ARFs, PP2C, starch synthase, FLC, PIF1, AGL80, and WRKY32, are involved mainly in plant hormone signal transduction, starch and sucrose metabolism, and flowering regulation and may be involved in early flowering. This study serves as a reference and theoretical basis for kenaf production and provides insights into the effects of DNA methylation on plant growth and development.DNA methylation regulates key biological processes in plants. In this study, kenaf seedlings were pretreated with the DNA methylation inhibitor 5-azacytidine (5-azaC) , and the results showed that pretreatment with 200 μM 5-azaC promoted flowering most effectively. To elucidate the underlying mechanism, phytohormone, adenosine triphosphate (ATP), and starch contents were determined, and genome-wide DNA methylation and transcriptome analyses were performed on anthers pretreated with 200 μM 5-azaC (5-azaC200) or with no 5-azaC . Biochemical analysis revealed that 5-azaC pretreatment significantly reduced indoleacetic acid (IAA) and gibberellic acid (GA) contents and significantly increased abscisic acid (ABA) and ATP contents. The starch contents significantly increased in response to 200 and 600 μM 5-azaC. Further genome-wide DNA methylation analysis revealed 451 differentially methylated genes (DMGs) with 209 up- and 242 downregulated genes. Transcriptome analysis showed 3,986 differentially expressed genes (DEGs), with 2,171 up- and 1,815 downregulated genes. Integrated genome-wide DNA methylation and transcriptome analyses revealed 72 genes that were both differentially methylated and differentially expressed. These genes, which included  Arabidopsis thaliana at all sites is performed by DNA METHYLTRANSFERASE 1 (MET1), a plant-specific CHROMOMETHYLASE 3 (CMT3), and DOMAINS REARRANGED METHYLTRANSFERASEs (DRMs)  . DNA mets (DRMs) . These Ds (DRMs) . For exaime gene . DNA demime gene . 5-Azacyime gene . Plants ime gene . Non-vered early . In addied early .Hibiscus cannabinus L.) is a valuable multipurpose natural fiber crop species. Kenaf biomass is used for paper, cordage, building materials, automotive interiors, carpet backing, animal bedding, and livestock forage  cultivar P3A was used as the plant material. Healthy P3A seeds were soaked for 10 min in 3% H2O2, washed three times in distilled water, and sown evenly in 15 plastic boxes (27 cm × 18 cm × 9 cm) that contained two layers of gauze, each plastic box was received with 300 seeds. The seeds were then treated with different concentrations of 5-azaC , with three replications. 5-azaC was added to each plastic box according to each corresponding concentration, and the seeds were cultivated under controlled conditions, i.e., 27°C in light for 10 h and 25°C in darkness for 14 h, with a relative humidity of 65–70% and a light intensity of 280 μmol/m2/s. After 5 days of 5-azaC pretreatment, 200 healthy seedlings were randomly selected from each plastic box, after which they were divided into two groups and then transplanted separately to the field at two locations: Nanning, Guangxi Province , and Xinyang, Henan Province . The average pH values of the topsoil in Xinyang (northern China) and Nanning (southern China) were 5.90 and 5.68, respectively, and the average organic matter contents were 18.12 and 20.81 g/kg, respectively. The average contents of total nitrogen, available phosphorus and available potassium in the two locations were 1.04 g/kg, 9.77 mg/kg, and 80.1 mg/kg, respectively (Xinyang) and 1.08 g/kg, 10.65 mg/kg, and 124.66 mg/kg, respectively (Nanning). The average temperatures of the two areas from June to September (which corresponds to the main development period of kenaf) in recent years were 23–31.3 and 25.7–33.5°C, respectively, with average precipitation amounts of 500 and 693.33 mm, respectively , gibberellic acid (GA), and abscisic acid (ABA) in kenaf anthers pretreated with different concentrations of 5-azaC were measured via enzyme-linked immunosorbent assay (ELISA). Endogenous hormones were extracted according to the method proposed by FspEI . FspEI can recognize 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) within CmCGG and mCHG sites and produce a double-stranded DNA break at a fixed distance from the modified cytosine. Accordingly, symmetrical DNA methylated sites were bidirectionally cleaved by FspEI to generate 32-base-long fragments. Two adapters were then added to the digested DNA by T4 DNA ligase , after which the ligation products were amplified via specific primers. The target DNA fragments were further purified and subjected to sequencing on an Illumina HiSeq XTM Ten platform.Genomic DNA was extracted from anthers via a DNA Secure Plant Kit  according to the manufacturer’s protocol. The DNA was then assessed via electrophoresis on agarose gels, and the quality and concentration of the DNA were determined via a NanoDrop 2000 spectrophotometer. Approximately 150–200 ng of DNA was used to construct MethylRAD libraries following a previous protocol . Briefly1 by Bowtie 2 (version 2.3.4.1), with the default parameters. The distribution of the methylated cytosine sites among kenaf chromosomes and different functional elements of the genome were evaluated. The relative expression level of each methylated site (CpG) was determined by a normalized read depth—reads per million . The changes in methylation level were assessed on the basis of the sequencing depth of each site in the relative quantitative methylation results by the R package DESeq. The different p-values and fold-changes (FCs) among different sites were calculated according to the sequencing data . Differentially methylated genes (DMGs) were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis.The raw reads were first subjected to quality filtering and adapter trimming. Reads with more than 8% ambiguous bases (N), reads of poor quality  or reads without enzyme restriction sites were removed by a custom Perl script. The reads with enzyme sites (enzyme reads) were subsequently aligned against the kenaf genomeThe total RNA from the anthers was extracted according to a modified cetyltrimethylammonium bromide (CTAB) RNA extraction protocol. The RNA purity and quantification were evaluated with a NanoDrop 2000 spectrophotometer , and the RNA integrity was assessed with an Agilent 2100 Bioanalyzer  . cDNA liTM Ten platform, and 150-bp paired-end reads were generated. The raw data in fastq format were first processed with Trimmomatic, and raw reads for each sample were generated. After reads containing adapters, reads containing poly-N bases and reads of low quality were removed from the raw data, clean reads were obtained for subsequent analyses. The clean reads were then mapped to the kenaf genome (see text footnote 1) by HISAT2.The libraries were sequenced on an Illumina HiSeq XP < 0.05 and FC > 2 or FC < 0.5 were set as the thresholds for significantly different expression. Hierarchical cluster analysis of differentially expressed genes (DEGs) was performed to explore gene expression patterns. GO functional enrichment and KEGG pathway enrichment analysis of the DEGs was performed with R on the basis of a hypergeometric distribution. Gene structure extension and novel transcript identification were performed by comparing the reference genome and the known annotated genes via Cuffcompare software.The fragments per kilobase of transcript per million mapped reads (FPKM) value of each gene was calculated with Cufflinks, and the read counts were obtained by the use of htseq-count. Differential gene expression was analyzed via the DESeq R package; 2 SnpEff was then used to annotate and predict the effects of variants on genes (such as amino acid changes).ASprofile was used to analyze the alternative splicing of differentially regulated transcript isoforms or exons. Single-nucleotide polymorphisms (SNPs) and insertions and deletions (INDELs) were called using SAMtools and BCF tools. The details are shown on the SAMtools webpage.Actin 3, Histone 3, and 18 SrRNA were used as endogenous reference genes for normalizing gene expression, and the relative gene expression was calculated via the 2–ΔΔCT method in this study  on a Bio-Rad CFX96 instrument (Bio-Rad Laboratories). Each 20 μL reaction mixture was composed of 1.0 μL of cDNA, 0.4 μL of primers , 10 μL ois study .P ≤ 0.05 for statistical significance.The data concerning the contents of IAA, GA, ABA, ATP, and starch are presented as the means ± standard errors (SEs) of three replicates. One-way variance analysis (ANOVA) was performed via SPSS Version 16  software. The means were analyzed via the least significant difference (LSD) test at The results showed that 5-azaC pretreatment could shift the flowering time of kenaf, and the effects of 5-azaC on flowering were closely related to the concentration used. Low-concentration (100 μM) and high-concentration (600 μM) pretreatment had no significant effects on flowering. Moderate concentrations (200 and 400 μM) resulted in early flowering, and the effects were significantly more obvious in Xinyang (northern China). In Nanning and Xinyang, pretreatment with 200 and 400 μM 5-azaC advanced start of the early blooming stage by 5–7 and 2–4 days  and the 5-azaC pretreatment significantly reduced the contents of IAA and GA in kenaf anthers  and sign3 of the NCBI (accession number: SRR11743644-SRR11743649).To obtain an overview of kenaf cytosine methylation patterns, three libraries were constructed from control (5-azaC0) anthers and those pretreated with 5-azaC (5-azaC200) separately and then sequenced on the MethylRAD sequencing platform. On average, 29.02 million raw reads were obtained for the 6 libraries. After the labels without the expected restriction sites were filtered and removed, the MethylRAD sequencing data (enzyme reads) comprised 5.18, 4.78, 4.96, 4.74, 5.00, and 4.90 million reads for 5-azaC0 and 5-azaC200. Finally, 0.79, 0.74, 0.74, 0.72, 0.77, and 0.69 million mapping reads were generated . The enzyme reads were submitted to the Sequence Read Archive (SRA)On average, 121,596 CCGG and 58,958 CCWGG DNA methylation sites with average depths of 4.26 and 4.29, respectively, were found for 5-azaC0, and on average, 118,747 CCGG and 57,674 CCWGG DNA methylation sites with average depths of 4.19 and 4.21, respectively, were found for 5-azaC200. These results showed that the level of DNA methylation at the CCGG sites was greater than that at the CCWGG sites in kenaf anthers, and the level of DNA methylation decreased by 2.29% after treatment with 200 μM 5-azaC compared with the control .This study is the first to systematically analyze the distribution of DNA methylation sites on each chromosome of kenaf. The distributions of CCGG methylation sites and CCWGG methylation sites across the chromosomes of kenaf were consistent, and most methylation sites were present on the chromosome 12, followed by chromosome 3. Chromosome 11 had the fewest methylation sites, followed by the chromosome 2. A map of the distribution of DNA methylation sites on each chromosome of kenaf is shown in The number and distribution of methylation sites within different gene elements of the kenaf genome were calculated. The results showed that the CCGG and CCWGG methylation sites lied mostly within intergenic regions, followed by downstream regions, upstream regions, introns and exon . The fewThe distribution of methylation sites was analyzed within 2-kb regions upstream and downstream of the transcription start site TSS; , transcrIn total, 862 CCGG-site DMSs and 323 CCWGG-site DMSs were identified in the 5-azaC0 and 5-azaC200 samples . There wIn addition, the methylation levels of DMGs were calculated . Among 1To further characterize the changes in DNA methylation levels in these two samples, we performed GO and KEGG enrichment analysis of the DMGs . As showKEGG enrichment analysis indicated that 31 DMGs  were highly enriched in 16 pathways. The main significant pathways included “signal transduction”, “translation”, “carbohydrate metabolism”, “lipid metabolism”, “nucleotide metabolism”, “biosynthesis of other secondary metabolites”, “amino acid metabolism”, “cell growth and death”, “transport and catabolism”, and so on. These are essential for plant growth and development and are suggested to be involved in the regulation of flowering .TM platform. An average of 49.42 million raw reads were obtained for the six libraries. After filtering was performed, 48.07 million clean reads were obtained on average in each of the 6 libraries, the average GC content was 46.16%, and their Q30 values were greater than 94%  in each of the experimental groups (5-azaC0 and 5-azaC200) and then sequenced on the Illumina HiSeq X Tenthan 94% . The comf 93.66% .r > 0.99) between the three biological replicates of 5-azaC0 and 5-azaC200 indicated that the RNA sequencing results were reliable.Additionally, we confirmed the reliability of the samples via correlation analyses based on FPKM values. The high similarity  and 1,815 (45.53%) of which were up- and downregulated, respectively, were identified from the comparison of 5-azaC200 and 5-azaC0  between 5-azaC200 and 5-azaC0 were identified on the basis of the following threshold values: log 5-azaC0 . These dGO and KEGG enrichment analysis was performed to better understand the functions of the DEGs whose expression was induced in response to 5-azaC pretreatment in terms of the regulation of kenaf flowering time . GO enriKEGG enrichment analysis revealed that 731 DEGs, including 405 and 326 of which were up- and downregulated, respectively, under 5-azaC200, were enriched in 23 pathways. Remarkably, the upregulated DEGs were enriched mainly in “carbohydrate metabolism”; “energy metabolism”; “nucleotide metabolism”; “cell growth and death”; “folding, sorting, and degradation”; “translation”; “transcription”; and “replication and repair”. The downregulated DEGs were enriched mainly in “lipid metabolism”, “biosynthesis of other secondary metabolites”, “metabolism of cofactors and vitamins”, and “xenobiotic biodegradation and metabolism” . These pIn addition, 19,256 transcription factors were distributed among 58 families. A total of 3,263 differentially expressed transcription factors (DTFs) were distributed across 57 families, including the bHLH, MYB, MADS, C2H2, WRKY, CO-like, and LFY families, whose members are closely associated with flowering . Among tauxin response factor 18 (ARF18), starch synthase, glutaredoxin family protein, serine/threonine-protein kinase CDL1, E3 ubiquitin-protein ligase LIN-1, ABC transporter G family member 25, indole-3-acetaldehyde oxidase, phosphatase 2C family protein, cytokinin dehydrogenase 7, cytochrome P450 83B1, and cytochrome P450 CYP72A219 in the first and third quadrants implied that DNA methylation may positively regulate gene expression. Conversely, the location of genes in the second and fourth quadrants implied that DNA methylation may negatively regulate gene expression. Examples of these genes include cytochrome P450, ethylene-responsive element-binding protein, mitogen-activated protein kinase, plastid developmental protein, cellulose synthase-like protein E1, ABC transporter G family, histone-lysine N-methyltransferase, and lipid-transfer protein. The reason about the appearance of two opposing regulatory modes, since the genomic location, underlying DNA sequence, and the type of DNA methylation is often reflective of its expression status  is the key enzyme of sucrose synthesis, which is strongly linked to plant growth, photosynthesis characteristics, and fruit ripening (6-Phosphofructokinase (PFK) is a key catalytic enzyme involved in fructose phosphorylation and plays an important role in fructose metabolism  and DELLA protein , the main signal factors in gibberellin pathway, plays a key role in the flower-forming process, which promotes the flowering of plants mainly by regulating  protein . The inh protein . Whether protein . In this protein . This suArabidopsis and Thlaspi with 5-azaC can bloomearly. Thus, the authors proposed that 5-azaC promotes flowering by demethylation of the flower-related gene activate the transcription, and they further suggested the transcription of kaurenoic acid hydroxylase, a key enzyme is required for GA biosynthesis , ethylene receptor (ETR), and phytochrome interacting factor 4 (pif4). Meanwhile, the remaining 31 genes were upregulated, including auxin response factors (ARFs), those encoding DELLA proteins, ABA receptor PYR/PYL family members, brassinosteroid (BR) signaling kinase, etc. Interestingly, genes such as PP2C, EIN3, ARFs, and SNPK2 were not only differentially expressed but also differently methylated.Increasing numbers of studies suggested that there is a close relationship between epigenetic regulation and plant hormone signaling. Phytohormone play an important role in chromatin compaction mediated by DNA methylation and histone modifications. At the same time, DNA methylation also regulates plant hormone signal transduction . Treatinynthesis . Here, cGIGANTEA (GI), the flowering site T gene (FT) and TWIN SISTER OF FT (TSF)  . PP2C, asduction . In the gulation . The levlowering . The accelopment . Our rescis-acting elements in the promoter region of a gene or functional regions of other transcription factors. TFs play a central role in regulating plant growth and development, defense responses, and biotic and abiotic stress responses , FLOWERING LOCUS M (FLM), MADS AFFECTING FLOWERING 1 (MAF1), MAF2 to MAF5 FLC-like proteins, AGAMOUS-LIKE 15 (AGL15), and AGL18. Factors that promote flowering include SOC1 and AGL24 (AGL15 and AGL18 genes in the anthers in the 5-azaC pretreatment group was only one-third of that in the control group. SVP can inhibit the late-flowering phenotype caused by FLM overexpression (SVP significantly increased under 5-azaC200 (by 4.57-fold). In addition, the genes encoding some DTFs such as MADS-box protein A, AGL62 and AGL80 were also differentially methylated. Thus, the level of DNA methylation might affect gene expression and play an important role in early flowering.MADS-box transcription factors have been shown to play a vital role in the regulation of floral organ development and flowering time . With thox genes . In termnd AGL24 . The exppression . In the MiWRKY12 gene can promote the expression of flowering-related genes such as CONSTANS (CO), thus leading to the early Miscanthus flowering  to cause early flowering of soybean  cycle  cycle . This sthttps://www.ncbi.nlm.nih.gov/sra/) of NCBI (accession numbers: SRR11743644-SRR11743649 and SRR10912354-SRR10912359).The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: Sequence Read Archive (SRA) (PC conceived the project and revised and edited the manuscript. ZL performed the experiments, analyzed the data, and wrote the manuscript. RL, MT, MK, SC, and YH revised and edited the manuscript. DL, WZ, ZH, and JY assisted in managing the materials. All the authors have read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


RANBP2 gene are associated with acute necrotizing encephalopathy type 1 (ANE1), a rare condition where patients experience a sharp rise in cytokine production in response to viral infection and undergo hyperinflammation, seizures, coma, and a high rate of mortality. Despite this, it remains unclear howRanBP2 and its ANE1-associated mutations contribute to pathology. Mounting evidence has shown that RanBP2 interacts with distinct viruses to regulate viral infection. In addition, RanBP2 may regulate innate immune response pathways. This review summarizes recent advances in our understanding of how mutations in RANBP2 contribute to ANE1 and discusses how RanBP2 interacts with distinct viruses and affects viral infection. Recent findings indicate that RanBP2 might be an important therapeutic target, not only in the suppression of ANE1-driven cytokine storms, but also to combat hyperinflammation in response to viral infections.Ran Binding Protein 2 (RanBP2 or Nucleoporin358) is one of the main components of the cytoplasmic filaments of the nuclear pore complex. Mutations in the  Ran Binding Protein 2 (RanBP2), also known as Nucleoporin 358 KDa (Nup358), is one of the main components of the cytoplasmic filaments of the nuclear pore complex (NPC) . RanBP2 Mutations in RanBP2 are associated with acute-necrotizing encephalopathy type 1 (ANE1), a pediatric neurological disease that manifests as an overproduction of cytokines (known as a “cytokine storm”) after viral infection ,38,39. TAcute necrotizing encephalopathy (ANE) was first described by Mizuguchi in 1995. It is a rare disorder that is triggered by viral infections such as influenza or parainfluenza and causes a loss of consciousness, seizures, coma, and rapidly progressing encephalopathy . Most caRANBP2 gene involving c.1880C>T: p.Thr585Met, c.2085C>T: p.Thr653Ile, or c.2094A>G: p.Ile656Val by the old nomenclature, which are also known as c.1754C>T: p.Thr585Met, c.1958C>T:p.Thr653Ile, or c.1966A>G: p.Ile656Val by the Human Genome Variation Society (HGVS) preferred nomenclature [RANBP2 gene, including c.2043G>C: p.Trp681Cys, c.4993A>G: p.Lys1665Glu, c.5249C>G: p.Pro1750Arg, c.3363G>T, p.Lys1121Asn, c.128A>T, p.Asp43Val, and c.1350A>T, p.Leu450Phe, have also been reported to be linked to ANE1 [RANBP2, the penetrance of these dominant mutations was found to be only approximately 40% in the initial report, and half of ANE1 cases experience recurrent episodes [RANBP2 gene is the most common ANE1-associated mutation . Similar to ANE cases, ANE1 predominantly affects young children, especially ages 1–4 years old   B with thpisodes) C.The majority of pathogens that trigger ANE1 are viruses, including the influenza virus (50.0%), parainfluenza virus (8.9%), human herpes virus 6 (7.1%), respiratory syncytial virus (5.4%), adenovirus (1.8%), rhinovirus (1.8%), and rotavirus (1.8%). In addition, it has been reported that bacteria mycoplasma (7.1%) can also induce ANE1, and only 16.1% of infectious workups in all reported ANE1 episodes were negative D.Of the The diagnostic criteria of ANE and ANE1 have been well described previously ,41,47,83RANBP2 are genetic factors that contribute to ANE1, their actual role in the pathogenetic mechanisms of this disease remains unclear.Although it has been known that missense mutations in RANBP2 gene show metabolic defects, especially in glucose metabolism [As described previously, RanBP2 has been implicated in several different cellular processes, including energy maintenance in neurons by perhaps regulating microtubule and/or mitochondrial function ,37,84,85tabolism . In additabolism . ANE muttabolism . DespiteRANBP2 gene in retinal ganglion cells show increased microglial activation [Another plausible theory, which is the most widely accepted, is that mutations in RanBP2 directly trigger a “cytokine storm” after infection. Previous reports showed that ANE patients had elevated concentrations of cytokines including proinflammatory cytokines such as interleukin 6 (IL6), tumor necrosis factor-α (TNF-α), IL10, interferon γ (IFNγ), soluble tumor necrosis factor (sTNF) receptor, and IL15 in their CSF and plasma ,93,94,95tivation , suggestIL6 mRNA. Our data is consistent with the model that Argonaute proteins first interact with the IL6 mRNA in the nucleus and accompany the mRNA as it traverses the nuclear pore. Then just after the completion of nuclear export, these Argonautes are sumoylated by RanBP2. This sumoylation event stabilizes Argonaute onto the IL6 mRNA, thereby enforcing its silencing. Our results suggest that, under certain circumstances, ANE1-associated mutations alter microRNA-dependent silencing, which eventually causes a massive secretion of cytokines. In agreement with this idea, RanBP2-depletion has been shown to reduce the number of P-bodies, which are cytoplasmic foci that have been implicated in microRNA-silencing [IL6 mRNA in human osteosarcoma cells [Recently, we documented that RanBP2 promotes the microRNA-mediated suppression of ANE1-associated cytokines, such as IL6 and TNF-α . We founilencing ,105. Morilencing . Despitema cells . Thus, iThere may be other ways in which mutations in RanBP2 trigger cytokine storms, possibly through the over activation of innate immune pathways. It is known that the production of cytokines requires the activation of innate immune pathways. These sense viral infections and stimulate downstream transcription factors, including interferon (IFN)-regulatory factor 3 (IRF3), IRF7, nuclear factor kappa-light-chain-enhancer of activated B cells , and signal transducer and activator of transcription (STAT) proteins . In geneRANBP2 mutations, additional environmental factors, such as pathogen infection, are required for ANE1 development [RANBP2 alter the viral infection process by impacting the interactions between RanBP2 and either viral proteins or anti-viral host factors. Indeed, RanBP2 has been reported to be required for the replication of a variety of viruses . Most humans are infected by HSV-1, and part of them present clinical symptoms, such as cold sores. In 2008, Hofemeister and O’Hare showed that HSV infection reduces the amount ofO-glycosylation in RanBP2, which may affect NPC function [UL36) interacts with RanBP2, thus allowing capsids to attach to the surface of the nuclear envelope [Herpes simplex viruses (HSVs) are double-stranded DNA viruses that belong to function . It has envelope . In addienvelope ,113. AltAdenoviruses are a family of nonenveloped double-stranded DNA viruses that infect a broad range of vertebrate species and cause respiratory, gastrointestinal, ocular, and urogenital diseases. Strunze et al. showed that during adenovirus infection, RanBP2 interacts with the kinesin-1 heavy-chain (KIF5C) to promote the transport of NPC components  away from the nucleus and into the cytoplasm. This transport is thought to disrupt the nuclear envelope and promote the transport of viral DNA from viral capsids, which are docked onto NPCs, to the exposed nucleoplasm .In another study, the authors proposed a model whereby RanBP2 serves as an assembly platform where the adenovirus genome becomes coated with nuclear transport receptors, facilitating its nuclear import . To datePoxviridae. VACV is known as a live, naturally attenuated vaccine used for eradicating smallpox caused by variola virus, another member of the Poxviridae family [Poxviridae viruses can cause ANE1 remains unknown as smallpox has been eradicated.Vaccinia virus (VACV) is a double-stranded DNA virus that belongs to the family of e family . VACV ene family . Viral fe family ,132. Alte family . Indeed,Papillomaviridae [Papillomaviruses are small, nonenveloped, icosahedral DNA viruses with an 8 kb circular double-stranded DNA genome, which belong to the family aviridae . Papilloaviridae ,135. Theaviridae ,142,143.aviridae . Insteadaviridae . CuriousCoronaviridae [Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV2) is an enveloped, single-stranded, positive-sense RNA virus with a genome of approximately 30-kb, which belongs to beta-coronavirus of the family aviridae ,145. SARSARS-CoV2 proteins interact with many human proteins including distinct nuclear transport receptors and nucleoporins . SARS-CoThe link between RanBP2 and cytokine storms suggest that it may play a role in the development of the COVID-19 disease ,153,154.Picornaviridae, which infects the upper respiratory tract and also leads to the majority of asthma exacerbations [pro) and its precursor, 3CD, both encoded by HRV. The degradation of these nucleoporins is thought to disrupt NPCs, enhance nuclear envelope permeability, and alter nucleocytoplasmic trafficking [Human rhinovirus (HRV) is a nonenveloped, single-stranded, positive-sense RNA virus belonging to the family rbations ,162,163.fficking ,120. To fficking . DespiteFlaviviridae family, which are single-stranded, positive-sense RNA viruses.Both hepatitis C virus (HCV) and Japanese encephalitis virus (JEV) belong to members of the Like VACV, HCV replicate in viral factories ,164. TheInterestingly, JEV infection also increased RanBP2 expression; however, the depletion of RanBP2 significantly increases JEV replication . To dateOrthomyxoviridae family. There are four type species of influenza virus, including Influenza A, B, C, and D. Influenza A is highly contagious to humans and causes seasonal flu epidemics [The influenza virus is a single-stranded, negative-sense RNA virus, which belongs to the pidemics . To dateLentivirus of the Retroviridae family. HIV-1 attacks the body’s immune system and can cause acquired immunodeficiency syndrome (AIDS) at the late stage of its infection. Upon entry into host cells, the HIV-1 virion releases viral RNA genomes into the cytoplasm, and then the single-stranded RNA genome is reverse transcribed into complementary DNA by reverse transcriptase encoded by HIV-1. The resulting viral DNA, which is associated with viral proteins, such as integrase, and host co-factors in the form of preintegration complex (PIC), is then imported into the nucleus and integrated into the host genome [Human immunodeficiency virus type-1 (HIV-1) is an enveloped, single-stranded, positive-sense RNA virus that belongs to the genus t genome ,167, allt genome . It has t genome ,127,128.t genome . AlthougRANBP2 cause ANE1 and summarizes recent advances in understanding the interactions between RanBP2 and distinct viruses. It has been known that viruses are the major environmental triggers for ANE1 in individuals with certain RANBP2 missense mutations. However, it remains unclear how ANE1 mutations in RANBP2 contribute to disease development. These mutations may directly impact the viral infection process, or act downstream in the modulation of immune cells, immune activation and/or cytokine production.Taken together, this review presents the progress that has been made towards understanding how dominant mutations in As detailed above, RanBP2 interacts with different viruses and plays a role in viral infection. Despite this, we have yet to have any reports that show whether/how ANE1-associated mutations alter RanBP2-virus interactions, or whether all the viruses interacting with RanBP2, as discussed above, cause ANE1. Future studies focusing on the interactions between ANE1-associated mutations, viruses, and host antiviral innate immune responses are needed to clarify the molecular mechanisms that cause this disease. Ultimately, this work will also help develop novel therapeutic strategies against ANE1 and similar pathological states caused by viral-induced cytokine storms.


The phosphoproteome was profiled to a depth of 2003 phosphopeptides, of which 207, 854, and 1335 were identified in MCF10A, MCF7, and MDA-MB-231 cell lines, respectively. Furthermore, 2450 phosphorylation sites were mapped to 855 distinct proteins, covering a wide range of functions. The identified proteins are associated with several diseases, mostly related to cancer. Among the phosphoproteins, we validated four enzymes associated with cancer and present only in sEVs isolated from MCF7 and MDA-MB-231 cell lines: ATP citrate lyase (ACLY), phosphofructokinase-M (PFKM), sirtuin-1 (SIRT1), and sirtuin-6 (SIRT6). With the exception of PFKM, the specific activity of these enzymes was significantly higher in MDA-MB-231 when compared with MCF10A-derived sEVs. This study demonstrates that sEVs contain functional metabolic enzymes that could be further explored for their potential use in early BC diagnostic and therapeutic applications.Small membrane-derived extracellular vesicles have been proposed as participating in several cancer diseases, including breast cancer (BC). We performed a phosphoproteomic analysis of breast cancer-derived small extracellular vesicles (sEVs) to provide insight into the molecular and cellular regulatory mechanisms important for breast cancer tumor progression and metastasis. We examined three cell line models for breast cancer: MCF10A , MCF7 , and MDA-MB-231 . To obtain a comprehensive overview of the sEV phosphoproteome derived from each cell line, effective phosphopeptide enrichment techniques IMAC and TiO Extracellular vesicles (EVs) derived from human cancer cell lines are involved in multiple biological processes in tumor biology, such as modulation of the microenvironment, angiogenesis, sustained growth, and tissue invasion ,4,5,6,7.In order to elucidate the molecular changes that coincide with cancer or discover new biomarkers, it is important to measure protein expression in EVs, followed by the identification of post-translationally modified proteins such as phosphorylated proteins. Protein phosphorylation is the most important post-translational modification that dominates signaling transduction, which plays an essential role for almost all cellular functions and is involved in major regulatory mechanisms of cell signaling networks . Recent Some studies reported the detection of phosphoproteins in biofluids for disease diagnosis ,19,20. FCell lines are commonly used as models in cancer biology since they are easily grown and relatively inexpensive, making them suitable for high-throughput testing and omics studies . Blood iThe aim of this study was to gain insight into the phosphoproteome of sEVs derived from the metastatic BC cell lines MDA-MB-231 and MCF7 and from the non-cancerous cell line MCF10A. Differences in phosphoproteomes were observed between the control cell line, MCF10A, and BC cell lines, MDA-MB-231 and MCF7. We selected and validated four phosphoenzymes: ATP citrate lyase (ACLY), phosphofructokinase-M (PFKM), sirtuin-1 (SIRT1), and sirtuin-6 (SIRT6) identified in both MDA-MB-231 and MCF7 that might be potentially relevant to BC diagnosis.8 cells.Breast cancer epithelial cell lines MDA-MB-231 (ATCC HTB-26) and MCF7 (ATCC HTB-22) and the non-tumorigenic breast epithelial cell line MCF10A (ATCC CRL-10317) were obtained from the American Type Culture Collection (ATCC) and used in this study. MDA-MB-231 and MCF10A cells were cultured as described in our previous work [g for 10 min. Apoptotic bodies were removed by centrifugation at 2000× g using a Sigma13190 rotor (MBI) for 20 min. Samples were then spun at 16,500× g for 1 h using an SW28 Ti rotor  to deplete microvesicles. To pellet sEVs, the same rotor was used to centrifuge samples for 3 h at 100,000× g. Collected sEVs were washed with PBS and centrifuged at 100,000× g for 1 h and finally resuspended in 200 μL PBS and stored at −80 °C. A Bradford protein assay kit  was used to assess sample protein concentrations. The average amount of EV protein from three replicate samples was roughly 22 ± 5, 27 ± 6 and 34 ± 6 μg for MCF10A, MCF7 and MDA-MB-231 cell lines, respectively. These samples were used for proteomic analysis.Small EVs were isolated from cell culture supernatant by UC, as described previously . ConditiThe average particle diameter and concentration of isolated EV samples were measured by the ZetaView PMX-110 . The camera shutter speeds were adjusted to 85 and 40. The instrument was calibrated and focused with 102 nm polystyrene beads .g, and the supernatant was then collected.Isolated sEVs were resuspended in the lysis buffer with a volume ratio of 4:1 (fraction/buffer) with a final concentration of 20 mM HEPES, pH 8.0, 5% glycerol, 0.1% n-Dodecyl-β-D-Maltoside, 0.2 mM DTT, 1.6 M urea, and 1/250 (v:v) phosphatase inhibitors  and gently vortexed for 3 min. The suspension was centrifuged for 1 min at 10,000× 2-phosphopeptide enrichment was carried out according to the manufacturer’s protocol . All enriched phosphopeptide samples were acidified by adding TFA , subsequently desalted , and dried in a vacuum evaporator.Protein samples obtained from the isolated sEVs were reduced, alkylated, digested, and desalted, as described previously . Phospho2O/1% formic acid (v/v), three microliters of sample was injected onto an in-house packed column and eluted for 65 min at a flow rate of 300 nL/min . The ESI+ parameters were set as follows: top speed mode, ion source temperature 250 °C, ion spray voltage 2.1 kV, and a full-scan MS (m/z 350–2000) resolution of 60,000. For collision-induced dissociation (CID), the automatic gain control (AGC) target was set to 5 × 105 for full scans and 1 × 104 for MS/MS scans. Precursor ions were filtered from +2 to +7 charge states within 2 m/z isolation windows. CID in the linear ion trap was performed at a normalized collision energy of 35%. For the higher-energy collision dissociation (HCD) method, the AGC target for precursor ions was set to 5 × 105 with an ion filling time of 150 ms. The highest intensity ions were isolated and fragmented with a normalized collision energy of 32% and detected at a mass resolution of 15,000. The AGC target for MS/MS was set to 5 × 104 with a maximum injection time of 150 ms and a dynamic exclusion of 30 s.To process samples, the Orbitrap Fusion mass spectrometer  coupled to an UltiMate 3000 nanoRSLC  was utilized, as previously described with modified instrument parameters ,7. FolloMS raw files were analyzed with MaxQuant (version 2.0.1.0)  and the MaxQuant output tables for protein groups, peptides, and phosphosites were used for all analyses in R . We onlyDisGeNET protein–disease annotations were retrieved with the disgenet2r R package . Cancer-All MS raw data were submitted to the PRIDE repository (Accession: PXD030424) at the European Bioinformatics Institute.Cells and sEVs were lysed by gentle vortexing in extraction buffer provided by the ACLY Activity Assay Kit . Supernatants were collected and used for the analyses of enzymatic activities. ACLY Activity Assay Kit was used according to the manufacturer’s instructions. The assays were performed at room temperature for 60 min.The phosphofructokinase activity was measured using kits from Sigma-Aldrich (Phosphofructokinase Activity Colorimetric Assay Kit MAK093) according to manufacturer’s instructions. The assays were performed at room temperature in 100 μL of reaction mixture. The phosphofructokinase activity was calculated via the standard curve of the NADH standard at known concentrations. The samples were mixed and incubated for 30 min, and the absorbance was monitored at 450 nm.+. Fluorescence intensities of SIRT1  and SIRT6  were measured using a microplate reader (FilterMax F3 and F5 Multi-Mode Microplate Readers from Molecular Devices). The activity of enzymes was calculated from the assay time between 5 and 10 min.The enzymatic activity of SITR1 and SIRT6 was assessed by using the Fluorescent Screening Assay Kit  following the manufacturer’s instructions. The protein extract from cells and EVs was obtained by treatment with Triton X-100 . The assay was performed in 96-well black microplate  with a reaction volume of 50 μL per well. Briefly, the reaction was started by incubating the protein extract with the reaction mixture containing an acetylated peptide substrate. Samples were incubated for 30 min at 23 °C. Control samples were prepared in absence of NADSDS electrophoresis and Western blot experiments were carried out as previously described .In this work, we carried out a systematic analysis of the phosphoproteome of sEVs derived from MCF10A, MCF7, and MDA-MB-231 cell lines to provide insight into the molecular mechanism of breast cancer. To evaluate the range of measured diameters of sEVs isolated by differential ultracentrifugation, nanoparticle tracking analysis was performed. Isolated vesicles, from the three cell lines, measured 50 to 300 nm in diameter, with an average size of about 125 nm A. Additi2 affinity chromatography. An overview of our experimental strategy is presented in 2 affinity chromatography from all sEVs , the probability of correct site analysis identified a total of 1613 class I (>75% confidence), 774 class II and III (25–75% confidence), and 63 class IV (<25% confidence) phosphosites, respectively [A total of three biological replicates were prepared from MCF10A-, MCF7-, and MDA-MB-231-derived sEVs and examined independently. EVs were buffered with phosphatase inhibitors, digested, and the phosphopeptides were enriched to enhance the identification of low-abundance phosphorylated proteins. In addition, we combined two techniques for the enrichments of phosphopeptides: Fe-IMAC and TiOall sEVs . In addiWe visualized sequence motifs for the phosphorylated amino acids for each cell line fraction . The majIn addition, we used the GPS 5.0 software  to prediTo determine whether identified phosphoproteins are linked to cancer diseases, we annotated proteins using the recent set of disease annotations from the DisGeNET database . PhosphoWe annotated and classified proteins using the KEGG pathway database. Pathways terms such as metabolic pathway, spliceosome, cell cycle, and viral carcinogenesis were highly abundant in MCF7- and MDA-MB-231-derived sEVs, compared with MCF10A-derived sEVs  Table S. InteresACLY, PKFM, SIRT1, and SIRT6 activities were detected in all extracted protein fractions from the three cell lines, as well as their sEVs . The spe2, and fragmentation methods, CID and HCD, were employed to maximize the purification and identification of phosphopeptides. We identified 2450 phosphorylation sites with 60 novel ones. These phosphorylation sites were mapped to 870 distinct proteins, covering a broad range of functions. Many identified phosphoproteins were unique to BC EVs encompassing a variety of signaling, metabolic, and regulatory pathways and cellular processes. The significant difference in the number of phosphoproteins identified from sEVs isolated from MCF10A and either MDA-MB-231 or MCF7 could be due to MCF10A producing a lower number of sEVs. This is supported by studies showing that MCF10A cells produce significantly fewer sEVs and less protein content than MDA-MB-231 [To understand the molecular and cellular regulatory mechanisms important for BC tumor progression and metastasis, we performed a phosphoproteomic analysis of BC-derived sEVs. In our study, we used sEVs from two breast cancer cell lines, MCF7 and MDA-MB-231, and a non-tumorigenic breast cell line, MCF10A. Two enrichment techniques, IMAC and TiOA-MB-231 . MoreoveIn this study, among all groups of kinases, CMGC, CAMKs, STE, AGC, and TKL were predicted to regulate the largest number of phosphorylation events in sEVs C. KinaseIn total, 266 phosphoproteins were associated with cancer in sEVs derived from MCF7 and MDA-MB-231 cell lines. Among them, 78 are related to breast cancer, suggesting that the cargo of phosphoproteins in sEVs may play an important role in cancer progression and metastasis. Moreover, these identified phosphoproteins could serve as biomarkers for BC diagnosis. Therefore, this study identified nine proteins  previousAdvancements in proteomics facilitate discovery of enzymes that can provide an attractive source for cancer biomarker tests. Due to their unique enzyme specificity and selectivity, enzymatic assays are a reliable, simple, and rapid diagnostic method . A phospACLY is a cytoplasmic homologous tetramer composed of four polypeptide chains and acts as a metabolic enzyme involved in fatty acid synthesis in rapidly proliferating cancer cells ,45,46. AExpression levels of phosphofructokinase-M (PFKM) are closely related to the occurrence and development of malignant tumors. To meet the metabolic demands of tumor cells in energy, the activity of PFKM in cancer cells is increased ,54,55,56Sirtuin-1 (SIRT1) is a class-III histone deacetylase (HDAC) enzyme involved in gene regulation, genome stability, apoptosis, autophagy, senescence, proliferation, aging, and tumorigenesis . This enSirtuin-6 (SIRT6) is involved in multiple molecular pathways related to DNA repair, glycolysis, gluconeogenesis, tumorigenesis, neurodegeneration, and cardiac hypertrophic responses . SIRT6 m2 enrichment and CID fragmentation methods were employed for peptide enrichment and fragmentation; the study was based on cell culture lysates and did not identify the four enzymes presented in our work.Previous studies have investigated the phosphoproteome of BC cell lines, as well as their EVs ,71. HoweThe objective of this study, along with previously published works ,7, was tIn this study, we conducted a phosphoproteomic analysis of breast cancer-derived extracellular vesicles from MCF10A, MCF7, and MDA-MB-231 cell lines. In total, 855 distinct phosphoproteins were identified collectively among the cell lines, covering a wide range of functions, most of which are related to cancer. Among these phosphoproteins, we validated four enzymes: ACLY, PFKM, SIRT1, SIRT6. The results demonstrate that the specific activity of PFKM in BC cancer cell lines was not statistically different from the non-cancerous cell line. In contrast, the three phosphorylated enzymes ACLY, SIRT1, and SIRT6 showed a significantly higher specific enzymatic activity in MDA-MB-231 in comparison to MCF10A-derived sEVs. These three enzymes might serve as a potential prognostic biomarker for BC. They have been previously proposed as therapeutic targets for cancer therapy. Thus, our findings justify further investigation of these enzymes as promising drug targets for BC treatment.


These marine alkaloids, particularly the hexabrominated compound, fistularin-3, shows promising docking interactions with predicted binding affinities  for the main protease (Mpro) (PDB ID: 6lu7), spike glycoprotein (PDB ID: 6VYB), nucleocapsid phosphoprotein (PDB ID: 6VYO), membrane glycoprotein (PDB ID: 6M17), and non-structural protein 10 (nsp10) (PDB ID: 6W4H), respectively, where it forms better interactions with the protein pockets than the native interaction. It also shows promising molecular dynamics, pharmacokinetics, and toxicity profiles. As such, further exploration of the antiviral properties of fistularin-3 against SARS-CoV-2 is merited.There have been more than 150 million confirmed cases of SARS-CoV-2 since the beginning of the pandemic in 2019. By June 2021, the mortality from such infections approached 3.9 million people. Despite the availability of a number of vaccines which provide protection against this virus, the evolution of new viral variants, inconsistent availability of the vaccine around the world, and vaccine hesitancy, in some countries, makes it unreasonable to rely on mass vaccination alone to combat this pandemic. Consequently, much effort is directed to identifying potential antiviral treatments. Marine brominated tyrosine alkaloids are recognized to have antiviral potential. We test here the antiviral capacity of fourteen marine brominated tyrosine alkaloids against five different target proteins from SARS-CoV-2, including main protease (M The 2019 novel coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has significantly impacted global health and economics .SARS-CoV-2 is the seventh coronavirus known to infect humans, but the only one, to date, that has caused a pandemic ,5. SARS-Host cell entry is the first step in the viral life cycle. The first step in the life cycle of SARS-CoV-2 is the attachment of the viral particle via the receptor-binding domain (RBD) of its S protein (see below) to the angiotensin-converting enzyme-2 (ACE-2) receptor on the plasma membrane of the pulmonary alveolar epithelial cells and capillary endothelial cells. In some instances, this ultimately leads to severe acute respiratory failure  8,9].,9.8,9].Similar to other coronaviruses (CoVs), the size of the SARS-CoV-2 genome is approximately 30 Kb and encodes four main structural proteins, including the Spike glycoprotein (S protein), Envelope (E) protein, Membrane (M) protein, and Nucleocapsid (N protein) . In addiCryptotethya crypta, in the early 1950s, a new era of the exploitation of bioactive marine natural products (MNPs) has emerged decane, as is the case with compound 3, showed the greatest interaction with Mpro, having the ability to occupy its four major pockets: S1, S2, S3 and S4.The SARs of this series of marine alkaloids, based on the results presented in 6, 7, 8 and 11. For the interaction, at least one amine must be a secondary amine, as is the case for compounds 9, 10 and 13. However, attaching the amide group to a long saturated aliphatic chain, as in compound 5, provides a better chance of occupying the spike glycoprotein.The presence of the tertiary amines is also not favorable, as is the case for compounds pro, the presence of the two amides connected to the unsaturated spirodecane increases the predicted binding affinities of the nucleocapsid phosphoprotein, membrane glycoprotein, and nsp10, as clearly shown by compounds 3 and 4. However, dissimilar to Mpro, the presence of the two terminal amines is not favorable for binding to the nucleocapsid phosphoprotein, membrane glycoprotein, or nsp10, as is the case for compounds 11 and 12. The presence of a terminal hydroxyl group on the other side of the compound increases the binding interactions in these compounds. The presence of alpha-beta unsaturated compounds, such as those in 6–10, decreases binding ability.Similar to M3 was the only compound that showed a combination between effective ADME/Tox properties and interactions with high S-scores in all five SARS-CoV-2 target proteins, it was selected for conducting a 100 ns MD simulation with the five target proteins. 3 with the five targets, respectively. This enabled the analysis of the stability of the simulated system throughout the 100 ns MD simulations. As expected, all complexes showed predicted, small RMSD fluctuations within only 2 Å, confirming their high stability throughout the whole simulation, where compound 3 showed the greatest stability with membrane glycoprotein (PDB ID: 6M17). Moreover, the radius of gyration was also consistent with high stability, with all fluctuations being within 0.05 nm.As compound 3 to quantify the strength of the interactions through computing non-bonded interaction energy. The least energy at −250 kJ/mol occurs when compound 3 binds to Mpro (PDB ID: 6LU7). Compound 3 has a higher stability with the other four SARS-CoV-2 proteins (~200 kJ/mol).1–5) that were recognized to have antiviral activities, shown in 6–14) from the French Polynesian marine sponge, Suberea ianthelliformis [https://pubchem.ncbi.nlm.nih.gov/, accessed on 28 June 2021) and saved as mdb files using MOE v.2019.01.The MOL2 files for the five bromotyrosine derived compounds , spike glycoprotein (PDB ID: 6VYB), nucleocapsid phosphoprotein (PDB ID: 6VYO), membrane glycoprotein (PDB ID: 6M17), and non-structural protein 10 , were retrieved from the Protein Data Bank . Their resolutions were 2.16 Å, 3.20 Å, 1.70 Å, 2.90 Å, and 1.80 Å, respectively. All water molecules were removed from these crystal structures with only main-chain amino acids retained. An AMBER (AMBER10:EHT) force field was used for the energy minimization of these five X-ray crystal structures using parameters suitable for proteins and nucleic acids (ff10) and small molecules (EHT). Protons were added by employing the 3D protonation feature in MOE v.2019.01; Asn, Gln and His flips were allowed during 3D protonation. Complexes were then refined to a RMS gradient of 0.1 Kcal/mol/Å.The X-ray crystal structures for the five target proteins from SARS-CoV-2, including the main protease (M1–14) were docked with the five SARS-CoV-2 target proteins, main protease , spike glycoprotein (PDB ID: 6VYB), nucleocapsid phosphoprotein (PDB ID: 6VYO), membrane glycoprotein (PDB ID: 6M17), and non-structural protein 10  using MOE v.2019.01. In addition, re-docking the target proteins’ co-crystallized ligands was performed for validation purposes except for 6M17. Docking validation figures are included in the The fourteen compounds in our screening library . The properties predicted here were lipophilicity, reported as Log Po/w (WLOGP); water solubility class; and Blood–brain barrier (BBB) penetration, in addition to medicinal chemistry parameters employing pan-assay interference alerts (PAINS) [http://biosig.unimelb.edu.au/pkcsm/prediction, accessed on 28 June 2021) to predict the safety of these small molecules upon ingestion in human and animal models, with respect to toxicological effects on hERG-I inhibition [The pharmacokinetic properties of the fourteen compounds in our screening library were calculated using the SWISS-ADME webtool ( (PAINS) ,40. Addihibition .3, Fistularin-3, displayed the best binding interactions and free energies with the five SARS-CoV-2 target proteins among the fourteen library compounds investigated. It also showed the best pharmacokinetics properties. Accordingly, it was subjected to 100 ns molecular dynamics investigation against the five SARS-CoV-2 target proteins. MD simulations were performed using the GROMACS 2021 software package with the CHARMM36 force field used for protein topology preparation and the official CHARMM General Force Field server (CGenFF) used for ligand topology preparation. The solvation method used was a dodecahedron box of common simple point charge (SPC) water model with explicit solvent periodic boundary conditions. Charge neutralization using sodium and chloride ions was performed for the five solvated complexes. These systems were subjected to energy minimization to resolve steric clashes or inappropriate geometry employing the steepest descent method of 5000 steps. System equilibration was also set to ensure a reasonable starting structure using NVT and equilibration under constant number of particles, volume, and temperature (NVT) for 100 ps using a Berendsen thermostat [Compound ermostat . Then, rermostat . Finallyermostat .3, the complexes were re-centered and rewrapped within unit cells using the trjconv function of GROMACS. The stabilities of trajectories were then determined throughout the 100 ns simulation using the radius of gyration and the root-mean-square deviation (RMSD) of the protein backbone referenced to its initial position at 10 ps intervals. Lastly, g_mmpbsa was employed using Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) binding free energy [After determining the trajectories of the five complexes resulting from the MD simulation of compound e energy  to calcu3), displayed the best docking scores with predicted binding affinities  for main protease (Mpro) (PDB ID: 6lu7), spike glycoprotein (PDB ID: 6VYB), nucleocapsid phosphoprotein (PDB ID: 6VYO), membrane glycoprotein (PDB ID: 6M17), and non-structural protein 10 (nsp10) (PDB ID: 6W4H), respectively, where it formed better interactions with the protein pockets than the native interaction. This was supported by very stable molecular dynamics simulations. In addition, it was found that compound 3 structurally complied with the previously reported structural and pharmacophoric requirements for efficient bio-target binding [3 [Fourteen structurally diverse brominated tyrosine alkaloids were comprehensively explored for their virtual antiviral potentials against five SARS-CoV-2 proteins. Among the tested compounds, the polybrominated alkaloid, fistularin-3 ( binding ,46. Consnding [3 ,49,50,51


Vitis vinifera includes a large number of cultivars that are further distinguished in biotypes and clones, and it is actually hard to differentiate them, even through complex molecular techniques. In this work, the plant materials of 56 putative Sangiovese and 14 putative Montepulciano biotypes, two of the most widespread black-berried Italian cultivars, were collected in different wine-growing areas of Italy distributed in 13 regions, from north to south. Firstly, the samples were analyzed using SSR markers to have proper varietal identification. According to the results, the genotypes belonged to three different cultivars: Sangiovese, Sanforte, and Montepulciano. Subsequently, the samples were investigated using AFLP, SAMPL, M-AFLP, and I-SSR molecular markers to estimate their intra-varietal genetic variability. The DNA marker-based method used turned out to be performing to bring out the geographic differences among the biotypes screened, and it can therefore be considered as a powerful tool available for all the grapevine varieties. Vitis vinifera L. is one of the oldest known fruit-producing crops that, over the centuries, has undergone strong domestication. This process led to obtaining plants with hermaphroditic flowers (self-fertile), good fruitfulness, propagation capacity, and most of all optimal grape quality for fresh consumption or winemaking [nemaking ,2. The tnemaking . Additionemaking ,5,6,7. Inemaking ,8,9. Mornemaking ,11.In Italy, the first wine-producing country in the world , Sangiovampelos and graphia, meaning vine description) and ampelometry (the measurement of vine organs) [Vitis species. The most commonly employed are RAPD (random amplified polymorphic DNA), MSAP (methylation-sensitive amplified polymorphism), RT (retrotransposon-based molecular markers), SNP (single-nucleotide polymorphism), IRAP (inter-retrotransposon-amplified polymorphism), AFLP (amplified fragment length polymorphism), M-AFLP (microsatellites amplified fragment length polymorphism), SAMPL (selective amplification of microsatellite polymorphic loci), I-SSR (inter simple sequence repeat), and ISTR (inverse sequence-tagged repeat), and they can also be combined to obtain much more comprehensive information [Varietal identification in viticulture has a long tradition. It has always been based mainly on ampelography (from the Greek words  organs) ,35. More organs) ,37. Howe organs) . Therefo organs) ,39,40, t organs) . The use organs) , and thuormation ,50,51,52Vitis species [This research aimed at exploring the genetic variability in a broad pool of biotypes allegedly belonging to Sangiovese, the most widespread, black-berried Italian grapevine cultivars , and Mon species , we wantVitis International Variety Catalogue code 10680 [Vitis International Variety Catalogue code 7136, registered as Maiolica), and 15 Montepulciano . According to the results obtained by the SSR marker-assisted screening at 11 microsatellite loci, the 78 samples analyzed can be divided into three variety clusters: 51 Sangiovese , 12 SanBy observing the SSR profiles  were validated as Sangiovese, while 12 samples  turned out to be Sanforte . This reThe true-to-type correspondence of all the 14 alleged Montepulciano samples was proven by microsatellites markers , and theThe samples belonging to Sangiovese, Sanforte, and Montepulciano grapevine varieties  were theA total of 1905 reproducible amplification products were obtained with the four molecular marker systems: 689 AFLPs, 650 SAMPLs, 503 M-AFLPs, and 63 I-SSRs. Of these, 972 (51.2%) were polymorphic: 326 (47.3%) AFLPs, 355 (54.6%) SAMPLs, 268 (53.2%) M-AFLPs, and 23 (36.5%) I-SSRs. The mean numbers of marker loci assayed per single experiment were 62.6, 59.1, 45.7, and 5.7 for AFLP, SAMPL, M-AFLP, and I-SSR markers, respectively.TOT) was 0.9056; the genetic similarity estimated within the groups (GSW) was 0.9431 for the 51 Sangiovese, 0.9541 for the 12 Sanforte, and 0.9463 for the 15 Montepulciano samples. According to the results, Sanforte was the most homogeneous grapevine variety. The genetic similarity estimated between the groups (GSB) was 0.9098 for Sangiovese–Sanforte, 0.8445 for Sangiovese–Montepulciano comparison, and 0.8227 for the Sanforte–Montepulciano comparisons. The data obtained showed that Sangiovese and Sanforte were genetically similar, and it was also found that Montepulciano stands closer to Sangiovese than to Sanforte.For all the possible pairwise comparisons of 51 Sangiovese (45 biotypes and 6 clones), 12 Sanforte (12 biotypes), and 15 Montepulciano samples (14 biotypes and 1 clone), a matrix based on Dice’s genetic similarity coefficient (GS) was constructed. The observed total genetic similarity (GSA UPGMA (unweighted pair group method with arithmetic mean) dendrogram based on genetic diversity  was obtaBy observing the lower part of the dendrogram, it can be noted that the 15 samples belonging to the Montepulciano cultivar are grouped according to their region of origin. In particular, there is a sharply defined separation between the samples from Abruzzo, where this grapevine variety is predominant with 56% of the total vineyard area  . The more complex Sangiovese sub-subcluster brings out pronounced and highly detailed geographical differences. In fact, the 51 samples are split into four main branches: Tuscany, Central Italy, Southern Italy, and Northern Italy. The largest branch is composed of biotypes originating from Tuscany, where this cultivar plays a central role in the regional wine industry and is spread on 64% of the total vineyard area . Within The GS matrix estimates and the dendrogram results agree with the principal component analysis (PCA) reported in Pst+AG/Mse+CAA with 31 polymorphic markers (51%), the primer combination of SAMPL As2/Mse+TGG with 32 polymorphic markers (54%), the primer combination M-AFLP I-SSR#02/M+AGG with 26 (55%), and the primer combination of I-SSR (TC)7ACGG with 4 polymorphic markers (44%)  .Vitis vinifera. In particular, the molecular markers used were effective in finding intra-varietal differences since they detect both some repeated regions (different from those of SSR markers) and not-repeated regions adjacent to these repeated regions, thus having a broad view of the grapevine genome. This in-depth genetic characterization has yielded interesting evidence: Sangiovese, Sanforte, and Montepulciano samples were separated quite exhaustively based on their geographical origin  allowed us to discriminate very efficiently the biotypes belonging to the same variety of l origin . GenotypIt is well-known that the grapevines, especially several historically more exploited varieties such as Sangiovese ,58,59, hOur findings are in agreement with the results of other research successfully conducted with the same DNA marker-based method on other important grapevine varieties, such as Malvasia Nera di Brindisi/Lecce, Malvasia di Candia, Negroamaro, Primitivo/Zinfandel, Grenache Noir/Garnacha Tinta/Cannonau, and Malvasia Istriana ,49,65, wVitis vinifera L. subsp. vinifera), belonging both to “Sangiovese grosso” and “Sangiovese piccolo” biotypes, were made available by some farm owners in different wine-growing districts located in 13 different regions along the Italian peninsula (Vitis vinifera L. subsp. vinifera) woody branch samples  on the Microlab Starlet Liquid Handling Workstation . Genomic DNA was stored undiluted in TE buffer , pH 8.00 at −20° C. DNA quantification was obtained by FLx800 TBI Fluorometer , and PicoGreen dsDNA quantification assay .Grapevine samples were genotyped using a set of 11 SSR loci, 6 core loci selected within GenRes 081 European Project  and other additional 5 , according to internationally accepted standards ,55,56. The microsatellite PCR reactions were performed on a Microlab Starlet Liquid Handling Workstation  using SSR forward (F) 6FAM-labeled primers , VIC-labeled primers , NED-labeled primers , PET-labeled primers  and SSR reverse (R) unlabeled primers, each at 5 pmol/µL , as reported by Meneghetti . Vitis vinifera microsatellite bin set of 11 standard loci.The microsatellite PCR contained 10 ng genomic DNA; 0.06 μL of VMC6E1, VrZAG79, and VrZAG62 primers (F + R); 0.09 μL of VMCNG4b9, VMC6F1, and VMC6G1 primers (F + R); 0.13 μL of VVS2, VVMD27, and VVMD7 primers (F + R); 0.19 μL of VVMD5 and VVMD28 primers (F + R); 1× PCR buffer ; 4 mM deoxyribonucleotide triphosphates (dNTPs) and 1 U of Taq DNA polymerase ; and ddH2O to final volume . The PCR was performed in a GeneAmp PCR System 9700  with the following conditions: 94 °C for 2 min, 35 cycles at 94 °C for 40 s, 55 °C for 60 s, and 72 °C for 60 s, a final extension at 72 °C for 60 min, and the last step at 4 °C to stop the reaction. SSR polymorphisms were detected on an ABI-3130XL Genetic Analyzer . The samples were prepared by combining 0.5 μL of labeled PCR, 8 μL Hi-Di™ Formamide, and 0.3 μL of GS500-LIZ size standard . The software used for the analyses was GeneMapper v.4.1  with a After having established the correct genetic identity, each of the 78 samples was renamed with an assigned code  compoundThe genetic variability within the same grapevine cultivar was investigated by AFLP (amplified fragment length polymorphism), SAMPL (selective amplification of microsatellite polymorphic loci), M-AFLP (microsatellites amplified fragment length polymorphism), and I-SSR (inter simple sequence repeat) molecular markers ,66.The restriction of DNA was performed by EcoRI, PstI, and MseI enzymes with EcoRI, PstI, and MseI adapters  matrix was created for AFLP, SAMPL, M-AFLP, and I-SSR markers and for each genotype. Molecular markers were defined by a standard ladder using the GeneMapper software with some reference DNA genotypes and automatically visualized using the software of ABI-3130XL capillary sequencer. TOT) estimates among individuals were calculated in all possible pairwise comparisons using Dice’s genetic coefficient by NTSYS software and UPGMA algorithm [W) and between (GSB) sample clusters and marker systems . The cluster analysis of GS  was performed according to the UPGMA algorithm using the NTSYS software [Genetic similarity (GSlgorithm ,49. GS wsoftware . The molVitis vinifera cultivars, especially those characterized by high morphological variability. It finds applications where SSR analysis is not sensitive enough, such as biotype intra-varietal screening or if more complex “omics” approaches cannot be used. The system based on the four AFLP, SAMPL, M-AFLP, and I-SSR molecular markers used turned out to be performing to bring out the geographic differences among the biotypes comprised in this research work. This method can be considered a powerful tool available for all


Its increasing incidence has led stroke to be the second leading cause of death worldwide. Despite significant advances in recanalization strategies, patients are still at risk for ischemia/reperfusion injuries in this pathophysiology, in which neuroinflammation is significantly involved. Research has shown that in the acute phase, neuroinflammatory cascades lead to apoptosis, disruption of the blood–brain barrier, cerebral edema, and hemorrhagic transformation, while in later stages, these pathways support tissue repair and functional recovery. The present review discusses the various cell types and the mechanisms through which neuroinflammation contributes to parenchymal injury and tissue repair, as well as therapeutic attempts made in vitro, in animal experiments, and in clinical trials which target neuroinflammation, highlighting future therapeutic perspectives. Stroke is the second leading cause of death and a major cause of disability worldwide , with anThe present paper provides an overview of the involvement of various cell types and pathways in neuroinflammation following acute ischemic stroke and a narrative review of preclinical and clinical studies performed so far (or that are underway) acting on neuroinflammatory mechanisms in an attempt to improve stroke outcome, highlighting promising future perspectives.The brain is more difficult to access by immune cells from the periphery due to the presence of the BBB , which hIt is, nonetheless, important to understand that the neuroinflammatory pathways discussed in the following sections are not merely damaging pathways but also include mechanisms which significantly contribute to reparative processes and enhance recovery after the ischemic insult ,19. In fThe inflammatory cascade is activated immediately after vessel occlusion by the modified shear stress on the endothelium and altered blood rheology of stagnant blood flow . P-selecActivation of the coagulation cascade leads to thrombin generation, which attracts additional monocytes and neutrophils, and induces the expression of adhesion molecules on endothelial cells through the activation of nuclear factor κB (NF-κB) . Thus, iThe acute oxygen and glucose deprivation also increases the production of reactive oxygen species (ROS) by cytosolic enzymes and mainly by mitochondria , with inThe resident immune cells of the brain, representing 5-20% of the glial population, are microglia . They arIn the resting state, microglial cells have a small cell soma and numerous processes in constant motion which monitor the microenvironment of the CNS ,32. The By expressing Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), and retinoic acid-inducible gene-1 (RIG-1)-like receptors (RLRs), microglia recognize pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), followed by induction of the nuclear factor-kappa B (NF-κB) and Toll/interleukin-1 receptor (TIR)-domain-containing adapter-inducing interferon-β(TRIF)-induced interferon regulatory factor-3 (IRF3) pathways ,38.Upon activation by endogenous stimuli generated following injury or infection, microglia retract their processes and take on an amoeboid shape ,40. AfteOnce activated, microglia adopt different phenotypes: microglia with enlarged cell bodies with short ramifications and amoeboid cell structures with rare ramifications have been described in the peri-infarct regions in the post-acute phase, while round-shaped microglia, characterizing highly activated cells, have been found near the ischemic core . Aside fOnce activated and polarized towards an M1 phenotype, microglia release tumor necrosis factor-alpha (TNF-α), inflammatory interleukins such as IL-1β, IL-6, other inflammatory cytokines, and reactive oxidative species (ROS) into the circulation ,58. The M2-polarized microglia, by producing anti-inflammatory factors , contribute to the resolution of inflammation  and haveBeing highly plastic cells, microglia can shift between different phenotypes ,65, as dTemporal analyses of microglial phenotypes in animal stroke models showed an increase in the M1 phenotype in the first 14 days post-stroke, while M2 phenotypes were detected 12 h after the ischemic insult and increased during the first 3 days, followed by a decline in their number . After transient middle cerebral artery occlusion in mice, microglial and macrophage infiltration peaks at 48-72 h after the ischemic insult . TransieIn humans, histological studies and, more recently, positron emission tomography studies have shown abundant activated microglia in the ischemic core within 24-48 h after stroke onset , which pAstrocytes are essential housekeeping cells supporting neuronal function by regulating the ion–water balance; removing excess neurotransmitters and waste products; secreting trophic factors such as fibroblast growth factor-2, brain-derived neurotrophic factor, and nerve growth factor; and, through their end feet, participating in the normal structure and function of the BBB . Following ischemia, the impaired expression of the excitatory amino acid transporter 2 (EAAT2) interferes with the clearance of glutamate by astrocytes , while cIn animal experiments, astrocytic activation started 4 h after the insult, peaked on day 4, and persisted for 28 days . An impoLeukocytes, mainly neutrophils , are amoThe various subtypes of leukocytes which infiltrate the brain parenchyma after an ischemic stroke contribute to cerebral injury in many ways. Adherence of leukocytes to the endothelium can block erythrocytes in the microvasculature (“clogging”), leading to the “no-reflow” phenomenon . In addiAmong the leukocytes infiltrating the brain are blood-derived monocytes/macrophages, which infiltrate the cerebral tissue in the hyperacute stage of stroke (<24 h) . They haNeutrophils start infiltrating the ischemic tissue after 30 min and peak between days 1 and 3, after which their number declines . In the + T cells, and CD8+ T cells may infiltrate the parenchyma within hours after stroke onset, peak by day 3, and aggravate ischemic injury [Lymphocytes have less important contributions in cerebral ischemic injury; the mechanisms are mainly related to the innate T cell functions . IL-17 sc injury ,103,104,c injury , while rc injury ,107 havec injury ,109. Thec injury . In the early post-stroke phase, Tregs act on peripheral leucocytes to inhibit the production of matrix metalloproteinase-9 (MMP-9) by neutrophils ,112, theDuring ischemia and energy failure, there is a build-up of hypoxanthine which, upon reperfusion, will be converted by hypoxanthine oxidase to xanthine and uric acid, with significant amounts of superoxide generated in the process . In addiIn response to hypoxia, and additionally stimulated by uric acid , endotheA series of molecules released by damaged cells  interacts with platelet TLR 4 and promotes platelet activation and thrombus formation . In addiAfter activation, the content of platelet granules is released and serves to further increase platelet activation (adenosine diphosphate) or act as pro-inflammatory cytokines (such as platelet factor 4 and β-thromboglobulin) to activate endothelial cells and recruit additional leukocytes and form platelet–neutrophil aggregates . PhosphaHowever, platelet granules also contain factors which prevent the aggravation of ischemia/reperfusion injuries, especially hemorrhagic transformation . PlateleCytokines are small polypeptides (8-26 kDa), normally expressed at very low levels, which regulate immune responses . VariousNecrotic neurons release DAMPs, or “hazard signals”, which induce TLR4 on microglia and stimulate microglial cells to produce inflammatory cytokines via the NF-κB pathway . M1-polarized microglia will produce pro-inflammatory cytokines such as IL-1β, IL-6, IL-18, and TNF-α . The perThe best-studied is the NLRP3 inflammasome, activated in two steps. First, DAMPs activate TLRs on immune cells, which induce the expression of inactive protein NLRP3 and pro-IL-1β via the NF-κB pathway ,145. ResIL-1 occurs in 3 forms: IL-1α, IL-1β, and IL-1ra . IL-1α iTumor necrosis factor (TNF)-α has two forms: a transmembrane form, involved in regulation of inflammation via cell–cell interactions (tmTNF-α), and a soluble form (sTNF-α), which increases the phagocytic and cytotoxic activity of macrophages and upregulates the expression of IL-1β and IL-6 ,157,158.Both IL-1β and TNF are required for the synthesis of IL-6, released by microglia, astrocytes, neurons, and endothelial cells . Within IL-8 also promotes inflammatory responses in cerebral ischemia, proven by the reduced infarct size and better outcome after blocking the cytokine and its receptors ,167.Interferon gamma (IFN-γ) is another cytokine able to modulate immune responses and produced by various cell types, such as monocytes, macrophages, and B and T lymphocytes, as well as natural killer cells . It can Other pro-inflammatory cytokines are IL-16, IL-17, IL-18, monocyte chemotactic protein 1 (MCP-1), and leukotrienes . In the subacute phase, M2-polarized microglia start producing anti-inflammatory cytokines, such as IL-10, IL-4, and transforming growth factor-beta (TGF-β), which will lead to inhibition of the inflammatory response .IL-10 is an anti-inflammatory cytokine produced by various cell types, such as monocytes, CD4, CD25, Foxp3 regulatory T cells, and mastocytes . IL-10 iWhile IFN-γ is a pro-inflammatory cytokine, interferon-β (IFN-β) has been used as immunomodulatory treatment in multiple sclerosis, where it reduces MMP-9 levels and diminishes BBB disruption ,175. In Transforming growth factor-beta (TGF-β), although rarely expressed in normal brain, has been found significantly upregulated after stroke, being produced by microglia, macrophages, neurons, and astrocytes . TGF-β3 IL-4 is able to polarize microglia toward the anti-inflammatory M2 phenotype , indirecN-terminal domain, a β-sheet subunit, and a C-terminal α-helix that are involved in cellular activation and leukocyte recruitment. Their main function is to control leukocyte migration into tissues during inflammation [-CC—two cysteine residues, immediately adjacent,-CXC—two cysteine residues separated by one amino acid,-CX3C—two cysteine residues separated by three amino acids.Chemokines, or chemotactic cytokines, are low-molecular-weight proteins (8-10 kDa) with an ammation . Dependiammation : -CC—twoChemokines are released by neurons, astrocytes, microglial cells, oligodendrocytes, and endothelial cells  and act Monocyte chemoattractant protein-1 (MCP-1) directly increases the permeability of the BBB by causing tight junction proteins to redistribute in endothelial cells  and alsoOther chemokines upregulated in the first 3 h after stroke are microglial response factor-1 (MRF-1), fractalkine (CX3CL1), and macrophage inflammatory protein 1 (MIP-1), which all contribute to the infiltration of the injured tissue with inflammatory cells and thus weaken the BBB . HoweverStromal cell-derived factor 1 (SDF-1), also known as CXC motif chemokine 12 (CXCL12), also seems to have a dual role. SDF-1 and its receptor were found upregulated in the penumbral area , and ratAlthough not inflammatory mediators, matrix metalloproteinases (MMPs) are tightly related to neuroinflammation and contribute significantly to BBB disruption, cerebral edema, and hemorrhagic transformation. MMPs are a family of proteolytic enzymes which have a catalytic zinc site, a pro-peptide region, a fibronectin binding site, and a transmembrane site . They caUpon activation of the constitutive MMPs, such as MMP-2, they fragment the tight junction proteins which “zip” endothelial cells, leading to a transient opening of the BBB 2 to 3 h after reperfusion ,197. AftOn the other hand, plasma levels of MMP-3 were found increased in patients with better functional and motor recovery , highligAlthough the central nervous system has a limited ability to regenerate, neurogenesis is still possible in the subventricular zone and the subgranular zone of the dentate gyrus , althougThe link between neuroinflammation and neurogenesis is complex, with the former being able to both suppress and promote neurogenesis . For exaHowever, since most of these newly formed neurons die within 2-5 weeks , it is iCentral administration of IGF-1 prevented white matter lesions after hypoxic-ischemic insult in rat brain , but theExtensive studies have reported on the neuroprotective effects of BDNF in various diseases of the CNS, from neurodegenerative ones to traumatic and vascular insults ,227. WitThe expression of nerve growth factor (NGF) and tyrosine kinase receptor A is rapidly upregulated following ischemia , promotiGlial-cell-line-derived neurotrophic factor (GDNF) acts by binding to the extracellular glycosylphosphatidylinositol (GPI)-linked receptor, GFRα1, and the transmembrane tyrosine kinase, c-Ret , the expOther proteins and peptides have also been shown to promote tissue repair after stroke. One such protein is annexin A1, a calcium- and phospholipid-binding protein, with anti-inflammatory actions mediated through the G-protein-coupled formyl peptide receptor type 2 , which pAlthough promising in preclinical studies, the clinical trials targeting stroke immunology have not shown clear benefits despite strategies addressing both innate and adaptive immune responses . The duaMinocycline, a highly lipophilic antibiotic agent able to cross the BBB, was shown to be able to inhibit microglial activation, diminish the magnitude of neuronal apoptosis , decreasAmong the receptors involved in microglial M1 polarization are TLR4 receptors . In precIn preclinical studies, the angiotensin II type 2 receptor agonist C21, administered 3 days after stroke onset, improved functional outcome by reducing neuroinflammation and shifting microglia toward the M2 phenotype ,270. Recent research focuses on influencing microglial polarization from M1 to M2 phenotype by using IL-4 or IL-33 in a preclinical setting ,272. Several trials have focused on targeting leukocyte adhesion and transmigration as an anti-inflammatory treatment in acute ischemic stroke. A murine ICAM-1 antibody, enlimomab, interferes with leukocyte adhesion and was shown in experimental studies to reduce infarct size ,274. HowA cellular adhesion molecule located on the leukocyte surface, L-selectin, has been targeted with a humanized monoclonal antibody (HuDREG200) alone or in combination with alteplase in a rabbit stroke model, but the results have not been disclosed . Another humanized antibody targeting neutrophil adhesion, Hu23F2G, or LeukArrest, was tested in the HALT study, a phase 3 clinical trial which, although it was shown to improve the modified Rankin score at 3 months in a phase 2 study, was prematurely stopped by the sponsor due to lack of benefit suggested by an interim analysis . Preclinical studies suggested that targeting CD11/CD18 for blocking neutrophil infiltration was beneficial only in transient ischemic stroke models and had no effect in permanent ones ,278. TheNatalizumab, an anti-CD49b antibody, used in the immunomodulatory treatment of multiple sclerosis, has been tested in preclinical studies in ischemic stroke with conflicting results: one study showed no effect , while fSince lymphocytes interfere in the delayed phase of ischemic injury, therapeutic strategies targeting these cells would theoretically have wide therapeutic windows. Preclinical studies suggested that blocking cerebral invasion of pro-inflammatory lymphocytes  would be neuroprotective ,286. IntRegulatory T cells (Tregs) have been traditionally viewed as anti-inflammatory lymphocytes, but their therapeutic manipulation in different stroke models yielded divergent results. Treg depletion led to increased infarct sizes in three studies ,289,290,Fingolimod, a drug approved for the treatment of multiple sclerosis, inhibits lymphocyte trafficking and infiltration of the central nervous system. Tested in models of transient and permanent focal ischemia, it did not influence lesion size and behavioral dysfunction . A pilot1. Due to the potential of oxidative stress to elicit inflammatory responses, antioxidant molecules could act as anti-inflammatory agents as well. Although promising in vitro and in preclinical studies, most clinical trials failed or yielded inconclusive results . A notab2. Statins, aside from their lipid-lowering effects, also exhibit anti-inflammatory properties by altering the release of interleukins and TNF-α  and inhi3. Vinpocetine, a derivative of the alkaloid vincamine available in many countries as dietary supplement to enhance memory and cognition, reduces the release of pro-inflammatory cytokines and chemokines from microglia, endothelial cells, and vascular smooth muscle cells . It can 4. RO27-3225, a melanocortin MC4 receptor agonist, reduces the expression of TNF-α, Bax, ERK, JNK, and caspase-3 and promotes improved functional recovery following ischemic stroke in gerbils . 5. Omega-3 polyunsaturated fatty acids prevent the release of cyclooxygenase 2, hypoxia-inducible factor 1α, and IL-1β, as well as the activation of NOS, thereby having therapeutic potential in stroke . 6. Pharmacological inhibition or genetic deletion of COX-2 limits the production of pro-inflammatory prostanoids and diminishes BBB damage in acute cerebral ischemia , but blo7. TNF-α receptor inhibition with R-7050 has recently been reported to decrease the activation of NF-κB and reduce levels of IL-6, thereby protecting against neurological deficits and edema in a rat model of permanent cerebral ischemia . 8. Inhibition of IL-1β receptor with recombinant human IL-1 receptor antagonist  was tested in the SCIL-STROKE trial based on the promising results of a previous trial in subarachnoid hemorrhage. Although the drug was safe and well-tolerated, it did not significantly improve the outcome of ischemic stroke patients .9. MicroRNAs, members of the small, noncoding RNA superfamily, have been much studied in recent years. They are single-stranded RNA molecules comprising 18–25 nucleotides which inhibit the expression of protein-coding genes by degrading or inhibiting the translation of target mRNA ,311. Mic10. Transplantation of mesenchymal stem cells provides neuroprotection and immunomodulation and stimulates neurogenesis, astrogenesis, oligodendrogenesis, and angiogenesis, as well as the formation of new synapses . They caMany reasons for the discrepancies between preclinical and clinical trials can be discussed.1. Most transient animal models of stroke use the mechanical artery occlusion, which causes thromboinflammation with subsequent microthrombosis, leading to brain injury. Clot-induced stroke models are less used, although they allow for determining the nature of the clot, whether platelet-rich or fibrin-rich . Using t2. In most animal models of transient arterial occlusion, complete occlusion is followed by complete reperfusion, a situation rarely achieved in human patients except for thrombectomized patients . 3. Many of the risk factors for stroke, such as smoking, age, alcohol consumption, stress, and age, cannot be adequately replicated in the laboratory, with most studies being performed on young animals. Moreover, although stroke predominates in aged women and inflammatory cells vary between genders, preclinical studies mainly use male animals . 4. One must not overlook the risk of infectious complications after stroke, which can significantly interfere with immunomodulatory drugs .Immunomodulatory therapy is a very appealing strategy for the treatment of acute ischemic stroke, both for patients treated with recanalization methods as well as for patients no longer eligible for reperfusion therapies. However, given the dual nature of neuroinflammation, further research is needed to establish the exact sequence and the stroke subtypes in which these strategies will yield positive results and lead to better functional outcomes. In our opinion, recanalization strategies will continue to be increasingly used for treating acute ischemic stroke, with extended therapeutic time windows through various methods  and a more careful selection of eligible patients (trying to identify the presence of penumbra through MRI techniques). Recanalization would be administered concomitantly with a cocktail of antioxidant molecules to diminish the magnitude of oxidative stress induced by reperfusion. In the subacute phase, neuroinflammatory pathways could be modulated to enhance the proliferation and migration of stem cells and improve recovery. From the studies performed so far, it appears that these strategies have an extended time window. It seems more plausible that using endogenous repair mechanisms, such as stem cell transplantation or genetically manipulated extracellular vesicle delivery, would have a better outcome than simply delivering exogenous molecules. To avoid rejection, autologous bone marrow cells could be harvested from the patient in the acute phase.