Ectopic Colonization and Immune Landscapes of Periodontitis Microbiota in Germ-Free Mice With Streptozotocin-Induced Type 1 Diabetes Mellitus.

A two-way relationship between diabetes and periodontitis has been discussed recently. Periodontitis microbiota might affect the immune homeostasis of diabetes, but the molecular mechanism of their interactions is still not clear. The aims of this study were to clarify the possible immune regulatory effects of periodontitis microbiota on diabetes and the correlation between immunomodulation and ectopic colonization. A model of germ-free mice with streptozotocin-induced type 1 diabetes mellitus (T1D), which was orally inoculated with mixed saliva samples for 2 weeks, was used in this study. Those mice were randomly divided into two groups, namely, SP (where the T1D mice were orally inoculated with mixed saliva samples from periodontitis patients) and SH (where the T1D mice were orally inoculated with mixed saliva samples from healthy subjects). Ectopic colonization of saliva microbiota was assessed using culture-dependent method and Sanger sequencing, and the composition of gut microbiota was analyzed using 16S rRNA gene sequencing. Changes in 15 types of immune cells and six cytokines either from the small intestine or spleen were detected by multicolor flow cytometry. The correlation between gut microbiota and immune cells was evaluated by redundancy analysis. Although periodontitis microbiota minorly colonized the lungs, spleens, and blood system, they predominantly colonized the gut, which was mainly invaded by Klebsiella. SH and SP differed in beta diversity of the gut bacterial community. Compared to SH, microbial alteration in small intestine occurred with an increase of Lacticaseibacillus, Bacillus, Agathobacter, Bacteroides, and a decrease of Raoultella in SP. More types of immune cells were disordered in the spleen than in the small intestine by periodontitis microbiota, mainly with a dramatical increase in the proportion of macrophages, plasmacytoid dendritic cells (pDCs), monocytes, group 3 innate lymphoid cells, CD4-CD8- T cells and Th17 cells, as well as a decline of αßT cells in SP. Cytokines of IFNγ, IL17, and IL22 produced by CD4 + T cells as well as IL22 produced by ILCs of small intestine rose in numbers, and the intestinal and splenic pDCs were positively regulated by gut bacterial community in SP. In conclusion, periodontitis microbiota invasion leads to ectopic colonization of the extra-oral sites and immune cells infiltration, which might cause local or systemic inflammation. Those cells are considered to act as a "bridge" between T1D and periodontitis.

[Role of M2 Macrophage Exosomes in Osteogenic Differentiation of Mouse Bone Marrow Mesenchymal Stem Cells under High-Glucose and High-Insulin].

OBJECTIVE: To study the role of M2 macrophage-derived exosomes (M2-exo) in osteogenic differentiation and Hedgehog signaling pathway of mouse bone marrow mesenchymal stem cells (BMSCs) under in vitro high-glucose and high-insulin conditions. METHODS: RAW 264.7 cells were induced toward M2 macrophage polarization and then M2-exo were extracted and identified. Immunofluorescence assay was performed to detect the internalization of M2-exo by BMSCs. BMSCs were divided into the normal control group (Control group), the high-glucose and high-insulin group (HGI group), and the HGI with M2-exo intervention group (HGI+M2e group). BMSCs in the Control group were cultured in osteogenic inductive medium with 5.5 mmol/L glucose, but no insulin or M2-exo. BMSCs in the HGI group were cultured in osteogenic inductive medium with 25 mmol/L glucose and 174 nmol/L insulin. BMSCs in the HGI+M2e group were cultured in the same medium as that of the HGI group, with the additional treatment of 6, 30, 60 µg/mL M2-exo, respectively. After osteogenic induction for 7 days and 14 days, alkaline phosphatase (ALP) staining and alizarin red staining were performed respectively to assess the osteogenic differentiation potential of BMSCs from different groups. In addition, BMSCs in the Control group, HGI group, and HGI+M2e group treated with 30 µg/mL M2-exo were examined with qPCR after osteogenic induction for 14 days and Western blot after osteogenic induction for 21 days to assess the osteogenesis and the expression of Hedgehog pathway-related genes and proteins. RESULTS: M2 macrophage polarization was induced successfully, with highly positive expression of CD206, the M2 polarization surface marker. The M2-exo had the typical structure of round or oval-shaped bilayered-membrane vesicles. The diameter distribution of M2-exo ranged from 50 to 125 nm (accounting for 99.14% of all M2-exo). M2-exo samples showed positive expression of exosomal markers CD9, CD63 and CD81 proteins. Immunofluorescence staining showed that M2-exo were taken up and internalized by BMSCs. After osteogenic induction for 7 days, the ALP activity of BMSCs in the HGI group was lower than that of the Control group. After interventions of 6 µg/mL, 30 µg/mL, and 60 µg/mL M2-exo, the ALP activity of the HGI+M2-exo group was significantly increased compared with that of the HGI group ( P<0.05). After osteogenic induction for 14 days, the number of mineralized nodules in the HGI group was lower than that in the Control group, and after intervention, only the HGI+M2e group treated with 30 µg/mL M2-exo showed higher level of mineralization than that in the HGI group ( P<0.05). qPCR analysis revealed that the expression levels of the osteogenesis-related genes, including Runx2, Alp and Ocn, and Hedgehog pathway-related genes, including Gli1, Smo and Ptch1, were downregulated in the HGI group, all being lower than those of the Control group to varying degrees, while 30 µg/mL M2-exo treatment could promote the up-regulation of these genes, showing significant difference in comparison with their expression levels in the HGI group ( P<0.05). In addition, Western blot analysis showed that the expression of the osteogenesis-related proteins, including RUNX2 and COL1A1, and GLI1, the Hedgehog signaling pathway protein, was down-regulated in the HGI group, while the expression of COL1A1 and GLI1 was up-regulated after 30 µg/mL M2-exo treatment, showing significant difference when compared with that of the HGI group ( P<0.05). CONCLUSION: High glucose and high insulin had inhibitory effect on the osteogenic differentiation potential of BMSCs. After intervention with M2-exo, the Hedgehog signaling pathway in BMSCs was activated and the osteogenic differentiation potential was enhanced, suggesting that M2-exo might have therapeutic potentials for the treatment of diabetic bone disease.

Egg yolk immunoglobulin (IgY) targeting SARS-CoV-2 S1 as potential virus entry blocker.

AIMS: COVID-19 pandemic caused by SARS-CoV-2 has become a public health crisis worldwide. In this study, we aimed at demonstrating the neutralizing potential of the IgY produced after immunizing chicken with a recombinant SARS-CoV-2 spike protein S1 subunit. METHODS AND RESULTS: E. coli BL21 carrying plasmid pET28a-S1 was induced with IPTG for the expression of SARS-CoV-2 S1 protein. The recombinant His-tagged S1 was purified and verified by SDS-PAGE, Western blot and biolayer interferometry (BLI) assay. Then S1 protein emulsified with Freund's adjuvant was used to immunize layer chickens. Specific IgY against S1 (S1-IgY) produced from egg yolks of these chickens exhibited a high titer (1:25,600) and a strong binding affinity to S1 (KD  = 318 nmol L-1 ). The neutralizing ability of S1-IgY was quantified by a SARS-CoV-2 pseudotyped virus-based neutralization assay with an IC50  value of 0.99 mg ml-1 . In addition, S1-IgY exhibited a strong ability in blocking the binding of SARS-CoV-2 S1 to hACE2, and it could partially compete with hACE2 for the binding sites on S1 by BLI assays. CONCLUSIONS: We demonstrated here that after immunization of chickens with our recombinant S1 protein, IgY neutralizing antibodies were generated against the SARS-CoV-2 spike protein S1 subunit; therefore, showing the potential use of IgY to block the entry of this virus. SIGNIFICANCE AND IMPACT OF THE STUDY: IgY targeting S1 subunit of SARS-CoV-2 could be a promising candidate for pre- and post-exposure prophylaxis or treatment of COVID-19. Administration of IgY-based oral preparation, oral or nasal spray may have profound implications for blocking SARS-CoV-2.

Beware of pharyngeal Fusobacterium nucleatum in COVID-19.

BACKGROUND: Fusobacterium nucleatum (F. n) is an important opportunistic pathogen causing oral and gastrointestinal disease. Faecalibacterium prausnitzii (F. p) is a next-generation probiotic and could serve as a biomarker of gut eubiosis/dysbiosis to some extent. Alterations in the human oral and gut microbiomes are associated with viral respiratory infection. The aim of this study was to characterise the oral and fecal bacterial biomarker (i.e., F. n and F. p) in COVID-19 patients by qPCR and investigate the pharyngeal microbiome of COVID-19 patients through metagenomic next-generation sequencing (mNGS). RESULTS: Pharyngeal F. n was significantly increased in COVID-19 patients, and it was higher in male than female patients. Increased abundance of pharyngeal F. n was associated with a higher risk of a positive SARS-CoV-2 test (adjusted OR = 1.32, 95% CI = 1.06 ~ 1.65, P < 0.05). A classifier to distinguish COVID-19 patients from the healthy controls based on the pharyngeal F. n was constructed and achieved an area under the curve (AUC) of 0.843 (95% CI = 0.688 ~ 0.940, P < 0.001). However, the level of fecal F. n and fecal F. p remained unaltered between groups. Besides, mNGS showed that the pharyngeal swabs of COVID-19 patients were dominated by opportunistic pathogens. CONCLUSIONS: Pharyngeal but not fecal F. n was significantly increased in COVID-19 patients, clinicians should pay careful attention to potential coinfection. Pharyngeal F. n may serve as a promising candidate indicator for COVID-19.

The preparation of N-IgY targeting SARS-CoV-2 and its immunomodulation to IFN-γ production in vitro.

Specific antibodies against SARS-CoV-2 structural protein have a wide range of effects in the diagnose, prevention and treatment of the COVID-19 epidemic. Among them, egg yolk immunoglobulin Y (IgY), which has high safety, high yield, and without inducing antibody-dependent enhancement, is an important biological candidate. In this study, specific IgY against the conservative nucleocapsid protein (NP) of SARS-CoV-2 was obtained by immunizing hens. Through a series of optimized precipitation and ultrafiltration extraction schemes, its purity was increased to 98%. The hyperimmune IgY against NP (N-IgY) at a titer of 1:50,000 showed strong NP binding ability, which laid the foundation of N-IgY's application targeting NP. In an in vitro immunoregulatory study, N-IgY (1 mg/mL) modulated NP-induced immune response by alleviating type II interferon secretion stimulated by NP (20 µg/mL). In summary, N-IgY can be mass produced by achievable method, which endows it with potential value against the current COVID-19 pandemic.

Detection of SARS-CoV-2 in fecal samples with different pretreatment methods and PCR kits.

BACKGROUND: Gastrointestinal symptoms are common in COVID-19 patients and SARS-CoV-2 RNA has been detected in the patients' feces, which could lead to fecal-oral transmission. Therefore, fecal sample testing with real-time RT-PCR is highly recommended as a routine test for SARS-CoV-2 infection. However, varying rates of detection in fecal sample have been reported. The aim of this study was to provide insights into the detection rates of SARS-CoV-2 in COVID-19 patients' fecal sample by using four real-time RT-PCR kits and two pretreatment methods (inactive and non-inactive). RESULTS: The detection rate of Trizol pretreatment group was slightly higher than that of Phosphate Buffered Saline (PBS) groups, showing that pretreatment and inactivation by Trizol had no influence to SARS-CoV-2 nucleic acid test (NAT) results. 39.29% detection rate in fecal sample by DAAN was obtained, while Bio-germ was 40.48%, Sansure 34.52%, and GeneoDx 33.33%. The former three kits had no significant difference. The DAAN kit detection rates of ORF1ab and N gene were nearly equal and Ct value distribution was more scattered, while the Bio-germ kit distribution was more clustered. The positive rate of SARS-COV-2 in fecal samples correlated with the severity of the disease, specifically, severe cases were less likely to be identified than asymptomatic infection in the DAAN group (adjusted OR 0.05, 95%CI = 0.00 ~ 0.91). CONCLUSIONS: Trizol should be of choice as a valid and safe method for pretreatment of fecal samples of SARS-CoV-2. All real-time RT-PCR kits assessed in this study can be used for routine detection of SARS-CoV-2 in fecal samples. While DAAN, with high NAT positive rate, could be the best out of the 4 kits used in this study. SARS-CoV-2 positive rate in fecal sample was related to the severity of illness.

EB virus-induced ATR activation accelerates nasopharyngeal carcinoma growth via M2-type macrophages polarization.

Chronic inflammation induced by persistent viruses infection plays an essential role in tumor progression, which influenced on the interaction between the tumor cells and the tumor microenvironment. Our earlier study showed that ATR, a key kinase participant in single-stranded DNA damage response (DDR), was obviously activated by Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC). However, how EBV-induced ATR activation promotes NPC by influencing inflammatory microenvironment, such as tumor-associated macrophages (TAMs), remains elusive. In this study, we showed that EBV could promote the expression of p-ATR and M2-type TAMs transformation in clinical NPC specimens. The expression of p-ATR and M2-type TAMs were closely correlated each other and involved in TNM stage, lymph node metastasis and poor prognosis of the patients. In addition, the expression levels of CD68+CD206+, Arg1, VEGF, and CCL22 were increased in EB+ CNE1 cells, and decreased when ATR was inhibited. In the nude mice, EBV-induced ATR activation promoted subcutaneous transplanted tumor growth, higher expression of Ki67 and lung metastasis via M2-type TAMs recruitment. Experimental data also showed that the polarization of M2, the declined tumor necrosis factor-α (TNF-α) and increased transforming growth factor-ß (TGF-ß) were associated with ATR. Meanwhile, ATR activation could promote PPAR-δ and inhibited c-Jun and p-JNK expression, then downregulate JNK pathway. Collectively, our current study demonstrated the EBV infection could activate the ATR pathway to accelerate the transition of TAMs to M2, suggesting ATR knockdown could be a potential effective treatment strategy for EBV-positive NPC.

Purification of HCC-specific extracellular vesicles on nanosubstrates for early HCC detection by digital scoring.

We report a covalent chemistry-based hepatocellular carcinoma (HCC)-specific extracellular vesicle (EV) purification system for early detection of HCC by performing digital scoring on the purified EVs. Earlier detection of HCC creates more opportunities for curative therapeutic interventions. EVs are present in circulation at relatively early stages of disease, providing potential opportunities for HCC early detection. We develop an HCC EV purification system (i.e., EV Click Chips) by synergistically integrating covalent chemistry-mediated EV capture/release, multimarker antibody cocktails, nanostructured substrates, and microfluidic chaotic mixers. We then explore the translational potential of EV Click Chips using 158 plasma samples of HCC patients and control cohorts. The purified HCC EVs are subjected to reverse-transcription droplet digital PCR for quantification of 10 HCC-specific mRNA markers and computation of digital scoring. The HCC EV-derived molecular signatures exhibit great potential for noninvasive early detection of HCC from at-risk cirrhotic patients with an area under receiver operator characteristic curve of 0.93 (95% CI, 0.86 to 1.00; sensitivity = 94.4%, specificity = 88.5%).

Oral Microbiome and SARS-CoV-2: Beware of Lung Co-infection.

The new coronavirus SARS-CoV-2, the cause of COVID-19, has become a public health emergency of global concern. Like the SARS and influenza pandemics, there have been a large number of cases coinfected with other viruses, fungi, and bacteria, some of which originate from the oral cavity. Capnocytophaga, Veillonella, and other oral opportunistic pathogens were found in the BALF of the COVID-19 patients by mNGS. Risk factors such as poor oral hygiene, cough, increased inhalation under normal or abnormal conditions, and mechanical ventilation provide a pathway for oral microorganisms to enter the lower respiratory tract and thus cause respiratory disease. Lung hypoxia, typical symptoms of COVID-19, would favor the growth of anaerobes and facultative anaerobes originating from the oral microbiota. SARS-CoV-2 may aggravate lung disease by interacting with the lung or oral microbiota via mechanisms involving changes in cytokines, T cell responses, and the effects of host conditions such as aging and the oral microbiome changes due to systemic diseases. Because the oral microbiome is closely associated with SARS-CoV-2 co-infections in the lungs, effective oral health care measures are necessary to reduce these infections, especially in severe COVID-19 patients. We hope this review will draw attention from both the scientific and clinical communities on the role of the oral microbiome in the current global pandemic.

Proliferation and odontogenic differentiation of human umbilical cord mesenchymal stem cells and human dental pulp cells co-cultured in hydrogel.

OBJECTIVE: The aim of this study was to evaluate the proliferation and odontogenic differentiation of human dental pulp cells (hDPCs) and human umbilical cord mesenchymal stem cells (hUCMSCs) in three-dimensional co-culture system which was established with the help of bone morphogenetic protein-2 (BMP-2) and hydrogel. METHODS: hDPCs and hUCMSCs were cultured in different concentrations of hydrogel to explore the more suitable concentrations for subsequent experiments. hUCMSCs and hDPCs induced by BMP-2 were co-cultured in the hydrogel. MTT assay was used to measure the cell viability. The differentiation into odontoblast-like cells were measured by the mRNA expression of dentin salivary phosphoprotein (DSPP), dentin matrix protein-1 (DMP-1), alkaline phosphatase and osteocalcin. Alizarin red staining was performed for the formation of mineralized nodules. RESULTS: hUCMSCs and hDPCs could grow and proliferate in hydrogel scaffold. The growth rate of cells in lower concentrations hydrogels were higher than that of high concentrations hydrogels (P < 0.05). The study showed that 0.25% hydrogel scaffold was more suitable for subsequent experiments than other groups. Compared with hUCMSCs-monoculture and hDPCs-monoculture, the co-culture groups exhibited more proliferative potential, alkaline phosphatase activity and mineralization nodule formation (P < 0.05). The mRNA expression in co-culture groups were higher than that of hUCMSCs-monoculture, closed to or even higher than that of hDPCs-monoculture. CONCLUSION: 0.25% hydrogel was the suitable concentration in co-culture system for subsequent experiments. The co-culture groups had stronger abilities of odontoblastic differentiation and mineralization than cells-monoculture groups, indicated that the co-culture conditions could regulate cell proliferation and differentiation within a certain range.

Covalent chemistry on nanostructured substrates enables noninvasive quantification of gene rearrangements in circulating tumor cells.

Well-preserved mRNA in circulating tumor cells (CTCs) offers an ideal material for conducting molecular profiling of tumors, thereby providing a noninvasive diagnostic solution for guiding treatment intervention and monitoring disease progression. However, it is technically challenging to purify CTCs while retaining high-quality mRNA.Here, we demonstrate a covalent chemistry-based nanostructured silicon substrate ("Click Chip") for CTC purification that leverages bioorthogonal ligation-mediated CTC capture and disulfide cleavage-driven CTC release. This platform is ideal for CTC mRNA assays because of its efficient, specific, and rapid purification of pooled CTCs, enabling downstream molecular quantification using reverse transcription Droplet Digital polymerase chain reaction. Rearrangements of ALK/ROS1 were quantified using CTC mRNA and matched with those identified in biopsy specimens from 12 patients with late-stage non-small cell lung cancer. Moreover, CTC counts and copy numbers of ALK/ROS1 rearrangements could be used together for evaluating treatment responses and disease progression.

Bio-Inspired NanoVilli Chips for Enhanced Capture of Tumor-Derived Extracellular Vesicles: Toward Non-Invasive Detection of Gene Alterations in Non-Small Cell Lung Cancer.

Tumor-derived extracellular vesicles (EVs) present in bodily fluids are emerging liquid biopsy markers for non-invasive cancer diagnosis and treatment monitoring. Because the majority of EVs in circulation are not of tumor origin, it is critical to develop new platforms capable of enriching tumor-derived EVs from the blood. Herein, we introduce a biostructure-inspired NanoVilli Chip, capable of highly efficient and reproducible immunoaffinity capture of tumor-derived EVs from blood plasma samples. Anti-EpCAM-grafted silicon nanowire arrays were engineered to mimic the distinctive structures of intestinal microvilli, dramatically increasing surface area and enhancing tumor-derived EV capture. RNA in the captured EVs can be recovered for downstream molecular analyses by reverse transcription Droplet Digital PCR. We demonstrate that this assay can be applied to monitor the dynamic changes of ROS1 rearrangements and epidermal growth factor receptor T790M mutations that predict treatment responses and disease progression in non-small cell lung cancer patients.

The rapid H2 release from AlH3 dehydrogenation forming porous layer in AlH3/hydroxyl-terminated polybutadiene (HTPB) fuels during combustion.

Although the motivation of AlH3 enhancing combustion were recognized in many research, the promotion mechanism have been rarely explored. Herein, a previously unreported porous layer mechanism when combustion were determined in HTPB/AlH3 fuels by SEM, thermo-analysis and a new simplified calculation method, owing to rapidly released gas phase H2 from AlH3 dehydrogenation exposing in melting layer. 5/10% 40-80 µm and 10% 80-200 µm AlH3-HTPB formulas show the regression rate increase by, 25.7%, 29.0% and 43.0% at Gox = 350 kg/m2·s, while by 57.2%, 42.0% and 44.2% enhancement at Gox = 150 kg/m2·s. The low AlH3 content (≤ 10%) promotes the regression rate obviously, while excess AlH3 content (≥ 20%) promotes slightly as a result of comprehensive factors combined by energy release, a certain porous layer mechanism, aggregated Al2O3 attached on the burning surface and the blocking effect of the gaseous released H2. A new model predicting the overlapping process of AlH3 dehydrogenation and Al oxidation in air atmosphere was developed by superimposing AlH3 dehydrogenation simulation and corresponding separated Al oxidation simulation. A 1.5th Avrami-Erofeev (A-E) simulation was proposed for Al passivation weight gain between 420 and 520 K with an activation energy of 124.92 kJ/mol and the pre-exponential of 10^12.35.

[Human umbilical cord mesenchymal stem cells co-cultured with dental pulp cells in vitro and its effect on cell biological behaviors].

PURPOSE: To investigated the effect of human umbilical cord mesenchymal stem cells (hUCMSCs) and human dental pulp cells (hDPCs) on cell biological behaviors by co-culture system in vitro. METHODS: hUCMSCs and hDPCs were obtained by primary culture. A culture system of hUCMSCs and hDPCs induced by BMP2 was established in vitro. hUCMSCs and hDPCs were co-cultured at the ratio of 1:1, 1:5 and 5:1. The optimum ratio of each group was selected to further experiment. The formation of calcium nodule was stained by alizarin red staining at 21 day. The expression of DSPP，ALP，DMP1，OCN，VEGF，HGF and Nanog gene was detected by real-time quantitative PCR at 7 day and 14 day. 1:1 group and hUCMSCs, hDPCs group were selected for alizarin red staining at 21 day according to PCR results. Statistical analysis was performed using SPSS 21.0 software package. RESULTS: Calcified nodules formation in 1:1 group was significantly higher than in hUCMSCs group (P<0.05), close to that in hDPCs. qPCR showed that the mRNA expression of DSPP, ALP, DMP1, OCN, VEGF and HGF in 1:1 group was significantly higher than that in hUCMSCs (P<0.05); mRNA expression of Nanog in 1:1 group was significantly lower than in hUCMSCs group (P<0.05). The results of alizarin red staining showed that the OD value of 1:1 group was significantly higher than that of hUCMSCs group (P<0.05). CONCLUSIONS: The cells can be induced to differentiate into odontoblastoid-like cells and the mRNA expression of angiogenic factors was stimulated by hUCMSCs co-culure wih hDPCs.

Recent Developments in Spectroscopic Techniques for the Detection of Explosives.

Trace detection of explosives has been an ongoing challenge for decades and has become one of several critical problems in defense science; public safety; and global counter-terrorism. As a result, there is a growing interest in employing a wide variety of approaches to detect trace explosive residues. Spectroscopy-based techniques play an irreplaceable role for the detection of energetic substances due to the advantages of rapid, automatic, and non-contact. The present work provides a comprehensive review of the advances made over the past few years in the fields of the applications of terahertz (THz) spectroscopy; laser-induced breakdown spectroscopy (LIBS), Raman spectroscopy; and ion mobility spectrometry (IMS) for trace explosives detection. Furthermore, the advantages and limitations of various spectroscopy-based detection techniques are summarized. Finally, the future development for the detection of explosives is discussed.

Glycan Stimulation Enables Purification of Prostate Cancer Circulating Tumor Cells on PEDOT NanoVelcro Chips for RNA Biomarker Detection.

A glycan-stimulated and poly(3,4-ethylene-dioxythiophene)s (PEDOT)-based nanomaterial platform is fabricated to purify circulating tumor cells (CTCs) from blood samples of prostate cancer (PCa) patients. This new platform, phenylboronic acid (PBA)-grafted PEDOT NanoVelcro, combines the 3D PEDOT nanosubstrate, which greatly enhances CTC capturing efficiency, with a poly(EDOT-PBA-co-EDOT-EG3) interfacial layer, which not only provides high specificity for CTC capture upon antibody conjugation but also enables competitive binding of sorbitol to gently release the captured cells. CTCs purified by this PEDOT NanoVelcro chip provide well-preserved RNA transcripts for the analysis of the expression level of several PCa-specific RNA biomarkers, which may provide clinical insights into the disease.

[Effect of bone morphogenetic protein 2 and dexamethason on proliferation and differentiation of human dental pulp cells in vitro].

Objective: To investigate the effect of bone morphogenetic protein 2 (BMP-2) and dexamethason (DXM) on proliferation and differentiation of human dental pulp cells in vitro. Methods: Primary human dental pulp cells were cultured in vitro by tissue culture method. The 3rd generation cells were used to identify cell phenotype for vimentin and cytokeratin by immunocytochemistry staining. The 3-5 generations of human dental pulp cells were randomly divided into 4 groups: 100 ng/mL BMP-2 (group A), 1×10 -8 mol/L DXM (group B), and both 100 ng/mL BMP-2 and 1×10 -8 mol/L DXM (group C) were added; neither BMP-2 nor DXM was added in group D as control group. The cell growth curve was drawn at 1, 3, 5, and 7 days after culture. The expressions of osteo/dentanogenic genes including alkaline phosphatase (ALP), dentin sialophoshoprotein (DSPP), and dentin matrix protein 1 (DMP-1) were detected by RT-PCR analysis at 5 and 7 days after culture, the ratio between the positive staining area and the total area by ALP staining at 14 days, and absorbance ( A) value at 562 nm by alizarin red staining at 21 days after culture. Results: Human dental pulp cells were successfully isolated and cultured, which were long fusiform and showed a positive reaction for vimentin and a negative reaction for cytokeratin. The growth curve indicated that cells increased with the extending of incubation time, reached a peak at 5 days, then reduced at 7 days to the level at 3 days. At 5 days after culture, the cells were significantly more in groups A, B, and C than group D ( P<0.05), in group C than group A ( P<0.05), and in group A than group B ( P<0.05). RT-PCR analysis showed that the mRNA expressions of ALP, DSPP, and DMP-1 at 5 days were significantly higher in groups A, B, and C than group D ( P<0.05), and in group C than groups A and B ( P<0.05), but no significant difference was found between groups A and B ( P>0.05); the mRNA expression of DSPP in groups A, B, and C was significantly higher than that in group D ( P<0.05), but there was no significant difference in mRNA expressions between other groups at 7 days ( P>0.05). At 14 days, positive staining in varying degrees was observed in each group, especially in group C; the ratio between the positive staining area and the total area was significantly higher in group C than groups A, B, and D ( P<0.05), and in groups A and B than group D ( P<0.05), but there was no significant difference between groups A and B ( P>0.05). At 21 days, there were a variety of mineralized nodules in groups A, B, and C in nonuniformly scattered or clustered distribution, but no mineralized nodules were observed in group D. The A values of mineralized nodules showed significant difference between groups ( P<0.05). Conclusion: BMP-2 may be more effective in promoting proliferation of human dental pulp cells than DXM. Combined application of BMP-2 and DXM can remarkably promote the proliferation and differentiation of human dental pulp cells.

[miRNA profile of the human dental pulp cells during odontoblast differentiation induced by BMP-2].

PURPOSE: To screen and verify the differentially expressed microRNAs (miRNAs) during the differentiation of human dental pulp cells (hDPCs) to odontoblasts induced by BMP-2. METHODS: The isolated hDPCs were cultured in vitro and induced by BMP-2. The levels of ALP, DMP-1 and DSPP were quantified by quantitative real-time polymerase chain reaction (qRT-PCR). The potential characteristics of hDPCs were investigated by miRNA microarray and highly expressed miRNAs were selected with bio-information software for predicting target genes and their biological functions. Then the results were validated using qRT-PCR analysis for the selected miRNAs. Statistical analysis was performed using SPSS 18.0 software package. RESULTS: The expression of ALP, DSPP, and DMP-1 showed significantly higher levels in BMP-2 induced groups compared to the control group(P<0.05). A total of 36 miRNAs were changed (i.e. 20 miRNAs were up-regulated and 16 were down-regulated). The results of qRT-PCR were consistent with the microarray results. GO categories revealed that they were mainly associated with biological process(37.8%), cellular component (29%), molecular function(33%), while the function of other 0.2% genes remained unknown. CONCLUSIONS: This study identified differential expression of miRNAs in BMP-2-induced odontoblastic differentiation of hDPCs, thus contributing to further investigations of regulatory mechanisms and biological effect of target genes in BMP-2-induced odontoblastic differentiation of hDPCs.