搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

Nuclear exclusion of the RNA- and DNA-binding protein TDP-43 can induce neurodegeneration in different diseases. Diverse processes have been implicated to influence TDP-43 mislocalization,including disrupted nucleocytoplasmic transport (NCT); however,the physiological pathways that normally ensure TDP-43 nuclear localization are unclear. The six-transmembrane enzyme glycerophosphodiester phosphodiesterase 2 (GDE2 or GDPD5) cleaves the glycosylphosphatidylinositol (GPI) anchor that tethers some proteins to the membrane. Here we show that GDE2 maintains TDP-43 nuclear localization by regulating the dynamics of canonical Wnt signaling. Ablation of GDE2 causes aberrantly sustained Wnt activation in adult neurons,which is sufficient to cause NCT deficits,nuclear pore abnormalities,and TDP-43 nuclear exclusion. Disruption of GDE2 coincides with TDP-43 abnormalities in postmortem tissue from patients with amyotrophic lateral sclerosis (ALS). Further,GDE2 deficits are evident in human neural cell models of ALS,which display erroneous Wnt activation that,when inhibited,increases mRNA levels of genes regulated by TDP-43. Our study identifies GDE2 as a critical physiological regulator of Wnt signaling in adult neurons and highlights Wnt pathway activation as an unappreciated mechanism contributing to nucleocytoplasmic transport and TDP-43 abnormalities in disease. Synopsis Nuclear exclusion of TDP-43 is observed in various pathologies,but the physiological mechanisms that ensure its nuclear localization are not well-known. This work shows that inhibition of persistent Wnt activation in neurons by GDE2 prevents TDP-43 nuclear exclusion. GDE2 inhibits canonical Wnt signaling in adult postmitotic neurons.Sustained activation of canonical Wnt signaling in neurons disrupts the nuclear pore complex,impairs nucleocytoplasmic transport,and results in TDP-43 nuclear exclusion.iPS neurons from patients with C9orf72 ALS show decreased GDE2 expression and increased activation of canonical Wnt signaling.Inhibition of Wnt activation mitigates TDP-43 dysfunction in C9orf72 iPS neurons. GDE2 maintains TDP-43 nuclear localization by inhibiting Wnt activation in neurons. View Publication

The neutralization of integrin α4 (Itga4) is currently used as treatment in multiple sclerosis. Although most studies have focused on its function on lymphocyte migration to the CNS,we have uncovered the importance of Itga4 for the generation of regulatory B cells in peripheral immune organs and their control of pathogenic T cell response and CNS pathology. Our study underscores the importance of looking at the dual role of B cells in CNS autoimmunity and provides important perspectives regarding the efficacy and side effects associated with Itga4 neutralization and other B cell-targeting therapies. View Publication

Delivering macromolecules across biological barriers such as the blood–brain barrier limits their application in vivo. Previous work has demonstrated that Toxoplasma gondii,a parasite that naturally travels from the human gut to the central nervous system (CNS),can deliver proteins to host cells. Here we engineered T. gondii ’s endogenous secretion systems,the rhoptries and dense granules,to deliver multiple large (>100 kDa) therapeutic proteins into neurons via translational fusions to toxofilin and GRA16. We demonstrate delivery in cultured cells,brain organoids and in vivo,and probe protein activity using imaging,pull-down assays,scRNA-seq and fluorescent reporters. We demonstrate robust delivery after intraperitoneal administration in mice and characterize 3D distribution throughout the brain. As proof of concept,we demonstrate GRA16-mediated brain delivery of the MeCP2 protein,a putative therapeutic target for Rett syndrome. By characterizing the potential and current limitations of the system,we aim to guide future improvements that will be required for broader application. Subject terms: Parasitology,Biologics,Synthetic biology View Publication

Metastasis is one of the leading causes of cancer-related death worldwide. Fascin,a protein that bundles actin filaments to produce protrusions in cancer cells,plays a significant role in the enhancement of cell migration. This protein has been shown that the overexpression of this protein is related to the appearance of different types of cancer,such as colorectal cancer. In this study,we conducted in silico screening of the Enamine library,a compound library with a broad chemical space. Using a ligand-based virtual screening approach based on the pharmacophore model of G2,we identified the predicted inhibitors. First,these compounds were validated by physicochemical analysis. Differential scanning calorimetry (DSF) was used to study the binding between the predicted compounds and fascin protein,followed by an F-actin bundling assay to determine which compounds inhibited the bundling function of fascin. Z1362873773,which exhibited binding to fascin and inhibited F-actin bundling,was further tested in cell cultures to assess its effects on cancer cell viability and migration as well as in organoid models to evaluate potential cytotoxicity. Finally,we established a protocol that can be applied to discover anti-fascin agents from diverse compound libraries. A new molecule has been identified with considerable fascin inhibitory and migration-arresting capacity,which may lead to the development of new therapies to treat cancer. The online version contains supplementary material available at 10.1038/s41598-025-96457-x. Subject terms: Biochemistry,Biophysics,Cancer,Drug discovery,Molecular biology,Virtual drug screening View Publication

Suspension bath bioprinting,whereby bioinks are extruded into a yield stress bath with rapid recovery from shearing,has enabled the printing of low viscosity bioinks into constructs with high geometric complexity. Previous studies have often relied upon external stabilisation of the suspension bath (e.g. collagen) in order to culture soft materials without loss of printed structure. Here,we report a systematic investigation of suspension bath properties that support the printing,fusion,and culture of spheroid-based bioinks without added stabilisation. Specifically,agarose fluid gels of varied polymer concentrations and dilutions were produced and characterised morphologically and rheologically. Juvenile bovine chondrocytes or mesenchymal stromal cells (MSCs) were formed into spheroids of ∼150 µ m in diameter and investigated within agarose suspension baths either for their fusion in hanging drop cultures or as jammed bioinks. MSC spheroids were also printed when mixed with hydrogel microparticles to demonstrate additional versatility to the approach. Suspension baths of lower polymer concentrations and increased dilution enabled faster spheroid fusion; however,the most heavily diluted suspension bath was unable to maintain print fidelity. Other formulations supported the printing,fusion,and culture of spheroid-based inks,either as simple lines or more complex patterns. These findings help to inform the design of suspension baths for bioprinting and culture. View Publication

Eosinophils,a type of granulocyte derived from myeloid precursors in the bone marrow,are distinguished by their cytoplasmic granules. They play crucial roles in immunoregulation,tissue homeostasis,and host defense,while also contributing to the pathogenesis of various inflammatory diseases. Although long non-coding RNAs (lncRNAs) are known to be involved in eosinophilic conditions,their specific expression and functions within eosinophils have not been thoroughly investigated,largely due to the reliance on tissue homogenates. In an effort to address this gap,we analyzed publicly available high-throughput RNA sequencing data to identify lncRNAs associated with eosinophilic conditions. Among the identified lncRNAs,ITGB2 antisense RNA 1 (ITGB2-AS1) was significantly downregulated in blood eosinophils from patients with hypereosinophilia. To further explore its role in eosinophil biology,we generated a stable ITGB2-AS1 knockdown in the HL-60 cell line. Interestingly,ITGB2-AS1 deficiency led to impaired eosinophil differentiation,as evidenced by a reduction in cytoplasmic granules and decreased expression of key eosinophil granule proteins,including eosinophil peroxidase (EPX) and major basic protein-1 (MBP-1). Additionally,ITGB2-AS1-deficient cells exhibited compromised eosinophil effector functions,with reduced degranulation and impaired production of reactive oxygen species (ROS). These findings suggest that ITGB2-AS1 plays a pivotal role in eosinophil differentiation and function,positioning it as a novel regulator in eosinophil biology. View Publication

A human invitro cardiac tissue model would be a significant advancement for understanding,studying,and developing new strategies for treating cardiac arrhythmias and related cardiovascular diseases. We developed an invitro model of three-dimensional (3D) human cardiac tissue by populating synthetic filamentous matrices with cardiomyocytes derived from healthy wild-type volunteer (WT) and patient-specific long QT syndrome type 3 (LQT3) induced pluripotent stem cells (iPS-CMs) to mimic the condensed and aligned human ventricular myocardium. Using such a highly controllable cardiac model,we studied the contractility malfunctions associated with the electrophysiological consequences of LQT3 and their response to a panel of drugs. By varying the stiffness of filamentous matrices,LQT3 iPS-CMs exhibited different level of contractility abnormality and susceptibility to drug-induced cardiotoxicity. textcopyright 2013 Elsevier Ltd. View Publication

Several recent reports suggest that there is far more plasticity that previously believed in the developmental potential of bone-marrow-derived cells (BMCs) that can be induced by extracellular developmental signals of other lineages whose nature is still largely unknown. In this study,we demonstrate that bone-marrow-derived mesenchymal stem cells (MSCs) co-cultured with mouse proliferating or fixed (by paraformaldehyde or methanol) neural stem cells (NSCs) generate neural stem cell-like cells with a higher expression of Sox-2 and nestin when grown in NS-A medium supplemented with N2,NSC conditioned medium (NSCcm) and bFGF. These neurally induced MSCs eventually differentiate into beta-III-tubulin and GFAP expressing cells with neuronal and glial morphology when grown an additional week in Neurobasal/B27 without bFGF. We conclude that juxtacrine interaction between NSCs and MSCs combined with soluble factors released from NSCs are important for generation of neural-like cells from bone-marrow-derived adherent MSCs. View Publication

The CRISPR/Cas9 system is a rapid and customizable tool for gene editing in mammalian cells. In particular,this approach has widely opened new opportunities for genetic studies in neurological disease. Human neurons can be differentiated in vitro from hPSC (human Pluripotent Stem Cells),hNPCs (human Neural Precursor Cells) or even directly reprogrammed from fibroblasts. Here,we described a new platform which enables,rapid and efficient CRISPR/Cas9-mediated genome targeting simultaneously with three different paradigms for in vitro generation of neurons. This system was employed to inactivate two genes associated with neurological disorder (TSC2 and KCNQ2) and achieved up to 85% efficiency of gene targeting in the differentiated cells. In particular,we devised a protocol that,combining the expression of the CRISPR components with neurogenic factors,generated functional human neurons highly enriched for the desired genome modification in only 5 weeks. This new approach is easy,fast and that does not require the generation of stable isogenic clones,practice that is time consuming and for some genes not feasible. View Publication