Salvia miltiorrhiza Bunge, known as Danshen in traditional Chinese medicine, is a highly valued medicinal plant used to treat cardiovascular and cerebrovascular diseases. The dry roots and rhizomes of S. miltiorrhiza contain biologically active secondary metabolites of tanshinone and salvianolic acid. Given an assembled and annotated genome and a set of transcriptome data of S. miltiorrhiza, we analysed and identified the candidate genes that likely involved in the bioactive metabolite transportation of this medicinal plant, starting with the members of the ABC transporter family.ATP-binding cassette (ABC) transporters have been found to play important roles in metabolic transport in plant cells, influencing subcellular compartmentalisation and tissue distribution of these metabolic compounds. S. miltiorrhiza. All of these ABC genes were divided into eight subfamilies: 3ABCA, 31ABCB, 14ABCC, 2ABCD, 1ABCE, 7ABCF, 46ABCG, and 10 ABCI. Gene expression analysis revealed tissue-specific expression profiles of these ABC transporters. In particular, we found 18 highly expressed transporters in the roots of S. miltiorrhiza, which might be involved in transporting the bioactive compounds of this medicinal plant. We further investigated the co-expression profiling of these 18 genes with key enzyme genes involved in tanshinone and salvianolic acid biosynthetic pathways using quantitative reverse transcription polymerase chain reaction (RT-qPCR). From this RT-qPCR validation, we found that three ABC genes  and another gene (SmABCC1) co-expressed with the key biosynthetic enzymes of these two compounds, respectively, and thus might be involved in tanshinone and salvianolic acid transport in root cells. In addition, we predicted the biological functions of S. miltiorrhiza ABC transporters using phylogenetic relationships and analysis of the transcriptome to find biological functions.A total of 114 genes encoding ABC transporters were identified in the genome of S. miltiorrhiza and predict candidate transporters involved in bioactive compound transportation in this important medicinal plant. Using genome-wide identification, transcriptome profile analysis, and phylogenetic relationships, this research provides a new perspective on the critical functions of ABC transporters in S. miltiorrhiza.Here, we present the first systematic analysis of ABC transporters in The online version contains supplementary material available at 10.1186/s12864-021-07623-0. Salvia miltiorrhiza is a common medicinal plant used to treat inflammation and cardiovascular diseases because of its high quantities of biologically active hydrophilic salvianolic acid (SA) and lipophilic diterpenoids (tanshinones) in its roots or rhizomes [S. miltiorrhiza is an ideal model medicinal plant for studying secondary metabolic biosynthesis. GGPP is the biosynthetic precursor of tanshinone, which is catalysed by copalyl diphosphate synthase (CPS) to form copalyl diphosphate. Then a series of cytochrome P450 monooxygenases (CYP450s) catalyses downstream oxidation reactions. Ferruginol, the catalytic product of CYP76AH1, is an important intermediate product in the biosynthetic pathway of tanshinone [S. miltiorrhiza, but the transport and storage mechanisms of these compounds in plant cells have not yet been elucidated.rhizomes . S. miltnshinone . CYP76AHnshinone . SA biosX4GK(ST)), Walker B ((RK)X3GX3L(hydrophobic)3), ABC signature, Q-loop, D-loop and H-loop [ATP-binding cassette (ABC) transporters, one of the few gene families present in all domains of life, are involved in a wide range of biological processes and play key roles in the transmembrane transport of metabolites across biological membranes by hydrolysing ATP in plant cells . In mostd H-loop –8. In ged H-loop .Arabidopsis and rice) show that the plant genome contains a large number of ABC transporters compared to animals and other eukaryotes [Genome analyses of model plants . These 204 ABC transporters in S. miltiorrhiza (SmABCs) were verified by manually confirming the integrity of the conserved domains and motifs of ABC proteins. Ultimately, 114 genes encoding for ABC transporters were identified in the S. miltiorrhiza genome  which characterizes the diversity of ABC proteins  [The plant ABCA subfamily includes one full-sized and several half-sizedABC proteins. In D2-NBD2) , 12. TheD2-NBD2) , 27.SmABCA1–3) were annotated to be ABCAs in the S. miltiorrhiza genome  [Arabidopsis [The transporter genes of the ABCC subfamily were expressed in all organs and tissues of za Table . SmABCC2ana Fig. a. SmABCCCC4 Fig. a. CsABCCsaffron)  and AtABbidopsis , respectSmABCC4 in the leaves was higher under MeJA induction  regulation other than transport [Arabidopsis [The ABCE subfamily, conserved in eukaryotes and archaea, consists of a soluble protein with only two conserved NBDs and without any detectable TMD. In ransport , 58. AtAS. miltiorrhiza genome, and it was constitutively expressed in all plant organs  [The ABCG subfamily is the largest ABC protein subfamily in plants, including both full-sized and half-sized transporters. The NBD-TMD domains of this subfamily are arranged in opposite directions. Most of the characterised ABCGs are located in the plasma membrane , 61. SpTtABCG11) , plant htABCG11) , pathogetABCG11)  and kanatABCG11) . SeveraltABCG11) , lignin tABCG11) , and exitABCG11) .S. miltiorrhiza, comprised of 46 members  had tissue-specific expression profiles in this subfamily, all of which were highly expressed in the roots of S. miltiorrhiza  were highly expressed in all organs. By contrast, 11 genes showed low expression levels in all organs, including SmABCA3, SmABCB1, SmABCB3, SmABCC3, SmABCC6, SmABCC14, SmABCF2, SmABCG13, SmABCG33 and SmABCI2. Furthermore, a total of 46 genes were rarely expressed in all organs, including SmABCB6, SmABCB8, SmABCB12, SmABCB15-SmABCB17, SmABCB20-SmABCB23, SmABCB25-SmABCB27, SmABCB29, SmABCB31, SmABCC7, SmABCC9, SmABCC12, SmABCD2, SmABCF1, SmABCG2, SmABCG3, SmABCG5-SmABCG7, SmABCG9, SmABCG10, SmABCG14, SmABCG16-SmABCG18, SmABCG20-SmABCG23, SmABCG25, SmABCG26, SmABCG29-SmABCG31, SmABCG34, SmABCG36, SmABCG38, SmABCG39, SmABCG41 and SmABCI7. The expression of some genes showed tissue- or organ-dependent specificity. For example, 14 genes were highly expressed in the roots and root tissues, including SmABCA1, SmABCB2, SmABCB5, SmABCB9, SmABCB30, SmABCC2, SmABCC4, SmABCC11, SmABCC13, SmABCG1, SmABCG4, SmABCG11, SmABCG40 and SmABCI5. Although nine genes were expressed in the flowers, stems and leaves, they were not expressed in the roots and root tissues, such as SmABCB14, SmABCB24, SmABCC10, SmABCG12, SmABCG15, SmABCG19, SmABCG32, SmABCG45 and SmABCI6. The 13 genes were expressed more highly in the flowers, stems, leaves and roots but not in the three tissues of the root, including SmABCA2, SmABCB11, SmABCB19, SmABCC8, SmABCF7, SmABCG24, SmABCG27, SmABCG28, SmABCG37, SmABCG43, SmABCI1, SmABCI3 and SmABCI8. Moreover, 6 genes were highly expressed in the root rather than in other tissues, including SmABCB10, SmABCB13, SmABCB18, SmABCB28, SmABCG8 and SmABCG45. The different expression profiles of these ABC genes suggested that they might perform different gene functions in S. miltiorrhiza.The gene expression profiles of the 114 putative ABC transporters were detected from the transcriptome data generated from the different organs  and tissues  of  studies , 24 , 5 ABCBs , 4 ABCCs  and 8 ABCGs . Amongst these candidate ABC genes, we found that the expression patterns of SmABCG46, SmABCG40 and SmABCG4 were nearly identical to that of CYP76AH1 and SmCPS1, which are key enzyme genes involved in the biosynthetic pathway of tanshinone  and methyl jasmonate (MeJA) Fig. . Under teJA Fig. a. In ABAves Fig. b. For thves Fig. b, while ent Fig. a. Under oot Fig. a. In conves Fig. b. The exn YFP-PM , the subcis-elements in SmABC promoters, the 1500 bp DNA sequences upstream of the start codon (ATG) for each of the 114 SmABCs were screened using the Plant Cis-acting Regulatory DNA Elements (PLACE) (https://www.dna.affrc.go.jp/PLACE/). PLACE identified a total of 267 different cis-elements in the promoter of SmABC genes, and there were nine common cis-regulatory elements in all promoter regions of SmABC genes , ABCB (31 genes) and ABCC (14 genes) subfamily have the most members, whiles the ABCA, ABCD and ABCE subfamily have fewer members , ABCB 3 genes anand ABCC  genes su comosus , L. japoaponicus , and O. . sativa   have been reported to be involved in polar auxin transport as their mutants show reduced auxin transport [S. miltiorrhiza  may be homologous to those in Arabidopsis ABCB and thus might be involved in plant hormone transport  and one ABCC member (SmABCC1) members might be involved in transport of tanshinone and SA in S. miltiorrhiza, respectively  and one ABCC member (SmABCC1) to be the lead candidates involved in tanshinone and SA transport, respectively. The transporters identified in the ABCG and ABCC subfamilies might be involved in the transport of secondary metabolites of S. miltiorrhiza. In addition, the transporters might be involved in the transport of anthocyanins, auxin and metal resistance have been identified in several ABC subfamilies of S. miltiorrhiza. Our study outlined the ABC proteins in the S. miltiorrhiza genome and explained their possible transporting pathways for some compounds, laying an important foundation for further research on the metabolic regulation, synthetic biology and utilisation of these compounds in S. miltiorrhiza. Our analysis provides new insight into the diversity and the predicted function of the entire ABC transporters in S. miltiorrhiza compared with Arabidopsis. These results will provide new insights into the function of ABC transporters in S. miltiorrhiza.In this study, we identified and analysed ABC transporters in S. miltiorrhiza Bunge (line 99–3) was collected from the garden at the Institute of Medicinal Plant Development (IMPLAD) in Beijing. The plants were authenticated by Professor Yulin Lin of the IMPLAD using the morphological identification approach of the Flora of China. The 1-year-old S. miltiorrhiza seedlings were cultured in Hoagland basal salt medium   for 7 days, and then transferred to Hoagland medium containing ABA (10 mM) or MeJA (200 μM) for induction induction of 0 h (CK), 3 h and 12 h, respectively. N. benthamiana was grown in pots at 23 ± 2 °C under 16 h light/8 h dark photoperiod.S. miltiorrhiza genome with the ABC proteins in the Arabidopsis TAIR11 database  and identify ABC homologues in S. miltiorrhiza. HMMER (https://www.ebi.ac.uk/Tools/hmmer/) was used to characterise the topology of ABC proteins containing TMD and NBD domains. The TMD domain in these ABC proteins was identified by PF12698, PF06472 and PF00664, and the NBD domain was identified by PF00005 in Pfam database (https://pfam.xfam.org/). A protein with at least one NBD domain was predicted to encode an ABC transporter. The domain analysis also provided evidence for the subfamily classification of the ABC family in S. miltiorrhiza. The obtained S. miltiorrhiza ABC protein sequences were submitted to the MEME Web server (http://meme-suite.org/) to confirm the relationship between the conserved motifs and genes.BLAST was used to align all the proteins in the http://www.clustal.org/) was used to perform sequence alignment on the deduced amino acid sequence of S. miltiorrhiza ABC proteins, and then, MEGA 6 was used to construct a phylogenetic tree with 1000 bootstrap repeats through the neighbor-joining method. The maximum likelihood method was used to establish phylogenetic trees of S. miltiorrhiza ABC transporter subfamilies with the ABC transporters that have been functionally identified in Arabidopsis and other plants .Clustal X (S. miltiorrhiza (line 99–3) plants were grown in the medicinal plant garden of the Institute of Medicinal Plant Development. The transcriptome of different organs , root tissues  and leaves (with and without MeJA treatment) was sequenced by Illumina Hiseq 2000 technology in our previous studies [S. miltiorrhiza ABC genes were analysed with these transcriptome data. The candidate genes that were highly expressed in the roots of S. miltiorrhiza were selected for quantitative reverse transcription polymerase chain reaction (qRT-PCR) verification. studies , 24. TheS. miltiorrhiza and the expression patterns of these genes in the seedlings treated with ABA or MeJA were analysed by qRT-PCR. Total RNA was extracted from the flowers, stems, leave, roots and the root tissues including of periderm, phloem and xylem, all of which were isolated from the 2-year-old S. miltiorrhiza, and the roots/leave of 1-year-old seedlings treated with ABA (10 mM) or MeJA (200 μM) for 0 h (CK), 3 h and 12 h, according to the manufacturer’s instructions of the RNAprep Pure Plant Kit , and then reversed into cDNA using the Promega GoScript Reverse Transcription System . qRT-PCR was performed on an ABI PRISM 7500 real-time PCR system (Applied Biosys) using SYBR Premix Ex Taq™  with the following program: 95 °C for 30 s, 1 cycle; 95 °C for 5 s and 60 °C for 34 s, 40 cycles. The relative gene expression level was calculated using the 2−ΔΔCT method [SmActin gene (Genbank number HM231319.1) as an internal reference. The experiments were performed in three independent biological experiments with three technical replicates. qRT-PCR was performed to determine the relative expression levels of the 18 candidate ABC genes along with CYP98A14, SmRAS1, CYP76AH1 and SmCPS1, which were used as positive genes to calculate the correlation coefficient of co-expression for these ABC genes with the key enzyme-encoding genes involved in the biosynthesis of SAs and tanshinones. Tbtools [The relative expression levels of 18 selected SmABC genes in different organs/tissues of T method  with theSmABCG46 was cloned from S. miltiorrhiza using total RNA isolated from seedlings as a template for amplification. In order to perform subcellular localization analysis, the ORF of SmABCG46 was introduced into the pCAMBIA1300-Super-GFP vector using the Seamless Cloning and Assembly Kit  according to the manufacturer’s instructions. The full-length coding region of SmABCG46 (without stop codon) was fused with green fluorescent protein (GFP) in pCAMBIA1302 vector, and identified by sequencing. The expression vector was transiently introduced into Agrobacterium strain GV3101, and infiltrated into the leaves of N. benthamiana. After 48 or 72 h of infiltration, the GFP fluorescence of the gene was observed using a confocal laser scanning microscope . The acquisition software is LAS AF Lite 3.0. The pCAMBIA1300-Super plasmid was transformed into tobacco leaves as a positive control. The location of plasma membrane was determined by the fluorescence of YFP-PM [The candidate gene of f YFP-PM .S. miltiorrhiza [cis-elements of promoters for each gene were identified by PLACE Web Signal Scan-PLACE (https://www.dna.affrc.go.jp/PLACE/).All the promoter sequences (1500 bp upstream of start codon “ATG”) of the SmABC transporters were extracted from the draft genome of iorrhiza  accordinAdditional file 1: Figure S1. Conserved motifs of SmABC proteins. The motif in the SmABC proteins was identified by using Multiple Em for Motif Elicitation (MEME). Ten conserved motifs were identified and displayed in different colorsAdditional file 2: Table S1. The ABC transporters that have been functionally identified from other plants used for phylogenetic tree analysis in this study (sheet 1) and the accession numbers of SmABCs deposited in the GenBank (sheet 2)Additional file 3: Figure S2. qRT-PCR validation of the 18 selected candidate transporter genes involved in bioactive compound transportation in S. miltiorrhiza. SmActin was used as an internal control. Each gene has three biological replicates and three technical replicatesAdditional file 4: Table S2. The cis-elements identified in the promoter sequences of all the SmABC genesAdditional file 5: Table S3. Comparative analysis of ABC proteins between S. miltiorrhiza and other plant speciesAdditional file 6: Table S4. Primers used in this study


G-Quadruplex (G4) DNA (G4 DNA) and RNA (G4 RNA) are secondary nucleic acid structures that have multiple roles in vital cellular processes. G4 DNA- and RNA-binding proteins and unwinding helicases associate with and regulate G4s during virtually all processes that involve DNA and RNA. DEAH-Box helicase 36 (DHX36), a member of the large DExD/H box helicase family, enzymatically unwinds both G4 DNA and G4 RNA. By exerting its G4 helicase function, DHX36 regulates transcription, genomic stability, telomere maintenance, translation and RNA metabolism. This review will provide an overview of G4s and DHX36, including DHX36’s potential role in neuronal development and neurodegeneration. We conclude with a discussion of the possible functions of G4s and DHX36 in the aging brain. Guanine-rich nucleic acid structures called G-quadruplexes G4) found in DNA and RNA motifs have become a focal point for the investigation of how secondary structures in nucleic acids affect cellular processes , 2. As n found inDHX36 , a highly conserved member of the DExD/H box helicase family, binds with and unfolds G4s, thereby changing how G4 structures influence DNA- and RNA-dependent processes. DHX36, a major G4 helicase, unwinds both G4 DNA and G4 RNA, and understanding the impact on both structures makes it an interesting research target, considering the role of G4s in a variety of processes . Recent >X>G>X>G>X>G>X, wherein each guanine (>G) is interspaced by a linking nucleotide sequence (>X) . DH. DH19]. Due to the ubiquity of G4 structures in promoters, promoter-enhancement regions, and oncogenes, much of helicase study focuses on how these enzymes unwind DNA and RNA and their impact on cancer tumorigenesis . HelicasDHX36 is a proto-oncogene. The long non-coding RNA, FLJ39051, that contains G4 RNA, binds to and inhibits DHX36, leading to enhanced colon cancer cell migration . Since sNevertheless, DHX36 is implicated as a regulating factor in breast cancer tumor suppression, and is positively associated with increased patient survival. Using a xenograft tumor model in mice, researchers showed that lentiviral knockdown of DHX36 increases tumor growth and cancer cell invasion, with a concurrent reduction of cellular apoptosis . TherefoIn vivo and in vitro, DHX36 binds to the telomerase RNA component, and this binding depends on the presence of the G4 RNA structure in the 5′-region of the telomerase RNA [Telomeres have a central role in aging by protecting the ends of chromosomes from damage during cellular mitosis . In vivorase RNA . DHX36 rrase RNA . Telomerrase RNA . In agrerase RNA . Intrigurase RNA . It wouldhx36 tamoxifen-induced knock-out cellular system, DHX36-deficient cells displayed deficits in IFN production and had higher vulnerability to RNA virus infections [Helicase enzymes, including DHX36, have an active role in innate cellular immune response, acting as cytoplasmic sensors of viral nucleic acids . Among ofections . Thus, DViruses, such as coronaviruses, hijack the host machinery for their replication, and helicases are no exception. Coronaviruses encode their own RNA helicases, but they still hijack the host helicases to regulate virus proliferation. For example, the host helicases, including DHX36, may in fact positively modulate SARS-CoV-2 replication .Many G4 DNA helicases are linked to human diseases with some degree of brain pathology, often severe. For example, an expansion of a non-coding GGGGCC repeat in the gene C9ORF72 is associated with 10% of familial cases of frontotemporal dementia and amyotrophic lateral sclerosis. The repeat forms G4 DNA, G4 RNA, and G4 DNA-RNA hybrids, leading to an accumulation of ribonucleoprotein complexes and increased intranuclear stress . G4s areDHX36 knock-out causes lethality in embryos that show signs of cellular degeneration at the early embryonic stage E7.5 . Is therAs described above, many G4 DNA helicases are linked to human diseases, which are characterized by accelerated aging. Cellular senescent phenotypes arise from genomic instability, telomere dysfunction, inflammation, dysregulated transcription, translation, and mRNA metabolism that are the result of the presence of defective or absent helicases. For example, in knockdown assays, the closely related helicase DHX9 that unwinds G4 DNA, G4 RNA, and R-loops causes premature senescence in human fibroblasts. These cells have significant morphological differences, changing cell shape from a small and spindle shape to an enlarged and irregularly shaped appearance. Positive staining of the widely used senescent marker SA β-gal was also observed when compared to controls .As humans age, declines occur in the systems for controlling protein production quality and mitochondrial function and dynamic processes regulating cellular homeostasis. Stress granule formation, maintenance and clearance processes may become compromised and lead to promotion of aging-related disease and dysfunction. For example, G4s have long been recognized as factors in stress granule assembly . IntriguAtg7expression in neurons and astrocytes, suggesting a mechanism by which G4 DNA downregulates autophagy. Another example is G4-associated transcription of the Brca1 gene [Brca1 downregulation and DNA damage [Brca1 in astrocytes and microglia, indicating differences in DNA damage and repair pathways between brain cell types [As a multifunctional helicase, DHX36 may participate in controlling aging and cellular senescence processes in neurons. Previously, we showed that G4 DNA is important in regulating autophagy, an essential cellular survival and maintenance process [ca1 gene . In neurA damage . IntriguAlthough the interaction of DHX36 and G4 structures has been characterized across a variety of biological processes, much work is still needed to understand the mechanistic roles controlled by this dynamic relationship. As reviewed here, G4 DNA, G4 RNA, and DHX36 together define behavior for many genes at transcriptional and post-transcriptional levels. DHX36 may be an important genome instability hotspot worth deeper investigation in age-associated diseases. Though much attention has been given to the G4 structures and DHX36 interactivity in cancer tumorigenesis  and vira


The beneficial effects of coffee on human diseases are well documented, but the molecular mechanisms of its bioactive compounds on cancer are not completely elucidated. This is likely due to the large heterogeneity of coffee preparations and different coffee-based beverages, but also to the choice of experimental models where proliferation, differentiation and immune responses are differently affected. The aim of the present study was to investigate the effects of one of the most interesting bioactive compounds in coffee, i.e., caffeine, using a cellular model of melanoma at a defined differentiation level. A preliminary in silico analysis carried out on public gene-expression databases identified genes potentially involved in caffeine’s effects and suggested some specific molecular targets, including tyrosinase. Proliferation was investigated in vitro on human melanoma initiating cells (MICs) and cytokine expression was measured in conditioned media. Tyrosinase was revealed as a key player in caffeine’s mechanisms of action, suggesting a crucial role in immunomodulation through the reduction in IL-1β, IP-10, MIP-1α, MIP-1β and RANTES secretion onto MICs conditioned media. The potent antiproliferative effects of caffeine on MICs are likely to occur by promoting melanin production and reducing inflammatory signals’ secretion. These data suggest tyrosinase as a key player mediating the effects of caffeine on melanoma. Coffea arabica L. and Coffea canephora Pierre ex Froehner), tea leaves (Camellia sinensis (L.) Kuntze) and cocoa powder. Caff acts primarily as an adenosine receptor (ADORA) antagonist and as a non-competitive acetylcholinesterase (ACHE) inhibitor [A large body of evidence points to a positive correlation between habitual coffee consumption and the reduced risk of several diseases including type 2 diabetes and neurological, neurodegenerative and cardiovascular diseases ,2,3. In nhibitor . A furthnhibitor .Malignant melanoma, one of the most aggressive forms of skin cancer, is an immunogenic tumor characterized by resistance to target therapy , great pWe have previously demonstrated that theophylline  possesses potent antiproliferative, differentiative and immunomodulatory activities against MICs . TherefoTo investigate the effects of Caff on melanoma, we performed an in silico analysis of CMT, which allows the screening of several potential targets. Starting from literature data available on Pubmed, 26 CMT were selected and further analyzed in the GDS1375 dataset .n = 45) and in nevi (n = 18) biopsies (p < 0.001). For each gene, the calculated fold change in melanoma vs. controls is indicated. ROC curve analyses demonstrated that eight genes (in bold in p ≤ 0.0001). In particular, the AUC was 0.89 for DNA2 (DNA replication helicase/nuclease 2), 0.87 for MAPK1 (mitogen-activated protein kinase 1), 0.96 for PTK2B (protein tyrosine kinase 2 beta), 0.94 for ADORA2 (adenosine A2a receptor), 0.95 for MAOA (monoamine oxidase A), 0.98 for PARP1 (poly (ADP-ribose) polymerase (1), 0.94 for TYR (tyrosinase), and 0.86 for HDAC1 (histone deacetylase 1).Whereas 14 genes showed no significant differences , a totalDNA2, MAOA, PARP1, TYR, and HDAC1 in the IST Online database  shows specificity of expression in both cutaneous and uveal melanoma . HoweverIn order to validate the in silico results, we investigated the effects of Caff on in vitro models of BRAF-mutated MICs. Treating Mel1 and Mel3 cells with increased concentrations of Caff significantly reduced cell proliferation in a dose-dependent manner, with a strong and significant reduction in cellular density after 2 mM Caff treatments . The abil-3,4-dihydroxyphenylalanine (l-DOPA) and represents the rate-limiting enzyme of melanin synthesis [Based on the in silico results described above, the effects of Caff on melanin production in MICs were investigated. It is known that TYR (EC 1.14.18.1) catalyzes the oxidation of tyrosine to ynthesis . A two-fCXCL10), macrophage inflammatory protein 1-α (MIP-1α or CCL3), and MIP-1β (CCL4) levels in CM were significantly reduced after Caff treatment compared to control in both cell lines, whereas regulated and normal T cell expressed and secreted (RANTES or CCL5) levels were reduced only in Mel3 cells  and TYR expression were further analyzed. The combined expression of TYR with cytokines/chemokines secretion modulated in MICs was subject to PCA analysis carried out by the “Dimensionality reduction” tool in GEPIA2. Finally, all previously identified CMT  and Caff-modulated cytokines/chemokines were uploaded to the online software GOnet. Interestingly, we found that all studied Caff targets were enriched in several biological processes , suggestSeveral studies demonstrate that coffee induces beneficial effects on many human diseases, including cardiovascular, neurodegenerative and cancerous ones ,2,3,26. DNA2, MAPK1, PTK2B, ADORA2A, MAOA, PARP1, TYR, and HDAC1, whose expression was significantly changed in melanoma specimens compared to nevi, and with highly interesting AUC. When these data have been validated on other databases, allowing the evaluation of larger numbers of patients, only PARP1 and TYR have been confirmed as the most significantly related, among the CMT, to severity and survival. Moreover, low TYR expression in BRAF-mutated melanoma subtypes showed a significant association with longer survival. Interestingly, a role of brain tyrosinase has been involved in age-dependent neuromelanin production in the pathogenesis of Parkinson’s disease [A potentially beneficial effect of Caff on melanoma cells, a highly malignant skin cancer, has been reported in several investigations ,38. Neve disease , a neuro disease .TYR mRNA in peripheral blood relates to the occurrence of melanoma metastases [TYR levels are associated with poor clinical outcome. Therefore, the use of molecules that have melanin-inducing effects, such as Caff, appears to be an interesting and intriguing field of investigation.These observations prompted us to investigate in depth the role of TYR, the key enzyme in melanin synthesis, in two human melanoma-initiating cell lines, namely Mel1 and Mel3, previously characterized as suitable melanoma cell models for experimentation ,40. Bothtastases . Neverthtastases . This obTaking advantage of the MICs model, the effects of Caff were studied to evaluate the secretion of 12 cytokine/chemokines known as important immunoregulatory signals, to understand how cancer stem cells can communicate with the TME. The case of cutaneous melanoma is very interesting as it is considered one of the human cancers whose growth and progression can be positively affected by immunotherapy, which, on the other hand, requires novel approaches aimed at minimizing immune-related toxicity , such asCaffeine (Caff), bovine serum albumin (BSA), buffer saline (PBS), glutamine, synthetic melanin, penicillin  and streptomycin (10 mg/mL) and all other reagents were obtained from Sigma-Aldrich .https://www.ncbi.nlm.nih.gov/sites/GDSbrowser?acc=GDS1375 (accessed on 10 October 2020)) reports the expression profiles of 45 primary malignant melanoma and 18 benign skin nevi, and normal skin samples [Caffeine Molecular Targets (CMT) were derived from the literature ,60,61,62 samples . http://ist.medisapiens.com/) (accessed on 15 October 2020) and GEPIA2 (http://gepia2.cancer-pku.cn/#index) (accessed on 20 March 2021). These databases are useful for expression profiles of different genes in both cancer and normal tissues. The scatter plots were obtained from IST Online database according to a previously reported procedure [Genes selected from the CMT, as the most relevant, were subjected to GEO analysis and those having AUC ≥ 0.85 were further studied. The validation of the expression levels of selected genes were verified by two independent databases and online resources, IST Online  in complete culture medium, and treated with 1 mM and 2 mM Caff for 72 h. Cells were harvested and counted with a Neubauer modified chamber, after Trypan Blue staining for cytotoxicity evaluation. Long-term effects of Caff on proliferation were established by spheroid formation assay, as described previously [3 cells/mL) and exposed to Caff for a week. Spheroids were photographed (Olympus BH-2 LH50A) and their number and size (percentage area covered by spheroids) were quantified with ImageJ® (v. 1.5d) software . Data are expressed as a percentage of the control (100%) [Two patient-derived BRAF-mutated human melanoma-initiating cell lines (MICs), namely Mel1 and Mel3, were isolated as melanospheres and cultured in a suitable medium, as described previously . For proeviously . Brieflyl (100%) .For melanin evaluation, Mel1 and Mel3 cells, treated or not with Caff for 72 h, were washed twice in PBS, collected by centrifugation and counted. Melanin was extracted from cells with 1 mL of 1 M NaOH at 75 °C for 1 h. Pigment content was determined by absorption at 475 nm and compared with the standard curve for synthetic melanin.Conditioned media (CM) of Mel1 and Mel3 cells untreated and treated with Caff, were collected and centrifuged to remove cell debris. CM were analyzed with a magnetic-bead-based multiplex immunoassay, i.e., xMAP technology on the Luminex platform, carried out according to the manufacturer’s guidelines . Cytokines/chemokines were measured with a custom Bio-Rad Bio-Plex human cytokine reagent kit for IP-10, monocyte chemoattractant protein-1, MIP-1α, MIP-1β, RANTES, IL-1β, IL-10, IL-12, IL-17, IL-1ra, TNF-α, and IFN-γ. Data were acquired on the Bio-Rad Bio-Plex X200 reader equipped with Bio-Plex Pro™ Wash Station and analyzed using Bio-Plex Software Manager™ 6.1 .Dimensionality reduction by PCA analysis was carried out by the specific tool available at the GEPIA2 database .https://tools.dice-database.org/GOnet/ (accessed on 20 January 2021)) [Gene Ontology (GO) analysis  of selected caffeine targets was obtained using the GOnet database (y 2021)) . Proteint-test, the significance threshold was set at p ≤ 0.05. GraphPad PRISM 5 software  was sued. Generation of ROC curves from expression data reported in the GDS1375 dataset and calculation of AUC data were performed as previously described [Results from in vitro experiments are expressed as means of three independent experiments ± standard deviation (SD). The statistical significance of differences was determined by Student’s escribed ,69.In silico screening of genes potentially involved in the effects of Caff, validated by in vitro analysis with two MICs models exposed to caffeine to evaluate their behaviors, confirms a key role of TYR and melanin, possibly acting through an immunomodulatory action, on melanoma growth and evolution. This opens new viewpoints to explain the powerful antineoplastic effects of caffeine.


Background: Fibroblast growth factor receptor 2 (FGFR2) is frequently altered in tumors and one of the top therapeutic targets in cholangiocarcinoma (CHOL) with FGFR2 fusions. Although there have been several studies on individual tumors, a comprehensive analysis of FGFR2 genetic aberrations and their simultaneous clinical implications across different tumors have not been reported.Methods: In this study, we used the large comprehensive datasets available, covering over 10,000 tumor samples across more than 30 cancer types, to analyze FGFR2 abnormal expression, methylation, alteration (mutations/fusions and amplification/deletion), and their clinical associations.Results: Alteration frequency, mutation location distribution, oncogenic effects, and therapeutic implications varied among different cancers. The overall mutation rate of FGFR2 is low in pancancer. CHOL had the highest mutation frequency, and fusion accounted for the major proportion. All these fusion aberrations in CHOL were targetable, and an FDA-approved drug was approved recently. Uterine corpus endometrial carcinoma (UCEC) had the highest number of FGFR2 mutations, and the most frequently mutated positions were S252W and N549K, where the functional impact was oncogenic, but targeted therapy was less effective. Additionally, DNA methylation was associated with FGFR2 expression in several cancers. Moreover, FGFG2 expression and genetic aberrations showed clinical associations with patient survival in several cancers, indicating their potential for application as new tumor markers and therapeutic targets.Conclusions: This study showed the full FGFR2 alteration spectrum and provided a broad molecular perspective of FGFR2 in a comprehensive manner, suggesting some new directions for clinical targeted therapy of cancers. The fibroblast growth factor receptor 2 (FGFR2) gene is located at chromosome 10q26 and encodes two major isoforms, FGFR2b and FGFR2c, which act as FGF receptors with different functional domains and ligand specificities . The FGFFGFR2 alterations have been reported in multiple cancers. Enhanced FGFR2 signaling, mediated by FGFR2 alterations containing genetic amplification, mutation, and fusion, has been observed in several cancers and is associated with tumorigenesis  and UCECs. Additionally, FGFR2 fusion aberrations are commonly observed in CHOL  patterns and their impact on gene expression. Finally, survival association analysis was conducted to investigate the aberration patterns and potential clinical significance of FGFR2 across different cancer types. In general, these findings highlight the critical role of FGFR2 in oncogenesis and provide a potential therapeutic target for cancers.Previous studies on FGFR2 in tumors are limited to individual tumor types and/or to limited sample sizes. In this analysis, we fill this gap in a comprehensive manner by taking advantage of the large datasets from TCGA. We systematically profiled abnormal FGFR2 mRNA expression profiles between tumors and normal tissues. The TCGA and GTEx gene expression data of FGFR2 were downloaded and recomputed from raw RNA-Seq data by the UCSC Xena project (http://xena.ucsc.edu/) based on a standard pipeline  database was used to compare pipeline  to make pipeline . They coplatform . After lnd so on . The datDatabase , which cFGFR2 alterations among different cancers , cBioportal , GSCALite (http://bioinfo.life.hust.edu.cn/web/GSCALite/), and Kaplan-Meier Plotter (http://kmplot.com/) were also used.The statistical analyses were analyzed by Student's FGFR2 expression has been reported in various cancer types , bladder urothelial carcinoma (BLCA), pancreatic adenocarcinoma (PAAD), KIRP, head and neck squamous cell carcinoma (HNSC), lung adenocarcinoma (LUAD), KIRC, esophageal carcinoma (ESCA), COAD, and PRAD  frequency in all TCGA cancer types was ~3.3% . However, the frequencies among different cancer types were quite variable , in which fusion was more common . Other cancer types that also had FGFR2 fusion but had much lower fusion rates included LUSC (0.41%), breast invasive carcinoma (BRCA) (0.28%), ovarian serous cystadenocarcinoma (OV) (0.17%), liver hepatocellular carcinoma (LIHC) (0.54%), and THCA (0.4%). Moreover, several cancer types mainly had FGFR2 mutations but relatively few amplifications and/or deep deletions, such as UCEC, SKCM, BLCA, LUSC, COADREAD, LUAD, and so on . Tumors with dominant FGFR2 amplification included STAD (4.09%), OV (1.88%), pheochromocytoma and paraganglioma (PCPG) (1.12%), ESCA (0.55%), and PAAD (0.54%). Deep deletion was more common in brain lower grade glioma (LGG), mesothelioma (MESO), and sarcoma (SARC) . Although the FGFR2 alteration frequencies of the two lung cancer subtypes (LUSC and LUAD) were similar, LUSC had FGFR2 fusion, but LUAD did not. Cancer types, including kidney chromophobe (KICH), KIRP, TGCT, and THYM, had neither FGFR2 CNVs nor FGFR2 mutations (The variable . FGFR2 autations .FGFR2 somatic mutations across 32 cancer types. Nearly one-third of the mutations (26 of 79 mutations) in the Pkinase_Tyr domain were accompanied by FGFR2 copy gain, shallow deletion and deep deletion, while ~40% of the mutations (62 of 152 mutations) in the other function-unknown domain had amplification, gain, and shallow deletion. Mutations in the Ig_2 domains and I-set domains rarely had concurrent CNVs  for all tumor samples and 2.4%  for all patients across the 32 cancer types. The most common cancer types with FGFR2 mutations were CHOL (12.3%), UCEC (10.5%), SKCM (8.8%), and STAD (4.2%). In contrast, ESCA, KICH, KIRP, MESO, PAAD, SARC, TGCT, and THYM showed almost no FGFR2 mutations  and the Pkinase_Tyr domain (83 samples), followed by the I-set domain , the I-set domain , and the Ig_2 domain (21 samples). Fusions (22 samples) were also observed in FGFR2 somatic mutations. The location distribution of these FGFR2 somatic mutations was quite different among different cancer types. Mutations in UCEC, COADREAD, STAD, and BLCA were most commonly located in the other domain, the functions of which were rarely known, especially for BLCA, which amounted to nearly three-fifths of all somatic mutations. Mutations in SKCM, BRCA, and LUSC were primarily in the Pkinase_Tyr domain. Meanwhile, fusion was found in UCEC, BRCA, STAD, LUSC, and CHOL, and especially in CHOL, fusion was the most common mutation, amounting to approximately three-fourths of all mutations , truncating (29 mutations), fusion (22 samples), and in-frame (four mutations). S252 in the other domain was the most frequently mutated position, which was observed in 26 samples . Mutations at this position almost exclusively occurred in UCEC samples (24/26). S252W is known to be oncogenic and may be targetable with selective FGFR-targeted inhibitors , BRCA (2), LUSC (2), PRAD (2), THCA (2), UCEC (2), LIHC (1), OV (1), STAD (1), and uveal melanoma (UVM) (1). CHOL harbored the highest number of fusion transcripts . The only other tumor with the FGFR2__BICC1 fusion transcript was LIHC (one FGFR2__BICC1). Most of these FGFR2 fusion transcripts belonged to the in-frame class, while KIAA1967_FGFR2 in LUSC belonged to the 5′ UTR-CDS class.Fusion genes produced by genomic-level cleavage and resplicing are often targets for tumor diagnostic treatment. Based on the TCGA Fusion Gene Database , we detenfidence . FGFR2 fFGFR2 mutations could be classified into five categories. As shown in FGFR2 mutations in UCEC were also distributed in the functional classes   .FGFR2 somatic mutations could be divided into four levels: level 1 (6 mutations), level 3B (16 mutations), level 4 (77 mutations), and level NA (210 mutations)  . Most oftations) . All levtations) .FGFR2 mRNA expression was compared across all TCGA cancer types and exhibited a broad spectrum . Most of the CNVs were shallow deletions , followed by gain (924 samples), amplification (73 samples), and deep deletion (31 samples). The most common tumors with FGFR2 CNVs were glioblastoma multiforme (GBM) (87.0%), KICH (76.9%), UCS (70.2%), LUSC (60.6%), and OV (60.0%). In contrast, acute myeloid leukemia (LAML) (2.0%), THCA (1.8%), and UVM (1.25%) exhibited very low frequencies of FGFR2 CNVs   . In addihylation .FGFR2 somatic mutations described above, 109 also harbored FGFR2 CNVs, of which 42 harbored gains, five harbored amplifications, 61 harbored shallow deletions, and one harbored a deep deletion. SKCM had the highest number of shallow deletions, and UCEC had the highest number of gains  and progression-free survival (PFS) in individual cancer types was analyzed. We found that decreased FGFR2 expression was associated with short patient OS in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), EAC, HNSC, KIRC, LUAD, and LUSC, while increased FGFR2 expression was associated with short patient OS in KIRP , and mutation took up a major portion in most cancer types. Targetable mutations were mainly observed in CHOL and could be targeted by pemigatinib, a selective competitive inhibitor of FGFR1, FGFR2, and FGFR3 approved by FDA recently. Genetic fusions were mainly detected in CHOL, followed by BRCA, LUSC, PRAD, THCA, and UCEC. BICC1 was found to be the most common partner gene of FGFR2. CHOL, UCEC, and SKCM are the three cancer types that harbor the highest frequencies of FGFR2 alteration. In CHOL, fusion accounted for the major proportion, while in UCEC and SKCM, mutation was the dominant alteration. Mutations in these two cancer types were most commonly located in the Pkinase_Tyr domain and the other function-unknown domain. S252W, the most common mutation in UCEC, is located in another domain of unknown function, and its role in targeted therapy implications is not well known. In addition, CHOL and UCEC had high FGFR2 expression, and SKCM had low FGFR2 expression, but in these cancer types, there was no survival association shown between FGFR2 expression and patient prognosis. In addition, other cancer types, including KICH, KIRP, TGCT, and THYM, had almost no FGFR2 alterations. Moreover, some common tumors with FGFR2 alterations, including BLCA, LUSC, COADREAD, and LUAD, had similar characteristics: an alteration frequency of ~2–4% and mutation as the most common type. Over half of the mutations in LUSC belonged to the likely oncogenic class and were level 3B and level 4. Conversely, certain cancer types, including OV, PCPG, ESCA, and PAAD, mainly harbor FGFR2 amplification but rarely mutations. Meanwhile, deep deletion was the dominant alteration type in LGG and MESO; however, in LGG, high expression of FGFR2 was observed, indicating that additional genetic alterations may contribute to high expression of FGFR2 in this cancer type.In our study, the FGFR2 fusions are observed mostly in cholangiocarcinoma, occurring in 10–16% of patients (FGFR2 alteration, and fusion accounted for the major proportion. Fusion types in CHOL included FGFR2__BICC1, BICC1__FGFR2, FGFR2__SHTN1, and FGFR2__CCDC186. The first two fusion types are known to be oncogenic, and the last two are likely oncogenic. Consistent with previous findings (BICC1 was found to be the most common partner gene of FGFR2. However, different fusion types did not seem to exhibit different effects on the therapeutic and prognostic implications of patients (FGFR2 fusions (FGFR2 fusions (A previous study reported that patients , 44, 45.findings , in our patients –50. Acco fusions , 25. Pem fusions , 52. Oth fusions . Debio 1 fusions , 28.FGFR2 mutations have been observed in ~12% of UCEC, suggesting an opportunity for targeted therapy (FGFR2 alteration, which was driven by a high proportion of mutations. The most frequently mutated positions in UCEC were S252W and N549K, and these two mutations are known to be oncogenic; however, they all belong to the level 4 class, which represents rare targeted therapy implications. Recently, several laboratory findings showed the potential implications of FGFR inhibitors in targeted therapy in UCEC (FGFR2-mutant UCEC (FGFR2 mutations are associated with poor prognosis in patients with UCEC (FGFR2 alterations and patient prognosis. This paradox could be due to insufficient sample size; more research needs to be conducted on this aspect.Frequent activating  therapy , 54. In  in UCEC , 56. Forant UCEC . Howeverith UCEC . HoweverFGFR2 expression in these two cancer types was associated with patient OS. Their FGFR2 alteration frequencies were ~3% and had similar alteration patterns, in which mutation was the most common alteration. However, LUSC featured FGFR2 fusion, which was not present in LUAD. The fusions in LUSC were divided into level 3B, and the remaining mutations were in level 4/level NA classes without targeted therapy implications. BGJ398, a pan-FGFR inhibitor, showed antitumor activity in LUSC (FGFR2 was found in LUSC tissues compared with corresponding normal tissues. However, most mutations in LUAD were in the unknown class, suggesting that more efforts are needed to further explore the roles of these unknown mutations, and they may also have critical functional roles in driving oncogenesis.LUSC and LUAD are two lung cancer subtypes.  in LUSC  and is eFGFR2 expression, methylation, alteration and their clinical associations. However, there were several limitations that need to be mentioned. First, even though this was a pancancer global analysis, it lacked an in-depth analysis of individual tumor types. Additionally, several rare tumor types did not harbor sufficient sample sizes, which made the full expression, methylation and alteration spectrum difficult to capture. Furthermore, the frequency of FGFR2 alteration across all tumor types was between 0 and 20%, and this low alteration frequency could make our analysis challenging. Furthermore, even though the TCGA and GTEx gene expression data of FGFR2 were re-computed from raw RNA-Seq data by the UCSC Xena project based on a standard pipeline to minimize differences from these two sources (In this study, we profiled 32 cancer types regarding  sources , 30, minFGFR2 abnormal expression, methylation, alteration, and their therapeutic and prognostic implications, covering 10,967 tumor samples across 32 cancer types. While several tumors with low alteration frequency are correlated with prognosis, others with high alteration frequency were not. Some alterations are found to be more involved in oncogenesis, while other alternations are found to participate more in targeted therapy. Genomic profiling may offer directions for their application in clinical targeted therapy.In conclusion, our study provided the first comprehensive pancancer view of The original contributions presented in the study are included in the article/YY and ZX: conception and design. JL, KH, and JH: writing, review, and/or revision of the manuscript. LZ: administrative, technical, or material support. All authors approved final version of 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.


GSEA results showed that high PLAGL2 expression was associated with cell migration, proliferation, actin cytoskeletal, and angiogenesis. To sum up, our findings indicated that PLAGL2 could serve as an independent prognostic biomarker and might be a potential therapeutic target for HGG, which should be further investigated.Pleomorphic adenoma gene like-2 (PLAGL2) has been implicated in the development and progression of diverse malignancies, including glioblastoma. An increasing number of studies have reported that dysregulated expression of PLAGL2 is a common phenomenon in different malignancies. However, the mechanism and biological functions of PLAGL2 in patients with high-grade glioma (HGG) remain unclear. In addition, the expression and clinical significance of PLAGL2 in HGG have not yet been reported. Herein, we investigated the expression patterns and prognostic values of PLAGL2 in patients with HGG by using various databases, including Tumor Immune Estimation Resource 2.0 (TIMER2.0), GENT2, ONCOMINE, GEPIA, Human Protein Atlas, and Gene Expression Omnibus datasets. In the present study, we analyzed the relationship between PLAGL2 mRNA expression and clinical parameters in 184 HGG cases and found that PLAGL2 presented positively high expression and was relevant to poor prognosis. Immunohistochemistry analysis confirmed the overexpression of PLAGL2 protein, which is mainly expressed in the nucleus of glioma. Additionally, a high level of expression of the PLAGL2 gene was associated with lower survival in progression-free survival and overall survival in GBM patients. The correlation analysis between PLAGL2 and immune infiltration related to the abundance of B cells, CD8 Malignant gliomas, encompassing a heterogeneous cluster of subtypes, are the most common primary central nervous system (CNS) tumors in both children and adults. Despite its low occurrence, glioma is considered one of the deadliest cancers worldwide, with the incidence at 5/100,000 each year . AccordiA member of the PLAG gene family, PLAGL2, is a putative transcription factor that includes C2H2 zinc finger domains on the N-terminus with the DNA binding function. PLAGL2 was initially identified to have high structural conserved and similar DNA binding affinity with PLAG1 . AberranAccording to our knowledge, bioinformatics methods have not been used to investigate the role of PLAGL2 in GBM. In this study, we investigated the expression patterns and prognostic values of PLAGL2 in patients with HGG by using various databases, including TIMER2.0, GENT2, ONCOMINE, GEPIA, HPA, and GEO datasets.A total of 184 surgically resected human glioma specimens were collected in the Department of Neurosurgery, the First Affiliated Hospital, Zhengzhou University, between 2011 and 2019. Among 184 patients, 115 (62.5%) were males and 69 (37.5%) were females. According to the WHO classification of CNS tumors , the colThe current study was approved by the Ethics Committees of the First Affiliated Hospital of Zhengzhou University before the clinical information was used for research purposes, and written informed consent was obtained from all participants. All study procedures were carried out in accordance with the ethical standards of the Helsinki Declaration.To block the endogenous peroxidase activity, the sections from 4% formalin-fixed and paraffin-embedded tissue blocks were dewaxed in xylene, rehydrated using graded concentration ethanol, and soaked in 0.3% hydrogen peroxide, which were cut into 5 μm-thick sections. Then, the tissue sections were rinsed in PBS. After having blocked in 10% goat serum  for nonspecific reactions, the sections were incubated with the anti-PLAGL2 polyclonal antibody  overnight at 4°C. Negative control slides were processed in parallel with a nonspecific IgG. After washing, the sections were incubated with horse reddish peroxidase (HRP)-labeled anti-rabbit secondary antibody  for 1 h at room temperature. The slides were subjected to staining with 3, 30-diaminobenzidine solution and briefly counterstained with hematoxylin. Finally, the sections were rehydrated with xylene and ethanol, mounted in mounting resin, and examined with a microscope.Two independent pathologists blinded to clinicopathological outcome calculated immunostaining reactions by multiplying the intensity and proportion of positive tumor cells. Briefly, the scores about the proportion of positive cells  and the scores about the staining intensity  were combined. All the cases were divided into low expression and high expression subjected to the total score (median). Any discrepancy in the scoring was resolved by discussion between two pathologists in less than 10% of the examined slides, and an agreement was reached by rescoring the sections combined with a third pathologist in a multi-viewer microscope.https://portals.broadinstitute.org/ccle) was downloaded from Gene Expression Omnibus  and was used to identify the alternation of the expression of PLAGL2 across various cancer types. The Affymetrix Human Genome U133 Plus 2.0 DNA microarray gene expressions of 84 CNS cell lines were downloaded from CCLE in March 2012. Robust multiarray average (RMA) normalization and gene expression values was performed. One-way ANOVA was used to compare the expression of PLAGL2 in different types of cancer cell lines , with a threshold set as such that P < 1E − 4, fold change >2, top gene rank 10%  public database under accession numbers GSE4290 and GSE53733 (The transcript level of PLAGL2 in glioma was ascertained by the ONCOMINE database (rank 10% . The mRNGSE53733 .http://gliovis.bioinfo.cnio.es/) (http://cancergenome.nih.gov/), which includes 29 cancer types, along with gene expression data and clinical information. Furthermore, a database (ID: mRNAseq 325) consisting of 325 glioma tissues with different grades (WHO I–IV) was downloaded from the CGGA  and Chinese Glioma Genome Atlas (CGGA) were both downloaded from Gliovis (nio.es/) . TCGA isthe CGGA . LGG andthe CGGA .http://ualcan.path.uab.edu) and GEPIA datasets (http://gepia.cancer-pku.cn/) of TCGA gene expression were used to analyze the expression of PLAGL2 in GBM tissues and normal tissues (The UALCAN ( tissues . Two onehttps://www.proteinatlas.org/), which contained the immunohistochemistry staining images of 153 clinical GBM patients and their survival information with 54 females and 99 males  dataset  is a public web database for comprehensive assessments of abundance of tumor-infiltrating immune cells (TIICs) according to gene expression profiles , compared with purity in GBM and assessed how PLAGL2 is correlated with immune cell markers including CD8+T cells, T cells, B cells, monocytes, M1 macrophages, M2 macrophages, neutrophils, DCs, Th1 cells, type 2 helper T cells (Th2), Tfh cells, type 17 helper T cells (Th17), and Treg.The Tumor Immune Estimation Resource (TIMER2.0) (profiles . We mainhttp://gent2.appex.kr/gent2/) revealed the gene expression in several types of cancer. The data from about 49,000 healthy and cancer individuals were analyzed in more than 30 different cancer types  web database revealed the interaction relationship of a gene and the performed gene function, physical interaction, pathway, co-expression, and localization (https://string-db.org/) was utilized to obtain the interaction network of related genes for PLAGL2, including direct  and indirect  associations. Thus, nodes represent genes, whereas links conduct connected networks (The GeneMANIA (lization . The STRnetworks .http://software.broadinstitute.org/gsea/msigdb)  . Gene se/msigdb) . Normalip < 0.05, **p < 0.01, and ***p < 0.001.Statistical analyses were performed by SPSS 22.0 software, and the results were presented as the mean ± standard deviation (SD). Student’s t-test was performed to detect significant differences between groups. The relationships of PLAGL2 expression and various clinicopathological features were estimated by chi-squared test. Analyses with more than two groups were performed by the one-way ANOVA test. The Kaplan–Meier with log-rank test was used to analyze the survival data. The correlation analysis between PLAGL2 expression and immune-related markers was performed using Pearson correlation. Univariate and multivariate Cox regression analyses were used to analyze the clinical prognostic parameters and related independent risk factors. The results were considered significant at *We performed pan-cancer analysis derived from TCGA by adopting Timer 2.0 and Gent2 so as to estimate the expression pattern of PLAGL2. According to Timer analysis, PLAGL2 was upregulated in bladder, breast, cervix, bile ducts, colorectal, esophagus, kidney, liver, lung, stomach, and CNS cancer compared with normal cases , whereasMoreover, we estimated the PLAGL2 expression data profiles of 72 paired cancers vs. normal tissues by utilizing the HG-U133 microarray (GPL570 platform) of the Gent2 database. The results showed that PLAGL2 was upregulated in several cancers, including adrenal gland, brain, breast, cervix, colon, esophagus, liver, lung, oral, ovary, and skin. Conversely, the upregulation of PLAGL2 was reported in the pharynx, small intestine, testes, stomach, head, and neck . CombiniNext, we used these respective cancer types in which significant upregulation was found for further investigation. In addition, we selected CCLE and HPA databases to further validate the PLAGL2 expression in the cell line. The CCLE analysis results indicated a certain degree of PLAGL2 expression in 84 CNS cell lines containing glioma cancer cell lines . In GBM https://www.oncomine.org/)  . Based one.org/)  (Rickmanne.org/) . Similarne.org/) . To furtne.org/) . Moreovene.org/) .IDH mutation and 1p/19q co-deletion status are well-known clinically relevant molecular markers in glioma. Herein, we analyzed the correlation between the mRNA levels of PLAGL2 and major clinical features of GBM patients in CGGA, including grade level, IDH mutation status, 1p/19q co-deletion status, gender, and age. As shown in p < 0.05), but not with gender, tumor locus, histology, cell origin, treatment regimen, and radiochemotherapy. Meanwhile, considering the GBM group, the analysis results showed that the expression of PLAGL2 was significantly correlated with gender (p < 0.01), but not with tumor locus and radiochemotherapy . Similarly, we explored the expression levels of PLAGL2 in the GEO database. GSE53733 experiments indicated that the expression of PLAGL2 in the short-term OS group was significantly higher than that in the long-term OS group (p < 0.05). Moreover, a low prognostic value was observed in high-grade stage groups (p = 0.001)  and progression-free survival (PFS) of HGG patients and explore the prognostic value of PLAGL2 in HGG, survival analysis was performed based on information of 184 HGG patients. As shown in OS group . MoreoveOS group . The GEN= 0.001) . Next, H= 0.001) . A highe= 0.001) . Further= 0.001) . The AUC= 0.001) . These f= 0.001) ..To further determine the prognostic value of PLAGL2 expression, we performed the univariate and multivariate Cox regression analyses based on OS and PFS of HGG patients. As shown in .+T cells, CD8+T cells, and DCs. Next, we then performed a correlation analysis between PLAGL2 and tumor-infiltrating immune cells, including B cells, CD4+T cells, CD8+T cells, macrophages, neutrophils, and DCs by the TIMER database in GBM patients to evaluate the immunotherapy effect. Tumor purity acted as a critical factor, which affected the analysis of immune infiltration in the analysis of the genomic approach. As shown in p = 0.0017), B cells , CD4+T cells , macrophages , neutrophils  and DCs , and negatively associated with CD8+T cells  in GBM. Additionally, we also investigated the immune infiltration status of PLAGL2 related to immune cells in LGG. Similarly, the purity , B cells , CD4+T cells , CD8+T cells , macrophages , neutrophils , and DCs  were significantly correlated with PLAGL2 in LGG  could affect a patient’s OS and regulate the tumor response to therapies . By comb2 in LGG . In gene+ T cells, CD4+ T cells, regulatory T cells, B cells, tumor-associated macrophages (TAMs), M1 and M2 macrophages, monocytes, neutrophils, DCs, and natural killer (NK) cells. Meanwhile, NK cells, T helper 1 (Th1), T helper 2 (Th2), follicular helper T (Tfh), T helper 17 (Th17), and regulatory T (Tregs) were also analyzed. The correlation analysis data were adjusted for tumor purity. An analysis of the TIMER database showed that PLAGL2 expression in GBM was significantly correlated with the expression of marker genes in CD8+T cells, general T cells, B cells, monocytes, M1 and M2 macrophages, neutrophils, DCs, NK cells, Th1, Th2, Tfh, Th17, and Treg ; T-cell marker, CD3D  and CD2 ; B-cell marker, CD79B ; monocyte marker, CD14  and CD115 ; M1 macrophage marker, CCL5 , IL18  and COX ; M2 macrophage marker, CD163 , neutrophil marker, CD11b ; DC marker, HLA-DQB1  and BDCA4 , and NK cell marker, NCR3 .Next, we comprehensively clarified the correlation of PLAGL2 and the status of infiltrating immune cells in GBM based on a cohort of immunological makers using the TIMER2.0 database. The gene markers of immune cells were used to analyze and identify the immune cells, including CD8and Treg . Interesp < 0.001); Th2 marker, CD14 , GATA3 , STAT6 , STAT5A ; Tfh marker, BCL6 ; Th17 marker, STAT3 ; Treg marker, FOXP3 , STAT5B , and TGFB . However, PLAGL2 expression did not reveal any significant correlation with CD4+ T cells and TAMs. In summary, a comprehensive analysis indicated that PLAGL2 expression was strongly correlated with infiltration of immune cells in GBM.Moreover, the expression of PLAGL2 was associated with the expression of markers of specific subsets of T cells in GBM, which included the Th1 marker, TBX2  cells, including Th1 (TBX2), Th2 , Tfh (BCL6), Th17(STAT3) and Tregs , which also had a potential influence in regulating the tumor immunity infiltration of T-helper cells for PLAGL2.Our results also revealed that PLAGL2 expression was associated with infiltration levels of immune cells in GBM. There was a strong positive association of PLAGL2 with the expression and infiltration of immune cells, including B cells, CD4et al. (So far, the role of PLAGL2-induced oncogenesis was still somewhat unclear. PLAGL2 impeded the differentiation of neural stem cells through WNT/β-catenin signaling . NIH3T3 et al.  found thet al. .PLAGL2 was distinctly expressed in different tumors with tissue specificity. The contrary PLAGL2 function attributed to different modulatory mechanisms and contributing to glioma tumorigenesis needs further investigation. The enrichment plots of GSEA analysis showed that PLAGL2 expression was positively associated with the activation of genes of cell migration, proliferation, actin cytoskeletal, and angiogenesis assembly in clinical specimens. Thus, it suggested that PLAGL2 expression could be associated with the progression and development of glioma. Interestingly, GSEA analysis showed that PLAGL2 expression was positively correlated with the VEGFA-VEGFR2 signaling pathway. To the best of our knowledge, gliomas are highly vascular tumors, which require a longstanding therapeutic approach for the suppression of angiogenesis . Thus, fThe present study had some limitations. Firstly, multiple databases could cause some bias because of the differences in sample size. In addition, the study only supplied the mRNA level of PLAGL2, regardless of protein or its posttranslational levels. Finally, the molecular mechanism and effect for clinical translational therapy should be further investigated.In conclusion, this was the first study that evaluated the upregulated expression of PLAGL2 in gliomas compared with normal brain tissue. Additionally, the overexpression of PLAGL2 was correlated with adverse clinicopathological characteristics, such as tumor grade. Our study also demonstrated that PLAGL2 was an independent prognostic indicator for PFS and OS in glioma clinical specimens. Furthermore, the present study also furthered the understanding of the differential mechanisms positively associated with PLAGL2 expression in the progression of gliomas, which may serve as a favorable prognostic marker in the diagnosis and prognostic of gliomas. Yet, the precise mechanism of PLAGL2 action on gliomas angiogenesis needed to be further investigated. We hoped that our findings could have significant implications for diagnosis and therapies designed to treat patients with HGG and prevent HGG.


Aichivirus C whilst the strains identified in domestic and wild ruminants grouped into the species Aichivirus B. The complete VP1 gene was obtained for chamois and goat KoVs. Interestingly, upon phylogenetic analysis the strains grouped together with a KoV of ovine origin within a distinct genetic type (B3) of the species Aichivirus B.Since the first identification in 1989 in humans, kobuviruses (KoVs) have been identified from a wide range of animal species including carnivores, rodents, birds, ungulates, rabbits, and bats. Several studies have described the identification of genetically related KoVs in the fecal virome of domestic and wild animals suggesting a mutual exchange of viruses. By screening a total of 231 fecal samples from wild and domestic ungulates, KoVs RNA was detected in wild boars , chamois , and goats . On phylogenetic analysis of the partial RdRp sequence, the wild boar strains clustered within the species  Kobuvirus within the family Picornaviridae (Kobuviruses (KoVs) are small ~30–32 nm), icosahedral, non-enveloped viruses with a single stranded positive sense RNA genome of 8.2–8.4 kb in length, classified in the genus aviridae . The vir nm, icosKobuvirus is currently classified into six established species, Aichivirus A to F, and 20 genetic types [https://talk.ictvonline.org]. Aichivirus A includes a total of ten genetic types (Aichivirus A1–A10) identified in humans (A1) (Aichivirus B comprises KoVs detected in cattle (B1) (Aichivirus C are classified KoVs detected in swine (C1)  (Aichivirus A-2 (formerly canine kobuvirus) in dogs and wild canids (Aichivirus C-1 (formerly porcine kobuvirus) in pigs and wild boars (Aichivirus B-1 (formerly bovine kobuvirus) and Aichivirus C-2 (formerly caprine kobuvirus) in domestic and wild ruminants  , canids ans (A1) , rodentsans (A1) –17, domeans (A1) , and birans (A1) ; Aichivitle (B1) , ferret tle (B1) , and shetle (B1) ; within ine (C1)  and in gits (E1) . Althougd canids –24, Aichld boars , 26, Aicuminants . A molecuminants  has docuuminants , systema2. It is an alpine area characterized by the concomitant presence of domestic ruminant semi-rural farms and abundant ungulates wildlife. In this setting, the population of wild ungulates, composed primarily by wild boar (Sus scrofa), red deer (Cervus elaphus), and chamois (Rupicapra rupicapra), may occasionally enter in contact with domestic small ruminants through the use of pastures during seasonal transhumance or in the surrounding of backyard farms. Between September 2017 and December 2019, using a convenience sampling strategy, a total of 231 fecal samples was obtained from domestic and wild ungulates. Briefly, 95 fecal specimens were collected from 77 goats and 18 sheep, respectively, in 14 caprine and 3 ovine small farms from 16 municipalities in the Valle d'Aosta region  and FASTA (http://www.ebi.ac.uk/fasta33) with default values were used to find homologous hits. Multiple alignments were performed using the commercially available Geneious software package version 9.1.6 . Phylogenetic tree (neighbor joining and Kimura 2-parameter model) with bootstrap analysis  were constructed by using the MEGA software package, version X  fecal suspension using TRIzol LS  procedure. KoVs RNA was detected by RT-PCR using broadly reactive primer pair, UNIV-kobu-F/UNIV-kobu-R  designedersion X .KoVs RNA was detected in a total of 6 fecal samples with an overall prevalence of 2.6% (6/231). Among the five ungulate species investigated in this study, viral RNA was found in wild boars , chamois , and goats , whilst all the samples collected from red deer and sheep resulted negative . The twoHowever, none significant association was found between the detection of KoVs RNA and age. Also, on the basis of the positivities obtained, a geographical clustering between wild and domestic animals was not observed.MW307937-8), shared 98.6% nt identity to each other and displayed a closed relatedness (94.0–96.1% nt identity) to porcine KoV sequences detected from swine enteric samples with or without diarrhea (Aichivirus C (MW307939-40) and the goat strains Goat-23/ITA and Goat-24/ITA (accessions MW307941-2) displayed the highest sequence identity (97.0–98.2% nt) to KoVs detected in calves and goats in previous surveys performed in an Italian study in Abruzzo, Southern Italy . The mean nt identity within this group was 88.7%. A second group, sharing more than 85.5% nt identity (mean identity 86.4%) comprised KoVs detected from ferrets (Aichivirus B-2). Group 3 included a strain of ovine origin, TB3/HUN/2009 (Aichivirus B-3). Identity among these VP1 sequences was higher than 90% nt (mean identity 93.2%).Partial RdRp sequences were determined from the KoV positive samples. By sequence analysis, two wild boar strains, WB-15/ITA and WB-28/ITA  were first identified in wild boars in Hungary  than in goats without enteritis , although the difference was not statistically significant. The possible role of Aichivirus B-1 as enteric pathogen involved in neonatal calf diarrhea has been hypothesed .The data that supports the findings of this study are openly available in the GenBank database at For this study ethical statement was not required . Faecal samples from wild ruminants were collected during the routinely monitoring activities performed by the National Reference Centre for Wild Animal Diseases (CeRMAS – IZS PLV), whilst samples from sheep and goats were collected only for veterinary diagnostic purposes.BDM, FDP, and SR: conceptualization. BDM, FDP, AP, PF, VS, and IM: methodology. AP, SR, PF, VS, FDP, IM, and RO: investigation. BDM, FDP, VM, and FM: writing—original draft preparation. BDM, SR, and RO: funding acquisition. All authors have read and agreed to the published version of 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.


RECQL gene. The affected individuals had short stature, progeroid facial features, a hypoplastic nose, xeroderma, and skin photosensitivity and were homozygous for the same missense mutation in RECQL1 (p.Ala459Ser), located within its zinc binding domain. Biochemical analysis of the mutant RECQL1 protein revealed that the p.A459S missense mutation compromised its ATPase, helicase, and fork restoration activity, while its capacity to promote single-strand DNA annealing was largely unaffected. At the cellular level, this mutation in RECQL1 gave rise to a defect in the ability to repair DNA damage induced by exposure to topoisomerase poisons and a failure of DNA replication to progress efficiently in the presence of abortive topoisomerase lesions. Taken together, RECQL1 is the fourth member of the RecQ family of helicases to be associated with a human genome instability disorder.Despite being the first homolog of the bacterial RecQ helicase to be identified in humans, the function of RECQL1 remains poorly characterized. Furthermore, unlike other members of the human RECQ family of helicases, mutations in RECQL1 have not been associated with a genetic disease. Here, we identify 2 families with a genome instability disorder that we have named RECON (RECql ONe) syndrome, caused by biallelic mutations in the   Escherichia coli RecQ. Although bacteria and yeast only express one RecQ-type helicase, higher eukaryotes possess multiple homologs that are highly conserved. The RecQ helicase family has 5 known homologs in humans: RECQL , WRN, BLM, RECQL4, and RECQL5. Strikingly, biallelic mutations in 3 of 5 of these RecQ homologs — WRN, BLM, and RECQL4 — have been associated with rare genetic disorders in humans, characterized by chromosomal instability and cancer predisposition , which is a progeroid syndrome associated with premature aging, type 2 diabetes, osteoporosis, cataracts, graying and loss of hair, skin atrophy, and cancer predisposition. Cells from individuals with WS characteristically undergo premature replicative senescence associated with increased telomere erosion and have been reported to be hypersensitive to replication stress–inducing genotoxic agents, such as camptothecin (CPT), methylmethane sulfonate (MMS), and G-quadruplex (G4) stabilizers , RAPADILINO syndrome, and Baller-Gerold syndrome (BGS). While patients with these disorders display some unique clinical features, e.g., poikiloderma (RTS) or craniosynostosis (BGS), the vast majority of affected individuals exhibit a constellation of developmental abnormalities, such as growth retardation, aplastic/absent thumbs, aplastic/absent patellae, aplastic/absent bones in the forearms, skin hypo-/hyperpigmentation, cleft palate, and an increased predisposition for developing cancer  of RECQL1 (p.Arg455Cys and p.Met458Lys) that completely abolished its helicase activity. Moreover, functional analysis of conserved cysteine residues within this domain demonstrated that cysteine residues 453, 475, and 478 are critical for RECQL1 to hydrolyze ATP and unwind dsDNA but not to promote SSA  syndrome. Notably, individuals from both affected, but seemingly unrelated, families carried a homozygous missense mutation  in the ZBD of RECQL1. Clinically, this RECQL mutation was associated with a distinct disorder characterized by striking progeroid facial features, a tiny, pinched nose, apparent skin photosensitivity, xeroderma, and slender, elongated thumbs. Functionally, the p.A459S mutation significantly compromised the ATPase/helicase activity of RECQL1 and modestly affected its ability to bind DNA. However, its capacity to promote SSA was unaffected. At the cellular level, this mutation gave rise to a modest defect in repairing abortive topoisomerase 1 (TOP1) and topoisomerase 2 (TOP2) cleavage complexes and a reduced ability to replicate in the presence of CPT- or ETOP-induced DNA damage. Furthermore, cells expressing the p.A459S mutant RECQL1 also failed to efficiently restart replication following exposure to HU and MMS, indicating that the role that RECQL1 plays in restarting damaged replication forks is not just limited to those associated with abortive TOP1/2 complexes. Taken together, these data suggest that, while it is not essential , a weight of 22.5 kg , and a head circumference of 50 cm . A negative z score represents the number of standard deviations below the population mean for each clinical measurement. The proband’s dysmorphic features included a round face, redness of eyes, a tiny, pinched nose with anteverted nares, prominent premaxilla, and a smooth philtrum  was a female evaluated at age 9 years, 8 months for short stature, dysmorphism, and dryness of the skin and eyes. She was the second of 5 children born to parents of Middle Eastern descent, who were double first cousins . The prephiltrum . Her thuphiltrum .z score, –1.02), a weight of 13.9 cm , and a head circumference of 47.8 cm . She had dysmorphism including a somewhat aged (progeroid) appearance with little subcutaneous fat, red eyes, a tiny, pinched nose with anteverted nares, prominent premaxilla, and a smooth philtrum , examined at 4 years of age, presented with similar clinical features. The pregnancy and delivery were uncomplicated, and the girl’s developmental milestones were age appropriate. Physical examination revealed a height of 96.4 cm , a weight of 13.5 kg , and a head circumference of 47 cm . Dysmorphic features included a mask senile-like face (progeroid), deep-set eyes, absent lower lid eyelashes, small ears with small, attached earlobes, a prominent nasal bridge with a tiny, pinched nose with hypoplastic alae nasi, prominent premaxilla, thin lips, and a smooth philtrum , a weight of 22.1 kg , and a head circumference of 50.8 cm   was a female and the fourth of 6 children born to parents of Middle Eastern descent with no known consanguinity . She wasphiltrum . The pro, –2.92) . NeitherRECQL  as the primary candidate (https://doi.org/10.1172/JCI147301DS1). Moreover, this variant is not found in the gnomAD or Greater Middle East (GME) variome databases, nor in our local databases of approximately 10,000 exomes. It is predicted to be pathogenic by several bioinformatics algorithms including combined annotation-dependent depletion (CADD) , MutationTaster , PolyPhen-2 , and deleterious annotation of genetic variants using neural networks (DANN) . Notably, monoallelic alteration of the adjacent amino acid 458 (p.Met458Lys) has previously been linked with an increased predisposition for developing breast cancer. Moreover, functional studies of this mutation revealed that it significantly compromised the helicase activity of RECQL1  of family A. After exclusion of nonauthentic variants and inconsistent mutations, data mining identified a predicted pathogenic variant in andidate . This vaf RECQL1 . All 3 aTo determine what impact the p.A459S mutation had on RECQL1 protein stability, we performed Western blotting on whole-cell extracts generated from lymphoblastoid cell lines (LCLs) derived from RECQL1 patient III-2 from family A (RECQL1-P1-1) and RECQL1 patient III-4 from family B (RECQL1-P2). We observed no obvious differences in the expression of RECQL1 protein in LCLs from the 2 affected patients when compared with the expression of RECQL1 in LCLs from 2 normal individuals . MoreoveGiven that it had been previously shown that mutating the residue preceding alanine-459 (methionine-458) to a lysine abolished helicase activity , this prKinetics analysis of RECQL1-dependent unwinding of the 19 bp forked DNA duplex showed a 2.6-fold reduction in the rate catalyzed by the p.A459S mutant compared with WT at a protein concentration of 0.3 nM , A–C. UsRECQL1, like the other human RecQ helicases, shows significantly compromised helicase activity on longer duplex DNA substrates, suggesting that the enzyme lacks processivity , 19. ThiAltogether, the findings from these helicase studies demonstrate that the p.A459S amino acid substitution in the ZBD of RECQL1 compromised its DNA unwinding activity, a result that was consistent with those of previous studies assessing the impact of other RECQL1 ZBD mutations on its helicase activity , 17.Since RECQL1 has been previously shown to restore replication forks that have been regressed , we usedPurified human recombinant RECQL1 protein is also known to display potent strand annealing activity . To deteKD for RECQL1-A459S compared with WT RECQL1 , the Michaelis constant (Km), and the maximum velocity (Vmax) for ATP hydrolysis. In kinetics assays with an ATP concentration of 1 mM, WT RECQL1 had a nearly 3-fold higher kcat for ATP hydrolysis in the presence of the M13mp18 ssDNA effector compared with RECQL1-A459S (max) and a 2-fold greater Km compared with RECQL1-A459S  carrying a hypomorphic homozygous truncating mutation in MRE11A , followiMRE11A . We obseMRE11A . ImportaMRE11A .2 cells). Quantification of Rad51 foci in S/G2 cells by fluorescence microscopy revealed that, although the cells containing an empty vector or expressing mutant RECQL1 had higher levels of spontaneous Rad51 than did the cells complemented with WT RECQL1  were transiently exposed to CPT or ETOP, and, following the induction of DNA damage, early and late time points were set to examine the activation of the DDR by Western blotting using phosphorylated ATM (p-ATM), p-SMC1, p-Nbs1, p-Chk1, p-RPA2, and p-H2AX as markers. Despite some mild variability in the level of the DDR induced by CPT or ETOP, both RECQL1-mutant cell lines showed robust activation of the ATM-dependent DDR  have yet to be linked to an inherited chromosomal instability syndrome. Here, we describe the identification of a human chromosomal instability disorder, which we named RECON syndrome (RECql ONe), caused by biallelic missense mutations of RECQL. Notably, although it has been suggested that the function of RECQL1 may be redundant with other RecQ helicases such as BLM , which is caused by mutations in the fork remodeling factor SMARCAL1 , 42. ThiInterestingly, in addition to RECQL1, other disease-associated DNA helicases involved in replication fork processing and homologous recombination, such as DNA2, WRN, BLM, FANCJ, and FANCM , have alRECQL are the underlying cause of an inherited chromosomal instability syndrome that shares some clinical and cellular phenotypic similarities with WS. Thus, to our knowledge, RECQL1 represents the fourth RecQ helicase to be implicated in genetically inherited disease characterized by genome stability defects.In summary, we demonstrate that mutations in Further information can be found in Supplemental Methods.We performed exome analysis using DNA extracted from whole blood of the proband in family A . Exonic RECQL variant was amplified by conventional PCR of genomic DNA derived from the probands and all available parents and siblings. PCR amplicons were analyzed by Sanger sequencing. The genomic DNA primer sequences used for RECQL PCR amplification were as follows: RECQL forward, 5′-TTAGCCTATAAAGGTTTACAAAACA-3′ and RECQL reverse, 5′-GGTAATATTATGGCAGTTATAGGAAGC-3′. The primers used to sequence RECQL cDNA were as follows: RECQL forward, 5′-TGCAGGTCGAGATGACATGA-3′ and RECQL reverse, 5′-AGCATGTTTGCAGCCTTCTTC-3′.An amplicon containing the RECQL gene variant data are available in the NCBI’s ClinVar archive (accession number SCV001364447.1).The l-glutamine, and 5% penicillin-streptomycin antibiotics (Merck). Primary fibroblasts were immortalized with a lentivirus expressing human telomerase reverse transcriptase (hTERT) that was generated by transfecting 293FT cells (Thermo Fisher Scientific) with the plasmids pLV-hTERT-IRES-hygro (Addgene no. 85140), psPax2 (Addgene no. 12260), and pMD2.G (Addgene no. 12259). Selection was performed using hygromycin (Thermo Fisher Scientific) at 70 μg/mL. All LCLs were routinely grown in RPMI-1640 (Thermo Fisher Scientific) supplemented with 10% FCS, 5% l-glutamine, and 5% penicillin-streptomycin. RECQL-KO HeLa cells were routinely grown in DMEM supplemented with 10% FCS, 5% l-glutamine, and 5% penicillin-streptomycin. Fibroblast and HeLa cell complementation was carried out using the pLVX-IRES-Neo lentiviral vector (Takara Bio) encoding 2xHA-tagged RECQL. All cell lines were routinely tested for mycoplasma.Dermal primary fibroblasts were grown from skin-punch biopsies and maintained in DMEM (Thermo Fisher Scientific) supplemented with 20% FCS, 5% Camptothecin (Merck), etoposide (Merck), hydroxyurea (MilliporeSigma), and methylmethane sulfonate (MilliporeSigma) were used as indicated.2 were added to the lysis buffer. Anti-RECQL1 antibody  or a nonspecific IgG (Dako) coupled with protein A agarose beads  were used to isolate protein complexes from 5 mg whole-cell extract.Whole-cell extracts were prepared and subjected to immunoblotting as previously described . Immunob2, 300 mM sucrose, and 0.5% Triton X-100) and then fixed with 4% paraformaldehyde for 10 minutes. Fixed cells were stained with primary antibodies against γ-H2AX , RAD51 , 53BP1 , RECQL1 , and mitosin , with secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 594 , and then with DAPI (VectaLabs). Images were visualized using a Nikon Eclipse Ni microscope with NIS-Elements software (Nikon Instruments) and captured using a 100× oil immersion objective.Cells were seeded onto coverslips 24 hours before extraction and fixation. Cytochalasin B  was added to the media for 24 hours to delete micronuclei. Cells were preextracted for 5 minutes on ice with ice-cold buffer  were incubated with the indicated concentrations of WT RECQL1 or RECQL1-A459S helicase proteins. Reactions were quenched with an equal volume of 2× stop dye  and incubated for an additional 15 minutes at 37°C to remove bound RECQL1 protein. Reaction products were resolved by electrophoresis on native 12% polyacrylamide gels at 200 V for 1.5 hours. Radiolabeled DNA products were visualized with a phosphoimager and quantified using ImageQuant software (Cytiva). For kinetics analysis of DNA unwinding, 19 bp or 31 bp forked duplex DNA substrates were incubated with the specified RECQL1 concentration and incubated for the indicated durations at 37°C. The rates of helicase-catalyzed DNA unwinding were determined by linear regression analysis.DNA helicase assays were performed as previously described . Briefly2) and quenched by addition of an equal volume of 2× stop dye  and incubated for an additional 15 minutes at 37 °C to remove bound RECQL1 protein. Reaction products were electrophoresed on native 12% polyacrylamide gels at 200 V for 1.5 hours. Radiolabeled products were visualized and quantified as described above.Strand annealing assays were performed as previously described . Briefly2, 0.1 mg/mL BSA, 2 mM DTT, 5% glycerol) in 100 μL reactions at 37°C. Aliquots of 20 μL were removed at the indicated times and were quenched by the addition of 10 μL 3X stop buffer , followed by incubation for at least 10 minutes at room temperature. Separate reactions containing either a reversed fork and replication fork structure without RECQL1 were incubated for 30 minutes at 37 °C followed by addition of the 3× stop buffer. Samples were immediately loaded onto an 8% polyacrylamide 1× TBE gel and electrophoresed for 4 hours at 200 V and 4°C. Gels were exposed to a phosphoimager screen overnight and then imaged using a Typhoon FLA 9500 imager (Cytiva). Gel images were quantified using ImageQuant TL software (Cytiva), and raw data were processed using Microsoft Excel. The percentage fork restoration was calculated by dividing the volume of the replication fork band by the total volume at each time point and subtracting the percentage of the fork restored in the no-enzyme control from each time point. Data representing 3 replicates of the assay were averaged and graphed.WT or A459S RECQL1 (20 nM) was incubated with 2 nM radiolabeled reversed fork substrate, 2 mM ATP, 15 mM phosphocreatine, and 30 u/mL creatine phosphokinase in reaction salts . Further approval for this research was obtained from the West Midlands, Coventry, and Warwickshire Research Ethics Committee .TH, VMP, AAL, and BAL identified the patients with the RECQL1 mutation and provided clinical information and photographs of the affected individuals. TH and VM analyzed the whole-exome sequencing data. KDB performed segregation analysis and established patient-derived LCLs. SSJ, SLC, and AD performed DNA fiber analysis. LJG, JJR, and RH performed flow cytometric, SCE, and chromosome breakage analyses. BLW performed growth and cytotoxicity assays on LCLs. GSM performed chromatin fractionation and immunofluorescence. SJS performed structural modeling. CNM and GLM generated the RECQL1 CRISPR-KO HeLa cell line. GSS generated complemented cell lines, carried out immunofluorescence and co-immunoprecipitation studies, and performed Western blotting. SD and JAS purified recombinant RECQL1 and performed in vitro protein biochemical assays. SD and AD performed cell-based DNA damage sensitivity assays and the comet assay. ANG generated RECQL1 expression vectors. GMCL and VR carried out C-Fusion 3D high-throughput imaging. GSS, SSJ, and RMB planned and supervised the study and wrote the manuscript.


High monocytes expressing pro-inflammatory cytokines, and second, infiltration of Ly6CLow monocytes with pro-resolving and pro-reparative function [High monocyte migration is driven by the presence of tissue CCL2 chemokine gradients and their interaction with their cognate receptor, CCR2 [LowCCR2High macrophages, known as monocyte-derived macrophages [LowCCR2Low macrophages deposited in the myocardial tissue during embryonic development [LowCCR2High and Ly6CLowCCR2Low) contribute to myocardial repair by clearance of dead tissue via efferocytosis and production of pro-reparative and pro-resolving mediators. Macrophage-derived cytokines play an essential role in the proliferation and activation of cardiac fibroblasts (fibroblast-myofibroblast conversion) that deposit collagen at the site of injury. This process of scar formation fulfills the immediate need to preserve the structural integrity of the myocardium, forestalling ventricular rupture [Myocardial infarction (MI) due to coronary artery stenosis compromises vascular endothelial integrity and increases vascular permeability ,2. Concufunction ,6,7. Ly6or, CCR2 —a group rophages ,10. Theselopment -13. Both rupture -16. Thus rupture -16. Hencin vivo [MI often leads to a substantial and irreversible loss of cardiomyocytes (sometimes upwards of 1 billion cells) ,3,16. Thin vivo -23. Althin vivo ,25. By ein vivo ,26-28. Tin vivo ,27,29,30In vivo and in vitro studies have shown that BM MSCs exert immunomodulatory effects on both innate and adaptive immunity [Neg, also known as N1, and CD206Pos, defined as N2 [in vitro that conditioned medium-derived from CMCs serves as a robust neutrophil chemoattractant, as well as an inhibitor of neutrophil apoptosis [The discovery that bone marrow mesenchymal stromal cells (BM MSCs) are intrinsically immune-privileged and ably suppress T cell proliferation continue to fuel scientific and clinical interests concerning their immuno-regulatory properties. immunity ,32. Saidimmunity ,34. Thouimmunity , Kang etimmunity . There iimmunity , injectied as N2 . In the ed as N2 , both N1ed as N2  leads uspoptosis . Althougpoptosis . It was High monocytes. Others have shown that MI itself results in the recruitment of Ly6CHigh monocytes, which follow a CCL2 chemokine gradient [Pos macrophages that contribute to myocardial repair [Low macrophages [Pos and CCR2Neg populations. In similar studies, Vagnozzi et al. [Pos CPCs or BM MNCs provokes the accumulation of CCR2Pos and CXC3R1Pos myocardial resident macrophages. Furthermore, the authors demonstrate that systemic depletion of macrophages prior to cell injection impairs cell therapy-induced myocardial repair [in vivo. We show that CMC-derived conditioned medium decreases pro-inflammatory, macrophage polarization in response to LPS and IFNγ, but induces a pro-resolving and pro-reparative program in response to IL-4 and IL-13 [in vivo studies to confirm whether this shift in macrophage polarization is responsible for the beneficial effects of CMC therapy. The primary function of macrophages is to remove necrotic tissue via efferocytosis [m vivo experiments.Notwithstanding, it is well recognized that macrophages are imperative for myocardial repair ,6,14. Ingradient ,10,38. Tl repair ,9,39. Werophages ; howeveri et al.  showed tl repair . Thus, tl repair ,40 suggend IL-13 ; howeverocytosis . We founocytosis . This isocytosis ,42. We hocytosis . Thus, oPosCD19PosIgDPosIgMLow mature phenotype [Pos regulatory T cells and also CD4Pos Th cells expressing IFNγ; indicating predominantly Th1-differentiated cells rather than Th2 or Th17 [in vitro indicate that BM MSCs are potent inhibitors of T cell proliferation, suggesting that cell therapy may also have an impact on adaptive immunity [Although the activation of innate immunity is a hallmark of tissue injury, recently, it was reported that the adaptive immune system is responsive to MI as well -46. Aftehenotype . At the  or Th17 ,49. Stud or Th17 ,44. Studimmunity -34. In oimmunity , we founimmunity . Hence, immunity ,44,49. Hin vivo studies elucidating changes in immune cell function in response to cell therapy. Furthermore, it would be imperative to design interventions aiming to understand how these changes affect myocardial repair after cell therapy. Although the Vagnozzi study [in vitro studies have characterized the impact of BM MSC-, CMC-, and CDC-derived secretomes on the phenotypic properties of various immune cell populations (i.e., neutrophils, dendritic cells, monocytes/macrophages, T and B cells [in vitro relate to the reparative potential of donor cells in vivo. Furthermore, one of the future challenges would be to identify how injection time after MI impacts the immune system. Currently, it is well known that MI elicits a temporally coordinated immune cell infiltration response, which undergoes dynamic changes over time until the process of replacement fibrosis concludes. The completion of scar formation culminates in the resolution of inflammation [in situ [Future studies should also address other important questions regarding the immunomodulatory properties of cell therapy. The cardiovascular field would benefit from zi study  demonstr B cells ,35,37,42ammation . An impoammation ,50. Ther[in situ . PerhapsStudies from us  and othe