Deep Brain Stimulation to Increase Generalized Arousal in Intact Mice and a Mouse Model of Traumatic Brain Injury

G protein-coupled receptors (GPCRs) are dynamic membrane proteins that bind extracellular molecules to transduce biological signals. Although GPCRs represent the largest class of targets for therapeutic agents, ligand-binding sites have been precisely defined for only a small percentage of the receptors in the human genome. A general cellbased photocrosslinking approach was developed to investigate the binding interfaces necessary for the formation of GPCR signaling complexes. Amber codon suppression was extended to facilitate the incorporation of photoactivatable unnatural amino acids, pbenzoyl- L-phenylalanine and p-azido-L-phenylalanine, into engineered GPCRs expressed in mammalian cells in culture. Proof-of-concept studies were carried out in chemokine receptors C-X-C chemokine receptor 4 (CXCR4) and C-C chemokine receptor 5 (CCR5), which are known HIV-1 co-receptors required for HIV-1 cellular entry in CD4+ cells. A cyclic peptide CXCR4-specific inhibitor, T140, photocrosslinked primarily to a specific site on CXCR4 and the result was reconciled with existing structural biology data. A small molecule drug, maraviroc, photocrosslinked to multiple sites on CCR5 and the results were extended to develop a computer homology model of the CCR5-maraviroc complex. In summary, the application of a novel targeted cellbased photocrosslinking strategy provided detailed information about receptor-ligand complexes in two chemokine receptors. The general approach described here using genetically-encoded photoreactive molecules to study the binding interactions between GPCRs and ligands represents a significant advance in the application of photocrosslinking reagents to address problems in biochemistry and pharmacology

Structure-activity relationships of synthetic analogs of jasmonic acid and coronatine on induction of benzo[c]phenanthridine alkaloid accumulation in Eschscholzia californica cell cultures

A facile test system based on the accumulation of benzo[c]phenanthridine alkaloids in Eschscholzia californica cell suspension culture (an indicator of defense gene activation) has been used to analyze a series of synthetic compounds for elicitor-like activity. Of the 200 jasmonic acid and coronatine analogs tested with this system, representative results obtained with 49 of them are presented here. The following can be summarized concerning structure-actvity relationships: there is a large degree of plasticity allowed at the C-3 of jasmonic acid in the activation of defense genes. The carbonyl moiety is not strictly required, but exocyclic double bond character appears necessary. The pentenyl side chain at C-2 cannot tolerate bulky groups at the terminal carbon and still be biologically active. Substitutions to the C-1' position are tolerated if they can potentially undergo beta-oxidation. Either an alkanoic acid or methyl ester is required at c-l, or a side chain that can be shortened by beta-oxidation or by peptidase hydrolysis. Coronatine and various derivatives thereof are not as effective as jasmonic acid, and derivatives in inducing benzo[c]phenanthridine alkaloid accumulation. Jasmonic acid rather than the octadecanoic precursors is therefore considered to be a likely signal transducer of defense gene activation in planta

Wirelessly Powered and Transmitting Current Sensing Device

As the rate of incidence of diabetes increases in the modern world, more accurate and reliable methods of glucose detection must be developed for patients with diabetes. Currently, 25.8 million people have diabetes in the United States alone and account for $174 billion in healthcare costs annually [1]. Devices that can be used without the need for as much patient interaction, regular replacement, or great patient expenses would be a large step forward in the ability of doctors and patients to effectively manage diabetes. To measure glucose concentrations in vivo, a biosensor is used to transduce glucose to an electrical current. Because of this, a device capable of converting an inputted current to a corresponding digital output, wirelessly transmitting the output to a computer, and operating completely on wireless powering was developed and tested. The device is able to take multiple measurements per second of an input current source within the range of 0 – 1mA at a resolution of about 4µA. This range can be reduced to smaller ranges with smaller resolutions within reason. For future work, the device will be tested with amperometric glucose sensors to create a fully, implantable wireless device

Implantable Device for Wireless Regulation of the Bladder through Pelvic Nerve Stimulation

Urinary incontinence (UI) is the involuntarily urination that usually effects older people or is the result of an injury. UI affects more than eleven million people and the cost of incontinence management in the United States in 2000 was $19.5 billion. Where conventional physical therapies have failed, pelvic nerve stimulation is a promising form of regulating the bladder long term. Piezoresistive pressure sensors consist of two variable resistance values and two known resistance values that are represented on a daughterboard. This unknown resistance represents the change in pressure. The filling and voiding of the bladder was characterized through acute surgeries. It was found that the pressure sensor successfully detects changes in the bladder. Based on the collected data an implantable package can be assembled for chronic surgeries. The package consists of a bionode to record and stimulate the pelvic nerve, a powernode to apply a voltage, and a daughterboard. The next phase of the research is to thoroughly test the implantable package by placing the rat inside a cavity that will wirelessly power the device to regulate the bladder. Future applications of this will be long term use in humans with the ability to control the stimulus through a smart phone application. This research can improve the overall health and quality of life of patients by giving them back control of their bodies

Quantitative analysis of the Hepatitis C Virus replication complex and identification of associated cellular factors

Hepatitis C virus (HCV) has a positive-strand RNA genome and is grouped into the family of Flaviviridae. Similar to other positive-stranded RNA viruses, HCV RNA replication takes place in the cytoplasm. The sites of viral replication are designated “membranous web” and represented by an accumulation of vesicular structures, which are induced by the viral non-structural proteins and probably originate from membranes of the Endoplasmic Reticulum. The aim of this work was to purify and characterize these viral replication complexes (RCs) in vitro and to identify potential host factors of viral replication. First a purification strategy for enzymatically active viral replication complexes was developed to determine associated cellular proteins by proteomics. Thereby, several potential host factors of viral replication were identified and the most reproducible, Annexin II (ANXA2) was further characterized. In immunofluorescence analyses, ANXA2 strongly colocalized to the sites of viral replication in all applicable cell lines supporting HCV replication, in HCV-transfected as well as in infected cells. In contrast, we found no obvious colocalization of HCV proteins with Annexin I, IV or V or with p11 (also denoted S100A10), a common cellular ligand of Annexin II. Specificity of the ANXA2-HCV interaction was further indicated by the lack of colocalization with replication sites of other positive-strand RNA viruses, namely Dengue virus and Semliki-Forest-Virus. By individual expression of the viral non-structural (NS) proteins we found that NS5A colocalized with Annexin II, indicating that NS5A might be involved in the recruitment of ANXA2. SiRNA-mediated silencing clearly reduced Annexin II levels but failed to block HCV replication. However, FACS analyses revealed a strong correlation of intracellular HCV and ANXA2 levels even in presence of ANXA2 siRNA, suggesting that Annexin II expression was induced by HCV, thereby counteracting siRNA-mediated knockdown. Still, ANXA2 silencing moderately reduced the number of HCV positive cells. Interestingly, the presence of replicating HCV sequences in HepG2 cells, harboring very little endogenous ANXA2, clearly induced Annexin II expression to detectable levels perfectly colocalizing with the viral NS proteins. However, the role and function of ANXA2 in the HCV life cycle has yet to be defined. In a second line of investigations, a detailed stoichiometric analysis of HCV RCs was performed. Thus, the ratio of non-structural proteins to RNA that is required for HCV RNA replication could be determined. Almost the entire negative- and positive-strand RNA but <5% of the non-structural proteins present in HCV-harboring cells were protected against nuclease and protease treatments. Nevertheless, this protease-resistant portion of NS proteins accounted for the full in vitro replicase activity. Therefore, only a minor fraction of the HCV non-structural proteins was actively involved in RNA synthesis. However, due to the high amounts present in replicon cells, this still represented a huge excess compared to the viral RNA. Based on the comparison of nuclease-resistant viral RNA to protease-resistant viral proteins, an active HCV replication complex probably consists of one negative-strand RNA, two to ten positive-strand RNAs, and several hundred non-structural protein copies. These might be required as structural components of the vesicular compartments that are the site of HCV replication

Movement and Distribution of Bacteria near Surfaces

Bacteria are found everywhere in nature, including within human/animal bodies, biomedical devices, industrial equipment, oceans and lakes. They can be found in planktonic state within a bulk liquid phase or attached to surfaces with the potential to form biofilms. In this study we are interested in the movement and distribution of bacteria near surfaces. The concentrations and fluid interactions of bacteria were characterized at various distances from a surface. Psuedomonas putida F1 was observed in a suspension near a surface. Bacteria movements were visualized with an inverted microscope at 40x magnification. P. putida F1 exhibited greater density in close proximity to the surface when compared to the bulk. Additionally, the ability to move in a direction normal to the surface was significantly reduced

A Proposal for a Wirelessly Powered, Implantable Pressure Sensor and Neural Stimulator for the Control of Urinary Incontinence

47 to 53 percent of women over the age of 20 suffer from urinary incontinence, often caused by childbirth-related damage to the pelvic nerve. This uncertainty of when bladder voiding will occur causes social anxiety and can compromise quality of life. This study explores one method to restore the ability to sense the need to urinate and prevent unwanted voiding. We propose a device to measure pressure due to bladder content as the difference between pressure in the bladder and pressure in the abdominal cavity. Integrated circuits, biocompatible packaging, and wireless radiofrequency powering allow for a fully implantable device to process the pressure data, stimulate the pelvic nerve, and stop stimulation on command. The device recognizes pressure spikes similar to those seen in the bladder prior to urinating and stimulates the pelvic nerve in acute surgeries. We hope to chronically implant the device soon to monitor long-term performance and effects of the device in vivo