Bisphenol a affects neurodevelopmental gene expression, cognitive function, and neuromuscular synaptic morphology in Drosophila melanogaster

Bisphenol A (BPA) is an environmentally prevalent endocrine disrupting chemical that can impact human health and may be an environmental risk factor for neurodevelopmental disorders. BPA has been associated with behavioral impairment in children and a variety of neurodevelopmental phenotypes in model organisms. We used Drosophila melanogaster to explore the consequences of developmental BPA exposure on gene expression, cognitive function, and synapse development. Our transcriptome analysis indicated neurodevelopmentally relevant genes were predominantly downregulated by BPA. Among the misregulated genes were those with roles in learning, memory, and synapse development, as well as orthologs of human genes associated with neurodevelopmental and neuropsychiatric disorders. To examine how gene expression data corresponded to behavioral and cellular phenotypes, we first used a predator-response behavioral paradigm and found that BPA disrupts visual perception. Further analysis using conditioned courtship suppression showed that BPA impairs associative learning. Finally, we examined synapse morphology within the larval neuromuscular junction and found that BPA significantly increased the number of axonal branches. Given that our findings align with studies of BPA in mammalian model organisms, this data indicates that BPA impairs neurodevelopmental pathways that are functionally conserved from invertebrates to mammals. Further, because Drosophila do not possess classic estrogen receptors or estrogen, this research suggests that BPA can impact neurodevelopment by molecular mechanisms distinct from its role as an estrogen mimic

Hypervirulent Clostridium difficile PCR-Ribotypes Exhibit Resistance to Widely Used Disinfectants

The increased prevalence of Clostridium difficile infection (CDI) has coincided with enhanced transmissibility and severity of disease, which is often linked to two distinct clonal lineages designated PCR-ribotype 027 and 017 responsible for CDI outbreaks in the USA, Europe and Asia. We assessed sporulation and susceptibility of three PCR-ribotypes; 012, 017 and 027 to four classes of disinfectants; chlorine releasing agents (CRAs), peroxygens, quaternary ammonium compounds (QAC) and biguanides. The 017 PCR-ribotype, showed the highest sporulation frequency under these test conditions. The oxidizing biocides and CRAs were the most efficacious in decontamination of C. difficile vegetative cells and spores, the efficacy of the CRAs were concentration dependent irrespective of PCR-ribotype. However, there were differences observed in the susceptibility of the PCR-ribotypes, independent of the concentrations tested for Virkon®, Newgenn®, Proceine 40® and Hibiscrub®. Whereas, for Steri7® and Biocleanse® the difference observed between the disinfectants were dependent on both PCR-ribotype and concentration. The oxidizing agent Perasafe® was consistently efficacious across all three PCR ribotypes at varying concentrations; with a consistent five Log10 reduction in spore titre. The PCR-ribotype and concentration dependent differences in the efficacy of the disinfectants in this study indicate that disinfectant choice is a factor for llimiting the survival and transmission of C. difficile spores in healthcare settings

Analysis of Imprinted Gene Expression in Normal Fertilized and Uniparental Preimplantation Porcine Embryos

In the present study quantitative real-time PCR was used to determine the expression status of eight imprinted genes (GRB10, H19, IGF2R, XIST, IGF2, NNAT, PEG1 and PEG10) during preimplantation development, in normal fertilized and uniparental porcine embryos. The results demonstrated that, in all observed embryo samples, a non imprinted gene expression pattern up to the 16-cell stage of development was common for most genes. This was true for all classes of embryo, regardless of parental-origins and the direction of imprint. However, several differentially expressed genes (H19, IGF2, XIST and PEG10) were detected amongst the classes at the blastocyst stage of development. Most interestingly and despite the fact that maternally and paternally expressed genes should not be expressed in androgenones and parthenogenones, respectively, both uniparental embryos expressed these genes when tested for in this study. In order to account for this phenomenon, we compared the expression patterns of eight imprinted genes along with the methylation status of the IGF2/H19 DMR3 in haploid and diploid parthenogenetic embryos. Our findings revealed that IGF2, NNAT and PEG10 were silenced in haploid but not diploid parthenogenetic blastocysts and differential methylation of the IGF2/H19 DMR3 was consistently observed between haploid and diploid parthenogenetic blastocysts. These results appear to suggest that there exists a process to adjust the expression status of imprinted genes in diploid parthenogenetic embryos and that this phenomenon may be associated with altered methylation at an imprinting control region. In addition we believe that imprinted expression occurs in at least four genes, namely H19, IGF2, XIST and PEG10 in porcine blastocyst stage embryos

Developmental potentials of bi and uniparental porcine embryos^*.

<p>*The number of replicates was 5.</p><p>†Those zygotes having two pronuclei (IVF and AG zygotes) or one large pronucleus or two pronuclei (PG zygotes) were selected after staining with Hoechst 33342.</p><p>‡The cells of blastocysts were counted on Day 7.</p>a–c<p>Values with different letters within each column are significantly different, <i>p</i><0.05.</p

Imprinted gene expression of haploid and diploid PG blastocysts.

<p>Haploid PG blastocysts were generated using the electrical activation method without cytochalasin D treatment. Zygotes possessing two polar bodies and a small pronucleus (presumed haploid) or with a polar body and a large pronucleus or two pronuclei (presumed diploid) were selected by Hoechst staining at 12 to 14 hr following parthenogenetic activation, respectively. Results for each sample were conducted in triplicate. Y-value is expressed as a relative fold change in mRNA levels in haploid PG blastocysts (n = 5) compared with that of the diploid ones (n = 5) defined as 1. The Data are presented as means ± SEM.</p

The methylation status of <i>IGF2/H19</i> DMR3 in porcine haploid and diploid PG blastocysts.

<p>The methylation patterns of DMR3 in porcine A; adult liver tissue (1×10⁵ cells), B; MII oocytes (n = 100), C; sperm (1×10⁶ sperm cells), D; IVF (n = 50), E; haploid PG (n = 100), and F; diploid PG (n = 50) blastocysts are shown. Individual circles indicate a CpG dinucleotide. Open and solid circles represent unmethylated and methylated CpGs, respectively. Each horizontal line represents one individual clone from three independently amplified PCR products.</p

Differential expression of XIST transcripts in individual in vivo blastocysts.

<p>Each value derived from transcripts of the <i>XIST</i> gene in <i>in vivo</i> blastocysts, after normalization relative to <i>β ACTIN</i> (internal control), were compared with that of one of 10 <i>in vivo</i> blastocysts defined as 1. Of these, the labeled No. 3 and No. 4 samples were determined their sex by SRY gene; ^F and ^M indicate female and male embryos, respectively.</p

Analysis of imprinted gene expression in in vivo derived and in vitro fertilized blastocysts.

<p>Y-value is expressed as a relative fold change in mRNA levels in <i>in vitro</i> blastocysts compared with that of the <i>in vivo</i> ones defined as 1, (n = 10). This data is presented as mean ± SEM (n = 5).</p

Relative expression levels of the eight imprinted genes in porcine MII oocytes and in diploid normal and uniparental embryos from the two-cell to the blastocyst stage.

<p>The relative levels of mRNA were quantified using qRT-PCR and then calculated with the 2^{−ΔΔ Ct} method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022216#pone.0022216-Livak1" target="_blank">[23]</a>. Five replicate samples were examined for each class. The values from transcripts of the imprinted genes in PG and AG blastocysts, after normalization relative to the <i>β ACTIN</i> (internal control) gene, were compared to those of IVF counterparts which were taken as a standard (1). This data is presented as mean ± SEM. The relative abundance of eight imprinted genes among the different types of embryos at each stage are shown; A; the 2-cell (n = 25), B; the 4-cell (n = 25), C; the 8∼16-cell (n = 15), D; the morula (n = 10), and E; the blastocyst stage (n = 5) of porcine embryos.</p