The Location of Biofilms on Chronic Prosthetic Joint Infections and the Ramifications for Clinical Practice.

Revision surgery is paramount to cure chronic prosthetic joint infections because these infections are associated with biofilms on prosthetics that conventional antibiotics cannot eradicate. However, there is a paucity of research on where in vivo biofilms are located on infected prosthetics. Consequently, the objective of this pilot study was to address this gap in knowledge by staining 5 chronically infected prosthetics, that were removed at the time of revision surgery, with methylene blue. Scanning electron microscopic images were then taken of the methylene blue-stained areas to visualize biofilms. The findings show that all chronically infected prosthetics had biofilms located on the bone-prosthetic interface, yet only 2 had biofilms also located on the prosthetic interface exposed to synovial fluid. Subsequently, this pilot study provides a pathophysiological understanding of why the current treatment paradigm for chronic periprosthetic joint infection requires a revision surgery and not debridement and an implant retention surgery.

Glucose Signaling Is Connected to Chromosome Segregation Through Protein Kinase A Phosphorylation of the Dam1 Kinetochore Subunit in Saccharomycescerevisiae.

The Dam1 complex is an essential component of the outer kinetochore that mediates attachments between spindle microtubules and chromosomes. Dam1p, a subunit of the Dam1 complex, binds to microtubules and is regulated by Aurora B/Ipl1p phosphorylation. We find that overexpression of cAMP-dependent protein kinase (PKA) catalytic subunits (i.e., TPK1, TPK2, TPK3) is lethal in DAM1 mutants and increases the rate of chromosome loss in wild-type cells. Replacing an evolutionarily conserved PKA site (S31) in Dam1p with a nonphosphorylatable alanine suppressed the high-copy PKA dosage lethality in dam1-1 Consistent with Dam1p as a target of PKA, we find that in vitro PKA can directly phosphorylate S31 in Dam1p and we observed phosphorylation of S31 in Dam1p purified from asynchronously growing yeast cells. Cells carrying high-copy TPK2 or a Dam1p phospho-mimetic S31D mutant displayed a reduction in Dam1p localization at the kinetochore, suggesting that PKA phosphorylation plays a role in assembly and/or stability of the Dam1 complex. Furthermore, we observed spindle defects associated with S31 phosphorylation. Finally, we find that phosphorylation of Dam1p on S31 is reduced when glucose is limiting as well as during α-factor arrest, conditions that inhibit PKA activity. These observations suggest that the PKA site of Dam1p participates in regulating kinetochore activity. While PKA is a well-established effector of glucose signaling, our work shows for the first time that glucose-dependent PKA activity has an important function in chromosome segregation.

Immunoelectron Microscopy for Visualization of Nanoparticles.

Immunoelectron microscopy (IEM) on a solid phase such as a carbon film is a fast and powerful way to detect and visualize surface antigens on nanoparticles by using a transmission electron microscope (TEM). Nanoparticles, in particular ones for medical applications, are often modified on the surface with soft materials to make them more soluble, less toxic, or targetable to cancerous tumors. Imaging the soft material on the surface of solid nanoparticles by electron microscopy is often a challenge. IEM can overcome this issue in cases where antibodies to any of the surface material are available, which is often the case for proteins, but also for commonly used materials such as polyethylene glycol (PEG). This effective procedure has been used traditionally for viruses and macromolecules, but it can be directly applied to nanoparticles.

Biological tissue and cell culture specimen preparation for TEM nanoparticle characterization.

This chapter outlines the procedures for ex vivo TEM preparation of nanoparticle-containing tissue or cell culture samples using an epoxy resin embedding method. The purpose of this procedure is to preserve the structure of tissue in a hardened epoxy block with minimal disruption of cellular structures, to aid in the meaningful analysis of in vivo or cell culture experiments. The process begins with hydrated tissue and ends with tissue that is virtually water-free and preserved in a static state within a plastic resin matrix. The resin mixture permeates the dehydrated tissue, making the sample firm enough to cut. Procedures are also given for fixing nanoparticle-containing cell culture samples.

SEM X-ray microanalysis of nanoparticles present in tissue or cultured cell thin sections.

Energy Dispersive X-ray (EDX) microanalysis is a technique used for identification of the elemental composition of a specimen. The detection of nanoparticles in tissue is a common problem of biodistribution and toxicity studies. High-resolution transmission electron microscopy (TEM) can be employed to detect nanoparticles based on morphology; however, TEM alone cannot conclusively identify nanoparticles. Indeed, micrographs are often ambiguous due to particle aggregation, contamination, or morphology change after cellular uptake. EDX can be used to confirm the composition and distribution of the nanoparticles through spectrum and elemental mapping. This protocol outlines the procedures for compositional identification of nanoparticles using an EDX spectrometer incorporated into a scanning electron microscopy (SEM) system. This protocol outlines sample preparation, EDX spectrum acquisition, elemental peak analysis and spectral mapping acquisition.

Lack of significant dermal penetration of titanium dioxide from sunscreen formulations containing nano- and submicron-size TiO2 particles.

Titanium dioxide (TiO(2)) is included in some sunscreen formulations to physically block ultraviolet radiation. A dermal penetration study was conducted in minipigs with three TiO(2) particles (uncoated submicron sized, uncoated nano-sized, and dimethicone/methicone copolymer-coated nanosized) applied 5% by weight in a sunscreen. These and control formulations were topically applied to minipigs at 2 mg cream/cm(2) skin (4 applications/day, 5 days/week, 4 weeks). Skin (multiple sites), lymph nodes, liver, spleen, and kidneys were removed, and the TiO(2) content was determined (as titanium) using inductively coupled plasma mass spectroscopy. Titanium levels in lymph nodes and liver from treated animals were not increased over the values in control animals. The epidermis from minipigs treated with sunscreens containing TiO(2) showed elevated titanium. Increased titanium was detected in abdominal and neck dermis of minipigs treated with uncoated and coated nanoscale TiO(2). Using electron microscopy-energy dispersive x-ray analysis, all three types of TiO(2) particles were found in the stratum corneum and upper follicular lumens in all treated skin samples (more particles visible with coated nanoscale TiO(2)). Isolated titanium particles were also present at various locations in the dermis of animals treated with all three types of TiO(2)-containing sunscreens; however, there was no pattern of distribution or pathology suggesting the particles could be the result of contamination. At most, the few isolated particles represent a tiny fraction of the total amount of applied TiO(2). These findings indicate that there is no significant penetration of TiO(2) nanoparticles through the intact normal epidermis.