RESUMO
This in vitro study evaluated bacterial cell proliferation and biofilm adhesion on titanium discs with and without antibacterial surface treatment to reduce the chances of peri-implant infections. Hexagonal boron nitride with 99.5% purity was converted to hexagonal boron nitride nanosheets via the liquid phase exfoliation process. The spin coating method was used for uniform coating of h-BNNSs over titanium alloy (Ti6Al4V) discs. Two groups of titanium discs were formed: Group I (n = 10) BN-coated titanium discs and Group II (n = 10) uncoated titanium discs. Two bacterial strains, Streptococcus mutans (initial colonizers) and Fusobacterium nucleatum (secondary colonizers), were used. A zone of inhibition test, microbial colony forming units assay, and crystal violet staining assay were used to evaluate bacterial cell viability. Surface characteristics and antimicrobial efficacy were examined by scanning electron microscopy with energy dispersion X-ray spectroscopy. SPSS (Statistical Package for Social Sciences) version 21.0 was used to analyze the results. The data were analyzed for probability distribution using the Kolmogorov-Smirnov test, and a non-parametric test of significance was applied. An inter-group comparison was done using the Mann-Whitney U test. A statistically significant increase was observed in the bactericidal action of BN-coated discs compared to uncoated discs against S. mutans, but no statistically significant difference was found against F. nucleatum.
RESUMO
BACKGROUND: Gene regulation in eukaryotes is a complex process entailing the establishment of transcriptionally silent chromatin domains interspersed with regions of active transcription. Imprinted domains consist of clusters of genes, some of which exhibit parent-of-origin dependent monoallelic expression, while others are biallelic. The Kcnq1 imprinted domain illustrates the complexities of long-range regulation that coexists with local exceptions. A paternally expressed repressive non-coding RNA, Kcnq1ot1, regulates a domain of up to 750 kb, encompassing 14 genes. We study how the Kcnq1 gene, initially silenced by Kcnq1ot1, undergoes tissue-specific escape from imprinting during development. Specifically, we uncover the role of chromosome conformation during these events. RESULTS: We show that Kcnq1 transitions from monoallelic to biallelic expression during mid gestation in the developing heart. This transition is not associated with the loss of methylation on the Kcnq1 promoter. However, by exploiting chromosome conformation capture (3C) technology, we find tissue-specific and stage-specific chromatin loops between the Kcnq1 promoter and newly identified DNA regulatory elements. These regulatory elements showed in vitro activity in a luciferase assay and in vivo activity in transgenic embryos. CONCLUSIONS: By exploring the spatial organization of the Kcnq1 locus, our results reveal a novel mechanism by which local activation of genes can override the regional silencing effects of non-coding RNAs.
RESUMO
Insight into how the mammalian genome is structured in vivo is key to understanding transcriptional regulation. This is especially true in complex domains in which genes are coordinately regulated by long-range interactions between cis-regulatory elements. The regulation of the H19/Igf2 imprinted region depends on the presence of several cis-acting sequences, including a methylation-sensitive insulator between Igf2 and H19 and shared enhancers downstream of H19. Each parental allele has a distinct expression pattern. We used chromosome conformation capture to assay the native three-dimensional organization of the H19/Igf2 locus on each parental copy. Furthermore, we compared wild-type chromosomes to several mutations that affect the insulator. Our results show that promoters and enhancers reproducibly co-localize at transcriptionally active genes, i.e. the endodermal enhancers contact the maternal H19 and the paternal Igf2 genes. The active insulator blocks traffic of the enhancers along the chromosome, restricting them to the H19 promoter. Conversely, the methylated inactive insulator allows the enhancers to contact the upstream regions, including Igf2. Mutations that either remove or inhibit insulator activity allow unrestricted access of the enhancers to the whole region. A mutation that allows establishment of an enhancer-blocker on the normally inactive paternal copy diminishes the contact of the enhancer with the Igf2 gene. Based on our results, we propose that physical proximity of cis-acting DNA elements is vital for their activity in vivo. We suggest that enhancers track along the chromosome until they find a suitable promoter sequence to interact with and that insulator elements block further tracking of enhancers.
