Chemotactic separation of enzymes.

We demonstrate a procedure for the separation of enzymes based on their chemotactic response toward an imposed substrate concentration gradient. The separation is observed within a two-inlet, five-outlet microfluidic network, designed to allow mixtures of active (ones that catalyze substrate turnover) and inactive (ones that do not catalyze substrate turnover) enzymes, labeled with different fluorophores, to flow through one of the inlets. Substrate solution prepared in phosphate buffer was introduced through the other inlet of the device at the same flow rate. The steady-state concentration profiles of the enzymes were obtained at specific positions within the outlets of the microchannel using fluorescence microscopy. In the presence of a substrate concentration gradient, active enzyme molecules migrated preferentially toward the substrate channel. The excess migration of the active enzyme molecules was quantified in terms of an enrichment coefficient. Experiments were carried out with different pairs of enzymes. Coupling the physics of laminar flow of liquid and molecular diffusion, multiphysics simulations were carried out to estimate the extent of the chemotactic separation. Our results show that, with appropriate microfluidic arrangement, molecular chemotaxis leads to spontaneous separation of active enzyme molecules from their inactive counterparts of similar charge and size.

Self-powered enzyme micropumps.

Non-mechanical nano- and microscale pumps that function without the aid of an external power source and provide precise control over the flow rate in response to specific signals are needed for the development of new autonomous nano- and microscale systems. Here we show that surface-immobilized enzymes that are independent of adenosine triphosphate function as self-powered micropumps in the presence of their respective substrates. In the four cases studied (catalase, lipase, urease and glucose oxidase), the flow is driven by a gradient in fluid density generated by the enzymatic reaction. The pumping velocity increases with increasing substrate concentration and reaction rate. These rechargeable pumps can be triggered by the presence of specific analytes, which enables the design of enzyme-based devices that act both as sensor and pump. Finally, we show proof-of-concept enzyme-powered devices that autonomously deliver small molecules and proteins in response to specific chemical stimuli, including the release of insulin in response to glucose.

DNA polymerase as a molecular motor and pump.

DNA polymerase is responsible for synthesizing DNA, a key component in the running of biological machinery. Using fluorescence correlation spectroscopy, we demonstrate that the diffusive movement of a molecular complex of DNA template and DNA polymerase enhances during nucleotide incorporation into the growing DNA template. The diffusion coefficient of the complex also shows a strong dependence on its inorganic cofactor, Mg2+ ions. When exposed to gradients of either nucleotide or cofactor concentrations, an ensemble of DNA polymerase complex molecules shows collective movement toward regions of higher concentrations. By immobilizing the molecular complex on a patterned gold surface, we demonstrate the fabrication of DNA polymerase-powered fluid pumps. These miniature pumps are capable of transporting fluid and tracer particles in a directional manner with the pumping speed increasing in the presence of the cofactor. The role of DNA polymerase as a micropump opens up avenues for designing miniature fluid pumps using enzymes as engines.

Dual stimuli-responsive, rechargeable micropumps via "host-guest" interactions.

We demonstrate a supramolecular approach to the fabrication of self-powered micropumps based on "host-guest" molecular recognition between α- and ß-cyclodextrin and trans-azobenzene. Both hydrogels and surface coatings based on host-guest partners were used as scaffolds to devise the micropumps. These soft micropumps are dual stimuli-responsive and can be actuated either by light or by introducing guest molecules. Furthermore, the micropumps can be recharged through reversible host-guest interaction.

Enzyme molecules as nanomotors.

Using fluorescence correlation spectroscopy, we show that the diffusive movements of catalase enzyme molecules increase in the presence of the substrate, hydrogen peroxide, in a concentration-dependent manner. Employing a microfluidic device to generate a substrate concentration gradient, we show that both catalase and urease enzyme molecules spread toward areas of higher substrate concentration, a form of chemotaxis at the molecular scale. Using glucose oxidase and glucose to generate a hydrogen peroxide gradient, we induce the migration of catalase toward glucose oxidase, thereby showing that chemically interconnected enzymes can be drawn together.

Fat body dSir2 regulates muscle mitochondrial physiology and energy homeostasis nonautonomously and mimics the autonomous functions of dSir2 in muscles.

Sir2 is an evolutionarily conserved NAD(+)-dependent deacetylase which has been shown to play a critical role in glucose and fat metabolism. In this study, we have perturbed Drosophila Sir2 (dSir2) expression, bidirectionally, in muscles and the fat body. We report that dSir2 plays a critical role in insulin signaling, glucose homeostasis, and mitochondrial functions. Importantly, we establish the nonautonomous functions of fat body dSir2 in regulating mitochondrial physiology and insulin signaling in muscles. We have identified a novel interplay between dSir2 and dFOXO at an organismal level, which involves Drosophila insulin-like peptide (dILP)-dependent insulin signaling. By genetic perturbations and metabolic rescue, we provide evidence to illustrate that fat body dSir2 mediates its effects on the muscles via free fatty acids (FFA) and dILPs (from the insulin-producing cells [IPCs]). In summary, we show that fat body dSir2 is a master regulator of organismal energy homeostasis and is required for maintaining the metabolic regulatory network across tissues.

Intelligent, self-powered, drug delivery systems.

Self-propelled nano/micromotors and pumps are considered to be next generation drug delivery systems since the carriers can either propel themselves ("motor"-based drug delivery) or be delivered ("pump"-based drug delivery) to the target in response to specific biomarkers. Recently, there has been significant advancement towards developing nano/microtransporters into proof-of-concept tools for biomedical applications. This review encompasses the progress made to date on the design of synthetic nano/micromotors and pumps with respect to transportation and delivery of cargo at specific locations. Looking ahead, it is possible to imagine a day when intelligent machines navigate through the human body and perform challenging tasks.

Fantastic voyage: designing self-powered nanorobots.

The use of swarms of nanobots to perform seemingly miraculous tasks is a common trope in the annals of science fiction.1 Although several of these remarkable feats are still very much in the realm of fiction, scientists have recently overcome many of the physical challenges associated with operating on the small scale and have generated the first generation of autonomous self-powered nanomotors and pumps. The motors can be directed by chemical and light gradients, pick up and deliver cargo, and exhibit collective behavior.

dSir2 deficiency in the fatbody, but not muscles, affects systemic insulin signaling, fat mobilization and starvation survival in flies.

Sir2 is an evolutionarily conserved NAD+ dependent protein. Although, SIRT1 has been implicated to be a key regulator of fat and glucose metabolism in mammals, the role of Sir2 in regulating organismal physiology, in invertebrates, is unclear. Drosophila has been used to study evolutionarily conserved nutrient sensing mechanisms, however, the molecular and metabolic pathways downstream to Sir2 (dSir2) are poorly understood. Here, we have knocked down endogenous dSir2 in a tissue specific manner using gene-switch gal4 drivers. Knockdown of dSir2 in the adult fatbody leads to deregulated fat metabolism involving altered expression of key metabolic genes. Our results highlight the role of dSir2 in mobilizing fat reserves and demonstrate that its functions in the adult fatbody are crucial for starvation survival. Further, dSir2 knockdown in the fatbody affects dilp5 (insulin-like-peptide) expression, and mediates systemic effects of insulin signaling. This report delineates the functions of dSir2 in the fatbody and muscles with systemic consequences on fat metabolism and insulin signaling. In conclusion, these findings highlight the central role that fatbody dSir2 plays in linking metabolism to organismal physiology and its importance for survival.

Efficacy of chloroquine and sulphadoxine-pyrimethamine either alone or in combination before introduction of ACT as first-line therapy in uncomplicated Plasmodium falciparum malaria in Jalpaiguri District, West Bengal, India.

OBJECTIVE: In India, till recently, Chloroquine was used as first-line therapy in areas with Chloroquine sensitive Plasmodium falciparum malaria cases. The National Vector Borne Disease Control Programme (NVBDCP) has introduced artemisinin combination therapy (ACT) as first-line option to treat all P. falciparum cases in the country. This study was carried out to ascertain the efficacy of Chloroquine and Sulphadoxine-Pyrimethamine, either alone or in combination, before the launch of ACT by NVBDCP. METHODS: A total of 300 P. falciparum malaria cases were enrolled randomly in three study arms, Chloroquine (CQ), Sulphadoxine-Pyrimethamine (SP) and Chloroquine plus Sulphadoxine-Pyrimethamine (CQ + SP). All patients were followed up for 28 days as per WHO (Assessment and Monitoring of Antimalarial Drug Efficacy for the Treatment of Uncomplicated Falciparum Malaria, Geneva, 2003) Protocol. Paired blood samples of treatment failure cases were collected and subjected to MSP 1, MSP 2 and GLURP genotyping for differentiation between re-infection and recrudescence. The data were analysed by Kaplan-Meier survival curve according to WHO standard procedures. RESULTS: The overall failure rate including both early treatment failure (ETF) and late treatment failure (LTF) of CQ, SP and CQ + SP were 61%, 14% and 8%, respectively, in the study area. Of 60 recurrent malaria cases, genotyping was successful in 49 cases, revealing that most of the (46/49; 94%) cases of recurrent malaria were due to recrudescence. CONCLUSION: In Jalpaiguri District the overall failure rate of CQ was 61% and of SP 14%, which was well above the WHO recommended cut-off threshold level (10%) for change of drug policy.

Substrate catalysis enhances single-enzyme diffusion.

We show that diffusion of single urease enzyme molecules increases in the presence of urea in a concentration-dependent manner and calculate the force responsible for this increase. Urease diffusion measured using fluorescence correlation spectroscopy increased by 16-28% over buffer controls at urea concentrations ranging from 0.001 to 1 M. This increase was significantly attenuated when urease was inhibited with pyrocatechol, demonstrating that the increase in diffusion was the result of enzyme catalysis of urea. Local molecular pH changes as measured using the pH-dependent fluorescence lifetime of SNARF-1 conjugated to urease were not sufficient to explain the increase in diffusion. Thus, a force generated by self-electrophoresis remains the most plausible explanation. This force, evaluated using Brownian dynamics simulations, was 12 pN per reaction turnover. These measurements demonstrate force generation by a single enzyme molecule and lay the foundation for a further understanding of biological force generation and the development of enzyme-driven nanomotors.