Physics of the Preparation and Observation of Specimens that Involve Cryoprocedures

With this introductory chapter we attempt a synthesis of old and new knowledge of the physical principles that govern cryomethods. Interface phenomena determine the increase or decrease of the number of particles observed in frozen-hydrated suspensions because they occupy the air-liquid interface according to their specific balance of hydrophobic versus hydrophilic properties. Biological macromolecules are surrounded by organised water, the hydration shell, that prevents them from sticking to each other. Partial or complete removal of these hydration shells by freeze-drying or freeze-substitution leads to collapses or aggregations. The solvent-induced aggregation is usually decreased by prior cross-linking with adequate chemical fixatives. A new finding is that aggregations are also decreased with lower temperatures. This al lows us, for example, to preserve DNA-containing plasma from coarse aggregation even in cases where it has not been previously crosslinked. When rapidly freezing a physiologically homogeneous population of bacterial cells without added cryoprotectants we find a 10-20 μm thick layer of cells without ice crystal formation. In deeper layers an increasing proportion of cells exhibits crystallization damage, although some cells are still well preserved. Treatment of cells with aqueous solutions of OsO4 and/or uranyl acetate leads to 10-20 % (w/w) heavy metal deposit. Direct staining of sections of resin embedded material results in 10 times more deposit. The location of these deposited metals is at first unknown and is best visualized through a comparison with totally unstained material. Sufficient contrast is achieved with the dark field, or the ratio-contrast mode of imaging. The latter has the advantage of giving less weight to thickness variations than does the former. When observing fully unstained thin sections by ratio contrast the influence of the surface reliefs is thereby virtually eliminated. With CTEM the dark field mode requires too great a dose to use it successfully on frozen-hydrated material. With STEM this is possible for both dark field and ratio contrast

Preservice teacher beliefs related to mathematics and language arts.

This study investigated the relationship between preservice teachers\u27 mathematical and language arts learning histories and beliefs about their (1) attribution of students\u27 achievement, (2) self-efficacy as future teachers, and (3) other subject-/topic-related perceptions. The belief most closely linked to the learning history was the self-efficacy as future teachers. The group with a less favourable mathematical learning history and a more favourable language arts learning history believed they were significantly less able to influence students\u27 effort, interest, and achievement in mathematics compared to language arts. In addition, when groups were compared, preservice teachers with a less favourable mathematical learning history believed they were significantly less able to influence students\u27 mathematical achievement compared to those with a more favourable mathematical learning history. The group of preservice teachers with a favourable learning history in both subjects consistently attributed effort or lack of effort as being more applicable in explaining students\u27 achievement. Both groups were found to share the belief that a successful mathematics student would probably be male while a successful language arts student would probably be female. (Abstract shortened by UMI.) Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1991 .K455. Source: Masters Abstracts International, Volume: 30-04, page: 1014. Thesis (M.Ed.)--University of Windsor (Canada), 1990

Low Temperature Embedding

The Lowicryl resins K4M and HM20 are methacrylate/acrylate based formulations which can re used for embedding biological material at low temperature in conjunction with either the progressive lowering of temperature (PLT) technique or with freeze-substitution. The resins are applicable over a very extended temperature range, approximately 210°K to 340°K. Even lower temperatures down to ca. 190°K can be reached with two new resins, K11M and HM23. Test embeddings of unfixed material after freeze-substitution have given promising results which could re useful for imnunocytochemical labeling. Lipid extraction is small or absent when the two new resins are used in combination with freeze-substitution

A third of the radiotracer dose: two decades of progress in pediatric [18^{18}F]fluorodeoxyglucose PET/CT and PET/MR imaging

OBJECTIVES To assess the evolution of administered radiotracer activity for F-18-fluorodeoxyglucose (18F-FDG) PET/CT or PET/MR in pediatric patients (0-16 years) between years 2000 and 2021. METHODS Pediatric patients (≤ 16 years) referred for 18F-FDG PET/CT or PET/MR imaging of the body during 2000 and 2021 were retrospectively included. The amount of administered radiotracer activity in megabecquerel (MBq) was recorded, and signal-to-noise ratio (SNR) was measured in the right liver lobe with a 4 cm$^{3}$ volume of interest as an indicator for objective image quality. Descriptive statistics were computed. RESULTS Two hundred forty-three children and adolescents underwent a total of 466 examinations. The median injected 18F-FDG activity in MBq decreased significantly from 296 MBq in 2000-2005 to 100 MBq in 2016-2021 (p < 0.001), equaling approximately one-third of the initial amount. The median SNR ratio was stable during all years with 11.7 (interquartile range [IQR] 10.7-12.9, p = 0.133). CONCLUSIONS Children have benefited from a massive reduction in the administered 18F-FDG dose over the past 20 years without compromising objective image quality. CLINICAL RELEVANCE STATEMENT Radiotracer dose was reduced considerably over the past two decades of pediatric F-18-fluorodeoxyglucose PET/CT and PET/MR imaging highlighting the success of technical innovations in pediatric PET imaging. KEY POINTS • The evolution of administered radiotracer activity for F-18-fluorodeoxyglucose (18F-FDG) PET/CT or PET/MR in pediatric patients (0-16 years) between 2000 and 2021 was assessed. • The injected tracer activity decreased by 66% during the study period from 296 megabecquerel (MBq) to 100 MBq (p < 0.001). • The continuous implementation of technical innovations in pediatric hybrid 18F-FDG PET has led to a steady decrease in the amount of applied radiotracer, which is particularly beneficial for children who are more sensitive to radiation

Is the kinetoplast DNA a percolating network of linked rings at its critical point?

In this work we present a computational study of the Kinetoplast genome, modelled as a large number of semiflexible unknotted loops, which are allowed to link with each other. As the DNA density increases, the systems shows a percolation transition between a gas of unlinked rings and a network of linked loops which spans the whole system. Close to the percolation transition, we find that the mean valency of the network, i.e. the average number of loops which are linked to any one loop, is around 3 as found experimentally for the Kinetoplast DNA. Even more importantly, by simulating the digestion of the network by a restriction enzyme, we show that the distribution of oligomers, i.e. structures formed by a few loops which remain linked after digestion, quantitatively matches experimental data obtained from gel electrophoresis, provided that the density is, once again, close to the percolation transition. With respect to previous work, our analysis builds on a reduced number of assumptions, yet can still fully explain the experimental data. Our findings suggest that the Kinetoplast DNA can be viewed as a network of linked loops positioned very close to the percolation transition, and we discuss the possible biological implications of this remarkable fact.Comment: 13p, 5f, merged S

Inherent Dynamics of the Acid-Sensing Ion Channel 1 Correlates with the Gating Mechanism

A combination of computational and experimental approaches reveals the dynamics of ASIC1 gating, involving a deformation of the channel that triggers “twist-to-open” motions of the channel pore

Crystal Structure of Diedel, a Marker of the Immune Response of Drosophila melanogaster

Background: The Drosophila melanogaster gene CG11501 is up regulated after a septic injury and was proposed to act as a negative regulator of the JAK/STAT signaling pathway. Diedel, the CG11501 gene product, is a small protein of 115 residues with 10 cysteines. Methodology/Principal Findings: We have produced Diedel in Drosophila S2 cells as an extra cellular protein thanks to its own signal peptide and solved its crystal structure at 1.15 A ˚ resolution by SIRAS using an iodo derivative. Diedel is composed of two sub domains SD1 and SD2. SD1 is made of an antiparallel b-sheet covered by an a-helix and displays a ferredoxin-like fold. SD2 reveals a new protein fold made of loops connected by four disulfide bridges. Further structural analysis identified conserved hydrophobic residues on the surface of Diedel that may constitute a potential binding site. The existence of two conformations, cis and trans, for the proline 52 may be of interest as prolyl peptidyl isomerisation has been shown to play a role in several physiological mechanisms. The genome of D. melanogaster contains two other genes coding for proteins homologous to Diedel, namely CG43228 and CG34329. Strikingly, apart from Drosophila and the pea aphid Acyrthosiphon pisum, Diedel-related sequences were exclusively identified in a few insect DNA viruses of the Baculoviridae and Ascoviridae families. Conclusion/Significance: Diedel, a marker of the Drosophila antimicrobial/antiviral response, is a member of a small famil

Molecular Modeling of Mechanosensory Ion Channel Structural and Functional Features

The DEG/ENaC (Degenerin/Epithelial Sodium Channel) protein family comprises related ion channel subunits from all metazoans, including humans. Members of this protein family play roles in several important biological processes such as transduction of mechanical stimuli, sodium re-absorption and blood pressure regulation. Several blocks of amino acid sequence are conserved in DEG/ENaC proteins, but structure/function relations in this channel class are poorly understood. Given the considerable experimental limitations associated with the crystallization of integral membrane proteins, knowledge-based modeling is often the only route towards obtaining reliable structural information. To gain insight into the structural characteristics of DEG/ENaC ion channels, we derived three-dimensional models of MEC-4 and UNC-8, based on the available crystal structures of ASIC1 (Acid Sensing Ion Channel 1). MEC-4 and UNC-8 are two DEG/ENaC family members involved in mechanosensation and proprioception respectively, in the nematode Caenorhabditis elegans. We used these models to examine the structural effects of specific mutations that alter channel function in vivo. The trimeric MEC-4 model provides insight into the mechanism by which gain-of-function mutations cause structural alterations that result in increased channel permeability, which trigger cell degeneration. Our analysis provides an introductory framework to further investigate the multimeric organization of the DEG/ENaC ion channel complex