Sample Preparation Methodologies for In Situ Liquid and Gaseous Cell Analytical Transmission Electron Microscopy of Electropolished Specimens

AbstractIn recent years, an increasing number of studies utilizingin situliquid and/or gaseous cell scanning/transmission electron microscopy (S/TEM) have been reported. Because of the difficulty in the preparation of suitable specimens, these environmental S/TEM studies have been generally limited to studies of nanoscale structured materials such as nanoparticles, nanowires, or sputtered thin films. In this paper, we present two methodologies which have been developed to facilitate the preparation of electron-transparent samples from conventional bulk metals and alloys forin situliquid/gaseous cell S/TEM experiments. These methods take advantage of combining sequential electrochemical jet polishing followed by focused ion beam extraction techniques to create large electron-transparent areas for site-specific observation. As an example, we illustrate the application of this methodology for the preparation ofin situspecimens from a cold-rolled Type 304 austenitic stainless steel sample, which was subsequently examined in both 1 atm of air as well as fully immersed in a H2O environment in the S/TEM followed by hyperspectral imaging. These preparation techniques can be successfully applied as a general procedure for a wide range of metals and alloys, and are suitable for a variety ofin situanalytical S/TEM studies in both aqueous and gaseous environments.</jats:p

Fomitiporia mediterranea M. Fisch., the historical Esca agent: a comprehensive review on the main grapevine wood rot agent in Europe

International audienceFomitiporia mediterranea M. Fisch. (Fmed) is a basidiomycete first described in 2002, and was considered up to then as part of Fomitiporia punctata (P. Karst) Murrill. This fungus can degrade lignocellulosic biomass, causing white rot and leaving bleached fibrous host residues. In Europe Fmed is considered the main grapevine wood rot (Esca) agent within the Esca disease complex, which includes some of the most economically important Grapevine Trunk Diseases (GTDs). This review summarises and evaluates published research on Fmed, on white rot elimination by curettage or management by treatments with specific products applied to diseased grapevines, and on the relationship between wood symptoms and Grapevine Leaf Stripe Disease (GLSD) in the Esca disease complex. Information is also reviewed on the fungus biology, mechanisms of pathogenicity, and their possible relationships with external foliar symptoms of the Esca disease complex. Information on Fmed control strategies is also reviewed

The sodium iodide symporter (NIS) as theranostic gene: its emerging role in new imaging modalities and non-viral gene therapy

Cloning of the sodium iodide symporter (NIS) in 1996 has provided an opportunity to use NIS as a powerful theranostic transgene. Novel gene therapy strategies rely on image-guided selective NIS gene transfer in non-thyroidal tumors followed by application of therapeutic radionuclides. This review highlights the remarkable progress during the last two decades in the development of the NIS gene therapy concept using selective non-viral gene delivery vehicles including synthetic polyplexes and genetically engineered mesenchymal stem cells. In addition, NIS is a sensitive reporter gene and can be monitored by high resolution PET imaging using the radiotracers sodium [ 124 I]iodide ([ 124 I]NaI) or [ 18 F]tetrafluoroborate ([ 18 F]TFB). We performed a small preclinical PET imaging study comparing sodium [ 124 I]iodide and in-house synthesized [ 18 F]TFB in an orthotopic NIS-expressing glioblastoma model. The results demonstrated an improved image quality using [ 18 F]TFB. Building upon these results, we will be able to expand the NIS gene therapy approach using non-viral gene delivery vehicles to target orthotopic tumor models with low volume disease, such as glioblastoma

The HOPE fixation technique - a promising alternative to common prostate cancer biobanking approaches

<p>Abstract</p> <p>Background</p> <p>The availability of well-annotated prostate tissue samples through biobanks is key for research. Whereas fresh-frozen tissue is well suited for a broad spectrum of molecular analyses, its storage and handling is complex and cost-intensive. Formalin-fixed paraffin-embedded specimens (FFPE) are easy to handle and economic to store, but their applicability for molecular methods is restricted. The recently introduced Hepes-glutamic acid-buffer mediated Organic solvent Protection Effect (HOPE) is a promising alternative, which might have the potential to unite the benefits of FFPE and fresh-frozen specimen. Aim of the study was to compare HOPE-fixed, FFPE and fresh-frozen bio-specimens for their accessibility for diagnostic and research purposes.</p> <p>Methods</p> <p>10 prostate cancer samples were each preserved with HOPE, formalin, and liquid nitrogen and studied with in-situ and molecular methods. Samples were H&E stained, and assessed by immunohistochemistry (i.e. PSA, GOLPH2, p63) and FISH (i.e. <it>ERG </it>rearrangement). We assessed DNA integrity by PCR, using control genes ranging from 100 to 600 bp amplicon size. RNA integrity was assessed through qRT-PCR on three housekeeping genes (TBP, GAPDH, β-actin). Protein expression was analysed by performing western blot analysis using GOLPH2 and PSA antibodies.</p> <p>Results</p> <p>Of the HOPE samples, morphologic quality of H&E sections, immunohistochemical staining, and the FISH assay was at least equal to FFPE tissue, and significantly better than the fresh-frozen specimens. DNA, RNA, and protein analysis of HOPE samples provided similar results as compared to fresh-frozen specimens. As expected, FFPE-samples were inferior for most of the molecular analyses.</p> <p>Conclusions</p> <p>This is the first study, comparatively assessing the suitability of these fixation methods for diagnostic and research utilization. Overall, HOPE-fixed bio-specimens combine the benefits of FFPE- and fresh-frozen samples. Results of this study have the potential to expand on contemporary prostate tissue biobanking approaches and can serve as a model for other organs and tumors.</p

Practical Aspects of Electrochemical Corrosion Measurements during in situ Analytical TEM of Austenitic Stainless Steel in Aqueous Media

AbstractThe capability to perform liquid in situ transmission electron microscopy (TEM) experiments provides an unprecedented opportunity to examine the real-time processes of physical and chemical/electrochemical reactions during the interaction between metal surfaces and liquid environments. This work describes the requisite steps to make the technique fully analytical, from sample preparation, through modifications of the electrodes, characterization of electrolytes, and finally to electrochemical corrosion experiments comparing in situ TEM to conventional bulk cell and microcell configurations.</jats:p

Wood degradation in grapevine diseases

International audienceThe major Grapevine Trunk Diseases (GTD) gather diseases associated with either one particular fungal species, e.g., eutypiose, or with fungal species complexes, e.g., Botryosphaeriae dieback. GTD-associated fungi are part of different fungal classes, depending on their substrate degradation mechanisms or on their lifestyles. They are identified in grapevine wood and they lead to wood typical symptoms. They are sometimes associated to foliar symptoms too, despite not found in aerial parts, probably acting through fungal toxins or through physiological disorders, such as non-functional wood vessels. Grapevine is a liana, and as such shows structural and chemical differences compared to forest tree wood. However, knowledge on wood-degrading fungi growing on tree wood can contribute to a better understanding of grapevine wood adaptation mechanisms to fungal attack. For example, wood density, xylem vessels size and number, or lignin content and composition can play a role in wood sensitivity to fungi. Also, some fungal detoxification mechanisms against wood extractibles are host-specific. In a first part of this chapter, structure and chemical composition of grapevine wood are described, pointing out properties that might play a role in fungal growth. In a second part, GTD main associated fungi and their known mechanisms for wood degradation or wood chemical detoxification are discussed. Extracellular and intracellular enzymatic and non-enzymatic mechanisms are considered. These mechanisms, well described for their role in fungal adaptation to tree wood, have been much less studied for GTD associated fungi. Further studies about those mechanisms would contribute to understand fungal adaptation to grapevine

Wood degradation by Fomitiporia mediterranea M. Fischer: Physiologic, metabolomic and proteomic approaches

Fomitiporia mediterranea (Fmed) is one of the main fungal species found in grapevine wood rot, also called “amadou,” one of the most typical symptoms of grapevine trunk disease Esca. This fungus is functionally classified as a white-rot, able to degrade all wood structure polymers, i.e., hemicelluloses, cellulose, and the most recalcitrant component, lignin. Specific enzymes are secreted by the fungus to degrade those components, namely carbohydrate active enzymes for hemicelluloses and cellulose, which can be highly specific for given polysaccharide, and peroxidases, which enable white-rot to degrade lignin, with specificities relating to lignin composition as well. Furthermore, besides polymers, a highly diverse set of metabolites often associated with antifungal activities is found in wood, this set differing among the various wood species. Wood decayers possess the ability to detoxify these specific extractives and this ability could reflect the adaptation of these fungi to their specific environment. The aim of this study is to better understand the molecular mechanisms used by Fmed to degrade wood structure, and in particular its potential adaptation to grapevine wood. To do so, Fmed was cultivated on sawdust from different origins: grapevine, beech, and spruce. Carbon mineralization rate, mass loss, wood structure polymers contents, targeted metabolites (extractives) and secreted proteins were measured. We used the well-known white-rot model Trametes versicolor for comparison. Whereas no significant degradation was observed with spruce, a higher mass loss was measured on Fmed grapevine culture compared to beech culture. Moreover, on both substrates, a simultaneous degradation pattern was demonstrated, and proteomic analysis identified a relative overproduction of oxidoreductases involved in lignin and extractive degradation on grapevine cultures, and only few differences in carbohydrate active enzymes. These results could explain at least partially the adaptation of Fmed to grapevine wood structural composition compared to other wood species, and suggest that other biotic and abiotic factors should be considered to fully understand the potential adaptation of Fmed to its ecological niche. Proteomics data are available via ProteomeXchange with identifier PXD036889