Methyl Jasmonate (MeJA) could promote the opening of sorghum florets, but the molecular mechanism remains unclear.We aimed to investigate the molecular mechanism of exogenous MeJA in promoting the opening of sorghum florets.Hybrid sorghum Aikang-8 was selected as the test material in this study. Sorghum plants of uniform growth with approximately 20%-25% florets open were selected and treated with 0, 0.5 and 2.0 mmol/L of MeJA. Totally there were 27 samples with lodicules removed were obtained at different time points and used for the transcriptome analysis using the BGISEQ_500RS platform.LOC-1 (LOC8063704) and LOC-2 (LOC8076139) could induce the earlier flowering of Arabidopsis thaliana.The results showed the sorghum florets opened earlier than the control after the treatment with exogenous MeJA, and the promotive effect increased along with the increase of exogenous MeJA concentration. The number of differentially expressed genes (DEGs) in plasma cells increased with the increase of MeJA concentration, whether up- or down-regulated, after the exogenous MeJA treatment. Besides, the number of metabolic pathways was also positively correlated with the concentration of MeJA. GO and KEGG analysis suggested the DEGs were mainly enriched in starch and sucrose metabolism-related pathways , plant hormone signal transduction pathways , energy metabolic pathway  and the α-linolenic acid metabolic pathway . Functional analysis of target genes showed that two genes named The results of this study suggest that exogenous MeJA treatments could induce the up- or down- regulation of genes related to starch and sucrose metabolism, -linolenic acid metabolism and plant hormone signal transduction pathways in the plasma cells of sorghum florets, thereby promoting the opening of sorghum florets. So the research of floret opening became increasingly important in sorghum for the growing demand of people. Different species and varieties, and even the flowering time of spikelet at different positions on the same spike in one day are also different, which are affected by the characteristics of varieties and external environment factors . At pres unknown .Sorghum bicolor L. Moench and Sorghum Sudanesis (Piper) Stapf was significantly stimulated by immersing into 2 mM methyl jasmonate (MeJA) solution [Floret opening in grass plants is mainly caused by the swelling of a pair of lodicules at the base of the floret after water absorption . In thissolution . So it iRNA-seq is an very effective tool for studying the regulatory mechanism in plants, such as wheat , rice 1, cotton S. bicolor L. Moench) cultivar Aikang-8 was purchased from Shandong Ruiyou Agricultural Science and Technology Development Co., Ltd. in China and used to conduct the experiment. Aikang-8 seeds were planted in the training base of the Jiangxi Agricultural Engineering College On May 1st and May 10th for two times. Each time Aikang-8 was planted for 40 m2 with normal cultivation managements. The MeJA  was purchased from BOMEI (China). It was diluted into 10.0 mmol/L mother liquor with 1.0% ethanol, followed by further dilutions with 1.0% ethanol to 0.5 and 2.0 mmol/L. 1.0% alcohol was used for the blank control. Arabidopsis thaliana of wild-type was planted in small pots with a uniformly mixed mixture of peat, vermiculite, perlite by the ratio of 2:7:1 in growth chamber, respectively. The humidity was set at 60% and the temperature was set at 20–22°C. The photoperiod was 24h and the illumination intensity was set 80–200 μmol·M-2·S-1. Agrobacterium-mediated genetic transformation method was used to infect the inflorescence of Arabidopsis thaliana for 2–3 times. Arabidopsis plants were moved to dark environment for 16-24h after the infection. Arabidopsis seeds were harvested and used for the screening of transgenic plants.In the study, sorghum  and 20:30 (2.5h) were labeled as SorgHM_1 and SorgHM_2, respectively; samples treated with 0.5 mmol/L MeJA at 19:00 (1h), 20:30 (2.5h) and 22:30 (4.5h) were labeled as SorgLM_1, SorgLM_2 and SorgLM_3, respectively; and samples treated with 0 mmol/L MeJA at 19:00 (1h), 20:30 (2.5h), 22:30 (2.5h) and 0:30 (6.5h) were labeled as SorgCK_1, SorgCK_2, SorgCK_3 and SorgCK_4, respectively. Each sample was repeated three times. Therefore, there were 27 samples were obtained with lodicules removed, followed by the storage in cold-proof RNase-free tubes at -80°C. The weight of each sample was greater than 0.13 g.Total RNAs of lodicules were extracted using pBIOZOL method, and RNA concentrations were measured using Nanodrop 2000, followed by RNA integrity test using 1.0% agarose gels. Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit) was used to detect the concentrations, RIN values, 28S/18S values and fragment sizes of the total RNAs. The results indicated that 27 samples were qualified for library construction. The same amount of RNA from each of the three replicates at each time point were collected and uniformly mixed into nine samples for the sequencing and cDNA library construction. Nine libraries were named libSorgHM_1, libSorgHM_2, libSorgLM_1, libSorgLM_2, libSorgLM_3, libSorgCK_1, libSorgCK_2, libSorgCK_3 and libSorgCK_4, respectively. Library was constructed as follow procedures: Magnetic beads with oligo (dTs) were used to enrich for mRNAs having polyA tails. A DNA probe was hybridized with the rRNA, and RNaseH was used to selectively digest DNA/RNA hybrids. Then, the DNA probe was digested using DNaseI. Target RNA was obtained through purification, and interruption buffer was used to obtain RNA fragments. This was followed by reverse transcription using a random N6 primer. Double-stranded DNA was obtained through the synthesis of double-stranded cDNA. The 5’-end of the synthesized double-stranded DNA was phosphorylated, with an “A” tail at its 3’ end. A connector with a “T” bulge at its 3’ end was ligated as well. The ligated product was PCR amplified using specific primers. The PCR product was heated and denatured into single strands. The obtained single-stranded DNA was circularized using a bridge primer to obtain a single-stranded circular DNA library for further sequencing. Library construction and sequencing were performed by BGI, China.https://www.ncbi.nSorgLM.nih.gov/genome/?term=Sorghum%20bicolor%20) [The filtration of raw reads was performed using SOAPnuke (v1.5.2)  by BGI, olor%20) . The Mapolor%20) ,23. The 2ratio| values ≥ 1. The up- and down- regulation modes of the corresponding samples were described and analyzed. The DEG sequences were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database using BLAST (parameter: -p blastx-e1e-5-m8) [p-value < 0.05 were considered as significant enrichments.Clean reads were mapped to the reference sequence using Bowtie2 , and gen1e-5-m8) . An enri1e-5-m8)  and GO t® Select Master Mix (2X) reagents (ABI Inc.) and qRT-PCR. GAPDH (AK064960) was selected and used as the reference gene. The relative expression analysis of selected genes was calculated using the 2−ΔΔCt method with triplicate for each sample [The extracted RNAs were used as templates for reverse transcription with a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) and several DEGS were randomly selected. The expression levels of genes were verified using SYBRh sample ,30.LOC-1 and LOC-2 were cloned with cDNA by PCR method using 50 μl reaction system, containing 10×PCR Buffer 5 μl, MgCl2 (25 mM) 3 μl, dNTP mixture (2.5 mM) 4 μl, forward primer (10 μM) 1 μl, reverse primer (10 μM) 1 μl, Taq (5U/μl) 0.5μl, cDNA 2μl, ddH2O 3.5μl. After the purification, PCR products were linked to pMD19-T Vector and transformed into DH5α according to the optimized procedure. At the same time, vector pCAMBIA1300 and pBI121 were digested with EcoRI and HindIII simultaneously. Klenow fragment of pCAMBIA1300 and small fragment of PBI121 were recycled and linked to construct an integrated expression vector pCAMBIA1300-BI containing 35S promoter, GUS gene and terminator. Then the GUS gene was replaced with two target gene LOC-1 and LOC-2, which were added CaMV35s promoter and Nos terminator at 5’ and 3’ end, respectively. The constructed plasmid was transformed into Arabidopsis thaliana to verify its functions [Two target genes unctions .After the treatment at 18:00 PM, the florets did not open until 19:00 after treatment with the three MeJA concentrations. At 20:30 (2.5h), a large number of sorghum florets started to open under the treatment of 2.0 mmol/L MeJA, while other sorghum florets didn’t open in abundance. At 22:30 (4.5h), a large number of sorghum florets started to open under the treatment of 0.5 mmol/L MeJA while the control sorghum florets (0 mmol/L MeJA) still didn’t open in abundance. At 0:30 (6.5h), a large number of control sorghum florets started to open . BesidesAs shown in As shown in LOC8075740, LOC8056099, LOC8064482, LOC8058060, LOC110437037 and LOC8069151 were involved in starch and sucrose metabolism, and LOC8084355 was involved in hormone signal transduction pathways and LOC110429834 was involved in the α-linoleic acid metabolic pathway and LOC8072504 was involved in the regulation of plant circadian rhythm.To further verify the reliability of the transcriptome results, 17 genes with significantly different expression levels were selected  for fluoBased on the results of DEGs detection, biological pathway classification and enrichment analysis was performed in KEGG. Total of 29,135 unigenes were used to blast in the KEGG database, and finally 20,803 (70.96%) unigenes were annotated . In the At the same time point, the number of enriched metabolic pathways and DEGs increased along with the increase of MeJA concentration. For example, the number of enriched metabolic pathways and DEGs in SorgCK_1-VS-SorgHM_1 were greater than that in group SorgCK_1-VS-SorgLM_1. And the number of enriched metabolic pathways and DEGs in group SorgCK_2-VS-SorgHM_2 was greater than that in group SorgCK_2-VS-SorgLM_2.In order to dig out the pathways which were most closely associated with spikelets opening, screening was performed with the following rules: 1) total number of DEGs > 3,000; 2) number of enriched metabolic pathways > 100; and 3) Q value < 0.05. Finally four comparison groups met the criteria, including SorgCK_3-VS-SorgLM_3, SorgCK_2-VS-SorgHM_2, SorgLM_1-VS-SorgLM_3 and SorgLM_3-VS-SorgLM_3. Totally nine metabolic pathways were enriched, including alpha-Linolenic acid metabolism, glycerolipid metabolism, glycerophospholipid metabolism, plant hormone signal transduction, anthocyanin biosynthesis, phenylpropanoid biosynthesis, starch and sucrose metabolism, limonene and pinene degradation, and circadian rhythm—plant .LOC8084842 and LOC8065295) that catalyze the binding of JA and Ile. The expression level of LOC8084842 significantly increased after the treatment with 2.0 mmol/L MeJA for 2 hr, and the score reached to 7.6. In addition, 10 genes, including OsJAZ13 , OsJAZ10 , OsJAZ8 (LOC8062740), OsJAZ2 (LOC8079184), OsJAZ9 (LOC8059788) and OsJAZ6 (LOC8077075), were found to be correlated with the JAZ protease activity. These genes could promote the degradation of JAZ, and significant differences were observed after the treatments of 2.0 mmol/L MeJA for 2 hr and 0.5 mmol/L for 4.5 hr compared with the controls. The difference score for LOC8072010 was 6.4. Seven genes, including LOC8069641, LOC8057408, LOC110436805, LOC8055721, LOC110433436, LOC8059158 and LOC8060491, were found to be correlated with bHLH zip transcription factors. These genes promoted MYC2 transcription initiation and the difference score reached 2.0 compared with the controls when treated with 2.0 mmol/L MeJA for 2 hr and 0.5 mmol/L MeJA for 4.5 hr. All results indicated that exogenous MeJA could induce the differential expression of these important genes, including JAR1, JAZM and MYC2. This demonstrated the important regulatory function of exogenous MeJA in sorghum floret opening at the molecular level.JA and isoleucine (Ile) could form a conjugate  when catalyzed by jasmonic acid-amido synthetase (JAR1), which induces the binding of the Jas motif in the jasmonate ZIM domain and COI1. The JAZ motif was then degraded, which was followed by the dissociation of the NINJA-TPL and transcription factor MYC2, resulting in the transcription of JA signaling-related genes –34. On tLOC8085653) and four genes related to the activity of lecithin A2 (EC: 3.1.1.4) . The differential expression score of LOC8085653 in SorgCK_2-VS-SorgHM_2 was 5.4, which was the most remarkable differential expression among the five groups. Besides, three genes related with lipoxygenase activity were also found, including LOC8082015, LOC8055177 and LOC8065835. All these genes were up-regulated in the α-linolenic acid metabolic pathway, positively regulating the biosynthesis of JA.The JAs are a class of fatty acid derivatives which play important regulatory roles in plant growth and development, mechanical damage and the induction of defense-related gene expression. JAs are mainly biosynthesized through the α-linolenic acid metabolic pathway. Lecithin on the cell membrane is catalyzed to release the precursor linolenic acid for the synthesis of JA, which is then oxidized to 13S-hydroperoxylinolenic acid through catalysis by lipoxygenase . 13S-Hydroperoxylinolenic acid subsequently forms 12S,13S-epoxylinolenic acid, followed by cyclization to form 12-oxo-phyto-dienoicacid, which is then subject to be catalyzed to form MeJA or other complexes/metabolites . As showLOC110437037, LOC8069151, LOC8064663 and LOC8060513 were related to the AGPase activity and five genes LOC8066439, LOC8081295, LOC8066807, LOC8068976 and LOC8071331 were related to GBSS and GBE activity levels  were significantly upregulated to promote the degradation of starch. Two target genes named LOC-1, playing an important role in the starch and sucrose metabolic pathway, and LOC-2, playing an important role in energy metabolic pathway, were selected and cloned, then transformed into wild-type Arabidopsis thaliana  into uridine diphosphate glucose (UDPG) and fructose, followed by a series of biochemical reactions to form starch . AGPase y levels . Expressthaliana . The resc plants . In summMa1, Ma2, Ma3, Ma4, Ma5 and Ma6 [OsDAD1, OSAOS1, OSAOC and OSOPR7, signal transduction related genes OsJAR1 and OsCOI1b, and 13 genes of the OsJAZ family, except OsJAZ5 and OsJAZ15, in lodicules cells were significantly up-regulated compared with the expressions at 18 h before floret open [Sorghum is a short-day tropical species with the characters of substantial photoperiod sensitivity and delayed flowering in long days . Sorghum and Ma6 ,39, but  and Ma6 . DocumenIn this study, RNA-seq method was used to explore transcriptome changes in lodicule cells during sorghum floret opening after exogenous MeJA treatments. 0.5 and 2.0 mmol/L MeJA were applied to treat sorghum florets and the results showed the number of DEGs annotated and functionally metabolic pathways increased along with the increase of MeJA concentration. Functional enrichment analysis of DEGS showed plant hormone signal transduction and starch and sucrose metabolism were closely related with the open of sorghum florets. Thus, the results in this study showed MeJA could induce various physiological metabolisms in lodicules of sorghum florets, thereby promoting the floret open in advance. The results of this study are consistent with the previous reports, further confirming the regulatory mechanism we proposed in gramineal crops.DAD1, LOX, AOS, AOC and OPR3. As shown in 2ratio = 3.0 as the criterion, significant expression differences were observed under the high concentration of 2.0 mmol/L treatment, including DAD1 activity related genes LOC8057399 (log2ratio = 3.5), LOC8085653 (log2ratio = 5.4) and LOC8055636 (log2ratio = 4.2), AOS activity related genes LOC8056861 (log2ratio = 3.1), 12-oxo-dienoic acid reductase related genes LOC8070772 (log2ratio = 3.9) and LOC8070773 (log2ratio = 3.3), OPC-8:0 coenzyme A ligase related gene LOC8063048 (log2ratio = 5.0). Interestingly, 19 genes including LOC8057399, LOC8082061, LOC80770776 and LOC8068239 and etc., were significantly upregulated under the treatment of 2.0 mmol/L, while no expressions were found under the treatment of 0.5 mmol/L. These 19 genes were actively involved in the biosynthesis of JA, and high expression levels of these genes could further increase the content of JA, which was favorable to the open of sorghum florets.JA biosynthesis begins with the release of the precursor α-linolenic acid from lecithin on the cell membrane, followed by a cascade of reactions catalyzed by multiple enzymes, including LOC8062786, LOC8083641, LOC8059386, LOC8056732, LOC110436805, LOC110429687, LOC8055721, LOC8055143, LOC8068250 and LOC110430094 were only differentially expressed under the 2.0 mmol/L MeJA treatment and these genes were barely expressed under the 0.5 mmol/L MeJA treatment, which suggested that 2.0 mmol/L MeJA treatment induced the expression of these genes to promote the open of sorghum florets by JA signal transduction.The JA signal transduction pathway starts with the JAZ complex, followed by enzyme catalysis by JAR1, JAZ and MYC2 to induce JA signals. As shown in LOC-1 and LOC-2 were cloned and transformed into Arabidopsis thaliana, overexpression of these two genes resulted the earlier flowering in transgenic Arabidopsis plants. This was not reported before.In this study, four genes correlated with AGPase, which is a rate-limiting enzyme in starch synthesis and metabolism, were all downregulated, while four genes correlated with AMY and AMS were significantly upregulated to promote the degradation of starch. This result suggested that exogenous MeJA induced the up-regulation of starch synthesis related genes and the down-regulation of starch degradation related genes, thereby promoting the degradation of starch in lodicules cells into sucrose, maltose or other sugars, which caused the decrease of the osmotic potentials in lodicule cells. This further allowed lodicule cells to absorb water and swell, resulting in floret open. Previous research reported that the altered expression patterns of genes involved in glycolysis/gluconeogenesis pathway could cause early flowering in rice ,41. In aIn conclusion, floret opening is initiated by water absorption and lodicule swelling that pushes the chaff out. This process requires the loosening of lodicule cell walls and the reduction of the solute potential in lodicule cells. The results of this study showed that exogenous MeJA could promote the degradation of starch and the reduction of the solute potential in sorghum lodicule cells at the molecular level , but theS1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


The sex ratio  was 2.5. The mean delay of diagnosis was 53.5 months. Oral aphthosis was constant. The frequency of the manifestations was: genital aphtosis 71.5%, pseudofolliculitis 84.6%, erythema nodosum 11.5%, positive pathergy test 50%, ocular disease 36.9%, venous thrombosis 30%, arterial disease 4.6%, joint damage 30.8%, neurological disease 19.2% and digestive disease 0.8%. The male gender was significantly associated with ocular involvement (p =0.02), venous disease (p =0.01) and occurrence of relapses (p =0.01). The mean follow up was 68.5 ± 77.3 months. The poor survival prognostic factors were male gender, ocular involvement, venous disease, cardiovascular disease, a duration of follow up ≤12 months and a diagnostic delay ≤ 24 months.  Behçet’s disease (BD) is a systemic vasculitis characterized by buccal and/or genital aphthosis with or without systemic manifestations. Its prognosis depends on clinical forms. Although BD seems to be the most common form of vasculitis in Tunisia , no reprType of the study: this study is a single-center, retrospective, descriptive and longitudinal study. We retained all patients with BD admitted to an Internal Medicine Department in Tunisia over a period of 26 years.Inclusion criteria: we retained all patients with confirmed BD responding to the International Criteria for BD [aphtosis of the mouth (two points); b) genital aphtosis (two points); c) skin damage (one point): pseudofolliculitis, erythema nodosum, papulo-pustular lesions and/or acneiform nodules not explained by corticosteroids and cutaneous aphtosis. d) Positive pathergic test (one point): it was considered positive if an aseptic papuleor pustule (superior than 2 mm) was objectified at the point of the bite. e) Eye damage (two points): in case of ocular abnormalities in ophthalmic examination, ophthalmological exploration was supplemented with retinal fluoresce in angiography. Visual field, visual evoked potentials and/or the ocular echography were practiced according to the clinical context.a for BD . A positmajor ocular lesions: uveal involvement  and/or retinal vasculitis (RV). Minor ocular lesions: conjunctival ulcerations, keratoconjunctivitis, episcleritis and / or scleritis.We included: Complications and ocular sequelaeVascular involvement (One point): it was confirmed by the imaging data. We included venous thrombosis: superficial vein thrombosis, deep vein thrombosis (DVT), pulmonary embolism (PE) and /or cerebral venous thrombosis (CVT). Arterial injury: thrombosis and/or arterial aneurysms.Neurological impairment (one point): retained in front of somatic neurological examination, neuroimaging and / or cerebro-spinal fluid.non-parenchymal lesions: they included CTV, arterial thrombosis and/or cerebral arterial aneurysms; neurological sequelae: objective neurological disorders persisting during the evolution and that could be a source of neurological handicap.We included: a) parenchymatous lesions retained on the cerebro-medullary magnetic resonance imagery sequences by hypo-signal lesions in T1 sequence and in hyper-signal in T2 and T2 flair sequence; b) Non inclusion criteria: we have not included the patients with confirmed BD but had never been hospitalized for BD and the patients to whom the diagnosis was rejected during the evolution and whose medical files were lost and/or unworkable.Modalities of evaluation: we defined different modalities of evaluation: a) a patient lost sight of was defined by a follow-up period necessarily longer than a month. b) A patient lost sight of out of hand was defined by a duration of follow-up obligatorily lower than one month. c) An improvement or remission was defined by subjective and/or objective good evolution of the clinical signs with a complete and/ or partial remission. d) A complete remission was defined by a complete resolution with disappearance of any sign of the disease, in the absence of any relapse during the follow-up period. e) A partial remission was defined by a significant but incomplete decrease in the signs of the disease, while maintaining a risk of subsequent relapse during the observation period. f) A relapse was defined by the reappearance of clinical signs after a favourable evolution. g) A stabilization was defined by a stability that no longer progressed toward improvement or worsening with a risk of relapse during the follow-up period. h) An aggravation was defined by an exacerbation of clinical lesions with appearance of complications.Statistical analysis: all data were analysed by the software SPSS 19.0. Statistical analysis contained absolute frequencies and relative frequencies. We calculated averages, medians and standard deviations and we determined the extreme values for the quantitative variables. For the analytic study, the comparison between two averages was made with the Student test for independent series, and by the non-parametric test of Mann and Whitney in case of low effectives. The comparison in percentages on independent series were made by the chi-Square test of Pearson and in case of significance of chi-square test and its non-validity, with exact bilateral Fisher test. The study of the binding was carried out by the Kaplan Meier method. Survival prognostic factors was performed in univaried analysis by comparing survival curves with Log rank test. In all statistical tests, the level of significance is 0.05.Epidemiological characteristics: we included 130 patients. Disease incidence was of five new cases per year. Patients were Tunisian in 97.7% of the cases and Algerian in two cases. The mean age at first disease manifestation was 30.3 years ± 8.8 (extremes 12-58 years). The mean age at diagnosis was 34.6 years ± 9.4 (extremes 15-65 years). Five patients were aged less than 16 years. Sex ratio (M/F) was 2.5.Onset disease: buccal aphthosis was inaugural in 55.4% of the cases. Genital and buccal aphthoses were associated in 25.4% of the cases. Severe organ involvement was revealed in 27 BD cases (20.8%). Mean diagnosis delay was 53.5 months ± 65.2. Mean diagnosis delay for 72 patients with inaugural buccal aphthosis was 58.1 months ± 79.6 and 49.5 months ± 60.5 for other patients.Cutaneous mucosal manifestations: buccal aphthosis was constantly observed among patients. Evolutive genital aphthosis was observed in 71.5% of the cases. Genital scars were noted in 45.4% of patients. Two patients had peri-analaphthosis. Skin hypersensitivity was noted in 46.9% of the cases. Pathergy test was performed in 87.7% of cases and was positive in 50% of the cases. Skin damages were observed in 87.7% of the cases. Pseudofolliculitis and erythema nodosum (EN) were observed in 84.6% and 11.5% of the cases, respectively.Ocular manifestations: they were observed in 36.9% of the cases and inaugural in 10.8% of the cases. Ocular involvement was discovered in 6.2% of the cases. Complications and sequelae were diagnosed in respectively 47.9% and 6.2% of the cases. Patients were definitely blind at the moment of diagnosis in 8.3% of the cases. Uveal involvement was the most frequent lesion (77.1%) followed by retinal vasculatis (62.5%). Hyalitis was diagnosed in 15 patients (31.3%) and was associated to posterior uveitis in six cases. Seven patients (14.6%) had retinal hemorrhage.Vascular manifestations: they were noted in 31.5% of cases. It was about 35 cases of venous manifestations in 35 cases, arterial manifestations in two cases and both in four cases. Deep venous thrombosis(DVT) was the most frequent vascular manifestations (78%).Venous manifestations: venous impairment was observed in 95.1% of vascular manifestations and 30% of BD patients. It revealed the diagnosis in 20.7% of the cases. Venous manifestation appeared during BD evolution in 9.2% of cases with a mean delay of 45.8 months ± 45.9. There were DVT in 32 cases and superficial vein thrombosis in 32 cases. The locations of DVT were the lower limbs (84.4%), the vena cava (28.1%), the upper limbs (9.4%), pulmonary embolism (PE) (9.4%), cerebral venous thrombosis (6.2%) and Budd Chiari syndrome (3.1%).Arterial impairment: it was observed in 14.6% of vascular manifestations and 4.6% of patient with BD and revealed BD in three cases. All patients were male. Arterial manifestations were as follows: isolated arterial aneurysms (n=1), isolated arterial thrombosis (n=4), and both manifestations (n=4). Pulmonary artery was the most frequent disease localization (n=5).Neurological manifestations: central nervous system disorders was reported in 19.2% and was inaugural in two cases. They were caused by parenchymatal lesions (88%). Psychiatric manifestations were observed in 36% of the cases with neuro-BD (6.9% of patients with BD).Cardiac manifestations: they were inaugural in one case. Three patients had pericardial effusion and coronary involvement.Other manifestations: joint damage and digestive disease were diagnosed in respectively 30.8% and 0.8% cases.Biology: biological inflammatory disorders were observed in 77 patients (59.2%). HLA typing was performed in 47 patients (32.2%) and was positive in 48.9% of the cases. Only one patient had a combined deficiency of proteins S and C.Therapeutic means in Behcet´s disease: the different therapies used for BD are summarized in Anticoagulant therapy and platelet aggregation inhibitors: all patients with DVT and /or arterial thrombosis were treated with anticoagulant therapies. The mean period of treatment was 60.9 months with extremes 2 to 218. Two patients had hemorrhagic complications secondary to vitamin K overdose.Evolution modalities: twenty-two patients (16.9%) were lost sight of the out of hand. The mean follow-up was 68.5 ± 77.3 months (5.7 years), and extremes were 6 days to 26 years. Among 130 patients, 48.5% noted an improvement. It was about vascular manifestations (53.6%), cutaneous and mucosal manifestations (46.9%) and ocular manifestations (25%). Complete and partial remission were observed in respectively 26.2% and 22.3%. Disease stabilization and a worsening were observed in respectively 28.5 and 4.6%. The mortality rate in our study was 1.5%. A fatal vasculo-BD occurred in two cases. The relapse was observed in 42.3%. The mean number of relapses per patient during BD evolution was 7 ± 6. The maximum number of relapses was 14. The mean period between BD diagnosis and first disease relapse was 34 months ± 53. Definitive sequelae were observed in 10% of cases (n=13) and were mainly secondary to ocular and neurological involvement.Gender: women developed significantly more frequently EN (p=0.03) than men. Male gender was significantly associated with ocular involvement (p=0.02), venous manifestations (p=0.01), corticosteroid treatment (p=0.007), immunosuppressive therapy (p=0.01), anticoagulation (p=0.009) and relapses (p=0.001). Age: no difference in epidemiological, clinical, biological, diseaseevolution and prognosis in terms of age was observed among patients. b) Types of manifestations: patients with ocular involvement developed less frequent biological inflammatory disorders (p=0.001) and vascular manifestations (p=0.016). Remission is rarely observed compared with patients without ocular BD (p=0.001). Biological inflammatory disorders were more frequently observed in patients with angio-BD (p=0.003). Patients with articular manifestations develop significantly less relapses than patient with other organ involvement. (p=0.007). HLA profile: no difference in the type of manifestations and HLAB51 profile was observed among the patients. d) Prognostic factors: two cases of death were noted in our series. We were unable to identify prognostic factors of mortality. We studied the survival events of patients without relapses and survival events without aggravation and thus released the prognostic factors of survival that can influence the evolutionary profile and the survival of these patients. We compared the two subgroups of patients: 1) group1 (n = 74): patients who had no relapses or worsening of the disease (n=51) and patients who had only isolated mucocutaneous relapses (n=23); group 2 (n = 29): patients who have relapsed ocular, vascular, articular, and neurological manifestations and patients who have had a worsening of their disease. According to Kaplan Meier analysis, one- three- five-10-and 20 years the survival rates with-neither relapses nor worsening were observed in respectively 84%, 70%, 67%, 61% and 61% of the cases. The disease was stabilized after 8 years of evolution (volution . These pOur study was conducted during a long period of 26 years. A few works have been focused on BD characteristics and survival factor prognosis without relapse nor worsening. A better knowledge of different manifestations and prognosis factors of BD, are a key for early diagnosis and better management to improve the function and prognosis and guarantee a better quality of life for North African patients. However, retrospective and single center characteristics were the limitations of this study. Selection biais can also explain the important prevalence of multisystemic forms. BD seems to be the most frequent vasculitis in Tunisia . In a NoCutaneous-mucosal manifestations were inaugural in many studies , 13, andCentral nervous system involvement in BD is characterized by diffuse involvement with brain stem predilection observed in 21.4% to 70%of the cases. Hemispherical and medullary localizations were observed in respectively 14% to 19.8% and in 2.6% to 14% of the cases , 23. Theet al. [Cardiac manifestations incidence was comparable to the literature , 12. Caret al. . Pericaret al. , 27. Theet al. , 29. In et al. . Howeveret al. .BD has an important polymorphism and a relatively high incidence in the Maghreb. Poor survival prognostic factors are male gender, ocular involvement, venous disease, cardiovascular disease, a duration of follow up ≤12 months and a diagnostic delay ≤24 month. Behind the therapeutical progress, prognosis improvement is very important by shortening the diagnosis delay, multidisciplinary staff participation, and intensive treatment of the functional involvement and regular monitoring of patients. Education is important for the treatment of BD to guarantee patients´ compliance.BD is an inflammatory, systemic, chronic disease that evolves by relapses;The clinical presentation is polymorphic and associated with a bucco-genital aphthosis with various systemic manifestations. The positive diagnosis is often difficult especially in the absence of aphthosis;The therapeutic management remains controversial, due to the scarcity of therapeutic trials and the absence of standardized criteria for measuring the evolution. Its evolution is unpredictable. Its prognosis varies according to its clinical presentation. The neurological and ocular manifestations are responsible for a progressive deterioration of the functional prognosis. The vascular and digestive disorders condition the vital prognosis.The male gender is significantly associated with ocular involvement, venous disease and occurrence of relapses;The poor survival prognostic factors are male gender, ocular involvement, venous disease, cardiovascular disease, a duration of follow up ≤12 months and a diagnostic delay ≤24 months.


Humic substance (HS)-based biostimulants show potentials as sustainable strategies for improved crop development and stress resilience. However, cellular and molecular mechanisms governing the agronomically observed effects of HS on plants remain enigmatic. Here, we report a global metabolic reprogramming of maize leaves induced by a humic biostimulant under normal and nutrient starvation conditions. This reconfiguration of the maize metabolism spanned chemical constellations, as revealed by molecular networking approaches. Plant growth and development under normal conditions were characterized by key differential metabolic changes such as increased levels of amino acids, oxylipins and the tricarboxylic acid (TCA) intermediate, isocitric acid. Furthermore, under starvation, the humic biostimulant significantly impacted pathways that are involved in stress-alleviating mechanisms such as redox homeostasis, strengthening of the plant cell wall, osmoregulation, energy production and membrane remodelling. Thus, this study reveals that the humic biostimulant induces a remodelling of inter-compartmental metabolic networks in maize, subsequently readjusting the plant physiology towards growth promotion and stress alleviation. Such insights contribute to ongoing efforts in elucidating modes of action of biostimulants, generating fundamental scientific knowledge that is necessary for development of the biostimulant industry, for sustainable food security. This advances the understanding of regulatory network rules and mechanistic events in the cellular and chemical space of the plant under consideration, which, in turn, provides greater impetus for the translation of fundamental knowledge to actionable programs in the field + = 556.766, and [M − H]− = 554.2615, to ensure high mass accuracy (1–3 mDa) of analytes. For downstream structural elucidation, the MS analyses were set to result in both unfragmented and fragmented experiments through collision-induced dissociation (MSE), where the fragmentation patterns were obtained by alternating the collision energy from 10 to 50 eV. For targeted analysis, a triple quadrupole mass spectrometry platform, LCMS-8050 , equipped with an ESI source and ultra-fast liquid chromatography (UFLC) as the front-end, was utilized. A multiple reaction monitoring (MRM) method was used for absolute quantification of the targeted metabolites (amino acid and hormones)  source, was used for untargeted analysis. The MS detector was set to acquire centroid data in both positive and negative ionisation modes. The MS conditions used were as follows: the source temperature was set at 120 °C, desolvation temperature at 450 °C, capillary voltage 2.5 kV, sampling and extraction cones at 30 V and 4 V, respectively, cone gas flow at 50 L hormones) : descripApexTrack algorithm [m/z variable pairs, with m/z peak intensity for each sample. The following parameters were used for data processing: retention time (Rt) range of 1–17 min, a 100–1100 Da mass range, intensity threshold of 50, mass tolerance of 0.05 Da, and an Rt window of 0.2 min for both polarities. Normalization was then performed by using total ion intensities of each defined peak; prior to calculating intensities, the software performs patented modified Savitzky–Golay smoothing and integration. Only data matrices with noise levels below 50% (MarkerLynx metrics) were used for downstream data analysis strategies. The data matrices generated from MassLynx were exported into the SIMCA-15.0 software  for statistical modelling. Some of the computed chemometrics models were included principal component analysis (PCA). The latter is an unsupervised method that aims at data dimensionality reduction and generates a model that reveals clusters, trends, and similarities between treatment groups [The UHPLC-qTOF-MS raw data were processed using MassLynx XS™ software’s MarkerLynx application . This application makes use of the patented lgorithm  to perfot groups . SuperviR1 significant components) were considered. Furthermore, different metrics and tests were used for model validation, which included an evaluation of explained and predicted variation (cumulative R2 and Q2), the analysis of variance testing of cross-validated predictive residuals , the receiver operating characteristic (ROC) curves, response permutation tests (with n = 100) and predictive testing. Thus, to ensure reliable results, only thoroughly validated and (preferably) parsimonious models were considered in this study. Quantitative analysis  was performed using average integrated peak areas and concentrations for untargeted and targeted metabolites, respectively.Before building the chemometrics models PLS-DA), data pre-treatment  was applied to normalize the variances and correct heteroscedasticity ,52. A nohttps://www.reifycs.com/AbfConverter/, accessed on 21 April 2021) and then uploaded into the Mass Spectrometry-Data Independent AnaLysis (MS-DIAL) software. The MS-DIAL data-processing program makes use of a deconvolution algorithm to perform mass spectral deconvolution of data-independent acquisition (DIA) data, thus making it applicable for the extensive untargeted metabolomics analysis of both DIA and data-dependent acquisition (DDA) centroid datasets [https://gnps.ucsd.edu/, accessed on 28 April 2021) using the WinSCP server for molecular networking.All the raw vendor  format MS/MS data were first converted to ‘analysis base file’ (ABF) format using the Reifys Abf converter software , thus allowing for molecular networks to be created [m/z) and coloured by means of pie charts based on the differential changes in the metabolite levels under different treatment conditions. The MolNetEnhancer networks, on the other hand, were coloured based on the classes such that nodes present in the same class had the same colour while grey nodes represented the non-matched metabolites. The fragmentation spectra of all the putatively annotated metabolites matched to the GNPS spectral libraries were manually validated using the metabolite annotation workflow described below.A feature-based molecular network (FBMN) was generated for both the negative and positive mode data by uploading the respective feature quantification table, MGF file and a metadata file describing the properties of the sample file . The MS/MS (fragmentation) spectra were clustered using the MS-Cluster algorithm with a precursor ion mass tolerance of 0.05 Da and fragment ion mass tolerance of 0.05 Da to create the consensus spectra. A network was generated where the lines/edges connecting the nodes were filtered to have a cosine score above 0.7 and a minimum of 4 corresponding fragment ions. This approach builds on the assumption that molecules which are structurally related give rise to similar fragmentation patterns when subjected to MS created ,54. The https://www.plantcyc.org/, accessed on 15 March 2021), Dictionary of Natural Product (DNP) , Chemspider , and Kyoto Encyclopedia of Genes and Genomes (KEGG) , to putatively assign compound names to the MF; (iii) structural elucidation was performed based on the fragmentation patterns by examining the MS1 and MSE spectra of the metabolites; and (iv) putative annotations of metabolites were also compared to the available literature, considering their respective chromatographic elution profiles on a reverse-phase column. In the current study, metabolites were putatively annotated to level 2 of the Metabolomics Standards Initiative (MSI) [Metabolite features were annotated based on the following criteria: (i) molecular formula (MF) from full-scan accurate mass data, filtered through heuristic rules such as mass differences, nitrogen rules, restrictions of element numbers, isotopic fit and rings-and-double-bond equivalent; (ii) the calculated, filtered elemental composition predictions were searched against bioinformatics tools or databases such as PlantCyc (ve (MSI) .http://metamapp.fiehnlab.ucdavis.edu/, accessed on 30 April 2021). MetaMapp-encoded chemical structures of all the identified metabolites were retrieved from the PubChem and KEGG databases, and the p-values and fold changes were obtained from OPLS-DA-derived descriptive statistics (All annotated and targeted metabolites  were useatistics . A TanimUnderstanding the modes of action involved in biostimulant-mediated growth promotion and stress resilience is one of the critical steps necessary for the full implementation and integration of biostimulants into agricultural practices. Thus, this present study intended to decode a metabolic choreography that defines the effects of an HS-based biostimulant on maize plants, under normal and starved conditions, in a greenhouse setting. Although further investigation may be needed to build on our findings, the model derived from this metabolomics study suggests that the HS-biostimulant induced a metabolic reprogramming in maize plants towards growth promotion and the alleviation of starvation stress. Molecular networking approaches aided in characterizing the HS-altered chemical space. In more detail, a wide and coordinated range of metabolic processes was involved in the response of maize plants to HS treatments. Impacted metabolic pathways included amino acid metabolism, phenylalanine metabolism, and alpha-linolenic acid metabolism, among others, involving a spectrum of metabolite classes such as amino acids, phytohormones, lipids, HCA compounds and flavonoids which are involved in growth promotion and nutrient stress alleviation. Furthermore, metabolic network analysis revealed some qualitative characteristics of HS effects on maize metabolism under nutrient starvation: a complex structural interconnectivity between altered metabolites involved in stress alleviation and metabolite hubs depicting possible biochemical regulatory mechanisms, which can be investigated further. These HS-induced multilayered metabolic reconfigurations in maize plants could generally be linked to morphophysiological data such as chlorophyll content, nutrient assimilation, and changes in biomass. The knowledge generated from this work provides a morphophysiological and metabolomic gateway to the mechanisms underlying the effects of HS-biostimulant on plants. Such insights lay a foundation for advancement of the biostimulant industry and incorporation of these formulations in agronomic practices, for sustainable food security.


Solanum lycopersium) under salinity stress conditions. This metabolomics study postulates that Si-based biostimulants could alleviate salinity stress in tomato plants through modulation of the primary metabolism involving changes in the tricarboxylic acid cycle, fatty acid and numerous amino acid biosynthesis pathways, with further reprogramming of several secondary metabolism pathways such as the phenylpropanoid pathway, flavonoid biosynthesis pathways including flavone and flavanol biosynthesis. Thus, the postulated hypothetical framework, describing biostimulant-induced metabolic events in tomato plants, provides actionable knowledge necessary for industries and farmers to, confidently and innovatively, explore, design, and fully implement Si-based formulations and strategies into agronomic practices for sustainable agriculture and food production.The ongoing unpredictability of climate changes is exponentially exerting a negative impact on crop production, further aggravating detrimental abiotic stress effects. Several research studies have been focused on the genetic modification of crop plants to achieve more crop resilience against such stress factors; however, there has been a paradigm shift in modern agriculture focusing on more organic, eco-friendly and long-lasting systems to improve crop yield. As such, extensive research into the use of microbial and nonmicrobial biostimulants has been at the core of agricultural studies to improve crop growth and development, as well as to attain tolerance against several biotic and abiotic stresses. However, the molecular mechanisms underlying the biostimulant activity remain enigmatic. Thus, this study is a liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics approach to unravel the hypothetical biochemical framework underlying effects of a nonmicrobial biostimulant (a silicon-based formulation) on tomato plants ( Excessive amounts of salt ions in the rhizosphere could occur naturally from processes such as mineral weathering and gradual withdrawal from the ocean, or because of irrigation malpractices and overuse of fertilisers . In. In37]. p-value rankings, with a cut off at 0.0005. DSPC graphical models allow discovering weighted connectivity between large numbers of metabolites, where several associations of metabolites can be discovered. On the network, very strong correlations were discovered between the flavonoid quercetin and vitexin, rutin and coumaroyl derivatives, and phenolic acids feruloyl-, coumaroyl- and sinapoyl derivatives  and the lowest negative between phenolic compounds and organic acids A. As disivatives B. This cIn general, correlation analysis can be used as proxy to describe a given physiological state of the system of interest because the correlation matrix and networks can change with the steady-state concentrations of metabolites ,41. HencIn metabolomics studies, correlation analysis provides a powerful view to understand biological systems where not only individual metabolites are connected, but also their interconnections, hence providing a more comprehensive understanding of the metabolome of the system in question . The gloAlthough some amino acids including alanine, leucine, glutamine and methionine appear to have been downregulated (red) in response to salt stress A, othersThe computed network-based correlation analysis provides a complementary tool to the statistical and multivariate analysis of metabolomics datasets for identification of metabolite alterations in different physiological states ,43. As sThe application of Si-based biostimulants on tomato plants under salt stress conditions induced shifts in primary metabolism, reflected through the reorganisation of several pathways involving fatty acids-, organic acids-, sugar- and amino acids metabolism C. PrimarZea mays and Swertia chirayita, respectively, where phosphogluconate and the enzymes involved in the PPP were enhanced upon Si formulation inclusion.Si-biostimulant application to salinity-stressed plants evidently alter the content of different organic compounds and sugars, e.g., reprogramming of ascorbate and alderate metabolism, sugar metabolism pathways and the TCA cycle in this current study . Under sOn the other hand, arabinose levels were relatively lower in stressed plants compared to the non-stressed and were marginally decreased post-biostimulant treatment . Under sIn addition to sugars, several studies have reported stress-induced modifications in the levels of organic acids including most commonly malic acid, citric acid and succinic acid ,56,57. OArabidopsis thaliana Pooled quality control (QC) samples were used for assessment of the reliability and reproducibility of the data generated, as well as for corrections of the non-linear signals. A QC sample was injected after each 10 randomised sample injections to accurately monitor the deviations caused by the instrument. In addition, 6 injections of a QC sample were done at the beginning and the end of each batch to ensure equilibration of the system and reduce bias in the measurements. The blank samples (consisting of 50% methanol) were also randomly run to detect any background noise.Centroid raw data acquired from both positive and negative ESI modes was visualised and pre-processed using MassLynx XSTM 4.1 , generating a data matrix of paired retention time (Rt) and m/z variables, with their respective peak intensities. For data processing, the MarkerLynx software parameters were set to process the m/z domain of 100–1200 Da within the retention time (Rt) range of 0.9–14 min. Mass tolerance of 0.05 Da and intensity threshold counts of 100 were set for both positive and negative data, with a Rt window of 0.2 min. Prior to computation of peak intensities, MarkerLynx software was set to execute a modified Savitzky-Golay smoothing and integration, retaining only data matrices with the noise levels below 10% (as determined by MarkerLynx) for subsequent data analysis. After data extraction (by MarkerLynx), a mandatory data scrutiny was meticulously done, including assessment of the number of extracted features , applying the 80% rule  and monitoring the quality of data and stability of the analysis using QC samples. Data transformation methods—centering, scaling or transformation—were ‘exploratively’ employed to put all variables on equal footing, minimise variable redundancy and adjust for measurement errors.The MassLynx generated data matrices were exported to SIMCA  software, version 15  and a web-based data analysis tool, MetaboAnalyst version 4.0  for data mining and interpretation. The approach followed in this study was a chemometric approach: (i) firstly, exploration of the data using unsupervised chemometrics methods, such as principal component analysis (PCA), to reduce the dimensionality of the data, evaluate the structures and characteristics of the data ; (ii) following unsupervised modelling, and based on the insights extracted from it, supervised multivariate hybrid machine learning (ML)/classical statistical methods such as orthogonal projection to latent structures-discriminant analysis (OPLS-DA) were applied. This allowed for sample classification  as well as the identification and selection of variables (metabolite features) underlying the discrimination between groups or classes. Different metrics and tests were applied for model validation, and these include evaluation of explained and predicted variation (cumulative R2 and Q2), cross-validation analysis of variance (CV-ANOVA) and permutation testing.k-fold cross-validation is the most common method for model evaluation and selection in machine learning, where a dataset is iterated k times. In each iteration, the dataset is divided into k parts: validation and training subsets. This implies that, in the case of a 7-fold CV, for a fixed number of components, the Y values of all individuals of each subset are predicted using a sub-model computed with the 6 other subsets. Results of this k-fold cross-validation procedure are summarised by quality parameters, such as R2 and Q2 metrics in this case. R2 indicates the explained variation  and Q2 refers to predicted variation  [Prior to computing chemometric and ML models, pre-treatment methods such as data transformation and Pareto-scaling were applied to normalise and adjust all the variables to a comparable footing. An adjusted non-linear iterative partial least square (NIPALS) algorithm was used to manage the missing values, with a correction factor of 3.0 and a default threshold of 50% . A sevene model) ,118.https://www.reifycs.com/AbfConverter (accessed on 6 June 2021)) then uploaded into the Mass Spectrometry-Data Independent AnaLysis (MS-DIAL) software ). The MS-DIAL data-processing program uses the deconvolution algorithm to perform mass spectral deconvolution of data-independent acquisition (DIA) data, thus making it applicable for the extensive untargeted metabolomics analysis of both DIA and data-dependent acquisition (DDA) centroid datasets [https://gnps.ucsd.edu/ (accessed on 21 June 2021)) using the WinSCP server for molecular networking.The raw MS/MS data was converted to ‘analysis base file’ (ABF) format using the Reifys Abf converter software (datasets . The dathttps://cytoscape.org/ (accessed on 30 September 2021)) network visualization tool/software (version 3.8.2), where the nodes were labelled with the precursor mass (m/z) and coloured based on metabolite class. The individual nodes, representative of measured ions, were also coloured to display colour-coded pie charts illustrating relative ion abundance across different treatments. The edges were coloured based on the type of interaction: Kyoto Encyclopaedia of Genes and Genomes (KEGG) reactant repair (krp) and the Tanimoto chemical similarity (tmsim) between the metabolites. The fragmentation spectra of all the putatively annotated metabolites matched to the GNPS spectral libraries were manually validated as described in the next session.The respective feature quantification table, MGF file and a metadata file describing the properties of the sample file  were uploaded for generation of a feature-based molecular network (FBMN). The MS/MS fragmentation spectra were assembled using the MS-Cluster algorithm with a precursor ion mass tolerance of 0.05 Da and fragment ion mass tolerance of 0.05 Da. A network was generated where the lines/edges connecting the nodes were filtered to have a cosine score above 0.7 and a minimum of 4 corresponding fragment ions. The MN spectra were then searched against the spectral libraries housed in GNPS where the same parameters  were used for metabolite annotation. To improve the chemical structural annotations, the generated molecular network data was enhanced with MolNetEnhancer then visualised on the Cytoscape (https://www.genome.jp/kegg/ (accessed on 18 October 2020)), Dictionary of Natural Product (DNP), ChemSpider (http://www.chemspider.com/ (accessed on 12 April 2020)) [https://www.metaboanalyst.ca/ (accessed on 17 August 2021)) [http://metamapp.fiehnlab.ucdavis.edu/ (accessed on 30 August 2021)) and visualized on Cytoscape v3.8.1 for a global view of the metabolic changes across different treatments.The chemometrically and statistically selected variables  from data modelling were confidently annotated through a multistep workflow, and metabolites were annotated to the level 2 as classified by the Metabolomics Standard Initiative (MSI) . Thus, the main steps followed for metabolite annotation in this study included: (i) computation of the molecular formula (MF) based on mass accuracy and the golden heuristic rules integrated in MarkerLynx formula generator algorithms  and element number restrictions); (ii) the computed MF were manually and automatically searched against various databases such as KEGG (l 2020))  and an it 2021)) , which aIdentifying and delving into the regulatory mechanisms of biostimulants in improving plant stress tolerance will help fine-tune the most effective application rates, areas, concentrations, and biostimulant-plant specificities which may collectively yield the highest impact on stress protection. Fundamental knowledge and understanding of such mechanisms are pivotal for the development and generation of biostimulants, where synergies of microbial and/or nonmicrobial mechanisms are functionally and complementarily designed, offering the potential to overcome specific and general stresses to a spectrum of plant species. A comprehensive and systematic approach to discover the mode of action of biostimulants has been proposed—metabolomics. This multidisciplinary omics technique can be used to interrogate cellular biochemistry, providing a window to understand cellular and molecular language in the context of biostimulant-plant interactions. As such, metabolomics reveals mechanisms that define biostimulant activity in stress alleviation. Meta-analysis of the Si-biostimulant-mitigated salt stress alleviation was conducted in this study.The predictive models derived show positive modulations in the primary and secondary mechanisms of Si-biostimulant-treated plants under salt stress conditions, including reconfigurations in the metabolite profiles of amino acids, fatty acids, organic acids, and phenolics. The analysis found that Si-biostimulant application induces accumulation of primary metabolites in such treated plants, which collectively replenish the energy for several metabolic reactions, modulate stress signalling waves that lead to specialised plant stress responses, act as osmotic and photosynthetic protectants and are precursors for the more defence-oriented secondary metabolic reactions. Moreover, Si-biostimulant is postulated to confer salt stress tolerance via accumulation of phenolics, as indicated by the timely decrease in their content which points to stress alleviation. These specialised metabolites define a huge part of secondary metabolism with defence-related roles including ROS scavenging, activation of defence-related proteins and antioxidant enzymes, and inhibition of ROS-generating enzymes. Such metabolite reprogramming spans several biostimulant-impacted primary metabolism pathways including the TCA cycle, amino acid- and fatty acid biosynthesis pathways, the PPP and secondary metabolic mechanisms such as the phenylpropanoid pathway, flavonoid biosynthesis pathway, and other secondary metabolite biosynthesis pathways.As illuminating as the current study is, emphasis of future studies for an expanded and hypothesis testing could be extended to (i) a targeted analysis focused on the proposed mechanisms that define a nonmicrobial biostimulant activity, (ii) development of integrated omics technologies  research for a systems biology approach towards biostimulants and (iii) synergistic effects of microbial and/or nonmicrobial biostimulant formulations against a plethora of biotic and abiotic stresses.


Following the completion of the treatment, gene expression of inflammatory mediators , protein expression of MMP-2, MMP-9 and their respective inhibitors- TIMP-1 and TIMP-2, and gelatinolytic activity of MMP-2 and MMP-9 were determined. At the protein level, cadmium incites a differential effect on the expression and activity of gelatinases and their endogenous inhibitors in an exposure-dependent manner. Results also show that the administered cadmium dose elicits an inflammatory response until week 10 that slightly diminishes after 4 weeks. This study provides evidence of cadmium-induced imbalance in the MMP-TIMP system in the cardiac tissue. This imbalance may be mediated by cadmium-induced inflammation that could contribute to various cardiovascular pathologies.The aim of this study was to evaluate the role of chronic cadmium exposure in modulating cardiac matrix metalloproteinases (MMPs) in the heart of rats. Adult male Sprague-Dawley rats were exposed to 15 ppm CdCl Since the discovery of cadmium in 1817 , this elEven at low doses, cadmium has been reported to impart its toxic effects on other organ systems including the nervous system , immune In the context of cardiovascular dysfunction, a hallmark of chronic cadmium exposure is the induction of fibrosis . CriticaTherefore, this study aims to evaluate the role of cadmium in modulating cardiac MMPs in the heart of rats after cadmium exposure.All experimental procedures performed are in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals  and were approved by the Institutional Animal Care and Use Committee of Qatar University . The samples obtained in this study were acquired from the animals employed in a previous study from our research group .2  in drinking water for 10 weeks followed by 4 weeks of normal drinking water. The administered cadmium exposure dose was selected based on previous literature reporting that a dose of 15 ppm of CdCl2 is a non-carcinogenic dose  male Sprague-Dawley rats were obtained from Laboratory Animal Research Center, Qatar University and housed in individually ventilated cages (IVC) under standard conditions . Animals were randomly assigned to one of two regimes for 14 weeks- 1) control group or 2) cadmium treatment (Cd-treated) . The connic dose  leading nic dose . The calnic dose  for the nic dose .ad libitum access to standard rodent chow and drinking water during the experiment. Animals were sacrificed at weeks 5, 10 and 14 under anaesthesia with sodium thiopentone . Heart tissue was dissected, cleaned from residual blood, immersed in liquid nitrogen, and preserved in the tissue archive at −80°C until further analysis.Animals received Frozen heart samples were weighed to approximately 20–30 mg and homogenized by sonication at 40% amplitude in pulses of 5 s. Heart tissue was homogenized in radioimmunoprecipitation assay (RIPA) cell lysis buffer to extract total proteins in combination with cocktail protease inhibitor and TRIzol reagent to extract RNA.Sonicated heart tissue samples were lysed for crude protein by incubating in RIPA cell lysis buffer  containing cocktail protease inhibitor . Protein extraction for MMP activity assay included EDTA in the cell lysis buffer. Cellular debris was pelleted by centrifugation for 10,000 rpm, 10 min at 4°C. The supernatant containing total crude protein concentration was estimated using Quick Start™ Bradford Protein assay  as per instructions in the user’s manual provided .The protein expression of gelatinases i.e., MMP-2 & MMP-9  and their respective inhibitors-TIMP-1 & TIMP-2  in response to cadmium treatment were analyzed by Western blotting. Equal amounts of crude protein was loaded and resolved by reducing SDS-PAGE. Separated proteins were electroblotted to polyvinylidene difluoride (PVDF) membrane . Electroblots were washed with TBST , blocked and probed with primary antibodies for the targets overnight at 4°C. Detection was done using compatible secondary HRP-conjugated antibodies  and incubated in ECL solution for a minute. Immunoreactive bands were visualized using ECL  under SynGene Gel documentation system. Protein expressions was evaluated by densitometric analysis using Image studio Lite (Ver 5.2) and normalized against the corresponding expression of GAPDH. Analysis was presented as a fold change compared to the respective controls.2 and 0.02% Triton X-100] at 37°C until a clear lysis zone is observed. The gel was quick stained in 0.10% Coomassie brilliant blue R250 for 5 min and destained till contrast between the lysis bands and blue gel background was visible. The zymographs were documented using the SynGel Documentation system. The proteolytic activity was analyzed using ImageJ and presented as a fold change compared to the control.Gelatinolytic activity of MMP-9 and MMP-2 was determined by gelatin zymography. Equal amounts of crude protein (40 µg) were mixed with sample loading buffer  and were loaded into a 9% acrylamide:bis-arcylamide gel containing 0.1% gelatin  as substrate. Non-reducing electrophoresis was done. After electrophoresis, the gel was washed twice in renaturing buffer containing 2.5% Triton X-100 at 37°C and incubated in developing buffer [50 mM Tris-HCl (pH 7.8), 0.2 M NaCl, 5 mM CaClTotal RNA was isolated from frozen heart samples using TRIzol reagent  according to the manufacturer’s instructions as previously described . Total R-α, NF-κB and GAPDH individually . Quantification was done by the ΔΔCt method wherein results were compared against both calibrator (control group) and normalizer  to obtain the ΔΔCt value . Extracted total RNA was reverse transcribed to cDNA using High-Capacity cDNA reverse transcriptase kit  as per instructed in user’s manual. Total cDNA was diluted by 4-fold in RNase-free water. The gene expression assay was performed using Quant Studio 6 Flex Real Time PCR system (Applied Biosystems). Amplification plots were obtained for each target using specific primers for IL-1β, IL-6, IL-10, TNFCt value . This vapost-hoc test using GraphPad Prism (ver.8.4) for Windows . p-value < 0.05 was considered as statistically significant.The results were presented as mean ± S.E.M and analyzed by two-way ANOVA followed by Tukey’s To evaluate the level of expression of MMPs, 40 µg of crude protein was separated in 9% SDS-PAGE. Protein expression was evaluated by probing electroblots specific for MMP-2 and MMP-9. By western blotting, only MMP-2 was detectable at approximately 64 kDa. It was observed that MMP-2 increased by more than 2-fold at week 5, slightly decreased at week 10 and reaches a maximum fold change of more than 4-fold at week 14 . In compProtein expression of TIMPs was assessed by separating 50 µg of crude protein in 12% SDS-PAGE and probing immunoblots against TIMP-1 and TIMP-2. The expression of TIMP-1 showed limited changes at the level of the protein in comparison to the control group . Less thActivity of MMPs were evaluated by gelatine zymography indicated by visualization of bands of lysis corresponding to the molecular weight of MMP-2 and MMP-9. After 24 h incubation, activity corresponding to the proenzyme form of MMP-2 at 72 kDa was observed. Densitometric analysis of the detected area of lysis shows that the activity of pro-MMP-2 does not increase by more than 1-fold at all time points in comparison to their respective control group .p < 0.05). Also, the decrease in activity from week 10 to week 14 was found to be statistically significant (p < 0.01). In regard to proenzyme MMP-2 (p < 0.05).On extension of the incubation period to more than 60 h, the activity of proenzyme form of MMP-9 and both proenzyme and active enzyme form of MMP-2 was detected . Quantifme MMP-2 , the actme MMP-2 , there i, IL-6, IL-10, NF-κB and TNF-α were assessed and represented as a fold change  play a vital role in both extracellular matrix turnover and inflammatory response in normal physiology. A dysfunction in their expression and activity, specifically gelatinases has been widely implicated in various cardiovascular pathologies . The curatinases . Similarin vitro study on cadmium treated U-937 cells showed that cadmium doses (1.0–50.0 µM) did not have any effect of TIMP-1 levels however there was an alteration in the MMP-9/TIMP-1 expression at the level of the gene albeit not statistically significant (One of the strategies to regulate and modulate the expression of MMPs is by means of TIMPs. The inhibitory relationship of the TIMPs on their preferential MMPs was clearly reflected in the protein expression levels of TIMP-1 and TIMP-2. Furthermore, the expression of TIMP-2 can be inversely correlated with the expression of MMP-2 wherein an elevation in the expression of TIMP-2 decreased the expression of MMP-2. These results demonstrate the effect of cadmium on the expression of TIMPs and hence the inhibition of MMPs. An nificant . The stunificant , cadmiumnificant . This rein vitro study in ECV304 cells suggested that NF-κB was activated by the cadmium treatment via phosphorylation and degradation of the NF-κB inhibitor, IκBα (It is well-documented that IL-1β, IL-6 and TNF-α cause inflammation, therefore changes in gene expression of these cytokines are indicative of inflammation . In the or, IκBα . In the or, IκBα .TNF-α plays a pro-inflammatory role in local and systemic inflammation when secreted by activated macrophages in response to injury to amplify and prolong the inflammatory response . A studyin vivo and in vitro (in vivo and in vitro (Extensive research exploring the impact of cadmium along the inflammation axis have reported the production and upregulation of IL-6, TNF-α and IL-1β both in vitro . In thisin vitro . For thein vitro . Recent in vitro .The present study did not include the role of chronic cadmium exposure on the kidneys and its relation to the described cardiac dysfunction has not been examined. On the other hand, several prior studies have reported that cadmium at the same dose has resulted in renal and cardiac injuries . The autThe findings of this study provides considerable insight in understanding that despite the withdrawal of cadmium exposure, the impact of the exposure still persists in the heart. It must be noted that to the best of our knowledge, this study is the first to evaluate the impact of cadmium exposure on the heart after withdrawal of the treatment. This study shows that cadmium alters the expression and activity of gelatinases in the heart. Also, cadmium significantly stimulates an inflammatory response by the upregulation in the expression of pro-inflammatory cytokines that is still slightly sustained after withdrawal of the treatment. The sustained expression of cytokines can be attributed to the feedback regulation of MMPs to counter-regulate the inflammatory response and promote resolution . Alterat


With many improvements on the culture protocols, current brain organoids could self-organize into a complicated three-dimensional organization that mimics most of the features of the real human brain at the molecular, cellular, and further physiological level. However, lacking positional information, an important characteristic conveyed by gradients of signaling molecules called morphogens, leads to the deficiency of spatiotemporally regulated cell arrangements and cell–cell interactions in the brain organoid development. In this review, we will overview the role of morphogen both in the vertebrate neural development in vivo as well as the brain organoid culture in vitro Understanding human brain development and relevant neurological diseases have been one of the most important challenges in current biology. Considering human ethics, it is difficult to directly work on the live human brains. The brain specimens taken from the corpses cannot be sufficient to examine the dynamic development and function of the brain, therefore, animal models such as mice are widely used. However, there are a lot of nonnegligible differences between the brain of animal models and humans. For example, the surface of the mouse brain is smoother, while the neocortex is largely expanded and highly folded in humans due to the contribution of ventricular radial glial cells (vRGCs) and outer radial glial cells (oRGCs) , therefore, there are still some challengeable limitations for modeling complicated human structures such as the brain. For example, the concentration gradients of different growth factors and morphogens, which are important for the cell fate decision and regional specification in human brain development, are hard to completely recapitulate in the limited monolayer system. Besides, the cell–cell interactions and cell migration during neurogenesis would be easily lost in such systems.In 1975, Martin and Evans have successfully formed an embryoid body using isolated single teratocarcinoma cells that can differentiate into almost all of the cell types in vitro, which can be regarded as the beginning of the in vitro culture system. Since then, the advance in pluripotent stem cell research including embryonic stem cell  developed by Sasai Lab  secrets several BMP antagonists including noggin  glycoproteins in canonical HH signaling. It plays key roles in the various important developmental process like the development of vertebrate limbs, brain, muscle, gastrointestinal tract as well as the heart , is expressed in the anterior border of the neural plate (ANB), which becomes the anterior end of the neural tube after neural tube closure, therefore antagonize the Wnt8B expressing in the presumptive midbrain and posterior forebrain and help form the global gradient of Wnt signaling along the A-P axis  expression in a dose-dependent manner (Rhinn et al. Although there are still many arguments on whether Wnt and FGF pattern A-P neural patterning through concentration gradient (Green et al. . For example, BMP signals transposed to the serum-free culture medium of ESCs could promote non-neural cell differentiation (Wiles and Johansson The success in organoid research was attributed to the early exploration of the morphogens as microenvironmental cues in many in vitro models, especially the ESCs and later the iPSCs. Early in 1981, Martin and his colleagues have already obtained the mouse ESCs from the inner cell mass of a blastocyst. The human ESCs were later successfully derived in 1998 (Thomson et al. brachyury in a dose-dependent manner (Kubo et al. . Therefore, different doses of Nodal/activin were then used to induce mesendoderm-derived structures such as heart, muscle, lung, intestine and so on in vitro (Kattman et al. Besides BMP, the addition of other morphogens such as Nodal/activin also helps direct cell differentiation from a pluripotent stem cell. An intractable problem for researching ESCs at the beginning is that it is hard to define primitive and definitive endoderm since their gene expression has a large overlapping. However, the addition of Nodal/activin, a vital factor in mesendoderm specification Schier , could bbrachyury induction (Watanabe et al. Wnts also play specific roles in cell fate specification. Adding Wnt1/Wnt3a to the ESC culture media could restrict neural differentiation of the ESC and enhance the The role of FGF in cell fate specification is more complicated as it comprises three main downstream signalings (Mossahebi-Mohammadi et al. Over the past two decades, multiple studies have improved the morphology of the aggregates and the organoids with different bioengineered strategies to achieve better reproducibility (Fedorchak et al. With sufficient comprehension of the role of different morphogens at different developmental stages both in vivo and in vitro. Many attempts using 3D culture systems together with different morphogen addition were done to recapitulate a more precise structure of the brain in vitro.3D neuroepithelial cyst model is a simple approach to uniformly generate neural plate progenitors that resemble dorsal neural tubes and responsive to different morphogens such as Shh (Meinhardt et al. SFEBq is another mature system applying self-organizing theory to generate complicated and well-organized 3D structures created by Yoshiki Sasai Lab (Eiraku et al. The sequential addition of specific morphogens together with improving 3D culture systems do help recapitulate several precise brain structures. However, these methods all directly supply the morphogen through the medium, the enriched morphogens and other intrinsic signals surrounding the organoids during the 3D culture may compromise the construction of the morphogen concentration gradient, therefore disrupt the positional information (Corrò et al. First, the generation of assembloids, a co-culture system of different kinds of organoids (Andersen et al. Second, the 3D co-culture strategy could imitate the organizer to reproduce a more complete self-organization process Fig. . This coThird, a powerful approach for creating complex biomolecular gradients is the use of the microfluidic system Fig. C, which With such a short history of less than 10 years, brain organoid technology has brought us too much surprise to strengthen our understanding of human brain development and the relevant brain diseases. Many problems appear at the early stage of this technology such as lack of reproducibility, lack of cell-type specificity, heterogeneity and uncontrolled size have been largely improved in these years with the advance in engineering and stem cell biology. For example, the mixing of extracellular matrix and other additives with the biomaterial could produce a cultural environment that is more similar to the actual human brain and generate a high-reproducible brain organoid (Yin et al. As we discussed in this review, lacking accurate positional information is a nonnegligible problem in organoid research. In 2013, Sasai has already raised the idea that providing 3D self-organization cell cultures with positional information corresponding to morphogens presented during embryogenesis is the next generation of organogenesis Sasai , offerinHowever, there are still many obstacles that might not be solved only by providing spatial cues. For example, the absence of a vascular system restricts the growth and maturation of the organoid due to the limited oxygen and nutrients diffused from the culture medium, especially for the deep cells inside. Luckily, there are many successful attempts to establish vascular systems in brain organoids. Besides transplantation to the adult mouse brain to provide a vascularized physiological environment (Mansour et al. Also, such assembloid technology could be applied to supplement other missing non-neural cells in the human brain such as microglia and endothelial cells. Alternatively, a recent study reported that the organoid without SMAD inhibition at the early stage could contain several mesodermal progenitors that are able to differentiate into mature microglia (Ormel et al. Moreover, bioengineering research also promotes functional reproduction in the organoid. Early in this century, scientists have already found that geometry and patterning could improve the activity of cultured neuron networks (Nam et al. Overall, the brain organoid is a cutting-edge technology in the research of the human brain. Combining advanced research strategies and techniques, we could have an opportunity to better understand the human brain from broad perspectives not only limited to genetic, molecular and cellular level but also biomechanics. Establishing appropriate morphogen gradients have been suggested as a promising and effective approach to provide the positional information during human neural organoid culture, which largely improve the patterning and the maturation of the organoids. Although there are still many challenges, we strongly believe after replenishing those missing puzzles, this technology will bring us more surprise.


The TNF receptor-interacting protein kinases (RIPK)-1 and 3 are regulators of extrinsic cell death response pathways, where RIPK1 makes the cell survival or death decisions by associating with distinct complexes mediating survival signaling, caspase activation or RIPK3-dependent necroptotic cell death in a context-dependent manner. Using a mass spectrometry-based screen to find new components of the ripoptosome/necrosome, we discovered the protein-arginine methyltransferase (PRMT)-5 as a direct interaction partner of RIPK1. Interestingly, RIPK3 but not RIPK1 was then found to be a target of PRMT5-mediated symmetric arginine dimethylation. A conserved arginine residue in RIPK3  was identified as the evolutionarily conserved target for PRMT5-mediated symmetric dimethylation and the mutations R486A and R486K in human RIPK3 almost completely abrogated its methylation. Rescue experiments using these non-methylatable mutants of RIPK3 demonstrated PRMT5-mediated RIPK3 methylation to act as an efficient mechanism of RIPK3-mediated feedback control on RIPK1 activity and function. Therefore, this study reveals PRMT5-mediated RIPK3 methylation as a novel modulator of RIPK1-dependent signaling. TNF receptor-interacting protein kinases (RIPK)-1 and -3 are the master regulators of programmed cell death signaling. The concerted action of the RIP kinases downstream to the death receptors, interferon-alpha receptor (IFNαR) and Toll like receptor (TLR) regulate cell death/survival decisions during development and inflammation  catalyze transfer of one or two methyl groups to arginine residues, giving rise to monomethyl-arginine, asymmetric dimethyl-arginine (aDMA) or symmetric dimethylarginine (sDMA). The family of mammalian PRMTs consists of nine members sorted into three groups: Type I methyltransferases  generate aDMA by adding two methyl groups to the same terminal nitrogen of the arginine residue, Type II (PRMT5 and 9) mark each of the terminal nitrogen atoms of arginine with one methyl group, hence generating sDMA and the type III member PRMT7 catalyzes mono-methylation of arginine residues . Tight  in ref. . Interes in ref. . Other i in ref. . PRMT5 a in ref. , PKCι-metivation . Moreovetivation .Recent evidence indicate that the switch between pro-survival and pro-death functions of RIPK1 is regulated by a panel of modifications including phosphorylation and ubiquitination  and the Ripk1−/− MEFs were rescued by the expression of doxycycline (dox)-inducible FLAG-tagged RIPK1 and single clonal cell lines were isolated and stably labeled by isotopes. Dox-induced expression of RIPK1 was detectable in both clones analyzed and RIPK1 was efficiently enriched by FLAG-tag-based immunoprecipitation. Immunoblots with anti-FLAG and anti-RIPK1 antibodies indicate that there is no detectable RIPK1 expression and enrichment in the non-induced cells  model system of induced expression of epitope-tagged RIPK1. To identify ripoptosome interactors by a non-biased screen, we applied SILAC-based MS analysis. After the enrichment of FLAG-RIPK1-associated proteins from differentially SILAC-labeled control and TNF + SM + zVAD Fig.  treated Since the RIPK1 interactors included the core methylosome components PRMT5 and WDR77, which induce symmetric dimethylation of arginine residues, we monitored arginine methylation of RIPK1 after co-expression of PRMT5 by using a symmetric di-methyl arginine motif (SdmArg) antibody. Despite clear association of RIPK1 and PRMT5 in the pulldown, there was no symmetric dimethylation detectable for GST-RIPK1 enriched using glutathione beads Fig. . We thenhttp://www.phosphosite.org) documents the existence of arginine methylation sites in both human and mouse RIPK3 outside the kinase domain  and methylation-deficient mutants (R486K or R486A) of RIPK3. After transduction and selection of cell lines for uniform transduction, the selected empty vector and RIPK3 expression lines showed similar expression levels of GFP expressed from the bidirectional promotor demonstrating uniform transduction and similar expression levels of WT and mutant RIPK3 expression [Ripk1−/− MEFs, HEK293T cells, and PANC-1 cells were cultured in DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS) and 1% penicillin/streptomycin. The cell lines were maintained at 37 °C and with 5% CO2 in a humidified atmosphere.3T3-immortalized  Germany). Ripk1−/MLKL (#14993), αSdmArg (#13222), RIP3 (#13526), Caspase-8 (#4927), pS166-RIPK1 (#65746), pS358-MLKL (#91689), pS345-MLKL (#37333), pS227-RIPK3 (#93654) phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#4370) and pS473-Akt (#4060), pS536-NFkB p65 (#93H1), IkBα (#9242) antibodies were from Cell Signaling Technology (CST). Further antibodies used were against GST , RIPK1 , GAPDH , EF2 , AKT1/2/3 , ERK2 , FLAG , PRMT5 Polyclonal antibody  and GFP . The secondary antibodies used were anti-rabbit IgG-HRP (Conformation Specific) , mouse TrueBlot® ULTRA , goat anti-mouse IgG (H + L)-HRP  and goat anti-rabbit IgG (H + L)-HRP .The following reagents at given concentrations were used for cell treatments: Doxycycline , recombinant human TNFα , Birinapant/Smac Mimetics , pan-caspase inhibitor zVAD-fmk , PF3644022 , GSK591 , LLY-283 , Nec-1 , PP2 , NSA , GSK’872 .Dox-inducible pSERS retroviral vector as described previously  was convHEK293T cells were transfected using polyethylenimine . Transfected cells were maintained in antibiotic-free DMEM media supplemented with 10% FCS for 12–16 h followed by providing with complete DMEM media. Transfected cells were analyzed between 20 and 36 h post-transfection. To monitor the effect of pharmacological inhibitors on methylation and interaction in transfected cells, cells were treated with inhibitors for the last 6 h prior to cell lysis.Ripk1−/− MEFs were stably rescued with Doxycycline inducible retroviral vectors expressing mouse RIPK1. For preparation of viral supernatants, 7.5 million Ecopack-HEK293T cells were seeded in 10 cm plates and transfected overnight with 5 µg each of pCL-Eco and pSERS11 based retroviral vectors using PEI, in antibiotic-free medium with 10% FCS. Medium was changed to fresh complete media supplemented with 1X non-essential amino acids. Virus-containing supernatants were harvested five times from transfected Ecopack-HEK293 cells (Clontech/Takara), over a period of three days post-transfection and filtered with 0.45 µm filters. 1.2 × 105Ripk1−/− MEF cells were seeded per well in a six well plate and were cultured for 4 days with viral supernatants in the presence of 8 µg/ml polybrene. RIPK1 expression was monitored by intracellular staining and flow cytometry analysis using an Accuri-C6 cytometer (BD Biosciences).To generate a rescue model for RIPK3, PANC-1 cells (RIPK3-deficient) were transduced with lentiviral vectors expressing WT/mutant human RIPK3 or pLBID-MCS-GFP-P2A-Puro empty vector. ViraPower Lentiviral Expression System (Invitrogen) was used to package pLBID lentiviral vectors expressing FLAG-hRIPK3 or methylation site mutants or empty vector. Viral supernatants were collected 48 h post-transfection and filtered through 0,45 µm filters as described previously . PANC-1 7 cells/ml and were fixed with 3-volumes of 4% PFA. After 30 min at room temperature, fixed cells were washed and permeabilized for 30 min with ice-cold 90% methanol. After 2× washes with PBS, cells were blocked with 4% BSA on ice for 30 min. After 1 h incubation at room temperature with anti-RIPK1 antibody  cells were incubated with 1:700 diluted secondary antibodies  for 30 min. Samples were washed with PBS and analyzed with Flow cytometry using an Accuri-C6 cytometer (BD Biosciences).Cells were suspended in PBS at a concentration of 10Ripk1−/− MEFs. These clones were metabolically labeled with light  and medium  non-radioactive isotope amino acids. SILAC Protein Quantitation Kit (Trypsin), DMEM  was used for generating light labeled cell lines and for generation of medium labeled cell lines, L-Lysine-2HCL,4,4,5,5-D4 (ThermoFisher Scientific) and 13C-labeled L-Arginine HCl  were individually purchased. Cells were cultured in respective medium supplemented with 200 µg/ml of L-Proline for at least 10 passages for achieving incorporation of each respective labels. Cells were seeded at a density of 3 × 106 cells in 10 cm plates and were treated the next day with doxycycline (1 µg/ml) for 4 h, followed by treatment of light-labeled cells with only DMSO control and of medium-labeled cells with TNF + SM + zVAD for 2 h. FLAG-RIPK1 bound complexes were enriched from 1.5 mg of protein lysate per sample. Pre-cleared lysates were incubated with 40 µl of 50% ANTI-FLAG® M2 affinity gel  on a rotor for 3 h at 4 °C. After immunoprecipitation, Anti-FLAG beads were pooled from the two differentially treated light and medium labeled samples for each clonal cell line. Proteins were eluted from pooled beads with 0.5 mg/ml FLAG peptide  by shaking at 1000 rpm for 1 h at 4 °C. Samples were separated on a 10% SDS-PAGE gel and in-gel digested overnight with Trypsin/Lys-C protease mix and resulting peptides were desalted using C18-stage tip. The purified peptides from each sample were analyzed by mass spectrometry in technical duplicates. Samples were analyzed on the Evosep One system using an in-house packed 15 cm, 150 μm i.d. capillary column with 1.9 μm Reprosil-Pur C18 beads  using the pre-programmed gradients for 60 samples per day (SPD). The column temperature was maintained at 60 °C using an integrated column oven  and interfaced online with the Orbitrap Exploris 480 MS. Spray voltage was set to 2.0 kV, funnel RF level at 40, and heated capillary temperature at 275 °C. Full MS resolutions were set to 60,000 at m/z 200 and full MS AGC target was 300 with an IT of 22 ms. Mass range was set to 350−1400. Full MS scan was followed by top 10 DDA scans, using 1.3 m/z isolation window, 45,000 resolution, 86 ms injection time, AGC target of 200 and normalized collision energy was set at 30%. All data were acquired in profile mode using positive polarity and peptide match was set to off, and isotope exclusion was on. The intensity of each peptide was normalized with intensity of RIPK1 in respective light and medium-labeled samples. SILAC ratios were then calculated by comparing the intensities of medium-labeled peptides to intensities of light-labeled peptides  as per manufacturer protocol. Transfected cells were cultured for 48 h. Cells were then re-transfected overnight with mRIPK3 and hRIPK3 expression vectors. The next day, fresh media was provided to the transfected cells and plates were washed and frozen 24 h post-transfection.Two siRNAs targeting human PRMT5  were used for knockdown experiments in HEK293T cells. The siRNA sequences are provided in Supplementary Table Cells were lysed directly in cell culture plates with 2× SDS sample buffer containing 10% SDS, 1.5 M TRIS pH 8.8, 5% Glycerol, 2.5% 2-Mercaptoethanol, and Bromophenol Blue. The samples were then boiled for 5 min at 95 °C. Denatured protein lysates were run on SDS-PAGE (7.5–16% gradient) gels and immunoblotted to Nitrocellulose blotting membrane . Western blots were blocked with 5% powdered skim milk in PBS with 0.1% Tween 20 for 1 h at room temperature. After three washes with PBS containing 0.1% Tween 20, membranes were incubated overnight with the primary antibody at 4 °C followed by 1 h incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature. Blots were developed with an ECL detection kit, WESTAR NOVA 2.0 , and the digital chemiluminescence images were taken by a Luminescent Image Analyser LAS-3000 (Fujifilm).3VO4, 10 mM b-glycerophosphate, 50 mM Sodium Fluoride, 5 mM Na-pyrophosphate, 1% Triton X-100, 1 mM benzamidine, 2 µg/ml leupeptin, 0.1% 2-Mercaptoethanol, 0.27 M Sucrose, 0.2 mM PMSF, 1 µg/ml Pepstatin A, 1X Protease Inhibitor Cocktail , Phosphatase Inhibitor Cocktail . Lysates were then cleared by centrifugation and the beads were blocked with 1% BSA to avoid non-specific binding of PRMT5 to Glutathione beads. For the enrichment of the fusion proteins, pre-cleared lysates were incubated with Monoclonal ANTI-FLAG® M2 affinity gel  or Protino Glutathione Agarose 4B  for 16 h at 4 °C. The beads were then washed 4× with immunoprecipitation wash buffer containing 1X TBS, 50 mM Sodium fluoride, 1% Triton X-100, 1 mM Na3VO4. The beads were boiled in 2× SDS sample buffer for 5 minutes at 95 °C and the eluted protein complexes were analyzed by Western blotting.GST-pulldowns (GST-PD) and FLAG-IPs were performed as described previously , 58. Celt-tests were used to calculate the statistical significance of the viability assays. This data is presented in Supplementary Table All Immunoblot results presented in the figures are representative results from at least three independent experiments. The calculations and statistical analyses were performed and graphs were plotted using Microsoft Excel. Two-tailed unpaired Supplementary Information LegendSupplementary Figures S1-S8Supplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Information_Original Immunoblot data