Aspects of poriferan (Suberites domuncula) apoptosis and innate immune system and evolutionary implications

Survivin, a unique member of the family of inhibitors of apoptosis (IAP) proteins, orchestrates intracellular pathways during cell division and apoptosis. Its central regulatory function in vertebrate molecular pathways as mitotic regulator and inhibitor of apoptotic cell death has major implications for tumor cell proliferation and viability, and has inspired several approaches that target survivin for cancer therapy. Analyses in early-branching Metazoa so far propose an exclusive role of survivin as a chromosomal passenger protein, whereas only later during evolution the second, complementary antiapoptotic function might have arisen, concurrent with increased organismal complexity. To lift the veil on the ancestral function(s) of this key regulatory molecule, a survivin homologue of the phylogenetically oldest extant metazoan taxon (phylum Porifera) was identified and functionally characterized. SURVL of the demosponge Suberites domuncula shares significant similarities with its metazoan homologues, ranging from conserved exon/intron structures to the presence of localization signal and protein-interaction domains, characteristic of IAP proteins. Whereas sponge tissue displayed a very low steady-state level, SURVL expression was significantly up-regulated in rapidly proliferating primmorph cells. In addition, challenge of sponge tissue and primmorphs with cadmium and the lipopeptide Pam3Cys-Ser-(Lys)4 stimulated SURVL expression, concurrent with the expression of newly discovered poriferan caspases (CASL and CASL2). Complementary functional analyses in transfected HEK-293 revealed that heterologous expression of poriferan survivin in human cells not only promotes cell proliferation but also augments resistance to cadmium-induced cell death. Taken together, these results demonstrate both a deep evolutionary conserved and fundamental dual role of survivin, and an equally conserved central position of this key regulatory molecule in interconnected pathways of cell cycle and apoptosis. Additionally, SDCASL, SDCASL2, and SDTILRc (TIR-LRR containing protein) may represent new components of the innate defense sentinel in sponges. SDCASL and SDCASL2 are two new caspase-homolog proteins with a singular structure. In addition to their CASc domains, SDCASL and SDCASL2 feature a small prodomain NH2-terminal (effector caspases) and a remarkably long COOH- terminal domain containing one or several functional double stranded RNA binding domains (dsrm). This new caspase prototype can characterize a caspase specialization coupling pathogen sensing and apoptosis, and could represent a very efficient defense mechanism. SDTILRc encompasses also a unique combination of domains: several leucine rich repeats (LRR) and a Toll/IL-1 receptor (TIR) domain. This unusual domain association may correspond to a new family of intracellular sensing protein, forming a subclass of pattern recognition receptors (PRR).Survivin, ein einzigartiges Mitglied der Familie von Apoptose-Inhibitor (IAP) Proteinen, dirigiert und reguliert interzelluläre Abläufe während der Zellteilung und Apoptose. Seine zentrale, regulierende Funktion als mitotischer Regulator und Inhibitor des apoptotischen Zelletods in molekularen Prozessen, hat Auswirkungen auf das Tumorwachstum und die Lebensfähigkeit und lieferte mehrere Ansätze um Survivin als Zielmolekül für die Krebs-Therapie zu verwenden. Analysen in niederen Metazoen implizieren bis jetzt eine exklusive Rolle des Survivins als „chromosomales Passenger Protein“, wohingegen die zweite, ergänzende, antiapoptotische Funktion, als Begleitumstand mit der größer werdenden Komplexität der Organismen nur später während der Entwicklung entstanden sein kann. Um die ursprüngliche Funktion dieses regulatorischen Schlüsselmoleküls untersuchen zu können, wurde ein Survivin-Homolog aus Schwämmen, dem phylogenetisch ältesten, noch existierenden Metazoen-Taxon (Unterabteilung Porifera) identifiziert und funktionell charakterisiert. Das SURVL des Schwammes Suberites domuncula (Hormschwämme/Demospongia) zeigt deutliche Ähnlichkeiten zu seinen Metazoen-Homologen, unter anderem konservierte Exon/Intron-Strukturen, die Anwesenheit des Lokalisationssignals und Domänen der Protein-Wechselwirkung, welche charakteristisch für IAP Proteine sind. Während Schwammgewebe ein sehr niedriges Grundniveau der SURVL-Expression zeigte, wurde sie in schnell wachsenden Primmorphzellen deutlich hoch reguliert. Außerdem stimulierte die Inkubation des Schwammgewebes und der Primmorphe mit Cadmium und dem Lipopeptid pam3cys-ser-(Lys)4 die SURVL Expression, simultan zu der Expression der kürzlich entdeckten Schwamm-Caspasen (CASL und CASL2). Ergänzende Funktionsanalysen in transfizierten HEK-293 Zellen offenbarten, dass die heterologe Expression des Porifera-Survivins in menschlichen Zellen nicht nur das Zellwachstum fördert, sondern auch den Widerstand gegen durch Cadmium induzierten Zelletod erhöht. Zusammen gefasst demonstrieren diese Resultate sowohl eine tiefe, grundlegende, evolutionär konservierte Doppelrolle des Survivins, als auch eine ebenso konservierte zentrale Position dieses regulatorischen Schlüsselmoleküls in miteinander verbundenen Prozessen des Zell-Zyklus und der Apoptose. Zusätzlich, können SDCASL, SDCASL2, und SDTILRC (TIR-LRR enthaltendes Proteins) neue Bestandteile des angeborenen Verteidigungssystems in Schwämmen repräsentieren. SDCASL und SDCASL2 sind zwei neue Caspase-Homologe mit einer einzigartigen Struktur. Zusätzlich zu ihren CASC Domänen, zeichnen sich SDCASL und SDCASL2 durch eine kleine NH2-terminale Prodomäne (effector caspases) und eine bemerkenswert lange COOH-terminale Domäne aus, die eine oder mehrere funktionelle Bindedomänen für doppelsträngige RNA (dsrm) enthält. Dieser neue Caspase Prototyp kann eine caspase-spezifische Kopplung charakterisieren, die Pathogenerkennung und Apoptose verbindet, und könnte einen sehr effizienten Abwehrmechanismus darstellen. SDTILRc umfasst ebenfalls eine einzigartige Kombination von Domänen: mehrere Leucin-reiche Wiederholungen (LRR) und eine Toll/IL-1 Rezeptor (TIR) Domäne. Diese ungewöhnliche Domänenkombination könnte einer neuen Familie des interzellularen Erkennungsproteins entsprechen, die eine Subklasse von Mustererkennungsrezeptoren(PRR) bildet

Comparing medium pressure dielectric barrier discharge (DBD) plasmas and classic methods of surface cleaning/activation of pure Mg for biomedical applications

Pure magnesium (Mg), being a lightweight biodegradable material, is increasingly attracting attention for its use in biomedical applications. However, surface contaminations arising from material machining can result in a deleterious degradation behavior thus a poor implant biocompatibility performance. Therefore, this paper represents a comparative study tackling the surface chemistry, cytocompatibility and degradation of Mg discs subjected to classical grinding and chemical treatments and to unconventional medium pressure (5 kPa) plasmas operated in Ar and NH3/He. Results reveal that both plasmas remove more than 40% of the carbonaceous contaminations while introducing surface oxygen (and nitrogen) containing functionalities. However, grinding only removes 34% of the initial surface carbon. Despite being the most efficient in eliminating the organic layer, the chemical treatment leads to an excessive surface oxidation. In vitro tests involving mesenchymal stem cells reveal that plasma-treated samples outperform their ground and chemically cleaned counterparts in terms of cellsurface affinity as more spread out cells with significantly bigger areas are detected, suggesting higher cell attachment. Moreover, plasma does not alter the degradation rate of Mg discs, thus providing a striking insight for their application in tissue engineering. Overall, one can conclude that the eco-friendly and economical subatmospheric plasma is an effective alternative synergistically cleaning and improving the cytocompatibility of Mg surfaces

Influence of Magnesium Alloy Degradation on Undifferentiated Human Cells

Background Magnesium alloys are of particular interest in medical science since they provide compatible mechanical properties with those of the cortical bone and, depending on the alloying elements, they have the capability to tailor the degradation rate in physiological conditions, providing alternative bioresorbable materials for bone applications. The present study investigates the in vitro short-term response of human undifferentiated cells on three magnesium alloys and high-purity magnesium (Mg). Materials and Methods The degradation parameters of magnesium-silver (Mg2Ag), magnesium-gadolinium (Mg10Gd) and magnesium-rare-earth (Mg4Y3RE) alloys were analysed after 1, 2, and 3 days of incubation in cell culture medium under cell culture condition. Changes in cell viability and cell adhesion were evaluated by culturing human umbilical cord perivascular cells on corroded Mg materials to examine how the degradation influences the cellular development. Results and Conclusions The pH and osmolality of the medium increased with increasing degradation rate and it was found to be most pronounced for Mg4Y3RE alloy. The biological observations showed that HUCPV exhibited a more homogeneous cell growth on Mg alloys compared to high-purity Mg, where they showed a clustered morphology. Moreover, cells exhibited a slightly higher density on Mg2Ag and Mg10Gd in comparison to Mg4Y3RE, due to the lower alkalinisation and osmolality of the incubation medium. However, cells grown on Mg10Gd and Mg4Y3RE generated more developed and healthy cellular structures that allowed them to better adhere to the surface. This can be attributable to a more stable and homogeneous degradation of the outer surface with respect to the incubation time

SEM images and the corresponding EDX analysis of magnesium alloys and hp Mg.

<p>Analysis was performed after 72 h pre-incubation time applying 10 KeV accelerating voltage.</p

Confocal fluorescence microscopy of focal adhesion and actin cytoskeleton in HUCPV cells cultured for 24 h on Mg2Ag, Mg10Gd, Mg4Y3RE and Mg on 48 h pre-incubated.

<p>Focal contacts were stained with vinculin monoclonal antibody (green); actin filaments were stained with a mixture of anti-mouse secondary antibody (FITC) and TRITC-conjugated phalloidin (red); nuclei were stained with DAPI (blue). Images at 40x were merged displaying the triple labeling (yellow areas obtained from the overlapping of red and green labelling).</p

Weight % of O, C, P and Mg quantified out of the EDX mapping with 10 keV on Mg, Mg2Ag, Mg10Gd and Mg4Y3Re after 72 h of pre-incubation in culture medium and in cell culture conditions.

<p>Weight % of O, C, P and Mg quantified out of the EDX mapping with 10 keV on Mg, Mg2Ag, Mg10Gd and Mg4Y3Re after 72 h of pre-incubation in culture medium and in cell culture conditions.</p

FITC intensity quantification for living HUCPV cultured for 24 h on Mg, Mg2Ag, Mg10Gd and Mg4Y3RE at 24 h, 48 h and 72 h pre-incubation states.

<p>Measurements were obtained from images taken form 3 samples of each material at each pre-incubation condition after LIVE/DEAD assay. Significance level was set at p < 0.05; n = 3 per group.</p

Fluorescence images of HUCPV cultured on Mg, Mg2Ag, Mg10Gd and Mg4Y3RE.

<p>Fluorescence LIVE (green)/RED (dead) staining was performed after 24 h of cell culture on 24 h, 48 h and 72 h pre-incubated samples. Monochrome images were taken in large image mode (4x4) at 20x magnification.</p

Confocal fluorescence microscopy of focal adhesion and actin cytoskeleton in HUCPV cells cultured for 24 h on Mg2Ag, Mg10Gd, Mg4Y3RE and Mg on 24 h (a) and 48 h (b) pre-incubated samples.

<p>Focal contacts were stained with vinculin monoclonal antibody (green); actin filaments were stained with a mixture of anti-mouse secondary antibody (FITC) and TRITC-conjugated phalloidin (red); nuclei were stained with DAPI (blue). Images at 20x were merged displaying the triple labeling (yellow areas obtained from the overlapping of red and green labelling).</p