Carious lesions are bacteria-caused destructions of the mineralised dental tissues, marked by the simultaneous activation of immune responses and regenerative events within the soft dental pulp tissue. While major molecular players in tooth decay have been uncovered during the past years, a detailed map of the molecular and cellular landscape of the diseased pulp is still missing. In this study we used single-cell RNA sequencing analysis, supplemented with immunostaining, to generate a comprehensive single-cell atlas of the pulp of carious human teeth. Our data demonstrated modifications in the various cell clusters within the pulp of carious teeth, such as immune cells, mesenchymal stem cells (MSC) and fibroblasts, when compared to the pulp of healthy human teeth. Active immune response in the carious pulp tissue is accompanied by specific changes in the fibroblast and MSC clusters. These changes include the upregulation of genes encoding extracellular matrix (ECM) components, including COL1A1 and Fibronectin (FN1), and the enrichment of the fibroblast cluster with myofibroblasts. The incremental changes in the ECM composition of carious pulp tissues were further confirmed by immunostaining analyses. Assessment of the Fibronectin fibres under mechanical strain conditions showed a significant tension reduction in carious pulp tissues, compared to the healthy ones. The present data demonstrate molecular, cellular and biomechanical alterations in the pulp of human carious teeth, indicative of extensive ECM remodelling, reminiscent of fibrosis observed in other organs. This comprehensive atlas of carious human teeth can facilitate future studies of dental pathologies and enable comparative analyses across diseased organs.
Humans ; Dental Pulp ; Fibronectins ; Extracellular Matrix/pathology* ; Dental Caries ; Sequence Analysis, RNA

PURPOSE: This study was designed to identify novel fusion transcripts (FTs) and their functional significance in colorectal cancer (CRC) lines. MATERIALS AND METHODS: We performed paired-end RNA sequencing of 28 CRC cell lines. FT candidates were identified using TopHat-fusion, ChimeraScan, and FusionMap tools and further experimental validation was conducted through reverse transcription-polymerase chain reaction and Sanger sequencing. FT was depleted in human CRC line and the effects on cell proliferation, cell migration, and cell invasion were analyzed. RESULTS: One thousand three hundred eighty FT candidates were detected through bioinformatics filtering. We selected six candidate FTs, including four inter-chromosomal and two intrachromosomal FTs and each FT was found in at least one of the 28 cell lines. Moreover, when we tested 19 pairs of CRC tumor and adjacent normal tissue samples, NFATC3–PLA2G15 FT was found in two. Knockdown of NFATC3–PLA2G15 using siRNA reduced mRNA expression of epithelial–mesenchymal transition (EMT) markers such as vimentin, twist, and fibronectin and increased mesenchymal–epithelial transition markers of E-cadherin, claudin-1, and FOXC2 in colo-320 cell line harboring NFATC3–PLA2G15 FT. The NFATC3–PLA2G15 knockdown also inhibited invasion, colony formation capacity, and cell proliferation. CONCLUSION: These results suggest that that NFATC3–PLA2G15 FTs may contribute to tumor progression by enhancing invasion by EMT and proliferation.
Cadherins ; Cell Line* ; Cell Movement ; Cell Proliferation ; Claudin-1 ; Colorectal Neoplasms* ; Computational Biology ; Fibronectins ; Humans ; RNA* ; RNA, Messenger ; RNA, Small Interfering ; Sequence Analysis, RNA* ; Vimentin

Acanthamoeba spp. are free-living protozoa that are opportunistic pathogens for humans. Cysteine proteases of Acanthamoeba have been partially characterized, but their biochemical and functional properties are not clearly understood yet. In this study, we isolated a gene encoding cysteine protease of A. castellanii (AcCP) and its biochemical and functional properties were analyzed. Sequence analysis of AcCP suggests that this enzyme is a typical cathepsin L family cysteine protease, which shares similar structural characteristics with other cathepsin L-like enzymes. The recombinant AcCP showed enzymatic activity in acidic conditions with an optimum at pH 4.0. The recombinant enzyme effectively hydrolyzed human proteins including hemoglobin, albumin, immunoglobuins A and G, and fibronectin at acidic pH. AcCP mainly localized in lysosomal compartment and its expression was observed in both trophozoites and cysts. AcCP was also identified in cultured medium of A. castellanii. Considering to lysosomal localization, secretion or release by trophozoites and continuous expression in trophozoites and cysts, the enzyme could be a multifunctional enzyme that plays important biological functions for nutrition, development and pathogenicity of A. castellanii. These results also imply that AcCP can be a promising target for development of chemotherapeutic drug for Acanthamoeba infections.
Acanthamoeba castellanii ; Acanthamoeba ; Cathepsin L ; Cathepsins ; Cysteine Proteases ; Cysteine ; Fibronectins ; Genes, vif ; Humans ; Hydrogen-Ion Concentration ; Lysosomes ; Sequence Analysis ; Trophozoites ; Virulence

To investigate the effect of a novel EZH2 inhibitor GSK126 on cell growth, apoptosis and migration of prostate cancer cells.Prostate cancer PC-3 and DU145 cells were treated with GSK126 at different doses. Cell growth was detected by sulforhodamine assay. Cell apoptosis was assayed by Annexin V-/PI kit. Transwell chamber and wound healing assays were conducted to detect cell migration. The mRNA level was detected by quantitative PCR, and protein expression was detected by Western blot analysis.GSK126 showed significant effect on cell growth and apoptosis when the dose was higher than 50 μmol/L. Wound healing assay revealed that scratch space in PC-3 cells was significantly increased in a dose-dependent manner in GSK126-treated groups[(247.2±24.4),(347.2±19.2) and (410.5±18.1) μm in low, medium and high dose (5.0, 20.0, 50.0 μmol/L), respectively] as compared with the control group[(171.3±17.8) μm](all<0.05). Transwell assay showed that migrated PC-3 cells in control group was 322.0±17.9,while those in GSK126-treated groups were 198.3±15.4 (low),82.7±6.2 (medium) and 30.2±4.1 (high), and the differences between the control group and GSK126-treated groups were significant(all<0.05). In addition, GSK126 up-regulated E-cadherin mRNA expression and down-regulated N-cadherin and Vimentin mRNA expression, whereas had no significant effect on Snail, Fibronectin and VEGF-A mRNA expression. The protein expression of E-cadherin was elevated but VEGF-A protein did not change in GSK126-treated groups. Similar results were exhibited in DU145 cell.GSK126 can significantly inhibit cell migration and invasion in prostate cancer PC-3 and DU145 cells, which may be resulted from its effect on epithelial-mesenchymal transition. GSK126 may be used as a potential anti-prostate cancer dug in clinic.
Apoptosis ; drug effects ; Cadherins ; analysis ; drug effects ; metabolism ; Cell Line, Tumor ; drug effects ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Down-Regulation ; drug effects ; Drug Screening Assays, Antitumor ; methods ; Enhancer of Zeste Homolog 2 Protein ; analysis ; drug effects ; metabolism ; Fibronectins ; analysis ; drug effects ; metabolism ; Humans ; Indoles ; pharmacology ; Male ; Prostatic Neoplasms ; chemistry ; genetics ; physiopathology ; Pyridones ; pharmacology ; RNA, Messenger ; Up-Regulation ; drug effects ; Vascular Endothelial Growth Factor A ; analysis ; drug effects ; Vimentin ; analysis ; drug effects ; metabolism

Select 106 pregnant women with threatened preterm labor between 28 and 37 weeks of gestation. They have been treated continually by the preventing preterm labor therapy including antibiotics and magnesium sulfate. Cervicovaginal fetal fibronectin (fFN) and C-reactive protein (CRP) in serum were detected before treatment and after 3-day and 7-day treatment respectively. 100 normal pregnant women were included as control group. (1) The fFN and CRP had significant differences between study group and control group (P<0.05). (2) The fFN and CPR were different compared in the treatment period (P<0.05). Fetal fibronectin and CRP can be used can be used in therapeutic effect evaluation of threatened preterm labor.
C-Reactive Protein ; analysis ; Female ; Fibronectins ; blood ; Humans ; Infant, Newborn ; Obstetric Labor, Premature ; diagnosis ; prevention & control ; Pregnancy

PURPOSE: Cancer tissue may display a wide spectrum of expression phenotypes of epithelial-mesenchymal transition (EMT)-related proteins. The purpose of this study was to investigate the clinical significance of EMT phenotypes in breast cancer. METHODS: We evaluated the expression pattern of the EMT-related proteins E-cadherin and fibronectin in samples from 1,495 patients with invasive breast carcinoma (IBC) on tissue microarrays using immunohistochemistry to investigate the clinical significance of EMT phenotypes in IBC. EMT phenotypes were divided into complete type (E-cadherin-negative/fibronectin-positive), incomplete type (hybrid type, E-cadherinpositive/fibronectin-positive; null type, E-cadherin-negative/fibronectin-negative), and wild-type (E-cadherin-positive/fibronectin-negative). We analyzed the correlation of EMT phenotype with clinicopathological factors and patient survival. RESULTS: Loss of E-cadherin was observed in 302 patients (20.2%), and fibronectin was expressed in the cancer cells of 354 patients (23.7%). In total, 64 (4.3%), 290 (19.4%), 238 (15.9%), and 903 (60.4%) samples were categorized as complete, hybrid, null, and wild-type, respectively. The complete EMT phenotype exhibited significant associations with young age (p=0.017), advanced pT (p<0.001) and pN (p<0.001) stages, higher histological grade (p<0.001), lymphovascular invasion (p<0.001), and triple negativity (p<0.001). Patients with complete and hybrid EMT phenotypes had poorer overall survival (OS) and disease-free survival (DFS) than those with the wild-type phenotype (OS, p=0.001; DFS, p<0.001). In multivariate analysis, the hybrid EMT phenotype was an independent prognostic factor for DFS in patients with IBC (p=0.032). CONCLUSION: EMT phenotypes exhibited significant associations with clinicopathological factors indicating aggressive biologic behavior and poor outcome in patients with IBC.
Breast Neoplasms* ; Breast* ; Cadherins ; Disease-Free Survival ; Epithelial-Mesenchymal Transition* ; Fibronectins ; Humans ; Immunohistochemistry ; Multivariate Analysis ; Phenotype* ; Prognosis

Cellular fibronectin (cFn) is a type of bioactive non-collagen glycoprotein regarded as the main substance used to maintain periodontal attachment. The content of cFn in some specific sites can reflect the progress of periodontitis or peri-implantitis. This study aims to evaluate the expression of cFn messenger RNA (mRNA) in tissues of adult periodontitis and peri-implantitis by real-time fluorescent quantitative polymerase chain reaction (PCR) and to determine its clinical significance. A total of 30 patients were divided into three groups of 10: healthy, adult periodontitis and peri-implantitis. Periodontal tissue biopsies (1 mm×1 mm×1 mm) from each patient were frozen in liquid nitrogen. Total RNA was extracted from these tissues, and the content, purity and integrity were detected. Specific primers were designed according to the sequence, and the mRNA expression levels of cellular fibronectin were detected by real-time PCR. The purity and integrity of the extracted total RNA were both high, and the specificity of amplified genes was very high with no other pollution. The mRNA expression of cFn in the adult periodontitis group (1.526±0.441) was lower than that in the healthy group (3.253±0.736). However, the mRNA expression of cFn in the peri-implantitis group (3.965±0.537) was significantly higher than that in the healthy group. The difference revealed that although both processes were destructive inflammatory reactions in the periodontium, the pathomechanisms were different and the variation started from the transcription level of the cFn gene.
Adult ; Alveolar Bone Loss ; metabolism ; Female ; Fibronectins ; analysis ; genetics ; Gingiva ; metabolism ; Humans ; Male ; Middle Aged ; Peri-Implantitis ; metabolism ; Periodontal Attachment Loss ; metabolism ; Periodontal Index ; Periodontal Pocket ; metabolism ; Periodontitis ; metabolism ; Periodontium ; metabolism ; RNA, Messenger ; analysis ; Real-Time Polymerase Chain Reaction ; Transcription, Genetic ; genetics ; Young Adult

BACKGROUNDIn addition to hematopoietic effect, the erythropoietin is known as a multifunctional cytokine with anti-fibrosis and organ-protective activities. The purpose of this study was to evaluate the effect of recombinant human erythropoietin (rhEPO) on hepatic fibrosis and hepatic stellate cells (HSCs).

METHODSCarbon tetrachloride (CCl(4)) induced hepatic fibrosis mice models were used for in vivo study and HSCs line for in vitro study. CCl(4) and rhEPO (0, 200 or 1000 U/kg) was injected intraperitoneally in BALB/c mice three times a week for 4 weeks. Immunohistochemistry and immunoblotting were performed to evaluate expressions of transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (α-SMA), and fibronectin in explanted liver. Immunoblotting of α-SMA, phophorylated Smad-2 and Smad-2/3 was performed in HSCs treated with TGF-β1 and/or rhEPO.

RESULTSExpressions of TGF-β1, α-SMA, and fibronectin were increased in CCl(4) injected mice livers, but significantly attenuated by co-treatment with CCl(4) and rhEPO. Co-treatment of rhEPO markedly suppressed fibrosis in Masson's trichrome compared with treatment of only CCl(4). TGF-β1 increased phosphorylated α-SMA, Smad-2 expressions in HSCs, which were decreased by rhEPO co-treatment.

CONCLUSIONSTreatment of rhEPO effectively suppressed fibrosis in CCl(4)-induced liver fibrosis mice models. Anti-fibrosis effect of rhEPO could be related to inhibition of TGF-β1 induced activation of HSCs.

Animals ; Carbon Tetrachloride ; toxicity ; Cells, Cultured ; Erythropoietin ; pharmacology ; therapeutic use ; Fibronectins ; analysis ; Hepatic Stellate Cells ; drug effects ; Liver Cirrhosis, Experimental ; metabolism ; prevention & control ; Male ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; pharmacology ; Smad2 Protein ; metabolism ; Transforming Growth Factor beta ; antagonists & inhibitors ; physiology

Granular cell ameloblastoma is a rare, benign neoplasm of the odontogenic epithelium. A case of massive granular cell ameloblastoma in a 44-year-old Thai female is reported. Histopathological features displayed a follicular type of ameloblastoma with an accumulation of granular cells residing within the tumor follicles. After treatment by partial mandibulectomy, the patient showed a good prognosis without recurrence in a 2-year follow-up. To characterize the granular cells in ameloblastoma, we examined the expression of basement membrane (BM) proteins, including collagen type IV, laminins 1 and 5 and fibronectin using immunohistochemistry. Except for the granular cells, the tumor cells demonstrated a similar expression of BM proteins compared to follicular and plexiform ameloblastomas in our previous study, whereas the granular cells showed strong positivity to laminins 1 and 5 and fibronectin. The increased fibronectin expression in granular cells suggests a possibility of age-related transformation of granular cells in ameloblastoma.
Adult ; Ameloblastoma ; chemistry ; pathology ; Basement Membrane ; chemistry ; Cell Adhesion Molecules ; analysis ; Collagen Type IV ; analysis ; Female ; Fibronectins ; analysis ; Humans ; Keratinocytes ; chemistry ; pathology ; Laminin ; analysis ; Mandibular Neoplasms ; chemistry ; pathology ; Membrane Proteins ; analysis

OBJECTIVETo study the effects of interleukin-1β (IL-1β) on transdifferentiation of human embryonic lung fibroblasts to myofibroblasts and the effects of lentinan on the transdifferentiation.

METHODSThe human embryonic lung fibroblasts were cultured in vitro, and fibroblasts were treated with different concentrations of IL-1β and lentinan. The proliferation activity of the human embryonic lung fibroblasts was evaluated by the Cell Counting Kit-8 (CCK-8). The expression of α-smooth muscle actin (α-SMA) protein was measured by immunocytochemistry. The levels of fibronectin (FN), typeⅠcollagen (ColⅠ) and α-SMA mRNA were detected by RT-PCR.

RESULTSCompared with the untreated control group, the absorbance value of cell proliferation, α-SMA protein levels, FN, ColⅠand α-SMA mRNA expression were significantly up-regulated after different concentrations of IL-1β (0.1, 1, 10 ng/mL) treatment for 48 hrs (P<0.01). Lentinan treatment inhibited up-regulation of the cell proliferation activity, α-SMA protein levels, FN, ColⅠand α-SMA mRNA expression induced by IL-1β in a dose-independent manner (P<0.01).

CONCLUSIONSLentinan can suppress human embryonic lung fibroblast proliferation, fibroblast-myofibroblast transdifferentiation and extra cellular matrix synthesis induced by IL-1β.

Actins ; analysis ; genetics ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cell Transdifferentiation ; Cells, Cultured ; Fibroblasts ; cytology ; drug effects ; Fibronectins ; analysis ; genetics ; Humans ; Interleukin-1beta ; pharmacology ; Lentinan ; pharmacology ; Myofibroblasts ; cytology