Design of Antibacterial Prochelators to Target Drug-Resistant Bacteria

Transition metals such as iron and copper are valued in biology for their redox activities because they are able to access various oxidation states. However, these transition metals are also implicated in a number of human disease states and play a role in bacterial infections. The ability to manipulate and monitor metal ions has vast implications on the fields of biology and human health. As such, the research described here covers two related goals: to manipulate metals in specific biological circumstances and to visualize this disturbance in cellular metal homeostasis.

Antibiotic resistance necessitates the development of drugs that exploit new mechanisms of action such as the disruption of metal homeostasis. In order to manipulate metals at the site of bacterial infection, two prochelators were developed around a β-lactam core such that the active chelator is released in the presence of bacteria that produce the resistance-causing β-lactamase enzyme. Both prochelators display enhanced activity toward resistant bacteria compared to clinical antibiotics.

Fluorescent sensors are a powerful tool for detecting small concentrations of biological analytes. Two analogs of a ratiometric fluorescent sensor were designed and synthesized to monitor cellular concentrations of copper and iron. These sensors were found to operate as designed in vitro; however the fluorescence intensity necessary for quantification of cellular metal pools has not yet been achieved.

STAR 3 randomized controlled trial to compare sensor-augmented insulin pump therapy with multiple daily injections in the treatment of type 1 diabetes: research design, methods, and baseline characteristics of enrolled subjects.

BACKGROUND: Sensor-augmented pump therapy (SAPT) integrates real-time continuous glucose monitoring (RT-CGM) with continuous subcutaneous insulin infusion (CSII) and offers an alternative to multiple daily injections (MDI). Previous studies provide evidence that SAPT may improve clinical outcomes among people with type 1 diabetes. Sensor-Augmented Pump Therapy for A1c Reduction (STAR) 3 is a multicenter randomized controlled trial comparing the efficacy of SAPT to that of MDI in subjects with type 1 diabetes. METHODS: Subjects were randomized to either continue with MDI or transition to SAPT for 1 year. Subjects in the MDI cohort were allowed to transition to SAPT for 6 months after completion of the study. SAPT subjects who completed the study were also allowed to continue for 6 months. The primary end point was the difference between treatment groups in change in hemoglobin A1c (HbA1c) percentage from baseline to 1 year of treatment. Secondary end points included percentage of subjects with HbA1c < or =7% and without severe hypoglycemia, as well as area under the curve of time spent in normal glycemic ranges. Tertiary end points include percentage of subjects with HbA1c < or =7%, key safety end points, user satisfaction, and responses on standardized assessments. RESULTS: A total of 495 subjects were enrolled, and the baseline characteristics similar between the SAPT and MDI groups. Study completion is anticipated in June 2010. CONCLUSIONS: Results of this randomized controlled trial should help establish whether an integrated RT-CGM and CSII system benefits patients with type 1 diabetes more than MDI.

Thermodynamic analysis of ligand-induced changes in protein thermal unfolding applied to high-throughput determination of ligand affinities with extrinsic fluorescent dyes.

The quantification of protein-ligand interactions is essential for systems biology, drug discovery, and bioengineering. Ligand-induced changes in protein thermal stability provide a general, quantifiable signature of binding and may be monitored with dyes such as Sypro Orange (SO), which increase their fluorescence emission intensities upon interaction with the unfolded protein. This method is an experimentally straightforward, economical, and high-throughput approach for observing thermal melts using commonly available real-time polymerase chain reaction instrumentation. However, quantitative analysis requires careful consideration of the dye-mediated reporting mechanism and the underlying thermodynamic model. We determine affinity constants by analysis of ligand-mediated shifts in melting-temperature midpoint values. Ligand affinity is determined in a ligand titration series from shifts in free energies of stability at a common reference temperature. Thermodynamic parameters are obtained by fitting the inverse first derivative of the experimental signal reporting on thermal denaturation with equations that incorporate linear or nonlinear baseline models. We apply these methods to fit protein melts monitored with SO that exhibit prominent nonlinear post-transition baselines. SO can perturb the equilibria on which it is reporting. We analyze cases in which the ligand binds to both the native and denatured state or to the native state only and cases in which protein:ligand stoichiometry needs to treated explicitly.

Identification of fluorescent beads using a coded aperture snapshot spectral imager.

We apply a coded aperture snapshot spectral imager (CASSI) to fluorescence microscopy. CASSI records a two-dimensional (2D) spectrally filtered projection of a three-dimensional (3D) spectral data cube. We minimize a convex quadratic function with total variation (TV) constraints for data cube estimation from the 2D snapshot. We adapt the TV minimization algorithm for direct fluorescent bead identification from CASSI measurements by combining a priori knowledge of the spectra associated with each bead type. Our proposed method creates a 2D bead identity image. Simulated fluorescence CASSI measurements are used to evaluate the behavior of the algorithm. We also record real CASSI measurements of a ten bead type fluorescence scene and create a 2D bead identity map. A baseline image from filtered-array imaging system verifies CASSI's 2D bead identity map.

An energy-efficient data delivery scheme for delay-sensitive traffic in wireless sensor networks

We propose a novel data-delivery method for delay-sensitive traffic that significantly reduces the energy consumption in wireless sensor networks without reducing the number of packets that meet end-to-end real-time deadlines. The proposed method, referred to as SensiQoS, leverages the spatial and temporal correlation between the data generated by events in a sensor network and realizes energy savings through application-specific in-network aggregation of the data. SensiQoS maximizes energy savings by adaptively waiting for packets from upstream nodes to perform in-network processing without missing the real-time deadline for the data packets. SensiQoS is a distributed packet scheduling scheme, where nodes make localized decisions on when to schedule a packet for transmission to meet its end-to-end real-time deadline and to which neighbor they should forward the packet to save energy. We also present a localized algorithm for nodes to adapt to network traffic to maximize energy savings in the network. Simulation results show that SensiQoS improves the energy savings in sensor networks where events are sensed by multiple nodes, and spatial and/or temporal correlation exists among the data packets. Energy savings due to SensiQoS increase with increase in the density of the sensor nodes and the size of the sensed events. © 2010 Harshavardhan Sabbineni and Krishnendu Chakrabarty.

Bioluminescence imaging of glucose in tissue surrounding polyurethane and glucose sensor implants.

BACKGROUND: The bioluminescence technique was used to quantify the local glucose concentration in the tissue surrounding subcutaneously implanted polyurethane material and surrounding glucose sensors. In addition, some implants were coated with a single layer of adipose-derived stromal cells (ASCs) because these cells improve the wound-healing response around biomaterials. METHODS: Control and ASC-coated implants were implanted subcutaneously in rats for 1 or 8 weeks (polyurethane) or for 1 week only (glucose sensors). Tissue biopsies adjacent to the implant were immediately frozen at the time of explant. Cryosections were assayed for glucose concentration profile using the bioluminescence technique. RESULTS: For the polyurethane samples, no significant differences in glucose concentration within 100 μm of the implant surface were found between bare and ASC-coated implants at 1 or 8 weeks. A glucose concentration gradient was demonstrated around the glucose sensors. For all sensors, the minimum glucose concentration of approximately 4 mM was found at the implant surface and increased with distance from the sensor surface until the glucose concentration peaked at approximately 7 mM at 100 μm. Then the glucose concentration decreased to 5.5-6.5 mM more than 100 μmm from the surface. CONCLUSIONS: The ASC attachment to polyurethane and to glucose sensors did not change the glucose profiles in the tissue surrounding the implants. Although most glucose sensors incorporate a diffusion barrier to reduce the gradient of glucose and oxygen in the tissue, it is typically assumed that there is no steep glucose gradient around the sensors. However, a glucose gradient was observed around the sensors. A more complete understanding of glucose transport and concentration gradients around sensors is critical.

Intravital microscopy evaluation of angiogenesis and its effects on glucose sensor performance.

An optical window model for the rodent dorsum was used to perform chronic and quantitative intravital microscopy and laser Doppler flowmetry of microvascular networks adjacent to functional and non-functional glucose sensors. The one-sided configuration afforded direct, real-time observation of the tissue response to bare (unmodified, smooth surface) sensors and sensors coated with porous poly-L-lactic acid (PLLA). Microvessel length density and red blood cell flux (blood perfusion) within 1 mm of the sensors were measured bi-weekly over 2 weeks. When non-functional sensors were fully implanted beneath the windows, the porous coated sensors had two-fold more vasculature and significantly higher blood perfusion than bare sensors on Day 14. When functional sensors were implanted percutaneously, as in clinical use, no differences in baseline current, neovascularization, or tissue perfusion were observed between bare and porous coated sensors. However, percutaneously implanted bare sensors had two-fold more vascularity than fully implanted bare sensors by Day 14, indicating the other factors, such as micromotion, might be stimulating angiogenesis. Despite increased angiogenesis adjacent to percutaneous sensors, modest sensor current attenuation occurred over 14 days, suggesting that factors other than angiogenesis may play a dominant role in determining sensor function.

Improving Indwelling Glucose Sensor Performance: Porous, Dexamethasone-Releasing Coatings that Modulate the Foreign Body Response

Inflammation and the formation of an avascular fibrous capsule have been identified as the key factors controlling the wound healing associated failure of implantable glucose sensors. Our aim is to guide advantageous tissue remodeling around implanted sensor leads by the temporal release of dexamethasone (Dex), a potent anti-inflammatory agent, in combination with the presentation of a stable textured surface.

First, Dex-releasing polyurethane porous coatings of controlled pore size and thickness were fabricated using salt-leaching/gas-foaming technique. Porosity, pore size, thickness, drug release kinetics, drug loading amount, and drug bioactivity were evaluated. In vitro sensor functionality test were performed to determine if Dex-releasing porous coatings interfered with sensor performance (increased signal attenuation and/or response times) compared to bare sensors. Drug release from coatings monitored over two weeks presented an initial fast release followed by a slower release. Total release from coatings was highly dependent on initial drug loading amount. Functional in vitro testing of glucose sensors deployed with porous coatings against glucose standards demonstrated that highly porous coatings minimally affected signal strength and response rate. Bioactivity of the released drug was determined by monitoring Dex-mediated, dose-dependent apoptosis of human peripheral blood derived monocytes in culture.

The tissue modifying effects of Dex-releasing porous coatings were accessed by fully implanting Tygon® tubing in the subcutaneous space of healthy and diabetic rats. Based on encouraging results from these studies, we deployed Dex-releasing porous coatings from the tips of functional sensors in both diabetic and healthy rats. We evaluated if the tissue modifying effects translated into accurate, maintainable and reliable sensor signals in the long-term. Sensor functionality was accessed by continuously monitoring glucose levels and performing acute glucose challenges at specified time points.

Sensors treated with porous Dex-releasing coatings showed diminished inflammation and enhanced vascularization of the tissue surrounding the implants in healthy rats. Functional sensors with Dex-releasing porous coatings showed enhanced sensor sensitivity over a 21-day period when compared to controls. Enhanced sensor sensitivity was accompanied with an increase in sensor signal lag and MARD score. These results indicated that Dex-loaded porous coatings were able to elicit a favorable tissue response, and that such tissue microenvironment could be conducive towards extending the performance window of glucose sensors in vivo.

The diabetic pilot animal study showed differences in wound healing patters between healthy and diabetic subjects. Diabetic rats showed lower levels of inflammation and vascularization of the tissue surrounding implants when compared to their healthy counterparts. Also, functional sensors treated with Dex-releasing porous coatings did not show enhanced sensor sensitivity over a 21-day period. Moreover, increased in sensor signal lag and MARD scores were present in porous coated sensors regardless of Dex-loading when compared to bare implants. These results suggest that the altered wound healing patterns presented in diabetic tissues may lead to premature sensor failure when compared to sensors implanted in healthy rats.