Risk Factors for Recurrence Following Arthroscopic Bankart Repair - A Systematic.

BACKGROUND: Recurrent instability remains a major source of morbidity following arthroscopic Bankart repair. Many risk factors and predictive tools have been described, but there remains a lack of consensus surrounding individual risk factors and their contribution to outcomes. PURPOSE: To systematically review the literature to identify and quantify risk factors for recurrence following arthroscopic Bankart repair. METHODS: A literature search was performed using the PubMed/Medline databases based on the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Studies were included if they evaluated risk factors for recurrent instability following arthroscopic Bankart repair. RESULTS: Overall, 111 studies were included in the analysis, including a total of 19,307 patients and 2,750 episodes of recurrent instability with 45 risk factors described. Age at operation was reported by 60 studies, with 35 finding increased risk at younger ages. Meta-analysis showed a two-fold recurrence rate of 27.0% (171/634) for patients under 20 years old compared to 13.3% (197/1485) for older patients (p<0.001). Seventeen studies completed multivariable analysis, 13 of which were significant (Odds Ratio 1.3-14.0). Glenoid bone loss was evaluated by 39 studies, with 20 finding an increased risk. Multivariable analysis in 9 studies found OR ranging from 0.7-35.1; 6 were significant. Off-track Hill-Sachs lesions were evaluated in 21 studies (13 significant), with 3 of 4 studies that conducted multivariable analysis finding a significant association with odds ratio of 2.9-8.9 of recurrence. The number of anchors used in repair was reported by 25 studies, with 4 finding increased risk with fewer anchors. Pooled analysis demonstrated a 25.0% (29/156) risk of recurrence with 2 anchors compared to 18.1% (89/491) with 3 or more anchors (p=0.06). Other frequently described risk factors included glenohumeral joint hyperlaxity (46% of studies reporting a significant association), number of preoperative dislocations (31%), contact sport participation (20%), competitive sport participation (46%), patient sex (7%), and concomitant SLAP tear (0%). CONCLUSION: Younger age, glenoid bone loss, and off-track Hill-Sachs lesions, are established risk factors for recurrence following arthroscopic Bankart repair. Other commonly reported risk factors included contact and competitive sports participation, number of fixation devices, and patient sex.

Gene Therapy and Spinal Fusion: Systematic Review and Meta-Analysis of the Available Data.

OBJECTIVE: To analyze the extant literature describing the application of gene therapy to spinal fusion. METHODS: A systematic review of the English-language literature was performed. The search query was designed to include all published studies examining gene therapy approaches to promote spinal fusion. Approaches were classified as ex vivo (delivery of genetically modified cells) or in vivo (delivery of growth factors via vectors). The primary endpoint was fusion rate. Random effects meta-analyses were performed to calculate the overall odds ratio (OR) of fusion using a gene therapy approach and overall fusion rate. Subgroup analyses of fusion rate were also performed for each gene therapy approach. RESULTS: Of 1179 results, 35 articles met criteria for inclusion (all preclinical), of which 26 utilized ex vivo approaches and 9 utilized in vivo approaches. Twenty-seven articles (431 animals) were included in the meta-analysis. Gene therapy use was associated with significantly higher fusion rates (OR 77; 95% confidence interval {CI}: [31, 192]; P < 0.001); ex vivo strategies had a greater effect (OR 136) relative to in vivo strategies (OR 18) (P = 0.017). The overall fusion rate using a gene therapy approach was 80% (95% CI: [62%, 93%]; P < 0.001); overall fusion rates were significantly higher in subjects treated with ex vivo compared to in vivo strategies (90% vs. 42%; P = 0.011). For both ex vivo and in vivo approaches, the effect of gene therapy on fusion was independent of animal model. CONCLUSIONS: Gene therapy may augment spinal fusion; however, future investigation in clinical populations is necessary.

SIDT2 Inhibits Phosphorothioate Antisense Oligonucleotide Activity by Regulating Cellular Localization of Lysosomes.

Phosphorothioate (PS)-modified antisense oligonucleotide (ASO) drugs enter cells through endocytic pathways where a majority are entrapped within membrane-bound endosomes and lysosomes, representing a limiting step for antisense activity. While late endosomes have been identified as a major site for productive PS-ASO release, how lysosomes regulate PS-ASO activity beyond macromolecule degradation remains not fully understood. In this study, we reported that SID1 transmembrane family, member 2 (SIDT2), a lysosome transmembrane protein, can robustly regulate PS-ASO activity. We showed that SIDT2 is required for the proper colocalization between PS-ASO and lysosomes, suggesting an important role of SIDT2 in the entrapment of PS-ASOs in lysosomes. Mechanistically, we revealed that SIDT2 regulates lysosome cellular location. Lysosome location is largely determined by its movement along microtubules. Interestingly, we also observed an enrichment of proteins involved in microtubule function among SIDT2-binding proteins, suggesting that SIDT2 regulates lysosome location via its interaction with microtubule-related proteins. Overall, our data suggest that lysosome protein SIDT2 inhibits PS-ASO activity potentially through its interaction with microtubule-related proteins to place lysosomes at perinuclear regions, thus, facilitating PS-ASO's localization to lysosomes for degradation.

N1-methylpseudouridine found within COVID-19 mRNA vaccines produces faithful protein products.

Synthetic mRNA technology is a promising avenue for treating and preventing disease. Key to the technology is the incorporation of modified nucleotides such as N1-methylpseudouridine (m1Ψ) to decrease immunogenicity of the RNA. However, relatively few studies have addressed the effects of modified nucleotides on the decoding process. Here, we investigate the effect of m1Ψ and the related modification pseudouridine (Ψ) on translation. In a reconstituted system, we find that m1Ψ does not significantly alter decoding accuracy. More importantly, we do not detect an increase in miscoded peptides when mRNA containing m1Ψ is translated in cell culture, compared with unmodified mRNA. We also find that m1Ψ does not stabilize mismatched RNA-duplex formation and only marginally promotes errors during reverse transcription. Overall, our results suggest that m1Ψ does not significantly impact translational fidelity, a welcome sign for future RNA therapeutics.

The RNA m6A Reader YTHDF2 Maintains Oncogene Expression and Is a Targetable Dependency in Glioblastoma Stem Cells.

Glioblastoma is a universally lethal cancer driven by glioblastoma stem cells (GSC). Here, we interrogated N 6-methyladenosine (m6A) mRNA modifications in GSCs by methyl RNA immunoprecipitation followed by sequencing and transcriptome analysis, finding transcripts marked by m6A often upregulated compared with normal neural stem cells (NSC). Interrogating m6A regulators, GSCs displayed preferential expression, as well as in vitro and in vivo dependency, of the m6A reader YTHDF2, in contrast to NSCs. Although YTHDF2 has been reported to destabilize mRNAs, YTHDF2 stabilized MYC and VEGFA transcripts in GSCs in an m6A-dependent manner. We identified IGFBP3 as a downstream effector of the YTHDF2-MYC axis in GSCs. The IGF1/IGF1R inhibitor linsitinib preferentially targeted YTHDF2-expressing cells, inhibiting GSC viability without affecting NSCs and impairing in vivo glioblastoma growth. Thus, YTHDF2 links RNA epitranscriptomic modifications and GSC growth, laying the foundation for the YTHDF2-MYC-IGFBP3 axis as a specific and novel therapeutic target in glioblastoma. SIGNIFICANCE: Epitranscriptomics promotes cellular heterogeneity in cancer. RNA m6A landscapes of cancer and NSCs identified cell type-specific dependencies and therapeutic vulnerabilities. The m6A reader YTHDF2 stabilized MYC mRNA specifically in cancer stem cells. Given the challenge of targeting MYC, YTHDF2 presents a therapeutic target to perturb MYC signaling in glioblastoma.This article is highlighted in the In This Issue feature, p. 211.

Identification of a Xist silencing domain by Tiling CRISPR.

Despite essential roles played by long noncoding RNAs (lncRNAs) in development and disease, methods to determine lncRNA cis-elements are lacking. Here, we developed a screening method named "Tiling CRISPR" to identify lncRNA functional domains. Using this approach, we identified Xist A-Repeats as the silencing domain, an observation in agreement with published work, suggesting Tiling CRISPR feasibility. Mechanistic analysis suggested a novel function for Xist A-repeats in promoting Xist transcription. Overall, our method allows mapping of lncRNA functional domains in an unbiased and potentially high-throughput manner to facilitate the understanding of lncRNA functions.

Publisher Correction: N6-methyladenosine RNA modification regulates embryonic neural stem cell self-renewal through histone modifications.

In the version of this article initially published online, there were errors in URLs for www.southernbiotech.com, appearing in Methods sections "m6A dot-blot" and "Western blot analysis." The first two URLs should be https://www.southernbiotech.com/?catno=4030-05&type=Polyclonal#&panel1-1 and the third should be https://www.southernbiotech.com/?catno=6170-05&type=Polyclonal. In addition, some Methods URLs for bioz.com, www.abcam.com and www.sysy.com were printed correctly but not properly linked. The errors have been corrected in the PDF and HTML versions of this article.

N6-methyladenosine RNA modification regulates embryonic neural stem cell self-renewal through histone modifications.

Internal N6-methyladenosine (m6A) modification is widespread in messenger RNAs (mRNAs) and is catalyzed by heterodimers of methyltransferase-like protein 3 (Mettl3) and Mettl14. To understand the role of m6A in development, we deleted Mettl14 in embryonic neural stem cells (NSCs) in a mouse model. Phenotypically, NSCs lacking Mettl14 displayed markedly decreased proliferation and premature differentiation, suggesting that m6A modification enhances NSC self-renewal. Decreases in the NSC pool led to a decreased number of late-born neurons during cortical neurogenesis. Mechanistically, we discovered a genome-wide increase in specific histone modifications in Mettl14 knockout versus control NSCs. These changes correlated with altered gene expression and observed cellular phenotypes, suggesting functional significance of altered histone modifications in knockout cells. Finally, we found that m6A regulates histone modification in part by destabilizing transcripts that encode histone-modifying enzymes. Our results suggest an essential role for m6A in development and reveal m6A-regulated histone modifications as a previously unknown mechanism of gene regulation in mammalian cells.

Exomic and Epigenomic Analyses in a Pair of Monozygotic Twins Discordant for Cryptorchidism.

Cryptorchidism represents one of the most common human congenital anomalies. In most cases, its etiology remains unclear and seems to be multifactorial. In the present study, a pair of monozygotic twins discordant for cryptorchidism was identified. Twin zygosity was confirmed by microsatellite genotyping. Whole exome sequencing and methylated DNA immunoprecipitation sequencing (MeDIP-Seq) of DNA extract from leucocytes were performed to, respectively, evaluate their exomes and epigenomes. No differences in exome sequencing data were found between the twins after validation. MeDIP-Seq analysis detected 5,410 differentially hypermethylated genes and 2,383 differentially hypomethylated genes. Bioinformatic analysis showed that these genes belonged to several biological processes and signaling pathways, including regulation of actin cytoskeleton, which has been previously implicated in the etiology of cryptorchidism. The findings of the present study suggest that non-genetic factors might contribute to the pathogenesis of cryptorchidism.

Messenger RNA Methylation Regulates Glioblastoma Tumorigenesis.

Messenger RNA (mRNA) modification provides an additional layer of gene regulation in cells. In this issue of Cancer Cell, Zhang et al. report that ALKBH5, a demethylase of the mRNA modification N6-methyladenosine, regulates proliferation and self-renewal of glioblastoma stem-like cells by modulating pre-mRNA stability and expression of the FOXM1 gene.

SMARCAD1 Contributes to the Regulation of Naive Pluripotency by Interacting with Histone Citrullination.

Histone citrullination regulates diverse cellular processes. Here, we report that SMARCAD1 preferentially associates with H3 arginine 26 citrullination (H3R26Cit) peptides present on arrays composed of 384 histone peptides harboring distinct post-transcriptional modifications. Among ten histone modifications assayed by ChIP-seq, H3R26Cit exhibited the most extensive genomewide co-localization with SMARCAD1 binding. Increased Smarcad1 expression correlated with naive pluripotency in pre-implantation embryos. In the presence of LIF, Smarcad1 knockdown (KD) embryonic stem cells lost naive state phenotypes but remained pluripotent, as suggested by morphology, gene expression, histone modifications, alkaline phosphatase activity, energy metabolism, embryoid bodies, teratoma, and chimeras. The majority of H3R26Cit ChIP-seq peaks occupied by SMARCAD1 were associated with increased levels of H3K9me3 in Smarcad1 KD cells. Inhibition of H3Cit induced H3K9me3 at the overlapping regions of H3R26Cit peaks and SMARCAD1 peaks. These data suggest a model in which SMARCAD1 regulates naive pluripotency by interacting with H3R26Cit and suppressing heterochromatin formation.

Update: Mechanisms Underlying N6-Methyladenosine Modification of Eukaryotic mRNA.

Eukaryotic mRNA undergoes chemical modification both at the 5' cap and internally. Among internal modifications, N6-methyladensone (m6A), by far the most abundant, is present in all eukaryotes examined so far, including mammals, flies, plants, and yeast. m6A modification has an essential role in diverse biological processes. Over the past few years, our knowledge relevant to the establishment and function of this modification has grown rapidly. In this review, we focus on technologies that have facilitated m6A detection in mRNAs, the identification of m6A methylation enzymes and binding proteins, and potential functions of the modification at the molecular level.

Rnf25/AO7 positively regulates wnt signaling via disrupting Nkd1-Axin inhibitory complex independent of its ubiquitin ligase activity.

Wnt signaling components have been shown to control key events in embryogenesis and to maintain tissue homeostasis in the adult. Nkd1/2 and Axin1/2 protein families are required for feedback regulation of Wnt signaling. The mechanisms by which Nkd1 and Nkd2 exhibit significant differences in signal transduction remain incompletely understood. Here we report that Rnf25/AO7, a previously identified E3 ubiquitin ligase for Nkd2, physically interacts with Nkd1 and Axin in an E3 ligase-independent manner to strengthen Wnt signalling. To determine the biological role of Rnf25 in vivo, we found that the renal mesenchymal cell, in which rnf25 was knocked-down, also exhibited more epithelial characters than MOCK control. Meanwhile, the transcriptional level of rnf25 was elevated in three separate tumor tissues more than that in paracarcinomatous tissue. Depletion of Rnf25 in zebrafish embryos attenuated transcriptions of maternal and zygotic Wnt target genes. Our results indicated that Rnf25 might serve as a molecular device, controlling the different antagonizing functions against canonical Wnt signaling between Nkd1 and Nkd2 cooperated with Axin.

Genome-wide detection of high abundance N6-methyladenosine sites by microarray.

N(6)-methyladenosine (m(6)A), the most abundant internal RNA modification, functions in diverse biological processes, including regulation of embryonic stem cell self-renewal and differentiation. As yet, methods to detect m(6)A in the transcriptome rely on the availability and quality of an m(6)A antibody and are often associated with a high rate of false positives. Here, based on our observation that m(6)A interferes with A-T/U pairing, we report a microarray-based technology to map m(6)A sites in mouse embryonic stem cells. We identified 72 unbiased sites exhibiting high m(6)A levels from 66 PolyA RNAs. Bioinformatics analyses suggest identified sites are enriched on developmental regulators and may in some contexts modulate microRNA/mRNA interactions. Overall, we have developed microarray-based technology to capture highly enriched m(6)A sites in the mammalian transcriptome. This method provides an alternative means to identify m(6)A sites for certain applications.

nRIP-seq: a technique to identify RNA targets of an RNA binding protein on a genome-wide scale.

Native RNA immunoprecipitation (nRIP) coupled with high-throughput sequencing (nRIP-seq) is a powerful technique that allows transcriptome-wide identification of the entire subset of coding and noncoding RNAs associated with a particular protein. Since this technology is carried out in a native condition without cross-linking, nRIP-seq detects RNAs that bind a protein directly or indirectly through a larger RNA-protein complex. Here, we use the interaction between RNA and chromatin modifiers, Polycomb proteins, as an example to describe this method. Using nRIP-seq, we provide a snapshot of Ezh2, a Polycomb component, and RNA interaction in mouse embryonic stem cells.

Functional analysis of long noncoding RNAs in development and disease.

Once viewed as part of the "dark matter" of genome, long noncoding RNAs (lncRNAs), which are mRNA-like but lack open reading frames, have emerged as an integral part of the mammalian transcriptome. Recent work demonstrated that lncRNAs play multiple structural and functional roles, and their analysis has become a new frontier in biomedical research. In this chapter, we provide an overview of different lncRNA families, describe methodologies available to study lncRNA-protein and lncRNA-DNA interactions systematically, and use well-studied lncRNAs as examples to illustrate their functional importance during normal development and in disease states.

N6-methyladenosine modification destabilizes developmental regulators in embryonic stem cells.

N(6)-methyladenosine (m(6)A) has been identified as the most abundant internal modification of messenger RNA in eukaryotes. m(6)A modification is involved in cell fate determination in yeast and embryo development in plants. Its mammalian function remains unknown but thousands of mammalian mRNAs and long non-coding RNAs (lncRNAs) show m(6)A modification and m(6)A demethylases are required for mammalian energy homeostasis and fertility. We identify two proteins, the putative m(6)A MTase, methyltransferase-like 3 (Mettl3; ref. ), and a related but uncharacterized protein Mettl14, that function synergistically to control m(6)A formation in mammalian cells. Knockdown of Mettl3 and Mettl14 in mouse embryonic stem cells (mESCs) led to similar phenotypes, characterized by lack of m(6)A RNA methylation and lost self-renewal capability. A large number of transcripts, including many encoding developmental regulators, exhibit m(6)A methylation inversely correlated with mRNA stability and gene expression. The human antigen R (HuR) and microRNA pathways were linked to these effects. This gene regulatory mechanism operating in mESCs through m(6)A methylation is required to keep mESCs at their ground state and may be relevant to thousands of mRNAs and lncRNAs in various cell types.

Regulation of Mammalian Gene Dosage by Long Noncoding RNAs.

Recent transcriptome studies suggest that long noncoding RNAs (lncRNAs) are key components of the mammalian genome, and their study has become a new frontier in biomedical research. In fact, lncRNAs in the mammalian genome were identified and studied at particular epigenetic loci, including imprinted loci and X-chromosome inactivation center, at least two decades ago-long before development of high throughput sequencing technology. Since then, researchers have found that lncRNAs play essential roles in various biological processes, mostly during development. Since much of our understanding of lncRNAs originates from our knowledge of these well-established lncRNAs, in this review we will focus on lncRNAs from the X-chromosome inactivation center and the Dlk1-Dio3 imprinted cluster as examples of lncRNA mechanisms functioning in the epigenetic regulation of mammalian genes.