Neutrophil Extracellular Traps have DNAzyme activity that drives bactericidal potential.

The mechanisms of bacterial killing by neutrophil extracellular traps (NETs) are unclear. DNA, the largest component of NETs is believed to merely be a scaffold with minimal antimicrobial activity through the charge of the backbone. Here, we report that NETs DNA is beyond a scaffold and produces hydroxyl free radicals through the spatially concentrated G-quadruplex/hemin DNAzyme complexes, driving bactericidal effects. Immunofluorescence staining showed colocalization of G-quadruplex and hemin in extruded NETs DNA, and Amplex UltraRed assay portrayed its peroxidase activity. Proximity labeling of bacteria revealed localized concentration of radicals resulting from NETs bacterial trapping. Ex vivo bactericidal assays revealed that G-quadruplex/hemin DNAzyme is the primary driver of bactericidal activity in NETs. NETs are DNAzymes that may have important biological consequences.

Transient Induced Molecular Electronic Spectroscopy (TIMES) for study of protein-ligand interactions.

We present a method, Transient Induced Molecular Electronic Spectroscopy (TIMES), to detect protein-ligand interactions without any protein engineering or chemical modification. We developed a physics model for the TIMES signal and mathematically formulated the problem to attain physical insight of protein-ligand interactions without any disturbances by molecular probes, fluorescent labels, or immobilization of molecules. To demonstrate the functionality of this method, we have used the TIMES signals to find the dissociation constants for the affinity of reactions, the shear-stress dependent adsorption time of molecules on surface, and other interesting features of protein-ligand interaction in native conditions. As a unique tool, TIMES offers a simple and effective method to investigate fundamental protein chemistry and drug discoveries.

Self-Assembled Pico-Liter Droplet Microarray for Ultrasensitive Nucleic Acid Quantification.

Nucleic acid detection and quantification technologies have made remarkable progress in recent years. Among existing platforms, hybridization-based assays have the advantages of being amplification free, low instrument cost, and high throughput, but are generally less sensitive compared to sequencing and PCR assays. To bridge this performance gap, we developed a quantitative physical model for the hybridization-based assay to guide the experimental design, which leads to a pico-liter droplet environment with drastically enhanced performance and detection limit several order above any current microarray platform. The pico-liter droplet hybridization platform is further coupled with the on-chip enrichment technique to yield ultrahigh sensitivity both in terms of target concentration and copy number. Our physical model, taking into account of molecular transport, electrostatic intermolecular interactions, reaction kinetics, suggests that reducing liquid height and optimizing target concentration will maximize the hybridization efficiency, and both conditions can be satisfied in a highly parallel, self-assembled pico-liter droplet microarray that produces a detection limit as low as 570 copies and 50 aM. The pico-liter droplet array device is realized with a micropatterned superhydrophobic black silicon surface that allows enrichment of nucleic acid samples by position-defined evaporation. With on-chip enrichment and oil encapsulated pico-liter droplet arrays, we have demonstrated a record high sensitivity, wide dynamic range (6 orders of magnitude), and marked reduction of hybridization time from >10 h to <5 min in a highly repeatable fashion, benefiting from the physics-driven design and nanofeatures of the device. The design principle and technology can contribute to biomedical sensing and point-of-care clinical applications such as pathogen detection and cancer diagnosis and prognosis.

Nucleic Acid Aptamers: An Emerging Tool for Biotechnology and Biomedical Sensing.

Detection of small molecules or proteins of living cells provides an exceptional opportunity to study genetic variations and functions, cellular behaviors, and various diseases including cancer and microbial infections. Our aim in this review is to give an overview of selected research activities related to nucleic acid-based aptamer techniques that have been reported in the past two decades. Limitations of aptamers and possible approaches to overcome these limitations are also discussed.

Tapping a bacterial enzymatic pathway for the preparation and manipulation of synthetic nanomaterials.

We present a spherical micelle generated in a three-step sequence in which a farnesyl-pantetheine conjugate is phosphorylated, adenylated, and phosphorylated once more to generate a farnesyl-CoA amphiphile that self-assembles into spherical micelles. A sphere-to-fibril morphological switch is achieved by enzymatically transferring the farnesyl group of the farnesyl-CoA micelle onto a peptide via phosphopantetheinyl transferase to generate a peptide amphiphile. Each step in the sequence is followed with characterization by HPLC, MS, TEM, and DLS. This system offers an entry into cofactor-mediated peptide decoration by extending the principles of bioresponsive polymeric materials to sequential enzyme cascades.

Oil-encapsulated nanodroplet array for bio-molecular detection.

Detection of low abundance biomolecules is challenging for biosensors that rely on surface chemical reactions. For surface reaction based biosensors, it require to take hours or even days for biomolecules of diffusivities in the order of 10(-10-11) m2/s to reach the surface of the sensors by Brownian motion. In addition, often times the repelling Coulomb interactions between the molecules and the probes further defer the binding process, leading to undesirably long detection time for applications such as point-of-care in vitro diagnosis. In this work, we designed an oil encapsulated nanodroplet array microchip utilizing evaporation for pre-concentration of the targets to greatly shorten the reaction time and enhance the detection sensitivity. The evaporation process of the droplets is facilitated by the superhydrophilic surface and resulting nanodroplets are encapsulated by oil drops to form stable reaction chamber. Using this method, desirable droplet volumes, concentrations of target molecules, and reaction conditions (salt concentrations, reaction temperature, etc.) in favour of fast and sensitive detection are obtained. A linear response over 2 orders of magnitude in target concentration was achieved at 10 fM for protein targets and 100 fM for miRNA mimic oligonucleotides.

Enzyme-directed assembly of a nanoparticle probe in tumor tissue.

Enzyme-directed assembly in vivo: A targeting strategy is demonstrated, which leads to an active accumulation of nanoparticles by virtue of an assembly event specific to endogenous, enzymatic biochemical signals associated with tumor tissue. The viability of this approach is examined through a proof-of-concept study showing enzyme-directed particle targeting and accumulation in human xenograft tumors in mice following intravenous injection, and the retention of particles is demonstrated within tumors for extended periods of time.

Controlling and switching the morphology of micellar nanoparticles with enzymes.

Micelles were prepared from polymer-peptide block copolymer amphiphiles containing substrates for protein kinase A, protein phosphatase-1, and matrix metalloproteinases 2 and 9. We examine reversible switching of the morphology of these micelles through a phosphorylation-dephosphorylation cycle and study peptide-sequence directed changes in morphology in response to proteolysis. Furthermore, the exceptional uniformity of these polymer-peptide particles makes them amenable to cryo-TEM reconstruction techniques lending insight into their internal structure.

Smart lipids for programmable nanomaterials.

Novel, responsive liposomes are introduced, assembled from DNA-programmed lipids allowing sequence selective manipulation of nanoscale morphology. Short, single-stranded DNA sequences form polar head groups conjugated to hydrophobic tails. The morphology of the resulting lipid aggregates depends on sterics and electronics in the polar head groups and, therefore, is dependent on the DNA hybridization state. The programmability, specificity, and reversibility of the switchable system are demonstrated via dynamic light scattering, transmission electron microscopy, and fluorescence microscopy.

Association between lysyl oxidase polymorphisms and oral submucous fibrosis in older male areca chewers.

OBJECTIVE: Areca use is the major cause for oral squamous cell carcinoma and oral submucous fibrosis (OSF) in South Asians. Lysyl oxidase (LOX) is a copper-activated enzyme critical for collagen cross-linking and organization of extracellular matrix. The presence of a G to A polymorphism at nucleotide 473 caused a non-conservative Arg158Gln change in the LOX amino acid sequence. OSF is a precancerous lesions characterized by the accumulation of collagen in oral submucosa. The aim of this study was to investigate the relationship between LOX Arg158Gln polymorphism and the risk of OSF. METHOD: PCR-restriction fragment length polymorphisms and direct sequencing was utilized to compare LOX polymorphic allelotype in male areca-chewing controls (n = 216) and OSF (n = 83) patients. RESULTS: There was a borderline of statistically significant difference in Arg158Gln genotype lying between control and OSF patients. However, the G/A+A/A of LOX Arg158Gln in OSF patients older than 50 year was statistically significantly higher than controls older than 50 year (odd's ratio: 4.48; 95% CI = 1.58-12.67). CONCLUSION: The elder OSF patients were increased in LOX Arg158Gln. Our findings may suggest a potential application in risk population selection using LOX polymorphism for preventive intervention of OSF genesis in a subset of areca chewers.

Association of expression aberrances and genetic polymorphisms of lysyl oxidase with areca-associated oral tumorigenesis.

PURPOSE: Areca nut use is the major cause of oral squamous cell carcinoma (OSCC) in Southern Asians. Areca nut contains a high level of free copper ions. Lysyl oxidase (LOX) is a copper-activated enzyme critical for extracellular matrix organization. Contradictory evidence has been put forward to suggest that LOX may be either an oncogenic or a suppressive element. This study investigated the oncogenic significance of LOX in areca-associated OSCC. EXPERIMENTAL DESIGN: The expression assays and polymorphism analysis were done to know the clinicopathologic implications of LOX status in OSCC. Knockdown and overexpression experiments were conducted to know the phenotypic effects of LOX on OSCC cells. RESULTS: Up-regulation of LOX mRNA and LOX protein expression in OSCCs relative to adjacent oral mucosa was found. Precancerous lesions had the highest LOX mRNA expression. Areca nut extract up-regulated LOX expression in oral epithelial cells. Knockdown of LOX induced cellular migration and invasion, but it reduced the anchorage-independent growth and xenographic tumorigenesis of OSCC cells. The reduction of migration and invasion by LOX overexpression was partially rescued by blockage of LOX activity. The Arg158Gln polymorphism was associated with earlier clinical stage of OSCC. Wild-type LOX overexpression induced anchorage-independent growth in OSCC cells, but this was not for LOXArg158Gln overexpression. CONCLUSION: LOX exerts oncogenic roles in areca-associated OSCC. This potential could be affected by the existence of LOX propeptide domain or genetic polymorphism.