Detection of Gut Inflammation and Tumors Using Photoacoustic Imaging

Catalyst Volume 10

Abstract:

Photoacoustic imaging is a technique in which contrast agents absorb photon energy and emit signals that can be analyzed by ultrasound transducers. This method allows for unprecedented depth imaging that can provide a non-invasive alternative to current diagnostic tools used to detect internal tissue inflammation.¹ The Rice iGEM team strove to use photoacoustic technology and biomarkers to develop a noninvasive method of locally detecting gut inflammation and colon cancer. As a first step, we genetically engineered Escherichia coli to express near-infrared fluorescent proteins iRFP670 and iRFP713 and conducted tests using biomarkers to determine whether expression was confined to a singular local area.

Introduction:

In photoacoustic imaging, laser pulses of a specific, predetermined wavelength (the excitation wavelength) activate and thermally excite a contrast agent such as a pigment or protein. The heat makes the contrast agent contract and expand producing an ultrasonic emission wavelength longer than the excitation wavelength used. The emission wavelength data are used to produce 2D or 3D images of tissues that have high resolution and contrast.²

The objective of this photoacoustic imaging project is to engineer bacteria to produce contrast agents in the presence of biomarkers specific to gut inflammation and colon cancer and ultimately to deliver the bacteria into the intestines. The bacteria will produce the contrast agents in response to certain biomarkers and lasers will excite the contrast agents, which will emit signals in local, targeted areas, allowing for a non-invasive imaging method. Our goal is to develop a non-invasive photoacoustic imaging delivery method that uses engineered bacteria to report gut inflammation and identify colon cancer. To achieve this, we constructed plasmids that have a nitric-oxide-sensing promoter (soxR/S) or a hypoxia-sensing promoter (narK or fdhf) fused to genes encoding near-infrared fluorescent proteins or violacein with emission wavelengths of 670 nm (iRFP670) and 713 nm (iRFP713). Nitric oxide and hypoxia, biological markers of gut inflammation in both mice and humans, would therefore promote expression of the desired iRFPs or violacein.^3,4

Results and Discussion

Arabinose

To test the inducibility and detectability of our iRFPs, we used pBAD, a promoter that is part of the arabinose operon located in E. coli.⁵ We formed genetic circuits consisting of the pBAD expression system and iRFP670 and iRFP713 (Fig. 1a). AraC, a constitutively produced transcription regulator, changes form in the presence of arabinose sugar, allowing for the activation of the pBAD promoter.

View fullsize

Fluorescence levels emitted by the iRFPs increased significantly when placed in wells containing increasing concentrations of arabinose (Figure 2). This correlation suggests that our selected iRFPs fluoresce sufficiently when promoters are induced by environmental signals. The results of the arabinose assays showed that we successfully produced iRFPs; the next steps were to engineer bacteria to produce the same iRFPs under nitric oxide and hypoxia.

Nitric Oxide

The next step was to test the nitric oxide induction of iRFP fluorescence. We used a genetic circuit consisting of a constitutive promoter and the soxR gene, which in turn expresses the SoxR protein (Figure 1b). In the presence of nitric oxide, SoxR changes form to activate the promoter soxS, which activates the expression of the desired gene. The source of nitric oxide added to our engineered bacteria samples was diethylenetriamine/nitric oxide adduct (DETA/NO).

Figure 3 shows no significant difference of fluorescence/OD600 between DETA/NO concentrations. This finding implies that our engineered bacteria were unable to detect the nitric oxide biomarker and produce iRFP; future troubleshooting includes verifying promoter strength and correct sample conditions. Furthermore, nitric oxide has an extremely short half-life of a few seconds, which may not be enough time for most of the engineered bacteria to sense the nitric oxide, limiting iRFP production and fluorescence.

Hypoxia

We also tested the induction of iRFP fluorescence with the hypoxia-inducible promoters narK and fdhf. We expected iRFP production and fluorescence to increase when using the narK and fdhf promoters in anaerobic conditions (Figure 1c and d).

However, we observed the opposite result. A decreased fluorescence for both iRFP constructs in both promoters was measured when exposed to hypoxia (Figure 4). This finding suggests that our engineered bacteria were unable to detect the hypoxia biomarker and produce iRFP; future troubleshooting includes verifying promoter strength and correct sample conditions.

Future Directions

Further studies include testing the engineered bacteria co-cultured with colon cancer cells and developing other constructs that will enable bacteria to sense carcinogenic tumors and make them fluoresce for imaging and treatment purposes.

Violacein has anti-cancer therapy potential

Violacein is a fluorescent pigment for in vivo photoacoustic imaging in the near-infrared range and shows anti-tumoral activity⁶. It has high potential for future work in bacterial tumor targeting. We have succeeded in constructing violacein using Golden Gate shuffling⁷ and intend to use it in experiments such as the nitric oxide and hypoxia assays we used for iRFP670 and 713.

Invasin can allow for targeted cell therapy

Using a beta integrin called invasin, certain bacteria are able to invade mammalian cells.^8-9 If we engineer E. coli that have the beta integrin invasion as well as the genetic circuits capable of sensing nitric oxide and/or hypoxia, we can potentially allow the E. coli to invade colon cells and release contrast agents for photoacoustic imaging or therapeutic agents such as violacein only in the presence of specific biomarkers.¹⁰ Additionally, if we engineer the bacteria that exhibit invasin to invade colon cancer cells only and not normal cells, then this approach would potentially allow for a localized targeting and treatment of cancerous tumors. This design allows us to create scenarios with parameters more similar to the conditions observed in the human gut as we will be unable to test our engineered bacteria in an actual human gut.

Acknowledgements:

The International Genetically Engineered Machine (iGEM) Foundation (igem.org) is an independent, non-profit organization dedicated to education and competition, the advancement of synthetic biology, and the development of an open community and collaboration.

This project would not have been possible without the patient instruction and generous encouragement of our Principal Investigators (Dr. Beth Beason-Abmayr and Dr. Jonathan Silberg, BioSciences at Rice), our graduate student advisors and our undergraduate team. I would also like to thank our iGEM collaborators.

This work was supported by the Wiess School of Natural Sciences and the George R. Brown School of Engineering and the Departments of BioSciences, Bioengineering, and Chemical and Biomolecular Engineering at Rice University; Dr. Rebecca Richards-Kortum, HHMI Pre-College and Undergraduate Science Education Program Grant #52008107; and Dr. George N. Phillips, Jr., Looney Endowment Fund.

If you would like to know more information about our project and our team, please visit our iGEM wiki at 2016.igem.org/Team:Rice.

References

1. Ntziachristos, V. Nat Methods. 2010, 7, 603-614.
2. Weber, J. et al. Nat Methods. 2016, 13, 639-650.
3. Archer, E. J. et al. ACS Synth. Biol. 2012, 1, 451–457.
4. Hӧckel, M.; Vaupel, P. JNCI J Natl Cancer Inst. 2001, 93, 266−276.
5. Guzman, L. M. et al. J of Bacteriology. 1995, 177, 4121-4130.
6. Shcherbakova, D. M.; Verkhusha, V. V. Nat Methods. 2013, 10, 751-754.
7. Engler, C. et al. PLOS One. 2009, 4, 1-9.
8. Anderson, J. et al. Sci Direct. 2006, 355, 619–627
9. Arao, S. et al. Pancreas. 2000, 20, 619-627.
10. Jiang, Y. et al. Sci Rep. 2015, 19, 1-9.

A Fourth Neutrino? Explaining the Anomalies of Particle Physics

Catalyst Volume 10

Abstract

The very first neutrino experiments discovered that neutrinos exist in three flavors and can oscillate between those flavors as they travel through space. However, many recent experiments have collected anomalous data that contradicts a three neutrino flavor hypothesis, suggesting instead that there may exist a fourth neutrino, called the sterile neutrino, that interacts solely through the gravitational force. While there is no conclusive evidence proving the existence of a fourth neutrino flavor, scientists designed the IceCube laboratory at the South Pole to search for this newly hypothesized particle. Due to its immense size and sensitivity, the IceCube laboratory stands as the most capable neutrino laboratory to corroborate the existence of these particles.

Introduction

Neutrinos are subatomic, ubiquitous, elementary particles that are produced in a variety of ways. Some are produced from collisions in the atmosphere between different particles, while others result from the decomposition and decay of larger atoms.^1,3 Neutrinos are thought to play a role in the interactions between matter and antimatter; furthermore, they are thought to have significantly influenced the formation of the universe.³ Thus, neutrinos are of paramount concern in the world of particle physics, with the potential of expanding our understanding of the universe. When they were first posited, neutrinos were thought to have no mass because they have very little impact on the matter around them. However, decades later, it was determined that they have mass but only interact with other matter in the universe through the weak nuclear force and gravity.²

Early neutrino experiments found that measuring the number of neutrinos produced from the sun resulted in a value almost one third of the predicted value. Coupled with other neutrino experiments, these observations gave rise to the notion of neutrino flavors and neutrino flavor oscillations. There are three flavors of the standard neutrino: electron (ve), muon (vμ), and tauon (v𝜏). Each neutrino is a decay product that is produced with its namesake particle; for example, ve is produced alongside an electron during the decay process.⁹ Neutrino oscillations were also proposed after these results, stating that if a given type of neutrino is produced during decay, then at a certain distance from that spot, the chance of observing that neutrino with the properties of a different flavor becomes non-zero.² Essentially, if ve is produced, then at a sufficient distance, the neutrino may become either vμ or v𝜏. This is caused by a discrepancy in the flavor and mass eigenstates of neutrinos.

In addition to these neutrino flavor states, there are also three mass eigenstates, or states in which neutrinos have definite mass. Through experimental evidence, these two different states represent two properties of neutrinos. As a result, neutrinos of the same flavor can be of different masses. For example, two electron neutrinos will have the same definite flavor, but not necessarily the same definite mass state. It is this discrepancy in the masses of these particles that actually leads to their ability to oscillate between flavors with the probability function given by the formula P(ab) = sin2(2q)sin2(1.27Dm2LvEv-1), where a and b are two flavors, q is the mixing angle, Dm is the difference in the mass eigenstate values of the two different neutrino flavors, L is the distance from source to detector, and E is the energy of the neutrino.⁶ Thus, each flavor is a different linear combination of the three states of definite mass.

The equation introduces the important concept of the mixing angle, which defines the difference between flavor and mass states and accounts for neutrino flavor oscillations. Thus, if the mixing angle were zero, this would imply that the mass states and and flavor states were the same and therefore no oscillations could occur. For example, all muon neutrinos produced at a source would still be muon neutrinos when P(mb) = 0. On the other hand, at a mixing angle of π/4, when P(mb) = 1, all muon neutrinos would oscillate to the other flavors in the probability function.⁹

Anomalous Data

Some experimental data has countered the notion of three neutrino flavor oscillations.³ If the experimental interpretation is correct, it would point to the existence of a fourth or even an additional fifth mass state, opening up the possibility of other mass states that can be taken by the hypothesised sterile neutrino. The most conclusive anomalous data arises from the Liquid Scintillator Neutrino Detector (LSND) Collaboration and MiniBooNE. The LSND Collaboration at Los Alamos National Laboratory looked for oscillations between vm neutrinos produced from muon decay and ve neutrinos. The results showed a lower-than-expected probability of oscillation.⁶ These results highly suggest either an oscillation to another neutrino flavor. A subsequent experiment at Fermilab called the mini Booster Neutrino Experiment (MiniBooNE) again saw a discrepancy between predicted and observed values of ve appearance with an excess of ve events.⁷ All of these results have a low probability of fit when compared to the standard model of particle physics, which gives more plausibility to the hypothesis of the existence of more than three neutrino flavors.

GALLEX, an experiment measuring neutrino emissions from the sun and chromium-51 neutrino sources, as well as reactor neutrino experiments gave inconsistent data that did not coincide with the standard model’s predictions for neutrinos. This evidence merely suggests the presence of these new particles, but does not provide conclusive evidence for their existence.^4,5 Thus, scientists designed a new project at the South Pole to search specifically for newly hypothesized sterile neutrinos.

IceCube Studies

IceCube, a particle physics laboratory, was designed specifically for collecting data concerning sterile neutrinos. In order to collect conclusive data about the neutrinos, IceCube’s vast resources and acute precision allow it to detect and register a large number of trials quickly. Neutrinos that come into contact with IceCube’s detectors are upgoing atmospheric neutrinos and thus have already traversed the Earth. This allows a fraction of the neutrinos to pass through the Earth’s core. If sterile neutrinos exist, then the large gravitational force of the Earth’s core should cause some muon neutrinos that traverse it to oscillate into sterile neutrinos, resulting in fewer muon neutrinos detected than expected in a model containing only three standard mass states, and confirming the existence of a fourth flavor.³

For these particles that pass upward through IceCube’s detectors, the Earth filters out the charged subatomic particle background noise, allowing only the detection of muons (the particles of interest) from neutrino interactions. The small fraction of upgoing atmospheric neutrinos that enter the ice surrounding the detector site will undergo reactions with the bedrock and ice to produce muons. These newly created muons then traverse the ice and react again to produce Cherenkov light, a type of electromagnetic radiation, that is finally able to be detected by the Digital Optical Modules (DOMs) of IceCube. This radiation is produced when a particle having mass passes through a substance faster than light can pass through that same substance.⁸

In 2011-2012, a study using data from the full range of DOMs, rather than just a portion, was conducted.⁸ This data, along with other previous data, were examined in order to search for conclusive evidence of sterile neutrino oscillations in samples of atmospheric neutrinos. Experimental data were compared to a Monte Carlo simulation. For each hypothesis of the makeup of the sterile neutrino, the Poissonian log likelihood, a probability function that finds the best correlation of experimental data to a hypothetical model, was calculated. Based on the results shown in Figure 2, no evidence points towards sterile neutrinos.⁸

Conclusion

Other studies have also been conducted at IceCube, and have also found no indication of sterile neutrinos. Although there is strong evidence against the existence of sterile neutrinos, this does not completely rule out their existence. These experiments have focused only on certain mixing angles and may have different results for different mixing angles. Also, if sterile neutrinos are conclusively found to be nonexistent by IceCube, there is still the question of why the anomalous data appeared at LSND and MiniBooNE. Thus, IceCube will continue sterile neutrino experiments at variable mixing angles to search for an explanation to the anomalies observed in the previous neutrino experiments.

References

1. Fukuda, Y. et al. Evidence for Oscillation of Atmospheric Neutrinos. Phys. Rev. Lett. 1998, 81, 1562.
2. Beringer, J. et al. Review of Particle Physics. Phys. Rev. D. 2012, 86, 010001.
3. Schmitz, D. W. Viewpoint: Hunting the Sterile Neutrino. Physics. [Online] 2016, 9, 94. https://physics.aps.org/articles/pdf/10.1103/Physics.9.94
4. Hampel, W. et al. Final Results of the 51Cr Neutrino Source Experiments in GALLEX. Phys. Rev. B. 1998, 420, 114.
5. Mention, G. et al. Reactor Antineutrino Anomaly. Phys. Rev. D. 2011, 83, 073006.
6. Aguilar-Arevalo, A. A. et al. Evidence for Neutrino Oscillations for the Observation of ve Appearance in a vμ Beam. Phys. Rev. D. 2001, 64, 122007.
7. Aguilar-Arevalo, A. A. et al. Phys. Rev. Lett. 2013, 110, 161801.
8. Aartsen, M. G. et al. Searches for Sterile Neutrinos with the IceCube Detector. Phys. Rev. Lett. 2016, 117, 071801.