Coronaviruses are positive-strand RNA viruses with 3′ polyadenylated genomes and subgenomic transcripts. The lengths of the viral poly(A) tails change during infection by mechanisms that remain poorly understood. Here, we use a splint-ligation method to measure the poly(A) tail length and poly(A) terminal uridylation and guanylation of the mouse hepatitis virus (MHV) RNAs. Upon infection of 17-CL1 cells with MHV, a member of the Betacoronavirus genus, we observe two populations of terminally uridylated viral transcripts, one with poly(A) tails ~44 nucleotides long and the other with poly(A) tails shorter than ~22 nucleotides. The mammalian terminal uridylyl-transferase 4 (TUT4) and terminal uridylyl-transferase 7 (TUT7), referred to as TUT4/7, add non-templated uracils to the 3′-end of endogenous transcripts with poly(A) tails shorter than ~30 nucleotides to trigger transcript decay. Here we find that depletion of the host TUT4/7 results in an increased replication capacity of the MHV virus. At late stages of infection, the population of uridylated subgenomic RNAs with tails shorter than ~22 nucleotides is reduced in the absence of TUT4/7 while the viral RNA load increases. Our findings indicate that TUT4/7 uridylation marks the MHV subgenomic RNAs for decay and delays viral replication. The mammalian terminal uridylyl-transferases TUT4 and TUT7 uridylate the mouse hepatitis virus subgenomic RNAs with poly(A) tails shorter than 22 nucleotides to promote their decay and therefore delay viral replication. During the course of the infection, the poly(A) tails change in length which is believed to contribute to viral replication. Typically, at 8 hours post-infection (hpi), the viral RNA (vRNA) poly(A) tails are ~60 nucleotides long and gradually decrease in size as the infection progresses. By 48 hpi, the poly(A) tails shorten to less than ~40 nucleotides3. However, little is known about the mechanisms regulating the vRNA poly(A) tail size.Coronaviruses have single-stranded RNA genomes which are used to generate subgenomic transcripts. Both the viral genome and the subgenomic RNAs (sgRNAs) are 3′ polyadenylated1. In addition to replicating the gRNA, nsp12 also synthesizes each complementary sgRNA starting from the 3′-end of the gRNA. In this way, the gRNA and all sgRNAs share the same 3′ UTR. While both the gRNA and the sgRNAs are translated, only the gRNA is incorporated into the viral particles. The contributions of vRNA 3′-end processing to the differential treatment of the genomic and subgenomic RNAs remains poorly understood.Once the virus infects the cells, the genomic RNA (gRNA) is translated into various proteins required for viral RNA processing, such as the viral RNA-dependent RNA polymerase nps124. Two mammalian TENTs, terminal uridylyl-transferase 4 (TUT4) and terminal uridylyl-transferase 7 (TUT7), hereafter referred to as TUT4/7 transfer U residues to the 3′-end of the influenza A virus mRNAs, but not to its genomic RNAs nor complementary RNAs5. The terminal uridylation of the influenza A mRNAs delays the replication of the virus, presumably by targeting the mRNAs for degradation. TUT4/7 are redundant proteins required for normal development in mammals; their depletion leads to aberrant cell differentiation and perinatal death8. In the cytoplasm, TUT4/7 uridylate transcripts with poly(A) tails shorter than 30 nucleotides, which are no longer bound and protected by poly(A) binding proteins (PABP)9. Terminal uridylation also plays a role in the innate immune response against viruses in C. elegance alongside siRNA targeting pathways5.Recent studies have shown that host terminal nucleotidyl-transferases (TENTs) can add non-templated nucleotides to the poly(A) tails of different viruses to affect their replication capacity12. TENT4A and TENT4B are non-canonical poly(A) polymerases that occasionally incorporate G residues while elongating the RNA 3′-end resulting in poly(A) tails with interspersed Gs13. The presence of G residues in the tail provides stability to the transcript by inhibiting the activity of deadenylases11. TENT4A and TENT4B have also been shown to promote the viral replication of the hepatitis A virus14. Thus, endogenous TENTs can also be co-opted by viruses.Viruses can use endogenous RNA-modifying enzymes to facilitate their replication as well. In the case of the hepatitis B virus, the terminal nucleotidyltransferase 4 A (TENT4A) and terminal nucleotidyltransferase 4B (TENT4B) extend the poly(A) tail of the vRNA promoting its stabilization and facilitating viral replicationIn this study, we investigate the role of TENTs in the replication of the mouse hepatitis virus (MHV), a member of the Betacoronavirus genus. To determine whether the poly(A) tails of the viral gRNA and sgRNAs are targets of TUT4/7 or the TENT4s, we used a splint-ligation protocol to capture the 3′-end of the vRNAs. Although we did not detect a high frequency of guanylated tails, the hallmark of TENT4s activity, we found two populations of uridylated vRNAs, one with poly(A) tails shorter than ~22 nucleotides and the other one with poly(A) tails longer than ~44 nucleotides. Uridylation of tails shorter than ~22 nucleotides occurred only on sgRNAs. Depletion of TUT4/7 reduced the levels of uridylation of short tails, increased the vRNA load, and enhanced viral replication. We propose that TUT4/7 directly target sgRNAs to promote their decay, which in turn limits the replication capacity of the virus.12. To determine whether the TENTs act upon MHV RNAs, we developed a splint-ligation protocol to capture terminal uridylation and terminal guanylation, the signature modifications of TUT4/7 and TENT4s, respectively  tails of vRNAs affecting the replication capacity of the virusesely Fig. . A peak ely Fig. .Fig. 1MHTo determine whether the uridylation profile changed during the MHV infection, we examined the virion RNA and the viral RNA at 24 and 48 hpi. While the virion RNA shows uridylation of tails ~55 to ~66 nucleotides long Fig. , the peaThe poly(A) profile of the virus also changes during infection Fig. . The polWe next sought to understand whether the uridylation profile depended on the type of vRNA. In addition to facilitating the determination of transcripts’ poly(A) tail lengths, our long-read library preparation protocol allowed us to simultaneously differentiate between different vRNAs and opened the possibility of examining the poly(A) tail profile for individual vRNA species. However, given that most of our reads did not extend beyond the spike sgRNA (S sgRNA), we were unable to differentiate between the gRNA and the S sgRNA; thus, we analyzed both these populations together, and we refer to them as gRNA/S. The poly(A) profile of the gRNA/S species showed a unimodal distribution throughout the infection, while the nucleocapsid sgRNA (N sgRNA) presented an accumulation of poly(A) tails shorter than ~22 nucleotides over the course of the infection Fig. . Other sWe then examined the uridylation profile of the different vRNAs to determine whether the uridylation profile was also dependent on the type of vRNA. As for the poly(A) profile, we observed that the gRNA/S species presented a unimodal uridylation distribution which remained centered at ~60 nucleotides as the infection progressed Fig. . The N s9. Thus, we speculated that TUT4/7 could be uridylating the vRNAs with poly(A) tails shorter than 22 nucleotides, potentially impacting the replication cycle of MHV. To determine whether TUT4/7-depletion affects viral replication, we performed plaque assays to quantify the production of viral particles from 17-CL1 control (shCTL) and TUT4/7-depleted (shTUT4/7) cells at 0, 8, 16, 24, 36, and 48 hpi  tails shorter than 30 nucleotideshpi Fig. . While shpi Fig. , shTUT4/hpi Fig. . Thus, a15. We hypothesized that depletion of TUT4/7 could facilitate the replication of the virus by altering the cellular response to pathogens, as previously reported19. To explore this possibility, we performed gene expression analysis of shCTL and shTUT4/7 cells before infection  signaling and a significant downregulation of genes involved in the interferon response Fig. . Thus, t22. While the complementary sgRNAs are being generated, the RNA-dependent RNA polymerase switches templates from a characteristic sequence immediately upstream of the sgRNAs’ open reading frames (ORFs) to the leader sequence at the 5′-end of the virus genome. The template switching results in the staggered decrease in the read coverage immediately upstream of the sgRNAs ORFs. In some instances, the template switching occurs in the vicinity of the leader sequence rather than precisely on the leader sequence itself, which accounts for the increase in 5′ coverage.Since TUT4/7 are known to promote RNA decay, we sought to determine whether they could affect the viral RNA load during infection. To address this point, we normalized viral RNA reads to the endogenous transcripts and plotted the normalized read coverage across the viral genome Fig. . The covWhen comparing the read coverage between control and mutant cells, we observed that at 24 hpi, there was less vRNA accumulation in the TUT4/7-depleted cells compared to controls, probably due to the activation of the IL33 pathway in the absence of TUT4/7 Fig. . HoweverTo gain mechanistic insight into the pathways that regulate the poly(A) tail dynamics of the virus, we monitored changes in 3′-end regulation during MHV infection in 17-CL1 shCTL and shTUT4/7 cells. Although differences between the endogenous shCTL and shTUT4/7 transcriptome were maintained throughout the infection  and uridylation profiles of the different vRNA species changed upon depletion of TUT4/7. The poly(A) profile of the gRNA/S species, which did not change during infection, also preserved their distribution after TUT4/7-depletion Fig. . In contFinally, we examined whether the uridylation profile of the different vRNA species was affected by the TUTs. We hypothesized that, as for the global distribution, the sgRNAs with uridylated short poly(A) tails would present a shift toward the uridylation of tails ~44 nucleotides long. Indeed, while the uridylation profile of the gRNA/S species was not affected in the shTUT4/7 cells compared to controls Fig. , the N s9. The accumulation of vRNAs in the absence of TUT4/7 is consistent with the role of these enzymes in mRNA decay. Upon TUTs depletion, these transcripts revert to poly(A) and uridylation profiles similar to those of the gRNA/S species. Thus TUT4/7 is a critical component of the pathways that shape the poly(A) profile of the most abundant sgRNA species, with implications on the replication capacity of the virus.Taken together, these results are consistent with a model in which all vRNA species have poly(A) tails longer than ~55 nucleotides, but only a subset of the most abundant sgRNA have their tails shortened as the infection progresses. These short poly(A) tails are uridylated by TUT4/7, which are known to promote transcript degradationThe high transmissibility of coronaviruses requires a multilayered approach to minimize infections and symptoms. Identification of endogenous factors that regulate viral RNA processing is critical for developing novel treatments. Here we found that coronaviruses poly(A) tails show two types of uridylation, one of which is dependent on the endogenous proteins TUT4/7. Depletion of these proteins promotes viral replication and increases viral RNA load, revealing an unexplored pathway in the RNA processing of coronaviruses.l-glutamine, and 1× MEM non-essential amino acids (Millipore Sigma). None of the cell lines were authenticated. Cell lines were tested for mycoplasma throughout the course of the experiments. Cells lines that tested negative for mycoplasma were propagated. Cell lines that tested positive were immediately discontinued and replaced with clean ones.The murine 17-CL1 cell line  was maintained in Dulbecco Modified Eagle Medium  supplemented with 10% Fetal Bovine Serum , L-glutamine (Invitrogen), and Sodium pyruvate (Invitrogen). The NCTC clone 1469 cell line  was maintained in DMEM medium with 10% FBS, https://horizondiscovery.com/en/gene-modulation/knockdown/shrna/products/gipz-lentiviral-shrna. The shRNA sense-targeting sequences used in this study are summarized in Supplementary Table 23. Briefly, cells were transiently transfected with psPAX2, a gift from Didier Trono , pMD2.G, a gift from Didier Trono , and pGIPZ shRNA vectors carrying shCTL, shTUT4 or shTUT7 using Lipofectamine 2000. Supernatants were collected 48 hours post-transfection, aliquoted, and stored at −80 °C. Titers were determined using digital droplet PCR to measure the number of lentiviral particles integrated into the transduced HEK293T genome. To generate 17-CL1 shCTL and shTUT4/7 stable lines, cells were seeded in 6-well plates . The next morning, cells were infected with a multiplicity of infection (MOI) of 20 for shCTL or 10 MOI for shTUT4, and 10 MOI for shTUT7 lentiviruses. The next day, the medium was replaced with 2 µg/ml puromycin-supplemented medium. Cells were maintained for 48 hours under selection to obtain the shCTL and shTUT4/7 stable lines. The reduction of Tut4 and Tut7 levels in shTUT4/7 cells was confirmed by qPCR.The mouse pGIPZ lentiviral shRNA vectors (ThermoScientific) were provided by the NIEHS Epigenetics Core facility; detailed information can be found on the website, Tut4 and Tut7 were measured by qPCR using SYBR green assays (Applied Biosystems). The qPCR protocol consisted of a denaturation step of 20 seconds at 95 °C followed by 40 cycles of 3 seconds at 95 °C and 30 seconds at 60 °C. The sequences of qPCR primers are shown in Supplementary Table Gapdh expression, and fold changes were calculated relative to 17-CL1 shCTL cells Ct values.Total RNA was extracted from infected and not infected 17-CL1 shCTL or shTUT4/7 cells using the RNeasy Mini Kit  or from MHV virion using TRIzol. First-strand cDNA synthesis was performed using SuperScript II reverse transcriptase according to the manufacturer’s protocol (Invitrogen). The expression levels of The MHV strain S was purchased from ATCC (Cat. No. VR-766). For MHV infection, 17-CL1 cells were seeded into 6-well plates  overnight and infected with 10 MOI MHV for 0, 8, 16, 24, 30, 36, 42, and 48 hours. The supernatants were collected for MHV plaque assay, and cells were harvested for RNA isolation.l-Glutamine, 100 units of penicillin and 0.1 mg of streptomycin) until plaques become visible. The cell monolayers were stained with 2% crystal violet in 10% ethanol for at least an hour. The stained monolayers were washed and air-dried, and clear plaques were counted manually under a light microscope.17-CL1 cells were used for the plaque assay with MHV titers expressed as PFUs/ml. Briefly, cell monolayers in 24-well plates  were adsorbed for an hour with a series of 10-fold dilutions of either MHV inoculum or cell culture supernatants. The infected monolayers were left for 3–4 days under 0.8% methylcellulose overlay , 1× Phusion polymerase buffer, 1 pg/µl of the vector, 1 mM dNTP mix and 1 µM of each primer in a total volume of 100 µl. The amplification reaction consisted of an initial denaturation at 95 °C for 3 minutes followed by 34 cycles of 30 seconds at 95 °C, 30 seconds at 58 °C, and 2 minutes at 72 °C. The final extension at 72 °C was maintained for 30 minutes, after which the reaction was cooled down to 12 °C. The PCR amplicon was purified using the QIAquick PCR purification kit . First, 500 µl of PB buffer was added to the PCR reaction, and the mix was transferred to the column. After spin down, the flow through was discarded, and the column was washed with 750 µl of PE buffer. The flow-through was removed again, and the column spun for 2 more minutes. The amplicon was eluted in 30 µl of elution buffer.In vitro transcribed RNAs were used as internal poly(A) standards. Barcoded oligos , 2 µl of T7 RNA polymerase mix , and 1 µg of the amplicon. The reaction was incubated for 2 hours at room temperature. To remove the amplicon, 20 µl of water, 5 µl of 10X Zymo DNase buffer, and 5 µl of Zymo DNase I were added to the in vitro transcription product, and the reaction was incubated for 15 minutes at room temperature. The Zymo kit  column purification protocol was used to purify the RNA. First, 50 µl of RNA binding buffer and 50 µl of 100% ethanol were added to the DNase-treated in vitro transcribed RNA. The mix was then transferred to a column, and the RNA was bound to the column by spinning down for 30 seconds. The column was washed with 400 µl of RNA prep buffer, then with 700 µl of wash buffer, followed by a final wash with 400 µl of wash buffer. After removing the flow through, the column was centrifuged for an additional 2 minutes, and then the RNA was eluted with 30 µl of nuclease-free water.E. coli Poly(A) polymerase for different amounts of time to achieve different poly(A) tail lengths. The poly(A) polymerization mix consisted of 1× E. coli poly(A) polymerase reaction buffer, ATP 1 mM, 0.5 µl of E. coli Poly(A) polymerase , and 10 µg of purified in vitro transcribed RNA in a total volume of 20 µl. The reaction was incubated for 10 to 30 minutes for the different barcodes to archive poly(A) tails of different lengths. The RNA was purified again using the Zymo kit column as described above, and an equimolar master mix of the different standards 50 ng/µl was prepared.For standards with poly(A) tails 16, 32, or 64 nucleotides long, the poly(A) tail was encoded on the barcoded oligos . To prepare the oligo(dT) beads, 20 µl of beads per sample were transferred to a low-binding tube and placed on a magnet to remove the storage buffer. The beads were washed with 40 µl of binding buffer  and resuspended in 40 µl of binding buffer. Then 5 µg of total RNA was diluted with nuclease-free water up to 40 µl and mixed with 40 µl of binding buffer, except for the virion where 1 µg of RNA was used. Diluted RNA was denatured by incubating at 65 °C for 2 minutes and immediately placed on ice. The denatured RNA was mixed with 40 µl of pre-equilibrated oligo(dT) beads and incubated for 5 minutes at room temperature. The supernatant was removed from the beads by incubating them on a magnetic rack for 5 minutes. The beads were washed twice with wash buffer . After final washing, the supernatant was removed, and beads were resuspended in 10 µl of elution buffer . The enriched mRNA was eluted by incubating at 80 °C for 2 minutes, and beads were separated using a magnetic stand.Viral RNA and host mRNA were purified from total RNA using Dynabeads™ Oligo(dT)A 100 nM mixture of different barcoded oligos was made to detect tail modifications. First, ten sets of 1 µM annealed upper and lower strand oligos were made as listed in Supplementary Table To detect the terminal RNA modifications, we generated direct RNA sequencing libraries using the protocol from Oxford Nanopore Technologies (Direct RNA Sequencing Kit- SQK-RNA002) with some minor changes to assemble the barcoded oligos that bind to the specific 3′ terminal nucleotides. A splint ligation was used to bind the RNA to the 3′ adaptors. For this, 3 µl of ligation buffer (6% PEG8000 in NEB 10× T4 DNA ligation buffer), 1.5 µl of T4 DNA ligase , 1 µl of 0.1 ng/µl RNA spike mix, and 1.5 µl of barcoded oligos mix (100 nM) , 8 µl of 5× first-strand buffer, 4 µl of 100 mM DTT, and 2 µl of SuperScript II reverse transcriptase . The reaction was then incubated at 50 °C for 50 minutes, followed by 10 minutes at 70 °C, and cooled at 4 °C.The resulting cDNA/RNA hybrid was purified using AMPure beads . First, 72 µl of AMPure beads were added to each reaction and incubated at room temperature for 5 minutes. The supernatant was then removed using a magnetic stand, and beads were washed with 150 µl of 70% ethanol by rotating the tubes twice 180° on the magnetic stand. The beads were resuspended in 20 µl of nuclease-free water and incubated at room temperature for 5 minutes. The eluate was separated from beads using the magnetic stand.The Nanopore adaptors with the motor protein (RMX) supplied with the Direct RNA Sequencing Kit (SQK-RNA002) were then ligated to the samples. The 20 µl of AMPure beads purified cDNA/RNA hybrid was mixed with 8 µl of ligation buffer (6% PEG8000 in NEB 10× T4 DNA ligation buffer), 6 µl of RMX-RNA adapter, 3 µl of nuclease-free water, and 3 µl of T4 DNA Ligase  for 40 µl of the total final volume. The reaction was incubated for 15 minutes at room temperature, and then 40 µl of AMPure beads were mixed with adaptor-ligated RNA and incubated at room temperature for 5 minutes. The supernatant was removed using a magnetic stand, and beads were washed twice with 150 µl of wash buffer (WSB from SQK-RNA002). After washing, beads were resuspended in 21 µl of elution buffer (EB from SQK-RNA002) and incubated at room temperature for 20 minutes. The eluate was separated from beads using a magnetic stand and kept aside.The libraries were prepared for loading into the Nanopore flow cells (R9.4.1) using the flow cell priming kit (EXP-FLP002) according to the manufacturer’s instructions. Briefly, 17.5 µl of nuclease-free water and 37.5 µl of RNA running buffer (RRB from SQK-RNA002) were added to the 21 µl of adaptor-ligated RNA samples and mixed well to prepare the loading mix. To prepare the flow cell, 30 µl of FLT solution was added to 1 ml of FB solution (from EXP-FLP002) at room temperature, and 800 µl of this solution was loaded to the flow cell through the priming port and incubated at room temperature for 5 minutes. Then, the spot-on port was gently opened, and 200 µl of the FLT-FB mix was loaded through the priming port. Finally, 75 µl of RNA library was loaded through the spot-on port, and both ports were closed after loading the library. Fast5 files were generated by the Nanopore MinKNOW software.25. Briefly, 1.0 µg of MHV virion RNA was used as a starting material and was subjected to decapping using 5U of mRNA decapping enzyme  and incubated at 37 °C for 1 hour in a 20 µl reaction. After incubation, RNA was purified using RNA Clean & Concentrator-25 kit  and eluted in 30 µl nuclease-free water. RNA was denatured at 95 °C for 5 minutes and quickly cooled on ice. Then 30 µl of decapped RNA was head to tail ligated using 10U of T4 RNA ligase I  in 60 µl reaction containing 1× T4 RNA ligase I buffer, 1 mM ATP, 40U of RNase inhibitor, and 10% PEG8000 and incubated for 16 hours at 16 °C. After incubation, circular RNA was purified using RNA Clean & Concentrator-25 kit and resuspended in 30 µl nuclease-free water. Next, 15 µl of purified, circular RNA was reverse transcribed using SuperScript II reverse transcriptase  with a gene-specific primer (5′-GTACGTACGGACGCCAATC-3′). The resulting circular cDNA was used to amplify the region spanning the poly(A) tail and terminal modifications of the MHV genome using AccuPrime Taq DNA polymerase . To this end, 5 µl of cDNA was used in a PCR reaction with forward primer (5′-GATAGAGAAGGTTGTGGCAGAC-3′) and reverse primer (5′-GCAGAGAACGAAAGTCAAGGA-3′). The reaction was heated at 94 °C for 3 min, followed by 35 cycles of 30 seconds at 94 °C, 30 seconds at 57.5 °C and 1 minute at 72 °C and a final extension at 72 °C for 7 minutes. The PCR product was purified using a QIAquick PCR purification kit . The resulting PCR amplicon was ligated into the PCR 2.1 vector using a Topo cloning kit , and the reaction was incubated at room temperature for 1 hour. The ligated reaction was transformed into OneShot TOP10 competent cells and plated onto ampicillins/X-gal containing LB-plates. White colonies were screened by Sanger sequencing.Circular RT-PCR was performed to determine the terminal modifications of the MHV virion genome following previously described protocols with some modifications26 and base called with Guppy version 5.0.10. The demultiplexed reads were mapped to the mouse transcriptome and the MHV genome using Minimap2 version 2.2027. The poly(A) tail length was determined using Nanopolish28 version 0.13.3, and the terminal modifications were assigned according to the barcodes associated with each modification. The poly(A) tail frequency plots showing uridylation levels were obtained by normalizing the number of uridylated reads of any given length by the total number of reads, in contrast to only the total number of uridylated reads. This allows a direct comparison of the frequency of uridylated transcripts and all transcripts at each poly(A) tail length across plots. Differential gene expression was determined using DESeq229. Only transcripts with 180 reads or more across the 18 samples were considered for analysis. The ontology of the differentially regulated genes was obtained using the TopGO R package. Pathway analysis of differentially expressed genes at 0 hpi and 24 hpi was performed using Ingenuity Pathway Analysis (IPA) (http://www.ingenuity.com/index.html). The read count matrix file obtained from DESeq2 was used as an input for core analysis. The graphical summaries of the pathway obtained from IPA were exported and manually curated using Inkscape. Measurements in every case were taken from distinct samples.Direct RNA-seq libraries were demultiplexed with DeePlexiCon version 1.2.010-transformed, and a two-way ANOVA with multiple comparison tests was performed for the statistical analysis using GraphPad Prism version 8.2.1. For DESeq differential gene expression analysis, three biological replicates were used. Transcripts with adjusted p values lower than 0.05 were considered differentially expressed.For the statistical data analysis of qPCR data, a one-way ANOVA with multiple comparison tests was performed using GraphPad Prism version 8.2.1. Four replicates were used for each condition. For the plaque assay time course between 8 and 48 hours, samples were taken from infections of different plates/wells . To control for batch effects, infections were performed on three different days, with 2–3 infections each day. For each biological replicate, we performed two technical replicates. The average of the technical replicates for each biological replicate was used for the statistical analysis. A similar strategy was used for the time course between 24 and 48 hours, this time using four biological replicates. The data were logFurther information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


To address the regional lymph node (RLN) distribution and the long‐term efficacy in unilateral nasopharyngeal carcinoma (NPC), providing elective irradiation for RLN with intensity‐modulated radiotherapy (IMRT).The involvement of clinical data of 136 patients with unilateral NPC, who underwent IMRT from November 2003 to December 2020 were analyzed retrospectively. The therapeutic effect and failure pattern of RLN metastasis were evaluated.Of 57.1% patients have bilateral RLN metastasis. The rate of contralateral RLNs metastasis is lower than that of ipsilateral RLNs. Contralateral RLN metastasis mainly occurs in level VIIa (39.0%) and II (38.2%). While level IVa is only 0.7%, and none of RLN metastasis was found in level IVb and Va. The median follow‐up was 70 months, and the 3‐, 5‐and 10‐year regional recurrence‐free survival (RRFS) were 94.1%, 93.1%, and 93.1%, respectively.Routine prophylactic irradiation may not include contralateral lower neck LN and level Va for N0‐1 unilateral NPC. The inclusion criteria were: (1) pathologically and initially confirmed unilateral NPC; (2) >18 years old; (3) received the IMRT; (4) the performance status (PS) of 0 or 1; (5) without distant metastasis; (6) informed consent form related to treatment signed. Exclusion criteria: (1) metachronous or synchronous malignant tumors; (2) pregnancy or lactation; (3) mental disorders. Retrospective ethical approval was obtained from the institutional ethics committee of the Sun Yat‐sen University Cancer Center, with the approval number YB2019‐169‐01.2.2All patients underwent MRI or electronic nasopharyngoscope (ENS) before treatment. Unilateral NPC was defined as the primary tumor that was confined to one side and did not exceed the midline of the nasopharynx.All patients were examined by electronic nasopharyngoscopy and MRI. The criteria for RLN metastasis2.3All patients received the head and neck MR examination with a scan range from the suprasellar cistern to the inferior margin of the sternal end of the clavicle. Patients underwent MRI by a 1.5‐T MRI unit  or 3‐T MRI unit . T1‐ and T2‐weighted fast spin echo images in the axial were obtained before injection of contrast material. Enhanced T1WI axial sequences were performed 40 seconds after intravenous Gd‐DTPA  injection at a dose of 0.1 mmol/kg. In order to obtain more accurate diagnostic results, MRI should be completed within a reasonable time frame, and efforts should be made to shorten the scan time while ensuring image quality.2.4All patients were treated with IMRT which was based on the consensus guideline2.52, cisplatin 60 mg/m2, fluorouracil 500 mg/m2 continuous venous perfusion for 120 h), three to four times in total. Concurrent chemotherapy: a weekly regimen containing platinum, including cisplatin (30 mg/m2), nedaplatin (30 mg/m2), four to six times in total; concurrent chemotherapy, including cisplatin weekly regimen (30 mg/m2), four to six times in total and cisplatin three‐week regimen (80 mg/m2), three to four times in total.Induction chemotherapy: TPF  and the corresponding confidence interval (CI) were calculated. p < 0.05 was considered statistically significant.Statistical Package for the Social Sciences (SPSS) software version 24.0 was used for all statistical analyses. To test the homogeneity of variance, the continuous variables with normal distribution and homogeneity of variance were tested using the 33.1The baseline characteristics of 136 unilateral NPC patients are shown in Table 3.2The total rate of RLN metastasis was 92.6% (126/136), of which the contralateral RLN metastasis rate was 60.3% (76/126), and the bilateral RLN metastasis rate was 57.9% (73/126). In each LN region, the contralateral metastasis rate was lower than that of the ipsilateral site, and the contralateral metastatic LNs mainly occurred in level VIIa (41.9%) and II (38.2%)  and IIa  metastasis, contralateral level IIa metastasis was associated with ipsilateral level IIa  and IIb  metastasis, and contralateral level IIb was associated with ipsilateral level IIa , IIb  and III  metastasis. Multivariate analysis showed that contralateral level VIIa metastasis was associated with ipsilateral level VIIa  and IIa  metastasis, contralateral level IIa metastasis was associated with ipsilateral level IIa  and IIb  metastasis, and contralateral level IIb was associated with ipsilateral level IIa  and III  metastasis.The correlation between contralateral RLNs and ipsilateral ones was further analyzed and the results are shown in Table 3.4p > 0.05, Table The location of the primary focus of unilateral NPC was not significantly correlated with contralateral RLN metastasis, and had no significant correlation with T stage and tumor volume . Figure 3.6A total of 21 cases had recurrent disease, including six cases of local recurrence, 8 cases of regional recurrence and 11 cases of distant metastasis Table . Regiona4Tomita et al.At present, there are few studies on RLN distribution of unilateral NPC. Tomita et al.p < 0.05). Contralateral level III also had a moderate risk of RLN metastasis, with an incidence of approximately 8.1%. Therefore, it is recommended that unilateral NPC should be given preventive irradiation in contralateral level VIIa, II and III.Previous literatureIn this study, the rate of RLN metastasis in the contralateral level IVa was lower (0.7%), and similar results were observed in previous studiesCompared with other head and neck squamous cell carcinomas, patients with NPC are more likely to have metastasis in level Va (11.1%–25%),Retropharyngeal LNs are one of the first levels of metastases from NPC. Previous studies on 3100 NPC casesThe 5‐year RRFS was 93.1% in the present study, which is similar to that in previous studies (91%–92.5%).Several limitations should be taken into consideration. First, late toxicity and the cause of death are not detailed enough over the long‐term. Therefore, we failed to confirm the relevant evaluation and analysis. Second, this is a retrospective study with a small sample size. The results of the study need to be verified by further prospective clinical studies.5In each RLN region, the metastasis rate of contralateral RLNs was lower than that of the ipsilateral rate for unilateral NPC. The metastasis of RLNs for unilateral NPC were from the upper neck to the lower neck. Metastasis in contralateral lower neck RLNs  were at low‐risk areas, and level Va was rare. Therefore, upper neck RLNs needed to be included in CTV, while reduced prophylactic irradiation in contralateral lower neck LNs and contralateral level Va could be recommended for N0‐1 patients.Feifei Lin: Conceptualization ; data curation ; formal analysis (lead); investigation ; methodology ; project administration ; resources ; software (lead); supervision ; validation ; visualization (lead); writing – original draft (lead); writing – review and editing . Zichen Qiu: Data curation ; formal analysis ; investigation ; methodology ; resources ; software ; validation ; visualization ; writing – original draft . Dehuan Xie: Conceptualization ; data curation ; investigation ; resources ; software ; validation ; writing – original draft . Xiong Zhou: Data curation ; investigation ; methodology ; software ; validation ; visualization . Lei Wang: Formal analysis ; investigation ; software ; validation . Zheng Wu: Investigation ; project administration ; software ; writing – review and editing . Wanqin Cheng: Investigation ; methodology ; resources ; software . Shaowen Lyu: Methodology ; resources ; software . Yong Su: Conceptualization (lead); data curation ; formal analysis (lead); investigation ; methodology ; project administration (lead); resources ; supervision (lead); validation ; visualization ; writing – review and editing (lead). Yalan Tao: Conceptualization (lead); data curation ; formal analysis (lead); investigation ; methodology ; project administration (lead); resources ; supervision ; validation (lead); visualization ; writing – original draft .The authors declare that they had no funding for this study.The authors declare that they have no competing interests.Retrospective ethical approval was obtained from the institutional ethics committee of the Sun Yat‐sen University Cancer Center, with the approval number YB2019‐169‐01. Written informed treatment consents were required from all patients with respect to chemotherapy and/or radiotherapy.


Tilia cordata and tentatively named cytorhabdovirus tiliae. The assumed genome organization is 3′-N-P-P3-M-G-p6-p6’-L-5′. The N gene encodes the putative nucleoprotein (59.1 kDa), P encodes the phosphoprotein (34.7 kDa), P3 encodes the movement protein (23.1 kDa), M encodes the matrix protein (23.1 kDa), G encodes a glycoprotein (64.4 kDa), and L encodes the viral RNA polymerase (247 kDa). P6 and P6’ are overlapping open reading frames (ORFs), which may encode gene products of 7.9 and 9.5 kDa, respectively, of unknown functions.Here, we report the coding-complete sequence  of a novel cytorhabdovirus detected in  Plant viruses can be introduced from tree nurseries into different ecosystems, spread within them, and invade neighboring ecosystems through their vectors. As the epidemiological, ecological, and financial consequences of virus-infected nursery trees cannot be estimated, tested virus-free planting material is therefore highly recommended.Tilia cordata tree showing chlorotic spots consistent with a viral infection was collected from a German tree nursery in 2016. Total RNAs were extracted from 0.3 g leaf tissue using the InviTrap spin plant RNA minikit , followed by removal of remaining DNA with rDNase , and RNA purification using a NucleoSpin RNA clean-up kit (Macherey-Nagel). The rRNA depletion was performed using the RiboMinus plant kit for transcriptome sequencing (RNA-Seq) , followed by cDNA synthesis with the Maxima H minus double-stranded cDNA synthesis kit (Thermo Scientific) primed with random hexamers. Library preparation with the Nextera XT DNA library preparation kit , RNA sequencing , and de novo assembly of contigs using BayersHammer  were identified by BLASTx and tBLASTx (TGTTTCAGGTTTTGCAGG-3′) and Cy_Ti_6’_R (5′-GATTGGAGTAAGCAGCTC-3′) or Cy_Ti_6_F (5′-TCCGATTACAAAGATCCGAC-3′) and Cy_Ti_L_R (5′-GCCTTTTCTTGATATGAGCAG-3′).A leaf sample of a  tBLASTx  against  tBLASTx  was used tBLASTx  6, 7). , 7. TiliTilia cordata revealed identities of 12 to 22% , ERS14351145 (SAMPLE_ID), PRJEB57650 (STUDY_ID), and GCA_948193075 (ASSEMBLY_ACC). Sanger sequences has been deposited in GenBank with OQ361387 and OQ361388.The complete annotated genome sequence of the new cytorhabdovirus tentatively named cytorhabdovirus tiliae has been deposited in European Nucleotide Archive with the following numbers:


Perceived health is a well-known, low-cost measure in public health, and has been used in several studies on individuals with impairment. Although many studies have related impairment to self-rated health (SRH), few have considered the origin and degree of limitation of the impairment. This study examined whether physical, hearing, or visual impairments—when analyzed according to origin  and degree of limitation (with or without)—are associated with the SRH status.This cross-sectional study used data of 43,681 adult individuals from the Brazilian National Health Survey . The outcome SRH was dichotomized into poor  or good (including the good and very good responses). Crude and adjusted (for socio-demographic characteristics and chronic diseases history) prevalence ratios (PR) estimates were evaluated using Poisson regression models with the robust variance estimator.Poor SRH prevalence was estimated at 31.8% (95%CI:31.0–33.0) among the non-impaired population, 65.6% (95%CI:60.6–70.0) among individuals with physical impairment, 50.3% (95%CI:45.0–56.0) for people with hearing impairment, and 55.3% (95%CI:51.8–59.0) for the visually impaired. Individuals with congenital physical impairment—with or without limitations—presented the strongest association with the poorest SRH status. Participants with non-limiting, congenital hearing impairment showed a protective factor to poor SRH (PR = 0.40 95%CI: 0.38–0.52). Individuals with acquired visual impairment with limitations demonstrated the strongest association with poor SRH (PR = 1.48 95%CI:1.47–1.49). Among the impaired population, middle-aged participants showed a stronger association with poor SRH than older adult participants.Impairment is associated with poor SRH status, especially among people with physical impairment. The origin and degree of limitation of each type of impairment differently impacts SRH among the impaired population.The online version contains supplementary material available at 10.1186/s12889-023-15445-w. Currently, approximately 15% of the world’s population suffers from some form of impairment or considerable functional difficulties . Data frdisability and impairment according to the World Health Organization (WHO) definitions: disability is an umbrella term for impairments, activity limitation, and participation restriction. It denotes the negative aspects of the interaction between an individual  and his/her contextual factors . Impairments are problems in body function and structure, such as a significant deviation or loss [In this study, we use the terms  or loss .Measurement of the health status of several populations has been performed using what is known as self-rated health (SRH)—which is essentially a single question: “How do you evaluate your health?” Respondents are given a scale to classify their health as very good, good, regular, poor, or very poor. SRH is a low-cost, easy-to-apply tool that considers the contextual frameworks of an individual’s health status, such as cultural and psychosocial aspects, social participation, health behaviors, and lifestyle . DespiteInvestigations regarding the health status of populations with impairment using the SRH tool have been gaining space in the national and international literature. Visually impaired, hearing impaired, and physically impaired individuals has been found associated with poor SRH –16. AnotIn this context, a systematic review has demonstrated that individuals with any type of acquired impairment and a high degree of limitation due to this impairment have worse SRH compared to the general population , 18, 19.Studies on congenital and acquired impairments have argued that an individual’s behavior and self-concept of impairment, as well as their age and time of exposure to the impairment, can influence the SRH status. Thus, people with congenital impairments, and who have adaptive behaviors, are associated with better SRH compared to those with impairment acquired later in life. Furthermore, aging in this population increases the association with poor SRH status , 21, 22.Meanwhile, very few studies have focused on middle-aged populations with impairments. A few authors have argued that more demanding work activities can lead to limited physical functions. In this population, promoting self-care and preventing physical decline can improve health status and reduce poor SRH scores among older individuals , 24.Given this scenario, the research hypothesis of this study is that the SRH status among individuals with physical or sensory impairments varies based on origin  and limitation .We believe that classifying SRH into these strata can facilitate clarifying epidemiological phenomena, such as the social determinants of health, participation in social activities, and how these individuals use health services. The data collected against this local backdrop can lend support to public health promotion and prevention campaigns that better target these populations and their heterogeneous needs.Thus, this study aims to assess whether physical, hearing, or vision impairment, when analyzed according to origin  and degree of limitation (with or without), are associated with the SRH status.This is a cross-sectional study conducted using the data from the Brazilian NHS collected in 2013 by the Brazilian Institute of Geography and Statistics (IBGE), in partnership with the Ministry of Health and the Oswaldo Cruz Foundation (Fiocruz) . StructuThe NHS is a household survey with probabilistic sampling aimed at estimating the Brazilian population. It was developed as a Master Sample of the IBGE’s Integrated Home Survey System. Cluster sampling was used and the stratification of the Primary Sampling Units (PSU)—that is, geographic regions—was divided into three stages: (i) PSU census sectors; (ii) PSU households; and (iii) an adult resident respondent (aged 18 years or more) per household. Households and residents were selected via simple random sampling , 26. DetThe SRH status—that is, the outcome of this study—was obtained through self-reports. The participants answered the question: “In general, how would you assess your health: very good, good, regular, poor, or very poor?” For the analyses, the SRH outcome was dichotomized into poor  or good (including the good and very good responses) to facilitate interpretation. Dichotomizing the variables to include the regular response in the bad category made the conclusions more conservative than a three-category classification (in which the regular response was a separate category). Sensitivity analysis considering two other scenarios of how to categorize the SRH outcome was performed. Scenario 2 is the binarization of SRH into poor (very poor + poor + regular) and not poor (very good + good); and scenario 3 is considering SRH into three categories: poor (very poor + poor), regular and not poor (very good + good).Three exposure factors were considered: physical impairment, hearing impairment, and visual impairment  measured through self-reports. These factors were analyzed in two ways:As a binary factor (having or not having impairment): individuals who answered positively to the question “Do you have a physical impairment?” were considered impaired. For sensory impairments, those who answered positively to the questions: “Do you have a hearing impairment?” or “Do you have a visual impairment?” were considered impaired.As a factor with five categories: no impairment, a congenital impairment without limitations, a congenital impairment with limitations, an acquired impairment without limitations, and an acquired impairment with limitations. To measure limitation, the following question was asked: “To what extent does this impairment limit your usual activities?” To answer, respondents were given a 5-point Likert Scale. In this study, limitations were categorized as “limited”  and “minimally limited/not limited.” As for the question about the origin of their impairment, the participants answered the following question: “Were you born with this impairment or was it acquired?” (answered as born or acquired).For adjustment, socio-demographic variables were used: sex , age (considered quantitatively), skin color , education  and employment . Additionally, a health variable for chronic diseases was also included, namely Diabetes mellitus, cardiovascular disease , lung disease, cancer, arthritis, and depression. This variable was constructed using answers to questions such as: “Have you ever had a medical diagnosis of ___?,” to which the possible answers were “yes” or “no.” Thus, the variable of chronic diseases was categorized into: no disease, one disease, two diseases, and three or more diseases.The population studied were described using absolute and relative frequencies. Owing to the complexity of the sample, the relative frequencies and all other results were weighted . To evaluate association between impairment and SRH, Poisson regression models with robust variance estimation were adjusted. For each type of impairment, two models were considered: one with the impairment as a binary factor, and another with impairment as a five-categories factor , 28. As A total of 60,202 individuals, aged 18 years old or older, responded to the Brazilian NHS survey. From these, we excluded 578 participants who had more than one impairment; a population of 59,624 remained. Of these, 9,958 did not answer questions regarding education and ethnicity and 7,637 did not answer questions about chronic disease. These participants were also excluded from the study, resulting in a final sample of 43,681 participants.The distributions of impairment and SRH did not differ between study and excluded populations (p = 0.52 and p = 0.71). Additionally, there was no significant difference between the prevalence of poor SRH comparing the included and excluded populations for all populations , except for the visual impairment population  had an average age of 44.20 years (SD = 15.73). The prevalence of physical impairment was estimated at 1.44% (95% CI: 1.25–2.00), hearing impairment at 1.77% (95% CI: 1.57–2.00), and visual impairment at 4.70% (95% CI: 4.32–5.00).We used the SRH outcome in its binary format (poor or not poor). Comparing with the SRH in its five categories original format , we observed that the “poor” category of the binary version predominantly comprised the “regular” response ; the “not poor” category predominantly comprised the “good” responses  regardless of the group—whether impaired or not  among the non-impaired population, and at 65.6% (95%CI:60.6–70.0) among individuals with physical impairment, 50.3% (95%CI:45.0–56.0) among individuals with hearing impairment, and 55.3% (95%CI:51.8–59.0) among those with visual impairments. Furthermore, for the three types of impairment, there are certain imbalances regarding the socio-demographic characteristics and chronic diseases between the groups of impaired participants and the group of people with no impairments  “acquired with limitations”; 0.10% (95% CI: 0.06–0.15) “congenital with limitations”; 0.40% (95% CI: 0.31–1.00) “acquired without limitations”; and 0.20% (95% CI: 0.07–0.20) “congenital without limitations”  attributed to losses “acquired with limitations,” 0.06% (95% CI: 0.03–0.09) to “congenital with limitations,” 1.60% (95% CI: 1.45–2.00) to “acquired without limitations,” and 0.13% (95% CI: 0.09–0.20) to “congenital without limitations”  “acquired with limitations”; 0.20% (95% CI: 0.11–0.25) “congenital with limitations”; 3.70% (95% CI: 3.30–4.00) “acquired without limitations”; and 0.36% (95% CI: 0.27–0.48) “congenital without limitations”  impairments were found to be significantly associated with poor health perception. Poor SRH prevalence was estimated at 31.8% (95%CI:31.0–33.0) among the non-impaired population; 65.6% (95%CI:60.6–70.0) for persons with physical impairment; 50.3% (95%CI:45.0–56.0) for individuals with hearing impairment; and 55.3% (95%CI:51.8–59.0) for those with visual impairments.Our results concur with those of other studies that indicated that individuals with impairments tend to report a poorer SRH compared to individuals with no impairments , 30. ThoFurthermore, the associations between impairment and SHR differ based on age; younger people with impairments are more likely to rate their health as poor compared to older people. In general, individuals below age 60 with physical impairments—acquired or congenital—had poorer SRH status compared to their seniors. The literature shows that individuals with physical impairments constitute the largest unattended subpopulation, presenting a 75% higher chance of having unmet health needs, not only related to physical architectural barriers , but assIn this study, visual acquired impairments in older aadults with limitations showed strong association with poor SRH, probably because lack of vision is associated with loss of autonomy in daily life activities. Additionally, adults below age 60 with physical impairments showed a stronger association with poor SRH compared to their older counterparts. Subjects with congenital physical impairments with no limitations and aged 60 or more showed a strong association with poor SRH. Even though adjustments by age and clinical history were made in this study, probably there are other associated factors that lead to poor SRH in physically impaired individuals—even those with congenital physical impairments and no limitations. Further studies are required to investigate this association.Individuals with congenital hearing impairment with limitations were not associated with poor SRH; congenital hearing impairment with no limitations seemed to be a protective factor against poor SRH. This can be explained by the fact that congenital hearing impaired individuals develop other mechanisms to communicate than speaking and hearing. Additionally, after access to hearing aids and/or rehabilitation, they develop coping strategies to integrate with the community, such as sign language, orofacial reading, etc. Moreover, hearing impairment is a type of “silent impairment” that does not impede physical mobility and integration into the community, even though, at times, spoken messages may not be understood , 32.Similarly, it is believed that people with congenital impairments that do not limit daily activities are capable of developing optimal functionality . They soimpairment was used to define changes in body functions caused by structural deterioration and restriction [Several studies have indicated the relationship between poor SRH status and older disabled populations –37. Thestriction . A signitriction . We hypotriction .Additionally, it is significant to consider the conceptual model that guides this study , and cerThis study considered the origin  and degree of limitation caused by the impairment. Additionally, the dataset used is from a representative sample of the Brazilian adult population in which health status was measured through an SRH tool. SRH assessments are widely used in health surveys worldwide, and are significant predictors of mortality , 6, 9, mSince 2002, Brazil has been following the National Health Policy for People with Disabilities, which is focused on the inclusion of people with disabilities in the entire service network of the Unified Health System (SUS) and recognizes the need to implement the process of responses to the complex issues that involve healthcare for people with disabilities in Brazil. Therefore, this study uses actualized data of the Brazilian disabled population according to age range, origin, and grade of self-perceived limitation and self-rated heath. It can serve as a guide to other middle-income countries with similar demographic characteristics to plan policies for disease prevention and health promotion.In closing, we believe that this study has filled a gap in the investigations regarding the SRH status of impaired populations, according to type, origin, and degree of limitation. This type of research is still in infancy; however, it is highly relevant in the current domestic as well global scenarios because it addressed a crucial theme associated with health policies of nations.Moreover, as demographic profile worldwide have been changing, disabilities and impairments have been increasing. Therefore, population-based studies are necessary because they facilitate simultaneous evaluation of health scenarios of specific countries, and enable comparison of different populations worldwide. Furthermore, the data were given a weighted analysis and yielded results that can be extrapolated to the entire Brazilian population.Additionally, information about the impairments was obtained through self-reports and depended on the personal understanding of each participant and their values, conception of health, and culture , 22. In In Brazil, physical and sensory impairments are associated with poor SRH, especially among adults below 60 years old. These association depend on the following factors: type of impairment ; whether congenital or acquired impairment; and whether impairment causes limitations. Among individuals with physical and visual impairments, acquired conditions that result in limitations were the strongest categories associated with poor SRH for both age categories—that is, below and above 60 years old. Regarding hearing impairment, associations differ: congenital without limitations presented as a protective factor to poor SRH; acquired—whether with or without limitation—presented as a risk factor. Our results suggest the significance of stratifying impairments to better understand their dynamic influence on SRH.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


Recently, increasing light has been shed on the NMD–virus interaction. This review summarizes the current scenario of NMD-mediated viral RNA degradation and classifies various molecular means by which viruses compromise the NMD-mediated antiviral defense for better infection in their hosts.The virus–host interaction is dynamic and evolutionary. Viruses have to fight with hosts to establish successful infection. Eukaryotic hosts are equipped with multiple defenses against incoming viruses. One of the host antiviral defenses is the nonsense-mediated mRNA decay (NMD), an evolutionarily conserved mechanism for RNA quality control in eukaryotic cells. NMD ensures the accuracy of mRNA translation by removing the abnormal mRNAs harboring pre-matured stop codons. Many RNA viruses have a genome that contains internal stop codon(s) (iTC). Akin to the premature termination codon in aberrant RNA transcripts, the presence of iTC would activate NMD to degrade iTC-containing viral genomes. A couple of viruses have been reported to be sensitive to the NMD-mediated antiviral defense, while some viruses have evolved with specific  Saccharomyces cerevisiae, Drosophila melanogaster, Arabidopsis thaliana, and humans . On. On18]. Drosophila melanogaster, Caenorhabditis elegans, Tetrahymena thermophile, Schizosaccarhomyces pombe, and so on ,6047,60]features ,62,63. Ahost NMD ,65. Stil viruses . Here, U viruses . In addi for NMD .Caulimoviridae possessing a circular double-stranded DNA genome. The virus replicates via reverse transcription of 35S pre-genomic RNA under the control of the transactivator protein (TAV) . Re. ReCoronThe genome of (+) RNA viruses is the template for translation to produce viral proteins, but it also is recruited into viral replicating compartments as the template for viral RNA replication. Thus, there must be a competition for viral gRNA between viral RNA translation and replication. NMD is dependent on RNA translation. Balistreri and colleagues found that the knockdown of NMD proteins  markedly increased the concentration of SFV RNA and proteins, and the mutation or deletion of viral replicase enhanced NMD response . Accordirotavirus circumvented the NMD pathway in a strain-independent manner [Recent evidence reported that non-structural protein from t manner . Viral nt manner .trans-acting viral proteins, viruses have evolved to counteract the NMD-mediated antiviral defense via cis-acting RNA elements. These RNA elements include some RNA structures involved in viral non-canonical RNA translation, such as leaky scanning, readthrough, frameshifting and reinitiating, and some specialized RNA sequences engaged in RNA: protein interaction to restrict UPF1 binding [cis-acting RNA elements directly interact with core NMD components to overwhelm RNA decay [Besides  binding . HoweverNA decay ,94,95.cis-acting element called RNA stability element (RSE) protects the RSV gRNA from host NMD. RSE restricts UPF1 to bind eRF3 by recruiting polypyrimidine tract binding protein 1 (PTBP1) [pol and src may also function as stability elements. Increasing the stability of viral RNAs in cytoplasm also helps vaccinia virus and other poxviruses to overcome RNA decay [Unspliced full-length RSV RNA is entirely resistant to NMD , but the (PTBP1) ,62. MutaNA decay . SimilarNA decay . PTBP1 rN. benthamiana [TCV has a (+) single-stranded gRNA, which can be used as a template to express p33 and its readthrough product p94, which are viral replicases. TCV transcribes to create two sgRNAs that are translated to produce viral CP and MP proteins. Both gRNA or sgRNAs of TCV are resistant to NMD in thamiana . A 51 ntMoloney murine leukemia virus (MoMLV) and Colorado tick fever virus (CTFV) protect sensitive transcripts from NMD [Readthrough elements from from NMD ,99. The from NMD . Insertifrom NMD . Furtherfrom NMD .Simian immunodeficiency virus, and WNV [Ribosomal frameshifting is the translation process where translating ribosomes move forward or step back by one or two nucleotides to escape in-frame stop codon, allowing ribosomes to translate continuously to produce a large fusion protein. Viruses commonly use programmed -1 ribosomal frameshifting to produce viral replicase. -1 frameshifting requires a slippery sequence just upstream of the in-frame stop codon and a structured RNA element several bases downstream of the slippery sequence. Ribosomal frameshifting has been demonstrated to be a means to increase the stability of PEMV2 RNA and its reporter transcript [ and WNV , which aAlternative splicing is tightly controlled on HIV-1 genomic RNA when generating multiple mRNAs from single pre-mRNA species. Although HIV-1 viral RNA possesses NMD-inducing features, it can evade NMD by hijacking UPF1 ,101. UPFcis- and trans-arm-based strategies to attenuate host antiviral immunity and facilitate viral replication [The ample scenarios of the host NMD–virus interaction have been rooted out to a substantial measure. The diverse families of viruses evolve multifarious tactics to protect viral genomes from RNA decay and antiviral silencing during the infection cycle. Evolutionarily, to cope with host antiviral actions, (+) RNA viruses and retroviruses deploy lication . AlthougHerpesvirus RNAs to NMD. Whether DNA viruses encode anti-NMD factors or have any specific RNA sequence or structure that could resist NMD remains unsolved. On the other hand, questions can be raised that most (+) RNA viruses can potentially be an NMD target, but a significant number are still undiscovered. All (+) RNA viruses reported to be NMD targets have a monopartite viral genome that contains one or more iTC. Quite a portion of plant viruses is segmented in their genome, such as the family of bromoviridea that has a tripartite RNA genome. It would be interesting to investigate whether segmented viral genomes have the potential for NMD? Moreover, little is known about viruses whose genome is double-stranded RNA. Thus, numerous efforts will be required to shed light on taxonomically different viruses to create a whole picture of the NMD–virus molecular interaction.Regarding DNA viruses that previously seemed unlikely to be associated with NMD, recent evidence shows that EBV and KSHV vigorously interact with host NMD components ,72. LongNMD is a translation-dependent pathway. Alternative translation initiation factors such as VPg, 3′ cap-independent translation enhancer (CITE), and an internal ribosomal entry site (IRES) employed by viruses in various circumstances inside host cells may potentially impact the modulating or reshaping decay pathway, which have been overlooked till now. Viruses deploy virally induced cellular microenvironment to avoid the antiviral response . What is


In recent years, several functions of different RNA molecules have been identified, as well as the ability of these molecules to regulate multiple processes in the cell, allowing the identification of RNA molecules as a large class of promising novel drug targets. Moreover, some small RNA molecules were recently approved in therapy or are being developed, because of their efficacy in treating diseases untreatable with classic drugs that target proteins; such as diseases caused by alternative spicing. Besides, small RNAs have the possibility to rapidly personalize treatments. Since 1998, eight Antisense oligonucleotides (ASO), four small interfering RNA (siRNA), one aptamer, and two messenger RNA (mRNA) vaccines were approved by the U.S. Food and Drug Administration (FDA) . These dTo analyse available experimental and computational methods and recent developments in the therapeutic RNA field, we have collected five contributions to the present Research Topic: Three original research articles and two review articles.Despite great advances, ranging from surgery to pharmaceutical sciences, it remains a challenge both to understand the development mechanisms of cancer and to identify specific and successful treatments for the disease. Four out of the five contributions to this Research Topic explore cancer from different perspectives, all of them focussing on the crucial role of RNA interactions at several levels.Yi and Yu discusses the regulation of Solute Carrier (SLC) superfamily transporters expression by microRNA molecules (miRNAs). This transporter family is responsible for nutrients and drug-dysregulated transport in cancer cells. Moreover, several miRNAs were found to regulate SLC expression. For these reasons, miRNA-mediated regulation of SLC transporters in cancer therapy can override chemoresistance and/or help therapies to prevent cancer cells from “feeding.”The review proposed by Yu et al. report the development of the Prediction of SM-miRNA Regulation pairs (PSRR) web server. The inhibition of miRNA by small molecules can be useful to regulate cellular processes acting on a single target. miRNAs can be targeted by small molecules. To identify miRNA targets for cancer therapies, the authors implement a web server that predicts the regulation relations between miRNAs and small molecules. Different predicting algorithms were implemented, but the random forest returns the best performances.Esposito et al. group describes a modified cell-SELEX approach for the isolation of RNA aptamers specific for hypoxia-related epitopes expressed on breast cancer cell surfaces. Owing to the central role of hypoxia in tumor progression, treatment failure, and negative prognosis, the authors developed a “Hypoxic cell-SELEX” methodology that relies on a simple and rapid system to reproduce in vitro the hypoxic conditions present in the tumor micro-environment. In this context, they report the identification and the biophysical characterization of the most represented aptamer with improved binding affinity for the hypoxic phenotype.The work of Cheng et al. propose an innovative nanobubble complex to be used in the sonodynamic treatment of Hepatocellular carcinoma. The treatment induces cellular apoptosis. The authors also evaluate the differentially expressed RNA  before and after treatment, and a subset of differentially expressed genes as potential targets for HCC treatment.ad hoc computational modeling pipelines for RNA structures have enabled the growth of RNA-based therapy. In this context, Mollica et al. summarize the recent advances and applications of therapeutic RNAs, and the structural viewpoint, the available strategies, and the computational and experimental analysis methods with a focus on dynamic and flexibility aspects and binding analysis.Finally, the recent advancements in experimental resolution and the Overall, the contributions highlight the interest in focussing on RNA both as a target and as a treatment, and the possibility of combining experimental and computational methods to increase the efficacy of the analysis. Besides, we believe that the study of RNA structures in conjunction with a biological and pharmacological characterization of RNA interactors and interactions could be an asset for future development in the field. The RNA structure is still not often considered in the design of new therapies, but some recent research emphasizes this aspect . We beliWe thank all Authors and Reviewers for their contributions to this Research Topic and we acknowledge the Frontiers in Molecular Biosciences Team for its support.


How can we study RNA that is in the process of being synthesized? For over half a century, researchers have developed diverse approaches to isolate and sequence nascent RNA and reveal layers of transcriptional control that are invisible when capturing only mature RNA . For exaHistorically, there are three major classes of nascent RNA-seq protocols. First, there are approaches that rely on nuclear run-on assays, such as global run-on-sequencing (GRO-seq) . During The Plant Cell, Min Liu and colleagues (In this issue of lleagues  systematlleagues ..The authors first tested reproducibility and sensitivity. Each method was highly reproducible within replicates. Across methods, GRO, pNET, and ChrNET were particularly well correlated, indicating that these methods may be more accurate at measuring transcriptional activity. pNET and ChrNET were also more sensitive and could identify the most actively transcribed genes, with the genes identified by ChrNET being the most reproducible. Similarly, for detecting long noncoding RNA, pNET and ChrNET were the most sensitive, but other methods rivaled these approaches in identifying active miRNA genes. Overall, ChrNET consistently matched or rose above competing methods for detecting nascent RNA and is cost-effective with reduced reads from rRNA and plastid RNA.For RNA Pol II stalling, all methods can detect RNA Pol II position except for CB RNA-seq, where RNA is fragmented before library preparation. The 5′ stalling detection was largely repeatable among methods, but 3′ stalling was less correlated, although pNET, ChrNET, and GRO still show positive correlations. However, it should be noted that pNET, ChrNET, and 3′CB might detect some terminating or arrested RNA Pol II, while GRO might detect more paused polymerases that have actively transcribed during the run-on assay.Finally, pNET and 3′CB could detect co-transcriptional splicing intermediates, with 3′CB showing particularly high sensitivity and accuracy. 3′CB revealed nearly double the number of predicted 5′ splice sites compared to pNET. Over 85% of these sites came from annotated sites. CB RNA-seq was also useful to identify candidates for recursive splicing, where two consecutive splicing steps are required to completely remove an intron. Overall, 3′CB and CB RNA-seq were the most effective tools to investigate splicing intermediates and splicing efficiency, respectively.When studying nascent RNA, there are numerous approaches to choose from, and each of these approaches shines in unique areas. Not all methods were tested here, although the authors provide some additional comparisons of other popular approaches by using published datasets. In summary, the in-depth trials and explanations provided offer useful advice when selecting an approach for nascent RNA sequencing.


This report aims to investigate a case of canine cutaneous melanoma with generalized metastasis and an unusual parietal bone metastasis, never reported before to the authors’ knowledge. The primary tumour was located on the skin of the carpal region and tumoral cells migrated towards the regional lymph node, reaching the general circulation through the blood and lymphatic streams. The histopathological examination showed a mixture of spindle, epithelioid and dendritic melanocytes, with variations in terms of pigmentation, a mitotic count of 10  for the primary tumour and higher for the metastasis. Immunohistochemistry was used to screen the expression of matrix metalloproteinases −2, −10 and the vascular endothelial growth factor (VEGF), all considered important invasiveness factors that facilitate the modelling of the extracellular matrix to enable invasiveness and metastasis. The results showed a strong positive reaction for VEGF and MMP-10, and a moderate positivity for MMP-2. This report suggests that metastasis in canine cutaneous melanomas may be present in various locations, such as the parietal bone, and is facilitated not only by the migratory abilities of melanocytes, but also by invasiveness factor expression, such as matrix metalloproteinases, MMP-2 and MMP-10, or VEGF.Melanocytic tumour anatomic location is considered an important prognostic indicator. The cutaneous forms are generally considered benign and may show various biological behaviours. This work reports a rare case of canine cutaneous melanoma showing parietal bone metastasis. Bone invasion in melanocytic tumours is often described in oral or visceral melanomas, but not in cutaneous forms. The patient  was initially presented for the surgical removal of a cutaneous tumour located on the skin of the carpal region of the right forelimb. Four months after, the patient returned with enlarged lymph nodes and acute respiratory failure. The patient was euthanized due to a decline in physical condition. The necropsy showed metastases in the affected forelimb, regional lymph node, splanchnic organs, parietal bone and meninges. Histopathological examination of tumour tissue samples revealed a mixture of pigmented and non-pigmented spindle and epithelioid melanocytes, while according to immunohistochemistry, the tumours showed a strong immunopositivity for VEGF and MMP-10, and a moderate positivity for MMP-2 expression. This case shows that cutaneous melanocytic tumours may show an aggressive malignant form with positive immunohistochemical reactions for multiple invasiveness factors. Melanocytic tumours originate primarily from melanocytes, cells dispersed within the basal layer of the epidermis, dermis, mucosae and eye structures ,2.In animals, the incidence of melanocytic tumours is higher for melanomas (76.9%) compared to melanocytomas 23.1%), while in dogs the occurrence represents 7% of all malignant neoplasms 3.1%, whi,5. MoreoCutaneous melanocytic tumours are generally considered benign neoplasms that can be treated with surgical excision. However, highly malignant forms have been described which in turn may be explained by the migratory nature of melanocytes and partly by the release of factors that act on the extracellular matrix degradation, allowing for invasiveness and metastasis ,7. MelanBesides the migratory features of melanocytes, the invasiveness of melanomas can be facilitated by other factors as well, such as enzymes that change the permeability of the extracellular matrix to allow for easy cellular infiltration. Matrix metalloproteinases are enzymes specialized in degrading the extracellular matrix components and basal membranes, facilitating the advance of the tumoral cells through the healthy tissue. They are classified into several types: collagenases , stromelysins , matrilysins , metallo-elastase , membrane-type MMPs , gelatinases  and others  [All these factors have been demonstrated to be expressed by different types of tumours, and it was suggested that they contribute to the reorganization of the extracellular matrix in order to facilitate invasion and metastasis. The purpose of this work was to investigate the expression of MMP-2, MMP-10 and VEGF in a case of canine cutaneous melanoma with accelerated progression. Moreover, to our knowledge, this is the first report of a canine cutaneous melanoma resulting in metastasis in the parietal bone.The patient  was initially presented for the surgical removal of a cutaneous tumour, which, after histopathological examination, was diagnosed as a melanoma. The primary tumour was located on the skin of the carpal region of the right forelimb, presenting a nodular shape and a diameter of about 2 cm, pigmented and well-circumscribed, with a calculated mitotic count of 10. Four months later, the patient returned with enlarged lymph nodes and acute respiratory failure. Euthanasia was recommended and performed using the following protocol based on the recommendations of the World Society for the Protection of Animals and the American Veterinary Medicine Association and agreed upon by the owner [Necropsy was performed and tissue samples were collected for histopathological examination, fixed in 10% formalin solution and processed using the paraffin embedding method as follows: embedding in paraffin using a Leica TP 1020 automatic tissue processor , Slee MPS paraffin embedding centre and a cold plate . Sectioning was done at 3 µm using a Slee Cut 5062 microtome. The slides were stained using the Masson trichrome staining method and mounted using a clear mounting medium.Further, the slides were evaluated using a Leica DM750 optical microscope equipped with a Leica ICC50HD camera . The mitotic count was calculated by analysing 10 random high-power fields = microscopic fields with magnification power ×400 (HPF).2O2 solution heated at 60 °C, for 60 min (to eliminate the melanin which would interfere with the diaminobenzidine (DAB) staining), treatment for the inactivation of peroxidase in a H2O2—methanol solution for 20 min, hydration with three successive alcohol baths at 90 °C, 80 °C and 50 °C, and distilled water for 5 min each. The proteolytic treatment was performed in a pH 6.00 citrate buffer. Following this treatment, two phosphate-buffered saline (PBS) pH 7.4 washes (5′ each) and serum blocking for 20 min were performed. The primary antibodies, anti-VEGF , anti-MMP-10  and anti-MMP-2  were applied overnight at 4 °C. The next day, the formalin-fixed paraffin-embedded (FFPE) sections were washed twice in PBS (5′ each), and then incubated with the secondary antibody at room temperature for 30 min. To visualize the specific immunoreactivity, the FFPE sections were incubated with diaminobenzidine (DAB) and then counterstained with haematoxylin for 5 min. Next, the slides were dehydrated , cleared (two successive baths in xylene for 15 min each) and mounted with a clear mounting media. Negative control sections were also performed, where primary antibodies were omitted and replaced with phosphate-buffered saline instead. MMP-2, -10 and VEGF immunoreactivity were scored as previously described [Immunohistochemistry was performed on paraffin-embedded tissue samples using the detection kit, Novolink Max Polymer Detection system (Leica). The immunohistochemical protocol was described by authors in a previous study  and sligescribed : negativNecropsy showed metastases had formed in the carpal and metacarpal regions of the affected limb, the suprascapular lymph node, which was enlarged, the internal lymph nodes, kidneys a, lungs The immunohistochemistry showed a strong positive reaction for VEGF a and MMPThe evolution of the neoplastic disease in the next 4 months was rapid, although studies report that cutaneous melanocytic tumours rarely result in distal metastasis and are more likely to affect only the regional lymph nodes [In this case, the metastasis was generalized, of different sizes and shapes and intensely pigmented from a macroscopical point of view. The suprascapular lymph node of the right side was transformed into a tumoral mass, with tumoral emboli inside the lymphatic vessels and sinuses indicating a lymphatic pathway. This is considered the most common path for the formation of metastasis, since tumoral cells that access the extracellular matrix, beneath the basal membrane, will have access to the lymphatic vessels .The metastases were located on all serosa and were infiltrative, affecting the underlying organs. In one study, serosal metastases are mentioned as a frequent finding in melanotic disease . In the The cardiac metastases were located perivascularly, with infiltrative areas that followed the connective stroma of the myocardium. The same perivascular pattern of melanotic metastases was also seen in the renal parenchyma, suggesting perivascular migration. Tumoral emboli were observed both in the renal blood vessels and inside the glomeruli, accounting for the circulatory pathway. All four paths were considered  as possible mechanisms for the formation of metastases .Although the pigmentation degree was similar in the primary and secondary tumours from a macroscopical point of view, histologically, it was possible to observe that the metastases contained areas with tumoral cells that were either less pigmented or amelanotic. A similar pattern was described by Smith et al. in 2002 .Pulmonary metastasis was considered common, given the fact that this location is one of the most frequent sites for secondary tumours for cutaneous melanomas .The most surprising metastatic site was the parietal bone and to our knowledge, this is the first report of a canine cutaneous melanoma metastasizing in this location. The melanocytes were infiltrating the meninges from within the bone. Osseous metastasis is most commonly found in oral melanomas and affects the mandible or maxillary bones. However, some studies mention this location both in humans ,17 and dThe immunohistochemistry showed a strong positive reaction for VEGF, suggesting an angiogenic phenotype, which could be an indicator of rapid growth, but also easy future metastasis . InteresAlthough cutaneous melanocytic tumours are usually considered benign because of their localization, the highly malignant forms can show invasive behaviour, with the contribution of various factors that change the characteristics of the extracellular matrix, such as matrix metalloproteinases. These enzymes can allow for a rapid dispersion of tumoral cells, even inside bones that have no physical contact with the primary tumour. This process may follow any of the known metastasis pathways , which means that even the sampling of sentinel lymph nodes that shows negative results may not be sufficient for a positive prognosis. Moreover, the release of factors  that stimulate neoangiogenesis may contribute to and accelerate the formation of tumoral emboli.


UNC-18, is mostly restricted to the nervous system of the nematode Caenorhabditis elegans, where it is important for synaptic transmission. Moreover, unc-18 null mutants do not display the lethality phenotype associated with (a) loss of all Drosophila and mouse orthologs of unc-18 and (b) with complete elimination of synaptic transmission in C. elegans. We investigated whether a previously uncharacterized unc-18 paralog, which we named uncp-18, may be able to explain the restricted expression and limited phenotypes of unc-18 null mutants. A reporter allele shows ubiquitous expression of uncp-18. Analysis of uncp-18 null mutants, unc-18 and uncp-18 double null mutants, as well as overexpression of uncp-18 in an unc-18 null mutant background, shows that these 2 genes can functionally compensate for one another and are redundantly required for embryonic viability. Our results indicate that the synaptic transmission defects of unc-18 null mutants cannot necessarily be interpreted as constituting a null phenotype for SM protein function at the synapse.Sec1/Munc18 (SM) proteins are important regulators of SNARE complex assembly during exocytosis throughout all major animal tissue types. However, expression of a founding member of the SM family,  BrieflyA total of 20 late L4 hermaphrodite from an unstarved plate were picked to individual fresh plate and were incubated for 2 days at 20°C. These plates were then scored, and all the hatched larvae were removed until the individual worms were stop laying progeny. The same protocol was used to determine the hatching percentage; the total number of larvae were compared to the number unhatched eggs.unc-18; uncp-18 double mutants, 4 individual youn adult stage unc-18; uncp-18/+ hermaphrodites were picked to individual fresh plate. After a few hours at 20°C, about 16 fresh eggs were isolated from each plate and followed during the next days to determine whether they hatched into viable larvae.To score embryonic viability of Worms were anesthetized using 100 mM of sodium azide and mounted on 5% agarose on glass slides. All images were acquired using a Zeiss confocal microscope (LSM 980). Image reconstructions were performed using Zen software tools. Maximum intensity projections of representative images were shown.UNC-18 protein sequences show close sequence similarity with a previously uncharacterized and unnamed gene, T07A9.10. Protein sequence comparison of T07A9.10 and other members of the SM family of proteins reveals that T07A9.10 clusters together with UNC-18, the DrosophilaUNC-18 homolog Rop, and their vertebrate orthologs STXBP1 (Munc18-1), STXBP2 (Munc18-2), and STXBP3 (Munc18-3). SM protMunc18-3), clusterMunc18-3). Phylogeparalogs .unc-18 ortholog STXBP1 are among the most frequent causes of epilepsy and encephalopathy  on protein stability and function have been investigated in detail  animals show GFP expression in an apparently ubiquitous pattern in all cell types  animals show GFP expression throughout the nervous and the intestine, as previously reported with a fosmid-based reporter  the unc-18 null phenotype (locomotory defects and synaptic transmission defects) is the result of lowering of the SM protein dosage (UNC-18 + UNCP-18), rather than a complete loss of SM protein function in the nervous system, or, (b), unc-18 and uncp-18 may have different biochemical functions at the synapse, but overexpression of UNCP-18 is able to substitute for the biochemical activity that UNC-18 normally fulfills.We next asked whether the locomotory defects of  rescued . Since uunc-18/uncp-18 function, we generated unc-18(e81); uncp-18(syb6377) double null mutant animals. Our initial expectation was that such animals would arrest development at the first larval stage, like other synaptic transmission mutants (unc-18(e81) and heterozygous for uncp-18(syb6377) are fully viable, we were unable to identify double homozygous viable larval or adult offspring. We carefully followed the progeny of individual unc-18(e81); uncp-18(syb6377)/+ animals and found that these animals produce about 25% inviable offspring (unc-18; uncp-18 double null mutant embryos. The morphology of inviable embryos appears highly disorganized (To further investigate the possibility of redundant  mutants . Howeverffspring , indicatrganized . We viderganized . We haverganized . Other SUNC-18 and UNCP-18 operate synergistically during embryonic development. Given this embryonic synergy, it appears conceivable that UNC-18 and UNCP-18 may also have redundant functions at the synapse and, therefore, that the role of SM proteins at the C. elegans synapse was previously underappreciated.We conclude that iyad180_Supplementary_DataClick here for additional data file.


Cytoprotective heme oxygenases derivatize heme to generate carbon monoxide, ferrous iron, and isomeric biliverdins, followed by rapid NAD(P)H-dependent biliverdin reduction to the antioxidant bilirubin. Recent studies have implicated biliverdin IXβ reductase (BLVRB) in a redox-regulated mechanism of hematopoietic lineage fate restricted to megakaryocyte and erythroid development, a function distinct and non-overlapping from the BLVRA (biliverdin IXα reductase) homologue. In this review, we focus on recent progress in BLVRB biochemistry and genetics, highlighting human, murine, and cell-based studies that position BLVRB-regulated redox function (or ROS accumulation) as a developmentally tuned trigger that governs megakaryocyte/erythroid lineage fate arising from hematopoietic stem cells. BLVRB crystallographic and thermodynamic studies have elucidated critical determinants of substrate utilization, redox coupling and cytoprotection, and have established that inhibitors and substrates bind within the single-Rossmann fold. These advances provide unique opportunities for the development of BLVRB-selective redox inhibitors as novel cellular targets that retain potential for therapeutic applicability in hematopoietic (and other) disorders. HemogloCYB5A) [Although erythrocytes express BLVRB as a non-physiological methemoglobin reductase , subsequCYB5A) ,29. ThisCYB5A) .Crystal structures of both BLVRA  and BLVR+-dependent short chain oxidoreductases [+’s 2′-phosphate group and the guanidinium side chains of Arg35 and Arg78, and a hydrogen bond to the hydroxyl of Thr12 [BLVRB’s overall structure is similar to that of the NAD(P)ductases . NADPH iof Thr12 . Ser111,of Thr12 , is at tof Thr12 .The reduction of substrates requires protonation and a hydride transfer, with protonation being the first step . While sUgt1a1 (UDP glucuronosyltransferase member A1) deficiency [Predicated on in vitro, human, and murine models ,32,39 (sficiency . Pre-clii) for FMN of ~0.7 μM (erythrosin extra bluish) and 1.8 μM (phloxine B) [The crystal structures for BLVRB in complex with various inhibitors have been solved ,32,40, aoxine B) . However+/NADH ratio is also controlled by glycolytic and mitochondrial activities that are dynamically regulated during differentiation or reprogramming [+/NADH redox state may also have a role in driving pluripotent stem cell fate.The bioenergetic requirements of self-renewing, quiescent hematopoietic stem cells (HSC) within the hypoxic marrow niche are distinct from those during differentiation and lineage commitment ,43,44, cgramming ; thus, tlow, exhibit low mitochondrial potential (ΔΨμ), and are dependent on anaerobic glycolysis. This adapted phenotype contrasts with that of lineage commitment that results in a metabolically-active bioenergetic phenotype manifested by ROShigh subsets and the conversion to oxidative phosphorylation [FoxOs [HSCs are heterogeneous, with the subset selectivity regulated by environmental stressors such as inflammation  and aginrylation ,53. ROS rylation , althougrylation ,55, whicn [FoxOs  or cysten [FoxOs .Adaptive mechanisms for cellular antioxidant handling are temporally and differentially regulated during lineage speciation. Cumulative evidence further suggests that lineage bias is differentially regulated by metabolic adaptations and substrate(s) , chronolS111L) was genetically associated with both clonal and non-clonal disorders of thrombocytosis. As outlined above (+ (αIIBβ3) megakaryocytes. Furthermore, thrombopoietin-differentiated hematopoietic (CD34+) stem cells acquired the CD41+ phenotype primarily within the ROShigh subset, consistent with a requisite developmental ROS signal during terminal megakaryocytopoiesis [The first evidence linking BLVRB redox activity to hematopoietic cell fate originated from a large-scale genetic screen of cohorts with thrombocytosis (high platelet counts) , in whiced above , the criopoiesis ,63. Sincopoiesis , ROS acchox-dependent NADPH oxidase activity regulates ROS production and is required for megakaryocyte differentiation [FoxO family of transcription factors [111L redox mutation in humans, a limited cohort analysis demonstrated reciprocal effects on hemoglobin (progressive anemia) and platelet counts (exaggerated thrombocytosis) in S111LBLVRB subsets, suggesting pathogenetically relevant consequences in the modulation of human blood cell counts [ROS as a priming signal for accelerated hematopoietic differentiation has been previously proposed . Recent ntiation . Low int factors . The pre factors , there a factors ,65. Bili factors . Diverge factors ,67,68. Il counts .BLVRB-deficient pluripotent stem cells (PSCs) imputed potentially synergistic mechanisms for BLVRB metabolic functions, coupled to defective bioenergetics and glutamate TCA (tricarboxylic acid) entry [Studies using d) entry . Heme syd) entry . Thus, Td) entry ,70. DownBLVRB-deficient PSCs connects heme generation and degradation with anaplerotic entry into the TCA cycle. Evidence for shunted glutamine was demonstrated using isotopomeric tracings at the initial α-ketoglutarate TCA entry point. The shunted glutamine response presumably functions as an adaptive means to limit the heme generation, and results in glutamine-restricted defects in oxygen consumption under both basal and maximal respiration. Downstream effects would result in decreased heme-containing electron transport protein (ETC) proteins displaying acquired ETC defects, ROS accumulation, and functionally defective TCA cycle NAD+/NADH generation [Dysregulated TCA glutamine utilization in neration . Previouneration . These iBLVRB-deficient PSCs is a presumed adaptation either for bioenergetics or for redox homeostasis through the PPP. Interestingly, dichotomous results are evident in the presence of PPP inhibition, which is responsible not only for the generation of NADPH reducing equivalents, but also the ribose-5-phosphate that is required during riboneogenesis [The BLVRB-deficient glutamine bioenergetics defect was found to be associated with enhanced glucose utilization and was largely reversible using glucose as a substrate. Energy metabolism switches from glycolysis to oxidative phosphorylation with cellular differentiation, and the preferential utilization of glycolytic pathways is a common bioenergetic feature of PSCs despite inefficient ATP generation ,73. Thusogenesis . BLVRB-dogenesis ,76. DiffBlvrb at the intersection of ROS-generating and stress hematopoietic pathways, with a further suggestion for lineage effects on erythroid/megakaryocyte partitioning independent of other heme degradation pathway genes [Blvrb-deficient (−/−Blvrb) mice, including necropsy at 8-, 26-, and 52-weeks, revealed no organ pathology (including hemograms and chemistries) in either male or female mice. These results are consistent with the non-requisite functions of BLVRB in humans. In contrast, differences in hematopoietic recovery were evident in −/−Blvr exposed to 5FU (5-fluoruracil) stress, with evidence that −/−Blvr mice demonstrated > 2-fold exaggerated platelet counts at peak recovery (Day 14) compared to a control. The exaggerated rebound thrombocytosis in −/−Blvr mice was unaccompanied by exaggerated RPs% (reticulated platelets), suggesting that the thrombopoietic response was not due to enhanced proplatelet formation; bone marrow histology established that exaggerated thrombopoiesis was accompanied by a ~5-fold expansion of megakaryocytes [−/−Blvr mice at Day 28 excluded the long-term post-stress consequences on bone marrow (BM) function. Consistent with previous data using in-vitro-differentiated megakaryocytes, exaggerated (~3-fold) expansion of the bone marrow ROShighCD41+ fraction was evident in −/−Blvr compared to +/+Blvrb mice. These results  demonstocytosis , it is aocytosis . Thus, sopoiesis ,80.−/−Blvr-stressed mice displayed erythroid repopulation defects in recovering bone marrows and spleens. −/−Blvr spleens were smaller in size, and splenic histology was most striking for the relative loss of red pulp constituents (erythrocytes and macrophages), with general preservation of the white pulp (lymphoid). A defective erythroid repopulation was caused by oxidant mishandling as documented by the identification of a 4-hydroxynonenal (4-HNE) lipid peroxidation product, a marker of oxidative stress or redox imbalance [Blvrb as a dominant erythroid transition gene in single-cell RNA hematopoietic studies [Blvrb displayed coordinate expression with erythrocyte Rhd (Rhesus Blood group D) antigen and reciprocal expression with megakaryocyte platelet factor 4 (Pf4). No correlation was seen with myeloid myeloperoxidase (Mpo), most consistent with a bifurcating effect restricted to erythroid/megakaryocyte lineage speciation. Among heme degradation genes , only Blvrb displays temporally distinct expression patterns during megakaryocytopoiesis and erythropoiesis [Blvrb expression peaks during early MK development, and displays late and sustained induction during erythroid development. While the metabolic control of hematopoiesis and stem cell biology is an active area of investigation, no other redox-regulated protein with stress-associated divergent effects on megakaryocyte and erythroid fates has been described [In contrast to enhanced megakaryocytopoiesis, mbalance . These d studies ,82. Blvropoiesis ; thus, Bescribed . Furtherescribed .−/−Blvr mice display phenotypic features that mirror the hematopoietic defect(s) previously described in human [Hmox1 deficiency. These observations emphasize the critical role of an intact heme degradation pathway in both basal and stress hematopoiesis. Heme-containing proteins are present in the majority of eukaryotic cells and tissues, although erythrocyte heme encompasses ~80% of the organismal heme in mammals. Heme preponderance in erythrocytes presumably accounts for the hematopoiesis-restricted phenotype(s) demonstrable in murine models of heme degradation pathway deficiency [HMOX1 deficiency in humans limits firm genotype/phenotype conclusions [−/−Hmox1 mice demonstrates defective iron utilization, with the development of age-dependent anemia and progressive splenic fibrosis [−/−Hmox1 deficiency is also evident, and supported by phenotypic rescue of the hematopoietic defect using macrophage-restricted bone marrow transplantation [−/−Blvrb mice. While the phenotype of Hmox1−-deficient mice is well-established, additional information has been obtained using heterozygous Hmox-deficient models. Haploinsufficient +/−Hmox1 mice display a progressively restricted phenotype that is manifest by a disrupted response to acute stress in stem cells and progenitors [−/−Blvrb mice, although megakaryocytopoietic effects were not described in +/−Hmox1 mice [Hmox- or Blvrb-deficient mice are summarized in in human  and muriin human  Hmox1 deficiency ,86,87. Tclusions . In contantation ,89. No cgenitors , with ev−/−Blvra mice display baseline plasma oxidation differences that are different compared to wild-type controls. −/−Blvra plasma displayed (1) increased ratios of cholesterol ester hydroperoxides to cholesteryl esters (CE-OOH:CE), and (2) increased levels of α-tocopherol. Both phenotypic defects presumably result from the impaired production of the bilirubin IXα antioxidant. −/−Blvra mice also had evidence for increased red cell oxidative stress, as judged by defects in erythroid peroxiredoxin 2 activity [Blvra in stress hematopoiesis (if any) have not been described. Importantly, these collective data establish for the first time that the Hmox and Blvrb pathways are mutually exclusive stress pathways linked to the overlapping heme catabolic step (note that an intact Hmox/Blvra pathway cannot rescue the −/−Blvrb stress phenotype). These divergent and non-redundant pathways of heme degradation are consistent with the distinct and non-overlapping substrate specificities used for redox activity and reductase functions.Recently reported activity ; in geneSimilar to the metabolic requirements of lineage-committed hematopoietic cells, enhanced metabolic and mitochondrial activity is seen in actively proliferating cancer cells, evident by a redox imbalance and ROS accumulation that impacts the cellular viability . To survEvidence supporting the pathophysiological significance of BLVRB in cancer originates predominantly from unbiased proteomics and gene expression profiling. Higher BLVRB expression compared to normal tissues has been reported in numerous types of cancer, including esophageal carcinoma , acute lVarious studies focusing on the development of blood-based biomarkers have identified elevated BLVRB in the serum of cancer patients compared to healthy controls, including pancreatic ,111 and Blvrb deficiency that display comparable phenotypes of exaggerated proplatelet formation. Whether or not the phenotypic manifestations require an ancillary stress signal is suggested and should become more evident with future studies. Of relevance, murine models of Hmox deficiency highlight the importance of the heme degradation pathway in hematopoiesis. A number of parallel advances provide additional reinforcement for anticipated research progress in this area: (1) availability of BLVRB crystal structures, (2) scalable methods for isomeric BV generation and purification as BLVRB-restricted substrates, (3) availability of Blvrb-deficient mice, and (4) new progress on the development of BLVRB-selective redox inhibitors. Unlike BLVRA, which has a multidomain structure encompassing putative DNA binding and kinase domains [BLVRB maintains a historically enigmatic role in biology, variably characterized as a flavin reductase, methemoglobin reductase, or overlooked as a “fetal” biliverdin reductase based largely on the restricted pattern(s) of isomeric BV substrates found in early fetal development. Recent renewed interest in BLVRB and the heme degradation pathway provides unique opportunities for further research, predicated on (1) initial studies in humans in which a redox mutation was causally associated with enhanced platelet counts in two distinct human disorders of thrombocytosis, additionally supported by (2) subsequent models of murine  domains ,120,121,