Neural progenitor cell-derived nanovesicles promote hair follicle growth via miR-100.

BACKGROUND: Accumulating evidence shows that mesenchymal stem cell-derived extracellular vesicles (EVs) hold great promise to promote hair growth. However, large-scale production of EVs is still a challenge. Recently, exosome-mimetic nanovesicles (NV) prepared by extruding cells have emerged as an alternative strategy for clinical-scale production. Here, ReNcell VM (ReN) cells, a neural progenitor cell line was serially extruded to produce NV. RESULTS: ReN-NV were found to promote dermal papilla cell (DPC) proliferation. In addition, in a mouse model of depilation-induced hair regeneration, ReN-NV were injected subcutaneously, resulting in an acceleration of hair follicle (HF) cycling transition at the site. The underlying mechanism was indicated to be the activation of Wnt/ß-catenin signaling pathway. Furthermore, miR-100 was revealed to be abundant in ReN-NV and significantly up-regulated in DPCs receiving ReN-NV treatment. miR-100 inhibition verified its important role in ReN-NV-induced ß-catenin signaling activation. CONCLUSION: These results provide an alternative agent to EVs and suggest a strategy for hair growth therapy.

Toxicity Assessment of 4 Azo Dyes in Zebrafish Embryos.

Azo dyes are used widely as color additives in food, drugs, and cosmetics; hence, there is an increasing concern about their safety and possible health hazards. In the present study, we chose 4 azo dyes tartrazine, Sunset Yellow, amaranth, and Allura red and evaluated their developmental toxicity on zebrafish embryos. At concentration levels of 5 to 50 mM, we found that azo dyes can induce hatching difficulty and developmental abnormalities such as cardiac edema, decreased heart rate, yolk sac edema, and spinal defects including spinal curvature and tail distortion. Exposure to 100 mM of each azo dye was completely embryolethal. The median lethal concentration (LC50), median effective concentration (EC50), and teratogenic index (TI) were calculated for each azo dye at 72 hours postfertilization. For tartrazine, the LC50 was 47.10 mM and EC50 value was at 42.66 mM with TI ratio of 1.10. For Sunset Yellow, the LC50 was 38.93 mM and EC50 value was at 29.81 mM with TI ratio of 1.31. For amaranth, the LC50 was 39.86 mM and EC50 value was at 31.94 mM with TI ratio of 1.25. For Allura red, the LC50 was 47.42 mM and EC50 value was 40.05 mM with TI ratio of 1.18. This study reports the developmental toxicity of azo dyes in zebrafish embryos at concentrations higher than the expected human exposures from consuming food and drugs containing azo dyes.

Interleukin-10 polymorphisms affect the key periodontal pathogens in Chinese periodontitis patients.

Interleukin-10 (IL-10) polymorphisms have been shown to affect IL-10 production. This study investigated the influences of IL-10 polymorphisms on the susceptibility to chronic periodontitis (CP) and aggressive periodontitis (AP), and their possible role in the quantity of subgingival bacteria Aggregatibacter Actinomycetemcomitans and Porphyromonas gingivalis. 92 CP patients, 83 AP patients and 91 periodontal healthy controls were recruited. Serum IL-10 concentration was analyzed by enzyme-linked immunosorbent assay (ELISA). Gene polymorphisms were determined by multiplex SNaPshot technique. Bacteria were quantified by real-time polymerase chain reaction with TaqMan MGB probes. Taking into account age, gender and periodontal status, IL-10-592 AA, -819 TT and ATA/ATA genotype occurred more frequently in patients with CP than in healthy controls. In CP cases, higher quantity of subgingival A. actinomycetemcomitans and lower serum IL-10 levels could be detected in homozygous ATA/ATA carriers. These findings indicate that variants in IL-10 promoter gene were not only associated with predisposition to chronic periodontitis but also affected the subgingival number of A. Actinomycetemcomitans in a Chinese Han population.

The mechanism of Jurkat cells apoptosis induced by Aggregatibacter actinomycetemcomitans cytolethal distending toxin.

Cytolethal distending toxin (CDT) which is produced by Aggregatibacter actinomycetemcomitans causes apoptosis in lymphocytes. But the specific mechanism is not clear. The aim of our research was to investigate the effect and mechanism during this process. The wild-type CdtA, CdtB, CdtC (CdtAW, CdtBW, CdtCW) and mutant CdtB (CdtBM) were expressed and purified respectively and the purity of each subunit was examined by BandScan software. And the type I deoxyribonuclease and PI-3,4,5-triphosphate (PI-3,4,5-P3, PIP3) phosphatase activity were detected by DNA agarose gel electrophoresis and enzyme-linked immunosorbent assay respectively. The cell apoptosis rates were analyzed by flow cytometry. The morphological changes of apoptosis cells were observed by confocal laser scanning microscopy. The protein expression of Bax and Bcl-2 was examined by western blot. Differentially expressed apoptosis-related proteins were identified based on isobaric tags for relative and absolute quantitation technology. In the present study we found that: (i) recombinant wild-type CdtA, CdtB and CdtC (CdtAW, CdtBW, CdtCW) and mutant CdtB (CdtBM) were correctly expressed and the purity of each protein was higher than 80%, (ii) the average apoptosis rate in wild-type CDT (CDTW) treated groups was 50.54%, which was significantly higher than the controls (4.71%) and mutant CDT (CDTM) treated groups (5.58%) (p < 0.05), (iii) morphological changes of apoptosis were observed in CDTW treated cells, (iv) the expression of Bax protein was significantly increased in CDTW treated cells, while Bcl-2 protein expression was significantly decreased, (v) 17 apoptosis-related proteins were expressed differentially, among which 10 proteins (SMNDC1, TNFRSF10B, UBE2I, ITM2A, CASP3, P53, EIF1, TCF3, HMGN5, CASP8) were up-regulated and 7 proteins (RRM2, TPX2, KIF11, NUCKS1, TOP2A, XRCC1, PTPLAD1, RRM2) were down-regulated, (vi) one possible apoptotic pathway [Ubc9 (UBE2I)/P53/DR5 (TNFRSF10B)/Caspase-8 (CASP8)/ Caspase-3 (CASP3)] was selected and partially proved.

Differential Expression of Proteins Associated with the Hair Follicle Cycle - Proteomics and Bioinformatics Analyses.

Hair follicle cycling can be divided into the following three stages: anagen, catagen, and telogen. The molecular signals that orchestrate the follicular transition between phases are still unknown. To better understand the detailed protein networks controlling this process, proteomics and bioinformatics analyses were performed to construct comparative protein profiles of mouse skin at specific time points (0, 8, and 20 days). Ninety-five differentially expressed protein spots were identified by MALDI-TOF/TOF as 44 proteins, which were found to change during hair follicle cycle transition. Proteomics analysis revealed that these changes in protein expression are involved in Ca2+-regulated biological processes, migration, and regulation of signal transduction, among other processes. Subsequently, three proteins were selected to validate the reliability of expression patterns using western blotting. Cluster analysis revealed three expression patterns, and each pattern correlated with specific cell processes that occur during the hair cycle. Furthermore, bioinformatics analysis indicated that the differentially expressed proteins impacted multiple biological networks, after which detailed functional analyses were performed. Taken together, the above data may provide insight into the three stages of mouse hair follicle morphogenesis and provide a solid basis for potential therapeutic molecular targets for this hair disease.

Histone deacetylase 8 suppresses osteogenic differentiation of bone marrow stromal cells by inhibiting histone H3K9 acetylation and RUNX2 activity.

Bone marrow stromal cells (BMSCs) are multipotent progenitor cells with capacities to differentiate into the various cell types and hold great promise in regenerative medicine. The regulatory roles of histone deacetylases (HDACs) in osteoblast differentiation process have been increasingly recognized; however, little is known about the precise roles of HDAC8 in the osteogenic differentiation of BMSCs. Herein we aimed to investigate the roles of HDAC8 in the osteogenic differentiation of rat BMSCs by pharmacological and genetic manipulations in vitro. During osteogenic differentiation of BMSCs, pharmacological inhibition of HDAC8 by HDAC inhibitor valproic acid (VPA) promoted the level of histone H3 lysine 9 acetylation (H3K9Ac) and significantly enhanced the expression of osteogenesis-related genes Runx2, Osterix, osteocalcin (OCN), osteopontin (OPN) and alkaline phosphatase (ALP). Similarly, knockdown of HDAC8 using short interfering RNA triggered H3K9Ac and enhanced osteogenic differentiation of BMSCs, largely phenocopied the effects of VPA-mediated HDAC8 depletion. However, enforced expression of HDAC8 significantly reduced the level of H3K9Ac and inhibited osteogenic differentiation of BMSCs, which can be attenuated by VPA addition. Mechanistically, HDAC8 suppressed osteogenesis-related genes expression by removing the acetylation of histone H3K9, thus leading to transcriptional inhibition during osteogenic differentiation of BMSCs. Importantly, we found that HDAC8 physically associated with Runx2 to repress its transcriptional activity and this association decreased when BMSCs underwent osteogenic differentiation. Taken together, these results indicate that epigenetic regulation of Runx2 by HDAC8-mediated histone H3K9 acetylation is required for the proper osteogenic differentiation of BMSCs.

miR-1271 regulates cisplatin resistance of human gastric cancer cell lines by targeting IGF1R, IRS1, mTOR, and BCL2.

Numerous studies showed that drug resistance of gastric cancer cells could be modulated by the abnormal expression of microRNAs (miRNAs) which target multiple cell signaling pathways. The possible function of miR-1271 in the formation of cisplatin resistance in gastric cancer cells has been investigated in this study. miR-1271 was significantly down-regulated in gastric cancer tissues and various gastric cancer cell lines. Moreover, it was down-regulated in the cisplatin-resistant gastric cancer cell line SGC7901/cisplatin (DDP) and the down-regulation of miR-1271 in SGC7901/DPP cells was accompanied by the up-regulation of insulin-like growth factor 1 receptor (IGF1R)/insulin receptor substrate 1 (IRS1) pathway-related proteins, i.e., IGF1R, IRS1, serine/threonine-protein kinase mTOR (mTOR), and the apoptosis regulator Bcl-2 (BCL2), compared with the parental SGC7901 cells. Over-expression of miR-1271 sensitized SGC7901/DDP cells to cisplatin. Changes in the luciferase activity of reporter constructs harboring the 3'-untranslated region of the above proteins in SGC7901/DDP cells suggested that IGF1R, IRS1, mTOR, and BCL2 were target genes of miR-1271. Enforced miR-1271 expression repressed the protein levels of its targets, inhibited proliferation of SGC7901/DDP cells, and sensitized SGC7901/DDP cells to DDP-induced apoptosis. Overall, on the basis of the results of our study, we proposed that miR-1271 could regulate cisplatin resistance in human gastric cancer cells, at least partially, via targeting the IGF1R/IRS1 pathway.

Prevalence and distribution of Aggregatibacter actinomycetemcomitans and its cdtB gene in subgingival plaque of Chinese periodontitis patients.

BACKGROUND: Aggregatibacter actinomycetemcomitans (A.actinomycetemcomitans) is an important periodontal pathogen that can participate in periodontitis and other non-oral infections. The cytolethal distending toxin (Cdt) is among the virulence factors produced by this bacterium. This study was to elucidate the distribution of A.actinomycetemcomitans and the prevalence of its cdtB gene in Chinese subjects. METHODS: A total of 255 subgingival samples were obtained from 30 subjects. Samples were collected from periodontal healthy sites as well as shallow, moderate and deep pockets. The absolute quantity of A.actinomycetemcomitans and cdtB gene were determined by real-time polymerase chain reaction. RESULTS: A.actinomycetemcomitans was detected in 92 of 105 (87.6%) samples of aggressive periodontitis (AgP) patients, in 73 of 79 (92.4%) samples of chronic periodontitis ( CP) patients and in 5 of 71 (7.0%) samples of periodontal healthy subjects. The cdtB gene was detected in 72 sites (78.3%) with AgP infected with A.actinomycetemcomitans, 54 sites (74.0%) with CP infected with A.actinomycetemcomitans and none in healthy sites infected with A.actinomycetemcomitans. In addition, quantity of A.actinomycetemcomitans and cdt gene in samples from deep pockets were significant larger than moderate, shallow and healthy sites (P < 0.05). In comparison to CP, AgP patients were infected with increased numbers of cdt genotype in deep pockets (P < 0.05). CONCLUSION: This study suggests that the cdtB gene are prevalent in A.actinomycetemcomitans, and the distribution of cdt genotype strain may be correlated with AgP and serious periodontal inflammation.

A new functional site W115 in CdtA is critical for Aggregatibacter actinomycetemcomitans cytolethal distending toxin.

Aggregatibacter actinomycetemcomitans, a specific pathogen of localized aggressive periodontitis, produces a cytolethal distending toxin (CDT) that arrests eukaryotic cells irreversibly in G0/G1 or G2/M phase of the cell cycle. Although structural studies show that the aromatic patch region of CdtA plays an important role in its biological activity, the functional sites of CdtA have not been firmly established. In this study, site-specific mutagenesis strategy was employed for cdtA point mutations construction so as to examine the contributions of individual amino acids to receptor binding and the biological activity of holotoxin. The binding ability was reduced in CdtA(Y181A)BC holotoxin and the biological function of CDT was not weaken in CdtA(Y105A)BC, CdtA(Y125A)BC, CdtA(F109A)BC and CdtA(S106N)BC holotoxin suggesting that these sites were not critical to CDT. But the binding activity and cell cycle arrest ability of holotoxin complexes were inhibited in CdtA(W115G)BC. And this site did not affect the holotoxin assembly by size exclusion chromatography. Therefore, W115 might be a critical site of CdtA binding ability. These findings suggest that the functional sites of CdtA are not only in the aromatic patch region. W115, the new functional site is critical for receptor binding and cell cycle arrest, which provides potential targets for pharmacological disruption of CDT activity.

Effects of aging on endotoxin tolerance induced by lipopolysaccharides derived from Porphyromonas gingivalis and Escherichia coli.

BACKGROUND: Periodontitis is a bacterially induced chronic inflammatory disease. Exposure of the host to periodontal pathogens and their virulence factors induces a state of hyporesponsiveness to subsequent stimulations, termed endotoxin tolerance. Aging has a profound effect on immune response to bacteria challenge. The aim of this study was to explore the effects of aging on endotoxin tolerance induced by Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) and Escherichia coli (E. coli) LPS in murine peritoneal macrophages. METHODOLOGY/PRINCIPAL FINDINGS: We studied the cytokine production (TNF-α and IL-10) and Toll-like receptor 2, 4 (TLR2, 4) gene and protein expressions in peritoneal macrophages from young (2-month-old) and middle-aged (12-month-old) ICR mice following single or repeated P. gingivalis LPS or E. coli LPS stimulation. Pretreatment of peritoneal macrophages with P. gingivalis LPS or E. coli LPS resulted in a reduction in TNF-α production and an increase in IL-10 production upon secondary stimulation (p<0.05), and the markedly lower levels of TNF-α and higher levels of IL-10 were observed in macrophages from young mice compared with those from middle-aged mice (p<0.05). In addition, LPS restimulations also led to the significantly lower expression levels of TLR2, 4 mRNA and protein in macrophages from young mice (p<0.05). CONCLUSIONS/SIGNIFICANCE: Repeated LPS stimulations triggered endotoxin tolerance in peritoneal macrophages and the ability to develop tolerance in young mice was more excellent. The impaired ability to develop endotoxin tolerance resulted from aging might be related to TLR2, 4 and might lead to the incontrollable periodontal inflammation in older adults.

Cloning and characterization of ribosomal protein S29, a deltamethrin resistance associated gene from Culex pipiens pallens.

The 40S ribosomal protein S29 (RPS29) was shown differentially expressed in deltamethrin (DM)-susceptible and resistant Culex pipiens pallens previously. Here, we cloned RPS29 from this mosquito. An open reading frame (ORF) of 171 bp was found to encode a putative protein with 56 amino acid residues, which shares more than 90% identities with other mosquito RPS29 genes. Moreover, quantitative real-time PCR analysis demonstrated that the transcriptional level of RPS29 was significantly up-regulated both in DM-resistant Cx. p. pallens and C6/36 cells compared with the susceptible ones. Over-expression of RPS29 showed a slight decrease of cell viability in DM-susceptible C6/36 cells under DM treatment and knockdown of RPS29 by siRNA comparably increased cell viability in DM-resistant cells. Our study provides evidence that RPS29 might be associated with fitness cost of DM resistance in mosquitoes for the first time.

prag01, a novel deltamethrin-resistance-associated gene from Culex pipiens pallens.

The prag01 gene (GenBank accession no. EU073017) was cloned from Culex pipiens pallens. An open reading frame of 270 bp was found to encode a putative 89-amino-acid protein which has the highest homology with Culex quinquefasciatus and Anopheles funestus. Real-time quantitative PCR analysis demonstrated that the transcription level of prag01 gene in deltamethrin-resistant strain was 1.65-fold higher than in deltamethrin-susceptible strain of C. pipiens pallens. Overexpression of prag01 gene in the mosquito C6/36 cells showed better prolification than the cells with empty vector when treated by deltamethrin. Our data for the first time approved that prag01 gene might play some role in the development of deltamethrin resistance in C. pipiens pallens.

[Gene cloning of cdtB from Actinobacillus actinomycetemcomitans and bioactivity test of the recombinant protein CdtB in vitro].

OBJECTIVE: To construct the prokayotic expression vector pET-15b-cdtB containing the cdtB gene from Actinobacillus actinomycetemcomitans (Aa) and to test the bioactivity of this recombinant CdtB in vitro. METHODS: The toxic cytolethal distending toxin (CDT) subunit encoding gene cdtB was amplified by PCR. Through restriction endonuclease digestion, gene cdtB and vector pET-15b were ligated to form pET-15b-cdtB expression system which was transformed into competent cells Escherichia coli BL21 (DE3). Protein expression was induced by isopropyl-beta-D-thiogalactoside and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Supercoiled plasmid pET-32a DNA was incubated with purified recombinant CdtB protein in vitro to view any changes in the electrophoretic mobility of the plasmid pET-32a DNA band. RESULTS: PCR testing results of pET-15b-cdtB transformed cells demonstrated that all strains contained cdtB gene. The DNA sequence was blast with cdtB gene from GenBank and 99% homology was obtained. Both of SDS-PAGE and Western blotting confirmed that recombinant CdtB was obtained. After incubated with the purified recombinant CdtB in vitro, the supercoiled plasmid pET-32a DNA was observed relaxing by 1% agarose gel electrophoresis test. CONCLUSIONS: The recombinant plasmid pET-15b-cdtB was successfully constructed and the recombinant CdtB protein which has the DNaseI-like activity was obtained.

Differential regulation of proteoglycan-4 expression by IL-1α and TGF-ß1 in rat condylar chondrocytes.

Proteoglycan 4 (PRG4) is a multifaceted glycoprotein that mediates boundary lubrication of articular cartilage and its dysregulation is associated with impaired lubrication and cartilage destruction in multiple synovial joints. However, the spatiotemporal expression of PRG4 and the associated regulatory networks remain largely unknown in the mandibular condylar cartilage that is responsible for homeostasis and functions of the temporomandibular joint. We here investigated the possible regulatory effects of the interleukin-1α (IL-1α) or/and transforming growth factor-ß1 (TGF-ß1) on the expression of PRG4 in primary chondrocytes that were isolated from the superficial layer of the condylar cartilage of the 20-day-old male Sprague-Dawley rats. Both IL-1α and TGF-ß1 have been implicated in joint destruction and repair. Treatment of primary chondrocytes for 24 h with recombinant human (rh) IL-1α (10 ng/ml) resulted in pronounced reduction in the expression levels of PRG4 mRNA and protein, whereas stimulation with rhTGF-ß1 (10 ng/ml) significantly increased the expression levels, as measured by RT-PCR and ELISA, respectively. Moreover, rhTGF-ß1 was capable to antagonize the inhibitory effects on the PRG4 expression caused by rhIL-1α and robustly restored its abundance in the cultured condylar chondrocytes. Taken together, our data indicate that PRG4 is synthesized and secreted by condylar cartilage chondrocytes and its expression is differentially regulated by IL-1α and TGF-ß1. The rhIL-1α-mediated PRG4 repression is reversible and potently antagonized by rhTGF-ß1 in condylar chondrocytes. The observed up-regulation of PRG4 upon rhTGF-ß1 treatment further supports the therapeutic application of rhTGF-ß1 in the treatment of temporomandibular joint osteoarthritis.

Cloning and characterization of myosin regulatory light chain (MRLC) gene from Culex pipiens pallens.

Myosin regulatory light chain (MRLC) (GenBank accession no. DQ140391) was cloned from Culex pipiens pallens. An open reading frame (ORF) of 630 bps was found to encode a putative 210 amino acids protein which shows 73% similarity with myosin regulatory light chain of Gryllotalpa orientalis. Real-time quantitative PCR analysis demonstrated that the transcription level of MRLC in deltamethrin-resistant strain (DR-strain) was 4.08-fold higher than in deltamethrin-susceptible strain (DS-strain) of C. pipiens pallens. Over-expression of MRLC in Aedes albopictus C6/36 cells conferred protection against deltamethrin based on tritiated methyl tritiated thymidine ((3)H-TdR) incorporation assay. These results indicate that MRLC may be a potential cause of deltamethrin resistance in C. pipiens pallens.

Glycogen branching enzyme: a novel deltamethrin resistance-associated gene from Culex pipiens pallens.

The glycogen branching enzyme (GBE) gene has been cloned from Culex pipiens pallens. An open reading frame of 2,070 bp was found to encode a putative 689 amino acid protein. The deduced amino acid sequence shares 82% and 60% identity with GBE genes from Aedes aegypti and Homo sapiens. Transcript expression of GBE was determined by real-time polymerase chain reaction in deltamethrin-susceptible and deltamethrin-resistant strains of the Culex mosquito. The results demonstrated that this gene has 19.7-fold higher expression in deltamethrin-resistant strain. Unexpectedly, the following study showed that cell proliferation decreased obviously in the GBE overexpression group compared to the empty vector control or the unrelated gene when treated by deltamethrin in the mosquito C6/36 cells whose mechanism is still unexplained. Our study provides the first direct evidence that GBE gene may play some role in the development of deltamethrin resistance in C. pipiens pallens.