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Find video protocols related to scientific articles indexed in Pubmed.
Enzymatically synthesized inorganic polymers as morphogenetically active bone scaffolds: application in regenerative medicine.
Int Rev Cell Mol Biol
PUBLISHED: 11-08-2014
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In recent years a paradigm shift in understanding of human bone formation has occurred that starts to change current concepts in tissue engineering of bone and cartilage. New discoveries revealed that fundamental steps in biomineralization are enzyme driven, not only during hydroxyapatite deposition, but also during initial bioseed formation, involving the transient deposition and subsequent transformation of calcium carbonate to calcium phosphate mineral. The principal enzymes mediating these reactions, carbonic anhydrase and alkaline phosphatase, open novel targets for pharmacological intervention of bone diseases like osteoporosis, by applying compounds acting as potential activators of these enzymes. It is expected that these new findings will give an innovation boost for the development of scaffolds for bone repair and reconstruction, which began with the use of bioinert materials, followed by bioactive materials and now leading to functional regenerative tissue units. These new developments have become possible with the discovery of the morphogenic activity of bioinorganic polymers, biocalcit, bio-polyphosphate and biosilica that are formed by a biogenic, enzymatic mechanism, a driving force along with the development of novel rapid-prototyping three-dimensional (3D) printing methods and bioprinting (3D cell printing) techniques that may allow a fabrication of customized implants for patients suffering in bone diseases in the future.
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Dispacamide E and other bioactive bromopyrrole alkaloids from two Indonesian marine sponges of the genus Stylissa.
Nat. Prod. Res.
PUBLISHED: 08-12-2014
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Chemical investigation of methanolic extracts of the two Indonesian marine sponges Stylissa massa and Stylissa flabelliformis yielded 25 bromopyrrole alkaloids including 2 new metabolites. The structures of all isolated compounds were unambiguously elucidated based on extensive 1D and 2D NMR, LR-MS and HR-MS analyses. All isolated compounds were assayed for their antiproliferative and protein kinase inhibitory activities. Several of the tested compounds revealed selective activity(ies) which suggested preliminary SARs of the isolated bromopyrrole alkaloids.
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Hierarchical structure and cytocompatibility of fish scales from Carassius auratus.
Mater Sci Eng C Mater Biol Appl
PUBLISHED: 07-10-2014
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To study the structure and the cytocompatibility of fish scales from Carassius auratus, scanning electron microscopy (SEM) was used to observe the morphology of fish scales treated with different processing methods. Based on varying morphologies and components, the fish scales can be divided into three regions on the surface and three layers in vertical. The functions of these three individual layers were analyzed. SEM results show that the primary inorganic components are spherical or cubic hydroxyapatite (HA) nanoparticles. The fish scales have an ~60° overlapped plywood structure of lamellas in the fibrillary plate. The plywood structure consists of co-aligned type I collagen fibers, which are parallel to the HA lamellas. X-ray diffraction (XRD), thermogravimetric analysis/differential scanning calorimetry (TGA/DSC) and Fourier transform infrared (FTIR) analysis indicate that the main components are HA and type I collagen fibers. MC3T3-E1 cell culture results show a high cytocompatibility and the ability to guide cell proliferation and migration along the scale ridge channels of the fish scales. This plywood structure provides inspiration for a structure-enhanced composite material.
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Do surgical patellar interventions restore patellar kinematics in fixed-bearing, cruciate-retaining total knee arthroplasty?: An in vitro study.
J Arthroplasty
PUBLISHED: 07-10-2014
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Despite different surgical patellar interventions, the decision how to treat the patella during TKA remains controversial. The purpose of this study was to quantify the effect of different reconstructive patellar interventions on patellar kinematics during TKA using optical computer navigation. We implanted ten navigated TKAs in full body specimens. During passive motion, the effect of different surgical patellar interventions on patellar kinematics was analysed. A contrarily tilt behaviour was observed in the TKA group without patellar intervention compared to the natural knee. Lateral release led to similar tilt values (P<0.05). All surgical interventions led to a 3 to 5mm medial shift of the patella (P<0.05). None of the analysed surgical patellar interventions could restore natural patellar kinematics after TKA.
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Clinical and radiological long-term outcome after posterior cruciate ligament reconstruction and nonanatomical popliteus bypass.
Int Orthop
PUBLISHED: 06-22-2014
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The purpose of this study was to analyse the long-term outcome of patients treated for combined posterior cruciate ligament (PCL) and posterolateral corner injuries by combined PCL reconstruction and popliteus bypass according to Mueller or refixation of the popliteus tendon.
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Transient ablation of regulatory T cells improves antitumor immunity in colitis-associated colon cancer.
Cancer Res.
PUBLISHED: 06-06-2014
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Regulatory T cells (Treg) are supportive to cancer development in most tissues, but their role in colitis-associated colon cancer (CAC) remains unclear. In this study, we investigated the role of CD4(+)Foxp3(+) Treg in a mouse model of CAC and in patients with colon cancer. These Treg were increased strongly in number in a mouse model of CAC and in the peripheral blood of patients with colon cancer, exhibiting an activated phenotype as defined by elevated expression of GARP, CD103, CTLA-4, and IL10, along with an increased suppressive effect on the proliferation and Th1 cytokine expression of CD4(+)CD25(-) responder T cells ex vivo. Transient ablation of CD4(+)Foxp3(+) Treg during tumor development in the CAC model suppressed tumor outgrowth and distribution, accompanied by an increased number of CD8(+)IFN?/granzyme B-producing effector T cells. Conversely, inactivation of IL10 in Treg did not elevate the antitumor response but instead further boosted tumor development. Our results establish a tumor-promoting function for Treg during CAC formation, but they also suggest that a selective, transient ablation of Treg can evoke antitumor responses, with implications for immunotherapeutic interventions in patients with CAC.
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Glycoprotein 130 receptor signaling mediates ?-cell dysfunction in a rodent model of type 2 diabetes.
Diabetes
PUBLISHED: 05-08-2014
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Dysregulated glucagon secretion accompanies islet inflammation in type 2 diabetes. We recently discovered that interleukin (IL)-6 stimulates glucagon secretion from human and rodent islets. IL-6 family cytokines require the glycoprotein 130 (gp130) receptor to signal. In this study, we elucidated the effects of ?-cell gp130 receptor signaling on glycemic control in type 2 diabetes. IL-6 family cytokines were elevated in islets in rodent models of this disease. gp130 receptor activation increased STAT3 phosphorylation in primary ?-cells and stimulated glucagon secretion. Pancreatic ?-cell gp130 knockout (?gp130KO) mice showed no differences in glycemic control, ?-cell function, or ?-cell mass. However, when subjected to streptozotocin plus high-fat diet to induce islet inflammation and pathophysiology modeling type 2 diabetes, ?gp130KO mice had reduced fasting glycemia, improved glucose tolerance, reduced fasting insulin, and improved ?-cell function. Hyperinsulinemic-euglycemic clamps revealed no differences in insulin sensitivity. We conclude that in a setting of islet inflammation and pathophysiology modeling type 2 diabetes, activation of ?-cell gp130 receptor signaling has deleterious effects on ?-cell function, promoting hyperglycemia. Antagonism of ?-cell gp130 receptor signaling may be useful for the treatment of type 2 diabetes.
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Enzyme-based biosilica and biocalcite: biomaterials for the future in regenerative medicine.
Trends Biotechnol.
PUBLISHED: 05-07-2014
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The oldest animals on Earth, sponges, form both the calcareous and the siliceous matrices of their spicules enzymatically. Until recently, it has been neglected that enzymes play crucial roles during formation of these biominerals. This paradigm shift occurred after the discovery that the enzyme silicatein, which catalyzes the polycondensation of silica, and the enzyme carbonic anhydrase (CA), which catalyzes the formation of bicarbonate (HCO3(-)/CaCO3), produce solid amorphous bioglass or biocalcite. This suggests that in mammals, biosilica and biocalcite can act anabolically during hydroxyapatite (HA) synthesis and bone formation. Biosilica and biocalcite are thus promising candidates for the fabrication of biomaterials for regenerative medicine.
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Engineering a morphogenetically active hydrogel for bioprinting of bioartificial tissue derived from human osteoblast-like SaOS-2 cells.
Biomaterials
PUBLISHED: 05-02-2014
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Sodium alginate hydrogel, stabilized with gelatin, is a suitable, biologically inert matrix that can be used for encapsulating and 3D bioprinting of bone-related SaOS-2 cells. However, the cells, embedded in this matrix, remain in a non-proliferating state. Here we show that addition of an overlay onto the bioprinted alginate/gelatine/SaOS-2 cell scaffold, consisting of agarose and the calcium salt of polyphosphate [polyP·Ca(2+)-complex], resulted in a marked increase in cell proliferation. In the presence of 100 ?m polyP·Ca(2+)-complex, the cells proliferate with a generation time of approximately 47-55 h. In addition, the hardness of the alginate/gelatin hydrogel substantially increases in the presence of the polymer. The reduced Young's modulus for the alginate/gelatin hydrogel is approximately 13-14 kPa, and this value drops to approximately 0.5 kPa after incubation of the cell containing scaffolds for 5 d. In the presence of 100 ?m polyP·Ca(2+)-complex, the reduced Young's modulus increases to about 22 kPa. The hardness of the polyP·Ca(2+)-complex containing hydrogel remains essentially constant if cells are absent in the matrix, but it drops to 3.2 kPa after a 5 d incubation period in the presence of SaOS-2 cells, indicating that polyP·Ca(2+)-complex becomes metabolized, degraded, by the cells. The alginate/gelatine-agarose system with polyP·Ca(2+)-complex cause a significant increase in the mineralization of the cells. SEM analyses revealed that the morphology of the mineral nodules formed on the surface of the cells embedded in the alginate/gelatin hydrogel do not significantly differ from the nodules on cells growing in monolayer cultures. The newly developed technique, using cells encapsulated into an alginate/gelatin hydrogel and a secondary layer containing the morphogenetically active, growth promoting polymer polyP·Ca(2+)-complex opens new possibilities for the application of 3D bioprinting in bone tissue engineering.
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Malaria parasite infection compromises control of concurrent systemic non-typhoidal Salmonella infection via IL-10-mediated alteration of myeloid cell function.
PLoS Pathog.
PUBLISHED: 05-01-2014
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Non-typhoidal Salmonella serotypes (NTS) cause a self-limited gastroenteritis in immunocompetent individuals, while children with severe Plasmodium falciparum malaria can develop a life-threatening disseminated infection. This co-infection is a major source of child mortality in sub-Saharan Africa. However, the mechanisms by which malaria contributes to increased risk of NTS bacteremia are incompletely understood. Here, we report that in a mouse co-infection model, malaria parasite infection blunts inflammatory responses to NTS, leading to decreased inflammatory pathology and increased systemic bacterial colonization. Blunting of NTS-induced inflammatory responses required induction of IL-10 by the parasites. In the absence of malaria parasite infection, administration of recombinant IL-10 together with induction of anemia had an additive effect on systemic bacterial colonization. Mice that were conditionally deficient for either myeloid cell IL-10 production or myeloid cell expression of IL-10 receptor were better able to control systemic Salmonella infection, suggesting that phagocytic cells are both producers and targets of malaria parasite-induced IL-10. Thus, IL-10 produced during the immune response to malaria increases susceptibility to disseminated NTS infection by suppressing the ability of myeloid cells, most likely macrophages, to control bacterial infection.
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Biosilica-loaded poly(?-caprolactone) nanofibers mats provide a morphogenetically active surface scaffold for the growth and mineralization of the osteoclast-related SaOS-2 cells.
Biotechnol J
PUBLISHED: 04-27-2014
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Bioprinting/3D cell printing procedures for the preparation of scaffolds/implants have the potential to revolutionize regenerative medicine. Besides biocompatibility and biodegradability, the hardness of the scaffold material is of critical importance to allow sufficient mechanical protection and, to the same extent, allow migration, cell-cell, and cell-substrate contact formation of the matrix-embedded cells. In the present study, we present a strategy to encase a bioprinted, cell-containing, and soft scaffold with an electrospun mat. The electrospun poly(?-caprolactone) (PCL) nanofibers mats, containing tetraethyl orthosilicate (TEOS), were subsequently incubated with silicatein. Silicatein synthesizes polymeric biosilica by polycondensation of ortho-silicate that is formed from prehydrolyzed TEOS. Biosilica provides a morphogenetically active matrix for the growth and mineralization of osteoblast-related SaOS-2 cells in vitro. Analysis of the microstructure of the 300-700 nm thick PCL/TEOS nanofibers, incubated with silicatein and prehydrolyzed TEOS, displayed biosilica deposits on the mats formed by the nanofibers. We conclude and propose that electrospun PCL nanofibers mats, coated with biosilica, may represent a morphogenetically active and protective cover for bioprinted cell/tissue-like units with a suitable mechanical stability, even if the cells are embedded in a softer matrix.
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Bioactive and biodegradable silica biomaterial for bone regeneration.
Bone
PUBLISHED: 04-15-2014
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Biosilica, a biocompatible, natural inorganic polymer that is formed by an enzymatic, silicatein-mediated reaction in siliceous sponges to build up their inorganic skeleton, has been shown to be morphogenetically active and to induce mineralization of human osteoblast-like cells (SaOS-2) in vitro. In the present study, we prepared beads (microspheres) by encapsulation of ?-tricalcium phosphate [?-TCP], either alone (control) or supplemented with silica or silicatein, into the biodegradable copolymer poly(d,l-lactide-co-glycolide) [PLGA]. Under the conditions used, ?5% ?-TCP, ?9% silica, and 0.32?g/mg of silicatein were entrapped into the PLGA microspheres (diameter?800?m). Determination of the biocompatibility of the ?-TCP microspheres, supplemented with silica or silicatein, revealed no toxicity in the MTT based cell viability assay using SaOS-2 cells. The adherence of SaOS-2 cells to the surface of silica-containing microspheres was higher than for microspheres, containing only ?-TCP. In addition, the silica-containing ?-TCP microspheres and even more pronounced, a 1:1 mixture of microspheres containing ?-TCP and silica, and ?-TCP and silicatein, were found to strongly enhance the mineral deposition by SaOS-2 cells. Using these microspheres, first animal experiments with silica/biosilica were performed in female, adult New Zealand White rabbits to study the effect of the inorganic polymer on bone regeneration in vivo. The microspheres were implanted into 5mm thick holes, drilled into the femur of the animals, applying a bilateral comparison study design (3 test groups with 4-8 animals each). The control implant on one of the two hind legs contained microspheres with only ?-TCP, while the test implant on the corresponding leg consisted either of microspheres containing ?-TCP and silica, or a 1:1 mixture of microspheres, supplemented with ?-TCP and silica, and ?-TCP and silicatein. The results revealed that tissue/bone sections of silica containing implants and implants, composed of a 1:1 mixture of silica-containing microspheres and silicatein-containing microspheres, show an enhanced regeneration of bone tissue around the microspheres, compared to the control implants containing only ?-TCP. The formation of new bone induced by the microspheres is also evident from measurements of the stiffness/reduced Young's modulus of the regenerated bone tissue. The reduced Young's modulus of the regenerating bone tissue around the implants was markedly higher for the silica-containing microspheres (1.1MPa), and even more for the 1:1 mixture of the silica- and silicatein-containing microspheres (1.4MPa), compared to the ?-TCP microsphere controls (0.4MPa). We propose that based on their morphogenetic activity on bone-forming cells in vitro and the results of the animal experiments presented here, silica/biosilica-based scaffolds are promising materials for bone repair/regeneration.
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Macrophage-restricted interleukin-10 receptor deficiency, but not IL-10 deficiency, causes severe spontaneous colitis.
Immunity
PUBLISHED: 03-18-2014
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Interleukin-10 (IL-10) is a pleiotropic anti-inflammatory cytokine produced and sensed by most hematopoietic cells. Genome-wide association studies and experimental animal models point at a central role of the IL-10 axis in inflammatory bowel diseases. Here we investigated the importance of intestinal macrophage production of IL-10 and their IL-10 exposure, as well as the existence of an IL-10-based autocrine regulatory loop in the gut. Specifically, we generated mice harboring IL-10 or IL-10 receptor (IL-10R?) mutations in intestinal lamina propria-resident chemokine receptor CX3CR1-expressing macrophages. We found macrophage-derived IL-10 dispensable for gut homeostasis and maintenance of colonic T regulatory cells. In contrast, loss of IL-10 receptor expression impaired the critical conditioning of these monocyte-derived macrophages and resulted in spontaneous development of severe colitis. Collectively, our results highlight IL-10 as a critical homeostatic macrophage-conditioning agent in the colon and define intestinal CX3CR1(hi) macrophages as a decisive factor that determines gut health or inflammation.
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Interleukin-10 receptor signaling in innate immune cells regulates mucosal immune tolerance and anti-inflammatory macrophage function.
Immunity
PUBLISHED: 03-18-2014
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Intact interleukin-10 receptor (IL-10R) signaling on effector and T regulatory (Treg) cells are each independently required to maintain immune tolerance. Here we show that IL-10 sensing by innate immune cells, independent of its effects on T cells, was critical for regulating mucosal homeostasis. Following wild-type (WT) CD4(+) T cell transfer, Rag2(-/-)Il10rb(-/-) mice developed severe colitis in association with profound defects in generation and function of Treg cells. Moreover, loss of IL-10R signaling impaired the generation and function of anti-inflammatory intestinal and bone-marrow-derived macrophages and their ability to secrete IL-10. Importantly, transfer of WT but not Il10rb(-/-) anti-inflammatory macrophages ameliorated colitis induction by WT CD4(+) T cells in Rag2(-/-)Il10rb(-/-) mice. Similar alterations in the generation and function of anti-inflammatory macrophages were observed in IL-10R-deficient patients with very early onset inflammatory bowel disease. Collectively, our studies define innate immune IL-10R signaling as a key factor regulating mucosal immune homeostasis in mice and humans.
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Characterization and osteogenic activity of a silicatein/biosilica-coated chitosan-graft-polycaprolactone.
Acta Biomater
PUBLISHED: 03-17-2014
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Several attempts have been made in the past to fabricate hybrid materials that display the complementary properties of the polyester polycaprolactone (PCL) and the polysaccharide chitosan (CHS) for application in the field of bone regeneration and tissue engineering. However, such composites generally have no osteogenic activity per se. Here we report the synthesis of a chitosan-graft-polycaprolactone (CHS-g-PCL) and its subsequent characterization, including crystallinity, chemical structure and thermal stability. Upon surface-functionalization of CHS-g-PCL with osteogenic biosilica via the surface-immobilized enzyme silicatein, protein adsorption, surface morphology and wettability were assessed. Finally, the cultivation of osteoblastic SaOS-2 cells on the surface-functionalized CHS-g-PCL was followed by analyses of cell viability, mineral deposition and alkaline phosphatase activity. These characterizations revealed a composite that combines the versatile properties of CHS-g-PCL with the osteogenic activity of the silicatein/biosilica coating and, hence, represents an innovative alternative to conventionally used CHS/PCL composites for biomedical applications, where stable bone-material interfaces are required.
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Regulatory T cells and T-cell-derived IL-10 interfere with effective anti-cytomegalovirus immune response.
Immunol. Cell Biol.
PUBLISHED: 03-05-2014
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Cytomegalovirus (CMV) establishes lifelong chronic infection in its host with mostly asymptomatic or only mild disease, but under immunosuppressive conditions the virus can reactivate and infection can cause life-threatening disease. CMV has evolved several mechanisms to escape from host's immunity, allowing persistence of the virus. Until now, it remains elusive whether regulatory T cells (Tregs) have an impact on insufficient host immune response against the virus in this context. In the present study, we provide evidence that CD4(+)Foxp3(+) naturally occurring Tregs (nTregs) as well as CD4(+)Foxp3(-)IL-10(+)-induced Tregs (iTregs) interfere with an effective anti-mouse CMV (mCMV) immune response. Depletion of Foxp3(+) Tregs by using DEREG mice resulted in enhanced T-cell activation as measured by the expression of CD62L, granzyme B and interferon (IFN)-? and was associated with reduced viral titers in salivary glands, the organ where mCMV mainly persists. Moreover, we identified CD4(+)Foxp3(-) T cells to produce elevated levels of the immunosuppressive cytokine IL-10 at early time points during mCMV infection. Analysis of T-cell activation and viral replication in mCMV-infected IL-10(flox/flox) × CD4-cre mice and IL-10(flox/flox) × FIC mice revealed that T-cell-specific inactivation of IL-10, but not Foxp3(+) Treg-specific IL-10 ablation alone, resulted in elevated IFN-? production by T cells associated with a significant decrease in viral loads in salivary glands. Thus, our data illustrate a crucial role for CD4(+)Foxp3(+) nTregs as well as IL-10-producing CD4(+)Foxp3(-) iTregs in the regulation of appropriate T-cell responses and viral clearance during mCMV infection.Immunology and Cell Biology advance online publication, 29 July 2014; doi:10.1038/icb.2014.62.
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Extracellular Vesicles from Neural Stem Cells Transfer IFN-? via Ifngr1 to Activate Stat1 Signaling in Target Cells.
Mol. Cell
PUBLISHED: 02-25-2014
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The idea that stem cell therapies work only via cell replacement is challenged by the observation of consistent intercellular molecule exchange between the graft and the host. Here we defined a mechanism of cellular signaling by which neural stem/precursor cells (NPCs) communicate with the microenvironment via extracellular vesicles (EVs), and we elucidated its molecular signature and function. We observed cytokine-regulated pathways that sort proteins and mRNAs into EVs. We described induction of interferon gamma (IFN-?) pathway in NPCs exposed to proinflammatory cytokines that is mirrored in EVs. We showed that IFN-? bound to EVs through Ifngr1 activates Stat1 in target cells. Finally, we demonstrated that endogenous Stat1 and Ifngr1 in target cells are indispensable to sustain the activation of Stat1 signaling by EV-associated IFN-?/Ifngr1 complexes. Our study identifies a mechanism of cellular signaling regulated by EV-associated IFN-?/Ifngr1 complexes, which grafted stem cells may use to communicate with the host immune system.
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Isoquercitrin and polyphosphate co-enhance mineralization of human osteoblast-like SaOS-2 cells via separate activation of two RUNX2 cofactors AFT6 and Ets1.
Biochem. Pharmacol.
PUBLISHED: 02-25-2014
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Isoquercitrin, a dietary phytoestrogen, is a potential stimulator of bone mineralization used for prophylaxis of osteoporotic disorders. Here we studied the combined effects of isoquercitrin, a cell membrane permeable 3-O-glucoside of quercetin, and polyphosphate [polyP], a naturally occurring inorganic polymer inducing bone formation, on mineralization of human osteoblast-like SaOS-2 cells. Both compounds isoquercitrin and polyP induce at non-toxic concentrations the mineralization process of SaOS-2 cells. Co-incubation experiments revealed that isoquercitrin (at 0.1 and 0.3?M), if given simultaneously with polyP (as Ca(2+) salt; at 3, 10, 30 and 100?M) amplifies the mineralization-enhancing effect of the inorganic polymer. The biomineralization process induced by isoquercitrin and polyP is based on two different modes of action. After incubation of the cells with isoquercitrin or polyP the expression of the Runt-related transcription factor 2 [RUNX2] is significantly upregulated. In addition, isoquercitrin causes a strong increase of the steady-state-levels of the two co-activators of RUNX2, the activating transcription factor 6 [ATF6] and the Ets oncogene homolog 1 [Ets1]. The activating effect of isoquercitrin occurs via a signal transduction pathway involving ATF6, and by that, is independent from the induction cascade initiated by polyP. This conclusion is supported by the finding that isoquercitrin upregulates the expression of the gene encoding for osteocalcin, while polyP strongly increases the expression of the Ets1 gene and of the alkaline phosphatase. We show that the two compounds, polyP and isoquercitrin, have a co-enhancing effect on bone mineral formation and in turn might be of potential therapeutic value for prevention/treatment of osteoporosis.
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Effects of water-filtered infrared-A and of heat on cell death, inflammation, antioxidative potential and of free radical formation in viable skin--first results.
J. Photochem. Photobiol. B, Biol.
PUBLISHED: 02-18-2014
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The effects of water-filtered infrared-A (wIRA) and of convective heat on viability, inflammation, inducible free radicals and antioxidative power were investigated in natural and viable skin using the ex vivo Bovine Udder System (BUS) model. Therefore, skin samples from differently treated parts of the udder of a healthy cow were analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, by prostaglandin E2 (PGE2) measurement and by electron spin resonance (ESR) spectroscopy. Neither cell viability, the inflammation status, the radical status or the antioxidative defence systems of the skin were significantly affected by wIRA applied within 30 min by using an irradiance of 1900 W m(-2) which is of relevance for clinical use, but which exceeded the maximum solar IR-A irradiance at the Earth's surface more than 5 times and which resulted in a skin surface temperature of about 45 °C without cooling and of about 37 °C with convective cooling by air ventilation. No significant effects on viability and on inflammation were detected when convective heat was applied alone under equivalent conditions in terms of the resulting skin surface temperatures and exposure time. As compared with untreated skin, free radical formation was almost doubled, whereas the antioxidative power was reduced to about 50% after convective heating to about 45 °C.
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Protective mucosal immunity mediated by epithelial CD1d and IL-10.
Nature
PUBLISHED: 02-17-2014
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The mechanisms by which mucosal homeostasis is maintained are of central importance to inflammatory bowel disease. Critical to these processes is the intestinal epithelial cell (IEC), which regulates immune responses at the interface between the commensal microbiota and the host. CD1d presents self and microbial lipid antigens to natural killer T (NKT) cells, which are involved in the pathogenesis of colitis in animal models and human inflammatory bowel disease. As CD1d crosslinking on model IECs results in the production of the important regulatory cytokine interleukin (IL)-10 (ref. 9), decreased epithelial CD1d expression--as observed in inflammatory bowel disease--may contribute substantially to intestinal inflammation. Here we show in mice that whereas bone-marrow-derived CD1d signals contribute to NKT-cell-mediated intestinal inflammation, engagement of epithelial CD1d elicits protective effects through the activation of STAT3 and STAT3-dependent transcription of IL-10, heat shock protein 110 (HSP110; also known as HSP105), and CD1d itself. All of these epithelial elements are critically involved in controlling CD1d-mediated intestinal inflammation. This is demonstrated by severe NKT-cell-mediated colitis upon IEC-specific deletion of IL-10, CD1d, and its critical regulator microsomal triglyceride transfer protein (MTP), as well as deletion of HSP110 in the radioresistant compartment. Our studies thus uncover a novel pathway of IEC-dependent regulation of mucosal homeostasis and highlight a critical role of IL-10 in the intestinal epithelium, with broad implications for diseases such as inflammatory bowel disease.
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A modified version of the combined in-diffusion/abrasive peeling technique for measuring diffusion of strongly sorbing radionuclides in argillaceous rocks: a test study on the diffusion of caesium in Opalinus Clay.
Appl Radiat Isot
PUBLISHED: 02-06-2014
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A filter free diffusion set-up was developed for measuring the diffusion of strongly sorbing radionuclides in indurated argillaceous rocks such as Opalinus Clay (OPA) that normally disintegrate when contacted with a solution. Small bore cores drilled parallel to the bedding plane and embedded in epoxy resin were found to be stable and could be used for performing in-diffusion measurements. The method was tested with the diffusion of caesium, spiked with caesium-134, in Opalinus Clay. The profile of Cs in the clay sample was determined with a modified version of the abrasive peeling technique. The diffusion parameters obtained for caesium were in fair agreement with those determined earlier using the classical through-diffusion technique where stainless steel filters were used to confine the samples.
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Cytotoxic and protein kinase inhibiting nakijiquinones and nakijiquinols from the sponge Dactylospongia metachromia.
J. Nat. Prod.
PUBLISHED: 01-30-2014
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Chemical investigation of the sponge Dactylospongia metachromia afforded five new sesquiterpene aminoquinones (1-5), two new sesquiterpene benzoxazoles (6 and 7), the known analogue 18-hydroxy-5-epi-hyrtiophenol (8), and a known glycerolipid. The structures of all compounds were unambiguously elucidated by one- and two-dimensional NMR and by MS analyses, as well as by comparison with the literature. Compounds 1-5 showed potent cytotoxicity against the mouse lymphoma cell line L5178Y with IC50 values ranging from 1.1 to 3.7 ?M. When tested in vitro for their inhibitory potential against 16 different protein kinases, compounds 5, 6, and 8 exhibited the strongest inhibitory activity against ALK, FAK, IGF1-R, SRC, VEGF-R2, Aurora-B, MET wt, and NEK6 kinases (IC50 0.97-8.62 ?M).
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Efficacy of an abbreviated induction regimen of amphotericin B deoxycholate for cryptococcal meningoencephalitis: 3 days of therapy is equivalent to 14 days.
MBio
PUBLISHED: 01-30-2014
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Cryptococcal meningoencephalitis is an urgent global health problem. Induction regimens using 14 days of amphotericin B deoxycholate (dAmB) are considered the standard of care but may not be suitable for resource-poor settings. We investigated the efficacy of conventional and abbreviated regimens of dAmB for cryptococcal meningoencephalitis in both murine and rabbit models of cryptococcal meningoencephalitis. We examined the extent to which immunological effectors contribute to the antifungal effect. We bridged the results to humans as a first critical step to define regimens suitable for further study in clinical trials. There were significant differences in the murine plasma-versus-cerebrum dAmB concentration-time profiles. dAmB was detectable in the cerebrum throughout the experimental period, even following the administration of only three doses of 3 mg/kg. dAmB induced a fungistatic effect in the cerebrum with a 2- to 3-log10 CFU/g reduction compared with controls. The effect of 3 days of therapy was the same as that of daily therapy for 14 days. There was no evidence of increased numbers of CD3(+) CD4(+) or CD3(+) CD8(+) cells in treated mice to account for the persistent antifungal effect of an abbreviated regimen. The administration of dAmB at 1 mg/kg/day for 3 days was the same as daily therapy in rabbits. The bridging studies suggested that a human regimen of 0.7 mg/kg/day for 3 days resulted in nearly maximal antifungal activity in both the cerebrum and cerebrospinal fluid. An abbreviated regimen (3 days of therapy) of dAmB appears to be just as effective as conventional induction therapy for cryptococcal meningoencephalitis.
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Long-lasting significant functional improvement in chronic severe spinal cord injury following scar resection and polyethylene glycol implantation.
Neurobiol. Dis.
PUBLISHED: 01-28-2014
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We identified a suitable biomatrix that improved axon regeneration and functional outcome after partial (moderate) and complete (severe) chronic spinal cord injury (SCI) in rat. Five weeks after dorsal thoracic hemisection injury the lesion scar was resected via aspiration and the resulting cavity was filled with different biopolymers such as Matrigel™, alginate-hydrogel and polyethylene glycol 600 (PEG) all of which have not previously been used as sole graft-materials in chronic SCI. Immunohistological staining revealed marked differences between these compounds regarding axon regeneration, invasion/elongation of astrocytes, fibroblasts, endothelial and Schwann cells, revascularization, and collagen deposition. According to axon regeneration-supporting effects, the biopolymers could be ranked in the order PEG>alginate-hydrogel>Matrigel™. Even after complete chronic transection, the PEG-bridge allowed long-distance axon regeneration through the grafted area and for, at least, 1cm beyond the lesion/graft border. As revealed by electron microscopy, bundles of regenerating axons within the matrix area received myelin ensheathment from Schwann cells. The beneficial effects of PEG-implantation into the resection-cavity were accompanied by long-lasting significant locomotor improvement over a period of 8months. Following complete spinal re-transection at the rostral border of the PEG-graft the locomotor recovery was aborted, suggesting a functional role of regenerated axons in the initial locomotor improvement. In conclusion, scar resection and subsequent implantation of PEG into the generated cavity leads to tissue recovery, axon regeneration, myelination and functional improvement that have not been achieved before in severe chronic SCI.
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The marine sponge-derived inorganic polymers, biosilica and polyphosphate, as morphogenetically active matrices/scaffolds for the differentiation of human multipotent stromal cells: potential application in 3D printing and distraction osteogenesis.
Mar Drugs
PUBLISHED: 01-10-2014
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The two marine inorganic polymers, biosilica (BS), enzymatically synthesized from ortho-silicate, and polyphosphate (polyP), a likewise enzymatically synthesized polymer consisting of 10 to >100 phosphate residues linked by high-energy phosphoanhydride bonds, have previously been shown to display a morphogenetic effect on osteoblasts. In the present study, the effect of these polymers on the differential differentiation of human multipotent stromal cells (hMSC), mesenchymal stem cells, that had been encapsulated into beads of the biocompatible plant polymer alginate, was studied. The differentiation of the hMSCs in the alginate beads was directed either to the osteogenic cell lineage by exposure to an osteogenic medium (mineralization activation cocktail; differentiation into osteoblasts) or to the chondrogenic cell lineage by incubating in chondrocyte differentiation medium (triggering chondrocyte maturation). Both biosilica and polyP, applied as Ca²? salts, were found to induce an increased mineralization in osteogenic cells; these inorganic polymers display also morphogenetic potential. The effects were substantiated by gene expression studies, which revealed that biosilica and polyP strongly and significantly increase the expression of bone morphogenetic protein 2 (BMP-2) and alkaline phosphatase (ALP) in osteogenic cells, which was significantly more pronounced in osteogenic versus chondrogenic cells. A differential effect of the two polymers was seen on the expression of the two collagen types, I and II. While collagen Type I is highly expressed in osteogenic cells, but not in chondrogenic cells after exposure to biosilica or polyP, the upregulation of the steady-state level of collagen Type II transcripts in chondrogenic cells is comparably stronger than in osteogenic cells. It is concluded that the two polymers, biosilica and polyP, are morphogenetically active additives for the otherwise biologically inert alginate polymer. It is proposed that alginate, supplemented with polyP and/or biosilica, is a suitable biomaterial that promotes the growth and differentiation of hMSCs and might be beneficial for application in 3D tissue printing of hMSCs and for the delivery of hMSCs in fractures, surgically created during distraction osteogenesis.
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Effect of Bioglass on Growth and Biomineralization of SaOS-2 Cells in Hydrogel after 3D Cell Bioprinting.
PLoS ONE
PUBLISHED: 01-01-2014
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We investigated the effect of bioglass (bioactive glass) on growth and mineralization of bone-related SaOS-2 cells, encapsulated into a printable and biodegradable alginate/gelatine hydrogel. The hydrogel was supplemented either with polyphosphate (polyP), administered as polyP•Ca2+-complex, or silica, or as biosilica that had been enzymatically prepared from ortho-silicate by silicatein. These hydrogels, together with SaOS-2 cells, were bioprinted to computer-designed scaffolds. The results revealed that bioglass (nano)particles, with a size of 55 nm and a molar ratio of SiO2?CaO?P2O5 of 55?40?5, did not affect the growth of the encapsulated cells. If silica, biosilica, or polyP•Ca2+-complex is co-added to the cell-containing alginate/gelatin hydrogel the growth behavior of the cells is not changed. Addition of 5 mg/ml of bioglass particles to this hydrogel significantly enhanced the potency of the entrapped SaOS-2 cells to mineralize. If compared with the extent of the cells to form mineral deposits in the absence of bioglass, the cells exposed to bioglass together with 100 µmoles/L polyP•Ca2+-complex increased their mineralization activity from 2.1- to 3.9-fold, or with 50 µmoles/L silica from 1.8- to 2.9-fold, or with 50 µmoles/L biosilica from 2.7- to 4.8-fold or with the two components together (100 µmoles/L polyP•Ca2+-complex and 50 µmoles/L biosilica) from 4.1- to 6.8-fold. Element analysis by EDX spectrometry of the mineral nodules formed by SaOS-2 revealed an accumulation of O, P, Ca and C, indicating that the mineral deposits contain, besides Ca-phosphate also Ca-carbonate. The results show that bioglass added to alginate/gelatin hydrogel increases the proliferation and mineralization of bioprinted SaOS-2 cells. We conclude that the development of cell-containing scaffolds consisting of a bioprintable, solid and cell-compatible inner matrix surrounded by a printable hard and flexible outer matrix containing bioglass, provide a suitable strategy for the fabrication of morphogenetically active and biodegradable implants.
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Spinal cord injury - there is not just one way of treating it.
F1000Prime Rep
PUBLISHED: 01-01-2014
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In the last century, research in the field of spinal cord trauma has brought insightful knowledge which has led to a detailed understanding of mechanisms that are involved in injury- and recovery-related processes. The quest for a cure for the yet generally incurable condition as well as the exponential rise in gained information has brought about the development of numerous treatment approaches while at the same time the abundance of data has become quite unmanageable. Owing to an enormous amount of preclinical therapeutic approaches, this report highlights important trends rather than specific treatment strategies. We focus on current advances in the treatment of spinal cord injury and want to further draw attention to arising problems in spinal cord injury (SCI) research and discuss possible solutions.
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Biocalcite, a multifunctional inorganic polymer: Building block for calcareous sponge spicules and bioseed for the synthesis of calcium phosphate-based bone.
Beilstein J Nanotechnol
PUBLISHED: 01-01-2014
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Calcium carbonate is the material that builds up the spicules of the calcareous sponges. Recent results revealed that the calcium carbonate/biocalcite-based spicular skeleton of these animals is formed through an enzymatic mechanism, such as the skeleton of the siliceous sponges, evolutionarily the oldest animals that consist of biosilica. The enzyme that mediates the calcium carbonate deposition has been identified as a carbonic anhydrase (CA) and has been cloned from the calcareous sponge species Sycon raphanus. Calcium carbonate deposits are also found in vertebrate bones besides the main constituent, calcium phosphate/hydroxyapatite (HA). Evidence has been presented that during the initial phase of HA synthesis poorly crystalline carbonated apatite is deposited. Recent data summarized here indicate that during early bone formation calcium carbonate deposits enzymatically formed by CA, act as potential bioseeds for the precipitation of calcium phosphate mineral onto bone-forming osteoblasts. Two different calcium carbonate phases have been found during CA-driven enzymatic calcium carbonate deposition in in vitro assays: calcite crystals and round-shaped vaterite deposits. The CA provides a new target of potential anabolic agents for treatment of bone diseases; a first CA activator stimulating the CA-driven calcium carbonate deposition has been identified. In addition, the CA-driven calcium carbonate crystal formation can be frozen at the vaterite state in the presence of silintaphin-2, an aspartic acid/glutamic acid-rich sponge-specific protein. The discovery that calcium carbonate crystals act as bioseeds in human bone formation may allow the development of novel biomimetic scaffolds for bone tissue engineering. Na-alginate hydrogels, enriched with biosilica, have recently been demonstrated as a suitable matrix to embed bone forming cells for rapid prototyping bioprinting/3D cell printing applications.
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Cellular effects of bacterial N-3-Oxo-dodecanoyl-L-Homoserine lactone on the sponge Suberites domuncula (Olivi, 1792): insights into an intimate inter-kingdom dialogue.
PLoS ONE
PUBLISHED: 01-01-2014
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Sponges and bacteria have lived together in complex consortia for 700 million years. As filter feeders, sponges prey on bacteria. Nevertheless, some bacteria are associated with sponges in symbiotic relationships. To enable this association, sponges and bacteria are likely to have developed molecular communication systems. These may include molecules such as N-acyl-L-homoserine lactones, produced by Gram-negative bacteria also within sponges. In this study, we examined the role of N-3-oxododecanoyl-L-homoserine lactone (3-oxo-C12-HSL) on the expression of immune and apoptotic genes of the host sponge Suberites domuncula. This molecule seemed to inhibit the sponge innate immune system through a decrease of the expression of genes coding for proteins sensing the bacterial membrane: a Toll-Like Receptor and a Toll-like Receptor Associated Factor 6 and for an anti-bacterial perforin-like molecule. The expression of the pro-apoptotic caspase-like 3/7 gene decreased as well, whereas the level of mRNA of anti-apoptotic genes Bcl-2 Homolog Proteins did not change. Then, we demonstrated the differential expression of proteins in presence of this 3-oxo-C12-HSL using 3D sponge cell cultures. Proteins involved in the first steps of the endocytosis process were highlighted using the 2D electrophoresis protein separation and the MALDI-TOF/TOF protein characterization: ? and ? subunits of the lysosomal ATPase, a cognin, cofilins-related proteins and cytoskeleton proteins actin, ? tubulin and ? actinin. The genetic expression of some of these proteins was subsequently followed. We propose that the 3-oxo-C12-HSL may participate in the tolerance of the sponge apoptotic and immune systems towards the presence of bacteria. Besides, the sponge may sense the 3-oxo-C12-HSL as a molecular evidence of the bacterial presence and/or density in order to regulate the populations of symbiotic bacteria in the sponge. This study is the first report of a bacterial secreted molecule acting on sponge cells and regulating the symbiotic relationship.
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IFN-?-Mediated Induction of an Apical IL-10 Receptor on Polarized Intestinal Epithelia.
J. Immunol.
PUBLISHED: 12-23-2013
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Cytokines secreted at sites of inflammation impact the onset, progression, and resolution of inflammation. In this article, we investigated potential proresolving mechanisms of IFN-? in models of inflammatory bowel disease. Guided by initial microarray analysis, in vitro studies revealed that IFN-? selectively induced the expression of IL-10R1 on intestinal epithelia. Further analysis revealed that IL-10R1 was expressed predominantly on the apical membrane of polarized epithelial cells. Receptor activation functionally induced canonical IL-10 target gene expression in epithelia, concomitant with enhanced barrier restitution. Furthermore, knockdown of IL-10R1 in intestinal epithelial cells results in impaired barrier function in vitro. Colonic tissue isolated from murine colitis revealed that levels of IL-10R1 and suppressor of cytokine signaling 3 were increased in the epithelium and coincided with increased tissue IFN-? and IL-10 cytokines. In parallel, studies showed that treatment of mice with rIFN-? was sufficient to drive expression of IL-10R1 in the colonic epithelium. Studies of dextran sodium sulfate colitis in intestinal epithelial-specific IL-10R1-null mice revealed a remarkable increase in disease susceptibility associated with increased intestinal permeability. Together, these results provide novel insight into the crucial and underappreciated role of epithelial IL-10 signaling in the maintenance and restitution of epithelial barrier and of the temporal regulation of these pathways by IFN-?.
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Modulation of the Initial Mineralization Process of SaOS-2 Cells by Carbonic Anhydrase Activators and Polyphosphate.
Calcif. Tissue Int.
PUBLISHED: 11-04-2013
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Ca-phosphate/hydroxyapatite (HA) crystals constitute the mineral matrix of vertebrate bones, while Ca-carbonate is the predominant mineral of many invertebrates, like mollusks. Recent results suggest that CaCO3 is also synthesized during early bone formation. We demonstrate that carbonic anhydrase-driven CaCO3 formation in vitro is activated by organic extracts from the demosponge Suberites domuncula as well as by quinolinic acid, one component isolated from these extracts. Further results revealed that the stimulatory effect of bicarbonate (HCO3 (-)) ions on mineralization of osteoblast-like SaOS-2 cells is strongly enhanced if the cells are exposed to inorganic polyphosphate (polyP), a linear polymer of phosphate linked by energy-rich phosphodiester bonds. The effect of polyP, administered as polyP (Ca(2+) salt), on HA formation was found to be amplified by addition of the carbonic anhydrase-activating sponge extract or quinolinic acid. Our results support the assumption that CaCO3 deposits, acting as bio-seeds for Ca-carbonated phosphate formation, are formed as an intermediate during HA mineralization and that the carbonic anhydrase-mediated formation of those deposits is under a positive-negative feedback control by bone alkaline phosphatase-dependent polyP metabolism, offering new targets for therapy of bone diseases/defects.
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TGF-? signalling is required for CD4? T cell homeostasis but dispensable for regulatory T cell function.
PLoS Biol.
PUBLISHED: 10-01-2013
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TGF-? is widely held to be critical for the maintenance and function of regulatory T (T(reg)) cells and thus peripheral tolerance. This is highlighted by constitutive ablation of TGF-? receptor (TR) during thymic development in mice, which leads to a lethal autoimmune syndrome. Here we describe that TGF-?-driven peripheral tolerance is not regulated by TGF-? signalling on mature CD4? T cells. Inducible TR2 ablation specifically on CD4? T cells did not result in a lethal autoinflammation. Transfer of these TR2-deficient CD4? T cells to lymphopenic recipients resulted in colitis, but not overt autoimmunity. In contrast, thymic ablation of TR2 in combination with lymphopenia led to lethal multi-organ inflammation. Interestingly, deletion of TR2 on mature CD4? T cells does not result in the collapse of the T(reg) cell population as observed in constitutive models. Instead, a pronounced enlargement of both regulatory and effector memory T cell pools was observed. This expansion is cell-intrinsic and seems to be caused by increased T cell receptor sensitivity independently of common gamma chain-dependent cytokine signals. The expression of Foxp3 and other regulatory T cells markers was not dependent on TGF-? signalling and the TR2-deficient T(reg) cells retained their suppressive function both in vitro and in vivo. In summary, absence of TGF-? signalling on mature CD4? T cells is not responsible for breakdown of peripheral tolerance, but rather controls homeostasis of mature T cells in adult mice.
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Cytotoxic rocaglamide derivatives from Aglaia duppereana.
Z. Naturforsch., C, J. Biosci.
PUBLISHED: 09-27-2013
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Phytochemical investigation of Aglaia duppereana flowers led to the isolation of a new rocaglamide derivative and twelve known congeners. The structure of the new compound was unambiguously elucidated by spectroscopic techniques (1D- and 2D-NMR, HRESIMS). The isolated compounds exhibited a potent cytotoxic activity against mouse lymphoma (L5178Y) cells with EC50 values ranging from 5.1 to 54.8 nM.
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Neuroprotective intervention by interferon-? blockade prevents CD8+ T cell-mediated dendrite and synapse loss.
J. Exp. Med.
PUBLISHED: 09-02-2013
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Neurons are postmitotic and thus irreplaceable cells of the central nervous system (CNS). Accordingly, CNS inflammation with resulting neuronal damage can have devastating consequences. We investigated molecular mediators and structural consequences of CD8(+) T lymphocyte (CTL) attack on neurons in vivo. In a viral encephalitis model in mice, disease depended on CTL-derived interferon-? (IFN-?) and neuronal IFN-? signaling. Downstream STAT1 phosphorylation and nuclear translocation in neurons were associated with dendrite and synapse loss (deafferentation). Analogous molecular and structural alterations were also found in human Rasmussen encephalitis, a CTL-mediated human autoimmune disorder of the CNS. Importantly, therapeutic intervention by IFN-? blocking antibody prevented neuronal deafferentation and clinical disease without reducing CTL responses or CNS infiltration. These findings identify neuronal IFN-? signaling as a novel target for neuroprotective interventions in CTL-mediated CNS disease.
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Interferon-dependent IL-10 production by Tregs limits tumor Th17 inflammation.
J. Clin. Invest.
PUBLISHED: 08-06-2013
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The capacity of IL-10 and Tregs in the inflammatory tumor microenvironment to impair anticancer Th1 immunity makes them attractive targets for cancer immunotherapy. IL-10 and Tregs also suppress Th17 activity, which is associated with poor prognosis in several cancers. However, previous studies have overlooked their potential contribution to the regulation of pathogenic cancer-associated inflammation. In this study, we investigated the origin and function of IL-10-producing cells in the tumor microenvironment using transplantable tumor models in mice. The majority of tumor-associated IL-10 was produced by an activated Treg population. IL-10 production by Tregs was required to restrain Th17-type inflammation. Accumulation of activated IL-10+ Tregs in the tumor required type I IFN signaling but not inflammatory signaling pathways that depend on TLR adapter protein MyD88 or IL-12 family cytokines. IL-10 production limited Th17 cell numbers in both spleen and tumor. However, type I IFN was required to limit Th17 cells specifically in the tumor microenvironment, reflecting selective control of tumor-associated Tregs by type I IFN. Thus, the interplay of type I IFN, Tregs, and IL-10 is required to negatively regulate Th17 inflammation in the tumor microenvironment. Therapeutic interference of this network could therefore have the undesirable consequence of promoting Th17 inflammation and cancer growth.
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Cryptochrome in sponges: a key molecule linking photoreception with phototransduction.
J. Histochem. Cytochem.
PUBLISHED: 08-06-2013
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Sponges (phylum: Porifera) react to external light or mechanical signals with contractile or metabolic reactions and are devoid of any nervous or muscular system. Furthermore, elements of a photoreception/phototransduction system exist in those animals. Recently, a cryptochrome-based photoreceptor system has been discovered in the demosponge. The assumption that in sponges the siliceous skeleton acts as a substitution for the lack of a nervous system and allows light signals to be transmitted through its glass fiber network is supported by the findings that the first spicules are efficient light waveguides and the second sponges have the enzymatic machinery for the generation of light. Now, we have identified/cloned in Suberites domuncula two additional potential molecules of the sponge cryptochrome photoreception system, the guanine nucleotide-binding protein ? subunit, related to ?-transducin, and the nitric oxide synthase (NOS)-interacting protein. Cryptochrome and NOSIP are light-inducible genes. The studies show that the NOS inhibitor L-NMMA impairs both morphogenesis and motility of the cells. Finally, we report that the function of primmorphs to produce reactive nitrogen species can be abolished by a NOS inhibitor. We propose that the sponge cryptochrome-based photoreception system, through which photon signals are converted into radicals, is coupled to the NOS apparatus.
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Carbonic anhydrase: a key regulatory and detoxifying enzyme for Karst plants.
Planta
PUBLISHED: 07-17-2013
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Karstification is a rapid process during which calcidic stones/limestones undergo dissolution with the consequence of a desertification of karst regions. A slow-down of those dissolution processes of Ca-carbonate can be approached by a reforestation program using karst-resistant plants that can resist alkaline pH and higher bicarbonate (HCO3 (-)) concentrations in the soil. Carbonic anhydrases (CA) are enzymes that mediate a rapid and reversible interconversion of CO2 and HCO3 (-). In the present study, the steady-state expression of a CA gene, encoding for the plant carbonic anhydrase from the parsley Petroselinum crispum, is monitored. The studies were primarily been performed during germination of the seeds up to the 12/14-day-old embryos. The CA cDNA was cloned. Quantitative polymerase chain reaction (qPCR) analysis revealed that the gene expression level of the P. crispum CA is strongly and significantly affected at more alkaline pH in the growth medium (pH 8.3). This abolishing effect is counteracted both by addition of HCO3 (-) and by addition of polyphosphate (polyP) to the culture medium. In response to polyP, the increased pH in the vacuoles of the growing plants is normalized. The effect of polyP let us to propose that this polymer acts as a buffer system that facilitates the adjustment of the pH in the cytoplasm. In addition, it is proposed that polyP has the potential to act, especially in the karst, as a fertilizer that allows the karstic plants to cope with the adverse pH and HCO3 (-) condition in the soil.
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Bioactive polyketides and alkaloids from Penicillium citrinum, a fungal endophyte isolated from Ocimum tenuiflorum.
Fitoterapia
PUBLISHED: 07-12-2013
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Chemical investigation of the endophytic fungus Penicillium citrinum cultured on white beans or on rice led to the isolation of two new alkaloids (1 and 2), along with fourteen known polyketides (6-12, 14-20) and four known alkaloids (3-5, and 13). The structures of the isolated compounds were determined by extensive analysis of the 1D, 2D NMR, and MS data, and by comparison with the literature. Compound 13, which had been previously obtained only by chemical synthesis, was isolated as a natural product for the first time, while compound 6 was firstly reported as a fungal metabolite. A re-isolation of sclerotinin A (14) revealed it to be a diastereoisomeric mixture (14a and 14b), whose stereochemistry was proposed for the first time based on ROESY experiment. All isolated compounds were evaluated for their cytotoxic and antibacterial activities. Compounds 12 and 17 showed significant cytotoxicity against the murine lymphoma cell line L5178Y with IC50 values of 1.0, and 0.78 ?g/ml, respectively, while compounds 5, 11, and 15 were moderately active against Staphylococcus aureus ATCC 29213 (MIC 64 ?g/ml).
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Induction of carbonic anhydrase in SaOS-2 cells, exposed to bicarbonate and consequences for calcium phosphate crystal formation.
Biomaterials
PUBLISHED: 07-11-2013
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Ca-phosphate/hydroxyapatite crystals constitute the mineralic matrix of vertebrate bones, while Ca-carbonate dominates the inorganic matrix of otoliths. In addition, Ca-carbonate has been identified in lower percentage in apatite crystals. By using the human osteogenic SaOS-2 cells it could be shown that after exposure of the cells to Ca-bicarbonate in vitro, at concentrations between 1 and 10 mm, a significant increase of Ca-deposit formation results. The crystallite nodules formed on the surfaces of SaOS-2 cells become denser and larger in the presence of bicarbonate if simultaneously added together with the mineralization activation cocktail (?-glycerophosphate/ascorbic acid/dexamethasone). In parallel, with the increase of Ca-deposit formation, the expression of the carbonic anhydrase-II (CA-II) gene becomes upregulated. This effect, measured on transcriptional level is also substantiated by immunohistological studies. The stimulatory effect of bicarbonate on Ca-deposit formation is prevented if the carbonic anhydrase inhibitor acetazolamide is added to the cultures. Mapping the surface of the Ca-deposit producing SaOS-2 cells by scanning electron microscopy coupled with energy-dispersive X-ray analysis revealed an accumulation of the signals for the element carbon and, as expected, also for phosphorus. Finally, it is shown that ortho-phosphate and hydrolysis products of polyphosphate inhibit CA-II activity, suggesting a feedback regulatory system between the CA-driven Ca-carbonate deposition and a subsequent inactivation of this process by ortho-phosphate. Based on the presented data we suggest that Ca-carbonate deposits act as bioseeds for a downstream Ca-phosphate deposition process. We propose that activators for CA, especially for CA-II, might be beneficial for the treatment of bone deficiency diseases.
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Monocyte-derived dendritic cells perform hemophagocytosis to fine-tune excessive immune responses.
Immunity
PUBLISHED: 06-11-2013
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Because immune responses simultaneously defend and injure the host, the immune system must be finely regulated to ensure the hosts survival. Here, we have shown that when injected with high Toll-like receptor ligand doses or infected with lymphocytic choriomeningitis virus (LCMV) clone 13, which has a high viral turnover, inflammatory monocyte-derived dendritic cells (Mo-DCs) engulfed apoptotic erythroid cells. In this process, called hemophagocytosis, phosphatidylserine (PS) served as an "eat-me" signal. Type I interferons were necessary for both PS exposure on erythroid cells and the expression of PS receptors in the Mo-DCs. Importantly, hemophagocytosis was required for interleukin-10 (IL-10) production from Mo-DCs. Blocking hemophagocytosis or Mo-DC-derived IL-10 significantly increased cytotoxic T cell lymphocyte activity, tissue damage, and mortality in virus-infected hosts, suggesting that hemophagocytosis moderates immune responses to ensure the hosts survival in vivo. This sheds light on the physiological relevance of hemophagocytosis in severe inflammatory and infectious diseases.
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T cell-derived IL-10 determines leishmaniasis disease outcome and is suppressed by a dendritic cell based vaccine.
PLoS Pathog.
PUBLISHED: 06-01-2013
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In the murine model of Leishmania major infection, resistance or susceptibility to the parasite has been associated with the development of a Th1 or Th2 type of immune response. Recently, however, the immunosuppressive effects of IL-10 have been ascribed a crucial role in the development of the different clinical correlates of Leishmania infection in humans. Since T cells and professional APC are important cellular sources of IL-10, we compared leishmaniasis disease progression in T cell-specific, macrophage/neutrophil-specific and complete IL-10-deficient C57BL/6 as well as T cell-specific and complete IL-10-deficient BALB/c mice. As early as two weeks after infection of these mice with L. major, T cell-specific and complete IL-10-deficient animals showed significantly increased lesion development accompanied by a markedly elevated secretion of IFN-? or IFN-? and IL-4 in the lymph nodes draining the lesions of the C57BL/6 or BALB/c mutants, respectively. In contrast, macrophage/neutrophil-specific IL-10-deficient C57BL/6 mice did not show any altered phenotype. During the further course of disease, the T cell-specific as well as the complete IL-10-deficient BALB/c mice were able to control the infection. Furthermore, a dendritic cell-based vaccination against leishmaniasis efficiently suppresses the early secretion of IL-10, thus contributing to the control of parasite spread. Taken together, IL-10 secretion by T cells has an influence on immune activation early after infection and is sufficient to render BALB/c mice susceptible to an uncontrolled Leishmania major infection.
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CD4+ T cell-derived IL-10 promotes Brucella abortus persistence via modulation of macrophage function.
PLoS Pathog.
PUBLISHED: 06-01-2013
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Evasion of host immune responses is a prerequisite for chronic bacterial diseases; however, the underlying mechanisms are not fully understood. Here, we show that the persistent intracellular pathogen Brucella abortus prevents immune activation of macrophages by inducing CD4(+)CD25(+) T cells to produce the anti-inflammatory cytokine interleukin-10 (IL-10) early during infection. IL-10 receptor (IL-10R) blockage in macrophages resulted in significantly higher NF-kB activation as well as decreased bacterial intracellular survival associated with an inability of B. abortus to escape the late endosome compartment in vitro. Moreover, either a lack of IL-10 production by T cells or a lack of macrophage responsiveness to this cytokine resulted in an increased ability of mice to control B. abortus infection, while inducing elevated production of pro-inflammatory cytokines, which led to severe pathology in liver and spleen of infected mice. Collectively, our results suggest that early IL-10 production by CD25(+)CD4(+) T cells modulates macrophage function and contributes to an initial balance between pro-inflammatory and anti-inflammatory cytokines that is beneficial to the pathogen, thereby promoting enhanced bacterial survival and persistent infection.
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Enzyme-accelerated and structure-guided crystallization of calcium carbonate: Role of the carbonic anhydrase in the homologous system.
Acta Biomater
PUBLISHED: 05-13-2013
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The calcareous spicules from sponges, e.g. from Sycon raphanus, are composed of almost pure calcium carbonate. In order to elucidate the formation of those structural skeletal elements, the function of the enzyme carbonic anhydrase (CA), isolated from this species, during the in vitro calcium carbonate-based spicule formation, was investigated. It is shown that the recombinant sponge CA substantially accelerates calcium carbonate formation in the in vitro diffusion assay. A stoichiometric calculation revealed that the turnover rate of the sponge CA during the calcification process amounts to 25 CO2s(-1)×molecule CA(-1). During this enzymatically driven process, initially pat-like particles are formed that are subsequently transformed to rhomboid/rhombohedroid crystals with a dimension of ?50?m. The CA-catalyzed particles are smaller than those which are formed in the absence of the enzyme. The Martens hardness of the particles formed is ?4GPa, a value which had been determined for other biogenic calcites. This conclusion is corroborated by energy-dispersive X-ray spectroscopy, which revealed that the particles synthesized are composed predominantly of the elements calcium, oxygen and carbon. Surprising was the finding, obtained by light and scanning electron microscopy, that the newly formed calcitic crystals associate with the calcareous spicules from S. raphanus in a highly ordered manner; the calcitic crystals almost perfectly arrange in an array orientation along the two opposing planes of the spicules, leaving the other two plane arrays uncovered. It is concluded that the CA is a key enzyme controlling the calcium carbonate biomineralization process, which directs the newly formed particles to existing calcareous spicular structures. It is expected that with the given tools new bioinspired materials can be fabricated.
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Silicateins--a novel paradigm in bioinorganic chemistry: enzymatic synthesis of inorganic polymeric silica.
Chemistry
PUBLISHED: 03-19-2013
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The inorganic matrix of the siliceous skeletal elements of sponges, that is, spicules, is formed of amorphous biosilica. Until a decade ago, it remained unclear how the hard biosilica monoliths of the spicules are formed in sponges that live in a silica-poor (<50??M) aquatic environment. The following two discoveries caused a paradigm shift and allowed an elucidation of the processes underlying spicule formation; first the discovery that in the spicules only one major protein, silicatein, exists and second, that this protein displays a bio-catalytical, enzymatic function. These findings caused a paradigm shift, since silicatein is the first enzyme that catalyzes the formation of an inorganic polymer from an inorganic monomeric substrate. In the present review the successive steps, following the synthesis of the silicatein product, biosilica, and resulting in the formation of the hard monolithic spicules is given. The new insight is assumed to open new horizons in the field of biotechnology and also in biomedicine.
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A comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N mouse strains.
Genome Biol.
PUBLISHED: 03-18-2013
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The mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as the International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms.
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Flexible minerals: self-assembled calcite spicules with extreme bending strength.
Science
PUBLISHED: 03-16-2013
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Silicatein-? is responsible for the biomineralization of silicates in sponges. We used silicatein-? to guide the self-assembly of calcite "spicules" similar to the spicules of the calcareous sponge Sycon sp. The self-assembled spicules, 10 to 300 micrometers (?m) in length and 5 to 10 ?m in diameter, are composed of aligned calcite nanocrystals. The spicules are initially amorphous but transform into calcite within months, exhibiting unusual growth along [100]. They scatter x-rays like twinned calcite crystals. Whereas natural spicules evidence brittle failure, the synthetic spicules show an elastic response, which greatly enhances bending strength. This remarkable feature is linked to a high protein content. With nano-thermogravimetric analysis, we measured the organic content of a single spicule to be 10 to 16%. In addition, the spicules exhibit waveguiding properties even when they are bent.
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T-cell-derived, but not B-cell-derived, IL-10 suppresses antigen-specific T-cell responses in Litomosoides sigmodontis-infected mice.
Eur. J. Immunol.
PUBLISHED: 03-13-2013
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IL-10, a cytokine with pleiotropic functions is produced by many different cells. Although IL-10 may be crucial for initiating protective Th2 responses to helminth infection, it may also function as a suppressive cytokine preventing immune pathology or even contributing to helminth-induced immune evasion. Here, we show that B cells and T cells produce IL-10 during murine Litomosoides sigmodontis infection. IL-10-deficient mice produced increased amounts of L. sigmodontis-specific IFN-? and IL-13 suggesting a suppressive role for IL-10 in the initiation of the T-cell response to infection. Using cell type-specific IL-10-deficient mice, we dissected different functions of T-cell- and B-cell-derived IL-10. Litomosoides sigmodontis-specific IFN-?, IL-5, and IL-13 production increased in the absence of T-cell-derived IL-10 at early and late time points of infection. In contrast, B-cell-specific IL-10 deficiency did not lead to significant changes in L. sigmodontis-specific cytokine production compared to WT mice. Our results suggest that the initiation of Ag-specific cellular responses during L. sigmodontis infection is suppressed by T-cell-derived IL-10 and not by B-cell-derived IL-10.
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Principles of biofouling protection in marine sponges: a model for the design of novel biomimetic and bio-inspired coatings in the marine environment?
Mar. Biotechnol.
PUBLISHED: 03-09-2013
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The process of biofouling of marine structures and substrates, such as platforms or ship hulls, proceeds in multiple steps. Soon after the formation of an initial conditioning film, formed via the adsorption of organic particles to natural or man-made substrates, a population of different bacterial taxa associates under the formation of a biofilm. These microorganisms communicate through a complex quorum sensing network. Macro-foulers, e.g., barnacles, then settle and form a fouling layer on the marine surfaces, a process that globally has severe impacts both on the economy and on the environment. Since the ban of tributyltin, an efficient replacement of this antifouling compound by next-generation antifouling coatings that are environmentally more acceptable and also showing longer half-lives has not yet been developed. The sponges, as sessile filter-feeder animals, have evolved antifouling strategies to protect themselves against micro- and subsequent macro-biofouling processes. Experimental data are summarized and suggest that coating of the sponge surface with bio-silica contributes to the inhibition of the formation of a conditioning film. A direct adsorption of the surfaces by microorganisms can be impaired through poisoning the organisms with direct-acting secondary metabolites or toxic peptides. In addition, first, compounds from sponges have been identified that interfere with the anti-quorum sensing network. Sponge secondary metabolites acting selectively on diatom colonization have not yet been identified. Finally, it is outlined that direct-acting secondary metabolites inhibiting the growth of macro-fouling animals and those that poison the multidrug resistance pump are available. It is concluded that rational screening programs for inhibitors of the complex and dynamic problem of biofilm production, based on multidisciplinary studies and using sponges as a model, are required in the future.
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Metazoan circadian rhythm: toward an understanding of a light-based zeitgeber in sponges.
Integr. Comp. Biol.
PUBLISHED: 03-08-2013
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In all eukaryotes, the 24-h periodicity in the environment contributed to the evolution of the molecular circadian clock. We studied some elements of a postulated circadian clock circuit in the lowest metazoans, the siliceous sponges. First, we identified in the demosponge Suberites domuncula the enzyme luciferase that generates photons. Then (most likely), the photons generated by luciferase are transmitted via the biosilica glass skeleton of the sponges and are finally harvested by cryptochrome in the same individual; hence, cryptochrome is acting as a photosensor. This information-transduction system, generation of light (luciferase), photon transmission (through the siliceous spicules), and photon reception (cryptochrome), all occur in the same individual. Therefore, we propose that this photoreception/phototransduction process might function as a nerve-cell-like signal transmitting system. This was corroborated by the fact that S. domuncula reacts to different wavelengths of light, originating from the sponge environment, with a differential gene expression of the transcription factor SOX. Recently, we succeeded in demonstrating that in sponges a light/dark controlled gene is expressed, which encodes for nocturnin, a protein showing poly(A)-specific 3-exoribonuclease activity. Quantitative real-time polymerase chain reaction analyses revealed that primmorphs, 3D cell aggregates of sponge cells, after transfer from light to dark, show a 10-fold increased expression of the nocturnin gene. In contrast, the expression level of the gene encoding glycogenin decreases in the dark by three- to four-fold. It is concluded that sponges are provided with the molecular circadian clock protein nocturnin which is highly expressed in the dark. This finding together with the proposed light-transduction and spicule-based signaling system strongly supports the view that already the lowest metazoans, the sponges, have elements of a circadian rhythm, characteristic of higher metazoans.
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Silicatein conjugation inside nanoconfined geometries through immobilized NTA-Ni(II) chelates.
Chem. Commun. (Camb.)
PUBLISHED: 02-08-2013
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The chemical modification and bioconjugation processes inside confined geometries by His-tagged silicatein promote sensitive changes in the polarity and surface charge density that mainly contribute to the ionic current rectification properties of the single conical nanopores.
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Biosilica aging: from enzyme-driven gelation via syneresis to chemical/biochemical hardening.
Biochim. Biophys. Acta
PUBLISHED: 02-02-2013
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The distinguished property of the siliceous sponge spicules is their enzyme (silicatein)-catalyzed biosilica formation. The enzymatically formed, non-structured biosilica product undergoes a molding, syneresis, and hardening process to form the species-specifically shaped, hard structured skeletal spicules. Besides of silicatein, a silicatein-associated protein, silintaphin-2, is assumed to be involved in the process of biosilica formation in vivo.
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The silicatein propeptide acts as inhibitor/modulator of self-organization during spicule axial filament formation.
FEBS J.
PUBLISHED: 02-02-2013
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Silicateins are crucial enzymes that are involved in formation of the inorganic biosilica scaffold of the spicular skeleton of siliceous sponges. We show that silicatein acquires its structure-guiding and enzymatically active state by processing of silicatein from pro-silicatein to the mature enzyme. A recombinant propeptide (PROP) of silicatein from the siliceous demosponge Suberites domuncula was prepared, and antibodies were raised against the peptide. In sponge tissue, these antibodies reacted with both surface structures and the central region of the spicules. Using phage display expression, spicule-binding 12-mer peptides were identified that are rich in histidine residues. In the predicted tertiary structure of PROP, these histidine residues are only present in the ?-helical region. The recombinant PROP was found to inhibit self-assembly of silicatein molecules. By light scattering, it was shown that, in the presence of 4 m urea, the recombinant silicatein is obtained in the mono/oligomeric form with a hydrodynamic radius of 4 nm, while lower urea concentrations promote self-aggregation and assembly of the protein. Finally, it is shown that the enzymatic activity of silicatein is abolished by PROP in silicatein samples that predominantly contain mono- or oligomeric silicatein particles, but the enzyme is not affected if present in the filamentous aggregated form. It is concluded that the functions of silicatein, acting as a structural template for its biosilica product and as an enzyme, are modulated and controlled by its propeptide.
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Self-healing, an intrinsic property of biomineralization processes.
IUBMB Life
PUBLISHED: 01-31-2013
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The sponge siliceous spicules are formed enzymatically via silicatein, in contrast to other siliceous biominerals. Originally, silicatein had been described as a major structural protein of the spicules that has the property to allow a specific deposition of silica onto their surface. More recently, it had been unequivocally demonstrated that silicatein displays a genuine enzyme activity, initiating and maintaining silica biopolycondensation at low precursor concentrations (<2 mM). Even more, as silicatein becomes embedded into the biosilica polymer, formed by the enzyme, it retains its functionality to enable a controlled biosilica deposition. The protection of silicatein through the biosilica mantel is so strong that it conserves the functionality of the enzyme for thousands of years. The implication of this finding, the preservation of the enzyme function over such long time periods, is that the intrinsic property of silicatein to display its enzymatic activity remains in the biosilica deposits. This self-healing property of sponge biosilica can be utilized to engineer novel hybrid materials, with silicatein as a functional template, which are more resistant toward physical stress and fracture. Those hybrid materials can even be used for the fabrication of silica dielectrics coupled to optical nanowires.
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Bacterial sensors based on biosilica immobilization for label-free OWLS detection.
N Biotechnol
PUBLISHED: 01-21-2013
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In the last years, a new group of enzymes, the so-called silicateins, have been identified and characterized, which form the axial filaments of the spicules of the siliceous sponges, consisting of not only amorphous silica among others. These enzymes are able to catalyze the polycondensation and deposition of silica at mild conditions. Silicateins can be expressed in Escherichia coli. The recombinant proteins are expressed on the surface of the cell wall and are able to catalyze the formation of a polysilicate net around the bacterial cells providing the possibility for further attachment to the surface of SiO2 containing sensor chips. With this mild immobilization process it is now possible to prepare novel microbial sensors based on Optical Waveguide Lightmode Spectroscopy. In the present study, the immobilization of silicatein modified E. coli BL21AI cells onto the SiO2-type chips was optimized (buffer concentration, pH, temperature, reaction time, and so on) and then the biological properties, in particular the inhibitory effect of stressors/environmental pollutants on the novel bacterial sensor were studied in real time. The effect of oxidative stress was investigated by exposing the sensors containing biosilica-immobilized E. coli BL21AI cells to various concentrations of hydrogen peroxide. The effect of antibiotics was tested using chloramphenicol (CAP) which is effective against a variety of Gram-positive and Gram-negative bacteria and penicillin G which destroys the bacterial cell wall. In addition, the inhibition by carbofuran (CF) pesticide was also tested. CF is a highly toxic compound which inhibits cholinesterase activity. According our results we can conclude that the novel bacterial sensor consisting of the silicatein modified E. coli BL21AI cells immobilized on OWLS sensor surface could be an effective tool to detect the presence of different type of pollutants in real time measurement. However penicillin G and CF are not specifically inhibitors of E. coli strain, but some inhibitory effect could be still determined beside the well expressed signals for H2O2 and CAP obtained with the novel microbial sensor.
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Development of a morphogenetically active scaffold for three-dimensional growth of bone cells: biosilica-alginate hydrogel for SaOS-2 cell cultivation.
J Tissue Eng Regen Med
PUBLISHED: 01-12-2013
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Polymeric silica is formed from ortho-silicate during a sol-gel formation process, while biosilica is the product of an enzymatically driven bio-polycondensation reaction. Both polymers have recently been described as a template that induces an increased expression of the genes encoding bone morphogenetic protein 2 (BMP-2) and osteoprotegerin in osteoblast-related SaOS-2 cells; simultaneously or subsequently the cells respond with enhanced hydroxyapatite formation. In order to assess whether the biocompatible polymeric silica/biosilica can serve as a morphogenetically active matrix suitable for three-dimensional (3D) cell growth, or even for 3D cell bioprinting, SaOS-2 cells were embedded into a Na-alginate-based hydrogel. Four different gelatinous hydrogel matrices were used for suspending SaOS-2 cells: (a) the hydrogel alone; (b) the hydrogel with 400 ?m ortho-silicate; (c) the hydrogel supplemented with 400 ?m ortho-silicate and recombinant silicatein to allow biosilica synthesis to occur; and (d) the hydrogel with ortho-silicate and BSA. The SaOS-2 cells showed an increased growth if silica/biosilica components were present in the hydrogel. Likewise intensified was the formation of hydroxyapatite nodules in the silica-containing hydrogels. After an incubation period of 2 weeks, cells present in silica-containing hydrogels showed a significantly higher expression of the genes encoding the cytokine BMP-2, the major fibrillar structural protein collagen 1 and likewise of carbonic anhydrase. It is concluded that silica, and to a larger extent biosilica, retains its morphogenetic/osteogenic potential after addition to Na-alginate-based hydrogels. This property might qualify silica hydrogels to be also used as a matrix for 3D cell printing. Copyright © 2013 John Wiley & Sons, Ltd.
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The deep-sea natural products, biogenic polyphosphate (Bio-PolyP) and biogenic silica (Bio-Silica), as biomimetic scaffolds for bone tissue engineering: fabrication of a morphogenetically-active polymer.
Mar Drugs
PUBLISHED: 01-10-2013
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Bone defects in human, caused by fractures/nonunions or trauma, gain increasing impact and have become a medical challenge in the present-day aging population. Frequently, those fractures require surgical intervention which ideally relies on autografts or suboptimally on allografts. Therefore, it is pressing and likewise challenging to develop bone substitution materials to heal bone defects. During the differentiation of osteoblasts from their mesenchymal progenitor/stem cells and of osteoclasts from their hemopoietic precursor cells, a lineage-specific release of growth factors and a trans-lineage homeostatic cross-talk via signaling molecules take place. Hence, the major hurdle is to fabricate a template that is functioning in a way mimicking the morphogenetic, inductive role(s) of the native extracellular matrix. In the last few years, two naturally occurring polymers that are produced by deep-sea sponges, the biogenic polyphosphate (bio-polyP) and biogenic silica (bio-silica) have also been identified as promoting morphogenetic on both osteoblasts and osteoclasts. These polymers elicit cytokines that affect bone mineralization (hydroxyapatite formation). In this manner, bio-silica and bio-polyP cause an increased release of BMP-2, the key mediator activating the anabolic arm of the hydroxyapatite forming cells, and of RANKL. In addition, bio-polyP inhibits the progression of the pre-osteoclasts to functionally active osteoclasts. Based on these findings, new bioinspired strategies for the fabrication of bone biomimetic templates have been developed applying 3D-printing techniques. Finally, a strategy is outlined by which these two morphogenetically active polymers might be used to develop a novel functionally active polymer.
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Deep metazoan phylogeny: when different genes tell different stories.
Mol. Phylogenet. Evol.
PUBLISHED: 01-08-2013
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Molecular phylogenetic analyses have produced a plethora of controversial hypotheses regarding the patterns of diversification of non-bilaterian animals. To unravel the causes for the patterns of extreme inconsistencies at the base of the metazoan tree of life, we constructed a novel supermatrix containing 122 genes, enriched with non-bilaterian taxa. Comparative analyses of this supermatrix and its two non-overlapping multi-gene partitions (including ribosomal and non-ribosomal genes) revealed conflicting phylogenetic signals. We show that the levels of saturation and long branch attraction artifacts in the two partitions correlate with gene sampling. The ribosomal gene partition exhibits significantly lower saturation levels than the non-ribosomal one. Additional systematic errors derive from significant variations in amino acid substitution patterns among the metazoan lineages that violate the stationarity assumption of evolutionary models frequently used to reconstruct phylogenies. By modifying gene sampling and the taxonomic composition of the outgroup, we were able to construct three different yet well-supported phylogenies. These results show that the accuracy of phylogenetic inference may be substantially improved by selecting genes that evolve slowly across the Metazoa and applying more realistic substitution models. Additional sequence-independent genomic markers are also necessary to assess the validity of the phylogenetic hypotheses.
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Aaptamine derivatives from the Indonesian sponge Aaptos suberitoides.
J. Nat. Prod.
PUBLISHED: 01-02-2013
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Four new aaptamine derivatives (1-4) along with aaptamine (5) and three related compounds (6-8) were isolated from the ethanol extract of the sponge Aaptos suberitoides collected in Indonesia. The structures of the new compounds were unambiguously determined by one- and two-dimensional NMR and by HRESIMS measurements. Compounds 3, 5, and 6 showed cytotoxic activity against the murine lymphoma L5178Y cell line, with IC(50) values ranging from 0.9 to 8.3 ?M.
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The enzyme carbonic anhydrase as an integral component of biogenic Ca-carbonate formation in sponge spicules.
FEBS Open Bio
PUBLISHED: 01-01-2013
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The inorganic scaffold of the spicules, the skeletal elements of the calcareous sponges, is formed of calcium carbonate (CaCO3). The growth of the approximately 300-?m large spicules, such as those of the calcareous sponge Sycon raphanus used in the present study, is a rapid process with a rate of about 65 ?m/h. The formation of CaCO3 is predominantly carried out by the enzyme carbonic anhydrase (CA). The enzyme from the sponge S. raphanus was isolated and prepared by recombination. The CA-driven deposition of CaCO3 crystallites is dependent on temperature (optimal at 52 °C), the pH value of the reaction assay (7.5/8.0), and the substrate concentration (CO2 and Ca(2+)). During the initial phase of crystallite formation, ?40 ?m large round-shaped deposits are formed that remodel to larger prisms. These crystal-like prisms associate to each other and form either rope-/bundle-like aggregates or arrange perfectly with their smaller planes along opposing surfaces of the sponge spicule rays. The CA-dependent CaCO3 deposition can be inhibited by the CA-specific inhibitor acetazolamide. The Michaelis-Menten constant for the CA-driven mineralization has been determined to be around 8 mM with respect to CaCO3. The deposits formed have a Martens hardness of ?5 GPa. The data presented here highlights for the first time that calcite deposition in the sponge system is decisively controlled enzymatically. This data will contribute to the development of new strategies applicable for the fabrication of novel biomaterials.
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IL-27 promotes IL-10 production by effector Th1 CD4+ T cells: a critical mechanism for protection from severe immunopathology during malaria infection.
J. Immunol.
PUBLISHED: 12-28-2011
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Infection with the malaria parasite, Plasmodium, is characterized by excessive inflammation. The establishment of a precise balance between the pro- and anti-inflammatory responses is critical to guarantee control of the parasite and survival of the host. IL-10, a key regulatory cytokine produced by many cells of the immune system, has been shown to protect mice against pathology during acute Plasmodium0 chabaudi chabaudi AS model of malaria. However, the critical cellular source of IL-10 is still unknown. In this article, we demonstrate that T cell-derived IL-10 is necessary for the control of pathology during acute malaria, as mice bearing specific deletion of Il10 in T cells fully reproduce the phenotype observed in Il10(-)(/)(-) mice, with significant weight loss, decline in temperature, and increased mortality. Furthermore, we show that IFN-?(+) Th1 cells are the main producers of IL-10 throughout acute infection, expressing high levels of CD44 and ICOS, and low levels of CD127. Although Foxp3(+) regulatory CD4(+) T cells produce IL-10 during infection, highly activated IFN-?(+) Th1 cells were shown to be the essential and sufficient source of IL-10 to guarantee protection against severe immune-mediated pathology. Finally, in this model of malaria, we demonstrate that the generation of protective IL10(+)IFN-?(+) Th1 cells is dependent on IL-27 signaling and independent of IL-21.
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Derivation of the propagation equations for higher order aberrations of local wavefronts.
J Opt Soc Am A Opt Image Sci Vis
PUBLISHED: 12-24-2011
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From the literature the analytical calculation of local power and astigmatism of a wavefront after refraction and propagation is well known; it is, e.g., performed by the Coddington equation for refraction and the classical vertex correction formula for propagation. Recently the authors succeeded in extending the Coddington equation to higher order aberrations (HOA). However, equivalent analytical propagation equations for HOA do not exist. Since HOA play an increasingly important role in many fields of optics, e.g., ophthalmic optics, it is the purpose of this study to extend the propagation equations of power and astigmatism to the case of HOA (e.g., coma and spherical aberration). This is achieved by local power series expansions. In summary, with the results presented here, it is now possible to calculate analytically the aberrations of a propagated wavefront directly from the aberrations of the original wavefront containing both low-order and high-order aberrations.
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Induction of regulatory T cells by a murine ?-defensin.
J. Immunol.
PUBLISHED: 12-14-2011
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?-Defensins are antimicrobial peptides of the innate immune system produced in the skin by various stimuli, including proinflammatory cytokines, bacterial infection, and exposure to UV radiation (UVR). In this study we demonstrate that the UVR-inducible antimicrobial peptide murine ?-defensin-14 (mBD-14) switches CD4(+)CD25(-) T cells into a regulatory phenotype by inducing the expression of specific markers like Foxp3 and CTLA-4. This is functionally relevant because mBD-14-treated T cells inhibit sensitization upon adoptive transfer into naive C57BL/6 mice. Accordingly, injection of mBD-14, comparable to UVR, suppresses the induction of contact hypersensitivity and induces Ag-specific regulatory T cells (Tregs). Further evidence for the ability of mBD-14 to induce Foxp3(+) T cells is provided using DEREG (depletion of Tregs) mice in which Foxp3-expressing cells can be depleted by injecting diphtheria toxin. mBD-14 does not suppress sensitization in IL-10 knockout mice, suggesting involvement of IL-10 in mBD-14-mediated immunosuppression. However, unlike UVR, mBD-14 does not appear to mediate its immunosuppressive effects by affecting dendritic cells. Accordingly, UVR-induced immunosuppression is not abrogated in mBD-14 knockout mice. Together, these data suggest that mBD-14, like UVR, has the capacity to induce Tregs but does not appear to play a major role in UVR-induced immunosuppression. Through this capacity, mBD-14 may protect the host from microbial attacks on the one hand, but tame T cell-driven reactions on the other hand, thereby enabling an antimicrobial defense without collateral damage by the adaptive immune system.
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B cell-derived IL-10 does not regulate spontaneous systemic autoimmunity in MRL.Fas(lpr) mice.
J. Immunol.
PUBLISHED: 12-09-2011
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B cells contribute to the pathogenesis of chronic autoimmune disorders, like systemic lupus erythematosus (SLE), via multiple effector functions. However, B cells are also implicated in regulating SLE and other autoimmune syndromes via release of IL-10. B cells secreting IL-10 were termed "Bregs" and were proposed as a separate subset of cells, a concept that remains controversial. The balance between pro- and anti-inflammatory effects could determine the success of B cell-targeted therapies for autoimmune disorders; therefore, it is pivotal to understand the significance of B cell-secreted IL-10 in spontaneous autoimmunity. By lineage-specific deletion of Il10 from B cells, we demonstrated that B cell-derived IL-10 is ineffective in suppressing the spontaneous activation of self-reactive B and T cells during lupus. Correspondingly, severity of organ disease and survival rates in mice harboring Il10-deficient B cells are unaltered. Genetic marking of cells that transcribe Il10 illustrated that the pool of IL-10-competent cells is dominated by CD4 T cells and macrophages. IL-10-competent cells of the B lineage are rare in vivo and, among them, short-lived plasmablasts have the highest frequency, suggesting an activation-driven, rather than lineage-driven, phenotype. Putative Breg phenotypic subsets, such as CD1d(hi)CD5(+) and CD21(hi)CD23(hi) B cells, are not enriched in Il10 transcription. These genetic studies demonstrated that, in a spontaneous model of murine lupus, IL-10-dependent B cell regulation does not restrain disease and, thus, the pathogenic effects of B cells are not detectably counterbalanced by their IL-10-dependent regulatory functions.
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Complex structures - smart solutions: Formation of siliceous spicules.
Commun Integr Biol
PUBLISHED: 10-17-2011
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The siliceous skeletal elements of the sponges, the spicules, represent one of the very few examples from where the molecule toolkit required for the formation of an extracellular mineral-based skeleton, has been elucidated. The distinguished feature of the inorganic matrix, the bio-silica, is its enzymatic synthesis mediated by silicatein. Ortho-silicate undergoes in the presence of silicatein a polycondensation reaction and forms bio-silica under release of reaction water. The protein silicatein aggregates non-covalently to larger filaments, a process that is stabilized by the silicatein-associated protein, silintaphin-1. These structured clusters form the axial filament that is located in the center of the spicules, the axial canal. Surprisingly it has now been found that the initial axial orientation, in which the spicules grow, is guided by cell processes through evagination. The approximately two µm wide cell extensions release silicatein that forms the first organic axial filament, which then synthesizes the inner core of the siliceous spicule rods. In parallel, the radial growth of the spicules is controlled by a telescopic arrangement of organic layers, into which bio-silica and ortho-silicate are deposited. Hence, the formation of a mature siliceous spicule is completed by a centrifugal accretion of bio-silica mediated by the silicatein in the axial filament, and a centripetal bio-silica deposition catalyzed by the extra-spicular silicatein. Finally this contribution highlights that for the ultimate determination of the spicule shapes, their species-specific morphologies, bio-silica hardens during a process which removes reaction water. The data presented can also provide new blueprints for the fabrication of novel biomaterials for biomedical applications. 
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SDF-1/CXCL12: its role in spinal cord injury.
Int. J. Biochem. Cell Biol.
PUBLISHED: 09-26-2011
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The chemokine stromal cell-derived factor 1 (SDF-1/CXCL12), is not only the most ancient, but also one of the most potent chemotactic factors. Orchestrating the migration of cells as well as promoting axon outgrowth in the presence of myelin inhibitors, SDF-1 is fundamental to central nervous system development, homeostasis and traumatic injury. SDF-1 attracts endogenous stem/precursor cells and immune cells to the injury site and, upon local infusion, enhances axonal sprouting following spinal cord injury. Together these features make SDF-1 a very exciting molecule for spinal cord repair.
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Significant clinical, neuropathological and behavioural recovery from acute spinal cord trauma by transplantation of a well-defined somatic stem cell from human umbilical cord blood.
Brain
PUBLISHED: 09-08-2011
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Stem cell therapy is a potential treatment for spinal cord injury and different stem cell types have been grafted into animal models and humans suffering from spinal trauma. Due to inconsistent results, it is still an important and clinically relevant question which stem cell type will prove to be therapeutically effective. Thus far, stem cells of human sources grafted into spinal cord mostly included barely defined heterogeneous mesenchymal stem cell populations derived from bone marrow or umbilical cord blood. Here, we have transplanted a well-defined unrestricted somatic stem cell isolated from human umbilical cord blood into an acute traumatic spinal cord injury of adult immune suppressed rat. Grafting of unrestricted somatic stem cells into the vicinity of a dorsal hemisection injury at thoracic level eight resulted in hepatocyte growth factor-directed migration and accumulation within the lesion area, reduction in lesion size and augmented tissue sparing, enhanced axon regrowth and significant functional locomotor improvement as revealed by three behavioural tasks (open field Basso-Beattie-Bresnahan locomotor score, horizontal ladder walking test and CatWalk gait analysis). To accomplish the beneficial effects, neither neural differentiation nor long-lasting persistence of the grafted human stem cells appears to be required. The secretion of neurite outgrowth-promoting factors in vitro further suggests a paracrine function of unrestricted somatic stem cells in spinal cord injury. Given the highly supportive functional characteristics in spinal cord injury, production in virtually unlimited quantities at GMP grade and lack of ethical concerns, unrestricted somatic stem cells appear to be a highly suitable human stem cell source for clinical application in central nervous system injuries.
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Biosilica-based strategies for treatment of osteoporosis and other bone diseases.
Prog. Mol. Subcell. Biol.
PUBLISHED: 08-31-2011
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Osteoporosis is a common disease in later life, which has become a growing public health problem. This degenerative bone disease primarily affects postmenopausal women, but also men may suffer from reduced bone mineral density. The development of prophylactic treatments and medications of osteoporosis has become an urgent issue due to the increasing proportion of the elderly in the population. Apart from medical/hormonal treatments, current strategies for prophylaxis of osteoporosis are primarily based on calcium supplementation as a main constituent of bone hydroxyapatite mineral. Despite previous reports suggesting an essential role in skeletal growth and development, the significance of the trace element silicon in human bone formation has attracted major scientific interest only rather recently. The interest in silicon has been further increased by the latest discoveries in the field of biosilicification, the formation of the inorganic silica skeleton of the oldest still extant animals on Earth, the sponges, which revealed new insights in the biological function of this element. Sponges make use of silicon to build up their inorganic skeleton which consists of biogenously formed polymeric silica (biosilica). The formation of biosilica is mediated by specific enzymes, silicateins, which have been isolated, characterized, and expressed in a recombinant way. Epidemiological studies revealed that dietary silicon reduces the risk of osteoporosis and other bone diseases. Recent results allowed for the first time to understand the molecular mechanism underlying the protective effect of silicic acid/biosilica against osteoporosis. Biosilica was shown to modulate the ratio of expression of two cytokines involved in bone formation-RANKL and osteoprotegerin. Hence, biosilica has been proposed to have a potential in prophylaxis and therapy of osteoporosis and related bone diseases.
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The unique invention of the siliceous sponges: their enzymatically made bio-silica skeleton.
Prog. Mol. Subcell. Biol.
PUBLISHED: 08-31-2011
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Sponges are sessile filter feeders that, among the metazoans, evolved first on Earth. In the two classes of the siliceous sponges (the Demospongiae and the Hexactinellida), the complex filigreed body is stabilized by an inorganic skeleton composed of amorphous silica providing them a distinct body shape and plan. It is proposed that the key innovation that allowed the earliest metazoans to form larger specimens was the enzyme silicatein. This enzyme is crucial for the formation of the siliceous skeleton. The first sponge fossils with body preservation were dated back prior to the "Precambrian-Cambrian" boundary [Vendian (610-545 Ma)/Ediacaran (542-580 Ma)]. A further molecule required for the formation of a hard skeleton was collagen, fibrous organic filaments that need oxygen for their formation. Silicatein forming the spicules and collagen shaping their morphology are the two organic components that control the appositional growth of these skeletal elements. This process starts in both demosponges and hexactinellids intracellularly and is completed extracellularly where the spicules may reach sizes of up to 3 m. While the basic strategy of their formation is identical in both sponge classes, it differs on a substructural level. In Hexactinellida, the initial silica layers remain separated, those layers bio-fuse (bio-sinter) together in demosponges. In some sponge taxa, e.g., the freshwater sponges from the Lake Baikal, the individual spicules are embedded in an organic matrix that is composed of the DUF protein. This protein comprises clustered stretches of amino acid sequences composed of pronounced hydrophobic segments, each spanning around 35 aa. We concluded with the remark of Thompson (1942) highlighting that "the sponge-spicule is a typical illustration of the theory of bio-crystallisation to form biocrystals ein Mittelding between an inorganic crystal and an organic secretion." Moreover, the understanding of the enzymatic formation of the spicules conferred sponge biosilica a considerable economical actuality as a prime raw material of this millennium.
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Molecular biomineralization: toward an understanding of the biogenic origin of polymetallic nodules, seamount crusts, and hydrothermal vents.
Prog. Mol. Subcell. Biol.
PUBLISHED: 08-31-2011
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Polymetallic nodules and crusts, hydrothermal vents from the Deep Sea are economically interesting, since they contain alloying components, e.g., manganese or cobalt, that are used in the production of special steels; in addition, they contain rare metals applied for plasma screens, for magnets in hard disks, or in hybrid car motors. While hydrothermal vents can regenerate in weeks, polymetallic nodules and seamount crusts grow slowly. Even though the geochemical basis for the growth of the nodules and crusts has been well studied, the contribution of microorganisms to the formation of these minerals remained obscure. Recent HR-SEM (high-resolution scanning electron microscopy) analyses of nodules and crusts support their biogenic origin. Within the nodules, bacteria with surface S-layers are arranged on biofilm-like structures, around which Mn deposition starts. In crusts, coccoliths represent the dominant biologically formed structures that act as bio-seeds for an initial Mn deposition. In contrast, hydrothermal vents have apparently an abiogenic origin; however, their minerals are biogenically transformed by bacteria. In turn, strategies can now be developed for biotechnological enrichment as well as selective dissolution of metals from such concretions. We are convinced that the recent discoveries will considerably contribute to our understanding of the participation of organic matrices in the enrichment of those metals and will provide the basis for feasibility studies for biotechnological applications.
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How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.