Trusted CI SLATE Engagement

Final report of the Trusted CI SLATE Engagement.NSF #1547272NSF #1724821Ope

The khmer software package: enabling efficient nucleotide sequence analysis

The khmer package is a freely available software library for working efficiently with fixed length DNA words, or k-mers. khmer provides implementations of a probabilistic k-mer counting data structure, a compressible De Bruijn graph representation, De Bruijn graph partitioning, and digital normalization. khmer is implemented in C++ and Python, and is freely available under the BSD license at https://github.com/dib-lab/khmer/

The khmer software package: enabling efficient nucleotide sequence analysis [version 1; referees: 2 approved, 1 approved with reservations]

The khmer package is a freely available software library for working efficiently with fixed length DNA words, or k-mers. khmer provides implementations of a probabilistic k-mer counting data structure, a compressible De Bruijn graph representation, De Bruijn graph partitioning, and digital normalization. khmer is implemented in C++ and Python, and is freely available under the BSD license at https://github.com/dib-lab/khmer/

Development of a Novel Imaging Methodology for Quantitative Analysis of the Mouse Cortical Vasculature

This thesis describes a novel imaging methodology for visualization and quantitative analysis of the vascular topology of different cortical regions of the mouse brain in 3D. The brain is perfused with a fluorescent contrast agent, and rendered fully transparent via optical clearing. This procedure enables images through the whole cortical depth to be obtained with a 2-photon microscope without sectioning. The Allen Reference Atlas (ARA) (Lein et al., 2007) is registered to the 2-photon data for delineation of the cortical regions. Quantitative metrics are then extracted from the different regions using an automatic vessel segmentation algorithm. These metrics are compared with those obtained by other investigators to validate this technique, and are found to be in agreement. Since this methodology possesses the resolution to visualize vessels of all sizes, and provides reasonable estimates of quantitative parameters, it shows strong potential for quantitative analysis of normal and abnormal cortical vascular architecture.MAS

Vessel signal as a function of diameter and cortical depth.

<p>(A) Vessel signal as a function of diameter. Signal is normalized for the e<i>x</i> and <i>in vivo</i> data by calculating the mean signal of all vessels above 10 μm diameter in each of the 4 images. The signal for each vessel is calculated separately for each image. The mean signal for vessels above 10 μm diameter is given an arbitrary value of 1, and the signal for all vessels is calculated relative to this normalized value. Smaller vessels have a weaker signal <i>ex vivo</i> compared to <i>in vivo</i>, likely due to the larger PSF <i>ex vivo</i>. (B) Capillary signal as a function of cortical depth. The <i>in vivo</i> signal is constant for the first several hundred microns, before decreasing quickly with depth (characteristic attenuation length of 171 ± 15 μm). In contrast, the <i>ex vivo</i> signal maintains its strength through the cortical thickness. The lines in Figs A and B are fits to the data, and the ribbons surrounding the lines are the 95% confidence intervals.</p

FWHM of signal along optical axis and x-axis versus depth for beads embedded in agar.

<p>The beads were 0.5 μm diameter yellow-green fluorescent beads (excitation peak 505 nm; emission peak 515 nm) and were embedded in fructose-cleared 1% low melting point agar. Imaging was performed using 2PFM at an excitation wavelength of 800 nm. The FWHM was calculated by fitting a Gaussian to the signal profile along either the optical or x-axis for these beads. Prior to fitting the Gaussian, the image of the beads was blurred by a Gaussian with FWHM 1.5 μm, as per the vascular images on which vessel tracking was performed. Since the slope of the x-axis was not statistically different from 0 (p = 0.8136), only the PSF along the optical axis was assumed to change with depth when performing vessel tracking. The ribbons surrounding the straight lines represent the 95% confidence interval.</p

The impact of vessel shadowing on capillary signal.

<p>(A) <i>In vivo</i> (B) <i>Ex vivo</i>. The shadowing artifact is noticeably absent <i>ex vivo</i> (no difference in signal between shadowed/unshadowed vessels), but significant <i>in vivo</i> for depths below 0.6 mm.</p

3D morphological analysis of the mouse cerebral vasculature: Comparison of <i>in vivo</i> and <i>ex vivo</i> methods

<div><p><i>Ex vivo</i> 2-photon fluorescence microscopy (2PFM) with optical clearing enables vascular imaging deep into tissue. However, optical clearing may also produce spherical aberrations if the objective lens is not index-matched to the clearing material, while the perfusion, clearing, and fixation procedure may alter vascular morphology. We compared <i>in vivo</i> and <i>ex vivo</i> 2PFM in mice, focusing on apparent differences in microvascular signal and morphology. Following <i>in vivo</i> imaging, the mice (four total) were perfused with a fluorescent gel and their brains fructose-cleared. The brain regions imaged <i>in vivo</i> were imaged <i>ex vivo</i>. Vessels were segmented in both images using an automated tracing algorithm that accounts for the spatially varying PSF in the <i>ex vivo</i> images. This spatial variance is induced by spherical aberrations caused by imaging fructose-cleared tissue with a water-immersion objective. Alignment of the <i>ex vivo</i> image to the <i>in vivo</i> image through a non-linear warping algorithm enabled comparison of apparent vessel diameter, as well as differences in signal. Shrinkage varied as a function of diameter, with capillaries rendered smaller <i>ex vivo</i> by 13%, while penetrating vessels shrunk by 34%. The pial vasculature attenuated <i>in vivo</i> microvascular signal by 40% 300 μm below the tissue surface, but this effect was absent <i>ex vivo</i>. On the whole, <i>ex vivo</i> imaging was found to be valuable for studying deep cortical vasculature.</p></div

Ratio of <i>ex vivo</i>: <i>in vivo</i> vessel diameters as a function of <i>in vivo</i> vessel diameter.

<p>For each vessel, the ratio of its diameter <i>ex vivo</i> (after correction for refractive index mismatch) to that <i>in vivo</i> was computed. In this figure are the ratios computed for all vessels pooled together from the four mice.</p

Ultrasound detection of abnormal cerebrovascular morphology in a mouse model of sickle cell disease based on wave reflection

Sickle cell disease (SCD) is associated with a high risk of stroke, and affected individuals often have focal brain lesions termed silent cerebral infarcts. The mechanisms leading to these types of injuries are at present poorly understood. Our group has recently demonstrated a non-invasive measurement of cerebrovascular impedance and wave reflection in mice using high-frequency ultrasound in the common carotid artery. To better understand the pathophysiology in SCD, we used this approach in combination with micro-computed tomography to investigate changes in cerebrovascular morphology in the Townes mouse model of SCD. Relative to controls, the SCD mice demonstrated the following: (i) increased carotid artery diameter, blood flow and vessel wall thickness; (ii) elevated pulse wave velocity; (iii) increased reflection coefficient; and (iv) an increase in the total number of vessel segments in the brain. This study highlights the potential for wave reflection to aid the non-invasive clinical assessment of vascular pathology in SCD