3D-Printing for Analytical Ultracentrifugation

Analytical ultracentrifugation (AUC) is a classical technique of physical biochemistry providing information on size, shape, and interactions of macromolecules from the analysis of their migration in centrifugal fields while free in solution. A key mechanical element in AUC is the centerpiece, a component of the sample cell assembly that is mounted between the optical windows to allow imaging and to seal the sample solution column against high vacuum while exposed to gravitational forces in excess of 300,000 g. For sedimentation velocity it needs to be precisely sector-shaped to allow unimpeded radial macromolecular migration. During the history of AUC a great variety of centerpiece designs have been developed for different types of experiments. Here, we report that centerpieces can now be readily fabricated by 3D printing at low cost, from a variety of materials, and with customized designs. The new centerpieces can exhibit sufficient mechanical stability to withstand the gravitational forces at the highest rotor speeds and be sufficiently precise for sedimentation equilibrium and sedimentation velocity experiments. Sedimentation velocity experiments with bovine serum albumin as a reference molecule in 3D printed centerpieces with standard double-sector design result in sedimentation boundaries virtually indistinguishable from those in commercial double-sector epoxy centerpieces, with sedimentation coefficients well within the range of published values. The statistical error of the measurement is slightly above that obtained with commercial epoxy, but still below 1%. Facilitated by modern open-source design and fabrication paradigms, we believe 3D printed centerpieces and AUC accessories can spawn a variety of improvements in AUC experimental design, efficiency and resource allocation.Comment: 25 pages, 6 figure

Tumor-associated endothelial cells display GSTP1 and RARβ2 promoter methylation in human prostate cancer

BACKGROUND: A functional blood supply is essential for tumor growth and proliferation. However, the mechanism of blood vessel recruitment to the tumor is still poorly understood. Ideally, a thorough molecular assessment of blood vessel cells would be critical in our comprehension of this process. Yet, to date, there is little known about the molecular makeup of the endothelial cells of tumor-associated blood vessels, due in part to the difficulty of isolating a pure population of endothelial cells from the heterogeneous tissue environment. METHODS: Here we describe the use of a recently developed technique, Expression Microdissection, to isolate endothelial cells from the tumor microenvironment. The methylation status of the dissected samples was evaluated for GSTP1 and RARβ2 promoters via the QMS-PCR method. RESULTS: Comparing GSTP1 and RARβ2 promoter methylation data, we show that 100% and 88% methylation is detected, respectively, in the tumor areas, both in epithelium and endothelium. Little to no methylation is observed in non-tumor tissue areas. CONCLUSION: We applied an accurate microdissection technique to isolate endothelial cells from tissues, enabling DNA analysis such as promoter methylation status. The observations suggest that epigenetic alterations may play a role in determining the phenotype of tumor-associated vasculature

Sedimentation coefficient distributions from a rectangular cell.

Sedimentation coefficient distributions calculated from the data in Fig 4 for rectangular (magenta) and sectorial (blue) geometry.</p

Picture of a 12 mm pathlength centerpiece printed of ABS-like resin MicroFine Green.

Sedimentation velocity data of BSA at 50,000 rpm collected with this centerpiece installed into a standard cell assembly without gaskets are shown in Fig 3B.</p

Radial concentration distribution in a sedimentation equilibrium experiment with enhanced green fluorescent protein in a “prime gray” photopolymer centerpiece.

<p>Data were acquired with the absorbance detection sequentially at rotor speeds of 15,000 rpm (purple), 24,000 rpm (blue), and 35,000 rpm (cyan) (symbols, only every 5^th data point shown). A global model (lines) results in an apparent molar mass of 29.7 kDa with a root-mean-square deviation (rmsd) of 0.0032 OD₄₈₉, and residuals as shown in the lower plot.</p

Sedimentation velocity experiment in rectangular cell.

<p>Sedimentation velocity analysis of bovine serum albumin sedimenting at 50,000 rpm in acrylic centerpieces with a sector-shaped (A) and rectangular shaped (B) solution column. The protein sample was identical in both. The upper panel shows the sedimentation boundaries (points, for clarity, only every 2^nd data point of every 2^nd scan is shown), along with the best-fit <i>c</i>(<i>s</i>) profiles (solid lines). Below are the residuals of the fit as bitmap and overlay plot. The <i>c</i>(<i>s</i>) distribution for both data sets are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155201#pone.0155201.g005" target="_blank">Fig 5</a>.</p

Temporal evolution of radial concentration profiles in a sedimentation velocity experiment with bovine serum albumin in a “prime gray” photopolymer centerpiece.

<p>Panel A: Absorbance data acquired at a rotor speeds of 50,000 rpm at a series of time points (symbols, only every 3^rd data point of every 2^nd scan shown, with color temperature indicating progress of time). The <i>c</i>(<i>s</i>) fit (lines) results in an rmsd of 0.0065 OD₂₈₀, with the residuals shown in the small plots as residuals bitmap and superposition. Panel B: The corresponding <i>c</i>(<i>s</i>) distribution (magenta), and for comparison the <i>c</i>(<i>s</i>) distribution from a control in the same run using a standard Epon centerpiece (black); microgreen (green); Xtreme white (blue dashed); in-house clear (cyan dotted).</p

Fluorescence optical data in a 3D printed carbonate centerpiece.

<p>A centerpiece featuring a 3 mm deep sector-shaped well at the top was used, with filling and venting holes, and an embossed seal. The focal depth of the fluorescence optics was 2.0 mm. (A) Shown are sedimentation profiles acquired with 561 nm excitation for 46 nM mCherry [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155201#pone.0155201.ref057" target="_blank">57</a>] dissolved in phosphate buffered saline (dots), and best-fit c(s) sedimentation coefficient distribution with adjustments for characteristic signals of fluorescence detection [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155201#pone.0155201.ref056" target="_blank">56</a>] (solid lines). The plot appended below shows the residuals of the fit. (B) Corresponding sedimentation coefficient distribution showing a main peak at 2.68 S and diffusional boundary broadening corresponding to a species of 26.9 kDa.</p