Current research activities are listed below.
Engineering Non-Natural Fluorinases for the Synthesis of Therapeutic and Diagnostic Organofluorines
Using a combination of chemistry-inspired rational engineering and evolution in the laboratory, we have developed two new biocatalytic approaches to incorporating fluorine into organic molecules. We have engineered new enzymes for in vivo and in vitro synthesis of organosilicon compounds, a class of molecules with medical applications such as positron emission tomography (PET) imaging through site-specific 18F-labeling. Ours is the first biocatalyst capable of carbon-silicon bond formation. The technology provides access to organosilicon compounds that are not readily accessible otherwise, opening new opportunities for fluorine-incorporation and labeling studies that are biologically relevant. This breakthrough was published in 2016 in Science. We have also developed a method for introducing trifluoromethyl groups to organic molecules biocatalytically. This transformation is unknown in nature and will find applications in the preparation of pharmaceuticals and agrochemicals.
Inhalation Dosimetry for Ultrafine Particles
This project had, as the primary focus, the development new low cost sensors capable of assessment of the dose to different regions of the airways when fine particles are inhaled. Present ambient air quality measurements focus on exposure rather than dose. The primary exposure measurement today is PM2.5, the mass concentration of particles smaller than 2.5 µm aerodynamic diameter. Air quality standards are written in terms of this metric, which also serves as the primary measure of fine particle exposure in many research studies. Many particles in the size range, when inhale remain suspended in the air and are exhaled without depositing. Thus, PM2.5 measurements report an amount particulate matter that is larger than deposited in the airways.
Nonetheless, a statistical links between PM2.5 exposure and number of adverse health outcomes are well-established. What is not clear, is an appropriate measure of dose in the effort to understand the origins or causes of adverse health outcomes. Numerous studies have suggested alternative metrics. In studies of the potential health consequences of workplace exposures in the burgeoning nanotechnology industry have suggested that deposited surface area may be a more relevant measure of dose to sensitive regions of airways, at least for some varieties of engineered nanoparticles. A number of instruments have been developed of lung- deposited surface area, using the rate relationship between particle charging and surface area to infer the area of particles that may deposit in a system in which deposition efficiency is intentionally biased to match the deposition profile of the respiratory tract. Several other studies have measured the aerosol number concentration in order to infer the number of fine particles that might deposit within the airways, and have found strong associations with short- term responses, on the order of hours or less, particularly with respect to a heart rate variability. The preponderance of data on fine particle exposures report PM2.5; lesser amounts provide other measures of exposure. Optical dust sensors (low cost optical particle counters) are increasingly being used to provide a surrogate for PM2.5 though they are insensitive to ultrafine particles, even in the rare instances when they account for significant aerosol mass. Condensation particle counter measurements of the number concentration are fairly common in air quality studies.
The working hypothesis of this study is that data that enables estimation of the dose in terms of these different metrics that is delivered to different regions of the airways will enhance the ability of epidemiological and other health effects studies to examine physiological mechanisms behind adverse health outcomes and to discriminate between different sources of airborne fine particles. Furthermore, the power of such studies will be greatly enhanced if instruments can be made sufficiently small, unobtrusive, and low cost that they can be deployed as personal dosimeters in cohort studies, or in dense networks in community health studies. All three measures of dose of inhaled fine particles can be provided today using a combination of particle size distribution data obtained by commercially available differential mobility analyzers (DMAs) and established lung deposition models that predict the inhaled-particle deposition efficiency inmdifferent regions of the human airways as a function of particle size. Present DMAs are, however, far to large, complex, and expensive to satisfy the latter constraints.
This study has examined how this class of measurements could be adapted to meet the needs of the health effects research community. Using atmospheric simulations of major pollution events and validated models of DMAs and other aerosol instruments, we have demonstrated that biases in estimation of regional airway deposition using size distribution data are minimal, even when the instruments have much lower size resolution than is needed for many atmospheric science application. Moreover, we have examined the biases that associated with present-day fine particle exposure measurements. Only size-resolved measurements provide a useful correlation with all three metrics.
The ability to make measurements at relatively low size resolution relaxes many design constrains, opening the door to dramatic reductions in instrument cost. By applying these simplified design specifications to the opposed migration aerosol classifier (OMAC), a new form of differential mobility analyzer that allows instrument miniaturization, this project has focused on the development of fine aerosol particle dosimetry instruments that will enable the aforementioned improvements in the health effects of fine particle exposures. Over the past year, we have demonstrated that an OMAC that was designed based on computational simulations performs as predicted, and explored several alternative approaches to the design of the low cost, size-resolving fine particle sensor. In side-by-side comparisons with a conventional, high resolution scanning mobility particle sizer (SMPS), a new, low-size-resolution OMAC effectively captured rapid transients in exposure and dose of fine airborne particles.
Additional refinements are being implemented in new low-cost designs, that will further simplify the instruments toward our objective of enabling scientifically valid measurement of exposure and dose of ultrafine particles in personal monitors and community air quality monitors.
Toward a rapid test of antibiotic resistance
The goal of this work is to answer fundamental scientific questions to develop a point of care (POC) test of antibiotic susceptibility that is amenable to limited-resource settings (LRS).
Ideally, an antibiotic susceptibility test (AST) should be able to be performed within a single doctor visit. To achieve such a short (30 min) phenotypic test requires determining a pathogen’s susceptibility after only a short antibiotic exposure, performing all of the sample-handling steps and using an ultra-fast assay to acquire the final readout in a very short time frame.
We have had great success meeting these requirements. Primarily using clinical isolates and clinical samples provided by our collaborators at UCLA, we have validated digital single molecule counting as a superior way to detect antibiotic susceptibility. We first used digital PCR (dPCR) to test whether assessing DNA replication of the target pathogen via digital single-molecule counting could be used to shorten the required antibiotic exposure time, thus decreasing the overall time of the assay. We have found that partitioning bacterial chromosomal DNA into many small volumes during dPCR enabled AST via (i) precise quantification and (ii) a measure of how antibiotics affect the states of macromolecular assembly of bacterial chromosomes. This digital AST (dAST) determined susceptibility of clinical isolates from urinary tract infections (UTI) after just 15 min of exposure for all four antibiotic classes relevant to UTI. This work was published in Angewandte Chemie (Schoepp et al. 2016).
Our work proves our digital AST method can work directly on clinical samples for several types of antibiotics. We also show in this article that we have discovered a new mechanism for rapidly detecting beta-lactam antibiotic susceptibility: chromosome segregation. More recently, we have demonstrated that our dAST method can yield a sample-to-answer result from a clinical urine sample in as few as 30 min! In this work, published in Science Translational Medicine (Schoepp et al. 2017), we demonstrate that we can shorten the time of antibiotic exposure to 15 min, the time for sample prep to 3 min, and that we can then quantify with digital LAMP in 6 min.
Thus far, the work being done with JIMEM support is laying a strong foundation to develop a rapid, point-of-care AST and strengthen global antibiotic stewardship.
Thinner, radiopaque bioresorbable vascular scaffolds for the treatment of coronary heart disease
Bioresorbable vascular scaffolds (BVSs) are a promising new treatment for coronary heart disease (CHD), the leading cause of death in the world (>7 million/year). Unlike permanent metal stents, BVSs are transient implants; they support the artery for the requisite 6 months, at which time the blood vessel tissue has filled the scaffold and the epithelium has covered its luminal surface. Within 2 years, the BVS is completely resorbed and the treated vessels are observed to regain vasomotion and vasodilation (virtually eliminating angina symptoms, which are prevalent and persistent in stented patients). The transient character of the BVS overcomes the most dreaded complication associated with metal stents—late stent thrombosis. Surgeons advocate two main improvements to foster wider adoption and serve a broader cross-section of patients: reduce the BVS profile to enable treatment of smaller arteries and more complex lesions, and increase x-ray opacity to permit visualization during and after implantation.
We proposed to tackle the dual challenge of a thinner yet more radio-opaque BVS by reinforcing it with inorganic nanotubes that have high x-ray scattering power. Specifically, we will focus on tungsten disulfide (WS2) nanotubes (NT) that confer a substantial increase in strength, have shown low toxicity in vitro, and have radio-opacity comparable to platinum, the current standard for radio-opaque markers. The key to a thinner, radio-opaque BVS made from PLLA-WSNT is the control of the nanocomposite morphology during processing: extrusion to create a largely amorphous tubular preform; tube expansion to obtain a uniform wall thickness, laser-cutting to create the struts and rings of the scaffold (Fig. 1A), and crimping onto a balloon catheter (Fig. 1C) prior to deployment via inflation of the balloon (Fig. 1D). We will apply insight into the interplay of these processing steps to create a nanocomposite that has the potential to address both of the most pressing clinical needs in BVS—reduced scaffold profile and increased radio-opacity.
FIGURE: Processing of BVS: (A) struts (along z) and rings (along θ) are created by laser cutting the expanded tube (B); the “as cut” scaffold” is crimped onto a balloon catheter (C); the scaffold is deployed (D) via inflation of the balloon after it is positioned at the lesion.
Targeted Cellular Agents
The Shapiro Laboratory is developing methods to use penetrant forms of energy, such as ultrasound and magnetic fields, to control cellular function. This work entails engineering proteins and genetic circuits that can convert these forms of energy into cellular signals such as gene expression. One recent example of our work, funded in part by the Jacobs Institute, showed that bacteria could be engineered to respond remotely to MRI-guided focused ultrasound signals mediated by temperature.
Related Publication on Ultrasonic Control of Cellular Function:
Piraner DI, Abedi MH, Moser BA, Lee-Gosselin A, Shapiro M. G.* Tunable thermal bioswitches for in vivo control of microbial therapeutics. Nature Chemical Biology 13, 75-80 (2017).
FIGURE: Remote control of bacterial agents using focused ultrasound. (a) Illustration of the in vitro focused ultrasound experiment: focused ultrasound is used to heat a target area of a bacterial culture lawn through a tofu phantom (depicted as translucent) under MRI guidance, followed by fluorescent imaging. (b) MRI-based temperature map of the bacterial specimen during steady-state ultrasound application, overlaid on a raw grayscale MRI image of the phantom. (c) Fluorescent image of the region targeted by ultrasound, showing activation consistent with a bacterial construct expressing GFP under the control of TlpA36 and RFP regulated by TcI. (d) Illustration of the in vivo experiment, in which focused ultrasound is used to activate subcutaneously-injected bacterial agents at a specific anatomical site. (e) Representative thresholded fluorescence map of a mouse injected subcutaneously in both left and right hindlimbs with E. coli expressing GFP under the control of TlpA36, following ultrasound activation at only the right hindlimb. Scale bars 2 mm (b and c) and 1 cm (e).
The objective of our Jacobs Institute research is to elucidate the strategies used by methicillin-resistant Staphylococcus aureus (MRSA) to establish and sustain infection in the skin. Each year in the United States, roughly 90,000 people suffer invasive MRSA infections, and more than 20,000 die. Because the preferred niche of S. aureus is the human host, the pathogen has developed a powerful arsenal of mechanisms for manipulating the mammalian immune system. Staph now accounts for almost one-third of skin and bloodstream infections.
Over the past year, we have discovered a protein that increases the severity of skin infection by MRSA. This protein has not previously been associated with infection. Our current research is directed toward elucidating the mechanism by which the protein contributes to virulence, and to finding ways to treat infection by inhibiting the action of the protein.
FIGURE: Fluorescence image of MRSA infection in the mouse skin. Bacterial cells and proteins can be independently stained to provide unique insight into the nature of the infection.
Electrostatics of Polyelectrolyte Self Assembly
Many biomolecules are charged polymers, or polyelectrolytes – for example, the genetic material RNA and DNA have negatively charged backbones, while many intrinsically disordered proteins have mixed charges along their backbone. The charge on these biomolecules results in a wide range of self-assembly behaviors, from viral assembly to the formation of membraneless organelles, and can be leveraged to produce nanoparticles for the medical delivery of biomolecules. In this reporting period, we used our group’s variational theory of charged macromolecules to study the thermodynamics of polyelectrolyte phase separation in the presence of salt, which is always present under physiological conditions. The variational theory is able to describe how chain structure varies under changing solution conditions, and we show how failure to do so in previous theories leads to significant overestimates of the driving force for phase separation. Our work demonstrates how appropriately describing chain structure is critical to accurately predicting the complexation of polyelectrolytes.
FIGURE: Phase boundaries for symmetric solutions of polyecations and polyanions with added salt; dashed lines demarcate the metastability limit. We compare our theory (black), which accounts for changing chain structure over different concentrations, to the commonly-used fg-RPA (green) which assumes a Gaussian chain structure at all length scales, and rods (red). Note that the fg-RPA suggests phase separations can persist even up to the limit where the solution is very nearly all salt (blue dashed line). Inset shows the overly dilute low-concentration branch predicted by the fg-RPA.