miR-486-3p regulates CyclinD1 and promotes fluoride-induced osteoblast proliferation and activation.

Fluoride is a persistent environmental pollutant, and its excessive intake contributes to skeletal and dental fluorosis. The mechanisms underlying fluoride-induced abnormal osteoblast proliferation and activation, which are related to skeletal fluorosis, have not yet been fully clarified. As important epigenetic regulators, microRNAs (miRNAs) participate in bone metabolism. On the basis of our previous miRNA-seq results and bioinformatics analysis, this study investigated the role and specific molecular mechanism of miR-486-3p in fluoride-induced osteoblast proliferation and activation via CyclinD1. Herein, in the fluoride-challenged population, we observed that miR-486-3p expression decreased while CyclinD1 and transforming growth factor (TGF)-ß1 increased, and miR-486-3p level correlated negatively with the expression of CyclinD1 and TGF-ß1 genes. Further, we verified that sodium fluoride (NaF) decreases miR-486-3p expression in human osteoblasts and overexpression of miR-486-3p reduces fluoride-induced osteoblast proliferation and activation. Meanwhile, we demonstrated that miR-486-3p regulates NaF-induced upregulation of CyclinD1 by directly targeting its 3'-untranslated region (3'-UTR). In addition, we observed that NaF activates the TGF-ß1/Smad2/3/CyclinD1 axis and miR-486-3p mediates transcriptional regulation of CyclinD1 by TGF-ß1/Smad2/3 signaling pathway via targeting TGF-ß1 3'-UTR in vitro. This study, thus, contributes significantly in revealing the mechanism of miR-486-3p-mediated CyclinD1 upregulation in skeletal fluorosis and sheds new light on endemic fluorosis treatment.

Reduced Graphene Oxide Nanohybrid-Assembled Microneedles as Mini-Invasive Electrodes for Real-Time Transdermal Biosensing.

A variety of nanomaterial-based biosensors have been developed to sensitively detect biomolecules in vitro, yet limited success has been achieved in real-time sensing in vivo. The application of microneedles (MN) may offer a solution for painless and minimally-invasive transdermal biosensing. However, integration of nanostructural materials on microneedle surface as transdermal electrodes remains challenging in applications. Here, a transdermal H2 O2 electrochemical biosensor based on MNs integrated with nanohybrid consisting of reduced graphene oxide and Pt nanoparticles (Pt/rGO) is developed. The Pt/rGO significantly improves the detection sensitivity of the MN electrode, while the MNs are utilized as a painless transdermal tool to access the in vivo environment. The Pt/rGO nanostructures are protected by a water-soluble polymer layer to avoid mechanical destruction during the MN skin insertion process. The polymer layer can readily be dissolved by the interstitial fluid and exposes the Pt/rGO on MNs for biosensing in vivo. The applications of the Pt/rGO-integrated MNs for in situ and real-time sensing of H2 O2 in vivo are demonstrated both on pigskin and living mice. This work offers a unique real-time transdermal biosensing system, which is a promising tool for sensing in vivo with high sensitivity but in a minimally-invasive manner.

Aberrant methylation-induced dysfunction of p16 is associated with osteoblast activation caused by fluoride.

Chronic exposure to fluoride continues to be a public health problem worldwide, affecting thousands of people. Fluoride can cause abnormal proliferation and activation of osteoblast and osteoclast, leading to skeletal fluorosis that can cause pain and harm to joints and bones and even lead to permanent disability. Nevertheless, there is no recognized mechanism to explain the bone lesions of fluorosis. In this work, we performed a population study and in vitro experiments to investigate the pathogenic mechanism of skeletal fluorosis in relation to methylation of the promoter of p16. The protein coded by the p16 gene inhibits cdk (cyclin-dependent kinase) 4/cdk6-mediated phosphorylation4 of retinoblastoma gene product and induces cell cycle arrest. The results showed that hypermethylation of p16 and reduced gene expression was evident in peripheral blood mononuclear cells of patients with fluorosis and correlated with the level of fluoride exposure. Studies with cell cultures of osteoblasts revealed in response to sodium fluoride (NaF) treatment, there was an induction of p16 hypermethylation and decreased expression, leading to increased cell proliferation, a longer S-phase of the cell cycle, and development of skeletal fluorosis. Further, the methylation inhibitor, 5-aza-2-deoxycytidine, reversed the p16 hypermethylation and expression in response to NaF. These results reveal a regulatory role of p16 gene methylation on osteoblasts activation during the development of skeletal fluorosis.

Enhancing Multistep DNA Processing by Solid-Phase Enzyme Catalysis on Polyethylene Glycol Coated Beads.

Covalent immobilization of enzymes on solid supports provides an alternative approach to homogeneous biocatalysis by adding the benefits of simple enzyme removal, improved stability, and adaptability to automation and high-throughput applications. Nevertheless, immobilized (IM) enzymes generally suffer from reduced activity compared to their soluble counterparts. The nature and hydrophobicity of the supporting material surface can introduce enzyme conformational change, spatial confinement, and limited substrate accessibility, all of which will result in loss of the immobilized enzyme activity. In this work, we demonstrate through kinetic studies that flexible polyethylene glycol (PEG) moieties modifying the surface of magnetic beads improve the activity of covalently immobilized DNA replication enzymes. PEG-modified immobilized enzymes were utilized in library construction for Illumina next-generation sequencing (NGS) increasing the read coverage across AT-rich regions.

Microplastics cause reproductive toxicity in male mice through inducing apoptosis of spermatogenic cells via p53 signaling.

BACKGROUND: Studies on male reproductive toxicity of microplastics are still scarce and the precise mechanism is not distinct. METHODS: C57BL/6 male mice were given oral gavage treatments treated with 5 µm (MPs) and 80 nm (NPs) polystyrene microplastics every day for 60 consecutive days in a row at dosages of 0, 10 and 40 mg/kg/d. The major damage of MPs and NPs were assessed by the assays in vivo and in vitro. Transcriptome sequencing was applied to screen the key involved pathways. RESULTS: In the 10 mg/kg/d NPs group, there was an increase in testicular organ coefficient, and in the 40 mg/kg/d MPs group, an increase in epididymal weight was observed. Vacuolization of spermatogenic cell layer, interstitial congestion, and germ cell apoptosis were found in the testes of MPs and NPs treatment mice at different dose groups. Higher apoptosis rate was observed in GC-2 cells after MPs and NPs treatment at different concentrations. Transcriptome analysis suggested that p53 pathway might be the key signal pathway of the cell apoptosis, and the expressions of p53 and other markers of cell apoptosis were indeed altered after exposure to MPs and NPs. CONCLUSIONS: MPs and NPs can cause reproductive toxicity in male mice through inducing apoptosis of spermatogenic cells via p53 signaling pathway, indicating MPs and NPs exposure be an unnegligible risk factor for reproductive health in male mice.

[The efficacy and safety of continuous erythropoietin receptor activator in dialytic patients with chronic renal anemia: an open, randomized, controlled, multi-center trial].

OBJECTIVE: To evaluate the efficacy and safety of continuous erythropoietin receptor activator (C.E.R.A.) once every 4 weeks by subcutaneous administration on hemoglobin (Hb) maintenance in dialytic patients with chronic renal anemia who had been treated with stable dose of erythropoietin (EPO). METHODS: This was an open, randomized, controlled, multi-center trial. All the hemodialysis or peritoneal dialytic patients in EPO maintenance treatment received subcutaneous EPO-ß during the 6-week pre-treatment period to maintain Hb level between 100 g/L and 120 g/L. Eligible patients were randomized (2:1) to accept either C.E.R.A. once every 4 weeks by subcutaneous administration (C.E.R.A. group, n = 187) or subcutaneous EPO-ß 1-3 times weekly (EPO group, n = 94) for 28 weeks (including 20-week dose titration period and 8-week efficacy evaluation period). The starting dose of C.E.R.A. was converted according to the dose of EPO-ß administered in the week preceding the first study drug administration. The primary outcome was the change of Hb level between the baseline and that in the efficacy evaluation period. RESULTS: Totally 253 patients completed the whole 28-week treatment. The change of baseline-adjusted mean Hb was +2.57 g/L for C.E.R.A. group and +1.23 g/L for EPO group, resulting in a treatment difference of 1.34 g/L (95%CI -1.11 - 3.78 g/L). Since the lower limit of 95%CI was greater than the pre-defined non-inferiority margin -7.5 g/L (P < 0.0001), C.E.R.A. once every 4 weeks by subcutaneous administration was clinically non-inferior to EPO regarding the maintenance of stable Hb level. The proportion of patients maintaining Hb level within the range of 100-120 g/L through efficacy evaluation period was similar between the two groups (69.0% for C.E.R.A. group vs 68.9% for EPO group, P > 0.05). The overall incidence of adverse events was similar between the C.E.R.A.(41.7%) and EPO (46.2%) groups (P > 0.05). The safety findings were in accordance with the patients' primary diseases rather than the administration. CONCLUSIONS: Conversion from EPO to C.E.R.A. once every 4 weeks by subcutaneous injection could maintain the Hb in target level in dialytic patients with renal anemia, and it was non-inferior to EPO. In general, subcutaneous administration of C.E.R.A. is well tolerated in dialytic patients with chronic renal anemia.

Let-7c-5p Regulates CyclinD1 in Fluoride-Mediated Osteoblast Proliferation and Activation.

Endemic fluorosis is caused by the intake of high environmental fluoride, which causes dental and skeletal fluorosis. Osteoblast proliferation and activation is closely related to skeletal fluorosis and is tightly regulated by the cell cycle. Several biological processes, including bone metabolism and osteoblast proliferation and activation, are regulated by a type of noncoding RNA called microRNAs (miRNAs). However, the understanding of miRNA functions in skeletal fluorosis is limited. Based on our previous miRNA sequencing results and bioinformatics analysis, we investigated the function of the miRNA let-7c-5p to regulate CyclinD1 in fluoride-induced osteoblast proliferation and activation. We designed population experiments as well as in vitro studies using 5-Ethynyl-2'-deoxyuridine (EdU), flow cytometry, immunofluorescence, dual-luciferase reporters, and chromatin immunoprecipitation. The population-based analysis showed a decrease in let-7c-5p expression as fluoride exposure increased. In addition, let-7c-5p levels were negatively correlated with CyclinD1 and Wnt9a (another let-7c-5p target). We verified in vitro that let-7c-5p participates in the fluoride-induced proliferation and activation of human osteoblasts by directly targeting CyclinD1. Furthermore, we demonstrated that let-7c-5p regulates CyclinD1 expression via the Wnt/ß-catenin signaling pathway. This study demonstrated the participation of let-7c-5p in fluoride-induced proliferation and activation of human osteoblasts by regulation of CyclinD1 expression at the post-transcriptional and transcriptional levels.

miR-122-5p Mediates Fluoride-Induced Osteoblast Activation by Targeting CDK4.

Chronic intake of fluoride, existing in the environment, may cause endemic fluorosis, which is characterized by the occurrence of skeletal and dental fluorosis. However, the pathogenesis of fluorosis has not yet been elucidated. Abnormal osteoblast proliferation and activation have a pivotal role in bone turnover disorders which are linked to skeletal fluorosis. MicroRNAs are involved in fundamental cellular processes, including cell proliferation. Based on our previous study, population study and in vitro experiments were designed to understand the effect of miR-122-5p on osteoblast activation in skeletal fluorosis through targeting cyclin-dependent kinase 4 (CDK4). In human populations with coal-burning type fluoride exposure, the results showed that miR-122-5p was downregulated but CDK4 expression was upregulated and miR-122-5p was negatively correlated with CDK4 expression. Furthermore, in human osteoblasts treated with sodium fluoride, we demonstrated that miR-122-5p mediated osteoblast activation of skeletal fluorosis via upregulation of the CDK4 protein. In support of this, dual-luciferase reporter assay showed that miR-122-5p modulated CDK4 protein levels by targeting its 3'-untranslated region. These findings show, for the first time, that miR-122-5p may be involved in the cause and development of skeletal fluorosis by targeting CDK4.

Liquid-like Polymer Coating as a Promising Candidate for Reducing Electrode Contamination and Noise in Complex Biofluids.

Biosensors that can automatically and continuously track fluctuations in biomarker levels over time are essential for real-time sensing in biomedical and environmental applications. Although many electrochemical sensors have been developed to quickly and sensitively monitor biomarkers, their sensing stability in complex biofluids is disturbed by unavoidable nonspecific adhesion of proteins or bacteria. Recently, various substrate surface modification techniques have been developed to resist biofouling, yet functionalization of electrodes in sensors to be anti-biofouling is rarely achieved. Here, we report an integrated three-electrode system (ITES) modified with a "liquid-like" polydimethylsiloxane (PDMS) brush that can continuously and stably monitor reactive oxygen species (ROS) in complex fluids. Based on the slippery "liquid-like" coating, the modified ITES surface could prevent the adhesion of various liquids as well as the adhesion of proteins and bacteria. The "liquid-like" coating does not significantly affect the sensitivity of the electrode in detecting ROS, while the sensing performance could remain stable and free of bacterial attack even after 3 days of incubation with bacteria. In addition, the PDMS brush-modified ITES (PMITES) could continuously record ROS levels in bacterial-rich fluids with excellent stability over 24 h due to the reduced bacterial contamination on the electrode surface. This technique offers new opportunities for continuous and real-time monitoring of biomarkers that will facilitate the development of advanced sensors for biomedical and environmental applications.

Good Manufacturing Practices production and analysis of a DNA vaccine against dental caries.

AIM: To prepare a clinical-grade anti-caries DNA vaccine pGJA-P/VAX and explore its immune effect and protective efficacy against a cariogenic bacterial challenge. METHODS: A large-scale industrial production process was developed under Good Manufacturing Practices (GMP) by combining and optimizing common unit operations such as alkaline lysis, precipitation, endotoxin removal and column chromatography. Quality controls of the purified bulk and final lyophilized vaccine were conducted according to authoritative guidelines. Mice and gnotobiotic rats were intranasally immunized with clinical-grade pGJA-P/VAX with chitosan. Antibody levels of serum IgG and salivary SIgA were assessed by an enzyme-linked immunosorbent assay (ELISA), and caries activity was evaluated by the Keyes method. pGJA-P/VAX and pVAX1 prepared by a laboratory-scale commercial kit were used as controls. RESULTS: The production process proved to be scalable and reproducible. Impurities including host protein, residual RNA, genomic DNA and endotoxin in the purified plasmid were all under the limits of set specifications. Intranasal vaccination with clinical-grade pGJA-P/VAX induced higher serum IgG and salivary SIgA in both mice and gnotobiotic rats. While in the experimental caries model, the enamel (E), dentinal slight (Ds), and dentinal moderate (Dm) caries lesions were reduced by 21.1%, 33.0%, and 40.9%, respectively. CONCLUSION: The production process under GMP was efficient in preparing clinical-grade pGJA-P/VAX with high purity and intended effectiveness, thus facilitating future clinical trials for the anti-caries DNA vaccine.

[Evaluation of the efficacy of sodium fluoride dental protective agent combined with pit and fissure sealant in prevention of dental caries in preschool children].

PURPOSE: To explore the effect of sodium fluoride tooth protector combined with pit and fissure sealing to prevent caries in preschool children. METHODS: Two hundred preschool children who were treated with pit and fissure closure from January 2014 to September 2014 were selected as subjects. According to random number table method, 100 cases were divided into the combined group and the control group. Children in the control group were treated with pit and fissure sealant for prevention of caries, while children in the combined group were treated with sodium fluoride tooth protector combined with pit and fissure sealant for caries prevention. The incidence of dental caries, proximal caries, mean DMFT, and expulsion rate of the sealants were compared between the two groups at 1 year and 2 years. Data analysis was performed using SPSS 16.0 software package for data analysis. RESULTS: At 1 year of follow-up, there was no significant difference in the incidence of dental caries, proximal caries, and mean DMFT between the two groups (P>0.05). The incidence of caries and mean DMFT in the combined group were significantly lower than those in the control group at 2 years of follow-up (P<0.05). The incidence of proximal caries in the combined group was not significantly different from that in the control group (P>0.05). The retension rate of the pit and fissure sealant in the combined group was significantly higher than that in the control group (P<0.05). There was no significant difference in the partial shedding rate and complete expulsion rate between the two groups (P>0.05). CONCLUSIONS: Sodium fluoride tooth protector combined with pit and fissure sealing has better anti-caries effect than the use of pit and fissure sealant alone in preschool children.

Histone Deacetylation in the Promoter of p16 Is Involved in Fluoride-Induced Human Osteoblast Activation via the Inhibition of Sp1 Binding.

Chronic fluorosis is a systemic condition which principally manifests as defects in the skeleton and teeth. Skeletal fluorosis is characterized by aberrant proliferation and activation of osteoblasts, however, the underlying mechanisms of osteoblast activation induced by fluoride are not fully understood. Therefore, we investigated the pathogenic mechanism of human primary osteoblast proliferation and activation in relation to histone acetylation of the promoter p16, a well-known cell cycle regulation-related gene. The results showed that sodium fluoride (NaF) induced deacetylation and decreased expression of the p16 gene via inhibition of specificity protein 1 (Sp1) binding to its response element, which accounts for NaF increasing cell viability and promoting proliferation in human primary osteoblasts. These results reveal the regulatory mechanism of histone acetylation of the p16 gene on osteoblast activation in skeletal fluorosis.

The effect of elemental content on the risk of dental fluorosis and the exposure of the environment and population to fluoride produced by coal-burning.

Endemic fluorosis is a geochemical disease that affects thousands of people. Growing evidence from domestic and foreign studies indicate that fluorosis is associated with an abnormal level of the elements (such as F, Ca, Fe, Mg, Cu, Zn, P) in the environment and a population exposed to fluoride. To study the effect of the elemental content on the risk of dental fluorosis, the content of 25 elements in the environment produced by coal-burning and a population exposed to fluoride was determined. The results show that an abnormal level of various elements (including F, Al, Se, Zn, Cu, Fe, Mo, Mn, B, V, Ca, Mg, and P) in the population exposed to fluoride, which is related to the increasing or decreasing of the corresponding elements in the environment. Subsequent univariate and multivariate regression analyses show that high levels of F, Al, As, Pb and Cr were a risk factor for dental fluorosis, but not Se, Zn, Cu, B, Ca and P which were a protective factor for dental fluorosis. This study can provide a scientific basis for a further understanding of the causes of health damage caused by fluoride and the improvement of targeted prevention strategies.

High-throughput metabolomic approach revealed the acupuncture exerting intervention effects by perturbed signatures and pathways.

Metabolomics can capture global changes and the overall physiological status in biochemical networks and pathways in order to elucidate sites of perturbations. High-throughput metabolomics and acupuncturology have similar characteristics such as entirety, comprehensiveness and dynamic changes, and can identify potential candidates for acupuncture effects and provide valuable information towards understanding therapy mechanisms. Saliva has recently gained popularity as a potential tool for biomarker monitoring, as its composition may potentially reflect plasma metabolite levels and, therefore, may be used as an indicator of the physiological state. However, the underlying mechanism of acupuncture, remains largely unknown, which hinders its widespread use. Acupuncture would produce unique characterization of metabolic perturbations. In this study, UPLC/ESI-HDMS in high-accuracy mode coupled with pattern recognition analysis was carried out to investigate the mechanism and saliva metabolite biomarkers for acupuncture treatment at 'Zusanli' acupoint (ST-36) as a case study. Putative metabolite identifications for these ions were obtained through a mass-based database search. As a result, the top canonical pathways including phenylalanine metabolism, alanine, aspartate and glutamate metabolism, d-glutamine and d-glutamate metabolism, and steroid hormone biosynthesis pathways were acutely perturbed. 26 differential metabolites were identified by chemical profiling, and may be useful to clarify the physiological basis and mechanism of ST-36. More importantly, network construction has led to the integration of metabolites associated with the multiple perturbation pathways. These results provide useful insights into biomarker discovery utilizing metabolomics as an efficient and cost effective platform. This study opens new possibilities for the selection of saliva as a source of metabolite biomarkers representative of specific disorders.

Salivary proteomics in biomedical research.

Proteins that are important indicators of physiological or pathological states, can provide information for the identification of early and differential markers for disease. Saliva, contains an abundance of proteins, offers an easy, inexpensive, safe, and non-invasive approach for disease detection, and possesses a high potential to revolutionize the diagnostics. Discovery of salivary biomarkers could be used to scrutinize health and disease surveillance. The impact of human saliva proteome analysis in the search for clinically relevant disease biomarkers will be realized through advances made using proteomic technologies. The advancements of emerging proteomic techniques have benefited biomarker research to the point where saliva is now recognized as an excellent diagnostic medium for the detection of disease. This review presents an overview of the value of saliva as a credible diagnostic tool and we aim to summarize the proteomic technologies currently used for global analysis of saliva proteins and to elaborate on the application of saliva proteomics to the discovery of disease biomarkers, and discuss some of the critical challenges and perspectives in this field.

Saliva metabolomics opens door to biomarker discovery, disease diagnosis, and treatment.

Metabolomics is the systematic study of the unique chemical fingerprints of low molecular weight endogenous metabolites or metabolite profiles in a biological sample. Metabolites that are important indicators of physiological or pathological states can provide information for the identification of early and differential markers for disease and help to understand its occurrence and progression. Analysis of these key biomarkers has become an important role to monitor the state of biological organisms and is a widely used diagnostic tool for disease. Metabolomic analyses are propelling the field of medical diagnostics forward at unprecedented rates because of its ability to reliably identify metabolites that are at the metabolic level in concentration. These advancements have benefited biomarker research to the point where saliva is now recognized as an excellent diagnostic medium for the detection of disease. Saliva contains a large array of metabolites, many of which can be informative for the detection of diseases. Salivary diagnostics offer an easy, inexpensive, safe, and noninvasive approach for disease detection. Discovery of salivary biomarkers that could be used to scrutinize health and disease surveillance has addressed its diagnostic value for clinical applications. Availability of emerging metabolomic techniques gives optimism that saliva can eventually be placed as a biomedium for clinical diagnostics. Comprehensive salivary metabolome will be an important resource for researchers who are studying metabolite chemistry, especially in the fields of salivary diagnostics, and will be helpful for analyzing and hence identifying corresponding disease-related salivary biomarkers. This review presents an overview of the value of saliva as a credible diagnostic tool, the discovery of salivary biomarkers, and the development of salivary diagnostics now and in the future. In particular, proof of principle has been demonstrated for salivary biomarker research.