Vivek Verma

Surface-bound casein modulates the adsorption and activity of kinesin on SiO2 surfaces.

Conventional kinesin is routinely adsorbed to hydrophilic surfaces such as SiO(2). Pretreatment of surfaces with casein has become the standard protocol for achieving optimal kinesin activity, but the mechanism by which casein enhances kinesin surface adsorption and function is poorly understood. We used quartz crystal microbalance measurements and microtubule gliding assays to uncover the role that casein plays in enhancing the activity of surface-adsorbed kinesin. On SiO(2) surfaces, casein adsorbs as both a tightly bound monolayer and a reversibly bound second layer that has a dissociation constant of 500 nM and can be desorbed by washing with casein-free buffer. Experiments using truncated kinesins demonstrate that in the presence of soluble casein, kinesin tails bind well to the surface, whereas kinesin head binding is blocked. Removing soluble casein reverses these binding profiles. Surprisingly, reversibly bound casein plays only a moderate role during kinesin adsorption, but it significantly enhances kinesin activity when surface-adsorbed motors are interacting with microtubules. These results point to a model in which a dynamic casein bilayer prevents reversible association of the heads with the surface and enhances association of the kinesin tail with the surface. Understanding protein-surface interactions in this model system should provide a framework for engineering surfaces for functional adsorption of other motor proteins and surface-active enzymes.

Beta-phase enhancement in polyvinylidene fluoride through filler addition: comparing cellulose with carbon nanotubes and clay

The beta-phase in polyvinylidene fluoride (PVDF) is responsible for its ferro-, piezo- and pyroelectric properties, key for memory, sensing and actuation applications. Filler addition as a route to enhance the smart beta-phase in PVDF is presented in this study. PVDF composites with varying concentrations of microcrystalline cellulose, carbon nanotubes and kaolinite clay were prepared by solution-sonication method and subsequently cast into thin standing composite films. Crystallinity of PVDF and its composites were found to be similar. Phase evolution studies showed that addition of microcrystalline cellulose yields a beta-phase fraction comparable to carbon nanotubes and significantly higher than that of clay. The mechanism of interaction between microcrystalline cellulose and PVDF is also proposed.

Enhancing beta-phase in PVDF through physicochemical modification of cellulose

The piezo-, pyro- and ferroelectric properties of polyvinylidene fluoride (PVDF) are strongly associated with its β-phase having an all-trans conformation that can be enhanced via filler addition. Hypothesizing an interaction between -OH groups of cellulose and PVDF, alkali treatment, ball milling and acid hydrolysis of cellulose were carried out to improve the availability of surface -OH groups. β-phase development in PVDF composites was studied with respect to addition of physicochemically altered variants of cellulose at different concentrations. Our study establishes cellulose nanowhiskers produced via acid hydrolysis as the optimal filler for PVDF.

The role of casein in supporting the operation of surface bound kinesin

Microtubules and associated motor proteins such as kinesin are envisioned for applications such as bioseparation and molecular sorting to powering hybrid synthetic mechanical devices. One of the challenges in realizing such systems is retaining motor functionality on device surfaces. Kinesin motors adsorbed onto glass surfaces lose their functionality or ability to interact with microtubules if not adsorbed with other supporting proteins. Casein, a milk protein, is commonly used in microtubule motility assays to preserve kinesin functionality. However, the mechanism responsible for this preservation of motor function is unknown. To study casein and kinesin interaction, a series of microtubule motility assays were performed where whole milk casein, or its αs1 and αs2, β or κ subunits, were introduced or omitted at various steps of the motility assay. In addition, a series of epifluorescence and total internal reflection microscopy (TIRF) experiments were conducted where fluorescently labeled casein was introduced at various steps of the motility assay to assess casein-casein and casein-glass binding dynamics. From these experiments it is concluded that casein forms a bi-layer which supports the operation of kinesin. The first tightly bound layer of casein mainly performs the function of anchoring the kinesin while the second more loosely bound layer of casein positions the head domain of the kinesin to more optimally interact with microtubules. Studies on individual casein subunits indicate that β casein was most effective in supporting kinesin functionality while κ casein was found to be least effective.

Crosslinking of agarose bioplastic using citric acid

We report chemical crosslinking of agarose bioplastic using citric acid. Crosslinking was confirmed using Fourier transform infrared (FTIR) spectroscopy. The effects of crosslinking on the tensile strength, swelling, thermal stability, and degradability of the bioplastic were studied in detail. The tensile strength of the bioplastic films increased from 25.1MPa for control films up to a maximum of 52.7MPa for citric acid crosslinked films. At 37°C, the amount of water absorbed by crosslinked agarose bioplastic was only 11.5% of the amount absorbed by non-crosslinked controls. Thermogravimetric results showed that the crosslinked samples retain greater mass at high temperature (>450°C) than control samples. Moreover, while the crosslinked films were completely degradable, the rate of degradation was lower compared to non-crosslinked controls.

Agarose bioplastic based drug delivery system for surgical and wound dressings

We have developed an agarose‐based biocompatible drug delivery vehicle. The vehicle is in the form of thin, transparent, strong and flexible films. The biocompatibility and haemocompatibility of the films is confirmed using direct and indirect contact biological assay. Contact angle measurement exhibits hydrophilic nature of the films, and protein adsorption test shows low protein adsorption on the film surface. Drugs, antibiotics and antiseptics, retain their potency after their incorporation into the films. Our bioplastic films can be a versatile medium for drug delivery applications, especially as wound and surgical dressings where a fast drug release rate is desired.

Click chemistry route to covalently link cellulose and clay

An efficient method for covalently linking of cellulose and clay using a click chemistry based strategy is reported. Azide and alkynyl derivatives of silane were synthesized and used for silanization of cellulose and clay respectively. Functionalized cellulose and clay were then coupled using Cu(I) catalyzed azide–alkyne cycloaddition reaction, resulting in a covalent linkage between them. Successful synthesis of the silane derivates was established using Fourier transform infrared (FTIR) and nuclear magnetic resonance. Silanization of cellulose and clay with azide and alkynyl derivatives and the formation of a triazole linkage were confirmed using FTIR.Graphical Abstract

Micro- and nanofabrication processes for hybrid synthetic and biological system fabrication

The application of micro- and nanofabrication processes to the development of hybrid synthetic and biological systems may enable the production of new devices such as controllable transporters, gears, levers, micropumps, or microgenerators powered by biological molecular motors. However, engineering these hybrid devices requires fabrication processes that are compatible with biological materials such as kinesin motor proteins and microtubules. In this paper, the effects of micro- and nanofabrication processing chemicals and resists on the functionality of casein, kinesin, and microtubule proteins are systematically examined to address the important missing link of the biocompatibility of micro- and nanofabrication processes needed to realize hybrid system fabrication. It is found that both casein, which is used to prevent motor denaturation on surfaces, and kinesin motors are surprisingly tolerant of most of the processing chemicals examined. Microtubules, however, are much more sensitive. Exposure to the processing chemicals leads to depolymerization, which is partially attributed to the pH of the solutions examined. When the chemicals were diluted in aqueous buffers, a subset of them no longer depolymerized microtubules and in their diluted forms still worked as resist removers. This approach broadens the application of micro- and nanofabrication processes to hybrid synthetic and biological system fabrication.

Microtubule asters as templates for nanomaterials assembly.

Self organization of the kinesin-microtubule system was implemented as a novel template to create percolated nanofiber networks. Asters of microtubule seeds were immobilized on glass surfaces and their growth was recorded over time. The individual aster islands became interconnected as microtubules grew and overlapped, resulting in a highly percolated network. Cellulose nanowhiskers were used to demonstrate the application of this system to nanomaterials organization. The size distribution of the cellulose nanowhiskers was comparable to that of microtubules. To link cellulose nanowhiskers to microtubules, the nanowhiskers were functionalized by biotin using cellulose binding domains. Fluorescence studies confirmed biotinylation of cellulose nanowhiskers and binding of cellulose nanowhiskers to biotinylated microtubules.

Preparation and characterization of maghemite nanoparticles from mild steel for magnetically guided drug therapy

Maghemite (γ-Fe2O3) nanoparticles for therapeutic applications are prepared from mild steel but the existing synthesis technique is very cumbersome. The entire process takes around 100 days with multiple steps which lack proper understanding. In the current work, maghemite nanoparticles of cuboidal and spheroidal morphologies were prepared from mild steel chips by a novel cost effective oil reduction technique for magnetically guided intravascular drug delivery. The technique developed in this work yields isometric sized γ-Fe2O3 nanoparticles in 6 h with higher saturation magnetization as compared to the existing similar solid state synthesis route. Mass and heat flow kinetics during the heating and quenching steps were studied with the help of Finite element simulations. Qualitative and quantitative analysis of the γ-Fe2O3 phase is performed with the help of x-ray diffraction, transmission electron microscope and x-ray photoelectron spectroscopy. Mechanism for the α-Fe2O3 (haematite) to γ-Fe2O3 (maghemite) phase evolution during the synthesis process is also investigated.Graphical AbstractMaghemite (γ-Fe2O3) nanoparticles were prepared bya novel cost effective oil reduction technique as mentioned below in the figure. The raw materials included mild steel chips which is one of the most abundant engineering materials. These particles can be used as ideal nanocarriers for targeted drug delivery through the vascular network.