Super-resolution imaging and estimation of protein copy numbers at single synapses with DNA-PAINT

In the brain, the strength of each individual synapse is defined by the complement of proteins present or the "local proteome." Activity-dependent changes in synaptic strength are the result of changes in this local proteome and posttranslational protein modifications. Although most synaptic proteins have been identified, we still know little about protein copy numbers in individual synapses and variations between synapses. We use DNA-point accumulation for imaging in nanoscale topography as a single-molecule super-resolution imaging technique to visualize and quantify protein copy numbers in single synapses. The imaging technique provides near-molecular spatial resolution, is unaffected by photobleaching, enables imaging of large field of views, and provides quantitative molecular information. We demonstrate these benefits by accessing copy numbers of surface AMPA-type receptors at single synapses of rat hippocampal neurons along dendritic segments

The Effects of Negative Legacies on the Adjustment of Parentally Bereaved Children and Adolescents

This is a report of a qualitative analysis of a sample of bereaved families in which one parent died and in which children scored in the clinical range on the Child Behavior Check List. The purpose of this analysis was to learn more about the lives of these children. They were considered to be at risk of developing emotional and behavioral problems associated with the death. We discovered that many of these “high risk” children had a continuing bond with the deceased that was primarily negative and troubling for them in contrast to a comparison group of children not at risk from the same study. Five types of legacies, not mutually exclusive, were identified: health related, role related, personal qualities, legacy of blame, and an emotional legacy. Coping behavior on the part of the surviving parent seemed to make a difference in whether or not a legacy was experienced as negative

Construction and Assembly of the Wire Planes for the MicroBooNE Time Projection Chamber

In this paper we describe how the readout planes for the MicroBooNE Time Projection Chamber were constructed, assembled and installed. We present the individual wire preparation using semi-automatic winding machines and the assembly of wire carrier boards. The details of the wire installation on the detector frame and the tensioning of the wires are given. A strict quality assurance plan ensured the integrity of the readout planes. The different tests performed at all stages of construction and installation provided crucial information to achieve the successful realisation of the MicroBooNE wire planes.Comment: 24 pages, 22 figures, accepted for publication as Technical Report in JINS

Direct Visualization of Single Nuclear Pore Complex Proteins Using Genetically-Encoded Probes for DNA-PAINT

The nuclear pore complex (NPC) is one of the largest and most complex protein assemblies in the cell and, among other functions, serves as the gatekeeper of nucleocytoplasmic transport. Unraveling its molecular architecture and functioning has been an active research topic for decades with recent cryogenic electron microscopy and super-resolution studies advancing our understanding of the architecture of the NPC complex. However, the specific and direct visualization of single copies of NPC proteins is thus far elusive. Herein, we combine genetically-encoded self-labeling enzymes such as SNAP-tag and HaloTag with DNA-PAINT microscopy. We resolve single copies of nucleoporins in the human Y-complex in three dimensions with a precision of circa 3 nm, enabling studies of multicomponent complexes on the level of single proteins in cells using optical fluorescence microscopy

Retinal Architecture in ​\u3cem\u3eRGS9-\u3c/em\u3e and ​\u3cem\u3eR9AP\u3c/em\u3e-Associated Retinal Dysfunction (Bradyopsia)

Purpose To characterize photoreceptor structure and mosaic integrity in subjects with RGS9- and R9AP-associated retinal dysfunction (bradyopsia) and compare to previous observations in other cone dysfunction disorders such as oligocone trichromacy. Design Observational case series. Methods setting: Moorfields Eye Hospital (United Kingdom) and Medical College Wisconsin (USA). study population: Six eyes of 3 subjects with disease-causing variants in RGS9 or R9AP. main outcome measures: Detailed retinal imaging using spectral-domain optical coherence tomography and confocal adaptive-optics scanning light ophthalmoscopy. Results Cone density at 100 μm from foveal center ranged from 123 132 cones/mm2to 140 013 cones/mm2. Cone density ranged from 30 573 to 34 876 cones/mm2 by 600 μm from center and from 15 987 to 16,253 cones/mm2 by 1400 μm from center, in keeping with data from normal subjects. Adaptive-optics imaging identified a small, focal hyporeflective lesion at the foveal center in both eyes of the subject with RGS9-associated disease, corresponding to a discrete outer retinal defect also observed on spectral-domain optical coherence tomography; however, the photoreceptor mosaic remained intact at all other observed eccentricities. Conclusions Bradyopsia and oligocone trichromacy share common clinical symptoms and cannot be discerned on standard clinical findings alone. Adaptive-optics imaging previously demonstrated a sparse mosaic of normal wave-guiding cones remaining at the fovea, with no visible structure outside the central fovea in oligocone trichromacy. In contrast, the subjects presented in this study with molecularly confirmed bradyopsia had a relatively intact and structurally normal photoreceptor mosaic, allowing the distinction between these disorders based on the cellular phenotype and suggesting different pathomechanisms

Can Standard Cosmological Models Explain the Observed Abell Cluster Bulk Flow?

Lauer \& Postman (LP) observe that all Abell clusters with redshifts less than 15,000\kms\ appear to be participating in a bulk flow of 689 km s$^{-1}$ with respect to the Cosmic Microwave Background. We find this result difficult to reconcile with all popular models for large-scale structure formation that assume Gaussian initial conditions. This conclusion is based on Monte-Carlo realizations of the LP data, drawn from large Particle-Mesh $N$-body simulations. We have taken special care to treat properly the longest-wavelength components of the power spectra. Bulk flows with amplitude as large as that reported by LP are not uncommon in the Monte-Carlo datasets. However, the $\chi^2$ of the observed bulk flow, taking into account the anisotropy of the error ellipsoid, is much more difficult to match in the simulations. The models examined are ruled out at confidence levels between 94\% and 98\%. Any model that has {\it intrinsic} flows of less than 480\kms\ on the scales probed by LP scales can be ruled out at a similar level.Comment: Submitted to ApJ. 31 pages of uuencoded compressed postscript (810 kbytes); figures included. Also available via anonymous ftp to eku.ias.edu in /pub/strauss/warpfire/warpfire.ps.