Tumor-to-stroma cd8+ t cells ratio combined with cancer-associated fibroblasts: an innovative approach to predicting lymph node metastases of cervical cancer.

PURPOSE: Precise identification of lymph node metastases is vital for the management of cervical cancer. However, the existing diagnostic methods for lymph node metastases have certain drawbacks. In this study, we aim to explore the expression of cancer-associated fibroblasts (CAFs) and tumor-to-stroma CD8+ T cells ratio (CD8+ T cells T:S ratio) and its association with lymph node metastases of cervical cancer. METHODS: Hundred and ten cervical cancer tissues and 39 biopsy tissues from patients were investigated immunocytochemically for the expression of CAFs and CD8+ T cells. The statistical correlation analysis was carried out using the SPSS system. RESULTS: A strong and statistically significant negative correlation (r= - 0.690; P < 0.001) was observed between CAF density and CD8+ T cells T:S ratio. Not only were CAFs density and CD8+ T cells T:S ratio correlated with lymph node metastases respectively (P < 0.001), but the combination of them also significantly correlated with lymph node metastases (P < 0.001). Then, we constructed the combined diagnosis model (Logit (P) = - 4.446 + 0.300 × CAFs + 0.752 × CD8+ T cells T:S Ratio) of cervical cancer lymph node metastases. ROC curves analysis showed that the ROC curves areas for CAFs, CD8+ T cells T:S ratio, and a combination of both are 0.879, 0.747, and 0.951. Then, the prediction model was verified by biopsy specimens and consistent results were obtained. CONCLUSIONS: The combination of CAF density and CD8+ T cells T:S ratio has a significant predictive value for lymph node metastases in patients with cervical cancer.

Hypoxia-induced ZEB1 promotes cervical cancer immune evasion by strengthening the CD47-SIRPα axis.

BACKGROUND: The dynamic interaction between cancer cells and tumour-associated macrophages (TAMs) in the hypoxic tumour microenvironment (TME) is an active barrier to the effector arm of the antitumour immune response. Cancer-secreted exosomes are emerging mediators of this cancer-stromal cross-talk in the TME; however, the mechanisms underlying this interaction remain unclear. METHODS: Exosomes were isolated with ExoQuick exosome precipitation solution. The polarizing effect of TAMs was evaluated by flow cytometry, western blot analysis, immunofluorescence staining and in vitro phagocytosis assays. Clinical cervical cancer specimens and an in vivo xenograft model were also employed. RESULTS: Our previous study showed that hypoxia increased the expression of ZEB1 in cervical squamous cell carcinoma (CSCC) cells, which resulted in increased infiltration of TAMs. Here, we found that hypoxia-induced ZEB1 expression is closely correlated with CD47-SIRPα axis activity in CSCC, which enables cancer cells to evade phagocytosis by macrophages and promotes tumour progression. ZEB1 was found to directly activate the transcription of the CD47 gene in hypoxic CSCC cells. We further showed that endogenous ZEB1 was characteristically enriched in hypoxic CSCC cell-derived exosomes and transferred into macrophages via these exosomes to promote SIRPα+ TAM polarization. Intriguingly, exosomal ZEB1 retained transcriptional activity and reprogrammed SIRPα+ TAMs via activation of the STAT3 signalling pathway in vitro and in vivo. STAT3 inhibition reduced the polarizing effect induced by exosomal ZEB1. Knockdown of ZEB1 increased the phagocytosis of CSCC cells by macrophages via decreasing CD47 and SIRPα expression. CONCLUSIONS: Our results suggest that hypoxia-induced ZEB1 promotes immune evasion in CSCC by strengthening the CD47-SIRPα axis. ZEB1-targeted therapy in combination with CD47-SIRPα checkpoint immunotherapy may improve the outcomes of CSCC patients in part by disinhibiting innate immunity.

A novel APC mutation associated with Gardner syndrome in a Chinese family.

Gardner syndrome (GS) is a specific form of familial adenomatous polyposis (FAP), which manifests as colorectal polyps, multiple osteomas and soft tissue tumors, and in the oral cavity as osteomas of the jaws, odontomas, and abnormal tooth counts. The underlying cause of GS is attributed to mutations in the APC gene. Mutations in this gene disrupt the normal functioning of the protein and lead to the development of GS. To further investigate GS, a family affected by the syndrome was selected from Dongguan, Guangdong Province. The family members underwent a comprehensive survey, which involved collecting clinical data and peripheral venous blood samples. The samples were then used for genetic analysis. Whole exome sequencing (WES) and Sanger sequencing techniques were utilized to screen and identify specific mutation sites in the APC gene. The clinical findings for the GS family included the presence of gastrointestinal polyps and odontomas. After analyzing the genetic sequencing results, a novel mutation site c.4266dupA on the APC gene was found in the patients, which leading to the APC protein truncation. As a result of this study, it is suggested that odontoma may be an early indicator of GS. Additionally, the identification of this novel mutation site in the APC gene expands the known spectrum of genetic mutations associated with the disease. This discovery has significant implications for the early diagnosis of GS, thus enabling timely intervention to reduce the risk of developing colon cancer and other related diseases.

A comprehensively prognostic and immunological analysis of chloride intracellular channel protein 5 (CLIC5) in pan-cancer and identification in ovarian cancer.

CLIC5 encoded protein associates with actin-based cytoskeletal and is increasingly thought to play significant roles in human cancers. We use TCGA and GEO to explore CLIC5 expression differences, mutation and DNA methylation, TMB, MSI, and immune cell infiltration. We verified the mRNA expression of CLIC5 in human ovarian cancer cells by real-time PCR and detected the expression of CLIC5 as well as immune marker genes in ovarian cancer by immunohistochemistry. The pan-cancer analysis showed that CLIC5 is highly expressed in several malignant tumors. In some cancers, CLIC5 expression in tumor samples is associated with poorer overall survival. For example, patients with ovarian cancer with high expression of CLIC5 have a poor prognosis. CLIC5 mutation frequency increased in all tumor types. The CLIC5 promoter is hypomethylated in most tumors. CLIC5 was associated with tumor immunity and different immune cells of different tumor types, such as CD8 + T cells, tumor-associated fibroblasts, macrophages, etc. CLIC5 was positively correlated with various immune checkpoints, and TMB and MSI were correlated with dysregulation of CLIC5 in tumors. The expression of CLIC5 in ovarian cancer was detected by qPCR and IHC, and the results were consistent with the bioinformatics results. There were a strong positive correlation between CLIC5 expression and M2 macrophage (CD163) infiltration and a negative correlation with CD8 + T-cell infiltration. In conclusions, our first pan-cancer analysis offered a detailed grasp of the cancerogenic functions of CLIC5 in a variety of malignancies. CLIC5 participated in immunomodulation and performed a crucial function in the tumor microenvironment.

Effectiveness of telehealth interventions on depression symptoms after stroke: A systematic review and meta-analysis.

BACKGROUND: Depression symptoms are common after stroke, and affect survivors' recovery of neurological function, ability to return to society, and quality of life. Telehealth has been shown to improve depression symptoms and quality of life among patients post-stroke. However, evidence from clinical trials has not previously been systematically synthesized. OBJECTIVE: This study aimed to systematically evaluate the effectiveness of telehealth interventions in reducing depression symptoms among patients post-stroke. METHODS: Following the PRISMA guidelines, we conducted a meta-analysis of randomized control trials of telehealth interventions for post-stroke depression symptoms. The quality of included studies was assessed using the Cochrane risk of bias tool. RevMan 5.4 software was used for the meta-analysis. Data were synthesized by fixed (I2 ≤ 50 %) or random (I2 > 50 %) effects models based on a heterogeneity test. RESULTS: In total, ten studies with 1717 participants were included, eight of which were eligible for the meta-analysis. There were no significant differences in efficacy between the telehealth and control groups for depression symptoms (standardized mean difference [SMD] = -0.16, 95 % confidence interval [CI] -0.67 to 0.36; P = .54), quality of life (SMD = 0.00, 95%CI -0.18 to 0.18; P = .99), limb function (SMD = 0.46, 95%CI -0.26 to 1.18; P = .21), and daily living ability (SMD = 0.38, 95%CI -1.39 to 2.15; P = .67). The telemedicine group had significantly lower anxiety scores than the control group (SMD = -1.05, 95%CI -1.22 to -0.89; P < .001). LIMITATIONS: The number of randomized controlled trials (RCTs) included in the review was relatively small. CONCLUSIONS: This study suggests that telehealth interventions have comparable effects to usual nursing care in improving depression symptoms after a stroke. However, large-scale, high-quality RCTs are needed to further explore the potential of telehealth interventions in improving mental health among patients post-stroke.

A novel single-base deletion of the RUNX Family Transcription Factor 2 gene associated with cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is a rare, autosomal dominant hereditary disorder characterized by skeletal malformations and dental abnormalities. The purpose of this study was to explore the functional role of a novel mutation in the pathogenesis of CCD. Genomic DNA was extracted from peripheral blood mononuclear cells collected from family members of a Chinese patient with CCD. An analysis of their RUNX Family Transcription Factor 2 (RUNX2) gene sequences was performed by PCR amplification and Sanger sequencing. The function of the mutant RUNX2 was studied by bioinformatics, real-time PCR, western blotting, and subcellular localization analysis. Sanger sequencing identified a novel single-base deletion (NM_001024630.4:c.132delG;NP_001019801.3: Val45Trpfs* 99) in the RUNX2 gene present in the Chinese patient with CCD. In vitro, functional studies showed altered protein localization and increased expression of mutant RUNX2 mRNA and mutant Runt-related transcription factor 2 (RUNX2). Luciferase reporter assay demonstrated that the novel RUNX2 mutations significantly increased the transactivation activity of RUNX2 on the osteocalcin gene promoter. In conclusion, we identified a patient with sporadic CCD carrying a novel deletion/frameshift mutation of the RUNX2 gene and performed screening and functional analyses to determine the cause of the CCD phenotype. This study provides new insights into the pathogenesis of CCD.3.

Identification of a novel missense heterozygous mutation in the KDF1 gene for non-syndromic congenital anodontia.

OBJECTIVES: KDF1 is a recently identified gene related to tooth development, but it has been little studied. To date, only three cases have been reported in which KDF1 mutations are related to tooth development, including two ectodermal dysplasia cases accompanied by tooth loss and one non-syndromic case with tooth agenesis. However, no KDF1 mutations have been reported as associated with non-syndromic anodontia. Here, the aim was to investigate the genetic etiology of this condition and explore the functional role of a novel KDF1 mutation in a Chinese patient with non-syndromic anodontia. MATERIALS AND METHODS: Pathogenic variants were identified by whole-exome and Sanger sequencing. Meanwhile, we conducted a literature review of the reported KDF1 mutations and performed an in vitro functional analysis of four anodontia-causing KDF1 mutations (one novel and three known). RESULTS: We identified a novel de novo missense mutation (c.911 T > A, p.I304N) in the KDF1 gene in a Chinese patient with severe non-syndromic anodontia. In vitro functional studies showed altered mRNA and protein expression levels of the mutant KDF1. CONCLUSIONS: Our results are the first report of KDF1 missense mutation causing non-syndromic anodontia. CLINICAL RELEVANCE: This study not only further supports the important role of KDF1 in non-syndromic congenital anodontia, but also expands the spectrum of KDF1 mutations and will contribute to the genetic diagnosis and counselling of families with anodontia.

Case Report: A Novel Compound Heterozygous Mutation of the FRMD4A Gene Identified in a Chinese Family With Global Developmental Delay, Intellectual Disability, and Ataxia.

Background: FERM domain-containing protein 4A (FRMD4A) is a scaffolding protein previously proposed to be critical in the regulation of cell polarity in neurons and implicated in human intellectual development. Case Presentation: We report a case of a 3-year-old boy with corpus callosum anomaly, relative macrocephaly, ataxia, and unexplained global developmental delay. Here, compound heterozygous missense mutations in the FRMD4A gene [c.1830G>A, p.(Met610Ile) and c.2973G>C, p.(Gln991His)] were identified in the proband, and subsequent familial segregation showed that each parent had transmitted a mutation. Conclusions: Our results have confirmed the associations of mutations in the FRMD4A gene with intellectual development and indicated that for patients with unexplained global developmental delay, the FRMD4A gene should be included in the analysis of whole exome sequencing data, which can contribute to the identification of more patients affected by this severe phenotypic spectrum.

Sequential Application of Calcium Phosphate and ε-Polylysine Show Antibacterial and Dentin Tubule Occluding Effects In Vitro.

In this study, ε-polylysine and calcium phosphate precipitation (CPP) methods were employed to induce antibacterial effects and dentin tubule occlusion. Antibacterial effects of ε-polylysine were evaluated with broth dilution assay against P. gingivalis. CPP solution from MCPM, DCPD, and TTCP was prepared. Four concentrations of ε-polylysine(ε-PL) solutions (0.125%, 0.25%, 0.5%, 1%) were prepared. Dentin discs were prepared from recently extracted human third molars. Dentin discs were incubated with P. gingivalis (ATCC 33277) bacterial suspension (ca. 105 bacteria) containing Brain Heart Infusion medium supplemented with 0.1 g/mL Vitamin K, 0.5 mg/mL hemin, 0.4 g/mL L-cysteine in anaerobic jars (37 °C) for 7 days to allow for biofilm formation. P. g-infected dentin specimens were randomly divided into four groups: CPP + 0.125% ε-PL, CPP + 0.25% ε-PL, CPP + 0.5% ε-PL, CPP + 1% ε-PL. On each dentin specimen, CPP solution was applied followed by polylysine solution with microbrush and immersed in artificial saliva. Precipitate formation, antibacterial effects, and occlusion of dentinal tubules were characterized in vitro over up to 72 h using scanning electron microscopy. ε-PL showed 34.97% to 61.19% growth inhibition levels against P. gingivalis (P. g) after 24 h of incubation. On P. g-infected dentin specimens, DCPD + 0.25% ε-PL, and DCPD + 0.5% ε-PL groups showed complete bacterial inhibition and 78.6% and 98.1% dentin tubule occlusion, respectively (p < 0.001). The longitudinal analysis on fractured dentin samples in DCPD and TTCP groups revealed deeply penetrated hydroxyapatite-like crystal formations in dentinal tubules after 72 h of incubation in artificial saliva.

Functional Analysis of Ectodysplasin-A Mutations in X-Linked Nonsyndromic Hypodontia and Possible Involvement of X-Chromosome Inactivation.

BACKGROUND: Mutations of the Ectodysplasin-A (EDA) gene are generally associated with syndrome hypohidrotic ectodermal dysplasia or nonsyndromic tooth agenesis. The influence of EDA mutations on dentinogenesis and odontoblast differentiation has not been reported. The aim of this study was to identify genetic clues for the causes of familial nonsyndromic oligodontia and explore the underlying mechanisms involved, while focusing on the role of human dental pulp stem cells (hDPSCs). MATERIALS AND METHODS: Candidate gene sequences were obtained by PCR amplification and Sanger sequencing. Functional analysis was conducted, and the pathogenesis associated with EDA mutations in hDPSCs was investigated to explore the impact of the identified mutation on the phenotype. Capillary electrophoresis (CE) was used to detect X-chromosome inactivation (XCI) in the blood of female carriers. RESULTS: In this study, we identified an EDA mutation in a Chinese family: the missense mutation c.1013C>T (Thr338Met). Transfection of hDPSCs with a mutant EDA lentivirus decreased the expression of EDA and dentin sialophosphoprotein (DSPP) compared with transfection of control EDA lentivirus. Mechanistically, mutant EDA inhibited the activation of the NF-κB pathway. The CE results showed that symptomatic female carriers had a skewed XCI with a preferential inactivation of the X chromosome that carried the normal allele. CONCLUSIONS: In summary, we demonstrated that EDA mutations result in nonsyndromic tooth agenesis in heterozygous females and that, mechanistically, EDA regulates odontogenesis through the NF-κB signalling pathway in hDPSCs. Due to the large heterogeneity of tooth agenesis, this study provided a genetic basis for individuals who exhibit similar clinical phenotypes.

Case Report: A Novel COL1A1 Missense Mutation Associated With Dentineogenesis Imperfecta Type I.

Background: Osteogenesis imperfecta (OI) is a clinical and genetic disorder that results in bone fragility, blue sclerae and dentineogenesis imperfecta (DGI), which is mainly caused by a mutation in the COL1A1 or COL1A2 genes, which encode type I procollagen. Case Report: A missense mutation (c.1463G > C) in exon 22 of the COL1A1 gene was found using whole-exome sequencing. However, the cases reported herein only exhibited a clinical DGI-I phenotype. There were no cases of bone disease or any other common abnormal symptom caused by a COL1A1 mutation. In addition, the ultrastructural analysis of the tooth affected with non-syndromic DGI-I showed that the abnormal dentine was accompanied by the disruption of odontoblast polarization, a reduced number of odontoblasts, a reduction in hardness and elasticity, and the loss of dentinal tubules, suggesting a severe developmental disorder. We also investigated the odontoblast differentiation ability using dental pulp stem cells (DPSCs) that were isolated from a patient with DGI-I and cultured. Stem cells isolated from patients with DGI-I are important to elucidate their pathogenesis and underlying mechanisms to develop regenerative therapies. Conclusion: This study can provide new insights into the phenotype-genotype association in collagen-associated diseases and improve the clinical diagnosis of OI/DGI-I.

Exosomes from LPS-Stimulated hDPSCs Activated the Angiogenic Potential of HUVECs In Vitro.

BACKGROUND: Exosomes from human dental pulp stem cells (hDPSCs) were indicated to play a positive role in vascular regeneration processes. But the angiogenic capabilities of exosomes from inflammatory hDPSCs and the underlying mechanism remain unknown. In this study, the inflammatory factor lipopolysaccharide (LPS) was used to stimulate hDPSCs, and exosomes were extracted from these hDPSCs. The proangiogenic potential of exosomes was examined, and the underlying mechanism was studied. METHOD: Exosomes were isolated from hDPSCs with or without LPS stimulation (N-EXO and LPS-EXO) and cocultured with human umbilical vein endothelial cells (HUVECs). The proangiogenic potential of exosomes was evaluated by endothelial cell proliferation, migration, and tube formation abilities in vitro. To investigate the proangiogenic mechanism of LPS-EXO, microRNA sequencing was performed to explore the microRNA profile of N-EXO and LPS-EXO. Gene Ontology (GO) analysis was used to study the functions of the predicted target genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was used to estimate the signaling pathways associated with the inflammation-induced angiogenesis process. RESULT: Compared to the uptake of N-EXO, uptake of LPS-EXO activated the angiogenic potential of HUVECs by promoting the proliferation, migration, and tube formation abilities in vitro. The mRNA expression levels of vascular endothelial growth factor (VEGF) and kinase-insert domain-containing receptor (KDR) in the LPS-EXO group were significantly higher than those in the N-EXO group. MicroRNA sequencing showed that 10 microRNAs were significantly changed in LPS-EXO. Pathway analysis showed that the genes targeted by differentially expressed microRNAs were involved in multiple angiogenesis-related pathways. CONCLUSION: This study revealed that exosomes derived from inflammatory hDPSCs possessed better proangiogenic potential in vitro. This is the first time to explore the role of exosomal microRNA from hDPSCs in inflammation-induced angiogenesis. This finding sheds new light on the effect of inflammation-stimulated hDPSCs on tissue regeneration.

Antibacterial Effect of the Natural Polymer ε-Polylysine Against Oral Pathogens Associated with Periodontitis and Caries.

Antimicrobials are important adjuncts in the treatment of caries and periodontitis. However, increased bacterial resistance and hypersensitivity reactions to commonly used antimicrobials have led to an increasing demand for safe and natural substances. The objective of this study was to investigate the antibacterial effects of ε-polylysine against oral pathogens Streptococcus mutans and Porphyromonas gingivalis. Broth dilution assay, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) analyses were performed to explore the antibacterial effect of ε-polylysine against S. mutans strain ATCC25175 and P. gingivalis strain ATCC332277. For the test solution, ε-polylysine was added to the bacterial suspension to prepare 0.125%, 0.25%, 0.5% and 1% ε-polylysine solutions diluted in broth medium. All four concentrations demonstrated complete inhibition of S. mutans and significantly reduced viable cell counts of P. gingivalis after 24 h. From starting inoculum of 9.15 log CFU/mL, P. gingivalis cell counts reduced to 4.01 log CFU/mL in the 0.125% ε-polylysine treatment group. SEM, CLSM, and the LIVE/DEAD bacterial assay of ε-polylysine application on P. gingivalis biofilm-dentin specimens revealed bacterial cell membrane disruption and irregular cell morphologies. The results indicated satisfactory antibacterial efficacy of ε-polylysine against P. gingivalis and S. mutans in liquid medium and as an application on biofilm-dentin specimens.

Genome-wide identification of long noncoding RNAs and their competing endogenous RNA networks involved in the odontogenic differentiation of human dental pulp stem cells.

BACKGROUND: Long noncoding RNAs (lncRNAs) play an important role in the multiple differentiations of mesenchymal stem cells (MSCs). However, few studies have focused on the regulatory mechanism of lncRNAs in the odontogenic differentiation of human dental pulp stem cells (hDPSCs). METHODS: hDPSCs were induced to differentiate into odontoblasts in vitro, and the expression profiles of lncRNAs, microRNAs (miRNAs), and messenger RNAs (mRNAs) in differentiated and undifferentiated cells were obtained by microarray. Bioinformatics analyses including Gene Ontology (GO) analysis, pathway analysis, and binding site prediction were performed for functional annotation of lncRNA. miRNA/odontogenesis-related gene networks and lncRNA-associated ceRNA networks were constructed. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to verify the expression of selected genes. RNA fluorescence in situ hybridization (FISH), qRT-PCR, and western blot analysis were used to explore the location and function of lncRNA-G043225. Dual-luciferase reporter assay was performed to confirm the binding sites of miR-588 with G043225 and Fibrillin 1 (FBN1). RESULTS: We identified 132 lncRNAs, 114 miRNAs, and 172 mRNAs were differentially expressed. GO analysis demonstrated that regulation of the neurogenic locus notch homolog (Notch), Wnt, and epidermal growth factor receptor (ERBB) signaling pathways and activation of mitogen-activated protein kinase (MAPK) activity were related to odontogenic differentiation. Pathway analysis indicated that the most significant pathway was the forkhead box O (FoxO) signaling pathway, which is related to odontogenic differentiation. Two odontogenesis-related gene-centered lncRNA-associated ceRNA networks were successfully constructed. The qRT-PCR validation results were consistent with the microarray analysis. G043225 mainly locating in cytoplasm was proved to promote the odontogenic differentiation of hDPSCs via miR-588 and FBN1. CONCLUSION: This is the first study revealing lncRNA-associated ceRNA network during odontogenic differentiation of hDPSCs using microarray, and it could provide clues to explore the mechanism of action at the RNA-RNA level as well as novel treatments for dentin regeneration based on stem cells.

[Structural characteristics of the deciduous teeth of Tibetan miniature pigs].

OBJECTIVE: To explore the histological structure of the deciduous teeth and the tooth germs of Tibetan miniature pigs for studies of dental tissue diseases and tooth regeneration. METHODS: The structure of the deciduous teeth of Tibetan miniature pigs was observed by X-ray. The ultrastructure of the enamel and dentin of deciduous teeth was characterized by scanning electron microscopy. The jaws and teeth were three-dimensionally reconstructed using Mimics software based on Micro-CT scanning of the deciduous teeth. Image J software was used to calculate the gray value and the mineralization density of the deciduous teeth. Hisotological structure of the tooth germ and the pulp tissue of Tibetan miniature pigs was observed using HE staining. RESULTS: The deciduous teeth of Tibetan miniature pigs were composed of enamel, dentin and medullary pulp tissue. The permanent tooth germ were formed during the deciduous dentition. The enamel and dentin ultrastructure of deciduous teeth were consistent with that of human deciduous teeth. The enamel and dentin mineralization densities were 2.47±0.09 g/cm3 and 1.72±0.07 g/cm3, respectively. The pathological structures of tooth germ and pulp tissue were similar to those of human teeth, and the pulp tissue of the deciduous teeth was in an undifferentiated state. CONCLUSIONS: The deciduous teeth of Tibetan miniature pig have similar anatomy, ultrastructure and histopathological structure to human teeth and can serve as a good animal model for studying human dental tissue diseases and the mechanisms of tooth regeneration.

Effects of vascular endothelial growth factor and insulin growth factor­1 on proliferation, migration, osteogenesis and vascularization of human carious dental pulp stem cells.

The present study aimed to investigate the effects of vascular endothelial growth factor (VEGF) and insulin­like growth factor­1 (IGF­1) on the proliferation, migration and differentiation of human carious dental pulp stem cells (hCDPSCs), and to elucidate the underlying mechanism(s). Cell counting kit­8 assay was used to detect the effect of different concentrations of IGF­1 and VEGF on the proliferation of hCDPSCs. Transwell assay was used to detect the migratory ability of the hCDPSCs. Alizarin red and alkaline phosphatase (ALP) staining were used to detect the osteogenic ability of hCDPSCs, whereas the angiogenic ability of the hCDPSCs was tested by tube formation assay. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blotting were used to detect the expression levels of associated genes and proteins. IGF­1 (100 ng/ml) or VEGF (25 ng/ml) alone were revealed to be able to promote proliferation and migration of hCDPSCs; however, the combined use of IGF­1 and VEGF enhanced this effect when compared with the use of either agent in isolation. Alizarin red and ALP staining revealed that the use of either VEGF or IGF­1 alone did not result in any significant effects, whereas their use in combination promoted the osteogenic differentiation of hCDPSCs. In addition, the RT­qPCR and western blotting analyses revealed that the expression levels of Runt­related transcription factor 2 (RUNX2), bone sialoprotein (BSP) and ALP were increased upon combined treatment of the cells with VEGF and IGF­1. The expression levels of VEGF and plateletderived growth factor (PDGF) in hCDPSCs were enhanced upon treatment with either VEGF or IGF­1 in isolation, with greater effects observed when VEGF and IGF­1 were added in combination, indicating that VEGF and IGF­1 may exert a synergistic role in these events. Further experiments revealed that the combination of VEGF and IGF­1 led to an activation of the AKT signaling pathway. The proliferation and angiogenesis of hCDPSCs were also shown to be more effective compared with treatment with either VEGF or IGF­1 in isolation. Taken together, the present study has demonstrated that the combined use of VEGF and IGF­1 leads to an increase in the proliferation, migration, osteogenesis and angiogenesis of hCDPSCs and, furthermore, these signaling molecules may mediate their effects via activation of the AKT signaling pathway.

Vacuolar protein sorting 4B regulates the proliferation and odontoblastic differentiation of human dental pulp stem cells through the Wnt-ß-catenin signalling pathway.

Our previous studies have revealed that a dominant mutation in vacuolar protein sorting 4B (VPS4B), a member of the AAA ATPase family, causes dentin dysplasia type I. The purpose of the present study was to investigate the roles of VPS4B in human dental pulp stem cells (hDPSCs) and to elucidate the underlying molecular mechanisms. In this study, we found that VPS4B was highly expressed in the dental pulp cells of the mouse molar tooth germ, and the expression of VPS4B increased significantly during the odontoblastic differentiation of hDPSCs. VPS4B downregulation inhibited the proliferation, migration, and odontoblastic differentiation of hDPSCs. Moreover, treatment with lithium chloride, an agonist of the Wnt-ß-catenin signalling pathway, partially reversed the VPS4B knockdown-driven suppression of proliferation and of odontoblastic differentiation of hDPSCs. Collectively, our findings indicate that VPS4B, via Wnt-ß-catenin signalling, acts as a regulator of the proliferation and differentiation of hDPSCs. Our results suggest potential therapeutic avenues for dentin formation and regenerative endodontics in patients with dentin dysplasia type I.

Anion- and Solvent-Induced Single-Crystal-to-Single-Crystal Transformation within an Iron(II) Triazole System: a Promising Luminescent Probe for CrO42- and Cyano-Containing Molecules.

Anion- and solvent-induced single-crystal-to-single-crystal transformation within an iron(II) triazole system has been generated from {[Fe(TPPT)2Cl2]·CHCl3} n (1a) to [Fe(TPPT)(C2O4)0.5Cl(H2O)] n (1b). Luminescence studies indicated that the resultant 1b can be considered as a promising luminescent probe for CrO42- and cyano molecules.

Wedelolactone exhibits anti-fibrotic effects on human hepatic stellate cell line LX-2.

Wedelolactone is a major coumarin of Eclipta prostrata, which is used for preventing liver damage. However the effects of wedelolactone on hepatic fibrosis remained unexplored. The purpose of this study was to demonstrate the anti-fibrotic effects of wedelolactone on activated human hepatic stellate cell (HSC) line LX-2 and the possible underlying mechanisms by means of MTT assay, Hoechst staining, as well as real-time quantitative PCR and western blot. The results showed that wedelolactone reduced the cellular viability of LX-2 in a time and dose-dependent manner. After treatment of wedelolactone, the expressions of collagen I and α-smooth muscle actin, two biomarkers of LX-2 activation, were remarkably decreased. The apoptosis of LX-2 cells was induced by wedelolactone accompanied with the decreasing expression of anti-apoptotic Bcl-2 and increasing expression of pro-apoptotic Bax. In addition, phosphorylated status of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was up-regulated, but not in p38. Moreover, wedelolactone significantly repressed the level of phosphorylated inhibitor of nuclear factor κB (IκB) and p65 in nucleus in spite of tumor necrosis factor-α stimulation. In conclusion, wedelolactone could significantly inhibit the activation of LX-2 cells, the underlying mechanisms of which included inducing Bcl-2 family involved apoptosis, up-regulating phosphorylated status of ERK and JNK expressions, and inhibiting nuclear factor-κB (NF-κB) mediated activity. Wedelolactone might present as a useful tool for the prevention and treatment of hepatic fibrosis.

Cytologic diagnosis of a metastatic oligodendroglioma in a pleural effusion. A case report.

BACKGROUND: Extraneural metastasis of oligodendroglioma is extremely rare and is diagnosed primarily by biopsy or autopsy and very occasionally by fine needle cytologic examination. We report a case of metastatic oligodendroglioma diagnosed by cytologic examination of a pleural effusion. Such a diagnosis has not been reported before. CASE: A 64-year-old woman developed anemia and bilateral pleural effusion 7 years after an operation for an oligodendroglioma over the left frontal lobe. Cytologic examination of the pleural effusion showed aggregates of atypical polygonal cells containing round, hyperchromatic nuclei and scanty, granular cytoplasm in Liu's and Papanicolaou stain and cell blocks. Immunohistochemical staining of the tumor cells revealed a positive reaction for antibodies to glial fibrillary acidic protein, S-100 and Olig2. Pleural biopsy confirmed the cytologic diagnosis of pleural effusion. A pathologic fracture of the right humeral and femoral bones was noted 1 month later, and the specimen also showed infiltrating oligodendroglioma cells in bone tissue. CONCLUSION: To the best of our knowledge, this is the first metastatic oligodendroglioma diagnosed by pleural cytology. Fine needle cytology can provide a reliable and rapid way to detect an extracranial metastatic oligodendroglioma in different organs.