Conservative, gravitational self-force for a particle in circular orbit around a Schwarzschild black hole in a Radiation Gauge

This is the second of two companion papers on computing the self-force in a radiation gauge; more precisely, the method uses a radiation gauge for the radiative part of the metric perturbation, together with an arbitrarily chosen gauge for the parts of the perturbation associated with changes in black-hole mass and spin and with a shift in the center of mass. We compute the conservative part of the self-force for a particle in circular orbit around a Schwarzschild black hole. The gauge vector relating our radiation gauge to a Lorenz gauge is helically symmetric, implying that the quantity h_{\alpha\beta} u^\alpha u^\beta (= h_{uu}) must have the same value for our radiation gauge as for a Lorenz gauge; and we confirm this numerically to one part in 10^{13}. As outlined in the first paper, the perturbed metric is constructed from a Hertz potential that is in term obtained algebraically from the the retarded perturbed spin-2 Weyl scalar, \psi_0 . We use a mode-sum renormalization and find the renormalization coefficients by matching a series in L = \ell + 1/2 to the large-L behavior of the expression for the self-force in terms of the retarded field h_{\alpha\beta}^{ret}; we similarly find the leading renormalization coefficients of h_{uu} and the related change in the angular velocity of the particle due to its self-force. We show numerically that the singular part of the self-force has the form f_{\alpha} \propto < \nabla_\alpha \rho^{-1}>, the part of \nabla_\alpha \rho^{-1} that is axisymmetric about a radial line through the particle. This differs only by a constant from its form for a Lorenz gauge. It is because we do not use a radiation gauge to describe the change in black-hole mass that the singular part of the self-force has no singularity along a radial line through the particle and, at least in this example, is spherically symmetric to subleading order in \rho.Comment: 21 pages, 2 figure

Synergistic assembly of human pre-spliceosomes across introns and exons

Most human genes contain multiple introns, necessitating mechanisms to effectively define exons and ensure their proper connection by spliceosomes. Human spliceosome assembly involves both cross-intron and cross-exon interactions, but how these work together is unclear. We examined in human nuclear extracts dynamic interactions of single pre-mRNA molecules with individual fluorescently tagged spliceosomal subcomplexes to investigate how cross-intron and cross-exon processes jointly promote pre-spliceosome assembly. U1 subcomplex bound to the 5\u27 splice site of an intron acts jointly with U1 bound to the 5\u27 splice site of the next intron to dramatically increase the rate and efficiency by which U2 subcomplex is recruited to the branch site/3\u27 splice site of the upstream intron. The flanking 5\u27 splice sites have greater than additive effects implying distinct mechanisms facilitating U2 recruitment. This synergy of 5\u27 splice sites across introns and exons is likely important in promoting correct and efficient splicing of multi-intron pre-mRNAs

A General Mechanism for Competitor-induced Dissociation of Molecular Complexes

The kinetic stability of non-covalent macromolecular complexes controls many biological phenomena. Here we find that physical models of complex dissociation predict that competitor molecules will, in general, accelerate the breakdown of isolated bimolecular complexes by occluding rapid rebinding of the two binding partners. This prediction is largely independent of molecular details. We confirm the prediction with single-molecule fluorescence experiments on a well-characterized DNA strand dissociation reaction. Contrary to common assumptions, competitor-induced acceleration of dissociation can occur in biologically relevant competitor concentration ranges and does not necessarily imply ternary association of competitor with the bimolecular complex. Thus, occlusion of complex rebinding may play a significant role in a variety of biomolecular processes. The results also show that single-molecule colocalization experiments can accurately measure dissociation rates despite their limited spatiotemporal resolution

Single-Molecule Studies of Origin Licensing Reveal Mechanisms Ensuring Bidirectional Helicase Loading

Loading of the ring-shaped Mcm2–7 replicative helicase around DNA licenses eukaryotic origins of replication. During loading, Cdc6, Cdt1, and the origin-recognition complex (ORC) assemble two heterohexameric Mcm2–7 complexes into a head-to-head double hexamer that facilitates bidirectional replication initiation. Using multi-wavelength single-molecule fluorescence to monitor the events of helicase loading, we demonstrate that double-hexamer formation is the result of sequential loading of individual Mcm2–7 complexes. Loading of each Mcm2–7 molecule involves the ordered association and dissociation of distinct Cdc6 and Cdt1 proteins. In contrast, one ORC molecule directs loading of both helicases in each double hexamer. Based on single-molecule FRET, arrival of the second Mcm2–7 results in rapid double-hexamer formation that anticipates Cdc6 and Cdt1 release, suggesting that Mcm-Mcm interactions recruit the second helicase. Our findings reveal the complex protein dynamics that coordinate helicase loading and indicate that distinct mechanisms load the oppositely oriented helicases that are central to bidirectional replication initiation.National Institutes of Health (U.S.) (NIH grant GM52339)National Institutes of Health (U.S.) (NIH grant R01 GM81648)G. Harold and Leila Y. Mathers FoundationNational Institutes of Health (U.S.) (NIH Pre-Doctoral Training Grant (GM007287))Howard Hughes Medical Institute (Investigator

Nerve-targeted probes for fluorescence-guided intraoperative imaging.

A fundamental goal of many surgeries is nerve preservation, as inadvertent injury can lead to patient morbidity including numbness, pain, localized paralysis and incontinence. Nerve identification during surgery relies on multiple parameters including anatomy, texture, color and relationship to surrounding structures using white light illumination. We propose that fluorescent labeling of nerves can enhance the contrast between nerves and adjacent tissue during surgery which may lead to improved outcomes. Methods: Nerve binding peptide sequences including HNP401 were identified by phage display using selective binding to dissected nerve tissue. Peptide dye conjugates including FAM-HNP401 and structural variants were synthesized and screened for nerve binding after topical application on fresh rodent and human tissue and in-vivo after systemic IV administration into both mice and rats. Nerve to muscle contrast was quantified by measuring fluorescent intensity after topical or systemic administration of peptide dye conjugate. Results: Peptide dye conjugate FAM-HNP401 showed selective binding to human sural nerve with 10.9x fluorescence signal intensity (1374.44 ± 425.96) compared to a previously identified peptide FAM-NP41 (126.17 ± 61.03). FAM-HNP401 showed nerve-to-muscle contrast of 3.03 ± 0.57. FAM-HNP401 binds and highlight multiple human peripheral nerves including lower leg sural, upper arm medial antebrachial as well as autonomic nerves isolated from human prostate. Conclusion: Phage display has identified a novel peptide that selectively binds to ex-vivo human nerves and in-vivo using rodent models. FAM-HNP401 or an optimized variant could be translated for use in a clinical setting for intraoperative identification of human nerves to improve visualization and potentially decrease the incidence of intra-surgical nerve injury