Mobilization of genomic islands of Staphylococcus aureus by temperate bacteriophage

The virulence of Staphylococcus aureus, in both human and animal hosts, is largely influenced by the acquisition of mobile genetic elements (MGEs). Most S. aureus strains carry a variety of MGEs, including three genomic islands (νSaα, νSaβ, νSaγ) that are diverse in virulence gene content but conserved within strain lineages. Although the mobilization of pathogenicity islands, phages and plasmids has been well studied, the mobilization of genomic islands is poorly understood. We previously demonstrated the mobilization of νSaβ by the adjacent temperate bacteriophage ϕSaBov from strain RF122. In this study, we demonstrate that ϕSaBov mediates the mobilization of νSaα and νSaγ, which are located remotely from ϕSaBov, mostly to recipient strains belonging to ST151. Phage DNA sequence analysis revealed that chromosomal DNA excision events from RF122 were highly specific to MGEs, suggesting sequence-specific DNA excision and packaging events rather than generalized transduction by a temperate phage. Disruption of the int gene in ϕSaBov did not affect phage DNA excision, packaging, and integration events. However, disruption of the terL gene completely abolished phage DNA packing events, suggesting that the primary function of temperate phage in the transfer of genomic islands is to allow for phage DNA packaging by TerL and that transducing phage particles are the actual vehicle for transfer. These results extend our understanding of the important role of bacteriophage in the horizontal transfer and evolution of genomic islands in S. aureus

The Influence of Spin-Labeled Fluorene Compounds on the Assembly and Toxicity of the Aβ Peptide

The deposition and oligomerization of amyloid β (Aβ) peptide plays a key role in the pathogenesis of Alzheimer's disease (AD). Aβ peptide arises from cleavage of the membrane-associated domain of the amyloid precursor protein (APP) by β and γ secretases. Several lines of evidence point to the soluble Aβ oligomer (AβO) as the primary neurotoxic species in the etiology of AD. Recently, we have demonstrated that a class of fluorene molecules specifically disrupts the AβO species. Methodology/Principal Findings To achieve a better understanding of the mechanism of action of this disruptive ability, we extend the application of electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels in the Aβ peptide to investigate the binding and influence of fluorene compounds on AβO structure and dynamics. In addition, we have synthesized a spin-labeled fluorene (SLF) containing a pyrroline nitroxide group that provides both increased cell protection against AβO toxicity and a route to directly observe the binding of the fluorene to the AβO assembly. We also evaluate the ability of fluorenes to target multiple pathological processes involved in the neurodegenerative cascade, such as their ability to block AβO toxicity, scavenge free radicals and diminish the formation of intracellular AβO species. Conclusions Fluorene modified with pyrroline nitroxide may be especially useful in counteracting Aβ peptide toxicity, because they posses both antioxidant properties and the ability to disrupt AβO species

Differentiation of Embryonic Stem Cells into Cardiomyocytes in a Microfluidic System

The differentiation process of murine embryonic stem cells into cardiomyocytes was investigated with a compliant microfluidic platform which allows for versatile cell seeding arrangements, optical observation access, long-term cell viability, and programmable uniaxial cyclic stretch. Specifically, two environmental cues were examined with this platform—culture dimensions and uniaxial cyclic stretch. First, the cardiomyogenic differentiation process, assessed by a GFP reporter driven by the α-MHC promoter, was enhanced in microfluidic devices (µFDs) compared with conventional well-plates. The addition of BMP-2 neutralizing antibody reduced the enhancement observed in the µFDs and the addition of exogenous BMP-2 augmented the cardiomyogenic differentiation in well plates. Second, 24 h of uniaxial cyclic stretch at 1 Hz and 10% strain on day 9 of differentiation was found to have a negative impact on cardiomyogenic differentiation. This microfluidic platform builds upon an existing design and extends its capability to test cellular responses to mechanical strain. It provides capabilities not found in other systems for studying differentiation, such as seeding embryoid bodies in 2D or 3D in combination with cyclic strain. This study demonstrates that the microfluidic system contributes to enhanced cardiomyogenic differentiation and may be a superior platform compared with conventional well plates. In addition to studying the effect of cyclic stretch on cardiomyogenic differentiation, this compliant platform can also be applied to investigate other biological mechanisms.Singapore-MIT Alliance for Research and TechnologyAmerican Heart AssociationNational Science Foundation (U.S.) (Science and Technology Center (EBICS): Emergent Behaviors of Integrated Cellular Systems, Grant CBET-0939511)International Research & Development Program (Grant number 2009-00631

Evidence of effectiveness: How much can we extrapolate from existing studies?

Drug development can be a science of extrapolation if the use of a drug exposure-response relationship is embraced and implemented through mechanistically oriented pharmacokinetic (PK)-pharmacodynamic (PD) modeling analysis and clinical trial simulation. The traditional requirement of at least 2 adequate and well-controlled phase III studies by the US Food and Drug Administration for drug approval can be waived in certain situations, substantially reducing the resources and time. In this article, the authors introduce a real drug development case where the chance for this exemption was maximized by actively using PK-PD modeling followed by clinical trial simulation, resulting in faster and more economical introduction of a new dosage regimen to patients