Harmonic and anharmonic vibrational computations for biomolecular building blocks: Benchmarking DFT and basis sets by theoretical and experimental IR spectrum of glycine conformers.

Advanced vibrational spectroscopic experiments have reached a level of sophistication that can only be matched by numerical simulations in order to provide an unequivocal analysis, a crucial step to understand the structure-function relationship of biomolecules. While density functional theory (DFT) has become the standard method when targeting medium-size or larger systems, the problem of its reliability and accuracy are well-known and have been abundantly documented. To establish a reliable computational protocol, especially when accuracy is critical, a tailored benchmark is usually required. This is generally done over a short list of known candidates, with the basis set often fixed a priori. In this work, we present a systematic study of the performance of DFT-based hybrid and double-hybrid functionals in the prediction of vibrational energies and infrared intensities at the harmonic level and beyond, considering anharmonic effects through vibrational perturbation theory at the second order. The study is performed for the six-lowest energy glycine conformers, utilizing available "state-of-the-art" accurate theoretical and experimental data as reference. Focusing on the most intense fundamental vibrations in the mid-infrared range of glycine conformers, the role of the basis sets is also investigated considering the balance between computational cost and accuracy. Targeting larger systems, a broad range of hybrid schemes with different computational costs is also tested.

Potential safety concerns of volatile constituents released from coffee-ground-blended single-use biodegradable drinking straws: A chemical space perspective.

Incorporating spent coffee grounds into single-use drinking straws for enhanced biodegradability also raises safety concerns due to increased chemical complexity. Here, volatile organic compounds (VOCs) present in coffee ground straws (CGS), polylactic acid straws (PLAS), and polypropylene straws (PPS) were characterized using headspace - solid-phase microextraction and migration assays, by which 430 and 153 VOCs of 10 chemical categories were identified by gas chromatography - mass spectrometry, respectively. Further, the VOCs were assessed for potential genetic toxicity by quantitative structure-activity relationship profiling and estimated daily intake (EDI) calculation, revealing that the VOCs identified in the CGS generally triggered the most structural alerts of genetic toxicity, and the EDIs of 37.9% of which exceeded the threshold of 0.15 µg person-1 d-1, also outnumbering that of the PLAS and PPS. Finally, 14 VOCs were prioritized due to their definite hazards, and generally higher EDIs or detection frequencies in the CGS. Meanwhile, the probability of producing safer CGS was also illustrated. Moreover, it was uncovered by chemical space that the VOCs with higher risk potentials tended to gather in the region defined by the molecular descriptor related to electronegativity or octanol/water partition coefficient. Our results provided valuable references to improve the chemical safety of the CGS, to promote consumer health, and to advance the sustainable development of food contact materials.

Detection of trace chlorine pollutants in recycled pulp using gas chromatography-tandem mass spectrometry with response surface-optimized carbon structure online catalysis.

RATIONALE: Chlorinated aromatics and alkanes are widely used for their flame retardancy, but they need to be monitored when used in recycled pulp. This paper reports the use of palladium acetate/activated carbon (Pa/Ac) activated by nitric acid as an online catalyst to determine chlorinated aromatics and chlorinated alkanes in recycled paper products using gas chromatography-tandem mass spectrometry (GC-MS/MS), which significantly improves the sensitivity of the method and remarkably lowers the detection limits. METHODS: The Pa/Ac catalyst was prepared using a self-made catalytic device and used as key to the online catalytic conversion of target chlorinated aromatic hydrocarbons and chlorinated alkanes for GC-MS/MS analysis. The response surface model was used to optimize catalytic conditions. Then GC-MS/MS in the multireaction monitoring mode with online catalysis was applied for the analysis of polychlorinated biphenyls, polychlorinated terphenyls, polychlorinated naphthalene, and chlorinated paraffins (CP) in recycled paper products. RESULTS: Compared with traditional methods, the Pa/Ac catalyst can transform chlorinated aromatic hydrocarbons into aromatic hydrocarbons through dechlorination hydrogenation, thus lowering the detection limit of the GC-MS/MS method significantly. It can transform paraffin chloride into the corresponding alkane to better distinguish short-chain, medium-chain, or long-chain CPs. Online catalytic conversion significantly improved the sensitivity and reproducibility (88.7%-113.1%) of the method. Tissue samples with various concentration levels of chlorinated aromatics and chlorinated alkanes were tested. The linearity range of the reduced target compounds in the reduction product solution was 0.02-1.00 µg/ml (R2  > 0.995). The quantitative detection limit was 0.03-0.05 µg/kg, and relative standard deviation was less than 6.9%. CONCLUSION: This study was the first to introduce the Pa/Ac catalytic device as an online catalytic unit in the determination of chlorinated aromatics and chlorinated alkanes using the GC-MS/MS method. The target compounds were converted into alkanes and aromatic hydrocarbons with unchanged carbon structures, and the method could achieve a low detection limit with no need for high-end methods such as GC-chemical ionization ion source (CI)-MS or high-resolution mass spectrometry. These methods are suitable for the determination of chlorine pollutants in recycled paper and its raw materials.

Accounting for molecular flexibility in photoionization: case of tert-butyl hydroperoxide.

tert-Butyl hydroperoxide (tBuOOH) is a common intermediate in the oxidation of organic compounds that needs to be accurately quantified in complex gas mixtures for the development of chemical kinetic models of low temperature combustion. This work presents a combined theoretical and experimental investigation on the synchrotron-based VUV single photon ionization of gas-phase tBuOOH in the 9.0 - 11.0 eV energy range, including dissociative ionization processes. Computations consist of the determination of the structures, vibrational frequencies and the energetics of neutral and ionic tBuOOH. The Franck-Condon spectrum for the tBuOOH+ (X+) + e- ← tBuOOH (X) + hν transition is computed, where special treatment is undertaken because of the flexibility of tBuOOH, in particular regarding the OOH group. Through comparison of the experimental mass-selected threshold photoelectron spectra with explicitly correlated coupled cluster calculations and Franck-Condon simulations that account for the flexibility of the molecule, an estimation of the ionization energy is given. The appearance energy of the only fragment observed within the above-mentioned energy range, identified as the tert-butyl C4H9+, is also reported. Finally, the signal branching ratio between the parent and the fragment ions is provided as a function of photon energy, essential to quantify tBuOOH in gas-phase oxidation/combustion experiments via advanced mass spectrometry techniques.

Modulation of π character upon complexation captured by molecular rotation spectra.

Two configurations of the furan-CF3Cl complex have been observed by high-resolution rotational spectroscopy. One is characterized by a dominant Cl lone pair⋯π*aromatic interaction and the other is stabilized by a C-Cl···π-CC- halogen bond. This is the first rotational spectroscopic evidence, to the best of our knowledge, that shows how a complexation with a partner like CF3Cl (the weak lone pair belt of Cl, to be more specific) can modulate both the aromatic π* and diene π characters of a heteroaromatic molecule in the formation of non-covalent interactions. The results emphasize the partner molecules' role in modulating the π electron structure and will not only deepen our understanding on non-covalent interactions but also lead to better designs of heteroaromatic-based drugs and materials.

A novel dealloying strategy for fabricating nanoporous Ag via ζ'-AgGa alloy.

A mild strategy for fabricating nanoporous silver (np-Ag) pieces was reported via preparation of Ag-Ga alloys in relatively low temperature and subsequent electrochemical dealloying in nitric acid (HNO3) aqueous solution. After selectively etching Ga out of the Ag-Ga alloy, a typical three-dimensional (3D) bicontinuous nanoporous structure with a pore size of ∼67.21-159.33 nm was observed. A series of studies have shown that the addition of sodium dodecyl sulfate (SDS) results in minimum pore size. The coarsen exponent (n) is 1.61, and the activation energy was calculated to be 27.04 kJ mol-1. Theζ'-AgGa alloy prepared at low temperature can be used as the precursor for the preparation of fine np-Ags, and this method provides a new strategy for the industrial production of dealloyed np-Ag.

PD-1 expression on uveal melanoma induces tumor proliferation and predicts poor patient survival.

INTRODUCTION: Uveal melanoma is one of the most common primary intraocular malignant tumors with poor prognosis and limited treatments. Programmed cell death receptor-1 (PD-1) blockade represents the primary treatment strategy of immune checkpoint inhibition; however, there is a lack of studies on whether PD-1 expression in primary (ocular) uveal melanoma affects tumor progression. METHODS: PD-1 expression in 82 cases of primary (ocular) uveal melanoma was detected by immunohistochemistry. The clinical significance of PD-1 expression was evaluated using univariate and multivariate analysis. PD-1 overexpression and knockdown studies were conducted in C918 and Mum-2B cell lines to analyze the effect of PD-1 expression on tumor cell proliferation and intracellular cell signaling transduction. real-time qPCR (RT-qPCR) and western blot analysis were performed to investigate the gene expression level. CCK8 assays were performed to examine the cell proliferation ability. RESULTS: High expression of primary (ocular) intratumor PD-1 was associated with poor patient survival. Moreover, PD-1 expression was correlated with the largest tumor diameter. PD-1 expression and optic nerve invasion were independent prognostic risk factors. PD-1 overexpression in uveal melanoma cell lines promoted tumor cell proliferation, while knockdown of PD-1 inhibited cell proliferation capacity. CONCLUSION: Our study established the role of PD-1 in the progression of uveal melanoma and provided a new potential treatment selection for uveal melanoma.

Toward accurate prediction of amino acid derivatives structure and energetics from DFT: glycine conformers and their interconversions.

This work provides the accurate reference data for structural and energetic properties relevant for computational studies of amino acids and polypeptides. Glycine due to its small size allows for detailed theoretical explorations of its whole conformational space. The reference energies are computed at the CCSD(T)/CBS+CV level on the best estimated geometries of all local minima and the transition states. For the minima, we complete the set of reference structures reported in Phys. Chem. Chem. Phys., 2013, 15, 10094, by determining the CCSD(T)/(CBS+CV)MP2 quality geometries for the two highest energy conformers VIIp/tcc and VIIIn/gtc. These data stand as the reference to asses reliability and accuracy of less expensive computational models, with particular focus on dispersion-corrected hybrid and double-hybrid DFT approaches, considering several basis sets of double- and triple-ζ quality. Based on results for minima the B2LYP-D3(BJ)/aug-cc-pVTZ level is set as the reference for transition states geometries. Considering accuracy of both single point energies and structural parameters B2PLYP(-D3BJ) and DSDPBEP86 in conjunction with aug-cc-pVTZ basis set can be recommended for reference studies of amino-acids and small poly-peptides, while B3LYP(-D3(BJ)) shown to be the most robust from considered hybrid DFT approaches. Graphical abstract TOC: Benchmarking DFT for amino acid analogues: highly accurate characterization of energies and structures from full glycine conformational space.

Butyrophilin-Like 9 (BTNL9) Suppresses Invasion and Correlates with Favorable Prognosis of Uveal Melanoma.

BACKGROUND Uveal melanoma (UM) is the most common intraocular malignancy, and the prognosis of patients with advanced stage of UM is very dismal. The T cell receptor ectopic expression of butyrophilin-like 9 (BTNL9) has been observed in several types of cancers, but the expression and clinical significance of BTNL9 in UM is unclear. MATERIAL AND METHODS In our study, we detected the expression of BTNL9 in 6 pairs of UM tissues and adjacent tissues using quantitative real-time polymerase chain reaction (qRT-PCR), and further investigated BTNL9 expression with immunohistochemistry (IHC) in a retrospective cohort consisted of 62 UM patients. The correlations between BNTL9 expression and clinicopathological factors were analyzed with Fisher's test, and the prognostic significance of BTNL9 was evaluated with univariate analysis and multivariate analysis. Using experiments in vitro, we investigated the function of BTNL9 in UM proliferation and invasion. RESULTS BTNL9 mRNAs in adjacent tissues were remarkably higher than in UM tissues. The percentages of BTNL9 low expression and high expression were 56.45% and 43.55%, respectively. High expression of BTNL9 was significantly associated with favorable prognosis of UM. BTNL9 expression was identified as a prognostic biomarker predicting better outcome of UM patients. Moreover, BTNL9 could suppress invasion instead of proliferation in melanoma cell line. CONCLUSIONS BTNL9 was a favorable prognostic factor of UM and it could suppress invasion of UM, suggesting that BTNL9 detection could help stratify high-risk patients with UM after operation and guide more precise surveillance and treatment.

Association of rs11801299 and rs1380576 polymorphisms at MDM4 with risk, clinicopathological features and prognosis in patients with retinoblastoma.

BACKGROUND: rs11801299 and rs1380576, two novel polymorphisms in MDM4 gene, have been investigated in several different cancer types. However, the role of these two polymorphisms in retinoblastoma (RB) remains unclear. METHODS: A total of 126 patients with primary RB and 148 age-/gender-matched controls were included in this retrospective study. The frequency of rs11801299 and rs1380576 were determined between RB patients and controls. The association of these two polymorphisms with clinicopathological characteristics, prognosis were further evaluated. RESULTS: AA genotype at rs11801299 was significantly associated with an increased risk of developing RB (OR = 2.06, 95%CI 1.09-3.90). The possibility of developing RB was also significantly increased in individuals with A allele at rs11801299 (OR = 1.49, 95%CI 1.06-2.08). RB patients carrying AA genotype and A allele at rs11801299 were more likely to have tumor invasion and poor differentiation. As for rs1380576, a significantly lower risk of developing RB was observed in patients with G allele (CG + GG) compared with wild-type CC genotype (OR = 0.59, 95%CI 0.36-3.95). RB patients with GG genotype or G allele had a lower risk of developing highly aggressive cancer. Kaplan-Meier curves and log-rank results revealed that RB patients carrying AA genotype or A allele (AA + GA) at rs11801299 had significantly poorer prognosis. Multivariate COX analysis showed that the rs11801299 G allele was associated with decreased survival but was not an independent prognostic factor. CONCLUSION: rs11801299 was significantly associated with RB risk, pathological differentiation, tumor aggressiveness and poor prognosis.

Upregulation of BCL2 19 kD Protein-Interacting Protein 3 (BNIP3) is Predictive of Unfavorable Prognosis in Uveal Melanoma.

BACKGROUND BCL2 19 kD protein-interacting protein 3 (BNIP3) is a BH3-containing protein of the BCL-2 family; it can regulate cell death, autophagy, and cytoprotection. The upregulation of BNIP3 has been reported to relate to progression and poor prognosis in different cancer types. However, the clinical significance of BNIP3 in uveal melanoma (UM) is still unknown. MATERIAL AND METHODS In our study, 47 patients with UM were enrolled; the expression of BNIP3 was detected with immunohistochemistry. According to BNIP3 immunohistochemical scores, the patients were divided into BNIP3 high- and low-expression subgroups. The correlation between the expression of BNIP3 and clinicopathological factors was evaluated with Fisher's test; the associations with survival rates were analyzed with log-rank test. The independent prognostic factors were identified with the Cox-regression model. RESULTS BNIP3 was mainly localized in the cytoplasm, and high expression of BNIP3 accounted for 31.9% (15/47) of the patients in our study. High expression of BNIP3 was demonstrated to be significantly associated with more pigment (P=0.018) and deeper scleral invasion (P=0.013). High expression of BNIP3 was also correlated with lower overall survival rate (P=0.006). Multivariate analysis confirmed positive ciliary body involvement and lymphatic infiltration as independent prognostic factors. CONCLUSIONS High expression of BNIP3 was significantly associated with poor prognosis of patients with UM, indicating that BNIP3 detection could help stratify high-risk patients and identify new therapies targeting BNIP3 as a promising approach to treat UM.

Accurate geometries for "Mountain pass" regions of the Ramachandran plot using quantum chemical calculations.

Unusual local arrangements of protein in Ramachandran space are not well represented by standard geometry tools used in either protein structure refinement using simple harmonic geometry restraints or in protein simulations using molecular mechanics force fields. In contrast, quantum chemical computations using small poly-peptide molecular models can predict accurate geometries for any well-defined backbone Ramachandran orientation. For conformations along transition regions-ϕ from -60 to 60°-a very good agreement with representative high-resolution experimental X-ray (≤1.5 Å) protein structures is obtained for both backbone C-1 -N-Cα angle and the nonbonded O-1 …C distance, while "standard geometry" leads to the "clashing" of O…C atoms and Amber FF99SB predicts distances too large by about 0.15 Å. These results confirm that quantum chemistry computations add valuable support for detailed analysis of local structural arrangements in proteins, providing improved or missing data for less understood high-energy or unusual regions.

A Functional Polymorphism (rs937283) in the MDM2 Promoter Region is Associated with Poor Prognosis of Retinoblastoma in Chinese Han Population.

The effect of single nucleotide polymorphisms (SNPs) at MDM2 has been investigated in several cancer types. Three MDM2 SNPs(rs937283, rs2270744 and rs769412) have previously been suggested to be positively correlated with cancer. In this study, we aimed to explore the association of rs937283, rs2270744 and rs769412 polymorphisms with retinoblastoma (RB) risk, clinicopathological characteristics, and prognosis. Compared with wild-type genotype AA at rs937283, individuals carrying AG and GG genotype had a significantly increased risk for developing RB (OR = 1.86, 95% CI 1.13-3.08; OR = 2.48, 95% CI 1.10-5.62, respectively). RB patients with allele G at rs937283 were more susceptible to invasion and high tumor aggression (OR = 2.42, 95% CI 1.43-4.11; OR = 2.15, 95% CI 1.27-3.64, respectively). Kaplan-Meier curves and log-rank results revealed that RB patients harboring genotype GG and G allele at rs937283 had worse survival (P < 0.02 and P < 0.01, respectively). In addition, the A to G substitution at rs937283 significantly enhanced the transcription activity of the MDM2 gene in vitro. In vivo, we found that MDM2 mRNA and protein were overexpressed in individuals who carried the G allele at rs937283. This study suggested that the MDM2 rs937283 polymorphism is a novel functional SNP both in vitro and in vivo as well as a biomarker for poor prognosis in RB.

[Gene therapy for tongue carcinoma mediated by oral administration of attenuated Salmonella harboring tip30 and IFN-γ genes in nude mouse model].

PURPOSE: To observe the effect of gene therapy mediated by oral administration of attenuated Salmonella carrying tip30 and IFN-γ genes in human tongue carcinoma nude mouse model. METHODS: 25 four-week-old BALB/C male nude mice were divided randomly into 5 groups based on the differently harboring genes in the recombinant attenuated Salmonella typhimuriums SL7207, which included blank group (PBS), control group (SL7207), IFN treatments group (SL7207-pCI-IFN), tip30 treatment group (SL7207-pCI-tip30) and the combination of IFN and tip30 treatment group (SL7207-pCI-tip30/IFN). On 10d after submandibular subcutaneous injection of Tca8113 cells into the mice, the recombinant attenuated Salmonella typhimuriums were orally administrated 3 times at 1 week interval. The treatment effect indicators included the growth curve, the tumor inhabitation rate, the survival rate, the apoptosis typical DNA ladder and the protein expressions of tip30 and IFN-γ in tumor cells. Statistical analysis was performed with SPSS15.0 software package. RESULTS: The growth curve, the tumor inhabitation rate and the survival rate indicated that the tumor in the PBS group constantly grew up and induced the animal death in earlier time, while the SL7207 treatment had a slight inhibiting effect compared with PBS group. However, the SL7207-pCI-IFN group and the SL7207-pCI-tip30 had a moderate inhibiting effect compared with the SL7207 group, while the combination of IFN and tip30 had the strongest inhibiting effect. The protein expressions of tip30 and IFN-γ were detected in tumor cells while the typical DNA ladders of apoptosis were observed only in the tip30 gene transfer groups (SL7207-pCI-tip30 and SL7207-pCI-tip30/IFN). CONCLUSIONS: The recombinant attenuated Salmonella typhimurium possesses a capacity of gene transfer in vivo through oral administration. The combination of the harboring tip30 and IFN-γ genes have synergistic inhibiting tumor effect for tongue carcinoma.

Using attenuated Salmonella typhi as tumor targeting vector for MDR1 siRNA delivery.

OBJECTIVE: To investigate the feasibility of using attenuated Salmonella typhi as an in vivo delivery vector for multidrug-resistance gene (MDR1) small interference RNA (siRNA) in a mouse model bearing human tongue squamous cell cancer. This technique may represent a novel and effective route for the in vivo administration of siRNA against malignant tumors. METHODS: The cisplatin (DDP)-resistant human tongue squamous cell carcinoma cell line Tca8113/DDP, which highly expresses the MDR1 gene, was established by exposure to gradually increasing concentrations of cisplatin. A plasmid MDR1 siRNAwas constructed and transformed into attenuated Salmonella typhi strain SL7207. Tca8113/ DDP cells were infected with recombinant salmonella and expression of the MDR1 gene encoding P-glycoprotein (P-gp) product was detected. Tca8113/DDP tumor-bearing nude mice were established by inoculation by gavage administration of recombinant salmonella and were simultaneously injected intraperitoneally with cisplatin. Tumor growth was observed. RESULTS: Recombinant salmonella-bearing MDR1 siRNA expression plasmids can infect Tca8113/DDP cells in vitro and suppress P-gp expression and reverse DDP tolerance in Tca8113/DDP cells. Oral administration of recombinant salmonella in tumor-bearing nude mice can suppress tumor proliferation and enhance the therapeutic effect of DDP. CONCLUSION: Attenuated Salmonella typhi is expected to act as an in vivo targeting delivery vector for siRNA in tumor tissues.

[Clinical evaluation of two light cured pit and fissure sealants for preventing pit and fissure caries in children].

PURPOSE: To evaluate the clinical effect of two light cured pit and fissure sealants for preventing pit and fissure caries. METHODS: 416 health permanent teeth in 162 children aged 6 to 8 years old children were divided into two groups, enameloplasty sealant technique (EST) and cup-shaped brush sealant technique (CST) were used in two groups, respeitively. 90 and 112 contralateral healthy teeth were used as self controls respectively. The rate of the sealant reservation and the incidence of caries were compared in the two groups, and the clinical effect of the two groups was compared using SPSS10.0 software package for Chi-square test. RESULTS: After 3 years, the rate of the sealant reservation and the incidence of caries of EST were 85.4% and 0.9% respectively, the rate of the sealant reservation and the incidence of caries of CST were 63.8% and 6.7% respectively. Statistical analysis revealed a significant difference (P<0.05) between the two groups. CONCLUSION: EST and CST both have good clinical effects, While EST is superior to CST in preventing pit and fissure caries.

[Primary culture and identification of mouse odontoblast-like cells].

PURPOSE: To culture primary mouse odontoblast and to provide a base for study on inducing ES cells to odontoblast. METHODS: Lower incisor germs were removed from 1-week-old mouse. The dental papillae were isolated in microscope, and the dental papillae cells were dispersed using 0.25% collagenase and 0.25% trypsin. The cell clones, which had similar morphology to odontoblasts, were selected for further culturing. The primary cultured cells were identified by light and electron microscopes and mRNA expression of mouse dentin sialophoprotein. RESULTS: The cultured cells had the same morphology and ultrastructure. They were rich in Golgi's complex, ribosome and rough endoplasmic reticulum. These cells expressed DSPP at mRNA levels. CONCLUSION: The cultured cells were mouse odontoblast-like cells. The method could be used for the study of odontal cells in vitro.

[A three-year clinical evaluation of ceramic optimized polymer posterior inlays].

PURPOSE: To evaluate the clinical results of ceramic optimized polymer (Targis) posterior inlays. METHODS: 345 type I cavities were divided into two groups: 170 in Targis inlays group (42 premolars and 128 molars) and 175 in control group (composite resin inlays, 45 premolars and 130 molars). The clinical effects of Targis inlays and composite resin inlays in posterior teeth were compared in marginal discoloration, marginal adaption, secondary caries, anatomic shape integrity, fractures of tooth and color match 3 years later. The data were analyzed using Chi-square test. RESULTS: 3 years after treatment, 166 Targis inlays were followed up, 2 had marginal discoloration, 158 had good marginal adaption, 162 had anatomic shape integrity, 1 had secondary caries, 161 had color match, and 5 had fractures of teeth. 169 composite resin inlays were followed up, 27 had marginal discoloration, 134 had good marginal adaption, 150 had anatomic shape integrity, 20 had secondary caries, 164 had color match,5 had fractures of teeth. The marginal discoloration, marginal adaption, secondary caries, anatomic shape integrity of Targis inlays were better than that of composite resin inlays (P < 0.05), but fractures of teeth was not different between inlays in the two group (P > 0.05). CONCLUSION: Targis posterior inlays is a good newly developed prosthesis.

[Induction of mouse embryonic stem cells forming odontoblast-like cells by co-culture with pulp fibroblast].

PURPOSE: To investigate the methods of induction of mouse embryonic stem cells to differentiate into odontoblast-like cells by co-culture with pulp fibroblast. METHODS: By suspension culture, embryonic stem cells were induced to form embryoid bodies. Then the cells from embryoid bodies were co-cultured with pulp fibroblast in Transwell system for differentiation toward odontoblast-like cells. RESULTS: By RT-PCR analysis, embryoid body cells gave rise to the cell population expressing DSPP, a specific odontoblast cell marker, after 10 days of co-culture with pulp fibroblast and the expression of DSPP was enhanced after 15 days of co-culture. On the other hand, the expression of DSPP was not detected in the isolately cultured embryoid body cells. CONCLUSIONS: Embryonic stem cells can be induced into odontoblast-like cells by co-culture with pulp fibroblast. Supported by Science and Technology Development Fund of Shanghai Municipality (Grant No. 03ZR14027).