Changepoint Analysis for Single-Molecule Polarized Total Internal Reflection Fluorescence Microscopy Experiments

The experimental study of individual macromolecules has opened a door to determining the details of their mechanochemical operation. Motor enzymes such as the myosin family have been particularly attractive targets for such study, in part because some of them are highly processive and their “product” is spatial motion. But single-molecule resolution comes with its own costs and limitations. Often, the observations rest on single fluorescent dye molecules, which emit a limited number of photons before photobleaching and are subject to complex internal dynamics. Thus, it is important to develop methods that extract the maximum useful information from a finite set of detected photons. We have extended an experimental technique, multiple polarization illumination in total internal reflection fluorescence microscopy (polTIRF), to record the arrival time and polarization state of each individual detected photon. We also extended an analysis technique, previously applied to FRET experiments, that optimally determines times of changes in photon emission rates. Combining these improvements allows us to identify the structural dynamics of a molecular motor (myosin V) with unprecedented detail and temporal resolution

Twirling of Actin by Myosins II and V Observed via Polarized TIRF in a Modified Gliding Assay

The force generated between actin and myosin acts predominantly along the direction of the actin filament, resulting in relative sliding of the thick and thin filaments in muscle or transport of myosin cargos along actin tracks. Previous studies have also detected lateral forces or torques that are generated between actin and myosin, but the origin and biological role of these sideways forces is not known. Here we adapt an actin gliding filament assay in order to measure the rotation of an actin filament about its axis (“twirling”) as it is translocated by myosin. We quantify the rotation by determining the orientation of sparsely incorporated rhodamine-labeledactin monomers, using polarized total internal reflection (polTIRF) microscopy. In order to determine the handedness of the filament rotation, linear incident polarizations in between the standard s- and p-polarizations were generated, decreasing the ambiguity of our probe orientation measurement four-fold. We found that whole myosin II and myosin V both twirl actin with a relatively long (~ µm), left-handed pitch that is insensitive to myosin concentration, filament length and filament velocity

Twirling of actin by myosins II and V observed via polarized TIRF in a modified gliding assay

The force generated between actin and myosin acts predominantly along the direction of the actin filament, resulting in relative sliding of the thick and thin filaments in muscle or transport of myosin cargos along actin tracks. Previous studies have also detected lateral forces or torques that are generated between actin and myosin, but the origin and biological role of these sideways forces is not known. Here we adapt an actin gliding filament assay in order to measure the rotation of an actin filament about its axis (twirling) as it is translocated by myosin. We quantify the rotation by determining the orientation of sparsely incorporated rhodamine-labeled actin monomers, using polarized total internal reflection (polTIRF) microscopy. In order to determine the handedness of the filament rotation, linear incident polarizations in between the standard s- and p-polarizations were generated, decreasing the ambiguity of our probe orientation measurement four-fold. We found that whole myosin II and myosin V both twirl actin with a relatively long (micron), left-handed pitch that is insensitive to myosin concentration, filament length and filament velocity

\u3cem\u3eγ\u3c/em\u3e-ray Spectroscopy of \u3csup\u3e166\u3c/sup\u3eHf: X(5) in \u3cem\u3eN\u3c/em\u3e \u3e 90?

Excited states in 166Hf were populated in the β+/∈ decay of 166Ta and studied through off-beam γ –ray spectroscopy at the Yale moving tape collector. New coincidence data found no support for two previously reported excited 0+ states and led to a substantially revised level scheme. Similarities between the revised level scheme of 166Hf and the X(5) critical point symmetry are discussed, and the extent of X(5) behavior in this mass region is explored through the W and Os isotopes. Among X(5) candidates with N \u3e 90, good agreement is observed for most energies and interband B(E2) strengths, while all exhibit similar disagreements with other key observables, in particular, yrast B(E2) values and spacing in the excited K = 0+ sequence

Revisiting anomalous \u3cem\u3eB\u3c/em\u3e(\u3cem\u3eE\u3c/em\u3e2;4\u3csup\u3e+\u3c/sup\u3e\u3csub\u3e1\u3c/sub\u3e→2\u3csup\u3e+\u3c/sup\u3e\u3csub\u3e1\u3c/sub\u3e)/\u3cem\u3eB\u3c/em\u3e(\u3cem\u3eE\u3c/em\u3e2;2\u3csup\u3e+\u3c/sup\u3e\u3csub\u3e1\u3c/sub\u3e→0\u3csup\u3e+\u3c/sup\u3e\u3csub\u3e1\u3c/sub\u3e) values in \u3csup\u3e98\u3c/sup\u3eRu and \u3csup\u3e180\u3c/sup\u3ePt

Recently, a set of nine nonmagic nuclei with anomalous values of the B(E2) ratio B4/2 ≡ B(E2; 4+1 → 2+1)/B(E2; 2+1 → 0+1) were identified. Such values are outside the range allowed by current collective models. In the present work, the B(E2; 4+1 → 2+1) values for two of these nuclei, 98Ru and 180Pt, were re-measured to determine if the current literature values for these nuclei are correct. 98Ru was studied in a 27Al(98Ru,98Ru∗) Coulomb excitation experiment in inverse kinematics, while the lifetime of the 4+1 state in 180Pt was measured in a 122Sn(62Ni, 4n)180Pt recoil distance method (RDM) experiment. For both nuclei, the remeasured B4/2 values are well above 1, removing the deviations from collective models

Inferring hidden Markov models from noisy time sequences: a method to alleviate degeneracy in molecular dynamics

We present a new method for inferring hidden Markov models from noisy time sequences without the necessity of assuming a model architecture, thus allowing for the detection of degenerate states. This is based on the statistical prediction techniques developed by Crutchfield et al., and generates so called causal state models, equivalent to hidden Markov models. This method is applicable to any continuous data which clusters around discrete values and exhibits multiple transitions between these values such as tethered particle motion data or Fluorescence Resonance Energy Transfer (FRET) spectra. The algorithms developed have been shown to perform well on simulated data, demonstrating the ability to recover the model used to generate the data under high noise, sparse data conditions and the ability to infer the existence of degenerate states. They have also been applied to new experimental FRET data of Holliday Junction dynamics, extracting the expected two state model and providing values for the transition rates in good agreement with previous results and with results obtained using existing maximum likelihood based methods.Comment: 19 pages, 9 figure

Spin-rotor Interpretation of Identical Bands and Quantized Alignment in Superdeformed A ≈\approx 190 Nuclei

The ``identical'' bands in superdeformed mercury, thallium, and lead nuclei are interpreted as examples of orbital angular momentum rotors with the weak spin-orbit coupling of pseudo-$SU(3)$ symmetries and supersymmetries.Comment: 15 pages, revtex 3.0, 7 figures available upon request from [email protected]

Triplet energy differences and the low lying structure of Ga 62

Background: Triplet energy differences (TED) can be studied to yield information on isospin-non-conserving interactions in nuclei. Purpose: The systematic behavior of triplet energy differences (TED) of T=1, J\u3c0=2+ states is examined. The A=62 isobar is identified as having a TED value that deviates significantly from an otherwise very consistent trend. This deviation can be attributed to the tentative assignments of the pertinent states in Ga62 and Ge62. Methods: An in-beam \u3b3-ray spectroscopy experiment was performed to identify excited states in Ga62 using Gamma-Ray Energy Tracking In-Beam Nuclear Array with the S800 spectrometer at NSCL using a two-nucleon knockout approach. Cross-section calculations for the knockout process and shell-model calculations have been performed to interpret the population and decay properties observed. Results: Using the systematics as a guide, a candidate for the transition from the T=1, 2+ state is identified. However, previous work has identified similar states with different J\u3c0 assignments. Cross-section calculations indicate that the relevant T=1, 2+ state should be one of the states directly populated in this reaction. Conclusions: As spins and parities were not measurable, it is concluded that an unambiguous identification of the first T=1, 2+ state is required to reconcile our understanding of TED systematics

Isomeric Decay of \u3csup\u3e208\u3c/sup\u3eRa

Low-energy excited states of 208Ra were investigated using the 182W(30Si, 4n) reaction at the Wright Nuclear Structure Laboratory of Yale University. Fusion evaporation recoils were selected using the gas-filled spectrometer SASSYER. Delayed γ rays, following isomeric decays, were detected at the focal plane of SASSYER with a small array of three clover Ge detectors. Transitions following a proposed J π = 8+ isomer were observed, and the half-life was measured

MicroRNA inhibition using antimiRs in acute human brain tissue sections

Antisense inhibition of microRNAs is an emerging preclinical approach to pharmacoresistant epilepsy. A leading candidate is an "antimiR" targeting microRNA-134 (ant-134), but testing to date has used rodent models. Here, we develop an antimiR testing platform in human brain tissue sections. Brain specimens were obtained from patients undergoing resective surgery to treat pharmacoresistant epilepsy. Neocortical specimens were submerged in modified artificial cerebrospinal fluid (ACSF) and dissected for clinical neuropathological examination, and unused material was transferred for sectioning. Individual sections were incubated in oxygenated ACSF, containing either ant-134 or a nontargeting control antimiR, for 24 h at room temperature. RNA integrity was assessed using BioAnalyzer processing, and individual miRNA levels were measured using quantitative reverse transcriptase polymerase chain reaction. Specimens transported in ACSF could be used for neuropathological diagnosis and had good RNA integrity. Ant-134 mediated a dose-dependent knockdown of miR-134, with approximately 75% reduction of miR-134 at 1 μmol L-1 and 90% reduction at 3 μmol L-1 . These doses did not have off-target effects on expression of a selection of three other miRNAs. This is the first demonstration of ant-134 effects in live human brain tissues. The findings lend further support to the preclinical development of a therapy that targets miR-134 and offer a flexible platform for the preclinical testing of antimiRs, and other antisense oligonucleotide therapeutics, in human brain