Silver enhancement of Nanogold particles during freeze substitution for electron microscopy

Recent advances in rapid freezing and fixation by freeze substitution have allowed structural cell biologists to apply these reliable modes of sample preparation to a wide range of specimens and scientific problems. Progress in electron tomography has produced cellular images with resolution approaching 4 nm in 3D, but our ability to localize macromolecules in these well-fixed, well-resolved samples has remained limited. When light fixation and low temperature embedding are employed with appropriate resins, immuno-localizations can recognize antigens at a section's surface, but labelling is therefore confined, not throughout the section's depth. Small, electron-dense markers, like Nanogold®, will often enter a living cell, serving as reliable tracers for endocytic activity, but these markers are usually too small to be visible in the context of a cell. We have developed a method for the silver enhancement of Nanogold particles that works during freeze substitution in organic solvents at low temperature. Here, we describe the development of this method, based on in vitro tests of reagents and conditions. We then show results from application of the method to an in vivo system, using Nanogold to track the internalization of immunoglobulin by neonatal murine intestinal epithelium, a specific example of receptor-mediated membrane traffic

A freeze substitution fixation-based gold enlarging technique for EM studies of endocytosed nanogold-labeled molecules

We have developed methods to locate individual ligands that can be used for electron microscopy studies of dynamic events during endocytosis and subsequent intracellular trafficking. The methods are based on enlargement of 1.4 nm Nanogold attached to an endocytosed ligand. Nanogold, a small label that does not induce misdirection of ligand–receptor complexes, is ideal for labeling ligands endocytosed by live cells, but is too small to be routinely located in cells by electron microscopy. Traditional pre-embedding enhancement protocols to enlarge Nanogold are not compatible with high pressure freezing/freeze substitution fixation (HPF/FSF), the most accurate method to preserve ultrastructure and dynamic events during trafficking. We have developed an improved enhancement procedure for chemically fixed samples that reduced auto-nucleation, and a new pre-embedding gold enlarging technique for HPF/FSF samples that preserved contrast and ultrastructure and can be used for high-resolution tomography. We evaluated our methods using labeled Fc as a ligand for the neonatal Fc receptor. Attachment of Nanogold to Fc did not interfere with receptor binding or uptake, and gold-labeled Fc could be specifically enlarged to allow identification in 2D projections and in tomograms. These methods should be broadly applicable to many endocytosis and transcytosis studies

IBPP Research Associates: Space and Extreme Environments

Per M. Ephimia Morphew. President of the Society for Human Performance in Extreme Environments previously online at http://www.hpee.org, colleagues at the Institute of Biomedical Problems in Russia (IBMP) are engaged in an isolation experiment pertaining to space exploration that is two months underway and preliminary findings are reported [in the article entitled First Two Months of Simulated Isolation Passed.] IBPP commentary includes a discussion of human factors and political psychology as parameters of space

Characterization of microtubule-associated protein tau isoforms and Alzheimer’s disease-like pathology in normal sheep (Ovis aries):Relevance to their potential as a model of Alzheimer’s disease

Alzheimer’s disease is a chronic neurodegenerative disease that accounts for up to 80% of all dementias. Characterised by deteriorations of memory and cognitive function, the key neuropathological features are accumulations of β-amyloid and hyperphosphorylated tau, as ‘plaques’ and ‘tangles’, respectively. Despite extensive study, however, the exact mechanism underlying aggregate formation in Alzheimer’s disease remains elusive, as does the contribution of these aggregates to disease progression. Importantly, a recent evaluation of current Alzheimer’s disease animal models suggested that rodent models are not able to fully recapitulate the pathological intricacies of the disease as it occurs in humans. Therefore, increasing attention is being paid to species that might make good alternatives to rodents for studying the molecular pathology of Alzheimer’s disease. The sheep (Ovis aries) is one such species, although to date, there have been few molecular studies relating to Alzheimer’s disease in sheep. Here, we investigated the Alzheimer’s disease relevant histopathological characteristics of 22 sheep, using anti-β-amyloid (Abcam 12267 and mOC64) and phosphorylation specific anti-tau (AT8 and S396) antibodies. We identified numerous intraneuronal aggregates of both β-amyloid and tau that are consistent with early Alzheimer’s disease-like pathology. We confirmed the expression of two 3-repeat (1N3R, 2N3R) and two 4-repeat (1N4R, 2N4R) tau isoforms in the ovine brain, which result from the alternative splicing of two tau exons. Finally, we investigated the phosphorylation status of the serine396 residue in 30 sheep, and report that the phosphorylation of this residue begins in sheep aged as young as 2 years. Together, these data show that sheep exhibit naturally occurring β-amyloid and tau pathologies, that reflect those that occur in the early stages of Alzheimer’s disease. This is an important step towards the validation of the sheep as a feasible large animal species in which to model Alzheimer’s disease. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00018-022-04572-z

Identification of the major proteins of an immune modulating fraction from adult Fasciola hepatica released by Nonidet P40

Fasciola hepatica NP-40 released antigens (FhTeg) exhibit potent Th1 immunosuppressive properties in vitro and in vivo. However, the protein composition of this active fraction, responsible for Th1 immune modulatory activity, has yet to be resolved. Therefore, FhTeg, a Nonidet P-40 extract, was subjected to a proteomic analysis in order to identify individual protein components. This was performed using an in house F. hepatica EST database following 2D electrophoresis combined with de novo sequencing based mass spectrometry. The identified proteins, a mixture of excretory/secretory and membrane-associated proteins, are associated with stress response and chaperoning, energy metabolism and cytoskeletal components. The immune modulatory properties of these identified protein(s) is discussed and HSP70 from F. hepatica is highlighted as a potential host immune modulator for future study

Characterising STEM Ways of Thinking in Engineering Design (ED)-based tasks

Investigating students\u27 thinking in routine classroom tasks, especially in science and engineering, is crucial. Given the rising interest in STEM Ways of Thinking (SWoT), in this exploratory study, we focus on two multi-week Engineering Design tasks within an undergraduate physics laboratory. Given that the term \u27ways of thinking\u27 has varied interpretations, we aim to further the discourse by identifying four SWoTs: Design Thinking, Physics Concepts, Mathematical Constructs, and Metacognitive Reflection. Analyzing discussions from 14 student-groups reveals notable differences in how students solve an instructor-assigned challenge given earlier in the semester and a student-generated challenge later in the semester. Students considered physics concepts more frequently and combined mathematical and physics concepts in more detail in the latter task. Our findings underscore the value of small-group discussions in understanding and operationalizing SWoT. We acknowledge the need for diverse frameworks and believe our study can benefit educators and researchers exploring similar strategies

FcRn-mediated antibody transport across epithelial cells revealed by electron tomography

The neonatal Fc receptor (FcRn) transports maternal IgG across epithelial barriers, thereby providing the fetus or newborn with humoral immunity before its immune system is fully functional. In newborn rats, FcRn transfers IgG from milk to blood by apical-to-basolateral transcytosis across intestinal epithelial cells. The pH difference between the apical (pH 6.0–6.5) and basolateral (pH 7.4) sides of intestinal epithelial cells facilitates the efficient unidirectional transport of IgG, because FcRn binds IgG at pH 6.0–6.5 but not at pH 7 or more. As milk passes through the neonatal intestine, maternal IgG is removed by FcRn-expressing cells in the proximal small intestine (duodenum and jejunum); remaining proteins are absorbed and degraded by FcRn-negative cells in the distal small intestine (ileum). Here we use electron tomography to make jejunal transcytosis visible directly in space and time, developing new labelling and detection methods to map individual nanogold-labelled Fc within transport vesicles and simultaneously to characterize these vesicles by immunolabelling. Combining electron tomography with a nonperturbing endocytic label allowed us to conclusively identify receptor-bound ligands, resolve interconnecting vesicles, determine whether a vesicle was microtubule-associated, and accurately trace FcRn-mediated transport of IgG. Our results present a complex picture in which Fc moves through networks of entangled tubular and irregular vesicles, only some of which are microtubule-associated, as it migrates to the basolateral surface. New features of transcytosis are elucidated, including transport involving multivesicular body inner vesicles/tubules and exocytosis through clathrin-coated pits. Markers for early, late and recycling endosomes each labelled vesicles in different and overlapping morphological classes, revealing spatial complexity in endo-lysosomal trafficking

Predatory Organisms with Untapped Biosynthetic Potential:Descriptions of Novel Corallococcus Species C. aberystwythensis sp. nov., C. carmarthensis sp. nov., C. exercitus sp. nov., C. interemptor sp. nov., C. llansteffanensis sp. nov., C. praedator sp. nov., C. sicarius sp. nov., and C. terminator sp. nov

Corallococcus spp. are common soil-dwelling organisms which kill and consume prey microbes through the secretion of antimicrobial substances. Two species of Corallococcus have been described previously (Corallococcus coralloides and Corallococcus exiguus). A polyphasic approach was taken to characterise antimicrobial, biochemical and phenotypic properties of eight Corallococcus spp. strains and the two type strains. We also report here the genome sequence of the C. exiguus type strain (DSM 14696T). The genomes of the eight candidate strains, C. exiguus DSM 14696T and C. coralloides DSM 2259T, had an average nucleotide identity below 95% and digital DNA-DNA hybridisation scores less than the 70% lower bound for species identity, indicating they belong to distinct species. All ten strains, including the two type strains, were thoroughly characterised, including biochemical analysis of their fatty acid methyl esters, substrate utilisation and sugar assimilation. Each strain gave a distinct profile of properties, which together with their genomic differences supports the proposal of the eight candidate strains as novel species: Corallococcus exercitus sp. nov. (AB043AT = DSM 108849T = NBRC 113887T), Corallococcus interemptor sp. nov. (AB047AT = DSM 108843T = NBRC 113888T), Corallococcus aberystwythensis sp. nov. (AB050AT = DSM 108846T = NBRC 114019T), Corallococcus praedator sp. nov. (CA031BT = DSM 108841T = NBRC 113889T), Corallococcus sicarius sp. nov. (CA040BT = DSM 108850T = NBRC 113890T), Corallococcus carmarthenensis sp. nov. (CA043DT = DSM 108842T = NBRC 113891T), Corallococcus llansteffanensis sp. nov. (CA051BT = DSM 108844T = NBRC 114100T) and Corallococcus terminator sp. nov. (CA054AT = DSM 108848T = NBRC 113892T)

Mechanics Cognitive Diagnostic: Mathematics skills tested in introductory physics courses

Physics instructors and education researchers use research-based assessments (RBAs) to evaluate students\u27 preparation for physics courses. This preparation can cover a wide range of constructs including mathematics and physics content. Using separate mathematics and physics RBAs consumes course time. We are developing a new RBA for introductory mechanics as an online test using both computerized adaptive testing and cognitive diagnostic models. This design allows the adaptive RBA to assess mathematics and physics content knowledge within a single assessment. In this article, we used an evidence-centered design framework to inform the extent to which our models of skills students develop in physics courses fit the data from three mathematics RBAs. Our dataset came from the LASSO platform and includes 3,491 responses from the Calculus Concept Assessment, Calculus Concept Inventory, and Pre-calculus Concept Assessment. Our model included five skills: apply vectors, conceptual relationships, algebra, visualizations, and calculus. The deterministic inputs, noisy \u27and\u27 gate\u27\u27 (DINA) analyses demonstrated a good fit for the five skills. The classification accuracies for the skills were satisfactory. Including items from the three mathematics RBAs in the item bank for the adaptive RBA will provide a flexible assessment of these skills across mathematics and physics content areas that can adapt to instructors\u27 needs