Prenatal and early life exposure to air pollution induced hippocampal vascular leakage and impaired neurogenesis in association with behavioral deficits.

Exposure to traffic-related air pollution (TRAP) is associated with a range of neurodevelopmental disorders in human populations. In rodent models, prenatal TRAP exposure increased depressive behaviors and increased brain microglial activity. To identify cellular mechanisms, we examined adult neurogenesis and the blood-brain barrier (BBB) in relation to cognition and motivated behaviors in rats that were exposed to a nano-sized TRAP subfraction from gestation into adulthood. At age 5 months, exposed male rats had 70% fewer newly generated neurons in the dentate gyrus (DG) of the hippocampus. Microglia were activated in DG and CA1 subfields (35% more Iba1). The BBB was altered, with a 75% decrease of the tight junction protein ZO-1 in the CA1 layer, and twofold more iron deposits, a marker of microhemorrhages. The exposed rats had impaired contextual memory (novel object in context), reduced food-seeking behavior, and increased depressive behaviors (forced swim). Deficits of de novo neurogenesis were inversely correlated with depressive behavior, whereas increased microbleeds were inversely correlated with deficits in contextual memory. These findings give the first evidence that prenatal and early life exposure to TRAP impairs adult hippocampal neurogenesis and increases microbleeds in association with behavioral deficits.

A hippocampus to prefrontal cortex neural pathway inhibits food motivation through glucagon-like peptide-1 signaling.

The hippocampus and the medial prefrontal cortex (mPFC) are traditionally associated with regulating memory and executive function, respectively. The contribution of these brain regions to food intake control, however, is poorly understood. The present study identifies a novel neural pathway through which monosynaptic glutamatergic ventral hippocampal field CA1 (vCA1) to mPFC connectivity inhibits food-motivated behaviors through vCA1 glucagon-like peptide-1 receptor (GLP-1R). Results demonstrate that vCA1-targeted RNA interference-mediated GLP-1R knockdown increases motivated operant responding for palatable food. Chemogenetic disconnection of monosynaptic glutamatergic vCA1 to mPFC projections using designer receptors exclusively activated by designer drugs (DREADDs)-mediated synaptic silencing ablates the food intake and body weight reduction following vCA1 GLP-1R activation. Neuropharmacological experiments further reveal that vCA1 GLP-1R activation reduces food intake and inhibits impulsive operant responding for palatable food via downstream communication to mPFC NMDA receptors. Overall these findings identify a novel neural pathway regulating higher-order cognitive aspects of feeding behavior.

Designer germanium quantum dot phototransistor for near infrared optical detection and amplification.

We demonstrated a unique CMOS approach for the production of a high-performance germanium (Ge) quantum dot (QD) metal-oxide-semiconductor phototransistor. In the darkness, low off-state leakage (Ioff ∼ 0.27 pA µm(-2)), a high on-off current ratio (Ion/Ioff ∼ 10(6)), and good switching behaviors (subthreshold swing of 175 mV/dec) were measured on our Ge-QD phototransistor at 300 K, indicating good hetero-interfacial quality of the Ge-on-Si. Illumination makes a significant enhancement in the drain current of Ge QD phototransistors when biased at both the on- and off-states, which is a great benefit from Ge QD-mediated photoconductive and photovoltaic effects. The measured photocurrent-to-dark-current ratio (Iphoto/Idark) and the photoresponsivities from the Ge QD phototransistor are as high as 4.1 × 10(6) and 1.7 A W(-1), respectively, under an incident power of 0.9 mW at 850 nm illumination. A superior external quantum efficiency of 240% and a very fast temporal response time of 1.4 ns suggest that our Ge QD MOS phototransistor offers great promise as optical switches and transducers for Si-based optical interconnects.

Formation of Ge quantum dots array in layer-cake technique for advanced photovoltaics.

We report a simple and manageable growth method for placing dense three-dimensional Ge quantum dot (QD) arrays in a uniform or a graded size distribution, based on thermally oxidizing stacked poly-SiGe in a layer-cake technique. The QD size and spatial density in each stack can be modulated by conditions of the Ge content in poly-Si(1-x)Ge(x), oxidation, and the underlay buffer layer. Size-dependent internal structure, strain, and photoluminescence properties of Ge QDs are systematically investigated. Optimization of the processing conditions could be carried out for producing dense Ge QD arrays to maximize photovoltaic efficiency.

Development of a high-performance liquid chromatographic method for bioanalytical applications with sulpiride.

An improved HPLC method using a silica gel column with fluorescence detection (excitation at 300 nm and emission at 365 nm) was developed for the determination of sulpiride concentrations in plasma. Analysis of sulpiride in plasma samples was simplified by a one-step liquid-liquid extraction after alkaline treatment of only 1 ml of plasma. The low limit of quantitation was 20 ng/ml with a coefficient of variation of less than 20%. A linear range was found from 20 to 1500 ng/ml. This HPLC method was validated with the precision for inter-day and intra-day runs being 0.36-8.01% and 0.29-5.25%, respectively, and the accuracy (standard deviation of mean, SD) for inter-day and intra-day runs being -1.58 to 5.02% and -2.14 to 5.21%, respectively. Bioequivalence of the two products was evaluated in 12 normal healthy male volunteers in a single-dose, two-period, two-sequence, two-treatment cross-over study. Sulpiride plasma concentrations were analyzed with this validated HPLC method. Results demonstrated that the two tablet formulations of sulpiride appear to be bioequivalent.

Universal SNP genotyping assay with fluorescence polarization detection.

The degree of fluorescence polarization (FP) of a fluorescent molecule is a reflection of its molecular weight (Mr). FP is therefore a useful detection methodfor homogeneous assays in which the starting reagents and products differ significantly in Mr. We have previously shown that FP is a good detection method for the single-base extension and the 5'-nuclease assays. In this report, we describe a universal, optimized single-base extension assay for genotyping single nucleotide polymorphisms (SNPs). This assay, which we named the template-directed dye-terminator incorporation assay with fluorescence polarization detection (FP-TDI), uses four spectrally distinct dye terminators to achieve universal assay conditions. Even without optimization, approximately 70% of all SNP markers tested yielded robust assays. The addition of an E. coli ssDNA-binding protein just before the FP reading significantly increased FP values of the products and brought the success rate of FP-TDI assays up to 90%. Increasing the amount of dye terminators and reducing the number of thermal cycles in the single-base extension step of the assay increased the separation of the FP values benveen the products corresponding to different genotypes and improved the success rate of the assay to 100%. In this study the genomic DNA samples of 90 individuals were typed for a total of 38 FP-TDI assays (using both the sense and antisense TDI primers for 19 SNP markers). With the previously described modifications, the FP-TDI assay gave unambiguous genotyping data for all the samples tested in the 38 FP-TDI assays. When the genotypes determined by the FP-TDI and 5'-nuclease assays were compared, they were in 100% concordance for all experiments (a total of 3420 genotypes). The four-dye-terminator master mixture described here can be used for assaying any SNP marker and greatly simplifies the SNP genotyping assay design.

Genotyping single-nucleotide polymorphisms by the invader assay with dual-color fluorescence polarization detection.

BACKGROUND: The PCR-Invader assay is a robust, homogeneous assay that has been shown to be highly sensitive and specific in genotyping single-nucleotide polymorphism (SNP) markers. In this study, we introduce two changes to improve the assay: (a) we streamline the PCR-Invader method by assaying both alleles for each SNP in one reaction; and (b) we reduce the cost of the method by adopting fluorescence polarization (FP) as the detection method. METHODS: PCR product was incubated with Invader oligonucleotide and two primary probes at 93 degrees C for 5 min. Signal probes corresponding to the cleaved flaps of the primary probes [labeled with fluorescein and 6-carboxytetramethylrhodamine (TAMRA) dye] and Cleavase VIII enzyme (a flap endonuclease) were then added to the mixture. This reaction mixture was incubated at 63 degrees C for 5 min. FP measurements were made with a fluorescence plate reader. RESULTS: Eighty-eight individuals were genotyped across a panel of 10 SNPs, using PCR product as template, for a total of 880 genotypes. An average "no call" rate of 3.2% was observed after first round of experiments. PCR products were remade in those samples that failed to produce any genotype in the first round, and all gave clear-cut genotypes. When the genotypes determined by the PCR-Invader assay and template-directed dye-terminator incorporation assay with FP were compared, they were in 100% concordance for all SNP markers and experiments. CONCLUSIONS: The improvements introduced in this study make PCR-Invader assay simpler and more cost-effective, and therefore more suitable for high-throughput genotyping.

Immunoperoxidase quantitation of 4-aminobiphenyl- and polycyclic aromatic hydrocarbon-DNA adducts in exfoliated oral and urothelial cells of smokers and nonsmokers.

Immunoperoxidase methods using two antibodies were developed for detection and quantitation of DNA damage in single cells. A monoclonal antibody that recognizes 4-aminobiphenyl (4-ABP)-DNA adducts was initially tested on liver tissues of BALB/c mice treated with 4-ABP, then applied to the detection of adducts in oral mucosa and exfoliated urothelial cells of smokers and nonsmokers. Levels of 4-ABP-DNA in exfoliated urothelial cells were elevated in each of 20 smokers (mean relative staining intensity, 517 +/- 137) compared with age-, race-, and sex-matched nonsmokers (313 +/- 79; P < 0.0005). Significantly higher damage levels were also observed in oral mucosa cells of smokers compared with nonsmokers (552 +/- 157 versus 326 +/- 101; P < 0.0005). A polyclonal antiserum that recognizes benzo(a)pyrene and structurally related polycyclic aromatic hydrocarbon (PAH) diol epoxide-DNA adducts was also applied to the same study samples after validation by staining of 10T1/2 cells treated with (+/-)-trans-anti-benzo(a)pyrene diol epoxide. Smokers had higher levels of PAH-DNA damage in oral mucosa and exfoliated urothelial cells than nonsmokers (oral mucosa cells, 684 +/- 107 versus 370 +/- 83; P < 0.0005; urothelial cells, 689 +/- 72 versus 495 +/- 57; P < 0.0005). A similar 2-3-fold range in relative staining was found in smokers and nonsmokers for both 4-ABP- and PAH-DNA, suggesting the importance of individual differences in capacity to metabolize the carcinogens and/or repair damaged DNA. Significant correlations were found among the biomarkers in both cell types. This noninvasive method, requiring small numbers of cells and with a relatively low cost, will be useful for monitoring DNA damage in large-scale molecular epidemiology studies.

Inward growth of colonic adenomatous polyps.

BACKGROUND & AIMS: Most colon cancers arise from polypoid adenomas, but how these benign lesions develop into malignant neoplasms is not understood. This study examined the migration of epithelial cells within human adenomatous polyps by determining the distribution of proliferating and apoptotic cells and immunoreactivity to transforming growth factor beta (TGF-beta). METHODS: Sections of surgically resected normal (n = 10) and adenomatous (n = 22) formalin-fixed tissue were examined for proliferating cells and TGF-beta isoenzymes 1-3 by immunohistochemistry and apoptotic cells by terminal deoxyuridine nick end-labeling. RESULTS: The distribution of proliferating, apoptotic, and TGF-beta immunoreactive cells was strikingly reversed in adenomatous polyps compared with normal mucosa. Proliferating cells were located in the base of normal colonic crypts and TGF-beta immunoreactive and apoptotic cells near or at the luminal surface, corresponding to the normal migration of colonocytes. In adenomas, increased numbers of proliferating cells were mainly located at the luminal surface and TGF-beta immunoreactive and apoptotic cells were located principally at the crypt base. CONCLUSIONS: This distribution suggests that cell migration in adenomas is not toward the lumen but instead inward toward the polyp base.

Immunoperoxidase detection of 8-hydroxydeoxyguanosine in aflatoxin B1-treated rat liver and human oral mucosal cells.

An immunoperoxidase method using a monoclonal antiserum that recognizes 8-hydroxydeoxyguanosine has been developed for detection and quantitation of oxidative damage in single cells. The method was initially applied to cultured cells treated with H2O2 or aflatoxin B1 and then to cryostat liver sections of rats treated with aflatoxin B1. To demonstrate that the method has sufficient sensitivity for detection of damage in human samples, oral mucosal cells from a total of 12 pairs of smokers and nonsmokers were analyzed. Mean staining intensity of oral cells of smokers was 1.6-fold higher than in nonsmokers. The immunoperoxidase method, requiring a small number of cells and eliminating the need for isolation of DNA, will be useful for evaluation of oxidative damage in a wide range of biological samples.

Determination of stereospecificity of benzo[a]pyrene diolepoxide-DNA antisera with site-specifically modified oligonucleotides.

Antisera developed against benzo[a]pyrene diolepoxide (BPDE)-DNA adducts are sensitive tools for detection of DNA adducts in human samples. All antisera currently used for biomonitoring studies were produced against DNA or guanosine modified with racemic anti-BPDE. Using a non-competitive enzyme-linked immunosorbent assay (ELISA), Venkatachalam and Wani (Carcinogenesis, 15, 565-572, 1994) recently tested polyclonal and monoclonal (5D2) antisera for cross-reactivity against oligonucleotides containing (+)-and (-)-trans-anti-BPDE-N2-guanine or N6-adenine adducts and showed different stereospecificity for the two antisera. Because of the importance of antiserum specificity in human biomonitoring studies, we have tested several monoclonal (Mab 5D11 and 5D2) and polyclonal (Pab #29) antisera developed against racemic anti-BPDE-DNA adducts, and Mab 8E11 developed against anti-BPDE-guanosine adducts. Stereoisomeric anti-BPDE-modified oligonucleotide adducts in the sequence 5'-d(CC-AT-CG*CTACC)-3' where G* = anti-BPDE-N2-dG with (+)-trans, (-)-trans, (+)-cis and (-)-cis adduct stereochemistry at the C10 position of anti-BPDE were tested by competitive ELISA. Two structurally related 5-methylchrysene diolepoxide adducts with G* = (+)- and (-)-trans-anti-5-MeCDE-N2-dG in the same oligonucleotide were also tested. While Mab5D2 had the highest affinity for the (-)-trans-anti-BPDE-modified oligomer, Mab 5D11 and 8E11 and Pab #29 recognized the (+)-trans-anti-BPDE-modified oligomer better than the (-)-trans-anti-BPDE modified oligomer. Mab 5D11 and Pab #29 recognized racemic anti-BPDE-modified DNA adducts better than trans-anti-BPDE-modified oligonucleotides; however, Mab 8E11 showed similar sensitivity to racemic anti-BPDE-DNA adducts and (+)- and (-)-trans-anti-BPDE-modified oligomers. All antisera exhibited lower reactivities with both 5-MeCDE modified oligomers. Because of their sensitive detection of (+)-trans-anti-BPDE-dG adducts, the primary adduct produced in vivo, Mab 8E11 and 5D11 and Pab #29 are appropriate for measurement of most adducts formed in humans.

Immunoperoxidase detection of polycyclic aromatic hydrocarbon-DNA adducts in oral mucosa cells of smokers and nonsmokers.

An immunoperoxidase method using a polyclonal antiserum which recognizes benzo(a)pyrene and structurally related polycyclic aromatic hydrocarbon diol epoxide-DNA adducts has been developed for the detection and quantitation of DNA damage in single cells. The method was used initially on 10T1/2 cells treated with [3H]anti-benzo(a)pyrene diol epoxide then applied to the detection of adducts in oral mucosa cells of smokers and nonsmokers. Levels of DNA damage were elevated in each of 16 smokers (mean relative staining, 503 +/- 104) compared to 16 age-, race- and sex-matched nonsmokers (251 +/- 82; P < 0.0001). There was an approximately 3-fold range in relative staining in both smokers (252 +/- 125 to 663 +/- 189) and nonsmokers (157 +/- 72 to 431 +/- 269) suggesting the importance of individual differences in capacity to metabolize the carcinogens and/or repair damaged DNA. This noninvasive method, requiring small numbers of cells, will be useful for routine monitoring of DNA damage in intervention studies as well as for biofeedback in smoking cessation programs.