7196 research outputs found
Sort by
CellSIUS provides sensitive and specific detection of rare cell populations from complex single cell RNA-seq data
No full textWe develop CellSIUS (Cell Subtype Identification from Upregulated gene Sets) to fill a methodology gap for rare cell population identification for scRNA-seq data. CellSIUS outperforms existing algorithms for specificity and selectivity for rare cell types and their transcriptomic signature identification in synthetic and complex biological data. Characterization of a human pluripotent cell differentiation protocol recapitulating deep-layer corticogenesis using CellSIUS reveals unrecognized complexity in human stem cell-derived cellular populations. CellSIUS enables identification of novel rare cell populations and their signature genes providing the means to study those populations in vitro in light of their role in health and disease
The EBI2-oxysterol axis promotes the development of intestinal lymphoid structures and colitis
No full textThe gene encoding for Epstein-Barr virus-induced G-protein coupled receptor 2 (EBI2) is a risk gene for inflammatory bowel disease (IBD). Together with its oxysterol ligand 7α,25-dihydroxycholesterol, EBI2 mediates migration and differentiation of immune cells. However, the role of EBI2 in the colonic immune system remains insufficiently studied.
We found increased mRNA expression of EBI2 and oxysterol synthesizing enzymes (CH25H, CYP7B1) in the inflamed colon of patients with ulcerative colitis and mice with acute or chronic dextran sulfate sodium (DSS) colitis. Accordingly, we detected elevated extraintestinal levels of 25-hydroxylated oxysterols, including 7α,25-dihydroxycholesterol in mice with acute colonic inflammation. Knockout of EBI2 or CH25H did not affect severity of DSS colitis; however, inflammation was decreased in male EBI2-/- mice in the IL-10 colitis model.
The colonic immune system comprises mucosal lymphoid structures, which accumulate upon chronic inflammation in IL-10-deficient mice and in chronic DSS colitis. However, EBI2-/- mice formed significantly less colonic lymphoid structures at baseline and showed defects in inflammation-induced accumulation of lymphoid structures.
In summary, we report induction of the EBI2-7α,25-dihydroxycholesterol axis in colitis and a role of EBI2 for the accumulation of lymphoid tissue during homeostasis and inflammation. These data implicate the EBI2-7α,25-dihydroxycholesterol axis in IBD pathogenesis
Multifunctional carrier based on halloysite/laponite hybrid hydrogel for kartogenin delivery
No full textA novel carrier system based on halloysite nanotubes (HNT), for the potential intraarticular delivery of kartogenin (KGN)
by means laponite (Lap) hydrogel (HNT/KGN/Lap), is developed. The drug was first loaded into HNT, and the hybrid composite obtained was used as filler for laponite hydrogel. Both the filler and the hydrogel were thoroughly investigated by several techniques and the hydrogel morphology was imaged by transmission electron microscopy. Furthermore, the gelating ability of laponite in the presence of the filler and the rheological properties of the hybrid hydrogel were also investigated. The kinetic release of kartogenin from HNT and HNT/Lap hybrid hydrogel was studied both in
physiological conditions and in ex vivo synovial fluid. In the last case, the kinetic results highlighted that HNT carrier can effectively release KGN in a sustained manner for at least 38 days. Finally, a preliminary biological assays showed that the HNT/KGN/Lap hybrid hydrogel did not exhibit any cytotoxic effect
Orthogonal redundant monitoring of a new continuous fluid bed dryer for pharmaceutical processing by means of mass and energy balance calculations and spectroscopic techniques
No full textIn line with the ongoing shift from batch- to continuous pharmaceutical production of solid oral dosage forms, a novel continuous fluid bed drying system was developed. The forced-feed nature of the Glatt GPCG2 CM fluid bed dryer allows continuous, first-in-first-out drying of wet granulate materials based on its compartmentalized, rotating fluidizing chamber. The design ensures a narrow residence time distribution with limited risk of carryover from one compartment to the adjacent ones. Drying efficiency can be adapted through temperature, air flow and especially dryer rotation speed, a process parameter that is unique to the described system and allows fast and accurate process control.
The presented work aims to introduce the dryer’s functionalities in detail, and to demonstrate that the compartmentalized, rotating fluid bed chambers facilitates a stable, continuous drying behavior, which in turn ensures robust and repeatable residual moisture contents (loss-in-drying; LOD) of the discharged granules. Furthermore, a thorough mass- and energy balance (MEB) for the prediction of granules LOD is derived, based on the constantly logged process values provided by the granulating and drying unit. The resulting formulas are applied to two independent test-experiments, in order to demonstrate that precise LOD prediction in real-time is achievable by MEB as an orthogonal PAT method to common Near Infrared Spectroscopy (NIRS). On average, MEB results differed by 0.36 % LOD (absolute) from offline reference analyses, and by 0.61 % LOD from predictions made with an in-house available NIRS method. Furthermore, a good correlation between the observed (based on inlet- and exhaust air temperature measurements) and the expected thermal energy loss (caused by evaporation) in the dryer was found. Since the derived MEB is solely based on physical principles, it is product independent and can be transferred to any other material to be processed on the described equipment
A lipoprotein lipase --GPI-anchored high density lipoprotein binding protein 1 fusion lowers triglycerides in mice: implications for managing familial chylomicronemia syndrome
No full textLipoprotein lipase (LPL) is central to triglyceride metabolism. Severely compromised LPL activity causes familial chylomicronemia syndrome (FCS), which is associated with very high plasma triglyceride levels and increased risk of life-threatening pancreatitis. Currently, no approved pharmacological intervention can acutely lower plasma triglycerides in FCS. Low yield, high aggregation, and poor stability of recombinant LPL have thus far prevented development of enzyme replacement therapy. Recently, we showed that LPL monomers form 1:1 complexes with the LPL transporter glycosylphosphatidylinositol anchored high density lipoprotein binding protein 1 (GPIHBP1) and solved the structure of the complex. In the present work, we further characterized the monomeric LPL/GPIHBP1 complex, and its derivative, the LPL-GPIHBP1 fusion protein, with the goal of contributing to the development of an LPL enzyme replacement therapy. Fusion of LPL to GPIHBP1 increased yields of recombinant LPL, prevented LPL aggregation, stabilized LPL against spontaneous inactivation, and made it resistant to inactivation by the LPL antagonists angiopoietin-like protein 3(ANGPTL3) or ANGPTL4. The high stability of the fusion protein enabled us to identify LPL amino acids that interact with ANGPTL4. Additionally, the LPL-GPIHBP1 fusion protein exibited high enzyme activity in in vitro assays. Importantly, both intravenous and subcutaneous administrations of the fusion protein lowered triglycerides in several mouse strains without causing adverse effects. These results indicate that the LPL-GPIHBP1 fusion protein has potential for use as a therapeutic for managing FC
Dynamic behaviors of α-synuclein and tau in the cellular context: New mechanistic insights and therapeutic opportunities in neurodegeneration
No full textα-Synuclein (αS) and tau have a lot in common. Dyshomeostasis and aggregation of both proteins are central in the pathogenesis of neurodegenerative diseases: Parkinson's disease, dementia with Lewy bodies, multi-system atrophy and other ‘synucleinopathies’ in the case of αS; Alzheimer's disease, frontotemporal dementia, progressive supranuclear palsy and other ‘tauopathies’ in the case of tau. The aggregated states of αS and tau are found to be (hyper)phosphorylated, but the relevance of the phosphorylation in health or disease is not well understood. Both tau and αS are typically characterized as ‘intrinsically disordered’ proteins, while both engage in transient interactions with cellular components, thereby undergoing structural changes and context-specific folding. αS transiently binds to (synaptic) vesicles forming a membrane-induced amphipathic helix; tau transiently interacts with microtubules forming an ‘extended structure’. The regulation and exact nature of the interactions are not fully understood. Here we review recent and previous insights into the dynamic, transient nature of αS and tau with regard to the mode of interaction with their targets, the dwell-time while bound, and the cis and trans factors underlying the frequent switching between bound and unbound states. These aspects are intimately linked to hypotheses on how subtle changes in the transient behaviors may trigger the earliest steps in the pathogenesis of the respective brain diseases. Based on a deeper understanding of transient αS and tau conformations in the cellular context, new therapeutic strategies may emerge, and it may become clearer why existing approaches have failed or how they could be optimized
Pharmacological characterization of a novel 5-hydroxybenzothiazolone (5-HOB) derived β2 adrenoceptor agonist with functional selectivity for anabolic effects on skeletal muscle resulting in a wider cardiovascular safety window in preclinical studies
No full textThe anabolic effects of β2-adrenoceptor (β2-AR) agonists on skeletal muscle have been demonstrated in various species, including man. However, the clinical use of β2-AR agonists for skeletal muscle wasting conditions has been limited by their undesired cardiovascular effects. Here, we describe the preclinical pharmacological profile of a novel 5-hydroxybenzothiazolone (5-HOB) derived β2-AR agonist, (R)-7-(2-(1-(4-butoxyphenyl)-2-methylpropan-2-ylamino)-1-hydroxyethyl)-5-hydroxybenzo[d]thiazol-2(3H)-one, in comparison to formoterol as a representative β2-AR agonist which has been well characterized both clinically and preclinically. In vitro, 5-HOB has nanomolar affinity for the human β2-AR and functional selectivity over the β1-AR and β3-AR. 5-HOB also shows potent agonistic activity at the β2-AR in primary skeletal muscle myotubes isolated from rats, dogs, monkeys and humans, and induces hypertrophy of skeletal muscle myotubes. When compared to formoterol, 5-HOB demonstrates comparable full-agonist activity on cAMP production in skeletal muscle cells and skeletal muscle tissue derived membranes, in contrast a greatly reduced intrinsic activity was determined in cardiomyocytes and cell membranes prepared from the rat heart. In addition, 5-HOB shows weak effects on chronotropy in rabbit sinoatrial nodes, inotropy in guinea pig left atria, and vascular relaxation in rat aortic rings when compared to formoterol. In vivo, 5-HOB significantly increases hind limb muscle weight in rats at doses which do not affect heart weight and ejection fraction, unlike formoterol which has similar effects in both tissues. Furthermore, changes in cardiovascular parameters after bolus subcutaneous treatment in rats and rhesus monkeys are significantly lower with 5-HOB when compared to formoterol. In conclusion, the pharmacological profile of 5-HOB indicates that this compound has superior tissue selectivity compared to the conventional β2-AR agonist formoterol in preclinical studies, and supports the notion that such tissue-selective agonists should be investigated for the safe treatment of muscle wasting conditions without cardiovascular limiting effects
YAP, but Not RSPO-LGR4/5, Signaling in Biliary Epithelial Cells Promotes a Ductular Reaction in Response to Liver Injury
No full textThe liver has an intrinsically high capacity to regenerate involving several distinct cellular niches. Hepatic progenitor cells (HPCs) transiently appear around the portal vein following damage to support liver regeneration. Despite the clinical relevance of HPC-mediated liver regeneration, the regulatory mechanisms controlling HPC response are poorly understood. Liver organoids can be clonally expanded in vitro and differentiated into both hepatocytes and biliary cells, thereby displaying characteristics of bipotential HPCs. Here, we performed a CRISPR loss-of-function (LOF) screen in liver organoids, highlighting pathways regulating HPC expansion in vitro, followed by in vivo validation using drug-induced HPC response in transgenic mice. We show that the YAP/Hippo pathway is essential for HPC growth in vitro and liver-specific YAP1 LOF impairs HPC response in vivo. While Rspo-mediated Wnt/β-Catenin signalling is required for liver organoid formation and growth in vitro, blocking RSPO-induced Wnt/β-Catenin activation by Lgr4/5 LOF in vivo does not affect HPC response. Detailed spatiotemporal analysis of Wnt/β-Catenin reporter mice and single cell RNA sequencing (scRNA-Seq) of EpCam+ cells isolated during HPC response indicate absence of Wnt/β-Catenin signalling and no expression of the Rspo-Lgr4/5-Znrf3/Rnf43 module components, whereas YAP signalling is prominent in EpCam+ cells. However, periportal hepatocytes show Axin2, Lgr4 and Lgr5 expression during HPC response and Lgr4/5 LOF impairs resolution of HPC response during recovery, suggesting that Wnt/β-Catenin signalling in periportal hepatocytes is important for HPC response resolution and portal tract repair. Our data highlight time-sensitive requirement of growth signalling pathways in initiating and controlling key regulatory pathways of HPC response and liver regeneration
Defining the Dosage Strength for Labelling of DPIs: Use, Limitations and Relevance of in-vitro Data
No full textNo abstrac
PathwayMap: Molecular Pathway Association with Self-Normalizing Neural Networks
No full textDrug discovery suffers from high attrition because compounds initially deemed as promising can later show ineffectiveness or toxicity resulting from a poor understanding of their activity profile. In this work, we describe a deep self-normalizing neural network model for the prediction of molecular pathway association and evaluate its performance, showing an AUC ranging from 0.69 to 0.91 on a set of compounds extracted from ChEMBL and from 0.81 to 0.83 on an external data set provided by Novartis. We finally discuss the applicability of the proposed model in the domain of lead discovery. A usable application is available via PlayMolecule.org