Genetic studies demonstrate that proper m6A-methylation is required for mouse brain development and function. Revealing landscapes of m6A-methylation in the cerebral cortex at different developmental stages will help to understand the biological meaning of epitranscriptomic regulation. Here, we depict the temporal-specific m6A-methylation status in mouse embryonic and postnatal cortices using methylated RNA immunoprecipitation (MeRIP) sequencing. We identified unique m6A binding motifs in stage-specific RNAs and found that more RNA transcripts are temporally methylated in embryonic cortices than in postnatal ones. Moreover, we found that cortical transcription factors and genes associated with neurological disorders are broadly as well specifically methylated at m6A sites. Our study highlights the importance of epitranscriptomic regulation in the developing cortex and provides a fundamental reference for future mechanistic examinations of m6A methylation-mediated gene expression regulation in normal brain development and neurological disorders.N Proper development of the mammalian cerebral cortex is crucial for its morphogenesis and function. In the mouse embryonic cortex, neural stem cells and neural progenitors proliferate and differentiate properly to set up the architectural foundation of the cortical size and shape ,2. In th6-methyladenosine (m6A) modification, have been reported to regulate RNA splicing, stability, translation rate and localization [6A-methylation is required for normal cortical development. Genetic deletion of m6A methyltransferase Mettl14 results in abnormal proliferation and differentiation of neural progenitor cells in the embryonic cortex [6A-methylation is critical for maturation and function of postnatal and adult brains, as m6A-methylation has been shown to regulate dopamine transmission, sensory experience, learning and response to brain injury [6A-methylation is also associated with neurological disorders, such as Alzheimer’s disease, glioblastoma and major depression [6A regulation are still unclear, understanding comprehensive profiles of RNA transcripts methylated by m6A will facilitate uncovering RNA methylation-mediated gene expression regulation and function.Accumulating studies have demonstrated essential roles of genetic and epigenetic factors in controlling proper cortical development ,9. For ilization ,14. In pc cortex ,16,17. Mn injury ,19,20. Apression ,22,23,246A-methylation status in the developing cerebral cortex, we conduct methylated RNA immunoprecipitation (MeRIP) sequencing of RNAs collected from mouse embryonic and postnatal cortices. We identify unique m6A binding sites in temporally expressed RNAs and reveal specific m6A patterns carried by RNAs encoding transcription factors and disease-risk genes in the nervous system. Our results have provided strong evidence and useful reference of the importance of m6A-methylation in cortical development.In this study, to reveal mWild-type C57BL/6 mice were purchased from Wushi company . All animal experiments and euthanasia were approved (A2019009) and performed in accordance with the guidelines of Animal Care and Use, Committee of School of Medicine, Huaqiao University.TM Plus Reagent . Total RNA was extracted using the same reagent according to the manufacturer’s instructions. In general, embryonic-stage RNA was extracted from three E12.5–13 mouse embryos from one litter, and postnatal-stage RNA was extracted from three P14 mice from one litter. About 300 μg RNA was used for MeRIP, and all RNA samples were treated with DNase to remove genomic DNA.Mice were sacrificed by cervical dislocation and their cerebral cortex was removed and immediately digested with RNAiso6A-MeRIP reaction was performed according to a previous description [6A-seq). A portion of fragmented RNA was saved as input before immunoprecipitation (IP). The remaining fragmented RNA was incubated with an m6A-specific antibody  at 4 °C for 2 h, followed by addition of Immobilized Recombinant Protein A  for immunoprecipitation. After 2-h incubation at 4 °C, beads were extensively washed three times with IP Buffer . Immunoprecipitated RNA was recovered through elution with m6A nucleotide followed by ethanol precipitation. Due to the recognition feature of the m6A antibody, the detection of methylated sites might contain both m6A and m6A(m) [The mcription . Brieflyd m6A(m) . Additio6A-MeRIP were used for library construction with the NEBNext Ultra RNA library Prep kit from Illumina and were sequenced with Illumina HiSeq 4000. Library preparation and high-throughput sequencing were performed by Novogene . The m6A-seq data (NCBI GEO: GSE141938) was analyzed with reference to the previous standardized pipeline [6A-seq data was cleaned by trimming the adapter using trim galore. The cleaned reads were aligned to the house mouse (Mus musculus) genome UCSC mm10 using Bowtie2 with default parameters and were then converted to a bam file by samtools. The PCR duplicates in the aligned data were removed by the MarkDuplicates function of Picard (version 1.95) with parameters, REMOVE_DUPLICATES = Ture. After filtering the duplicates, samtools was used to sort the bam file according to the coordinates. The m6A peaks were detected by MACS (Version 1.4.0rc 2) with parameters, -format = “BAM” -gsize = 282000000 -tsize = 101 -nomodel -shiftsize =150 -to-large False -w -S. The resulting m6A-seq data from m6A-antibody immunoprecipitation samples at E12.5–13 and P14 were normalized to their respective input RNA data before being used for the following analysis.For high-throughput sequencing, purified RNA fragments from mpipeline . Briefly6A motif analysis. Briefly, we set the peak-harboring genome interval as detected data, while the peak-removed exon interval was set as background data. The detected data and background data were compared for motif analysis using HOMER with the following orders: -gc, -RNA, -len 6, -size given, -homer2, -p 2, -S 25, -dumpFasta. Each sample was independently measured, followed by intra-group comparison in E12.5–13 triplicates and P14 triplicates, respectively.Hypergeometric Optimization of Motif EnRichment (HOMER) was used for m6A peaks along the whole transcript [6A peaks, the Volcano maps of transcript levels and the Venn analysis of targeted genes were all performed using the Omic Share tools, a free online platform for data analysis (http://www.omicshare.com/tools).The integrative genomics viewer (IGV) tool was used for visualization of manscript . Notably10 , while the length of columns or the size of the circles indicated the gene counts. A full list of all selected terms of the biological process, cellular components, molecular functions and KEGG pathway category are provided in the The DAVID tool  was usedAll data are shown as mean ± SEMT. One-way analysis of variance (ANOVA) with post hoc contrasts were used for statistical analysis. The results were considered significant at probability level less than 0.05.6A-methylation is essential for cortical development, we suspected that m6A-methylation profiles should reflect the dynamic process in developing cortices. Because mouse cortices at embryonic days 12.5 to 13 (E12.5–E13), termed here as embryonic stage (E-stage), and cortices at postnatal day 14 (P14), termed as postnatal stage (P-stage), consisted of more neural progenitors and mature neurons, respectively, we performed MeRIP sequencing (MeRIP-Seq) using cortical tissues from E12.5–E13 and P14 mouse brains   .6A motif in temporal-specific cortical RNAs, we applied a sensitive and unbiased tool, Hypergeometric Optimization of Motif EnRichment (HOMER), for motif discovering. We detected the most confident motif, GGGACA, which was highly enriched in m6A peaks from triplicated E-stage cortices , and identified 2970 RNA transcripts from E-stage neural progenitors displaying higher expression levels than those from P-stage cortices, while 5416 transcripts showed higher expression from P-stage cortices than those from E-stage ones A. We detectively B,C. Thes aspects D,E.6A, the amounts of m6A sites were calculated according to their distribution region in RNAs with different changes in their transcription level , coding sequence (CDS) and 3′UTR  were generally higher than of E-special and P-special m6A sites in targeted regions of both changed and none-changed RNAs , termed as E-SMRs, 18% postnatal stage specifically methylated RNAs, named P-SMRs, and 56% continuously methylated RNAs, termed as CMRs, at both stages D. Notablp < 0.001, peak score > 500, FEs > 2.0)  score, which is calculated as peak-reads of immunoprecipitation samples versus those of relative input samples. We detected 3061 CMRs, 605 E-SMRs and 531 P-SMRs (s > 2.0) E. Moreovs > 2.0) F. These 6A binding sites within transcribed RNAs, we first analyzed the number of m6A binding sites in them. In E-stage cortices, one single m6A peak was detected in 58% RNAs, two m6A peaks in 26% RNAs, three m6A peaks in 10% RNAs, and four or more m6A peaks in 6% RNAs  in 47.02% and 45.85% RNAs from E-stage and P-stage cortices, respectively, and in untranslated regions (UTRs) in 36.62% and 37.12% RNAs from E-stage and P-stage cortices, respectively , downstream of CDS (2 kb), near stop codon , 3′UTR and transcriptional termination site   and 3′UTR regions  B. Additi regions . Collect6A binding sites show enrichment in the NSC and 3′UTR regions, and within these regions, more m6A binding sites are detected in RNAs from P-stage than those from E-stage cortices (6A binding sites in the NSC and 3′UTR regions. We first performed a gene ontology (GO) analysis on E-SMRs and P-SMRs  and found that enriched categories for E-SMRs are associated with DNA-replication and RNA-synthesis, and those for P-SMRs are enriched in cell membrane construction and assembly of specific neuronal cell bodies, dendrites and synapses B. In add6A-methylation. We first analyzed E-SMRs with m6A binding sites in the NSC and 3′UTR and identified 27 enriched pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis   . We detenscripts A. Moreovpathways . We foun pathway . Collect6A-methylation status in P-SMRs with m6A binding sites in the NSC and 3′UTR using the KEGG analysis. We observed that neural signaling transmissions, such as neuroactive ligand-receptor interaction pathways and multi-chemical synapses, are specifically enriched in P-SMRs (2+) and the calcium signaling pathway , and 3 megalencephaly-related E-SMRs   . MoreoveR region B.6A sites located near stop codon and in the 3′UTR, and the remaining 3 P-SMRs harbored one m6A site near stop codon , Parkinson’s disease (Dnajc6) and major depression disorder (Htr2a and Htr5a) . Htr5a cop codon .6A sites in the NSC or 3′UTR, Nin displayed three sites, and the rest of the CMRs contained one m6A site in the NSC or 3′UTR  in cortices at each stage is in accordance with previous studies of the mouse cerebellum and cortex [6A is likely achieved by binding to specific sites in transcribed RNAs specifically functioning in neural progenitors and mature neurons. Finally, while m6A-methylation occurs in the coding sequence in an RNA, it appears more frequently near the stop codon and in the 3′ UTRs. Our finding is consistent with previous reports of m6A binding sites [Besides the cerebral cortex, ms system ,40,41. Dl cortex , but als neurons . Moreoved cortex . Our finng sites .6A-methylated RNAs display distinct functions in embryonic and postnatal cortices. While genes specifically methylated in embryonic cortices function in cell differentiation, DNA repair and cell cycle, those in postnatal cortices play roles in transport and synapse assembly. Genes methylated at both stages function mostly in chromatin modification, nucleus and membrane structures, which represent fundamental cellular functions. Similarly, m6A-modified RNAs in the cerebellum are functionally enriched in cell cycle and DNA repair at early developing stages and changed to signal transduction and synaptic plasticity at later developing stages [We also found that mg stages . These s6A methyltransferases, such as METTL3 and METTL14 or demethylase FTO (fat mass and obesity associated protein), have demonstrated their important roles in controlling cortical development and regulating brain cognitive functions [6A sites, even though the position and number of methylation sites are different among them. Moreover, Par3 and Par6 are involved in the hippo-YAP signaling pathway and function in controlling polarity of cortical neural progenitors [6A-methylation in these and other key genes in the hippo pathway. Furthermore, we found that major genes associated with neurological disorders also are methylated at the m6A site. Even though genetic mutations in these genes are associated with disorders, such as microcephaly-related genes, it is unclear why m6A-methylation seems to be a common event in them and how it may modulate proper expression and translation of these disease genes.Genetic studies of depleting either munctions ,43,44. Ounctions ,46,47. Wgenitors ,49. Our 6A-methylation in the mouse developing cortex and revealed the basic rules of m6A-methylation in different genes highly expressed in the cortex. Our finding has provided an informative reference for future mechanistic examinations of m6A methylation-mediated gene expression regulation in normal brain development and neurological disorders.Our study has uncovered an image of m


HBB) gene, is one of the most common genetic diseases in the world. The HBB −28(A>G) mutation is one of the five most common mutations in Chinese patients with β-thalassemia. However, few studies have been conducted to understand how this mutation affects the expression of pathogenesis-related genes, including globin genes, due to limited homozygote clinical materials. Therefore, we developed an efficient technique using CRISPR/Cas9 combined with asymmetric single-stranded oligodeoxynucleotides (assODNs) to generate a K562 cell model with HBB −28(A>G) named K562–28(A>G). Then, we systematically analyzed the differences between K562–28(A>G) and K562 at the transcriptome level by high-throughput RNA-seq before and after erythroid differentiation. We found that the HBB −28(A>G) mutation not only disturbed the transcription of HBB, but also decreased the expression of HBG, which may further aggravate the thalassemia phenotype and partially explain the more severe clinical outcome of β-thalassemia patients with the HBB −28(A>G) mutation. Moreover, we found that the K562–28(A>G) cell line is more sensitive to hypoxia and shows a defective erythrogenic program compared with K562 before differentiation. Importantly, all abovementioned abnormalities in K562–28(A>G) were reversed after correction of this mutation with CRISPR/Cas9 and assODNs, confirming the specificity of these phenotypes. Overall, this is the first time to analyze the effects of the HBB −28(A>G) mutation at the whole-transcriptome level based on isogenic cell lines, providing a landscape for further investigation of the mechanism of β-thalassemia with the HBB −28(A>G) mutation.β-thalassemia, caused by mutations in the human hemoglobin β ( HBB) gene  gene . ApproxiBB) gene . MutatioBB) gene . As for BB) gene .HBB −28(A>G) mutation is one of the five most common HBB mutations carried by β-thalassemia patients in China (HBB −28(A>G) mutation, adenine (A) base located at 28 base pairs upstream from the cap site is mutated to guanine (G), disrupting the binding of transcription factor of ATA box and decreasing the RNA expression of HBB (HBB −28(A>G) mutation to understand how this mutation affects gene expression at the transcriptome level although correcting the HBB −28(A>G) mutation with the base editing (BE) system has also been reported in human iPS cells and reconstituted embryos . It shows that hemopoiesis, heme biosynthesis, response to oxidative stress, and other cellular activity pathways were directly or indirectly enriched by differentially expressed genes related to β-thalassemia  was used to compare control samples with patient samples carrying a novel lassemia , suggestlassemia . Howeverlassemia . Howeverlassemia . Therefolassemia .HBB −28(A>G) mutation on erythroid differentiation and how it affects genome-wide gene expression without confounding factors, such as different genetic background in allogenic samples, we used the CRISPR/Cas9 gene-editing system combined with asymmetric single-stranded oligodeoxynucleotides (assODNs) to generate the disease model of isogenic K562 cell lines (–28(A>G) showed no detectable HBB gene expression, enabling comparative studies in the same genetic background. We found that HBB was transcriptionally prevented by the mutation, and the K562–28(A>G) cell line is more sensitive to hypoxia and present a defective erythrogenic program when compared with wild-type K562 before erythroid differentiation by Gene Ontology (GO) and KEGG analysis. Interestingly, HBG showed a lower rate of induction in K562–28(A>G) when compared with wild-type K562 after erythroid differentiation. Taken together, our study is the first to analyze the effects of the HBB −28(A>G) mutation at the whole-transcriptome level based on isogenic cell lines. The unraveled molecular biomarkers and signaling pathways that affected in K562–28(A>G) cell line may be further investigated to explore the study of the mechanism of β-thalassemia in the future.In order to explore the specific impact of the ll lines  and thenTwo 20-bp sgRNAs were chosen containing the −28(A>G) site and the cutting sites were about 3 and 9 bp before the mutation site, respectively. The sequence shown in The 127-nt asymmetric single-stranded oligodeoxynucleotides (ssODNs) templates were designed by overlapping the CRISPR/Cas9 cleavage site with 36 bp on the PAM-distal side and a 91-bp extension on the PAM-proximal side of the break  and wereHBB −28 mutation, 1 × 106 cells were collected and resuspended in 100 μL electroporation solution (Buffer R) at a final density of 1 × 107 cells/mL, and then 3 μg ss ODNs and 5 μg Cas9/sgRNA (Addgene 48138) were added into the 100 μL resuspended cells. Electroporation was completed by the Neon Transfection System (Invitrogen). Electroporated cells were slowly loaded into growth medium without antibiotics and cultured in 5% CO2 at 37°C. 48 h after transfection, flow cytometry was used to isolate the single cell into 96-well plates. 1 week later, we characterized the single cell clone by PCR and Sanger sequencing. The resistant colonies were picked and expanded until we generated the expected cell line.To generate the –28(A>G) were cultured in glutamine-minus RPMI 1640 medium (Gibco) with 10% FBS in the presence of 1 mM sodium butyrate for 7 days to induce the erythroid differentiation in humidified atmosphere of 5% CO2 at 37°C (2 at 37°C.K562 cells were obtained from the American Tissue Culture Collection. The cells of K562 and K562 at 37°C . The conTM Reagent (Invitrogen). Single-stranded cDNA was synthesized with the oligo(dT) primer using the PrimeScriptTM RT reagent Kit with gDNA Eraser (Takara), the obtained cDNAs were analyzed by real-time PCR, using the indicated primers. The HBB primers were 5′- GCTCGGTGCCTTTAGTGATG -3′ (forward) and 5′- GCACACAGACCAGCACGTT -3′ (reverse); for HBG, 5′- GGAAGATGCTGGAGGAGAAACC -3′ (forward) and 5′- GTCAGCACCTTCTTGCCATGTG -3′ (reverse). The cycling conditions were 95°C denaturation for 10 min, 95°C for 15 s, annealing and extension at 60°C for 40 s, 40 cycles on the ABI step one machine.Total RNA was isolated from the cells by the TRIzol–28(A>G), K562cor−28(A > G)] and induced cells , we used the tetramethylbenzidine (TMB) ELISA Kit (Invitrogen): 1 × 105 cells from each of the test and control groups were resuspended in phosphate buffered saline (PBS) and mixed with an equal volume of the TMB solution and then kept at room temperature for 30 minutes. Then add a double volume of TMB stop solution to the wells. Brown-blue wells were regarded as positive and read at 450 nm by Biotek epoch. Each treatment was performed in triplicate, and each experiment was repeated three times.To compare the hemoglobin expression of the uninduced cells .FITC-conjugated CD235ab (BioLegend) was used as the erythroid-specific surface marker. About 5 × 10There are more than 65 million 100 bp pair-ended reads per sample. Prior to assembly, raw reads were filtered by SOAPnuke  with theWe used Samtools  to sort p < 0.05.The StringTie  suite of1, which were collected in the molecular signatures database (MSigDB)  of the DEGs  . Meanwhithe DEGs .To compare the expression levels of transcription regulator genes between pre- and post-induction K562 cells, we collected a comprehensive transcription factor annotation from AnimalTFDB 3.0  and iRegHBB −28(A>G) mutation affects gene expression at the genome-wide level, an isogenic human cell line carrying this mutation was generated. The diagram of the generating mutant cell line is shown, including transfection, CRISPR/Cas9 editing, single cell sorting, cell characterization, and expansion (HBB −28(A>G) mutation, sgRNA mediating DNA cleavage 3 bp aside from the −28 mutation site and assODNs with 36 bp on the PAM-distal side and 91 bp on the PAM-proximal side of the cutting site were used (HBB −28(A>G) mutation that showed a single peak at the mutation site by sanger sequencing and named it K562–28(A>G) (HBB −28(A>G) is generated by a large deletion of the other allele, we then performed CRISPR/Cas9-mediated gene correction on this K562–28(A>G) cell clone and obtained heterozygosity on the mutation site in our heterozygous correction clone K562cor−28(A > G)-het  and, thus, confirming the homozygosity of K562–28(A>G). In order to reconfirm the functional effect of the HBB −28(A>G) mutation, we used qRT-PCR and ELISA to detect the expression of HBB mRNA and HBB protein, respectively. In agreement with the sequencing result, the expression of both HBB mRNA and HBB protein were undetectable in the K562–28(A>G) cell line. In contrast, wild-type K562 shows a considerable expression of HBB mRNA and HBB protein even without erythroid differentiation (HBB −28(A>G) was successfully generated in which the expression of HBB was eliminated.To study how the xpansion . As prevxpansion  or asymmxpansion  as a repere used . K562, a–28(A>G) . In orde)cor-het , suggestntiation . These r–28(A>G) cells appear to have irregular morphology and spontaneous cell death compared to the wild-type counterpart, suggesting loss of HBB expression by −28(A>G) mutation may compromise cell viability in normal culture condition (–28(A>G) and its control K562. Pairwise Pearson correlation analysis revealed high similarity between replicates from K562–28(A>G) and K562 cells, indicating high reproducibility of our data (–28(A>G) and K562 cell are similar on a transcriptome level, suggesting that the mutation may not affect the cell identity (–28(A>G) compared to K562 (–28(A>G) had a higher expression of genes related to the mitochondrial electron transport chain, such as MT-CO1 and MT-ND, implying that the mutant cell line may need more energy and be subject to oxidative stress (–28(A>G) (–28(A>G) mutant cell line  (−28(A > G). In agreement with previous results, we observed the hypoxia-related genes, such as HMOX1 and SRGN on the PPI network, and the GATA family, HOXD10 and SPIC, which are well-known regulators of erythroid differentiation and hypoxia (–28(A>G) (–28(A>G) and the core regulators were the GATA family, HOXD10 and SPIC.Many K562ondition . To undeour data . Overallidentity . We cond to K562 . Interese stress . To furte stress  was down–28(A>G) . Furtherell line . Consist–28(A>G) . To furt hypoxia  were the–28(A>G) . The reg–28(A>G) . Taken tin vitro. In order to investigate the effect of the HBB −28(A>G) mutation during erythroid differentiation, we induced erythroid differentiation using sodium butyrate, confirmed the differentiation efficiency (–28(A>G) mutant cell line. Pairwise Pearson correlations represented in the matrix indicated that the isogenic cell lines showed high similarities up to 90%, and the samples were clustered together depending on their differentiation conditions (–28(A>G) and K562 when compared to undifferentiated samples (–28(A>G) and K562 (–28(A>G) and K562. GO analysis showed that erythrocytes take up carbon dioxide and release oxygen pathway and O2/CO2 exchange in the erythrocytes pathway were enriched in mutant cell lines , HBG (γ-globin), and HBE (ε-globin). HBB, HBG, and HBE are β-globin-like and all capable of forming a tetramer with α-globin. They are located within a gene cluster on chromosome 11, and their expression is coordinated by the same locus control region (LCR) as other regulatory DNA elements (HBG may be upregulated to compensate the loss of HBB. To study the compensatory gene expression of globin genes in K562–28(A>G) after differentiation [K562–28(A>G)-dif], the expression of HBB, HBA, and HBG was analyzed by RNA-seq in isogenic cell lines of K562–28(A>G) and K562. As expected, IGV analysis showed that HBB expression was induced but undetectable in K562–28(A>G) after differentiation (–28(A>G) after differentiation -dif when compared to those in K562-dif. However, HBG expression in K562–28(A>G)-dif was two-fold lower by FPKM than that in K562-dif , and its expression level in K562–28(A>G)-dif after differentiation was negligible when compared to K562-dif control  than that in K562 . Expression of other positive regulators, including GATA1, BGL3, and NFE2 (–28(A>G). In contrast, negative regulators BCL11A and ZBTB7A were upregulated in K562–28(A>G) compared to K562 before differentiation (–28(A>G) and K562 suggest the erythroid differentiation is generally normal in K562–28(A>G), but the mutation of HBB with −28(A>G) may affect the HBG expression through a network of transcription factors.elements . In β-thntiation . In contntiation . We notiK562-dif . The exp control . In agre in K562 . We furtpression , is founand NFE2 , were drntiation . The simHBB −28(A>G) mutation during erythroid differentiation, we identified the DEGs  within K562–28(A>G)-dif and K562-dif. The number of upregulated and downregulated DEGs in K562–28(A>G) were 158 and 740, respectively (–28(A>G)-dif were enriched in pathways related to stress-response and hematopoiesis disorder, such as regulation of the apoptosis process, negative regulation of leukocyte activation, myeloid leukocyte cytokine production, negative regulation of blood coagulation, negative regulation of hemostasis, and negative regulation of hemopoiesis. Meanwhile, downregulated DEGs in K562–28(A>G)-dif were enriched in oxygen-related pathways, including oxygen transport; erythrocytes take up carbon dioxide and release oxygen and O2/CO2 exchange in erythrocytes (–28(A>G)-dif reveal the oxygen transport and blood circulation were enriched -dif (–28(A>G)-dif (–28(A>G) after differentiation and transcription factors, such as GATA, HOX, and ZNF families, may play important roles as core regulators.To understand the effect of the ectively . With thhrocytes . Pathwayenriched . And PDE targets . MoreoveA>G)-dif  while thA>G)-dif . InteresA>G)-dif . Taken t–28(A>G) as mentioned before and used a homozygous correction cell line K562cor−28(A > G) to perform transcriptome comparison (cor−28(A > G) had a high relationship with K562 (cor−28(A > G) and K562 (cor−28(A > G) (–28(A>G) are reversed in corrected K562corG)–28 and not caused by editing process.As it took weeks for the generation of K562 mutant cells by CRISPR/Cas9 and clone selection, it is possible that the differential gene expression is due to secondary effects of the editing process, similar to studies of patient samples . In ordemparison . As expeith K562 . Importaand K562 , indicat > G)cor . The gen > G)cor . In summ–28(A>G) of HBB with the –28(A>G) mutation using CRISPR/Cas9 and a 127 nt assODN. This cell-based disease model was used to study how the HBB −28(A>G) mutation affected the cellular function on the transcriptome level before and after erythroid differentiation.In our study, we successfully generated the cell line K562HBB −28(A>G) mutation prevented the transcription of HBB gene. Analysis of enriched pathways suggested the PI3K pathway as well as the JAK-STAT pathway, which play important roles in erythroid differentiation, were disrupted in K562–28(A>G) before erythroid differentiation. The PI3K-Akt signaling pathway is a significant pathway that controls many cellular processes known as cell division, autophagy, survival, and differentiation (–28(A>G). Many clinical manifestations observed in β-thalassemia are attributed to the chronic hypoxic environment due to pathologic erythrocyte production, and our data suggest hematopoietic precursors may also be subject to oxidative stress before differentiation.Our results showed the ntiation . Moreove–28(A>G) (–28(A>G) after induction, suggesting the defective PI3K pathway may be caused by a lack of activators in undifferentiated K562–28(A>G). Other pathways, such as cell adhesion, pluripotency of stem cells, platelet activation, and Notch pathway, were also coactivated in differentiated K562 and K562–28(A>G) samples, indicating mutation of HBB −28(A>G) did not block the pathways required for differentiation. Nevertheless, consistent with data from undifferentiated K562–28(A>G), oxygen-related pathways were downregulated in differentiated K562–28(A>G)  during erythroid differentiation  , a findi–28(A>G) . Interes–28(A>G) . In both–28(A>G) , 4. The –28(A>G) . GATA-1 in human . Interesntiation , which mHBG in adults could partially reverse the severity of symptoms in sickle disease and β-thalassemia, it is important to understand the coordinated regulation between HBB and HBG (HBG was decreased in K562–28(A>G). ZBTB7A and BCL11A were two major repressors of HBG by directly binding the HBG gene promoters , paradoxical with the results of decreased fold induction of HBG in a mutant cell line. Although expression of BCL11A was lower in K562 when compared to K562–28(A>G) before differentiation, its expression increased dramatically during differentiation  mutation. However, these results may require further validation in clinical samples or alternative models.As improving the levels of  and HBG . In thisromoters . Expressntiation . Our uncHBB −28(A>G) mutation.Last, we not only used CRISPR/Cas9 to generate mutation, but also corrected the mutation with the same strategy. In one aspect, reversed abnormalities in corrected cell line confirmed the specificities of these phenotypes. In another aspect, the efficient editing results indicate our gene editing strategy using assODNs is powerful and provide means for gene editing treatment of –28(A>G) cell line generated with CRISPR/Cas9 and assODNs is a valuable model to evaluate the β-thalassemia with homozygous mutation of HBB with −28(A>G). It provides us with the first transcriptome data for mechanistic study on the effects of HBB −28(A>G), and the mutant cell line generated in our study can serve as a cellular disease model to screen lentivirus vectors in potential gene therapies or to evaluate the gene-editing strategies targeting HBB −28(A>G) mutation.In summary, we show the isogenic K562https://db.cngb.org/cnsa/ of CNGBdb with accession code CNP0000981 for more details.The datasets for this study can be found in the CNSA. Please see the YG, CL, and XZ conceived and designed the study. JL performed experiment from LG, TL, GD, and WO. ZZ and H-XS performed bioinformatics analysis. JL, ZZ, YG, and CL wrote the manuscript with the inputs from all authors. All authors contributed to the article and approved the submitted version.All authors were employed by BGI-Shenzhen which is a non-profit organization. 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.


AppNL-G-F/NL-G-F with amyloid pathology, rTg4510 with tauopathy, and SOD1G93A with motor neuron disease by magnetic activated cell sorting. In parallel, gene expression patterns of the human precuneus with early Alzheimer’s change (n = 11) and control brain (n = 14) were also analyzed by RNA sequencing. We found that a substantial reduction of homeostatic microglial genes in rTg4510 and SOD1G93A microglia, whereas DAM genes were uniformly upregulated in all mouse models. The reduction of homeostatic microglial genes was correlated with the degree of neuronal cell loss. In human precuneus with early AD pathology, reduced expression of genes related to microglia- and oligodendrocyte-specific markers was observed, although the expression of DAM genes was not upregulated. Our results implicate a loss of homeostatic microglial function in the progression of AD and other neurodegenerative diseases. Moreover, analyses of human precuneus also suggest loss of microglia and oligodendrocyte functions induced by early amyloid pathology in human.Microglia-mediated neuroinflammation has been implicated in the pathogenesis of Alzheimer’s disease (AD). Although microglia in aging and neurodegenerative disease model mice show a loss of homeostatic phenotype and activation of disease-associated microglia (DAM), a correlation between those phenotypes and the degree of neuronal cell loss has not been clarified. In this study, we performed RNA sequencing of microglia isolated from three representative neurodegenerative mouse models, The online version of this article (10.1186/s40478-020-01099-x) contains supplementary material, which is available to authorized users. Alzheimer’s disease (AD) is the most common neurodegenerative disease that causes dementia, neuropathologically characterized by the accumulation of amyloid β (Aβ), phosphorylated Tau, and neuronal dystrophy and loss . NeuroinSeveral transcriptomic studies using single nucleus analysis examined the molecular aspects of neuroinflammation in the prefrontal or entorhinal cortex of AD , 26, 49;AppNL-G-F/NL-G-F mice that display an amyloid pathology [G93A mice with motor neurodegeneration [AppNL-G-F/NL-G-F mice do not show neuronal death, whereas rTg4510 and SOD1G93A mice show a substantial loss of neurons. We found that most homeostatic microglial genes were downregulated in rTg4510 and SOD1G93A mice, and were correlated with the degree of neuronal cell loss. In contrast, DAM genes were uniformly upregulated in all disease models, and this alteration was not correlated with neurodegeneration. Moreover, precunei of individuals with early AD pathology show downregulation of some microglial genes linked to homeostatic functions and genes linked to oligodendrocyte function.In this study, we first performed comparative gene expression analysis of isolated microglia from the three mouse models of neurodegenerative diseases: athology , rTg4510athology , and SODneration . DespiteOLIG1/2 mRNA levels in non-AD and AD brain as internal controls. The use of human postmortem brain tissue was approved by the Ethics Committee of Research Institute of Environmental Medicine, Nagoya University and Tokyo Metropolitan Institute. Brain tissues for RNA preparation were immediately frozen using liquid nitrogen and stored at − 80 °C before use.The postmortem brains from 25 individuals (non-AD = 14 and AD = 11) were obtained by autopsy with informed consent and diagnosed by a neuropathologist in the Brain Bank for Aging Research, Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology. All subjects with early AD pathology did not have apparent family history of dementia, therefore, were recognized as sporadic cases. The subjects were neuropathologically grouped according to the neurofibrillary tangle staging of Braak and Braak [App/NL-G-F+ mice (C57BL/6-App < tm3(NL-G-F)Tcs >), carrying App gene with humanized Aβ sequence , Swedish (KM670/671NL), Beyreuther/Iberian (I716F), and Arctic (E693G) mutations, were previously established by a knock-in strategy [AppNL-G-F/NL-G-F and wild-type mice were obtained by crossbreeding, and were maintained as inbred lines. Tg(Camk2a-tTA)1Mmay Fgf14Tg(tetO-MAPT*P301L)4510Kha/J mice were previously established [G93A gene (B6.Cg-Tg (SOD1*G93A)1Gur/J) on the C57BL/6 background were obtained from Jackson Laboratory  [Heterozygous strategy . Homozygablished . A parenablished . tTA actME, USA) . GenotypME, USA) , 23, 37.All mice were maintained under a standard specific pathogen-free environment  with free access to food and water throughout experiments. Animals were treated in compliance with the guidelines established by the Institutional Animal Care and Use Committee of Nagoya University and National Institutes for Quantum and Radiological Science and Technology.Magnetic-activated cell sorting (MACS) of microglia in brain or spinal cord is performed as described elsewhere . In brieAppNL-G-F/NL-G-F and 7-month-old rTg4510 mice and lumbar spinal cords of 5-month-old SODG93A mice together with the corresponding wild-type or control mice, respectively. For the RNA-seq of the human samples, total RNA was prepared from precuneus of frozen postmortem brain using mirVana™ miRNA Isolation Kit  according to the manufacturer instructions. The total RNA was qualified by using Agilent 2100 Bioanalyzer . Libraries were prepared by using TruSeq mRNA or TruSeq Stranded mRNA , and, from these libraries, 151-nt paired-end reads were sequenced on the HiSeq X Ten with the HiSeq X Reagent Kits (Illumina) and the NovaSeq 6000 with the NovaSeq Reagent Kits (Illumina).For the RNA-seq of the mouse samples, total RNA was extracted from MACS-isolated microglia of each models using an RNeasy Mini Kit . The RNAs were sampled from cerebral cortices of 8-month-old https://support.illumina.com/sequencing/sequencing_software/igenome.html). The Illumina adapter sequences, as well as low quality bases , were trimmed from 3′-ends of the sequencing reads with the Trim Galore v0.5.0 . The reads were qualified with FastQC v0.11.8 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) before and after trimming. The processed reads were mapped on the reference genome assembly with HISAT2 v2.1.0 [https://cran.r-project.org/web/packages/pheatmap/).The reference genome assemblies and gene annotations, the mouse genome version UCSC mm10 and the human genome version UCSC hg38, were retrieved from iGenomes  according to the manufacture’s protocol. Relative similarities of gene expression profiles of the samples were represented by a plot of the principal component analysis (PCA). The PCA analysis was performed with the prcomp function in R by using CPM values of the individual genes.ACTB primer, forward, 5′-CTGGAACGGTGAAGGTGACA-3′; reverse, 5′-CGGCCACATTGTGAACTTTG-3′; human HBEGF primer, forward, 5′-GGACCCATGTCTTCGGAAAT-3′; reverse, 5′-CCCATGACACCTCTCTCCAT-3′; mouse Actb primer, forward, 5′-CGGACTCATCGTACTCCTGCTT-3′; reverse, 5′-TTGGCCTCACTGTCCACCTT-3′; mouse P2ry12 primer, forward, 5′-CATTGACCGCTACCTGAAGACC-3′; reverse, 5′-GCCTCCTGTTGGTGAGAATCATG-3′; mouse Sall1 primer, forward, 5′-TGTCAAGTTCCCAGAAATGTTCCA-3′; reverse, 5′-ATGCCGCCGTTCTGAATGA-3′; mouse Apoe primer, forward, 5′-GAACCGCTTCTGGGATTACCTG-3′; reverse, 5′- GCCTTTACTTCCGTCATAGTGTC-3′; mouse Itgax (Cd11c) primer, forward, 5′-CTGGATAGCCTTTCTTCTGCTG-3′; reverse, 5′-GCACACTGTGTCCGAACTCA-3′; mouse Hbegf primer, forward, 5′-ACCAGTGGAGAATCCCCTATAC-3′; reverse, 5′-GCCAAGACTGTAGTGTGGTCA-3′.Total RNA was extracted from MACS-sorted microglia using the RNeasy Micro Kit (Qiagen) according to the manufacturer’s instructions. Complementary DNA (cDNA) from MACS sorted cells was generated and amplified from 2.5 or 5 ng of total RNA by using the PrimeScript™ RT reagent Kit   and 1/50 of the yield was amplified with the SYBR Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa Bio) using the Thermal Cycler Dice Real Time System II or III (TaKaRa Bio). The thermocycle protocol was as follows: 1 cycle at 95 °C for 30 s, 40 cycles at 95 °C for 5 s and 60 °C for 30 s, and a dissociation stage of 95 °C for 15 s, 60 °C for 30 s, and 95 °C for 15 s. Actin was used for normalization. The primers used for real-time RT-PCR were as follows: human Immunofluorescence analysis was performed as described previously . In brieq-value was calculated for multiple testing correction of the p-values with the q-value package v2.24.1 [t test with the Bonferroni method.For RNA sequencing data, differential expression analysis between two groups with replicates was performed with edgeR v3.24.3  implemen v2.24.1  in R. TPAppNL-G-F/NL-G-F mice with amyloid pathology [G93A mice modeling inherited ALS [AppNL-G-F/NL-G-F mice did not show neuronal loss or neurofibrillary tangles (NFTs) [G93A mice exhibited robust neurodegeneration. Therefore, we used the AppNL-G-F/NL-G-F mouse as a mild model and rTg4510 and SOD1G93A mice as severe models of neurodegenerative diseases. We analyzed gene expression in microglia isolated by magnetic-activated cell sorting (MACS) from cerebral cortex of AppNL-G-F/NL-G-F and rTg4510 mice or lumbar spinal cord of SOD1G93A mice at the middle-to-late disease stage  . In contAppNL-G-F/NL-G-F, rTg4510, and SOD1G93A microglia, respectively  Fig. a. AnalysAPOE)- and TREM2-dependent manner [AppNL-G-F/NL-G-F mice show neuroinflammation and an amyloid pathology without neuronal cell death, whereas rTg4510 and SOD1G93A mice show robust neuroinflammation with neuronal cell death. A heat map of the 68 gene expression profiles illustrates changes in expression of homeostatic microglial genes associated with the degree of neuronal cell loss  [Itgax     were unchanged, expressions of BCO2, CAPN3, small G protein-associated genes (RASGRP3 and RAPGEF5), PACC1 , and P2RX7 were significantly decreased in AD precuneus compared with controls  were downregulated  were not altered in the precuneus of AD brain  was significantly decreased in human AD precuneus  revealed that 12 genes  were commonly altered in AppNL-G-F/NL-G-Fand rTg4510 cortical microglia, and only one gene (HBEGF/Hbegf) was significantly decreased in AD precuneus as well as AppNL-G-F/NL-G-Fand rTg4510 cortical microglia  and deregulated genes of cortical microglia of AppNL-G-F/NL-G-F or rTg4510 mice . We found that deregulated 32 genes, consisting of 8 upregulated and 24 downregulated, were common among AD precuneus, AppNL-G-F/NL-G-F and rTg4510 , and interferon-induced genes , indicating neuroinflammatory changes in AD precuneus and AD mice (Table S1j). In human precuneus of AD brains and AppNL-G-F/NL-G-F mice, we found 44 deregulated genes (3 upregulated and 41 downregulated), including TNFSF10 and SLC6A8, whereas in AD precuneus and rTg4510 mice, we found 13 deregulated genes (2 upregulated and 11 downregulated) including PTPRD. These results suggest that some deregulated genes are common between microglia of different AD mouse models and human sporadic AD precuneus, and that AppNL-G-F/NL-G-F and rTg4510 cortical microglia may represent different neuroinflammatory aspects relevant to AD pathologies.To understand the neuroinflammatory nature of early amyloid pathology in AD, we generated expression profiles of commonly altered genes in the precuneus of human sporadic AD brain and cortical microglia isolated from ice Fig. e. We comOur gene expression analysis of isolated microglia from mice of three neurodegenerative disease models including AD revealed that downregulation of homeostatic microglial genes and robust upregulation of DAM genes. More importantly, the extent of downregulation of homeostatic microglial genes was correlated with the degree of neuronal cell loss. In addition, a gene expression profile of human precuneus of early AD pathology shows reduced expression of some microglial homeostatic genes without induction of DAM genes, suggesting that the microglial signature in human early AD has limited similarity to that found in AD mice.G93A mice exhibited more robust downregulation in homeostatic microglial genes than that of AppNL-G-F/NL-G-F mice, which exhibit neuroinflammation without neuronal loss  indicates neuroinflammatory changes in early AD precuneus. We also found downregulation of several microglial markers and DAM genes in early AD precuneus . Although the slight elevation of astrocytic and microglia activation markers in early AD precuneus was not statistically significant, the upregulation of chemokine and proinflammatory genes  in early AD precuneus. In recent years, increasing evidence suggests that white matter abnormalities are also an important component of AD [Another striking finding was the robust downregulation of genes linked to oligodendrocytes  compared with rTg4510 microglia (45 genes). AppNL-G-F/NL-G-F mice specifically shared 44 deregulated genes with AD precuneus, and TNFSF10, one of the upregulated genes, is implicated in neuroinflammation and Aβ accumulation [SLC6A8 is one of the 41 downregulated genes, and it mediates inflammation by regulating creatine uptake [PTPRD was decreased in both AD precuneus and rTg4510 mice; an association was reported between the rs560380 polymorphism in PTPRD and NFT burden [AppNL-G-F/NL-G-F and rTg4510 cortical microglia represent different aspects of AD pathologies.Comparative analysis of the gene expression profiles of AD precuneus and microglia isolated from e uptake . We alsoicroglia 6 genes cMicroglial gene signature in mice revealed that a loss of homeostatic microglia function is associated with the degree of neuronal cell loss. In humans, our evaluation of the precuneus of early AD pathology also suggests a loss of microglia and oligodendrocyte function induced by early amyloid pathology. Results from the present study indicate a correlation between glial phenotypes and severity of neurodegeneration, and also provide important resources to better understand the role of glial dysfunction in AD progression.Additional file 1: Table S1 Assembled multiple supplementary tables containing data for RNA sequencing and clinical and neuropathological data of human subjects. Table S1a. A list of differentially expressed genes in isolated-microglia from AppNL-G-F/NL-G-F mice. Table S1b. A list of differentially expressed genes in isolated-microglia from rTg4510 mice. Table S1c. A list of differentially expressed genes in isolated-microglia from SOD1G93A mice. Table S1d. Changes in homeostatic microglial markers in each mouse model of neurodegenerative disease. Table S1e. Changes in DAM markers in each mouse model of neurodegenerative disease. Table S1f. Clinical and neuropathological data of human subjects. Table S1g. A list of differentially expressed genes in the precuneus of AD brain. Table S1h. A list of differentially expressed genes enriched in neurons in the precuneus of AD brain. Table S1i. Changes in the genes defined as risk factors for AD in the AD precuneus and microglia isolated from AppNL-G-F/NL-G-F and rTg4510 mice. Table S1j. A list of commonly upregulated genes in the precuneus of AD brain and isolated-microglia from AppNL-G-F/NL-G-F mice and/or rTg4510 mice. Table S1k. A list of commonly downregulated genes in the precuneus of AD brain and isolated-microglia from AppNL-G-F/NL-G-F mice and/or rTg4510 mice.


The integrins function as the primary receptor molecules for the pathogenic infection of foot-and-mouth disease virus (FMDV) in vivo, while the acquisition of a high affinity for heparan sulfate (HS) of some FMDV variants could be privileged to facilitate viral infection and expanded cell tropism in vitro. Here, we noted that a BHK-adapted Cathay topotype derivative (O/HN/CHA/93tc) but not its genetically engineered virus (rHN), was able to infect HS-positive CHO-K1 cells and mutant pgsD-677 cells. There were one or three residue changes in the capsid proteins of O/HN/CHA/93tc and rHN, as compared with that of their tissue-originated isolate (O/HN/CHA/93wt). The phenotypic properties of a set of site-directed mutants of rHN revealed that E83K of VP1 surrounding the fivefold symmetry axis was necessary for the integrin-independent infection of O/HN/CHA/93tc. L80 in VP2 was essential for the occurrence of E83K in VP1 during the adaptation of O/HN/CHA/93wt to BHK-21 cells. L80M in VP2 and D138G in VP1 of rHN was deleterious, which could be compensated by K83R of VP1 for restoring an efficient infection of integrin-negative CHO cell lines. These might have important implications for understanding the molecular and evolutionary mechanisms of the recognition and binding of FMDV with alternative cellular receptors. Aphthovirus of the Picornaviridae family . Seven immunologically and genetically distinguishable serotypes  have been reported, and multiple subtypes occur in each serotype with varying geographical distributions [1–3, 3Cpro, 3Dpol) [Foot-and-mouth disease virus (FMDV) is the etiological agent of a highly contagious vesicular disease in domestic and wild cloven-hoofed animals, mostly cattle, swine, sheep and goats, and many species of ruminants ,2. The vibutions . The viributions . The ORF, 3Dpol) . The non, 3Dpol) .The fundamental initial stage in the life cycle of FMDV is represented by the recognition and binding of receptors on the cell surface that enable virus attachment and entry via the endocytic pathways . αVβ3 haRemarkably, clusters of conserved mutations at or adjacent to the classical RGD motif in the G–H loop of VP1 (130–165 residues) and compensatory replacements  around the VP1 G–H loop of FMDV would ablate integrin interaction that exhibits the non-RGD binding capacity to infect the target cells (reviewed in ). Sa-CarFor our initial study, we were concerned that a genetically engineered virus of Cathay topotype of FMDV serotype O (rHN) with a high affinity for heparin was insufficient to initiate an integrin-independent entry into HS-positive CHO-K1 cells and mutant pgsD-677 cells ,32. It wN-acetylglucosaminyl and glucuronyltransferase deficient, CRL-2244) were purchased from the American Type Culture Collection  and were grown in F-12K nutrient mixture (Gibco) with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma). All cells in culture were incubated at 37 °C in a humidified chamber containing 5% CO2.BSR-T7/5 cells (which can stably express T7 RNA polymerase) were kindly provided by Prof. Karl-Klaus Conzelmann  and were cultivated in Glasgow minimal essential medium  supplemented with 10% fetal bovine serum , 4% tryptose phosphate broth (BD-Bacto) and 1 mg/mL G418 (Sigma). BHK-21 cells were obtained from the China Center for Type Culture Collection  and were maintained in Dulbecco’s modified Eagle’s medium  containing 10% FBS and 2 mM L-glutamine (Gibco). Two integrin-negative CHO cell lines . O/HN/CHA/93tc is a cell-adapted derivative after nine passages of BHK-21 cells cytolytically infected with O/HN/CHA/93wt . AdditioHere, pOFS was used as the original backbone for the construction of site-directed mutated full-length cDNA clones. The introduction of amino acid mutations in the VP2 and VP1 coding regions of FMDV were carried out by one-step overlap extension PCR with five pairs of primers , using aNot I (New England Biolabs), purified by the JetQuick PCR product purification spin kit (Genomed) and transfected into subconfluent monolayers of cultured cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.BSR-T7/5 cells were seeded in G418-free medium. The plasmid cDNAs were linearized with The supernatants of transfected cells were serially passaged in BHK-21 cells up to 20 times , following three freeze–thaw cycles. The total RNAs of the harvested viruses were extracted by using the RNeasy mini kit (Qiagen). The RT-PCR products were amplified with a pair of primers . The DN2. Finally, the cultured cells were fixed with cold acetone/methanol  and stained with 0.2% crystal violet (Sigma) at 48 h or 72 h post infection [The confluent monolayers of BHK-21, CHO-K1 and pgsD-677 cells were cultured in six-well plates. Serial 10-fold dilutions of virus stocks (200 μL/well) were inoculated onto the surface of cells for 1 h of incubation. Then, the inocula were removed, 2 mL of overlay medium containing 0.6 gum tragacanth  and 1% FBS were added and the cells were routinely incubated at 37 °C in 5% COnfection . The titx mg/mL, x = −4~0; Sigma) or phosphate-buffered saline  at room temperature for 10 min. Monolayers of BHK-21 cells and two CHO cell lines were refreshed with PBS (containing 1 mM CaCl2 and 0.5 mM MgCl2), pre-treated with an RGD-containing peptide (VR-17: 141-VPNLRGDLQVLAQKVAR-157 [The appropriate virus concentrations (10–50 PFU/100 μL) were prepared in DMEM and mixed with equal volumes of soluble heparin sodium  were inoculated with the sample preparations of the specific viruses at a multiplicity of infection (MOI) of 10 for 1 h adsorption at 4 °C. The virus suspensions were then removed and the inoculated cells were washed with ice-cold PBS and incubated in fresh medium at 37 °C. At the appropriate times, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Tween 20 in PBS and blocked with 1% bovine serum albumin. After washing three times with PBS, the fixed cells were incubated with guinea pig anti-FMDV (serotype O) polyclonal antibody , and clathrin heavy chain monoclonal antibody  or caveolin-1 polyclonal rabbit antibody  overnight at 4 °C. The cells were washed again with PBS and incubated with the goat anti-guinea pig IgG H&L , and goat anti-mouse IgG H&L  or goat anti-rabbit IgG H&L  for 1 h at 37 °C. Following immunofluorescence, the antibody-incubated cells were washed with PBS, nuclei-stained with DAPI  for 5 min at room temperature, then washed, mounted and viewed under a Leica TCS SP8 confocal microscope. The images were captured digitally and processed by using Adobe Photoshop software.M2080L+K1083E+G1138D and rHNM2080L+G1138D) were examined by standard plaque assays on BHK-21 cells and two CHO cell lines. As expected, the formation of plaques was observed on CHO-K1 and pgsD-677 cells infected with rHNM2080L+G1138D, but not rHNM2080L+K1083E+G1138D  and its BHK-adapted derivative (O/HN/CHA/93tc) of Cathay topotype of FMDV serotype O were used to perform plaque assays on BHK-21, CHO-K1 and pgsD-677 cells. As a result, O/HN/CHA/93wt was unable to produce plaques on HS-positive CHO-K1 cells and mutant pgsD-677 cells, whereas O/HN/CHA/93tc formed plaques on these two integrin-negative CHO cell lines . ComparaE+G1138D . In addi21 cells . TherefoM2080L+K1083E+G1138D  and rHN  could compensate the deleterious effects of L80M in VP2 and D138G in VP1 of rHN for the production of viral plaques on CHO-K1 and pgsD-677 cells  bind to soluble αVβ6 with relatively higher affinity than αVβ3 for virus adsorption and penetration [The flexibility of non-RGD-dependent receptor usage reflects quasispecies dynamics of FMDV populations within various tissue-specific cells from susceptible host species ,42,43,44etration . The ligetration ,49, but etration ,50. TherM2080L and rHNG1138D in the integrin-independent infection of BHK-21 and CHO cell lines  surrounding the pentamer axis were crucial in the response to the integrin-independent infection of a few genetic and engineered FMDVs  in BHK-21 and CHO cell lines. The presence of E83K in VP1 might have co-evolved with L80 in VP2, and the introduction of K83R in VP1 might compensate for the deleterious effects of L80M in VP2 and D138G in VP1 upon the interaction of FMDV with alternative cellular receptors. These data prompt efforts to interpret the alternative receptor usage of FMDV involved in genetic heterogeneity, viral pathogenesis and antigenic variation.


Furthermore, the association of hnRNP-F with the phosphoinositide 3‑kinase (PI3K)/protein kinase B (AKT) signalling pathway was confirmed by western blotting after bioinformatic analysis. HnRNP-F expression was significantly decreased by treatment with the PI3K/AKT signalling pathway inhibitor LY294002, whereas hnRNP-F knockdown did not significantly affect PI3K or AKT expression, suggesting that hnRNP-F is likely a downstream target of the PI3K/AKT pathway. Forkhead box O1 (FOXO1) is a molecule downstream of PI3K/AKT and can be inhibited by phosphorylation. In addition, chromatin immunoprecipitation (ChIP) and luciferase reporter assays indicated that FOXO1 expression was negatively correlated with hnRNP-F expression as FOXO1 was found to bind to the promoter region of hnRNP-F mRNA and inhibit its transcription. To sum up, our findings suggest that hnRNP-F expression is regulated by the PI3K/AKT-mediated phosphorylation of FOXO1, with phosphorylation inhibiting FOXO1, which subsequently allows hnRNP-F to promote proliferation. This finding is a novel discovery in BC and could help reveal the mechanism of BC progression.Our previous study showed that heterogeneous nuclear ribonucleoprotein F (hnRNP-F) could induce epithelial-mesenchymal transition and metastasis in bladder cancer (BC), however, the role and mechanism of hnRNP-F in mediating the proliferative ability of BC cells remain unclear. HnRNP-F promoted the proliferation of BC cells by using BC cell lines and cell counting kit-8 (CCK8), colony formation and flow cytometry assays  Bladder cancer is one of the most common malignancies in adults, with high morbidity and mortality rates worldwide The pathological proliferation of cancer cells involves a large number of genes and proteins and multiple signalling pathways, such as the p21, MAPK, and phosphoinositide 3‑kinase (PI3K)/protein kinase B (AKT) signalling pathways Heterogeneous ribonucleoprotein F (hnRNP-F) belongs to the hnRNP family, which is involved in multiples aspects of translational regulation, including pre- and mature mRNA transcription, mRNA stabilization, posttranscriptional modificaiton and alternative splicing To explore the role and mechanism of hnRNP-F in mediating the proliferative ability of BC cells, in this study, we first show that hnRNP-F could promote the proliferation of cells in BC EJ and UMUC3 cells. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis based on 403 BC dataset from The Cancer Genome Atlas (TCGA) indicated that the PI3K/AKT pathway is significantly enriched in BC tumour tissue samples with up-regulated hnRNP-F expression. The further results showed that hnRNP-F is likely a downstream target of the PI3K/AKT pathway, and FOXO1 could bind to the promoter region of hnRNP-F mRNA and inhibit its transcription. Our findings provide novel insights into the function and mechanisms of PI3K/AKT/FOXO1-mediated regulation of hnRNP-F in BC pathogenesis, indicating that hnRNP-F could affect BC proliferation and therefore cancer progression.A rabbit polyclonal antibody against hnRNP-F was acquired from Novus Biologicals , and rabbit monoclonal antibodies against AKT, p-AKT, PI3K, p-PI3K, FOXO1, and p-FOXO1 were acquired from Cell Signaling Technology (CST) . An IgG rabbit polyclonal antibody, IgG mouse polyclonal antibody, Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L) and Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) were purchased from Proteintech . HRP-conjugated goat anti-rabbit IgG (H+L) and HRP-conjugated goat anti-mouse IgG (H+L) antibodies and antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were also purchased from Proteintech .2 atmosphere. The cell lines were authenticated by short tandem repeat (STR) profiling upon receipt and were propagated for <6 months after resuscitation. The PI3K/AKT signal pathway inhibitor LY294002 was purchased from Procell  and was used according to the instruction.The human BC cell lines 5637, T24, EJ and UMUC3 were cultured in RPMI-1640 medium  supplemented with 10% foetal bovine serum  and 1% penicillin/streptomycin at 37 °C in a humidified incubator in a 5% COProtein lysates derived from BC cells were separated by 12% SDS-PAGE gels and then transferred to 0.45-μm PVDF membranes. After being blocked with 10% skim milk for 30 minutes at room temperature, the membranes were incubated with antibodies against hnRNP-F, AKT, p-AKT, PI3K, p-PI3K, FOXO1, p-FOXO1  and GAPDH (1:5000) at 4 °C overnight, followed by an incubation with appropriate HRP-conjugated secondary antibodies (1:5000). Immunoreactivity was visualized by ECL  and an enhanced chemiluminescence  detection system for blot visualization. The protein levels were normalized to those of GAPDH.RT-qPCR was performed according to the instructions of a standard SYBR Green PCR kit  on an Applied Biosystems 7500 Real-Time PCR system . GAPDH was used for normalization. The primer sequences used in our study are shown in To stably suppress the expression of hnRNP-F in the EJ and UMUC3 cell lines, a lentivirus encoding a short hairpin RNA (shRNA) targeting hnRNP-F (sh-hnRNP-F) with the sequence 5'-GGUACAUUGAGGUGUUCAATT-3' was constructed . After transduction with the sh-hnRNP-F virus or a negative control virus, cells were screened using 5 µg/ml puromycin . The transfection efficiency was assessed by western blotting performed as described in subsection 2.3.2 atmosphere at 37 °C. The absorbance was measured at a wavelength of 450 nm, with a reference wavelength of 570 nm. The cell proliferation assay was performed on days 1, 2, 3, 4, 5 and 6.The proliferation of EJ and UMUC3 cells was evaluated by using CCK8  according to the manufacturer's instructions. In brief, 10 μl of CCK8 solution was added to the culture medium and incubated for 2 h in a 5% COEJ and UMUC3 cells (600 cells per well) were added to 6-well plates and cultured for 14 days. The resultant colonies were washed with PBS, fixed with 4% paraformaldehyde for 30 min, and stained with Giemsa . The colonies were photographed with an inverted microscope  and counted.The cell cycles of EJ and UMUC3 cells were examined by flow cytometry. Cells in log-phase growth were harvested and fixed with 70% ice-cold alcohol overnight. The cell pellets were treated with 100 µg/ml RNase, stained with 50 µg/ml propidium iodide (PI) and incubated for 30 min, after which time they were analysed by flow cytometry .EJ and UMUC3 cells transfected with pENTER or pENTER-hnRNP-F were collected, and ChIP assays were performed with the ChIP-IT High Sensitivity® Kit  according to the manufacturer's instructions. In brief, BC cells were fixed and sonicated to shear DNA into fragments of 200~1000 bp, and the following immunoprecipitation antibodies were then added: anti-FOXO1 (CST) and rabbit IgG . Specific primers  were useA dual-luciferase reporter assay was used to investigate the relationship between FOXO1 and hnRNP-F. In brief, the firefly luciferase reporter vector pENTE-hnRNP-F and a Renilla luciferase plasmid (pRL-TK) were transiently co-transfected into EJ and UMUC3 cells pretreated with FOXO1-specific or control shRNA. PRL-TK was used to normalize for transfection efficiency. After transfection activity, luciferase activity was measured by using a dual-luciferase reporter assay kit  according to a previously reported protocol https://cancergen-ome.nih.gov/). The data were used for KEGG enrichment analysis (https://www.kegg.jp/kegg/pathway). Furthermore, the promoter sequence of hnRNP-F was identified by using ALGGEN in PubMed database.The RNA-seq raw count expression profile and fragments per kilobase million (FPKM)-normalized expression profile of 403 samples from the BC (BLCA) dataset were obtained from The Cancer Genome Atlas (TCGA) programme data portal (P <0.05 was considered significant.Data are presented as the mean±standard deviation (SD), and all experiments were repeated three times. Student's t-test was used to analyse continuous data. All statistical analyses were performed with SPSS 20.0 software . P<0.01, Figure P<0.01, Figure The expression levels of hnRNP-F in human BC cell lines were quantified by western blot and RT-qPCR assays. HnRNP-F protein level was significantly increased in EJ and UMUC3 cells compared with 5637 and T24 cells . In contrast, the percentage of S-phase cells was significantly decreased in the knockdown cells (P<0.01)  and UMUC3 (P<0.001) cell lines. In contrast, the proliferation of sh-FOXO1+sh-hnRNP-F cells was significantly lower than that of sh-FOXO1+con-hnRNP-F cells in both the EJ (P<0.001) and UMUC3 (P<0.001) cell lines (Figure To investigate the relationship between FOXO1 and hnRNP-F, the roles of FOXO1 in the proliferation and cell cycle progression of BC cells mediated by hnRNP-F and affected by PI3K/AKT were investigated by CCK8, colony formation and flow cytometry assays. Upon treatment with LY294002, the proliferation of sh-FOXO1+con-hnRNP-F cells was significantly more vigorous than that of con-FOXO1+con-hnRNP-F cells in both the EJ (s Figure A. Furthes Figure B. In adds Figure C. In sums Figure .In this study, to explore the role and potential mechanism of hnRNP-F in affecting the proliferative ability of BC cells, firstly, hnRNP-F was found to be up-regulated in human BC EJ and UMUC3 cell lines by western blot and RT-qPCR assays. The knockdown of hnRNP-F could inhibit proliferation and delay cell cycle progression in EJ and UMUC-3 cells. Mechanistically, hnRNP-F expression was significantly decreased by treatment with the PI3K/AKT signalling pathway inhibitor LY294002, whereas hnRNP-F knockdown did not significantly affect PI3K or AKT expression, suggesting that hnRNP-F is likely a downstream target of the PI3K/AKT pathway. Furthermore, the ChIP and luciferase reporter assays indicated that FOXO1 expression was negatively correlated with hnRNP-F expression as FOXO1 was found to bind to the promoter region of hnRNP-F mRNA and inhibit its transcription.th most common form of cancer worldwide, with an estimated 549,393 new cases and 199, 922 deaths occurring globally in 2018 BC is the 10HnRNP-F expression was reported to be increased in BC tissue, and increased hnRNP-F expression was associated with a poor prognosis in BC patients in our previous studies in vitro and in vivoin vitro was inhibited when hnRNP-F was depleted. This effect of hnRNP-F depletion on cell proliferation prompted us to further explore the role of hnRNPF in cell cycle progression. Our flow cytometry results showed that in EJ and UMUC3 cells, the percentages of G1-phase cells were increased and the percentages of S-phase cells were decreased when hnRNP-F expression was knocked down, indicating that hnRNP-F promotes the G1/S phase transition in BC cells.HnRNP-F can affect cell proliferation in cervical cancer by modulating telomerase function through interactions with hTERC and the telomerase holoenzyme PI3K/AKT signalling has been shown to play pivotal roles in the proliferation and invasion of cancer cells Aberrant activation of the PI3K/AKT pathway has been identified in a wide range of cancers FOXO1 is a molecule downstream of PI3K/AKT that has been shown act as a tumour suppressor in BC in vivo. Second, the genetic polymorphism factors were not considered in this study. We could not rule out that the possibility that could affect the association.The current study was subject to several limitations that need to be considered when interpreting our findings. First, our findings is based on the experiments conducted in human BC EJ and UMUC-3 cells and needs to be further confirmed by animal experiments In summary, our findings demonstrate that hnRNP-F promotes the proliferation of BC cells. Furthermore, FOXO1 expression is negatively correlated with hnRNP-F expression as FOXO1 binds to the promoter region of hnRNP-F to inhibit its transcription. Mechanistically, hnRNP-F transcription is regulated by the PI3K/AKT-mediated phosphorylation of FOXO1, which is a novel discovery in BC and could be helpful for revealing the mechanism of BC progression.Supplementary table S1.Click here for additional data file.


The accurate retrieval of synaptic vesicle (SV) proteins during endocytosis is essential for the maintenance of neurotransmission. Synaptophysin (Syp) and synaptobrevin-II (SybII) are the most abundant proteins on SVs. Neurons lacking Syp display defects in the activity-dependent retrieval of SybII and a general slowing of SV endocytosis. To determine the role of the cytoplasmic C terminus of Syp in the control of these two events, we performed molecular replacement studies in primary cultures of Syp knockout neurons using genetically encoded reporters of SV cargo trafficking at physiological temperatures. Under these conditions, we discovered, 1) no slowing in SV endocytosis in Syp knockout neurons, and 2) a continued defect in SybII retrieval in knockout neurons expressing a form of Syp lacking its C terminus. Sequential truncations of the Syp C-terminus revealed a cryptic interaction site for the SNARE motif of SybII that was concealed in the full-length form. This suggests that a conformational change within the Syp C terminus is key to permitting SybII binding and thus its accurate retrieval. Furthermore, this study reveals that the sole presynaptic role of Syp is the control of SybII retrieval, since no defect in SV endocytosis kinetics was observed at physiological temperatures. The correct formation of synaptic vesicles (SVs) by endocytosis after their activity-dependent fusion is essential for the maintenance of neurotransmission. To be functionally competent, SVs must be packaged with a specific complement of lipids and proteins in a defined stoichiometry , 2. Mostin vitro and in vivo . This mutant is almost identical to one that failed to rescue SV endocytosis kinetics during stimulation in Syp knockout neurons . This truncation is at the position of a disease-related frame-shift mutation in Syp, which rescued SV endocytosis kinetics but not SybII retrieval  or the empty mCer vector. The first mutant was truncated at amino acid K242, retaining 22% of C-terminal amino acids . Following stimulation, the kinetics of the fluorescence decay reflects the speed of SybII-pHluorin retrieval, since endocytosis is rate limiting when compared with SV acidification  fused to its intraluminal C terminus . At restFig. 1ication , 27 but  but A. Fication .via 300 action potentials delivered at 10 Hz. Experiments were performed at 37 °C, to ensure that any observed effects were physiologically relevant. Stimulation of Syp knockout neurons expressing wild-type mCer-Syp resulted in an increase in SybII-pHluorin fluorescence due to SV exocytosis, which returned to baseline after termination of the stimulus (C and D). In contrast, the SybII-pHluorin response failed to return to baseline in Syp knockout neurons (mCer), indicating impaired retrieval (C and D). Furthermore, these neurons displayed a significantly larger evoked SybII-pHluorin peak, due to perturbed SybII retrieval during stimulation . Expression of mCer-Syp-T22 failed to rescue the increase in evoked peak height , consistent with previous work , suggesting a role for the Syp C-terminus in SybII retrieval both during and after neuronal activity. In contrast, expression of mCer-Syp-T60 fully rescued both the evoked peak height and retrieval kinetics of SybII-pHluorin (D and E). Therefore, the Syp C terminus performs a key role in the activity-dependent trafficking of SybII with a region between K242 and P276 essential for this function.Syp knockout neurons were cotransfected with SybII-pHluorin and mCer-Syp mutants, with SV recycling evoked stimulus , C and Detrieval  . Therefore, deletion of the Syp C terminus has no impact on SV recycling kinetics.The fact that Syp-pHluorin-T22 displayed unaltered activity-dependent trafficking suggests that SV recycling is also unaffected by loss of the C terminus. To confirm this, we monitored SV recycling using the reporter, vGLUT-pHluorin , which wD–F), even though the recovery kinetics were surprisingly faster at room temperature. Furthermore, mCer-Syp-T22 was unable to rescue either parameter (D–F). Since these defects were absent at physiological temperatures, it suggests that the only role for Syp at central nerve terminals is the control of SybII retrieval during SV endocytosis.This result was surprising, since a slowing in SV endocytosis has been observed in Syp knockout neurons , 23, 24.arameter , D–F. SiA). The extent of SybII binding was examined by western blotting. GST-Syp-C-T22 displayed no SybII binding over background GST levels (B and C), as predicted from its inability to rescue SybII-pHluorin trafficking. In contrast, GST-Syp-C-T60 displayed strong binding to SybII (B and C), in agreement with the rescue of SybII-pHluorin retrieval.The ability of mCer-Syp-T60, but not mCer-Syp-T22, to rescue activity-dependent SybII-pHluorin trafficking suggests that the region between T22 and T60 contains a SybII interaction site. Therefore, we determined whether the Syp C terminus with these truncations could bind to SybII. To achieve this, the Syp C terminus was fused to glutathione-S-transferase (GST) to generate affinity columns, which were then incubated with nerve terminal lysates A. The exT levels , B and Cto SybII , B and CB and C), indicating that GST-Syp-C-T22 must contain part of the SybII interaction site. Surprisingly, full-length Syp C terminus (GST-Syp-C-FL) displayed no binding to SybII over background levels (B and C). Therefore, SybII only interacts with Syp if the distal portion of the C terminus is removed.We next determined whether the region between the T22 and T60 truncations was sufficient to bind SybII by generating a fusion protein encompassing this sequence (GST-Syp-C-22-60). This fusion protein did not bind to SybII , B and Cd levels , B and CD) revealed that removal of seven amino acids C terminal to T22 (QPAPGDA) was sufficient to ablate SybII binding, suggesting this region is essential for the interaction (E and F). Therefore, a cryptic SybII interaction site resides within the first 26 amino acids of the Syp C terminus (residues 219–244). This site is occluded by the full sequence, suggesting sybII interactions are controlled by the distal Syp C-terminus. To test this, we synthesized a peptide identical to this distal region (Syp270–308) and examined its ability to modulate SybII binding to either GST-Syp-C-FL or GST-Syp-C-T60. In the absence of peptide, GST-Syp-C-T60 bound SybII while GST-Syp-C-FL did not, as observed previously (G and H). In the presence of Syp270–308, SybII binding to GST-Syp-C-T60 was retained, suggesting it did not interfere with the interaction. Interestingly, Syp270–308 facilitated an interaction between GST-Syp-C-FL and SybII (G and H), significantly increasing binding of SybII. This suggests that the distal region of Syp is key to revealing a cryptic SybII interaction site within the C terminus.Further truncation studies D revealeeraction , E and Feviously , G and Hnd SybII , G and HI). Both full-length (residues 1–116) and the cytoplasmic domain (1–90) of His-SybII bound to GST-Syp-C-T60 (J and K). However, deletion of the SybII SNARE motif (J and K). Therefore, the SybII SNARE motif is the interaction interface for the cryptic Syp binding domain.To determine the region of SybII that interacts with the Syp cryptic interaction site, sequential truncations of His-tagged SybII were performed and their ability to be extracted from bacterial lysates by GST-Syp-C-T60 was determined I. Both fyp-C-T60 , J and Kresidues –116 and domain 1–0 of His-RE motif , 29, 30 RE motif , J and Kvia a cryptic interaction site.Syp is reported to control both the activity-dependent trafficking of SybII and SV endocytosis kinetics , 23, 24.Two mutants were chosen for this study. The T22 mutant mimics a truncation that slowed SV endocytosis kinetics during stimulation in Syp knockout neurons , whereasvia their transmembrane domains, since removal of the Syp C terminus in either yeast two-hybrid assays , except foetal bovine serum  and papain . Nitrocellulose membranes and molecular weight markers were from BioRad . Primary antibodies were from Abcam  unless specified. All other reagents were from Sigma-Aldrich .CTCGAGTCAAGGAGACAGGCTGGGCCGCCCC; reverse primer AAGCTTTTACATCTGATTGGAGAAGGAGGTG (restriction sites underlined). The Syp C terminus was truncated by adding a stop codon after amino acids K237 (T22) A244 (T29), G249 (T35), and G268 (T60). GST-Syp-C-22-60 was generated using the forward primer TAAGCACTCGAGCAACCGGCACCCGGGGACGCCTACG and reverse primer TGCTTAAAGCTTAAGGCTGGTAGCCGCCCTGAGGCCC. Syp270–308 (mouse residues 270–308) was generated by BioServUK Ltd . Mouse SybII (residues 1–116) was cloned into a pQE-30 vector  using BamHI and SalI enzymes  and (1–90) were generated by adding a stop codon after amino acids R30 and W90.Syp-pHluorin was from Prof. Leon Lagnado (University of Sussex), vGLUT-pHluorin from Prof. Robert Edwards , SybII-pHluorin from Prof. Gero Miesenboeck (University of Oxford), and mCer-Syp was generated as described . TruncatAll animal work was performed in accordance with the UK Animal (Scientific Procedures) Act 1986, under Project and Personal Licence authority approved by the University of Edinburgh Animal Welfare and Ethical Review Body (Home Office project licence—7008878). Animals were killed by schedule 1 procedures in accordance with UK Home Office Guidelines; adults were killed by cervical dislocation followed by exsanguination, embryos were killed by decapitation followed by destruction of the brain. Syp knockout mice  were mai4 cells on poly-D-lysine and laminin-coated 25 mm coverslips. Cells were transfected on 7 to 8 days in vitro (DIV) with Lipofectamine 2000  was continuously perfused at either 37 °C or 24 °C . After 180 s cultures were perfused with alkaline imaging buffer (50 mM NH4Cl substituted for 50 mM NaCl) to reveal total pHluorin fluorescence. Images were captured using an AxioCam 506 mono camera (Zeiss), with pHluorin or mCer vectors visualized at either 500 nm or 430 nm excitation (long-pass emission filter >520 nm). Each experimental condition was sampled on the same day, within the same set of primary cultures.Primary cultures were used at 13–16 DIV. Live fluorescence imaging was performed on a Zeiss Axio Observer D1 or Z1 inverted epifluorescence microscope  with a Zeiss EC Plan Neofluar 40x/1.30 oil immersion objective. Cultures were mounted in an imaging chamber with embedded parallel platinum wires  and stimulated with 300 action potentials delivered at 10 Hz . Imaging buffer , beads were washed in 20 mM Tris (pH 7.4) and boiled in SDS sample buffer. The released proteins were separated by SDS-PAGE for western blotting analysis . IRDye secondary antibodies  and Odyssey blocking PBS buffer were from LI-COR Biosciences . Blots were visualized using a LiCOR Odyssey fluorescent imaging system, with band densities quantified using either LiCOR Image Studio Lite software (version 5.2) or Image J (version 1.52). The SybII band was normalized to the GST fusion protein band revealed by Ponceau-S staining . Where indicated, Syp270–308 was incubated with GST fusion proteins for 1 h, before washing and addition of nerve terminal lysate.Isolated nerve terminals were prepared from rat brains of both sexes . GST fust-test was used, for more than two groups, a one-way ANOVA was performed with a post-hoc Tukey test when comparing all conditions and a Dunnett test when comparing to one condition (both corrected for multiple comparisons).Statistical analysis was performed in Graph Pad Prism 6.0. Sample size (n) for neuronal cultures was individual coverslips and for synaptosomes, individual experiments. All data are presented as mean values ±standard error of the mean (SEM). For comparisons between two groups, a Student’s All relevant data are contained within the article.The authors declare that they have no conflicts of interest with the contents of this article.


A40R gene, which encodes a protein with unknown immune function. The innate immune responses triggered by MVA-B ΔA40R in infected human macrophages, in comparison to parental MVA-B, revealed an increase in the mRNA expression levels of interferon (IFN)-β, IFN-induced genes, and chemokines. Compared to priming with DNA-B (a mixture of DNA-gp120 plus DNA-GPN) and boosting with MVA-B, mice immunized with a DNA-B/MVA-B ΔA40R regimen induced higher magnitude of adaptive and memory HIV-1-specific CD4+ and CD8+ T-cell immune responses that were highly polyfunctional, mainly directed against Env. and of an effector memory phenotype, together with enhanced levels of antibodies against HIV-1 gp120. Reintroduction of the A40R gene into the MVA-B ΔA40R genome (virus termed MVA-B ΔA40R-rev) promoted in infected cells high mRNA and protein A40 levels, with A40 protein localized in the cell membrane. MVA-B ΔA40R-rev significantly reduced mRNA levels of IFN-β and of several other innate immune-related genes in infected human macrophages. In immunized mice, MVA-B ΔA40R-rev reduced the magnitude of the HIV-1-specific CD4+ and CD8+ T cell responses compared to MVA-B ΔA40R. These results revealed an immunosuppressive role of the A40 protein, findings relevant for the optimization of poxvirus vectors as vaccines.Development of a safe and efficacious vaccine against the HIV/AIDS pandemic remains a major scientific goal. We previously described an HIV/AIDS vaccine based on the modified vaccinia virus Ankara (MVA) expressing HIV-1 gp120 and Gag-Pol-Nef (GPN) of clade B (termed MVA-B), which showed moderate immunogenicity in phase I prophylactic and therapeutic clinical trials. Here, to improve the immunogenicity of MVA-B, we generated a novel recombinant virus, MVA-B ΔA40R, by deleting in the MVA-B genome the vaccinia virus (VACV)  The acquired immune deficiency syndrome (AIDS) pandemic caused by the human immunodeficiency virus (HIV)-1 is spreading worldwide, with high impact and severity in human health. In spite of active antiretroviral therapy (ART), in 2017, an estimated 1.8 million individuals became newly infected with HIV-1 and 940,000 people died from AIDS-related illnesses worldwide, according to the Joint United Nations Programme on HIV/AIDS. Therefore, the discovery of an effective vaccine against HIV/AIDS that could control the infection and disease progression should be one of the main priorities of the developed world.An effective vaccine against HIV/AIDS should stimulate both humoral and cellular immune responses to multiple HIV-1 viral antigens, including structural and regulatory proteins, and induce strong, broad, polyfunctional, and durable T- and B-cell responses . For exaPreviously, we constructed a recombinant MVA expressing HIV-1 gp120 (engineered to be produced as a cell-released product) and Gag-Pol-Nef  antigens from clade B (termed MVA-B) . MVA-B hA41L, B16R, C6L, K7R, and N2L (either as single deletions or in combination), have been associated with a significant enhancement in the magnitude, breadth, polyfunctionality, and durability of the HIV-1-specific T-cell and humoral immune responses in immunized mice, when compared to parental MVA-B  expressing different HIV-1 antigens have been widely used in several human clinical trials in the last few years, proving that they are safe and immunogenic, inducing HIV-1-specific humoral and cellular immune responses ,67,68. IAmong the different approaches developed to enhance the immune response induced by poxvirus vectors, one promising strategy is the removal of the VACV genes that antagonize the immune system, as the virus genome still contains several genes that interfere with host immune responses ,40. SeveA40R gene and tested its role on antigen-specific immune responses in vivo after deleting the MVA A40R gene in the vector backbone of MVA-B, an HIV/AIDS vaccine candidate that expresses HIV-1IIIB GPN as an intracellular polyprotein and HIV-1BX08 gp120 as a cell-released product from HIV-1 clade B isolate [A40R was controversial. On one hand, it has been proposed that the VACV gene A40R encodes a type II membrane glycoprotein that is expressed early during infection on the cell surface, but is not incorporated into IMV or EEV particles [A40R gene from the VACV strain WR resulted in a modest attenuation after intradermal inoculation of mice, which could perhaps reflect an immunomodulatory role for this protein [In this work we described the immunosuppressive function of MVA  isolate . Previouarticles . These aarticles  and, in  protein . On the  protein . Althoug protein . HoweverA40R gene was generated from the HIV/AIDS vaccine candidate MVA-B (termed MVA-B ΔA40R). It is of note that the amino acid sequence of WR A40 protein differs from MVA A40 protein, in which the last five amino acids at the C-terminus are substituted with another 14 unrelated ones. The results showed that A40R deletion had no effect on virus growth, demonstrating that MVA A40R gene is not essential for VACV life cycle, in contrast to what has been suggested by others [A40R gene was to study the innate immune responses triggered in THP-1 human macrophages infected with MVA-WT, parental MVA-B, and MVA-B ΔA40R deletion mutant. The results showed that, compared to parental MVA-B, MVA-B ΔA40R significantly enhanced the expression of several genes involved in the type I IFN signaling pathway, such as IFN-β, IFIT1, and IFIT2, as well as the viral dsRNA sensor MDA-5, and the pro-inflammatory chemokine MIP-1α, suggesting that MVA A40 protein could have an immunomodulatory role, blocking innate immune responses during virus infection. The enhanced levels of these mRNAs in MVA-WT versus MVA-B were likely due to a suppressive effect mediated by expression of the HIV-1 antigens. Moreover, since it has been described that innate immune responses play a critical role in the control and resolution of HIV-1 infection, providing signals for the efficient priming of the adaptive branch of immune response [Consequently, to evaluate whether the VACV A40 protein has an immunomodulatory role, an MVA recombinant vector lacking the MVA y others . As the y others ,20,23,72response , the enhC6L, C6L-K7R, and A41L-B16R MVA genes), where a pattern of GPN-specific CD8+ T-cell immune responses was mainly induced [To further define whether MVA A40 could impair the immune system in vivo, a DNA prime/MVA boost immunization protocol was performed in mice to compare adaptive and memory immune responses to HIV-1 antigens induced by parental MVA-B and the deletion mutant MVA-B ΔA40R. Results showed that the DNA-B/MVA-B ΔA40R immunization group presented significantly enhanced magnitude of the overall adaptive and memory HIV-1-specific CD4+ and CD8+ T cells expressing CD107a, IFN-γ, TNF-α, and/or IL-2 compared to DNA-B/MVA-B. These results further suggest the immunosuppressive role of MVA A40 protein, as deletions of well-known immunosuppressive VACV genes from MVA or NYVAC vectors expressing HIV-1 antigens have produced similar results ,76,77,78 induced ,20,21, M induced . The bioThe analysis of the gp120-specific humoral immune responses at the adaptive and memory phases showed that DNA-B/MVA-B ΔA40R immunization induced higher levels of total IgG, IgG1, IgG2a, and IgG3 anti-gp120 antibodies than DNA-B/MVA-B. This enhancement in antibodies against gp120 may have been mediated by the increase in innate immune responses and HIV-1-specific CD4+ T helper cells triggered by the MVA-B ΔA40R deletion mutant, and it could be a positive immune parameter, as it has been described that anti-HIV-1 V2-specific IgG1 or IgG3 antibodies are able to drive ADCC correlated with efficacy in the RV144 phase III clinical trial .Moreover, evaluation of the Th1 or Th2 response by analyzing the ratio of IgG2a/IgG1 or IgG3/IgG1 antibodies showed that all the viruses induced higher levels of IgG1 than IgG2a or IgG3 antibodies, indicating a Th2 response. However, MVA-B ΔA40R induced in the adaptive and memory phases a ratio of IgG2a/IgG1 significantly higher than MVA-B , indicating that MVA-B ΔA40R may induce a more pronounced shift towards a Th1 response than MVA-B. According to current thinking, any therapeutic vaccination approach against HIV-1 should stimulate the induction of cytotoxic T lymphocytes (CTLs) and Th1 cells. It has been demonstrated that Th1 cells are more resistant to HIV-1 replication than Th2 cells ,83,84, aA40R gene, and to confirm our previously suggested immunosuppressive role exerted by the MVA A40R gene, a revertant virus termed MVA-B ΔA40R-rev was generated. Due to the lack of detection (using Western blot or immunofluorescence assays) of A40 protein when expressed from its natural locus in MVA or MVA-B viruses, we decided to reintroduce the MVA A40R gene into the HA locus of the MVA-B ΔA40R under the control of a stronger sE/L viral promoter. A similar strategy was used previously to generate NYVAC-based revertant viruses, which allowed us to demonstrate the important role of NFκB activation by VACV in enhancing neutrophil migration and HIV-1-specific T-cell responses [To finally demonstrate that the in vivo effects triggered by MVA-B ΔA40R in immunized mice were due to the deletion of the MVA esponses ,78. Moreesponses . In factesponses  and Ebolesponses , and thiA40R gene at higher mRNA levels (5–7-fold) than MVA-B or MVA-WT, allowing the amplification of the A40 protein signal during viral infection. In the MVA-B or MVA-WT viruses, the protein levels of A40 were very low since the rabbit polyclonal antibody anti-A40 used could not detect the A40 protein by Western blot or by immunofluorescence, while in MVA-B ΔA40R-rev-infected cells the A40 protein was readily detected. This was confirmed by Western blot analysis with a multiplicity of infection (MOI) of 5 PFU/cell. For these experiments, we used the same antibody that was described in the first report of the A40 protein from the WR strain [A40R gene was inserted into the MVA HA locus (A56R gene) controlled by a stronger VACV promoter than its naturally occurring promoter, the MVA A40 protein was clearly detectable by Western blot and immunofluorescence assays using the anti-A40 antibody recognizing the VACV WR A40 protein, indicating that the MVA A40 protein was stable, correctly expressed, and detected in the cell membrane, as previously described with the WR A40 protein [The MVA-B ΔA40R-rev expressed the MVA R strain . The WesR strain . The expR strain  and reinR strain . AlthougR strain  was main protein .A40R gene and of A40 protein, strongly suggest the immunosuppressive function of A40 by blocking the type I IFN signaling pathway.Importantly, the RT-qPCR experiments showed that mRNA levels of IFN-β, IFIT1, IFIT2, MDA-5, TNF-α, MIP-1α, RANTES, and RIG-I in MVA-B ΔA40R-rev–infected human THP-1 macrophages were significantly lower than those induced by MVA-B ΔA40R or MVA-B. However, mRNA levels of the constitutive cellular gene, HPRT, and the heterologous HIV-1 gp120 gene were not affected, suggesting that A40 blocks specifically innate immune sensing genes. The clear decrease in the levels of IFN-β, IFN-induced genes, proinflammatory cytokines, and chemokines induced by the revertant virus MVA-B ΔA40R-rev, expressing high mRNA levels of the MVA Interestingly, the enhancing effect on adaptive HIV-1-specific CD4+ and CD8+ T-cell immune responses observed with MVA-B ΔA40R was restored, in general, to levels similar or lower to those induced by MVA-B when we immunized mice with MVA-B ΔA40R-rev, either in DNA/MVA or in MVA/MVA regimens, further confirming in vivo the immunosuppressive function of MVA A40 protein.A40R gene has an immunosuppressive role, with its deletion enhancing innate immune responses in cell cultures, and adaptive and memory HIV-1-specific CD4+ and CD8+ T-cell and humoral immune responses in vivo, while its overexpression inhibited innate responses in human macrophages and adaptive HIV-1-specific CD4+ and/or CD8+ T-cell immune responses in immunized mice. Thus, deletion of the A40R gene has an immunomodulatory role in VACV infection and ablation of this gene provides an important strategy for the optimization of MVA vectors as vaccines.Overall, our findings revealed that MVA