Biomedical subjects

Aravind Srinivasan

Publications and source records attributed to Aravind Srinivasan.

9 recordsLinked to original sources

PubMed2006

The catalytic machinery of chondroitinase ABC I utilizes a calcium coordination strategy to optimally process dermatan sulfate.

The chondroitinases are bacterial lyases that specifically cleave chondroitin sulfate and/or dermatan sulfate glycosaminoglycans. One of these enzymes, chondroitinase ABC I from Proteus vulgaris, has the broadest substrate specificity and has been widely used to depolymerize these glycosaminoglycans. Biochemical and structural studies to investigate the active site of chondroitinase ABC I have provided important insights into the catalytic amino acids. In this study, we demonstrate that calcium, a divalent ion, preferentially increases the activity of chondroitinase ABC I toward dermatan versus chondroitin substrates in a concentration-dependent manner. Through biochemical and biophysical investigations, we have established that chondroitinase ABC I binds calcium. Experiments using terbium, a fluorescent calcium analogue, confirm the specificity of this interaction. On the basis of theoretical structural models of the enzyme-substrate complexes, specific amino acids that could potentially play a role in calcium coordination were identified. These amino acids were investigated through site-directed mutagenesis studies and kinetic assays to identify possible mechanisms for calcium-mediated processing of the dermatan substrate in the active site of the enzyme.

Calcium↗

PubMed2006

Novel silicon-carbon fullerene-like nanostructures: an Ab initio study on the stability of Si54C6 and Si60C6 clusters.

Silicon fullerene like nanostructures with six carbon atoms on the surface of Si60 cages by substitution, as well as inside the cage at various symmetry orientations have been studied within the generalized gradient approximation to density functional theory. Full geometry optimizations have been performed without any symmetry constraints using the Gaussian 03 suite of programs and the LANL2DZ basis set. Thus, for the silicon atom, the Hay-Wadt pseudopotential with the associated basis set are used for the core electrons and the valence electrons, respectively. For the carbon atom, the Dunning/Huzinaga double zeta basis set is employed. Electronic and geometric properties of the nanostructures are presented and discussed in detail. It was found that optimized silicon-carbon fullerene like nanostructures have increased stability compared to bare Si60 cage and the stability depends on the orientation of carbon atoms, as well as on the nature of bonding between silicon and carbon atoms and also on the carbon-carbon bonding.

Drug Stability↗

PubMed2005

Exploiting the reaction flexibility of a type III polyketide synthase through in vitro pathway manipulation.

A synthetic metabolic pathway has been constructed in vitro consisting of the type III polyketide synthase from Streptomyces coelicolor and peroxidases from soybean and Caldariomyces fumago (chloroperoxidase). This has resulted in the synthesis of the pentaketide flaviolin and its dimeric derivative, and a wide range of pyrones and their coupled derivatives with flaviolin, as well as their halogenated derivatives. The addition of acyl-CoA oxidase to the pathway prior to the polyketide synthase resulted in unsaturated pyrone side chains, further broadening the product spectrum that can be achieved. The approach developed in this work, therefore, provides a new model to exploit biocatalysis in the synthesis of complex natural product derivatives.

Acyl Coenzyme A↗

PubMed2004

Bacterial P450-catalyzed polyketide hydroxylation on a microfluidic platform.

The incorporation of a multicomponent, cofactor-dependant P450 into a microfluidic biochip is demonstrated. The PikC hydroxylase Streptomyces venezuelae was incorporated into a PDMS-based microfluidic channel. The enzyme was immobilized to Ni-NTA agarose beads via in situ attachment following the addition of the beads to the microchannel. The enzyme loading was approximately 6 microg per mg of beads resulting in a microchannel loading of 10.7 mg/mL. This high enzyme loading enabled the rapid hydroxylation of the macrolide YC-17 to methymycin and neomethymycin in about equal amounts with a conversion of >90% at a flow rate of 70 nL/min. This high reactivity allowed rapid hydroxylation reactions to be performed with short residence times, which is critical for complex enzymes with limited inherent stability.

Bioreactors↗

PubMed2004

Modelling disease outbreaks in realistic urban social networks.

Most mathematical models for the spread of disease use differential equations based on uniform mixing assumptions or ad hoc models for the contact process. Here we explore the use of dynamic bipartite graphs to model the physical contact patterns that result from movements of individuals between specific locations. The graphs are generated by large-scale individual-based urban traffic simulations built on actual census, land-use and population-mobility data. We find that the contact network among people is a strongly connected small-world-like graph with a well-defined scale for the degree distribution. However, the locations graph is scale-free, which allows highly efficient outbreak detection by placing sensors in the hubs of the locations network. Within this large-scale simulation framework, we then analyse the relative merits of several proposed mitigation strategies for smallpox spread. Our results suggest that outbreaks can be contained by a strategy of targeted vaccination combined with early detection without resorting to mass vaccination of a population.

Contact Tracing↗

PubMed2004

Visual experience during phacoemulsification under topical versus retrobulbar anesthesia: results of a prospective, randomized, controlled trial.

PURPOSE: To compare the subjective visual experience of cataract patients during phacoemulsification and intraocular lens implantation under topical anesthesia (TA) vs retrobulbar anesthesia (RA). DESIGN: Prospective, randomized, controlled trial. METHODS: Three hundred six cataract patients eligible for phacoemulsification and intraocular lens implantation were randomized to receive either TA or RA during surgery by one of three surgeons. The surgeons were familiar with both anesthetic techniques and operated on the patients using the technique to which the patients were randomized. A masked interviewer conducted in-person interviews with the patients using a standardized questionnaire about their intraoperative visual experience and their reaction to their visual experience between 30 minutes and 4 hours after the surgery. RESULTS: Two patients (one in each group) had intraoperative posterior capsule rupture and were excluded from analysis. There was no statistically significant difference between TA (n = 154) and RA (n = 150) groups, except that more males compared with females had TA (P = .03). More patients who had TA reported perception of light (P < .001) and colors (P < .001) and a change (either increase or decrease) in light brightness during the course of the surgery (P < .001). There was no statistically significant difference in the proportions of patients who saw movements, flashes, instruments, or the surgeon or medical staff during the operation and who found their visual sensations frightening in the two groups. However, 10.4% in the TA group and 9.3% in the RA group found their visual experience frightening. CONCLUSIONS: More patients undergoing cataract surgery under TA compared with RA reported perception of light, colors, and a change in light brightness. A significant proportion of patients in both groups found the visual experience frightening.

Administration, Topical↗

PubMed2003

Multienzyme catalysis in microfluidic biochips.

The attachment of enzymes to glass microfluidic channels has been achieved using a highly reactive poly(maleic anhydride-alt-alpha-olefin) (PMA)-based coating that is supplied to the microchannel in a toluene solution. The PMA reacts with 3-aminopropyltriethoxysilane groups linked to the glass surface to form a matrix that enables additional maleic anhydride groups to react with free amino groups on enzymes to give a mixed covalent-noncovalent immobilization support. Using a simple T-channel microfluidic design, with reaction channel dimensions of 200 microm wide (at the center), 15 microm deep, and 30 mm long giving a reaction volume of 90 nL, soybean peroxidase (SBP) was attached at an amount up to 0.6 microg/channel. SBP-catalyzed oxidation of p-cresol was performed in aqueous buffer (with 20% [v/v], dimethylformamide) containing H(2)O(2), with microfluidic transport enabled by electroosmotic flow (EOF). Michaelis-Menten kinetics were obtained with K(m) and V(max) values of 0.98 mM and 0.21 micromol H(2)O(2) converted/mg SBP per minute, respectively. These values are nearly identical to nonimmobilized SBP kinetics in aqueous-DMF solutions in 20-microL volumes in 384-well plates and 5-mL reaction volumes in 20-mL scintillation vials. These results indicate that SBP displays intrinsically native activity even in the immobilized form at the microscale, and further attests to the mild immobilization conditions afforded by PMA. Bienzymic and trienzymic reactions were also performed in the microfluidic biochip. Specifically, a combined Candida antarctica lipase B-SBP bienzymic system was used to convert tolyl acetate into poly(p-cresol), and an invertase-glucose oxidase SBP trienzymic system was used to take sucrose and generate H(2)O(2) for SBP-catalyzed synthesis of poly(p-cresol).

Catalysis↗

PubMed2003

Microfluidic peroxidase biochip for polyphenol synthesis.

An enzyme-containing microfluidic biochip has been developed for the oxidative polymerization of phenols. The biochip consists of a simple T-junction with two feed reservoirs 20 mm apart and a microreaction channel 30 mm long. The channel is 15 microm deep and 200 microm wide at the center, giving a reaction volume of 90 nL. The biochip was fabricated using conventional photolithographic methods on a glass substrate etched using a HF-based solution. Fluid transport was enabled using electroosmotic flow. Soybean peroxidase was used as the phenol oxidizing catalyst, and in the presence of p-cresol and H(2)O(2), essentially complete conversion of the H(2)O(2) (the limiting substrate) occurred in the microchannel at a flow rate of ca. 290 nL/min. Thus, peroxidase was found to be intrinsically active even upon dramatic scale-down as achieved in microfluidic reactors. These results were extended to a series of phenols, thereby demonstrating that the microfluidic peroxidase reactor may have application in high-throughput screening of phenolic polymerization reactions for use in phenolic resin synthesis. Finally, rapid growth of poly(p-cresol) on the walls of the microreaction channel could be performed in the presence of higher H(2)O(2) concentrations. This finding suggests that solution-phase peroxidase catalysis can be used in the controlled deposition of polymers on the walls of microreactors.

Bioreactors↗

PubMedDate not supplied

Chip-based polyketide biosynthesis and functionalization.

We demonstrate construction and novel compound synthesis from a synthetic metabolic pathway consisting of a type III polyketide synthase (PKS) known as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS) from Streptomyces coelicolor and soybean peroxidase (SBP) in a microfluidic platform. THNS immobilized to Ni-NTA agarose beads is prepacked into a microfluidic channel, while SBP is covalently attached to the walls of a second microfluidic channel precoated with a reactive poly(maleic anhydride) derivative. The result is a tandem, two-step biochip that enables the synthesis of novel polyketide derivatives. The first microchannel, consisting of THNS, results in the conversion of malonyl-CoA to flaviolin in yields up to 40% with a residence time of 6 min. This conversion is similar to that obtained in several-milliliter batch reactions after 2 h. Linking this microchannel to the SBP microchannel results in biflaviolin synthesis. During the course of this work, we discovered that the substrate specificity of THNS could be manipulated by simply changing the reaction pH. As a result, the starter acyl-CoA specificity can be broadened to yield a series of truncated pyrone products. When combined with variations in the ratio of acyl-CoA and malonyl-CoA (extender substrate) feed rates, high yields of the pyrone products could be achieved, which is further structurally diversified from self- and cross-coupling in the SBP microchannel. The ability to rapidly evaluate the effects of reaction conditions and synthetic multienzyme pathways on a microfludic platform provides a new paradigm for performing metabolic pathway engineering, namely, the reconstruction of pathways for use in new compound discovery.

Catalysis↗