BackgroundThe Hippo signaling pathway involves a kinase cascade that controls phosphorylation of the effector proteins YAP and TAZ, leading to regulation of cell growth, tissue homeostasis, and apoptosis. Morusin, a compound extracted from Morus alba, has shown potential in cancer therapy by targeting multiple signaling pathways, including the PI3K/Akt/mTOR, JAK/STAT, MAPK/ERK, and apoptosis pathways. This study explores the effects of morusin on YAP activation and its implications for apoptosis resistance.ResultsOur investigation revealed that morusin induces transient YAP activation, characterized by the dephosphorylation of YAP at S127 and nuclear localization, followed by gradual rephosphorylation in multiple cancer cells. Notably, this activation occurs independently of the canonical Hippo pathway and involves the LATS1/2, MINK1, and MAPK pathways during the YAP inactivation stage. Furthermore, morusin-induced stress granule formation was significantly impaired in YAP/TAZ-depleted cells, suggesting a role in apoptosis resistance. Additionally, the expression of constitutively active MINK1 maintained YAP activation and reduced apoptosis, indicating that prolonged YAP activation can enhance resistance to cell death.ConclusionsThese findings suggest that YAP/TAZ are crucial in resistance to morusin-induced apoptosis, and targeting YAP/TAZ could enhance the anti-cancer efficacy of morusin. Our study provides new insights into the molecular mechanisms of morusin, highlighting potential therapeutic strategies against cancer by disrupting apoptosis resistance.

BackgroundBioengineering of human teeth for replacement is an appealing regenerative approach in the era of gene therapy. Developmentally regulated transcription factors hold promise in the quest because these transcriptional regulators constitute the gene regulatory networks driving cell fate determination. Atonal homolog 1 (Atoh1) is a transcription factor of the basic helix-loop-helix (bHLH) family essential for neurogenesis in the cerebellum, auditory hair cell differentiation, and intestinal stem cell specification. The functional versatility of Atoh1 prompted us to test the possibility that Atoh1 may intersect the dental pulp stem cell (DPSC) gene regulatory network governing odontogenic differentiation.MethodsWe isolated DPSCs from human dental pulps and treated the cells with a replication-deficient adenoviral vector to achieve robust ectopic expression of Atoh1, following which the growth and odontogenic differentiation profiles of DPSCs were characterized.ResultsDPSCs harboring the Atoh1 expression vector exhibited an approximately 3,000-fold increase in the expression of Atoh1 compared to the negative control, leading to increased DPSC proliferation in the growth medium (P < 0.05). In the odontogenic medium, Atoh1 caused an early induction of BMP2 (P < 0.001) followed by a late induction of BMP7 (P < 0.01) and increased Wnt signaling (P < 0.01). The increased BMP/Wnt signaling led to up to 8-fold increased expression of the master osteogenic transcription factor Osterix (P < 0.005) while exhibiting no significant effect on Runx2 or Dlx5, which are abundantly expressed in DPSCs. Atoh1 stimulated expression of type I collagen (P < 0.005) and small integrin-binding ligand, N-linked glycoproteins (SIBLINGs) such as bone sialoprotein (P < 0.001), dentin matrix protein 1 (P < 0.05), dentin sialophosphoprotein (P < 0.005), and osteopontin (P < 0.001), resulting in increased dentin matrix mineralization (P < 0.05). The odontogenic phenotype is associated with metabolic remodeling marked by enhanced glycolytic flux and attenuated mitochondrial metabolic enzyme activities.ConclusionsAtoh1, despite being a proneural transcription factor in development, possesses a novel odontogenic function upon ectopic expression in DPSCs. This in vitro study demonstrates a novel odontogenic mechanism mediated by ectopic expression of the transcription factor Atoh1 in human DPSCs. The finding may offer an innovative strategy for gene-based regeneration of the pulp-dentin complex.

Oxytocin function is associated with a range of human traits and is often indexed by common polymorphisms of the receptor gene OXTR. Little is known however about the functional significance of these polymorphisms. To examine the effects of common polymorphisms of OXTR on transcription expression in human neural cells. The impact of four common OXTR SNPs (rs1042778, rs4686302, rs2254298 and rs237887) on OXTR gene expression were tested in human neuroblastoma cell line, SH-SY5Y, a commonly used cell line for neurological disease. SNPs were chosen as having robust evidence for associations with complex human traits after consideration of linkage patterns across OXTR. The expression level of GG genotype of rs1042778 was significantly lower than TT genotypes. None of the other SNPs were related to functional transcription. OXTR polymorphisms showing robust associations with complex human traits are not reliably associated with changes in transcription of OXTR. Increasing cooperation between behavioral and biological scientists is needed to bridge the gap between human trait and functional biological studies to improve our understanding of oxytocin and other important mammalian neuroendocrine processes.

BackgroundDuring the latter stages of their development, mammalian oocytes under dramatic chromatin reconfiguration, transitioning from a non-surrounded nucleolus (NSN) to a surrounded nucleolus (SN) stage, and concomitant transcriptional silencing. Although the NSN-SN transition is known to be essential for developmental competence of the oocyte, less is known about the accompanying molecular changes. Here we examine the changes in the transcriptome and DNA methylation during the NSN to SN transition in mouse oocytes.ResultsTo study the transcriptome and DNA methylation dynamics during the NSN to SN transition, we used single-cell (sc)M&T-seq to generate scRNA-seq and sc-bisulphite-seq (scBS-seq) data from GV oocytes classified as NSN or SN by Hoechst staining of their nuclei. Transcriptome analysis showed a lower number of detected transcripts in SN compared with NSN oocytes as well as downregulation of 576 genes, which were enriched for processes related to mRNA processing. We used the transcriptome data to generate a classifier that can infer chromatin stage in scRNA-seq datasets. The classifier was successfully tested in multiple published datasets of mouse models with a known skew in NSN: SN ratios. Analysis of the scBS-seq data showed increased DNA methylation in SN compared to NSN oocytes, which was most pronounced in regions with intermediate levels of methylation. Overlap with chromatin immunoprecipitation and sequencing (ChIP-seq) data for the histone modifications H3K36me3, H3K4me3 and H3K27me3 showed that regions gaining methylation in SN oocytes are enriched for overlapping H3K36me3 and H3K27me3, which is an unusual combination, as these marks do not typically coincide.ConclusionsWe characterise the transcriptome and DNA methylation changes accompanying the NSN-SN transition in mouse oocytes. We develop a classifier that can be used to infer chromatin status in single-cell or bulk RNA-seq data, enabling identification of altered chromatin transition in genetic knock-outs, and a quality control to identify skewed NSN-SN proportions that could otherwise confound differential gene expression analysis. We identify late-methylating regions in SN oocytes that are associated with an unusual combination of chromatin modifications, which may be regions with high chromatin plasticity and transitioning between H3K27me3 and H3K36me3, or reflect heterogeneity on a single-cell level.

BackgroundInactivation or mutations of FAM20C causes human Raine Syndrome, which manifests as lethal osteosclerosis bone dysplasia or non-lethal hypophosphatemia rickets. However, it is only hypophosphatemia rickets that was reported in the mice with Fam20c deletion or mutations. To further investigate the local and global impacts of Fam20c mutation, we constructed a knock-in allele carrying Fam20c mutation (D446N) found in the non-lethal Raine Syndrome. The Fam20cD446N allele replaced the WT Fam20c by 3.6Kb Col1a1-Cre to get the conditional knock-in mice, and by Hprt-cre to get conventional knock-in mice, respectively.ResultsThe radiology, serum biochemistry and immunohistochemistry indicated that all conditional and most conventional Fam20cD446N knock-in mice displayed hypophosphatemic rickets with the increased Fgf23 and deceased Dmp1 expression, which survived to adulthood. However, a few conventional Fam20cD446N knock-in mice died before weaning with the osteosclerotic X-ray radiography, though micro-CT assay displayed a reduced mineral density and increased porosity in the osteosclerotic tibia. Our results suggested that hypophosphatemia rickets was the predominant phenotype in both conditional and conventional Fam20c deficient mice, while the lethal osteosclerotic phenotype occasionally took place in the conventional Fam20c mutant mice.ConclusionThis finding also implicated that the osteosclerotic features resulting from Fam20c deficiency could be a semblance on the basis of rickets, which is most likely triggered by the alterations in the systems other than skeleton.

Cystic echinococcosis (CE) is a worldwide zoonotic public health issue. The reasons for this include a lack of specific therapy options, increasing antiparasitic drug resistance, a lack of control strategies, and the absence of an approved vaccine. The aim of the current study is to develop a multiepitope vaccine against CE by in-silico identification and using different Antigen B subunits. The five Echinococcus granulosus antigen B (EgAgB) subunits were examined for eminent antigenic epitopes, and then the best B-cell and Major Histocompatibility Complex MHC-binding epitopes were predicted. Most significant epitopes were combined to create an effective multi-epitope vaccine, which was then validated by testing its secondary and tertiary structures, physicochemical properties, and molecular dynamics (MD) modelling. A multi-epitope vaccine construct of 483 amino acid sequences was designed. It contains B-cell, Helper T Lymphocyte (HTL), and Cytotoxic T Lymphocyte (CTL) epitopes as well as the appropriate adjuvant and linker molecules. The resultant vaccinal construct had a GDT-HA value of 0.9725, RMSD of 0.299, MolProbity of 1.891, Clash score of 13.1, Poor rotamers of 0.9, and qualifying features with Rama favoured of 89.9. It was also highly immunogenic and less allergic. The majority of the amino acids were positioned in the Ramachandran plot’s favourable area, and during the molecular dynamic simulation at 100 ns, no notable structural abnormalities were noticed. The resultant construct was significantly expressed and received good endorsement in the pIB2-SEC13-mEGFP expressional vector. In conclusion, the current in-silico multi-epitope vaccine may be evaluated in-vitro, in-vivo, and in clinical trials as an immunogenic vaccine model. It can also play a vital role in preventing this zoonotic parasite infection.

Alanine-rich, alpha-helical type I antifreeze proteins (AFPs) in fishes are thought to have arisen independently in the last 30 Ma on at least four occasions. This hypothesis has recently been proven for flounder and sculpin AFPs, which both originated by gene duplication and divergence followed by substantial gene copy number expansion. Here, we examined the origins of the cunner (wrasse) and snailfish (liparid) AFPs. The cunner AFP has arisen by a similar route from the duplication and divergence of a GIMAP gene. The coding region for this AFP stems from an alanine-rich region flanking the GTPase domain of GIMAPa. The AFP gene has remained in the GIMAP gene locus and has undergone amplification there along with the GIMAPa gene. The AFP gene originated after the cunner diverged from its common ancestor with the closely related spotty and ballan wrasses, which exhibit similar gene synteny but lack AFP genes. Snailfish AFPs have also recently evolved because they are confined to a single genus of this family. In these AFP-producing species, the AFP locus does not share any similarity to functional genes. Instead, it is replete with repetitive DNAs and transposons, several stretches of which could encode alanine tracts with a dominant codon (GCC) that matches the bias observed in the AFP genes. All four known instances of type I AFPs occurring in fishes are independent evolutionary events that occurred soon after the onset of Northern Hemisphere Cenozoic glaciation events. Collectively, these results provide a remarkable example of convergent evolution to one AFP type.

In recent years, chondrocytes have been found to contain hemoglobin, which might be an alternative strategy for adapting to the hypoxic environment, while the potential mechanisms of that is still unclear. Here, we report the expression characteristics and potential associated pathways of hemoglobin in chondrocytes using single-cell RNA sequencing (scRNA-seq). We downloaded data of normal people and patients with osteoarthritis (OA) from the Gene Expression Omnibus (GEO) database and cells are unbiased clustered based on gene expression pattern. We determined the expression levels of hemoglobin in various chondrocyte subpopulations. Meanwhile, we further explored the difference in the enriched signaling pathways and the cell-cell interaction in chondrocytes of the hemoglobin high-expression and low-expression groups. Specifically, we found that SPP1 was closely associated with the expression of hemoglobin in OA progression. Our findings provide new insights into the distribution characteristics of hemoglobin in chondrocytes and provide potential clues to the underlying role of hemoglobin in OA and the mechanisms related to that, providing potential new ideas for the treatment of OA.

BackgroundCellular senescence is a key driver of decreased bone formation and osteoporosis. Leptin (LEP) has been implicated in cellular senescence and osteogenic differentiation. The aim of this study was to investigate the mechanisms by which LEP mediates cellular senescence and osteogenic differentiation.MethodsC3H10T1/2 cells were treated with etoposide to induce cellular senescence, which was assessed by β-galactosidase staining. Quantitative real-time PCR and western blotting were used to measure the levels of senescence markers p21 and p16, as well as osteogenic differentiation-related genes ALP, COL1A1, and RUNX2. Alkaline phosphatase (ALP) staining and alizarin red S staining were performed to evaluate osteogenic differentiation. The NF-κB pathway and O-GlcNAcylation were assessed by western blotting.ResultsEtoposide treatment increased the number of senescent cells and the levels of p21 and p16, along with elevated LEP expression. These effects were reversed by LEP knockdown. Additionally, LEP knockdown increased ALP staining density and osteoblast mineralization nodules, as well as the mRNA and protein levels of ALP, COL1A1, and RUNX2, indicating that LEP knockdown promoted osteogenic differentiation in C3H10T1/2 cells. Mechanistically, LEP knockdown inactivated the NF-κB pathway by inhibiting the nuclear translocation of p65. Furthermore, OGT was found to promote O-GlcNAcylation of LEP at the S50 site.ConclusionOur findings demonstrated that O-GlcNAcylation of LEP inactivated the NF-κB pathway by reducing LEP protein levels, thereby inhibiting cellular senescence and promoting osteogenic differentiation in C3H10T1/2 cells. This study may provide a novel therapeutic target for the treatment of osteoporosis.