Biology subjects

Shepherd, S.

Publications and source records attributed to Shepherd, S..

6 recordsLinked to original sources

bioRxiv2023

Designer DNA NanoGripper

DNA has shown great biocompatibility, programmable mechanical properties, and structural addressability at the nanometer scale, making it a versatile material for building high precision nanorobotics for biomedical applications. Herein, we present design principle, synthesis, and characterization of a DNA nanorobotic hand, called the "NanoGripper", that contains a palm and four bendable fingers as inspired by human hands, bird claws, and bacteriophages evolved in nature. Each NanoGripper finger has three phalanges connected by two flexible and rotatable joints that are bendable in response to binding to other entities. Functions of the NanoGripper have been enabled and driven by the interactions between moieties attached to the fingers and their binding partners. We showcase that the NanoGripper can be engineered to interact with and capture various objects with different dimensions, including gold nanoparticles, gold NanoUrchins, and SARS-CoV-2 virions. When carrying multiple DNA aptamer nanoswitches programmed to generate fluorescent signal enhanced on a photonic crystal platform, the NanoGripper functions as a sensitive viral biosensor that detects intact SARS-CoV-2 virions in human saliva with a limit of detection of [~] 100 copies/mL, providing RT-PCR equivalent sensitivity. Additionally, we use confocal microscopy to visualize how the NanoGripper-aptamer complex can effectively block viral entry into the host cells, indicating the viral inhibition. In summary, we report the design, synthesis, and characterization of a complex nanomachine that can be readily tailored for specific applications. The study highlights a path toward novel, feasible, and efficient solutions for the diagnosis and therapy of other diseases such as HIV and influenza. One-sentence summaryDesign, synthesis, characterization, and functional showcase of a human-hand like designer DNA nanobot

bioengineering↗

bioRxiv2023

Muscle of obese insulin-resistant humans exhibits losses in proteostasis and attenuated proteome dynamics that are improved by exercise training.

We examined muscle proteostasis in obese insulin-resistant (OIR) individuals to determine whether endurance exercise could positively influence proteome dynamics in this population. Male OIR (n = 3) and lean, healthy controls (LHC; n = 4) were recruited and underwent a 14-d measurement protocol of daily deuterium oxide (D2O) consumption and serial biopsies of vastus lateralis muscle. The OIR group then completed 10-weeks of high-intensity interval training (HIIT), encompassing 3 sessions per week of cycle ergometer exercise with 1 min intervals at 100 % maximum aerobic power (Wmax) interspersed by 1 min recovery periods. The number of intervals per session progressed from 4 to 8, and during weeks 8-10 the 14-d measurement protocol was repeated. The abundance and turnover rates of 880 and 301 proteins, respectively, were measured. OIR and LHC muscle exhibited 352 differences (p < 0.05, false discovery rate (p < 0.05) differences in protein turnover. OIR muscle was enriched with markers of metabolic stress, protein misfolding and components of the ubiquitin-proteasome system, and the turnover rate of many of these proteins was less compared to LHC muscle. HIIT altered the abundance of 53 proteins and increased the turnover rate of 22 proteins (p < 0.05) in OIR muscle and tended to restore proteostasis, evidenced by increasing muscle protein turnover rates and normalizing proteasome composition in OIR participants. In conclusion, obesity and insulin resistance are associated with compromised muscle proteostasis, which can be partially restored by endurance exercise.

physiology↗

bioRxiv2022

Target Recycling Amplification Process for Digital Detection of Exosomal MicroRNAs Through Photonic Resonator Absorption Microscopy

Exosomal microRNAs (miRNAs) have considerable potential as pivotal biomarkers to monitor cancer development, dis-ease progression, treatment effects and prognosis. Here, we report an efficient target recycling amplification process (TRAP) for the digital detection of exosomal miRNAs using photonic resonator absorption microscopy (PRAM). Through toehold-mediated DNA strand displacement reactions, we achieve multiplex digital detection with sub-attomolar sensitivity in 20 minutes, robust selectivity for single nucleotide variants, and a broad dynamic range from 1 aM to 1 pM. We then applied our TRAP system to quantify miRNA in exosomal total RNAs isolated from human cancer cell lines. Compared with traditional qRT-PCR methods, TRAP showed similar accuracy in profiling exosomal miRNAs derived from cancer cells, but also exhibited at least 31-fold and 61-fold enhancement in the limits of miRNA-375 and miRNA-21 detection, respectively. The TRAP approach is ideal for exosomal or circulating miRNA biomarker quantification, where the miRNAs are present in low concentrations or sample volume, with potentials for frequent, low-cost, and minimally invasive point-of-care testing.

bioengineering↗

bioRxiv2022

Photonic-Plasmonic Coupling Enhanced Fluorescence Enabling Digital-Resolution Ultrasensitive Protein Detection

Assays utilizing molecular fluorophores are common throughout life science research and diagnostic testing, although detection limits are generally limited by weak emission intensity, thus requiring many labeled target molecules to combine their output to achieve signal-to-noise greater than the background. Here, we describe how the synergistic coupling of plasmonic and photonic resonance modes can significantly boost the emission from fluorescent dye molecules without increasing the illumination intensity while utilizing a microscopy approach with a broad field of view. By optimally matching the resonant modes of a plasmonic fluor (PF) nanoparticle and a photonic crystal (PC) surface with the absorption and emission spectrum of the PFs fluorescent dye, we observe a 52-fold improvement in signal intensity, enabling individual PFs to be observed and digitally counted, using an approach in which one PF tag represents detection of one target molecule. The photonic amplification from the PF can be attributed to the strong near-field enhancement due to the cavity-induced activation of the PF, PC band structure-mediated improvement in collection efficiency of emitted photons, and increased rate of spontaneous emission. We demonstrate the applicability of the method by dose-response characterization of a sandwich immunoassay for human interleukin-6, a biomarker commonly used to assist diagnosis of cancer, inflammation, sepsis, and autoimmune disease. We achieve a limit of detection of 10 fg/ml, representing a capability three orders of magnitude lower than standard immunoassays.

bioengineering↗

bioRxiv2022

Spectral Interferences Impede the High-Resolution Mass Analysis of Recombinant Adeno-Associated Viruses

Recombinant adeno-associated viruses (rAAVs) are the leading platform for in vivo delivery of gene therapies, with several already approved for clinical use. However, the heterogeneity and structural complexity of these viral particles render them challenging targets to characterize. Orbitrap-based native mass spectrometry (MS) is a method capable of directly characterizing intact megadalton protein assemblies. Here we used such an approach to characterize four different preparations of rAAV8 (two empty and two filled) differing in both their transgene and relative capsid protein isoform (i.e. VP1, VP2 and VP3) content. Interestingly, in native MS measurements of these samples, we observe complicated, unusual, and dramatically different spectral appearances between the four rAAV preparations that cannot be rationalized or interpreted using conventional approaches (i.e. charge state deconvolution). By combining high-resolution native MS, single particle charge detection MS, and spectral simulations, we reveal that these unexpected features result from a combination of stochastic assembly-induced heterogeneity and divergent gas phase charging behaviour between the four rAAV preparations. Our results stress the often-neglected heterogeneity of rAAVs, but also highlight the pitfalls of standard high-resolution mass analysis for such particles. Finally, we show that charge detection MS and spectral simulations can be used to tackle these challenges.

biochemistry↗

bioRxiv2022

Updated Variant Curation Expert Panel Criteria and Pathogenicity Classifications for 251 Variants for RYR1-related Malignant Hyperthermia Susceptibility

The ClinGen malignant hyperthermia susceptibility (MHS) variant curation expert panel specified the ACMG/AMP criteria for RYR1-related MHS and a pilot analysis of 84 variants was published. We have now classified an additional 251 variants for RYR1-related MHS according to current ClinGen standards and updated the criteria where necessary. Criterion PS4 was modified such that individuals with multiple RYR1 variants classified as pathogenic (P), likely pathogenic (LP) or variant of uncertain significance (VUS) were not considered as providing evidence for pathogenicity. Critera PS1 and PM5 were revised to consider LP variants at the same amino acid residue as providing evidence for pathogenicity at reduced strength. Finally, PM1 was revised such that if PS1 or PM5 are used PM1, if applicable, should be downgraded to supporting. Of the 251 RYR1 variants, 42 were classified as P/LP, 16 as B/LB, and 193 as VUS. The primary driver of 176 VUS classifications was insufficient evidence supporting pathogenicity, rather than evidence against pathogenicity. Functional data supporting PS3/BS3 was identified for only 13 variants. Based on the posterior probabilities of pathogenicity and variant frequencies in gnomAD, we estimated the prevalence of individuals with RYR1-related MHS pathogenic variants to be between 1/300 and 1/1,075, considerably higher than current estimates. We have updated ACMG/AMP criteria for RYR1-related MHS and classified 251 variants. We suggest that prioritization of functional studies is needed to resolve the large number of VUS classifications and allow for appropriate risk assessment. RYR1-related MHS pathogenic variants are likely to be more common than currently appreciated.

genetics↗