Faculty Bibliography

The most frequently mutated oncogene in cancer is KRAS, which uses alternative fourth exons to generate two gene products (KRAS4A and KRAS4B) that differ only in their C-terminal membrane-targeting region¹. Because oncogenic mutations occur in exons 2 or 3, two constitutively active KRAS proteins-each capable of transforming cells-are encoded when KRAS is activated by mutation². No functional distinctions among the splice variants have so far been established. Oncogenic KRAS alters the metabolism of tumour cells³ in several ways, including increased glucose uptake and glycolysis even in the presence of abundant oxygen⁴ (the Warburg effect). Whereas these metabolic effects of oncogenic KRAS have been explained by transcriptional upregulation of glucose transporters and glycolytic enzymes^3-5, it is not known whether there is direct regulation of metabolic enzymes. Here we report a direct, GTP-dependent interaction between KRAS4A and hexokinase 1 (HK1) that alters the activity of the kinase, and thereby establish that HK1 is an effector of KRAS4A. This interaction is unique to KRAS4A because the palmitoylation-depalmitoylation cycle of this RAS isoform enables colocalization with HK1 on the outer mitochondrial membrane. The expression of KRAS4A in cancer may drive unique metabolic vulnerabilities that can be exploited therapeutically.

Thermoresponsive hydrogels are used for an array of biomedical applications. Lower critical solution temperature (LCST)-type hydrogels have been observed in nature and extensively studied in comparison to upper critical solution temperature (UCST)-type hydrogels. Of the limited protein-based UCST-type hydrogels reported, none have been composed of a single coiled-coil domain. Here we describe a biosynthesized homopentameric coiled-coil protein capable of demonstrating a UCST. Microscopy and structural analysis reveal that the hydrogel is stabilized by molecular entanglement of protein nanofibers, creating a porous matrix capable of binding the small hydrophobic molecule, curcumin. Curcumin binding increases the α-helical structure, fiber entanglement, mechanical integrity, and thermostability, resulting in sustained drug release at physiological temperature. This work provides the first example of a thermoresponsive hydrogel comprised of a single coiled-coil protein domain that can be used as a vehicle for sustained release and, by demonstrating UCST-type behavior, shows promise in forging a relationship between coiled-coil protein phase behavior and that of synthetic polymer systems.

PURPOSE/OBJECTIVE:To improve precision of R1 (=1/T1) maps of fetal brain in utero in order to perform QUEnch-assiSTed (QUEST) MRI in which a significant anti-oxidant-induced reduction in R1 indicates oxidative stress. METHODS:C57BL/6 mouse fetuses in utero (E16.5) were gently and non-surgically isolated and secured using a homemade 3D printed clip. Using a commercial receive-only surface coil, brain maps of R1, an index sensitive to excessive and continuous free radical production, were collected using either conventional Cartesian or a non-Cartesian (periodically rotated overlapping parallel lines with enhanced reconstruction) progressive saturation sequences. Data were normalized to the shortest TR time to remove bias. To assess oxidative stress, brain R1 maps were acquired on the lipopolysaccharide (LPS) model of preterm birth ± rosiglitazone (ROSI, which has anti-oxidant properties); phosphate buffered saline (PBS) controls ± ROSI were similarly studied. RESULTS:The highest quality R1 maps were generated by a combination of the 3D printed clip, surface coil detection, non-Cartesian sequence, and normalization scheme ensuring minimal fetal movement, good detection sensitivity, reduced motion artifacts, and minimal baseline variations, respectively. In the LPS group, the combined caudate-putamen and thalamus region R1 was reduced (p < 0.05) with ROSI treatment consistent with brain oxidative stress; no evidence for oxidative stress was found in pons region. In the PBS control group, brain R1's did not change with ROSI treatment. CONCLUSION/CONCLUSIONS:The improved sensitivity and reproducibility of the combined approaches described herein enabled first time demonstration of regional oxidative stress measurements of fetal brain in utero using QUEST MRI.

Engineered proteins provide an interesting template for designing fluorine-19 (¹⁹F) magnetic resonance imaging (MRI) contrast agents, yet progress has been hindered by the unpredictable relaxation properties of fluorine. Herein, we present the biosynthesis of a protein block copolymer, termed "fluorinated thermoresponsive assembled protein" (F-TRAP), which assembles into a monodisperse nanoscale micelle with interesting ¹⁹F NMR properties and the ability to encapsulate and release small therapeutic molecules, imparting potential as a diagnostic and therapeutic (theranostic) agent. The assembly of the F-TRAP micelle, composed of a coiled-coil pentamer corona and a hydrophobic, thermoresponsive elastin-like polypeptide core, results in a drastic depression in spin-spin relaxation ( T₂) times and unaffected spin-lattice relaxation ( T₁) times. The nearly unchanging T₁ relaxation rates and linearly dependent T₂ relaxation rates have allowed for detection via zero echo time ¹⁹F MRI, and the in vivo MR potential has been preliminarily explored using ¹⁹F magnetic resonance spectroscopy (MRS). This fluorinated micelle has also demonstrated the ability to encapsulate the small-molecule chemotherapeutic doxorubicin and release its cargo in a thermoresponsive manner owing to its inherent stimuli-responsive properties, presenting an interesting avenue for the development of thermoresponsive ¹⁹F MRI/MRS-traceable theranostic agents.

Volumetric analysis of brain ventricle (BV) structure is a key tool in the study of central nervous system development in embryonic mice. High-frequency ultrasound (HFU) is the only non-invasive, real-time modality available for rapid volumetric imaging of embryos in utero. However, manual segmentation of the BV from HFU volumes is tedious, time-consuming, and requires specialized expertise. In this paper, we propose a novel deep learning based BV segmentation system for whole-body HFU images of mouse embryos. Our fully automated system consists of two modules: localization and segmentation. It first applies a volumetric convolutional neural network on a 3D sliding window over the entire volume to identify a 3D bounding box containing the entire BV. It then employs a fully convolutional network to segment the detected bounding box into BV and background. The system achieves a Dice Similarity Coefficient (DSC) of 0.8956 for BV segmentation on an unseen 111 HFU volume test set surpassing the previous state-of-the-art method (DSC of 0.7119) by a margin of 25%.

PURPOSE/OBJECTIVE:To investigate the feasibility of measuring the subtle disruption of blood-brain barrier (BBB) using DCE-MRI with a scan duration shorter than 10 min. METHODS:) in the estimation of vascular permeability-surface area product (PS). Numerical simulation studies were carried out to investigate how the reduction in scan time affects the accuracy in estimating contrast kinetic parameters. DCE-MRI studies of the rat brain were conducted with Fisher rats to confirm the results from the simulation. Intracranial F98 glioblastoma models were used to assess areas with different levels of permeability. In the normal brain tissues, the Patlak model (PM) and EPM were compared, whereas the 2-compartment-exchange-model (TCM) and EPM were assessed in the peri-tumor and the tumor regions. RESULTS:was high as in the gray matter, the bias in PM-PS (>900%) were larger than that in EPM-PS (<42%). The animal study also showed similar results, where the PM parameters were more sensitive to the scan duration than the EPM parameters. It was also demonstrated that, in the peri-tumor region, the EPM parameters showed less change by scan duration than the TCM parameters. CONCLUSION/CONCLUSIONS:The results of this study suggest that EPM can be used to measure PS with a scan duration of 10 min or less.