Molecular Imaging in Synthetic Biology, and Synthetic Biology in Molecular Imaging

Biomedical synthetic biology is an emerging field in which cells are engineered at the genetic level to carry out novel functions with relevance to biomedical and industrial applications. This approach promises new treatments, imaging tools, and diagnostics for diseases ranging from gastrointestinal inflammatory syndromes to cancer, diabetes, and neurodegeneration. As these cellular technologies undergo pre-clinical and clinical development, it is becoming essential to monitor their location and function in vivo, necessitating appropriate molecular imaging strategies, and therefore, we have created an interest group within the World Molecular Imaging Society focusing on synthetic biology and reporter gene technologies. Here, we highlight recent advances in biomedical synthetic biology, including bacterial therapy, immunotherapy, and regenerative medicine. We then discuss emerging molecular imaging approaches to facilitate in vivo applications, focusing on reporter genes for noninvasive modalities such as magnetic resonance, ultrasound, photoacoustic imaging, bioluminescence, and radionuclear imaging. Because reporter genes can be incorporated directly into engineered genetic circuits, they are particularly well suited to imaging synthetic biological constructs, and developing them provides opportunities for creative molecular and genetic engineering

Transcranial Focused Ultrasound Generates Skull-Conducted Shear Waves: Computational Model and Implications for Neuromodulation

Focused ultrasound (FUS) is an established technique for non-invasive surgery and has recently attracted considerable attention as a potential method for non-invasive neuromodulation. While the pressure waves in FUS procedures have been extensively studied in this context, the accompanying shear waves are often neglected due to the relatively high shear compliance of soft tissues. However, in bony structures such as the skull, acoustic pressure can also induce significant shear waves that could propagate outside the ultrasound focus. Here, we investigate wave propagation in the human cranium by means of a finite-element model that accounts for the anatomy, elasticity, and viscoelasticity of the skull and brain. We show that, when a region on the scalp is subjected to FUS, the skull acts as a waveguide for shear waves that propagate with a speed close to 1500 m/s, reaching off-target structures such as the cochlea. In particular, when a sharp onset of FUS is introduced in a zone proximal to the intersection of the parietal and temporal cranium, the bone-propagated shear waves reach the inner ear in about 40 μs, leading to cumulative displacements of about 1 μm. We further quantify the effect of ramped and sharp application of FUS on the cumulative displacements in the inner ear. Our results help explain the off-target auditory responses observed during neuromodulation experiments and inform the development of mitigation and sham control strategies

Genetically engineered sensors for non-invasive molecular imaging using MRI

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2008.Cataloged from PDF version of thesis.Includes bibliographical references (p. 126-138).Technologies that provide information about the concentrations or activities of specific molecules in living subjects have the potential to greatly advance science and medicine. Magnetic resonance imaging (MRI) is a tool uniquely suited to this task because of its ability to image deep inside tissues at relatively high spatial and temporal resolution. However, the range of molecular phenomena currently accessible to MRI is limited by a lack of suitable molecular sensors. Most efforts to create such sensors have focused on synthetic contrast agents, whose complicated structures make them difficult to engineer, synthesize and deliver to target tissues. If MRI sensors could instead be made of proteins, a number of these difficulties could be mitigated. Here, we describe two platforms for the development of protein-based molecular sensors for MRI. The first is based on the heme domain of the bacterial cytochrome P450-BM3, which produces changes in TI contrast in MRI in response to small molecule binding. We developed a high-throughput assay that allowed us to evolve this protein into a sensor of the neurotransmitter dopamine (DA). We then used it to image DA release from cultured cells and cocaine-induced changes in DA transport in the brains of living rats. The second platform is based on the human iron storage protein ferritin (Ft), which enhances T2 contrast in MRI upon self-aggregation. We developed a system to express self-assembled Ft nanoparticles incorporating multiple surface functionalities, and used it to create a sensor for protein kinase A activity. Our results provide a proof of concept for two novel platforms for protein-based MRI sensor development, and highlight some key advantages of this approach over the synthetic methods used previously.by Mikhail G. Shapiro.Ph.D

Ultrasonic Neuromodulation Causes Widespread Cortical Activation via an Indirect Auditory Mechanism

Ultrasound has received widespread attention as an emerging technology for targeted, non-invasive neuromodulation based on its ability to evoke electrophysiological and motor responses in animals. However, little is known about the spatiotemporal pattern of ultrasound-induced brain activity that could drive these responses. Here, we address this question by combining focused ultrasound with wide-field optical imaging of calcium signals in transgenic mice. Surprisingly, we find cortical activity patterns consistent with indirect activation of auditory pathways rather than direct neuromodulation at the ultrasound focus. Ultrasound-induced activity is similar to that evoked by audible sound. Furthermore, both ultrasound and audible sound elicit motor responses consistent with a startle reflex, with both responses reduced by chemical deafening. These findings reveal an indirect auditory mechanism for ultrasound-induced cortical activity and movement requiring careful consideration in future development of ultrasonic neuromodulation as a tool in neuroscience research

Non-invasive imaging using reporter genes altering cellular water permeability

Non-invasive imaging of gene expression in live, optically opaque animals is important for multiple applications, including monitoring of genetic circuits and tracking of cell-based therapeutics. Magnetic resonance imaging (MRI) could enable such monitoring with high spatiotemporal resolution. However, existing MRI reporter genes based on metalloproteins or chemical exchange probes are limited by their reliance on metals or relatively low sensitivity. Here we introduce a new class of MRI reporters based on the human water channel aquaporin 1. We show that aquaporin overexpression produces contrast in diffusion-weighted MRI by increasing tissue water diffusivity without affecting viability. Low aquaporin levels or mixed populations comprising as few as 10% aquaporin-expressing cells are sufficient to produce MRI contrast. We characterize this new contrast mechanism through experiments and simulations, and demonstrate its utility in vivo by imaging gene expression in tumours. Our results establish an alternative class of sensitive, metal-free reporter genes for non-invasive imaging

Localization of Microscale Devices In Vivo using Addressable Transmitters Operated as Magnetic Spins

The function of miniature wireless medical devices, such as capsule endoscopes, biosensors and drug-delivery systems, depends critically on their location inside the body. However, existing electromagnetic, acoustic and imaging-based methods for localizing and communicating with such devices suffer from limitations arising from physical tissue properties or from the performance of the imaging modality. Here, we embody the principles of nuclear magnetic resonance in a silicon integrated-circuit approach for microscale device localization. Analogous to the behaviour of nuclear spins, the engineered miniaturized radio frequency transmitters encode their location in space by shifting their output frequency in proportion to the local magnetic field; applied field gradients thus allow each device to be located precisely from its signal’s frequency. The devices are integrated in circuits smaller than 0.7 mm3 and manufactured through a standard complementary-metal-oxide-semiconductor process, and are capable of sub-millimetre localization in vitro and in vivo. The technology is inherently robust to tissue properties, scalable to multiple devices, and suitable for the development of microscale devices to monitor and treat disease

Nanoscale Heat Transfer from Magnetic Nanoparticles and Ferritin in an Alternating Magnetic Field

Recent suggestions of nanoscale heat confinement on the surface of synthetic and biogenic magnetic nanoparticles during heating by radio frequency-alternating magnetic fields have generated intense interest because of the potential utility of this phenomenon for noninvasive control of biomolecular and cellular function. However, such confinement would represent a significant departure from the classical heat transfer theory. Here, we report an experimental investigation of nanoscale heat confinement on the surface of several types of iron oxide nanoparticles commonly used in biological research, using an all-optical method devoid of the potential artifacts present in previous studies. By simultaneously measuring the fluorescence of distinct thermochromic dyes attached to the particle surface or dissolved in the surrounding fluid during radio frequency magnetic stimulation, we found no measurable difference between the nanoparticle surface temperature and that of the surrounding fluid for three distinct nanoparticle types. Furthermore, the metalloprotein ferritin produced no temperature increase on the protein surface nor in the surrounding fluid. Experiments mimicking the designs of previous studies revealed potential sources of the artifacts. These findings inform the use of magnetic nanoparticle hyperthermia in engineered cellular and molecular systems