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Preservation of ultrastructural features in biological samples for electron microscopy (EM) is a challenging task that is routinely accomplished through chemical fixation or high-pressure freezing coupled to automated freeze substitution (AFS) using specialized devices. However, samples from clinical (e.g. “biobanking” of bulk biopsies) and preclinical (e.g. whole mouse tissues) specimens are often not specifically prepared for ultrastructural analyses but simply immersed in liquid nitrogen before long-term cryo-storage. We demonstrate that ultrastructural features of such samples are insufficiently conserved using AFS and developed a simple, rapid, and effective method for thawing that does not require specific instrumentation. This procedure consists of dry ice-cooled pre-trimming of frozen tissue and aldehyde fixation for 3 h at 37 °C followed by standard embedding steps. Herein investigated tissues comprised human term placentae, clinical lung samples, as well as mouse tissues of different composition (brown adipose tissue, white adipose tissue, cardiac muscle, skeletal muscle, liver). For all these tissues, we compared electron micrographs prepared from cryo-stored material with our method to images derived from directly prepared fresh tissues with standard chemical fixation. Our protocol yielded highly conserved ultrastructural features and tissue-specific details, largely matching the quality of fresh tissue samples. Furthermore, morphometric analysis of lipid droplets and mitochondria in livers of fasted mice demonstrated that statistically valid quantifications can be derived from samples prepared with our method. Overall, we provide a simple and effective protocol for accurate ultrastructural and morphometric analyses of cryo-stored bulk tissue samples. Supplementary Information The online version contains supplementary material available at 10.1007/s00418-020-01952-z.

Burn injuries initiate numerous processes such as heat shock response, inflammation and tissue regeneration. Reliable burn models are needed to elucidate the exact sequence of local events to be able to better predict when local inflammation triggers systemic inflammatory processes. In contrast to other ex vivo skin culture approaches, we used fresh abdominal skin explants to introduce contact burn injuries. Histological and ultrastructural analyses confirmed a partial-thickness burn pathology. Gene expression patterns and cytokine production profiles of key mediators of the local inflammation, heat shock response, and tissue regeneration were analyzed for 24 h after burn injury. We found significantly increased expression of factors involved in tissue regeneration and inflammation soon after burn injury. To investigate purely inflammation-mediated reactions we injected lipopolysaccharide into the dermis. In comparison to burn injury, lipopolysaccharide injection initiated an inflammatory response while expression patterns of heat shock and tissue regeneration genes were unaffected for the duration of the experiment. This novel ex vivo human skin model is suitable to study the local, early responses to skin injuries such as burns while maintaining an intact overall tissue structure and it gives valuable insights into local mechanisms at the very beginning of the wound healing process after burn injuries.

Gut hyperpermeability can be caused by either apoptosis of the intestinal epithelium or altered status, permeability or porosity of tight junctions. This project aims to elucidate these mechanisms in the early phase of sepsis. Eighteen male wild type mice were randomized to two groups. All mice received one single gavage of fluorescein isothiocyanate (FITC) dextran 30 min before intervention. One group (n = 10) underwent cecal ligation and puncture to induce sepsis. The other group (n = 8) was sham operated. Septic animals exhibited significantly increased permeability for FITC 8 h post-operatively. Significantly increased serum interleukin-6, tumor-necrosis-factor-alpha and interleukin-1-beta confirmed sepsis. Septic animals showed significant bowel wall inflammation of ileum and colon samples. PCR revealed significantly increased expression of claudin-2 and decreased expressions of claudin-4, tight-junction-protein-1 and occludin-1 resembling increased permeability of tight junctions. However, these alterations could not be confirmed at the protein level. Light microscopy revealed significant dilatation of intercellular spaces at the basal sections of intestinal epithelial cells (IEC) in septic animals confirmed by increased intercellular spaces at the level of tight junctions and adherens junctions in electron microscopy (TEM). In small angle X-ray scattering no increase in number or size of nanopores could be shown in the bowel wall. HOECHST staining and PCR of ileum samples for apoptosis markers proofed no relevant differences in intestinal epithelial cell apoptosis between the groups. Intestinal hyperpermeability in septic animals was most likely caused by alterations of the intercellular contacts and not by apoptosis or increased size/number of nanopores of intestinal epithelial cells in this murine model of early sepsis.

Chordoma is a rare tumor of the bone derived from remnants of the notochord with pronounced chemoresistance. A common feature of the notochord and chordoma cells is distinct vacuolization. Recently, the notochord vacuole was described as a lysosome-related organelle. Since lysosomes are considered as mediators of drug resistance in cancer, we were interested whether they may also play a role in chemoresistance of chordoma. We characterized the lysosomal compartment in chordoma cell lines by cytochemistry, electron microscopy (ELMI) and mutational analysis of genes essential for the physiology of lysosomes. Furthermore, we tested for the first time the cytotoxicity of chloroquine, which targets lysosomes, on chordoma. Cytochemical stainings clearly demonstrated a huge mass of lysosomes in chordoma cell lines with perinuclear accumulation. Also vacuoles in chordoma cells were positive for the lysosomal marker LAMP1 but showed no acidic pH. Genetic analysis detected no apparent mutation associated with known lysosomal pathologies suggesting that vacuolization and the huge lysosomal mass of chordoma cell lines is rather a relict of the notochord than a result of transformation. ELMI investigation of chordoma cells confirmed the presence of large vacuoles, lysosomes and autophagosomes with heterogeneous ultrastructure embedded in glycogen. Interestingly, chordoma cells seem to mobilize cellular glycogen stores via autophagy. Our first preclinical data suggested no therapeutically benefit of chloroquine for chordoma. Even though, chordoma cells are crammed with lysosomes which are according to their discoverer de Duve "cellular suicide bags". Destabilizing these "suicide bags" might be a promising strategy for the treatment of chordoma.

The placental barrier is crucial for the supply of nutrients and oxygen to the developing fetus and is maintained by differentiation and fusion of mononucleated cytotrophoblasts into the syncytiotrophoblast, a process only partially understood. Here transcriptome and pathway analyses during differentiation and fusion of cultured trophoblasts yielded p53 signaling as negative upstream regulator and indicated an upregulation of autophagy-related genes. We further showed p53 mRNA and protein levels decreased during trophoblast differentiation. Reciprocally, autophagic flux increased and cytoplasmic LC3B-GFP puncta became more abundant, indicating enhanced autophagic activity. In line, in human first trimester placenta p53 protein mainly localized to the cytotrophoblast, while autophagy marker LC3B as well as late autophagic compartments were predominantly detectable in the syncytiotrophoblast. Importantly, ectopic overexpression of p53 reduced levels of LC3B-II, supporting a negative regulatory role on autophagy in differentiating trophoblasts. This was also shown in primary trophoblasts and human first trimester placental explants, where pharmacological stabilization of p53 decreased LC3B-II levels. In summary our data suggest that differentiation-dependent downregulation of p53 is a prerequisite for activating autophagy in the syncytiotrophoblast.

Plants cope with cadmium (Cd) stress by complexation with phytochelatins (Pc), metallothioneins and glutathione and sequestration within vacuoles. Especially glutathione was found to play a major role in Cd detoxification as Cd shows a high binding affinity towards thiols and as glutathione is a precursor for Pc synthesis. In the present study, we have used an immunohistochemical approach combined with computer-supported transmission electron microscopy in order to measure changes in the subcellular distribution of glutathione during Cd-stress in mesophyll cells and cells of different glandular trichomes (long and short stalked) of Cucurbita pepo L. subsp. pepo var. styriaca Greb. Even though no ultrastructural alterations were observed in leaf and glandular trichome cells after the treatment of plants with 50 µM cadmium chloride (CdCl2) for 48 h, all cells showed a large decrease in glutathione contents. The strongest decrease was found in nuclei and the cytosol (up to 76%) in glandular trichomes which are considered as a major side of Cd accumulation in leaves. The ratio of glutathione between the cytosol and nuclei and the other cell compartments was strongly decreased only in glandular trichomes (more than 50%) indicating that glutathione in these two cell compartments is especially important for the detoxification of Cd in glandular trichomes. Additionally, these data indicate that large amounts of Cd are withdrawn from nuclei during Cd exposure. The present study gives a detailed insight into the compartment-specific importance of glutathione during Cd exposure in mesophyll cells and glandular trichomes of C. pepo L. plants.

Telomeres and their changes in length throughout the life span of cells have been intensively investigated in different organisms. Telomere length is assumed to control replicative senescence in mammalian cells. However, only very few data are available on the developmental dynamics of plant telomeres. Here, changes of telomere length and DNA-protein structure of Arabidopsis thaliana telomeres were analysed in different stages of development, with the main focus resting on the transition from pre-senescent to senescent leaves. The lengths of the telomeres, ranging from ca. 2.0 to 6.5 kb, do not significantly change during plant development indicating that telomere length is not involved in differentiation and replicative senescence nor in post-mitotic senescence of A. thaliana. In dedifferentiated cultured cells a slight increase in length can be determined. The nucleoprotein structure of the telomeric DNA was investigated by gel mobility shift assays, with synthetic oligonucleotides and nuclear protein extracts derived from four defined stages of post-mitotic leaf senescence. In all four stages, a highly salt-resistant DNA-protein complex was formed with the double-stranded as well as with the single-stranded G-rich telomeric DNA. An additional DNA-protein complex was identified in nuclear protein extracts isolated from plants in the transition stage from pre-senescence to senescence. The protein components of the DNA-protein complexes were analysed on native PAGE and SDS-PAGE gels. A protein of 67 kDa (ATBP1) bound to the telomeric DNA in all developmental stages. An additional protein of merely 22 kDa (ATBP2) was associated via protein-protein interaction with ATBP1 to form a higher-order complex exclusively during the onset of senescence. DNA interaction of this higher-order protein complex seems to be restricted to double-stranded telomeric DNA. The defined period of ATBP1/ATBP2 complex formation with the telomeric DNA probably indicates that ATBP2 is involved in the onset of post-mitotic leaf senescence by either disturbing an established or establishing an additional function exhibited by the telomeres in the interphase nuclei.

Triacylglycerol (TG) accumulation caused by adipose triglyceride lipase (ATGL) deficiency or very low-density lipoprotein (VLDL) loading of wild-type (Wt) macrophages results in mitochondrial-mediated apoptosis. This phenotype is correlated to depletion of Ca(2+) from the endoplasmic reticulum (ER), an event known to induce the unfolded protein response (UPR). Here, we show that ER stress in TG-rich macrophages activates the UPR, resulting in increased abundance of the chaperone GRP78/BiP, the induction of pancreatic ER kinase-like ER kinase, phosphorylation and activation of eukaryotic translation initiation factor 2A, the translocation of activating transcription factor (ATF)4 and ATF6 to the nucleus and the induction of the cell death executor CCAAT/enhancer-binding protein homologous protein. C16:0 ceramide concentrations were increased in Atgl-/- and VLDL-loaded Wt macrophages. Overexpression of ceramide synthases was sufficient to induce mitochondrial apoptosis in Wt macrophages. In accordance, inhibition of ceramide synthases in Atgl-/- macrophages by fumonisin B1 (FB1) resulted in specific inhibition of C16:0 ceramide, whereas intracellular TG concentrations remained high. Although the UPR was still activated in Atgl-/- macrophages, FB1 treatment rescued Atgl-/- macrophages from mitochondrial dysfunction and programmed cell death. We conclude that C16:0 ceramide elicits apoptosis in Atgl-/- macrophages by activation of the mitochondrial apoptosis pathway.