respectively . In total, 2440 transcripts showed altered expression in EMD−/y cells during the transition from day 1 to day 2 of myogenic differentiation , with 1836 transcripts also altered during wildtype differentiation and 604 transcripts uniquely altered in EMD−/y cells. Of the 2204 transcripts differentially expressed during S54F differentiation days 0 and 1, 1542 transcripts were also altered during wildtype differentiation and 662 transcripts were uniquely altered in S54F cells . In total, 2738 transcripts showed altered expression in S54F cells during the transition from day 1 to day 2 of myogenic differentiation , with 1681 transcripts also altered during wildtype differentiation and 1057 transcripts uniquely altered in S54F cells. Of the 1866 transcripts differentially expressed during Δ95–99 differentiation days 0 and 1, 1152 transcripts were also altered during wildtype differentiation and 714 transcripts were uniquely altered in Δ95–99 cells . In total, 1897 transcripts showed altered expression in Δ95–99 cells during the transition from day 1 to day 2 of myogenic differentiation , with 1054 transcripts also altered during wildtype differentiation and 843 transcripts uniquely altered in Δ95–99 cells. Of the 1405 transcripts differentially expressed during Q133H differentiation days 0 and 1, 805 transcripts were also altered during wildtype differentiation and 600 transcripts were uniquely altered in Q133H cells . In total, 1261 transcripts showed altered expression in Q133H cells during the transition from day 1 to day 2 of myogenic differentiation , with 674 transcripts also altered during wildtype differentiation and 587 transcripts uniquely altered in Q133H cells. To identify genes implicated in the impaired differentiation of EDMD, transcripts differentially expressed between wildtype progenitors and each EDMD-causing emerin mutant progenitor line and EMD−/y cells were compared during these transitions. This identified 64 transcripts and 40 transcripts differentially expressed in each EDMD mutant and EMD−/y cells during transitions day 0 to day 1 and day 1 to day 2, respectively  in an EMDactivity , histoneactivity , and inhactivity . Collectactivity ,35,36,38The genome undergoes dynamic reorganization during stem cell differentiation and development to regulate the coordinated temporal expression of differentiation genes ,43,44. I−/y myogenic progenitors [HDAC3 is the only reported binding partner that is altered in all four “special” EDMD-causing emerin mutations  . This higenitors . It will−/y progenitors. Δ95–99 myogenic progenitors were similar to EMD−/y cells in their cell cycle exit. We predict this failure of M179, S54F, and Q133H progenitors to exit the cell cycle is caused by binding to germ cell-less (GCL). GCL is a transcriptional repressor that binds the DP3 subunit of the E2F–DP3 heterodimer to inactivate E2F–DP3-mediated transcription [Emerin mutants M179, S54F, and Q133H all showed an increased number of cells cycling 24 h after differentiation induction compared to EMDcription  and inhicription . We predcription .Entry into the cell cycle is regulated by the G1/S checkpoint, which is also called the restriction point ,57. Pass−/y mice is the primary driving force for passage through the restriction point in EMD−/y mice, as first described by Melcon and colleagues [−/y mice, which have surprisingly mild phenotypes [−/y progenitors expressing wildtype emerin. GCL binding sites at the nuclear envelope would not be saturated because HDAC3 and GCL compete with each other for emerin binding. In this model there would be insufficient recruitment of GCL to alter the dynamics of E2F–DP-mediated transcription activation in the presence of high levels of Rb and hyperphosphorylated Rb in EMD−/y cells. Δ95–99 fails to bind GCL and HDAC3, so this mutant behaves just like the EMD−/y parental cell line. In M179-, S54F-, or Q133H-expressing EMD−/y progenitors, these mutants retain their ability to bind GCL but fail to bind HDAC3. Thus, emerin can bind more GCL, thereby releasing more GCL from E2F–DP to further increase the expression of E2F–DP-regulated genes that allow for passage through the restriction point.In our model, increased hyperphosphorylated Rb in EMDlleagues . Here, ienotypes ,28. In o−/y cells alters Rb phosphorylation dynamics will also need to be tested.We predict HDAC3 also plays a role in repressing transcription of cell cycle genes, but it is unclear whether it acts with Rb or GCL to repress E2F-regulated transcripts in cycling cells or if it acts to stably repress E2F-regulated chromatin in permanently arrested cells, like myotubes. Our data would suggest the latter. Thus, it will be important to test whether HDAC3 association with emerin plays a role in the compensatory mechanisms predicted to help stabilize repressed chromatin containing E2F-regulated genes, and if this is required for permanent cell cycle arrest. It will also be important to determine the dynamics of GCL and HDAC3 competition for emerin binding and how this affects passage through the restriction point. Whether expression of wildtype emerin in EMD−/y cells. S54F and M179 were similar to EMD−/y cells in differentiation index and myotube formation. Both S54F and M179 bind normally to lamin A, whereas Q133H and Δ95–99 disrupt binding to lamin A [−/y cells that may involve binding to and repressing an unknown partner at the inner nuclear membrane. In this functional redundancy model, expression of the Q133H and Δ95–99 mutants in the EMD−/y cells specifically alters binding to this unknown partner and more of the partner is now active to negatively repress myogenic differentiation. Alternatively, release of this partner from emerin now allows it to bind to Lap2β or Lap1 and displaces other binding partners on these proteins that negatively impact myogenic differentiation.Emerin mutants Q133H and Δ95–99 had less MyHC expression after 48 h and formed fewer myotubes after 72 h of differentiation than EMD lamin A . We spec lamin A , which m lamin A ,75, whic lamin A , suggest lamin A ,29 and L lamin A . Lap2β i lamin A . Both th lamin A ,77. It iYap1) and Taz (Wwtr1), primarily regulate gene expression by binding Tead1–4 transcription factors. Together Yap, Taz, and Teads form the core of the Hippo signal transduction network, which includes the Hippo kinase cascade, comprising kinases Mst1 (Stk4), Mst2 (Stk3), Lats1, and Lats2 [The transcriptomic analysis of the EDMD-causing emerin mutants uncovered pathways implicated in myogenic differentiation. Members of the Hippo family play vital roles in skeletal muscle development , organ snd Lats2 ,81,82. Ynd Lats2 ,84,85. Ind Lats2 ,85,86. Snd Lats2 ,88,89. Ynd Lats2 . Our anand Lats2 . TherefoThere is increasing evidence suggesting extracellular matrix (ECM) components are essential signaling mediators in the satellite cell niche, both for the maintenance of satellite cell identity and regulation of its activation ,92. HepaApelin is locally produced by skeletal muscle fibers in response to exercise and acts locally to improve muscle metabolism and function . Apelin These studies show, for the first time, that emerin mutations found in EDMD patients cause impaired myogenic differentiation. The impaired differentiation is likely caused by loss of HDAC3 binding, since these mutants share in their failure to bind HDAC3. The use of these individual mutants allowed for a robust approach to identifying pathways implicated in the impaired differentiation in EDMD. Using this approach, we narrowed the implicated pathways to four to six pathways. Future studies to independently test each of these pathways will be needed to confirm their involvement in the impaired differentiation seen in EDMD.