Activity

The realization of the vast potential of digital PCR (dPCR) to provide extremely accurate and sensitive measurements in the clinical setting has thus far been hindered by challenges such as assay robustness and high costs. Here we introduce a lossless and contamination-free dPCR technology, termed CLEAR-dPCR, which addresses these challenges by completing the dPCR sample preparation, PCR, and readout all in one tube. Optical clearing of the droplet dPCR emulsion was combined with emerging light-sheet fluorescence microscopy, to acquire a three-dimensional (3D) image of a half million droplets sealed in a tube in seconds. CLEAR-dPCR provides ultrahigh-throughput readout results in situ and fundamentally eliminates the possibility of either sample loss or contamination. This approach exhibits improved accuracy over existing dPCR platforms and enables a greatly increased dynamic range to be comparable to that of real-time quantitative PCR.Allostery is a fundamental regulatory mechanism of protein function. Despite notable advances, understanding the molecular determinants of allostery remains an elusive goal. Our current knowledge of allostery is principally shaped by a structure-centric view, which makes it difficult to understand the decentralized character of allostery. We present a function-centric approach using deep mutational scanning to elucidate the molecular basis and underlying functional landscape of allostery. We show that allosteric signaling exhibits a high degree of functional plasticity and redundancy through myriad mutational pathways. Residues critical for allosteric signaling are surprisingly poorly conserved while those required for structural integrity are highly conserved, suggesting evolutionary pressure to preserve fold over function. Our results suggest multiple solutions to the thermodynamic conditions of cooperativity, in contrast to the common view of a finely tuned allosteric residue network maintained under selection.Ionotropic glutamate receptors (iGluRs) are key molecules for synaptic signaling in the central nervous system, which makes them promising drug targets. Intensive efforts are being devoted to the development of subunit-selective ligands, which should enable more precise pharmacologic interventions while limiting the effects on overall neuronal circuit function. However, many AMPA and kainate receptor complexes in vivo are heteromers composed of different subunits. Despite their importance, little is known about how subunit-selective ligands affect the gating of heteromeric iGluRs, namely their activation and desensitization properties. RSV inhibitor Using fast ligand application experiments, we studied the effects of competitive antagonists that block glutamate from binding at part of the four subunits. We found that UBP-310, a kainate receptor antagonist with high selectivity for GluK1 subunits, reduces the desensitization of GluK1/GluK2 heteromers and fully abolishes the desensitization of GluK1/GluK5 heteromers. This effect is mirrored by subunit-selective agonists and heteromeric receptors that contain binding-impaired subunits, as we show for both kainate and GluA2 AMPA receptors. These findings are consistent with a model in which incomplete agonist occupancy at the four receptor subunits can provide activation without inducing desensitization. However, we did not detect significant steady-state currents during UBP-310 dissociation from GluK1 homotetramers, indicating that antagonist dissociation proceeds in a nonuniform and cooperativity-driven manner, which disfavors nondesensitizing occupancy states. Besides providing mechanistic insights, these results have direct implications for the use of subunit-selective antagonists in neuroscience research and envisioned therapeutic interventions.The regrowth of severed axons is fundamental to reestablish motor control after spinal-cord injury (SCI). Ongoing efforts to promote axonal regeneration after SCI have involved multiple strategies that have been only partially successful. Our study introduces an artificial carbon-nanotube based scaffold that, once implanted in SCI rats, improves motor function recovery. Confocal microscopy analysis plus fiber tracking by magnetic resonance imaging and neurotracer labeling of long-distance corticospinal axons suggest that recovery might be partly attributable to successful crossing of the lesion site by regenerating fibers. Since manipulating SCI microenvironment properties, such as mechanical and electrical ones, may promote biological responses, we propose this artificial scaffold as a prototype to exploit the physics governing spinal regenerative plasticity.During DNA replication, replicative DNA polymerases may encounter DNA lesions, which can stall replication forks. One way to prevent replication fork stalling is through the recruitment of specialized translesion synthesis (TLS) polymerases that have evolved to incorporate nucleotides opposite DNA lesions. Rev1 is a specialized TLS polymerase that bypasses abasic sites, as well as minor-groove and exocyclic guanine adducts. Lesion bypass is accomplished using a unique protein-template mechanism in which the templating base is evicted from the DNA helix and the incoming dCTP hydrogen bonds with an arginine side chain of Rev1. To understand the protein-template mechanism at an atomic level, we employed a combination of time-lapse X-ray crystallography, molecular dynamics simulations, and DNA enzymology on the Saccharomyces cerevisiae Rev1 protein. We find that Rev1 evicts the templating base from the DNA helix prior to binding the incoming nucleotide. Binding the incoming nucleotide changes the conformation of the DNA substrate to orient it for nucleotidyl transfer, although this is not coupled to large structural changes in Rev1 like those observed with other DNA polymerases. Moreover, we found that following nucleotide incorporation, Rev1 converts the pyrophosphate product to two monophosphates, which drives the reaction in the forward direction and prevents pyrophosphorolysis. Following nucleotide incorporation, the hydrogen bonds between the incorporated nucleotide and the arginine side chain are broken, but the templating base remains extrahelical. These postcatalytic changes prevent potentially mutagenic processive synthesis by Rev1 and facilitate dissociation of the DNA product from the enzyme.