Effects of single and combined low frequency electromagnetic fields and simulated microgravity on gene expression of human mesenchymal stem cells during chondrogenesis

Introduction: Low frequency electromagnetic fields (LF-EMF) and simulated microgravity (SMG) have been observed to affect chondrogenesis. A controlled bioreactor system was developed to apply LF-EMF and SMG singly or combined during chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in 3D culture. Material and methods: An external motor gear SMG bioreactor was combined with magnetic Helmholtz coils for EMF (5 mT;15 Hz). Pellets of hMSCs (+/- TGF-beta 3)were cultured (P5) under SMG, LF-EMF, LF-EMF/SMG and control (1 g) conditions for 3 weeks. Sections were stained with safranin-O and collagen type II. Gene expression was evaluated by microarray and real-time polymerase chain reaction analysis. Results: Simulated microgravity application significantly changed gene expression;specifically, COLXA1 but also COL2A1, which represents the chondrogenic potential, were reduced (p < 0.05). Low frequency electromagnetic fields application showed no gene expression changes on a microarray basis. LF-EMF/SMG application obtained significant different expression values from cultures obtained under SMG conditions with a re-increase of COL2A1, therefore rescuing the chondrogenic potential, which had been lowered by SMG. Conclusions: Simulated microgravity lowered hypertrophy but also the chondrogenic potential of hMSCs. Combined LF-EMF/SMG provided a rescue effect of the chondrogenic potential of hMSCs although no LF-EMF effect was observed under optimal conditions. The study provides new insights into how LF-EMF and SMG affect chondrogenesis of hMSCs and how they generate interdependent effects

WePass Störe am Eisernen Tor Wiederherstellung der Durchgängkeit an den Staustufen Iron Gate 1 & 2 in der Unteren Donau

Das Einzugsgebiet der 2.857 km langen Donau ist mit einer Gesamtfläche von 801.463 km² - rund 10% des europäischen Festlands - das zweitgrößte Flusseinzugsgebiet in Europa. Es erstreckt sich über 19 Länder und ist damit das internationalste Flusseinzugsgebiet der Welt. Über 100 Fischarten sind in der Donau nachgewiesen, darunter vier hochgradig gefährdete Störarten, die charakteristischen Flaggschiffarten der Donau (SCHIEMER ET AL. 2004). Die diadromen Donaufischarten (z. B. Störe (Acipenser spp.), Pontischer Maifisch (Alosa immaculata), Asowscher Maifisch (Alosa tanaica) und Schwarzmeerforelle (Salmo labrax)) sind zwingend darauf angewiesen ihren Lebenszyklus im Süß- und Salzwasser zu vollziehen. Einige Arten wandern bzw. wanderten dabei über sehr große Distanzen, beispielsweise der Beluga-Stör (Huso huso) vom Schwarzen Meer bis zu den Laichgründen >2.500 km ins Landesinnere (SCHMALL & FRIEDRICH 2014). Aber auch potamodrome Arten migrieren in der Donau über weite Strecken; für Döbel (Leuciscus cephalus), Barbe (Barbus barbus) und Nase (Chondrostoma nasus) sind z. B. Wanderungen über 169, 318 und 446 km nachgewiesen (STEINMANN ET AL. 1937, VAN TREECK ET AL. 2022). Die Fischartenzusammensetzung und der Fischbestand sind wichtige Indikatoren für die Gewässerqualität der Donau. Die Ergebnisse des jüngsten Joint Danube Survey 4 zeigen, dass die Fischartengemeinschaft entlang des gesamten Flusslaufs bedroht ist. Andererseits ist die Vielfalt der Fischtaxa immer noch auf einem Niveau, als dass wirksame Gewässerentwicklungsmaßnahmen dazu beitragen können den ökologischen Zustand bzw. das ökologische Potential zu verbessern und die Ziele der EG-WRRL zu erreichen (BĂNĂDUC ET AL. 2014, ICPDR 2021)

WEPASS Project - Making the Iron Gate Dams passable for migratory fish

European rivers are obstructed by more than one million barriers that have resulted in excessive loss of river continuity. On the main course of the Danube River there are 83 longitudinal continuity interruptions, out of which 65 dams are used for hydropower (ICPDR 2022a). The Iron Gate Hydropower and Navigation System is one of the largest river engineering projects undertaken in Europe, with the dams mainly built to provide hydropower and flood protection, and to facilitate navigation along the Danube. These infrastructures represent introduced barriers to fish migration. Hence, ensuring passage opportunities for fish at the Iron Gate dams is considered to be of major importance for the conservation of migratory fish populations in the Danube River basin. Restoration of river continuity at these sites would reopen an additional 900 km for migration up to the Gabčikovo dam, providing suitable habitats and spawning grounds along the Danube and its tributaries. Knowledge about fish behavior and movements in the vicinity of these river infrastructures is required to build effective up- and downstream passage facilities to allow the migration of fish species. To gain insight in the approach routes and aggregation areas a refined approach to acoustic telemetry is employed to support migration facilitation

Diagnosis and management of Cornelia de Lange syndrome:first international consensus statement

Cornelia de Lange syndrome (CdLS) is an archetypical genetic syndrome that is characterized by intellectual disability, well-defined facial features, upper limb anomalies and atypical growth, among numerous other signs and symptoms. It is caused by variants in any one of seven genes, all of which have a structural or regulatory function in the cohesin complex. Although recent advances in next-generation sequencing have improved molecular diagnostics, marked heterogeneity exists in clinical and molecular diagnostic approaches and care practices worldwide. Here, we outline a series of recommendations that document the consensus of a group of international experts on clinical diagnostic criteria, both for classic CdLS and non-classic CdLS phenotypes, molecular investigations, long-term management and care planning

Fibroblast-like cells change gene expression of bone remodelling markers in transwell cultures

Introduction Periprosthetic fibroblast-like cells (PPFs) play an important role in aseptic loosening of arthroplasties. Various studies have examined PPF behavior in monolayer culture systems. However, the periprosthetic tissue is a three-dimensional (3D) mesh, which allows the cells to interact in a multidirectional way. The expression of bone remodeling markers of fibroblast-like cells in a multilayer environment changes significantly versus monolayer cultures without the addition of particles or cytokine stimulation. Gene expression of bone remodeling markers was therefore compared in fibroblast-like cells from different origins and dermal fibroblasts under transwell culture conditions versus monolayer cultures. Methods PPFs from periprosthetic tissues (n = 12), osteoarthritic (OA) synovial fibroblast-like cells (SFs) (n = 6), and dermal fibroblasts (DFs) were cultured in monolayer (density 5.5 × 103/cm2) or multilayer cultures (density 8.5 × 105/cm2) for 10 or 21 days. Cultures were examined via histology, TRAP staining, immunohistochemistry (anti-S100a4), and quantitative real-time PCR. Results Fibroblast-like cells (PPFs/SFs) and dermal fibroblasts significantly increased the expression of RANKL and significantly decreased the expression of ALP, COL1A1, and OPG in multilayer cultures. PPFs and SFs in multilayer cultures further showed a higher expression of cathepsin K, MMP-13, and TNF-α. In multilayer PPF cultures, the mRNA level of TRAP was also found to be significantly increased. Conclusion The multilayer cultures are able to induce significant expression changes in fibroblast-like cells depending on the nature of cellular origin without the addition of any further stimulus. This system might be a useful tool to get more in vivo like results regarding fibroblast-like cell cultures

Dental and Orthopaedic Implant Loosening: Overlap in Gene Expression Regulation

Objectives: Endoprosthetic loosening still plays a major role in orthopaedic and dental surgery and includes various cellular immune processes within peri-implant tissues. Although the dental and orthopaedic processes vary in certain parts, the clinical question arises whether there are common immune regulators of implant loosening. Analyzing the key gene expressions common to both processes reveals the mechanisms of osteoclastogenesis within periprosthetic tissues of orthopaedic and dental origin. Methods: Donor peripheral blood mononuclear cells (PBMCs) and intraoperatively obtained periprosthetic fibroblast-like cells (PPFs) were (co-)cultured with [± macrophage-colony stimulating factor (MCSF) and Receptor Activator of NF-κB ligand (RANKL)] in transwell and monolayer culture systems and examined for osteoclastogenic regulations [MCSF, RANKL, osteoprotegerin (OPG), and tumor necrosis factor alpha (TNFα)] as well as the ability of bone resorption. Sequencing analysis compared dental and orthopaedic (co-)cultures. Results: Monolayer co-cultures of both origins expressed high levels of OPG, resulting in inhibition of osteolysis shown by resorption assay on dentin. The high OPG-expression, low RANKL/OPG ratios and a resulting inhibition of osteolysis were displayed by dental and orthopaedic PPFs in monolayer even in the presence of MCSF and RANKL, acting as osteoprotective and immunoregulatory cells. The osteoprotective function was only observed in monolayer cultures of dental and orthopaedic periprosthetic cells and downregulated in the transwell system. In transwell co-cultures of PBMCs/PPFs profound changes of gene expression, with a significant decrease of OPG (20-fold dental versus 100 fold orthopaedic), were identified. Within transwell cultures, which offer more in vivo like conditions, RANKL/OPG ratios displayed similar high levels to the original periprosthetic tissue. For dental and orthopaedic implant loosening, overlapping findings in principal component and heatmap analysis were identified. Conclusions: Thus, periprosthetic osteoclastogenesis may be a correlating immune process in orthopaedic and dental implant failure leading to comparable reactions with regard to osteoclast formation. The transwell cultures system may provide an in vivo like model for the exploration of orthopaedic and dental implant loosening

Impact of Periprosthetic Fibroblast-Like Cells on Osteoclastogenesis in Co-Culture with Peripheral Blood Mononuclear Cells Varies Depending on Culture System

Co-culture studies investigating the role of periprosthetic fibroblasts (PPFs) in inflammatory osteoclastogenesis reveal contrary results, partly showing an osteoprotective function of fibroblasts and high OPG expression in monolayer. These data disagree with molecular analyses of original periosteolytic tissues. In order to find a more reliable model, PPFs were co-cultivated with peripheral blood mononuclear cells (PBMCs) in a transwell system and compared to conventional monolayer cultures. The gene expression of key regulators of osteoclastogenesis (macrophage colony-stimulating factor (MCSF), receptor activator of NF-&#954;B ligand (RANK-L), osteoprotegerin (OPG), and tumor necrosis factor alpha (TNF&#945;)) as well as the ability of bone resorption were analyzed. In monolayer co-cultures, PPFs executed an osteoprotective function with high OPG-expression, low RANK-L/OPG ratios, and a resulting inhibition of osteolysis even in the presence of MCSF and RANK-L. For transwell co-cultures, profound changes in gene expression, with a more than hundredfold decrease of OPG and a significant upregulation of TNF&#945; were observed. In conclusion, we were able to show that a change of culture conditions towards a transwell system resulted in a considerably more osteoclastogenic gene expression profile, being closer to findings in original periosteolytic tissues. This study therefore presents an interesting approach for a more reliable in vitro model to examine the role of fibroblasts in periprosthetic osteoclastogenesis in the future

Immobilization of Denosumab on Titanium Affects Osteoclastogenesis of Human Peripheral Blood Monocytes

Immobilization of proteins has been examined to improve implant surfaces. In this study, titanium surfaces were modified with nanofunctionalized denosumab (cDMAB), a human monoclonal anti-RANKL IgG. Noncoding DNA oligonucleotides (ODN) served as linker molecules between titanium and DMAB. Binding and release experiments demonstrated a high binding capacity of cDMAB and continuous release. Human peripheral mononuclear blood cells (PBMCs) were cultured in the presence of RANKL/MCSF for 28 days and differentiated into osteoclasts. Adding soluble DMAB to the medium inhibited osteoclast differentiation. On nanofunctionalized titanium specimens, the osteoclast-specific TRAP5b protein was monitored and showed a significantly decreased amount on cDMAB-titanium in PBMCs + RANKL/MCSF. PBMCs on cDMAB-titanium also changed SEM cell morphology. In conclusion, the results indicate that cDMAB reduces osteoclast formation and has the potential to reduce osteoclastogenesis on titanium surfaces

Affirmative Cue Words in Task-Oriented Dialogue

We present a series of studies of affirmative cue words-a family of cue words such as "okay" or "alright" that speakers use frequently in conversation. These words pose a challenge for spoken dialogue systems because of their ambiguity: They may be used for agreeing with what the interlocutor has said, indicating continued attention, or for cueing the start of a new topic, among other meanings. We describe differences in the acoustic/prosodic realization of such functions in a corpus of spontaneous, task-oriented dialogues in Standard American English. These results are important both for interpretation and for production in spoken language applications. We also assess the predictive power of computational methods for the automatic disambiguation of these words. We find that contextual information and final intonation figure as the most salient cues to automatic disambiguation. © 2012 Association for Computational Linguistics.Fil: Gravano, Agustin. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hirschberg, Julia. Columbia University; Estados UnidosFil: B?nu?, Stefan. Institute Of Informatics Slovak Academy Of Sciences; Eslovaqui