Phaseolus vulgaris) is an important staple crop worldwide. In this study, we aimed to unravel the components of the RNA-guided silencing pathway in this non-model plant, taking advantage of the availability of two genome assemblies of Andean and Meso-American origin. We identified six PvDCLs, thirteen PvAGOs, 10 PvDRBs, 5 PvRDRs, in both genotypes, suggesting no recent gene amplification or deletion after the gene pool separation. In addition, we identified one PvNRPD1 and one PvNRPE1 encoding the largest subunits of Pol IV and Pol V, respectively. These genes were categorized into subgroups based on phylogenetic analyses. Comprehensive analyses of gene structure, genomic localization, and similarity among these genes were performed. Their expression patterns were investigated by means of expression models in different organs using online data and quantitative RT-PCR after pathogen infection. Several of the candidate genes were up-regulated after infection with the fungus Colletotrichum lindemuthianum.RNA silencing serves key roles in a multitude of cellular processes, including development, stress responses, metabolism, and maintenance of genome integrity. Dicer, Argonaute (AGO), double-stranded RNA binding (DRB) proteins, RNA-dependent RNA polymerase (RDR), and DNA-dependent RNA polymerases known as Pol IV and Pol V form core components to trigger RNA silencing. Common bean ( Small RNAs have regulatory roles in a multitude of biological processes, including stress responses, development, metabolism, and maintenance of genome integrity, in a sequence-specific manner . AlthougArabidopsis thaliana, dsRNAs are processed into specifically-sized sRNA duplexes by one of the four DCL (AtDCL1–4) proteins. dsRNA processing, called dicing, is facilitated by one of the six dsRNA-binding proteins  that interact with specific DCLs [A. thaliana, dsRNA may be synthesized by one of the six RDRs (AtRDR1–6) that copy single-stranded RNA (ssRNA) to initiate a new round of RNA silencing. These small RNAs are then incorporated into AGO-containing RNA-induced silencing complexes (RISCs) that guide small RNAs to their targets by sequence complementarity resulting in target RNA degradation, translational inhibition, or heterochromatin formation [A. thaliana genome encodes 10 AGO proteins (AGO1-10), with various functions such as implication in the RdDM pathway (AGO4) or viral defense (AGO2). RNA silencing is triggered by double-stranded RNA (dsRNA), and the generation and function of the small RNAs depend on key protein families such as Dicer-like (DCLs), Argonautes (AGOs), and RNA-dependent RNA polymerases (RDRs) . The RNAfic DCLs . dsRNA ms AtRDR1– that copRdDM requires specialized transcriptional machinery centered on two plant-specific RNA polymerase II (Pol II)-related enzymes called Pol IV and Pol V . Pol II,A. thaliana, rice, tomato, soybean, and Medicago truncatula present four, eight, seven, five, and six DCL genes, respectively [AGO gene family has expanded from three members in green algae [Arabidopsis, 17 in maize, 19 in rice, 25 in tomato, 22 in soybean, 27 in Brassica napus, and 11 in potato and coffee [AGO gene family is an ancient and probably continuous process. This could mirror a functional diversification of AGO and DCL proteins, presumably reflecting expanding small RNA-directed regulatory pathways [B. napus, respectively [The availability of an increasing number of plant genomes shows that there is a large variation in the number of gene members of the core families encoding key components of RNA silencing. For example, ectively ,13,14,15en algae  to 6 in d coffee ,21,22. Pd coffee . These pectively ,13,15,24P. vulgaris) is the most important grain legume for direct human consumption in the world, particularly in developing countries where it constitutes an important source of protein and essential micronutrients [C. lindemuthianum, is one of the most widespread and economically important diseases [P. vulgaris is not only a major pulse crop, but is also an ideal model for crop evolutionary studies because of its complex evolution, which led to two major gene pools known as the Andean and Meso-American gene pools [AGO and DCL genes have been analyzed in the Andean G19833 genotype leading to the identification of 15 PvAGO genes and 6 PvDCL genes [Common bean , we wanted to address the evolution of these genes on a short time scale. Their expression patterns were investigated in different organs using online data and after infection with the fungus C. lindemuthianum by quantitative RT-PCR. The identification of these core components to trigger RNA silencing in this non-model plant species of worldwide economic relevance pave the way for further investigation.The aims of this study were to identify and characterize, by in silico analysis, the genes involved in RNA silencing, including P. vulgaris genome assemblies and annotation data were downloaded from Phytozome (v10.0)  and from BAT93 genome data repository [http://denovo.cnag.cat/genomes/bean/, accessed on 1 December 2020), respectively.G19833 (v1.0) and BAT93 (v10) pository   from Phytozome (v10.0) and Arabidopsis (TAIRv10) , (4) contigs resulting from G19833 RNA-seq velvet assembly [http://pfam.xfam.org/, accessed on 1 December 2020) was used to confirm each candidate gene by checking the presence of the typical domain of each family. DCL proteins should have an N-terminal helicase domain (DExD/H-box and helicase-C subdomains) followed by DUF283 , PAZ (Piwi-Argonaute-Zwille), tandem RNase III domains, and one or two C-terminal double-stranded RNA binding domains (dsRBDs) [In order to identify  tBLASTn  search wSTn [DCL , AGO [17AGO [RDR , DRB [15nd NRPD2  gene seqnd NRPD2 , includiediction , (2) Glying Sim4 , (3) siming Sim4  on G. maassembly ,39 alignassembly . Finally(dsRBDs) . AGOs shA. thaliana, soybean, and/or M. truncatula. The prefix PvA or PvM was added for sequences originating from G19833 (Andean) or BAT93 (Meso-American), respectively.Candidate proteins were named based on their phylogenetic proximity to known members in M. truncatula [The complete sequence of each putative AGO, DCL, RDR, and DRB proteins were aligned using Muscle , and theuncatula , and RDRuncatula .PvA_AGO, PvA_DCL, PvA_RDR, PvA_DRB, PvA_NRPD, PvA_NRPE gene on G19833 chromosomes was determined by tBLASTn searching against the G19833 genome. Molecular weights (Mol. Wt.) and isoelectric points (pI) were determined using the Pepstats program from EMBOSS [The location of each m EMBOSS  analysism EMBOSS . The numm EMBOSS .https://www.ncbi.nlm.nih.gov/sra?linkname=bioproject_sra_all&from_uid=41439 (accessed on 1 December 2020), for 11 different organs including: roots_10DAP (days after planting), trifoliates_19DAP, young_pods, Leaves_10DAP, stem_10DAP, stem_19DAP, nodules_19DAP, roots_19DAP, mature_pods, flower_buds, flowers [RNA-seq data from G19833 genotype, were downloaded at  flowers . RNA-seq flowers ) in the  flowers ). Then f flowers ) to run C. lindemuthianum were carried out as previously described in Richard et al. [Infections of the common bean Andean landrace JaloEEP558 with the incompatible strain 100 of d et al. . A time-PvAGO1, PvAGO2a, PvDCL2a, PvDCL2b, PvAGO4a, PvAGO4b, and PvAGO4c were performed using the gene-specific primers listed in PvUkn1, PvUkn2, PvIDE, and PvAct11) [−ΔΔCt on three technical replicates and two biological replicates [t-tests between each mean value  and the corresponding mean value at t = 0 hpi.Total RNA extraction and quantitative RT-PCR (RT-qPCR) experiments were performed as described in Richard et al. . The expPvAct11)  . For paralogs, a letter  was used as the suffix. The same nomenclature was used for all genes involved in RNA silencing described in this study. Dicer-like 1–4 occurred as monophyletic groups containing DCLs from P. vulgaris, G. max, M. truncatula, and A. thaliana. Our manual annotation allowed us to identify PvM_DCL2c that was not present in the automatic annotation of BAT93 assembly. In P. vulgaris, for both BAT93 and G19833, DCL1, DCL3, and DCL4 occurred as single-copy genes, while DCL2 had three paralogs  (DCL genes in the common bean presented high levels of protein identity between BAT93 and G19833 (> 97% protein identity). PvA_DCL2a and PvA_DCL2b were separated by 2.5 kb on chromosome 6, while PvA_DCL2c was located on chromosome 8  are located in a syntenic region  , Table 1mosome 8 . Despiteeparated , such theparated . Manual c region . Indeed,her side . Amplifie copies ; howevere copies .P. vulgaris genome generated 13 full-length AGO genes recovered from both G19833 and BAT93 genomes  organized in a tandem array on chromosome 8 and separated by ∼20 kb  and BAT93 (PvM_AGO), testifying to the absence of recent gene duplication or deletion for this AGO gene family  out of the 6 AtRDR  closely linked on chromosome 3 and separated by 10 kb  as well as the DRB4 gene family (two members). A clear ortholog of AtHYL1, a key interactor of DCL1 in miRNA biogenesis [PvA/M_HYL1, was identified on common bean chromosome 9 , 11 (PvA_NRPE1), and 9 (PvA_NRPD2/NRPE2)  .DRB genes , two AGO genes , one DCL gene (PvA_DCL2c), and one RDR gene (PvA_RDR1a). Cluster 3 corresponds to genes that are highly expressed and comprises four AGO genes , two DRB genes , one DCL gene (PvA_DCL1), as well as PvA_NRPE1 and PvA_NRPD2. In particular, PvA_AGO1 seems to be highly expressed in all tested organs. Finally, the remaining 20 genes correspond to genes presenting an intermediary expression level . Significantly, temporal gene expression analysis revealed that DCL2a and DCL2b are both ca. nine-fold up-regulated after infection compared to the mock control at 72 hpi. Similarly, a clear upregulation is observed at 72 hpi for both AGO4a and AGO2a, and also for AGO4b but with a lower extend , DCL (6), DRB (10), RDR (5), NRPD1 (1), NRPE1 (1), and NRPD2/NRPE2 (1) genes in both G19833 (Andean) and BAT93 (Meso-American) genomes, suggesting that no recent gene duplication/deletion occurred after gene pool divergence. Indeed, for each of the 37 genes analyzed in the present study, orthologs presenting a high percentage of protein identity (> 94%) were unambiguously identified between BAT93 and G19833  genome [GmDCL4a/GmDCL4b and GmDCL1a/GmDCL1b correspond to such homoeologous gene pairs, while GmDCL3 is present as a single copy. By contrast, GmDCL2a and GmDCL2b are locally duplicated, separated by 5 kb, on chromosome 9. In soybean, the age of this GmDCL2a/GmDCL2b duplication was estimated to be 19.4 Mya [Pv Gm common ancestor, a locally duplicated pair of DCL2 genes were present. In agreement with this, we found in common bean two DCL2 genes, Pv_DCL2a and Pv_DCL2b, organized in tandem array in the corresponding syntenic region with soybean , pigeonpea (Cajanus cajan), and each of the two genomes composing the allotetraploid groundnut (Arachis duranensis and Arachis ipaensis) [DCL2 genes in specific legume species could lead to their functional diversification and probably reflect their functional importance. In Mt, a nodule-specific role for DCL2 has been proposed [DCL2 genes regulate traits such as seed color via the production of 22 nucleotide siRNA from long inverted repeats [GmDCL2 paralogs exhibited a wide range of transcriptional changes in response to stress, suggesting DCL2s may play an important role in stress response [Pv_DCL2a and Pv_DCL2b, mildly expressed in most organs  genome . GmDCL4a19.4 Mya , indicat19.4 Mya , and the19.4 Mya ,59. Cons soybean . In the ed in Mt . Howeverpaensis) . These iproposed , while i repeats . In anotresponse . Congruet organs , are up-uthianum .Pv, while 15 AGO genes were reported in a previous analysis performed on the G19833 genome. Indeed, compared to de Sousa Cardoso et al. [Pv AGO10 gene clearly corresponds to two Gm orthologs . In addition to AGO10, expansion was also observed for AGO2 (two members) and AGO4 (three members) in the Pv genome. AtAGO4 primarily binds 24-nt, repeat and heterochromatin-associated siRNAs, and functions in RNA-directed DNA methylation [AtAGO2 functions in antibacterial immunity [PvA_AGO1 seems to be highly expressed in all common bean-tested organs. In agreement with our results, AGO1 expression is detected in many organs, such as leaves, roots, and flowers, in Arabidopsis [B. napus [Salvia miltiorrhiza [We identified 13 AGO genes in o et al. , our mano et al. . Within o et al. . For exao et al. . More pro et al. . In commo et al. . Each Pvrthologs , stronglhylation , while Aimmunity . In commbidopsis , rice [1bidopsis , B. napuB. napus , and theiorrhiza .Verticillium dahliae [Verticillium longisporum [Magnaporthe oryzae [Botrytis cinerea [C. lindemuthianum. However, there is growing evidence that this is a common mechanism exploited by fungal pathogens to promote their infection [C. lindemuthianum, although not yet identified.Functional analysis of genes involved in RNA silencing revealed that most of them play multiple roles, not only in growth and development but also in immune defense against pathogens ,67,68,69 dahliae , Verticigisporum , Magnapoe oryzae , and Bot cinerea . The imp cinerea ,76,77,78 cinerea . At presnfection . ConsequC. lindemuthianum (in an incompatible context). Whereas expression levels of PvA_AGO2a, PvA_AGO4a, and PvA_DCL2 (a and b) is low to moderate without any biotic stress  infection, an increased expression of AGO4 has been reported, whereas no change in expression was observed for AGO1 [AGO2 expression after infection was reported in Arabidopsis after infection by the biotrophic bacteria P. syringae, in the oil crop B. napus infected by the fungal necrotrophic Sclerotinia sclerotiorum, and in the cowpea Vigna unguiculata infected by CPSMV [DCL2 transcripts increases at the local site of infection by the CLRDV (Cotton leafroll dwarf virus) in a resistant genotype cotton Gossypium hirsutum [PvA_DCL2a and Pv_DCL2b was observed in incompatible interaction with C. lindemuthianum. This suggests that in the common bean, an increased expression of specific genes involved in RNA silencing, acting in both the miRNA and the siRNA pathways, could counteract the infectious process of C. lindemuthianum. However, how these genes could regulate resistance in the common bean requires further investigation. To investigate the contribution of some of the genes involved in RNA silencing in the defense response in common bean, we performed quantitative RT-PCR-based expression analysis on leaves of resistant bean plants inoculated with the hemibiotrophic fungus c stress , a stronc stress . On the  pathway . AGO2 fu pathway . In the for AGO1 ,83. Moreby CPSMV ,82,84. Chirsutum . This isAGO, DCL, RDR, DRB, NRPD1, NRPE1, and NRPD2 genes in the common bean genome. For example, taking advantage of the work presented here, we would like to utilize virus-induced gene silencing (VIGS) to silence NRPD1 and NRPE1, in order to gain insight into the mechanisms involved in the unusual methylation pattern observed for NLR genes in the common bean [This work will further provide a solid foundation for future functional analysis of mon bean ,87,88. F


MAGI1 is a cytoplasmic scaffolding protein initially identified as a component of cell-to-cell contacts stabilizing cadherin-mediated cell–cell adhesion in epithelial and endothelial cells. Clinical-pathological and experimental evidence indicates that MAGI1 expression is decreased in some inflammatory diseases, and also in several cancers, including hepatocellular carcinoma, colorectal, cervical, breast, brain, and gastric cancers and appears to act as a tumor suppressor, modulating the activity of oncogenic pathways such as the PI3K/AKT and the Wnt/β-catenin pathways. Genomic mutations and other mechanisms such as mechanical stress or inflammation have been described to regulate MAGI1 expression. Intriguingly, in breast and colorectal cancers, MAGI1 expression is induced by non-steroidal anti-inflammatory drugs (NSAIDs), suggesting a role in mediating the tumor suppressive activity of NSAIDs. More recently, MAGI1 was found to localize at mature focal adhesion and to regulate integrin-mediated adhesion and signaling in endothelial cells. Here, we review MAGI1′s role as scaffolding protein, recent developments in the understanding of MAGI1 function as tumor suppressor gene, its role in endothelial cells and its implication in cancer and vascular biology. We also discuss outstanding questions about its regulation and potential translational implications in oncology.  MAGI1 undergoes mutations, gene rearrangements, and methylation. MAGI1 is also regulated post-transcriptionally by miRNAs, or at the protein level by degradation or phosphorylation. Other mechanisms such as mechanical stress or inflammation also regulate MAGI1 expression. This is an exciting journey for a gene that has not yet entered the full spotlight .MAGI1 , discovered in 1998 and named BAP1 (brain-specific angiogenesis inhibitor 1-associated protein) . LocatedHere, we summarize some of the most important findings about MAGI1, in particular concerning its emerging role as vascular regulator and tumor suppressor, along with the mechanisms that regulate its expression. We end up listing a number of open questions that have no clear answers yet, and that we believe deserve further attention.membrane-associated guanylate kinases) scaffold proteins, that play essential roles during development, cell–cell communication, and cellular signal transduction. Some of their diverse functions include the regulation of cellular processes such as cell polarity, tight junction (TJ) formation, neuronal synaptic transmission, cell proliferation, and apoptosis. Accordingly, mutations or changes in their expression are linked to defects in cell–cell adhesion, cell polarity, cell proliferation, and development. In mammals, the MAGUK family englobe 22 members classified phylogenetically into 8 sub-families, which vary in size, domain organization, localization, and biological functions. Despite their differences, all the MAGUKs share a well conserved core structure, which is comprised of one or multiple PDZ domains (except for CACNB) in the amino-terminal region, a catalytically inactive guanylate kinase (GUK) domain and a Src Homology 3 (SH3) domain (except for the MAGI subfamily). In contrast to other members of this family, MAGI1 has some distinct features: (1) the GUK domain is located in the amino-terminal region rather than at the carboxyl end; (2) its SH3 domain is replaced by two WW domains situated downstream of the GUK domain (that function in a similar fashion as the SH3 domain), and (3) it contains five PDZ domains instead of the usual one or three . The and ZO-1 , howeverand ZO-1 . Regardiand ZO-1 . Neverthand ZO-1 .N-methyl-d-aspartate receptors (NMDARs) and neuronal cell adhesion proteins, and was termed S-SCAM [All MAGUKs localize to regions of cell–cell contact, such as TJs in epithelial cells and synaptic junctions in neurons, where they orchestrate the assembly of multiprotein complexes via their protein–protein interaction domains ,13. The d S-SCAM , while Md S-SCAM  and the d S-SCAM . There iMAGI1 was first identified in the mouse, as a protein interacting with K-RasB. MAGI1 mRNA undergoes alternative splicing to produce three transcripts that encode proteins with a unique carboxyl-terminal region, named MAGI1-a, MAGI1-b, MAGI1-c. These different isoforms differ in length, cellular localization, and tissue distribution. While MAGI1-a is the shortest variant, encoding for a protein of 1139 amino acids, MAGI1-b and MAGI1-c encode for proteins of 1171 and 1374 amino acids, respectively. In addition, the carboxyl-terminal region of MAGI1-c contains 251 amino acids forming a three bipartite nuclear localization signal (NLS)  , JAM4 2, and ESAMAGI1 also binds to synaptopodin and α-actinin-4 in glomerular podocytes through its second WW and fifth PDZ domain, respectively . These fMAGI1, 2, and 3 have also been described to interact with a diversity of other ligands from the nervous system, including NMDA receptors, neuroligin, BAI1, activin type II receptor, δ-catenin, the muscle-specific tyrosine kinase (MuSK) (reviewed in ), atroph3) [ts-src cells [Due to the critical role of MAGI1 in regulating cell–cell contacts, it is not surprising that its loss influences the adhesion, proliferation, invasiveness, and metastasis of different tumor cell types. Disruption of cadherin junctional complexes is the hallmark of cancer progression and has been extensively associated with invasiveness and metastasis . The sta3) . Kotelevrc cells . In addirc cells .MAGI1 is downregulated in various cancers and acts as a tumor suppressor; in several cases by modulating direct or indirectly PTEN activity. For example, Zhang et al. demonstrated that transfection of MAGI1 in HepG2 cells inhibits cell migration and invasion by upregulating the expression of PTEN in hepatocellular carcinoma (HCC) . In addiMAGI2 contribute to prostate carcinogenesis by driving AKT phosphorylation and altering PI3K signaling, thus disrupting PTEN signaling [Other MAGI family members have also been described to regulate cell migration and invasion via PTEN modulation. MAGI2 was found to inhibit cell migration and proliferation in different HCC cell lines by diminishing the phosphorylation of FAK and AKT , two impignaling .N-cadherin/vimentin pathway [MAGI has been also found to modulate other signaling pathways such as the Wnt/β-catenin, the PI3K/AKT or the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathways. Three studies performed in glioma cells showed similar outcomes; the study of Lu et al. demonstrated that MAGI1 played an essential role during glioma progression; its silencing in different glioma cell lines enhanced proliferation and inhibited apoptosis, increased Wnt/β-catenin signaling, enhanced AKT phosphorylation, and reduced E-cadherin and PTEN expression. Conversely, overexpression of MAGI1 significantly inhibited tumor growth in vivo . Another pathway . MAGI3 w pathway . Likewis pathway .+/HER2- breast cancers (BCs), relative to HER2+ and triple negative (TN) BCs. More specifically, within this subset, high MAGI1 expression was associated with better prognosis, while low MAGI1 levels correlated with higher histological grade, more aggressive phenotype, inflammation, and worse prognosis. Experimentally, MAGI1 downregulation in ER+ BC cell lines promoted cell proliferation and survival in vitro and enhanced primary tumor growth and lung metastasis formation in vivo [Recently, MAGI1 was reported to be expressed at higher levels in estrogen receptor (ER) in vivo . MAGI1 d in vivo .Jia et al. showed that MAGI1 downregulation promoted the activation of pathways involved in metastasis and EMT in gastric cancer cells. In a panel of different gastric cancer cell lines, MAGI1 knockdown significantly enhanced migration and invasion of these cells by altering the expression of MMPs and EMT-related molecules via inhibiting the MAPK/ERK signaling pathway . MAGI1 was also shown to be regulated by oncogenic viruses, playing an important role in the consequent neoplastic transformation of the cells. There is increasing evidence of a link between the tumorigenic potential of certain viruses and its capacity to functionally inactivate certain TJ-associated proteins . NotablyWhile significant insights have been made in confirming the critical role of MAGI1 as tumor suppressor in different cancer types, research involving MAGI1 functions in endothelial cells (ECs) remains limited. The vascular endothelium, located at the interface between tissues and blood, plays a crucial role in the regulation of vascular permeability, thrombus formation, oxidative stress, and angiogenesis . These f [A first indication that MAGI1 may be involved in regulating angiogenesis was suggested when MAGI1 was cloned in 1998 and characterized as a protein interacting with brain-specific angiogenesis inhibitor 1 (BAI1) , whose e . A recen .Additional studies have pointed towards a role of MAGI1 in regulating ECs adhesion, their strength and dynamics, and eventually angiogenesis. The study of Sakurai et al. reported that MAGI1 mediated Rap1 activation , and enhanced vascular endothelial (VE)-cadherin-dependent endothelial cell–cell adhesion, while MAGI1 depletion impaired it . SimilarMAGI1 has been also reported to regulate endothelial nitric oxide synthase (eNOS) expression and vascular nitric oxide (NO) production through the cyclic AMP-dependent protein kinase/AMP-activated protein kinase (PKA/AMPK) signaling pathway in response to fluid shear stress , the friDifferent components of cell junctions, including some direct MAGI1 interacting partners, have been described to modulate ECs permeability and leukocyte extravasation. Under physiological conditions, the endothelium is characterized by low permeability to solutes and leukocytes . Under cThe podocyte of kidney glomeruli shares many cell biological characteristics with neuronal dendrites: both are postmitotic, polarized, and highly ramified cell types. They also share the expression of various molecules working for signal transduction, transmembranous transport, and intercellular contacts. They display the same cytoskeletal organization, with actin and some expression-restricted actin-binding molecules, such as synaptopodin, localized in podocyte foot processes and neuronal dendritic spines. In addition, podocytes express a wide variety of neurotransmitter receptors . Evi2+-activated K+ (BKCa) channels [MAGI2 expression or mutations have been associated with glomerular disorders such as nephrotic syndrome in mice and humans [At the glomerulus, MAGI1 is present in podocytes at the slit diaphragm (SD), the structure responsible for the glomerular filtration barrier that prevents serum proteins from leaking into the urine . There, channels . In micechannels . MAGI2 achannels ,101,102.channels ,101, whichannels . In addid humans ,105,106.d humans ).Drosophila melanogaster, Caenorhabditis elegans, or in Zebrafish) suggest an association of MAGI1 in the brain with memory and learning, but the mechanisms are still unknown [MAGI1 copy number variations and polymorphism are associated with schizophrenia, bipolar disorders, and depressive episodes [MAGI2 is highly expressed in the brain, where it is localized at both excitatory and inhibitory synapses ,108. Mic unknown . Genome-episodes ,113,114.PTEN and MAGI2. These rearrangements included two independent inversions and two long-range intrachromosomal inversions [MAGI2 rearrangements with frame shift have been also identified in malignant melanoma [MAGI2 expression by miR-101  reduces PTEN activity and AKT activation [MAGI2, leading to loss of PTEN stability [MAGI2 expression either via somatic genomic events or post-transcriptional regulation (by micro-RNAs) appears to affect PTEN activity [MAGI2 mRNA gene expression levels were found significantly downregulated in a cohort of clinical PCa samples and cell lines, however, in this case, the downregulation of MAGI2 was not correlated to PTEN mRNA expression, suggesting that different genomic events caused distinct chromosomal aberrations in each gene [MAGI2 was also found to be epigenetically silenced in cervical cancer [MAGI3. A study performed on whole-exome sequences of DNA from human BC tissues identified a recurrent MAGI3-AKT3 fusion enriched in triple-negative BC lacking estrogen and progesterone receptors and ERBB2 expression [Genomic mutations, gene rearrangements, promoter methylation, and microRNAs (miRNAs) have been described to alter the expression of MAGI subfamily members in different cell types. MAGI1-specific mechanisms that regulate its expression, and in particular cause or contribute to its downregulation in cancer cells, remain largely to be elucidated. With the aim to identify novel somatic events involved in the progression of prostate cancer (PCa), Berger et al. sequenced PCa samples and found genetic rearrangements disrupting both versions . In addimelanoma ,117, anomelanoma . A studytivation , while itability . MicroRNtability  and metatability . Dysreguactivity  in severach gene . MAGI2 wl cancer . DNA seqpression . The reapression , togethepression .MAGI1 mediates tumor metastasis through the c-Myb/miR-520h/MAGI1 signaling pathway in renal cell carcinoma [MAGI1 genes were also found to be associated with metachronous gastric cancer development [MAGI1 displayed promoter methylation as well as decreased expression in ATC compared with normal thyroid tissue [MAGI1 was reported among genes aberrantly methylated in acute lymphoblastic leukemia [MAGI1 gene [MAGI1 mutations were found in in parathyroid carcinoma, a rare endocrine malignancy [MAGI1 copy number variations were found associated with bipolar affective disorder and schizophrenia [MAGI1 in bipolar cases versus control subjects have also been reported [Regarding MAGI1, a recent report revealed that arcinoma . Somaticelopment , and in d tissue . Aberrand tissue . In thisleukemia . Next geGI1 gene . Similarlignancy , and in lignancy . As alreophrenia . In addireported , findingMAGI1 also seems to be regulated by other genomic-independent mechanisms. During apoptosis induced by Fas/Fas ligand, staurosporine, and UV irradiation in different models , MAGI1 i+/HER2− BC, and it was shown to be upregulated by estrogen/ER and necessary for estrogen/ER signaling [+ BC, MAGI1 mutations or copy number alterations are rare , indicating that in this particular case, DNA alterations are not the major cause of MAGI1 downregulation in this BC subtype. Reduced MAGI1 expression in ER+/HER2− BC correlates with inflammation, based both on human transcriptomic and experimental data. Furthermore, MAGI1 was shown to be downregulated by the prostaglandin E2 (PGE2)/Cyclooxygenase-2 (COX-2) axis. Experimentally, the inflammatory mediator PGE2 downregulates MAGI1 in MCF7 cells, while COX-2 inhibition (with the selective COX-2 inhibitor NS-398) upregulates its expression [2 play a critical role in cancer development and progression [2 synthesis, is overexpressed in several cancers including BC  [MAGI1 is highly expressed in ERignaling . An impopression . Chronicgression . COX-2, iewed in ), and PGpression . A signiogenesis ). In CRC(COXIBs) . The sel(COXIBs) ).2 [In glioma cells, the mechanisms that contribute to MAGI3 downregulation remains unknown. COX-2 was shown to correlate with glioma grade and is a negative prognostic marker . The use2 , howeverMAGI1 is emerging as a protein exerting multiple, essential cellular functions and implicated in important human pathologies, particularly in cancer and atherosclerosis. In this review, we have highlighted the current knowledge on MAGI1 functions in epithelial, endothelial, and cancer cells . The usiewed in ,61. Preciewed in . The bioiewed in . Animal iewed in ,149; howiewed in ,151. Regiewed in . Howeveriewed in . Anecdotiewed in . It is ier cells ,61. A beMAGI2 have similarly been described in Crohn’s disease, ulcerative colitis, and celiac disease [Different MAGI subfamily members have been described to play a negative role in inflammation in other human diseases. Many studies have linked MAGI1 to different chronic inflammatory diseases, such as inflammatory bowel disease (IBD) , psorias disease ,161. MAG disease ).While MAGI1 loss seems a common feature in many different cancers and inflammatory diseases, ECs activation and consequent MAGI1 upregulation contribute to the pathogenesis of atherosclerosis . A betteMAGI1 colocalizes to mature focal adhesions in ECs and regulates integrin-dependent cell adhesion and migration . Many qu+ BC [While it is clear that MAGI1 expression is reduced during cancer progression, it is not clear yet whether MAGI1 levels may be used to stratify cancer patients for prognostic purposes and, more importantly, for therapeutic decisions. As mentioned before, MAGI1 could serve as a prognostic marker for the identification of patients at high-risk of relapse in ER+ BC . MAGI1 i+ BC , while i+ BC  and glio+ BC , low MAG+ BC  and sugg+ BC . In addi+ BC . Identif+ BC .Could MAGI1 become a direct therapeutic target to treat human pathologies? In conditions where MAGI1 expression or function is decreased, this may not be an option, as it would require increasing protein expression. Should we, however, learn how to pharmacologically induce MAGI1 expression? MAGI1 may become a “reverse target” or a rational for considering such drugs. To this regard, the observed induction of MAGI1 expression by NSAIDs is consistent with the tumor suppressive capacities of both MAGI1 and NSAIDs. In the context of vascular diseases, and in view of the essential roles of MAGI1 in maintaining basic cellular and tissue functions , systemic pharmacological MAGI1 inhibition might not represent a feasible option, as it will be overshadowed by massive and generalize unwanted effects. In addition, due to its important role in regulating permeability, MAGI1 inhibition may cause increased EC permeability, which may enhance atherosclerosis formation .In conclusion, more experimental studies at molecular, cellular, and tissue levels in different contexts and cell types will be necessary in order to fully understand MAGI’s roles, regulation, prognostic value, and therapeutic relevance. We believe that additional information about MAGI1 will be revealed in the upcoming years, and it will be important not only to look at additional cancer fields or cell types, but also to re-evaluate existing studies about MAGI1 with greater attention—and maybe a different perspective—to fully understand MAGI1 implication and significance in both, cancer progression and vascular biology.


Coffea genotypes, C. canephora cv. Conilon Clone 153 and C. arabica cv. Icatu, grown at normal (aCO2) or elevated (eCO2) CO2 concentrations of 380 or 700 ppm, respectively, under well-watered (WW), moderate (MWD), or severe (SWD) water deficit conditions, in order to assess coffee responses to drought and how eCO2 can influence such responses. Primary metabolites were analyzed with a gas chromatography time-of-flight mass spectrometry metabolomics platform (GC-TOF-MS). A total of 48 primary metabolites were identified in both genotypes , with differences recorded in both genotypes. Increased metabolite levels were observed in CL153 plants under single and combined conditions of aCO2 and drought (MWD and SWD), as opposed to the observed decreased levels under eCO2 in both drought conditions. In contrast, Icatu showed minor differences under MWD, and increased levels  only under SWD at both CO2 concentration conditions, although with a tendency towards greater increases under eCO2. Altogether, CL153 demonstrated large impact under MWD, and seemed not to benefit from eCO2 in either MWD and SWD, in contrast with Icatu.Climate change scenarios pose major threats to many crops worldwide, including coffee. We explored the primary metabolite responses in two  Coffea arabica L. (Arabica coffee) and Coffea canephora Pierre ex Froehner (Robusta coffee) [C. arabica, with impacts on suitable cultivation areas, yield, and bean quality, as well as increased pest and disease incidence and economic losses [Coffee is an important agricultural commodity worldwide that provides income and employment to millions of people in developing countries. Coffee is cultivated in over 80 countries throughout the tropics with ca. 99% of the production based on  coffee) ,2. Estim coffee) . In thisc losses ,6,7,8,9.2 concentration, coupled with rising air temperatures , together with more frequent and prolonged drought events associated with unpredictable rainfall patterns [2 concentration (eCO2) on several crop plants, usually associated with increased photosynthetic activity [2 was recently reported to increase coffee plant resilience to heat stress [2 on plants subject to water deficits can be species-dependent [2 can clearly reduce drought impact on plant photosynthesis, growth, and yields [Coffea spp. as well, where eCO2 mitigated the negative impacts of drought on photosynthetic performance and components [One of the major components of global climate change is an increase in air COpatterns . Neverthactivity ,13. Thatactivity ,15,16,17activity , ultimatt stress ,20,21, wt stress  and beant stress . Additioependent , studiesd yields ,26,27). mponents ,30,31,32Under field conditions, plants are often exposed to a combination of different environmental changes. In this case, the plant response to combined changing conditions should be regarded not as a sum of each one applied independently, but as a unique response that can reflect a positive  or negative  interaction between them ,34,35. A2 at the physiological, biochemical, and molecular level [2 might influence drought response and plant resilience in Coffea spp. To date, GC-MS is the most widely accepted analytical technology used in post-genomic plant metabolomics studies [C. canephora cv. Conilon Clone 153 (CL153) and C. arabica cv. Icatu (Icatu), were grown under normal atmospheric (aCO2) or elevated (eCO2) air CO2 concentration, coupled with either well-watered conditions (WW), or moderate (MWD) or severe (SWD) water deficit conditions. Our findings provide important and timely evidence regarding the role of eCO2 in the response to drought in these species, contributing to improvements in knowledge regarding coffee plant performance in the face of ongoing and future global climate changes.Complementing the previous studies of our team, and regarding the coffee plant response to single and combined exposure to drought and/or eCOar level ,29,32,44 studies ,45,46,47The GC-TOF-MS analysis identified 48 primary metabolites in CL153 and Icatu leaves, including 23 amino acids and derivatives, 10 organic acids, 11 sugars, and 4 other metabolites .2 (WW plants), and MWD (both CO2 concentrations). However, under SWD, both genotypes showed a large (and similar) number of significant changes under both aCO2 or eCO2. Additionally, in CL153 SWD plants under eCO2, those changes reflected mostly a decrease in metabolite levels (as opposed to Icatu)  c. Lower D or SWD d, whereaith aCO2 d.2 led to a significant increase in aspartate, glutamate, methionine, pyroglutamate, 2-oxoglutarate, citrate, quinate, and glucose (up to 5-fold)  in most amino acids (γ-aminobutyric acid (GABA), glutamate, histidine, isoleucine, lysine, pyroglutamate, threonine, tryptophan, tyramine, tyrosine, and valine). A similar response was observed under SWD with significant increases in amino acids (up to 11-fold), together with a significant increase in glucose (5-fold), and a significant decline in sucrose, citrate, and phosphoric acid. Interestingly, CL153 plants grown under eCO2 and subjected to MWD and SWD conditions , histidine (only at MWD), proline (only at MWD), pyroglutamate, and serine. Nevertheless, under eCO2, significant increases in glucose , and in fructose (4-fold), trehalose (5-fold), tryptophan, (up to 7-fold), and valine (4-fold) were observed under SWD. Significant reductions were observed regarding pyruvate (under MWD), and in citrate, glycerate, and threonate under SWD.In CL153 plants, the effect of single exposure to eCO 5-fold) a. Additi 5-fold) b. Under nditions c showed 2 to aCO2 for each water deficit treatment in regards to amino acid levels, particularly in MWD .Furthermore, a global reduction in relative metabolite abundance was observed when comparing eCOy in MWD d. Still,C. arabica cv. Icatu showed a different pattern of metabolite variation when compared with the C. canephora genotype CL153. In Icatu plants, the single eCO2 effect promoted significant changes in a low number of metabolites   a. Furthentration b. Most snditions b,c. Over2 (2), putrescine, glycerate, threonate , and a significant decrease in arginine and fructose (in MWD under eCO2).In opposition to what was observed in CL153, comparison between the combined effect of MWD and SWD in plants grown under eCO2 d led to 2 concentration conditions could only be observed for MWD plants.Unsupervised principal component analysis (PCA) was applied to GC-TOF-MS relative primary metabolite data to identify major sources of variation in the data . The CL12 and eCO2.In Icatu plants b, a cleaCoffea spp. genotypes, a supervised partial least square discrimination analysis (PLS-DA) was performed  valine and isoleucine, arginine, GABA, lysine, phenylalanine, trehalose, tryptophan, and tyrosine for CL153 plants, and arginine, BCAAs, fructose, GABA, lysine, proline, tryptophan, and tyrosine for Icatu plants.The score plots revealed a high number of highly correlated metabolites responsible for the cluster of SWD samples in the negative quadrants of component 1 for both genotypes. These metabolites include mostly amino acids and sugars that significantly increased in plants exposed to single and combined drought and eCO2 concentration (eCO2) and water deficit (MWD and SWD) conditions. Unsupervised exploratory PCA revealed differential metabolite responses to single and combined stresses; this effect was clearer in Icatu plants, and PLS-DA analyses allowed us to further identify stress-responsive metabolites whose levels significantly varied upon single and/or combined exposure to drought and eCO2.GC-TOF-MS analysis allowed for the characterization of the primary metabolome of CL153 and Icatu leaves under single and combined exposure to increasing CO2 concentrations. This is also in agreement with the reported drought resilience of this genotype under both CO2 concentrations [2 concentrations, and under MWD only at aCO2, whereas in CL153 an increase was only observed under SWD and eCO2. BCAAs are well-known to accumulate under abiotic stress conditions and play the role of osmolytes during osmotic stress [The significant increase in CL153 alone of most amino acids upon single exposure to MWD indicated that Icatu was not affected by MWD, and thus, supports previous studies reporting its strong drought resilience . Howevertrations . The BCAc stress ,52,53. Bc stress ,55.2), and Icatu (under aCO2 and eCO2). Lysine catabolism can channel electrons to the mitochondrial electron transport chain in addition to feeding the operation of the tricarboxylic acid (TCA) cycle under carbon starvation [Lysine, a product of the aspartate pathway, also significantly increased in response to SWD in CL153 plants , and maintenance of the cellular redox balance [C. canephora clones  subjected to drought-stress revealed an accumulation of proline with increasing exposure to soil drying, followed by a decline upon re-watering [C. canephora than in a susceptible genotype. Proline levels were also found to increase in C. arabica cvs. Catuaí and Mundo Novo, C. canephora cv. Apoatã, and a graft of Mundo Novo shoot on Apoatã root in response to drought [2 concentrations and in both genotypes. Increased levels of arginine were suggested to contribute to drought tolerance [C. arabica cv. Catuaí during grain expansion [Metabolites from the glutamate family pathway significantly increased in Icatu, including the osmolytes proline and GABA, under SWD irrespective of CO balance ,42,43. Awatering . Moreovewatering  showed t drought . Argininolerance . Argininolerance . TCA cycolerance . In factxpansion , which h2; tryptophan and tyrosine also increased in SWD under eCO2 conditions. The accumulation of these metabolites is a valuable source of carbon-skeletons for the phenylpropanoid pathway and further biosynthesis of secondary metabolites [In Icatu, amino acids derived from the shikimate pathway  accumulated under single exposure to SWD under aCOabolites ,64. Trypabolites .2. Several drought experiments have shown a correlation between resistance to dehydration and the levels of putrescine—a plant growth regulator that helps to balance chlorophyll concentrations—and the accumulation of osmolytes and soluble phenolic compounds under drought conditions [A significant increase in putrescine occurred in Icatu under the interaction of SWD and eCOnditions . 2 conditions), which might have contributed to dehydration tolerance [Osmoprotective sugars were also affected by drought and a significant increase was observed in fructose in Icatu and in glucose, trehalose, and sucrose in CL153 , contrasting with the decreases observed in CL153 under eCO2, which is in line with the high tolerance of Icatu plants to water deficit under both CO2 concentrations [The metabolite responses of each genotype to water deficit and eCO earlier ,32, main2 concentration, and a greater tolerance than CL153 under aCO2. This resilience of Icatu was associated with the preservation of photosynthetic performance  and the maintenance or reinforcement of several chloroplast components , and RuBisCO activities) [2) was further associated with a wide abundance of proteins related to the photosynthetic apparatus , which was not observed in CL153 [Physiological and biochemical studies demonstrated an absence of impact on Icatu plants in most parameters under SWD, irrespective of COivities) ,44. Suchin CL153 .C. canephora plants of Clone 120 (drought-tolerant) and Clone 109 (drought-sensitive), subjected to repetitive cycles of drought conditions for 14 days, revealed an oxidative stress response on the sensitive clone, presumably leading to programmed cell death, while acclimation of the tolerant genotype seems to be related to antioxidant secondary metabolites and ABA response [Limited photochemical energy use can generate ROS leading to damage to the photosynthetic apparatus  ,68. Therresponse .Coffea canephora Pierre ex A. Froehner cv. Conilon Clone 153 (CL153) and C. arabica L. cv. Icatu Vermelho  were used, as described in [−2 s−1 at the upper part of the plant), relative humidity (70% ± 2%), photoperiod (12 h), and either ambient  or elevated  air CO2 concentration. Plants were grown without restriction of nutrients [Plants of two cropped genotypes (Brazil) from the two main coffee-producing species, ribed in . Brieflyutrients  or root n = 6 to 8) and maintained under well-irrigated conditions (WW), or exposed to moderate (MWD) and severe water deficit (SWD), as described in [pd), as WW: Ψpd > −0.35 MPa; MWD −1.5 < Ψpd < 2.5 MPa; and SWD Ψpd < 3.5 MPa, corresponding to ca. 80% (WW), 25% (MWD), or 10% (SWD) of maximal pot water availability, respectively [pd was determined immediately after leaf excision in 5–6 plants per treatment, using a pressure chamber .Within each chamber, plants were divided into three groups  under MWD conditions were exposed to total water withholding in the last five days of the four-week period, in order to further force the reduction of Ψpd values, which did not shift below −0.6 MPa until that stage.When the required ΨFor metabolomic analysis, newly matured leaves from the upper third part (well illuminated) were collected, immediately frozen in liquid nitrogen, and stored at −80 °C.–1 ribitol in water), as the internal standard, were added to each tube. Samples were vortex-mixed and incubated in a shaker  for 15 min at 70 °C and 950 rpm, and subsequently centrifuged . The supernatant was collected, mixed with 375 μL chloroform and 750 μL water, and vortex-mixed. After centrifugation , 150 mL of the polar aqueous/methanol phase was evaporated to dryness using a centrifugal concentrator for 3 h at 30 °C , and stored at −80 °C. Dried polar extracts were derivatized with 40 μL of 20 mg mL−1 methoxyamine hydrochloride in pyridine, followed by 70 μL of N-methyl-N-trimethylsilyltrifluoroacetamide (TMS derivatization) and 20 μL mL−1 of a mixture of fatty acid methyl esters (FAMES). Biological variations were controlled by analyzing quality control (QC) standards; namely, FAMES internal standard markers and a QC standard solution of 41 pure reference compounds, throughout the analysis. After GC-TOF-MS analysis, samples were subsequently evaluated using AMDIS . Primary metabolites were annotated using the TagFinder software [http://gmd.mpimp-golm.mpg.de/, accessed on 25 June 2021) [Primary metabolites were extracted and derivatized following a well-established protocol . Frozen software  and a rene 2021) ,79. Meta2 concentration (aCO2 or eCO2) and three levels of water availability . The experiment was implemented using a completely randomized design, with six to eight plants per treatment in individual pots.Plants from each coffee genotype were subjected to six treatment combinations, reflecting the single and combined exposure to two COStatistical analyses were performed in R and R Studio software ,49. R Pa10 transformed for heatmap plotting.The relative abundance of primary metabolite levels was normalized to the internal standard (ribitol) and the fresh weight/dry weight ratio of the samples. Within each genotype, one-way ANOVA at a 95% confidence level was used to assess differences between treatments. Subsequently, fold-changes between conditions and the control for each metabolite were determined and LogPrincipal component analysis (PCA) was applied using unit variance scaling, and to further maximize the discrimination between the sample groups, supervised partial least squares discriminant analysis (PLS-DA) was performed using the leave-one-out cross-validation embedded in the “mixOmics” package.2 clearly reduced metabolite changes under MWD in Icatu (but not in CL153), resulting in metabolic profiles close to those of their WW plants. These findings are relevant in terms of the coffee plant response to future climate change scenarios (particularly in regard to the role of eCO2), and also highlight the need for complementary and integrative studies, from ecophysiology to omics, to better clarify the role of specific metabolites in the resilience of this important tropical crop.Overall, our metabolite profiling findings showed significant drought impacts on two widely traded coffee species. Notably, eCO


Background: Liver hepatocellular carcinoma (LIHC) is the third leading cause of cancer-related death and the sixth most common solid tumor worldwide. In the tumor microenvironment, the cross-talk between cancer cells, immune cells, and stromal cells exerts significant effects on neoplasia and tumor development and is modulated in part by chemokines. Chemokine (C-C motif) ligands (CCL) can directly target tumor cells and stromal cells, and they have been shown to regulate tumor cell proliferation, cancer stem-like cell properties, cancer invasiveness and metastasis, which directly and indirectly affect tumor immunity and influence cancer progression, therapy and patient outcomes. However, the prognostic values of chemokines CCL in LIHC have not been clarified.Methods: In this study, we comprehensively analyzed the relationship between transcriptional chemokines CCL and disease progression of LIHC using the ONCOMINE dataset, GEPIA, UALCAN, STRING, WebGestalt, GeneMANIA, TRRUST, DAVID 6.8, LinkedOmics, TIMER, GSCALite, and Open Targets. We validated the protein levels of chemokines CCL through western blot and immunohistochemistry.Results: The transcriptional levels of CCL5/8/11/13/15/18/20/21/25/26/27/28 in LIHC tissues were significantly elevated while CCL2/3/4/14/23/24 were significantly reduced. A significant correlation was found between the expression of CCL14/25 and the pathological stage of LIHC patients. LIHC patients with low transcriptional levels of CCL14/21 were associated with a significantly poor prognosis. The functions of differentially expressed chemokines CCL were primarily related to the chemokine signaling pathway, cytokine–cytokine receptor interactions, and TNF-α signaling pathway. Our data suggested that RELA/REL, NFKB1, STAT1/3/6, IRF3, SPI1, and JUN were key transcription factors for chemokines CCL. We found significant correlations among the expression of chemokines CCL and the infiltration of six types of immune cells  and immune checkpoints . The western blot and immunohistochemistry results showed that protein expression levels of CCL5 and CCL20 were upregulated in LIHC. CCL5 and CCL20 were significantly correlated with the clinical outcome of patients with LIHC, and could be negatively regulated by some drugs or small molecules.Conclusions: Our results may provide novel insights for the potential suitable targets of immunological therapy and prognostic biomarkers for LIHC. Primary liver cancer is one of the most common malignant tumors of the digestive system. According to the latest statistics from the WHO, there are 906,000 newly diagnosed cases and 830,000 deaths worldwide in 2020, making it the sixth most common tumor in the world and the third cancer-related death in humans . Liver hThe chemokine system contains genes encoding 50 chemokine ligands and 20 homologous chemokine receptors . ChemokiAlthough some studies have characterized the general expression profile and functional mechanism of some chemokine CCL in LIHC, determining the appropriate chemokine CCL as a therapeutic target and prognostic biomarker of LIHC is still an enormous issue that needs urgent attention. In this study, we will use the current rapidly developing sequencing technology and various databases, combined with bioinformatics analysis, to conduct an in-depth and comprehensive discussion on the expression of chemokines CCL in LIHC, and then evaluate them as potential therapeutic targets and prognostic markers. What we have done is to provide some additional recommendations for clinically related treatment strategies of LIHC, which are conducive to the prognosis and long-term survival of patients.www.oncomine.org), an online database that integrates RNA and DNA-seq from Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), and published literature sources, was used to evaluate the expression of chemokines CCL in LIHC  is currently a valuable and highly cited resource for gene expression analysis based on tumor and normal samples from TCGA and Genotype-Tissue Expression (GTEx) databases  is a comprehensive, user-friendly, and interactive web resource for analyzing cancer OMICS data. It is built on PERL-CGI with high quality graphics using javascript and CSS  is an online database for searching known protein interaction relationships, which aims to collect, score, and integrate all publicly available sources of protein–protein interaction (PPI) data, and to complement these with computational predictions of potential functions  is a functional enrichment analysis web tool, which has on average 26,000 unique users from 144 countries and territories per year according to Google Analytics  is a flexible, user-friendly web interface for generating hypotheses about gene function, analyzing gene lists and prioritizing genes for functional assays (GeneMANIA (l assays .https://david.ncifcrf.gov/home.jsp) is a comprehensive set of functional annotation tools for investigators to understand biological meaning behind large list of genes , cellular components (CC), and molecular function (MF) were included in the GO enrichment analysis.DAVID 6.8 (of genes . In the https://www.grnpedia.org/trrust/) a manually curated database of human and mouse transcriptional regulatory networks. The current version of TRRUST contains 8,444 transcription factor (TF)-target regulatory relationships of 800 human TFs. The TRRUST database can provide information of mode of regulation (TRRUST (gulation . The Fishttps://cistrome.shinyapps.io/timer/) is a comprehensive resource for systematical analysis of immune infiltrates across diverse cancer types  is a web-based analysis platform for gene set cancer analysis. Genomic aberrations influence clinical responses to treatment and are potential biomarkers for drug screening  supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin, in 5% CO®20 (TBST) for 3 times, and further incubated with secondary antibodies  for 1 h at RT, and developed using ECL solutions (Beyotime).LIHC tissues and adjacent normal tissues were obtained at Shanghai General Hospital from 2016 to 2018 and used in our study. Written informed consent was obtained from each patient. This study was approved by ethics committee of Shanghai General Hospital. Protein extracted from tissues or cell cultures were using RIPA buffer (Thermo Fisher Scientific) mixed with PMSF  for 30 min on ice, and then centrifuged at 12,000 rpm for 10 min at 4°C. Protein lysates were separated using SDS-PAGE and transferred to PVDF membranes. After incubating with 5% Bovine serum albumin (BSA) for 1 h at room temperature (RT), the membranes were incubated with primary antibodies   overnight at 4°C, washed with Tris Buffered Saline with Tweenn = 5; adjacent tissue, n = 5).All formalin-fixed paraffin-embedded tissues were prepared for use. After oven-drying, dewaxing, antigen retrieval, incubating with endogenous peroxidase, and blocking treatments, the slides were incubated with primary antibodies overnight at 4°C, followed by incubating with HRP-conjugated secondary antibodies at RT for 1 h. The stain was then visualized by incubation in DAB and counterstained with hematoxylin. Finally, the slides were covered with resinene and then the staining intensity was observed under the microscope  and CCL3(p = 0.0007) in LIHC were reduced with fold changes of −3.398 and −3.255, respectively (CCL5 and CCL21 were significantly upregulated in LIHC (p = 0.0012) and 2.395 (p<0.0001) in the datasets of Wurmbach , CCL14(p = 1.77E-14), and CCL23(p = 3.65E-09) were significantly downregulated in LIHC compared to normal liver tissue, while the transcriptional levels of CCL15(p = 6.00E-15), CCL18(p = 4.68E-02), and CCL20(p = 1.04E-07) were significantly upregulated (p = 6.04E-04) and CCL24(p = 2.41E-02) were reduced remarkably and CCL8(p = 5.52E-05), CCL11(p = 2.04E-09), CCL13(p = 2.36E-08), CCL25(p = 1.70E-03), CCL26(p = 1.89E-08), CCL27(p = 1.80E-02), and CCL28(p = 2.97E-06) were elevated significantly in LIHC tumors compared with normal liver samples. Combining the results of the two databases, we have excluded those chemokines CCL whose expressions were not significantly different, including CCL1, CCL7, CCL12, CCL16, CCL17, CCL19, and CCL22.We also used the UALCAN database to evaluate the expression levels of chemokines CCL in LIHC tissues and normal liver tissues. Consistent with the ONCOMINE data, the transcriptional levels of egulated . In addiTo identify chemokines CCL associated with tumorigenesis and progression in LIHC, we further explored the correlation between the expression of differentially expressed chemokines CCL and the pathological stage of LIHC patients. As expected, the expression of CCL14, CCL25, CCL26, and CCL28 were obviously related to the pathological stage , and witCCL14 (p = 0.021) and CCL21 (p = 0.044) in LIHC patients were significantly associated with better disease-free survival (DFS). More importantly, we also proved that LIHC patients with high CCL14 and CCL23 transcription levels have longer overall survival (OS).We then used the GEPIA database to evaluate the correlation between the expression of differentially expressed chemokines CCL and the clinical prognosis of LIHC patients. As shown in h CCL14 and CCLd CCL23 transcrTo better understand the potential interactions between differentially expressed chemokines CCL, we used the STRING database to construct a PPI network . The resCCL2, CCL3, CCL4, CCL5, CCL11, CCL13, CCL20, CCL21, and CCL26. This finding indicated that a total of 9 transcription factors  were involved in the transcriptional regulation of chemokines CCL . CCL14 expression was negatively associated with the infiltration of B cells, CD4+ T cells, macrophages, neutrophils and dendritic cells . In contrast, CCL20 expression was positively associated with the infiltration of B cells, CD4+ T cells, macrophages, neutrophils and dendritic cells . In addition, there was a positive correlation between CCL21 expression and the infiltration of B cells, CD8+ T cells, CD4+ T cells, macrophages, and dendritic cells . CCL25 expression was negatively associated with the infiltration of macrophages . CCL27 expression was positively associated with the infiltration of CD8+ T cells  and dendritic cells . Except for B cells, the expression of CCL28 was positively correlated with the infiltration of CD8+ T cells, CD4+ T cells, macrophages, neutrophils and dendritic cells . We also assessed the association of differentially expressed chemokines CCL and immune cells infiltration using multivariable Cox proportional hazard model. As shown in p = 0.001), macrophages (p = 0.005), dendritic cells (p = 0), CCL2 expression (p = 0.04), CCL5 expression (p = 0.01), and CCL20 expression (p = 0.017) were significantly correlated with the clinical outcome of patients with LIHC.In the tumor microenvironment, chemokines CCL are related to the inflammatory response and the regulation of immune cells, which affect the prognosis of patients with LIHC. Therefore, regarding the association between differential expression of chemokines CCL and immune cell infiltration, we performed a comprehensive exploration using the TIMER database. The expression of CCL2, CCL4, CCL5, CCL8, CCL11, CCL13, CCL18, CCL23, and CCL26 were positively associated with the infiltration of B cells, CD8ic cells , while tic cells . The ex, programmed cell death-Ligand 1 , and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). In addition, CCL20 was also positively associated with PD-1 and CTLA-4 .p value . In the results, the GO BP category indicated that the disease progression of LIHC was related to chemokine-mediated signaling pathway, chemotaxis, cellular response to interferon-gamma, cellular response to interferon-1, and immune response. The extracellular space, extracellular region, and cell were the most highly enriched items in the GO CC category. Furthermore, the chemokine activity and chemokine receptor binding were primarily enriched items in the GO MF category. In addition, in the enrichment analysis of the KEGG pathway, the differentially expressed chemokines CCL and their neighboring genes were mainly enriched in chemokine signaling pathway, cytokine-cytokine receptor interaction, nuclear factor kappa B (NF-κB) signaling pathway, toll-like receptor (TLR) signaling pathway, and tumor necrosis factor (TNF) signaling pathway. It was worth noting that CCL5, and CCL20 were key regulators of the TNF signaling pathway.To explore the specific mechanism of differentially expressed chemokines CCL and their neighboring genes in LIHC, we used DAVID 6.8 for functional enrichment analysis, which makes the most meaningful enrichment based on the significance of the p value . In the). Similarly, the expression level of CCL20 was also significantly increased in Huh7, HepG2, and Hep3B cells compared to L02 cells . Furthermore, the results of tissue samples have demonstrated that both CCL5 and CCL20 were markedly upregulated in HCC tumor tissues than in adjacent tissues . Coincidentally, we also showed that the expressions of CCL5 and CCL20 were appreciably increased in HCC tumor tissues compared to adjacent tissues by immunohistochemistry .To test the protein expression levels of CCL5 and CCL20, we performed western blot and immunohistochemistry. As expected, CCL5 was dramatically upregulated in HCC cell lines, including Huh7, HepG2, HCC-LM3, and Hep3B, compared to human liver normal cell line L02 . Simila02 cells . Furthe tissues . Coincihemistry .). Among the GDSC database, the results indicated that there were 3 and 1 drugs or small molecules that could target the expression of CCL5 and CCL20, respectively . Among them, CCL5 was negatively regulated by VNLG/124, KIN001-260, and ATRA, while CCL20 was negatively regulated by Trametinib. These analyses provided potential strategies for the clinical treatment of aberrant chemokines CCL expression in patients with LIHC.To further determine the disease caused by the aberrant expression of CCL5, and CCL20, we performed an analysis based on the Open Targets database. The findings showed that these chemokines CCL were significantly related to gastrointestinal disease, endocrine system disease, and the disease of cell proliferation disorder . Among ectively . Among LIHC is considered the prototype of “inflammation-associated cancer,” because it usually originates in chronically inflamed tissue . It is wCCL5/8/11/13/15/18/20/21/25/26/27/28; downregulation of CCL2/3/4/14/23/24), the transcriptional expression of CCL25, CCL26, and CCL28 were obviously elevated with the aggravation of tumor malignancy while CCL14 was reduced. Moreover, we have demonstrated that the high transcription levels of CCL14 and CCL21 in LIHC patients were significantly associated with better DFS, and high CCL14 and CCL23 transcription levels also brought longer OS. Therefore, all the findings proved that the differential expression of chemokines CCL was significantly associated with the progression and prognosis of LIHC.Since the pathological stage of LIHC is critical to the prognosis of patients, we first explored the relationship between aberrantly expressed chemokines CCL and pathological stage in LIHC. Among the 18 aberrantly expressed genes in LIHC tissue compared with normal liver tissue , which played an important role in tumorigenesis .+ tumor infiltrating T cells and macrophages, which may ultimately affect the clinical outcome of cancer patients . RELA post-translational modification, particularly phosphorylation, has been proven to be critical for abnormal NF-κB activation  were involved in the transcriptional regulation of chemokines CCL  are mainly mRNA levels. However, gene expression is regulated at multiple levels, including epigenetics, transcription, translation and post-translation. Especially in tumor-related research, we should also pay attention to the impact of gene protein expression levels on disease progression. Secondly, bioinformatics analysis is relatively mature today, which provides a broader perspective for the potential mechanisms and therapeutic targets of disease progression, whereas the experimental verifications on this basis are necessary, which is also the most critical limitation of our research. Finally, small sample size verification in this study is far from sufficient, and we look forward to large-sample clinical cohort studies and biological validation In summary, our research findings hope to provide potential prognostic biomarkers for LIHC patients, and provide new insights for their therapeutic targets and clinical medications, which may help guide treatment strategies.


PMP22, GJB1, MPZ, and MFN2 as the most frequently involved genes. Other genes, such as BSCL2, MORC2, HINT1, LITAF, GARS, and autosomal dominant GDAP1 are responsible for only a minority of CMT cases. (2) Methods: we present here our records of CMT patients harboring a mutation in one of these rare genes . We studied 17 patients from 8 unrelated families. All subjects underwent neurologic evaluation and genetic testing by next-generation sequencing on an Ion Torrent PGM (Thermo Fischer) with a 44-gene custom panel. (3) Results: the following variants were found: BSCL2 c.263A > G p.Asn88Ser (eight subjects), MORC2 c.1503A > T p.Gln501His (one subject), HINT1 c.110G > C p.Arg37Pro (one subject), LITAF c.404C > G p.Pro135Arg (two subjects), GARS c.1660G > A p.Asp554Asn (three subjects), GDAP1 c.374G > A p.Arg125Gln (two subjects). (4) Expanding the spectrum of CMT phenotypes is of high relevance, especially for less common variants that have a higher risk of remaining undiagnosed. The necessity of reaching a genetic definition for most patients is great, potentially making them eligible for future experimentations.(1) Background: Charcot–Marie–Tooth disease (CMT) is the most frequent form of inherited chronic motor and sensory polyneuropathy. Over 100 CMT causative genes have been identified. Previous reports found  PMP22, GJB1, MPZ, and MFN2 as the most frequently involved genes [GDAP1 and HSPB1) could frequently be involved, suggesting that genetic distribution could possibly be influenced by geographical features [BSCL2, MORC2, HINT1, LITAF, GARS1, and autosomal dominant GDAP1 are responsible of only a minority of CMT cases, with the consequence that the corresponding phenotypic presentations are less known and less defined.Charcot–Marie–Tooth disease (CMT) is the most frequent form of inherited chronic motor and sensory polyneuropathies and one of the most frequent genetic neuromuscular disorders, with a prevalence of 1:2500 ,4. The ced genes ,7,8,9. Ofeatures ,12,13,14features . Other gBSCL2, encoding for Seipin, an integral membrane protein, has been associated with an autosomal dominant transmitted form of distal hereditary motor neuropathy (dHMNtype V) and axonal CMT (CMT2D)   and withfrequent . Recentlfrequent . BSCL2 porldwide ,23,24,25orldwide . Clinicaorldwide ,21,22,25orldwide ,22  that is involved in manifold transcriptional and signaling pathways [HINT1  encodes pathways . Autosompathways ,31. Disepathways . In mostpathways   is involved in an endolysosomal pathway that is required to maintain the homeostasis of late endosomes and lysosomes . LITAF pas CMT1C   localized enzyme of the aminoacyl-tRNA synthetase (aaRS) family [) family ,39. Defe) family  and lead) family  (Table 1GDAP1) is an integral mitochondrial membrane protein. It is mainly expressed in neurons of both central and peripheral nervous systems, and has the role of maintaining the normal functioning, structure, and movement of the mitochondria [GDAP1 have been associated with demyelinating, intermediate, and axonal CMT [Ganglioside-induced differentiation-associated protein 1 .We present here our records of CMT patients harboring a mutation in one of these rare genes  with a 44-gene custom panel see , and we BSCL2  (patients 6.2 and 6.3) underwent genetic testing that revealed the same variant in both: their neurological examination was normal, but the younger one complained of mild difficulty in fine hands’ movements and sporadic intentional tremor.The proband (patient 6.1) is a 53-year-old male that reported no family history suggestive of neuromuscular disease. For a couple of years, he had been complaining of a no better defined “pain” in the lower limbs and of a reduction of hands’ strength . At neurological examination, he could not stay on heels and presented hypotrophy of tenar and leg muscles, with mild hyposthenia of antero-lateral leg muscles. Deep tendon reflexes were reduced/absent in the upper limbs and normal in the lower limbs. Sensory examination was normal. A neurophysiological evaluation showed an axonal polyneuropathy . GeneticGDAP1 , a 52-year-old male, referred of a walking impairment since the age of 43, that gradually worsened during the years. He did not report any family history. Clinical evaluation showed bilateral steppage, severe lower limbs’ muscles hypotrophy, severe hyposthenia of the leg muscles (MRC 2), and mild hyposthenia of the calf muscles (MRC 4). He presented also hyposthenia of the proximal lower limbs’ muscles and was able to squat only with support. Deep tendon reflexes were very brisk, except for the Achille’s tendon reflexes (absent).The second patient (patient 8.1), a 50-year-old male, presented, since the age of 3, with a reduction of visual acuity. At the age of 20, he was diagnosed with optic nerve subatrophy and neurosensorial hypoacusia. At the age of 24, he developed a sensory–motor polyneuropathy. A neurological evaluation showed left steppage, reduction of lower limbs muscles’ tonus and trophism, mild hyposthenia of the anterolateral leg muscles (worse on the left side), and brisk reflexes in the upper limbs, with bilateral Hoffman’s sign and hypopallestesia from the anterior superior iliac spine.Nerve conduction studies revealed an axonal polyneuropathy in both patients .BSCL2, MORC2, HINT1, GARS1, and autosomal dominant GDAP1) are responsible for a CMT2 phenotype, types of CMT for which genetic definition is less frequently reached. Some peculiar features that could be associated with these variants could help clinicians direct the genetic testing. For example, in our BSCL2 patients, we found the already reported pyramidal signs and neurosensorial hypoacusia. In addition, we reported pigmentosus retinitis with bilateral cataract in patient 1.3 and proximal UL weakness in pt. 2.1. Pigmentosus retinitis has been associated with PHARC syndrome [ATP6 [MTMR2 gene variants [MORC2 VUS patient had no particular abnormal characteristics, with the exception of an increase in CSF protein level that initially led to a CIDP misdiagnosis. As this gene is considered to play a role in the development of the nervous system [LITAF and GARS1 patients, we confirmed the presence of foot deformities in the first and a prominent distal upper limb involvement in the second. Peculiar was the phenotype of the two patients with the GDAP1 variant, who presented with proximal/asymmetric lower limb weakness, clinical signs of first motoneuron involvement, and optic and acoustic nerve disorders.CMT genetic confirmation has always represented a challenge. Classically, clinicians could use some algorithms, based on NCV and type of inheritance, to guide the genetic testing . In thissyndrome  and withme [ATP6  and MTMRvariants . Recentlvariants . The hypvariants . The MORs system , it coulPMP22 duplication, CMT1A) [Expanding the spectrum of CMT phenotypes is of high relevance, especially as it allows identifying less common mutations that have a higher risk of remaining undiagnosed. A better knowledge of the clinical manifestation of these rare mutations could be crucial to correctly address genetic testing, in particular considering that NGS and WES are still not widespread, especially in some health systems. Moreover, now that a phase III clinical trial (PLEO-CMT) is available for CMT patients with a specific mutation  , the nec


N-Propargylaminesare useful synthetic scaffoldsfor the synthesis of bioactive molecules, and in addition, they possessimportant pharmacological activities. We obtained several neuroprotectivemolecules, chiral 1,2-amino alcohols and 1,2-diamines, able to reduceby almost 70% the rotenone and oligomycin A-induced damage in SH-SY5Ycells. Furthermore, some molecules assessed also counteracted thetoxicity evoked by the Ser/Thr phosphatase inhibitor okadaic acid.Before extrapolating these data to preclinical studies, we analyzethe molecules through an in silico prediction systemto detect carcinogenicity risk or other toxic effects. In light ofthese promising results, these molecules may be considered as a leadfamily of neuroprotective and relatively safe compounds. N-Propargylaminesand N-propargylamidesare synthetic scaffolds widely used by organic chemistry for the preparationof complex bioactive compounds,1 such as1,2-amino alcohols,2 β-amino acids,3 or polyhydroxylated heterocycles,4 among others. In this context, we notice that some contributionsin the literature report that the N-propargylaminemoiety possesses some biochemical activities involved in controllingthe cellular redox state, mainly by inhibiting nitric oxide synthaseenzymes.5 Reportedly, these molecules weredemonstrated to be involved in protein kinase C (PKC) and MAPK activation,7 inhibition of monoamine oxidases (MAO)8 or cysteine proteases,9 and inductionof neurotrophic factors.10 Thus, N-propargylamine substructures appear in many drugs withneuroprotective properties used for central nervous system diseases.Some examples are the marketed drugs rasagiline7 or selegiline11 and the drugcandidate for Parkinson’s disease treatment, ladostigil13 . There was atleast one structural alert for the micronucleus assay found, classifyingcompounds as Class I substances. In the carcinogenicity and mutagenicitydiscriminant analysis, there was nongenotoxic carcinogenicity, whetherit fired a structural alert for genotoxic carcinogenicity (Alkyl carbamateand thiocarbamate structure).We use in silicopredictions to assess the toxicity and cytochromeP450 isoform 3A4 metabolism of the compounds with Toxtree softwarev 3.1.0.Finally,each of the six compounds results in a class 2 persistentchemical due to its more than two rings. However, further experimentsneed to be developed to test whether these alerts certainly happen.in vitro models, SH-SY5Y cells, which are used to studyneuronal function and neurodegenerative diseases.Then, to test whether these compounds are not toxic, we use reliable Schemes S1 and S2, Supporting Information).23 Trimethylsilane(TMS)-protected N-propargylamides 2a–e and 5 were treated with tetrabutylammoniumfluoride, which removes the TMS group. Thereby, it furnished compounds 3a–e and 6 in good yields(Supporting Information), resulting inspectroscopic and analytical data according to their structure .The preparationof the compounds was accomplished according towhat was previously described , which inhibit complexesI and V of the mitochondrial electron transport chain, respectively,in SH-SY5Y cells, conditions that result in the generation of reactiveoxygen species (ROS) and impair the ATP synthesis. Thus, cells arein an oxidative stress environment, typically found in several neurodegenerativediseases. As shown in 24 was significantly reduced (37%), and the presence of compounds,tested at 0.3 μM, decreased in most cases such loss of cellviability in a significant manner. The best compound was 3d, which maintained the cell viability up to 76% with respect to abasal situation, similar to the well-known antioxidant drug melatoninused as the standard.25Obtained results reveal that only molecule 26 Theirinhibition results in the hyperphosphorylation of selected biologicaltargets, including tau protein, which in turn leads to its self-aggregationin the so-called neurofibrillary tangles, one of the principal hallmarksof AD. The administration of OA to neuronal cultures is a well-describedAD in vitro model, in which tauopathy is the sourceof neuronal damage. In this scenario, cells reduced their viabilityafter the incubation with OA to 38%; the loss of neuron viabilitywas counteracted by the administration of compounds 3a, 3b, 3d, 3e, or 4e at 0.3 μM, analogously to the protection provided by the anti-ADdrug memantine.27In the second test, we exposedSH-SY5Y cells to 15 nM okadaic acid(OA); this marine biotoxin is a selective inhibitor of phosphoproteinphosphatases, mainly PP1 and PP2A.N-propargylamides have shownpotential neuroprotective properties against two toxic stimuli relatedto neurodegeneration at sub-micromolar concentrations. These resultsprompt us to continue the study of chiral propargylamides as new chemicalentities with promising biological activities for the treatment ofneurodegenerative diseases.In summary, five


Down syndrome (DS) is one of the most frequent genomic disorders around the globe (∼1:700 births). During the COVID-19 pandemic, it has been recognized that children with DS are patients with a greater risk of presenting SARS-CoV-2 infection-related poor outcomes. Nonetheless, a few cases with DS and SARS-CoV-2 infection have been reported. Our aim is to describe the unfavorable clinical course of a child with DS infected with SARS-CoV-2 virus.  Female, 2 years old, karyotype 47,XX,+21[30], previously diagnosed with a cyanotic congenital heart disease  who started with diarrhea, developed shortness of breath, and cyanosis and was admitted to the hospital presenting low-oxygen saturation (33%) requiring invasive mechanical ventilation support. The patient tested positive for SARS-CoV-2 infection. During hospitalization, the patient presented hypotension, anuria, retarded capillary filling, and metabolic acidosis; management with vasoactive drugs was needed. Nonetheless, the patient developed respiratory and cardiac failure, acute renal injury (AKIN-III), and septic shock. After 24 days of hospitalization, the patient died. IFNAR1, IFNAR2, IFNGR2, and IL10RB) located at 21q22.11. Additionally, overexpression of TMPRSS2 at the pulmonary level, located also at 21q22.3, could be related with an increased susceptibility for the development of SARS-CoV-2 infection in DS patients. Multiple organ failure observed in the patient presented could be related to the triple gene dose of four interferon receptors ( In March 2020, the World Health Organization declared the novel coronavirus (COVID-19) outbreak a global pandemic. Since then, some cases have shown an unfavourable clinical course of disease.Down syndrome (DS) is the most frequent genomic disorder worldwide (∼1:700 births), with a high susceptibility to severe acute respiratory infections .Homo sapiens chromosome 21) [Down syndrome is caused by complete or partial trisomy of chromosome 21  . Its clisome 21) , 5. Nonesome 21) , 7.Our objective was to describe the clinical course of a child with DS and congenital heart disease who developed multiple organ failure associated with SARS-CoV-2 infection.A 2-year-old female with Down syndrome phenotype  was admitted to the Pediatric Hospital, National Medical Center “Siglo XXI” in Mexico city. During the neonatal period, she had been diagnosed with tricuspid atresia and infundibular pulmonary stenosis (type Ib).Six days before hospital admission, the patient had started experiencing abdominal pain and watery diarrhoea and was treated with oral electrolytes. Three days later, she presented with shortness of breath, cyanosis, and fever. The symptoms worsened in the following days.On admission, the patient had very low-oxygen saturation (33%). Mechanical ventilatory support was initiated. Concomitantly, a SARS-CoV-2 infection test (qRT-PCR) was performed, and vitamin D and corticosteroids were administered.3/µl; leukocytes, 9.0 × 103/µl; neutrophils, 2.36 × 103/µl; sodium, 143 mEq/l; potassium, 5.20 mEq/L; calcium, 9.48 mg/dL; phosphorus, 5.68 mg/dL; and magnesium, 2.26 mg/dL. Metabolic acidosis with severe hypoxaemia was noted in the arterial blood gas test . Serum lactate level was elevated (10 mmol/L). No relevant data were observed on chest radiographs. Considering these findings, vasopressors and platelet transfusion therapy were initiated. Positive results for SARS-CoV-2 infection were reported.The patient was admitted to the paediatric intensive care unit, with hypotension, tachycardia, oliguria, cyanosis, cold extremities, weak peripheral pulses, and a delay in the capillary refill time (>2 s). Laboratory test results were as follows: haemoglobin, 22.8 g/dl; hematocrit, 74.5%; platelets, 31 × 10On the third day of hospitalisation, a right pulmonary systemic fistula was created for palliative treatment because of recurrent hypoxic crises and as refractory hypoxemia had been observed. Cephalothin and amikacin were used as prophylaxis for the procedure. An echocardiogram performed after the operation showed mild left ventricular systolic dysfunction . No other alterations were reported.Three days after surgery, hypotension accompanied by an increase in C-reactive protein and erythrocyte sedimentation rate was observed. The patient manifested signs of septic shock accompanied by acute kidney injury (AKIN-III). Vasopressor treatment was adjusted. On chest X-rays, the presence of diffuse alveolar infiltrates in both lungs was seen.Pseudomonas aeruginosa was isolated from the central venous catheter, and specific antibiotic treatment was implemented. On day 17 of hospitalisation, the patient required an increase in ventilatory and vasopressor support. Repeat test for SARS-CoV-2 was positive. Regardless of these and other manoeuvres, the patient did not respond and suffered progressive oxygen desaturation, metabolic and respiratory acidosis, refractory septic shock, and multiple organ failure . The patient died 24 days after hospitalization.To our knowledge, there are few clinical reports detailing the evolution of SARS-CoV-2 infection in children with DS. The patient presented here had an unfavourable clinical course after infection with SARS-CoV-2. Other authors have also reported adverse outcomes in children with DS after SARS-CoV-2 infection. For instance, Kantar et al.  reportedIn a recent report by Newmann et al. , the cliOn the other hand, Krishnan et al.  reportedIn general, two mechanisms have been proposed to explain the global transcriptional imbalance seen in DS patients: (i) the effect of gene dose and (ii) the modification of gene expression following an epigenetic model, for example, micro-RNAs and changes in chromatin conformation. In both scenarios, there would be a direct and indirect effect on the cellular, tissue, organic, and systemic homeostasis of affected individuals –4. NonetIFNAR1, IFNAR2, IFNGR2, and IL10RB, located at 21q22.11. The overexpression of IFN receptor transcripts leads to hypersensitivity of this pathway in pulmonary tissue after SARS-CoV-2 infection, with the subsequent stimulation of immune cells to increase cytokine production in patients with DS [In a recent analysis by Espinosa et al. , a theor with DS .TMPRSS2  at the pulmonary level could be related to a possible increase in the susceptibility for severe COVID-19 disease in patients with DS. This takes into consideration that this gene encodes type II transmembrane serine protease, which is important in the labelling of the viral protein “Spike (S)” and for entry into the host's target cell through interaction with angiotensin converting enzyme 2 (ACE2) [TPMRSS2. Moreover, these authors also described a downregulation of the NLRP3 gene, which is a key component for fighting other infections, including those caused by bacterial microorganisms [Pseudomonas aeruginosa.In our clinical report, complementing the perspective described by , we must2 (ACE2) . In this2 (ACE2) , throughrganisms . InteresWe know that there may be no single explanation for how the interaction between SARS-CoV-2 and trisomy host cells would affect the clinical course of the patient presented here; however, we must consider the participation of different regulatory sequences of the immune response, such as miRNAs, specifically miR-155 and miR-125b, both located at 21q21, since they are overexpressed in memory B-cells of tonsillar tissue in patients with DS . This leACE2/TMPRSS2 in the development of acute respiratory distress syndrome associated with COVID-19, we must remember the previously reported downregulation of ACE2 in the respiratory epithelium of affected patients during the SARS-CoV pandemic, where it was associated with progressive damage to lung and heart tissues [ACE2 by acting as a negative regulator [ACE2 and be associated with the respiratory failure seen in our patient? [Returning to the context of the interaction between miRNAs and  tissues . In expeegulator . This lepatient? .http://explorer.trisome.org/transcriptome/), we realised that the KDM5B transcript, which encodes for a specific histone demethylation (H3K4) called JARID1B, is differentially expressed in DS subjects compared to healthy individuals [Finally, using the same research web portal used in a study by , TrisomEividuals . Recentlividuals . A limitThe different hypotheses, revised in the context of the present clinical report, aim to address the question of the increased susceptibility to SARS-CoV-2 infection in patients with DS, and whether once the disease is established, the poor and uncontrolled immune response may result in an unfavourable clinical course affecting multiple organs and systems, ultimately leading to the death of patients. Furthermore, supporting our hypotheses, in a study by Huls et al. , it is m