Renal macrophages monitor and remove particles from urine to prevent tubule obstruction.

When the filtrate of the glomerulus flows through the renal tubular system, various microscopic sediment particles, including mineral crystals, are generated. Dislodging these particles is critical to ensuring the free flow of filtrate, whereas failure to remove them will result in kidney stone formation and obstruction. However, the underlying mechanism for the clearance is unclear. Here, using high-resolution microscopy, we found that the juxtatubular macrophages in the renal medulla constitutively formed transepithelial protrusions and "sampled" urine contents. They efficiently sequestered and phagocytosed intraluminal sediment particles and occasionally transmigrated to the tubule lumen to escort the excretion of urine particles. Mice with decreased renal macrophage numbers were prone to developing various intratubular sediments, including kidney stones. Mechanistically, the transepithelial behaviors of medulla macrophages required integrin ß1-mediated ligation to the tubular epithelium. These findings indicate that medulla macrophages sample urine content and remove intratubular particles to keep the tubular system unobstructed.

Dual-Stimuli-Responsive Modulation Organic Afterglow Based on N─H Proton Migration Mechanism.

Organic afterglow materials have significant applications in information security and flexible electronic devices with unique optical properties. It is vital but challenging to develop organic afterglow materials possessing controlled output with multi-stimuli-responsive capacity. Herein, dimethyl terephthalate (DTT) is introduced as a strong proton acceptor. The migration direction of N─H protons on two compounds Hs can be regulated by altering the excitation wavelength (Ex) or amine stimulation, thereby achieving dual-stimuli-responsive afterglow emission. When the Ex is below 300 nm, protons migrate to S1-2 DTT, where strong interactions induce phosphorescent emission of Hs, resulting in afterglow behavior. Conversely, when the Ex is above 300 nm, protons interact with the S0 DTT weakly and the afterglow disappears. In view of amine-based compounds with higher proton accepting capabilities, it can snatch proton from S1-2 DTT and redirect the proton flow toward amine, effectively suppressing the afterglow but obtaining a new redshifted fluorescence emission with Δλ over 200 nm due to the high polarity of amine. Moreover, it is successfully demonstrated that the applications of dual-stimuli-responsive organic afterglow materials in information encryption based on the systematic excitation-wavelength-dependent (Ex-De) behavior and amine selectivity detection.

A novel mutation in intron 1 of Wnt1 causes developmental loss of dopaminergic neurons in midbrain and ASD-like behaviors in rats.

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders with a strong genetic liability. Despite extensive studies, however, the underlying pathogenic mechanism still remains elusive. In the present study, we identified a homozygous mutation in the intron 1 of Wnt1 via large-scale screening of ASD risk/causative genes and verified that this mutation created a new splicing donor site in the intron 1, and consequently, a decrease of WNT1 expression. Interestingly, humanized rat models harboring this mutation exhibited robust ASD-like behaviors including impaired ultrasonic vocalization (USV), decreased social interactions, and restricted and repetitive behaviors. Moreover, in the substantia nigra compacta (SNpc) and the ventral tegmental area (VTA) of mutant rats, dopaminergic (DAergic) neurons were dramatically lost, together with a comparable decrease in striatal DAergic fibers. Furthermore, using single-cell RNA sequencing, we demonstrated that the decreased DAergic neurons in these midbrain areas might attribute to a shift of the boundary of the local pool of progenitor cells from the hypothalamic floor plate to the midbrain floor plate during the early embryonic stage. Moreover, treatments of mutant rats with levodopa could attenuate the impaired USV and social interactions almost completely, but not the restricted and repetitive behaviors. Our results for the first time documented that the developmental loss of DAergic neurons in the midbrain underlies the pathogenesis of ASD, and that the abnormal progenitor cell patterning is a cellular underpinning for this developmental DAergic neuronal loss. Importantly, the effective dopamine therapy suggests a translational significance in the treatment of ASD.

The RALF1-FERONIA interaction modulates endocytosis to mediate control of root growth in Arabidopsis.

The interaction between the receptor-like kinase (RLK) FERONIA (FER) and the secreted peptide RAPID ALKALINIZATION FACTOR1 (RALF1) is vital for development and stress responses in Arabidopsis Ligand-induced membrane dynamics affect the function of several RLKs, but the effects of the RALF1-FER interaction on the dynamics of FER and the ensuing effects on its functionality are poorly understood. Here, we show that RALF1 modulated the dynamics and partitioning of FER-GFP at the plasma membrane (PM). Moreover, FER was internalized by both clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE) under steady-state conditions. After RALF1 treatment, FER-GFP internalization was primarily enhanced via the CME pathway, raising FER-GFP levels in the vacuole. RALF1 treatment also modulated trafficking of other PM proteins, such as PIN2-GFP and BRI1-GFP, increasing their vacuolar levels by enhancing their internalization. Importantly, blocking CME attenuated RALF1-mediated root growth inhibition independently of RALF1-induced early signaling, suggesting that the RALF1 can also exert its effects via the CME pathway. These findings reveal that the RALF1-FER interaction modulates plant growth and development, and this might also involve endocytosis of PM proteins.

KDM4D enhances osteo/dentinogenic differentiation and migration of SCAPs via binding to RPS5.

OBJECTIVES: Stem cells of the apical papilla (SCAPs) provide promising candidates for dental pulp regeneration. Despite great advances in the transcriptional controls of the SCAPs fate, little is known about the regulation of SCAP differentiation. MATERIALS AND METHODS: Short hairpin RNAs and full-length RNA were used to deplete or overexpress lysine demethylase 4D (KDM4D) gene expression. Western blotting, real-time RT-PCR, alizarin red staining, and scratch migration assays were used to study the role of KDM4D and the ribosomal protein encoded by RPS5 in SCAPs. RNA microarray, chromatin Immunoprecipitation (ChIP), and co-immunoprecipitation (Co-IP) assays were performed to explore the underlying molecular mechanisms. RESULTS: KDM4D enhanced the osteo/dentinogenic differentiation, migration, and chemotaxis of SCAPs. The microarray results revealed that 88 mRNAs were differentially expressed in KDM4D-overexpressed SCAPs. ChIP results showed knock-down of KDM4D increased the level of H3K9me2 and H3K9me3 in CNR1 promoter region. There were 37 possible binding partners of KDM4D. KDM4D was found to combine with RPS5, which also promoted the osteo/dentinogenic differentiation, migration, and chemotaxis of SCAPs. CONCLUSIONS: KDM4D promoted the osteo/dentinogenic differentiation and migration potential of SCAPs in combination with RPS5, which provides a therapeutic clue for improving SCAPs-based dental tissue regeneration.

Insulin-like growth factor binding protein 5 accelerate the senescence of periodontal ligament stem cells.

Evidences have showed stem cell mediated tissue regeneration is a promising method for the treatment of periodontitis. Insulin-like growth factor binding proteins-5 (IGFBP5) is a member of the insulin growth factor (IGFs) family and plays a regulatory role in cell proliferation and differentiation. Our previous study showed that IGFBP5 can promote osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and enhance periodontal tissue regeneration mediated by PDLSCs. However, the function of IGFBP5 in the process of PDLSCs senescence remains unclear. The present study showed IGFBP5 mRNA level was highly expressed in passage-induced aged PDLSCs cells. IGFBP5 knockdown decreased the ratio of senescence associated ß-galactosidase (SA-ß-Gal) positive cells, enhanced the activity of TERT, and down-regulated the expression levels of P16, P21, P53 mRNA and protein. Overexpression of IGFBP5 increased the ratio of SA-ß-Gal positive staining PDLSCs, decreased the activity of telomerase TERT, and up-regulated the expression levels of P16, P21, P53 mRNA and protein related to PDLSCs senescence. In conclusion, IGFBP5 can accelerate the senescence of PDLSCs, indicating the potential target for maintaining the "young state" of stem cells.

MicroRNA-196a-5p overexpression in Wharton's jelly umbilical cord stem cells promotes their osteogenic differentiation and new bone formation in bone defects in the rat calvarium.

The peri-tooth root alveolar loss often does not have sufficient space for repair material transplantation and plasticity. Mesenchymal stem cell (MSC) sheets have an advantage in providing more extracellular matrix (ECM) and may prove to be a new therapeutic consideration for this bone defect repair. The identification of key regulators that stimulate MSCs' osteogenic potential and sheet-derived ECM deposition is the key to promoting its application. In this study, we found that inhibition or overexpression of miR-196a-5p led to a decline or enhancement, respectively, in the alkaline phosphatase (ALP) activity, mineralization, and the levels of osteogenic markers, Osteocalcin (OCN), Dentin Matrix Protein 1 (DMP1), Bone Sialoprotein (BSP), and Dentin Sialophosphoprotein (DSPP) of Wharton's jelly of umbilical cord stem cells (WJCMSCs) in vitro. Moreover, the 5,6-Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) analysis revealed inhibition of the WJCMSCs' proliferative ability upon miR-196a-5p overexpression. Characterization of the sheet formation by picrosirius red and Masson staining indicated that miR-196a-5p overexpression significantly promoted the collagen content in whole WJCMSC sheet-derived ECM. Furthermore, micro-CT and histopathology results indicated that the miR-196a-5p-overexpressed WJCMSC sheets significantly promoted new bone regeneration and rat calvarial bone defect closure 12 weeks following transplantation. The mRNA microarray analysis of miR-196a-5p-overexpressed WJCMSCs revealed 959 differentially expressed genes (DEGs) (34 upregulated and 925 downregulated). Moreover, 241 genes targeted by miR-196a-5p were predicted by using miRNA function websites of which only 19 predicted genes were consistent with the microarray revealed DEGs. Hence, one unrevealed downregulated DEG Serpin Family B Member 2 (SERPINB2) was investigated. And the deletion of SERPINB2 enhanced the ALP activity and mineralization of WJCMSCs in vitro. In conclusion, our study found that miR-196a-5p, as a key regulator, could repress the proliferation tendency, while stimulating osteogenic ability and WJCMSC sheet-derived ECM deposition, thus promoting new bone formation and rat calvarial bone defect closure. Furthermore, SERPINB2 is a key downstream gene involved in the miR-196a-5p-promoted WJCMSC osteogenesis.

PRMT6/LMNA/CXCL12 signaling pathway regulated the osteo/odontogenic differentiation ability in dental stem cells isolated from apical papilla.

Tooth loss and maxillofacial bone defect are common diseases, which seriously affect people's health. Effective tooth and maxillofacial bone tissue regeneration is a key problem that need to be solved. In the present study, we investigate the role of PRMT6 in osteo/odontogenic differentiation and migration capacity by using SCAPs. Our results showed that knockdown of PRMT6 promoted the osteo/odontogenic differentiation compared with the control group, as detected by alkaline phosphatase activity, alizarin red staining, and the indicators of osteo/odontogenic differentiation measured by Western blot. In addition, overexpression of PRMT6 inhibited the osteo/odontogenic differentiation potentials of SCAPs. Then, knockdown of PRMT6 promoted the migration ability and overexpression of PRMT6 inhibited the migration ability in SCAPs. Mechanically, we discovered that the depletion of PRMT6 promoted the expression of CXCL12 by decreasing H3R2 methylation in the promoter region of CXCL12. In addition, PRMT6 formed a protein complex with LMNA, a nuclear structural protein. Depletion of LMNA inhibited the osteo/odontogenic differentiation and CXCL12 expression and increased the intranucleus PRMT6 in SCAPs. To sum up, PRMT6 might inhibit the osteo/odontogenic differentiation and migration ability of SCAPs via inhibiting CXCL12. And LMNA might be a negative regulator of PRMT6. It is suggested that PRMT6 may be a key target for SCAP-mediated bone and tooth tissue regeneration.

LncRNA, PLXDC2-OT promoted the osteogenesis potentials of MSCs by inhibiting the deacetylation function of RBM6/SIRT7 complex and OSX specific isoform.

Bone regeneration and remodeling are complex physiological processes that are regulated by key transcription factors. Understanding the regulatory mechanism of key transcription factors on the osteogenic differentiation of mesenchymal stem cells (MSCs) is a key issue for successful bone regeneration and remodeling. In the present study, we investigated the regulatory mechanism of the histone deacetylase Sirtuin 7 (SIRT7) on the key transcription factor OSX and osteogenesis of MSCs. In this study, we found that SIRT7 knockdown increased ALP activity and in vitro mineralization and promoted the expression of the osteogenic differentiation markers DSPP, DMP1, BSP, OCN, and the key transcription factor OSX in MSCs. In addition, SIRT7 could associate with RNA binding motif protein 6 (RBM6) to form a protein complex. Moreover, RBM6 inhibited ALP activity, the expression of DSPP, DMP1, BSP, OCN, and OSX in MSCs, and the osteogenesis of MSCs in vivo. Then, the SIRT7/RBM6 protein complex was shown to downregulate the level of H3K18Ac in the OSX promoter by recruiting SIRT7 to the OSX promoter and inhibiting the expression of OSX isoforms 1 and 2. Furthermore, lncRNA PLXDC2-OT could associate with the SIRT7/RBM6 protein complex to diminish its binding and deacetylation function in the OSX promoter and its inhibitory function on OSX isoforms 1 and 2 and to promote the osteogenic potential of MSCs.

Initial respiratory support modality and outcome in preterm infants with less than 32 weeks of gestation in China: A multicentre retrospective cohort study.

BACKGROUND: For initial respiratory management, continuous positive airway pressure (CPAP) is increasingly used for preterm infants, especially for gestational age less than 32 weeks. However, neonatologists are concerned about the potential risks of CPAP support failure. OBJECTIVES: To examine the association between different initial respiratory support modalities and the outcomes of preterm infants at <32 weeks of gestation across multiple neonatal intensive care units (NICU) in China. METHODS: This study was carried out over a period of 12 months in 2018. Unadjusted relative risks (RR) for demographic and clinical characteristics were calculated for CPAP failure and CPAP success in the total cohort using log-linear model based on generalised estimating equations for clustered observations. RESULTS: Among 1560 preterm infants delivered at <32 weeks, the incidence of CPAP failure was 10.3%. After adjustment for demographic and clinical factors, the relative risk of mortality (RR 7.54, 95% CI 5.56, 10.44), pneumothorax (RR 9.85, 95% CI 2.89, 61.53), pulmonary haemorrhage (RR 7.78, 95% CI 4.51, 14.64) and BPD (RR 3.65, 95% CI 3.65, 4.51) were considerably higher for infants in the CPAP failure group than those in the CPAP-S group. However, the risk of poor outcomes in CPAP failure infants was similar to that of those in the initial mechanical ventilation (MV) group. CONCLUSIONS: Continuous positive airway pressure failure was associated with an increased risk of mortality and major morbidities, including BPD, pulmonary haemorrhage and pneumothorax, and was comparable to the risk associated with initial MV.

Synthesis of Novel α-Trifluoroanisole Derivatives Containing Phenylpyridine Moieties with Herbicidal Activity.

To find novel herbicidal compounds with high activity and broad spectrum, a series of phenylpyridine moiety-containing α-trifluoroanisole derivatives were designed, synthesized, and identified via nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). Greenhouse-based herbicidal activity assays revealed that compound 7a exhibited > 80% inhibitory activity against Abutilon theophrasti, Amaranthus retroflexus, Eclipta prostrate, Digitaria sanguinalis, and Setaria viridis at a dose of 37.5 g a.i./hm2, which was better than fomesafen. Compound 7a further exhibited excellent herbicidal activity against Abutilon theophrasti and Amaranthus retroflexus in this greenhouse setting, with respective median effective dose (ED50) values of 13.32 and 5.48 g a.i./hm2, both of which were slightly superior to fomesafen (ED50 = 36.39, 10.09 g a.i./hm2). The respective half-maximal inhibitory concentration (IC50) for compound 7a and fomesafen when used to inhibit the Nicotiana tabacum protoporphyrinogen oxidase (NtPPO) enzyme, were 9.4 and 110.5 nM. The docking result of compound 7a indicated that the introduction of 3-chloro-5-trifluoromethylpyridine and the trifluoromethoxy group was beneficial to the formation of stable interactions between these compounds and NtPPO. This work demonstrated that compound 7a could be further optimized as a PPO herbicide candidate to control various weeds.

GREM1 inhibits osteogenic differentiation, senescence and BMP transcription of adipose-derived stem cells.

Purpose: Adipose-derived stem cells (ADSCs) are ideal for cell-based therapies to support bone regeneration. It is vital to understand the critical genes and molecular mechanisms involved in the functional regulation of ADSCs for enhancing bone regeneration. In the present study, we investigated the Gremlin 1 (GREM1) effect on ADSCs osteogenic differentiation and senescence.Materials and methods: The in vitro ADSCs osteogenic differentiation potential was evaluated by determining alkaline phosphatase (ALP) activity, mineralization ability, and the expression of osteogenic markers. Cell senescence is determined by SA-ß-gal staining, telomerase assay, and the expression of aging markers.Results: GREM1 overexpression in ADSCs reduced ALP activity and mineralization, inhibited the expression of osteogenic related genes OCN, OPN, DSPP, DMP1, and BSP, and key transcription factors, RUNX2 and OSX. GREM1 knockdown in ADSCs enhanced ALP activity and mineralization, promoted the expression of OCN, OPN, DSPP, DMP1, BSP, RUNX2, and OSX. GREM1 overexpression in ADSCs reduced the percent SA-ß-Gal positive cells, P16 and P53 expressions, and increased telomerase activity. GREM1 knockdown in ADSCs increased the percentage of SA-ß-Gal positive cells, P16 and P53 expressions, and reduced telomerase activity. Furthermore, GREM1 reduced the mRNA expression levels of BMP2, BMP6, and BMP7.Conclusions: In summary, our findings suggested that GREM1 inhibited ADSCs senescence and osteogenic differentiation and antagonized BMP transcription.

SFRP2 enhances dental pulp stem cell-mediated dentin regeneration in rabbit jaw.

OBJECTIVES: Dental tissue-derived mesenchymal stem cell (MSC)-mediated tooth regeneration may be a useful therapeutic tool for repairing tooth loss. However, the low success rate of tooth regeneration restricts its clinical application. Identifying key factors for enhancing dentinogenesis in MSCs is crucial for promoting tooth regeneration. MATERIALS AND METHODS: Human dental pulp stem cells (DPSCs) were transfected with retrovirus to obtain SFRP2-over-expressing DPSCs. Alkaline phosphatase (ALP) activity assay, Alizarin red staining, quantitative analysis of calcium, and dentinogenesis-related genes were detected. Additionally, transplantation in a rabbit tooth extraction model was used to explore the role of SFRP2 in dentin regeneration. RESULTS: We found SFRP2 over-expression greatly enhanced ALP activity, and mineralization in DPSCs. Real-time RT-PCR revealed SFRP2 over-expression promoted the expressions of OSX, RUNX2, DSPP, DMP1, and BSP. Moreover, Micro CT analysis showed high-density calcification occurred to a much higher extent in SFRP2 over-expressing group compared to control group in vivo. Additionally, HE staining, immmunohistochemistry staining, and scanning electron microscopy results showed much more dentin-like tissue formed in SFRP2 over-expressing group compared to control group. CONCLUSIONS: Our findings revealed SFRP2 is an important regulator that enhances the dentinogenesis of DPSCs and dentin regeneration in the jaw, which may have clinical applications.

Depletion of EREG enhances the osteo/dentinogenic differentiation ability of dental pulp stem cells via the p38 MAPK and Erk pathways in an inflammatory microenvironment.

BACKGROUND: Epiregulin (EREG) is an important component of EGF and was demonstrated to promote the osteo/dentinogenic differentiation of stem cells from dental apical papilla (SCAPs). Whether EREG can stimulate the osteo/dentinogenic differentiation of dental pulp stem cells (DPSCs) in inflammatory environment is not clear. The purpose of the present study is to investigate the role of EREG on the osteo/dentinogenic differentiation ability of DPSCs in inflammatory environment. METHODS: DPSCs were isolated from human third molars. Short hairpin RNAs (shRNAs) were used to knock down EREG expression in DPSCs. Recombinant human EREG (rhEREG) protein was used in the rescue experiment. TNF-α was employed to mimic the inflammatory environment in vitro. Alkaline phosphatase (ALP) staining, Alizarin red staining, quantitative calcium analysis, and real-time RT-PCR were performed to detect osteo/dentinogenic differentiation markers and related signalling pathways under normal and inflammatory conditions. RESULTS: EREG depletion promoted the ALP activity and mineralization ability of DPSCs. The expression of BSP, DMP-1, and DSPP was also enhanced. Moreover, 50 ng/mL rhEREG treatment decreased the osteo/dentinogenic differentiation potential of DPSCs, while treatment with 10 ng/mL TNF-α for 4 h increased the expression of EREG in DPSCs. Conversely, EREG knockdown rescued the impaired osteo/dentinogenic differentiation ability caused by TNF-α treatment. Further mechanistic studies showed that EREG depletion activated the p38 MAPK and Erk signalling pathways in DPSCs under normal and inflammatory conditions. CONCLUSIONS: Our results demonstrated that EREG could inhibit the osteo/dentinogenic differentiation potential of DPSCs via the p38 MAPK and Erk signalling pathways. Under inflammatory environment, EREG depletion enhanced osteo/dentinogenic differentiation potential of DPSCs by improving the expression of p-p38 MAPK and p-Erk.

Homeobox C8 inhibited the osteo-/dentinogenic differentiation and migration ability of stem cells of the apical papilla via activating KDM1A.

Enhancing the functions of mesenchymal stem cells (MSCs) is considered a potential approach for promoting tissue regeneration. In the present study, we investigate the role of HOXC8 in regulating differentiation and migration by using stem cells of the apical papilla (SCAPs). Our results showed that overexpression of HOXC8 suppressed the osteo-/dentinogenic differentiation, as detected by measuring alkaline phosphatase activity, in vitro mineralization, and the expressions of dentin sialophosphoprotein, dentin matrix acidic phosphoprotein 1, bone sialoprotein, runt-related transcription factor 2, and osterix in SCAPs, and inhibited in vivo osteo-/dentinogenesis of SCAPs. In addition, knockdown of HOXC8 promoted the osteo-/dentinogenic differentiation potentials of SCAPs. Mechanically, HOXC8 enhanced KDM1A transcription by directly binding to its promoter. HOXC8 and KDM1A also inhibited the migration and chemotaxis abilities of SCAPs. To sum up, HOXC8 negatively regulated the osteo-/dentinogenic differentiation and migration abilities of SCAPs by directly enhancing KDM1A transcription and indicated that HOXC8 and KDM1A could serve as potential targets for enhancing dental MSC mediated tissue regeneration.

Enhanced transmission through sub-wavelength aperture in specific frequency band by using topology optimized metamaterials.

Aiming at achieving metamaterials (MTM)-based enhanced transmission through the sub-wavelength aperture on a metallic isolating plate in specific frequency band, the topology optimization method for MTM microstructure design was proposed. The MTM was inserted in the sub-wavelength aperture and perpendicular to the isolating plate. A piecewise preset function was employed to describe the expected enhanced and non-enhanced transmission frequency band. The transmission coefficient of the waveguide system with the designed MTM was mapped to a step mapping function. In the topology optimization of the MTM configuration, matching the mapping function to the preset function was chosen as the design objective. Three designs aiming at different specific enhanced transmission frequency band were carried out. The design satisfied the demand for the specific enhanced transmission frequency band, which was also validated by experiment.

Epiregulin promotes osteogenic differentiation and inhibits neurogenic trans-differentiation of adipose-derived mesenchymal stem cells via MAPKs pathway.

Mesenchymal stem cells (MSCs) exists low efficiency to trans-differentiate into other germinal layer cell types. One key issue is to discover the effect of important factor on MSCs differentiation abiltiy. In this study, we investigated the role and mechanism of epiregulin (EREG) on the osteogenic differentiation and neurogenic trans-differentiation in adipose-derived stem cells (ADSCs). We discovered that the depletion of EREG inhibited the osteogenic differentiation in vitro. And 25 ng/mL recombinant human epiregulin protein (rhEREG) effectively improved the osteogenic differentiation of EREG-depleted-ADSCs. Depletion of EREG promoted the formation of neural spheres, and increased the expressions of nestin, ßIII-tubulin, NeuroD, NCAM, TH, and NEF in ADSCs. Then, 25 ng/mL rhEREG significantly inhibited these neurogenic differentiation indicators. Inhibition of p38 MAPK, JNK, or Erk1/2 signaling pathway separately, blocked the rhEREG-enhanced osteogenic differentiation ability and the rhEREG-inhibited neurogenic trans-differentiation ability of ADSCs. In conclusions, EREG promoted the osteogenic differentiation and inhibited the neurogenic trans-differentiation potentials of ADSCs via MAPK signaling pathways.

FAM96B inhibits the senescence of dental pulp stem cells.

Dental pulp stem cells (DPSCs) are considered a remarkable source for the regeneration of dental pulp tissues, but their therapeutic effectiveness remains limited, especially in elderly people. Previous studies found that senescence has a negative effect on the proliferation and differentiation potential of DPSCs. Moreover, numerous long non-coding RNA (lncRNA) and messenger RNA were significantly differentially regulated in DPSCs from young and elderly donors. However, the changes in DPSCs protein during senescence have not been addressed. In this study, differences in DPSC protein expression profiles and coexpression of protein and lncRNA were analyzed using proteomics and bioinformatics. The results showed 75 upregulated proteins and 69 downregulated proteins in DPSCs from elderly donors. Vasopressin-regulated water reabsorption, Parkinson's disease, Alzheimer's disease, and protein export were the top four functional pathways associated with DPSCs. High mobility group N1 (HMGN1), HMGN2, UCHL1, and the family with sequence similarity 96 member B homeobox gene (FAM96B) were associated with DPSCs senescence. Then, we investigated FAM96B function in DPSCs. After FAM96B depletion, telomerase reverse transcriptase (TERT) activity decreased, but the number of senescence-associated ß-galactosidase (SA-ß-gal) positive cells and the protein levels of p16, p53 were significantly increased. Gain-of-function assays suggested that FAM96B overexpression was positively correlated with TERT activity, but negatively correlated with the number of SA-ß-gal positive cells and the protein levels of P16 and P53. Moreover, after FAM96B overexpression, the results showed a significant increase in alkaline phosphatase activity and an enhanced mineralization ability of DPSCs. The reverse-transcription polymerase chain reaction results also showed that dentin sialophosphoprotein and osteocalcin were expressed at greater levels. The carboxyfluorescein succinimidyl ester (CFSE) results displayed that FAM96B increased the proliferation potential of DPSCs. Our study revealed candidate proteins that might be related to DPSCs senescence and provided information to elucidate the mechanism of the biological changes in DPSCs' aging. Moreover, FAM96B was demonstrated to play an important role in suppressing DPSCs senescence and promoting osteogenic differentiation and proliferation.

Distal-less homeobox 5 promotes the osteo-/dentinogenic differentiation potential of stem cells from apical papilla by activating histone demethylase KDM4B through a positive feedback mechanism.

Understanding the mechanism of osteo-/dentinogenic differentiation is beneficial for jaw bone and dental tissue regeneration. DLX5 is highly expressed in dental tissue-derived mesenchymal stem cells (MSCs) and is upregulated by lysine-specific demethylase 4B (KDM4B), enabling it to regulate osteo-/dentinogenic differentiation, while the function of DLX5 in osteo-/dentinogenesis has not been thoroughly elucidated to date. Therefore, we investigated DLX5 function using stem cells from apical papilla (SCAPs). SCAPs were obtained from the human wisdom tooth. Alkaline phosphatase (ALP) assay, Alizarin red staining (ARS), quantitative analysis of calcium, osteo-/dentinogenesis-related gene expression and in vivo transplantation were used to determine the osteo-/dentinogenic differentiation potential. Luciferase and ChIP assays were used to investigate the physical relationship between DLX5 and KDM4B. DLX5 and KDM4B were upregulated during osteogenic induction and were induced by BMP4 in SCAPs. Next, we found that DLX5 enhanced ALP activity, mineralization in vitro, and the expression of dentin sialophosphoprotein (DSPP), dentin matrix acidic phosphoprotein 1 (DMP1), osteopontin (OPN), and the key transcription factor osterix (OSX). Moreover, transplant experiments showed that DLX5 promoted osteo-/dentinogenesis in vivo. Interestingly, DLX5 enhanced KDM4B transcription by directly binding with its promoter. In addition, KDM4B upregulated DLX5 in SCAPs. These results indicate that DLX5 and KDM4B are positive effectors of BMP signaling and regulate each other via a positive feedback mechanism. DLX5 enhanced osteo-/dentinogenic differentiation via upregulated KDM4B in SCAPs, suggesting that activation of the DLX5/KDM4B signaling pathway might serve as an intrinsic mechanism that promotes tissue regeneration mediated by dental-derived MSCs.

IGFBP5 enhances the dentinogenesis potential of dental pulp stem cells via JNK and ErK signalling pathways.

BACKGROUND: Dental stem cell transplantation has become a new method for tooth tissue regeneration. However, its molecular mechanism of the dentinogenic differentiation is still unclear, limited its application. Our previous studies found that insulin-like growth factor-binding protein 5 (IGFBP5) can promote the osteogenic differentiation of periodontal ligament stem cells and the regeneration of periodontal tissues. This study aims to clarify the effect and mechanism of IGFBP5 on the dentinogenesis of dental pulp stem cells (DPSCs). OBJECTIVE AND METHODS: Lentiviral IGFBP5 shRNA was used to knock-down of IGFBP5. And recombinant human IGFBP5 protein (rhIGFBP5) was used to treat DPSCs. Alkaline phosphatase (ALP) staining, Alizarin red staining, quantitative calcium analysis, real-time RT-PCR and Western Blot were used to detect dentinogenic differentiation markers and related signalling pathways. Transplantation in nude mice was used to detect the dentin regeneration in vivo. RESULTS: Depletion of IGFBP5 inhibited ALP activity and the mineralisation and reduced the expressions of osteo/dentinogenic differentiation markers BSP, DMP-1 and DSPP in DPSCs. 0.05 ng/mL rhIGFBP5 promoted ALP activity, the mineralisation and the expressions of BSP, DMP-1 and DSPP in DPSCs. In addition, 0.05 ng/mL rhIGFBP5 could promote DPSC-mediated dentin-like tissues formation in vivo. Western blot results showed that IGFBP5 activated JNK and Erk signalling pathways in DPSCs. Furthermore, inhibition of JNK pathway by SP600125, the expression of p-JNK and p-Erk was reduced, while inhibition of Erk pathway by PD98059, only p-Erk expression was decreased. CONCLUSIONS: Our results demonstrated that IGFBP5 could promote the dentinogenic differentiation and dentinogenesis potential of DPSCs via JNK and ErK signalling pathways.