Enhanced photoelectrocatalytic decomplexation of Ni-EDTA and simultaneous recovery of metallic nickel via TiO2/Ni-Sb-SnO2 bifunctional photoanode and activated carbon fiber cathode.

In order to enhance Ni-EDTA decomplexation and Ni recovery via photoelectrocatalytic (PEC) process, TiO2/Ni-Sb-SnO2 bifunctional electrode was fabricated as the photoanode and activated carbon fiber (ACF) was introduced as the cathode. At a cell voltage of 3.5 V and initial solution pH of 6.3, the TiO2/Ni-Sb-SnO2 bifunctional photoanode exhibited a synergetic effect on the decomplexation of Ni-EDTA with the pseudo-first-order rate constant of 0.01068 min-1 with 180 min by using stainless steel (SS) cathode, which was 1.5 and 2.4 times higher than that of TiO2 photoanode and Ni-Sb-SnO2 anode, respectively. Moreover, both the efficiencies of Ni-EDTA decomplexation and Ni recovery were improved to 98% from 86% and 73% from 41% after replacing SS cathode with ACF cathode, respectively. Influencing factors on Ni-EDTA decomplexation and Ni recovery were investigated and the efficiencies were favored at acidic condition, higher cell voltage and lower initial Ni-EDTA concentration. Ni-EDTA was mainly decomposed via ·OH radicals which generated via the interaction of O3, H2O2, and UV irradiation in the contrasted PEC system. Then, the liberated Ni2+ ions which liberated from Ni-EDTA decomplexation were eventually reduced to metallic Ni on the ACF cathode surface. Finally, the stability of the constructed PEC system on Ni-EDTA decomplexation and Ni recovery was exhibited.

The absence of muscle segment homeobox 2 leads to the pyroptosis of ameloblasts by inducing squamous epithelial hyperplasia in the enamel organ.

Muscle segment homeobox 2 (MSX2) has been confirmed to be involved in the regulation of early tooth development. However, the role of MSX2 has not been fully elucidated in enamel development. To research the functions of MSX2 in enamel formation, we used a Msx2-/- (KO) mouse model with no full Msx2 gene. In the present study, the dental appearance and enamel microstructure were detected by scanning electron microscopy and micro-computed tomography. The results showed that the absence of Msx2 resulted in enamel defects, leading to severe tooth wear in KO mice. To further investigate the mechanism behind the phenotype, we performed detailed histological analyses of the enamel organ in KO mice. We discovered that ameloblasts without Msx2 could secrete a small amount of enamel matrix protein in the early stage. However, the enamel epithelium occurred squamous epithelial hyperplasia and partial keratinization in the enamel organ during subsequent developmental stages. Ameloblasts depolarized and underwent pyroptosis. Overall, during the development of enamel, MSX2 affects the formation of enamel by regulating the function of epithelial cells in the enamel organ.

Effects of polystyrene microplastic on uptake and toxicity of copper and cadmium in hydroponic wheat seedlings (Triticum aestivum L.).

Microplastics are widespread in freshwater environments, their biological effects and combined effects of other pollutants have attracted extensive attention. In this study, we investigated the adsorption properties of heavy metals onto polystyrene (PS) microplastics as well as the bioavailability and toxicity of microplastics and heavy metals by hydroponic wheat seedlings experiment. Results showed that PS microplastics (0.5 µm, 100 mg/L) had no significant effect on wheat seedlings growth, photosynthesis, and reactive oxygen species (ROS) content. However, PS microplastics could adsorb copper and cadmium, with a predominantly chemisorption. The accumulation of copper and cadmium in wheat seedlings reduced in the presence of PS microplastics, which meant the toxic effect by heavy metals might be mitigated. Compared with single heavy metals treatments, the combination of PS microplastics and heavy metals increased chlorophyll content, enhanced photosynthesis and reduced the accumulation of ROS. These findings suggest that PS microplastics (0.5 µm, 100 mg/L) have a mitigating effect on the bioavailability and toxicity of copper and cadmium.

An ultrasensitive electrochemiluminescent biosensor for the detection of concanavalin A based on poly(ethylenimine) reduced graphene oxide and hollow gold nanoparticles.

A highly sensitive electrochemiluminescent (ECL) biosensor was designed for the detection of concanavalin A (ConA) based on glucose oxidase (GOx) as a recognition element by carbohydrate-lectin biospecific interaction, and poly(ethylenimine) (PEI) reduced graphene and hollow gold nanoparticles (HAuNPs) as supporting matrix and signal amplifier. The modification process and detection principle of the biosensor are briefly described as follows. First, PEI reduced graphene oxide with abundant amino groups was cast onto the surface of glassy carbon electrode to adsorb HAuNPs for improving the signal intensity in luminol/H2O2 ECL system. Next, GOx was further assembled onto the electrode by the interaction between Au and -NH2. In the presence of glucose in the detection solution, GOx catalyzed glucose to generate H2O2 in situ, which served as a co-reactant of luminol to enhance ECL signal of luminol. Based on the fact that ConA could result in a decrease in ECL signal when immobilized on the electrode, an ECL biosensor was prepared for the determination of ConA. The ECL signal intensity was linear with the logarithm of ConA concentration and the linear range was from 1.0 to 20 ng/mL with a low detection limit of 0.31 ng/mL (signal to noise ratio =3). This strategy led to a nearly 1000-fold improvement in detection limit for ConA assays compared with previously reported method, thus exhibiting a great potential application in sensitive bioassays of ConA.

Effect of ultraviolet aged polytetrafluoroethylene microplastics on copper bioavailability and Microcystis aeruginosa growth.

Microplastics (MPs) are ubiquitous in aquatic environments, which can act as carriers to affect the bioavailability of heavy metals. The aging process in the environment changes the physicochemical properties of MPs, thereby affecting their environmental behavior and co-toxicity with other pollutants. However, relevant research is limited. In this study, we compared the properties and Cu2+ adsorption capacity of pristine and aged polytetrafluoroethylene (PTFE) MPs and further explored the influence on copper bioavailability and bio-effects on Microcystis aeruginosa. Aging process induced surface oxidation and cracks of PTFE MPs, and decreased the stability of MPs in water by increasing zeta potential. PTFE MPs had a strong adsorption capacity for Cu2+ and increased the bioavailability of copper to microalgae, which was not affected by the aging process. Pristine and aged PTFE MPs adhered to cyanobacterium surfaces and caused shrinkage and deformation of cells. Inhibition of cyanobacterium growth, photosynthesis and reduction of total antioxidant capacity were observed in the treatment of PTFE MPs. Combined exposure of pristine MPs and Cu2+ had stronger toxic effects to cyanobacterium, and increased Microcystin-LR release, which could cause harm to aquatic environment. Aging reduced the toxic effects of PTFE MPs on microalgae. Furthermore, soluble exopolysaccharide (EPS) content was significantly higher in co-exposure of aged MPs and Cu2+, which could reduce the toxicity to cyanobacterium cells. These results indicate that aging process alleviates the toxicity to microalgae and environmental risks caused by PTFE MPs. This study improves understanding of the combined toxicity of aged MPs and metals in freshwater ecosystems.

Balance Evaluation Based on Walking Experiments with Exoskeleton Interference.

The impairment of walking balance function seriously affects human health and will lead to a significantly increased risk of falling. It is important to assess and improve the walking balance of humans. However, existing evaluation methods for human walking balance are relatively subjective, and the selected metrics lack effectiveness and comprehensiveness. We present a method to construct a comprehensive evaluation index of human walking balance. We used it to generate personal and general indexes. We first pre-selected some preliminary metrics of walking balance based on theoretical analysis. Seven healthy subjects walked with exoskeleton interference on a treadmill at 1.25 m/s while their ground reaction force information and kinematic data were recorded. One subject with Charcot-Marie-Tooth walked at multiple speeds without the exoskeleton while the same data were collected. Then, we picked a number of effective evaluation metrics based on statistical analysis. We finally constructed the Walking Balance Index (WBI) by combining multiple metrics using principal component analysis. The WBI can distinguish walking balance among different subjects and gait conditions, which verifies the effectiveness of our method in evaluating human walking balance. This method can be used to evaluate and further improve the walking balance of humans in subsequent simulations and experiments.

Multidynamic Osteogenic Differentiation by Effective Polydopamine Micro-Arc Oxide Manipulations.

Introduction: The nanostructural modification of the oral implant surface can effectively mimic the morphology of natural bone tissue, allowing osteoblasts to achieve both proliferation and differentiation capabilities at the bone interface of the dental implant. To improve the osteoinductive activity on the surface of titanium implants for rapid osseointegration, we prepared a novel composite coating (MAO-PDA-NC) by micro-arc oxidation technique and immersion method and evaluated the proliferation, adhesion, and osteogenic differentiation of osteoblasts on this coating. Methods: The coatings were prepared by micro-arc oxidation (MAO) technique and immersion method, and characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM) for different coatings; the loading of PDA was examined using Fourier transform infrared spectroscopy (FTIR); the ion release capacity of the coatings was determined by inductively coupled plasma emission spectrometry (ICP-OES); the interfacial bonding of the coatings was examined using nanoscratch experiment strength. The cytotoxicity of the coating was examined by live/dead staining kit; cell proliferation viability was examined by CCK-8 kit; adhesion and osteogenic effect of the coating were examined by immunofluorescence staining and RT-PCR; osteogenic differentiation was examined by alkaline phosphatase staining. Results: The surface morphology of titanium implants was modified by micro-arc oxidation technology, and a new MAO-PDA-NC composite coating was successfully prepared. The results showed that the MAO-PDA-NC coating not only optimized the physical and chemical properties of the titanium implant surface but also significantly stimulated the biological properties of osteoblast adhesion, proliferation, and osteogenic differentiation on the coating surface. Conclusion: These results show that MAO-PDA-NC composite coating can significantly improve the surface properties of titanium implants and achieve a stable bond between implant and bone tissue, thus accelerating early osseointegration.

Detection of SARS-CoV-2 Specific Antibodies in Saliva Samples.

Molecular assays on nasopharyngeal swabs act as a confirmatory test in coronavirus disease (COVID-19) diagnosis. However, the technical requirements of nasopharyngeal sampling and molecular assays limit the testing capabilities. Recent studies suggest the use of saliva for the COVID-19 diagnostic test. In this study, 44 patients diagnosed with COVID-19 in The Third People's Hospital of Shenzhen were enrolled. Saliva and serum specimens were obtained at different time points and the immunoglobulins against SARS-CoV-2 were measured. The results showed that saliva IgA presented a higher COI value than IgG and IgM. In matched saliva and serum samples, all saliva samples presented lower IgG levels than serum samples, and only one saliva sample presented a higher IgM level. The conversion rates of saliva IgA and the detection of viral nucleic acids were analyzed in the first and second weeks after hospitalization. The positive rates increased when combining saliva IgA and viral nucleic acid detection. In conclusion, our results provide evidence that saliva IgA could serve as a useful index for the early diagnosis of COVID-19.

Quantitative label-free optical technique to analyze the ultrastructure changes and spatiotemporal relationship of enamel induced by Msx2 deletion.

New advances in the molecular mechanism of enamel mineralization reveal the practical significance of regenerative medicine in clinical transformation. Muscle segment homeobox 2 (MSX2), a transcription factor, is recently reported to be closely associated with the amelogenesis imperfecta (AI). To elucidate the biomineralization framework of AI enamel, herein, Msx2 gene mutant mice are investigated by dual-mode noninvasive spectroscopic analytical techniques for the first time. Optical coherence tomography (OCT) records the depth-resolved structural information of mice teeth, where a dramatic decrease in enamel thickness and quality occurred in Msx2 deficient (Msx2-/- ) enamel. And it has the advantages of fast, noninvasive and low cost. Raman spectroscopy, a powerful molecular fingerprint tool, further witnesses an imbalance of inorganic and organic contents in Msx2-/- enamel. In addition, abnormal expression of MSX2 also influences the spatial distribution of phosphate in enamel according to the Raman spectral imaging. Therefore, OCT integrated with Raman spectroscopy provides the quantitative label-free optical parameters of both the physical structure and chemical component in mice enamel, which strengthens the understanding of the biomineralization process underlying the Msx2-related amelogenesis imperfect.

The transfer and decay of maternal antibodies against enterovirus A71, and dynamics of antibodies due to later natural infections in Chinese infants: a longitudinal, paired mother-neonate cohort study.

BACKGROUND: Since 1997, epidemics of hand, foot, and mouth disease associated with enterovirus A71 (EV-A71) have affected children younger than 5 years in the Asia-Pacific region, including mainland China. EV-A71 vaccines have been licensed for use in children aged 6-71 months in China, but not for infants younger than 6 months. We aimed to assess the dynamics of maternal EV-A71 antibodies to inform choice of potential vaccination strategies to protect infants younger than 6 months, because they have a substantial burden of disease. METHODS: We did a longitudinal cohort study with mother-neonate pairs in local hospitals in southern China during 2013-18. We collected cord blood from neonates and venous blood from mothers at delivery. We followed up and collected blood samples from the children at ages 2, 4, 6, 12, 24, and 36 months and tested for the presence of neutralising antibodies against EV-A71 with virus neutralisation assays. Seropositivity, or protective titre, was defined as a neutralisation antibody titre of 16 or higher. We estimated the seroprevalence, geometric mean titre (GMT), and transfer ratio of maternal antibodies. We used a binomial distribution to derive the 95% CIs of seroprevalence. Seropositivity between mothers and neonates was compared by use of an agreement (κ), while GMTs were compared by use of paired Student's t tests. FINDINGS: Between Sept 20, 2013, and Oct 14, 2015, 1054 mothers with 1066 neonates were enrolled. The EV-A71 GMT was similar among pairs of neonates (22·7, 95% CI 20·8-24·9) and mothers (22·1, 95% CI 20·2-24·1; p=0·20). The mean transfer ratio of maternal antibodies was 1·03 (95% CI 0·98-1·08). Although 705 (66%) of 1066 neonates acquired protective concentrations of EV-A71 antibodies from mothers, these declined rapidly, with a half-life of 42 days (95% CI 40-44). The time to loss of protective immunity was extended to 5 months in neonates with mothers who had titres of 128 or higher. By age 30 months, 28% of children had become seropositive because of natural infection. INTERPRETATION: EV-A71 maternal antibodies were efficiently transferred to neonates, but declined quickly to below the protective threshold, particularly among those whose mothers had low antibody titres. Our findings suggest that maternal vaccination could be explored to provide neonatal protection against EV-A71 through maternal antibodies. Catch-up vaccination between ages 6 months to 5 years could provide protection to the approximately 30-90% of children that have not had natural EV-A71 infection by that age. FUNDING: National Science Fund for Distinguished Young Scholars, National Natural Science Foundation of China.

Anticancer Effects and Mechanisms of Action of Plumbagin: Review of Research Advances.

Plumbagin (PLB), a natural naphthoquinone constituent isolated from the roots of the medicinal plant Plumbago zeylanica L., exhibited anticancer activity against a variety of cancer cell lines including breast cancer, hepatoma, leukemia, melanoma, prostate cancer, brain tumor, tongue squamous cell carcinoma, esophageal cancer, oral squamous cell carcinoma, lung cancer, kidney adenocarcinoma, cholangiocarcinoma, gastric cancer, lymphocyte carcinoma, osteosarcoma, and canine cancer. PLB played anticancer activity via many molecular mechanisms, such as targeting apoptosis, autophagy pathway, cell cycle arrest, antiangiogenesis pathway, anti-invasion, and antimetastasis pathway. Among these signaling pathways, the key regulatory genes regulated by PLB were NF-kß, STAT3, and AKT. PLB also acted as a potent inducer of reactive oxygen species (ROS), suppressor of cellular glutathione, and novel proteasome inhibitor, causing DNA double-strand break by oxidative DNA base damage. This review comprehensively summarizes the anticancer activity and mechanism of PLB.

RUNX2 contributes to TGF-ß1-induced expression of Wdr72 in ameloblasts during enamel mineralization.

The elaborate modulation of the transforming growth factor ß (TGF-ß) superfamily signaling network plays an essential role in tooth morphogenesis and differentiation. In our previous studies, we have demonstrated that TGF-ß1 promotes enamel mineralization and maturation using TGF-ß1 gene conditional knockout (TGF-ß1-cKO) mice. However, the specific regulatory mechanisms of TGF-ß1 during enamel development remain unclear. Furthermore, we have previously found that the expression of WD repeat-containing protein 72(WDR72)in mouse enamel epithelium is decreased significantly in the absence of TGF-ß1. Therefore, the aim of the present study was to investigate how TGF-ß1 affects amelogenesis by regulating the expression of Wdr72. Histological examination showed that the absence of TGF-ß1 in ameloblastic epithelial cells resulted in a reduction in enamel mineralization and a delay in enamel matrix protein absorption. TGF-ß1, Runt-related transcription factor 2(RUNX2) and WDR72 were revealed to be colocalized in ameloblasts by immunohistochemistry, and it was also found that the expression of Runx2 and Wdr72 was markedly different between TGF-ß1-cKO mice and wild type(TGF-ß1-WT)mice. In addition, the effect of exogenous TGF-ß1 on Wdr72 was more significant when RUNX2 was present than when RUNX2 was absent. Furthermore, we found that there were binding sites for RUNX2 on the promoter of Wdr72 and that Wdr72 expression was regulated by RUNX2. Collectively, our results suggest that TGF-ß1 affects enamel mineralization by modulating RUNX2 and thus affecting the expression of Wdr72.

[Knockdown of MSX2 gene inhibits the expression of enamel matrix proteins and the enamel mineralization in mouse ameloblasts].

Objective To study the effect of muscle segment homeodomain homeobox 2 (MSX2) on the expression of enamel matrix protein and the formation of enamel. Methods Immunohistochemical staining was used to detect the expression of MSX2 in mouse tooth embryos and its localization in ameloblasts. The short hairpin RNA (shRNA) of the MSX2 gene was designed and synthesized, and then the annealed double stranded DNA was constructed into the pGMLV-SC5 RNAi lentivirus vector, and finally it was packaged with lentivirus. The lentivirus was used to infect ameloblasts. Real-time fluorescent quantitative PCR was performed to screen the best interference fragment, and detect the mRNAs of amelogenin (Amelx), ameloblastin (Ambn), enamelin (Enam), amelotin (Amtn) and kallikrein 4 (Klk4). The embryos were isolated for 18.5 days and then infected with RNAi recombinant lentivirus targeting MSX2. The tooth germ was implanted under the renal capsule of the mouse. Ten weeks later, the tissue was harvested to separate and observe the tooth form and contour. Results MSX2 was expressed in the secretory phase and maturation phase of mouse ameloblasts, but the expression signal was weaker in the secretory phase and was stronger in the mature stage. The lentivirus of MSX2-shRNA targeting MSX2 gene we constructed inhibited the expression of Amelx and Klk4 mRNAs. The RNAi lentivirus targeting MSX2 gene infected the tooth enamel and led to a decrease in the degree of enamel mineralization. Conclusion The MSX2 gene is expressed in ameloblasts. The knockdown of MSX2 can inhibit the expression of enamel matrix proteins and the enamel mineralization.

Effective treatment of drug resistant recurrent breast tumors harboring cancer stem-like cells by staurosporine/epirubicin co-loaded polymeric micelles.

Breast cancer recurrence and resistance are associated with cancer stem-like cell (CSC) sub-populations. As conventional therapies fail to treat CSCs, institution of novel therapeutic strategies capable of eradicating both cancer cells and CSCs is central for achieving effective treatments with long-term survival. Here, we studied the ability of polymeric micelles cooperatively loading the cytotoxic drug epirubicin (Epi) and the CSC inhibitor staurosporine (STS) to treat breast tumors, particularly when tumors relapsed after chemotherapy. The STS/Epi-loaded micelles (STS/Epi/m) demonstrated potent therapeutic efficacy against both naïve orthotopic 4T1-luc breast tumors and their recurrent Epi-resistant counterparts, significantly prolonging survival. This efficacy enhancement of STS/Epi/m was correlated with the ability of the micelles to suppress the CSC-associated sub-populations of breast cancer, i.e. the aldehyde dehydrogenase-positive (ALDH+) population and the CD44+/CD24- fraction, in Epi-resistant cells and tumors. These results demonstrated STS/Epi/m as a promising strategy for effective management of breast cancer.

Seed germination of Caragana species from different regions is strongly driven by environmental cues and not phylogenetic signals.

Seed germination behavior is an important factor in the distribution of species. Many studies have shown that germination is controlled by phylogenetic constraints, however, it is not clear whether phylogenetic constraints or environmental cues explain seed germination of a genus from a common ancestor. In this study, seed germination under different temperature- and water-regimes [induced by different osmotic potentials of polyethylene glycol (PEG)] was investigated in the phylogenetically-related Caragana species that thrive in arid, semiarid, semihumid and humid environments. The results showed that the final percentage germination (FPG) decreased from 95% in species from arid habitats to 0% in species from humid habitats, but with no significant phylogenetic signal. Rather, the response of seed germination to temperature and PEG varied greatly with species from arid to humid habitats and was tightly linked to the ecological niche of the species, their seed coat structure and abscisic acid concentration. The findings are not consistent with the hypothesis that within a family or a genus, seed germination strategies can be a stable evolutionary trait, thus constraining interspecific variation, but the results clearly show that seed germination of Caragana species distributed across a range of habitats has adapted to the environment of that habitat.

[Preparation and application of polyclonal antibody against amelotin polypeptide].

OBJECTIVE: To prepare and identify the rabbit anti-mouse polyclonal antibody against amelotin polypeptide. METHODS: The polypeptide was synthesized based on the bioinformatics analysis of amelotin, and coupled with keyhole limpet hemocyanin (KLH) for immunization. The amelotin polypeptide-KLH was injected into New Zealand rabbits to prepare the polyclonal antibody. ELISA technology was used to detect the titer of the antibody. The specificity of the polyclonal antibody was identified by Western blotting. The expression of amelotin in the submandibular tissue of mice was observed by immunohistochemistry. RESULTS: ELISA showed that the titer of amelotin antibody was 1:1,000,000. Western blotting verified that the antibody had a high specificity. Immunohistochemistry indicated that amelotin was highly expressed in odontoprisis full-thick enamel of 3-day-old and 7-day-old mice, and also expressed in duct epithelial cytoplasm of submandibular glands of 7-day-old mice. CONCLUSION: The polyclonal antibody against amelotin polypeptide has been prepared successfully with high titer and high specificity.

Healing effect of bioactive glass ointment on full-thickness skin wounds.

This study aimed to investigate the effect of bioactive glasses on cutaneous wound healing in both normal rats and streptozotocin-induced diabetic rats. Bioactive glass ointments, prepared by mixing the sol-gel bioactive glass 58S (SGBG-58S), nanobioactive glass (NBG-58S) and the melt-derived 45S5 bioactive glass (45S5) powder with Vaseline (V) at 18% weight percentage, were used to heal full thickness excision wounds. Pure V was used as control in this study. Compared to SGBG-58S, NBG-58S consists of relatively dispersible nanoparticles with smaller size. The analysis of wound healing rate and wound healing time showed that bioactive glasses promoted wound healing. The ointments containing SGBG-58S and NBG-58S healed the wounds more quickly and efficiently than the ointment containing 45S5. Histological examination indicated that bioactive glasses promoted the proliferation of fibroblasts and growth of granulation tissue. Immunohistochemical staining showed that the production of two growth factors, VEGF and FGF2, which are beneficial to wound healing, was also stimulated during the healing process. Transmission electron microscope observations showed that fibroblasts in wounds treated with bioactive glasses contained more rough endoplasmic reticula and had formed new capillary microvessels by the seventh day. The effects of SGBG-58S and NBG-58S were better than those of 45S5. All results suggest that bioactive glasses, especially SGBG-58S and NBG-58S, can accelerate the recovery of skin wounds in both normal and diabetes-impaired healing models and have a great potential for use in wound repair in the future.

Distribution of amelotin in mouse tooth development.

Amelotin is expressed and secreted by ameloblasts in tooth development, but amelotin distribution during enamel development is not clear. In this report, we first investigated amelotin expression in developing teeth by immunohistochemistry. Amelotin was detected in the enamel matrix at the secretion and maturation stages of enamel development. Amelotin was also observed at Tomes' processes on the apical ends of secretory ameloblasts. We then compared amelotin gene expression with those of amelogenin, enamelin, and ameloblastin in the mandibles of postnatal mice by RT-PCR. The expression of amelotin was detected as early as in postnatal day 0 mandibles and amelotin was coexpressed with amelogenin, ameloblastin, and enamelin during tooth development. These data strongly suggest that amelotin is an enamel matrix protein expressed at the secretion and maturation stages of enamel development.

TGF-beta1 and TGFBR1 are expressed in ameloblasts and promote MMP20 expression.

Transforming growth factor-beta (TGF-beta) signaling exerts a wide spectrum of biological functions. To investigate TGF-beta signaling in amelogenesis, we initially assessed the expression of TGF-beta1 and TGF-beta receptor 1 (TGFBR1) in developing teeth by immunohistochemistry. Both TGF-beta1 and TGFBR1 were strongly expressed in secreting ameloblasts. Next, we studied the effects of TGF-beta signaling on the expression of MMP20 and KLK4 mRNA using ameloblast-lineage cells (ALC) in vitro. Our RT-PCR study showed that TGF-beta1, TGFBR1, and enamel matrix proteases (MMP20 and KLK4) were expressed in ALC. Following TGF-beta1 treatment, the expression of MMP20 mRNA, but not KLK4 mRNA, was significantly upregulated. To further confirm the TGF-beta signaling involvement in the MMP20 expression, we constructed the activated TGFBR1 vector and transfected the construct into ALC. The activated TGFBR1 notably promoted MMP20 expression, but had no obvious effects on the KLK4 mRNA expression. Our studies strongly suggest that TGF-beta signaling involved in amelogenesis is partially mediated by regulating the expression of MMP20 mRNA.