Unraveling Key m6A Modification Regulators Signatures in Postmenopausal Osteoporosis through Bioinformatics and Experimental Verification.

OBJECTIVE: Bone marrow mesenchymal stem cells (BMSCs) show significant potential for osteogenic differentiation. However, the underlying mechanisms of osteogenic capability in osteoporosis-derived BMSCs (OP-BMSCs) remain unclear. This study aims to explore the impact of YTHDF3 (YTH N6-methyladenosine RNA binding protein 3) on the osteogenic traits of OP-BMSCs and identify potential therapeutic targets to boost their bone formation ability. METHODS: We examined microarray datasets (GSE35956 and GSE35958) from the Gene Expression Omnibus (GEO) to identify potential m6A regulators in osteoporosis (OP). Employing differential, protein interaction, and machine learning analyses, we pinpointed critical hub genes linked to OP. We further probed the relationship between these genes and OP using single-cell analysis, immune infiltration assessment, and Mendelian randomization. Our in vivo and in vitro experiments validated the expression and functionality of the key hub gene. RESULTS: Differential analysis revealed seven key hub genes related to OP, with YTHDF3 as a central player, supported by protein interaction analysis and machine learning methodologies. Subsequent single-cell, immune infiltration, and Mendelian randomization studies consistently validated YTHDF3's significant link to osteoporosis. YTHDF3 levels are significantly reduced in femoral head tissue from postmenopausal osteoporosis (PMOP) patients and femoral bone tissue from PMOP mice. Additionally, silencing YTHDF3 in OP-BMSCs substantially impedes their proliferation and differentiation. CONCLUSION: YTHDF3 may be implicated in the pathogenesis of OP by regulating the proliferation and osteogenic differentiation of OP-BMSCs.

METTL3-mediated m6A modification of SOX4 regulates osteoblast proliferation and differentiation via YTHDF3 recognition.

N6-methyladenosine (m6A), the most prevalent internal modification in mRNA, is related to the pathogenesis of osteoporosis (OP). Although methyltransferase Like-3 (METTL3), an m6A transferase, has been shown to mitigate OP progression, the mechanisms of METTL3-mediated m6A modification in osteoblast function remain unclear. Here, fluid shear stress (FSS) induced osteoblast proliferation and differentiation, resulting in elevated levels of METTL3 expression and m6A modification. Through Methylated RNA Immunoprecipitation Sequencing (MeRIP-seq) and Transcriptomic RNA Sequencing (RNA-seq), SRY (Sex Determining Region Y)-box 4 (SOX4) was screened as a target of METTL3, whose m6A-modified coding sequence (CDS) regions exhibited binding affinity towards METTL3. Further functional experiments demonstrated that knockdown of METTL3 and SOX4 hampered osteogenesis, and METTL3 knockdown compromised SOX4 mRNA stability. Via RNA immunoprecipitation (RIP) assays, we further confirmed the direct interaction between METTL3 and SOX4. YTH N6-Methyladenosine RNA Binding Protein 3 (YTHDF3) was identified as the m6A reader responsible for modulating SOX4 mRNA and protein levels by affecting its degradation. Furthermore, in vivo experiments demonstrated that bone loss in an ovariectomized (OVX) mouse model was reversed through the overexpression of SOX4 mediated by adeno-associated virus serotype 2 (AAV2). In conclusion, our research demonstrates that METTL3-mediated m6A modification of SOX4 plays a crucial role in regulating osteoblast proliferation and differentiation through its recognition by YTHDF3. Our research confirms METTL3-m6A-SOX4-YTHDF3 as an essential axis and potential mechanism in OP.

Defining the Role of Extrachromosomal DNA Amplifications in Medulloblastoma.

Circular extrachromosomal DNA (ecDNA), a common mechanism of oncogene amplification, has been identified as a major contributor to intratumoral heterogeneity and patient outcomes. In a recent publication in Nature Genetics, Chapman and colleagues further explored the role of ecDNA in the context of medulloblastoma. Using whole-genome sequencing, they found that 18% of the patients carry ecDNA amplification across a 468 medulloblastoma patient cohort. The presence of ecDNA was associated with worse survival. Single-cell FISH imaging and multiomic sequencing revealed that ecDNA copy number displayed a cell-to-cell variability within the sample, contributing to tumor heterogeneity. Furthermore, through sequencing and CRISPRi experiments, the authors uncovered frequent enhancer rewiring events on ecDNA that drive proliferation.

A novel lncRNA GM15416 regulates osteoblast apoptosis and differentiation through the c-Fos/Fas axis and mitigates osteoporosis.

Osteoporosis (OP) is a common systemic bone disorder, and the programmed cell death of osteoblasts is closely linked to the development of osteoporosis. Previous studies have shown that c-fos can cause osteoblast apoptosis. Furthermore, it has been demonstrated that long non-coding RNA (lncRNA) plays a pervasive role in regulating the biology of osteoblasts. Nevertheless, the precise role and mechanism of long non-coding RNA (lncRNA) in relation to c-Fos at the transcriptional level in osteoblast cell death remain uncertain. Compared with normal osteoblasts, serum deprivation resulted in significant upregulation of the transcription factor c-Fos and apoptosis-related Fas proteins in osteoblasts. In addition, the expression of lncRNA GM15416 related to c-Fos was significantly increased. The results showed that overexpression of c-Fos leads to an increase in downstream Fas protein, which subsequently leads to osteoblast apoptosis and hinders osteogenesis. On the contrary, a decrease in lncRNA GM15416 expression leads to a decrease in c-Fos/Fas expression, which hinders osteoblast apoptosis and promotes osteogenesis. Our results suggest that lncRNA GM15416 exerts inhibitory effects on osteoblast apoptosis and acts as a preventive factor against osteoporosis. As a result, GM15416 emerges as an important lncRNA associated with osteoporosis and holds potential as a future therapeutic target.

Live-Cell Imaging Shows Uneven Segregation of Extrachromosomal DNA Elements and Transcriptionally Active Extrachromosomal DNA Hubs in Cancer.

Oncogenic extrachromosomal DNA elements (ecDNA) play an important role in tumor evolution, but our understanding of ecDNA biology is limited. We determined the distribution of single-cell ecDNA copy number across patient tissues and cell line models and observed how cell-to-cell ecDNA frequency varies greatly. The exceptional intratumoral heterogeneity of ecDNA suggested ecDNA-specific replication and propagation mechanisms. To evaluate the transfer of ecDNA genetic material from parental to offspring cells during mitosis, we established the CRISPR-based ecTag method. ecTag leverages ecDNA-specific breakpoint sequences to tag ecDNA with fluorescent markers in living cells. Applying ecTag during mitosis revealed disjointed ecDNA inheritance patterns, enabling rapid ecDNA accumulation in individual cells. After mitosis, ecDNAs clustered into ecDNA hubs, and ecDNA hubs colocalized with RNA polymerase II, promoting transcription of cargo oncogenes. Our observations provide direct evidence for uneven segregation of ecDNA and shed new light on mechanisms through which ecDNAs contribute to oncogenesis. SIGNIFICANCE: ecDNAs are vehicles for oncogene amplification. The circular nature of ecDNA affords unique properties, such as mobility and ecDNA-specific replication and segregation behavior. We uncovered fundamental ecDNA properties by tracking ecDNAs in live cells, highlighting uneven and random segregation and ecDNA hubs that drive cargo gene transcription.See related commentary by Henssen, p. 293.This article is highlighted in the In This Issue feature, p. 275.

Diagnostic Value of Next-Generation Sequencing in Periprosthetic Joint Infection: A Systematic Review.

Next-generation sequencing (NGS) has developed rapidly in the last decade and is emerging as a promising diagnostic tool for periprosthetic joint infection (PJI). However, its diagnostic value for PJI is still uncertain. This systematic review aimed to explore the diagnostic value of NGS for PJI and verify its accuracy for culture-negative PJI patients. We conducted this systematic review in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Medline, Embase, and Cochrane Library were searched to identify diagnostic technique studies evaluating the accuracy of NGS in the diagnosis of PJI. The diagnostic sensitivity, specificity, and positive and negative predictive values were estimated for each article. The detection rate of NGS for culture-negative PJI patients or PJI patients with antibiotic administration history was also calculated. Of the 87 identified citations, nine studies met the inclusion criteria. The diagnostic sensitivities and specificities of NGS ranged from 63% to 96% and 73% to 100%, respectively. The positive and negative predictive values ranged from 71% to 100% and 74% to 95%, respectively. The detection rate of NGS for culture-negative PJI patients in six studies was higher than 50% (range from 82% to 100%), while in three studies it was lower than 50% (range from 9% to 31%). Also, the detection rate of NGS for PJIs with antibiotic administration history ranged from 74.05% to 92.31%. In conclusion, this systematic review suggests that NGS may have the potential to be a new tool for the diagnosis of PJI and should be considered to be added to the portfolio of diagnostic procedures. Furthermore, NGS showed a favorable diagnostic accuracy for culture-negative PJI patients or PJI patients with antibiotic administration history. However, due to the small sample sizes of studies and substantial heterogeneity among the included studies, more research is needed to confirm or disprove these findings.

Single-cell multimodal glioma analyses identify epigenetic regulators of cellular plasticity and environmental stress response.

Glioma intratumoral heterogeneity enables adaptation to challenging microenvironments and contributes to therapeutic resistance. We integrated 914 single-cell DNA methylomes, 55,284 single-cell transcriptomes and bulk multi-omic profiles across 11 adult IDH mutant or IDH wild-type gliomas to delineate sources of intratumoral heterogeneity. We showed that local DNA methylation disorder is associated with cell-cell DNA methylation differences, is elevated in more aggressive tumors, links with transcriptional disruption and is altered during the environmental stress response. Glioma cells under in vitro hypoxic and irradiation stress increased local DNA methylation disorder and shifted cell states. We identified a positive association between genetic and epigenetic instability that was supported in bulk longitudinally collected DNA methylation data. Increased DNA methylation disorder associated with accelerated disease progression and recurrently selected DNA methylation changes were enriched for environmental stress response pathways. Our work identified an epigenetically facilitated adaptive stress response process and highlights the importance of epigenetic heterogeneity in shaping therapeutic outcomes.

Effect of palm oil consumption on plasma lipid concentrations related to cardiovascular disease: a systematic review and meta-analysis.

BACKGROUND AND OBJECTIVES: The precise association between palm oil consumption and lipid-related cardiovascular disease risk remains unclear. A systematic review was thus performed to assess whether palm oil consumption has a negative effect on plasma lipid-related cardiovascular disease marker levels. METHODS AND STUDY DESIGN: In June 2018, the electronic bibliographic databases PubMed, EMBASE (Ovid), the Cochrane Library (Ovid) and the Chinese National Knowledge Infrastructure were searched and a total of 11 eligible dietary intervention articles involving 961 volunteers were selected. Both random and fixed effect models were used to calculate pooled weighted mean differences (WMD). RESULTS: A total of 11 articles involving 547 participants met the inclusion criteria. The pooled analysis revealed that palm oil increased the concentration of high-density lipoprotein cholesterol (WMD: 0.15 mmol/L; p<0.00001). Palm oil consumption had no significant effects on blood total cholesterol (WMD: -0.01 mmol/L; p=0.82) and LDL-c (WMD: -0.05mmol/L; p=0.10) and triglyceride concentrations (WMD: 0.00 mmol/L; p=0.96), relative to the effects of unsaturated fatty acid consumption. Subgroup analyses revealed that palm oil has a beneficial effect on High-density lipoprotein cholesterol levels when more than 30% of total dietary energy was constituted by fat. CONCLUSIONS: This review revealed that palm oil does not induce increases in cardiovascular disease risk risk-related biomarkers relative to unsaturated fatty acids. Furthermore, larger-scale samples of human dietary intervention trials are required to increase the accuracy of meta-analyses.

Marrow stromal stem cell autologous transplantation in denervated fracture healing: an experimental study in rats.

OBJECTIVE: To investigate the influence of bone marrow stromal stem cell (BMSCs) transplantation on healing of fractures combined with central nerve injuries in rats. METHODS: Forty-eight healthy adult SD male rats were randomly divided into the following three groups (16 rats in each group): group A, simple (left) tibial fracture; group B, tibial fracture combined with T10 spinal cord transection (SCT); group C, tibial fracture combined with T10 SCT and BMSCs transplantation. The tibial fractures were stabilized with modular intramedullary nails and all operated hind limbs were further immobilized in plaster casts to prevent unequal load bearing. BMSCs were labeled with bromodeoxyuridine and implanted into the fractures of C group rats 2 days after creation of the model. The animals in B and C groups were evaluated by postoperative Tarlov scores. The fractured tibiae were evaluated separately radiographically (X-ray and CT) and immunohistochemically 1, 2, 3 and 4 weeks after injury to assess fracture healing. In addition, the wet weights of the left tibias were measured. RESULTS: All Tarlov score of the B and C group animals reached the requirements of the experiment. One, 2 and 3 weeks after surgery, the tibial callus widths in B and C group animals were significantly greater than those of group A rats (P < 0.05). At 4 weeks the tibial callus width in group C animals had decreased, but still differed significantly from that in group A rats (P < 0.05). One, 2, 3 and 4 weeks after surgery, the wet weights of B and C group tibias were significantly greater than those of group A (P < 0.05). Hematoxylin-eosin-stained sections showed bony union and increased bone trabecula in B and C groups and areas with particles positive for alkaline phosphatase staining were more abundant in groups B and C, especially in group C. CONCLUSION: Neural regulation plays an important role in fracture healing. Treatment with BMSCs has a positive effect on defective callus in rats that have been subjected to SCT.