Effects of water-soluble components of atmospheric particulates from rare earth mining areas in China on lung cancer cell cycle.

BACKGROUND: This study aims to investigate the effects of water soluble particulate matter (WSPM) on the viability and protein expression profile of human lung adenocarcinoma cell A549 in the Bayou Obo rare earth mining area, and explore the influence of WSPM on the A549 cell cycle. RESULTS: It was found that WSPM can inhibit the viability of A549 cells and induce cell arrest in the G2/M phase. Compared with controls, exposure to WSPM10 and WSPM2.5 induced 134 and 116 proteins to be differentially expressed in A549 cells, respectively. In addition, 33 and 31 differentially expressed proteins were further confirmed, and was consistent with the proteomic analysis. The most prominent enrichment in ribosome-associated proteins were presented. When RPL6, RPL13, or RPL18A gene expression was inhibited, A549 cells were arrested in the G1 phase, affecting the expression of Cyclin D1, p21, RB1, Cyclin A2, Cyclin B1, CDC25A, CDK2, CHEK2 and E2F1. Furthermore, the La3+, Ce3+, Nd3+ and F- in WSPM also inhibited the viability of A549 cells. After 24 h of exposure to 2 mM of NaF, A549 cells were also arrested in the G2/M phase, while the other three compounds did not have this effect. These four compounds affected the cell cycle regulatory factors in A549 cells, mainly focusing on effecting the expression of CDK2, CDK4, RB1, ATM, TP53 and MDM2 genes. These results are consistent with the those from WSPM exposure. CONCLUSIONS: These results revealed that WSPM from rare earth mines decreased the viability of A549 cells, and induced cell cycle G2/M phase arrest, and even apoptosis, which may be independent of the NF-κB/MYD88 pathway, and be perceived by the TLR4 receptor. The dysfunction of the cell cycle is correlated to the down-expression of ribosomal proteins (RPs). However, it is not the direct reason for the A549 cell arrest in the G2/M phase. La3+, Ce3+, and F- are probably the main toxic substances in WSPM, and may be regulate the A549 cell cycle by affecting the expression of genes, such as MDM2, RB1, ATM, TP53, E2F1, CDK2 and CDK4. These results indicate the importance for further research into the relationship between APM and lung cancer.

The construction of DNAzyme-based logic gates for amplified microRNA detection and cancer recognition.

Benefiting from the simple DNAzyme and a duplex-specific nuclease, a series of microRNA-stimulated DNAzyme logic gates were rationally assembled for the highly accurate detection of multiple low-abundant microRNA biomarkers that correspond to the specific aberrant microRNA expression patterns of cancer tissues, thus showing a great potential in early diagnosis.

An Ultrasensitive Diagnostic Biochip Based on Biomimetic Periodic Nanostructure-Assisted Rolling Circle Amplification.

Developing portable and sensitive devices for point of care detection of low abundance bioactive molecules is highly valuable in early diagnosis of disease. Herein, an ultrasensitive photonic crystals-assisted rolling circle amplification (PCs-RCA) biochip was constructed and further applied to circulating microRNAs (miRNAs) detection in serum. The biochip integrated the optical signal enhancement capability of biomimetic PCs surface with the thousand-fold signal amplification feature of RCA. The biomimetic PCs displayed periodic dielectric nanostructure and significantly enhanced the signal intensity of RCA reaction, leading to efficient improvement of detection sensitivity. A limit of detection (LOD) as low as 0.7 aM was obtained on the PCs-RCA biochip, and the LOD was 7 orders of magnitude lower than that of standard RCA. Moreover, the PCs-RCA biochip could discriminate a single base variation in miRNAs. Accurate quantification of ultralow-abundance circulating miRNAs in clinical serum samples was further achieved with the PCs-RCA biochip, and patients with the nonsmall cell lung carcinoma were successfully distinguished from healthy donors. The PCs-RCA biochip can detect bioactive molecules with ultrahigh sensitivity and good specificity, making it valuable in clinical disease diagnosis and health assessment.

Disruption of Gen1 Causes Congenital Anomalies of the Kidney and Urinary Tract in Mice.

Congenital anomalies of the kidney and urinary tract (CAKUT) are among the most common developmental defects in humans. Despite of several known CAKUT-related loci (HNF1B, PAX2, EYA1, etc.), the genetic etiology of CAKUT remains to be elucidated for most patients. In this study, we report that disruption of the Holliday Junction resolvase gene Gen1 leads to renal agenesis, duplex kidney, hydronephrosis, and vesicoureteral reflux (VUR) in mice. GEN1 interacts with SIX1 and enhances the transcriptional activity of SIX1/EYA1, a key regulatory complex of the GDNF morphogen. Gen1 mutation impairs Grem1 and Gdnf expression, resulting in excessive ureteric bud formation and defective ureteric bud branching during early kidney development. These results revealed an unidentified role of GEN1 in kidney development and suggested its contribution to CAKUT.

Detecting 5-methylcytosine using an enzyme-free DNA strand exchange reaction without pretreatment under physiological conditions.

We present here a novel and efficient method for 5mC detection using a DNA strand exchange reaction (SER) strategy. This enzyme-free method needs no pre-treatment of target DNAs and can be adapted to most of the target duplexes under physiological conditions. The high sequence selectivity of this method can distinguish 5mC from normal cytosine in an accurate manner.

N6-Methyladenine hinders RNA- and DNA-directed DNA synthesis: application in human rRNA methylation analysis of clinical specimens.

N6-Methyladenine (m6A) is the most abundant internal modification on mammalian mRNA. Very recently, m6A has been reported as a potentially important 'epigenetic' mark in eukaryotes. Until now, site-specific detection of m6A is technically very challenging. Here, we first reveal that m6A significantly hinders DNA- and RNA-directed DNA synthesis. Systematic investigations of 5'-triphosphates of a variety of 5-substituted 2'-deoxyuridine analogs in primer extension have been performed. In the current study, a quantitative analysis of m6A in the RNA or DNA context has been achieved, using Bst DNA polymerase catalyzed primer extension. Molecular dynamics study predicted that m6A in template tends to enter into and be restrained in the MGR region of Bst DNA polymerase, reducing conformational flexibility of the DNA backbone. More importantly, a site-specific determination of m6A in human ribosomal RNA (rRNA) with high accuracy has been afforded. Through a cumulative analysis of methylation alterations, we first reveal that significantly cancer-related changes in human rRNA methylation were present in patients with hepatocellular carcinoma.

Computational discovery and experimental verification of tyrosine kinase inhibitor pazopanib for the reversal of memory and cognitive deficits in rat model neurodegeneration.

Cognition and memory impairment are hallmarks of the pathological cascade of various neurodegenerative disorders. Herein, we developed a novel computational strategy with two-dimensional virtual screening for not only affinity but also specificity. We integrated the two-dimensional virtual screening with ligand screening for 3D shape, electrostatic similarity and local binding site similarity to find existing drugs that may reduce the signs of cognitive deficits. For the first time, we found that pazopanib, a tyrosine kinase inhibitor marketed for cancer treatment, inhibits acetylcholinesterase (AchE) activities at sub-micromolar concentration. We evaluated and compared the effects of intragastrically-administered pazopanib with donepezil, a marketed AchE inhibitor, in cognitive and behavioral assays including the novel object recognition test, Y maze and Morris water maze test. Surprisingly, we found that pazopanib can restore memory loss and cognitive dysfunction to a similar extent as donepezil in a dosage of 15 mg kg-1, only one fifth of the equivalent clinical dosage for cancer treatment. Furthermore, we demonstrated that pazopanib dramatically enhances the hippocampal Ach levels and increases the expression of the synaptic marker SYP. These findings suggest that pazopanib may become a viable treatment option for memory and cognitive deficits with a good safety profile in humans.

Novel amplex red oxidases based on noncanonical DNA structures: property studies and applications in microRNA detection.

G-triplex has recently been identified as a new secondary structure in G-rich sequences. However, its functions and biological roles remain largely unknown. This study first developed two kinds of Amplex Red oxidases, which were based on relatively new G-triplex structure and a common G-quadruplex one. A collection of DNA binding assays including circular dichroism (CD) spectroscopy, a CD melting assay, and a UV titration study were used to determine the G-triplex structure of G3 oligomer. The low intrinsic oxidative activity of hemin was significantly enhanced using G-triplex or G-quadruplex. Only one key guanine deletion from the G3 oligomer or G4 one could result in a much decreased Amplex Red oxidation activity. To the best of our knowledge, this is the first case reporting direct use of air as the oxidant for fluorescence generation based on DNAzyme strategies. Further mechanism studies demonstrated an involvement of on-site H2O2 generation from O2 and water and a following oxidation of Amplex Red to resorufin, causing a fluorescence enhancement. Furthermore, the newly developed oxidases have been effectively used in microRNA detection, using only one biotin-labeled probe and one small-molecule substrate. The conjugation of a target DNA to the G-triplex- or G-quadruplex-forming sequence enabled one to produce G-triplex or G-quadruplex by endonuclease in the presence of a slight amount of miRNA and amplify the signal of fluorescence from the oxidation of Amplex Red. Our findings of novel Amplex Red oxidases could potentially be used in a wide range of applications.

5-Methyldeoxycytidine enhances the substrate activity of DNA polymerase.

Here, we first demonstrated that 5-MedCTP could be incorporated into the synthetic DNA template by the exonuclease deficient Klenow fragment with a much higher efficiency than dCTP and 5-hydroxymethyl-dCTP. Further, we first conducted a comparable study of primer extension reaction using templates containing deoxycytidine (dC) or 5-methyldeoxycytidine (5-mdC) for incorporating different triphosphates. Based on our findings, 5-methyldeoxycytidine could enhance the substrate activity of the Klenow fragment (exo-) and this feature could potentially be used in DNA methylation analysis.

Systematic investigation of DNAs with modified cytosines under hot alkali treatment.

We have conducted the first systematic investigation of DNAs with modified cytosines, including 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), under hot alkali treatment.

The G-triplex DNA could function as a new variety of DNA peroxidase.

This study is the first to investigate the interactions of hemin with a G-triplex DNA of T1 using different DNA binding assays. The low intrinsic peroxidatic activity of hemin could be significantly enhanced using T1. Furthermore, much decreased oxidation enhancement by T2 or T3 with one key guanine mutation was observed, and the observed peroxidatic activity of T1 should be directly due to the G-triplex complexed with hemin.

Highly sensitive detection of telomerase based on a DNAzyme strategy.

The present study demonstrated a highly sensitive strategy for measuring telomerase activity in cell extracts. Furthermore, we applied the new strategy for in situ detection of telomerase at the cellular level in cancer cells, together with a normal cell as the negative control.

Simultaneously sensitive detection of multiple miRNAs based on a strand displacement amplification.

We reported an efficient strategy based on a strand displacement amplification for serum miRNA detection. In such a system, a multiplexed, sensitive and quick detection of miRNAs could be achieved through a combination of fluorescence labeled probes, a common primer and a polymerase. This could be potentially used in the clinical field to achieve early disease diagnosis and prognosis.

Sensitive analysis of DNA methyltransferase based on a hairpin-shaped DNAzyme.

The analysis of DNA methylation and MTase (methyltransferase) activity is important in epigenetic study. We have developed a novel strategy for sensitive analysis of MTase activity based on a hairpin shaped DNAzyme; 8-17 DNAzyme amplicon has been adopted and found to be very effective in such analysis.

Effect of probiotic supplementation in the first 6 months of life on specific antibody responses to infant Hepatitis B vaccination.

Probiotics have been shown to enhance specific immune responses to vaccines. We aim to assess the effect of probiotic supplementation on specific IgG antibody responses to Hepatitis B (HepB) vaccination in infants. Compared to controls, probiotic supplementation improved HepB surface antibody responses in subjects receiving monovalent doses of HepB vaccine at 0, 1 month and a DTPa-HepB combination vaccine at 6 months [placebo (n=28): 187.97 (180.70-195.24), probiotic (n=29): 345.70 (339.41-351.99)mIU/ml] (p=0.069), but not those who received 3 monovalent doses [placebo (n=68): 302.34 (296.31-308.37), probiotic (n=77): 302.06 (296.31-307.81)mIU/ml] (p=0.996). Probiotics may enhance specific antibody responses in infants receiving certain Hepatitis B vaccine schedules.

The role of vision in academic school performance.

PURPOSE: To determine whether presenting distance visual acuity is related to subsequent academic school performance in Singaporean children between 9 to 10 years of age. METHODS: Singapore children (n = 1143 children) were examined during their visits at ages 9 to 10 years (grades 3 to 4) as part of the Singapore Cohort Study of the Risk Factors for Myopia (SCORM) longitudinal study. Each child underwent an annual comprehensive eye examination, including the assessment of presenting logarithm of the minimum angle of resolution (LogMAR) distance visual acuity (VA). The individual marks of a nation-wide standard examination in grade 4 were used as the outcome measure for academic school performance. Children with any known eye disease, (such as media opacities) were excluded from the analysis. RESULTS: The mean presenting distance VA of the better eye was 0.10 and 0.08 when the children were in grades 3 and 4, respectively. There was a statistically significant difference for mean presenting VA with 9 and 10 year old boys scoring better (0.08 and 0.07) compared to girls (0.12 and 0.09) for the same ages, (p = 0.001 and p = 0.007), respectively. After adjusting for gender, ethnicity, school, reading, intelligence quotient and father's education, no significant relationships were found between average examination marks at the end of grade 4 and presenting VA obtained (better eye and worst eye) in grade 3 (p = 0.38 and p = 0.98) and 4 (p = 0.27 and p = 0.16). CONCLUSION: In our sample of Singaporean children without ocular disease, distance VA did not play a significant role in predicting academic school performance.

Atropine for the treatment of childhood myopia: effect on myopia progression after cessation of atropine.

PURPOSE: The aim of this study was to assess the effect on myopia progression after cessation of topical atropine treatment. DESIGN: Parallel-group, placebo-controlled, randomized, double-masked study. PARTICIPANTS: Four hundred children aged 6 to 12 years with refractive error of spherical equivalent -1.00 to -6.00 diopters (D) and astigmatism of -1.50 D or less. INTERVENTION: No intervention was administered. Subjects were followed up for 12 months after stopping treatment, which consisted of either 1% atropine or vehicle eyedrops once nightly for 2 years. Only 1 eye of each subject was chosen through randomization for treatment. MAIN OUTCOME MEASURES: The main efficacy outcome measures were change in spherical equivalent refraction as measured by cycloplegic autorefraction and change in ocular axial length as measured by ultrasonography. RESULTS: After cessation of atropine drops, the mean progression in the atropine-treated group was -1.14+/-0.80 D over 1 year, whereas the progression in placebo-treated eyes was -0.38+/-0.39 D (P<0.0001). However, after 3 years of participation in the trial (with 2 years on atropine treatment), eyes randomized to atropine have less severe myopia than other eyes. Spherical equivalent was -4.29+/-1.67 D in the atropine-treated eyes compared with -5.22+/-1.38 D in the placebo-treated eyes (P<0.0001). Spherical equivalents in atropine-untreated and placebo-untreated eyes were -5.00+/-1.62 D and -5.28+/-1.43 D, respectively. Over the 3 years, the increase in axial length of the atropine-treated eyes was 0.29+/-0.37 mm compared with 0.52+/-0.45 mm in the placebo-treated eyes (P<0.0001). After cessation of atropine, the amplitude of accommodation and near visual acuity returned to pretreatment levels. CONCLUSIONS: After stopping treatment, eyes treated with atropine demonstrated higher rates of myopia progression compared with eyes treated with placebo. However, the absolute myopia progression after 3 years was significantly lower in the atropine group compared with placebo.

Red-green colour blindness in Singaporean children.

PURPOSE: X-linked red-green colour blindness is the most common form of colour blindness. Various studies suggest that, worldwide, 2-8% of men are afflicted with this condition. The purpose of this study is to determine the prevalence of red-green colour blindness in Singaporean schoolchildren. METHOD: A total of 1249 children aged 13-15 years were screened using the Ishihara 24-plate edition book during the School Cohort study of the Risk factors for Myopia visit. RESULTS: A total of 1210 children (96.8%) managed to correctly identify at least 13 of the initial 15 plates and were deemed to have normal colour vision.Thirty-three children (32 boys, one girl) were only able to identify nine or less plates and were considered to be colour blind. Overall, 5.4% (95% confidence interval 3%, 7%) of Chinese, 4.9% (1%, 9%) of Malay and 4.9% (2%, 11%) of Indian boys were colour blind (P = 0.97). Classification plates 16-17 were useful in determining deutran or protan tendencies in only 14 (43%) of the 33 children identified as being colour blind. CONCLUSION: 5.3% of boys and 0.2% of girls were found to be colour blind in this Singapore-based study. Although the Ishihara test proved useful in identifying colour-blind children, other tests are required to accurately classify the types of red-green colour blindness in these children.