Peripheral blood mononuclear cells (PBMC) are an attractive source for the generation of osteoclasts in vitro, which is an important prerequisite for the examination of resorption and remodeling of ...biomaterials. In this study, different preparation methods are used to obtain cell populations with a rising content of CD14(+) monocytes. We wanted to address the question whether there is a correlation between content of CD14(+) cells in the preparation and functionality of formed osteoclasts.
PBMC obtained by density gradient centrifugation with and without further purification by plastic adherence or immunomagnetic separation of CD14(+) cells were seeded on both cell culture polystyrene and a calcium phosphate bone cement (CPC) and cultivated under stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B ligand (RANKL). Cell cultures were characterized by histological and fluorescent staining of multinucleated cells that were positive for tartrate-resistant acid phosphatase (TRAP) activity and the presence of actin rings, respectively. Furthermore, activities of osteoclast marker enzymes TRAP and carbonic anhydrase II (CA II) were quantified. For osteoclasts cultured on CPC, resorption pits were visualized using scanning electron microscopy (SEM).
Monocytes of all preparations were successfully differentiated into multinucleated osteoclasts showing TRAP and CA II activity on both cell culture plastic and CPC. Preparations involving an additional plastic adherence step exhibited only a minor increase of TRAP and CA II activity in the second week of cultivation. Furthermore, the number of resorption pits on CPC was reduced in these cultures compared with immunomagnetically enriched monocytes and preparations without additional plastic adherence steps. Optimal results with regard to yield, number of multinucleated osteoclasts, activity of TRAP and CA II, and resorption of CPC were obtained by simple density gradient centrifugation.
All examined monocyte preparation protocols were suitable for the generation of osteoclasts on both polystyrene and CPC. Highly purified monocytes are not mandatory to obtain functional osteoclasts for investigation of biomaterial resorption.
Osteocytes are of high importance in bone metabolism as they orchestrate bone remodeling, react to mechanosensory stimuli and have endocrine functions. In vitro investigations with osteocytes are ...therefore of high relevance for biomaterial and drug testing. The application of primary human cells instead of rodent osteocyte cell lines like MLOY4 and IDG SW3 is desirable but provides the challenge of isolating these cells, which are deeply embedded into the mineralized bone matrix. The present study describes an improved protocol for the isolation of human primary osteocytes. In contrast to an already established protocol, resting steps between the demineralization /digestion steps of the bone particles considerably improved the yield of osteocytes. Real-time polymerase chain reaction (PCR) analysis revealed the expression of typical osteocyte markers like osteocalcin, E11/podoplanin and dentin matrix protein 1 (DMP-1).
Controlled stability of proteins is a highly efficient mechanism to direct diverse processes in living cells. A key regulatory system for protein stability is given by the ubiquitin proteasome ...pathway, which uses E3 ligases to mark specific proteins for degradation. In this work, MYB56 is identified as a novel target of a CULLIN3 (CUL3)-based E3 ligase. Its stability depends on the presence of MATH-BTB/POZ (BPM) proteins, which function as substrate adaptors to the E3 ligase. Genetic studies have indicated that MYB56 is a negative regulator of flowering, while BPMs positively affect this developmental program. The interaction between BPMs and MYB56 occurs at the promoter of FLOWERING LOCUS T (FT), a key regulator in initiating flowering in Arabidopsis, and results in instability of MYB56. Overall the work establishes MYB transcription factors as substrates of BPM proteins, and provides novel information on components that participate in controlling flowering time in plants.
Proline residues provide a backbone switch in a polypeptide chain, which is controlled by the cis/trans isomerization about the peptidyl−prolyl bond. Phosphorylation of serine- and threonine-proline ...motifs has been shown to be a critical regulatory event for many proteins. The biological significance of these motifs has been further highlighted by the discovery of a novel and essential peptidyl−prolyl cis/trans isomerase Pin1. Pin1 is required for progression through mitosis via catalyzing the isomerization of phosphorylated Ser/Thr-Pro motifs specifically present in mitosis-specific phosphoproteins. However, little is known whether the phosphorylation regulates the conformational switch of the Ser/Thr-Pro bonds. Here, we report the synthesis and conformational characterization of a series of peptides that contain the phosphorylated or nonphosphorylated Ser/Thr-Pro motifs. Phosphorylation affected the rate of the cis to trans isomerization of the Thr/Ser-Pro bonds. As determined by a protease-coupled assay, the isomerization rate of phosphorylated Thr-Pro bond was found to be 8-fold slower than that of the nonphosphorylated analogue. Furthermore, studies of the pH dependence of the isomerization of the phosphopeptides reveal that both cis content and the rate constant of prolyl cis to trans isomerization are lower for the dianionic state of the phosphothreonine-containing peptides. These effects of phosphorylation are specific for phosphorylated Ser/Thr since neither phosphorylated Tyr nor glutamic acid was able to affect the prolyl isomerization. Finally, our experiments provide evidence that effective catalysis of cis/trans isomerization of phosphorylated Ser/Thr-Pro bonds by Pin1 is specific to the dianionic form of the substrate. Thus, our results demonstrate that protein phosphorylation specifically regulates the backbone dynamics of the Ser/Thr-Pro motifs and that Pin1 specifically isomerizes the certain conformation of the phosphorylated Ser/Thr-Pro motifs.
Uncertain timing and magnitudes of past mega-earthquakes continue to confound seismic risk appraisals in the Himalayas. Telltale traces of surface ruptures are rare, while fault trenches document ...several events at best, so that additional proxies of strong ground motion are needed to complement the paleoseismological record. We study Nepal's Pokhara basin, which has the largest and most extensively dated archive of earthquake-triggered valley fills in the Himalayas. These sediments form a 148-km2 fan that issues from the steep Seti Khola gorge in the Annapurna Massif, invading and plugging 15 tributary valleys with tens of meters of debris, and impounding several lakes. Nearly a dozen new radiocarbon ages corroborate at least three episodes of catastrophic sedimentation on the fan between ∼700 and ∼1700 AD, coinciding with great earthquakes in ∼1100, 1255, and 1344 AD, and emplacing roughly >5 km3 of debris that forms the Pokhara Formation. We offer a first systematic sedimentological study of this formation, revealing four lithofacies characterized by thick sequences of mid-fan fluvial conglomerates, debris-flow beds, and fan-marginal slackwater deposits. New geochemical provenance analyses reveal that these upstream dipping deposits of Higher Himalayan origin contain lenses of locally derived river clasts that mark time gaps between at least three major sediment pulses that buried different parts of the fan. The spatial pattern of 14C dates across the fan and the provenance data are key to distinguishing these individual sediment pulses, as these are not evident from their sedimentology alone. Our study demonstrates how geomorphic and sedimentary evidence of catastrophic valley infill can help to independently verify and augment paleoseismological fault-trench records of great Himalayan earthquakes, while offering unparalleled insights into their long-term geomorphic impacts on major drainage basins.
•We review the largest and most extensively dated archive of earthquake-triggered valley fills in the Lesser Himalayas.•Radiocarbon dates indicate at least three pulses of catastrophic sedimentation following great medieval Himalayan earthquakes.•Sedimentological features and geochemical analysis independently identify these aggradation events.•Evidence for long-runout (70 km) debris flows from High Himalayan Crystalline Sequence.
spp. cause infections (scedosporiosis) in both immunocompetent and immunocompromised individuals and may persistently colonize the respiratory tract in patients with cystic fibrosis (CF). They are ...less susceptible against azoles than are other molds, such as
spp., suggesting the presence of resistance mechanisms. It can be hypothesized that the decreased susceptibility of
spp. to azoles is also CYP51 dependent. Analysis of the
and
genomes revealed one
gene encoding the 14-α-lanosterol demethylase. This gene from 159 clinical or environmental
isolates and three
isolates has been sequenced and analyzed. The
CYP51 protein clustered with the group of known CYP51B orthologues and showed species-specific polymorphisms. A tandem repeat in the 5' upstream region of
like that in
could not be detected. Species-specific amino acid alterations in CYP51 of
,
,
, and
isolates were located at positions that have not been described as having an impact on azole susceptibility. In contrast, two of the three
specific amino acid changes (Y136F and G464S) corresponded to respective mutations in
CYP51A at amino acid positions 121 and 448 (Y121F and G448S, respectively) that had been linked to azole resistance.
Increasing research has incorporated bioactive glass nanoparticles (BGN) and electric field (EF) stimulation for bone tissue engineering and regeneration applications. However, their interplay and ...the effects of different EF stimulation regimes on osteogenic differentiation of human mesenchymal stem cells (hMSC) are less investigated. In this study, we introduced EF with negligible magnetic field strength through a well-characterized transformer-like coupling (TLC) system, and applied EF disrupted (4/4) or consecutive (12/12) regime on type I collagen (Col) coatings with/without BGN over 28 days. Additionally, dexamethasone was excluded to enable an accurate interpretation of BGN and EF in supporting osteogenic differentiation. Here, we demonstrated the influences of BGN and EF on collagen topography and maintaining coating stability. Coupled with the release profile of Si ions from the BGN, cell proliferation and calcium deposition were enhanced in the Col-BGN samples after 28 days. Further, osteogenic differentiation was initiated as early as d 7, and each EF regime was shown to activate distinct pathways. The disrupted (4/4) regime was associated with the BMP/Smad4 pathways that up-regulate Runx2/OCN gene expression on d 7, with a lesser effect on ALP activity. In contrast, the canonical Wnt/β-Catenin signaling pathway activated through mechanotransduction cues is associated with the consecutive (12/12) regime, with significantly elevated ALP activity and Sp7 gene expression reported on d 7. In summary, our results illustrated the synergistic effects of BGN and EF in different stimulation regimes on osteogenic differentiation that can be further exploited to enhance current bone tissue engineering and regeneration approaches.
The unique release mechanisms of silica from bioactive glass nanoparticles (BGN) were coupled with pulsatile electric field (EF) stimulation to support hMSC osteogenic differentiation, in the absence of dexamethasone. Furthermore, the interplay with consecutive (12/12) and disrupted (4/4) stimulation regimes was investigated. The reported physical, mechanical and topographical effects of BGN and EF on the collagen coating, hMSC and the distinct progression of osteogenic differentiation (canonical Wnt/β-Catenin and BMP/Smad) triggered by respective stimulation regime were not explicitly reported previously. These results provide the fundamentals for further exploitations on BGN composites with metal ions and rotation of EF regimes to enhance osteogenic differentiation. The goal is sustaining continual osteogenic differentiation and achieving a more physiologically-relevant state and bone constructs in vitro.
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One of the key challenges in osteochondral tissue engineering is to define specified zones with varying material properties, cell types and biochemical factors supporting locally adjusted ...differentiation into the osteogenic and chondrogenic lineage, respectively. Herein, extrusion-based core-shell bioprinting is introduced as a potent tool allowing a spatially defined delivery of cell types and differentiation factors TGF-β3 and BMP-2 in separated compartments of hydrogel strands, and, therefore, a local supply of matching factors for chondrocytes and osteoblasts. Ink development was based on blends of alginate and methylcellulose, in combination with varying concentrations of the nanoclay Laponite whose high affinity binding capacity for various molecules was exploited. Release kinetics of model molecules was successfully tuned by Laponite addition. Core-shell bioprinting was proven to generate well-oriented compartments within one strand as monitored by optical coherence tomography in a non-invasive manner. Chondrocytes and osteoblasts were applied each in the shell while the respective differentiation factors (TGF-β3, BMP-2) were provided by a Laponite-supported core serving as central factor depot within the strand, allowing directed differentiation of cells in close contact to the core. Experiments with bi-zonal constructs, comprising an osteogenic and a chondrogenic zone, revealed that the local delivery of the factors from the core reduces effects of these factors on the cells in the other scaffold zone. These observations prove the general suitability of the suggested system for co-differentiation of different cell types within a zonal construct.
Growth folds developed at rift margin transfer zones are mostly studied for their structural style and stratal architecture. The influence of the growth of these folds on the syn-rift sedimentary ...facies development, however, remains less documented. To address this, we investigated the syn-rift facies of the Lower Miocene Rudeis Formation in relation to the growth of structures within the North Baba transfer zone at the eastern margin of the Suez rift. Sedimentological analysis defined seven facies associations in three successive depositional systems: (i) gravel-rich submarine fans with up-dip thinning geometry, comprising upper, middle and lower fan facies associations, (ii) a gravitationally modified Gilbert-type fan delta, with tangential foreset and bottomset. Both fan systems pass gradationally into basinal marl-dominated facies, and (iii) shoreface-to-offshore. Paleocurrent measurements and the spatial facies variability indicate that the North Baba transfer zone was acting as the main sediment fairway conveying the sediments southwards from the adjacent rift shoulder to the Baba hangingwall syncline. Mapping of key stratal surfaces and growth structures helped in distinguishing four lower Rudeis stratal units, which provide a framework for studying the spatial and vertical relationships among the recognized facies. Deposition of these stratal units was influenced by progressive steepening and localized faulting of a fault-tip growth monocline above the North Baba transfer zone and a submerged intrabasinal high. These structures controlled the hangingwall gradient, bathymetry, flow pathways and facies relations, triggered multiple gravity flows and slumps, and created terraces on which patch reefs and localized shoreface deposits intermittently developed. This study reveals that the transfer zone was still acting as growth monocline even during the rift-climax. The coeval deposits were mainly sourced from a hinterland drainage catchment rather than local footwall scarps. They form progressively tilted clastic wedges that onlap and pinch out up-dip toward the growth monocline, unlike those observed at faulted margins where the facies commonly thicken toward the fault.
The early exhumation history of the Tauern Window in the European Eastern Alps and its surface expression is poorly dated and quantified, partly because thermochronological and provenance information ...are sparse from the Upper Austrian Northern Alpine Foreland Basin. For the first time, we combine a single‐grain double‐dating approach (Apatite Fission Track and U‐Pb dating) with trace‐element geochemistry analysis on the same apatites to reconstruct the provenance and exhumation history of the late Oligocene/early Miocene Eastern Alps. The results from 22 samples from the Chattian to Burdigalian sedimentary infill of the Upper Austrian Northern Alpine Foreland Basin were integrated with a 3D seismic‐reflection data set and published stratigraphic reports. Our highly discriminative data set indicates an increasing proportion of apatites (from 6% to 23%) with Sr/Y values <0.1 up‐section and an increasing amount of apatites (from 24% to 38%) containing >1,000 ppm light rare‐earth elements from Chattian to Burdigalian time. The number of U‐Pb ages with acceptable uncertainties increases from 40% to 59% up‐section, with mostly late Variscan/Permian ages, while an increasing number of grains (10%–27%) have Eocene or younger apatite fission track cooling ages. The changes in the apatite trace‐element geochemistry and U‐Pb data mirror increased sediment input from an ≥upper amphibolite‐facies metamorphic source of late Variscan/Permian age – probably the Ötztal‐Bundschuh nappe system – accompanied by increasing exhumation rates indicated by decreasing apatite fission track lag times. We attribute these changes to the surface response to upright folding and doming in the Penninic units of the future Tauern Window starting at 29–27 Ma. This early period of exhumation (0.3–0.6 mm/a) is triggered by early Adriatic indentation along the Giudicarie Fault System.
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