Vladimir Parpura, M.D., Ph.D - US grants

[unreadable] DESCRIPTION (provided by applicant): Astrocytes, a subtype of glial cell, exhibit a form of excitability based on intracellular Ca2+ variations. These intracellular calcium variations, i.e., oscillations can be evoked by neurotransmitters. The functional consequences of calcium oscillations in astrocytes are poorly understood. Since the level of intracellular calcium ions control glutamate release from astrocytes, we hypothesize that the frequency of calcium oscillations also control the amount of glutamate release from these cells. We will test this hypothesis in Specific Aim I. Calcium-dependent secretion in neurons can be modulated by the protein kinase A (PKA)- and protein kinase C (PKC)-dependent phosphorylation of secretory machinery downstream of calcium. Since astrocytes express the core proteins of the secretory machinery responsible for neuronal secretion, we hypothesize that glutamate release from astrocytes may also be modulated by PKA- and PKC-dependent phosphorylation at the site of secretory machinery. We will test this hypothesis in Specific Aim II. [unreadable] Specific Aim I: We will test the hypothesis that glutamate release from astrocytes is controlled by the frequency of calcium oscillations. [unreadable] Specific Aim Il: We will test the hypothesis that PKA and PKC modulate calcium-dependent glutamate release from astrocytes. [unreadable] This study will provide new and important information on how astrocytes communicate with neurons. Since astrocytes modulate synaptic transmission by releasing glutamate, this new insight into glial action has potential to change the way we think about central nervous system functions and dysfunctions [unreadable] [unreadable]

0617240
Mulchandani

The overall objective is to develop a novel microcantilever-based array immunosensor which is highly-sensitive, highly selective, precise, accurate, rapid, field-deployable, robust, low-cost and allows the simultaneous detection and quantification of small analytes. The proposed sensor will be based on the combination of the force measurement transduction mechanism and the receptor-ligand biological interaction recognition principle to realize the high sensitivity and selectivity. Trinitrotoluene (TNT), atrazine (ATZ), and biotin will be studied as model analytes. Single chain antibody fragment (scFv) against TNT and ATZ with low affinity and high off-rate for the respective analogs will be generated using directed evolution. The selected scFv will be immobilized on the microcantilever tip to which agarose beads functionalized with the analog will be suspended and the adhesive force measured. The introduction of the target analyte that has higher affinity and high on-rate to this system will result in the displacement of the attached bead and hence rupture of the adhesive force. The time required to rupture the linkage will be correlated to the concentration of the analyte. The knowledge gained and the sensors developed through this research will introduce novel biosensor design concepts for the applications in health care, homeland security, food monitoring and quality control, etc. The proposed effort will have impact on teaching and training of the next generation work force in the traditional areas of molecular biology and physics and the emerging multidisciplinary subject of sensors/biosensors, nanobiotechnology and microfabrication.

EEC-0552562
Alexander a. Balandin

This REU award for a Site at the University of California Riverside supports 12 undergraduate students in year one and 16 students in years two and three for 9 weeks in the summer and up to 30 weeks of academic year activity.

The objective of this program is to provide meaningful research opportunities to undergraduates from institutions that do not have major research operations. The REU Site will take a multidisciplinary approach to nanoscience and technology based on the research agenda of the Center for Nanoscale Science and Engineering (CNSE) at the university. The program provides not only stimulating research opportunities, but it also offers guidance on preparation for graduate school and the graduate experience, and information for community college students on how to transfer to a bachelors program. Also, the program will include a module regarding real world ethics in research.

The program provides the opportunity for undergraduates to integrate research into their education as well as increase the participation of underrepresented groups in science and engineering. The involvement of these students in exciting research enhances the likelihood that community college students will consider transferring to a bachelors program and undergraduate students will consider post-graduate study. Many of the participants will be drawn from nearby accredited Minority Institutions and Hispanic Serving Institutions, thus contributing to a rich and diverse research environment..

Scientific Impact
The aim of this research is to construct Cyberplasm, a micro-scale robot using principles of synthetic biology. This will be accomplished using a combination of cellular device integration, advanced microelectronics and biomimicry; an approach that mimics animal models; in the latter we will imitate some of the behavior of the marine animal the sea lamprey. Synthetic muscle will generate undulatory movements to propel the robot through the water. Synthetic sensors derived from yeast cells will be reporting signals from the immediate environment. These signals will be processed by an electronic nervous system. The electronic brain will, in turn, generate signals to drive the muscle cells that will use glucose for energy. All electronic components will be powered by a microbial fuel cell integrated into the robot body. This research aims to harness the power of synthetic biology at the cellular level by integrating specific gene ?parts? into bacteria, yeast and mammalian cells to carry out device like functions. Moreover this approach will allow the cells/bacteria to be "simplified" so that the input/output (I/O) requirements of device integration can be addressed. In particular we plan to use visual receptors to couple electronics to both sensation and actuation through light signals. In addition synthetic biology will be carried out at the systems level by interfacing multiple cellular /bacterial devices together, connecting to an electronic brain and in effect creating a multi-cellular biohybrid micro-robot, we named Cyberplasm. Motile function will be achieved by engineering muscle cells to have the minimal cellular machinery required for excitation/contraction coupling and contractile function. The muscle will be powered by mitochondrial conversion of glucose to ATP, an energetic currency in biological cells, hence combining power generation with actuation.

Broader Aspects
The development of Cyberplasm will impact the imagination of the general public, the private sector, and education in general. The robotics industry (in terms of biotech, healthcare, agriculture and healthcare) is worth billions of dollars annually. A hybrid bio/synthetic robot would completely revolutionize aspects of this industry allowing robots to operate with a whole new level of control and functionality. Amongst the fundamental issues that this research addresses is the integration of bacteria into fuel cells, as well as yeast and mammalian cells into engineered devices such as sensors and actuators, respectively. Moreover, we will address the I/O problem by developing mechanisms for these engineered cells to communicate with electronics. The knowledge to be gained (namely at the biology-electronics interface) will not only contribute to advance the field as such by laying a solid ground upon which novel concepts and developments can be built, but could have a far-reaching, longer term industrial impact in industries such as those healthcare, where biosensors and drug delivery systems could be vastly improved by harnessing the sensing capabilities and efficiency of such cellular machines. Owing to its ?cybernetic? nature, the project can be effectively used as a vehicle to foster the enthusiasm and interest of lay public and, specifically, for the teaching of science in general and synthetic biology to students, ranging from primary to secondary/high schools.

DESCRIPTION (provided by applicant): The enteric nervous system (ENS) resides in the gut wall and controls physiology of the alimentary tract. The majority of the ENS is composed of enteric glial cells (EGCs), which have been linked to a wide range of gut pathologies, e.g., inflammatory bowel disease, necrotizing enterocolitis and idiopathic constipation. Our preliminary data point to a novel hypothesis that EGCs support normal gut function through their cell-cell connectivity established via connexin 43; the down regulation of Cx43 leads to inflammatory morphological changes and reduced motility of the gut. To test this hypothesis we will use an innovative molecular genetics approach integrated with histology, fiber-optic colonoscopy, GI transit tests in vivo, and an ex vivo imaging method of an isolated colon. This proposed investigation will gather valuable information on the novel role of Cx43 in the function of the gut and ENS. These findings will be of general interest to neurobiology. Additionally, the findings will be relevant to translational medicine, since they could open the door for the development of a cause-directed treatment for constipation and various inflammatory bowel diseases. These potential new opportunities for intervention could greatly improve clinical practice in gastroenterology by increasing the survival rate of neonatal patients suffering from necrotizing enterocolitis and substantially improve the quality of life and survival in the other ENS/EGC related gut pathologies.

PROJECT SUMMARY/ABSTRACT This is application for support of the 49th annual ASN meeting to be held in Riverside, California from March 24-28, 2018. Previous NIH funding has been invaluable for supporting our scientific programs and for enhancing our ability to involve graduate students, postdoctoral researchers and underrepresented groups. To accommodate the breadth of neurochemistry and to provide in-depth analyses of particular topics, the ASN continues to build its scientific program around four interwoven, but distinct, themes. These themes have been selected to increase our understanding of the cellular and molecular basis of neural development and disease. These four themes are: 1) Building the Nervous System/Neurodevelopment, 2) Cellular Metabolism and Neurotransmission/Cognition, 3) Glial Function in Health and Disease, and 4) Neurodegeneration; each theme has one of the four Plenary/Presidential speakers. The ASN is strongly committed to the representation of women and racial/ethnic minorities at its meetings. One of the four Plenary/Presidential speakers is female, 40% of the Chairs/Co-Chairs of Symposia and Colloquia are female, and 9% of the Chairs/Co-Chairs are from racial/ethnic minorities; 43% of the Speakers of Symposia and Colloquia are female, while 7% are from racial/ethnic minorities. The ASN strongly supports young investigators in their development; 11% of the Chairs/Co-Chairs of Symposia and Colloquia and 5% of the Session Speakers are at the pre-faculty, i.e. post- doctoral/graduate student levels. The ASN meeting provides numerous opportunities for participants to exchange ideas and to form new collaborations because total expected attendance is ~ 300, and there are numerous opportunities incorporated in our program for participants to congregate informally. The Society also has several mechanisms to enhance the professional development of junior investigators during the meeting. We organize meetings with the Plenary/Presidential speakers, which gives attendees a chance to discuss topics directly with the speakers. The fees for these meetings are reduced for students and postdoctoral fellows. There is a mingle that included an educational/mentoring component, exclusively for students and postdoctoral fellows. This event has been repeatedly recognized as an outstanding opportunity to network. There is a function entitled ?Women in Neurochemistry? that is open to all attendees, and junior investigators have reduced fees. There are Oral Sessions, which are selected from abstracts submitted, with an emphasis on choosing presentations from graduate students and postdoctoral fellows. There are travel awards for outstanding graduate students, postdoctoral fellows and junior researchers to defray their costs of attendance. We host a job-posting site and students can meet with potential future mentors or colleagues during the meeting. In 2010, we added a program for visiting high-school students to engage these future scientists. From results of yearly exit surveys, we know that the annual ASN meeting will continue to provide an excellent venue for cutting edge neurochemistry and neurobiology, and for enhancing the careers of young investigators.

Abstract Astrocyte-neuron signaling, a.k.a. gliotransmission, can modulate synaptic transmission/plasticity at tripartite synapses. Among the processes regulated by gliotransmission are sleep-regulation, respiration, and learning/memory. Despite these roles of gliotransmission in such fundamental life processes, its mechanism is not understood. Elucidating this mechanism should provide insights into basic brain processes, and suggest interventions when they go awry. Two early studies of astrocyte-neuron signaling explored the hypothesis that astrocytic glutamate release acts on neuronal glutamate receptors, but they led to different conclusions regarding the mechanism. One study concluded that glutamate is not the messenger but instead suggested that gap junctions might mediate astrocyte-neuron signaling. The other study concluded that the signaling is mediated by Ca2+-dependent glutamate release from astrocytes, subsequently shown to occur by regulated exocytosis of glutamate-containing vesicles. Virtually nothing is known about the subcellular distribution/localization of astrocyte release sites. It is not clear if they are localized uniquely to the tripartite synaptic regions of astrocytes or more broadly. There is much debate about the relative roles of exocytosis vs. gap junction-mediated communication as critical for astrocyte-neuron signals. Our preliminary data point to a novel, unifying hypothesis that these two mechanisms are, in fact, mechanistically linked.