Amiloride-sensitive ion channels are formed from homo-or heteromeric combinations of subunits from the epithelial Na ؉ channel (ENaC)/degenerin superfamily, which also includes the acid-sensitive ion channel (ASIC) family. These channel subunits share sequence homology and topology. In this study, we have demonstrated, using confocal fluorescence resonance energy transfer microscopy and co-immunoprecipitation, that ASIC and ENaC subunits are capable of forming cross-clade intermolecular interactions. We have also shown that combinations of ASIC1 with ENaC subunits exhibit novel electrophysiological characteristics compared with ASIC1 alone. The results of this study suggest that heteromeric complexes of ASIC and ENaC subunits may underlie the diversity of amiloride-sensitive cation conductances observed in a wide variety of tissues and cell types where co-expression of ASIC and ENaC subunits has been observed.Amiloride-sensitive Na ϩ channels are formed from combinations of subunits from the epithelial Na ϩ channel (ENaC) 2 / degenerin superfamily, which includes ENaC, degenerin, and acid-sensitive ion channels (ASICs). Members of this superfamily of more than 60 identified subunits share a common membrane topology, with relatively short intracellular N and C termini (ϳ100 amino acids), two transmembrane-spanning ␣-helices, and a large extracellular loop (ϳ400 amino acids) (1). ENaC/degenerin subunits share 15-20% sequence identity across the entire superfamily. Within individual subfamilies, the sequence identity rises (ϳ30% identity for ENaCs, 30% for degenerins, and 45-60% for ASICs) (2).Amiloride-sensitive ion channels have been identified in a wide variety of cell lines and tissue types. ENaCs were initially isolated from the kidney, where Na ϩ reabsorption in the distal collecting duct is required for water reabsorption and concentration of the urine (3). Inhibition of ENaC with amiloride therefore leads to diuresis. ENaCs have since been identified in several other tissues, including vascular smooth muscle, oocytes, lymphocytes, neurons, osteoblasts, pancreas, testis, ovary, heart, lung, and urinary bladder (4 -11). ASIC subunits were initially identified in the brain and dorsal root ganglion by sequence homology with known ENaC subunits (12). They have since been identified in the central and peripheral nervous system and in the cardiac and skeletal myocytes (13-17). Mounting evidence indicates that ASIC and ENaC subunits are co-expressed in multiple tissues and cell types, including pheochromocytoma cells, osteoblasts, chondocytes, astrocytes, retina, lung, kidney, taste receptors, and dorsal root ganglion cells (Table 1) (9,14,(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34).The electrophysiological characteristics of cells expressing amiloride-sensitive cation conductances vary widely, and regulation of ENaCs and ASICs occurs through several mechanisms. Pharmacological inhibitors of channel function include small molecules, such as amiloride, or peptide toxins, such as psalmoto...

Rapid, specific, and sensitive detection of airborne bacteria, viruses, and toxins is critical for biodefense, yet the diverse nature of the threats poses a challenge for integrated surveillance, as each class of pathogens typically requires different detection strategies. Here, we present a laboratory-on-a-chip microfluidic device (LOC-DLA) that integrates two unique assays for the detection of airborne pathogens: direct linear analysis (DLA) with unsurpassed specificity for bacterial threats and Digital DNA for toxins and viruses. The LOC-DLA device also prepares samples for analysis, incorporating upstream functions for concentrating and fractionating DNA. Both DLA and Digital DNA assays are single molecule detection technologies, therefore the assay sensitivities depend on the throughput of individual molecules. The microfluidic device and its accompanying operation protocols have been heavily optimized to maximize throughput and minimize the loss of analyzable DNA. We present here the design and operation of the LOC-DLA device, demonstrate multiplex detection of rare bacterial targets in the presence of 100-fold excess complex bacterial mixture, and demonstrate detection of picogram quantities of botulinum toxoid.

Current single-cell RNA-sequencing approaches have limitations that stem from the microfluidic devices or fluid handling steps required for sample processing. We develop a method that does not require specialized microfluidic devices, expertise or hardware. Our approach is based on particle-templated emulsification, which allows single-cell encapsulation and barcoding of cDNA in uniform droplet emulsions with only a vortexer. Particle-templated instant partition sequencing (PIP-seq) accommodates a wide range of emulsification formats, including microwell plates and large-volume conical tubes, enabling thousands of samples or millions of cells to be processed in minutes. We demonstrate that PIP-seq produces high-purity transcriptomes in mouse–human mixing studies, is compatible with multiomics measurements and can accurately characterize cell types in human breast tissue compared to a commercial microfluidic platform. Single-cell transcriptional profiling of mixed phenotype acute leukemia using PIP-seq reveals the emergence of heterogeneity within chemotherapy-resistant cell subsets that were hidden by standard immunophenotyping. PIP-seq is a simple, flexible and scalable next-generation workflow that extends single-cell sequencing to new applications.

Many applications in pharmaceutical development, clinical diagnostics, and biological research demand rapid detection of multiple analytes (multiplexed detection) in a minimal volume. This need has led to the development of several novel array-based sensors. The most successful of these so far have been suspension arrays based on polystyrene beads. However, the 5 microm beads used for these assays are incompatible with most microfluidic chip technologies, mostly due to clogging problems. The challenge, then, is to design a detection particle that has high information content (for multiplexed detection), is compatible with miniaturization, and can be manufactured easily at low cost. DNA is a solid molecular wire that is easily produced and manipulated, which makes it a useful material for nanoparticles. DNA molecules are very information-rich, readily deformable, and easily propagated. We exploit these attributes in a suspension array sensor built from specialized recombinant DNA, Digital DNA, that carries both specific analyte-recognition units, and a geometrically encoded identification pattern. Here we show that this sensor combines high multiplexing with high sensitivity, is biocompatible, and has sufficiently small particle size to be used within microfluidic chips that are only 1 microm deep. We expect this technology will be the foundation of a broadly applicable technique to identify and quantitate proteins, nucleic acids, viruses, and toxins simultaneously in a minimal volume.