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Introduction: To investigate the physiological function of leptin in human dental pulp, and to determine the specific pathways implicated in its effect., Methods: Twenty-seven dental pulp samples were obtained from human third molars. Pulp samples were treated with or without human recombinant leptin. Leptin functional effect was analyzed in terms of regulation of the synthesis levels of DSPP and DMP-1, determined by immunoblot., Results: Leptin stimulated DMP-1 and DSPP synthesis in all human dental pulp specimens. The stimulatory effect of leptin on DMP-1 and DSPP synthesis was partially prevented by blocking mitogen-activated protein kinase (MAPK 1/3) and phosphatidylinositol 3 kinase (PI3K) pathways, respectively., Conclusions: The present study demonstrates the functional effect of leptin in human dental pulp stimulating the expression of DMP-1 and DSPP, both proteins implicated in dentinogenesis. Leptin stimulates DSPP expression via PI3K pathway and DMP-1 synthesis via MAPK 1/3 pathway. These results support the role of leptin in pulpal reparative response, opening a new research line that could have translational application to the clinic in vital pulp therapy procedures., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In this study, we attempted to localize the immunoreactivities of podoplanin/E11/gp38 and CD44, a counterpart possessing a high affinity to podoplanin/E11/gp38, as well as endomucin-immunoreactive blood vessels in the regions of odontoblast layers and the underlying sub-odontoblastic layers in murine tooth germs. Endomucin-reactive small blood vessels were scattered throughout the dental papillae of the tooth germs at postnatal day 1 but came to be localized close to the odontoblast/sub-odontoblastic layers until day 3. After postnatal day 5, small blood vessels were seen in odontoblast cell layers, while blood vessels with relatively larger diameters were seen forming in sub-odontoblastic layers. Immunoreactivities of podoplanin/E11/gp38 and CD44 were not detectable in the cells of dental papillae facing the inner enamel epithelium at postnatal day 1. However, at around postnatal days 3-5, podoplanin/E11/gp38 was localized in the odontoblast layer but not in the sub-odontoblastic layer, whereas CD44 was observed in the sub-odontoblastic layer but not in the odontoblast layer. The exclusive immunolocalization of podoplanin/E11/gp38 and CD44 in the odontoblast layers and sub-odontoblastic layers was seen after postnatal day 3 of the tooth germs, when the mesenchymal cells of dental papillae have already differentiated into mature odontoblasts at the cusp tip. Taken together, it seems likely that endomucin-reactive small blood vessels extended to the podoplanin/E11/gp38-positive odontoblast layers, whereas endomucin-reactive large blood vessels were already present in CD44-immmunopositive sub-odontoblastic layer, indicating the cellular regulation on the vascularization of endomucin-reactive endothelial cells during odontogenesis of the tooth germs.

A chemical approach was developed for identifying cell-surface markers for primary neural stem cells (NSCs). Using an in vitro coculture system of primary NSCs combined with metabolic labeling of sialoglycans with bioorthogonal functional groups, we selectively enriched and identified a list of cell-surface sialoglycoproteins that were more abundantly expressed in neural stem and progenitor cells.

Previously, a Sanger-based sialotranscriptome analysis of adult female Culex tarsalis was published based on ∼2000 ESTs. During the elapsed 7.5 years, pyrosequencing has been discontinued and Illumina sequences have increased considerable in size and decreased in price. We here report an Illumina-based sialotranscriptome that allowed finding the missing apyrase from the salivary transcriptome of C. tarsalis, to determine several full-length members of the 34-62 kDa family, when a single EST has been found previously, in addition to identifying many salivary families with lower expression levels that were not detected previously. The use of multiple libraries including salivary glands and carcasses from male and female organisms allowed for an unprecedented insight into the tissue specificity of transcripts, and in this particular case permitting identification of transcripts putatively associated with blood feeding, when exclusive of female salivary glands, or associated with sugar feeding, when transcripts are found upregulated in both male and female glands., (© 2018 Published by Elsevier Ltd.)

Yes-associated protein 1 (YAP1) transcriptional coactivator is a mediator of mechanosensitive signaling. Cementum, which covers the tooth root surface, continuously senses external mechanical stimulation. Cementoblasts are responsible for the mineralization and maturation of the cementum. However, the effect of YAP1 on cementoblast differentiation remains largely unknown. In this study, we initially demonstrated that YAP1 overexpression enhanced the mineralization ability of cementoblasts. YAP1 upregulated the mRNA and protein expression of several cementogenesis markers, such as alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and dentin matrix acidic phosphoprotein 1 (DMP1). The YAP1 overexpression group showed higher intensities of ALP and Alizarin red stain than the YAP1-knockdown group. Unexpectedly, a sharp increase in the expression of dentin sialophosphoprotein (DSPP) was induced by the overexpression of YAP1. Knockdown of YAP1 suppressed DSPP transcriptional activity. YAP1 overexpression activated Smad-dependent BMP signaling and slightly inhibited Erk1/2 signaling pathway activity. Treatment with specific BMP antagonist (LDN193189) prevented the upregulation of the mRNA levels of ALP, RUNX2, and OCN, as well as intensity of ALP-stained and mineralized nodules in cementoblasts. The Erk1/2 signaling pathway inhibitor (PD 98,059) upregulated these cementogenesis markers. Thus, our study suggested that YAP1 enhanced cementoblast mineralization in vitro. YAP1 exerted its effect on the cementoblast partly by regulating the Smad-dependent BMP and Erk1/2 signaling pathways., (© 2017 Wiley Periodicals, Inc.)

Undifferentiated odontogenic epithelium and dental papilla cells differentiate into ameloblasts and odontoblasts, respectively, both of which are essential for tooth development. These differentiation processes involve dramatic functional and morphological changes of the cells. For these changes to occur, activation of mitochondrial functions, including ATP production, is extremely important. In addition, these changes are closely related to mitochondrial fission and fusion, known as mitochondrial dynamics. However, few studies have focused on the role of mitochondrial dynamics in tooth development. The purpose of this study was to clarify this role. We used mouse tooth germ organ cultures and a mouse dental papilla cell line with the ability to differentiate into odontoblasts, in combination with knockdown of the mitochondrial fission factor, dynamin related protein (DRP)1. In organ cultures of the mouse first molar, tooth germ developed to the early bell stage. The amount of dentin formed under DRP1 inhibition was significantly larger than that of the control. In experiments using a mouse dental papilla cell line, differentiation into odontoblasts was enhanced by inhibiting DRP1. This was associated with increased mitochondrial elongation and ATP production compared to the control. These results suggest that DRP1 inhibition accelerates dentin formation through mitochondrial elongation and activation. This raises the possibility that DRP1 might be a therapeutic target for developmental disorders of teeth., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A series of novel sialoglycopolypeptides carrying N-glycolylneuraminic acid (Neu5Gc)-containing trisaccharides having α(2 → 3)- and α(2 → 6)-linkages in the side chains of γ-polyglutamic acid (γ-PGA) were designed as competitive inhibitors against equine influenza viruses (EIV), which critically recognize the Neu5Gc residue for receptor binding. Using horse red blood cells (HRBC) we successfully evaluated the binding activity of the multivalent Neu5Gc ligands to both equine and canine influenza viruses in the hemagglutination inhibition (HI) assay. Our findings show the multivalent α2,3-linked Neu5Gc-ligands (3a-c and 7) selectively inhibit hemagglutination mediated by both influenza viruses and display a strong inhibitory activity. Our results indicate that the multivalent Neu5Gc-ligands can be used as novel probes to elucidate the mechanism of infection/adhesion of Neu5Gc-binding influenza viruses.

This study investigated the damage and expression of dentine phosphoprotein (DPP) in guinea pig teeth by the administration of fluoride (F) or/and aluminum (Al). Fifty-two guinea pigs were divided randomly into four groups (control, F, Al, and F+Al). F (150 mg NaF/L) or/and Al (300 mg AlCl 3 /L) were added in their drinking water for 90 days. The levels of F ion, dentine sialophosphoprotein (DSPP) gene, and DPP protein in incisor and molar were determined, respectively. The results showed that the concentrations of F ion in F and F+Al groups were increased significantly. F induced the mottled enamel and irregular abrasion of teeth, which might occur as a consequence of depressed DSPP mRNA and DPP protein expression. Both the gene and protein expressions showed obvious decrease induced by Al, especially by F. There were no synergistic effects between F and Al, instead, Al inhibited the toxicity of F., (© 2017 Wiley Periodicals, Inc.)

Background: A devastating condition that leads to trauma-related morbidity, multiple rib fractures, remain a serious unmet clinical need. Systemic administration of mesenchymal stem cells (MSCs) has been shown to regenerate various tissues. We hypothesized that parathyroid hormone (PTH) therapy would enhance MSC homing and differentiation, ultimately leading to bone formation that would bridge rib fractures., Methods: The combination of human MSCs (hMSCs) and a clinically relevant PTH dose was studied using immunosuppressed rats. Segmental defects were created in animals' fifth and sixth ribs. The rats were divided into four groups: a negative control group, in which animals received vehicle alone; the PTH-only group, in which animals received daily subcutaneous injections of 4 μg/kg teriparatide, a pharmaceutical derivative of PTH; the hMSC-only group, in which each animal received five injections of 2 × 10 6 hMSCs; and the hMSC + PTH group, in which animals received both treatments. Longitudinal in vivo monitoring of bone formation was performed biweekly using micro-computed tomography (μCT), followed by histological analysis., Results: Fluorescently-dyed hMSCs were counted using confocal microscopy imaging of histological samples harvested 8 weeks after surgery. PTH significantly augmented the number of hMSCs that homed to the fracture site. Immunofluorescence of osteogenic markers, osteocalcin and bone sialoprotein, showed that PTH induced cell differentiation in both exogenously administered cells and resident cells. μCT scans revealed a significant increase in bone volume only in the hMSC + PTH group, beginning by the 4 th week after surgery. Eight weeks after surgery, 35% of ribs in the hMSC + PTH group had complete bone bridging, whereas there was complete bridging in only 6.25% of ribs (one rib) in the PTH-only group and in none of the ribs in the other groups. Based on the μCT scans, biomechanical analysis using the micro-finite element method demonstrated that the healed ribs were stiffer than intact ribs in torsion, compression, and bending simulations, as expected when examining bone callus composed of woven bone., Conclusions: Administration of both hMSCs and PTH worked synergistically in rib fracture healing, suggesting this approach may pave the way to treat multiple rib fractures as well as additional fractures in various anatomical sites.

Dendritic spines are the postsynaptic compartments of glutamatergic synapses in the brain. Their number and shape are subject to change in synaptic plasticity and neurological disorders including autism spectrum disorders and Parkinson's disease. The L-type calcium channel Ca V 1.3 constitutes an important calcium entry pathway implicated in the regulation of spine morphology. Here we investigated the importance of full-length Ca V 1.3 L and two C-terminally truncated splice variants (Ca V 1.3 42A and Ca V 1.3 43S ) and their modulation by densin-180 and shank1b for the morphology of dendritic spines of cultured hippocampal neurons. Live-cell immunofluorescence and super-resolution microscopy of epitope-tagged Ca V 1.3 L revealed its localization at the base-, neck-, and head-region of dendritic spines. Expression of the short splice variants or deletion of the C-terminal PDZ-binding motif in Ca V 1.3 L induced aberrant dendritic spine elongation. Similar morphological alterations were induced by co-expression of densin-180 or shank1b with Ca V 1.3 L and correlated with increased Ca V 1.3 currents and dendritic calcium signals in transfected neurons. Together, our findings suggest a key role of Ca V 1.3 in regulating dendritic spine structure. Under physiological conditions it may contribute to the structural plasticity of glutamatergic synapses. Conversely, altered regulation of Ca V 1.3 channels may provide an important mechanism in the development of postsynaptic aberrations associated with neurodegenerative disorders.

The small integrin binding ligands n-linked glycoproteins (SIBLINGs) have emerged as potential diagnostic and prognostic indices, and as key targets, in cancer therapy. Three members of the SIBLING family: bone sialoprotein (BSP); osteopontin (OPN); and dentin matrix protein1 (DMP1), bind and interact with specific matrix metalloproteinases (MMPs): BSP-MMP2; OPN-MMP3; DMP1-MMP9, in biochemical and biologic systems. The other two family members are dentin sialophosphoprotein (DSPP) and matrix extracellular phosphoglycoprotein (MEPE). The specific SIBLING-MMP pairing reported in some cancers have not been reported in prostate neoplasms. In this study, we investigated SIBLING-MMP expression and potential interaction in prostate neoplasms. Chi square analysis of immunohistochemistry results showed significant upregulation of OPN (X2=25.710/p<0.001), BSP (X2=19.546/p<0.001), and DSPP (X2=8.720/p=0.003) in prostate adenocarcinoma (pAdC). MEPE was significantly upregulated in benign prostate hyperplasia (BPH; X2=44.153/p<0.001). There were no significant differences in MMP expression between BPH and pAdC. Western blot analysis showed significantly elevated BSP and DSPP in prostate cancer-derived cells. Immunofluorescence studies confirmed BSP-MMP2, OPN-MMP3, and DMP1-MMP9 coexpression in two cancer-derived cell lines, whereas in situ proximity ligation assays confirmed potential BSP-MMP2, OPN-MMP3, and DMP1-MMP9 interactions in BPH and pAdC. Our reports provide evidence that SIBLING-MMP interaction may play a role in the progression of BPH to pAdC., Competing Interests: The authors have no conflict of interest pertaining to this study.

Cardiac progenitor cells (CPCs) from adult myocardium offer an alternative cell therapy approach for ischaemic heart disease. Improved clinical performance of CPCs in clinical trials requires a comprehensive definition of their biology and specific interactions with the environment. In this work we characterize specific human CPC surface markers and study some of their related functions. c-kit(pos) human CPCs (hCPCs) were characterized for cell surface marker expression, pluripotency, early and late cardiac differentiation markers and therapeutic activity in a rat model of acute myocardial infarction. The results indicate that hCPCs are a mesenchymal stem cell (MSC)-like population, with a similar immunoregulatory capacity. A partial hCPC membrane proteome was analysed by liquid chromatography-mass spectrometry/mass spectrometry and 36 proteins were identified. Several, including CD26, myoferlin and podocalyxin-like protein 1 (PODXL), have been previously described in other stem-cell systems. Suppression and overexpression analysis demonstrated that PODXL regulates hCPC activation, migration and differentiation; it also modulates their local immunoregulatory capacity. Therefore, hCPCs are a resident cardiac population that shares many features with hMSCs, including their capacity for local immunoregulation. Expression of PODXL appears to favour the immature state of hCPCs, while its downregulation facilitates their differentiation. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2013 John Wiley & Sons, Ltd.)

Dentin sialophosphoprotein (DSPP) is proteolytically processed into an NH2-terminal fragment called dentin sialoprotein (DSP) and a COOH-terminal fragment known as dentin phosphoprotein (DPP). These two fragments are believed to perform distinct roles in formation of bone and dentin. To investigate the functions of DPP in skeletal development, we generated transgenic mice to overexpress hemagglutinin (HA)-tagged DPP under the control of a 3.6 kb type I collagen (Col1a1) promoter (designated as Col1a1-HA-DPP). The Col1a1-HA-DPP transgenic mice were significantly smaller by weight, had smaller skeletons and shorter long bones than their wild type littermates, as demonstrated by X-ray radiography. They displayed reduced trabecular bone formation and narrower zones of proliferative and hypertrophic chondrocytes in the growth plates of the long bones. Histological analyses showed that the transgenic mice had reduced cell proliferation in the proliferating zone, but lacked obvious defects in the chondrocyte differentiation. In addition, the transgenic mice with a high level of transgene expression developed spontaneous long bone fractures. In conclusion, overexpressing DPP inhibited skeletal development, suggesting that the balanced actions between the NH2- and COOH-terminal fragments of DSPP may be required for normal skeletal development.

Selective exo-enzymatic labeling (or SEEL) uses recombinant glycosyltransferases and nucleotide-sugar analogues to allow efficient labeling of cell surface glycans. SEEL can circumvent many of the possible issues associated with metabolic labeling, including low incorporation of sugar precursors, and allows for sugars to be added selectively to different types of glycans by virtue of the inherent specificity of the glycosyltransferases. Here we compare the labeling of sialoglycoproteins in undifferentiated and differentiated human erythroleukemia cells (HEL) using SEEL using the sialyltransferases ST6Gal1 and ST3Gal1, which label N- and O-glycans, respectively. Our results show that the profile of glycoproteins detected varies between undifferentiated HEL cells and those differentiated to megakaryocytes, with a shift to more N-linked sialoglycoproteins in the differentiated cells. The efficiency of SEEL for both sialyltransferases in HEL cells was greatly increased with prior neuraminidase treatment highlighting the necessity for the presence of available acceptors with this labeling method. Following metabolic labeling or SEEL, tagged glycoproteins were enriched by immunoprecipitation and identified using mass spectrometry. The proteomic findings demonstrated that the detection of many glycoproteins is markedly improved by SEEL labeling, and that unique glycoproteins can be identified using either ST6Gal1 or ST3Gal1. Furthermore, this analysis enabled the identification of increased surface expression of several sialylated cell adhesion molecules, including the known megakaryocytic markers integrinβ3 and CD44, upon differentiation of HEL cells to adherent megakaryocytes., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Overexpression of the transmembrane sialomucin podocalyxin, which is known to play a role in lumen formation during polarized epithelial morphogenesis, is an independent indicator of poor prognosis in a number of epithelial cancers, including those that arise in the breast. Therefore, we set out to determine if podocalyxin plays a functional role in breast tumor progression., Methods: MCF-7 breast cancer cells, which express little endogenous podocalyxin, were stably transfected with wild type podocalyxin for forced overexpression. 4T1 mammary tumor cells, which express considerable endogenous podocalyxin, were retrovirally transduced with a short hairpin ribonucleic acid (shRNA) targeting podocalyxin for stable knockdown. In vitro, the effects of podocalyxin on collective cellular migration and invasion were assessed in two-dimensional monolayer and three-dimensional basement membrane/collagen gel culture, respectively. In vivo, local invasion was assessed after orthotopic transplantation in immunocompromised mice., Results: Forced overexpression of podocalyxin caused cohesive clusters of epithelial MCF-7 breast tumor cells to bud off from the primary tumor and collectively invade the stroma of the mouse mammary gland in vivo. This budding was not associated with any obvious changes in histoarchitecture, matrix deposition or proliferation in the primary tumour. In vitro, podocalyxin overexpression induced a collective migration of MCF-7 tumor cells in two-dimensional (2-D) monolayer culture that was dependent on the activity of the actin scaffolding protein ezrin, a cytoplasmic binding partner of podocalyxin. In three-dimensional (3-D) culture, podocalyxin overexpression induced a collective budding and invasion that was dependent on actomyosin contractility. Interestingly, the collectively invasive cell aggregates often contained expanded microlumens that were also observed in vivo. Conversely, when endogenous podocalyxin was removed from highly metastatic, but cohesive, 4T1 mammary tumor cells there was a decrease in collective invasion in three-dimensional culture., Conclusions: Podocalyxin is a tumor cell-intrinsic regulator of experimental collective tumor cell invasion and tumor budding.

Growing evidence links abnormal epigenetic control to the development of hematological malignancies. Accordingly, inhibition of epigenetic regulators is emerging as a promising therapeutic strategy. The acetylation status of lysine residues in histone tails is one of a number of epigenetic post-translational modifications that alter DNA-templated processes, such as transcription, to facilitate malignant transformation. Although histone deacetylases are already being clinically targeted, the role of histone lysine acetyltransferases (KAT) in malignancy is less well characterized. We chose to study this question in the context of acute myeloid leukemia (AML), where, using in vitro and in vivo genetic ablation and knockdown experiments in murine models, we demonstrate a role for the epigenetic regulators CBP and p300 in the induction and maintenance of AML. Furthermore, using selective small molecule inhibitors of their lysine acetyltransferase activity, we validate CBP/p300 as therapeutic targets in vitro across a wide range of human AML subtypes. We proceed to show that growth retardation occurs through the induction of transcriptional changes that induce apoptosis and cell-cycle arrest in leukemia cells and finally demonstrate the efficacy of the KAT inhibitors in decreasing clonogenic growth of primary AML patient samples. Taken together, these data suggest that CBP/p300 are promising therapeutic targets across multiple subtypes in AML.

Cardiovascular disease associated with oxidative stress, including atherosclerosis, is the leading cause of mortality worldwide. The accelerated proliferation and migration of vascular smooth muscle cells are the predominant characteristics of atherogenesis, and endothelial dysfunction is a major risk factor for the pathogenesis of atherosclerosis. Podocalyxin (PODXL), a type I member of the cluster of differentiation 34 family of sialomucins, functions as a pro-adhesive molecule. Emerging evidence has revealed the importance of micro (mi)RNAs in the cardiovascular system. The present study demonstrated that there was an inverse association between miRNA (miR)-125b and PODXL in human umbilical vein endothelial cells and human aortic vascular smooth muscle cells (HAVSMCs) treated with oxidized low‑density lipoprotein (LDL) and platelet derived growth factor. Additionally, miR-125b had a suppressive function in cell proliferation and migration, at least partially via targeting PODXL in the HAVSMCs. Furthermore, the data suggested that the functions of miR-125b in arteriosclerosis obliterans may be associated with transgelin, lectin-type oxidized LDL receptor-1, vascular endothelial-cadherin, intercellular adhesion molecule-1, interleukin-6 and monocyte chemotactic protein-1. In conclusion, miR-125b was found to be important in arteriosclerosis obliterans by suppressing the expression of PODXL and may serve as a potential therapeutic target for the treatment of arteriosclerosis obliterans.

Objective: Vascular calcification (VC) is inhibited by the glycoprotein osteoprotegerin (OPG). It is unclear whether treatments for type 2 diabetes are capable of promoting or inhibiting VC. The present study examined the effects of insulin and liraglutide on i) the production of OPG and ii) the emergence of VC, both in vitro in human aortic smooth muscle cells (HASMCs) and in vivo in type 2 diabetes., Design/methods: HASMCs were exposed to insulin glargine or liraglutide, after which OPG production, alkaline phosphatase (ALP) activity and levels of Runx2, ALP and bone sialoprotein (BSP) mRNA were measured. A prospective, nonrandomised human subject study was also conducted, in which OPG levels and coronary artery calcification (CAC) were measured in a type 2 diabetes population before and 16 months after the commencement of either insulin or liraglutide treatment and in a control group that took oral hypoglycemics only., Results: Exposure to insulin glargine, but not liraglutide, was associated with significantly decreased OPG production (11 913±1409 pg/10(4) cells vs 282±13 pg/10(4) cells, control vs 10 nmol/l insulin, P<0.0001), increased ALP activity (0.82±0.06 IU/10(4) cells vs 2.40±0.16 IU/10(4) cells, control vs 10 nmol/l insulin, P<0.0001) and increased osteogenic gene expression by HASMCs. In the clinical study (n=101), insulin treatment was associated with a significant reduction in OPG levels and, despite not achieving full statistical significance, a trend towards increased CAC in patients., Conclusion: Exogenous insulin down-regulated OPG in vitro and in vivo and promoted VC in vitro. Although neither insulin nor liraglutide significantly affected CAC in the present pilot study, these data support the establishment of randomised trials to investigate medications and VC in diabetes., (© 2015 European Society of Endocrinology.)

Objectives: This study aimed to investigate effects of neurotrophin receptor-mediated melanoma antigen-encoding gene homology (NRAGE) on proliferation and odontoblastic differentiation of mouse dental pulp cells (mDPCs)., Materials and Methods: Mouse dental pulp cells were infected with recombinant lentivirus to stably knockdown expression of NRAGE, and biological effects of NRAGE on the cells were detected. Proliferation and odontoblastic differentiation of mDPCs were observed. Simultaneously, mRNA and protein levels of NRAGE and nuclear factor-κB (NF-κB) protein expression were detected. Immunofluorescence assay was used to detect expression and location of NRAGE and NF-κB., Results: NRAGE mRNA and protein levels reduced significantly after mDPC odontoblastic induction. Knockdown of NRAGE inhibited the proliferation of mDPCs. However, knockdown of NRAGE enhanced their odontoblastic differentiation with up-regulated ALPase activity. It also promoted mineral nodule formation as well as mRNA and protein expressions of ALP, DSPP and DMP1. Protein levels of NF-κB/p50 significantly increased, whereas NF-κB/p105 protein expression decreased in the mDPC/shNRG group. Immunofluorescence revealed that relocation of NF-κB was similar to that of NRAGE during odontoblastic induction, in which NF-κB translocated from the cytoplasm to the nucleus., Conclusion: NRAGE is a potent regulator of proliferation and odontoblastic differentiation of mDPCs, which might be via the NF-κB signalling pathway., (© 2015 John Wiley & Sons Ltd.)

Introduction: Leptin, an inflammation-related adipokine, and its receptor (LEPR) are expressed in human dental pulp. Dentin sialophosphoprotein (DSPP) is involved in dentinogenesis and the dental pulp reparative response. The cell type expressing LEPR in dental human pulp and the resultant effect of the binding of leptin to LEPR remain unknown. This study describes the immmunohistochemical localization of LEPR and the effect of leptin on DSPP expression in human dental pulp., Methods: Twenty-five dental pulp specimens were obtained from freshly extracted caries-free and restoration-free human third molars. LEPR localization was examined by immunohistochemistry using the antihuman LEPR monoclonal antibody. The effect of leptin on DSPP expression was determined by immunoblot analysis and quantitative real-time polymerase chain reaction., Results: Immunoreactivity for LEPR concentrated in the odontoblast layer but was not evident in the central zone of the dental pulp. Leptin dose dependently stimulated DSPP expression. Western blot analysis revealed the presence of a protein with an apparent molecular weight of ∼00 kDa, the estimated molecular weight of DSPP. The expression of DSPP messenger RNA was confirmed by quantitative real-time polymerase chain reaction, and the size of the amplified fragments (298 bp) was confirmed by agarose gel electrophoresis., Conclusions: The present study shows that human dental pulp is immunoreactive for LEPR, with the immunoreactivity concentrated in the odontoblast layer, and that leptin stimulates, in a dose-dependent manner, DSPP protein and messenger RNA (mRNA) expression in human dental pulp. These findings further support the functional role of leptin in the dentin mineralization process and/or in dental pulp reparative and immune responses., (Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Metastatic disease is the leading cause of cancer mortality. Identifying biomarkers and regulatory mechanisms is important toward developing diagnostic and therapeutic tools against metastatic cancer. In this study, we demonstrated that podocalyxin-like 1 (PODXL) is overexpressed in breast tumor cells and increased in lymph node metastatic cancer. Mechanistically, we found that the expression of PODXL was associated with cell motility and invasiveness. Suppression of PODXL in MDA-MB-231 cells reduced lamellipodia formation and focal adhesion kinase (FAK) and paxillin phosphorylation. PODXL knockdown reduced the formation of invadopodia, such as inhibiting the colocalization of F-actin with cortactin and suppressing phosphorylation of cortactin and neural Wiskott-Aldrich syndrome protein. Conversely, overexpression of PODXL in MCF7 cells induced F-actin/cortactin colocalization and enhanced invadopodia formation and activation. Invadopodia activity and tumor invasion in PODXL-knockdown cells are similar to that in cortactin-knockdown cells. We further found that the DTHL motif in PODXL is crucial for regulating cortactin phosphorylation and Rac1/Cdc42 activation. Inhibition of Rac1/Cdc42 impeded PODXL-mediated cortactin activation and FAK and paxillin phosphorylation. Moreover, inhibition of PODXL in MDA-MB-231 cells significantly suppressed tumor colonization in the lungs and distant metastases, similar to those in cortactin-knockdown cells. These findings show that overexpression of PODXL enhanced invadopodia formation and tumor metastasis by inducing Rac1/Cdc42/cortactin signaling network., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Metastasis-mediated death remains a major challenge in cancer treatment due to the lack of identifiable biomarkers for early diagnosis. Identifying tumor-specific biomarkers is critical for the development of diagnostic and therapeutic tools. In the present study, we found that podocalyxin-like 1 (PODXL), a cell surface glycoprotein, was overexpressed in cancer tissues and was upregulated in lymph node metastatic tumor cells. The expression of PODXL was associated with the migratory ability of human oral squamous cell carcinoma (OSCC). Knockdown of PODXL by small hairpin RNA in the SAS OSCC cell line reduced tumor migration and invasion, and inhibited cell proliferation and colony formation. Suppression of PODXL resulted in downregulation of focal adhesion kinase (FAK) and paxillin phosphorylation. PODXL silencing inhibited filopodia formation, and suppressed F-actin and cortactin colocalization. In addition, PODXL expression was associated with the DNA methylation status, and treatment with the DNA methyltransferase inhibitor 5-aza-deoxycytidine increased the PODXL transcriptional level. Moreover, DNA microarray analysis data revealed that suppression of PODXL significantly affected subsets of genes associated with extracellular matrix organization, the epithelial-mesenchymal transition, and the expression of metastasis-related cytokines. Collectively, these data showed that the overexpression of PODXL may be associated with tumor aggressiveness and that PODXL could be a diagnostic biomarker for metastatic OSCC.

Our recent study suggested that allogenic tooth transplantation may affect the maintenance of dental pulp stem/progenitor cells. This study aims to elucidate the influence of allograft on the maintenance of dental pulp stem/progenitor cells following tooth replantation and allo- or auto-genic tooth transplantation in mice using BrdU chasing, immunohistochemistry for BrdU, nestin and Ki67, in situ hybridization for Dspp, transmission electron microscopy and TUNEL assay. Following extraction of the maxillary first molar in BrdU-labeled animals, the tooth was immediately repositioned in the original socket, or the roots were resected and immediately allo- or auto-grafted into the sublingual region in non-labeled or the same animals. In the control group, two types of BrdU label-retaining cells (LRCs) were distributed throughout the dental pulp: those with dense or those with granular reaction for BrdU. In the replants and autogenic transplants, dense LRCs remained in the center of dental pulp associating with the perivascular environment throughout the experimental period and possessed a proliferative capacity and maintained the differentiation capacity into the odontoblast-like cells or fibroblasts. In contrast, LRCs disappeared in the center of the pulp tissue by postoperative week 4 in the allografts. The disappearance of LRCs was attributed to the extensive apoptosis occurring significantly in LRCs except for the newly-differentiated odontoblast-like cells even in cases without immunological rejection. The results suggest that the host and recipient interaction in the allografts disturbs the maintenance of dense LRCs, presumably stem/progenitor cells, resulting in the disappearance of these cell types.

In the mouse embryo and differentiating embryonic stem cells, the hematopoietic, endothelial, and cardiomyocyte lineages are derived from Flk1+ mesodermal progenitors. Here, we report that surface expression of Podocalyxin (Podxl), a member of the CD34 family of sialomucins, can be used to subdivide the Flk1+ cells in differentiating embryoid bodies at day 4.75 into populations that develop into distinct mesodermal lineages. Definitive hematopoietic potential was restricted to the Flk1+Podxl+ population, while the Flk1-negative Podxl+ population displayed only primitive erythroid potential. The Flk1+Podxl-negative population contained endothelial cells and cardiomyocyte potential. Podxl expression distinguishes Flk1+ mesoderm populations in mouse embryos at days 7.5, 8.5, and 9.5 and is a marker of progenitor stage primitive erythroblasts. These findings identify Podxl as a useful tool for separating distinct mesodermal lineages., (© AlphaMed Press.)

Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are extracellular matrix proteins produced by odontoblasts involved in the dentin mineralization. The aim this study was to compare the distribution of DMP1 and DSPP in human sound dentin vs human sclerotic dentin. Sixteen sound and sixteen carious human molars were selected, fixed in paraformaldehyde and processed for immunohistochemical detection of DMP1 and DSPP by means of light microscopy, transmission electron microscopy (TEM) and high-resolution field emission in-lens scanning electron microscopy (FEI-SEM). Specimens were submitted to a pre-embedding or a post-embedding immunolabeling technique using primary antibodies anti DMP1 and anti-DSPP and gold-conjugated secondary antibodies. Other samples were processed for the detection of DMP1 and DSPP levels. Dentin from these samples was mechanically fractured to powder, then a protein extraction and a protein level detection assay were performed. DMP1 and DSPP were more abundant in carious than in sound samples. Immunohistochemical analyses in sclerotic dentin disclosed a high expression of DMP1 and DSPP inside the tubules, suggesting an active biomineralization of dentin by odontoblasts. Furthermore, the detection of small amounts of these proteins inside the tubules far from the carious lesion, as shown in the present study, is consistent with the hypothesis of a preventive defense of all dentin after a noxious stimulus has undermined the tooth.

Background: Membranous expression of the anti-adhesive glycoprotein podocalyxin-like (PODXL) has previously been found to correlate with poor prognosis in several major cancer forms. Here we examined the prognostic impact of PODXL expression in urothelial bladder cancer., Methods: Immunohistochemical PODXL expression was examined in tissue microarrays with tumours from two independent cohorts of patients with urothelial bladder cancer: n=100 (Cohort I) and n=343 (Cohort II). The impact of PODXL expression on disease-specific survival (DSS; Cohort II), 5-year overall survival (OS; both cohorts) and 2-year progression-free survival (PFS; Cohort II) was assessed., Results: Membranous PODXL expression was significantly associated with more advanced tumour (T) stage and high-grade tumours in both cohorts, and a significantly reduced 5-year OS (unadjusted HR=2.25 in Cohort I and 3.10 in Cohort II, adjusted HR=2.05 in Cohort I and 2.18 in Cohort II) and DSS (unadjusted HR=4.36, adjusted HR=2.70). In patients with Ta and T1 tumours, membranous PODXL expression was an independent predictor of a reduced 2-year PFS (unadjusted HR=6.19, adjusted HR=4.60) and DSS (unadjusted HR=8.34, adjusted HR=7.16)., Conclusion: Membranous PODXL expression is an independent risk factor for progressive disease and death in patients with urothelial bladder cancer.

Aim: To detect and quantify dentine sialoprotein (DSP) in the gingival crevicular fluid (GCF) of luxated teeth., Methodology: Eighteen subjects were enroled and distributed as follows. Group I (n = 6, positive control): subjects with primary second molar teeth undergoing physiological root resorption. Group II (n = 6, negative control): subjects with permanent mature maxillary central incisors. Subjects with a recent history (<1 week) of luxation injury were included in group III (n = 6, test group) and standardized digital radiographs with a superimposed mesh gauge were exposed at various time intervals. Percentage of radiographic root resorption (%RRR) was calculated. GCF was collected using microcapillary pipettes. DSP in the GCF was quantified using enzyme-linked immunosorbant assay. Group III was subjected to Spearman's rank test to establish the correlation between the concentration of DSP and %RRR at 6 weeks, 3 and 6 months., Results: Quantifiable amounts of DSP were released in the GCF of subjects in Group I and III. However, the protein was not detected in Group II. Detectable quantities of DSP were observed in the GCF of luxated teeth before any radiographic evidence of root resorption (base line radiograph). A positive correlation was established at 6 weeks (r = 0.795), 3 (r = 0.755) and 6 month (r = 0.837) between the release of DSP and %RRR (P < 0.05)., Conclusion: Dentine sialoprotein was released in the GCF of luxated teeth and its concentration correlated with the active and remission phases of this pathological process. Further investigation is required to establish a potentially noninvasive aid for diagnosing and monitoring root resorption., (© 2012 International Endodontic Journal.)

Chitosan/poly(DL-lactide-co-glycolide) (Ch/DL PLG) composite scaffolds were fabricated by freeze-drying lyophilization, and were evaluated and compared for use as a bone regeneration scaffold through measurements of the compression mechanical properties of the porous scaffolds. Also, In vitro cell culture of Sprague-Dawley rat's osteoblasts were used to evaluate the phenotype expression of cells in the scaffolds, characterizing the cellular adhesion, proliferation and alkaline phosphatase activity. The gene expression of osteocalcin, sialoprotein, alkaline phosphatase, Type I collagen and TGFβ1 were confirmed in the samples; moreover, it was confirmed, the mineralization by IR spectra and EDS analysis. Our results thus show that Ch/DL PLG scaffolds are suitable for biological applications.

Introduction: Homeodomain gene Distal-less-3 (Dlx3) plays an essential role in tooth development. The aim of this study was to investigate the effects of Dlx3 on proliferation and odontoblastic differentiation of human dental pulp cells (hDPCs)., Methods: Human DPCs were infected by recombinant lentivirus to overexpress Dlx3 stably, and the biological effects of Dlx3 on the hDPCs were investigated. Proliferation of the hDPCs was measured by direct cell counting and 5-ethynyl-2'-deoxyuridine incorporation assay. Odontogenic differentiation of hDPCs was evaluated by von Kossa staining and alkaline phosphatase activity assay. Important mineral genes such as dentin sialophosphoprotein (DSPP), dentin matrix acidic phosphoprotein 1 (DMP1), alkaline phosphatase (ALP), and nestin (Nes) were determined by real-time polymerase chain reaction. Western blot analysis was performed to determine the difference of expressions of protein of dentin sialophosphoprotein (DSP) and DMP1 with or without the presence of exogenous Dlx3., Results: Overexpression of Dlx3 decreased the proliferation ability of hDPCs. Dlx3 enhanced differentiation of hDPCs with promoting mineralization nodule formation and up-regulated the ALP activity as well as the expressions of mineralization-related genes including DSPP, DMP1, ALP, and Nes. Meanwhile, the protein levels of DSP and DMP1 significantly increased in the presence of exogenous Dlx3., Conclusions: Dlx3 is a potent regulator for proliferation and odontoblastic differentiation of hDPCs., (Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

FAM20C is highly expressed in bone and tooth. Previously, we showed that Fam20C conditional knock-out (KO) mice manifest hypophosphatemic rickets, which highlights the crucial roles of this molecule in promoting bone formation and mediating phosphate homeostasis. In this study, we characterized the dentin, enamel, and cementum of Sox2-Cre-mediated Fam20C KO mice. The KO mice exhibited small malformed teeth, severe enamel defects, very thin dentin, less cementum than normal, and overall hypomineralization in the dental mineralized tissues. In situ hybridization and immunohistochemistry analyses revealed remarkable down-regulation of dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein in odontoblasts, along with a sharply reduced expression of ameloblastin and amelotin in ameloblasts. Collectively, these data indicate that FAM20C is essential to the differentiation and mineralization of dental tissues through the regulation of molecules critical to the differentiation of tooth-formative cells.

To investigate the functions of recombinant human dentin phosphoprotein (rhDPP), we examined cell adhesion, viability and the odontoblastic differentiation activity of human dental pulp cells (hDPCs). Firstly, rhDPP was constructed using pBAD-HisA plasmid in Escherichia coli. Cell adhesion and viability of hDPCs by rhDPP was examined using a crystal violet assay and a MTT assay, ALP, mineralization activity and odontoblastic differentiation-related mRNA levels of hDPCs were measured to elucidate the odontoblastic differentiation effect of rhDPP on hDPCs. Initially, rhDPP significantly and dose-dependently increased hDPCs adhesion versus the untreated control (p < 0.05). Cell viability was also significantly increased by rhDPP at 5 days (p < 0.001). Furthermore, the odontoblastic differentiation effect of rhDPP was verified by measuring ALP activity, mineralization activity and the mRNA levels of odontoblastic differentiation markers. Taken together, rhDPP is expected to play an important role on hDPCs, thereby suggesting its potential use for tooth repair and regeneration.

Background: Podocalyxin-like 1 (PODXL) is a cell-adhesion glycoprotein and stem cell marker that has been associated with an aggressive tumour phenotype and adverse outcome in several cancer types. We recently demonstrated that overexpression of PODXL is an independent factor of poor prognosis in colorectal cancer (CRC). The aim of this study was to validate these results in two additional independent patient cohorts and to examine the correlation between PODXL mRNA and protein levels in a subset of tumours., Method: PODXL protein expression was analyzed by immunohistochemistry in tissue microarrays with tumour samples from a consecutive, retrospective cohort of 270 CRC patients (cohort 1) and a prospective cohort of 337 CRC patients (cohort 2). The expression of PODXL mRNA was measured by real-time quantitative PCR in a subgroup of 62 patients from cohort 2. Spearman's Rho and Chi-Square tests were used for analysis of correlations between PODXL expression and clinicopathological parameters. Kaplan Meier analysis and Cox proportional hazards modelling were applied to assess the relationship between PODXL expression and time to recurrence (TTR), disease free survival (DFS) and overall survival (OS)., Results: High PODXL protein expression was significantly associated with unfavourable clinicopathological characteristics in both cohorts. In cohort 1, high PODXL expression was associated with a significantly shorter 5-year OS in both univariable (HR = 2.28; 95% CI 1.43-3.63, p = 0.001) and multivariable analysis (HR = 2.07; 95% CI 1.25-3.43, p = 0.005). In cohort 2, high PODXL expression was associated with a shorter TTR (HR = 2.93; 95% CI 1.26-6.82, p = 0.013) and DFS (HR = 2.44; 95% CI 1.32-4.54, p = 0.005), remaining significant in multivariable analysis, HR = 2.50; 95% CI 1.05-5.96, p = 0.038 for TTR and HR = 2.11; 95% CI 1.13-3.94, p = 0.019 for DFS.No significant correlation could be found between mRNA levels and protein expression of PODXL and there was no association between mRNA levels and clinicopathological parameters or survival., Conclusions: Here, we have validated the previously demonstrated association between immunohistochemical expression of PODXL and poor prognosis in CRC in two additional independent patient cohorts. The results further underline the potential utility of PODXL as a biomarker for more precise prognostication and treatment stratification of CRC patients.

Introduction: Osmotic stress is one of the stimulations related to dental pain caused by caries or dentin hypersensitivity. The mechanism of osmotic-induced dental pain is not completely understood. The purpose of this study was to examine the responses of odontoblasts under sucrose-induced hyperosmotic stress., Methods: We used an odontoblast-lineage cell (OLC) line in our experiments. OLCs were stimulated with sucrose to produce hyperosmotic stress. The expressions of dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP 1) were detected by using reverse transcriptase polymerase chain reaction assay. The cell viability of OLCs was detected by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay. The responses accompanied with cell death were detected by using 4-6-diamidino-2-phenylindole staining, Western blotting of caspase-3, and annexin V assay. The expression of mitogen-activated protein kinases (MAPKs) was detected by using Western blot analysis., Results: DSPP and DMP 1 were not affected by hyperosmotic stress in OLCs. Cell viability decreased over 700 mOsm for 3 hours of cell culture. The shapes of cells and nuclei became irregular and vacuolar under hyperosmotic stress. The expression of cleaved caspase-3 was increased after treatment with hyperosmotic stress. Some propidium iodide-positive cells were detected in flow cytometry analysis. Phosphorylation of 3 MAPKs was induced by hyperosmotic stress. Inhibitors of 3 MAPKs inhibited the hyperosmotic stress-induced decline in cell viability at 500 and 700 mOsm., Conclusions: Hyperosmotic stress induces cell death of OLCs with sucrose through a MAPK pathway., (Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: Mutations in the gene ALPL in hypophosphatasia (HPP) reduce the function of tissue nonspecific alkaline phosphatase, and the resulting increase in pyrophosphate (PP(i)) contributes to bone and tooth mineralization defects by inhibiting physiologic calcium-phosphate (P(i)) precipitation. Although periodontal phenotypes are well documented, pulp/dentin abnormalities have been suggested in the clinical literature although reports are variable and underlying mechanisms remains unclear. In vitro analyses were used to identify mechanisms involved in HPP-associated pulp/dentin phenotypes., Methods: Primary pulp cells cultured from HPP subjects were established to assay alkaline phosphatase (ALP) activity, mineralization, and gene expression compared with cells from healthy controls. Exogenous P(i) was provided to the correct P(i)/PP(i) ratio in cell culture., Results: HPP cells exhibited significantly reduced ALP activity (by 50%) and mineral nodule formation (by 60%) compared with the controls. The expression of PP(i) regulatory genes was altered in HPP pulp cells, including reduction in the progressive ankylosis gene (ANKH) and increased ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). Odontoblast marker gene expression was disrupted in HPP cells, including reduced osteopontin (OPN), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), and matrix extracellular phosphoprotein (MEPE). The addition of P(i) provided a corrective measure for mineralization and partially rescued the expression of some genes although cells retained altered messenger RNA levels for PP(i)-associated genes., Conclusions: These studies suggest that under HPP conditions pulp cells have the compromised ability to mineralize and feature a disrupted odontoblast profile, providing a first step toward understanding the molecular mechanisms for dentin phenotypes observed in HPP., (Published by Elsevier Inc.)

Introduction: Human dental apical papilla cells (APCs) have mineralization potential, which plays a key role in the root development of young permanent teeth. Limited literature is available about APC mineralization in the presence of inflammatory cytokines. The purpose of this study was to investigate the effects of tumor necrosis factor-α (TNF-α) on APC mineralization., Methods: APC cultures were established with the enzymatic dissociation method in vitro. The viability of APCs treated with TNF-α was investigated using methyl-thiazol-tetrazolium assays. Cells were then cultured in osteo-/dentinogenic medium with TNF-α, and mineralization was assessed by alizarin red S staining. Bone sialoprotein (BSP) and dentin sialoprotein (DSP) were analyzed using immunocytochemistry. Mineralization genes such as BSP, dentin sialophosphoprotein (DSPP), osteocalcin (OCN), and dentin matrix acidicphosphoprotein-1 (DMP1) were determined with real-time polymerase chain reaction analyses., Results: The viability of cultured cells was higher with TNF-α concentrations of 10 ng/mL and 50 ng/mL than with 5 ng/mL or in the control group. Alizarin red S staining showed that APCs had a higher mineralization activity when the osteo-/dentinogenic culture medium contained 10 ng/mL TNF-α. Immunocytochemical detection showed that the expression of BSP and DSP was positive in APCs after they were induced in osteo-/dentinogenic medium. The expression of mineralization genes differed when treated with 10 ng/mL TNF-α (ie, the expression of DSPP mRNA increased on days 7 and 14, whereas the expression of DSPP mRNA decreased on day 21)., Conclusions: TNF-α may promote APC mineralization in short-term cultures and inhibit the mineralization in long-term cultures., (Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

CCR4 on T cells is suggested to mediate skin homing in mice. Our objective was to determine the interaction of CCR4, E-selectin ligand (ESL), and α(4)β(1) on memory and activated T cells in recruitment to dermal inflammation. mAbs to rat CCR4 were developed. CCR4 was on 5-21% of memory CD4 cells, and 20% were also ESL(+). Anti-TCR-activated CD4 and CD8 cells were 40-55% CCR4(+), and ∼75% of both CCR4(+) and CCR4(-) cells were ESL(+). CCR4(+) memory CD4 cells migrated 4- to 7-fold more to dermal inflammation induced by IFN-γ, TNF, TLR agonists, and delayed-type hypersensitivity than CCR4(-) cells. CCR4(+) activated CD4 cells migrated only 5-50% more than CCR4(-) cells to these sites. E-selectin blockade inhibited ∼60% of CCR4(+) activated CD4 cell migration but was less effective on memory cells where α(4)β(1) was more important. Anti-α(4)β(1) also inhibited CCR4(-) activated CD4 cells more than CCR4(+) cells. Anti-E-selectin reduced activated CD8 more than CD4 cell migration. These findings modify our understanding of CCR4, ESL, α(4)β(1), and dermal tropism. There is no strict relationship between CCR4 and ESL for skin homing of CD4 cells, because the activation state and inflammatory stimulus are critical determinants. Dermal homing memory CD4 cells express CCR4 and depend more on α(4)β(1) than ESL. Activated CD4 cells do not require CCR4, but CCR4(+) cells are more dependent on ESL than on α(4)β(1), and CCR4(-) cells preferentially use α(4)β(1). The differentiation from activated to memory CD4 cells increases the dependence on CCR4 for skin homing and decreases the requirement for ESL.

Introduction: Although several biological roles of SIRT1 have been reported, the expression and role of SIRT1 in human dental pulp cells (HDPCs) remain unknown. To identify the role of SIRT1 in HDPCS, we measured SIRT1 messenger RNA and protein levels during the odontoblastic differentiation of HDPCs. Additionally, we investigated the effects of SIRT1 overexpression and knockdown on the differentiation of HDPCs., Methods: The expression of markers for odontoblastic differentiation and angiogenesis was analyzed by alizarin red staining, reverse-transcriptase polymerase chain reaction, and Western blot analysis., Results: A transient increase in SIRT1 gene expression occurred in early odontogenesis, which peaked at 1 day and decreased thereafter. SIRT1 induction by resveratrol and by infection with adenovirus-SIRT1 (Ad-SIRT1), a SIRT1-expressing adenoviral vector, increased mineralization nodules and up-regulated messenger RNA expression of odontoblastic markers (ie, alkaline phosphatase, osteocalcin, dentin matrix protein-1, and dentin sialophosphoprotein) as well as angiogenic markers (ie, vascular endothelial growth factor, endothelial cell adhesion molecules such as vascular endothelial cadherin and platelet endothelial cell adhesion molecule 1, and fibroblast growth factor-2). In contrast, the inhibition of SIRT1 expression by sirtinol and SIRT1 small interfering RNAs decreased odontoblastic differentiation and down-regulated angiogenic factors., Conclusions: SIRT1 gene activation may provide therapeutic effects in pulp regeneration and dentin tissue engineering., (Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

We report the case of a 38-year-old woman diagnosed with Gitelman syndrome. A kidney biopsy showed abundant floating cells in the Bowman's space of the mildly cystic glomeruli, moderate tubulointerstitial changes and apparent intimal thickening of small arteries. These floating cells were immunohistologically identified as podocytes, by the expression of podocalyxin, vimentin, Wilms' tumor 1, synaptopodin and nephrin with positivities of 100%, 88.4%, 80.4%, 74.7% and 22.6%, respectively. In these phenotypes, nephrin expression was notably decreased in both detached and capillary-attached podocytes in comparison with normal control podocytes. Immunostaining of both detached and capillary-attached podocytes for Bax, Bcl-2, desmin, fibroblast-specific protein-1, α-smooth muscle actin and Ki-67 was negative, as were TUNEL assays. These results suggest that apoptosis and epithelial-mesenchymal transition were not the main cause of podocyte detachment in this patient. In addition, levels of urinary podocalyxin were not elevated, suggesting the detached podocytes were not excreted in the urine. To the best of our knowledge, this is the first report of severe intraglomerular non-apoptotic detachment of podocytes in Gitelman syndrome. This podocyte detachment may be associated with nephron obstruction and reduced nephrin expression.

Background: Human dental pulp cells (hDPCs) have the potency to proliferate and differentiate into odontoblasts and play an important role in dentine formation and reparation. The aim of the present study is to evaluate the effects of ginsenoside Rg1 on the proliferation and differentiation of hDPCs., Methods: hDPCs were incubated with different concentrations of ginsenoside Rg1 (0.1, 0.5, 2.5, 5, 10 and 20 μmol/L). The effects of ginsenoside Rg1 on the proliferative ability of hDPCs were evaluated by a fibroblast colony forming test, MTT assay and flow cytometry for cell cycle. The control group, osteogenic induction group, ginsenoside Rg1 (5 μmol/L) group and combination group were designed, and alkaline phosphatase (ALP) activity and FQ-PCR for gene expressions of dentine sialophosphoprotein (DSPP) and dentine matrix protein 1 (DMP1) were performed to evaluate the differentiation of hDPCs., Results: The proliferative ability of hDPCs in ginsenoside Rg1 was significantly enhanced (p < 0.05), especially in the ginsenoside Rg1 (5 μmol/L) group. ALP activity and gene expressions of DSPP and DMP1 were increased in the induction group, ginsenoside Rg1 group, and their combination group compared with the control group (p < 0.05)., Conclusions: The results indicate that ginsenoside Rg1 promotes the proliferation and differentiation of hDPCs., (© 2012 Australian Dental Association.)

Introduction: The lysyl oxidase (LOX) family is an emerging family of amine oxidases responsible for the formation of collagen fibrils in the extracellular matrix. To date, 5 LOX family genes have been identified in humans, encoding LOX and LOX-like proteins (LOXL, LOXL2, LOXL3, and LOXL4). The goal of this study was to evaluate the expression and function of the LOX family genes in odontoblastic differentiation of human dental pulp (HDP) cells., Methods: Expression of the LOX family genes was assessed by reverse transcriptase polymerase chain reaction analysis, and the amine oxidase activity of HDP cells was evaluated by peroxidase-coupled fluorometric assays. Mineral nodule formation and expression of odontoblastic marker genes were assessed in the presence and absence of specific small interfering RNAs (siRNAs) of the LOX family genes., Results: Among the LOX family genes, only LOX and LOXL showed prominent expression during odontoblastic differentiation of HDP cells. Suppression of LOX and LOXL expression by siRNA-induced interference substantially decreased the amine oxidase activity of the differentiating HDP cells. Furthermore, interference of LOX and LOXL expression inhibited mineral nodule formation and expression of odontoblastic marker genes during odontoblastic differentiation of HDP cells., Conclusions: These findings show for the first time that the LOX- and LOXL-mediated organization of collagen fibrils in extracellular matrices of HDP cells might be an important regulator for odontoblastic differentiation of HDP cells., (Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Previous studies have shown that histone deacetylase (HDAC) inhibitors stimulate osteoblast differentiation in vitro and bone formation in vivo. However, the effects of HDAC inhibitors on odontoblasts have not been elucidated. Therefore, in this study, we examined the effect of suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, on odontoblast differentiation using an MDPC23 odontoblast-like cell line. SAHA significantly enhanced matrix mineralization and the expression levels of odontoblast marker genes. SAHA increased the expression levels of nuclear factor I/C (Nfic) and dentin sialophosphoprotein (Dspp). Nfic bound directly to the Dspp promoter and stimulated Dspp transcription. SAHA increased both basal and Nfic-induced Dspp promoter activity. SAHA-induced Dspp promoter activity disappeared when mutations were introduced within the Nfic binding element of the Dspp promoter. Nfic knockdown by siRNA blocked SAHA stimulation of Dspp expression. These results indicate that SAHA enhances odontoblast differentiation and that SAHA increases Dspp expression, at least in part, by increasing the expression level of Nfic.

Objective: The aim of this study was to investigate the effects of resinous monomers on the odontogenic differentiation and mineralization potential of apical papilla stem cells (SCAP)., Methods: Cultures were established from developing third molars of healthy donors aged 14-18 years-old and were extensively characterized for proliferation rate, colony forming unit efficiency and expression of stem cell markers (STRO-1, CD146, CD34, CD45, CD105, CD117-c-Kit, CD24, CD90, Nanog, Oct3/4), in order to select those with enhanced stem cell and odontogenic differentiation properties. SCAP enriched cultures were then induced for odontogenic differentiation in the continuous presence of low concentrations (0.05-0.5 mM) of the monomers 2-hydroxy-ethyl-methacrylate-HEMA and triethylene-glycol-dimethacrylate-TEGDMA for 3 weeks (long-term exposure). Additionally, the effects of a single exposure (72 h) to higher concentrations of HEMA (2 mM) and TEGDMA (1 mM) were evaluated., Results: The results showed that both types of monomer-exposure significantly delayed the odontogenic differentiation and mineralization processes of SCAP cells. A down-regulation followed by recovery in the expression of differentiation markers, including dentin sialophosphoprotein-DSPP, bone sialoprotein-BSP, osteocalcin-OCN and alkaline phosphatase-ALP was recorded. This was accompanied by reduction of the mineralized matrix produced by monomer-treated-compared to non-treated contol cultures. Furthermore, a concentration-dependence was observed for both monomers during long-term exposure, whereas the effects of HEMA were evident at much lower concentrations compared to TEGDMA., Significance: These findings suggest that resinous monomers can delay the odontogenic differentiation of SCAP cells, potentially disturbing the physiological repair and/or developmental processes of human permanent teeth., (Copyright © 2012 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Glomerulonephritides together create a heterogenic group of supposedly immunologically mediated diseases of glomeruli. They still belong among the most frequent causes of chronic renal failure. Detection of podocytes in urine might serve as an important marker of glomerulonephritides activity. The aim of this study was to develop a novel flow cytometric method for the detection of podocyte fragments and podocytes in urine and assess its possible use in clinical practice. We placed emphasis on the improvement of pre-analytic phase. To suppress the autofluorescence of the background, blocking solutions and magnetic separation were used. An additional surface marker CD10 (nephrilysin) was used together with routinely used podocalyxin (PCX) in order to achieve better identification of podocytes. Based on the surface marker expression, three different element types were identified in the urine samples: PCX+/CD10+ elements (EL) (supposedly podocytes), PCX-/CD10+ EL (supposedly parietal epithelial cells) and PCX+ EL. We examined a total of 36 patients who underwent renal biopsy (non-glomerular nephropathy, MGN, FSGS, IgAN, AAV and MPGN) and 27 healthy controls. Negative results were found in non-glomerular nephropathy and in MGN. In patients with FSGS and IgAN, the levels of urine elements were slightly increased. The highest levels of all elements were found in AAV and MPGN. Our first results suggest that flow cytometric detection may distinguish between glomerular and nonglomerular diseases and that the levels of urine elements might correlate with the degree of glomerular destruction.

The Golgi apparatus is an intracellular organelle playing central roles in post-translational modification and in the secretion of membrane and secretory proteins. These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the cis-, medial-and trans-cisternae of the Golgi. While trafficking through the Golgi, proteins are sequentially modified with glycan moieties by different glycosyltransferases. Therefore, it is important to analyze the glycosylation function of the Golgi at the level of cisternae. Markers widely used for cis-, medial- and trans-cisternae/trans Golgi network (TGN) in Drosophila are GM130, 120 kDa and Syntaxin16 (Syx16); however the anti-120 kDa antibody is no longer available. In the present study, Drosophila Golgi complex-localized glycoprotein-1 (dGLG1) was identified as an antigen recognized by the anti-120 kDa antibody. A monoclonal anti-dGLG1 antibody suitable for immunohistochemistry was raised in rat. Using these markers, the localization of glycosyltransferases and nucleotide-sugar transporters (NSTs) was studied at the cisternal level. Results showed that glycosyltransferases and NSTs involved in the same sugar modification are localized to the same cisternae. Furthermore, valuable functional information was obtained on the localization of novel NSTs with as yet incompletely characterized biochemical properties.

Using the Bmp2 floxed/3.6Col1a1-Cre (Bmp2-cKO(od)) mouse model, we have observed severe defects in odontogenesis and dentin formation with the removal of the Bmp2 gene in early-polarizing odontoblasts. The odontoblasts in the Bmp2-cKO(od) do not mature properly and fail to form proper dentin with normal dentinal tubules and activate terminal differentiation, as reflected by decreased Osterix, Col1a1, and Dspp expression. There is less dentin, and the dentin is hypomineralized and patchy. We also describe an indirect effect of the Bmp2 gene in odontoblasts on formation of the vascular bed and associated pericytes in the pulp. This vascular niche and numbers of CD146+ pericytes are likely controlled by odontogenic and Bmp2-dependent VegfA production in odontoblasts. The complex roles of Bmp2, postulated to be both direct and indirect, lead to permanent defects in the teeth throughout life, and result in teeth with low quantities of dentin and dentin of poor quality.

Objective: To investigate the effects of specific RNA interference (RNAi) to Notch ligand Delta1 on proliferation and differentiation of human dental pulp stem cells (DPSC)., Methods: DPSC were infected by lentivirus vectors carrying Delta1-RNAi. DPSC were divided into three groups, DPSC/Delta1-RNAi group, DPSC/wt group and DPSC/vector group as control. Cell counting kit-8 (CCK-8) assay and flow cytometry were used to evaluate the proliferation of DPSC. Expression of proliferating cell nuclear antigen (PCNA) was examined with immunohistochemical staining. All groups were cultured in an odonto-inductive medium and were observed under microscope. The number of mineralization nodules was counted after Alizarin red staining. Alkaline phosphatase (ALP) activity and the expression of dentin sialophosphoprotein (DSPP) were detected by ALP activity assay and Western blotting., Results: Compared with DPSC/wt or DPSC/vector separately, proliferating rate and S-cycle of DPSC/Delta1-RNAi was significantly lower. The S phase and proliferation index (PI) decreased markedly from 22.32 ± 2.35 and 33.68 ± 4.19 (DPSC/Delta1-RNAi) to 5.44 ± 0.91 and 16.00 ± 6.07 (DPSC/wt). The PCNA staining of DPSC/Delta1-RNAi was evidently weaker. DPSC/Delta1-RNAi group had more calcified cell nodules than the other two control groups, and ALP activity and DSPP expression of DPSC/Delta1-RNAi group increased markedly., Conclusions: Delta1-RNAi induced by the lentivirus vectors may inhibit DPSC proliferation and differentiation. Notch-Delta signal pathway plays an important role in self-renewal and differentiation.

Ticks are mites specialized in acquiring blood from vertebrates as their sole source of food and are important disease vectors to humans and animals. Among the specializations required for this peculiar diet, ticks evolved a sophisticated salivary potion that can disarm their host's hemostasis, inflammation, and immune reactions. Previous transcriptome analysis of tick salivary proteins has revealed many new protein families indicative of fast evolution, possibly due to host immune pressure. The hard ticks (family Ixodidae) are further divided into two basal groups, of which the Metastriata have 11 genera. While salivary transcriptomes and proteomes have been described for some of these genera, no tick of the genus Hyalomma has been studied so far. The analysis of 2084 expressed sequence tags (EST) from a salivary gland cDNA library allowed an exploration of the proteome of this tick species by matching peptide ions derived from MS/MS experiments to this data set. We additionally compared these MS/MS derived peptide sequences against the proteins from the bovine host, finding many host proteins in the salivary glands of this tick. This annotated data set can assist the discovery of new targets for anti-tick vaccines as well as help to identify pharmacologically active proteins., (Published by Elsevier B.V.)

Cfr (cysteine-rich fibroblast growth factor receptor) is an Fgf (fibroblast growth factor)-binding protein without a tyrosine kinase. We have shown previously that Cfr is involved in Fgf18 signalling via Fgf receptor 3c. However, as Cfr is also known as Glg (Golgi apparatus protein)-1 or MG-160 and occurs in the Golgi apparatus, it remains unknown how the distribution of Cfr is regulated. In the present study, we performed a mutagenic analysis of Cfr to show that two distinct regions contribute to its distribution and stability. First, the C-terminal region retains Cfr in the Golgi apparatus. Secondly, the Cfr repeats in the extracellular juxtamembrane region destabilizes Cfr passed through the Golgi apparatus. This destabilization does not depend on the cleavage and secretion of the extracellular domain of Cfr. Furthermore, we found that Cfr with a GPI (glycosylphosphatidylinositol) anchor was predominantly expressed on the cell surface in Ba/F3 cells and affected Fgf18 signalling in a similar manner to the full-length Cfr, indicating that the interaction of Cfr with Fgfs on the cell surface is important for its function in Fgf signalling. These results suggest that the expression of Cfr in the Golgi apparatus and on the plasma membrane is finely tuned through two distinct mechanisms for exhibiting different functions.