Phenotypical and molecular methods identified the yeast as Arxula adeninivorans (figure), which is used in biotechnology3 as a transgenic organism and not previously identified as a pathogen. Because ...A adeninivorans is used in the production of coffee and tea, we screened the patient's household but did not find this yeast. Rare fungi such as scedosporium,4 exophiala, and A adeninivorans can be difficult to detect; in this case, the fungus was first misidentified as Stephanoascus ciferii.
The ubiquitin proteasome pathway in plants has been shown to be important for many developmental processes. The E3 ubiquitin-protein ligases facilitate transfer of the ubiquitin moiety to substrate ...proteins. Many E3 ligases contain cullin proteins as core subunits. Here, we show that Arabidopsis (Arabidopsis thaliana) AtCUL3 proteins interact in yeast two-hybrid and in vitro pull-down assays with proteins containing a BTB/POZ (broad complex, tramtrack, bric-a-brac/pox virus and zinc finger) motif. By changing specific amino acid residues within the proteins, critical parts of the cullin and BTB/POZ proteins are defined that are required for these kinds of interactions. In addition, we show that AtCUL3 proteins assemble with the RING-finger protein AtRBX1 and are targets for the RUB-conjugation pathway. The analysis of AtCUL3a and AtCUL3b expression as well as several BTB/POZ-MATH genes indicates that these genes are expressed in all parts of the plant. The results presented here provide strong evidence that AtCUL3a and AtCUL3b can assemble in Arabidopsis with BTB/POZ-MATH and AtRBX1 proteins to form functional E3 ligases.
Extrusion-based bioprinting, also known as 3D bioplotting, is a powerful tool for the fabrication of tissue equivalents with spatially defined cell distribution. Even though considerable progress has ...been made in recent years, there is still a lack of bioinks which enable a tissue-like cell response and are plottable at the same time with good shape fidelity. Herein, we report on the development of a bioink which includes fresh frozen plasma from full human blood and thus a donor/patient-specific protein mixture. By blending of the plasma with 3 w/v% alginate and 9 w/v% methylcellulose, a pasty bioink (plasma-alg-mc) was achieved, which could be plotted with high accuracy and furthermore allowed bioplotted mesenchymal stromal cells (MSC) and primary osteoprogenitor cells to spread within the bioink. In a second step, the novel plasma-based bioink was combined with a plottable self-setting calcium phosphate cement (CPC) to fabricate bone-like tissue constructs. The CPC/plasma-alg-mc biphasic constructs revealed open porosity over the entire time of cell culture (35 d), which is crucial for bone tissue engineered grafts. The biphasic structures could be plotted in volumetric and clinically relevant dimensions and complex shapes could be also generated, as demonstrated for a scaphoid bone model. The plasma bioink potentiated that bioplotted MSC were not harmed by the setting process of the CPC. Latest after 7 days, MSC migrated from the hydrogel to the CPC surface, where they proliferated to 20-fold of the initial cell number covering the entire plotted constructs with a dense cell layer. For bioplotted and osteogenically stimulated osteoprogenitor cells, a significantly increased alkaline phosphatase activity was observed in CPC/plasma-alg-mc constructs in comparison to plasma-free controls. In conclusion, the novel plasma-alg-mc bioink is a promising new ink for several forms of bioprinted tissue equivalents and especially gainful for the combination with CPC for enhanced, biofabricated bone-like constructs.
The application of biologically active metal ions to stimulate cellular reactions is a promising strategy to accelerate bone defect healing. Brushite-forming calcium phosphate cements were modified ...with low doses of Cu2+, Co2+ and Cr3+. The modified cements released the metal ions in vitro in concentrations which were shown to be non-toxic for cells. The release kinetics correlated with the solubility of the respective metal phosphates: 17–45 wt.-% of Co2+ and Cu2+, but <1 wt.-% of Cr3+ were released within 28days. Moreover, metal ion doping led to alterations in the exchange of calcium and phosphate ions with cell culture medium. In case of cements modified with 50mmol Cr3+/mol β-tricalcium phosphate (β-TCP), XRD and SEM analyses revealed a significant amount of monetite and a changed morphology of the cement matrix. Cell culture experiments with human mesenchymal stromal cells indicated that the observed cell response is not only influenced by the released metal ions but also by changed cement properties. A positive effect of modifications with 50mmol Cr3+ or 10mmol Cu2+ per mol β-TCP on cell behaviour was observed in indirect and direct culture. Modification with Co2+ resulted in a clear suppression of cell proliferation and osteogenic differentiation. In conclusion, metal ion doping of the cement influences cellular activities in addition to the effect of released metal ions by changing properties of the ceramic matrix.
•A brushite-forming calcium phosphate cement was modified by doping with bioactive Cu2+, Co2+ and Cr3+ ions.•The metal ions were integrated in the cement matrix, setting was not affected.•The modified cements released the metal ions in doses non-toxic for cells.•Modification with Cr3+ ions enhanced the biocompatibility of the cement.
Carbonate platforms form informative archives for paleoclimates and their internal structures can also hold crucial information about the tectonic history and carbonate evolution of the ocean basins. ...The Zhongsha atoll (Macclesfield Bank) forms the largest atoll system in the South China Sea with a surface area of 23500 km2. However, the internal structure and evolution of this atoll system is completely unknown. 2D multichannel seismic reflection data were acquired in 2017 over the Zhongsha atoll in the South China Sea to unravel the stratigraphy, geomorphology, depositional processes, and seismic facies of one of the world's largest atoll for the first time. This Neogene carbonate platform comprises more than 1 km thick carbonate sequence and overlies a metamorphic basement. The southeastern part of the atoll comprises a fault-controlled graben system, which was formed during the Cenozoic rifting stage of the South China Sea. Most of the faults trend NE-SW and E-W and terminate at or slightly above the top of Middle Miocene strata. Atolls and abundant organic reefs initiated on the positive relief and closely mimicked the underlying topography during the Early Miocene. Shallow-water carbonates continued growing through Middle Miocene to present times. Regional uplift led to subaerial exposure, termination of platform growth and karstification during the Miocene. We also reveal a number of fluid-flow features such as vertical sub-bottom venting features (chimneys and pipes), chaotic reflection zones, which provide the first evidence of active fluid venting in the area of Zhongsha atoll. The Neogene sedimentation history of Zhongsha atoll further provide an important paleoenvironmental context for future scientific drilling to better constrain the evolution of Asia Monsoon.
•First documentation of seismic stratigraphy and deposition of Zhongsha atoll (Macclesfield Bank), South China Sea.•Reefs and lagoons developed during the post-rift phase, whose onset is marked by a turning point around the time of the formation of reflector T50.•Three drowning events are identified to tentatively imply three episodes of fast sea floor spreading in the study region.
Microextrusion bioprinting, within the emerging field of biofabrication, holds great promise for developing in vitro tissue models or implantable constructs that mimic complexity of native tissues. ...Despite the considerable progress made fabricating cell-laden scaffolds, mono-material approaches often cannot reproduce complex tissue architecture or satisfy biomechanical requirements, a particular challenge for hard, mineralized bone tissue. Thus, in this work, we focus on the fabrication of high-strength, partly-mineralized composite bone substitutes via multi-channel (bio)printing of inorganic and organic (bio)inks – with the aim of synergistically integrating the advantages of both phases. A hydroxyapatite (HAp) matrix-mimicking, clinically approved, ready-to-use calcium phosphate cement (CPC) ink as a stiff, mechanically robust phase is combined with an organic eggwhite-functionalized bioink of alginate-methylcellulose (AlgMC + EW) delivering cells in a spatially defined distribution. As proven in a novel systematic assessment, cell-free CPC ink and cell-laden AlgMC + EW bioinks exhibited excellent co-printability and shape fidelity in simple, more complex and anatomically shaped biphasic designs. The mechanical properties of such biphasic constructs were tailored via adjusting the ratio of inorganic and organic component. The survival and fate of human mesenchymal stem cells (MSC) and primary human pre-osteoblasts (hOB) in bioprinted inorganic/organic biphasic structures in response to CPC and EW functionalization during a long-term cultivation were investigated, revealing a defined, cell-friendly bioink delivery which allows cells to migrate and proliferate, as well as proving great potential for 3D osteogenesis. In line with further suggested technological refinements, this creates a toolbox for targeted design and construction of possibly pre-vascularized bone structures in clinically relevant dimensions.
Biomimetic mineralization of collagen is an advantageous method to obtain resorbable collagen/hydroxy-apatite composites for application in bone regeneration. In this report, established procedures ...for mineralization of bovine collagen were adapted to a new promising source of collagen from salmon skin challenged by the low denaturation temperature. Therefore, in the first instance, variation of temperature, collagen concentration, and ionic strength was performed to reveal optimized parameters for fibrillation and simultaneous mineralization of salmon collagen. Porous scaffolds from mineralized salmon collagen were prepared by controlled freeze-drying and chemical cross-linking. FT-IR analysis demonstrated the mineral phase formed during the preparation process to be hydroxyapatite. The scaffolds exhibited interconnecting porosity, were sufficiently stable under cyclic compression, and showed elastic mechanical properties. Human mesenchymal stem cells were able to adhere to the scaffolds, cell number increased during cultivation, and osteogenic differentiation was demonstrated in terms of alkaline phosphatase activity.
Cell-based in vitro resorption assays are an important tool to simulate the in vivo biodegradation of resorbable bone graft materials and to predict their clinical performance. The present study ...analyses the activity of osteoclast-specific enzymes as potential surrogate measures for classical pit assay, which is not applicable on irregular structured materials. Osteoclasts derived from human peripheral blood mononuclear cells were cultivated on different surfaces: calcium phosphate bone cements (CPC), dentin discs, osteoblast-derived extracellular matrix (ECM) and tissue culture polystyrene as control. Pit formation on the resorbable materials was investigated and correlated with the activity of tartrate resistant acid phosphatase (TRAP), carbonic anhydrase II (CAII) and cathepsin K (CTSK). Furthermore, the relation between intra- and extracellular enzyme activities was examined for TRAP and CTSK during resorption of the different materials. Resorbed area of CPC correlated with intracellular TRAP activity and intracellular CAII activity. Highest resorption was detected at around pH 7.2. Resorbed area on dentin correlated with the extracellular CTSK activity and extracellular TRAP activity and was maximal at around pH 6.8. Osteoclasts cultivated on cell-derived mineralised ECM showed a good correlation between both extracellular TRAP and CTSK activity and the release of calcium ions. Based on these data a different regulation of TRAP and CTSK secretion is hypothesised for the resorption of inorganic calcium phosphate compared to the resorption of collagenous mineralised matrix.
The three additive manufacturing techniques fused deposition modeling, gel plotting and melt electrowriting were combined to develop a mimicry of the tympanic membrane (TM) to tackle large TM ...perforations caused by chronic otitis media. The mimicry of the collagen fiber orientation of the TM was accompanied by a study of multiple funnel-shaped mimics of the TM morphology, resulting in mechanical and acoustic properties similar to those of the eardrum. For the different 3D printing techniques used, the process parameters were optimized to allow reasonable microfiber arrangements within the melt electrowriting setup. Interestingly, the fiber pattern was less important for the acousto-mechanical properties than the overall morphology. Furthermore, the behavior of keratinocytes and fibroblasts is crucial for the repair of the TM, and an in vitro study showed a high biocompatibility of both primary cell types while mimicking the respective cell layers of the TM. A simulation of the in vivo ingrowth of both cell types resulted in a cell growth orientation similar to the original collagen fiber orientation of the TM. Overall, the combined approach showed all the necessary parameters to support the growth of a neo-epithelial layer with a similar structure and morphology to the original membrane. It therefore offers a suitable alternative to autologous materials for the treatment of chronic otitis media.
Millions of people worldwide suffer from chronic middle ear infections. Although the tympanic membrane (TM) can be reconstructed with autologous materials, the grafts used for this purpose require extensive manual preparation during surgery. This affects not only the hearing ability but also the stability of the reconstructed TM, especially in the case of full TM reconstruction. The synthetic alternative presented here mimicked not only the fibrous structure of the TM but also its morphology, resulting in similar acousto-mechanical properties. Furthermore, its high biocompatibility supported the migration of keratinocytes and fibroblasts to form a neo-epithelial layer. Overall, this completely new TM replacement was achieved by combining three different additive manufacturing processes.
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