Leica Reichert Ultracut

Background: During neuromuscular junction (NMJ) development, synapses are produced in excess. By sensing the activity-dependent release of ACh, adenosine, and neurotrophins, presynaptic receptors prompt axonal competition and loss of the unnecessary axons. The receptor action is mediated by synergistic and antagonistic relations when they couple to downstream kinases (mainly protein kinases A and C (PKA and PKC)), which phosphorylate targets involved in axonal disconnection. Here, we directly investigated the involvement of PKA subunits and PKC isoforms in synapse elimination. Methods: Selective PKA and PKC peptide modulators were applied daily to the Levator auris longus (LAL) muscle surface of P5–P8 transgenic B6.Cg-Tg (Thy1-YFP) 16 Jrs/J (and also C57BL/6J) mice, and the number of axons and the postsynaptic receptor cluster morphology were evaluated in P9 NMJ. Results: PKA (PKA-I and PKA-II isozymes) acts at the pre- and postsynaptic sites to delay both axonal elimination and nAChR cluster differentiation, PKC activity promotes both axonal loss (a cPKCβI and nPKCε isoform action), and postsynaptic nAChR cluster maturation (a possible role for PKCθ). Moreover, PKC-induced changes in axon number indirectly influence postsynaptic maturation. Conclusions: PKC and PKA have opposed actions, which suggests that changes in the balance of these kinases may play a major role in the mechanism of developmental synapse elimination.

undefined Garcia, undefined Balañà, undefined Lanuza, undefined Tomàs, undefined Cilleros-Mañé, undefined Just-Borràs, undefined Tomàs. Cells, 8 (11), 1304, 2019.

Nonalcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver diseases with an increasing prevalence due to rising rates of obesity, metabolic syndrome and type II diabetes. Untreated NAFLD may progress to steatohepatitis (NASH) and ultimately liver cirrhosis. NAFLD is characterized by lipid accumulation, and when sufficient excess lipids are obtained, irreversible liver injury may follow. Perilipin 5 (PLIN5), a known lipid droplet coating protein and triglyceride metabolism regulator, is highly expressed in oxidatively modified tissues but it is still unclear how it affects NAFLD/NASH progress. We here studied how PLIN5 affects NAFLD development induced by a 30-week high-fat diet (HFD) administration in wild type and PLIN5 knock out (Plin5−/−) mice. The disruption of PLIN5 induced differences in lipid metabolism during HFD feeding and was associated with reduced hepatic fat accumulation. Surprisingly, Plin5−/− mice showed mitigated activation of the NLR family pyrin domain-containing 3 (NLRP3) inflammasome, leading to minor hepatic damage. We conclude that PLIN5 is a pleiotropic regulator of hepatic homeostasis in NASH development. Targeting the PLIN5 expression appears critical for protecting the liver from inflammatory activation during chronic NAFLD.

Anastasia Asimakopoulou, Kathrin M. Engel, Nikolaus Gassler, Thilo Bracht, Barbara Sitek, Eva M. Buhl, Stavroula Kalampoka, Manuela Pinoé-Schmidt, Josef van Helden, Jürgen Schiller, Ralf Weiskirchen. Cells, 9 (6), 1346, 2020.

Aging depicts one of the major challenges in pharmacology owing to its complexity and heterogeneity. Thereby, advanced glycated end-products modify extracellular matrix proteins, but the consequences on the skin barrier function remain heavily understudied. Herein, we utilized transmission electron microscopy for the ultrastructural analysis of ribose-induced glycated reconstructed human skin (RHS). Molecular and functional insights substantiated the ultrastructural characterization and proved the relevance of glycated RHS beyond skin aging. In particular, electron microscopy mapped the accumulation and altered spatial orientation of fibrils and filaments in the dermal compartment of glycated RHS. Moreover, the epidermal basement membrane appeared thicker in glycated than in non-glycated RHS, but electron microscopy identified longitudinal clusters of the finest collagen fibrils instead of real thickening. The stratum granulosum contained more cell layers, the morphology of keratohyalin granules decidedly differed, and the stratum corneum lipid order increased in ribose-induced glycated RHS, while the skin barrier function was almost not affected. In conclusion, dermal advanced glycated end-products markedly changed the epidermal morphology, underlining the importance of matrix–cell interactions. The phenotype of ribose-induced glycated RHS emulated aged skin in the dermis, while the two to three times increased thickness of the stratum granulosum resembled poorer cornification.

Roberta Balansin Rigon, Sabine Kaessmeyer, Christopher Wolff, Christian Hausmann, Nan Zhang, Michaela Sochorová, Andrej Kováčik, Rainer Haag, Kateřina Vávrová, Martina Ulrich, Monika Schäfer-Korting, Christian Zoschke. International Journal of Molecular Sciences, 19 (11), 3521, 2018.

Increased oxidative stress and mitochondrial damage are observed in protein aggregation diseases, such as age-related macular degeneration (AMD). We have recently reported elevated levels of oxidative stress markers, damaged mitochondria, accumulating lysosomal lipofuscin and extracellular drusen-like structures in the retinal pigment epithelial cells (RPE) of the dry AMD-resembling NFE2L2/PGC1α double knockout (dKO) mouse model. Here, we provide evidence of a disturbance in the autolysosomal machinery handling mitochondrial clearance in the RPE cells of one-year-old NFE2L2/PGC1α-deficient mice. Confocal immunohistochemical analysis revealed an upregulation of autophagosome marker microtubule-associated proteins 1A/1B light chain 3B (LC3B) as well as numerous mitophagy markers, such as PTE-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase (PARKIN) together with damaged mitochondria. However, we detected no evidence of increased autolysosome formation in transmission electron micrographs or of colocalization of lysosomal marker LAMP2 (lysosome-associated membrane protein 2) and the mitochondrial marker ATP synthase β in confocal micrographs. Interestingly, we observed an upregulation of late autolysosomal fusion Ras-related protein (Rab7) in the perinuclear space of RPE cells together with autofluorescence aggregates. Our results reveal that there is at least a relative decrease of mitophagy in the RPE cells of NFE2L2/PGC1α dKO mice. This further supports the hypothesis that mitophagy is a putative therapy target in AMD-like pathology.

Iswariyaraja Sridevi Gurubaran, Johanna Viiri, Ali Koskela, Juha M.T. Hyttinen, Jussi J. Paterno, Gréta Kis, Miklós Antal, Arto Urtti, Anu Kauppinen, Szabolcs Felszeghy, Kai Kaarniranta. International Journal of Molecular Sciences, 21 (6), 1976, 2020.

Human breast adenocarcinoma cells (MCF7) grow in three-dimensional culture as spheroids that represent the structural complexity of avascular tumors. Therefore, spheroids offer a powerful tool for studying cancer development, aggressiveness, and drug resistance. Notwithstanding the large amount of data regarding the formation of MCF7 spheroids, a detailed description of the morpho-functional changes during their aggregation and maturation is still lacking. In this study, in addition to the already established role of gap junctions, we show evidence of tunneling nanotube (TNT) formation, amyloid fibril production, and opening of large stable cellular bridges, thus reporting the sequential events leading to MCF7 spheroid formation. The variation in cell phenotypes, sustained by dynamic expression of multiple proteins, leads to complex networking among cells similar to the sequence of morphogenetic steps occurring in embryogenesis/organogenesis. On the basis of the observation that early events in spheroid formation are strictly linked to the redox homeostasis, which in turn regulate amyloidogenesis, we show that the administration of N-acetyl-l-cysteine (NAC), a reactive oxygen species (ROS) scavenger that reduces the capability of cells to produce amyloid fibrils, significantly affects their ability to aggregate. Moreover, cells aggregation events, which exploit the intrinsic adhesiveness of amyloid fibrils, significantly decrease following the administration during the early aggregation phase of neutral endopeptidase (NEP), an amyloid degrading enzyme.

Laura Pulze, Terenzio Congiu, Tiziana A. L. Brevini, Annalisa Grimaldi, Gianluca Tettamanti, Paola D’Antona, Nicolò Baranzini, Francesco Acquati, Federico Ferraro, Magda de Eguileor. International Journal of Molecular Sciences, 21 (15), 5400, 2020.

Recent studies performed on the invertebrate model Hirudo verbana (medicinal leech) suggest that the T2 ribonucleic enzyme HvRNASET2 modulates the leech’s innate immune response, promoting microbial agglutination and supporting phagocytic cells recruitment in challenged tissues. Indeed, following injection of both lipoteichoic acid (LTA) and Staphylococcus aureus in the leech body wall, HvRNASET2 is expressed by leech type I granulocytes and induces bacterial aggregation to aid macrophage phagocytosis. Here, we investigate the HvRNASET2 antimicrobial role, in particular assessing the effects on the Gram-negative bacteria Escherichia coli. For this purpose, starting from the three-dimensional molecule reconstruction and in silico analyses, the antibacterial activity was evaluated both in vitro and in vivo. The changes induced in treated bacteria, such as agglutination and alteration in wall integrity, were observed by means of light, transmission and scanning electron microscopy. Moreover, immunogold, AMPs (antimicrobial peptides) and lipopolysaccharide (LPS) binding assays were carried out to evaluate HvRNASET2 interaction with the microbial envelopes and the ensuing ability to affect microbial viability. Finally, in vivo experiments confirmed that HvRNASET2 promotes a more rapid phagocytosis of bacterial aggregates by macrophages, representing a novel molecule for counteracting pathogen infections and developing alternative solutions to improve human health.

Nicolò Baranzini, Laura Pulze, Marcella Reguzzoni, Rossella Roncoroni, Viviana Teresa Orlandi, Gianluca Tettamanti, Francesco Acquati, Annalisa Grimaldi. International Journal of Molecular Sciences, 21 (24), 9722, 2020.

This study aimed to clarify whether the light condition-dependent changes in the redox state and subcellular distribution of glutathione were similar in the dicotyledonous model plant Arabidopsis (wild-type, ascorbate- and glutathione-deficient mutants) and the monocotyledonous crop species wheat (Chinese Spring variety). With increasing light intensity, the amount of its reduced (GSH) and oxidized (GSSG) form and the GSSG/GSH ratio increased in the leaf extracts of both species including all genotypes, while far-red light increased these parameters only in wheat except for GSH in the GSH-deficient Arabidopsis mutant. Based on the expression changes of the glutathione metabolism-related genes, light intensity influences the size and redox state of the glutathione pool at the transcriptional level in wheat but not in Arabidopsis. In line with the results in leaf extracts, a similar inducing effect of both light intensity and far-red light was found on the total glutathione content at the subcellular level in wheat. In contrast to the leaf extracts, the inducing influence of light intensity on glutathione level was only found in the cell compartments of the GSH-deficient Arabidopsis mutant, and far-red light increased it in both mutants. The observed general and genotype-specific, light-dependent changes in the accumulation and subcellular distribution of glutathione participate in adjusting the redox-dependent metabolism to the actual environmental conditions.

Anna Gasperl, Eszter Balogh, Ákos Boldizsár, Nadine Kemeter, Richard Pirklbauer, Stefan Möstl, Balázs Kalapos, Gabriella Szalai, Maria Müller, Günther Zellnig, Gábor Kocsy. International Journal of Molecular Sciences, 22 (2), 607, 2021.

Age-related macular degeneration (AMD) is the most prevalent form of irreversible blindness worldwide in the elderly population. In our previous studies, we found that deficiencies in the nuclear factor, erythroid 2 like 2 (NFE2L2) and peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) genes caused AMD-like pathological phenotypes in mice. In the present work, we show hijacked epithelial-mesenchymal transition (EMT) due to the common loss of PGC-1α and NFE2L2 (double knock-out, dKO) genes in aged animals. The implanted area was assessed by histology, immunohistochemistry and transmission electron microscopy. Confocal microscopy revealed altered regions in the filamentous actin ring. This contrasted with hexagonal RPE morphology in wild-type mice. The ultrastructural RPE features here illustrated loss of apical microvilli, alteration of cell-cell contact, loss of basal in-folding with deposits on Bruch’s membrane, and excessive lipofuscin deposition in dKO samples. We also found the expression of epithelial-mesenchymal transition transcription factors, such as Snail, Slug, collagen 1, vimentin and OB-cadherin, to be significantly different in dKO RPEs. An increased immunoreactivity of senescence markers p16, DEC1 and HMGB1 was also noted. These findings suggest that EMT and senescence pathways may intersect in the retinas of dKO mice. Both processes can be activated by damage to the RPE, which may be caused by increased oxidative stress resulting from the absence of NFE2L2 and PGC-1α genes, important for antioxidant defense. This dKO model may provide useful tools for studying AMD pathogenesis and evaluating novel therapies for this disease.

Janusz Blasiak, Ali Koskela, Elzbieta Pawlowska, Mikko Liukkonen, Johanna Ruuth, Elisa Toropainen, Juha M. T. Hyttinen, Johanna Viiri, John E. Eriksson, Heping Xu, Mei Chen, Szabolcs Felszeghy, Kai Kaarniranta. International Journal of Molecular Sciences, 22 (4), 1684, 2021.

Biomineralization is the process by which living organisms generate organized mineral crystals. In human cells, this phenomenon culminates with the formation of hydroxyapatite, which is a naturally occurring mineral form of calcium apatite. The mechanism that explains the genesis within the cell and the propagation of the mineral in the extracellular matrix still remains largely unexplained, and its characterization is highly controversial, especially in humans. In fact, up to now, biomineralization core knowledge has been provided by investigations on the advanced phases of this process. In this study, we characterize the contents of calcium depositions in human bone mesenchymal stem cells exposed to an osteogenic cocktail for 4 and 10 days using synchrotron-based cryo-soft-X-ray tomography and cryo-XANES microscopy. The reported results suggest crystalline calcite as a precursor of hydroxyapatite depositions within the cells in the biomineralization process. In particular, both calcite and hydroxyapatite were detected within the cell during the early phase of osteogenic differentiation. This striking finding may redefine most of the biomineralization models published so far, taking into account that they have been formulated using murine samples while studies in human cell lines are still scarce.

Andrea Sorrentino, Emil Malucelli, Francesca Rossi, Concettina Cappadone, Giovanna Farruggia, Claudia Moscheni, Ana J. Perez-Berna, Jose Javier Conesa, Chiara Colletti, Norberto Roveri, Eva Pereiro, Stefano Iotti. International Journal of Molecular Sciences, 22 (9), 4939, 2021.

Given the anatomical simplicity and the extraordinary ability to regenerate missing parts of the body, Cnidaria represent an excellent model for the study of the mechanisms regulating regenerative processes. They possess the mesoglea, an amorphous and practically acellular extracellular matrix (ECM) located between the epidermis and the gastrodermis of the body and tentacles and consists of the same molecules present in the ECM of vertebrates, such as collagen, laminin, fibronectin and proteoglycans. This feature makes cnidarians anthozoans valid models for understanding the ECM role during regenerative processes. Indeed, it is now clear that its role in animal tissues is not just tissue support, but instead plays a key role during wound healing and tissue regeneration. This study aims to explore regenerative events after tentacle amputation in the Mediterranean anemone Anemonia viridis, focusing in detail on the reorganization of the ECM mesoglea. In this context, both enzymatic, biometric and histological experiments reveal how this gelatinous connective layer plays a fundamental role in the correct restoration of the original structures by modifying its consistency and stiffness. Indeed, through the deposition of collagen I, it might act as a scaffold and as a guide for the reconstruction of missing tissues and parts, such as amputated tentacles.

Maria Giovanna Parisi, Annalisa Grimaldi, Nicolò Baranzini, Claudia La Corte, Mariano Dara, Daniela Parrinello, Matteo Cammarata. International Journal of Molecular Sciences, 22 (11), 5971, 2021.

The hierarchical structure of bone and intrinsic material properties of its two primary constituents, carbonated apatite and fibrillar collagen, when being synergistically organized into an interpenetrating hard-soft composite, contribute to its excellent mechanical properties. Lamellar bone is the predominant structural motif in mammalian hard tissues; therefore, we believe the fabrication of a collagen/apatite composite with a hierarchical structure that emulates bone, consisting of a dense lamellar microstructure and a mineralized collagen fibril nanostructure, is an important first step toward the goal of regenerative bone tissue engineering. In this work, we exploit the liquid crystalline properties of collagen to fabricate dense matrices that assemble with cholesteric organization. The matrices were crosslinked via carbodiimide chemistry to improve mechanical properties, and are subsequently mineralized via the polymer-induced liquid-precursor (PILP) process to promote intrafibrillar mineralization. Neither the crosslinking procedure nor the mineralization affected the cholesteric collagen microstructures; notably, there was a positive trend toward higher stiffness with increasing crosslink density when measured by cantilever-based atomic force microscopy (AFM) nanoindentation. In the dry state, the average moduli of moderately (X51; 4.8 ± 4.3 GPa) and highly (X76; 7.8 ± 6.7 GPa) crosslinked PILP-mineralized liquid crystalline collagen (LCC) scaffolds were higher than the average modulus of bovine bone (5.5 ± 5.6 GPa).

Brian Wingender, Yongliang Ni, Yifan Zhang, Curtis Taylor, Laurie Gower. Materials, 11 (7), 1257, 2018.

Elephant endotheliotropic herpesvirus (EEHV) can cause a devastating haemorrhagic disease in young Asian elephants worldwide. Here, we report the death of two young Asian elephants after suffering from acute haemorrhagic disease due to EEHV-1A infection. We detected widespread distribution of EEHV-1A in various organs and tissues of the infected elephants. Enveloped viral particles accumulated within and around cytoplasmic electron-dense bodies in hepatic endothelial cells were detected. Attempts to isolate the virus on different cell cultures showed limited virus replication; however, late viral protein expression was detected in infected cells. We further showed that glycoprotein B (gB) of EEHV-1A possesses a conserved cleavage site Arg-X-Lys/Arg-Arg that is targeted by the cellular protease furin, similar to other members of the Herpesviridae. We have determined the complete 180 kb genome sequence of EEHV-1A isolated from the liver by next-generation sequencing and de novo assembly. As virus isolation in vitro has been unsuccessful and limited information is available regarding the function of viral proteins, we have attempted to take the initial steps in the development of suitable cell culture system and virus characterization. In addition, the complete genome sequence of an EEHV-1A in Europe will facilitate future studies on the epidemiology and diagnosis of EEHV infection in elephants.

Selvaraj Pavulraj, Kathrin Eschke, Adriane Prahl, Michael Flügger, Jakob Trimpert, Petra B. van den Doel, Sandro Andreotti, Sabine Kaessmeyer, Nikolaus Osterrieder, Walid Azab. Microorganisms, 7 (10), 396, 2019.

Growth of mutant gdhA Pasteurella multocida B:2 was inhibited by the accumulation of a by-product, namely ammonium in the culture medium during fermentation. The removal of this by-product during the cultivation of mutant gdhA P. multocida B:2 in a 2 L stirred-tank bioreactor integrated with an internal column using cation-exchange adsorption resin for the improvement of cell viability was studied. Different types of bioreactor system (dispersed and internal) with resins were successfully used for ammonium removal at different agitation speeds. The cultivation in a bioreactor integrated with an internal column demonstrated a significant improvement in growth performance of mutant gdhA P. multocida B:2 (1.05 × 1011 cfu/mL), which was 1.6-fold and 8.4-fold as compared to cultivation with dispersed resin (7.2 × 1010 cfu/mL) and cultivation without resin (1.25 × 1010 cfu/mL), respectively. The accumulation of ammonium in culture medium without resin (801 mg/L) was 1.24-fold and 1.37-fold higher than culture with dispersed resin (642.50 mg/L) and culture in the bioreactor integrated with internal adsorption (586.50 mg/L), respectively. Results from this study demonstrated that cultivation in a bioreactor integrated with the internal adsorption column in order to remove ammonium could reduce the inhibitory effect of this by-product and improve the growth performance of mutant gdhA P. multocida B:2.

Siti Nur Hazwani Oslan, Joo Shun Tan, Sahar Abbasiliasi, Ahmad Ziad Sulaiman, Mohd Zamri Saad, Murni Halim, Arbakariya B. Ariff. Microorganisms, 8 (11), 1654, 2020.