NCAM 180 Acting via a Conserved C-Terminal Domain and MLCK Is Essential for Effective Transmission with Repetitive Stimulation

SummaryNCAM 180 isoform null neuromuscular junctions are unable to effectively mobilize and exocytose synaptic vesicles and thus exhibit periods of total transmission failure during high-frequency repetitive stimulation. We have identified a highly conserved C-terminal (KENESKA) domain on NCAM that is required to maintain effective transmission and demonstrate that it acts via a pathway involving MLCK and probably myosin light chain (MLC) and myosin II. By perfecting a method of introducing peptides into adult NMJs, we tested the hypothesized role of proteins in this pathway by competitive disruption of protein-protein interactions. The effects of KENESKA and other peptides on MLCK and MLC activation and on failures in both wild-type and NCAM 180 null junctions supported this pathway, and serine phosphorylation of KENESKA was critical. We propose that this pathway is required to replenish synaptic vesicles utilized during high levels of exocytosis by facilitating myosin-driven delivery of synaptic vesicles to active zones or their subsequent exocytosis

Photo-strobo-acoustic Imaging at the Microscale by Laser-Induced Ultrasound

The combination of microfluidic technology and optical fluids characterization techniques has been recently applied to produce lab-on-a-chip systems. In the present work, bringing together the imaging technique called photoacoustic imaging (PAI) and microfluidic technology were implemented to obtain micro-scale imaging. Laser-induced ultrasound signals were measured from microdroplets produced in a simple T-junction microfluidic system. Single pulse laser images were produced as a result of the combination of the aforementioned techniques, allowing to obtain of geometrical information of the microdroplets and its spatial position.Comment: Manuscript in proces

Compact shock wave generating device for drug delivery

Genetic Engineering and Biotechnology News reported that "the burgeoning markets that surround biopharmaceuticals, RNA interference screening, and stem cell research are limited by the lack of a silver bullet for successful gene transfer. Because stable transfection is hard to achieve in primary cell lines, this application continues to be an important untapped niche in the transfection market." Inventors developed a microdevice creating shock waves from the reaction of nanoenergetic materials of fuel and oxidizer in nanoscale. The shock waves then permeabilize target cells allowing delivery of genetic material into the cells. The characteristics of the shock wave that can be controlled include pulse intensity, and pulse duration. The tunability of the shock waves allows the device to be adapted for use in a wide range of applications. DNA and nanoparticle delivery have been demonstrated. As compared to existing cell transfection products, this device achieves a significantly greater transfection success rate, significantly greater cell survival rate, and should cost less than most, if not all other methods. The invention was compared with commercially available chemical-based transfections (SiPort NeoFx, SiPort Amine, Lipofectamine 2000, Lipofectamine LTX, Transit LT1), and electroporation. The prototype of the invention produced transfection and survivability rates in excess of 99% while none of the existing transfection methods resulted in a rate greater than 10%, and the survivability of those transfected cells ranged from 0% to 80%. This device has the potential to revolutionize cell transfection, as the shock waves are particularly good at making the cell membranes porous, while at the same time the shock waves are gentle and do not cause catastrophic damage during the transfection, so cells survive. Potential Areas of Applications: * Cell Transfection * Shockwave drug delivery for killing cancer cells * Precision drug delivery of imaging particles * Fragmentation of kidney stones * Destruction of plaques Patent Status: Prototype tested and patent application 12/253,706 published Inventor(s): Dr. Shubhra Gangopadhyay, Ph.D.; Dr. Steven Apperson, Ph.D.; Dr. Luis Polo-Parada, Ph.D.; Dr. Keshab Gangopadhyay, Ph.D.; Dr. Andrey Bezmelnitsyn, Ph.D. Contact Info: Dr. Wayne McDaniel, Ph.D. ; [email protected] ; 573-884-330

Novel Nanostructured Organosilicate Nanoparticle Coatings for Chem-Bio Sensing [abstract]

Plenary speakerWe present novel nanostructured organosilicate particulate based films and demonstrate that these materials have a great potential for chemical-biological sensor development. With unprecedented high surface areas (> 1400 m2/g) and optical transparency together with its easy surface functionalization, these materials can be readily interfaced with existing immunoassays for the rapid and trace detection of both chemical and biological warfare agents. The ultra high surface area associated with these films stems from its unique nanostructure consisting of nanoparticles (2-5nm) in a “raspberry” structure in combination with interconnected nanopores (3-10nm). This unique nanostructure has been exploited to immobilize high areal density of sensor probes to improve the sensing performance. Two orders of magnitude increase in binding density was achieved when fluorescently tagged protein A molecules were immobilized upon these surfaces compared to flat substrates (glass and Silicon). Our on-going work applies these materials to develop platforms for multiplexed sensitive detection of biological and chemical agents at point of care for both army and civilian use

Development and characterization of fluorescent dye-doped nanoparticles with enhanced fluorescence intensity and photostability [abstract]

Nanoscience Poster SessionWe report the development of fluorescent dye doped organosilicate nanoparticles (DOSNPs) synthesized from poly-methylsilsesquioxane(PMSSQ), resulting in high fluorescence intensity and excellent photostability. The surface modified DOSNPs have hydrophilic surfaces and hydrophobic cores that enhance water-solubility and protect the dyes from oxidation and phtobleaching. These DOSNPs show superior properties over conventional dyes such as high fluorescence intensity due to approximately hundred dye molecules per particle and photostability demonstrating 7% and 76% fluorescence decay under continuous excitation for rhodamine 6G (R6G) DOSNP and R6G molecules, respectively, and have potential to be used in many areas, for example, imaging, sensing and solar cells. DOSNPs, when conjugated to anti-fibronectin antibodies, increased sensitivity of detection by approximately 600 fold relative to individual dye molecules conjugated to antibody. The DOSNPs are being applied to the development of diagnostic devices to be used in the detection of drugs, metabolites and pathogens

Knockdown of embryonic myosin heavy chain reveals an essential role in the morphology and function of the developing heart

The expression and function of embryonic myosin heavy chain (eMYH) has not been investigated within the early developing heart. This is despite the knowledge that other structural proteins, such as alpha and beta myosin heavy chains and cardiac alpha actin, play crucial roles in atrial septal development and cardiac function. Most cases of atrial septal defects and cardiomyopathy are not associated with a known causative gene, suggesting that further analysis into candidate genes is required. Expression studies localised eMYH in the developing chick heart. eMYH knockdown was achieved using morpholinos in a temporal manner and functional studies were carried out using electrical and calcium signalling methodologies. Knockdown in the early embryo led to abnormal atrial septal development and heart enlargement. Intriguingly, action potentials of the eMYH knockdown hearts were abnormal in comparison with the alpha and beta myosin heavy chain knockdowns and controls. Although myofibrillogenesis appeared normal, in knockdown hearts the tissue integrity was affected owing to apparent focal points of myocyte loss and an increase in cell death. An expression profile of human skeletal myosin heavy chain genes suggests that human myosin heavy chain 3 is the functional homologue of the chick eMYH gene. These data provide compelling evidence that eMYH plays a crucial role in important processes in the early developing heart and, hence, is a candidate causative gene for atrial septal defects and cardiomyopathy

Organosilicate nanoparticles and its applications in chem-biosensors, electronics, multifunctional coatings and textiles

This invention reports a novel technique for the rapid and cost-efficient synthesis of organosilicate nanoparticles (OSNPs) that have been successfully applied as individual building blocks for various applications. Doping these nanoparticles with fluorescent dyes results in highly fluorescent, biocompatible, water soluble nanoparticles with demonstrated long term photostability and with surface groups that can be readily used to attach various biological moieties. Fluorescent intensity of dye doped OSNPs (22.4 ± 5.3 nm) is shown to be 200 times brighter with 94% of the initial fluorescence intensity retained than the constituent dyes under continuous excitation for 10 minutes. In contrast, under identical test conditions, individual dye molecules retained only 58% of the initial fluorescence demonstrating that these nanoparticles have excellent utility in lifesciences research, forensics, chemical - biological sensors and biological imaging applications. Through our patented technology of novel bottom up fabrication technique, these nanoparticles have been used to fabricate highly porous transparent films. Optically smooth hydrophobic films with low refractive indices (as low as 1.048) and high surface areas (as high as 1325 m2/g) can be achieved on large area substrates. These unique materials can be readily interfaced with existing immunoassays in the form of inexpensive dip-stick assays for the sensitive detection of chemical and biological warfare agents or novel diagnostic strips for point of care applications. Our preliminary evaluation of these coatings in combination with dye doped OSNPs for construction of diagnostic immunoassays gave ~180 fold enhancement in fluorescence signal enhancement compared to traditional (microscope glass slide and fluorescent dye molecules) based assays. OSNPs used as filler elements within sol-gel based coatings have been shown to greatly enhance their structural stability, flexibility and wear resistance. Crack-free coatings (with thicknesses exceeding 30 microns)/novel multifunctional electrospun fibers have been successfully achieved by employing OSNP fillers (up to 75% by weight) within sol-gel compositions. POTENTIAL AREAS OF APPLICATIONS: *Chemical Biological sensors *Medical Diagnostics *Multifunctional coatings *Next generation Chemical-Biological protection textiles (Soldier technologies) PATENT STATUS: Non provisional patent application on file INVENTOR(S): Sangho Bok; Venumadhav Korampally; Luis Polo-Parada; Vamsi Mamidi; Keshab Gangopadhyay; William R. Folk; Purnendu K. Dasgupta and Shubhra Gangopadhyay CONTACT INFO: Wayne McDaniel, Ph.D.; [email protected] ; 573-884-330

Shock Wave Based Cell Transfection and Fluorescent Organosilicate Nanoparticles for Targeted Drug Delivery [abstract]

Nanoscience Poster SessionNanotechnology is a multidisciplinary field that has applications in life sciences, alternative energy, national defense, and electronics. In the field of medicine, nanotechnology may enable intelligent drug delivery using multifunctional nanoparticles. Here, we show two technologies that are envisioned to work in tandem to enable targeted detection and treatment. First, a shock wave generator used for cell transfection and drug/particle delivery is presented. Then, fluorescent dye/drug encapsulated organosilicate nanoparticles (OSNP) with functionalized surfaces for targeted delivery are described. The shock wave generator has been successfully used to deliver various molecules and nanoparticle to inside of the cells with very high efficiency and low cell damage. These include dextran (77 kDa), naked plasmid, and dye-doped organosilicate nanoparticles into several types of cells lines including T47-D, HL-60, and MCF-7, and also into tissues including entire chicken heart (at developmental stage 20-30) and chicken spinal cord. Dye doped organosilicate nanoparticle surfaces conjugated to antibodies have been successfully used in immunofluorescence assays. Close examination of the nanostructure of these particles reveal its unique nanoporous structure. These nanoparticles are currently under investigation for drug encapsulation and sustained release. The implication of these technologies is that the OSNP can be used as targeted drug carriers, and the shock wave generator can be used to deliver the OSNP into cells to which the particles attach. The research on shock wave micro-transfector system has been funded by the National Science Foundation Grant Opportunities for Academic Liason with Industries program

Tropomyosin 1: multiple roles in the developing heart and in the formation of congenital heart defects

Tropomyosin 1 (TPM1) is an essential sarcomeric component, stabilising the thin filament and facilitating actin's interaction with myosin. A number of sarcomeric proteins, such as alpha myosin heavy chain, play crucial roles in cardiac development. Mutations in these genes have been linked to congenital heart defects (CHDs), occurring in approximately 1 in 145 live births. To date, TPM1 has not been associated with isolated CHDs. Analysis of 380 CHD cases revealed three novel mutations in the TPM1 gene; IVS1 + 2T > C, I130V, S229F and a polyadenylation signal site variant GATAAA/AATAAA. Analysis of IVS1 + 2T > C revealed aberrant pre-mRNA splicing. In addition, abnormal structural properties were found in hearts transfected with TPM1 carrying I130V and S229F mutations. Phenotypic analysis of TPM1 morpholino-treated embryos revealed roles for TPM1 in cardiac looping, atrial septation and ventricular trabeculae formation and increased apoptosis was seen within the heart. In addition, sarcomere assembly was affected and altered action potentials were exhibited. This study demonstrated that sarcomeric TPM1 plays vital roles in cardiogenesis and is a suitable candidate gene for screening individuals with isolated CHDs