Retinoblastomas are the most common primary ocular malignancies in the pediatric population. They are known to undergo metastasis to distant sites like the bone marrow, lymph nodes, and skeleton, but liver metastasis is rare. 18F-fluorodeoxyglucose positron emission tomography-computed tomography is an interesting imaging technique that allows for staging for disease metastasis and plays a crucial role in treatment planning and prognostication of retinoblastoma patients with suspected distant metastasis. The case report highlights this very fact by demonstrating the case of a one-year-old boy with retinoblastoma that had metastasized to the skull bones and even the liver. Retinoblastoma is the most common intraocular pediatric malignancy in the world, with the majority of the patients being under five years of age at the time of presentation. The five-year survival rates are 95% in developed countries, but retinoblastoma can be very lethal if left untreated. The tumor mostly remains limited to the ocular bulb, with distant metastasis being quite rare. In the event that distant metastasis does occur, it is mostly in the marrow, lymph nodes, or the cortical skeleton, with visceral metastasis being very uncommon -3. RetinA one-year-old boy  from a low socio-economic stratum, presented to the pediatric oncology outpatient clinic with complaints of multiple swellings in his scalp as noticed by the parents. The history was negative for head trauma. However, it was significant for a history of retinoblastoma of the left eye, which was diagnosed at the age of two months. The child had undergone an enucleation of the left eye, at a different hospital. Unfortunately, the parents did not have any documentation with them as to the exact nature of the procedure whether any other imaging had been done prior to the enucleation, or if any chemotherapy/radiotherapy had been given to the child. According to them, the child was completely healthy up to two months ago when he first noticed a small swelling on his scalp, which they did not take seriously at the time. However, when the child’s swelling kept on gradually increasing in size and was accompanied by the appearance of other new scalp swellings, they became concerned and decided to get the child evaluated by a local pediatrician, who then referred them to a tertiary-level hospital for further evaluation. Upon evaluating the patient, there was a high level of suspicion for skeletal metastasis being present given the propensity of retinoblastomas to metastasize to the skeleton. With this in mind, it was decided to do a whole-body 18F-fluorodeoxyglucose positron emission tomography-computed tomography (18F-FDG PET-CT) to evaluate the extent of metastatic disease for staging purposes. Unfortunately, the 18F-FDG PET-CT revealed multiple hypermetabolic lesions not only in the skull bones  but also in the liver  extension, and distant metastasis . The livRetinoblastomas are the most common primary ocular malignancy in children and tends to very rarely metastasize. However, when it does metastasize the prognosis is guarded for these patients. Hepatic metastasis is rare in retinoblastoma and portend a poor prognosis. This case report highlights the importance of PET-CT in detecting distant metastasis in patients with advanced stage disease and may very well be the first case report demonstrating the role of 18F-FDG PET-CT in a retinoblastoma patient with both hepatic and skeletal metastasis.


CORO1A) and its protein with clinical features of combined immunodeficiency characterized by T-cell lymphopenia ranging from the severe phenotype to the mild phenotype, recurrent infections, and lymphoproliferative disorders. However, the effects of CORO1A protein disruption on actin-dependent functions in primary cells have not been fully delineated.To date, fewer than 20 patients have been identified as having germline biallelic mutations in the coronin-1A gene (CORO1A gene in combination with a de novo heterozygous microdeletion of chromosome 16p11.2 and also to provide evidence of the pathogenicity of this gene mutation.We sought to characterize the underlying defects of actin-dependent cellular functions in a female patient with combined immunodeficiency caused by a novel missense variant in the To identify the genetic defect, next-generation sequencing followed by Sanger confirmation and array comparative genomic hybridization were performed. Western blot and quantitative PCR tests were used to assess the effects on the protein. Flow cytometry and live microscopy were performed to investigate cellular motility and immune cell counts and function.CORO1A hemizygous variant c.19C>T, p. A7C induces significant decreases in cellular levels of the CORO1A protein while leaving mRNA concentrations unaffected. The observed mutation resulted in impaired natural killer cell cytotoxicity and platelet calcium signaling. In addition, primary granulocytes and mesenchymal cells showed significant defects in motility.We demonstrated that the CORO1A gene as a key player in actin cytoskeleton dynamics and cell signaling. Our findings expand the clinical spectrum regarding CORO1A protein deficiency and confirm the importance of a personalized therapeutic approach for each patient.Collectively, we added new data about the  This list has recently been enriched with the syndrome caused by CORO1A biallelic mutations. Since the first patient with this TCORO1A knockout mice demonstrated that this molecule is an essential regulator of T-cells’ actin dynamics, homeostasis, and trafficking and that its absence leads to a calcium-signaling defect downstream from the TCR receptor and stunted T-lymphocyte egress from the thymus.The CORO1A gene is located on the 16p11.2 chromosome; it encodes eponymous protein belonging to the coronin family.,CORO1A has been investigated predominantly in lymphocytes, whereas in other hematopoietic cells—platelets, neutrophils, and macrophages—its role remains less well delineated. Recent studies in mouse models showed that the absence of CORO1A affects platelets’ spreading, aggregation, and thrombus formation,CORO1A gene and a de novo heterozygous microdeletion of the 16p11.2 chromosome, leading to combined immunodeficiency and underlying defects in cell motility and calcium signaling.Herein, we describe a female patient with a novel missense mutation in the A-E). Immunologic evaluation demonstrated low IgG levels, and intravenous immunoglobulin substitution was initiated. This reduced the number of respiratory tract infections but did not improve the patient’s skin disease. She was also diagnosed with autism spectrum disorder.The female patient was born to nonconsanguineous healthy parents. Her early physical and cognitive development was normal. Beginning at age 6 years, she experienced recurrent respiratory tract infections—including 3 cases of pneumonia-chronic polypoid sinusitis and severe chickenpox that required hospitalization. At age 12 years, she developed disseminated papillomatous lesions  and progressive ulcerating dermatitis on 1 foot and leg, which were resistant to systemic and topical antibiotics and steroids , A-E. Im+ cells, 0.69 × 109/L) with low mitogen responses, absence of CD4+CD45RA+ naive T-cells, normal CD4+ and CD8+ effector memory cells levels, and increased CD3/TCRab+CD4–CD8– cell levels (17.8%)  .Table II+ and CD8+ lymphocytes, as well as CD163+ and CD68+ cells (F). A tissue sample was negative for bacteria, fungi (culture), herpetic and rubella viruses (PCR), and Mycobacterium spp (including nontuberculous Mycobacteria and Mycobacterium leprae [PCR]).Immunohistologic analysis of the patient’s skin lesion biopsy specimen showed granulomas with central necrosis and cellular infiltrate comprising CD48+ cells , F. A tiC and D).In an attempt to treat the patient’s skin granuloma, she was given the anti–TNF-α preparation , with only slight improvement of the lesion , C and DCORO1A (NM_007074.3) variant c.19C>T, p.Arg7Cys, which was subsequently confirmed by using Sanger sequencing. The patient’s father was a heterozygous carrier of the variant. The patient’s mother’s DNA sequence was determined to be of the wild type (A). The mutation was not found in the dbSNP, HGMD, or gnomAD databases, and it was predicted to be damaging or probably damaging by most in silico algorithms . Further analysis revealed a 2-fold coverage decrease in the CORO1A and ALDOA genes (C). Copy number analysis of the patient’s DNA using an exon-level oligonucleotide array (array comparative genomic hybridization) revealed a decrease in probe intensity in the 576-kb area on the short arm of chromosome 16, indicating a large heterozygous deletion (arr[hg38] 16p11.2(29613185_30189223) × 1), which included the whole CORO1A gene, as well as 26 other genes . Array comparative genomic hybridization did not detect any deletions in the patient’s mother’s DNA, thus confirming the de novo origin.Next-generation sequencing using a custom gene panel revealed the novel hemizygous ild type , A. The OA genes , C. Copy2), fludarabine (150 mg/m2), melphalan (140 mg/m2), horse antithymocyte globulin (100 mg/kg), rituximab (200 mg), abatacept (10 mg/kg), tocilizumab (8 mg/kg), and bortezomib (1.3 mg/m2) without posttransplant graft-versus-host disease prophylaxis. Neutrophil engraftment was detected on day 17 after HSCT and platelets engraftment on day 20 after transplantation. Three months after HSCT, the patient developed acute grade 2 skin graft-versus-host disease, which responded fully to treatment with cyclosporine A. The cyclosporine A treatment was discontinued after 7 months. The patient is currently at 12 months after HSCT, without immunosuppression and with full donor chimerism and good immune function (B and E).At age 16 years, the patient underwent haploidentical hematopoietic stem cell transplantation (HSCT) with TCRαβ/CD19 depletion from her unaffected mother.function ; in addifunction , B and EA and B).We attempted to verify the pathologic effects of the detected gene variant on CORO1A expression and primary cellular functions. The patient's COROA1 mRNA level was comparable to that of a healthy control; however, Western blot of her peripheral mononuclear cells revealed complete absence of the CORO1A protein , A and BCORO1A is required for normal NK cells’ lytic function,C). The degranulation defect was moderate, as complete recovery of degranulation was observed after phytohemagglutinin/IL-2 stimulation .Given that D and E). Interestingly, the MSCs also showed defects in cell motility despite generally significantly lower expression of CORO1A compared with that shown by hematopoietic cells. Recent studies have also shown that CORO1A plays an important role in fibroblast movement, not only via direct interaction with actin but also through several other mechanisms.We then performed a modified wound-healing assay with primary mesenchymal stem cells (MSCs) obtained from the patient’s bone marrow. The patient’s cells closed the defect significantly more slowly, requiring approximately 40 hours versus approximately 20 hours for the control , D and EF). These data correspond with the described defects in mouse neutrophils deficient in CORO1A.We also sought to assess the effect of protein loss on a different type of cellular motility, such as that seen in granulocytes. To assess their motility, we visualized granulocytes’ activity during thrombus formation in a flow chamber.CORO1A mutation on platelet function. Calcium response to various stimuli was decreased in the patient’s platelets versus in those of normal controls; it was completely restored after HSCT  identified in our patient has been reported previously in 2 patients with hemizygous CORO1A mutations and the T−B+NK+ SCID phenotype with an early onset of symptoms, including severe life-threatening infections and EBV-associated diffuse large B-cell lymphoma.,CORO1A mutations can lead to the development of SCID, it is also clear that alternative, milder phenotypes can develop from such mutations.Our functional experiments provide evidence of the damaging nature of the novel CORO1A mutations in combination with chromosome 16 deletion, our patient also had behavioral impairment.,21940615, PMID 21731881, and PMID 19914906). Therefore, the underlying molecular mechanisms of these cognitive problems and their definitive association with CORO1A deficiency remain unclear and require further in-depth investigation.Like the 2 subjects who had The function of CORO1A in various cell types remains elusive. Our data indicate that the absence of CORO1A causes significant defects in platelet calcium signaling, degranulation defects in NK cells that are indicative of CORO1A role in synapse formation, and attenuated motility in primary neutrophils and mesenchymal stromal cells. These data suggest that CORO1A is crucial in actin cytoskeletal reorganization in most, if not all, hematopoietic cell lineages.Assessing the direct role of the CORO1A protein in cytoskeletal reorganization in various cells remains a challenge. Recent studies have discussed CORO1A as a drug target for the treatment of multiple sclerosis, B-lineage leukemia, and other diseases, as well as a potentially emendable pathway by which to achieve long-term graft acceptance without compromising immunity to infections.www.jaci-global.org.For detailed methods, please see the Methods section in this article’s Online Repository at Supported by a grant from the Foundation for Support and Development in the Field of Pediatric Hematology, Oncology and Immunology "Science for Сhildren" (Foundation "Science for Сhildren").•CORO1A defect significantly attenuates primary granulocytes’ mobility.•CORO1A-deficient NK cells display decreased cytolytic function.•CORO1A deficiency leads to a defect in primary mesenchymal stromal cells motility, possibly owing to abnormal cytoskeletal reorganization.Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest.


Schizosaccharomyces pombe and Homo sapiens transcriptome data. We anticipate DeepEdit to be a powerful tool for the study of RNA editing from a new perspective.Single-molecule detection and phasing of A-to-I RNA editing events remain an unresolved problem. Long-read and PCR-free nanopore native RNA sequencing offers a great opportunity for direct RNA editing detection. Here, we develop a neural network model, DeepEdit, that not only recognizes A-to-I editing events in single reads of Oxford Nanopore direct RNA sequencing, but also resolves the phasing of RNA editing events on transcripts. We illustrate the robustness of DeepEdit by applying it to The online version contains supplementary material available at 10.1186/s13059-023-02921-0. RNA editing is a universal and critical post-transcriptional event discovered in diverse life forms –3. The dSchizosaccharomyces pombe strains with de novo RNA editing machinery for nanopore direct RNA sequencing. S. pombe lacks the ADAR-mediated A-to-I RNA editing system, which provides us with a “clean” background to study changes of nanopore electrical signals caused by A-to-I RNA editing. We introduced the human adenosine deaminase acting on RNA type 2 (hADAR2) gene into S. pombe chromosome II, resulting in a strain capable of A-to-I RNA editing, namely FY-ADAR2  detection of A-to-I RNA editing events on in vivo transcribed RNAs, we constructed To determine whether the hADAR2 protein can edit RNA in yeast cells, total RNA was extracted from FY-ADAR2 and FY-HFF1 and sequenced with Illumina Novaseq. The Illumina reads were mapped to the reference transcriptome for analysis. For FY-ADAR2, 11,615 and 11,496 A-to-G change sites, and 967 and 930 T-to-C change sites were detected in replicate experiments Fig. ” sectionP value distribution for the comparison of the random “A” sites (random “A” sites in FY-ADAR2 vs “A” counterparts in FY-HFF1) is flat, the edited “A” sites (edited “A” sites in FY-ADAR2 vs “A” counterparts in FY-HFF1) have a significant difference at the edited sites and nearby bases  . As a comparison, the electrical signals of random “A” sites in FY-ADAR2 samples were tested against those of the corresponding sites in the FY-HFF1 sample, which was regarded as the background electrical noise between the two samples. While the To make it possible to detect the editing events on adenosine residues in single nanopore sequencing reads, we first separated the edited and unedited reads for each editing site before model training. We found the base-calling errors were associated with A-to-I editing. The signal shifts caused by A-to-I editings may hinder the resolution by base-callers due to the unexperienced electrical signal values . We obseBased on these findings, we separated the nanopore reads mapped on editing sites in FY-ADAR2 samples into two groups, A-to-I edited reads (I-type reads) and unedited reads (A-type reads), for subsequent model training  than those of unedited sites  spanned a long distance (~ 3 kb)  Fig. e. Among RNA editing plays important roles in multiple human diseases, including neurological disorders –28 and cA recent study generated a model (Dinopore) for the site-level prediction of RNA editing events with aggregate nanopore read profiles . Their mS. pombe .The hADAR2 coding sequences were amplified from cDNA of 293 T cells with primer hADAR2-P5 and hADAR2-P3   was used to generate derived strains by transformation with various plasmids. FY-HFF1 and FY-ADAR2 strains were generated by transformation using the Lithium Acetate/PEG/Heat shock method [k method . Plasmid2 h-leu1- ura4-D18S. pombe strains FY-ADAR2 and FY-HFF1 were plated in EMM + ura plate [600) of 0.1. Cells in the mid-log phase were harvested for DNA and RNA extraction.ra plate  and growTotal RNA from FY-HFF1 and FY-ADAR2 strains was extracted using TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions. RNA integrity (RIN) was assessed using an Agilent 2100 Bioanalyzer. mRNA was purified from total RNA using poly-T oligo, and sequencing libraries were generated using TruSeq RNA Library Preparation Kit . The libraries were sequenced on an Illumina Novaseq platform and 150 bp PE reads were generated .https://github.com/najoshi/sickle) was used to remove the low-quality reads with options -l 50 -n -q 30. The workflow for the identification of A-to-I RNA editing sites is as described [S. pombe (GCF_000002945.1_ASM294v2_rna) downloaded from NCBI using Hisat2 [The raw Illumina sequencing data were first trimmed from 5′ end using Fastx_trimmer (version 0.0.13) with options -f11 -Q33. Replicate reads were removed using FastUniq , and theg Hisat2   , and theThe Oxford Nanopore direct RNA sequencing was performed by Nextomics Bioscience . The sequencing libraries were constructed using ONT SQK-RNA001 kit following the manufacturer’s protocol (Oxford). The libraries were sequenced on GridION platform with R9.4 flowcells.https://github.com/nanopore-wgs-consortium/NA12878). The direct RNA sequencing library was built using ONT SQK-RNA001 kit and sequenced on MinION or GridION using ONT R9.4 flowcells [The nanopore direct RNA sequencing data on human cell line GM12878 were downloaded from GitHub  with options –flowcell FLO-MIN106 –kit SQK-RNA001 – qscore_filtering –fast5_out. We employed Tombo  . Some editing sites were excluded due to the low read coverage in these regions. A total of 2318 editing sites were obtained, and 2318 unedited “A” sites randomly selected were used as control.Sites with reads coverage ≥ 30 in both FY-HFF1 and FY-ADAR2 samples were retained. For each site, the norm_mean values of nanopore bases mapped on this site were extracted from FY-HFF1 and FY-ADAR2, and KS-test was performed between the two samples using the extracted signal values. We compared the To distinguish the random base-calling errors from base-calling errors due to RNA editing events (ADAR2-specific errors), we used the following criteria to select ADAR2-specific errors (ASE): (1) the SNV that owned the most support reads was selected as the base-calling error for each site; (2) the base-calling error occurred in FY-ADAR2 samples and not in FY-HFF1 samples, or the error ratio  in FY-ADAR2 samples are at least twice that in FY-HFF1 samples; and (3) at least 5 nanopore reads support a base-calling error with error ratio ≥ 0.1.We separated the nanopore reads mapped to editing sites in FY-ADAR2 samples that are ADAR2-specific errors (ASEs). The nanopore reads harboring ASEs around editing sites were annotated as I-type reads and the remaining reads were annotated as A-type reads  and unedited sites (editing ratio = 0), the sites with mapped nanopore reads less than 30 were abandoned. A nanopore read was considered to be edited when having a predicted probability ≥ 0.9. The editing ratio of each site was calculated by counting the number of total mapped reads and that of predicted edited reads. When comparing the overlap of the detected editing sites between studies, we collected two atlases of RNA editing sites that were reported by previously published studies and conducted comparisons with the sites detected by our approach. From the first study by Ramaswami et al. , a totalAdditional file 1:Fig. S1. Implementation of hADAR2 editing system in S. pombe. Fig. S2. Confirmation of candidate A-to-I RNA editing sites by sanger sequencing. Fig. S3. Base-calling errors can be used to identify I-type reads in direct Nanopore reads. Fig. S4. Performance of models with different feature selection. Fig. S5. Challenge of reads mapping in repetitive elements by the short-read sequencing. Fig. S6. Changes in potential hydrogen bond interactions between bases induced by A-to-I RNA editing events in transcript NM_002794.5.Additional file 2:Table S1. Number of ADAR2-specific errors (ASEs) around editing sites in S.pombe. Table S2. Number of ASEs around random ‘A’ sites in S.pombe. Table S3. Primers used in experiments. Table S4. Predicted editing levels of sites collected from Ramaswami et al. Table S5. Predicted editing levels of sites collected from Peng et al. Table S6. Detected editing sites in Alu regions of 10 human transcripts.Additional file 3. Review history.


In vitro run-off transcription assays revealed FUS-enhanced RNA produced by a non-eukaryote polymerase. The activity also reduced the formation of R-loops between RNA products and their DNA template. Analysis by domain mutation and deletion indicated RNA-binding was required for activity. We interpret that FUS binds and sequesters nascent transcripts to prevent R-loops from forming with nearby DNA. DRIP-seq analysis showed that a knockdown of FUS increased R-loop enrichment near expressed genes. Prevention of R-loops by FUS binding to nascent transcripts has the potential to affect transcription by any RNA polymerase, highlighting the broad impact FUS can have on RNA metabolism in cells and disease.The protein FUS (FUSed in sarcoma) is a metazoan RNA-binding protein that influences RNA production by all three nuclear polymerases. FUS also binds nascent transcripts, RNA processing factors, RNA polymerases, and transcription machinery. Here, we explored the role of FUS binding interactions for activity during transcription.  A prominent member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family is FUS (FUsed in Sarcoma). FUS is best known for gene mutations leading to amyotrophic lateral sclerosis (ALS) or pediatric sarcomas , 2. FUS FUS affinity for RNA involves an RNA-recognition motif, Zinc finger domain, and the three disordered RGG/RG domains , 13, 14.We set out to better define the role that binding the RNA polymerase has in FUS activity on transcription. We anticipated direct binding to the polymerase and RNA processing factors would be required for FUS activity , 6, 12. in vitro run-off transcription of a linear DNA template (A).In our previous work, we observed FUS at concentrations found in the cell, >2 μM, greatly increased RNA Pol II transcription from a naked DNA template . We asketemplate A.A). Two stained bands could be seen: a larger band that was confirmed by DNase I digestion to be a linear DNA template (B and C), and a smaller band confirmed by RNaseA digestion to be the RNA transcript (D). The DNA template detected offered a normalization control for quantitative analysis of PAGE images.Accumulated products of run-off transcription were observed by PAGE and SYBR staining A. Two sttemplate , B and Canscript D. The DNB and D and E). Titrating buffer constituents present in the assay eliminated these as potential sources of activity (F). Increasing concentrations of FUS during T7 Pol transcription increased RNA products observed by PAGE . Sufficient concentration of FUS produced a shift of RNA to the well, which was reversed by proteinase K treatment to eliminate FUS aggregates formed (H). FUS tendency to aggregate has also been noted by previous studies  . Robust binding of T7 Pol could not be provoked by the linear DNA template, or an RNA known to cause FUS to bind RNA Pol II  (We investigated whether a direct interaction of FUS and T7 Pol could be observed through co-immunoprecipitation (co-IP). The protocol used for co-IP was successful in detecting FUS binding to protein interactors, including RNA Pol II and III, in previous studies , 21. T7 Figs. 1and S1I) , 16. We A). Fluorescence-based quantification with an RNA-specific dye also detected no activity from BSA . We also noted FUS activity was diminished after storage at −80 °C, but stored at room temperature, FUS activity remained stable up to 4 weeks, then no activity was seen at 12 weeks (A). The lack of effect on transcription by inactive FUS added support to the conclusion that the activity observed was specific.We next considered whether high protein concentrations affected transcription by non-specific molecular crowding. Increases to T7 Pol activity by molecular crowding with glycerol or PEG has been observed by previous studies , 23. ActFig. 212 weeks A. The laB–D, lanes “-N” and “-T”). We also found FUS incubated with RNase was restored to activity by an RNase inhibitor, indicating a contaminating RNA was not required (B). We investigated two RNA molecules, a non-specific competitor RNA, yeast tRNA, and an RNA, TET456, previously used to induce FUS binding to RNA Pol II (C). Titrating tRNA or TET456 RNA concentrations had no effect on T7 Pol or FUS activity (C–E). A repeat of these tests with single-stranded DNA also found the activity of both proteins unaffected (F).We considered if RNA in solution might alter FUS activity. Such an effect by RNA or DNA in solution could make the activity of FUS observed during run-off transcription difficult or impossible to observe in a cell, where concentrations of nucleic acids are high. No evidence was found of contaminant bacterial RNA in transcription assays. The 260/280 nm absorbance ratio measured for FUS was <0.6, and no contaminating nucleic acid was seen by SYBR stain . A nuclease that cleaves RNA:DNA hybrids, RNaseH, could eliminate signals detected, confirming specificity for the S9.6 antibody in this assay (A).By dot blot assay using the S9.6 antibody, RNA:DNA hybrids were found to arise early during run-off transcription and their level increased over time A. A nuclis assay , 28. Addis assay A.Figure A and S3B). Increasing concentration of FUS resulted in greater reduction in RNA:DNA hybrids (B). Samples were heated then cooled to reveal that RNA transcribed in the presence of FUS retained their full capacity to form an RNA:DNA hybrid . Dot blot assays could detect up to a 2-fold reduction in RNA:DNA hybrids resulted from the presence of FUS (C and S4A).The addition of FUS (2 μM) reduced RNA:DNA hybrids observed with the largest difference at 20 to 40 min in the assay A and S3B hybrids B. SampleA hybrid B, anneale of FUS C and S4AA). Neither FUS-LC or Delta-LC produced a significant increase in RNA transcript or reduced RNA:DNA hybrids . The lack of activity for Delta-LC and FUS-LC indicated that both self-assembly and RNA-binding, respectively, are required for FUS activity on T7 Pol transcription.We sought to identify FUS domains required for activity on T7 Pol transcription. We investigated truncations of FUS protein that lacked either the LC domain (Delta-LC) or the C-terminal RNA-binding domains (FUS-LC) A. NeitheA). Compared to FUS affinity for RNA, characterization of these substitutions by previous studies found RS1 and RS2 maintained similar affinity, RS3 affinity was reduced by 7-fold, and RS4 affinity was reduced by >30-fold (C and D). RS3 increased T7 Pol transcripts by >twofold, but less than that for FUS. RNA:DNA hybrids were reduced by RS3 a small but significant amount . RS4 activity on transcript production matched that of RS1 and RS2, and a small reduction of hybrids could be detected (S4D).We focused on the role of RNA-binding for activity by substituting serine for arginine residues in one  or all RS4) of the RGG/RG domains in FUS A. Compar>30-fold . In the >30-fold C and S4D of the RE). RS4 particles were not produced by incubation of RS3 and RS4 at 10 μM (F). Lastly, FUS and RS4 were tested with protein concentrations up to 100 μM, incubation at room temperature or 4 °C, and addition of RNA, but RS4 did not yield unambiguous particles, while FUS achieved particles ≥1 μm in diameter (G and H).Finally, inspection by microscopy was performed to assess any self-assembled particles made by FUS or RS proteins. A necessary change from conditions in run-off transcription assays was for proteins to be allowed to incubate at room temperature for 24 h. FUS, RS1, RS2, and RS3 proteins (2 μM) all produced visible particles up to 0.5 μm in diameter, but RS4 yielded no evidence of assemblies E. RS4 paat 10 μM F. Lastlydiameter , G and HA) . Real-time PCR analysis confirmed that positive control genomic sites, RPL13A and TFPT, known to contain R-loops was enriched by S9.6 pulldown, relative to a negative control site, EGR1. No enrichment was observed for lysates incubated with RNaseH  .We hypothesized the activity observed for T7 Pol transcription would also be found in human cells. To test this in human HEK293T/17 cells, we used a siRNA approach to knockdown FUS expression and measured R-loop enrichment genome-wide by DRIP next-generation sequencing approach, DRIP-seq, that immunoprecipitates R-loops with the S9.6 antibody , 29, 30.Fig. S5ig. S5C) . AnalysiA). A 2-fold increase in enrichment was observed at 29% (N = 5260) of protein coding genes  (B). In HEK293T/17 cells, a gene previously found by ChIP-seq and CLIP-seq analysis to be enriched for FUS is GAPDH  (D)  B. In HEK9% N = 520 of prot samples C 6). Th. ThA). Aples (C) D 6, 11,,A. A 2-f (C) (D) , 31, 32.D). Also, peaks for R-loop enrichment were primarily in intergenic and satellite DNA in SCR-treated cells (N = 5045 peaks), and consistent with published ChIP-seq and CLIP-seq data, peaks in siFUS-treated cells were mostly in protein-coding genes and promoters (N = 7247 peaks)  . Finally, FUS has been shown to have activity on RNA Pol III transcription .While an increase in R-loops caused by FUS knockdown was predicted by our T7 run-off transcription studies, the localization of R-loops observed by DRIP-seq were consistent with previous studies of FUS activity in cells. FUS binding specificity to RNA is degenerate but does prefer GC-rich sequences , 14, 33.Fig. S5ig. S5E) . DRIP-secription . Inspectin vitro run-off transcription.In conclusion, the analysis of DRIP-seq indicated widespread FUS activity preventing R-loops in human HEK293T/17 cells. FUS activity on R-loops was not limited to transcription by RNA Pol I, II, or III. The changes to R-loop enrichment that could be measured by DRIP-seq revealed a close similarity of FUS activity in human cells and that observed during Here we explore FUS activity on transcription that involves the potential for RNA transcripts to bind their DNA template, forming an R-loop. Our study followed the unexpected result that FUS increased the RNA yield from T7 Pol transcription. Subsequent analysis determined FUS reduced RNA:DNA hybrids that formed during transcription. Our model is that FUS increases RNA yield by binding nascent transcripts to prevent or delay R-loop formation, which would slow or block the RNA polymerase. In human cells, a reduction of FUS protein increased R-loop abundance. The R-loop enrichment was most prominent at expressed genes, notably those transcribed by RNA Pol I and II. These results indicate FUS bound to nascent RNA transcripts can influence transcription by preventing R-loops.B). Inspection of individual RGG domain contributions did not conclude that one could be mostly responsible for activity. The RS3 protein retained some activity on the amount of transcript produced, but activity on R-loops was indistinguishable from the RS4 protein, which has a >20-fold higher KD for RNA and no activity on product amounts (C). On the one hand, binding affinity is measured in steady-state conditions and transcription is a dynamic system. Specifically, the difference in RNA production in the presence of FUS is seen over 2 h of run-off transcription, while the difference in RNA:DNA hybrids is undetectable beyond 40 min. This can indicate effects of non-equilibrium or combined states in transcription and FUS activity .Our run-off transcription results suggest that by preventing R-loops, FUS addresses an obstacle faced by any RNA polymerase during transcription. R-loops have many effects on transcription and chromatin stability besides the simple stalling of the polymerase that we have studied here . PreviouFig. 1Like FUS activity found in T7 Pol transcription, our DRIP-seq analysis finds endogenous FUS is also involved in preventing R-loops in the cell. At least two studies anticipated FUS would play a role in either prevention or repair of R-loops. First, a study found that DNA damage where FUS was recruited had characteristics of R-loop-induced damage, and damage resulting from a loss of FUS was partly reduced by overexpressed RNASEH1 , 43, 44.in vitro assay results, the simplest explanation for R-loops observed at sites transcribed by RNA Pol I, II, and III is an activity mediated by binding RNA rather than the polymerase (B).The R-loops found at so many RNA Pol II genes after FUS knockdown in HEK293T/17 cells mirrors the enrichment of FUS previously found at promoters for most expressed genes in this cell line , 21. As lymerase . Ordinarlymerase . Failurelymerase , 47. Prolymerase B.In conclusion, the ability of FUS to influence transcription can be extended to include preventing R-loops by binding nascent RNA transcripts. FUS and R-loops have complex relationships with transcription and DNA stability. The mechanism we describe would not conflict with those that involve binding the polymerase, transcription factors, or RNA-processing machinery. It is noteworthy that FUS and R-loops are contributors to the same neurodegenerative diseases , 48, 49.E. coli BL21(DE3) cells. All FUS constructs were N-terminally fused to 6× His and MBP to improve purification and solubility. Protein expression was induced after cell growth reached an OD600 of 0.8 and then allowed to continue overnight at 17 °C with shaking (200 rpm). Purification was made using 1 to 2 g of frozen pellets of induced E. coli, lysates incubated with Ni-NTA Sepharose beads , and eluted in FUS-SEC buffer  with 250 mM imidazole and either 1.5 mM β-mercaptoethanol or 1 mM DTT added  cells and purification were done essentially as previously published (Plasmids for protein expression of T7(P266L) polymerase fused with a 6xHis tag were provided by A. Berman (University of Pittsburgh) . Expressublished . ProteinSize-exclusion chromatography (SEC) was performed with a 10/300 Superdex 200 column  for FUS using FUS-SEC buffer and for T7 Pol with T7-SEC buffer . FUS protein was stored at room temperature for up to 4 weeks or until activity appeared lost. T7 Pol activity was assessed by titrating protein into transcription assays as described below. The absence of nuclease activity was confirmed by comparing plasmid DNA and transcribed RNA incubated overnight with or without protein and electrophoresis using a Novex TBE-urea, 6% polyacrylamide gel  and stained with SYBR-Gold .DNA substrates used in T7 transcription assays were prepared from pcDNA3 vector plasmid and the gene inserted for RNA transcription was Flag-FUS . Bsu36I 2) with 2 mM NTP’s  and 40 U/μl RNasin Plus . T7 (P266L) polymerase  and anti-FUS (4H11) antibody  were immobilized by incubating together for 1 h at room temperature in phosphate buffered saline   and 0.1 mg/ml bovine serum albumin protein , then 1 h with Pierce Protein A/G agarose beads . Beads were washed 4 times with 0.1% NP-40 substitute  in tris-buffered saline (TBS). Beads with FUS were incubated with T7 Pol for 1 h at room temperature with rotation, then washed twice in high salt, twice in TBS with 0.1% NP-40 substitute, and one with TBS. Elution of protein bound to beads was incubated at 95 °C for 10 minutes in 2× NuPAGE LDS sample loading buffer  containing 4% lithium dodecyl-sulfate (LDS) and DTT (100 mM) added. Nucleic acids used were prepared as described earlier and RNA:DNA hybrids were detected and quantified by dot blot assay. From transcription assays, 10% of reactions were spotted on N+ membranes , dried and UV crosslinked. Blots were blocked with 5% nonfat dried milk (NFDM) in TBS-T, then incubated overnight at 4 °C with an anti-RNA:DNA hybrid antibody S9.6 against RNA:DNA hybrids (1/2000 dilution) in TBS-T with 2.5% NFDM. Blots were washed 5 min 4 times in TBS-T, incubated in secondary antibody  for 1 h at RT. Images were taken after incubation with SuperSignal West Pico PLUS . Images were acquired with a Bio-Rad ChemiDoc MP Imaging System. Densitometry was performed with Bio-Rad Image Lab Software v. 6.0.1.6 cells were harvested 72 h post-siRNA treatment, and cell pellets were frozen in liquid nitrogen. Pellets were later resuspended in 1.6 ml of TE buffer  that was added to include 0.6% sodium dodecyl sulfate (SDS) and 60 μg/ml Proteinase K. After Proteinase K digested lysates overnight at 37 °C, DNA was extracted using the MaXtract High Density tubes . DNA was spooled by glass Pasteur pipette, transferred, then ethanol precipitated.DRIP-seq assays were performed essentially as a previously published protocol . HEK293TDNA was digested overnight in TE buffer with 1× NEBuffer 2 , BSA (95 μg/ml), spermidine (1 mM), and 30 U each of restriction enzymes BsrGl, EcoRI, HindIII, SspI, and XbaI. Restriction digest was confirmed by agarose (0.8% w/v) gel electrophoresis. After restriction enzyme digestions, DNA was extracted by Phase Lock Gel  per manufacturer instructions. Negative control samples were digested by 40U RNaseH  per 10 μg DNA. 50 μg of DNA was incubated overnight at 4 °C with 20 μg S9.6 antibody and in 500 μl DRIP buffer . 5 μg of DNA was kept for input samples. Antibody precipitated with 100 μl Protein A/G beads  was washed twice in 700 μl DRIP buffer. Hybrids were eluted by incubating beads with 300 μl DRIP Elution buffer  for 45 min at 55 °C, then extracted by Phase Lock Gel. Samples were sonicated for 30 cycles (30 s on/30 s off) follow by ethanol precipitation.https://www.ncbi.nlm.nih.gov/geo/).Sample libraries were prepared and sequenced by Novogene Corporation Inc using a NovoSeq6000 with 150 base paired-end reads (8 G raw data per sample). Reads were trimmed using TrimGalore (version 0.6.6) and aligned to GRCh38 using STAR (version_2.7.8a), essentially as previously described. Bam files were processed using the deepTools2.0 suite (version 3.5.1) to normalize to 1× depth (reads per genome coverage) with bamCoverage, merge replicates by bigwigCompare, and tags binned by computeMatrix, which was then used to compute scaling factors and generate heat maps and profiles. Merged bigwig files were converted to bedgraph and then peaks by Macs2 (version 2.1.1.20160309). HOMER (version v4.11.1) was used to calculate GC-content and annotate peaks. Data was visualized, and figures were generated using Integrated Genomics Viewer (version 2.5.0). DRIP-seq data is available (GSE206740) from the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/).DRIP-seq data is available (GSE206740) from the Gene Expression Omnibus (This article contains The authors declare that they have no conflicts of interest with the contents of this article.


The thermostability studies and ultrastructural imaging of protein-free viral RNA cores suggest that Na+ keeps the encapsulated genome more open, allowing PDS and PhenDC3 to diffuse into the quasi-crystalline RNA and promote the formation and/or stabilization of GQs; the resulting conformational changes impair RNA unraveling and release from the virion. Preliminary reports have been published.Rhinoviruses (RVs) are the major cause of common cold, a respiratory disease that generally takes a mild course. However, occasionally, RV infection can lead to serious complications in patients debilitated by other ailments, e.g., asthma. Colds are a huge socioeconomic burden as neither vaccines nor other treatments are available. The many existing drug candidates either stabilize the capsid or inhibit the viral RNA polymerase, the viral proteinases, or the functions of other non-structural viral proteins; however, none has been approved by the FDA. Focusing on the genomic RNA as a possible target for antivirals, we asked whether stabilizing RNA secondary structures might inhibit the viral replication cycle. These secondary structures include G-quadruplexes (GQs), which are guanine-rich sequence stretches forming planar guanine tetrads via Hoogsteen base pairing with two or more of them stacking on top of each other; a number of small molecular drug candidates increase the energy required for their unfolding. The propensity of G-quadruplex formation can be predicted with bioinformatics tools and is expressed as a GQ score. Synthetic RNA oligonucleotides derived from the RV-A2 genome with sequences corresponding to the highest and lowest GQ scores indeed exhibited characteristics of GQs. In vivo, the GQ-stabilizing compounds, pyridostatin and PhenDC3, interfered with viral uncoating in Na HowevEnterovirus within the Picornaviridae family. Their icosahedral shell is built from 60 copies of each of the 4 capsid proteins (VP1 through VP4) and encloses a ~2.4 µm long ss (+) RNA genome of roughly 7200 nucleotides. The RNA is tightly folded to fit into the ~22 nm-diameter void in the protein shell; it carries a covalently linked 21-amino acid residue peptide (VPg) at the 5′ end and a poly-(A) tail at its 3′ end. Uptake into the cell occurs through receptor-mediated endocytosis. The receptor used depends on the RV species  and the RV-receptor group (minor or major). Receptors include the low-density lipoprotein receptor (LDLR) and related proteins, intercellular adhesion molecule 1 (ICAM-1), and cadherin-related family member 3 (CDHR3) 60 + ssRNA) and (empty) B-particles ([VP1VP2VP3]60) ,64. ProtTo confirm this result by using an orthogonal assay, HeLa cells were infected with native and PDS-treated RV-A2, and 30 min pi cell-associated viral material was released by freeze-thawing, as described above. The clarified cell extract was analyzed by sedimentation through preformed sucrose density gradients. Sucrose gradient ultracentrifugation separates native virions, A-particles, and empty B-particles . The virde-novo synthesized RV-A2  (+ and somewhat more flattened and prolate in Na+ (compare the insets), suggesting that Mg2+ and polyamines that are bound to the encapsidated genome [+ phosphate buffer led to the elongation and expansion into irregular rods  B. In thed genome ,70,73 welar rods A. No suc+ phosphate buffer, while more spherical objects with a diameter of ~25 nm were seen in K+ phosphate buffer . AFM scans of samples prepared as above yielded very similar images compare B,C. Agaie buffer C; this c+ phosphate buffer may be triggered by the refolding of metastable hairpin-like structures, involving part or all predicted QGRS, into PDS-stabilized GQs, determining the global structure of the RNA genome within the protein shell [+ might limit the access of PDS to these metastable regions, accounting for the different effects of Na+ and K+.The drastic shape change in the RNA induced by PDS and only observed in Nain shell ,75. The + and K+ phosphate buffers suggest that access of SYTO 82 to the RNA cores was restricted between 25 °C and 40 °C. This implies that the genome remains compact within this temperature range  to the fluorescence peak  likely results from disruption of the RNA tertiary structure, allowing SYTO 82 to bind newly exposed helices originally buried in the RNA core. The rise was ~2-fold (Na+) and ~1.2-fold (K+), suggesting a more extensive loss of tertiary organization in the Na+-phosphate buffer. The higher tertiary structure stability in K+ phosphate buffer is illustrated by the approximately 4 °C higher melting temperature Tm1. The following progressive drop in emission intensity is due to the unraveling of more stable secondary structures releasing the SYTO 82; the corresponding Tm2 for this transition was 60.5 °C in Na+ and 63 °C in the K+-containing buffer.To substantiate the above hypothesis, we carried out a melting analysis of the ex-virion RNA, similarly as described by Silvers et al. , exploitre range . The sub+ imposes a tighter tertiary structure on the viral genome, thereby limiting access of PDS to putative internal QGRS and preventing GQ formation/stabilization. If this is correct, a reduced number of PDS molecules should associate with the RV-A2 genome in a K+ phosphate buffer when compared to a Na+ phosphate buffer. Since assessing this figure is technically less demanding with encapsidated RNA, we quantified PDS levels in virions after incubation with PDS at 20 μM at 4 °C or at 34 °C for 4 h in Na+ or K+ phosphate buffers. The virus was extensively washed by ultrafiltration to remove all traces of free PDS. PDS remaining associated with the virus was then quantitated by mass spectrometry. In agreement with the above results, the number of PDS molecules was highest upon incubation in Na+ phosphate buffer at 34 °C , whereas the same pre-incubation in Na+ phosphate buffer resulted in a roughly 100-fold decrease (+).This result corroborates our hypothesis that Klization , prevental yield D; K+, whdecrease D, Na+.+ in PDS-mediated viral inhibition, as the Na+ concentration is highest in early endosomes  by a time-of-addition experiment similar to that described for Zika virus infection of Vero cells . The virruenberg ). This iruenberg .QGRS Mapper predicted conserved G-rich sequences that potentially fold into GQs in all RV genomes ; in the We chose RV-A2, one of the best-characterized RVs , for exp+ as well as Na+ buffers  strand comprising QGRS probably fold faster into alternative, long-lived metastable conformations (tens of microseconds for a hairpin ) than in+ but not K+ phosphate buffers, PDS induced large-scale conformational changes in protein-free viral RNA cores. At first glance, this appears paradoxical as K+ usually stabilizes GQ better than Na+  was found to critically regulate the binding of small molecules to the c-di-GMP riboswitch [+ than K+ [+ compared to Na+ furthermore suggests the presence of structure-stabilizing sites specifically chelating the larger K+. Structurally important K+-specific pockets have been described for ribozymes and ribosomal RNA [+ was also clearly evident when RV-A2 was incubated with PDS, and mass spectrometry revealed PDS accumulation only in the presence of Na+. Consequently, the latter also resulted in a profound drop in infectivity, as observed for viruses incubated with PDS or PhenDC3 in Na+ phosphate buffer. One might consider testing this ‘compactness’ via incubation of viral RNA cores in Na+ and K+ phosphate buffers, respectively, and assessing their sensitivity toward RNase digestion.In Naboswitch . Water m than K+ , which c than K+ . The subomal RNA ,103. The+ is comparable to the number of putative QGRS (11). Assuming that these QGRS exist as folded GQs, it is likely that the stabilizing effect of PDS prevents their unwinding. With an effective diameter of ~3.6 nm for the RNA Q-quadruplex [How does the binding of PDS affect RNA release from the capsid? The current model of enterovirus RNA exit favors the transient unfolding of the RNA to pass through one of the small pores  in the eadruplex , the GQ-adruplex . The stradruplex . Alternaive QGRS . Assuminadruplex ; our resadruplex ,17. Whil+ and low Na+ cytosolic environment, limiting the access of the compound to its target sequences within the viral RNA, as found for the ex-virion RNA. However, a complete block of PDS binding by high intracellular K+ is unlikely, based on recent life cell imaging of cellular mRNA GQs in the absence or presence of a variant of PDS [Time-of-drug-addition experiments showed that PDS had little consequence for RV-A2 protein production when added after the uncoating stage. All subsequent events required for viral reproduction occur in a high Kt of PDS . Previout of PDS , togethet of PDS  efficien+ but not K+ due to their differential impact on viral RNA compaction rather than GQ formation. While PDS has potential side effects that may preclude its direct use as a therapeutic, our data point to GQ stabilization as a novel therapeutic avenue for tackling rhinovirus infection. Finally, PDS did not interfere with RV-A2 binding to the host cell and likely preserved the immunogenic epitopes, making these weakly infectious particles attractive candidates in the development of attenuated vaccines.In summary, we have shown that targeting putative QGRS by GQ-stabilizing compounds specifically inhibits the uncoating of a common cold virus. We provide a mechanistic explanation for this result using biophysical and ultrastructural analyses. Strikingly, PDS uptake into the virus occurs in the presence of Na


Failure to restore the femoral offset of the native hip is a potential cause of dysfunctional hip arthroplasty. The aim of this study was to report our experience of using a modular head–neck adapter in revision THA, specifically analyzing its usefulness as a tool to correct a slightly diminished femoral offset.TM head–neck metal adapter was used. The preoperative and one year follow-up modified Merle d’Aubigné hip score was used to evaluate functional outcomes.This was a retrospective single-center study including all hip revisions performed at our institution from January 2017 to March 2022 where the BioBallOf a total of 34 cases included for revision, the head–neck adapter system was used specifically in six patients (17.6%) to increase femoral offset, retaining both the acetabular and femoral components. In this subgroup of patients, mean offset decrease after primary THA was 6.6 mm (4.0–9.1), equivalent to a mean 16.3% femoral offset reduction. The median modified Merle d’Aubigné score went from 13.3 preoperatively to 16.2 at one year follow-up.The use of a head–neck adapter is a safe and reliable procedure that may allow the surgeon to easily correct a slightly diminished femoral offset in a dysfunctional THA without the need to revise well-fixed prosthetic components. Total hip arthroplasty (THA) is one of the most successful surgeries, with well-reported survival rates. According to national registry reports, a hip replacement is expected to last 25 years in around 58% of patients , 2. HoweOne of the reasons for a dysfunctional hip arthroplasty is a failed restoration of the native femoral offset, which is essential to restore patients’ predisease hip biomechanics and abductor function. In fact, suboptimal restoration of femoral offset results in increased joint reaction forces caused by a reduction in abductor moment arm, which may eventually lead to limping and potential instability –7. FurthRetaining a well-fixed femoral stem significantly decreases not only the morbidity of the procedure but also blood loss, infection rates, and operating time –11. A heAccording to a recent systematic review by Novoa et al , the maiTM) was routinely used as a reference for determining the individual magnification factor [TM software . A femoral offset was defined as the distance from the center of rotation of the femoral head to a line bisecting the long axis of the femur [This was an observational retrospective single-center study including all hip revisions performed at our institution from January 2017 to March 2022 that required the use of a modular head–neck adapter system. All the procedures were performed by at least one of the hospital’s joint reconstruction surgeons. Digital templating was carried out in all cases. All preoperative digital radiographs of the pelvis were taken in an anterior–posterior orientation. Since external rotation of the femur usually leads to underestimation of femoral offset , patientn factor . Digitaln factor , 17, usihe femur . All revhe femur  hip scorTM  head–neck metal adapter was used in all cases. The BioBall™ is a titanium implant (TiAl6VA4) that comes in different lengths, is versatile enough to adapt to diverse Morse tapers, and is able to add 7.5° offset in any direction  or interquartile range (IQR), whereas qualitative variables were described by absolute frequencies and percentages. Analyses were made and plots were drawn using the SPSS® v. 20.0 statistical package .The study was reviewed by a Research Ethics Committee, which approved it under code HCB/2023/0067, and was conducted in accordance with the 1964 Helsinki Declaration. No funding was sought or secured in relation to this manuscript. At the time of publication, none of the authors disclosed any potential conflicts of interest. None of the authors received any financial support for the research, authorship, and/or publication of this article. The retrospective nature of this study made it unnecessary to obtain the participants’ informed consent. Data was codified, and patients’ anonymity was preserved at all times.n = 26, 76.5%) followed by head replacement (while retaining both cup and stem) in order to achieve a higher offset . There were two cases (5.9%) where the head–neck adapter was used during primary THA implantation. The adapter was not used in any case where all the prosthetic components had to be revised. An anterolateral Hardinge approach was used in 76.6% of cases (n = 26). As regards the implants’ characteristics, the most used neck length was 2XL (+ 10.5 mm) (30.3% of cases (n = 10)), followed by L (+ 3 mm) and 3XL (+ 14 mm) (27.3% (n = 9) and 18.2% (n = 6)), respectively. Regarding the taper, a 12/14 taper was used in 88.2% of cases (n = 30). The most frequently used head sizes were 36 mm (47.1% of patients (n = 16)) and 28 mm (44.1% of patients (n = 15)). The 7.5° offset adapter was used in 76.5% (n = 26) of patients. The head material was ceramics in 50% (n = 17) of cases, and dual-mobility heads were used in 41.2% of patients (n = 14). The characteristics of the patient sample are shown in Table A total of 34 cases were included for revision. Median patient age was 71.2 years; 61.8% (21 out of 34) of the sample were male. The most frequent indication for surgery was revision of the acetabular cup  specifically to increase femoral offset. Of these six patients, five presented with hip pain that improved after prosthetic revision. A total of five patients presented with walking difficulties, which resolved completely in three cases. There was one patient that reported no improvement following surgery and one patient with multiple sclerosis that progressively worsened in terms of function even though a certain degree of pain relief was achieved. All six patients were operated through an anterolateral Hardinge approach. A partial gluteus medius tear was found in all cases, which was repaired with direct transosseous suture. The median offset decrease after primary THA had been 6.6 mm (4.0–9.1), causing a median femoral offset reduction of 16.3% Figs.  and 3. TAn accurate restoration of native femoral offset is often regarded as an essential requirement to obtain an optimal prosthetic joint. However, a certain amount of controversy still persists among surgeons. It is well known that patients with decreased offset present with poorer joint function at one-year follow-up . Even thThe head–neck adapter has become a widely used tool, mainly in partial hip revisions where the cup is replaced and the adapter is used to restore the taper surface on a previously damaged cone. During the procedure, the existing taper is replaced by the adapter’s own taper, hence allowing retention of the primary stem. Moreover, the stability resulting from addition of a 7.5° offset may be used to address recurrent dislocation –27. For It has been reported that patients operated due to hip dysplasia are at a high risk of experiencing an excessively increased offset after THA. Indeed, dysplasia is typically accompanied by decreased femoral offset, which is usually excessively augmented when regular necks are used , 29. TheTM head–neck adapter to specifically correct a slightly diminished and hence suboptimal femoral offset. However, the present study is subject to some inherent limitations. Most importantly, because of its retrospective nature, certain biases may have influenced the results. The study’s retrospective design may have resulted in an inadequate control over confounding variables and limited the amount of information obtained. Secondly, it is impossible to ascertain whether the outcome of the procedure can be strictly attributed to the revision of the investigated component or whether it was also related to the way the periarticular soft tissues were managed. Given that all six cases presented with gluteus medius tears of various sizes, the authors acknowledge that the benefit might have been at least in part related to the direct tendon repair performed. Finally, the limited number of cases inherent in a single-center study on a low-prevalence condition requires the performance of further research to corroborate the findings obtained. All these limitations may affect the extent to which the results presented here can be generalized beyond the specific cases studied.To the best of our knowledge, this is the first study to report on the use of the BioballIn conclusion, a head–neck adapter is a safe and reliable tool that may allow the surgeon to easily correct a slightly diminished offset in a dysfunctional THA without the need to revise well-integrated prosthetic components. The use of the component might be considered whenever a revision of a decreased-offset arthroplasty is performed.


The rate by which the ribosome decodes an mRNA determines the final protein output of the mRNA. This can vary greatly between different mRNAs. Methods which allow accurate determination of translation rates of specific mRNAs require conditions that preserve native mRNA–ribosome interactions and thus cannot be denaturing-based. Complex tissues sometimes contain RNases that can breakdown the RNA. Here, we optimise methods in non-denaturing conditions enabling the application of methods that examine translational control to primary mouse tissue in an accurate and reproducible manner.The protein output of different mRNAs can vary by two orders of magnitude; therefore, it is critical to understand the processes that control gene expression operating at the level of translation. Translatome-wide techniques, such as polysome profiling and ribosome profiling, are key methods for determining the translation rates occurring on specific mRNAs. These techniques are now widely used in cell lines; however, they are underutilised in tissues and cancer models. Ribonuclease (RNase) expression is often found to be higher in complex primary tissues in comparison to cell lines. Methods used to preserve RNA during lysis often use denaturing conditions, which need to be avoided when maintaining the interaction and position of the ribosome with the mRNA is required. Here, we detail the cell lysis conditions that produce high-quality RNA from several different tissues covering a range of endogenous RNase expression levels. We highlight the importance of RNA integrity for accurate determination of the global translation status of the cell as determined by polysome gradients and discuss key aspects to optimise for accurate assessment of the translatome from primary mouse tissue. A multitude of mechanisms have now been identified that act upon mRNAs to control both protein output and specifically where within the cell mRNA translation takes place ,2,3,4. IApproaches that allow the analysis of the association of ribosomes with specific mRNAs are now being applied beyond cell lines into mouse models and patient samples ,6,7,8,9.The activity of these RNases can be controlled endogenously by ribonuclease inhibitor (RNH1) ,29. ThisWhen working with RNA, high-quality RNA is integral to the quality of the data output. Methods examining the engagement of the ribosomes with mRNAs requires preservation of the mRNA–ribosome interactions and for the mRNAs to be fully intact to migrate through the gradient at the correct speed. Degraded RNA can lead to an altered gradient profile and thus inaccurate determination of the translational landscape. For example, an intact mRNA bound by six elongating ribosomes would migrate through a sucrose gradient at a certain density; however, if this mRNA were cleaved by an endoribonuclease, resulting in two mRNA fragments each bound by fewer ribosomes, these would each migrate at a reduced density. Reagents such as RNAlater provide a means to retain RNA integrity immediately at the point of tissue harvesting. However, a key mechanism by which these reagents act involves denaturing the proteins, which renders any RNases present non-functional but also other proteins within the sample. Translational profiling methods require non-denaturing conditions to preserve the integrity of the ribosome. In this paper, we utilise a range of alternative methods to preserve the RNA integrity of mouse tissue samples and demonstrate how this varies across different tissues. We also demonstrate how these improved methodologies can then be utilised to interrogate the translatome accurately and consistently in mouse tissues.All experiments were performed in accordance with UK Home Office regulations and were approved by the University of Glasgow Animal Welfare and Ethical Review Board.For the liver, colon, and small intestine tissue samples, female C57BL/6 mice aged 6–7 weeks purchased from Charles River were used. These experiments were carried out under the UK Home Office project licence PP6345023. The mice (5 per cage) were allowed access to food and water ad libitum and were kept in a 12 h day/night cycle starting at 7:00 until 19:00. Rooms were kept at 21 °C at 55% humidity. These animals were culled in accordance with Schedule 1.For the lung and pancreas tissue, stock mice were housed in individually ventilated cages (IVCs) with access to standard irradiated diet and sterilised water pouches ad libitum. Mice were handled and assessed within sanitised laminar flow changing hoods. Some experimental mice were housed in conventional caging with access to diet and water ad libitum. Environmental enrichment was provided with nesting, fun tunnels, and wooden chews, with general cage maintenance being carried out by the Biological Services Unit at the Beatson Institute. These mice were bred and maintained in house. 2, with cervical dislocation as a secondary schedule 1. This was performed at 11 weeks for control lung tissue and at approximately 6 months post-induction with 1 × 107 pfu SPC-Cre for tumour tissue (mice approximately 34 weeks of age). Tumour-bearing mice were of the genotype KrasG12D/+, Myc−/+, and iRFP+. Lungs were flushed with PBS prior to sampling to remove residual red blood cells and then moved to a sterile surface sprayed with RNaseZAP  for dissecting out individual tumours. Both male and female mice were used, and when the amount of sample was limiting, samples were pooled to provide sufficient material for polysome gradient analysis.Work for the lung tissue samples was conducted under project licence number PE47BC0BF. Mice were culled with rising volume of COKRAS and Trp53. Female wild-type mice of approximately 100 days of age, bred on a mixed background, were used throughout these experiments. Following euthanasia, 70% ethanol was applied to the abdomen of the mice and the peritoneum was opened to inspect the internal organs. When collecting these pancreas tissue samples, dissection instruments were cleaned and sprayed with RNaseZAP  before removing small pieces of tissue (~30 mg), which were placed in RNAlater  or DNA/RNA Shield  and stored at −80 °C until further use.Work for the pancreas tissue was conducted under Project licence PP8411096. Following weaning, mice were ear notched and the tissue sent to Transnetyx  for DNA genotyping via real-time PCR to detect allelic alterations in genes of interest, including Tissue pieces were flash frozen in liquid nitrogen and stored at −80 °C until required. The CryoGrinder system , which ensures the samples remain frozen on nitrogen, was then used to grind the tissue with a mortar and pestle. To aliquot tissue in a manner that avoided the risk of thawing, empty 1.5 mL tubes were pre-marked, by comparing to a 1.5 mL tube with water in it; marks were made at 20 µL (for tests) or 200 µL (for sucrose gradients). Marked tubes were then chilled and kept on dry ice throughout dispensing—tipping the ground tissue up to the mark, which was performed in a cold room. Tissue aliquots and stocks were stored at −80 °C until required.2, 15 mM Tris-HCl pH 7.5, 100 µg/mL cycloheximide (the gradient buffer can be stored at −20 °C), 1% Triton X-100, 0.05% Tween-20, 0.5 mM DTT, 5 mM NaF, 1× cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail , 1× Protease Inhibitor Cocktail for plant cell and tissue extracts (Merck), 2% n-Dodecyl-beta-Maltoside Detergent . For lysis tests, 200 µL lysis buffer (with either 1000 U/mL Ribolock (Thermo Scientific), 1000 U/mL Superase.In , 1 mg/mL Heparin , 1000 U/mL, Murine RNase Inhibitor , or 10 mM Ribonucleoside Vanadyl Complex (New England Biolabs) was aliquoted and chilled on ice. A total of 20 µL of ground tissue sample was then tipped straight from dry ice into the lysis buffer. Tubes were immediately inverted to disperse the tissue fragments. Lysates were incubated on ice for 5 min with occasional inversion, then centrifuged at 13,000 rpm, 4 °C, for 5 min. A total of 150 µL of supernatant was retained and 1 mL TRIzol added (Invitrogen).Lysis buffer was made fresh each time and comprised 1× gradient buffer  was transferred to a new 1.5 mL Eppendorf tube. To precipitate the RNA, 2 µL glycogen (Roche) and 500 µL isopropanol were added and inverted to mix, then incubated for a minimum of 45 min on ice. Samples were then pelleted via centrifugation at 4 °C, 13,000 rpm for 30 min. Pellets were washed twice in ice-cold 750 µL 75% ethanol and spun at 4 °C, 13,000 rpm for 5 min each time. After the final wash removal, pellets were air-dried for 2 min then resuspended in 15µL RNase-free water.Total RNA concentration was determined via nanodrop and 0.5–1 µg was mixed with loading dye (New England Biolabs) and resolved via electrophoresis on a 1% agarose 1× TAE  with SYBR™ Safe DNA Stain  gel at 150 V for 20 min then visualised on a Biorad Chemidoc Imaging system (Blue imaging tray). One kb HyperLadder (Bioline) was loaded on each gel as a reference.Cell lysis was performed on 200 µL of tissue in 900 µL lysis buffer as described above. Tissue was lysed on ice for 5 min, centrifuged at 13,000 rpm, 4 °C, for 5 min and 800 µL of lysate transferred to a fresh tube. To obtain a corresponding total lysate sample, 40 µL of the lysate was taken and 1 mL TRIzol added. As a quality control, in parallel, 40 µL of tissue was taken and 1 mL TRIzol added directly to the tissue. This allows for the assessment of the RNA integrity within both the whole tissue and total lysate used for the gradients. A total of 600 µL of lysate was then loaded onto 10–50% sucrose gradients. This corresponds to 183 µg and 151 µg RNA for the liver tissue, 17 µg and 24 µg for the control lung tissue, 42 µg and 82 µg for lung tumour tissue, 56 µg and 69 µg for small intestine samples without inhibitors and 70 µg and 105 µg for small intestine samples treated with inhibitors. Gradients were spun at 38,000 rpm for 2 h at 4 °C in a Beckman Optima XPN90 centrifuge. Gradients were fractionated and collected using a Biocomp Gradient Station with attached Gilson FC203B fraction collector. Of note, with this machine, the final fraction remains in the gradient tube and is not run through the fractionator—this sample is manually retained as an additional final fraction (fraction 12); hence, there is an apparent extra fraction in the agarose gel RNA extractions compared to the gradient traces. Gradient fractions were collected into 7.7 M Guanidine HCl (3 mL) with glycogen (8 µL) and vortexed, then 4 mL of 100% ethanol was added, invert mixed, and precipitated at −20 °C overnight.x-axis, i.e., 0–1 on the gradient profile is denoted as fraction 1 on the agarose gel and 1–2 on the gradient profile is denoted as fraction 2 on the agarose gel. As detailed in the previous section, fraction 12 is the final fraction that remains in the gradient tube and is not run through the fractionator due to the nature of the machine used. RNA samples were stored at −80 °C.Gradient fractions were spun in a Beckman 5810R bench top at 4000 rpm, 4 °C, for 1 h. All the supernatant was removed, and pellets were resuspended in 320 µL RNase-free water. A total of 300 µL was transferred to a fresh 1.5 mL Eppendorf and 2 µL glycogen, 30 µL 3 M sodium acetate pH 5.2, and 900 µL 100% ethanol were added. Samples were inverted to mix and precipitated overnight at −20 °C. Samples were pelleted via centrifugation for 40 min, at 13,000 rpm and 4 °C. Pellets were washed twice with 750 µL 75% ethanol and centrifuged each time at 13,000 rpm for 5 min at 4 °C. Pellets were air-dried for 2 min and resuspended each in 25µL RNase-free water. Equal volumes of each fraction were loaded onto a 1% agarose/1× TAE/1:1000 SYBR™ Safe gel  to check RNA integrity across the fractions. Fraction numbers on the agarose gels reflect everything up to that number on the gradient trace e); the lower marker, 18S rRNA and 28 rRNA, are indicated on the traces. Of note, the RNA samples were run on a TapeStation after long term storage at −80 °C and following multiple freeze thaws, whereas the agarose gels were conducted close to the time of initial processing.For the agarose gels, RNA integrity values were calculated as the total signal of the 28S and 18S rRNA bands compared to the whole lane using Fiji software (version ImageJ2) . Then, tMouse RNA-seq data normalised as transcripts per million was obtained from Söllner et al., 2017 . RNASE4 and ANG being the highest in liver (RNASE1 dominating in the pancreas (RNASE2B has low expression in most cases but is elevated in the oesophagus (RAF1 (RNASE3) is expressed broadly across tissues, yet only weakly in the pancreas . Similarly, murine RNase inhibitor is a recombinant murine protein expressed and purified from an E. coli strain (New England Biolabs). Both inhibitors specifically target RNase A/B/C in a non-covalent manner with high affinity, with B and C being glycosylated forms of RNase A [In these tests, we added a range of different commonly available RNase inhibitors to the lysis buffer . Heparin RNase A ,17. Finae values obtained using the TapeStation [For the lysis of the tissue, aliquoted ground tissue was kept on dry ice and tipped directly into a 1.5 mL tube containing ice-cold lysis buffer, inverted, and lysed on ice. After centrifugation, RNA was isolated from the lysate using TRIzol. RNA integrity was determined by running the extracted RNA on an agarose gel. Through this method, the 28S (4.7 kb) rRNA and 18S (1.9 kb) rRNA bands are clearly visible and provide a strong indicator of RNA integrity a combination of clearly defined rRNA bands, the relative ratio between 28S and 18S rRNA (~2.0), and no smearing or additional banding being indicative of high RNA integrity ,45,46. IeStation . The aimHere, we have focused on tissues that cover a range of RNase activity levels. Comparing the lysis of the tissues directly in TRIzol to lysis buffer with no RNase inhibitors present, we observe a clear reduction in RNA integrity across the tissues . The extRNASE4 and ANG (First, the liver tissue was assessed and, despite the noted high expression of  and ANG , the liv and ANG . Althoug and ANG . This su and ANG . The sma and ANG D. Unlike and ANG . Of noteThe pancreas has very high RNase levels A,B, makiThere have been a range of next-generation sequencing techniques developed to allow detailed, high-resolution studies of mRNA translation, in particular polysome profiling  and riboHaving determined that liver tissue has consistent RNA integrity in non-denaturing conditions , we thenMycERT2 and KrasG12D and tumour tissue samples were collated 6 months post-induction to obtain large tumours [Lung tissue showed consistent RNA integrity in the presence of RNase inhibitors including RiboLock and murine RNase inhibitor , and whe tumours ,56. ThisGiven the variable RNA integrity of the small intestine, we next compared polysome profiles of small intestine lysed in the presence and absence of RNase inhibitor. To try to improve upon initial testing , we alsoFor RNA-based techniques, RNA integrity is important for ensuring the quality and reproducibility of downstream results. The quality of the RNA is even more important when examining translational control mechanisms which use polysome profiles and other methods for determining the association of ribosomes with mRNAs ,40. HereThe RNase inhibitors used in this study had differing degrees of success in improving RNA integrity. SUPERase.In only slightly improved integrity, while heparin improved integrity of the liver, lung, and colon, but was not sufficient in the small intestine. RiboLock and the murine RNase inhibitor led to the greatest and most consistent improvements in RNA integrity across the tissues tested in this study . SUPERasIt is aIso important to note that differences in tissue sample handling may be a key source of variability in RNA integrity. This can be an unpredictable source of variability as often when collecting samples from mice the person harvesting the tissue each time may differ, or the person processing the samples downstream may be different. This emphasises the importance of checking the RNA integrity of all samples and how including best practices from the very start of the sample handling may help improve consistency.Having determined optimal lysis conditions for high-quality RNA, we have demonstrated how these can be utilised to accurately examine the association of mRNA with ribosomes in liver, lung, and small intestine and how, without this key quality control, results could be misinterpreted. Polysome gradient profiles demonstrated how different tissues have very different levels of ribosome occupancy, highlighting the importance of being able to accurately study translation in a wide number of tissues.As for the pancreas, we show the difficulties in conducting RNA-based methodologies with this tissue. None of the non-denaturing based lysis methods tested in this study were sufficient to preserve both RNA and ribosome integrity ; thus, sRibosome profiling is another commonly used technique to study translational control, which differs from polysome profiling as it allows codon resolution of the ribosomes . This isThis study highlights the importance of checking RNA integrity of all tissues and all replicates due to the variability of RNase-mediated degradation, even when endeavouring to keep the tissue harvesting protocol as consistent as possible. There are a range of methods that could be applied to check the RNA integrity, including running the RNA on an agarose gel, TapeStation, or bioanalyzer or looking more closely at the integrity of specific mRNAs using methods such as Northern blotting. This will ensure quality results and meaningful interpretation. In addition, it is probable these optimisations and inhibitor combinations could also be readily applied to other RNase-rich tissues, such as the mouse spleen, and to human tissue.Overall, this study highlights the fundamental importance of RNA integrity for accuracy of downstream investigations and provides methods that consistently maintain RNA integrity in a range of different mouse tissues. These can now be utilised for accurate investigation of tissue samples with methodologies that require non-denaturing conditions.


Cardiovascular diseases (CVDs) are a group of diseases that have a major impact on global health and are the leading cause of death. A large number of chemical base modifications in ribonucleic acid (RNA) are associated with cardiovascular diseases. A variety of ribonucleic acid modifications exist in cells, among which adenosine deaminase-dependent modification is one of the most common ribonucleic acid modifications. Adenosine deaminase acting on ribonucleic acid 1 (Adenosine deaminase acting on RNA 1) is a widely expressed double-stranded ribonucleic acid adenosine deaminase that forms inosine (A-to-I) by catalyzing the deamination of adenosine at specific sites of the target ribonucleic acid. In this review, we provide a comprehensive overview of the structure of Adenosine deaminase acting on RNA 1 and summarize the regulatory mechanisms of ADAR1-mediated ribonucleic acid editing in cardiovascular diseases, indicating Adenosine deaminase acting on RNA 1 as a promising therapeutic target in cardiovascular diseases. Cardiovascular diseases (CVDs) represent the number one cause of death globally, accounting for nearly 50% of the 36 million deaths from non-communicable diseases worldwide each year . ResearcADAR1, ADAR2 and ADAR3 are the three most conserved members of the mammalian ADAR family . ADAR1 iThe general structure of ADAR protein includes the N-terminal double-stranded RNA binding domain and the C-terminal deaminase domain that catalyzes the substrate reaction . ADAR1 iThere are two subtypes of ADAR1, the full-length p150 and the shorter p110 . The strdrosophila genome (The ADAR2 protein encodes two major isoforms: ADAR2S (or ADAR2a) and ADAR2L (or ADAR2b) . ADAR2 ia genome .In general, ADAR1 is mainly responsible for the editing of repetitive sequences (such as primate Alu), and ADAR2 is mainly responsible for the editing of messenger RNA (mRNA) coding regions, performing site-specific editing . IsomersRNA editing is performed by A-to-I editors, which are members of the ADAR protein family . The regA-to-I editing not only occurs in the protein coding region of mRNAs, but also occurs in the non-coding region, especially the Alu region . CathepsMiRNA are short non-coding RNA molecules that regulate gene expression at the post-transcriptional level . MiRNA iIn addition to miRNA, long noncoding RNA (lncRNA) is also the transcription product of non-coding genes, which is usually more than 200 nucleotides (nt) in length . RNA ediHeart failure is characterized primarily by the inability of the heart to provide metabolic needs and organ/tissue perfusion . Some coCongenital heart disease (CHD) is currently the most common type of birth defect in the general population with an incidence of about 1% . Interesin vivo. Specifically, the mechanism involved in regulation is that the RNA-binding protein HuR preferentially binds to the edited CTSS3 ‘UTR, thus controlling the expression of CTSS mRNA  in human carotid plaque. In addition, the expression of ADAR1 and inosine in human carotid plaque mRNA was also enhanced. ENDOV can promote atherogenesis by enhancing the recruitment of monocytes into atherosclerotic lesions, possibly by increasing the influence of chemokine ligand 2 (CCL2) activation on these cells. Therefore, it is reasonable to speculate that the interaction between ADAR1, adenosine-inosine editing and ENDOV may play a pathogenic role in the development and progression of atherosclerosis .Viral myocarditis is a common clinical cardiovascular disease caused by viral infections, and the main pathological feature is myocardial inflammation. A large number of studies have shown that the direct damage of the virus to the cardiomyocytes and the myocardial injury caused by the excessive immune response eventually leads to cardiomyocyte dysfunction and reduced contractility . The maiAneurysm is a local lesion or injury of the arterial wall, resulting in the destruction of the structure and mechanical properties of the arterial wall. Abdominal aortic aneurysms (AAA) are due to rupture and dissection, with high morbidity and mortality . The swiMicroarray analysis of samples from patients with aortic aneurysms found elevated levels of ADAR1 and its editing target (CTSS) . At the Reports directly investigating ADAR1 in arrhythmia remain scarce. Previous studies have shown that the role of ADAR1 in calcium handling is not known, but ADAR1 editing of glutamate ionotropic receptor kainate type subunit 1 (GRIK1) and glutamate ionotropic receptor kainate type subunit 2 (GRIK2) has been shown to affect calcium channel permeability . Thus, lin vitro and in vitro  and end-stage PAD. ADAR1 can edit pri-microRNA in a microRNA-specific manner. Edited microRNAs promote pro-angiogenic function of vascular cells under ischemia/hypoxia conditions. Furthermore, miRNA editing enhanced angiogenesis in vitro . ADAR1-min vitro .RNA therapy refers to the use of RNA-based molecules to modulate biological pathways to treat specific diseases . Generalin vivo to target the repair of damaged fibrotic cardiomyocytes and restore the heart to normal function (Based on the mechanism of RNA editing linked to the pathophysiology of the cardiovascular system, targeted therapy for CVDs is a recent research hotspot. Many genetic diseases, including CVDs, are caused by single nucleotide changes . Over thfunction . The appCardiovascular diseases is the leading cause of death worldwide. The important point of our manuscript is that we summarize the mechanisms by which ADAR1 affects cardiac function and cardiovascular aspects as well as potential therapeutic targets . It is n


The YT521-B homology (YTH) domain-containing family proteins (YTHDFs), including YTHDF1, YTHDF2, and YTHDF3, are a class of cytoplasmic m6A-binding proteins defined by the vertebrate YTH domain, and exert extensive functions in regulating RNA destiny. Distinct expression patterns of the YTHDF family in specific cell types or developmental stages result in prominent differences in multiple biological processes, such as embryonic development, stem cell fate, fat metabolism, neuromodulation, cardiovascular effect, infection, immunity, and tumorigenesis. The YTHDF family mediates tumor proliferation, metastasis, metabolism, drug resistance, and immunity, and possesses the potential of predictive and therapeutic biomarkers. Here, we mainly summary the structures, roles, and mechanisms of the YTHDF family in physiological and pathological processes, especially in multiple cancers, as well as their current limitations and future considerations. This will provide novel angles for deciphering m6A regulation in a biological system.As the most abundant and conserved internal modification in eukaryote RNAs, N6-methyladenosine (m They cahylation , 9. More3 (eIF3) –16. They6A, which includes YTHDF1, YTHDF2, and YTHDF3. They regulate the translation and stability of target mRNAs to alter the expression of downstream molecules, thus affecting diverse biological processes , but with a lower affinity than m6A . The YTH domain is the basis of recognizing m on mRNA , 38. YTHthan m6A . The prithan m6A . The mRNications . Proteomications . Additioications . YTHDF2 ancement . Therefo6A mononucleotide (or m6A oligoribonucleotides) have been revealed . And the methyl group of m6A points to the ring of one aromatic residue   , 47, 48.ognition   (6A-containing circular RNAs (circRNAs) are also degraded by this pathway. Interestingly, under heat shock stress, nuclear-translocated YTHDF2 protects m6A motifs in the 5’ untranslated region (5’UTR) of stress-induced transcripts and activates cap-independent translation initiation  , 52. In itiation . The N-tications . YTHDF1 ications . YTHDF1 ications –57. Aparications . In addisubunits . Besidesh YTHDF2 . A recenh YTHDF2 .6A-modified mRNAs are first bound by the YTHDF3 or YTHDF3-YTHDF1 complex and then recognized by YTHDF2, thereby regulating the different fates of the targeted mRNA  mRNA and degrades IGF1R mRNA, hindering migration and invasion of trophoblast.Intriguingly, unlike the previous view that maternal mRNAs clearance and maternal-to-zygotic transition (MZT) are dependent on YTHDF2, Kontur et al. found that individual YTHDFs deletion does not prevent embryonic development, whereas double mutations of YTHDF2/YTHDF3 disrupts oogenesis and triple YTHDF depletion causes lethality in zebrafish , 73. Des4.26A-dependent deadenylation mechanisms , which have unlimited proliferation and pluripotent differentiation potential similar to human embryonic stem cells (ESCs) . YTHDF2 chanisms . While Ychanisms . In termnal axis . YTHDF1 nal axis . Importaan iPSCs . Similaran iPSCs .6A-YTHDF2-mediated decay of Notch1 mRNA is critical for the generation of the earliest hematopoietic stem/progenitor cells (HSPCs) during the endothelial-to-hematopoietic transition (EHT) in both zebrafish and mice embryos  are significantly regulated by YTHDF2. The m embryos , 83. Li  embryos . Therefo embryos . Interes embryos . Given tcapacity . While Ycapacity .6A-mediated self-renewal of mouse female germline stem cells (mFGSCs)  during regeneration after intestinal damage by driving a positive feedback loop of the YTHDF1/TCF4/WNT signaling axis . Similar(mFGSCs) .4.36A modification in an FTO-YTHDF2-dependent manner  . KnockouYTHDFs are also involved in a variety of brain disorders. For example, downregulated miR-421-3p in microglia after cerebral artery occlusion/reperfusion (MCAO/R) relieves the repression of YTHDF1, thereby promoting p65 mRNA translation, leading to aggravated inflammation and brain injury . Impairm4.5via the METTL3-m6A-LRP6 axis and the FTO-m6A-FAK axis, respectively (YTHDF1 promotes cardiomyocyte (CM) differentiation, whereas YTHDF3 does the opposite . YTHDF1,ectively , 123. YTectively . Furtherectively , 126. Anectively .4.66A-modified RNAs and degrade EBV cleavage gene transcripts  and influenza A virus (IAV) replication , Hepatitis B virus (HBV), Hepatitis C virus (HCV), Zika virus (ZIKV), and enterovirus 71 (EV71) –133. Fornscripts . Furthernscripts . Alternanscripts . During lication , 135. Molication –138.6A-dependent manner, thereby limiting viral replication  and Kaposi’s sarcoma-associated herpesvirus (KSHV) remains controversial. Evidence suggests that YTHDFs hinder HIV-1 replication in target cells contradicting previous views that YTHDFs increase viral transcript and protein levels –141. Spelication . Neverthlication . To ensulication . Additiolication . Insteadlication . Togethe4.76A-modified viral RNA, which protects viral RNA from RIG-I recognition, thereby inhibiting RIG-I activation and the downstream IFN signaling pathway  signaling pathway relies on the expression of IFN-stimulated genes (ISGs) to mediate a powerful innate antiviral immune response. YTHDF1-mediated upregulation of IFITM1, a subset of ISGs, initiates antiviral responses . Another pathway , 150. Inby ISG20 . Additioby ISG20 . Among tby ISG20 . MechaniInflammatory responses are also an important part of immunity. YTHDF1 counteracts the excessive and persistent development of inflammation in the septic response by promoting the expression of SOCS1, a negative regulator of macrophage-mediated inflammation . However+ T cells, macrophages, and dendritic cells (DCs) , in CRC  . Han et ls (DCs) . YTHDF1 ty in GC . Li et a, in CRC . To this, in CRC . However, in CRC  , YTHDF1 binds to the m6A site on the 5’UTR of EGFR mRNA and triggers EGFR translation, eventually resulting in HCC recurrence after IRFA (Studies have reported that YTHDF1 is an oncogene that is highly expressed and positively correlates with the pathology stage in hepatocellular carcinoma (HCC) , 169. YTt manner . In addit manner . Chi et t manner . YTHDF1 t manner . Hu et agression . Furthergression . FOXO3 igression . Importater IRFA .6A-dependent mechanism  and serpin family E member 2 (SERPINE2) to exert an inhibitory effect. Conversely, YTHDF2 is also considered a tumor-promoting factor in HCC  mRNA  phenotype and metastasis  mRNA . Additiotastasis . And PA2tastasis . The distastasis .In addition, YTHDF1 and YTHDF2 facilitate the advancement of intrahepatic cholangiocarcinoma (ICC) through increasing EGFR mRNA translation and IFIT2 mRNA decay, respectively , 189. Me5.1.2in vitro and in vivo. Mechanistically, YTHDF1 relies on m6A modification to promote the translation of frizzled7 (FZD7) and USP14, which transmit WNT/β-catenin signaling and AKT/ERK signaling, respectively (YTHDF1 mutations occur in approximately 7% of gastric cancer (GC) patients, and high expression of YTHDF1 is correlated with high-risk progression and poor prognosis in patients –193. YTHectively , 193. Inectively .in vitro  mRNA counteracts the effects of NUCB1 in halting pancreatic cancer growth and augmenting the antitumor with gemcitabine (GEM)  . Converspatients . And compatients . Notablypatients –207. A rpatients . In addipatients .5.1.46A-modified Rho/Rac guanine nucleotide exchange factor 2 (ARHGEF2) mRNA and activating RhoA signaling (6A of glutaminase 1 (GLS1) mRNA 3’UTR to promote its translation. And METTL3 deletion inhibits LDHA mRNA translation by reducing the binding of YTHDF1 to LDHA mRNA CDS, thereby hindering glycolysis and promoting 5-fluorouracil sensitivity in CRC cells , YTHDF1 may be a molecular target for diagnosis and treatment . Mechaniignaling . Furtherignaling . Chen etignaling . SpecifiRC cells . Interesy of CRC .6A-YTHDF2-dependent manner and boosts CRC progression  translation axis is a crucial pathway for stimulating glycolysis and tumorigenesis  flux for tumor growth  mRNA in an mr growth . The trar growth , 233. Hor growth . Furtherr growth . YTHDF2 ectively , 237. NecircASK1 . InterescircASK1 . In lungcircASK1 .6A-YAP mRNA, thereby aggravating and attenuating the malignancy behavior of NSCLC, respectively  mRNA promotes PCa proliferation in a YTHDF2-dependent manner of transcriptome degradation (YTHDF2 acts as a facilitator and is negatively regulated by miR-493-3p in prostate cancer (PCa) . Du et aNA decay . In addiand USP4 , 249. Thradation .5.3.36A site 5’UTR region of ATF3 mRNA to mitigate the resistance of breast cancer cells to tamoxifen  mRNA and stabilization of hexokinase 2 (HK2) mRNA  cells, YTHDF1 accelerates mK2) mRNA , 266. SpAS5 mRNA , 268. YTAS5 mRNA . YTHDF2 AS5 mRNA .5.3.6YTHDF1 and YTHDF2 modulate the negative regulator PHLPP2 and positive regulator mTORC2 of AKT respectively, which is unfavorable to the tumorigenicity of the AKT pathway in endometrial cancer (EC) . In addi5.45.4.16A-dependent manner  tissues compared to normal tissues . YTHDF1 t manner , 279. AmC growth . And thiC growth . Mechani5.4.26A-dependent fashion  is mostly attributed to the attack of the small T antigen of Merkel cell polyomavirus (MCPyV) . Meanwhi5.4.46A-modified mRNAs such as TNF receptor superfamily member 1b (TNFRSF1b) that are associated with the functional integrity of AML CSCs. Moreover, the AML1/ETO-HIF1α loop transactivates the YTHDF2 promoter to promote t  AML cell proliferation  T translocation partner gene . Notablyferation . Howeverferation . Interesferation  (Figure66A modification, there are still many mysteries about the YTHDF family that need to be discovered and solved in terms of structure, function, and treatment.Although it has been revealed that the YTHDF family is involved in a variety of biological processes as the “readers” of m6A sites on mRNAs, and why YTHDFs pair with different cooperating m6A regulators, has not been reached. In addition, YTHDFs can be localized in different cellular compartments and may re-enter the nucleus or transport out of the cell membrane, thus expanding the regulation of YTHDFs. The post-transcriptional modifications of YTHDFs and interactions of YTHDFs with other proteins also add to the structure and function complexity of YTHDFs. Therefore, the development of emerging technologies, the control of various conditions, and the change of different stimulus states are necessary to further investigations in the YTHDF family.The discussion of the structure and function of YTHDFs is partially doubtful due to the limitations of technology and conditions. The reason why YTHDFs select the same or different target mRNAs and min vitro to in vivo. However, the specific mutation of functional RNA binding sites of YTHDFs in mice needs to be further realized. In addition, one of the important purposes of experimental research is clinical transformation, so it is of great need to explore the application value of targeting YTHDFs in the clinic, especially in tumors. Many clinical-related studies have analyzed the expression profile of the m6A regulator in tumors and its association with the immune microenvironment, grading, staging, therapeutic effect, and prognosis. For example, the analysis of 162 HCC samples from the Zhou et al. and 177 HCC samples from the Nakagawa et al. showed that YTHDF1 was related to poor prognosis of HCC and YTHDF2 was related to HCC recurrence, respectively (At present, many experiments have successfully constructed the YTHDF1/2/3 genetic KO mouse model using different techniques. First, the whole-body YTHDF1/2/3 KO mice are generated directly based on CRISPR/Cas9 by deleting a certain exon or inducing the premature appearance of a stop codon , 213. Seectively , 182. YTectively . Interesectively . In addiectively . A similectively . MoreoveHowever, studies of YTHDFs are still in the preclinical stage and many issues need attention. First, the clinical application of YTHDFs in different diseases, alone or in combination with other targets, requires further investigation. Second, the effectiveness of YTHDFs in diagnosing and predicting prognosis may vary across disease types, grades, and stages. Most importantly, the specific molecules targeting YTHDFs have not yet been developed. So how can YTHDFs be used in clinical treatment? The expression of YTHDFs can be regulated by other strategies. Targeting upstream or metabolic mechanisms of YTHDFs is an alternative approach to indirectly regulate the levels of YTHDFs Figure 776A modification have been gradually and seriously excavated. By binding to m6A, the YTHDF family plays an important role in the regulation of various physiological and pathological processes, including embryonic development, stem cell fate, fat metabolism, neuromodulation, cardiovascular effect, viral infection, immunity, and especially in tumors. In particular, YTHDFs regulate multiple tumor phenotypes such as proliferation, metastasis, metabolism, drug resistance, and immunity. Additionally, YTHDFs can be used as biomarkers for the diagnosis, treatment, and predictors of prognosis evaluation. On-going explorations of YTHDFs in modeling disease progression are still warranted for a better and deeper understanding of epigenetic modifications.With multi-omics advancement, the roles of mLC collected the related papers and drafted the manuscript. YG made the figures and revised the manuscript. SX edited and revised the manuscript. JG designed the framework and revised the manuscript. JY, MW, and TL revised the manuscript. All authors contributed to the article and approved the submitted version.