MicroRNAs are regulators of gene expression. A wide-spread, yet not validated, assumption is that the targetome of miRNAs is non-randomly distributed across the transcriptome and that targets share functional pathways. We developed a computational and experimental strategy termed high-throughput miRNA interaction reporter assay (HiTmIR) to facilitate the validation of target pathways. First, targets and target pathways are predicted and prioritized by computational means to increase the specificity and positive predictive value. Second, the novel webtool miRTaH facilitates guided designs of reporter assay constructs at scale. Third, automated and standardized reporter assays are performed. We evaluated HiTmIR using miR-34a-5p, for which TNF- and TGFB-signaling, and Parkinson's Disease (PD)-related categories were identified and repeated the pipeline for miR-7-5p. HiTmIR validated 58.9% of the target genes for miR-34a-5p and 46.7% for miR-7-5p. We confirmed the targeting by measuring the endogenous protein levels of targets in a neuronal cell model. The standardized positive and negative targets are collected in the new miRATBase database, representing a resource for training, or benchmarking new target predictors. Applied to 88 target predictors with different confidence scores, TargetScan 7.2 and miRanda outperformed other tools. Our experiments demonstrate the efficiency of HiTmIR and provide evidence for an orchestrated miRNA-gene targeting.

These findings open a window of opportunity for the sensitive and specific early diagnosis of AS. Our results increase the potential for larger scale evaluation of an increased number of patients.

The human blood–brain barrier (BBB) represents the interface of microvasculature and the central nervous system, regulating the transport of nutrients and protecting the brain from external threats. To gain a deeper understanding of (patho)physiological processes affecting the BBB, sophisticated models mimicking the in vivo situation are required. Currently, most in vitro models are cultivated on stiff, semipermeable, and non-biodegradable Transwell® membrane inserts, not adequately mimicking the complexity of the extracellular environment of the native human BBB. To overcome these disadvantages, we developed three-dimensional electrospun scaffolds resembling the natural structure of the human extracellular matrix. The polymer fibers of the scaffold imitate collagen fibrils of the human basement membrane, exhibiting excellent wettability and biomechanical properties, thus facilitating cell adhesion, proliferation, and migration. Cultivation of human induced pluripotent stem cells (hiPSCs) on these scaffolds enabled the development of a physiological BBB phenotype monitored via the formation of tight junctions and validated by the paracellular permeability of sodium fluorescein, further accentuating the non-linearity of TEER and barrier permeability. The novel in vitro model of the BBB forms a tight endothelial barrier, offering a platform to study barrier functions in a (patho)physiologically relevant context.

In vitro model systems of the blood–brain barrier (BBB) play an essential role in pharmacological research, specifically during the development and preclinical evaluation of new drug candidates. Within the past decade, the trend in research and further development has moved away from models based on primary cells of animal origin towards differentiated models derived from human induced pluripotent stem cells (hiPSs). However, this logical progression towards human model systems from renewable cell sources opens up questions about the transferability of results generated in the primary cell models. In this study, we have evaluated both models with identical experimental parameters and achieved a directly comparable characterisation showing no significant differences in protein expression or permeability even though the achieved transendothelial electrical resistance (TEER) values showed significant differences. In the course of this investigation, we also determined a significant deviation of both model systems from the in vivo BBB circumstances, specifically concerning the presence or absence of serum proteins in the culture media. Thus, we have further evaluated both systems when confronted with an in vivo-like distribution of serum and found a notable improvement in the differential permeability of hydrophilic and lipophilic compounds in the hiPS-derived BBB model. We then transferred this model into a microfluidic setup while maintaining the differential serum distribution and evaluated the permeability coefficients, which showed good comparability with values in the literature. Therefore, we have developed a microfluidic hiPS-based BBB model with characteristics comparable to the established primary cell-based model.

Prodigiosenes are a family of red pigments with versatile biological activity. Their tripyrrolic core structure has been modified many times in order to manipulate the spectrum of activity. We have been looking systematically at prodigiosenes substituted at the C ring with alkyl chains of different lengths, in order to assess the relevance of this substituent in a context that has not been investigated before for these derivatives: Cu(II) complexation, DNA binding, self-activated DNA cleavage, photoinduced cytotoxicity and antimicrobial activity. Our results indicate that the hydrophobic substituent has a clear influence on the different aspects of their biological activity. The cytotoxicity study of the Cu(II) complexes of these prodigiosenes shows that they exhibit a strong cytotoxic effect towards the tested tumor cell lines. The Cu(II) complex of a prodigiosene lacking any alkyl chain excelled in its photoinduced anticancer activity, thus demonstrating the potential of prodigiosenes and their metal complexes for an application in photodynamic therapy (PDT). Two derivatives along with their Cu(II) complexes showed also antimicrobial activity against Staphylococcus aureus strains.

Human pluripotent stem cell‐derived cardiomyocytes (hPSC‐CMs) represent a valuable tool for in vitro modeling of the cardiac niche and possess great potential in tissue engineering applications. However, conventional polystyrene‐based cell culture substrates have adverse effects on cardiomyocytes in vitro due to the stress applied by a stiff substrate on contractile cells. Ultra‐high viscosity alginates offer a unique versatility as tunable substrates for cardiac cell cultures due to their biocompatibility, flexible biofunctionalization, and stability. In this work, we analyzed the effect of alginate substrates on hPSC‐CM maturity and functionality. Alginate substrates in high‐throughput compatible culture formats fostered a more mature gene expression and enabled the simultaneous assessment of chronotropic and inotropic effects upon beta‐adrenergic stimulation. Furthermore, we produced 3D‐printed alginate scaffolds with differing mechanical properties and plated hPSC‐CMs on the surface of these to create Heart Patches for tissue engineering applications. These exhibited synchronous macro‐contractions in concert with more mature gene expression patterns and extensive intracellular alignment of sarcomeric structures. In conclusion, the combination of biofunctionalized alginates and human cardiomyocytes represents a valuable tool for both in vitro modeling and regenerative medicine, due to its beneficial effects on cardiomyocyte physiology, the possibility to analyze cardiac contractility, and its applicability as Heart Patches.

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