Evaluation of Closed-system Transfer Devices in Reducing Potential Risk for Surface Contamination Following Simulated Hazardous-drug Preparation and Compounding.

Closed-system transfer devices mitigate occupational exposure risks associated with hazardous-drug handling. This study was conducted in a controlled laboratory to evaluate the effectiveness of a needle-free and a needle-based closed-system transfer device in minimizing surface contamination during simulated compounding, preparation, and administration. A needle-based and a needle-free closed-system transfer device underwent three trials per system. Each trial included reconstituting cyclophosphamide in a vial, withdrawing cyclophosphamide from the vial, and pushing cyclophosphamide into an intravenous bag. After every trial, wipe samples were collected from five sources: biological safety cabinet workbench (left and right sides), biological safety cabinet grill, biological safety cabinet airfoil, and technicians' gloves. Wipe samples were then analyzed using high-performance liquid chromatography with dual-mass spectrometry to measure cyclophosphamide concentrations. Surface contamination levels from 30 post-trial tests (15 per device) are reported, representing five different surface wipe samples from three trials for each device. Pre-trial samples of precleaned vials and work surfaces were obtained to ensure no cyclophosphamide contamination. Field blank samples were analyzed for quality-control purposes. Post-trial wipe sample analyses following each of the three needle- free trials did not detect cyclophosphamide on the biological safety cabinet workbench (both left/right), biological safety cabinet grill, biological safety cabinet airfoil, or the technician's gloves. For the needle-based closed-system transfer device, the wipe sample analyses after the first trial showed no contamination; however, cyclophosphamide was detected on the right biological safety cabinet workbench at concentrations of 0.223 ng/cm2 and 0.021 ng/cm2, respectively, following the second and third trials. No cyclophosphamide was found on the technician's gloves after any of the three needle- based closed-system transfer device trials. Based on surface contamination analyses, this study verified the ability of a needle-free closed-system transfer device in preventing the escape of cyclophosphamide during simulated compounding and preparation. Needle-free closed-system transfer devices warrant consideration for the handling of hazardous drugs.

Evaluation of Closed-system Transfer Devices in Reducing Potential Risk for Surface Contamination Following Simulated Hazardous-drug Preparation and Compounding.

Closed-system transfer devices mitigate occupational exposure risks associated with hazardous-drug handling. This study was conducted in a controlled laboratory to evaluate the effectiveness of a needle-free and a needle-based closed-system transfer device in minimizing surface contamination during simulated compounding, preparation, and administration. A needle-based and a needle-free closed-system transfer device underwent three trials per system. Each trial included reconstituting cyclophosphamide in a vial, withdrawing cyclophosphamide from the vial, and pushing cyclophosphamide into an intravenous bag. After every trial, wipe samples were collected from five sources: biological safety cabinet workbench (left and right sides), biological safety cabinet grill, biological safety cabinet airfoil, and technicians' gloves. Wipe samples were then analyzed using high-performance liquid chromatography with dual-mass spectrometry to measure cyclophosphamide concentrations. Surface contamination levels from 30 post-trial tests (15 per device) are reported, representing five different surface wipe samples from three trials for each device. Pre-trial samples of precleaned vials and work surfaces were obtained to ensure no cyclophosphamide contamination. Field blank samples were analyzed for quality-control purposes. Post-trial wipe sample analyses following each of the three needle- free trials did not detect cyclophosphamide on the biological safety cabinet workbench (both left/right), biological safety cabinet grill, biological safety cabinet airfoil, or the technician's gloves. For the needle-based closed-system transfer device, the wipe sample analyses after the first trial showed no contamination; however, cyclophosphamide was detected on the right biological safety cabinet workbench at concentrations of 0.223 ng/cm2 and 0.021 ng/cm2, respectively, following the second and third trials. No cyclophosphamide was found on the technician's gloves after any of the three needle- based closed-system transfer device trials. Based on surface contamination analyses, this study verified the ability of a needle-free closed-system transfer device in preventing the escape of cyclophosphamide during simulated compounding and preparation. Needle-free closed-system transfer devices warrant consideration for the handling of hazardous drugs.

C-Terminal Tail Polyglycylation and Polyglutamylation Alter Microtubule Mechanical Properties.

Microtubules are biopolymers that perform diverse cellular functions. Microtubule behavior regulation occurs in part through post-translational modification of both the α- and ß-subunits of tubulin. One class of modifications is the heterogeneous addition of glycine and/or glutamate residues to the disordered C-terminal tails (CTTs) of tubulin. Because of their prevalence in stable, high-stress cellular structures such as cilia, we sought to determine if these modifications alter microtubules' intrinsic stiffness. Here, we describe the purification and characterization of differentially modified pools of tubulin from Tetrahymena thermophila. We found that post-translational modifications do affect microtubule stiffness but do not affect the number of protofilaments incorporated into microtubules. We measured the spin dynamics of nuclei in the CTT backbone by NMR spectroscopy to explore the mechanism of this change. Our results show that the α-tubulin CTT does not protrude out from the microtubule surface, as is commonly depicted in models, but instead interacts with the dimer's surface. This suggests that the interactions of the α-tubulin CTT with the tubulin body contributes to the stiffness of the assembled microtubule, thus providing insight into the mechanism by which polyglycylation and polyglutamylation can alter microtubule mechanical properties.

In-Cell NMR within Budding Yeast Reveals Cytoplasmic Masking of Hydrophobic Residues of FG Repeats.

In-cell NMR spectroscopy is a powerful tool to determine the properties of proteins and nucleic acids within living cells. In-cell NMR can give site-specific measurements of interactions, modifications, and dynamics as well as their modulation by the cellular environment. In-cell NMR requires selective incorporation of heavy isotopes into a protein of interest, either through the introduction of exogenously produced protein to a cell's interior or the selective overexpression of a protein. We developed conditions to allow the use of Saccharomyces cerevisiae, which was chosen because of its genetic tractability, as a eukaryotic expression system for in-cell NMR. We demonstrate this technique using a fragment of S. cerevisiae Nsp1, an FG Nup. FG Nups are intrinsically disordered proteins containing phenylalanine (F)-glycine (G) repeats and form the selective barrier within the nuclear pore complex. Yeast FG Nups have previously been shown to be maintained in a highly dynamic state within living bacteria as measured by in-cell NMR. Interactions thought to stabilize this dynamic state are also present in the protein's native organism, although site specificity of interaction is different between the two cytosols.

Molecular Determinants of Tubulin's C-Terminal Tail Conformational Ensemble.

Tubulin is important for a wide variety of cellular processes including cell division, ciliogenesis, and intracellular trafficking. To perform these diverse functions, tubulin is regulated by post-translational modifications (PTM), primarily at the C-terminal tails of both the α- and ß-tubulin heterodimer subunits. The tubulin C-terminal tails are disordered segments that are predicted to extend from the ordered tubulin body and may regulate both intrinsic properties of microtubules and the binding of microtubule associated proteins (MAP). It is not understood how either interactions with the ordered tubulin body or PTM affect tubulin's C-terminal tails. To probe these questions, we developed a method to isotopically label tubulin for C-terminal tail structural studies by NMR. The conformational changes of the tubulin tails as a result of both proximity to the ordered tubulin body and modification by mono- and polyglycine PTM were determined. The C-terminal tails of the tubulin dimer are fully disordered and, in contrast with prior simulation predictions, exhibit a propensity for ß-sheet conformations. The C-terminal tails display significant chemical shift differences as compared to isolated peptides of the same sequence, indicating that the tubulin C-terminal tails interact with the ordered tubulin body. Although mono- and polyglycylation affect the chemical shift of adjacent residues, the conformation of the C-terminal tail appears insensitive to the length of polyglycine chains. Our studies provide important insights into how the essential disordered domains of tubulin function.

Fluorescence quantum yield measurements of fluorescent proteins: a laboratory experiment for a biochemistry or molecular biophysics laboratory course.

Fluorescent proteins are commonly used in cell biology to assess where proteins are within a cell as a function of time and provide insight into intracellular protein function. However, the usefulness of a fluorescent protein depends directly on the quantum yield. The quantum yield relates the efficiency at which a fluorescent molecule converts absorbed photons into emitted photons and it is necessary to know for assessing what fluorescent protein is the most appropriate for a particular application. In this work, we have designed an upper-level, biochemistry laboratory experiment where students measure the fluorescence quantum yields of fluorescent proteins relative to a standard organic dye. Four fluorescent protein variants, enhanced cyan fluorescent protein (ECFP), enhanced green fluorescent protein (EGFP), mCitrine, and mCherry, were used, however the methods described are useful for the characterization of any fluorescent protein or could be expanded to fluorescent quantum yield measurements of organic dye molecules. The laboratory is designed as a guided inquiry project and takes two, 4 hr laboratory periods. During the first day students design the experiment by selecting the excitation wavelength, choosing the standard, and determining the concentration needed for the quantum yield experiment that takes place in the second laboratory period. Overall, this laboratory provides students with a guided inquiry learning experience and introduces concepts of fluorescence biophysics into a biochemistry laboratory curriculum.