Nanotherapeutics using all-natural materials. Effective treatment of wound biofilm infections using crosslinked nanoemulsions.

Bacterial wound infections are a threat to public health. Although antibiotics currently provide front-line treatments for bacterial infections, the development of drug resistance coupled with the defenses provided through biofilm formation render these infections difficult, if not impossible, to cure. Antimicrobials from natural resources provide unique antimicrobial mechanisms and are generally recognized as safe and sustainable. Herein, an all-natural antimicrobial platform is reported. It is active against bacterial biofilms and accelerates healing of wound biofilm infections in vivo. This antimicrobial platform uses gelatin stabilized by photocrosslinking using riboflavin (vitamin B2) as a photocatalyst, and carvacrol (the primary constituent of oregano oil) as the active antimicrobial. The engineered nanoemulsions demonstrate broad-spectrum antimicrobial activity towards drug-resistant bacterial biofilms and significantly expedite wound healing in an in vivo murine wound biofilm model. The antimicrobial activity, wound healing promotion, and biosafety of these nanoemulsions provide a readily translatable and sustainable strategy for managing wound infections.

YY1 Is Required for Posttranscriptional Stability of SOX2 and OCT4 Proteins.

Yinyang1 (YY1) participates in protein-DNA, protein-RNA, and protein-protein interactions and regulates developmental processes and disease mechanisms. YY1 interactions regulate a range of important biological functions, including oocyte maturation, epithelial to mesenchymal transition, and vascular endothelial growth factor (VEGF) signaling. We tested the hypothesis that YY1 is required for inner cell mass (ICM) lineage commitment during preimplantation development. In this study, we document gene expression patterns and protein localization of key transcription factors in Yy1 global, tissue-specific, and dsRNA-mediated knockout/down embryos. YY1 protein was found in cells of preimplantation and peri-implantation embryos, and adult tissues where two isoforms are observed. In the absence of YY1, OCT4 and SOX2 protein were lost in the ICM during preimplantation and naive neuroectoderm during gastrulation stages, yet no difference in Oct4 or Sox2 mRNA levels was observed. The loss of OCT4 and SOX2 protein occurred specifically in cells that normally express both OCT4 and SOX2 protein. These observations support a role for YY1 meditating and/or regulating the interaction of OCT4 and SOX2 at a posttranscriptional level. Our results suggest that distinct mechanisms of YY1-mediated molecular regulation are present in the early embryo, and may offer insight to promote lineage commitment in in vitro cell lines.

cis-Regulatory control of Mesp1 expression by YY1 and SP1 during mouse embryogenesis.

BACKGROUND: Mesp1 is critical for early cardiomyocyte differentiation and heart development. We previously observed down-regulation of Mesp1 expression in YY1-ablated mouse embryonic hearts. However, how Mesp1 expression is mediated by YY1 is not well understood. RESULTS: We excised YY1 in the murine embryos using Sox2-cre and found that Mesp1 was down-regulated in the embryonic day (E) 7.5 mutant embryos. Also, YY1 activated the 6 kb Mesp1 regulatory element fused to a luciferase reporter. We identified two putative YY1 binding sites in the proximal promoter region of Mesp1 gene, and found that mutation of these sites significantly reduced YY1-induced activation of the Mesp1 promoter. We also uncovered one cognitive site for SP1, one of the earliest binding partners of YY1 identified. Mutation of this SP1 site repressed SP1-induced activation of the Mesp1 promoter. Moreover, YY1 and SP1 synergistically activated the Mesp1 promoter. Consistently, while Lacz expression driven by the wild-type 6 kb regulatory element of Mesp1 gene was robust in E7.5 mouse embryos, the mutation of these binding sites in the context of this 6 kb sequence substantially reduced the LacZ expression during embryogenesis. CONCLUSIONS: YY1 and SP1 independently and cooperatively govern the Mesp1 expression during embryogenesis.

Visceral endoderm expression of Yin-Yang1 (YY1) is required for VEGFA maintenance and yolk sac development.

Mouse embryos lacking the polycomb group gene member Yin-Yang1 (YY1) die during the peri-implantation stage. To assess the post-gastrulation role of YY1, a conditional knock-out (cKO) strategy was used to delete YY1 from the visceral endoderm of the yolk sac and the definitive endoderm of the embryo. cKO embryos display profound yolk sac defects at 9.5 days post coitum (dpc), including disrupted angiogenesis in mesoderm derivatives and altered epithelial characteristics in the visceral endoderm. Significant changes in both cell death and proliferation were confined to the YY1-expressing yolk sac mesoderm indicating that loss of YY1 in the visceral endoderm causes defects in the adjacent yolk sac mesoderm. Production of Vascular Endothelial Growth Factor A (VEGFA) by the visceral endoderm is essential for normal growth and development of the yolk sac vasculature. Reduced levels of VEGFA are observed in the cKO yolk sac, suggesting a cause for the angiogenesis defects. Ex vivo culture with exogenous VEGF not only rescued angiogenesis and apoptosis in the cKO yolk sac mesoderm, but also restored the epithelial defects observed in the cKO visceral endoderm. Intriguingly, blocking the activity of the mesoderm-localized VEGF receptor, FLK1, recapitulates both the mesoderm and visceral endoderm defects observed in the cKO yolk sac. Taken together, these results demonstrate that YY1 is responsible for maintaining VEGF in the developing visceral endoderm and that a VEGF-responsive paracrine signal, originating in the yolk sac mesoderm, is required to promote normal visceral endoderm development.

Yin-Yang1 is required for epithelial-to-mesenchymal transition and regulation of Nodal signaling during mammalian gastrulation.

The ubiquitously expressed Polycomb Group protein Yin-Yang1 (YY1) is believed to regulate gene expression through direct binding to DNA elements found in promoters or enhancers of target loci. Additionally, YY1 contains diverse domains that enable a plethora of protein-protein interactions, including association with the Oct4/Sox2 pluripotency complex and Polycomb Group silencing complexes. To elucidate the in vivo role of YY1 during gastrulation, we generated embryos with an epiblast specific deletion of Yy1. Yy1 conditional knockout (cKO) embryos initiate gastrulation, but both primitive streak formation and ingression through the streak is severely impaired. These streak descendants fail to repress E-Cadherin and are unable to undergo an appropriate epithelial to mesenchymal transition (EMT). Intriguingly, overexpression of Nodal and concomitant reduction of Lefty2 are observed in Yy1 cKO embryos, suggesting that YY1 is normally required for proper Nodal regulation during gastrulation. Furthermore, definitive endoderm is specified but fails to properly integrate into the outer layer. Although anterior neuroectoderm is specified, mesoderm production is severely restricted. We show that YY1 directly binds to the Lefty2 locus in E7.5 embryos and that pharmacological inhibition of Nodal signaling partially restores mesoderm production in Yy1 cKO mutant embryos. Our results reveal critical requirements for YY1 during several important developmental processes, including EMT and regulation of Nodal signaling. These results are the first to elucidate the diverse role of YY1 during gastrulation in vivo.

Yin-yang1 is required in the mammalian oocyte for follicle expansion.

The multifaceted polycomb group gene Yin-Yang1 (Yy1) has been implicated in a variety of transcriptional regulatory roles both as an activator and silencer of gene expression. Here we examine the role of Yy1 during oocyte growth by conditional deletion of the locus in the growing oocyte. Our results indicate that YY1 is required for oocyte maturation and granulosa cell expansion. In mutant oocytes, we observe severely reduced expression of both Gdf9 and Bmp15, suggesting a mechanism underlying the failure of granulosa cell expansion. Consequently, we observe infertility, failure of estrus cycling, and altered reproductive hormone levels in mutant females. Additionally, we find that YY1-deficient oocytes exhibit altered levels of several oocyte-specific factors, including Pou5f1, Figla, Lhx8, Oosp1, and Sohlh2. These results document YY1's involvement in folliculogenesis and ovarian function in the mouse and indicate that YY1 is required specifically in the oocyte for oocyte-granulosa cell communication.