Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ∼2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100 000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.

Dopamine (DA) is oxidized to the neurotoxic prooxidant species H2O2, OH•, and DA quinones. We tested whether dimethyl fumarate (DMF), an electrophile shown to induce a pleiotropic antioxidant response in nonneuronal cells, could reduce the toxicity of DA metabolites in neural cells. Treatment of the N18‐RE‐105 neuroblastoma‐retina hybridoma cell line with 30–150 µM dopamine led to cell death within 24 h, which increased steeply with dose, decreased with higher plating density, and was blocked by the H2O2‐metabolizing enzyme catalase. Pretreatment with DMF (30 µM, 24 h) significantly attenuated DA and H2O2 toxicity (40–60%) but not that caused by the calcium ionophore ionomycin. DMF treatment also elevated total intracellular GSH and increased activities of the antioxidant enzymes quinone reductase (QR), glutathione S‐transferase (GST), glutathione reductase, and the pentose phosphate enzyme glucose‐6‐phosphate dehydrogenase. To assess the protective efficacy of QR and GST, a stable cell line was constructed in which these enzymes were overexpressed. Cell death in the overexpressing line was not significantly different from that in a cell line expressing normal QR and GST activities, indicating that these two enzymes alone are insufficient for protection against DA toxicity. Although the relative importance of a single antioxidant enzyme such as QR or GST may be small, antioxidant inducers such as DMF may prove valuable as agents that elicit a broad‐spectrum neuroprotective response.

In the nervous system the influx of Ca2+ orchestrates multiple biochemical and electrical events essential for development and function. A major route for Ca2+ entry is through voltage-dependent calcium channels (VDCCs). It is becoming increasingly clear that the precise contribution VDCCs make to neuronal function depends not only upon their specific electrophysiological properties but also on their distribution over the nerve cell surface. One location where the presence of VDCCs may be critical is the dendritic spine, a structure known to be the major site of excitatory synaptic input. On spines, VDCCs are hypothesized to play an essential role in signal processing, learning, and memory. However, direct evidence for the presence of VDCCs on spines is lacking. Attempts to examine the distribution of VDCCs, or indeed any other components, on spines have been hampered since the size of many spines is close to the limits of resolution of conventional light microscopy. Using a new, biologically active, fluorescein conjugate of omega-conotoxin (Fl-omega-CgTx), a selective blocker of N-type VDCCs, and confocal microscopy, we have mapped the distributions of N-type VDCCs on live CA1 neurons in rat hippocampal slices. VDCCs were found on somata, throughout the dendritic arbor, and on dendritic spines in all hippocampal subfields. A comparison of three- dimensional reconstructions of structures labeled by Fl-omega-CgTx with those outlined by 1,1-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (Dil) or Lucifer yellow confirmed the presence of N-type VDCCs on dendritic spines. However, spine frequency on dendrites labeled with Fl- omega-CgTx was much lower than the spine frequency on dendrites labeled with Lucifer yellow or Dil, suggesting that some spines lack N-type VDCCs. These results offer the first direct evidence for the localization of any voltage-dependent channel on dendritic spines. The presence of N-type VDCCs on dendrites and their spines argues that these channels may participate in the generation of active Ca2+ conductances in distal dendrites, and is consistent with a role for spines as specialized compartments for concentrating Ca2+.

The switch from budding to filamentous growth is a key aspect of invasive growth and virulence for the fungal phytopathogen Ustilago maydis. The cyclic AMP (cAMP) signaling pathway regulates dimorphism in U. maydis, as demonstrated by the phenotypes of mutants with defects in protein kinase A (PKA). Specifically, a mutant lacking the regulatory subunit of PKA encoded by the ubc1 gene displays a multiple-budded phenotype and fails to incite disease symptoms, although proliferation does occur in the plant host. A mutant with a defect in a catalytic subunit of PKA, encoded by adr1, has a constitutively filamentous phenotype and is nonpathogenic. We employed serial analysis of gene expression to examine the transcriptomes of a wild-type strain and the ubc1 and adr1 mutants to further define the role of PKA in U. maydis. The mutants displayed changes in the transcript levels for genes encoding ribosomal proteins, genes regulated by the b mating-type proteins, and genes for metabolic functions. Importantly, the ubc1 mutant displayed elevated transcript levels for genes involved in phosphate acquisition and storage, thus revealing a connection between cAMP and phosphate metabolism. Further experimentation indicated a phosphate storage defect and elevated acid phosphatase activity for the ubc1 mutant. Elevated phosphate levels in culture media also enhanced the filamentous growth of wild-type cells in response to lipids, a finding consistent with PKA regulation of morphogenesis in U. maydis. Overall, these findings extend our understanding of cAMP signaling in U. maydis and reveal a link between phosphate metabolism and morphogenesis.Development and virulence are regulated by cyclic AMP (cAMP)/protein kinase A (PKA) and mitogen-activated protein (MAP) kinase signaling pathways in several fungi, including the corn smut pathogen Ustilago maydis (13,38,46). Mating of haploid U. maydis cells, which are nonpathogenic and yeast-like, leads to the formation of infectious, dikaryotic hyphae. This process is regulated by two unlinked mating-type loci, a and b. Cell recognition and fusion of conjugation tubes are controlled by the a mating-type locus encoding a pheromone (mfa)-pheromone receptor (pra) system that acts through a conserved MAP kinase module (8). Several components of the MAP kinase module have been identified, including the MAP kinase kinase kinase Ubc4, the MAP kinase kinase Fuz7/Ubc5, the MAP kinase Ubc3/Kpp2, and the putative adaptor protein Ubc2 (1,5,(46)(47)(48)50).