Preparation of large biological samples for high-resolution, hierarchical, synchrotron phase-contrast tomography with multimodal imaging compatibility

Imaging across different scales is essential for understanding healthy organ morphology and pathophysiological changes. The macro- and microscale three-dimensional morphology of large samples, including intact human organs, is possible with X-ray microtomography (using laboratory or synchrotron sources). Preparation of large samples for high-resolution imaging, however, is challenging due to limitations such as sample shrinkage, insufficient contrast, movement of the sample and bubble formation during mounting or scanning. Here, we describe the preparation, stabilization, dehydration and mounting of large soft-tissue samples for X-ray microtomography. We detail the protocol applied to whole human organs and hierarchical phase-contrast tomography at the European Synchrotron Radiation Facility, yet it is applicable to a range of biological samples, including complete organisms. The protocol enhances the contrast when using X-ray imaging, while preventing sample motion during the scan, even with different sample orientations. Bubbles trapped during mounting and those formed during scanning (in the case of synchrotron X-ray imaging) are mitigated by multiple degassing steps. The sample preparation is also compatible with magnetic resonance imaging, computed tomography and histological observation. The sample preparation and mounting require 24-36 d for a large organ such as a whole human brain or heart. The preparation time varies depending on the composition, size and fragility of the tissue. Use of the protocol enables scanning of intact organs with a diameter of 150 mm with a local voxel size of 1 μm. The protocol requires users with expertise in handling human or animal organs, laboratory operation and X-ray imaging

The connection between radio halos and cluster mergers and the statistical properties of the radio halo population

We discuss the statistical properties of the radio halo population in galaxy clusters. Radio bi-modality is observed in galaxy clusters: a fraction of clusters host giant radio halos while the majority of clusters do not show evidence of diffuse cluster-scale radio emission. The radio bi-modality has a correspondence in terms of dynamical state of the hosting clusters showing that merging clusters host radio halos and follow the well known radio--X-ray correlation, while more relaxed clusters do not host radio halos and populate a region well separated from that correlation. These evidences can be understood in the framework of a scenario where merger-driven turbulence re-accelerate the radio emitting electrons. We discuss the main statistical expectations of this scenario underlining the important role of upcoming LOFAR surveys to test present models.Comment: 11 pages, 2 figures, Invited talk at the conference "Diffuse Relativistic Plasmas", Bangalore, 1-4 March 2011; accepted for publication in the Journal of Astrophysics and Astronom

A Model for Damage Load and Its Implications for the Evolution of Bacterial Aging

Deleterious mutations appearing in a population increase in frequency until stopped by natural selection. The ensuing equilibrium creates a stable frequency of deleterious mutations or the mutational load. Here I develop the comparable concept of a damage load, which is caused by harmful non-heritable changes to the phenotype. A damage load also ensues when the increase of damage is opposed by selection. The presence of a damage load favors the evolution of asymmetrical transmission of damage by a mother to her daughters. The asymmetry is beneficial because it increases fitness variance, but it also leads to aging or senescence. A mathematical model based on microbes reveals that a cell lineage dividing symmetrically is immortal if lifetime damage rates do not exceed a threshold. The evolution of asymmetry allows the lineage to persist above the threshold, but the lineage becomes mortal. In microbes with low genomic mutation rates, it is likely that the damage load is much greater than the mutational load. In metazoans with higher genomic mutation rates, the damage and the mutational load could be of the same magnitude. A fit of the model to experimental data shows that Escherichia coli cells experience a damage rate that is below the threshold and are immortal under the conditions examined. The model estimates the asymmetry level of E. coli to be low but sufficient for persisting at higher damage rates. The model also predicts that increasing asymmetry results in diminishing fitness returns, which may explain why the bacterium has not evolved higher asymmetry

Quantitative principles of cis-translational control by general mRNA sequence features in eukaryotes.

BackgroundGeneral translational cis-elements are present in the mRNAs of all genes and affect the recruitment, assembly, and progress of preinitiation complexes and the ribosome under many physiological states. These elements include mRNA folding, upstream open reading frames, specific nucleotides flanking the initiating AUG codon, protein coding sequence length, and codon usage. The quantitative contributions of these sequence features and how and why they coordinate to control translation rates are not well understood.ResultsHere, we show that these sequence features specify 42-81% of the variance in translation rates in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Arabidopsis thaliana, Mus musculus, and Homo sapiens. We establish that control by RNA secondary structure is chiefly mediated by highly folded 25-60 nucleotide segments within mRNA 5' regions, that changes in tri-nucleotide frequencies between highly and poorly translated 5' regions are correlated between all species, and that control by distinct biochemical processes is extensively correlated as is regulation by a single process acting in different parts of the same mRNA.ConclusionsOur work shows that general features control a much larger fraction of the variance in translation rates than previously realized. We provide a more detailed and accurate understanding of the aspects of RNA structure that directs translation in diverse eukaryotes. In addition, we note that the strongly correlated regulation between and within cis-control features will cause more even densities of translational complexes along each mRNA and therefore more efficient use of the translation machinery by the cell

Impairment of Vowel Articulation as a Possible Marker of Disease Progression in Parkinson's Disease

Purpose: The aim of the current study was to survey if vowel articulation in speakers with Parkinson’s disease (PD) shows specific changes in the course of the disease. Method: 67 patients with PD (42 male) and 40 healthy speakers (20 male) were tested and retested after an average time interval of 34 months. Participants had to read a given text as source for subsequent calculation of the triangular vowel space area (tVSA) and vowel articulation index (VAI). Measurement of tVSA and VAI were based upon analysis of the first and second formant of the vowels /a/, /i/and /u / extracted from defined words within the text. Results: At first visit, VAI values were reduced in male and female PD patients as compared to the control group, and showed a further decrease at the second visit. Only in female Parkinsonian speakers, VAI was correlated to overall speech impairment based upon perceptual impression. VAI and tVSA were correlated to gait impairment, but no correlations were seen between VAI and global motor impairment or overall disease duration. tVSA showed a similar reduction in the PD as compared to the control group and was also found to further decline between first and second examination in female, but not in male speakers with PD. Conclusions: Measurement of VAI seems to be superior to tVSA in the description of impaired vowel articulation and its further decline in the course of the disease in PD. Since impairment of vowel articulation was found to be independent fro

Clusters of galaxies : observational properties of the diffuse radio emission

Clusters of galaxies, as the largest virialized systems in the Universe, are ideal laboratories to study the formation and evolution of cosmic structures...(abridged)... Most of the detailed knowledge of galaxy clusters has been obtained in recent years from the study of ICM through X-ray Astronomy. At the same time, radio observations have proved that the ICM is mixed with non-thermal components, i.e. highly relativistic particles and large-scale magnetic fields, detected through their synchrotron emission. The knowledge of the properties of these non-thermal ICM components has increased significantly, owing to sensitive radio images and to the development of theoretical models. Diffuse synchrotron radio emission in the central and peripheral cluster regions has been found in many clusters. Moreover large-scale magnetic fields appear to be present in all galaxy clusters, as derived from Rotation Measure (RM) studies. Non-thermal components are linked to the cluster X-ray properties, and to the cluster evolutionary stage, and are crucial for a comprehensive physical description of the intracluster medium. They play an important role in the cluster formation and evolution. We review here the observational properties of diffuse non-thermal sources detected in galaxy clusters: halos, relics and mini-halos. We discuss their classification and properties. We report published results up to date and obtain and discuss statistical properties. We present the properties of large-scale magnetic fields in clusters and in even larger structures: filaments connecting galaxy clusters. We summarize the current models of the origin of these cluster components, and outline the improvements that are expected in this area from future developments thanks to the new generation of radio telescopes.Comment: Accepted for the publication in The Astronomy and Astrophysics Review. 58 pages, 26 figure

The fatal trajectory of pulmonary COVID-19 is driven by lobular ischemia and fibrotic remodelling

BACKGROUND: COVID-19 is characterized by a heterogeneous clinical presentation, ranging from mild symptoms to severe courses of disease. 9-20% of hospitalized patients with severe lung disease die from COVID-19 and a substantial number of survivors develop long-COVID. Our objective was to provide comprehensive insights into the pathophysiology of severe COVID-19 and to identify liquid biomarkers for disease severity and therapy response. METHODS: We studied a total of 85 lungs (n = 31 COVID autopsy samples; n = 7 influenza A autopsy samples; n = 18 interstitial lung disease explants; n = 24 healthy controls) using the highest resolution Synchrotron radiation-based hierarchical phase-contrast tomography, scanning electron microscopy of microvascular corrosion casts, immunohistochemistry, matrix-assisted laser desorption ionization mass spectrometry imaging, and analysis of mRNA expression and biological pathways. Plasma samples from all disease groups were used for liquid biomarker determination using ELISA. The anatomic/molecular data were analyzed as a function of patients' hospitalization time. FINDINGS: The observed patchy/mosaic appearance of COVID-19 in conventional lung imaging resulted from microvascular occlusion and secondary lobular ischemia. The length of hospitalization was associated with increased intussusceptive angiogenesis. This was associated with enhanced angiogenic, and fibrotic gene expression demonstrated by molecular profiling and metabolomic analysis. Increased plasma fibrosis markers correlated with their pulmonary tissue transcript levels and predicted disease severity. Plasma analysis confirmed distinct fibrosis biomarkers (TSP2, GDF15, IGFBP7, Pro-C3) that predicted the fatal trajectory in COVID-19. INTERPRETATION: Pulmonary severe COVID-19 is a consequence of secondary lobular microischemia and fibrotic remodelling, resulting in a distinctive form of fibrotic interstitial lung disease that contributes to long-COVID. FUNDING: This project was made possible by a number of funders. The full list can be found within the Declaration of interests / Acknowledgements section at the end of the manuscript

Phylogeny, Diet, and Cranial Integration in Australodelphian Marsupials

Studies of morphological integration provide valuable information on the correlated evolution of traits and its relationship to long-term patterns of morphological evolution. Thus far, studies of morphological integration in mammals have focused on placentals and have demonstrated that similarity in integration is broadly correlated with phylogenetic distance and dietary similarity. Detailed studies have also demonstrated a significant correlation between developmental relationships among structures and adult morphological integration. However, these studies have not yet been applied to marsupial taxa, which differ greatly from placentals in reproductive strategy and cranial development and could provide the diversity necessary to assess the relationships among phylogeny, ecology, development, and cranial integration. This study presents analyses of morphological integration in 20 species of australodelphian marsupials, and shows that phylogeny is significantly correlated with similarity of morphological integration in most clades. Size-related correlations have a significant affect on results, particularly in Peramelia, which shows a striking decrease in similarity of integration among species when size is removed. Diet is not significantly correlated with similarity of integration in any marsupial clade. These results show that marsupials differ markedly from placental mammals in the relationships of cranial integration, phylogeny, and diet, which may be related to the accelerated development of the masticatory apparatus in marsupials

Implementation salvage experiences from the Melbourne diabetes prevention study

Background Many public health interventions based on apparently sound evidence from randomised controlled trials encounter difficulties when being scaled up within health systems. Even under the best of circumstances, implementation is exceedingly difficult. In this paper we will describe the implementation salvage experiences from the Melbourne Diabetes Prevention Study, which is a randomised controlled trial of the effectiveness and cost-effectiveness nested in the state-wide Life! Taking Action on Diabetes program in Victoria, Australia.Discussion The Melbourne Diabetes Prevention Study sits within an evolving larger scale implementation project, the Life! program. Changes that occurred during the roll-out of that program had a direct impact on the process of conducting this trial. The issues and methods of recovery the study team encountered were conceptualised using an implementation salvage strategies framework. The specific issues the study team came across included continuity of the state funding for Life! program and structural changes to the Life! program which consisted of adjustments to eligibility criteria, referral processes, structure and content, as well as alternative program delivery for different population groups. Staff turnover, recruitment problems, setting and venue concerns, availability of potential participants and participant characteristics were also identified as evaluation roadblocks. Each issue and corresponding salvage strategy is presented.Summary The experiences of conducting such a novel trial as the preliminary Melbourne Diabetes Prevention Study have been invaluable. The lessons learnt and knowledge gained will inform the future execution of this trial in the coming years. We anticipate that these results will also be beneficial to other researchers conducting similar trials in the public health field. We recommend that researchers openly share their experiences, barriers and challenges when conducting randomised controlled trials and implementation research. We encourage them to describe the factors that may have inhibited or enhanced the desired outcomes so that the academic community can learn and expand the research foundation of implementation salvage.<br /

Decay in survival motor neuron and plastin 3 levels during differentiation of iPSC-derived human motor neurons

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by mutations in Survival Motor Neuron 1 (SMN1), leading to degeneration of alpha motor neurons (MNs) but also affecting other cell types. Induced pluripotent stem cell (iPSC)-derived human MN models from severe SMA patients have shown relevant phenotypes. We have produced and fully characterized iPSCs from members of a discordant consanguineous family with chronic SMA. We differentiated the iPSC clones into ISL-1+/ChAT+ MNs and performed a comparative study during the differentiation process, observing significant differences in neurite length and number between family members. Analyses of samples from wild-type, severe SMA type I and the type IIIa/IV family showed a progressive decay in SMN protein levels during iPSC-MN differentiation, recapitulating previous observations in developmental studies. PLS3 underwent parallel reductions at both the transcriptional and translational levels. The underlying, progressive developmental decay in SMN and PLS3 levels may lead to the increased vulnerability of MNs in SMA disease. Measurements of SMN and PLS3 transcript and protein levels in iPSC-derived MNs show limited value as SMA biomarkers