MIR139 is a tumor suppressor and is commonly silenced in acute myeloid leukemia (AML). However, the tumor-suppressing activities of miR-139 and molecular mechanisms of MIR139-silencing remain largely unknown. Here, we studied the poorly prognostic MLL-AF9 fusion protein-expressing AML. We show that MLL-AF9 expression in hematopoietic precursors caused epigenetic silencing of MIR139, whereas overexpression of MIR139 inhibited in vitro and in vivo AML outgrowth. We identified novel miR-139 targets that mediate the tumor-suppressing activities of miR-139 in MLL-AF9 AML. We revealed that two enhancer regions control MIR139 expression and found that the polycomb repressive complex 2 (PRC2) downstream of MLL-AF9 epigenetically silenced MIR139 in AML. Finally, a genome-wide CRISPR-Cas9 knockout screen revealed RNA Polymerase 2 Subunit M (POLR2M) as a novel MIR139-regulatory factor. Our findings elucidate the molecular control of tumor suppressor MIR139 and reveal a role for POLR2M in the MIR139-silencing mechanism, downstream of MLL-AF9 and PRC2 in AML. In addition, we confirmed these findings in human AML cell lines with different oncogenic aberrations, suggesting that this is a more common oncogenic mechanism in AML. Our results may pave the way for new targeted therapy in AML. AF4, AF9, AF10, ENL, and ELL are most common [MLL to the AF9 gene in-frame and occurs in 30% of MLL-rearranged AMLs [Mixed-lineage leukemia (MLL) or Lysine Methyltransferase 2A (KMT2A) rearrangements occur in 10% of adult acute leukemias and in 35% of pediatric cases [t common . The mosged AMLs , 8.MIR139, encoding miR-139-5p and miR-139-3p, is downregulated and has been identified as a tumor suppressor in AML [MIR139 gene is located on chromosome-11 in intron-1 of PDE2A [MIR139 silencing in AML is largely unknown. Previously, we have found that miR-139-3p expression is induced by DNA interstrand-crosslinks [miR-139-5p controls the translation machinery through downregulation of Eif4g2 [MicroRNAs (miRNAs) are small non-coding RNAs of 19–22 nt in length and post-transcriptionally regulate mRNAs involved in critical cellular functions , 11. Forr in AML –14. The of PDE2A . Howeverosslinks . There iPolycomb-group (PcG) proteins have been implicated in silencing tumor suppressor genes in various cancers, including MLL-AF9 AML –19. One MIR139. We show that MIR139 is a critical tumor suppressor that is silenced by MLL-AF9 in AML. Furthermore, we have identified MIR139 targets that mediate its tumor suppressor activity in AML. Our study has elucidated the molecular silencing mechanism of MIR139 and provides insight into its tumor suppressor activity, offering potential targets of intervention for the poorly prognostic MLL-AF9 AML.Here, we have performed a comprehensive analysis of the tumor suppressor Mir139-deficient mice (Mir139KO) were generated using the CRISPR-Cas9 double-nicking strategy employed by the Cas9D10A mutant and 4 sgRNAs  flanking the Mir139 gene as described [C57BL/6 escribed . Enhancel-Glut (2 mM). AML cell line Kasumi-1 was cultured as above with 20% FBS and AML cell line TF-1 was cultured as above supplemented with human granulocyte-macrophage colony-stimulating factor  and sodium pyruvate (1%). The MLL-AF9 patient samples were cultured in Iscove’s Modified Dulbecco’s Medium, supplemented with FBS (20%), Pen-Strep (100 U/mL), IL-3 , IL-6 , TPO , FLT-3L , and SCF .For colony assays, 5000 MLL-AF9 cells were plated in MethoCult , supplemented with IL-3 (1:1000 supernatant), iIL-6 (10 ng/mL), granulocyte-macrophage colony-stimulating factor (GM-CSF) (10 ng/mL), and stem cell factor (SCF) (20 ng/mL), penicillin/streptomycin (100 U/mL) and neomycin 500 µg/mL and expanded for 7 days. DOT1L inhibitor SGC0946  or UNC1999  were dissolved in dimethyl sulfoxide (DMSO) and added to the medium at indicated concentrations. Human MLL-AF9 patients were diagnosed according to WHO criteria by cytomorphology, cytogenetics, molecular diagnostics and flow cytometric immunophenotyping. Mononuclear cells were isolated by Ficoll-Paque (density 1.077 g/mL) from peripheral blood or bone marrow. All included patients were positive for the t translocation and samples used for this study contained at least 84% AML blasts as defined by flow cytometry . The uset test, unless otherwise specified in the figure legends.Data are presented as mean ± SEM, unless otherwise specified in the figure legends. Data were compared with the two-tailed independent samples Student’s P < 2.74e−3), Hematological System Development and Function (P < 2.60e−3), and Hematological Diseases (P < 2.60e−3) were deregulated  HSPCs with MLL-AF9-expressing retrovirus. MLL-AF9 leukemia cells were positive for c-Kit, GR-1, CD16/CD32, and CD11b and negative for CD3 expression, suggesting a myeloid progenitor origin of the leukemia cells Fig. . As expeMIR139 is downregulated in AML patients including cases with MLL rearrangements and in THP-1 cells that express MLL-AF9 compared with normal HSPCs and myeloid cells including monocytes, macrophages, and neutrophils [Mir139 deletion collaborates with MLL-AF9 in AML outgrowth. Mir139KO mice developed normally and had expected HSPC counts and hematopoietic cell types in peripheral blood and BM [Mir139 influences genes that have potential effects on pathways other than those that are critical for hematopoiesis. We found that WT and Mir139KO HSPCs were indistinguishable at the transcriptome and proteome level, including Pde2a host gene expression  and proliferation (larger colonies) of MLL-AF9 cells -inducible miR-139-expressing MLL-AF9 clones (MLL-AF9-i139). DOX-treatment of MLL-AF9-i139 cells induced miR-139-5p (up to 130 Fold) and miR-139-3p (up to 100 Fold) expression  expansion, completely blocked colony outgrowth (LSK-MLL-AF9-i139) showed loss of cells upon miR-139 induction , a crucial co-factor for MLL-AF9 [Eed (three out of six sgRNAs) and Rbbp4 (five out of six sgRNAs), Dot1l (five out of six sgRNAs) and Mllt3 (four out of six sgRNAs) were lost  Hpgd (five out of six) and Eif4g2 (five out of six) were also lost, suggesting that expression of our identified miR-139-targets is essential for cell survival  or valporic acid (VPA), which are inhibitors of class-I and II histone deacetylases. We found that MLL-AF9 cells treated with either TSA or VPA, were significantly reduced in their colony-forming capacity compared with MLL-AF9-Mir139KO cells . As expected, H3K4me3 peaks overlapped with P and H3K27Ac peaks overlapped with enhancer region-1 (E1) and E2  and THP-1 (6.36%) are methylated  and E2 (60% and 87%) in Molm-13 cells and THP-1 cells, respectively  after 14 days of incubation , involved in stable transcriptional pausing of POL-II [Polr2m reduced colony outgrowth and cell viability of MLL-AF9 cells compared with MLL-AF9-139-KO cells  [MIR139   downstreMIR139 in MLL-AF9 leukemia. We describe that expression of miR-139 in MLL-AF9 cells caused apoptosis, abolished colony formation in vitro, and inhibited MLL-AF9 leukemia in vivo. Besides the previously published target Eif4g2 [Hpgd and Ptprt as novel miR-139-controlled targets of which depletion in MLL-AF9 AML at least in part phenocopied miR-139 expression. Our data suggest a mechanism of MIR139 silencing in MLL-AF9 AML in which PRC2 activity and recruitment of POLR2M at the E1, E2 inhibit MIR139 expression to promote oncogenesis , Cdkn2b (p15INK4B), Gata2 and Egr1, is a critical event in MLL-AF9 AML [Mir139 is a tumor suppressor that is targeted by PRC2 downstream of MLL-AF9. First, MLL-AF9 expression in HSPCs caused strong repression of Mir139 via PRC2. This is a critical step in oncogenic transformation as we demonstrated that miR-139 induction in MLL-AF9 cells caused apoptosis, loss of colony-forming capacity and inhibition of leukemogenesis in mice. We found that escaping AML cells in the DOX-treated mice have a miR-139 upregulation of only less than eightfold due to unknown reasons, in contrast to the 50-100-fold upregulation as found in vitro experiments. Apparently, an eightfold induction of miR-139 is not sufficient to cause apoptosis in the transplanted AML cells, allowing this aberrant sub-clone to give rise to full-blown leukemia. Second, genetic Mir139 deletion partly rescued the oncolytic effect of PRC2 inhibition in MLL-AF9 cells. Notably, the enhanced expansion capacity of MLL-AF9-Mir139KO cells compared with MLL-AF9 cells indicate that complete loss of Mir139 is a selective advantage for leukemia outgrowth in vitro. These results explain clinical data showing that AML patients with the lowest miR-139 expression levels have a poor prognosis [miR-139 expression in MLL-AF9 AML patient samples and other subtypes of AML, demonstrating direct silencing of Mir139 by PRC2 in AML, which is in agreement with previous findings in solid cancers [MIR139 silencing is a general mechanism in cancer and may be a key event in oncogenesis. Fourth, we identified Eif4g2, Ptprt, and Hpgd as miR-139 targets, which are involved in MLL-AF9 cell survival. Deregulation of these three genes by mutation, disruption of miRNA-mediated regulation or genomic aberrations are frequently found in different types of cancer [Eif4g2, caused by MIR139 silencing, enhances protein synthesis [Ptprt controls JAK/STAT signaling by dephosphorylation of STAT3, which is frequently dysregulated in human cancer, including AML [MIR139 silencing, STAT3β phosphorylation is potentially decreased, thereby contributing to MLL-AF9 oncogenesis. HPGD is an enzyme that converts PGE2 to 15-keto-PGE2 and has been associated with epithelial-to-mesenchymal transition and poor survival of patients with breast cancer and lung cancer [miR-139 expression in MLL-AF9-i139 cells induces loss of colony-forming capacity and apoptosis. Our gene expression data show that genes that inhibit cell cycle progression, such as Btg2 [, Cdkn1a [Cdkn2d [Bmf [Dusp1 [Rnasel [Trp53inp1 [Ypel3 [Zc3h12a [miR-139 expression and may explain this phenotype. Taken together, we conclude that MIR139 is silenced via PRC2 and that this silencing mechanism induces different pathways via miR-139 targets that contribute to cell survival and MLL-AF9 leukemogenesis.PRC2-mediated repression of tumor suppressor genes, including -AF9 AML , 23, 43. cancers , 45. Thif cancer –50. Incrynthesis , which iding AML , 52. Theding AML . Therefog cancer , 54. Fur as Btg2 , Cdkn1a , Cdkn1a , and Cdk [Cdkn2d , and genn2d [Bmf , Dusp1 [f [Dusp1 , Rnasel  [Rnasel , Trp53inrp53inp1 , Ypel3 [1 [Ypel3 , and Zc3[Zc3h12a  are upreMir139 silencing is independent of the transcriptional regulation of Pde2a. This may be explained by the high percentage of methylated CpGs within E1 and E2 in MLL-AF9 cells, whereas the upstream promoter region is relatively less methylated. PRC2 prefers binding to methylated DNA via AEBP2 [Mir139 silencing in MLL-AF9 cells. Thus, CpG methylation at these enhancers may be involved in the selective repression of Mir139, leaving the Pde2a host gene expression intact.Our data indicate that ia AEBP2 . AccordiPde2a expression and intron-1 splicing did not result in miR-139 expression in MLL-AF9 cells. These data suggest that in MLL-AF9 cells pri-miR-139 is degraded. We did not observe miR-139 induction upon PRC2 inhibition in MLL-AF9-E1KO cells and a reduced miR-139 induction in MLL-AF9-E2KO, suggesting that factors involved in miR-139 stability and processing may be recruited to the enhancers. Enhancer regions may play critical roles in the transcriptional regulation of miRNAs in cancer [MIR139. This interaction may occur under cellular stress conditions resulting in increased miR-139 expression and cell death to prevent oncogenic transformation.We found that n cancer . A recenn cancer . In agreMIR139 expression. POLR2M interacts with the POL-II complex to pause transcription [MIR139 expression in MLL-AF9.POL-II is the key factor responsible for the transcription of miRNAs . Althougcription , 68, 69.cription –72. Pauscription . TogetheMIR139 in AML. First, POLR2M depletion in MLL-AF9 patient samples and human AML cell lines induced the expression of pri-miR-139. Although the levels of the mature miR-139-5p and miR-139-3p are low compared with experiments with UNC1999-mediated repression of PRC2, this could be explained by the fact that the CRISPR-Cas9-mediated depletion of POLR2M by lentiviral transduction is an asynchronous process. This causes a heterogeneous induction of MIR139 expression and subsequent cell death, thereby underestimating miR-139 levels. Second, POLR2M-depleted MLL-AF9 cells were lost in our CRISPR-Cas9 screen and validation experiments, whereas POLR2M-depleted MLL-AF9-Mir139KO cells survived, indicating that POLR2M-controlled survival is specifically mediated by the silencing of MIR139. Third, we showed that POLR2M is bound to the E1 and E2 regions and TSS of MIR139. Depletion of POLR2M at these loci was observed as a result of UNC1999-mediated inhibition of PRC2 and coincides with induction of miR-139 expression and cell death.Our data demonstrate in different ways that POLR2M is a direct silencer of MIR139 in MLL-AF9 leukemia and report functionally relevant targets of miR-139-mediated cell death of MLL-AF9 cells. Furthermore, our findings reveal a PRC2-dependent POLR2M-mediated silencing mechanism of the MIR139 tumor suppressor in MLL-AF9 leukemia. The specific interaction of POLR2M with the POL-II complex is essential for transcriptional inhibition [MIR139 expression and eliminating AML. Together, our findings highlight the importance of POLR2M-mediated silencing of MIR139 in MLL-AF9 AML.In conclusion, we have identified key molecular mechanisms that control tumor suppressor hibition . TherefoSupplementary InformationSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Table 6


The Tripartite motif (TRIM) protein family, which contains over 80 members in human sapiens, is the largest subfamily of the RING-type E3 ubiquitin ligase family. It is implicated in regulating various cellular functions, including cell cycle process, autophagy, and immune response. The dysfunction of TRIMs may lead to numerous diseases, such as systemic lupus erythematosus (SLE). Lots of studies in recent years have demonstrated that many TRIM proteins exert antiviral roles. TRIM proteins could affect viral replication by regulating the signaling pathways of antiviral innate immune responses. Besides, TRIM proteins can directly target viral components, which can lead to the degradation or functional inhibition of viral protein through degradative or non-degradative mechanisms and consequently interrupt the viral lifecycle. However, new evidence suggests that some viruses may manipulate TRIM proteins for their replication. Here, we summarize the latest discoveries on the interactions between TRIM protein and virus, especially TRIM proteins’ role in the signaling pathway of antiviral innate immune response and the direct “game” between them. Ubiquitination is an extraordinary essential post-transcriptional modification process that functions in both innate and adaptive immune responses. The key enzymes required for the ubiquitination process are classified into ubiquitin-activating enzyme E1, ubiquitin-conjugating enzymes E2, and ubiquitin ligases E3  Figure . The E3 The tripartite motif (TRIM) proteins are a highly conserved superfamily of proteins, which is the largest subfamily of the RING-type E3 ubiquitin ligase family . TRIM prIn some cases, this domain can even exert the E3 ubiquitin ligase activity [such as TRIM18 ]. The foBesides the domains at amino-terminus, those at carboxyl-terminus are also diverse, including SPRY-associated domain (PRY), SPIa and the ryanodine receptor domain (SPRY), C-terminal subgroup one signature domain (COS), fibronectin type III repeats domain (FNIII), etc. According to the various domains in the carboxyl-terminal region, TRIM protein family members are divided into 11 subgroups from Class I to Class XI comprising about 80 members. Besides, there is an unclassified group lacking the RING domain (no RING) . TRIM23,via the lysosomal or proteasomal pathways  are sensed by pattern recognition receptors (PRRs) of innate immune cells, including retinoic acid-induced gene-I-like receptor (RLRs), Toll-like receptors (TLRs), and cytosolic DNA receptors . TriggerAt present, many studies have shown that there are mainly three innate immune signal pathways regulated by TRIM protein, namely, TLR signaling pathway, RLR signaling pathway, and cyclic GMP-AMP synthase (cGAS)- stimulator of IFN genes (STING) pathway.via adaptor molecule Myeloid differentiation primary response gene 88 (MyD88). TRAF6 catalyzes auto-ubiquitination. Ubiquitinated TRAF6 recognizes TAK1/MAP3K7-binding protein 2 (TAB2) and activates TGF-β-activated kinase 1 (TAK1), and ultimately results in activation of IκB kinase α/β/γ (IKKα/β/γ) and NF-κB (via adaptor molecule TRIF (TIR-domain-containing adaptor inducing IFNβ), but not MyD88, which leads to activation of transcription factors interferon regulatory factor 3 (IRF3) and interferon regulatory factor 7 (IRF7) through TNF receptor-associated factor 3 (TRAF3) and transforming growth factor-β- activated kinase 1/IκB kinase ϵ (TBK1/IKKϵ) , TLR3 subfamily, TLR4 subfamily, TLR5 subfamily, TLR7 subfamily , and TLR11 subfamily . TLR3, TLR7, TLR8, and TLR9 are mainly involved in viral recognition . Upon bind NF-κB . TLR3 isK1/IKKϵ) . A lot oK1/IKKϵ) . TRIM32 K1/IKKϵ) . TRIM38 K1/IKKϵ) , TRAF6 (K1/IKKϵ) , nucleosK1/IKKϵ) , TAB2/3 K1/IKKϵ) , and theK1/IKKϵ) . BesidesK1/IKKϵ) . Similar of TAK1 .RLR consists of three members: retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated protein 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2). RLRs harbor two N-terminal caspase recruitment domains (CARDs) excepting LGP2, a central DExD/H-box helicase domain and a C-terminal regulatory domain (RD). With the unique DExD/H-box RNA helicase activity, they could sense single- or double-stranded RNA from the virus to initiate immune response . When RIvia the proteasome -TRIM14-protein phosphatase PPP6C, which is essential for RIG-I K63 ubiquitination and activation of RIG-I-mediated innate antiviral immunity . During Studies have confirmed that the cGAS-STING signaling pathway mainly recognizes DNA viruses. The cGAS possessing anti-retroviral activity is essential for the recognition of DNA viruses. Upon recognizing viral DNA, cGAS produces the second messenger cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), which subsequently activates STING and induce type I interferon production . TRIM38 Besides, many TRIM proteins directly regulate the downstream interferon signaling pathway, which can work in coordination with the three signaling pathways mentioned above to precisely regulate the antiviral immune response.Upon viral invasion, the sensors in the cell membrane and the cytoplasm could recognize the virus to initiate a series of response mechanisms. TRIM proteins play a regulatory role in the innate viral immune response pathway and directly antagonize the virus. TRIM5α, which can inhibit the infection of retroviruses such as Human Immunodeficiency Virus 1 (HIV-1) and N-tropic murine leukemia virus (N-MLV), maybe the first and the most widely-studied protein in these aspects . AccordiFlaviviridae family, including Bovine viral diarrhea virus (BVDV), dengue virus serotype 2 (DENV2), and yellow fever virus (YFV)  OC43 replication  . Beyond lication . Furtherlication . C-termilication . Similarlication . TRIM22 lication . Besideslication . TRIM19,lication . TRIM19 lication . TRIM14 lication . TRIM52 lication .via endocytosis  NS1 protein could interact with the SPRY domain of TRIM25, interfering with TRIM25-mediated K63-linked polyubiquitination on the CARD domain of RIG-I to inhibit its signal transduction and preventing antiviral immune response . The samExcept for defensive, viruses can “domesticate” TRIM proteins for their replications as well. For example, TRIM21 is induced in JEV-infected human microglia cells CHME-3 to inhibit IRF3-mediated IFNβ production, thereby preventing antiviral immune response . EpsteinXenopus laevis nuclear factor 7 (XNF7)—was identified in 1991 (via the phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway , Scientific and Technological Research Project of Division of Science and Technology, Department of Education of Hebei Province (grant number are QN2018146 and QN2017105), and the Science and Technology Planning Project of Hebei Province (H2019206614) and The Sping Rain Program of Hebei Medical University (CYQD201812).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.


Staphylococcus aureus 17Gst354, isolated from a nasal swab. The strain possessed an additional 4,397-bp plasmid. Moreover, we induced and sequenced its temperate phage Staphylococcus phage vB_StaphS-IVBph354, which has a circular genome of 41,970 bp.We report the complete 2,783,931-bp circular genome sequence of the human methicillin-resistant strain  Staphylococcus aureus (MRSA) strain 17Gst354 was isolated from a nasal swab of a healthy Swiss farmer in 2017 (Switzerland) (The livestock-associated (LA) methicillin-resistant Staphylococcus phage vB_StaphS-IVB354 was purified from strain 17Gst354 through three successive single-plaque isolations after mitomycin C induction, as previously reported  and 3,044,780 paired-end short reads obtained previously (SRX6491214) were used to assemble the complete 2,783,931-bp chromosome  of strain 17Gst354 using Unicycler v0.4.4  type IVa(2B), as determined using SCCmecFinder v1.2 (https://cge.cbs.dtu.dk/services/SCCmecFinder/)], and blaZ (beta-lactamase), as well as 59 tRNAs and a set of six 5S, five 16S, and five 23S rRNAs. A 4,397-bp plasmid was also present. A BLASTN analysis  was determined using the coverage command of SAMtools v1.11  (attB site (5′-TGTATCCAAACTGG-3′) and a frameshift insertion (5′-GAGCGAAAGA-3′), which extend the corresponding ORF .Phage vB_StaphS-IVBph354 has a genome of 41,970 bp with a G+C content of 33% and includes 65 ORFs. Whole-genome alignment using EMBOSS Stretcher  showed t48634.1) , except SRX6491214 (strain 17Gst354) and SRX10584153 (Staphylococcus phage vB_StaphS-IVBph354). The PacBio reads are available under SRX10576957. The assemblies can be accessed under the GenBank accession numbers MW924889 (Staphylococcus phage vB_StaphS-IVBph354), CP073065 (strain 17Gst354), and CP073064 (17Gst354 plasmid).The Illumina reads are available under the following SRA accession numbers:


Further studies indicated that the PCV4 Cap NoLS exploited DDX21 to facilitate its nucleolar localization. In summary, our results firstly demonstrated that DDX21 binds directly to the NoLS of the PCV4 Cap thereby contributing to the nucleolar localization of the PCV4 Cap protein.Porcine circovirus type 4 (PCV4) is a newly emerging pathogen which might be associated with diverse clinical signs, including respiratory and gastrointestinal distress, dermatitis, and various systemic inflammations. The host cellular proteins binding to PCV4 capsid (Cap) protein are still not clear. Herein, we found that the PCV4 Cap mediated translocation of DEAD-box RNA helicase 21 (DDX21) to the cytoplasm from the nucleolus and further verified that the nucleolar localization signal (NoLS) of the PCV4 Cap bound directly to the DDX21. The NoLS of PCV4 Cap and  Circovirus in the family Circoviridae (Porcine circoviruses (PCVs) belong to non-enveloped viruses containing single-stranded circular DNA genomes (∼1.7–2.0 kb) within genus oviridae . Four geoviridae . PCV1 isoviridae . PCV3 waoviridae . PCV4, aoviridae . Since toviridae , indicatoviridae .Circoviruses depend on the host cellular replication machinery for viral genome synthesis owing to shortage of autonomous DNA polymerases . As for DEAD-box RNA helicases are a myriad of multifunctional enzymes that control multiple processes of RNA metabolism, such as transcription, processing, and modification . Sometim763GSRSNRFQNK772 residues at the CTD of DDX21 are essential for the PCV4 Cap/DDX21 interaction. In addition, the PCV4 Cap NoLS deployed DDX21 to promote its nucleolar localization. To sum up, these results firstly uncovered that DDX21 directly interacts with the PCV4 Cap NoLS and this interaction is critical for nucleolar retention of the PCV4 Cap protein.In this study, we found that DDX21 traffics to the cytoplasm from the nucleolus induced by the PCV4 Cap overexpression and the NoLS of the PCV4 Cap and Although DDX21 is known to be a nucleolar protein within the DEAD-box RNA helicase family, its localization can be altered upon certain types of stimulation . For theTo further explore the PCV4 Cap-induced DDX21 redistribution, Flag-PCV4-Cap-transfected PK-15 cell lysates were immunoprecipitated with anti-Flag beads and probed for DDX21 protein with anti-DDX21 mAb, showing that PCV4 Cap bound to the endogenous DDX21 protein . ConsistTo map the domain (mainly NoLS) in PCV4 Cap essential for binding to DDX21, plasmids GFP-PCV4-Cap-WT, -M1, and -M2 or Flag-gst-PCV4-Cap-WT, -M1, and -M2 were, respectively cotransfected into HEK293T cells together with Flag-DDX21. Reciprocal Co-IP and GST pull-down assays indicated that amino acids (aa) 1–37 (M2) and the full-length PCV4 Cap (WT) could bind to DDX21, whereas aa 38–228 (M1) of PCV4 Cap were not able to interact with DDX21 . These rDDX21 carries a N-terminal domain, a highly conserved central helicase domain, and a varied C-terminal domain . To iden1 and NLS Mapper2).1,2 A potential NoLS (763GSRSNRFQNK772) was predicted at the C-terminal of DDX21. Therefore, we cotransfected a series of mutants of GFP-DDX21-CTD, including GFP-DDX21-CTD-M7-(582–772 aa), GFP-DDX21-CTD-M8-(582–762 aa), and GFP-DDX21-CTD-M9-(763–772 aa) into HEK293T cells, and subjected to Co-IP and GST pull-down assays with Flag-PCV4-Cap or Flag-gst-PCV4-Cap. The results indicated that the mutants GFP-DDX21-CTD-M7-(582–772 aa) and GFP-DDX21-CTD-M9-(763–772 aa) bound with Flag-PCV4-Cap or Flag-gst-PCV4-Cap as well as the DDX21-CTD. However, GFP-DDX21-CTD-M8-(582–762 aa) mutant did not interact with PCV4 Cap  and a non-targeting control shRNA (shCON) were transferred via lentivirus-mediated shRNA to produce cells stably expressing GFP-shRNAs. The results demonstrated that the fluorescence intensity was remarkably reduced in the shDDX21-transfected PK-15 cells compared with that in the shCON-transfected cells (p > 0.05). The DDX21-silenced and control and DDX21-overexpressed PK-15 cells were transfected with GFP-PCV4-Cap for 24 h to investigate the subcellular localization of PCV4 Cap. Confocal imaging demonstrated that PCV4 Cap represented predominant nucleolar localization in shCON-transfected cells, and relocated to the nucleoplasm in DDX21-silenced cells, and restored nucleolar distribution in the DDX21-overexpressed cells  specific for ed cells . Westerned cells . Moreoveed cells . Taken tIn a silent state, DDX21, along with its binding ligands c-Jun, SIRT7, and WDR46, is distributed in the nucleolus . The nucThe NLSs of viral proteins are deemed as fundamental elements . Certain763GSRSNRFQNK772 of DDX21-CTD is crucial for interaction with the PCV4 Cap NoLS   supplemented with 10% fetal bovine serum (FBS) (Gibco). HEK293T cells  were maintained in Dulbecco’s modified Eagle medium (DMEM) (Gibco) as described elsewhere ,2021.Mouse monoclonal antibodies (mAbs) against GST (M0807-1), histone H3 (R1105-1), and β-actin (M1210-2), as well as rabbit polyclonal antibodies (pAbs) against Myc (R1208-1), Flag (0912-1), β-tubulin (0807-2), and GFP (SR48-02) were purchased from Huaan Biological Technology . Mouse anti-Flag (F1804) and anti-Myc (05–419) mAbs were obtained from Sigma-Aldrich . Rabbit mAb against DDX21 (ab182156) was obtained from Abcam . Anti-Flag affinity resin (A2220) for immunoprecipitation was purchased from Sigma-Aldrich. NP-40 cell lysis buffer  was obtained from Beyotime . Horseradish peroxidase (HRP)-labeled goat anti-mouse and anti-rabbit IgG or fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG were purchased from KPL . Alexa Fluor 546-conjugated donkey anti-rabbit IgG was obtained from Invitrogen (United States).Cap DNA fragments were amplified by polymerase chain reaction (PCR) from synthetic PCV4 genomic DNA (accession no. MK986820.1)  by Sangon Biotechnology . The NoLSs within capsid protein of different porcine circoviruses genotypes are listed in DDX21 (accession no. KX396051.1) and its truncated DDX21 variants amplified from PK-15 cells were cloned into vectors pCMV-Flag-N, pGEX-4T-1 , or pEGFP-C3 to obtain plasmids Flag-DDX21, GST-DDX21, GFP-DDX21-WT-(1–784 aa), GFP-DDX21-M1-(1–217 aa), GFP-DDX21-M2-(218–581 aa), GFP-DDX21-M3-(582–784 aa), GFP-DDX21-M4-(1–581 aa), GFP-DDX21-M5-(218–784 aa), GFP-DDX21-M6-(1–217 aa + 582–784 aa), GFP-DDX21-M7-(582–772 aa), GFP-DDX21-M8-(582–762 aa), or GFP-DDX21-M9-(763–772 aa). The detailed procedures for plasmids construction were conducted as described elsewhere (Full-length and truncated PCV4 86820.1) , and inslsewhere ,b. The pPK-15 cells were transfected with plasmids pcDNA3.0-PCV4-Cap or mCherry-PCV4-Cap. The cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min and then permeabilized with 0.1% Triton-X 100 for 10 min at room temperature. Mouse anti-PCV4 Cap pAb and rabbit anti-DDX21 mAb were used as primary antibodies, and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG and Alexa Fluor 546-conjugated donkey anti-rabbit IgG were used as secondary antibodies. Cellular nuclei were stained with 10 μg/ml 4′, 6′-diamidino-2-phenylindole  to obtain images using a Nikon Al confocal microscope.Cell lysate extracts prepared in lysis buffer after transfection were separated by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes  followed by being blocked in phosphate-buffered saline (PBS) containing 5% skimmed milk powder and 0.05% Tween 20. The membranes were then incubated with primary antibodies overnight at 4°C followed by incubation with HRP-labeled secondary antibodies at room temperature for 1.0 h. The membranes were then incubated with enhanced chemiluminescence reagent  and the immunoreactive protein bands were visualized using AI800 Images .g for 10 min. The supernatants were treated with protein A/G plus agarose  for 1.0 h at 4°C and then immunoprecipitated using anti-Flag beads. The beads were washed with NP-40 buffer and then revolved by the standard SDS-PAGE. For GST pull-down assays, the expression of His-sumo-PCV4-Cap, GST, or GST-DDX21 in Escherichia coli BL21 cells was induced using 0.2 mM isopropyl-β-D-thiogalactopyranoside (Amersham) for 16 h at 16°C. GST and GST-DDX21 were obtained through incubation with Pierce glutathione agarose beads  and were eluted with 1.0 ml of cold 1 × PBS per 10 mg of reduced glutathione. His-sumo-PCV4-Cap was purified using NTA agarose affinity resin  and eluted using an imidazole concentration gradient and used as the prey protein. Equal amount of purified GST or GST-DDX21 proteins which were immobilized on glutathione agarose beads, were incubated with the corresponding prey proteins at 4°C for 6.0 h. The precipitated proteins were washed with PBS, subjected to SDS-PAGE and western blotting using mouse mAbs against GST and His. The procedures for Co-IP and the unconventional GST pull-down assays were conducted as described elsewhere (For co-immunoprecipitation (Co-IP) assays, HEK293T cells transfected with the indicated plasmids for 48 h were lysed in NP-40 cell lysis buffer and centrifugated at 12,000 × lsewhere ,b.g for 5.0 min, the supernatants of the samples were collected as the cytoplasmic fractions, while the precipitate was lysed with strong lysis buffer and used as the nuclear fractions. Western blotting analysis was performed using mouse pAb anti-PCV4 Cap, mouse mAb against histone H3, and rabbit pAb against β-tubulin.Isolation of nuclear and cytoplasmic components was performed as stated in our previous study with moderate modifications . AccordiDDX21 knockdown was performed as previously described  were designed using siRNA design software and cloned into the lentivector pGreenPuro shRNA vector . After transfection and selection, an effective shDDX21 (targeting sequence: GCAGAGACTTCAGTGACATCA) and shCON, which was developed previously   assay according to the manufacturer’s protocol.escribed  with slieviously , were cot-test. p-values of < 0.05 were considered significant.All results are presented as means ± standard deviations (SD). Statistical analysis was performed using Student’s The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.JZ conceived, designed, performed the experiments, interpreted the data, and wrote the manuscript. JZ performed the experiments with assistance and advice from YW, YQ, YxW, XY, CL, YS, XF, and LH. JL revised the manuscript. All authors have read and approved the submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Ctnnb1OE-SEC) generated by using a BM-SEC-restricted Cre mouse line (Stab2-iCreF3) develop fatal anemia. While activation of Wnt-signaling in BM-SEC causes an increase in erythroblast subsets (PII–PIV), mature erythroid cells (PV) are reduced indicating impairment of terminal erythroid differentiation/reticulocyte maturation. Transplantation of Ctnnb1OE-SEC hematopoietic stem cells into wildtype recipients confirms lethal anemia to be caused by cell-extrinsic, endothelial-mediated effects. Ctnnb1OE-SEC BM-SEC reveal aberrant sinusoidal differentiation with altered EC gene expression and perisinusoidal ECM deposition and angiocrine dysregulation with de novo endothelial expression of FGF23 and DKK2, elevated in anemia and involved in vascular stabilization, respectively. Our study demonstrates that BM-SEC play an important role in the bone marrow microenvironment in health and disease.Within the bone marrow microenvironment, endothelial cells (EC) exert important functions. Arterial EC support hematopoiesis while H-type capillaries induce bone formation. Here, we show that BM sinusoidal EC (BM-SEC) actively control erythropoiesis. Mice with stabilized β-catenin in BM-SEC ( Niche crosstalk with Haematopoietic cells underlies normal haematopoiesis and myeloid disorders. Here the authors report a Stabilin2-Cre driver mouse with Cre-activity restricted to bone marrow sinusoidal endothelial cells, and that Stabilin2-Cre driven overactivation of b-catenin leads to erythroid differentiation defects and anaemia. Multiple cell types contribute to the BM niche, including mesenchymal stromal cell populations (MSC) and their progeny such as osteoblasts and perivascular stroma cells, but also macrophages as well as endothelial cells (ECs)3. The murine BM vasculature displays remarkable angiodiversity. Distinct vessel types within the BM niche comprise arterial endothelial cells (AEC), H-type capillary vessels, and BM sinusoidal (L-type) endothelial cells (BM-SEC) that contribute to osteogenesis, bone angiogenesis, and HSPC maintenance and differentiation via angiocrine signaling9. EC-derived stem cell factor (Scf) is selectively secreted by AEC, and genetic deletion of Scf in AEC, but not BM-SEC, significantly reduces functional HSPC10. H-type capillary vessels contribute to bone formation, vascularization, and regeneration via Notch signaling11. BM-SEC are the most abundant EC population in the BM, activate HSPC, and regulate BM trafficking4. E-type ECs and Apelin-expressing ECs are additional, specialized BM-EC subpopulations that control vascular development, or vascular regeneration and hematopoiesis after myeloablative injury, respectively13. The recent improvements of single-cell transcriptomics and refined flow cytometry sorting techniques have given new insights into the molecular profile of the BM vascular niche and the BM microenvironment (BMME), thereby identifying decisive gene signatures for AEC and BM-SEC14. The balance between AEC, H-type vessels and BM-SEC fluctuates; upon ageing, H-type vessels regress entailing reduced hematopoiesis and osteogenesis6. Notably, endothelial Notch activation can revert this process inducing arterialization and H-type capillary expansion11.Crosstalk between hematopoietic stem and progenitor cells (HSPCs) and cells of the bone marrow (BM) microenvironment is increasingly recognized to regulate normal hematopoiesis as well as development of myeloid disorders15. Patient-derived MSC were demonstrated to exhibit cytogenetic abnormalities that are distinct from their hematopoietic counterparts and to promote the malignant behavior of stem cells and their progeny in human myelodysplastic syndrome (MDS)16. In mice, for example, deletion of retinoic acid receptor-γ17 and activation or derepression of Wnt signaling in the BMME19 have been shown to promote the development of myeloproliferation or myelodysplasia. Regarding Wnt signaling, osteoblasts genetically engineered to specifically express a constitutively active mutant allele of Ctnnb1 using the α1(I)Collagen-Cre driver develop acute myeloid leukemia due to the upregulation of the Notch ligand Jagged 118. In the Apc haploinsufficiency MDS model in which Wnt signaling in the BMME causes ineffective hematopoiesis, the Mx1-Cre driver was used to target MSC20. Conflicting results, however, have been reported regarding targeting of other niche cells, especially EC by Mx1-Cre23. In analyzing published data on the regulation of HSPC interactions with the BM niche by Wnt-β-catenin signaling, Lane and Heidel therefore suggested to further dissect the specific contributions of MSC or EC potentially mediating Wnt-dependent myelodysplasia24. In order to be able to explore the distinct functions of these latter cellular participants within the BMME in vivo, however, the lack of suitable, highly specific Cre-driver mice constitutes a major obstacle. Unfortunately, most EC-targeted driver Cre mice target all EC as well as hematopoietic stem cells and part of their progeny22, and the EpoR-Cre driver used to target BM-SEC is expressed also in erythroid cells10.Despite these essential data on the involvement of BM-EC in normal hematopoiesis, evidence for the involvement of BM-EC in the pathogenesis of hematological disorders is scarce. Notably, emerging data suggest that alterations of the BM niche as opposed to cell-intrinsic factors do play a role in the pathogenesis of myeloid neoplasmsCre driver mouse under the control of the Stabilin-2 (Stab2) promotor. Stab2 is known to be specifically expressed by BM-SEC, but also by SEC of other organs, and recent studies confirmed Stab2 to be the gene most strongly overexpressed in BM-SEC versus AEC14. In contrast to the Stab2-iCre Founder 2 mouse (Stab2-iCreF2) reported previously26, the Stab2-iCre Founder 3 (Stab2-iCreF3) line shows restricted Cre activity in BM-SEC without reporter activity in HSPC and AEC. Here, we show that BM-SEC genetically engineered to specifically express a constitutively active mutant allele of Ctnnb1 using the Stab2-iCreF3 driver mouse line (Stab2-iCreF3tg/wtCtnnb1(Ex3)fl/wt) exhibit aberrant sinusoidal differentiation and cause lethal anemia due to a block in terminal erythroid differentiation.In order to comprehensively analyze the effects of BM-SEC-restricted activation of Wnt signaling within the BM niche, we generated a iCre driver mouse line was generated using the promoter of the Stabilin2 (Stab2) gene specifically expressed in SECs of the BM, the liver, the spleen and the lymph nodes (Stab2-iCreF3). In contrast to the Stab2-iCreF2 driver mice generated by us previously25, the reporter activity of Stab2-iCreF3;R26YFP mice was restricted to SECs with endothelial Stab2 expression in adult animals28. In comparison to SEC in liver, spleen, and lymph nodes , whereas embryonic day (E)17.5 embryos and P0 pups were YFP negative fl/fl mice29 (Stab2-iCreF3tg/wtCtnnb1(ex3)fl/wt [Ctnnb1OE-SEC])   of the BM of Ctnnb1OE-SEC mice further revealed accumulation of early erythroid progenitors and irregularly shaped reticulocytes outside the lumina of the sinusoids and an obvious lack of reticulocytes and mature erythrocytes beneath and inside the sinusoidal lumina  Fig. . Ctnnb1Orit Fig. . The mearit Fig.  indicatiund Fig. . BM histund Fig.  and smeaund Fig.  showed aina Fig. . Furtherice Fig. .Fig. 2AcCtnnb1OE-SEC mice developed excessive splenomegaly with strongly increased spleen weight as well as increased spleen to bodyweight ratio  of MEP in the BM, while numbers of HSC, ST-HSC, multipotent progenitors (MPP), GMP, and CMP were normal compared to Ctnnb1WT littermates  were present in the BM of Ctnnb1OE-SEC mice, while no differences were observed for CD11b+Gr1- cells (mostly monocytes). Ly6C+CD115+ cells (mostly monocytes/macrophages) were significantly increased in the BM of our mutant mice  to orthochromatic erythroblasts and immature reticulocytes (PIV). On the contrary, the numbers of mature erythroid cells (PV) were significantly reduced suggesting a block in erythroid differentiation between PIV and PV  and 1962 (PIV) genes, respectively , as well as of orthochromatic erythroblasts and immature reticulocytes (PIV) of ice Fig. . Remarkaice Fig. . On the lls Fig. .Ctnnb1OE-SEC stem and progenitor cells to cause fatal anemia, we performed transplantation experiments with HSC derived from Ctnnb1OE-SEC donor or control Ctnnb1WT donor mice (CD45.2) transplanted into lethally irradiated wildtype (CD45.1) recipients. Ctnnb1OE-SEC or Ctnnb1WT donor BM recipients did not show differences in survival until final examination at 6 months after transplantation  analysis demonstrating significant upregulation of Wnt target genes Axin2 and Apccd1, while Lef1 (p = 0.055) or TCF7 (p = 0.066) showed a tendency toward higher expression levels 2 and Fibroblast growth factor (FGF)23 were significantly upregulated  were significantly increased in 5-month-old Ctnnb1OE-SEC mice -SEC genes, induction of continuous EC and AEC genes, and increased deposition of perisinusoidal Collagen I, III, IV, and Fibronectin40. Whereas H-type vessels have been shown to be less permeable for cells and solutes as compared to L-type vessels and are surrounded by pericytes41, electron microscopy studies demonstrated that egress of BM cells into the bloodstream occurs particularly at sites where pericytes are absent42, such as in L-Type BM-SEC. Furthermore, a lower number of tight junctions in BM-EC might facilitate egress of BM cells into the bloodstream via interdigital sliding of EC junctions43. Structural and molecular changes in BM-SEC of Ctnnb1OE-SEC mice and subsequently in the BMME may contribute to the development of lethal anemia either by inhibiting terminal erythroid differentiation and/or by influencing the process of reticulocyte egress and maturation. We found numerous pathological, square-edged, polygonal reticulocytes within the BM stroma close to the abluminal surface of the BM sinuses and we detected immature reticulocytes with nuclear remnants in the circulation. Therefore, we propose that reticulocytes are released into the circulation in Ctnnb1OE-SEC mice before they have matured to the stage when they are normally released. These pathological reticulocytes obviously cannot accomplish definitive maturation into red blood cells, and thus may be prone to be easily destroyed. Furthermore, hematopoietic cells other than erythrocytes also managed to egress in Ctnnb1OE-SEC mice. Therefore, mechanisms other than impaired egress had to be considered to cause fatal anemia in Ctnnb1OE-SEC mice such as de novo secretion of angiocrine inhibitors or loss of angiocrine inducers of terminal erythroid differentiation and reticulocyte maturation.We show here that BM-SEC actively control erythropoiesis. β-catenin overactivation in BM-SEC induced a shift in vascular differentiation from EMCNCtnnb1OE-SEC mice. FGF23 was previously reported to be elevated in serum of MDS patients and cFGF23 correlated negatively with their hemoglobin levels36. While the expression of FGF23 is detected in many different tissues during development, the bone is recognized as the main source of circulating FGF23 during adulthood under normal health conditions44. Confirming our findings, upregulation of BM endothelial FGF23 as well as continuous EC markers Pecam1 and Apln was demonstrated after irradiation in H-type and E-type capillary vessels, but not BM-SEC45. Hitherto, there is no evidence for a direct link between FGF23 expression and Wnt-/β-catenin signaling in BM-SEC. In the heart, however, FGF23 was shown to activate β-catenin and to induce collagen and fibronectin expression in fibroblasts as was also seen in Ctnnb1OE-SEC mice47. Bone-derived FGF23 negatively regulates erythropoiesis in a paracrine manner and the FGF23 receptors and its coreceptor Klotho are expressed in erythroid precursor cells in a murine MDS model48. In the human erythroid cell line BEL-A in vitro, no direct effects of FGF23 on erythroblasts were detected when analyzing erythroid expansion, viability, apoptosis, and enucleation. Nevertheless, FGF23 may well contribute to the complex pathogenesis of lethal anemia in Ctnnb1OE-SEC mice in vivo in a direct or indirect manner or in concert with additional features instructed by aberrantly differentiated BM-SEC such as membrane charge, adhesion molecules, or other angiocrine factors. Therefore, FGF23 should be further investigated as a promising target using FGF23-deficient mice or anti-FGF23 antibodies in Ctnnb1OE-SEC mice and in murine and human MDS. In addition to FGF23, DKK2 was identified as an angiokine induced de novo in transdifferentiated BM-SEC in Ctnnb1OE-SEC mice. DKK2 is known to induce angiogenesis and to promote vascular stabilization and pericyte coverage during vessel maturation. Thus, we hypothesize that DKK2 may be involved in causing aberrant differentiation of BM-SEC to a tighter type of endothelium in an autocrine manner.Notably, we found increased levels of FGF23 and cFGF23 in the serum of 49. Here, we provide a mouse model to specifically study BM-SEC in vivo in this context and confirm BM-SEC as a significant BMME cell population controlling terminal erythropoiesis and reticulocyte maturation. Furthermore, Wnt signaling seems to be an important pathway in determining BM-SEC identity and functions as has similarly been shown for several EC subpopulations in other organs54. The Wnt signaling pathway and its downstream angiocrine target molecules such as FGF23 and DKK2 may also bear therapeutic potential in various types of anemia or in hematopoietic neoplasms. Therefore, BM-SEC need to be considered as main actors during normal functions and pathogenic alterations in the BMME.Altogether, our findings contribute to a better understanding of the regulatory function of heterogeneous cell populations within the BMME regarding hematopoiesis. Our study demonstrates that cell-specific alterations of BM-SEC can cause refractory anemia as in MDS-like disease. The BMME has recently been shown to comprise at least 17 different cell populations as analyzed by single-cell RNA-Seq technologyCtnnb1OE-SEC) Stab2-iCreF3tg/wt (Tg(Stab2-icre)1.3Cger)25 were crossed with Ctnnb1(ex3)fl/wt (Ctnnb1tm1Mmt)29 mice. Female and male animals aged 2–5 months were used in this study. Here Cre-mediated removal of exon 3 coded amino acids results in a dominant stable form of Ctnnb1 protein and subsequent activation of β-catenin pathway in target cells29. Cre-negative Ctnnb1(ex3)fl/wt siblings served as negative controls in experimental groups. Sinusoidal endothelial specificity of Cre activity was analyzed in crosses of Stab2-iCreF3tg/wt transgenic mice with R26YFPfl/fl (B6.129×1-Gt(ROSA)26Sortm1(EYFP)Cos/J) [JAX 006148]55 reporter animals. Female and male reporter animals were analyzed at the age of embryonic day (E)17.5 and older.All animals were housed under specific pathogen-free conditions in an animal facility (Heidelberg University) at a 12 h/12 h day/night cycle in individually ventilated plastic cages  with adjusted air temperature (21 °C) and 50% relative humidity. All mice were fed ad libitum with a standard rodent diet  and free access to water. The authors have complied with all relevant ethical regulations for animal testing and research. Namely, all animal experiments were performed in accordance with Federal Animal Regulations and were institutionally approved by the district government Karlsruhe and performed under institutional guidelines. To generate a sinusoidal endothelial subtype-specific Ctnnb1 gain-of-function model  and frozen in liquid nitrogen or fixed in 4% paraformaldehyde at 4 °C, followed by paraffin embedding according to routine protocols. Bones were fixed in 4% paraformaldehyde at 4 °C for 4 h and decalcified in 0.5 M EDTA at pH 7.4 for 24 h. Decalcified bones were immersed in 20% sucrose and 2% polyvinylpyrrolidone (PVP) in PBS for 24 h and finally embedded in OCT or 8% porcine gelatin, 20% Sucrose, and 2% PVP in PBSFor blood smear samples, 5 μL of whole blood sample mixed with up to 2.0 mg EDTA/mL was smeared uniformly across a microscopic slide using another slide at a 30-degree smearing angle and air-dried for 5 min. For differential cytomorphological assessment of BM, whole BM was spread onto glass slides and fixed using 100% methanol. Air-dried blood films and BM slides were stained with May-Grünwald/Giemsa stain and brilliant cresyl blue solutions according to standard protocols provided by the manufacturer. Paraffin-embedded tissues were sectioned in 3 μm. For hematoxylin & eosin, periodic acid-Schiff, and Prussian blue staining, formalin-fixed, paraffin-embedded samples were processed according to standard protocols provided by the manufacturer.RNA FISH was conducted using RNAscope Multiplex FISH version 2 assay  kits with mouse-specific probes against the positive control mouse Ppib (Cyclophilin B) gene, mouse Stab2-Channel 2 (4249 – 5075 NM_138673.2) and mouse FGF23-Channel 1 (2 – 1131 NM_022657.4) according to the manufacturer’s protocols.For immunofluorescence (IF), cryosections (8–14 μm) were air-dried, rehydrated in PBS, and blocked in 5% donkey serum in PBS for 30 min. Primary antibody was incubated for 2 h at room temperature or overnight at 4 °C. Sections were washed three times in PBS before incubation of appropriate Alexa-Fluor coupled secondary antibodies for 1 h at room temperature. Nuclei were counterstained with DAPI  or Toto3 iodide . Finally, sections were thoroughly washed in PBS before mounting with Dako fluorescent mounting medium (Dako).https://imagej.nih.gov/ij/). For quantification of IF images, RGB images were split into separate channels corresponding to stainings (including DAPI) in ImageJ. Images of a staining were uniformly visually adjusted using the “Multiply” and “Subtract Background” commands in ImageJ. Then, an automated threshold using the “Otsu” command was applied and the mean pixel signal intensity of the remaining area was analyzed.Sections were visualized with Axioskop 2 (Zeiss), ECLIPSE Ci microscope (Nikon), ECLIPSE Ni-E microscope (Nikon), or DMi8 (Leica). Processing of images was performed by NIS-Elements Imaging Software AR 5.02.00 (Nikon) and ImageJ 1.52e software  using protocols accredited according to norm ISO 15189 (German Accreditation Body DAkkS). BM cells were released from the bones by flushing murine tibia and fibula bones with FACS buffer (5% FCS in PBS), and cells were harvested from the flow-through of a 70-μm filter . Spleens were dissociated directly by passing the cells through a 40-μm filter. Livers were perfused via the portal vein with ice cold PBS, dissected, mechanically minced, digested at 38 °C in collagenase/GBSS  and filtered through a 250-μm filter . Then, 30 µL of peripheral blood was collected using EDTA-containing microtubes (Sarstedt), then suspended in PBS; the erythrocytes cells were lysed with RBC Lysis Buffer  by incubating them for 15 min at room temperature. Cell suspensions  were incubated for 30 min with 500 μg/mL whole mouse IgG (Jackson ImmunoResearch Laboratories) for blocking Fc receptors and stained for 30 min with titrated concentrations of fluorescent dye-labeled antibodies in FACS buffer (5% FCS in PBS) on ice. Dead cells were stained with either Sytox Blue (Invitrogen) or Fixable viability stain 780 (BD Horizon). Flow cytometry was performed using a BD Biosciences FACSCanto II flow cytometer, a BD Biosciences LSR Fortessa cell analyzer or sorted by BD Bioscience FACSAriaIII. All data were analyzed using FlowJo 10.7.1 (Tree Star Inc.) or FACS Diva software (BD Bioscience). Reticulocytes and immature reticulocyte fraction were measured with a Sysmex XN-9000 analyzer according to the manufacturer’s protocols.28. The following secondary antibodies were all purchased from Dianova and diluted 1:400: Alexa-Fluor 488 (donkey-anti-chicken (Cat. No. 703-545-155), donkey-anti-goat (Cat. No. 705-545-147), donkey-anti-rabbit (Cat. No. 711-545-152), donkey-anti-rat (Cat. No. 712-545-153), Alexa-Fluor 647 (donkey-anti-chicken (Cat. No. 703-605-155), donkey-anti-goat (Cat. No. 705-605-147), donkey-anti-rabbit (Cat. No. 711-605-152), donkey-anti-rat (Cat. No. 712-605-153), and Cy3 (donkey-anti-chicken (Cat. No. 703-165-155), donkey-anti-goat (Cat. No. 705-165-147), donkey-anti-rabbit (Cat. No. 711-165-152), donkey-anti-rat (Cat. No. 712-165-153). For FACS staining the following antibodies were used: monoclonal rat anti-CD3ε–APC–eFluor 780 , monoclonal rat anti-CD3ε–PE , monoclonal rat anti-CD3ε–BV510 , monoclonal rat anti-CD16/32–PE–Cy7 , monoclonal rat anti-CD34–eFluor660 , monoclonal rat anti-CD48-AF700 , monoclonal rat anti-Sca1–PerCP–Cy5.5 , monoclonal rat anti-CD19–BV421 , monoclonal rat anti-CD19–BV605 , monoclonal rat anti-CD19–PerCP–Cy5.5 , monoclonal rat anti-CD117–BV711 , monoclonal rat anti-CD150–BV605 , monoclonal rat anti-Gr1-APC , monoclonal rat anti-Gr1-PE , monoclonal rat anti-CD45-PE-Cy7 , monoclonal rat anti-CD45-APC , monoclonal rat anti-CD11b–PerCP–Cy5.5 , monoclonal rat anti-CD11b-PE , monoclonal rat anti-CD44–BV605 , monoclonal rat anti-Ter119–PE , monoclonal rat anti-Ter119–APC-Cy7 , monoclonal rat anti-CD71–PE-Cy7 , monoclonal rat anti-CD71–PE , monoclonal rat anti-CD45.1-BV785 , monoclonal rat anti-CD45.2-BV711 , monoclonal rat anti-CD45R-BV421 , monoclonal rat anti-IgM-FITC , monoclonal rat anti-MHCII-APC , monoclonal rat anti-Ly6C-APC-Cy7 , monoclonal rat anti-Ly6G–PerCP–Cy5.5 , monoclonal rat anti-CD115-BV605 , monoclonal rat anti-EMCN-PE .Primary antibodies: monoclonal rat anti-Endomucin , polyclonal chicken anti-GFP/YFP , polyclonal goat anti-CD32 , polyclonal rabbit anti-alpha smooth muscle Actin , monoclonal rat anti-VEGFR-3 , polyclonal rabbit anti-Cav1 , monoclonal rat anti-CD45 , monoclonal rat anti-Ter119 , polyclonal goat anti-c-kit-antibody , monoclonal rat anti-CD31 , polyclonal rabbit anti-Collagen type I , polyclonal rabbit anti-Collagen type III alpha 1 chain , polyclonal rabbit anti-Collagen IV , polyclonal rabbit anti-fibronectin , polyclonal goat anti-Podocalyxin , polyclonal rabbit anti-Glutamine Synthetase , polyclonal goat anti-Arginase I , polyclonal goat anti-Lyve1 , mouse anti–Stabilin-2 clone 3.1 antibody (dilution 1:700)-Kit+Sca-1+CD150+CD48-), ST-HSCs (Lin-Kit+Sca-1+CD150-CD48-), MPPs (Lin-Kit+Sca-1+CD150-CD48+), CMPs (Lin-Kit+Sca-1-CD34+CD16/32low), GMPs (Lin-Kit+Sca-1-CD34+CD16/32+), MEPs (Lin-Kit+Sca-1-CD34-CD16/32-). Erythroid progenitors were defined as: PII , PIII , PIV , PV . EMCN+ cells were defined as CD11b-EMCN+. Immune cells were defined as: CD3+ (Size/CD3+CD19-), CD19+ (Size/CD3-CD19+), CD11b+Gr- (Size/CD3-CD19-CD11b+Gr-), CD11b+Gr+ (Size/CD3-CD19-CD11b+Gr+), Pro-B Cells (CD43midCD45R+), Pre-B Cells (CD43-CD45R+IgM-), Immature B Cells (CD43-CD45RloIgM+), Mature B Cells (CD43-CD45RhiIgM+), Ly6G+ (Size/Ly6CmidLy6G+), Ly6CloCD115+ (Size/Ly6CloLy6G-CD115+), Ly6C+CD115+ (Size/Ly6C+Ly6G-CD115+).The lineage cocktail was composed of monoclonal rat anti-CD3ε-BV421 , monoclonal rat anti-CD4-BV421 , monoclonal rat anti-CD8-BV421 , monoclonal rat anti-CD11b-BV421 , monoclonal rat anti-CD19-BV421 , monoclonal rat anti-Gr-1-BV421 , and monoclonal rat anti-Ter119-BV421 . Sytox blue  and Fixable Viability Dye eFluor 780  were used for dead cell discrimination. All populations were gated for live single cells. The gating strategies are shown in Supplementary Fig. 9. Briefly, murine long bones were crushed in Ca2+/Mg2+ free PBS, digested with 0.7 mg/mL Collagenase A  for 45 min at 37 °C, filtered through a 40-μm mesh and incubated with 25 μL of EMCN-antibody  coated dynabeads  for 25 min at 4 °C under constant rotation. EMCN+ cells were then extracted by magnetic separation of dynabead-conjugated cells. Gene expression profiling was performed using arrays of mouse MoGene-2_0-st-type from Affymetrix. Biotinylated antisense complementary DNA (cDNA) was then prepared according to the standard labeling protocol with the GeneChip® WT Plus Reagent Kit (from Thermo Fisher Scientific). Afterwards, the hybridization on the chip was performed on a GeneChip Hybridization oven 640, dyed in the GeneChip Fluidics Station 450 with the GeneChip® Hybridization, Wash and Stain Kit and thereafter scanned with a GeneChip Scanner 3000. All of the equipment used was from the Affymetrix-Company.RNA was extracted from FACS-sorted erythroblastic cells, PIII, PIV, and BM-EC using InnuPrep RNA Mini Kit . ECs were isolated from murine BM as previously described by Kusumbe et al.57. The raw fluorescence intensity values were normalized applying quantile normalization and RMA background correction. One-way ANOVA was performed to identify differential expressed genes using a commercial software package SAS JMP15 Genomics, version 10, from SAS (SAS Institute). A false positive rate of a = 0.05 with FDR correction was taken as the level of significance.A Custom CDF Version 22 with ENTREZ-based gene definitions was used to annotate the arraysfgsea package58 ran under R v3.4.059. Pathways belonging to various cell functions such as cell cycle or apoptosis were obtained from public external databases . The gene set for angiocrine factors31 was defined merging published factors from Rafii et al.61 with Gene Ontology gene set cytokine activity filtered for murine genes63. Heatmaps were created with the ComplexHeatmap package64.GSEA was used to determine whether defined lists (or sets) of genes exhibit a statistically significant bias in their distribution within a ranked gene list using the http://www.ncbi.nlm.nih.gov/tools/primer-blast/). All the primers used in the present study are listed in Supplementary Table 65.RNA was extracted from primary ECs using E.Z.N.A. TM Total-RNA-Kit I . cDNA was synthesized with RevertAid H-Minus M-MuLV Reverse Transcriptase (Thermo Fisher Scientific). Quantitative PCR was performed on a qTOWER 3G touch thermal cycler  using innuMIX qPCR SyGreen Sensitive . Primers were designed using Primer-BLAST (3 Lin-Kit+Sca-1+CD150+CD48- cells from 10-week-old Ctnnb1OE-SEC mice (CD45.2) were sorted  and injected intravenously along with 1 × 105 Lin- BM cells sorted from CD45.1 mice into lethally irradiated  Pep Boy, B6 Cd45.1 recipients. Recipient mice were maintained on antibiotic water  for 14 days. Blood samples were analyzed 4, 8, 12, 16, 20, and 24 weeks post transplantation for Hb-content and donor chimerism. BM of recipient mice was isolated and analyzed for donor-derived HSPC by flow cytometry.For HSC sorting/enrichment, BM cells were stained with commercially prepared antibodies (see Supplementary Methods) and depleted with Dynabeads (Life technologies) according to the manufacturer’s instructions. In total, 1.2 × 10Singularized BM or spleen cell extracts were harvested in Iscove’s modified Dulbecco’s medium (Thermo Fisher Scientific) containing 2% FCS (Biochrom) plated in gridded 35-mm-diameter cell culture dishes (Sarstedt) at cell concentrations as suggested by the manufacturer, in triplicate. MethoCult™ GF M3434 (Stemcell Technologies) was used to stimulate mouse hematopoietic progenitor cells such as multi-potential granulocyte, erythroid, macrophage, megakaryocyte progenitor cells (CFU-GEMM), representing most colonies, but also BFU-E, granulocyte-macrophage progenitor cells . MethoCult™ M3334 was used for detection of CFU-E and MethoCult™ SF M3436 for BFU-E. Colonies were analyzed after 2 days (CFU-E), 10–12 days , and 10–14 days (BFU-E) with an Axio Vert.A1 microscope (Zeiss).After perfusion-fixation with 2% formaldehyde, 2% glutaraldehyde, 1 mM MgCl2, 1 mM CaCl2, in 100 mM Na-phosphate buffer, pH 7.2 bones (femur) were excised and immersion-fixed for 24 h at 4 °C, decalcified in 100 mM EDTA at neutral pH for 3 days at 4 °C. Medium was changed once daily. The softened samples were cut into pieces using razorblades and washed in deionized water. Next, treatment for conventional epoxy-resin embedding and TEM consisted of post-fixation in 1% osmium tetroxide, en-bloc staining with 1% uranylacetate, graded dehydration with ethanol and embedding in epoxide . Ultrathin sections at nominal thickness of 60 nm and contrast-stained with lead-citrate and uranylacetate were observed in Zeiss EM 910 at 100 kV (Zeiss) and micrographs taken with a slow scan CCD camera (TRS).67, established in the lab of Deborah E. Daniels and Jan Frayne, were cultured in StemSpan™ SFEM (Stemcell Technologies) containing 50 ng/mL SCF, 3 U/mL EPO, 1 μM dexamethasone, and 1 µg/mL doxycycline. To induce erythroid differentiation, expanding cells were transferred to primary medium supplemented with 1 µg/mL doxycycline for 4 days, and for a further 4 days without doxycycline. Cells were cultured with supplementation of either 100 ng/mL FGF23 (R&D Systems) or vehicle control for 48 h before sample collection for analysis. Cell viability was determined by trypan blue exclusion test. For FACS, aliquots of 2 × 105 cells were incubated with band 3 primary antibody  in PBS containing 1% (w/v) BSA (Park Scientific Ltd) and 2 mg/mL glucose (PBS-AG), followed by APC-secondary antibody (BioLegend), or with conjugate antibodies . Cells were analyzed on a BD LSR Fortessa flow cytometer. From day 6 of differentiation onwards cells were incubated with 5 μg/mL Hoechst 33342 nucleic acid stain (Merck) to distinguish the erythroblast and reticulocyte populations. Propidium iodide was used to exclude non-viable cells from analyses for band 3, CD36, and percentage reticulocyte measurements. Data were analyzed using FlowJo v10.6.1 (FlowJo LLC).BEL-A cells69. The source-object and object-detector distances were adjusted so that the sample filled the field of view. Imaging was performed using a tube voltage of 80 kV, tube current was set to 75 μA, and continuous image acquisition was carried out at 30 frames per second. A total of 990 projections were acquired per scan during a rotation of 360° within 33 s. Raw data were reconstructed with a filtered back projection algorithm with a matrix of 512 × 512 × 512  using a low pass filter (Shepp-Logan) to reduce image noise. The resulting digital imaging and communications in medicine data were analyzed using OsiriX imaging software .An industrial micro-CT  equipped with a multifocus transmission x-ray tube and a 14-bit direct amorphous silicon flat panel detector , in which the object is rotated around its horizontal axis in the cone beam, was used+ and Bilirubin . Serum iron levels were measured with Dimension Vista® 1500 analyzer (Siemens). FGF23, cFGF23, and EPO in plasma were detected by Mouse FGF23 DuoSet ELISA , Mouse/Rat FGF23 (C-Term) , and Mouse Erythropoietin/EPO Quantikine ELISA Kit  according to the manufacturer’s instructions.Relative hemoglobin values were analyzed based on photometric detection of cyanmethemoglobin. Plasma was collected was analyzed for the following routine parameters: LDH, Kt-test and Mann–Whitney U test were used. Differences between data sets with p < 0.05 were considered statistically significant. Data are presented as means with error bars indicating standard error. All experiments have been performed with a minimum of three age- and sex-matched biological replicates. Micrographs shown in the figures are representative selections of at least three different micrographs per animal of a minimum of three independent biological samples.Statistical analyses were performed with GraphPad Prism 9.1.2 (GraphPad) and SigmaPlot 11 Software (Systat Software GmbH). For pairwise comparisons the two-tailed unpaired Student’s Further information on research design is available in the Supplementary InformationReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2


Specifically, elevated IRE1 phosphorylation, Tumor Necrosis Factor (TNF) Receptor-Associated Factor-2 (TRAF2) expression, Apoptosis Signal-Regulating Kinase-1 (Ask1) phosphorylation, c-Jun N-Terminal Kinase (JNK) phosphorylation, and Noxa expression appeared in gefitinib-treated glioma cells. Genetic, pharmacological, and biochemical studies further indicated an active ROS/ER stress/Ask1/JNK/Noxa axis causing the glioma apoptosis induced by gefitinib. The findings suggest that ER-stress-based therapeutic targeting could be a promising option in EGFR inhibitor glioma therapy, and may ultimately achieve a better patient response.Adequate stress on the Endoplasmic Reticulum (ER) with the Unfolded Protein Response (UPR) could maintain glioma malignancy. Uncontrolled ER stress, on the other hand, predisposes an apoptosis-dominant UPR program. We studied here the proapoptotic actions of the Epidermal Growth Factor Receptor (EGFR) inhibitor gefitinib, with the focus on ER stress. The study models were human H4 and U87 glioma cell lines. We found that the glioma cell-killing effects of gefitinib involved caspase 3 apoptotic cascades. Three branches of ER stress, namely Activating Transcription Factor-6 (ATF6), Protein Kinase R (PKR)-Like ER Kinase (PERK), and Inositol-Requiring Enzyme 1 (IRE1), were activated by gefitinib, along with the elevation of intracellular free Ca While cancer cells are notorious for their high proliferation and quick spread, they are surrounded by hostile microenvironments such as hypoxia, nutrient deficiency, lactic acidosis, and oxidative stress. Intrinsic events from oncogenic activation, aerobic glycolysis, and high protein demands would disrupt cellular proteostasis, leading to Endoplasmic Reticulum (ER) stress. To overcome such obstacles and turn them into survival momentum, cancer cells possess several adaptive mechanisms. One of them is the ER stress-derived Unfolded Protein Response (UPR), which is crucial for their adaptation and survival ,2. ConveUPR is coordinated by three different signaling branches of transmembrane proteins resident on ER: Protein Kinase R (PKR)-Like ER Kinase (PERK), Inositol-Requiring Enzyme 1 (IRE1), and Activating Transcription Factor 6 (ATF6). Under a homeostatic ER, the lumenal domains of and PERK, IRE1, and ATF6 interact with ER abundant chaperone Glucose-Regulated Protein 78 (GRP78) and remain in an inactive state. Once misfolded proteins accumulate, GRP78 titrates misfolded proteins, liberating PERK, IRE1, ATF6. The liberated PERK initiates autophosphorylation and phosphorylates Eukaryotic Translation Initiation Factor-2α (eIF2α), leading to a greater expression of ATF4 and C/EBP Homologous Protein (CHOP). Activated IRE1, which possesses kinase and RNase activities, yields a spliced form of transcription factor X-Box Binding Protein-1 (XBP1) mRNA. IRE1 also recruits Tumor Necrosis Factor (TNF) Receptor-Associated Factor-2 (TRAF2) to activate the Apoptosis Signal-Regulating Kinase-1 (Ask1)/c-Jun N-Terminal Kinase (JNK) axis. When released from GRP78, ATF6 enters the Golgi apparatus, generating a cleaved fragment that is a transcription factor. Transcription factors ATF4, CHOP, XBP1, and ATF6 have shared and individual programs, governing the expression of a variety of genes involved in cell survival, angiogenesis, metastasis, immune surveillance, inflammation, autophagy, and apoptosis [In the central nerve system, the most aggressive malignancy is glioma, particularly the glioblastoma multiforme. Despite conventional and novel treatment strategies, patients with malignant glioma have a poor prognosis, with high recurrence rates . GeneticGlioma cells over-express markers of UPR, which contribute to proliferation, angiogenesis, resistance, and stem cell-like activity, and are correlated with a poor prognosis ,13,14. H2+  inducing apoptotic cell death in glioma cells are way higher than those inhibiting EGFR signaling (~5 μM). Once gefitinib treatment is effective, ER stress overrides survival machinery, predisposing cells to the apoptosis dominant program. Therefore, ER stress might well be valuable targets in combination therapy using gefitinib to combat malignant glioma.PTEN and one copy loss in TP53, but are not EGFRvIII variant. U87 cells are PTEN-deficient and TP53- and EGFR-wild type. Both cells are Isocitrate Dehydrogenase (IDH)-wild type [Human U87 glioblastoma cell line (ATCC HTB-14) and H4 neuroglioma cell line (ATCC HTB-148) purchased from American Type Culture Collection  were maintained in Dulbecco’s modified Eagle medium (DMEM), containing 10% fetal bovine serum (FBS) for propagation. All experiments were conducted on cells placed in DMEM containing 2% FBS. H4 cells harbor genetic mutations in ild type .Cells were plated onto 96-well plates 24 h prior to treatment. Cell viability was determined with an assay kit  . The optical absorbance at 570 and 600 nm was measured with a spectrophotometer.x 380 nm and Em 460 nm).Cells were plated onto 6-well plates 24 h prior to treatment. Caspase 3 activity was measured within isolated protein extracts (10 μg) according to instructions of the Caspase Fluorometric Assay kit . Fluorescent signals of released AMC moiety were measured with a fluorometer  for 30 min. Fluorescent signals were then measured in a fluorometer with excitation/emission at 495 nm/529 nm.2+, cells were loaded with Fura-2-Acetoxymethyl Ester . Fluorescent signals were measured in a fluorometer with dual excitation at 340 nm and 380 nm, with emission detected at 510 nm.Cells were plated onto 96-well plates 24 h prior to treatment. To detect intracellular free CaCells were plated onto 6-well plates 24 h prior to treatment. At the end of treatments, cells were homogenized in the Laemmli SDS buffer. Equal amounts of protein extracts were separated and analyzed in a standard SDS-PAGE, followed by reacting to horseradish peroxidase-labeled IgG, Enhanced Chemiluminescence (ECL) visualization, and densitometric measurement. Primary antibodies were used to recognize the following proteins: PARP-1 (1:1000), cleaved caspase 3 (1:1000), GRP78 (1:1000), ATF6 (1:1000), PERK (1:1000), phospho-PERK , eIF-2α (1:1000), phospho-eIF2α , ATF4 (1:1000), CHOP (1:2000), IRE1 (1:1000), phospho-IRE1 , TRAF2 (1:1000), Ask1 (1:1000), phospho-Ask1 , JNK (1:1000), phospho-JNK , ERK (1:1000), phospho-ERK , Akt (1:1000), phospho-Akt , NOX2 , NOX4 (1:1000), Noxa , and Glyceraldehyde-3-Phosphate Dehydrogenase .The siRNA against human IRE1 (sc-40705), Noxa (sc-37305), and control siRNA  were delivered to H4 cells using the INTERPERin siRNA transfection reagent .p < 0.05.Data were represented as mean ± standard deviation, and depicted with Sigma Plot 12.3 software. Statistical comparisons were analyzed using one-way or two-way analysis of variance, followed by Tukey or Dunnett post hoc test. Statistical significance was set at


Scientific Reports 10.1038/s41598-021-86936-2, published online 06 April 2021Correction to: The original version of this Article contained an error in the Acknowledgments section.“SS, NB and RC acknowledges the financial assistance received from SPARC proposal #754.”now reads:“SS, NB and RC acknowledge the financial assistance received from SPARC proposal #754 & DST Nanomission project (DST/NM/TUE/QM-11/2019).”The original Article has been corrected.


There is an error in the thirteenth paragraph of the Discussion section of this article . Prior thttps://infogram.com/covid-19-malta-superintendence-of-public-health-1h7k23gorvzv4xr) and the Malta Health Promotion & Disease Prevention Directorate (https://geosys-mt.maps.arcgis.com/apps/opsdashboard/index.html#/c17848aba0f94d79a4f57a7a584c4e8e) following the protocol outlined in the Methods section. A de-identified dataset underlying the clinical results is available from the Dryad repository (doi: 10.5061/dryad.mcvdnck12).The Data Availability statement for this paper is incorrect. The correct statement is: COVID-19 case data were sourced from the publicly available Superintendence of Public Health, Malta (


Cucumber green mottle mosaic virus (CGMMV) causes substantial global losses in cucurbit crops, especially watermelon. N6-methyladenosine (m6A) methylation in RNA is one of the most important post-transcriptional modification mechanisms in eukaryotes. It has been shown to have important regulatory functions in some model plants, but there has been no research regarding m6A modifications in watermelon.6A level in resistant watermelon after CGMMV infection using a colorimetric method. And the results found that the global m6A level significantly decreased in resistant watermelon after CGMMV infection. Specifically, m6A libraries were constructed for the resistant watermelon leaves collected 48 h after CGMMV infection and the whole-genome m6A-seq were carried out. Numerous m6A modified peaks were identified from CGMMV-infected and control (uninfected) samples. The modification distributions and motifs of these m6A peaks were highly conserved in watermelon transcripts but the modification was more abundant than in other reported crop plants. In early response to CGMMV infection, 422 differentially methylated genes (DMGs) were identified, most of which were hypomethylated, and probably associated with the increased expression of watermelon m6A demethylase gene ClALKBH4B. Gene Ontology (GO) analysis indicated quite a few DMGs were involved in RNA biology and stress responsive pathways. Combined with RNA-seq analysis, there was generally a negative correlation between m6A RNA methylation and transcript level in the watermelon transcriptome. Both the m6A methylation and transcript levels of 59 modified genes significantly changed in response to CGMMV infection and some were involved in plant immunity.We measured the global m6A patterns in the watermelon transcriptome and helps to clarify the roles and regulatory mechanisms of m6A modification in watermelon in early responses to CGMMV.Our study represents the first comprehensive characterization of mThe online version contains supplementary material available at 10.1186/s12870-021-03289-8. It plays multiple roles in RNA metabolism, including stability and degradation ). Differentially expressed genes (DEGs) were analyzed using the Cuffdiff program of Cufflinks [Charleston Gray) reference genome sequences and annotations were downloaded from the CuGenDB database (http://cucurbitgenomics.org/).RNA-seq reads were aligned to the watermelon  were reverse transcribed with random hexamers using M-MLV reverse transcriptase (Takara) to generate the cDNA for the IP and pre-IP samples. The cDNA was used as the template for the m6A-IP-qPCR and qRT-PCR. The enrichment of m6A methylation in specific gene regions was determined using the cycle threshold (CT) 2(-ΔCT) method [β-actin gene was used as an internal control [6A-IP-qPCR and qRT-PCR are listed in Supplementary file To validate mications , 44. The) method . The β-a control . The priAdditional file 1: Supplementary file 1 Figure S1. High Pearson correlation coefficient (PCC) for the abundance of confident peaks between biological replicates.Additional file 2: Supplementary file 2 Figure S2. Gene ontology (GO) analysis (GO: level 2) of the m6A-containing transcripts identified in m6A -seq in watermelon.Additional file 3: Supplementary file 3 Figure S3. Pie charts depicting the fraction of DMPs in four non-overlapping transcript segments.Additional file 4: Supplementary file 4 Figure S4. The phenotype of two watermelon germplasm (R1288 and S1252) plants infected CGMMV 20 d.Additional file 5: Supplementary file 5. Summary of m6A -seq information for CGMMV-infected and control samples.Additional file 6: Supplementary file 6. High-confidence m6A peaks identified for control samples and CGMMV-infected samples.Additional file 7: Supplementary file 7. Gene ontology (GO) analysis of the biological process, molecular function, and cellular component for the m6A -containing transcripts identified in m6A -seq in watermelon.Additional file 8: Supplementary file 8. Characteristics of differentially methylated peaks (DMPs) in CGMMV-infected and control samples.Additional file 9: Supplementary file 9. The m6A modification level of DEGs involved in immune responses and the production of siRNA involved in RNA interference in this study.Additional file 10: Supplementary file 10. DEGs identified in RNA-seq in this study.Additional file 11: Supplementary file 11. The response patterns of watermelon m6A elements to CGMMV infection in RNA-seq data.Additional file 12: Supplementary file 12. The differentially m6A methylated and expressed genes (DMEGs) identified in this study.Additional file 13: Supplementary file 13. Primers used for m6A-immunoprecipitation(IP)-qPCR (m6A -IP-qPCR) and Real-time quantitative PCR (qPCR).


Co-infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other viruses has been reported. We evaluated cell lines commonly used to isolate viruses and diagnose related diseases for their susceptibility to SARS-CoV-2. Although multiple kidney cell lines from monkeys were susceptible to SARS-CoV-2, we found many cell types derived from humans, dogs, minks, cats, mice, and chicken were not. We analyzed MDCK cells, which are most commonly used for surveillance and study of influenza viruses, and found that they were not susceptible to SARS-CoV-2. The low expression level of the angiotensin converting enzyme 2 receptor and lower receptor affinity to SARS-CoV-2 spike, which could be overcome by overexpression of canine angiotensin converting enzyme 2 in trans, strengthened the cellular barrier to productive infection. Moreover, a D614G mutation in the spike protein did not appear to affect SARS-CoV-2 cell tropism. Our findings should help avert inadvertent propagation of SARS-CoV-2 from diagnostic cell lines.  Coronavirus disease (COVID-19) has resulted in >70 million laboratory-confirmed cases and >1.6 million deaths in <1 year since the first case was confirmed. Co-infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other viruses, such as influenza virus, has been reported  of the viral population. Although furin cleavage site mutations were reported to decrease entry and infection efficiency to various degrees in lung epithelial cells (We used 3 virus stocks for our investigation. The SARS-CoV-2/USA-WA1/2020 (USA-WA1) viral strain was isolated from the specimen of the first confirmed case in the United States  and MDCK-hCK cells from the University of Wisconsin–Madison (https://www.wisc.edu). We obtained MDCK-NBL2, Vero E6, CV-1, A549, Crandell-Rees Feline Kidney (CRFK) cells, Mv1Lu, RD, Hep-2c, HeLa, and L20B cells from American Type Culture Collection (https://www.atcc.org); these cells were maintained at Division of Scientific Resources, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention . We obtained chicken embryo fibroblasts from Charles River Laboratories (https://www.criver.com). We obtained an additional 25 cell lines (https://www.quidel.com); these lines were preseeded in 24-well plates, except for CRFK and rhesus monkey kidney cells, which were obtained in T-75 flasks and seeded into 24-well plates in the laboratory 1 day before infection.We obtained MDCK-Atlanta, MDCK-London, and MDCK-SIAT1 cells from International Reagent Resources  in some figures. We then washed cells at 1–2 hpi and collected supernatants or cell lysates daily for up to 3 days for infectious virus titration and up to 5 days hpi for viral RNA quantification. We observed cytopathic effect and fluorescence signals for icSARS-CoV-2-mNG daily.5 median tissue culture infectious dose (TCID50)/egg and incubated them at 37°C for 3 days. Allantoic fluid was collected from individual eggs separately as E1 samples. We passaged 100 µL of each E1 sample into a corresponding egg and collected 24 E2 samples after 3 days of incubation. We also generated 24 E3 samples from passage of E2 samples in 24 eggs. We titrated all E1, E2, and E3 samples, as well as samples from cell lines, with TCID50 assay using VeroE6 cells; viral RNAs were quantified by real-time reverse transcription PCR (rRT-PCR) . We immunoblotted cell lysates and recombinant angiotensin-converting enzyme (ACE) 2 protein control  for ACE2 and β-actin using 1:500 polyclonal goat anti-human ACE2 AF933  and 1:1,000 monoclonal mouse anti-β-Actin AB8226  primary antibodies followed by Abcam 1:4,000 donkey anti-goat and 1:4,000 goat anti-mouse secondary antibodies . We developed immunoblots using ThermoFisher SuperSignal West Pico PLUS chemiluminescent substrate. Qualitative RT-PCR (qRT-PCR) was used to determine the relative mRNA ACE2 levels in different cell lines. Two sets of primers and probes (https://www.thermofisher.com). We used the comparative cycle threshold (ΔΔCt) method to quantify relative ACE2 gene expression. For each cell type and primer/probe set, we normalized ACE2 cycle threshold against 18S rRNA and then standardized to Vero E6.Cells were lysed in NP-40 lysis buffer and we determined protein concentrations using a Pierce BCA protein assay kit  as described elsewhere (https://www.sartorius.com) on Octet RED96 at 30°C with a shaking speed at 1,000 RPM. We corrected the data by subtracting reference sample and used 1:2 bivalent binding model with global fit to determine affinity constants.We used the ThermoFisher Expi293 Expression system to produce histidine-tagged ACE2 (ectodomain) proteins and purified them using HisTrap FF column  using the manufacturer’s protocol with program A024. We transfected 1.5 × 106 MDCK-SIAT1 cells with 10 µg DNA . One day posttransfection, we inoculated the cells with USA-WA1 or icSARS-CoV-2-mNG. We aligned ACE2 protein sequences for human (GenBank accession no. NP_001358344.1), African green monkey (accession no. AAY57872.1), rhesus macaque (accession no. ACI04564.1), mouse (accession no. NP_001123985.1), dog (accession no. XP_005641049.1), cat (accession no. NP_001034545.1), American mink (accession no. QPL12211), and chicken (accession no. XP_416822.2) using MUSCLE alignment in Geneious Prime software version 2019.2.3 (https://www.geneious.com).We generated constructs coexpressing full-length human ACE2 (hACE2) or canine ACE2 (cACE2) with mCherry2 protein (CMV promoter-ACE2-IRES-mCherry2) and transfected them into MDCK-SIAT1 cells through electroporation with the Lonza Nucleofector system (4 TCID50/well of a fluorescent reporter virus in which the open reading frame 7a gene was replaced by the mNeonGreen gene (icSARS-CoV-2-mNG), allowing successful infection to be visualized by a green fluorescence signal (4 TCID50/well of the wild type SARS-CoV-2/USA-WA1/2020 (USA-WA1) strain and titrated supernatants collected over 5 days. Consistent with the results from icSARS-CoV-2-mNG infection, all nonhuman primate cell lines except CV-1 cells supported productive virus replication, whereas all other cell lines failed to generate infectious virus . Viral titers in the allantoic fluid of E1, E2, and E3 eggs were below the limit of detection (101.5 TCID50/mL) even in E1 eggs (data not shown). We then used an rRT-PCR assay to quantify the viral RNA levels in the inoculum and allantoic fluid samples  of SARS-CoV-2/Massachusetts/VPT1/2020 (MA/VPT1), which encodes a spike with G614. In selecting cell lines for the subset, we included Vero E6 cells as a cell line that should support replication of MA/VPT1 given our previous findings with USA-WA1 (4 TCID50/well), CV-1, A549, Mv1Lu, MDCK-NBL-2, and MDCK-SIAT1, cell lines were not susceptible to this SARS-CoV-2 strain encoding spike G614. CRFK cells inoculated with MA/VPT1 generated virus titers slightly above the limit of detection at 1 dpi, after which titers decreased  or cACE2 (MDCK-cACE2) as determined by mCherry2 expression were efficiently infected by icSARS-CoV-2-mNG . We alsoD] = 19.5 nmol/L) 15-fold less efficiently than to hACE2 (KD = 1.30 nmol/L)  . The red nmol/L) . TherefoIn this study, we determined the SARS-CoV-2 susceptibility of >30 cell lines and derivatives and embryonated chicken eggs. Findings from our study corroborate and complement those from other susceptibility studies published in recent months , the lowWe expanded our examination to other clinically relevant cell lines used in diagnosis and isolation of a wide array of human viruses, particularly respiratory viruses . AlthougOverall, our study provides useful information on multiple cell lines and chicken eggs regarding their susceptibility to SARS-CoV-2. Of note, from a biosafety standpoint, humans can be co-infected with multiple pathogens. Specimens collected for testing and culture of other viruses may contain SARS-CoV-2; these data should help laboratories avoid inadvertent propagation. The data on canine ACE2 shed light on the relationship between SARS-CoV-2 susceptibility and ACE2 receptor affinity (species specificity) and expression level, suggesting that even ACE2 proteins with several substitutions at key residues that contact SARS-CoV-2 spike protein can still serve as functional receptors when expressed at high levels.


Multisystem inflammatory syndrome in children (MIS-C) is a severe postinfectious hyperinflammatory condition, which generally occurs 2–6 weeks after a typically mild or asymptomatic infection with SARS-CoV-2, the virus that causes COVID-19  . The medAmong the 70 children in this analysis who were fully vaccinated (with 2 doses), one syndrome-negative control patient had received a third dose. Among 102 MIS-C case-patients, five (5%) were fully vaccinated with 2 doses ≥28 days before hospitalization, and 97 (95%) were unvaccinated . OverallVE against MIS-C was 91% (95% CI = 78%–97%) .**** In During July–December 2021, a period of Delta variant predominance, a real-world evaluation of VE in 24 U.S. pediatric hospitals found that receipt of 2 doses of the Pfizer-BioNTech vaccine was associated with a high level of protection against MIS-C among patients aged 12–18 years who received their second vaccine dose ≥28 days before hospitalization. Most (95%) patients aged 12–18 years hospitalized with MIS-C were unvaccinated. No fully vaccinated patients with MIS-C required respiratory or cardiovascular life support, as opposed to 39% of unvaccinated MIS-C patients who did. A recent Overcoming COVID-19 hospital network investigation reported high VE (93% [95% CI = 83%–97%]) against COVID-19–related hospitalizations in persons aged 12–18 years , which can occur 2–6 weeks after SARS-CoV-2 infection, has remained uncharacterized.Estimated effectiveness of 2 doses of Pfizer-BioNTech vaccine against MIS-C was 91% (95% CI = 78%–97%). Among critically ill MIS-C case-patients requiring life support, all were unvaccinated.Receipt of 2 doses of Pfizer-BioNTech vaccine is highly effective in preventing MIS-C in persons aged 12–18 years. These findings further reinforce the COVID-19 vaccination recommendation for eligible children.


TNFR1 was found to cause skin inflammation in mice with epidermal keratinocyte-specific ablation of IKK2 or of both the NF-kB subunits RelA and cRel by triggering RIPK1-dependent, RIPK3-MLKL–mediated necroptosis of keratinocytes. Tumor necrosis factor receptor 1 (TNFR1) activates NF-κB–dependent pro-inflammatory gene expression, but also induces cell death by triggering apoptosis and necroptosis. Inhibition of inhibitor of NF-κB kinase (IKK)/NF-κB signaling in keratinocytes paradoxically unleashed spontaneous TNFR1-mediated skin inflammation in mice, but the underlying mechanisms remain poorly understood. Here, we show that TNFR1 causes skin inflammation in mice with epidermis-specific knockout of IKK2 by inducing receptor interacting protein kinase 1 (RIPK1)–dependent necroptosis, and to a lesser extent also apoptosis, of keratinocytes. Combined epidermis-specific ablation of the NF-κB subunits RelA and c-Rel also caused skin inflammation by inducing TNFR1-mediated keratinocyte necroptosis. Contrary to the currently established model that inhibition of NF-κB–dependent gene transcription causes RIPK1-independent cell death, keratinocyte necroptosis, and skin inflammation in mice with epidermis-specific RelA and c-Rel deficiency also depended on RIPK1 kinase activity. These results advance our understanding of the mechanisms regulating TNFR1-induced cell death and identify RIPK1-mediated necroptosis as a potent driver of skin inflammation. The skin constitutes an essential structural and immunological barrier protecting the organism from microbial challenges. The maintenance of a healthy skin homeostasis requires a proper balance between cell proliferation, differentiation, death, and a tightly regulated cross talk between epithelial and immune cells. Deregulation of this balance results in chronic inflammatory skin diseases, such as psoriasis. Whereas early studies of the mechanisms driving the pathogenesis of inflammatory skin diseases focused on the role of immune cells, recent studies in genetic mouse models have highlighted the importance of keratinocyte-intrinsic mechanisms in regulating skin immune homeostasis and inflammation . TNF is TNF binding to tumor necrosis factor receptor 1 (TNFR1) initiates the formation of a receptor-proximal signaling complex (termed complex I) composed of receptor interacting protein kinase 1 (RIPK1), TNFR1-associated death domain protein (TRADD), TNFR-associated factor 2 (TRAF2), and the E3 ubiquitin ligases cellular inhibitors of apoptosis (cIAP) 1 and 2. Ubiquitination of RIPK1 with K63-linked ubiquitin chains by cIAPs promotes the recruitment of the linear ubiquitin assembly complex (LUBAC), consisting of SHANK-associated RH domain-interacting protein (Sharpin), Heme-oxidized iron-regulatory protein 2 ubiquitin ligase-1 (HOIL-1), and HOIL-1–interacting protein (HOIP), as well as of the transforming growth factor-β-activated kinase 1 (TAK1)/TAK1-binding protein (TAB)1/2 complex. LUBAC ubiquitinates RIPK1 but also other proteins within the complex with linear Ub chains, which allows the recruitment of the IκB kinase (IKK) complex, consisting of the regulatory subunit NF-κB essential modulator (NEMO) and the catalytic subunits IKK2/IKKβ and IKK1/IKKα . IKK is Sharpincpdm/cpdm), by inhibiting TNFR1-mediated FADD-caspase-8–dependent keratinocyte apoptosis  develop severe psoriasis-like skin inflammation characterized by epidermal hyperplasia, altered keratinocyte differentiation, up-regulation of cytokines and chemokines, and accumulation of immune cells in the skin  also developed inflammatory skin lesions resembling the phenotype of IKK2E-KO mice , P4 and P8 with antibodies against activated cleaved caspase-3 (CC3) and with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). The skin of IKK2E-KO mice showed a progressive increase in the number of CC3 and TUNEL-positive cells in the epidermis  and found that TNFR1 ablation prevented keratinocyte death  conditions. These experiments showed that GF IKK2E-KO mice developed similar inflammatory skin lesions as seen in IKK2E-KO mice raised under specific pathogen-free conditions, characterized with increased epidermal thickness, elevated expression of keratin 6 (K6) and K14, reduced expression of K10, and increased number of dying keratinocytes , which lack IKK2 and FADD specifically in keratinocytes and RIPK3 in all cells. FADD deficiency prevents caspase-8–mediated apoptosis and RIPK3 deficiency prevents necroptosis; therefore, if death of keratinocytes would be important for the pathogenesis of the skin lesions, we would expect that IKK2E-KO FADDE-KORipk3−/− should not develop the pathology. Indeed, the triple mutant IKK2E-KO FADDE-KORipk3−/− mice were indistinguishable from their littermate controls and did not show skin lesions at the age of 7–8 d after birth . Epidermal keratinocyte-specific RIPK3 knockout fully recapitulated the effect of systemic RIPK3 deficiency, with IKK2E-KO RIPK3E-KO showing strongly ameliorated skin inflammation at P7-P8 but developing progressive mild skin lesions at the age of about 2–5 mo   . IKK2E-Kmed skin . In addiltration . Furtherce at P4 . In addiinocytes  and S2C.E-KORipk1D138N/D138N mice phenotype.IKK2Table S5 Relafl/flc-Relfl/fl mice with K14-Cre. These RelAE-KO c-RelE-KO mice were indistinguishable from their Relafl/flc-Relfl/fl littermate controls at birth but started to develop inflammatory skin lesions at around P4, which progressed to widespread inflammatory skin pathology by P8-P11. The inflammatory skin lesions of RelAE-KO c-RelE-KO mice resembled the phenotype of IKK2E-KO mice, although the pathology was generally milder   , which apoptosis . TherefoE-KO c-RelE-KO mice. Consistent with these results, we and others showed previously that inhibition of RIPK1 kinase activity rescued the embryonic lethality of Rela−/− mice . Animals received appropriate care and were euthanized when they reached pre-determined criteria based on the development of macroscopically visible skin lesions to minimize suffering. Mice were assigned at random to groups within specific genotypes. Mouse studies were performed in a blinded fashion. All animal procedures were conducted in accordance with European, national and institutional guidelines and protocols and were approved by local governmental authorities .kk2fl/fl , Faddfl/addfl/fl , K14-Cre, Keratin 6 , Keratin 10 , TUNEL (Promega)/Active caspase-3  staining was performed on paraffin, and F4/80  staining on cryo sections. The staining was visualized with Alexa-488 or Alexa-567 conjugated secondary antibody. Measurement of epidermal thickness and inflamed area was done as described before . Briefly2+ DMEM/Ham’s F12 medium  with 10% chelax-treated FCS and supplements. Cells were grown in the Ca2+ DMEM/Ham’s F12 medium. For cell death assay, 20–25,000 keratinocytes were seeded in collagen coated 96 well plates, stimulated with 20 ng/ml TNF with or without Z-VAD (20 μM) for 18 h and viability was assessed with WST-1 assay as per manufacturer’s instruction (Roche).Keratinocytes from newborn pups were isolated as described using dispase II . Briefly, the skin from newborn mice was incubated overnight at 4°C in dispase II. After incubation, epidermis was separated and placed in TrypLE  for 20 min followed by making single cell suspension using low CaIl1-b, Il-6, Il-24, Ccl3, and Ccl4 genes was performed with Sybr green. Hprt was used as reference genes. Primer sequences were used as described before  and purified on RNeasy Columns (QIAGEN). cDNA was prepared using Superscript III cDNA synthesis Kit (Life Technologies). qRT-PCR of d before . qRT-PCR4SCN) in phosphate buffer, pH 6.8 (0.1 M NH2HPO4 and 0.1 M KH2PO4) for 30 min on ice.Mouse epidermis was separated after incubating skin samples in 0.5 M ammonium thiocyanate (NHP ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005. Statistical analysis was performed using GraphPad Prism.Data shown in column graphs represent mean ± SEM. For statistical analysis of data from qRT-PCR, quantification of epidermal thickness and inflamed area, when data did not fulfill the criteria for Gaussian distribution, nonparametric Mann–Whitney test was performed. For the keratinocyte’s death experiments, multiple comparisons of groups were evaluated by ordinary or Kruskal–Wallis one-way ANOVAs with post-Dunn corrections. *