Protein-resistant silicones: incorporation of poly(ethylene oxide) via siloxane tethers

Silicones with enhanced protein resistance were prepared by introducing poly(ethylene oxide) (PEO) chains via siloxane tethers (a-c) of varying lengths. Three unique ambifunctional molecules (a-c) having the general formula alpha-(EtO)3Si(CH2)2-oligodimethylsiloxanen-block-poly(ethylene oxide)8-OCH3 (n = 0 (a), 4, (b), and 13 (c)) were prepared via regioselective Rh-catalyzed hydrosilylation. Nine films were subsequently produced by the H3PO4-catalyzed sol-gel cross-linking of a-c each with alpha,omega-bis(Si-OH)polydimethylsiloxane (P, Mn = 3000 g/mol) in varying ratios (1:1, 1:2, and 2:3 molar ratio a, b, or c to P). Films prepared with a 2:3 molar ratio (a-c to P) contained the least amount of un-cross-linked materials, which may migrate to the film surface. For this set of films, surface hydrophilicity and protein resistance increased with siloxane tether length (a-c). These results indicate that PEO was more effectively mobilized to the surface if incorporated into silicones via longer siloxane tethers.     

Leishmania donovani: effect of verapamil on in vitro susceptibility of promastigote and amastigote stages of Indian clinical isolates to sodium stibogluconate

Although pentavalent antimonials are the first-line drug for treatment of visceral leishmaniasis all over the world, yet, in India, increasing number of patients are being reported to be unresponsive to sodium stibogluconate. Verapamil, a calcium channel blocker, affects drug uptake by preventing its efflux and thereby accumulation within the cell. In the present study, effect of verapamil on in vitro susceptibility of both promastigote and amastigote stages of 15 clinical isolates and standard strain of Leishmania donovani to sodium stibogluconate was evaluated by detection of acid phosphatase. Amastigotes were found more susceptible to sodium stibogluconate than the promastigotes (p<0.05) and in the presence of verapamil, IC(50) value of sodium stibogluconate was reduced only for those isolates, which had a higher IC(50). Verapamil alone did not have any effect on the parasites. The results indicate that amastigotes are more susceptible to sodium stibogluconate than promastigotes and verapamil can reverse the in vitro drug resistance of L. donovani clinical isolates to sodium stibogluconate.     

Simultaneous increases in specific growth rate and specific lipid content of Chlorella vulgaris through UV‐induced reactive species

A challenge in algae‐based bio‐oil production is to simultaneously enhance specific growth rates and specific lipid content. We have demonstrated simultaneous increases in both the above in Chlorella vulgaris through reactive species (RS) induced under ultraviolet (UV) A and UVB light treatments. We postulated that the changes in photosystem (PS) stoichiometry and antenna size were responsible for the increases in specific growth rate. UVB treatment excited PSII, which resulted in a twofold to sevenfold increase in PSII/PSI ratio compared to control. An excited PSII caused a 2.7‐fold increase in the specific levels of superoxide and a twofold increase in the specific levels of hydroxyl radicals. We have established that the increased specific intracellular RS (si‐RS) levels increased the PSII antenna size by a significant 10‐fold as compared to control. In addition, the 8.2‐fold increase in specific lipid content was directly related to the si‐RS levels. We have also demonstrated that the RS induced under UVA treatment led to a 3.2‐fold increase in the saturated to unsaturated fatty acid ratio. Based on the findings, we have proposed and demonstrated a UV‐based strategy, which achieved an 8.8‐fold increase in volumetric lipid productivity. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:291–299, 2014     

Directed evolution of metabolic pathways

The modification of cellular metabolism is of biotechnological and commercial significance because naturally occurring metabolic pathways are the source of diverse compounds used in fields ranging from medicine to bioremediation. Directed evolution is the experimental improvement of biocatalysts or cellular properties through iterative genetic diversification and selection procedures. The creation of novel metabolic functions without disrupting the balanced intracellular pool of metabolites is the primary challenge of pathway manipulation. The introduction of coordinated changes across multiple genetic elements, in conjunction with functional selection, presents an integrated approach for the modification of metabolism with benign physiological consequences. Directed evolution formats take advantage of the dynamic structures of genomes and genomic sub-structures and their ability to evolve in multiple directions in response to external stimuli. The elucidation, design and application of genome-restructuring mechanisms are key elements in the directed evolution of cellular metabolic pathways.     

Differential susceptibility of a few members of the nasuta-albomicans complex of Drosophila to paraquat-induced lethality and oxidative stress

The evolution of karyotypically stabilized short-lived (SL) and long-lived (LL) cytoraces in the laboratory have been established and validated through our previous lifespan studies. In the present investigation, we examined the possible reason(s) for the differential longevity among selected members of SL and LL cytoraces, employing the well known paraquat (PQ) resistance bioassay. Exposure of these races to varying concentrations of PQ revealed relatively higher resistance among LL cytoraces than SL cytoraces, as evident by the lower incidence of mortality. Biochemical analysis for endogenous markers of oxidative stress revealed that LL-2 cytorace exhibited lower reactive oxygen species (ROS) and lipid peroxidation (LPO) levels, higher activity levels of superoxide dismutase (SOD), and coupled with higher levels of reduced glutathione (GSH) compared with the levels found in SL-2 cytorace. These findings suggest that the higher susceptibility of SL cytoraces to PQ challenge may be, at least in part, related to the higher endogenous levels of oxidative stress markers. Although the precise mechanisms responsible for the longer longevity among LL cytoraces of the nasuta-albomicans complex of Drosophila merits further investigation, our data suggest that the relatively longer lifespan may be related to the status of endogenous markers that renders them more resistant towards oxidative-stress-mediated lethality, as evident in the PQ assay.     

UVA‐induced reset of hydroxyl radical ultradian rhythm improves temporal lipid production in Chlorella vulgaris

We report for the first time that the endogenous, pseudo‐steady‐state, specific intracellular levels of the hydroxyl radical (si‐OH) oscillate in an ultradian fashion (model system: the microalga, Chlorella vulgaris), and also characterize the various rhythm parameters. The ultradian rhythm in the endogenous levels of the si‐OH occurred with an approximately 6 h period in the daily cycle of light and darkness. Further, we expected that the rhythm reset to a shorter period could rapidly switch the cellular redox states that could favor lipid accumulation. We reset the endogenous rhythm through entrainment with UVA radiation, and generated two new ultradian rhythms with periods of approximately 2.97 h and 3.8 h in the light phase and dark phase, respectively. The reset increased the window of maximum lipid accumulation from 6 h to 12 h concomitant with the onset of the ultradian rhythms. Further, the saturated fatty acid content increased approximately to 80% of total lipid content, corresponding to the peak maxima of the hydroxyl radical levels in the reset rhythm. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:673–680, 2014     

Development of Neurological Disease Is Associated with Increased Immune Activation in Simian Immunodeficiency Virus-Infected Macaques

Simian immunodeficiency virus (SIV) infection of macaques can result in central nervous system disorders, such as meningitis and encephalitis. We studied 10 animals inoculated with brain-derived virus from animals with SIV encephalitis. Over half of the macaques developed SIV-induced neurologic disease. Elevated levels of systemic immune activation were observed to correlate with viral RNA in the cerebral spinal fluid but not with plasma viral load, consistent with a role for SIV in the pathogenesis of neurologic disease.     

Ex vivo Culturing of Whole,Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab¹ has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)², was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine³ causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly⁴, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern⁵. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches⁶, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B1⁷, and that is dependent on the activity of the ubiquitin-proteasome system⁶. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.