Tensile Testing of Polyurea Crosslinked Silica Aerogel Impregnated Silicone and Amine Functionalized Polyoligomeric Silsesquioxane Crosslinked Polyimide Aerogel for Cryogenic Tank Applications.

Polymer-based thermally insulating vessels are an attractive option for future space exploratory missions as well as energy conserving terresterial applications as an alternative to the conventional metal-based structures. Aerogel due to its unique set of material properties, such as superior thermal insulation and light weight, makes it an attractive for future space applications. Room Temperature (RT) and Low Temperature (LT)evaluation of the mechanical properties of Polyurea Crosslinked Silica Aerogel (PCSA) impregnated RTV655 was performed under increasing concentrations of PCSA. The effect of PCSAparticles size on the tensile behavior of RTV655 was also assessed and compared with the tensile behavior of neat RTV655 at both RT and LT. Incorporated PCSAparticles ranges were varied from 90-425µm and the concentrations tested were 25%, 50%, and 75% weight to the RTV655. The tensile properties of amine functionalized Polyoligomeric Silsesquioxane (POSS) crosslinked polyimide (PI)aerogel strips were also evaluated at both RT and LT and the effect of geometry and flexibility of PI-based aerogels was compared with the results obtained from PCSAimpregnated RTV 655 samples

Single-particle HIV-1 capsid uncoating analysis to elucidate the role of the cofactor IP6 and the mode of action of the capsid-targeting drug Lenacapavir

The process and factors that trigger HIV-1 capsid uncoating remain an area of active research. Our group has previously reported an in-vitro single particle capsid uncoating assay using Total Internal Reflection Fluorescence (TIRF) microscopy. While the assay provided a unique advantage for investigating capsids at a single particle level, the method was constrained by two sample limitations caused by the necessity to include GFP within the Gag polyprotein: (1) the presence of a foreign insertion can affect maturation and reduce virus titre; and (2) availability of only a single proviral clone (WT) with GFP embedded in the sequence. Consequently, any alternative virus or mutant to be studied must be carefully engineered which would require extensive amount of cloning let alone validation prior to uncoating experimentation. To overcome above limitations, I have developed two different independent approaches with a motivation to broaden the capabilities of our uncoating assay so that it could be deployed to investigate various HIV groups, subgroups and their respective mutants. In the first method (CypA paint assay), I have fluorescently labelled capsid binding protein ‘CypA’ and utilised it to monitor capsid disassembly process. In the second method (GFP Vpr assay), I have loaded fluorescent protein (GFP/mScarlet) as a fluid marker inside virions by tagging it along with the viral accessory protein R (Vpr) and used the fluid marker to monitor the loss of capsid integrity. I have utilised the assays to elucidate the role of host factor inositol hexakisphosphate (IP6) in multiple stages of HIV-1 lifecycle. I show that IP6 is an important cofactor that (1) packages into immature lattice via a positively charged interface in the immature lattice and (2) drives the assembly and formation of a stable matured capsid via a positively charged interface in the matured capsid that is capable maintaining proper stability required for a successful infection. Furthermore, I have investigated the effect of various capsid targeting antivirals including newly reported ultrapotent GS-6207 and a peptide derived from host cofactor CSPF6. I show that drugs and CSPF6 peptide although they occupy a shared interface on HIV-1 capsid have differential effects on the capsid integrity and stability

Mechanical testing of cross-linked silica aerogel impregnated silicone for cryogenic tank applications

Cryogenic propellant tanks used for long duration space missions can experience self-pressurization due to solar radiation. Many technologies have been proposed as means of reducing and/or controlling the rate of vaporization to prevent pressure buildup. Room temperature vulcanizing silicones such as Sylgard-184 and RTV-655 are flexible materials that allow them to be molded into many shapes and geometries. While these materials are known for good thermal insulation, this property can be enhanced by adding components of a superior thermal insulator: aerogels at a various ratios. Aerogels are extremely lightweight materials when compared to silicones due to their porous nature. The highly porous nature of this material has been recognized to be the main reason behind its superior thermal insulating properties. The lightweight and unique thermal insulation capabilities of aerogels make this material useful for space applications. In this work a novel compound material is proposed which takes advantage of the flexibility of silicones and the excellent thermal insulation of aerogels. Ratios of aerogel particles to polymer background that were tested were at 25, 50, and 75 wt. % ratios The mechanical properties of the new material are tested using a bench top tensile tester at both room temperature and 77K. Results are presented that will help predict a compound that can be made with the correct proportions of aerogel to silicone to provide the flexibility and strength for cryogenic propellant tank applications in space. Copyright © 2012 by Jeffrey G. Marchetta

Microtubule Defects Influence Kinesin-Based Transport In Vitro

Microtubules are protein polymers that form molecular highways for long-range transport within living cells. Molecular motors actively step along microtubules to shuttle cellular materials between the nucleus and the cell periphery; this transport is critical for the survival and health of all eukaryotic cells. Structural defects in microtubules exist, but whether these defects impact molecular motor-based transport remains unknown. Here, we report a new, to our knowledge, approach that allowed us to directly investigate the impact of such defects. Using a modified optical-trapping method, we examined the group function of a major molecular motor, conventional kinesin, when transporting cargos along individual microtubules. We found that microtubule defects influence kinesin-based transport in vitro. The effects depend on motor number: cargos driven by a few motors tended to unbind prematurely from the microtubule, whereas cargos driven by more motors tended to pause. To our knowledge, our study provides the first direct link between microtubule defects and kinesin function. The effects uncovered in our study may have physiological relevance in vivo

Microtubule Defects Influence Kinesin-Based Transport In Vitro

Microtubules are protein polymers that form molecular highways for long-range transport within living cells. Molecular motors actively step along microtubules to shuttle cellular materials between the nucleus and the cell periphery; this transport is critical for the survival and health of all eukaryotic cells. Structural defects in microtubules exist, but whether these defects impact molecular motor-based transport remains unknown. Here, we report a new, to our knowledge, approach that allowed us to directly investigate the impact of such defects. Using a modified optical-trapping method, we examined the group function of a major molecular motor, conventional kinesin, when transporting cargos along individual microtubules. We found that microtubule defects influence kinesin-based transport in vitro. The effects depend on motor number: cargos driven by a few motors tended to unbind prematurely from the microtubule, whereas cargos driven by more motors tended to pause. To our knowledge, our study provides the first direct link between microtubule defects and kinesin function. The effects uncovered in our study may have physiological relevance in vivo

A lysine ring in HIV capsid pores coordinates IP6 to drive mature capsid assembly.

The HIV capsid self-assembles a protective conical shell that simultaneously prevents host sensing whilst permitting the import of nucleotides to drive DNA synthesis. This is accomplished through the construction of dynamic, highly charged pores at the centre of each capsid multimer. The clustering of charges required for dNTP import is strongly destabilising and it is proposed that HIV uses the metabolite IP6 to coordinate the pore during assembly. Here we have investigated the role of inositol phosphates in coordinating a ring of positively charged lysine residues (K25) that forms at the base of the capsid pore. We show that whilst IP5, which can functionally replace IP6, engages an arginine ring (R18) at the top of the pore, the lysine ring simultaneously binds a second IP5 molecule. Dose dependent removal of K25 from the pore severely inhibits HIV infection and concomitantly prevents DNA synthesis. Cryo-tomography reveals that K25A virions have a severe assembly defect that inhibits the formation of mature capsid cones. Monitoring both the kinetics and morphology of capsids assembled in vitro reveals that while mutation K25A can still form tubes, the ability of IP6 to drive assembly of capsid cones has been lost. Finally, in single molecule TIRF microscopy experiments, capsid lattices in permeabilised K25 mutant virions are rapidly lost and cannot be stabilised by IP6. These results suggest that the coordination of IP6 by a second charged ring in mature hexamers drives the assembly of conical capsids capable of reverse transcription and infection

Lipid Bilayers Are Long-Lived on Solvent Cleaned Plasma-Oxidized poly(dimethyl)siloxane (ox-PDMS)

<div><p>Although it is well known that phospholipids self-assemble on hydrophilic plasma-oxidized PMDS surfaces (ox-PDMS) to form cell membrane mimetic bilayers, the temporal stability of phospholipid membranes on these surfaces is unknown. Here we report that phospholipid bilayers remain stable on solvent-cleaned ox-PDMS for at least 132 hours after preparation. Absent solvent cleaning, the bilayers were stable for only 36 hours. We characterized the phospholipid bilayers, i) through quantitative comparative analysis of the fluorescence intensity of phospholipid bilayers on ox-PDMS and phospholipid monolayers on native PDMS and, ii) through measurements of the diffusive mobility of the lipids through fluorescence recovery after photobleaching (FRAP). The fluorescence intensity of the phospholipid layer remained consistent with that of a bilayer for 132 hours. The evolution of the diffusive mobility of the phospholipids in the bilayer on ox-PDMS over time was similar to lipids in control bilayers prepared on glass surfaces. Solvent cleaning was essential for the long-term stability of the bilayers on ox-PDMS. Without cleaning in acetone and isopropanol, phospholipid bilayers prepared on ox-PDMS surfaces peeled off in large patches within 36 hours. Importantly, we find that phospholipid bilayers supported on solvent-cleaned ox-PDMS were indistinguishable from phospholipid bilayers supported on glass for at least 36 hours after preparation. Our results provide a link between the two common surfaces used to prepare <i>in vitro</i> biomimetic phospholipid membranes—i) glass surfaces used predominantly in fundamental biophysical experiments, for which there is abundant physicochemical information, with ii) ox-PDMS, the dominant material used in practical, applications-oriented systems to build micro-devices, topographically-patterned surfaces, and biosensors where there is a dearth of information.</p></div

Quantification of the evolution of lipid bilayers on ox-PDMS as a function of time.

<p>A) The normalized area fraction of pixels with intensities consistent with lipid bilayers are represented by solid squares (■), the normalized area fraction of pixels with intensities consistent with lipid monolayers are represented by solid triangles (▲), and the normalized area fraction of pixels with indeterminate intensities are represented by solid circles (●). Standard deviations of the mean are represented by the corresponding error bars. The area fraction of bilayer was close to 100 percent for the duration of the 132-hour experiment. B) Plot of the histogram of pixel intensities (from <i>N</i> = 10 images at each time point) of lipid layers on ox-PDMS at 0 hours, 60 hours, and 132 hours after preparation. The peak of the distribution shifts with time signifying that the layer as a whole becomes less bright. The distributions show a longer right tail. The combination of movement of the peak of the distribution towards lower values and the development of a right tail should result in regions that are brighter when compared to the background, i.e. the mottled appearance of the layer. Note however, that the majority of pixels had intensities consistent with bilayers. The number of pixels classified as indeterminate increases slightly, and none of the pixels had intensities consistent with monolayers.</p

Representative confocal fluorescence images of lipid layers formed on PDMS, ox-PDMS, and glass immediately after preparation.

<p>A uniformly fluorescent lipid layer was present on all the surfaces after the fusion of SUVs. The layer on PDMS appeared to have a qualitatively lower intensity compared to the layers on ox-PDMS and glass. Unfused small unilamellar vesicles (SUV) appeared as bright compact spots on the lipid layers. The lower images are schematic diagrams of the configuration of the lipid molecules on the respective surfaces. Lipids form a monolayer on the PDMS, bilayers on ox-PDMS, and bilayers on glass. Scale bar 10 μm.</p