Scanning Electrochemical Microscopy of Human Bladder Cancer (T24) Cells

Scanning electrochemical microscopy (SECM) employs a biased ultramicroelectrode (few nm to 25 µm diameter) as a probe that is scanned over a sample to electrochemically characterize its physical properties and chemical reactivity with high temporal and spatial resolutions. In this dissertation, SECM was used to investigate the membrane responses of single live human bladder cancer (T24) cells to obtain insights into their topography, physiology and pathology. First, the membrane-impermeable ferrocene carboxylate was used as the SECM redox mediator to investigate the geometry and topography of these cells along with 3D finite elemental analysis (FEA) simulations. The use of 3D simulation models now allows characterization of asymmetric samples, previously restricted to a 2D axially symmetric geometry. Upon exposure to toxic and non-essential Cd2+ (mM range), the membrane permeability to the hydrophobic mediator, ferrocenemethanol, in T24 cells increased within minutes. Time-lapsed SECM was able to measure and quantify these changes in membrane permeability. Expanding on this work, T24 cells were exposed to low exposure (25 µM, up to 6 hours) or 1 hour of acute Cd2+ concentrations. Specifically, the permeation of hydrophilic redox mediators: ferrocene carboxylate, ferrocene dicarboxylate, and hexaamineruthenium(III), into the Cd2+-treated T24 cells confirmed membrane integrity loss. In conjunction with FEA, the membrane permeability coefficients were quantified. A correlation between cellular loss and membrane integrity was confirmed using MTT cell proliferation assays. Exposure to the trace essential Zn2+ (0-75 µM, 24 hours) did not significantly affect the cell membranes of T24 cells. Higher Zn2+-treatment led to cytotoxicity, where membrane integrity loss (increased permeation) and cellular death were observed (100-400 µM). The above discoveries suggested the Zn2+-induced apoptosis in these cells. This was confirmed by several apoptotic indicators, such as the externalization of phosphatidylserine and activation of caspases 3 and 7. Finally, to improve the spatial resolution of SECM, a fabrication method of nanometer-sized probes was successfully developed. This led to the reduction of Pt electrode disk diameters from 25 µm to 25 nm. Visualization of reactivity features of nanometer-sized samples using SECM is anticipated with this advancement

Germline Cas9 expression yields highly efficient genome engineering in a major worldwide disease vector, Aedes aegypti.

The development of CRISPR/Cas9 technologies has dramatically increased the accessibility and efficiency of genome editing in many organisms. In general, in vivo germline expression of Cas9 results in substantially higher activity than embryonic injection. However, no transgenic lines expressing Cas9 have been developed for the major mosquito disease vector Aedes aegypti Here, we describe the generation of multiple stable, transgenic Ae. aegypti strains expressing Cas9 in the germline, resulting in dramatic improvements in both the consistency and efficiency of genome modifications using CRISPR. Using these strains, we disrupted numerous genes important for normal morphological development, and even generated triple mutants from a single injection. We have also managed to increase the rates of homology-directed repair by more than an order of magnitude. Given the exceptional mutagenic efficiency and specificity of the Cas9 strains we engineered, they can be used for high-throughput reverse genetic screens to help functionally annotate the Ae. aegypti genome. Additionally, these strains represent a step toward the development of novel population control technologies targeting Ae. aegypti that rely on Cas9-based gene drives

Reachable set-based dynamic quantization for the remote state estimation of linear systems

We employ reachability analysis in designing dynamic quantization schemes for the remote state estimation of linear systems over a finite date rate communication channel. The quantization region is dynamically updated at each transmission instant, with an approximated reachable set of the linear system. We propose a set-based method using zonotopes and compare it to a norm-based method in dynamically updating the quantization region. For both methods, we guarantee that the quantization error is bounded and consequently, the remote state reconstruction error is also bounded. To the best of our knowledge, the set-based method using zonotopes has no precedent in the literature and admits a larger class of linear systems and communication channels, where the set-based method allows for a longer inter-transmission time and lower bit rate. Finally, we corroborate our theoretical guarantees with a numerical example.Comment: This manuscript was accepted for publication at the 62nd IEEE Conference on Decision and Control (CDC), 202

Fibulin-4 is essential for maintaining arterial wall integrity in conduit but not muscular arteries

Homozygous or compound heterozygous mutations in fibulin-4 (FBLN4) lead to autosomal recessive cutis laxa type 1B (ARCL1B), a multisystem disorder characterized by significant cardiovascular abnormalities, including abnormal elastin assembly, arterial tortuosity, and aortic aneurysms. We sought to determine the consequences of a human disease-causing mutation in FBLN4 (E57K) on the cardiovascular system and vascular elastic fibers in a mouse model of ARCL1B. Fbln4E57K/E57K mice were hypertensive and developed arterial elongation, tortuosity, and ascending aortic aneurysms. Smooth muscle cell organization within the arterial wall of large conducting vessels was abnormal, and elastic fibers were fragmented and had a moth-eaten appearance. In contrast, vessel wall structure and elastic fiber integrity were normal in resistance/muscular arteries (renal, mesenteric, and saphenous). Elastin cross-linking and total elastin content were unchanged in large or small arteries, whereas elastic fiber architecture was abnormal in large vessels. While the E57K mutation did not affect Fbln4 mRNA levels, FBLN4 protein was lower in the ascending aorta of mutant animals compared to wild-type arteries but equivalent in mesenteric arteries. We found a differential role of FBLN4 in elastic fiber assembly, where it functions mainly in large conduit arteries. These results suggest that elastin assembly has different requirements depending on vessel type. Normal levels of elastin cross-links in mutant tissue call into question FBLN4\u27s suggested role in mediating lysyl oxidase-elastin interactions. Future studies investigating tissuespecific elastic fiber assembly may lead to novel therapeutic interventions for ARCL1B and other disorders of elastic fiber assembly. 2017 © The Authors, some rights reserved

Tumor Angiogenesis as a Target for Dietary Cancer Prevention

Between 2000 and 2050, the number of new cancer patients diagnosed annually is expected to double, with an accompanying increase in treatment costs of more than $80 billion over just the next decade. Efficacious strategies for cancer prevention will therefore be vital for improving patients' quality of life and reducing healthcare costs. Judah Folkman first proposed antiangiogenesis as a strategy for preventing dormant microtumors from progressing to invasive cancer. Although antiangiogenic drugs are now available for many advanced malignancies (colorectal, lung, breast, kidney, liver, brain, thyroid, neuroendocrine, multiple myeloma, myelodysplastic syndrome), cost and toxicity considerations preclude their broad use for cancer prevention. Potent antiangiogenic molecules have now been identified in dietary sources, suggesting that a rationally designed antiangiogenic diet could provide a safe, widely available, and novel strategy for preventing cancer. This paper presents the scientific, epidemiologic, and clinical evidence supporting the role of an antiangiogenic diet for cancer prevention

Graphene Oxidation: Thickness Dependent Etching and Strong Chemical Doping

Patterned graphene shows substantial potential for applications in future molecular-scale integrated electronics. Environmental effects are a critical issue in a single layer material where every atom is on the surface. Especially intriguing is the variety of rich chemical interactions shown by molecular oxygen with aromatic molecules. We find that O2 etching kinetics vary strongly with the number of graphene layers in the sample. Three-layer-thick samples show etching similar to bulk natural graphite. Single-layer graphene reacts faster and shows random etch pits in contrast to natural graphite where nucleation occurs at point defects. In addition, basal plane oxygen species strongly hole dope graphene, with a Fermi level shift of ~0.5 eV. These oxygen species partially desorb in an Ar gas flow, or under irradiation by far UV light, and readsorb again in an O2 atmosphere at room temperature. This strongly doped graphene is very different than graphene oxide made by mineral acid attack.Comment: 15 pages, 5 figure

Live calcium imaging of Aedes aegypti neuronal tissues reveals differential importance of chemosensory systems for life-history-specific foraging strategies.

BackgroundThe mosquito Aedes aegypti has a wide variety of sensory pathways that have supported its success as a species as well as a highly competent vector of numerous debilitating infectious pathogens. Investigations into mosquito sensory systems and their effects on behavior are valuable resources for the advancement of mosquito control strategies. Numerous studies have elucidated key aspects of mosquito sensory systems, however there remains critical gaps within the field. In particular, compared to that of the adult form, there has been a lack of studies directed towards the immature life stages. Additionally, although numerous studies have pinpointed specific sensory receptors as well as responding motor outputs, there has been a lack of studies able to monitor both concurrently.ResultsTo begin filling aforementioned gaps, here we engineered Ae. aegypti to ubiquitously express a genetically encoded calcium indicator, GCaMP6s. Using this strain, combined with advanced microscopy, we simultaneously measured live stimulus-evoked calcium responses in both neuronal and muscle cells with a wide spatial range and resolution.ConclusionsBy coupling in vivo live calcium imaging with behavioral assays we were able to gain functional insights into how stimulus-evoked neural and muscle activities are represented, modulated, and transformed in mosquito larvae enabling us to elucidate mosquito sensorimotor properties important for life-history-specific foraging strategies

Stress, Motivation and Professional Satisfaction among Health Care Workers in HIV/AIDS Care and Treatment Centers in Urban Tanzania: A Cross-Sectional Study.

Shortages of health care workers (HCWs) represents a serious challenge to ensuring effective HIV care in resource-limited settings (RLS). Stress, motivation, and job satisfaction have been linked with HCW retention and are important in addressing HCW shortages. In this cross-sectional study HCW stress, motivation and perceived ability to meet patient needs were assessed in PEPFAR-supported urban HIV care and treatment clinics (CTCs) in Tanzania. A self-administered questionnaire measuring motivation, stress, and perceived ability to and meet patient needs was given to HCWs at 16 CTCs. Scales measuring HCW satisfaction, motivation, and stress were developed using principle components analysis. Hierarchical linear models were used to explore the association of HCW and site characteristics with reported satisfaction, stress, motivation, and ability to meet patients' needs.\ud Seventy-three percent (279) of HCWs completed the questionnaire. Most (73%) HCWs reported minimal/no work-related stress, with 48% reporting good/excellent motivation, but 41% also reporting feeling emotionally drained. Almost all (98%) reported feeling able to help their patients, with 68% reporting work as rewarding. Most reported receipt of training and supervision, with good availability of resources. In the multivariate model, direct clinical providers reported lower motivation than management (p < 0.05) and HCWs at medium-sized sites reported higher motivation than HCWs at larger sites (p < 0.05). HCWs at small and medium sites were more likely to feel able to help patients than those from larger sites (p < 0.05 and p < 0.001 respectively). Despite significant patient loads, HCWs in these PEPFAR-supported CTCs reported high levels of motivation, job satisfaction, ability to meet patients' needs, low levels of stress but significant emotional toll. Understanding the relationship between support systems such as strong supervision and training and these outcomes is critical in designing interventions to improve motivation, reduce stress and increase retention of HCWs

Do ultrafast exciton-polaron decoherence dynamics govern photocarrier generation efficiencies in polymer solar cells?

All-organic-based photovoltaic solar cells have attracted considerable attention because of their low-cost processing and short energy payback time. In such systems the primary dissociation of an optical excitation into a pair of photocarriers has been recently shown to be extremely rapid and efficient, but the physical reason for this remains unclear. Here, two-dimensional photocurrent excitation spectroscopy, a novel non-linear optical spectroscopy, is used to probe the ultrafast coherent decay of photoexcitations into charge-producing states in a polymer:fullerene based solar cell. The two-dimensional photocurrent spectra are interpreted by introducing a theoretical model for the description of the coupling of the electronic states of the system to an external environment and to the applied laser fields. The experimental data show no cross-peaks in the two-dimensional photocurrent spectra, as predicted by the model for coherence times between the exciton and the photocurrent producing states of 20\,fs or less