Biology subjects

Pearson, E.

Publications and source records attributed to Pearson, E..

4 recordsLinked to original sources

bioRxiv2024

Self-assembly of microstructured protein coatings with programmable functionality for fluorescent biosensors

Proteins, as genetically programmable functional macromolecules, hold immense potential as biocompatible self-assembling building blocks. Despite their versatility in building coating materials, it has been often hindered from programming their functionality genetically. In this study, we demonstrate a modular self-assembly of protein coatings that are genetically programmable for a biosensor application. We designed recombinant fusion protein building blocks to form microstructured coatings on diverse substrates, such as glass or polymer, through a thermally triggered liquid-liquid phase separation and an orthogonal high-affinity coiled-coil interaction. We incorporated fluorescence proteins into coatings and controlled protein density to enable fluorescence imaging and quantification in a low-resource setting. Then, we created a coating for a calcium biosensor using a genetically engineered calcium indicator protein. This protein coating served as the foundation for our smartphone-based fluorescence biosensor, which successfully measured free calcium concentrations in the millimolar range at which extracellular calcium homeostasis is maintained. Using this fluorescence biosensor, we were able to detect abnormal physiological conditions such as mild or moderate hypercalcemia. We envision that this modular and genetically programmable functional protein coating platform could be extended to the development of highly accessible, low-cost fluorescent biosensors for a variety of targets.

bioengineering↗

bioRxiv2022

Use of a novel knock-in allele of Pkd1 identifies nicotinamide nucleotide dehydrogenase as a mitochondrial binding partner of polycystin-1

The localization and function of Polycystin-1, the protein encoded by the gene most commonly mutated in autosomal dominant polycystic kidney disease, remains controversial. We have recently reported that its C-terminus is cleaved and traffics to the mitochondria rather than to the nucleus as had been previously described, and we found that absence of PC1 resulted in fragmented mitochondrial networks and increased mitochondrial membrane potential. Direct visualization of PC1 in mitochondria was only possible, however, after over-expression of recombinant, fluorescently labeled-PC1 in a cell culture system. To resolve the issue, we generated a new mouse model with three copies of the HA epitope and eGFP knocked-in frame into the endogenous mouse Pkd1 gene by CRISPR/Cas9. We show that the modified allele is fully functional but the eGFP-tagged protein cannot be detected without antibody amplification methods. We were, however, able to use nanobody-coupled beads and large quantities of tissue to isolate a PC1 interactome and verify nicotinamide nucleotide transhydrogenase (Nnt) as a mitochondrial partner, linking PC1 to regulation of reactive oxygen species levels in the mitochondria. Loss of Nnt function had no significant effect on renal cystic disease in Pkd1 mutants but treatment of young mice with early onset cystic disease with n-acetyl-cysteine (NAC) provided modest benefit only in the Nnt+/+ genetic background. These studies suggest that new methods and brighter tags will be required to track endogenous PC1, but this new mouse model will be a valuable resource for characterizing the protein interactome of endogenous PC1. The data also support our prior findings that the PC1 C-terminus localizes to mitochondria and regulates their function.

genetics↗

bioRxiv2019

A Subcutaneous Implant of Tenofovir Alafenamide Fumarate Causes Local Inflammation and Tissue Necrosis in Rabbits and Macaques

We describe the in vitro and in vivo evaluation of a subcutaneous reservoir implant delivering tenofovir alafenamide hemifumarate (TAF) for the prevention of HIV infection. These long-acting reservoir implants were able to deliver antiretroviral drug for over 90 days in vitro and in vivo. We evaluated the implants for implantation site histopathology and pharmacokinetics in plasma and tissues for up to 12 weeks in New Zealand White rabbits and rhesus macaque models. A dose-ranging study in rabbits demonstrated dose-dependent pharmacokinetics and local inflammation up to severe necrosis around the active implants. The matched placebos showed normal wound healing and fibrous tissue encapsulation of the implant. We designed a second implant with a lower release rate and flux of TAF and achieved a median cellular level of tenofovir diphosphate of 42 fmol per 106 rhesus macaque peripheral blood mononuclear cells at a dose of 10 {micro}g/kg/day. This dose and flux of TAF also resulted in adverse local inflammation and necrosis near the implant in rhesus macaques. Inflammation in the primates was markedly lower in the placebo group than the active implant. The histological inflammatory response to the TAF implant at 4 and 12 weeks in primates was graded as a severe reaction. Thus, while we were able to achieve sustained target dose we observed unacceptable inflammatory response locally at the implant tissue interface.

pharmacology and toxicology↗

bioRxiv2017

Novel Locus Influencing Retinal Venular Tortuosity Is Also Associated With Risk Of Coronary Artery Disease

Structural variation in retinal blood vessels is associated with global vascular health in humans and may provide a readily accessible indicator of several diseases of vascular origin. Increasing evidence suggests variation in retinal vasculature is highly heritable. This study aimed to identify genetic determinants of retinal vascular traits. We reported a meta-analysis of genome-wide association studies (GWAS) for quantitative retinal vascular traits derived using semi-automatic image analysis of digital retinal photographs from the Genetics of Diabetes Audit and Research in Tayside (GoDARTS) (n=1736) and the Orkney Complex Disease Study (ORCADES) (n=1358) cohorts. We identified a novel genome-wide significant locus at 19q13 (ACTN4/CAPN12) for retinal venular tortuosity (TortV), and one at 13q34 (COL4A2) for retinal arteriolar tortuosity (TortA); these two loci were subsequently confirmed in three independent cohorts (n=1413). In the combined analysis in ACTN4/CAPN12 the lead single nucleotide polymorphism (SNP) was rs1808382 (n=4507; Beta=-0.109; standard error (SE) =0.015; P=2.39x10-13) and in COL4A2 it was rs7991229 (n=4507; Beta=0.103; SE=0.015; P=4.66x10-12). Notably, the ACTN4/CAPN12 locus associated with retinal TortV is also associated with coronary artery disease and heart rate. Our findings demonstrate the contribution of genetics in retinal tortuosity traits, and provide new insights into cardiovascular diseases.\n\nAuthor SummaryRetinal vascular features are associated with wide range of diseases related to vascular health and provide an opportunity to understand early structural changes in vasculature which may help to predict disease risk. Emerging evidence indicates that retinal tortuosity traits are both associated with vascular health and highly heritable. However, the genetic architecture of retinal vascular tortuosity has not been investigated. We therefore performed a genome-wide association study on retinal arteriolar tortuosity (TortA) and retinal venular tortuosity trait (TortV) using data from two independent discovery cohorts of 3094 individuals of European-heritage. We found a novel associations at 19q13 (ACTN4/CAPN12) for TortV, and one at 13q34 (COL4A2) for TortA at discovery stage and validated in three independent cohorts. A significant association was subsequently found between lead SNPs at 19q13 and coronary artery disease, cardiovascular vascular risk factors and heart rate. We also performed genome-wide association studies for retinal vascular calibres and optic disc radius (ODradius) and replicated previously reported locus at 10q21.3 for ODradius. Our findings highlight genetic impacts on retinal venular tortuosity and it is association with cardiovascular disease. This may provide a molecular pathophysiological foundation for use of retinal vascular traits as biomarkers for cardiovascular diseases.

genomics↗