Biology subjects

Farrar, C. T.

Publications and source records attributed to Farrar, C. T..

5 recordsLinked to original sources

bioRxiv2024

Loss of spontaneous vasomotion precedes impaired cerebrovascular reactivity and microbleeds in a mouse model of cerebral amyloid angiopathy

BackgroundCerebral amyloid angiopathy (CAA) is a cerebral small vessel disease in which amyloid-{beta} accumulates in vessel walls. CAA is a leading cause of symptomatic lobar intracerebral hemorrhage and an important contributor to age-related cognitive decline. Recent work has suggested that vascular dysfunction may precede symptomatic stages of CAA, and that spontaneous slow oscillations in arteriolar diameter (termed vasomotion), important for amyloid-{beta} clearance, may be impaired in CAA. MethodsTo systematically study the progression of vascular dysfunction in CAA, we used the APP23 mouse model of amyloidosis, which is known to develop spontaneous cerebral microbleeds mimicking human CAA. Using in vivo 2-photon microscopy, we longitudinally imaged unanesthetized APP23 transgenic mice and wildtype littermates from 7 to 14 months of age, tracking amyloid-{beta} accumulation and vasomotion in individual pial arterioles over time. MRI was used in separate groups of 12-, 18-, and 24-month-old APP23 transgenic mice and wildtype littermates to detect microbleeds and to assess cerebral blood flow and cerebrovascular reactivity with pseudo-continuous arterial spin labeling. ResultsWe observed a significant decline in vasomotion with age in APP23 mice, while vasomotion remained unchanged in wildtype mice with age. This decline corresponded in timing to initial vascular amyloid-{beta} deposition ([~]8-10 months of age), although was more strongly correlated with age than with vascular amyloid-{beta} burden in individual arterioles. Declines in vasomotion preceded the development of MRI-visible microbleeds and the loss of smooth muscle actin in arterioles, both of which were observed in APP23 mice by 18 months of age. Additionally, evoked cerebrovascular reactivity was intact in APP23 mice at 12 months of age, but significantly lower in APP23 mice by 24 months of age. ConclusionsOur findings suggest that a decline in spontaneous vasomotion is an early, potentially pre-symptomatic, manifestation of CAA and vascular dysfunction, and a possible future treatment target.

neuroscience↗

bioRxiv2023

Development of a Synthetic Biosensor for Chemical Exchange MRI Utilizing In Silico Optimized Peptides

1Chemical Exchange Saturation Transfer (CEST) magnetic resonance imaging (MRI) has been identified as a novel alternative to classical diagnostic imaging. Over the last several decades, many studies have been conducted to determine possible CEST agents, such as endogenously expressed compounds or proteins, that can be utilized to produce contrast with minimally invasive procedures and reduced or non-existent levels of toxicity. In recent years there has been an increased interest in the generation of genetically engineered CEST contrast agents, typically based on existing proteins with CEST contrast or modified to produce CEST contrast. We have developed an in-silico method for the evolution of peptide sequences to optimize CEST contrast and showed that these peptides could be combined to create de novo biosensors for CEST MRI. A single protein, superCESTide 2.0, was designed to be 198 amino acids. SuperCESTide 2.0 was expressed in E. coli and purified with size-exclusion chromatography. The magnetic transfer ratio asymmetry (MTRasym) generated by superCESTide 2.0 was comparable to levels seen in previous CEST reporters, such as protamine sulfate (salmon protamine, SP), Poly-L-Lysine (PLL), and human protamine (hPRM1). This data shows that novel peptides with sequences optimized in silico for CEST contrast that utilizes a more comprehensive range of amino acids can still produce contrast when assembled into protein units expressed in complex living environments.

synthetic biology↗

bioRxiv2022

Protein Optimization Evolving Tool (POET) based on Genetic Programming

Proteins are used by scientists to serve a variety of purposes in clinical practice and laboratory research. To optimize proteins for greater function, a variety of techniques have been developed. For the development of reporter genes used in Magnetic Resonance Imaging (MRI) based on Chemical Exchange Saturation Transfer (CEST), these techniques have encountered a variety of challenges. Here we develop a mechanism of protein optimization using a computational approach known as "genetic programming". We developed an algorithm called Protein Optimization Evolving Tool (POET). Starting from a small library of literature values, use of this tool allowed us to develop proteins which produce four times more MRI contrast than what was previously state-of-the-art. Next, we used POET to evolve peptides that produced CEST-MRI contrast at large chemical shifts where no other known peptides have previously demonstrated contrast. This demonstrated the ability of POET to evolve new functions in proteins. Interestingly, many of the peptides produced using POET were dramatically different with respect to their sequence and chemical environment than existing CEST producing peptides, and challenge prior understandings of how those peptides function. This suggests that unlike existing algorithms for protein engineering that rely on divergent evolution, POET relies on convergent evolution.

synthetic biology↗

bioRxiv2020

Redesigned Reporter Gene for Improved Proton Exchange-based Molecular MRI Contrast

Reporter gene imaging allows for non-invasive monitoring of molecular processes in living cells, providing insights on the mechanisms underlying pathology and therapy. A lysine-rich protein (LRP) chemical exchange saturation transfer (CEST) MRI reporter gene has previously been developed and used to image tumor cells, cardiac viral gene transfer, and oncolytic virotherapy. However, the highly repetitive nature of the LRP reporter gene sequence leads to DNA recombination events and the expression of a range of truncated LRP protein fragments, thereby greatly limiting the CEST sensitivity. Here we report the use of a redesigned LRP reporter (rdLRP), aimed to provide excellent stability and CEST sensitivity. The rdLRP contains no DNA repeats or GC rich regions and 30% less positively charged amino-acids. RT-PCR of cell lysates transfected with rdLRP demonstrated a stable reporter gene with a single distinct band corresponding to full-length DNA. A distinct increase in CEST-MRI contrast was obtained in cell lysates of rdLRP transfected cells and in in vivo LRP expressing mouse brain tumors (p=0.0275, n=10).

bioengineering↗

bioRxiv2020

AI boosted molecular MRI for apoptosis detection in oncolytic virotherapy

Oncolytic virotherapy is a promising treatment for high mortality cancers1. Non-invasive imaging of the underlying molecular processes is an essential tool for therapy optimization and assessment of viral spread, innate immunity, and therapeutic response2, 3. However, previous methods for imaging oncolytic viruses did not correlate with late viral activity4 or had poor sensitivity and specificity5. Similarly, methods developed to image treatment response, such as apoptosis, proved to be slow, nonspecific, or require the use of radioactive or metal-based contrast agents6-8. To date, no method has been widely adopted for clinical use. We describe here a new method for fast magnetic resonance molecular imaging with quantitative proton chemical-exchange specificity to monitor oncolytic virotherapy treatment response. A deep neural network enabled the computation of quantitative biomarker maps of protein and lipid/macromolecule concentrations as well as intracellular pH in a glioblastoma multiforme mouse brain tumor model. Early detection of apoptotic response to oncolytic virotherapy, characterized by decreased cytosolic pH and protein synthesis, was observed in agreement with histology. Clinical translation was demonstrated in a normal human subject, yielding molecular parameters in good agreement with literature values9. The developed method is directly applicable to a wide range of pathologies, including stroke10, cancer11-13, and neurological disorders14, 15.

biophysics↗