Epstein–Barr virus (EBV) is a ubiquitous oncogenic virus that induces many cancers. N6-Methyladenosine (m6A) modification regulates many cellular processes. We explored the role of m6A in EBV gene regulation and associated cancers. We have comprehensively defined m6A modification of EBV latent and lytic transcripts. Furthermore, m6A modification demonstrated a functional role in regulation of the stability of viral transcripts. The methyltransferase METTL14 was induced at the transcript and protein levels, and knock-down of METTL14 led to decreased expression of latent EBV transcripts. METTL14 was also significantly induced in EBV-positive tumors, promoted growth of EBV-transformed cells and tumors in Xenograft animal models. Mechanistically, the viral-encoded latent oncoprotein EBNA3C activated transcription of METTL14, and directly interacted with METTL14 to promote its stability. This demonstrated that EBV hijacks METTL14 to drive EBV-mediated tumorigenesis. METTL14 is now a new target for development of therapeutics for treatment of EBV-associated cancers. Epstein–Barr virus (EBV) is a ubiquitous oncogenic virus that contributes to approximately 2% of all cancers by modulating a myriad of host cell activities. M6A is the most abundant RNA modification which is important for infection with HIV-1, HCV, Zika virus, KSHV and SV40 virus. The role of m6A modification and the associated enzymes in EBV infection and related oncogenic activities has not been explored. We investigated the role of m6A modification on EBV-infection and examined its effects on the proliferative activities of the virus which contributes to cancer. We showed that the m6A methyltransferase METTL14 is an important factor in EBV-induced oncogenesis. METTL14 was dramatically increased in EBV latently infected cells. Knock-down of METTL14 led to decreased latent viral gene expression. The essential EBV latent antigen EBNA3C was up-regulated by METTL14-mediated m6A modification, and its expression led to a feedback loop in up-regulation of METTL14 transcription, and the stability of its protein. We now provide a more comprehensive understanding of EBV and the host cell RNA processing machinery critical for regulation of EBV activities to maintain latent infection and its oncogenic properties. Epstein-Barr virus (EBV or HHV4), was the first human oncogenic virus discovered and isolated from a Burkitt’s lymphoma patient in 1964 . EBV exhN6 methylation of adenosine (m6A) is the most abundant RNA modification and it is distributed at translation start sites, CDS regions, and 3’ UTRs –7. DeposWe investigated the role of m6A modification on EBV-mediated cell transformation, and further examined its contribution to the tumorigenic activities of the virus. We demonstrated that m6A modification of the EBV epitranscriptome facilitated enhanced expression of its latent genes and repressed lytic gene expression. Furthermore, we showed that the m6A methyltransferase METTL14 is a critical factor required for EBV-induced oncogenesis. We also showed that METTL14 was dramatically increased in EBV latently infected cells and down-regulated during EBV lytic infection. Knock-down of METTL14 led to decreased latent gene expression and an increase in lytic gene expression. EBV latent antigen EBNA3C, which is critical for viral-mediated transformation of cells, was up-regulated by METTL14-mediated m6A modification, and its expression led to a feedback loop by which METTL14 transcription was also induced, as well as its protein stability. We now provide for the first time a comprehensive understanding of the EBV epitranscriptome, and the host cell RNA processing machinery critical for regulation of viral transcripts to maintain latent infection required for its oncogenic properties.To determine the role of m6A modifications in the EBV lifecycle, we first examined the viral epitranscriptome in EBV transformed lymphoblastoid cell lines (LcLs) and EBV-positive Burkitt's lymphoma, Akata cells, using methylated RNA immunoprecipitation followed by sequencing (MeRIP-seq). Biological replicates of ribo-RNA deleted mRNA of each cell type were prepared for MeRIP-seq followed by peak calling using the MeTPeak package on both strands of the genome. We monitored expression of some lytic genes in LcLs through RNA-seq and RT-qPCR . HoweverWe also investigated the levels at which the viral transcripts were modified during reactivation and induction of lytic replication. We detected m6A modification of EBV transcripts during lytic infection. TPA and Butyric acid were used to induce lytic reactivation. We observed a dramatic reduction of m6A modification of EBV transcripts both in reactivated LcLs and Akata cells  Tables. We identified an enrichment of m6A modifications of viral transcripts during EBV latent infection, and conversely a decrease in m6A modification of viral transcripts during lytic infection. Therefore, we hypothesized that m6A modification was important for latent antigen expression and stability in infected cells. One of the most important functions of m6A modification is its effects on mRNA stability. Therefore, we examined the role of m6A in regulating the mRNA stability expressed from several essential EBV genes.METTL14 is a major component of the methyltransferase complex required for m6A modification, and so we first determined binding of METTL14 to the transcripts modified with m6A. The target genes included 4 latent genes, EBNA1, EBNA3C, LMP1, LMP2A, and 4 lytic genes, BRLF1, gp350, BMRF1 and BGLF5. For input samples, similar results were observed that there were relatively higher levels of latent transcripts compared to lytic gene transcripts according to the results of our real-time PCR assays in latently infected LcLs. We showed the RNA levels of transcripts in LcL input samples in To directly demonstrate the role of METTL14 in regulating these viral genes, we knocked down METTL14 followed by m6A RIP-qPCR. The results showed that m6A modification of EBNA1, EBNA3C, LMP1, BRLF1, gp350 and BMRF1 in the absence of METTL14 was dramatically decreased in both LcLs and Akata cells . We furtTo investigate the role of specific m6A modification sites, we introduced 4 mutations to disrupt the DRACH motif   in the EWe identified a large number of m6A modifications in EBV transcripts, and more specifically a different profile of m6A modification during latent and lytic infection. Therefore, we wanted to determine whether m6A related enzymes were regulated on EBV infection. We first performed RT-qPCR to detect the expression of m6A modification enzymes including METTL3, METTL14, YTHDF1, YTHDF2, YTHDF3, FTO and ALKBH5 in B cells, LcLs, Akata EBV negative and EBV-positive Akata cells. The RT-qPCR results showed that METTL14 was significantly up-regulated in EBV-positive LcLs and Akata cells . YTHDF2 We further examined the protein levels of METTL14, YTHDF2, and ALKBH5 since there were differences in their RNA expression. EBV negative B cells, Akata EBV-, BL41 and HEK293T cells, and latently infected LcLs, BL41-EBV and HEK293T-BAC-EBV cells were used for analysis of the protein levels by Western blot. The expression of the demethylase ALKBH5 was significantly decreased while the m6A methyltransferase METTL14 was dramatically increased in EBV-positive LcLs, BL41-B95.8 and there is a modest increase in Akata and 293T-BAC-EBV cells . These rThe expression of METTL14, YTHDF2 and ALKBH5 was also monitored in several other EBV-infected cells. Latent Sav I, Sav III, Mutu I, and Mutu III as well as reactivated Sav I, Sav III, Mutu I, and Mutu III cells were used for analysis of the protein levels. BZLF1 was detected by western blot to confirm reactivation of EBV. We showed that METTL14 expression was increased in latency EBV III cells (Mutu III and Sav III), compared to latency I cells (Mutu I and Sav I)  for up to 12 hours. The protein level of METTL14 was decreased dramatically in the absence of EBNA3C . The resWe further examined whether EBNA3C can interact directly with METTL14. Co-immunoprecipitation (Co-IP) assays were performed in B-cells with physiologic expression of EBNA3C. To determine the association in B-cell lines, BJAB, EBNA3C stably expressing BJAB (BJAB7) and EBV-transformed (LcL) cells were used for the Co-IP experiment. We performed IP using an EBNA3C antibody or METTL14 specific antibody to immunoprecipitate the target proteins followed by western blot analyses. The Co-IP results showed that endogenous EBNA3C can physically associate with METTL14 and more importantly in EBV-transformed lymphoblastoid cell lines .To determine the domain of EBNA3C which can specifically interact with METTL14, Myc-tagged full length and specific regions of EBNA3C  were co-transfected into HEK293 cells with Flag-tagged METTL14 . The tarTo determine whether the EBNA3C residues EBNA3C100-200 or EBNA3CΔ100–200 (EBNA3C without 100-200aa residues) affect the protein expression, protein stabilities and the oncogene function of METTL14, we expressed EBNA3C100-200, EBNA3CΔ100–200, or EBNA3C in HEK293 cells. We found that only the full-length EBNA3C can upregulate the expression of METTL14. Neither the EBNA3C100-200 alone nor EBNA3C lacking the 100–200 residues up-regulated METTL14 expression . We alsoWe have shown that EBNA3C binds to METTL14, so it is expected that these two proteins can coexist in the same cellular compartment and colocalize with each other in cells. To determine the co-localization of EBNA3C and METTL14, Saos-2 and HEK293 cells were initially transfected with plasmids expressing Myc-tagged EBNA3C, and the cellular localization of EBNA3C and METTL14 was monitored by fluorescence microscopy. In the absence of EBNA3C, the METTL14 proteins were distributed around the nucleus as seen with some punctate dots . In cellTo further determine the co-localization of EBNA3C and METTL14 proteins in EBV-positive B-cells, immune-fluorescence assays were performed using specific antibodies against EBNA3C and METTL14 in order to examine the endogenous expression in different B-cell lines. The results corroborated the above results that EBNA3C co-localized with METTL14 in BJAB cells that can stably express EBNA3C, as well as in EBV-transformed LcLs . This waWe further investigated the expression and localization of METTL14 in EBV-negative and positive human tumor tissues. Immunohistochemistry for METTL14 expression showed that it was clearly up-regulated in EBV-positive Post-transplant lymphoproliferative disorders (PTLDs) tumor tissues and that EBNA3C signals were also clearly detected in the EBV-positive tissues compared to EBV negative tissues . We suppTo examine the effects of METTL14 on cell proliferation, Saos-2 and HEK293 cells were transfected with expression of EBNA3C and METTL14 and selected with G418 for two weeks. The expression of EBNA3C and METTL14 was confirmed by western blot . We moniTo investigate the functional role of METTL14 in EBV-associated tumorigenesis, we probed the effects of its knock-down in a xeno-transplant model in vivo. LcL shCr, LcL shMETTL14, LcL shEBNA3C and LcL shMETTL14 plus shEBNA3C were subcutaneously injected into NOD-SCID mice to assess the effect of knock-down METTL14 or EBNA3C on tumor growth. The results demonstrated that knock-down of METTL14 or EBNA3C led to a dramatic slowing of tumor growth compared to the control group . ImportaWe also investigated the functional role of specific m6A modification sites on EBNA3C for cell growth. We transfected the wild-type or EBNA3C mutant at the m6A sites into BJAB (EBV negative cells) and Raji cells (EBV-positive but without EBNA3C expression) . Cell grModification of RNA by m6A functions extensively in cellular processes linked to RNA metabolism. These include mRNA stability, translation, splicing and RNA transport . This moEBV latent infection allows the virus to persist in a mostly dormant state for the lifetime of the host and is associated with numerous cancers . EBV proModification of RNA by m6A is catalyzed by a methyltransferase complex composed of METTL3, METTL14, and WTAP , and mRNMETTL3-METTL14-WTAP-mediated m6A modification is associated with cancer  and the Studies in KSHV revealed that blocking m6A inhibits splicing of the pre-mRNA encoding replication transcription activator (RTA), a key KSHV lytic switch protein, and halts viral lytic replication . KnockdoIn this study, we have now shown for the first time that the host RNA m6A modification machinery can be targeted and manipulated specifically by a viral-encoded oncoprotein. This modulated latent and lytic viral gene expression enhancing the pro-oncogenic potential of EBV. The study now provides a deeper understanding of specific interactions between a potent oncogenic virus and host cells at the level of the viral epitranscriptome, and further suggests a critical role for viral mRNA modification in driving the oncogenic process. Furthermore, targeting METTL14 may be a critical strategy for controlling EBV-associated cancers.The University of Pennsylvania Institutional Animal Care and Use Committee reviewed and approved all the animal experiments (protocol 804549). All the experiments were carried out in accordance with the Laboratory Animal Welfare guidelines for the Care and Use provided by National Institutes of Health. The University of Pennsylvania CFAR Immunology Core provided B-cells from deidentified humans. The CFAR Immunology Core maintains an Institutional Review Board (IRB)-approved protocol in which Declaration of Helsinki protocols are strictly followed. Every donor gave written and informed consent at the CFAR Immunology Core. All patients provided written informed consent.The plasmid pA3M-EBNA3C and the LcLs, Akata, and BJAB cell lines, have been described previously . LcLs we2 followed by incubation at 94°C for 8 min. Successful fragmentation of mRNA with sizes close to 120 nucleotides was validated using a BioRad Geldoc Station . Before immunoprecipitation, 10 μg of anti-m6A antibody was incubated with 30 μl slurry of Pierce Protein A Agarose beads  in 250 μl PBS with 0.5% BSA at 4°C for 2 h. The beads were washed three times in cold PBS with 0.5% BSA. To isolate the m6A-containing fragments, 900 μg of fragmented total RNA was added to the antibody-bound beads in 250 μl IP buffer supplemented with RNasin Plus RNase inhibitor , and the mixture was mixed at 4°C for 2h. The beads were washed four times with 1 ml IP buffer before elution with 100 μl IP buffer supplemented with 6.67 mM of m6A salt . The mixture was incubated for 1 h at 4°C with continuous shaking and the eluate was collected. A second elution was carried out and the eluates were pooled together before purification with 2.5-fold ethanol and 1/10 volume of NaAC pH5.2. RNA was washed with 75% ethanol for twice and dissolved in RNase free H2O.Isolation of m6A-containing fragments was performed as previously described with minor modifications . BrieflyPurified eluate and Input samples were used for the preparation of libraries and sequencing at the Genome Sequencing Facility of the GATC core at the Washington University St. Louis. Approximately 300 ng of mRNA was used for RNA sequencing (RNA-Seq). The library was prepared using the TruSeq stranded mRNA kit  according to the manufacturer’s protocol. First, the elute-frag-prime stage was done at 80°C for 2 min to allow annealing without causing fragmentation . RNA washttps://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) to remove adapter sequences and low-quality bases . After trimming, the MeRIP-seq reads were aligned to EBV reference genome by the aligner HISAT2 [https://www.ncbi.nlm.nih.gov/nuccore/NC_007605.1). The m6A peaks were called by a graphical model-based peak calling method–MeTPeak [http://www.broadinstitute.org/igv).After sequencing, the first step is quality and adapter trimming. Trimming was conducted by Trim Galore . 3μg of METTL14  or IgG  was conjugated to protein A/G agarose beads  by incubation for overnight at 4°C in RIP buffer at 4°C overnight. Beads were washed with RIP buffer for three times, followed by DNA digestion at 37°C for 30 min and incubation with 50μg of proteinase K  at 55°C for 30min. Input and co-immunoprecipitated RNAs were recovered by TRIzol, extraction and analyzed by qPCR.RIP was performed as previously described . BrieflySaos-2 and HEK293 cells were transfected with jetPRIME  according to the manufacturer’s instructions. BJAB cells were transfected by electroporation  with Bio-Rad Gene Pulser II electroporator in 400 ul of serum-free medium. The cells were then transferred to complete RPMI 1640 media, which was preincubated to 37°C.Immunoprecipitation (IP) and Western blotting were performed as described previously . BrieflyNOD.CB17-PrkdcSCID (NOD/SCID) male mice were purchased from Jackson Laboratory  at 6 weeks of age and 6 million cells were subcutaneously injected. 3 weeks later, mice were sacrificed and the weight and volume of the tumor were measured.2O2 for 10 min, and permeabilized with 0.5% Triton X-100 at RT for 10 min. The slides were blocked in 5% bovine serum albumin (BSA) at RT for 30 min and incubated overnight with diluted primary antibody at 4°C, and then diluted HRP-labeled secondary antibody was added at room temperature for 60 min. After development with diaminobenzidine for 3 min, the slides were washed in water and counterstained with hematoxylin.Immunohistochemistry was performed as described previously . TissuesFor immunofluorescence, cells were seeded on glass coverslips in 24-well plates before transfection. After treatment, cells were fixed with 4% paraformaldehyde at 4°C for 60min and permeated with 0.2% Triton X-100 in PBS for 10min. Nuclei were visualized by staining with DAPI for 2 min. Images were acquired using a Fluoview FV300 confocal microscope and Fluoview software was used for image analysis.HEK293T cells were co-transfected with pGL4.1 plasmid with METTL14 promoter, pRL-TK , Myc-tagged EBNA3C, or empty plasmids. Cells were harvested and the dual-luciferase reporter assay was performed according to the manufacturer's instructions  48 hours after transfection. Luciferase value was read using a Cytation 5 .The total RNA extraction was performed using Trizol reagent  and treated with DNase I . cDNA was prepared with Superscript II reverse transcriptase kit  according to the manufacturer’s protocol. Quantitative real-time PCR analysis was performed by using SYBR green real-time master mix . The primers used are listed in Cells were collected and suspended in 1XPBS at a concentration of 1 million cells/ml. The CFSE solution was added to make a final concentration of 5μM. An equal volume of 1XPBS containing 5% FBS was added after 10 min incubation at room temperature. Cells were washed three times with 1XPBS containing 5% FBS and equally divided into several plates for incubation. 3 days later, cells were harvested, washed with ice-cold 1XPBS and resuspended in 5ml 1XPBS, then run on FACS Calibur cytometer  followed by analysis with FlowJo software .5 cells were seeded in 6-well plates and allowed to grow for 5 days. Cells were stained with 0.005% Crystal Violet overnight and scanned by PhosphorImager  and the area of the colonies measured by using Image J software . Three independent experiments were performed. The soft agar assays were performed using LcLs and Burkitt lymphoma cells. Briefly, 1 ml of 0.5% agar in RPMI media was poured into 6-well plates and set aside to solidify. 0.5 ml 0.3% agar/medium containing 2×105 cells were added to the plates as the middle layer. Then cells were covered with a top layer of another 1ml 0.5% agar/medium. After two weeks, colonies were stained with 0.005% crystal violet for 1 hour and scanned using a Licor Odyssey system . The number of colonies was counted using ImageJ software .HEK293 or Saos-2 cells were transfected with control vector, Myc-EBNA3C or Flag-METTL14 and allowed to grow in DMEM supplemented with 1mg/ml G418 . After two weeks of selection, 10t-test. All statistical tests were performed using Microsoft Office Excel. A P-value of 0.05 was considered to be a statistically significant difference. A P-value of 0.01 was considered to indicate highly statistical significance.Each experiment was repeated at least three times. The mean scores were examined by using Student’s S1 Fig(A-C) The transcription level of indicated genes in the input sample of LcLs. Viral gene transcription levels were detected and normalized to cellular control GAPDH. The relative amount of EBNA1 expression was set as 1. (D) RPKM: Reads Per Kilobase per Million mapped reads of indicated genes from RNA seq data of LcLs. DNA contamination: Detection of DNA in input RNA samples before reverse transcription.(TIF)Click here for additional data file.S2 Fig(A) DMSO or (B) Actinomycin D was used to inhibit transcription for 6 and 12 hours and the levels of METTL14 mRNA was determined. Experiments were independently repeated three times, and results are presented as mean±s.d. from the three experiments.(TIF)Click here for additional data file.S3 Fig(A), Akata EBV negative cell and Akata EBV-positive cells (B) were collected and total RNA extraction was performed using Trizol reagent and treated with DNase I, then cDNA was prepared with Superscript II reverse transcriptase kit. Genes transcription level was detected and normalized to a cellular control GAPDH RNA. ΔΔCt method was used to analyze qPCR data. Error bars represent standard deviation. Experiments were independently repeated three times, and results are presented as mean±s.d. from the three experiments. “**” represents p-value <0.01; “*” represents p-value <0.05.3 million B-cell, LcLs (TIF)Click here for additional data file.S4 Fig(A-F) The quantitation of METTL14 protein levels shown in (G-L) The quantitation of EBNA3C protein levels shown in Fold change means relative densities which were quantified using the Odyssey ImageQuant software. This was representative of experiments repeated for each panel with similar results. UI: uninduced; IN: induced. (TIF)Click here for additional data file.S5 Fig(A) LcLs with shRNA cr or shMETTL14 were treated with DMSO or TPA (20 ng/ml) and Butyric acid  for indicated time. Cells were harvested at various times  and EBNA1 primers were used for determination of viral copy number. (B) RIP using METTL3 antibody to detect the overall levels of METTL3 on viral genes in LcLs. Primers were designed for the indicated gene regions. (C) 5 million LcL shCr, LcL shMETTL3 (shM3) cells were collected, lysed and subjected to western blot with indicated antibodies. (D) The effects of the demethylase inhibitor on EBV latent and lytic gene expression. 5 million LcLs were treated with TPA and Butyric acid (IN) or DMSO (UI), with or without meclofenamic acid, for 48 hours. Cells were collected, lysed and subjected to western blot with indicated antibodies. UI: uninduced with drugs; IN: induced with drugs.(TIF)Click here for additional data file.S6 Fig(A-G) 5 million Saos-2 cells were transfected with control plasmids, Myc tagged EBNA2 (E2), LMP1 (L1), LMP2A (L2A), LMP2B (L2B), EBNA3A (E3A), EBNA3B (E3B) or EBNA3C (E3C). 48 hours later, cells were collected, lysed and subjected to western blot with indicated antibodies and METTL14 levels were quantitated. GAPDH (GAP) was used as the loading control. (H-I) 5 million BJAB, BJAB7 (B7), BJAB10 (B10), B cell, LcL shCr, and LcL shEBNA3C cells were collected, lysed and subjected to western blot with indicated antibodies and METTL14 levels were quantitated. (J-K) 5 million BJAB, BJAB7 (B7), BJAB10 (B10), B cell, LcL shCr, and LcL shEBNA3C cells were collected and total RNA was extracted with Trizol reagent. The cDNA was prepared with reverse transcriptase kit, and EBNA3C and METTL14 mRNA was detected by RT-qPCR. GAPDH (GAP) was set as an internal reference. (L-M) HEK293 and Saos-2 cells were transfected with the reporter constructs containing the wild-type METTL14 promoter and an increasing amount of Myc-EBNA3C. Cells were collected and lysed in lysis buffer at 48 hours post-transfection. Luciferase activity was measured according to the dual-luciferase reporter assay kit compared to pGL4 vector control. The cell lysate was resolved by 10% SDS-PAGE to monitor EBNA3C expression. GAPDH western blot was used as an internal loading control. Experiments were independently repeated three times, and results are presented as mean±s.d. from the three experiments. “**” represents p-value <0.01; “*” represents p-value <0.05.(TIF)Click here for additional data file.S7 FigHEK293 cells were transfected with an empty vector carrying a Flag tag. Flag antibody was used to do immunoprecipitations to exclude any non-specific binding in the control group. For blotting, Myc antibody was used to monitor the expression of EBNA3C and the possible pulled down EBNA3C truncates. Flag antibody was used to monitor the expression of Flag-associated proteins. METTL14 antibody was used to monitor the expression of METTL14 in different samples.(TIF)Click here for additional data file.S8 Fig(A) 30 million HEK293 cells were transfected with indicated plasmids and lysed with RIPA buffer 48 hours later, and samples were resolved by 10% SDS-PAGE and proteins were detected by the specific antibodies. (B-C) 10 million HEK293 cells were co-transfected with control plasmids, EBNA3C100-200 (B) and EBNA3CΔ100–200 (C), and Flag-METTL14 expression plasmids. 36 hours later, cells were treated with 40 μg/ml cycloheximide (CHX) for 0, 4, 8, 12 hours, and then cells were collected, lysed and subjected to western blot with indicated antibodies. (D-G) BJAB cells were transfected with control vector, Myc-EBNA3C, Myc-EBNA3C mutants, Flag-METTL14 or Myc-EBNA3C plus Flag-METTL14 and allowed to grow in DMEM supplemented with 1mg/ml G418. The same number of G418 selected cells were seeded to 6-well plates, collected and lysed in lysis buffer after 5 days culture. The lysates were subjected to western blots with indicated antibodies (D). 1X105 cells were stained with 5μM CFSE for 10 minutes at 37°C. The cells were washed, cultured, and harvested after culturing for 3 days. Flow cytometry was used to analyze CFSE-labeled cells (F). The transfected cells were assessed for their ability to promote colony formation using the soft agar assays (E and G).(TIF)Click here for additional data file.S9 Fig(A-D) HEK293 or Saos-2 cells were seeded on coverslips and transfected with Myc-EBNA3C using jetPRIME transfection reagent. The cells were fixed with 4% PFA, stained with specific antibodies. EBNA3C was detected by mouse anti-Myc (9E10) antibodies followed by the secondary anti-mouse Alexa Fluor 594. METTL14 were detected by rabbit anti-METTL14 antibodies, followed by anti-rabbit Alexa Fluor 488 as the secondary antibody. The nuclei were subsequently stained with DAPI, and the images were captured using an Olympus Fluoview confocal microscope.(TIF)Click here for additional data file.S10 Fig(A). 1X105 cells were stained with 5μM CFSE for 10 minutes at 37°C. The cells were washed, cultured, and harvested after culturing for 3 days. Flow cytometry was used to analyze CFSE-labeled cells (C). The transfected cells were assessed for their ability to promote colony formation using the soft agar assays (B and D).BJAB cells were transfected with control vector, Myc-EBNA3C, Flag-METTL14 or Myc-EBNA3C plus Flag-METTL14 and allowed to grow in DMEM supplemented with 1mg/ml G418. The same number of G418 selected cells were seeded to 6-well plates, collected and lysed in lysis buffer after 5 days in culture. The lysates were subjected to western blot with indicated antibodies (TIF)Click here for additional data file.S11 Figin vivo. 3 weeks later, mice were sacrificed and pictures were taken (A).The volume of the tumors was measured every 2 days (B).6 million LcL shCr, LcL shMETTL14, LcL shEBNA3C, and LcL shMETTL14 plus shEBNA3C were subcutaneously injected into NOD-SCID mice to assess the effect of knocking down METTL14 or EBNA3C on tumor growth (TIF)Click here for additional data file.S12 Fig(A) Mutations within the consensus site of four identified m6A sites in the EBNA3C gene were introduced into construct E3Cmut. The framed base pairs were considered as containing the DRACH motif. (B) The wild-type EBNA3C and mutant EBNA3C were transfected into BJAB cells and Raji cells. 48 hours later, the expression of wild-type EBNA3C and mutant EBNA3C were detected with western blot. (C-D) Wild-type EBNA3C or mutant EBNA3C were assessed for their ability to promote colony formation using the soft agar assays. Photomicrograph of representative colonies from three independent experiments is shown. (E) Wild-type EBNA3C or mutant EBNA3C were assessed for their ability to promote cell growth with CFSE staining assay. Experiments were independently repeated three times, and results are presented as mean±s.d. from the three experiments. “**” represents p-value <0.01; “*” represents p-value <0.05.(TIF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


Plasmodium vivax malaria. This drug is known to provoke acute hemolytic anemia in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Due to lack of data on G6PD deficiency, the use of primaquine has been limited in Africa. In the present study, G6PD deficiency was investigated in blood donors of various ethnic groups living in Nouakchott, a P. vivax endemic area in Mauritania.Primaquine is recommended by the World Health Organization (WHO) for radical treatment of -) and Mediterranean (B-) variants. Overall, 50 of 443 (11.3%) individuals (49 [11.8%] men and 1 [3.7%] woman) were phenotypically deficient. Amongst men, Black Africans had the highest prevalence of G6PD deficiency (15 of 100 [15%]) and White Moors the lowest . The most commonly observed G6PD allelic variants among 44 tested G6PD-deficient men were the African variant A- (202A/376G) in 14 (31.8%), the Mediterranean variant B- (563T) in 13 (29.5%), and the Betica-Selma A- (376G/968C) allelic variant in 6 (13.6%). The Santamaria A- variant (376G/542T) and A variant (376G) were observed in only one and two individuals, respectively. None of the expected variants was observed in 8 (18.2%) of the tested phenotypically G6PD-deficient men.Venous blood samples from 443 healthy blood donors recruited at the National Transfusion Center in Nouakchott were screened for G6PD activity using the CareStart G6PD deficiency rapid diagnostic test. G6PD allelic variants were investigated using DiaPlexC G6PD genotyping kit that detects African . It was mostly associated with either African or Mediterranean variants, in agreement with diverse Arab and Black African origins of the Mauritanian population. Plasmodium vivax [Plasmodium falciparum [Malaria remains a major public health burden in Mauritania with two-thirds of the population at risk. In the northern Saharan zone of the country, including Nouakchott, the capital city, malaria is predominantly caused by um vivax , 2, wherlciparum , 4.Plasmodium vivax infection is characterized by the occurrence of relapses several weeks or even months after the primary infection due to the persistent quiescent liver forms (hypnozoites). Radical cure of P. vivax malaria requires the elimination of both the blood stage parasites and hypnozoites. Primaquine, an 8-aminoquinoline, has long been the only available and effective drug capable of killing hypnozoites and, therefore, of preventing relapses [relapses . Tafenoqrelapses . Despiterelapses , 8.g6pd gene have been described and genetically characterized [- [- is characterized by the combination of A376G mutation and one of the following mutations: G202A, A542T, G680T, or T968C. In North Africa, g6pd gene with a point mutation (C563T), known as the “Mediterranean variant (B-),” also occurs [- variant is often associated with mild to moderate hemolytic anemia, as compared to other G6PD variants, although fatal reactions to primaquine have been recorded in a limited number of patients [G6PD deficiency is the most common genetic enzyme disorder in humans with 400 million affected persons worldwide. Currently, 217 mutations on the cterized , 10. In rized [- . The varo occurs . The G6Ppatients . The Medpatients . BecauseCurrent G6PD deficiency diagnostic methods include the fluorescent spot test , spectroP. vivax malaria in patients with normal G6PD activity [P. vivax malaria, screening for G6PD activity is required because primaquine is contraindicated in patients with G6PD deficiency. However, in almost all health facilities in the country, the G6PD status of P. vivax-infected malaria patients is not or cannot be determined at present, leading to non-use of primaquine. Moreover, there is no recent database on G6PD status in Mauritania. Therefore, the objective of this study was to assess the prevalence of different variants of G6PD deficiency in Mauritanian blood donors of different ethnic origins in Nouakchott, where P. vivax is endemic. The obtained data will be useful for decision making on the possible need for routine G6PD deficiency testing as part of P. vivax malaria control and elimination in the country.Since 2014, primaquine is recommended by the Mauritanian Ministry of Health for the radical treatment of activity . Before Hassaniya” derived from a Berber-influenced Arabic language, while each of Black African ethnic groups has its own language and cultural specificities [The study was conducted in 2016 at the National Blood Transfusion Center (CNTS) in Nouakchott . The Nouficities . Inter-eBetween May and December 2016, whole blood was collected in a 5-ml ethylenediaminetetraacetic acid (EDTA)-coated blood collection tube from 454 healthy blood donors and was immediately used for G6PD deficiency screening using CareStart rapid diagnostic test  according to the manufacturer’s instructions. About 200 μl of the remaining blood were spotted on Whatman 3MM filter paper for storage and DNA extraction.g6pd point mutations by a one-step multi-allelic specific PCR [-, C563T single mutation; Santamaria, A376G + A542T double mutations), class III , G202A + A376G double mutations; A-(680), A376G + G680T double mutations; A-(968) Betica-Selma, A376G + T968C double mutations), and class III-IV .DNA was extracted from samples with G6PD deficiency using Quick-DNA Miniprep Plus kit  according to the manufacturer’s instructions. G6PD variants were characterized using DiaPlexC G6PD genotyping kit . The kit provides a simple and rapid alternative to PCR and enzyme digestion-based assays and enables to detect six different ific PCR , 25: G20ific PCR , 26: claTwo microliters of total genomic DNA (25–50 ng) were added to 23 μl of master mixture containing pre-mix (12.5 μl), G6PD primer mixture , and nuclease-free ultrapure water (8.5 μl), as recommended by the manufacturer. PCR was performed for 30 cycles at an annealing temperature of 60°C, as recommended by the manufacturer. PCR products were analyzed by electrophoresis on 3% agarose gel, stained with GelGreen Nucleic Acid Gel Stain , and visualized under ultraviolet illumination. The following sizes of PCR products were obtained, depending on the presence of mutations: A376G, 103 bp; G202A, 157 bp; A542T, 241 bp; C563T, 220 bp; G680T, 388 bp; and T968C, 463 bp. The validity of each PCR amplification was confirmed by an internal control (947 bp). PCR was performed in a thermal cycler Eppendorf MasterCycler Personal .Data were analyzed using MedCalc statistical software version 16.4.3 . The chiThe study obtained ethical clearance from the ethical committee of the University of Nouakchott Al-Aasriya, and written informed consent was obtained from all participants.Overall, 454 unrelated individuals were enrolled during the study period. Data from 11 donors (2.4%) were excluded from analysis due to missing data . Data from the remaining 443 individuals were analyzed. χ2 = 1.6; df = 1; P = 0.8). The prevalence of G6PD deficiency by ethnic groups indicated that Black Africans  and black Moors  had similar frequencies of G6PD deficiency (P = 0.89). Compared to these two ethnic groups, the prevalence of G6PD deficiency in white Moors was 5.9% . Of 50 G6PD-deficient blood donors, 13 (26%) reported that they had a past history of malaria but were not able to remember the Plasmodium species and treatment they received.Among 443 screened study subjects, 50 (11.3%) were phenotypically deficient for G6PD using CareStart rapid diagnostic test . G6PD de- (202A/376G) in 14 of 44 (31.8%) individuals. Although the sample size was small, the African variant A- was observed more frequently in Black Moors than the other ethnic groups (10 Black Moors vs. 3 Black Africans and 1 White Moor). By contrast, in this small sample of G6PD-deficient Mauritanians, the severe Mediterranean variant B- (563T) was observed in 13 of 44 (29.5%) subjects and occurred more frequently in White Moors (8 White Moors vs. 3 Black Moor and 2 Black Africans). The moderate A- (376G/968C) allelic variant  was observed in 6 of 44 (13.6%) (5 Black Africans and 1 Black Moor) G6PD-deficient subjects. The severe Santamaria variant A- (376G/542T) was observed in only one White Moor. None of the expected allelic variants detectable by DiaPlexC G6PD genotyping kit was observed in 8 (18.2%) of the tested phenotypically G6PD deficient men from two different ethnic groups (5 Black Moors and 3 Black Africans). The allelic variant G680T was not found in our study population.G6PD allelic variants were assessed in 45 (44 men and 1 woman) of 50 phenotypically G6PD-deficient individuals. PCR failed in 5 of 50 samples, probably due to poor storage conditions leading to DNA degradation. G6PD variant A (376G), characterized by little or no clinical significance, was observed in three individuals (two men and one woman) and was associated with a faint color change in CareStart rapid diagnostic test. P. vivax is endemic, primaquine and more recently tafenoquine remain the only 8-aminoquinoline antimalarial drugs available for radical treatment. In Mauritania, several studies have confirmed that P. vivax is endemic in the northern Saharan zone, including Nouakchott [P. vivax infection than other ethnic groups, probably due to the high prevalence of positive Duffy blood group among them, even though P. vivax was also found in few Duffy-negative patients [P. vivax malaria. At present, artemisinin-based combination therapy (ACT) is the first-line drug to treat P. falciparum and P. vivax malaria. Primaquine administration has not been implemented in the country owing to concerns about its potential toxicity, including hemolytic anemia, in G6PD-deficient patients and the absence of a prior routine G6PD deficiency testing for the safe use of primaquine at health facilities.In countries where uakchott –3. Whitepatients , 28, 29.P. vivax in Mauritania. Our results showed that the prevalence of phenotypic G6PD deficiency using CareStart rapid diagnostic test is 11.3%. The mean prevalence of G6PD deficiency among countries with ongoing malaria transmission is estimated to be 8% [In the present study, G6PD deficiency was investigated in blood donors living in Nouakchott, an endemic area of to be 8% , 30. Theto be 8% , Sierra to be 8%  and Burkto be 8% . On the to be 8% , 35. How- (African variant) and G6PD B- (Mediterranean variant) found in 31.8% and 29.5% of G6PD-deficient blood donors, respectively. G6PD A- and G6PD B- are among the most common G6PD variants worldwide. They may be associated with different severity of clinical symptoms in the presence of antimalarial drugs such as primaquine, ranging from moderate (for G6PD A-) to severe (for G6PD B-) anemia. In sub-Saharan Africa, G6PD A- (202A/376G) variant is the most common variant [- (202A/376G) was the only variant observed [- (202A/376G) was also observed but the differences in proportions between ethnic groups were not significant [- was as high as 46% [- (376G/968C) was the most frequent allele associated with G6PD deficiency, with the prevalence of 10% and 7%, respectively [The study also revealed that the most common G6PD variants were G6PD A variant . For insobserved . In Malinificant . In someh as 46% , 35. Inv A- 202A/6G varianectively . The Medectively ,35.g6pd gene in these individuals could provide further information. In Tunisia, no mutation was identified in 6 of 56 (10.7%) G6PD-deficient screened subjects using PCR-restriction fragment length polymorphism (RFLP) [P. vivax-infected patients before primaquine is administered.Eight of 44 phenotypically deficient male blood donors showed none of the six expected mutations. One possible explanation is that G6PD deficiency in these ethnically different individuals is associated with other variants not detected using DiaPlexC G6PD genotyping kit and that the observed phenotype could be associated with different G6PD genotypes. Only sequencing of m (RFLP) . It is am (RFLP) –43. In tm (RFLP) –45. Low m (RFLP) –48. Thes- (563T) predominates, and black Africans in whom G6PD deficiency is mostly due to G6PD A- variant.Overall, the present study revealed heterogeneous prevalence of both African and Mediterranean variants of G6PD deficiency using phenotypic and molecular tests among study subjects belonging to different ethnic groups present in Mauritania. These findings were somewhat expected because Mauritania is located at the intersection between Maghreb and sub-Saharan African countries, and its ethnic composition is made up of Moors of Arab descent, in whom Mediterranean G6PD variant BP. vivax infection. Accurate and up-to-date mapping of the prevalence of G6PD deficiency and its variants is required to design appropriate strategies of P. vivax malaria elimination that includes mass administration of primaquine.The main limitation of the present study was the inability to link the observed phenotypic and genotypic G6PD deficiency with enzymatic activity determined by a reference method in the study population. Moreover, despite the fact that 454 healthy individuals were recruited, the sample size of the present study does not have an adequate power to compare differences according to sex, ethnicity, and genotyping data. Nonetheless, to our knowledge, this is the first report on the prevalence of G6PD deficiency and its molecular variants in Mauritania. Further genotyping studies are needed to confirm the presence of additional variants that may be associated with G6PD deficiency and determine further the frequencies of mutations in G6PD in the general population as well as in different ethnic groups in relation to P. vivax-infected patients in Mauritania to minimize the risk of clinically relevant and severe hemolytic anemia associated with different G6PD variants in the country. More studies on a larger population size, including a higher number of women, are being planned in areas where P. vivax transmission occurs in the country. Further evaluation of quantitative rapid diagnostic tests for G6PD is warranted, especially for females in whom the random X chromosome inactivation may affect the interpretation of qualitative tests.The observed prevalence of G6PD deficiency and molecular variants are in agreement with those observed in West and North African populations. The study highlights the need to implement adequate G6PD deficiency screening with a reliable rapid diagnostic method before prescribing primaquine for the radical treatment of


In particular, significant linkages between Argentina and Syria (D1), Brazil and Central/Eastern Europe (D2), USA and India (D2), and Brazil and Southern Europe (D3) were estimated, consistent with historical and epidemiological data.Hepatitis B virus (HBV) genotype D (HBV/D) is globally widespread, and ten subgenotypes (D1 to D10) showing distinct geographic distributions have been described to date. The evolutionary history of HBV/D and its subgenotypes, for which few complete genome sequences are available, in the Americas is not well understood. The main objective of the current study was to determine the full-length genomic sequences of HBV/D isolates from Brazil and frequency, origin and spread of HBV/D subgenotypes in the Americas. Complete HBV/D genomes isolated from 39 Brazilian patients infected with subgenotypes D1 (n = 1), D2 (n = 10), D3 (n = 27), and D4 (n = 1) were sequenced and analyzed together with reference sequences using the Bayesian coalescent and phylogeographic framework. A search for HBV/D sequences available in GenBank revealed 209 complete and 926 partial genomes from American countries , with the major circulating subgenotypes identified as D1 (26%), D2 (17%), D3 (36%), D4 (21%), and D7 (1%) within the continent. The detailed evolutionary history of HBV/D in the Americas was investigated by using different evolutionary time scales. Spatiotemporal reconstruction analyses using short-term substitution rates suggested times of the most recent common ancestor for the American HBV/D subgenotypes coincident with mass migratory movements to Americas during the 19 Despite the availability of an effective vaccine and potent antiviral treatment, hepatitis B virus (HBV) infection remains a major public health issue, affecting 257 million people worldwide , 2. HBV The origin and evolutionary history of HBV are still controversial and a wide range of HBV substitution rates have been estimated depending on the calibration approach used for the calculation , 21. StuThe study protocol was approved by the Brazilian Ethics Committee for Medical Research (CONEP nº 9604/2004) and the Ethics Committee of Oswaldo Cruz Institute (nº 1.358.935), and all patients signed informed consent before participation. All experiments were performed in accordance with the relevant guidelines and regulations.Thirty-nine retrospective HBsAg-positive serum samples (from 2003 to 2013) previously characterized as HBV/D-containing strains, collected from different geographical regions of Brazil , were selected for HBV complete genome amplification.et al., 1995 [5’-CCGGAAAGCTTGAGCTCTTCTTTTTCACCTCTGCCTAATCA-3’) and P2 (5’-CCGGAAAGCTTGAGCTCTTCAAAAAGTTGCATGGTGCTGG-3’), and the following PCR profile: denaturation at 94°C for 4 min followed by 10 cycles at 94°C for 40 s, 55°C for 1 min, and 72°C for 3 min; 10 cycles of 94°C for 40 s, 60°C for 1 min, and 72°C for 5 min; 10 cycles of 94°C for 40 s, 62°C for 1 min, and 72°C for 7 min; 10 cycles of 94°C for 40 s, 62°C for 1 min, and 72°C for 9 min; and a final extension step at 72°C for 10 min. The PCR assay was performed using Platinum Taq DNA polymerase and supplied reagents  in accordance with product instructions.HBV DNA was extracted from 0.2 mL serum using a High Pure Viral Nucleic Acid kit  according to the manufacturer’s instructions. The amplification of HBV complete genome sequences was attempted with a protocol modified from Günther l., 1995  using 4 http://hvdr.bioinf.wits.ac.za/serotyper/. Nucleotide sequences obtained during this study were deposited in the GenBank database under accession numbers MH724214–MH724252.PCR products were purified using the Wizard SV Gel and PCR Clean-Up System . HBV full-length genome sequences were determined via direct sequencing using a BigDye Terminator Kit v3.1 , and sequencing reactions analyzed on an ABI3730xl automated sequencer (Applied Biosystems). Inter and intragenotypic recombination were investigated using SimPlot v3.5.1 software , and RDPMultiple sequence alignment was performed using the Muscle program with 66 HBV complete genome sequences (39 sequences determined in this study and 27 reference sequences for HBV genotypes A to J) and subsequently subjected to Maximum Likelihood (ML) phylogenetic analysis. The ML phylogenetic tree was inferred with the online version of the PhyML program  under thTo identify the HBV/D subgenotypes circulating in the Americas and determine their proportions in individual countries, a search for HBV/D sequences from the continent available in GenBank by December 2018 was performed using the following queries (for each American country):((Hepatitis B virus) AND genotype D) AND country = "country name"((Hepatitis B virus) AND genotype D1) AND country = "country name"((Hepatitis B virus) AND genotype D2) AND country = "country name"((Hepatitis B virus) AND genotype D3) AND country = "country name"((Hepatitis B virus) AND genotype D4) AND country = "country name"((Hepatitis B virus) AND genotype D5) AND country = "country name"((Hepatitis B virus) AND genotype D6) AND country = "country name"((Hepatitis B virus) AND genotype D7) AND country = "country name"((Hepatitis B virus) AND genotype D8) AND country = "country name"((Hepatitis B virus) AND genotype D9) AND country = "country name"((Hepatitis B virus) AND genotype D10) AND country = "country name"Sequences without a specified subgenotype but more than 600 nt in length were submitted for HBV subgenotyping via phylogenetic analysis and added to the dataset.http://weizhongli-lab.org/cdhit_suite/cgi-bin/index.cgi?cmd=cd-hit-est) was used to group sequences with high similarity, and only one representative sequence of each group, prioritizing the one with the oldest collection date, maintained in the dataset.A total of 421 full-length genome sequences from HBV/D subgenotypes D1, D2, D3 and D4 available in GenBank in December 2018 with known country of origin and collection date were grouped into four datasets for each subgenotype and selected for phylogeographic analyses  [−6 s/s/y  [In order to investigate the phylogenetic signal of the datasets, we carried out a likelihood mapping analysis of 10,000 random sets of four sequences (quartets) using IQ-tree v1.6.11 software . In addi5 s/s/y)  and 3.7  x 10−6) .Spatial reconstruction was achieved by applying a reversible discrete Bayesian phylogeographic model . The Bayayw2, D2 as 90% ayw3 and 10% ayw4, D3 as 78% ayw2 and 22% ayw3, and the D4 strain as ayw2.Thirty-nine full-length HBV/D sequences from different Brazilian regions were successfully amplified and sequenced. All isolates had a genome size of 3,182 nt, with no evidence of in-phase deletion, insertion or recombination. The basal core promoter A1762T/G1764A and precore G1896A mutations were detected in 8/39 (20.5%) and 15/39 (38.5%) sequences, respectively. Using the search queries reported in the Methods section, 1,135 HBV/D sequences isolated in American countries were identified by December 2018 and downloaded from GenBank. Among these, 209 were complete genome and 926 were partial genome sequences. The 39 full-length sequenced genomes were additionally included in the dataset . HBV/D sequences were obtained from Argentina (n = 281), Brazil (n = 562), Canada (n = 49), Chile (n = 4), Colombia (n = 1), Cuba (n = 94), Haiti (n = 38), Martinique (n = 6), Mexico (n = 1), USA (n = 132) and Venezuela (n = 6). Subgenotypes D1 , D2 , D3 , D4  and D7  were identified and their distribution patterns throughout the continent presented in −5 to 3.27 × 10−4 s/s/y  complete genome sequences grouped into four datasets for subgenotypes D1, D2, D3 and D4. Ten D3 genomes sequenced here were excluded from analyses after using the online tool CD-HIT Suite to avoid bias from overrepresentation. Subgenotype D7 was not analyzed due to its recombinant nature (D/E intergenotypic genome) and the small number of available sequences. The phylogenetic signal tested for each dataset showed that the portion of randomly chosen quartets falling in the corners of the likelihood map was >90%  , thus su−4 s/s/y . The coePSP) = 0.98). From Syria, D1 migrated to Cuba (PSP = 0.58) and Brazil (PSP = 0.68), but it was not possible to determine the time of introduction, since only one D1 sequence was available from these countries. In Argentina, the most probable location for the origin of D1 was also Syria (PSP = 0.86) and its tMRCA calculated as 1982 (95% HPD: 1920–2006)  ; Fig 3A.20–2006) .PSP = 0.56). The majority of the Brazilian and Argentine sequences formed a single cluster (posterior probability (PP) = 0.99) whose tMRCA was dated back to 1969 (95% HPD: 1941–1984), with Central/Eastern Europe as the most probable source location (PSP = 0.81). On the other hand, sequences from USA grouped with Indian sequences, and at least two different introductions of this subgenotype seem to have occurred in USA. The main time of introduction was estimated between 1966 and 1978 from India (PSP = 0.99)  ( = 0.99) ; Fig 3B. = 0.99) .PSP = 0.54) as the putative origin of subgenotype D3. Viral sequences from Argentina, Brazil and USA segregated into different clusters, suggesting multiple introductions in these countries, with tMRCAs of the main clusters calculated as 1781 (95% HPD: 1589–1979), 1799 (95% HPD: 1615–1976) and 1965 (95% HPD: 1928–1997), respectively. In contrast, Canadian D3 sequences constituted a single monophyletic group whose tMRCA was dated back to 1855 (95% HPD: 1770–1980). Among the Caribbean D3 sequences, Martinique and Haiti clustered together with most sequences from Brazil, while Cuba showed a distinct dispersal pathway, grouping with viral sequences from South Asia (India and Pakistan)  (akistan) ; Fig 3C.akistan) .PSP = 0.33) as the most probable place of origin. All D4 sequences branched in country-specific monophyletic groups that probably arose between the middle 1800s and middle 1900s  calculated on the basis of heterochronous sampling calibration using ancient sequences [−6 s/s/y  estimated by means of external calibrations based on human migrations [Additionally, we tested alternative evolutionary time scales inferred by using informative priors based on two previously published substitution rates for HBV: (i) 1.18 x 10equences , and  throughout the continent . Interestingly, Cuba was the only country in which all five subgenotypes were identified, which may be attributable to interactions of the island with different countries over the years . Brazil Recent findings from two studies using ancient samples pointed out that HBV has been infecting humans for at least 7,000 years , 57. Owith and 20th centuries. Therefore, our results corroborate the concept that tip-dating analyses of modern heterochronous HBV sequences are probably more appropriate in dating recent dispersal events [In this study, we reconstructed the evolutionary history of HBV/D in the Americas by using substitution rates estimated from the sampling dates of the sequences or by using rates previously estimated for HBV as informative priors for calibration of time-scale. The time-scale reconstructions based on ancient heterochronous sequences  and extel events , 22. Conth century and the first half of the 20th century several thousand of people, mainly from Syria, Lebanon, Palestine, Turkey, and Egypt had migrated to Cuba. The introduction of D1 in Brazil was also suggested to have occurred from Syria. This proposal is reinforced by the fact that Brazil has the largest Arab colony outside the Middle East. It is estimated that about 7% of the Brazilian population is of Arab origin, the majority from Syria and Lebanon, and the migratory movement from these countries to Brazil was initially documented from the second half of the 19th century [th century, mainly originating from Syria and Lebanon [Our phylogeographic reconstruction showed that Syria is the most likely location for the origin of subgenotype D1. Syrian D1 sequences were not available when previous studies suggested that this subgenotype originated in Turkey , 26. In  century , 63. Phy Lebanon .th century (95% HPD: 1941–1984), with Central/Eastern Europe as the most probable place of origin. Although Latin America received the majority of immigrants from Southern Europe, there was also a considerable flow of migrants from Central, East and Southeastern European countries [Spatiotemporal reconstruction of subgenotype D2 suggested that this lineage originates in Central/Eastern Europe , in agreement with previous reports , highligountries . Betweenountries . On the ountries –69 and tountries .PSP = 0.54) as the root location of D3, and Brazil as the origin place of most dispersal pathways occurring from the late 18th century in the continent. Previous studies based on epidemiological and historical data have suggested that subgenotype D3 was transported to Brazil by European immigrants [Dispersal pathways of subgenotype D3 in the Americas were complex including different geographic regions and multiple introductions. Phylogeographic analysis suggested Southern Europe  Click here for additional data file.S1 FigML phylogenetic tree of 39 Brazilian HBV/D complete genomes recovered in this study plus 27 reference sequences. The sequences generated are denoted BR, followed by sample number and the geographic region of origin , and identified with the symbol ♦. Reference sequences are indicated by their accession number, followed by genotype. The numbers at nodes correspond to bootstrap values (1000 replicates) higher than 70%.(TIF)Click here for additional data file.S2 Fig(A) HBV/D1; (B) HBV/D2; (C) HBV/D3; (D) HBV/D4; The triangles show the distribution (percentages) of the data in the seven basins of attraction or quartet possibilities. The percentages in the corners and sides represent well resolved phylogenies, while the percentage in the center of the triangle represents unresolved phylogenies.(TIF)Click here for additional data file.S3 Fig2 values are given as an indicator of the degree to which evolution has been clock-like.Plots of the root-to-tip genetic distance against sampling time are shown for phylogenies estimated from four alignments: (A) HBV/D1; (B) HBV/D2; (C) HBV/D3 and (D) HBV/D4. Each dot represents a sequence. R(TIF)Click here for additional data file.


Similarly, higher contents of nutrients in grains, shoots and roots were observed for TB2 + CF: N , P  and K . Compared to CK, the total root length (41.1%), surface area (56.5%), root volume (54.2%) and diameter (78.4%) were the greatest for TB2 + CF, followed by BB2 + CF, OB2 + CF, TB1 + CF, BB1 + CF, OB1 + CF and CF, respectively. However, BB + CF and OB + CF treatments increased β-glucosidase and dehydrogenase, but not urease activity, as compared to the TB + CF amendment, while all enzyme activity decreased with the increased biochar levels. We concluded that the conversion of fruit peels and milk tea waste into biochar products contribute the benefits of environmental and economic issues, and should be tested as soil amendments combined with chemical fertilizers for the improvement of wheat growth and grain yield as well as soil fertility status under field conditions.The conversion of organic wastes into biochar via the pyrolysis technique could be used to produce soil amendments useful as a source of plant nutrients. In this study, we investigated the effects of fruit peels and milk tea waste-derived biochars on wheat growth, yield, root traits, soil enzyme activities and nutrient status. Eight amendment treatments were tested: no amendment (CK), chemical fertilizer (CF), banana peel biochar 1% (BB1 + CF), banana peel biochar 2% (BB2 + CF), orange peel biochar 1% (OB1 + CF), orange peel biochar 2% (OB2 + CF), milk tea waste biochar 1% (TB1 + CF) and milk tea waste biochar 2% (TB2 + CF). The results indicated that chlorophyll values, plant height, grain yield, dry weight of shoot and root were significantly ( Triticum aestivum L.) is considered as a key nutritional crop for feed throughout the worlds. Winter wheat and summer maize (Zea mays L.) are major cereal crops for food production in the Loess Plateau of China [4+-N) is easily taken up by the roots, and NO3−-N is available in a mobile form in the xylem and can be stored in the vacuoles of shoots and roots [2 environment [Wheat  is produced per day in Beijing and Shanghai . UnfortuTo manage environmental and economic issues as well as to improve soil quality and crop production in loess areas, most studies have focused on crop straw or organic manure-derived biochars assoil amendments in loess plateau areas. There is limited study on the use of biochar derived from the fruit peels and milk tea wastes for wheat growth, root traits and soil fertility status. In our study we used biochar based on fruit peels and milk tea wastes because these contain macro- and micronutrients in addition to serving as a carbon source. These organic materials could be the best alternative source for biochar production. Therefore, we used fruit and milk tea waste derived biochars (at a low pyrolysis temperature of 350 °C) as nutritional sources combined with inorganic fertilizers for the treatment of loess soil. The specific objectives were: (1) to compare the effects of different biochar amendments on wheat growth, yield and root traits; (2) to quantify their effects on the biochemical properties of loess soil.The morphology of biochars derived from banana and orange peels and milk tea waste was detected from SEM micrographs after the different biochar surfaces were pyrolyzed at a constant pyrolysis temperature of 350 °C for 2 h. The milk tea waste biochar (TB) had a greater surface area and a higher pore space than the banana peel biochar (BB) and orange peel biochar (OB) .p < 0.05) increased chlorophyll values, plant height (cm) and shoot and root dry weight (g pot−1) as compared to CF alone and CK treatments ( p < 0.01) correlated with grain yield, root traits and nutrients contents in grains, shoots and roots (Eight treatments were tested: no amendment (CK), chemical fertilizer (CF), banana peel biochar 1% (BB1 + CF), banana peel biochar 2% (BB2 + CF), orange peel biochar 1% (OB1 + CF), orange peel biochar 2% (OB2 + CF), milk tea waste biochar 1% (TB1 + CF) and milk tea waste biochar 2% (TB2 + CF). The application of different biochar amendments combined with inorganic fertilizers significantly (atments  A–E. The atments  D. As a pnd roots . Our finnd roots .p < 0.05) improved the grain yield over the chemical fertilizer alone and control treatments. The grain yield (g pot−1) was increased by CF and by co-application with biochar treatments as compared to CK (−1 or 58.5%) and BB2 + CF (8.4 g pot−1 or 51.9%). However, compared to the CK, other treatments also exhibited increased grain yield, BB1 + CF (36.3%), OB1 + CF (27.1%), OB2 + CF (35.3%), TB1 + CF (40.4%) and CF (26.6%). The maximum plant height was recorded for the TB1 + CF and TB2 + CF treatments (48.76 cm and 50.65 cm), followed by the BB2 + CF, OB2 + CF, BB1 + CF, OB1 + CF and CF treatments. The minimum plant height was recorded for CK (36.11 cm). A similar situation was observed for shoot and root dry weight, the highest shoot and root dry weight was recoded for TB2 + CF and BB2 + CF: TB2 + CF /BB2 + CF. Specifically, TB2 + CF/BB2 + CF equaled 5.21 g/4.70 g and 1.10 g/1.03 g for shoots and roots, respectively. Percentage increases in shoot and root dry biomass occurred for BB1 + CF (23.1% and 24.1%), for BB2 + CF (35.3% and 37.0%), for OB1 + CF (15.4% and 21.1%), for OB2 + CF (18.6% and 28.1%), for TB1 + CF (24.9% and 26.9%), for TB2 + CF (41.5 and 41.1%) and for CF (15.5% and 18.4%), compared with CK. Similar results were addressed by previous pot and field experiments using biochar amendments added to different crops [The biochar amendments apparently  improved by the biochar amendments and by CF alone (−1) but lowest for the control . The percentage increases in grains, shoots ands root were highest for TB2 + CF , followed by BB2 + CF , TB1 + CF , OB2 + CF , BB1 + CF , OB1 + CF  and CF  over CK. The P content was markedly (p < 0.05) increased for the co-application of BB + CF, OB + CF and TB + CF compared with CK (p < 0.05) difference in the K content values between the CF treatment alone and the treatments combined with different biochars  contents were significantly  influenced by the different biochars and rates (3), but had non-significant effects on surface area and root volume (24.7 cm2 and 1.3 cm3) in comparison to CK. In contrast, the co-application of different biochars and chemical fertilizers improved the root traits. Among all the treatments, the total root length ranged from 307 to 522 cm (3) and the lowest was recorded for the CK (1.03 cm3) under all treatment rates. This revealed that biochar or organic amendments with chemical fertilizer improved the crop root traits [p < 0.01) correlation between root traits and plant nutrients content  influenced by different biochar amendments (−1 soil h−1) and the lowest observed for the CK (268 mg p-nitrophenyl kg−1 soil h−1) treatments. The β-glucosidase activity was decreased with increasing biochar rates for all biochar amendments. The OB + CF treatment increased β-glucosidase activity as compared to BB + CF and TB + CF  positive correlation with soil NH4+-N and NO3−-N (Urease activity was accelerated with increasing biochar rates and it was increased by CF treatment as compared to over CK B. The mad NO3−-N . This cop < 0.05) impact dehydrogenase activity (−1 soil h−1) and the lowest was found for CK (20.5 mg Triphenyl formazan (TPF) kg−1 soil h−1). Similarly, the TB + CF amendment decreased dehydrogenase activity as compared to the BB + CF and OB + CF amendments. The OB + CF amendment increased dehydrogenase activity, indicating the slow immobilization of soil organic carbon (SOC) and increased soil microbial activity. Our results are in line with previous findings [3−- N and dehydrogenase activity  affected soil pH and soil organic carbon (SOC). Soil pH was slightly increased with increasing biochar rates, but showed non-significant (p < 0.05) differences from CK (The application of different biochar amendments combined with CF significantly ( from CK A. The hi−1) and minimum were observed in CK (8.8 g kg−1). The improved percentage of SOC compared to CK decreased in the order of TB2 + CF (66.3%) > BB2 + CF (59.7%) > TB1 + CF (55.4%) > OB2 + CF (52.7%) > BB1 + CF (48.2%) > OB1 + CF (39.1%) > CF (7.2%). The comparison between the biochars and their rates showed that TB + CF amendments increased SOC concentrations as compared to BB + CF and OB + CF rates. The carbon storage of biochar applications in soils depends on their labile natures [SOC concentrations were influenced by the application of different biochar and rates compared with the CF alone and CK B. The ma natures . In our  natures  and Zhu  natures  evaluate natures .4+-N and NO3−-N concentrations were significantly (p < 0.05) affected by the amendments as compared to CK. There was significant influence on NH4+-N concentrations in the CF group alone with the highest NH4+-N concentration (6.2 mg kg−1) treatment. The co-application of biochar and CF amendments resulted in decreased NH4+-N concentrations over CK group. The soil NH4+-N concentration decreased with the increase of biochar rates, with the maximum percentage decrease in BB2 + CF (66.6%) and the minimum in TB1 + CF (18.3%) in comparison to CK. In contrast, the soil NO3−-N concentrations were greater in biochar treatments as compared to the CF and CK groups. The NO3−-N concentrations decreased with the increase of the biochar levels, but still remained higher than the concentrations observed in the CF and CK groups. The NO3−-N concentrations ranged from 11.6 to 20.8 mg kg−1 among all treatments; the concentration was highest for TB1 + CF (20.8 mg kg−1) and lowest for CK (11.6 mg kg−1). Our results are consistent with the incubation study of Wang et al. [4+-N was improved following the treatment of CF alone over, as compared to the biochar and CK treatments. This might be because CF alone decreased the soil pH, which was slightly increased under co-applications of different biochars treatments. Esfandbod et al. [4+-N and NO3−-N concentrations depend on the pH and types of soil amendments.g et al.  and pot ration 6. mg kg−1 g et al. , and as g et al.  and Zhand et al.  and Lan d et al.  recognizp < 0.05) improved soil available P and K concentrations as compared to CK (−1 and 240 mg kg−1) and the lower concentrations were found in CK (25.1 mg kg−1 and 160 mg kg−1). Among all biochar treatments, a better performance was observed for the TB + CF treatments in comparison to the BB + CF and OB + CF treatments. Similar outcomes after biochar applications were reported in previous studies conducting short- and long- term experiments [p < 0.01) positive correlation among SOC, available P and K  and urease activity had a strong relation with each other, while soil pH, mineral N, glucosidase and dehydrogenase activities had weak relation with plant growth and root parameters . The PCA2 atmosphere with the temperature raised to 350 °C and maintained at required peak for 2 h to generate the biochars. All the biochars were stored in a desiccator for further use, and their basic properties are listed in Banana and orange peel waste and milk tea waste were respectively collected from the local fruit market of Yangling and a milk tea restaurant at Xi’an, Shaanxi, China, and subsequently shipped to the Soil Physics Laboratory of Northwest A&F University. The banana and orange peels and milk tea waste were washed with tap and distilled water four times. The fruit peels cut into small pieces of around 1 inch and air-dried for 12 days, then dried in an oven at 68 °C for 72 h and ground to pass through a 2 mm sieve. These raw materials were pyrolyzed in a laboratory scale using a Modified Muffle Furnace  system in presence of an NSurface soil (0–20 cm) samples were collected from a long-term experimental station of wheat and maize crop rotation an experimental station at Northwest A&F University (NWAFU), Yangling, Shaanxi, China . This station is classified as an Orthic Anthrosol with a silty clay loam in texture [−1. For each treatment, 5 kg of soil and the proper amendments were mixed manually and added into each pot according to the treatments. After amendment incorporation, the soil in each pot was moistened to about 70% water holding capacity (WHC) by adding tap water and 10 wheat seeds were sown in each pot. The all pots were properly arranged in a completely randomized block design under an artificial shed. The water loss checked every day and maintained a 70% WHC on daily basis up to wheat harvested. After 12 days, wheat plants were thinned and five plants were left in each pot until maturity to assess the grain yield. The wheat plants were uprooted from each pot, and the parameters of aboveground biomass and roots were recorded.The pot experiment was performed with a silty clay loam soil and designed as a randomized complete block, including eight treatments with three replicates under an artificial shed. The pot experiment included a control with no amendment (CK), chemical fertilizers (CF), and two levels of soil amendments with biochar derived from banana and orange peels and milk tea waste. In total, the eight treatments included (1) CK , (2) CF , (3) BB1 + CF , (4) BB2 + CF , (5) OB1 + CF , (6) OB2 + CF , (7) TB1 + CF  and (8) TB2 + CF . The chemical fertilizers nitrogen (N), phosphorus (P) and potassium (K) were applied as a source of urea, di-ammonium and sulfate of potash at the doses of 120, 80 and 60 kg ha−1) and root biomass were recorded. After the wheat crop was harvested at maturity, the biological yield was recorded by weighing the aboveground biomass of each replication of treatments. The shoots and roots were washed with running water and then washed with distilled water for the removal of adhered soil and biochar particles. Both shoots and roots were accurately dried with tissue paper to record the plant height as well as the shoot and root weight. They were then dried in an oven for 72 h at 68 °C until a constant weight was obtained. Cleaned wheat roots were used for root traits examination including total root length, root surface area, root volume and root diameter, using a Hewlett Packard scanner controlled by Win-RHIZO, 2007d  software, according to [Wheat growth, root traits and grain yield parameters such as chlorophyll values, plant height, biological and grain yields (g potrding to .w/v) soil-water extract), and biochar was applied in a 1:10 (biochar:water) ratio after 30 minutes of shaking at 180 rpm. Soil and biochar EC and pH were measured using a glass electrode (DDS-11AW EC meter) and a pH meter . Soil particle size was measured by using a Mastersizer 2000E  laser diffractometer. Total organic carbon (TOC), total nitrogen (TN) and C:N ratio of soil and biochars were determined using a CN analyzer . Soil organic carbon (SOC) was measured following wet digestion with sulfuric acid and potassium dichromate (H2SO4 + K2Cr2O7). Total nitrogen (TN), phosphorus (TP) and potassium (TK) contents of biochar and wheat grains, shoots and roots were analyzed colorimetrically after digestion with H2SO4 + HClO4 [4+-N and NO3−-N) for soil subsamples was extracted with 2 M of potassium chloride solution (KCl) with a 1:10 (soil: solution) ratio which was shaken for 1 h to determine the concentration of soil NH4+-N and NO3−-N using a continuous flow analyzer . Soil available phosphorus (AP) was determined using a 0.5 M NaHCO3 with a pH 8.5 extract followed by a visible light spectroscopic analysis of a blue colored complex using a UV-VIS spectrophotometer  [4OAc) extraction followed by emission using an atomic absorption spectrometer . Total concentrations of zinc (Zn), iron (Fe) and copper (Cu) in biochar samples were determined by acid digestion using nitric perchloric acid , and DTPA solution used for soil samples was determined for Zn, Fe and Cu concentrations using an atomic absorption spectrometer. Soil enzyme activities of β-glucosidase, urease and dehydrogenase were analyzed by using the colorimetric method and using  [Soil electrical conductivity (EC) and pH were measured in an aqueous soil extract in distilled water  . Soil avUV-2450) , as brieUV-2450) ,26. The p > 0.05). Patterns in the plant and soil data were investigated using a bivariate Pearson correlation test. CANOCO 5.0 software was used for redundancy analysis (RDA) and principle component analysis (PCA) to assess the overall differences between the chemical fertilizers alone and combined with different biochars, and to investigate the relationship between plant growth and grain yield parameters/root traits, as well as and the relationship among soil biochemical properties.All experimental data were analyzed using Microsoft Excel 2010 and SPSS 22 software , and all figures were produced using Origin Pro. 9.0 software . The pot experiment results were presented as the mean of triplicates. One-way analysis of variance (ANOVA) was used to evaluate the effects of the treatments. Mean comparison testing was performed using least significant difference (LSD) , but no significant effect on SOC was observed after wheat harvest. We recommend that the co-application of biochars with chemical fertilizers should be tested in long-term field experiments for the development of soil management strategies, maintained the soil fertility status for the reduction of economic and environment issues.


UBE2D1) RNA expression in different histological subtypes of non-small-cell lung cancer (NSCLC). A retrospective study was performed by using molecular, clinicopathological, and survival data in the Cancer Genome Atlas (TCGA)—Lung Cancer. Results showed that both lung adenocarcinoma (LUAD) (N = 514) and lung squamous cell carcinoma (LUSC) (N = 502) tissues had significantly elevated UBE2D1 RNA expression compared to the normal tissues . UBE2D1 RNA expression was significantly higher in LUAD than in LUSC tissues. Increased UBE2D1 RNA expression was independently associated with shorter OS  and RFS  in LUAD patients, but not in LUSC patients. DNA amplification was common in LUAD patients  and was associated with significantly upregulated UBE2D1 RNA expression. Based on these findings, we infer that UBE2D1 RNA expression might only serve as an independent prognostic indicator of unfavorable OS and RFS in LUAD, but not in LUSC.In this study, we investigated the potential prognostic value of ubiquitin-conjugating enzyme E2D1 ( Ubiquitination is a biological process, in which the targeting proteins were modified with ubiquitin for degradation . This prα- (TNFα-) stimulated receptor-interacting protein 1 (RIP1) ubiquitination and NF-kappaB activation [in vitro and increases tumor latency in vivo in cancer cells including mutant KRAS-driven tumors [The ubiquitin-conjugating enzyme E2D family constitutes three members, including UBE2D1, UBE2D2, and UBE2D3, which all belong to the E2s. These family members share over 88% sequence identity and thus have similar enzymic activity , 6. Prevtivation . One rectivation . Interrun tumors . These fUBE2D1 in the two major subtypes of non-small-cell lung cancer (NSCLC) lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) and its prognostic value in these subtypes.In this study, by using the data from the Cancer Genome Atlas (TCGA)—Lung Cancer, we examined the expression profile of https://xenabrowser.net/). In TCGA, molecular, clinicopathological, and over 10-year survival data of more than 500 LUAD or LUSC patients were recorded. Generally, in TCGA-LUAD, tumor tissues from 514 patients with primary tumors were collected for the RNA-seq study. 502 out of the 514 patients had intact OS data recorded. In TCGA-LUSC, tumor tissues from 501 patients with primary tumors were collected for the RNA-seq study. 494 out of the 501 patients had intact OS data recorded. Clinicopathological parameters of patients with primary LUAD, including age at diagnosis, gender, smoking history, clinical stage, nodal invasion, residual tumors, recurrence status, RFS in days, living status, and OS in days, were downloaded for survival-related analysis. Kaplan-Meier curves of OS and RFS were generated to examine the survival difference in patients with high/low UBE2D1 RNA expression.This study is a retrospective study using the data from TCGA, with access provided by the UCSC Xena browser , which were presented as gene-level thresholded GISTIC2-processed copy number data, were also downloaded to examine the association between UBE2D1 RNA expression and the CNAs in LUAD patients.The genomic data, including UBE2D1 expression at the protein level in normal respiratory epithelial tissues and LUAD and LUSC tissues was characterized by using IHC staining data in the Human Protein Atlas (HPA) (https://www.proteinatlas.org/) [as.org/) , 14. IHCas.org/) . Each coas.org/) .t-test was performed to examine the difference of UBE2D1 RNA expression. Receiver operating characteristic (ROC) analysis for death and recurrence detection was performed to identify the Youden index for UBE2D1 RNA expression (the cutoff to separate patients) in Kaplan-Meier curves of OS and RFS. The association between UBE2D1 RNA expression and the clinicopathological parameters in LUAD patients was assessed by using the chi-squared test by two-sided Fisher's exact test. The log-rank test was conducted to examine the significance of the difference between the curves. Univariate and multivariate Cox regression models were used to evaluate the prognostic significance of UBE2D1 RNA expression. p < 0.05 was considered statistically significant.Statistical analysis was conducted by using GraphPad Prism 6.0  or SPSS 19.0 software package . Welch's UBE2D1 expression in NSCLC tissues and in normal lung tissues. The comparison showed that both LUAD (N = 514) and LUSC (N = 502) tissues had significantly elevated UBE2D1 RNA expression compared to the normal tissues  had markedly higher UBE2D1 RNA expression compared to the living cases (N = 319) (N = 151) also had significantly increased UBE2D1 RNA expression compared to the patients without recurrence (N = 275)  and RFS (p = 0.0011) compared to the group with low expression  . BesidesN = 275) . In compN = 275) . Then, w Figures . In contUBE2D1 RNA expression and the clinicopathological parameters in LUAD patients is summarized in p = 0.04), nodal positive cases , cases with residual tumors , recurrence , and death  compared to the group with low UBE2D1 RNA expression.The association between UBE2D1 RNA expression were associated with unfavorable OS and RFS in LUAD  (p < 0.001)   and RFS < 0.001) .UBE2D1 dysregulation. Among 551 cases with both UBE2D1 CNAs and RNA-seq data, 88 cases had amplification (+1/+2) CNA, 295 had copy neutral (0) CNA, and 128 had deletion (−1/−2) CNA  CNA . DNA delal group .UBE2D1 RNA was significantly upregulated in both LUAD and LUSC tissues compared with normal tissues, its expression was even higher in LUAD tissues than in LUSC tissues. Interestingly, we observed that UBE2D1 RNA upregulation was associated with poor survival outcomes in LUAD patients, but not in LUSC patients. By performing univariate and multivariate analyses, we confirmed that increased UBE2D1 RNA expression was independently associated with shorter OS  and RFS  in LUAD patients. Therefore, we infer that UBE2D1 RNA expression might only serve as an independent prognostic indicator of unfavorable OS and RFS in LUAD, but not in LUSC.In this study, our data mining results showed that although P53 inactivation can lead to DDX3 loss, which subsequently results in Slug-suppressed E-cadherin expression via decreased MDM2-mediated Slug degradation [p53 inactivation also have poor survival outcomes [RAD51 and p21 and the upregulation of the oncogene SOX2 in LUAD [UBE2D1 upregulation is associated with poor OS and RFS in LUAD patients.Previous studies showed that UBE2Ds play a critical role in the ubiquitination and degradation of p53 , 8. P53 radation . The NSCoutcomes . P53 deg in LUAD . A recen in LUAD . In factUBE2D1 RNA expression in LUAD, the mechanisms underlying its dysregulation have not been explored. In this study, we examined the association between UBE2D1 RNA expression and the CNAs of its DNA, and the results showed that DNA amplification was common in LUAD patients  and was associated with significantly upregulated UBE2D1 RNA expression, compared to the copy neutral patients. These findings suggest that DNA amplification might be an essential cause of upregulated UBE2D1 in LUAD.Although we confirmed the potential prognostic value of UBE2D1 RNA expression might only serve as an independent prognostic indicator of unfavorable OS and RFS in LUAD, but not in LUSC. DNA amplification might be an essential cause of upregulated UBE2D1 RNA expression in LUAD.


Benefiting from extremely high specific surface area of porous carbon, plenty of active sites were adequately exposed. Meanwhile, unique anchoring structure between Co2P nanoparticles and amorphous carbon outerwear insured high charge transfer efficiency and superior stability of Co2P/C. Due to these favorable properties, low overpotential of 281 mV at 10 mA cm−2 and Tafel slope of 69 mV dec−1 were achieved in resultant Co2P/C catalyst. More significantly, it only exhibited a negligible overpotential increase after 30 h stability test, and these performances entirely preceded commercial RuO2 benchmark. In summary, we proposed a simple and feasible strategy to prepare metal phosphides wrapped with amorphous porous carbon outerwear for efficient and durable electrochemical water oxidation.Exploring highly active, enduringly stable, and low-cost oxygen evolution reaction catalysts continues to be a dominant challenge to commercialize renewable electrochemical water-splitting technology. High-active and earth-abundant cobalt phosphides are recently considered as promising candidates. However, the poor inherent electron transfer efficiency and instability hinder its further development. In this work, a novel approach was demonstrated to effectively synthesize Co The prepared Co2P/C presented a distinct encapsulation structure, in which Co2P nanoparticles were toughly wrapped by amorphous carbon outerwear and uniformly distributed in this amorphous porous carbon. This favorable structural feature insured the exposure of more active sites on carbon framework, sharply decreased the charge transfer resistance and improved catalytic stability. Consequently, excellent electrochemical properties were realized. To be specific, the result Co2P/C merely needs a low overpotential of 281 mV to reach the current density of 10 mA cm−2, under 291 mV for RuO2. Remarkably, a low Tafel slope of 69 mV dec−1 for Co2P/C verified its superior activity in comparison with RuO2 benchmark (160 mV dec−1). What's more, a negligible overpotential increase after 30 h stability test indicated superior electrocatalytic stability.In this work, we demonstrated a novel, low-cost and scalable approach to synthesize Co2P nanoparticles uniformly embedded in porous carbon support, as illustrated visually in Figure 2P/C catalyst, respectively. P was fixed in porous carbon after a carbonization process in N2. Thus, by means of Co2+ adsorption and subsequently annealing in Ar/H2, the Co2P nanoparticles uniformly wrapped in porous carbon structure were obtained. The fabricated catalyst was firstly revealed the crystalline composition via X-ray diffraction (XRD) analysis, as shown in Figure 2P  image shown in Figure 2P, respectively, strongly demonstrating that Co2P nanoparticles were successfully obtained. Meanwhile, distinct interface between the Co2P nanoparticle and amorphous carbon layer  image and corresponding element mapping of C, Co, and P for Co2P/C catalyst loaded on carbon fiber paper were displayed in Figures 2P/C catalyst was uniformly coated on carbon fiber paper, which can be also verified by low-magnification SEM image and EDS spectra given in Figures 2P nanoparticles and their uniform distribution in amorphous porous carbon support.A transmission electron microscopy (TEM) image was recorded to illustrate the morphology and distribution of Co Figures  further y Figure . These r2P/C and P/C samples (without Co species adsorption) both displayed two explicit characteristic D and G bands located at 1,350 cm−1 and 1,587 cm−1, which were associated with defects and graphitization , further revealing the excellent oxygen evolution reaction kinetics of the prepared porous Co2P/C catalyst.IR-corrected linear sweep voltammetry (LSV) measurements were first recorded in 1.0 M KOH solution to evaluate the electrocatalytic properties of porous Co2 Figure . Obvious2P/C sample showed a lower transfer resistance than P/C sample, indicating that introduction of Co caused partially graphitization of porous carbon support, which sharply decreased charge transfer resistance and improved the OER performance of Co2P/C. Besides, a similar transfer resistance of Co2P/C in comparison with the value of RuO2 also verifies that Co2P/C has comparable electrocatalytic properties relative to RuO2. Electrochemical double-layer capacitance (Cdl) was tested to address the effect of porous carbon structure  curves of Co2P/C and RuO2 electrodes at a fixed current density of 10 mA cm−2 were presented in Figure 2 electrode had a well-marked overpotential increase after 30 h V-T test; however, the Co2P/C exhibited a negligible overpotential rise (only ~5 mV) after the same durability test, displaying its superior electrochemical stability. These significant properties can be attributed to the novel and favorable structure of Co2P/C catalyst, in which the Co2P nanoparticle was strongly and uniformly anchored in amorphous porous carbon . Besides, Tafel plots indicated the Co2P/C sample had a faster oxygen evolution reaction kinetics in comparison with RuO2. More importantly, in contrast to the obvious overpotential increase of commercial RuO2, the Co2P/C sample almost maintained initial superior OER activity after a 30 h stability test. Apart from providing a promising electrocatalyst alternative, this work proposed a simple and cost-effective strategy to prepare metal phosphide encapsulated with porous carbon materials electrocatalyst for electrochemical energy conversion and storage.In summary, this work developed Co2 for 3 h. After naturally cooling to room temperature and a vigorously grinding, the resultant powder was soaked in 0.5 ml 3 M Co(NO3)2.6H2O solution and then strongly dispersed by an ultrasonic treatment to make Co2+ adsorb adequately and tightly. After that, the mixture was moved to air oven and dried in 60 80°C. Adjacently, the completely dried powder was transferred into tube furnace and annealed at 500°C 650°C for 2 h in Ar/H2 mixture gas (10%). After naturally cooling to room temperature and a strongly grinding, the Co2P catalyst uniformly wrapped in porous carbon support (Co2P/C) was obtained.To be specific, 1.0 g degreasing cotton was immersed into 32 ml deionized water dissolving 2 ml phosphoric acid. After an ultrasonic treatment for 5 min, the beaker was moved into air oven and dried in 100 120°C. Subsequently, the attained black blocks were placed in the tube furnace and annealed at 800°C in NX-ray diffraction (XRD) was carried out on Bruker AXS, D8 Advance X-ray powder diffractometer with Cu-Kα radiation . High-resolution transmission electronic microscope (HRTEM) was performed by JEOL 2010 (HT) operating at 200 kV. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) were carried out using a FEI Sirion FEG scanning electron microscope at accelerating voltage of 20 kV. Raman measurements were performed by laser confocal micro Raman spectrometers  with laser excited at 532 nm. X-ray photoelectron spectroscopy (XPS) measurements were done on a Thermo Scientific ESCALAB 250 Xi system (USA) with Al Kα (1486.6 eV) as the radiation source and all spectra were calibrated using the C 1s peak with a value of 284.6 eV.2P/C catalyst, 2.5 mg Co2P/C powder was dispersed into 0.25 mL isopropanol dissolving 5 uL nafion solution. After an ultrasonic concussion for 1 h, the mixed liquid was drip-coated on carbon fiber paper with a micropipette. Finally, the electrode sample were placed on heating stage and dried at 60°C for 1 h before performance measurements. For comparison with Co2P/C sample, 2.5 mg RuO2 and integrated P/C powder were also dispersed and coated on carbon fiber paper according to the same process. All electrochemical measurements were performed in an undivided three-electrode cell using an electrochemical workstation (Chenhua CHI 660D). A Pt foil and Ag/AgCl (saturated KCl) were used to record potential as the counter and reference electrode, respectively. The potential vs. the reference electrode was converted to the potential vs. reversible hydrogen electrode (RHE) by the equation below:To characterize electrochemical performance of Co2.Electrochemical measurements were taken in 40 ml 1.0 M KOH solution. Practical effective areas of all samples were 1 cmXX, CJ, and ZK designed and finished the synthesis and characterization analysis of materials and wrote the research paper. HW, DH, XS, CT, JL, and LH offered many valuable suggestions to materials preparation, data processing, and paper modification.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.


N1), 2-aminoethanol residue (N2), and 4-amino substituent (N4). Our investigations showed that the protonation mainly occurs on the N1 atom. Such protonation seems to be a major inhibitory factor in the thermal removal of 2-pyridynyl TPG by the “chemical switch” approach and decreases the aromaticity of the pyridine ring. We also discussed possible participation of N2 nitrogen in irreversible intramolecular cyclization under acidic conditions.1D and 2D NMR investigations as well as computational studies, including static quantum-mechanics calculations, density function theory formalism, and classical molecular dynamics, were applied to determine the protonation sites in the thermolabile protecting group (TPG) containing a 2-pyridynyl moiety within its structure. This protecting group has three possible sites for protonation: an azomethine (pyridinic) atom ( The lower the PA value is, the more stable the compound phosphoramidate for which anti-HIV activity was postulated [N2 atom and subsequent shortening of the bond between the C2 and N2 atoms was observed in an acidic environment. This observation correlates with already published crystallographic data [N2 bond is much shorter than the typical single bonding.From the above discussion, we conclude that protonation in the precursor is a key factor in the electron distribution and is strongly correlated with the thermolability of the 2-pyridinyl moiety. Based on a combined experimental and computational analysis, we have shown the preference for the protonation of the stulated . The elehic data , where tN2 atom in cyclization under acidic conditions and its correlation with the reversible process concluded in the recovery of 2-pyridinyl thermolytic devices.Understanding the electron distribution allows control of the stability of the thermolabile 2-Pyridinyl group during the irreversible processes involving protection and subsequent removal of the protective group. The data presented herein logically imply the role of the All reagents and solvents  were obtained from commercial suppliers and used without further purification. All other compounds used were synthesized or commercially available and dried before use when necessary . For TLC analysis, the precoated plates (Merck silica gel F254) were used, and for column chromatography silica gel Si 60, 35–70 mesh (Merck) was used. Mass spectra were recorded with ESI (electrospray ionization) technique, on mass spectrometer MicroTofQ Brucker Daltonic. All NMR spectra (1D and 2D) were carried out using Bruker Avance 400/500/700 MHz spectrometers. Spectral analysis was performed with Bruker's TOPSPIN/ MESTRANOVA simulation program. The 15N NMR spectra were measured in 5 mm sample tubes, at 20°C. Chemical shifts, on 15N predicted spectra, were determined using liquid NH3 as a primary reference.Compound for NMR studies, 4-amino-2-pyridinyl-N-benzylaminoethanol (4-APyBzOH) was synthesized according to previously reported method . 4-APyBzDensity functional calculations were executed, using the Gaussian 16 A.03 program, namely the B3LYP/6-31G, approach in the gaseous phase very tight criteria, keywords: Opt = vtight Int = ultrafine)  The vibrS1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.S8 Table(PDF)Click here for additional data file.S9 Table(PDF)Click here for additional data file.S10 Table(PDF)Click here for additional data file.S11 Table(PDF)Click here for additional data file.S12 Table(PDF)Click here for additional data file.S13 Table(PDF)Click here for additional data file.S14 Table(PDF)Click here for additional data file.S15 Table(PDF)Click here for additional data file.S16 Table(PDF)Click here for additional data file.S17 Table(PDF)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S3 Fig(TIFF)Click here for additional data file.S4 Fig(TIFF)Click here for additional data file.S5 Fig(TIFF)Click here for additional data file.S6 FigRadial distribution function of distances between the rotamer I and the water environment resulted from the classical molecular dynamics simulation .(TIFF)Click here for additional data file.S7 Fig3O+ with N1 within the adduct I–H3O+ ; XYZ coordinates of adducts I–H3O+  onsidering interaction of H3O+ with N2 are given below.SRC simulation considering interaction of H(TIFF)Click here for additional data file.S8 Fig(TIFF)Click here for additional data file.S9 Fig(TIFF)Click here for additional data file.S10 Fig(TIFF)Click here for additional data file.