Multimodal Single-Cell Analysis Reveals Physiological Maturation in the Developing Human Neocortex.

In the developing human neocortex, progenitor cells generate diverse cell types prenatally. Progenitor cells and newborn neurons respond to signaling cues, including neurotransmitters. While single-cell RNA sequencing has revealed cellular diversity, physiological heterogeneity has yet to be mapped onto these developing and diverse cell types.

Corrigendum: Fluidic Logic Used in a Systems Approach to Enable Integrated Single-Cell Functional Analysis.

[This corrects the article on p. 70 in vol. 4, PMID: 27709111.

Fluidic Logic Used in a Systems Approach to Enable Integrated Single-Cell Functional Analysis.

The study of single cells has evolved over the past several years to include expression and genomic analysis of an increasing number of single cells. Several studies have demonstrated wide spread variation and heterogeneity within cell populations of similar phenotype. While the characterization of these populations will likely set the foundation for our understanding of genomic- and expression-based diversity, it will not be able to link the functional differences of a single cell to its underlying genomic structure and activity.

Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex.

Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification.

A completely automated sample preparation instrument and consumable device for isolation and purification of nucleic acids.

Molecular diagnostic analysis and life science studies are dependent on the ability to effectively prepare samples for analysis. We report the development of a system that enables robust sample preparation of nucleic acids. To enable completely automated sample preparation, a consumable cartridge and consumable module system were developed to emulate every step of the sample preparation process.

An ultra-high temperature flow-through capillary device for bacterial spore lysis.

Rapid and specific characterization of bacterial endospores is dependent on the ability to rupture the cell wall to enable analysis of the intracellular components. In particular, bacterial spores from the bacillus genus are inherently robust and very difficult to lyze or solubilize. Standard protocols for spore inactivation include chemical treatment, sonication, pressure, and thermal lysis.

Surpassing specificity limits of nucleic acid probes via cooperativity.

The failure to correctly identify single nucleotide polymorphisms (SNPs) significantly contributes to the misdiagnosis of infectious disease. Contrary to the strategy of creating shorter probes to improve SNP differentiation, we created larger probes that appeared to increase selectivity. Specifically, probes with enhanced melting temperature differentials (>13x improvement) to SNPs were generated by linking two probes that consist of both a capture sequence and a detection sequence; these probes act cooperatively to improve selectivity over a wider range of reaction conditions.

Tentacle probe sandwich assay in porous polymer monolith improves specificity, sensitivity and kinetics.

Nucleic acid sandwich assays improve low-density array analysis through the addition of a capture probe and a specific label, increasing specificity and sensitivity. Here, we employ photo-initiated porous polymer monolith (PPM) as a high-surface area substrate for sandwich assay analysis. PPMs are shown to enhance extraction efficiency by 20-fold from 2 microl of sample.

Fabrication of porous polymer monoliths in microfluidic chips for selective nucleic acid concentration and purification.

Efficient and rapid isolation of nucleic acids is of significant importance in the field of genomics for a variety of applications. Current techniques for the isolation of specific nucleic acids or genes typically involve multiple rounds of amplification of the target sequence using polymerase chain reaction. Described here is a recent development in the fabrication and modification of porous polymer monoliths for the selective concentration and extraction of nucleic acids sequences.

Tentacle Probes: differentiation of difficult single-nucleotide polymorphisms and deletions by presence or absence of a signal in real-time PCR.

Background: False-positive results are a common problem in real-time PCR identification of DNA sequences that differ from near neighbors by a single-nucleotide polymorphism (SNP) or deletion. Because of a lack of sufficient probe specificity, post-PCR analysis, such as a melting curve, is often required for mutation differentiation.

Methods: Tentacle Probes, cooperative reagents with both a capture and a detection probe based on specific cell-targeting principles, were developed as a replacement for 2 chromosomal TaqMan-minor groove binder (MGB) assays previously developed for Yersinia pestis and Bacillus anthracis detection.

Microfluidic purification and preconcentration of mRNA by flow-through polymeric monolith.

Efficient and rapid isolation of mRNA is important in the field of genomics as well as in the clinical and pharmaceutical arena. We have developed UV-initiated methacrylate-based porous polymer monoliths (PPM) for microfluidic trapping and concentration of eukaryotic mRNA. PPM are cast-to-shape and are tunable for functionalization using a variety of amine-terminated molecules.

Tentacle probes: eliminating false positives without sacrificing sensitivity.

The majority of efforts to increase specificity or sensitivity in biosensors result in trade-offs with little to no gain in overall accuracy. This is because a biosensor cannot be more accurate than the affinity interaction it is based on. Accordingly, we have developed a new class of reagents based on mathematical principles of cooperativity to enhance the accuracy of the affinity interaction.

Microchip separations of protein biotoxins using an integrated hand-held device.

We report the development of a hand-held instrument capable of performing two simultaneous microchip separations (gel and zone electrophoresis), and demonstrate this instrument for the detection of protein biotoxins. Two orthogonal analysis methods are chosen over a single method in order to improve the probability of positive identification of the biotoxin in an unknown mixture. Separations are performed on a single fused-silica wafer containing two separation channels.

Hand-held microanalytical instrument for chip-based electrophoretic separations of proteins.

The design, fabrication, and demonstration of a hand-held microchip-based analytical instrument for detection and identification of proteins and other biomolecules are reported. The overall system, referred to as muChemLab, has a modular design that provides for reliability and flexibility and that facilitates rapid assembly, fluid and microchip replacement, troubleshooting, and sample analysis. Components include two independent separation modules that incorporate interchangeable fluid cartridges, a 2-cm-square fused-silica microfluidic chip, and a miniature laser-induced fluorescence detection module.

Repeated inhalation exposures to the bioactivated cytotoxicant naphthalene (NA) produce airway-specific Clara cell tolerance in mice.

Repeated exposures to bioactivated cytotoxicants such as naphthalene (NA) render the target population, Clara cells, resistant to further injury through a glutathione-dependent mechanism. The current studies were designed to test the hypothesis that the mechanism for tolerance is localized in Clara cells. We used three approaches to test this hypothesis.

Induction of tolerance to naphthalene in Clara cells is dependent on a stable phenotypic adaptation favoring maintenance of the glutathione pool.

Repeated exposures to the Clara cell cytotoxicant naphthalene (NA) result in target cell populations that become refractory to further injury. To determine whether tolerance occurs from specific adaptations favoring glutathione (GSH) resynthesis without broad shifts in cellular phenotype, mice were administered NA for 21 days. We found that gamma-glutamylcysteine synthetase (gamma-GCS) was induced in tolerant Clara cells by repeated exposures to NA.