Natalie Gunn

Sensing of protein molecules through nanopores: a molecular dynamics study

Solid-state nanopores have been shown to be suitable for single molecule detection. While numerous modeling investigations exist for DNA within nanopores, there are few simulations of protein translocations. In this paper, we use atomistic molecular dynamics to investigate the translocation of proteins through a silicon nitride nanopore. The nanopore dimensions and profile are representative of experimental systems. We are able to calculate the change in blockade current and friction coefficient for different positions of the protein within the pore. The change in ionic current is found to be negligible until the protein is fully within the pore and the current is lowest when the protein is in the pore center. Using a simple theory that gives good quantitative agreement with the simulation results we are able to show that the variation in current with position is a function of the pore shape. In simulations that guide the protein through the nanopore we identify the effect that confinement has on the friction coefficient of the protein. This integrated view of translocation at the nanoscale provides useful insights that can be used to guide the design of future devices.

Nanosensors for next generation drug screening

One promising path for future drug screening technologies is to examine the binding of ligands to target proteins at the single molecule level by passing them through nanometer sized pores and measuring the change in pore current during translocation. With the aim of evaluating such technologies we perform virtual experiments on the translocation of proteins through silicon nitride nanopores. These simulations consist of large scale, fully atomistic models of the translocation process that involve steering a test protein through the nanopore on a timescale of tens of nanoseconds. We make a comparison between theoretically expected and simulated values of the current drop that is seen when the protein occupies the pore. Details of the stability of the protein and the preservation of its function as measured by its secondary and tertiary structure will be presented to validate both the simulation results and the fundamental design of the proposed device. Finally, the results will be placed in the context of experimental work that combines nanofabrication and microuidics to create a high throughput, low cost, drug screening device.

A hybrid approach for automated mutation annotation of the extended human mutation landscape in scientific literature

As the cost of DNA sequencing continues to fall, an increasing amount of information on human genetic variation is being produced that could help progress precision medicine. However, information about such mutations is typically first made available in the scientific literature, and is then later manually curated into more standardized genomic databases. This curation process is expensive, time-consuming and many variants do not end up being fully curated, if at all. Detecting mutations in the literature is the first key step towards automating this process. However, most of the current methods have focused on identifying mutations that follow existing nomenclatures. In this work, we show that there is a large number of mutations that are missed by using this standard approach. Furthermore, we implement the first mutation annotator to cover an extended mutation landscape, and we show that its F1 performance is the same performance as human annotation (F1 78.29 for manual annotation vs F1 79.56 for automatic annotation).

A Novel Ultra-Stable, Monomeric Green Fluorescent Protein For Direct Volumetric Imaging of Whole Organs Using CLARITY

Recent advances in thick tissue clearing are enabling high resolution, volumetric fluorescence imaging of complex cellular networks. Fluorescent proteins (FPs) such as GFP, however, can be inactivated by the denaturing chemicals used to remove lipids in some tissue clearing methods. Here, we solved the crystal structure of a recently engineered ultra-stable GFP (usGFP) and propose that the two stabilising mutations, Q69L and N164Y, act to improve hydrophobic packing in the core of the protein and facilitate hydrogen bonding networks at the surface, respectively. usGFP was found to dimerise strongly, which is not desirable for some applications. A point mutation at the dimer interface, F223D, generated monomeric usGFP (muGFP). Neurons in whole mouse brains were virally transduced with either EGFP or muGFP and subjected to Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel (CLARITY) clearing. muGFP fluorescence was retained after CLARITY whereas EGFP fluorescence was highly attenuated, thus demonstrating muGFP is a novel FP suitable for applications where high fluorescence stability and minimal self-association are required.

Stage-based Variation in the Effect of Primary Tumor Side on All Stages of Colorectal Cancer Recurrence and Survival

Micro‐Abstract: Although the predictive and prognostic effect of primary tumor side in metastatic colorectal cancer is now widely accepted, it is poorly defined for early‐stage disease. In the present analysis of > 6500 patients, we found stage‐by‐stage differences in survival outcomes according to the primary tumor location, which was partially attributable to differences in survival after recurrence. However, the primary tumor location did not influence the benefit of adjuvant chemotherapy. Background: Multiple studies have defined the prognostic and potential predictive significance of the primary tumor side in metastatic colorectal cancer (CRC). However, the currently available data for early‐stage disease are limited and inconsistent. Materials and Methods: We explored the clinicopathologic, treatment, and outcome data from a multisite Australian CRC registry from 2003 to 2016. Tumors at and distal to the splenic flexure were considered a left primary (LP). Results: For the 6547 patients identified, the median age at diagnosis was 69 years, 55% were men, and most (63%) had a LP. Comparing the outcomes for right primary (RP) versus LP, time‐to‐recurrence was similar for stage I and III disease, but longer for those with a stage II RP (hazard ratio [HR], 0.68; 95% confidence interval [CI], 0.52–0.90; P < .01). Adjuvant chemotherapy provided a consistent benefit in stage III disease, regardless of the tumor side. Overall survival (OS) was similar for those with stage I and II disease between LP and RP patients; however, those with stage III RP disease had poorer OS (HR, 1.30; 95% CI, 1.04–1.62; P < .05) and cancer‐specific survival (HR, 1.55; 95% CI, 1.19–2.03; P < .01). Patients with stage IV RP, whether de novo metastatic (HR, 1.15; 95% CI, 0.95–1.39) or relapsed post–early‐stage disease (HR, 1.35; 95% CI, 1.11–1.65; P < .01), had poorer OS. Conclusion: In early‐stage CRC, the association of tumor side and effect on the time‐to‐recurrence and OS varies by stage. In stage III patients with an RP, poorer OS and cancer‐specific survival outcomes are, in part, driven by inferior survival after recurrence, and tumor side did not influence adjuvant chemotherapy benefit.

Optimization and 13 CH 3 methionine labeling of a signaling competent neurotensin receptor 1 variant for NMR studies

Neurotensin is a 13-residue peptide that acts as a neuromodulator of classical neurotransmitters such as dopamine and glutamate in the mammalian central nervous system, mainly by activating the G protein-coupled receptor (GPCR), neurotensin receptor 1 (NTS1). Agonist binding to GPCRs shifts the conformational equilibrium of the transmembrane helices towards distinct, thermodynamically favorable conformations that favor effector protein interactions and promotes cell signaling. The introduction of site specific labels for NMR spectroscopy has proven useful for investigating this dynamic process, but the low expression levels and poor stability of GPCRs is a hindrance to solution NMR experiments. Several thermostabilized mutants of NTS1 have been engineered to circumvent this, with the crystal structures of four of these published. The conformational dynamics of NTS1 however, has not been thoroughly investigated with NMR. It is generally accepted that stabilized GPCRs exhibit attenuated signaling, thus we thoroughly characterized the signaling characteristics of several thermostabilized NTS1 variants to identify an optimal variant for protein NMR studies. A variant termed enNTS1 exhibited the best combination of signaling capability and stability upon solubilization with detergents. enNTS1 was subsequently labeled with 13CH3-methionine in E. coli and purified to homogeneity in the absence of bound ligands. Using solution NMR spectroscopy we observed several well dispersed 13CH3-methionine resonances, many of which exhibited chemical shift changes upon the addition of the high affinity agonist peptide, NT8-13. Thus, enNTS1 represents a novel tool for investigating ligand induced conformational changes in NTS1 to gain insights into the molecular mechanisms underlying neurotensin signaling.