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 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

Direct detection and time-locked subsampling applied to pulsed electron paramagnetic resonance imaging

The application of direct time-locked subsampling (TLSS) to Fourier transform electron paramagnetic resonance (FT-EPR) spectroscopy at radio frequencies (rf) is described. With conventional FT-EPR spectroscopy, the high Larmor frequencies (L)often necessitate the use of intermediate frequency (IF) stages to down convert the received free induction decay (FID) signal to a frequency that can be acquired with common data acquisition technology. However, our research focuses on in vivo studies, and consequently utilizes a FT-EPR system with a L<SUB>f</SUB> of 300 MHz. This relatively low frequency L<SUB>f</SUB>, in conjunction with the advent of bandpass sampling analog-to-digital conversion and signal processing technologies, has enabled us to omit the I<SUB>F</SUB> stage in our FT-EPR system. With this in mind, TLSS techniques have been developed to directly sample the 300 MHz FID signal at a sampling rate of 80 MHz providing a signal bandwidth of 20 MHz. The required modifications to the data acquisition and processing system specific to this application are described. Custom software developed to control the EPR system setup, acquire the signals, and post process the data, is outlined. Data was acquired applying both coherent averaging and stochastic excitation sequences. The results of these experiments demonstrate digital down conversion of the 300 MHz FID signal to quadrature baseband. Direct FID TLSS eliminates many noise sources common in EPR systems employing traditional analog receiver techniques, such as the IF mixer stage in single channel systems, and the quadrature baseband mixer stage in dual channel systems

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

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

Physiological Recording in the MRI Environment (PRiME): MRI-Compatible Hemodynamic Recording System.

Hemodynamic recording during interventional cardiovascular procedures is essential for procedural guidance, monitoring patient status, and collection of diagnostic information. Recent advances have made interventions guided by magnetic resonance imaging (MRI) possible and attractive in certain clinical scenarios. However, in the MRI environment, electromagnetic interference (EMI) can cause severe distortions and artifacts in acquired hemodynamic waveforms. The primary aim of this paper was to develop and validate a system to minimize EMI on electrocardiogram (ECG) and invasive blood pressure (IBP) signals. A system was developed which incorporated commercial MRI compatible ECG leads and pressure transducers, custom electronics, user interface, and adaptive signal processing. Measurements were made on pediatric patients (N = 6) during MRI-guided catheterization. Real-time interactive scanning, which is known to produce significant EMI due to fast gradient switching and varying imaging plane orientations, was selected for testing. The effectiveness of the adaptive algorithms was determined by measuring the reduction of noise peaks, amplitude of noise peaks, and false QRS triggers. During real-time gradient-intensive imaging sequences, peak noise amplitude was reduced by 80% and false QRS triggers were reduced to a median of 0. There was no detectable interference on the IBP channels. A hemodynamic recording system front-end was successfully developed and deployed, which enabled high-fidelity recording of ECG and IBP during MRI scanning. The schematics and assembly instructions are publicly available to facilitate implementation at other institutions. Researchers and clinicians are provided a critical tool in investigating and implementing MRI guided interventional cardiovascular procedures