Glial Cell Line Research Articles

Impact of Chondrocyte Inflammation on Glial Cell Activation: The Mediating Role of Nitric Oxide.

This study investigates how the inflammatory response of ATDC5 murine chondrogenic cells influences the activity of C6 (rat) and GL261 (mouse) glial cell lines. Prior research suggested nitric oxide (NO) involvement in cartilage-immune crosstalk. The current study explores whether NO, produced by inflamed chondrocytes, mediates signaling between chondrocytes and glial cells. Pre-challenged ATDC5 cells with 250 ng/ml of lipopolysaccharide (LPS) were cocultured with GL261 or C6 glioma cells for 24 h with a transwell culture system. Cell viability was assessed using MTT assay. Gene and protein expression were evaluated by qRT-PCR and WB, respectively. Real-time reverse transcription-polymerase chain reaction (RT-qPCR) indicated statistically significant upregulation of LCN2, IL-6, TNF-α, IL-1β, and GFAP in glial cells following 24-h coculture with challenged ATDC5 cells. Suppression of LPS-induced NO production by aminoguanidine decreased LPS-mediated LCN2 and IL-6 expression in glioma cells. We identified also the involvement of the ERK1/2 and AKT signaling pathways in the glial neuroinflammatory response. This study demonstrates, for the first time, that NO produced by inflamed murine chondrocytes mediated pro-inflammatory responses in glial cells via ERK1/2 and AKT signaling, highlighting a potential mechanism linking cartilage NO to neuroinflammation and chronic pain in osteoarthritis.

STUDIES ON GLYCOGEN PHOSPHORYLASE AS A POTENTIAL MOLECULAR TARGET IN GLIOBLASTOMA

Abstract AIMS Glioblastoma (GB) is one of the most aggressive malignancies of the brain and spinal cord. Current standard of care has remained surgical resection of the tumour, followed by radiotherapy and/or chemotherapy. Crucial challenges in the effective treatment of GB include resistance to the main chemotherapeutic, temozolomide. Prognosis is poor with median survival remaining at 14.6 months. A now established hallmark of cancer is the ability of malignant cells to “modify, or reprogram, cellular metabolism”, such as glycolysis upregulation. This hallmark could suggest the potential of targeting a specific metabolic component within glycolysis, thus reduce energy production, resulting in possible apoptosis/suppressed proliferation. Glycogen phosphorylase (GP), an enzyme in glyconeolysis, could be inhibited for a therapeutic effect in GB. In this project, GP levels across the three different isoforms (PYGB, PYGL, and PYGM) and the effect of the GP inhibitor CP-91149 was investigated across three different GB cell lines. METHOD Cell viability of T98G, U251-MG, and U87-MG glioblastoma cell lines and SVGp19 human fetal glial cell line was measured after administration of CP-91149 for 24, 48, and 72 hours. Migration of T98G and U251 cells following treatment with CP-91149 was measured in a wound healing assay. Visualisation of the three GP isoforms was achieved through western blot and confirmed by immunocytochemistry. Flow cytometry was utilized to expand analysis on GP inhibition through assessing cell cycle before and after treatments. RESULTS Results showed CP-91149 had a significant dose-dependent effect in vitro on all cell lines. Inhibition of GP resulted in reduced cell migration, also in a dose-dependent manner. Western blot and immunocytochemistry analysis showed PYGL is the most predominant isoform of GP. CONCLUSION Future work in this project will aim to silence expression of GP in GB cell lines in order to compare the results with that yielded from cells treated with the GP inhibitor CP-91149.

Superior Tumor Cell Uptake by Mono- and Tri-Nuclear Rhodamine-Gadolinium(III) Agents.

The synthesis and characterization of a novel trinuclear rhodamine-Gd(III) complex, along with two analogous mononuclear rhodamine-Gd(III) complexes, are reported. All complexes displayed good selectivity in a human glioma cell line (T98G) when compared to a glial cell line (SVG p12), with low cytotoxicities. Superior tumor cell uptake for these Gd(III) complexes was observed at lower incubation concentrations compared to previously-reported delocalized lipophilic cations such as a rhodamine-lanthanoid(III) probe and Gd(III)-arylphosphonium complexes, with ca. 150 % and 250 % increases in Gd uptake, respectively.

Hyaluronic acid hybrid formulations optimised for 3D printing of nerve conduits and the delivery of the novel neurotrophic-like compound tyrosol to enhance peripheral nerve regeneration via Schwann cell proliferation

Peripheral nerve injuries, predominantly affecting individuals aged 20–40, pose significant healthcare challenges, with current surgical methods often failing to achieve complete functional recovery. This study focuses on the development of 3D printed hydrogel nerve conduits using modified hyaluronic acid (HA) for potentially enhancing peripheral nerve regeneration. Hyaluronic acid was chemically altered with cysteamine HCl and methacrylic anhydride to create thiolated HA (HA-SH) and methacrylated HA (HA-MA), achieving a modification degree of approximately 20 %. This modification was crucial to maintain the receptor interaction of HA. The modified HA was rigorously tested to ensure cytocompatibility in neuronal and glial cell lines. Subsequently, various 3D printed HA formulations were evaluated, focusing on improving HA’s inherent mechanical weaknesses. These formulations were assessed for cytotoxicity through direct contact and elution extract testing, confirming their safety over a 24-h period. Among the neurotrophic compounds tested, Tyrosol emerged as the most effective in promoting Schwann cell proliferation in vitro. The 3D printed HA system demonstrated proficiency in loading and releasing Tyrosol at physiological pH. The findings from this research highlight the promising role of 3D printed HA and Tyrosol in the field of nerve tissue engineering, offering a novel approach to peripheral nerve regeneration.

The anti-amoebic activity of Pinus densiflora leaf extract against the brain-eating amoeba Naegleria fowleri.

Naegleria fowleri invades the brain and causes a fatal primary amoebic meningoencephalitis (PAM). Despite its high mortality rate of approximately 97%, an effective therapeutic drug for PAM has not been developed. Approaches with miltefosine, amphotericin B, and other antimicrobials have been clinically attempted to treat PAM, but their therapeutic efficacy remains unclear. The development of an effective and safe therapeutic drug for PAM is urgently needed. In this study, we investigated the anti-amoebic activity of Pinus densiflora leaf extract (PLE) against N. fowleri. PLE induced significant morphological changes in N. fowleri trophozoites, resulting in the death of the amoeba. The IC50 of PLE on N. fowleri was 62.3±0.95 μg/ml. Alternatively, PLE did not significantly affect the viability of the rat glial cell line C6. Transcriptome analysis revealed differentially expressed genes (DEGs) between PLE-treated and non-treated amoebae. A total of 5,846 DEGs were identified, of which 2,189 were upregulated, and 3,657 were downregulated in the PLE-treated amoebae. The DEGs were categorized into biological process (1,742 genes), cellular component (1,237 genes), and molecular function (846 genes) based on the gene ontology analysis, indicating that PLE may have dramatically altered the biological and cellular functions of the amoeba and contributed to their death. These results suggest that PLE has anti-N. fowleri activity and may be considered as a potential candidate for the development of therapeutic drugs for PAM. It may also be used as a supplement compound to enhance the therapeutic efficacy of drugs currently used to treat PAM.

Immortalization and Characterization of GFAP-expressing Glial Cells from the Adult Mouse Hypothalamus, Cortex, and Brain Stem

The generation of astrocyte cell lines from the hypothalamus is key to study glial involvement in hypothalamic physiology, including energy homeostasis. As such, we immortalized astrocytes from the hypothalamus of an adult male CD-1 mouse using SV40 T-antigen to generate the mHypoA-Ast1 cell line. A comparative approach was taken with two other murine GFAP-expressing cell lines that were also generated in this study: a mixed glial cell line from the cortex (mCortA-G1) and an oligodendrocyte cell line from the brainstem (mBstA-Olig1), as well as an established microglial cell line (IMG). mHypoA-Ast1 cells express GFAP, alongside other astrocytic markers such as Aldh1l1, Aqp4, Glt1 and S100b, and express low levels of microglial, ependymal and oligodendrocyte markers. 100 ng/mL lipopolysaccharide (LPS) elevated mRNA levels of Il6, Il1b, Tnfα and Cxcl5 in mHypoA-Ast1 cells after 4 h, while 50 μM palmitate increased Il6 and Chop mRNA, demonstrating the ability of these cells to respond to inflammatory and nutrient signals. Interestingly, co-culture of mHypoA-Ast1 cells with mHypoE-N46 hypothalamic neuronal cells prevented the palmitate-mediated increase in orexigenic neuropeptide Agrp mRNA in mHypoE-N46 cells, suggesting that this cell line can alter neuronal responses to nutrients. In conclusion, we report mHypoA-Ast1 cells representing a functional astrocyte cell line from the adult mouse brain that can be used to study the complex interactions of hypothalamic cells, as well as dysregulation that may occur in disease states, providing a key tool for neuroendocrine research.

Toxicity of Large and Small Surface-Engineered Upconverting Nanoparticles for In Vitro and In Vivo Bioapplications.

In this study, spherical or hexagonal NaYF4:Yb,Er nanoparticles (UCNPs) with sizes of 25 nm (S-UCNPs) and 120 nm (L-UCNPs) were synthesized by high-temperature coprecipitation and subsequently modified with three kinds of polymers. These included poly(ethylene glycol) (PEG) and poly(N,N-dimethylacrylamide-co-2-aminoethylacrylamide) [P(DMA-AEA)] terminated with an alendronate anchoring group, and poly(methyl vinyl ether-co-maleic acid) (PMVEMA). The internalization of nanoparticles by rat mesenchymal stem cells (rMSCs) and C6 cancer cells (rat glial tumor cell line) was visualized by electron microscopy and the cytotoxicity of the UCNPs and their leaches was measured by the real-time proliferation assay. The comet assay was used to determine the oxidative damage of the UCNPs. An in vivo study on mice determined the elimination route and potential accumulation of UCNPs in the body. The results showed that the L- and S-UCNPs were internalized into cells in the lumen of endosomes. The proliferation assay revealed that the L-UCNPs were less toxic than S-UCNPs. The viability of rMSCs incubated with particles decreased in the order S-UCNP@Ale-(PDMA-AEA) > S-UCNP@Ale-PEG > S-UCNPs > S-UCNP@PMVEMA. Similar results were obtained in C6 cells. The oxidative damage measured by the comet assay showed that neat L-UCNPs caused more oxidative damage to rMSCs than all coated UCNPs while no difference was observed in C6 cells. An in vivo study indicated that L-UCNPs were eliminated from the body via the hepatobiliary route; L-UCNP@Ale-PEG particles were almost eliminated from the liver 96 h after intravenous application. Pilot fluorescence imaging confirmed the limited in vivo detection capabilities of the nanoparticles.

Multi-omics technologies and molecular biomarkers in brain tumor-related epilepsy.

Brain tumors are one of the leading causes of epilepsy, and brain tumor-related epilepsy (BTRE) is recognized as the major cause of intractable epilepsy, resulting in huge treatment cost and burden to patients, their families, and society. Although optimal treatment regimens are available, the majority of patients with BTRE show poor resolution of symptoms. BTRE has a very complex and multifactorial etiology, which includes several influencing factors such as genetic and molecular biomarkers. Advances in multi-omics technologies have enabled to elucidate the pathophysiological mechanisms and related biomarkers of BTRE. Here, we reviewed multi-omics technology-based research studies on BTRE published in the last few decades and discussed the present status, development, opportunities, challenges, and prospects in treating BTRE. First, we provided a general review of epilepsy, BTRE, and multi-omics techniques. Next, we described the specific multi-omics (including genomics, transcriptomics, epigenomics, proteomics, and metabolomics) techniques and related molecular biomarkers for BTRE. We then presented the associated pathogenetic mechanisms of BTRE. Finally, we discussed the development and application of novel omics techniques for diagnosing and treating BTRE. Genomics studies have shown that the BRAF gene plays a role in BTRE development. Furthermore, the BRAF V600E variant was found to induce epileptogenesis in the neuronal cell lineage and tumorigenesis in the glial cell lineage. Several genomics studies have linked IDH variants with glioma-related epilepsy, and the overproduction of D2HG is considered to play a role in neuronal excitation that leads to seizure occurrence. The high expression level of Forkhead Box O4 (FOXO4) was associated with a reduced risk of epilepsy occurrence. In transcriptomics studies, VLGR1 was noted as a biomarker of epileptic onset in patients. Several miRNAs such as miR-128 and miRNA-196b participate in BTRE development. miR-128 might be negatively associated with the possibility of tumor-related epilepsy development. The lncRNA UBE2R2-AS1 inhibits the growth and invasion of glioma cells and promotes apoptosis. Quantitative proteomics has been used to determine dynamic changes of protein acetylation in epileptic and non-epileptic gliomas. In another proteomics study, a high expression of AQP-4 was detected in the brain of GBM patients with seizures. By using quantitative RT-PCR and immunohistochemistry assay, a study revealed that patients with astrocytomas and oligoastrocytomas showed high BCL2A1 expression and poor seizure control. By performing immunohistochemistry, several studies have reported the relationship between D2HG overproduction and seizure occurrence. Ki-67 overexpression in WHO grade II gliomas was found to be associated with poor postoperative seizure control. According to metabolomics research, the PI3K/AKT/mTOR pathway is associated with the development of glioma-related epileptogenesis. Another metabolomics study found that SV2A, P-gb, and CAD65/67 have the potential to function as biomarkers for BTRE. Based on the synthesized information, this review provided new research perspectives and insights into the early diagnosis, etiological factors, and personalized treatment of BTRE.

1243 FOXM1*3 Overexpression Correlates to Staging of Astrocytic Glioma

INTRODUCTION: Diffuse gliomas are a group of primary brain tumors characterized by the presence of neoplastic glial-lineage cells. WHO 2021 classification of gliomas recognizes the mutational status of the isocitrate dehydrogenase-1(IDH-1,G395A) gene and the 1p/19q codeletion as identification of cell lineage and a prognosis marker. Transcription factors are binding proteins of genomic DNA and can activate or suppress gene expression. Forkhead box M1(FOXM1) is a transcription factor for Forkhead box(Fox) proteins, a superfamily which is defined by a conserved winged helix DNA binding domain. FOXM1 is a key regulator of both G1/S and G2/M phases of cell cycle and mitotic spindle integrity, it also plays an important role in angiogenesis, DNA damage repair and tissue regeneration. The human FOXM1 gene consists of 10 exons, two of them alternatively spliced. This splicing gives rise to three isoforms: FOXM1*1,*2 and *3. FOXM1*3 is the smallest isoform and is overexpressed in proliferative tissue such as malignant tumors. METHODS: A control group of 10 brain cortex, 22 A2, 13 O2, 11 A3, 3 O3, 6 A4 and 46 GBM with histological and molecular diagnosis of astrocytoma, oligodendroglioma and glioblastoma from 2011 to 2022 were included. The diagnosis correlated with patient outcome. mRNA FOXM1*3 and its housekeeping gen IPO8 was quantified by qPCR with Taqman probes. Data analysis was conducted by MannWhitney U test. RESULTS: There was a statistically significant difference between expression of mRNA FOXM1*3 in normal brain tissue compared to tumor tissue (p < 0.001). The transcript allowed differentiation between grade 2, 3 and 4 astrocytomas (p < 0.005), nevertheless there was no difference between astrocytic versus oligodendrocytic lineage, and grade 4 astrocytoma versus glioblastoma. CONCLUSIONS: The normalized expression of transcript FOXM1*3 allows differentiation of astrocytic gliomas grades.

Hyperglycemic stress induces oxidative damage of enteric glial cells by triggering redoxosomes/p66SHC activation.

Diabetic gastrointestinal dysfunction (DGD) is a serious complication of diabetic mellitus (DM), affecting the enteric nervous system (ENS), particular enteric glial cells (EGCs). This study aimed to elucidate the effects and underlying molecular mechanisms of hyperglycemic stress on EGCs in in vitro and in vivo models of DM. In in vitro studies, enteric glial cell line CRL-2690 was exposed to hyperglycemia stress, and cell viability, cell apoptosis and oxidative damage were assessed. In in vivo studies, STZ-induced diabetic mice were constructed, and cell apoptosis and oxidative damage of EGCs in the duodenum of DM mice were assessed. The results showed that hyperglycemic stress markedly induced oxidative damage of EGCs in in vitro and in vivo models of DM. This damage was found to be dependent on the activation of redoxosomes, which involved the phosphorylation of SRC and Vav2, the up-regulation of active RAC1-GTP, and the activation of NADPH oxidase (NOX). Moreover, inhibitors of redoxosomes, such as the RAC1 inhibitor NSC23766 and the NOX inhibitor VAS2870, effectively mitigated the hyperglycemic stress-induced oxidative damage of EGCs. Additionally, inhibition of p66SHC, a downstream target of redoxosomes, attenuated oxidative damage of EGCs under hyperglycemic stress. Our findings suggest that the redoxosomes/p66SHC signaling is involved in the oxidative damage of EGCs during the pathological process of DGD. This signaling cascade may represent a potential therapeutic target for the treatment of DGD.

Magnesium-lithium thin films for neurological applications–An in vitro investigation of glial cytocompatibility and neuroinflammatory response

Lithium (Li), a widely used drug for bipolar disorder management, is associated with many side effects due to systemic exposure. The localized delivery of lithium through implants could be an approach to overcome this challenge, for which biodegradable magnesium (Mg)-based materials are a promising choice. In this study, we focus on Mg-Li thin film alloys as potential Li-releasing implants. Therefore, we investigated the in vitro short-term corrosion behavior and cytocompatibility of two alloys, Mg-1.6wt%Li and Mg-9.5wt%Li. As glial cells are the key players of foreign body responses to implants, we used human glial cell lines for cytocompatibility studies, and a murine brain slice model for a more holistic view at the neuroinflammatory response. We found that Mg-1.6wt%Li corrodes approximately six times slower than Mg-9.5wt%Li. Microscopic analysis showed that the material surface (Mg-1.6wt%Li) is suitable for cell adhesion. The cytocompatibility test with Mg-1.6wt%Li and Mg-9.5wt%Li alloy extracts revealed that both cell types proliferated well up to 10 mM Mg concentration, irrespective of the Li concentration. In the murine brain slice model, Mg-1.6wt%Li and Mg-9.5wt%Li alloy extracts did not provoke a significant upregulation of glial inflammatory/ reactivity markers (IL-1β, IL-6, FN1, TNC) after 24 h of exposure. Furthermore, the gene expression of IL-1β (up to 3-fold) and IL-6 (up to 16-fold) were significantly downregulated after 96 h, and IL-6 downregulation showed a Li concentration dependency. Together, these results indicate the acute cytocompatibility of two Mg-Li thin film alloys and provide basis for future studies to explore promising applications of the material. Statement of significanceWe propose the idea of lithium delivery to the brain via biodegradable implants to reduce systemic side effects of lithium for bipolar disorder therapy and other neurological applications. This is the first in vitro study investigating Mg-xLi thin film degradation under physiological conditions and its influence on cellular responses such as proliferation, viability, morphology and inflammation. Utilizing human brain-derived cell lines, we showed that the material surface of such a thin film alloy is suitable for normal cell attachment. Using murine brain slices, which comprise a multicellular network, we demonstrated that the material extracts did not elicit a pro-inflammatory response. These results substantiate that degradable Mg-Li materials are biocompatible and support the further investigation of their potential as neurological implants.

Resveratrol Alleviates the Early Challenges of Implant-Based Drug Delivery in a Human Glial Cell Model.

Brain diseases are oftentimes life-threatening and difficult to treat. The local administration of drug substances using brain implants can increase on-site concentrations and decrease systemic side effects. However, the biocompatibility of potential brain implant materials needs to be evaluated carefully as implants can trigger foreign body reactions, particularly by increasing the microglia and astrocyte reactivity. To date, these tests have been frequently conducted in very simple in vitro models, in particular not respecting the key players in glial cell reactions and the challenges of surgical implantation characterized by the disruption of oxygen and nutrient supply. Thus, we established an in vitro model in which we treated human glial cell lines with reduced oxygen and glucose levels. The model displayed cytokine and reactive oxygen species release from reactive microglia and an increase in a marker of reactive astrocytes, galectin-3. Moreover, the treatment caused changes in the cell survival and triggered the production of hypoxia-inducible factor 1α. In this comprehensive platform, we demonstrated the protective effect of the natural polyphenol resveratrol as a model substance, which might be included in brain implants to ease the undesired glial cell response. Overall, a glial-cell-based in vitro model of the initial challenges of local brain disease treatment may prove useful for investigating new therapy options.

DNA methylation-mediated repression of microRNA-410 promotes the growth of human glioma cells and triggers cell apoptosis through its interaction with STAT3

This study's purpose was to confirm the observed underexpression of miRNA-410 in glioma tissues and several glioma cells by Quantitative RT-PCR. Our findings suggest that epigenetic alterations occurring at the promoter region of miR-410 may be responsible for the reduced expression of miR-410 in glioma. The occurrence of DNA methylation in the miR-410 promoter was verified to be more prevalent through glioma tissues contrasted to adjacent non-tumor brain tissues through the utilization of methylation-specific PCR and CpG bisulfite sequencing sites in the miR-410 promoter region. Accordantly, miR-410 expression in glioma cell lines was observed to be significantly lesser in comparison to that of the human fetal glial cell line. In addition, it was demonstrated through gain- and loss-of-function investigations that miR-410 exerts significant regulation over cell growth, cell cycle development, and glioma cell apoptosis. The findings of the Luciferase reporter assay and western blot analysis indicate that miR-410 has a direct effect on the 3’-UTR of signal transducer and activator of transcription 3 (STAT3), thereby inhibiting its expression within glioma cells. Besides, our clinical investigation indicates a negative association between miR-410 expression and STAT3 within the glioma tissues of humans. In aggregate, the data provided in this investigation indicates that miR-410 is subjected to underexpression via DNA methylation. Furthermore, it has been observed to perform its function as a tumor suppressor in glioma cells through direct targeting of STAT3. The previously mentioned results could potentially have significant implications for the advancement of a new therapeutic approach for treating glioma.

Cold atmospheric‐pressure plasma selectively inhibits glioblastoma via DNA damage and AKT dephosphorylation in vitro and in vivo

AbstractIn this work, the effects of cold atmospheric‐pressure plasma (CAP) on glioblastoma are evaluated comprehensively. After CAP treatment, U251 cell viability, migration, and invasion functions were inhibited, while an appropriate dose of CAP had no inhibitory effect on human brain glial cell line cells. Western blots indicated that expression of caspase‐3 was upregulated with ki‐67 expression downregulated. Moreover, mitochondrial membrane potential decreased, and energy metabolisms of U251 cells were influenced afterward. TUNEL assays and comet assays suggested the DNA damage of U251 cells after CAP treatment. Furthermore, as one of the DNA damage responses associated pathways, the AKT (AKT8 virus oncogene cellular homolog) signaling pathway was also indicated in the work. The findings raise great promise for clinical applications of CAP in glioblastoma treatments.

Simple design for membrane-free microphysiological systems to model the blood-tissue barriers

Microphysiological systems (MPS) incorporate physiologically relevant microanatomy, mechanics, and cells to mimic tissue function. Reproducible and standardized in vitro models of tissue barriers, such as the blood-tissue interface (BTI), are critical for next-generation MPS applications in research and industry. Many models of the BTI are limited by the need for semipermeable membranes, use of homogenous cell populations, or 2D culture. These factors limit the relevant endothelial-epithelial contact and 3D transport, which would best mimic the BTI. Current models are also difficult to assemble, requiring precise alignment and layering of components. The work reported herein details the engineering of a BTI-on-a-chip (BTI Chip) that addresses current disadvantages by demonstrating a single layer, membrane-free design. Laminar flow profiles, photocurable hydrogel scaffolds, and human cell lines were used to construct a BTI Chip that juxtaposes an endothelium in direct contact with a 3D engineered tissue. A biomaterial composite, gelatin methacryloyl and 8-arm polyethylene glycol thiol, was used for in situ fabrication of a tissue structure within a Y-shaped microfluidic device. To produce the BTI, a laminar flow profile was achieved by flowing a photocurable precursor solution alongside phosphate buffered saline. Immediately after stopping flow, the scaffold underwent polymerization through a rapid exposure to UV light (<300 mJ/cm2). After scaffold formation, blood vessel endothelial cells were introduced and allowed to adhere directly to the 3D tissue scaffold, without barriers or phase guides. Fabrication of the BTI Chip was demonstrated in both an epithelial tissue model and blood-brain barrier (BBB) model. In the epithelial model, scaffolds were seeded with human dermal fibroblasts. For the BBB models, scaffolds were seeded with the immortalized glial cell line, SVGP12. The BTI Chip microanatomy was analyzed post facto by immunohistochemistry, showing the uniform production of a patent endothelium juxtaposed with a 3D engineered tissue. Fluorescent tracer molecules were used to characterize the permeability of the BTI Chip. The BTI Chips were challenged with an efflux pump inhibitor, cyclosporine A, to assess physiological function and endothelial cell activation. Operation of physiologically relevant BTI Chips and a novel means for high-throughput MPS generation was demonstrated, enabling future development for drug candidate screening and fundamental biological investigations.

Architecture and regulation of a GDNF-GFRα1 synaptic adhesion assembly

Glial-cell line derived neurotrophic factor (GDNF) bound to its co-receptor GFRα1 stimulates the RET receptor tyrosine kinase, promoting neuronal survival and neuroprotection. The GDNF-GFRα1 complex also supports synaptic cell adhesion independently of RET. Here, we describe the structure of a decameric GDNF-GFRα1 assembly determined by crystallography and electron microscopy, revealing two GFRα1 pentamers bridged by five GDNF dimers. We reconsitituted the assembly between adhering liposomes and used cryo-electron tomography to visualize how the complex fulfils its membrane adhesion function. The GFRα1:GFRα1 pentameric interface was further validated both in vitro by native PAGE and in cellulo by cell-clustering and dendritic spine assays. Finally, we provide biochemical and cell-based evidence that RET and heparan sulfate cooperate to prevent assembly of the adhesion complex by competing for the adhesion interface. Our results provide a mechanistic framework to understand GDNF-driven cell adhesion, its relationship to trophic signalling, and the central role played by GFRα1.

Glioma Single-Cell Biomechanical Analysis by Cyclic Conical Constricted Microfluidics.

Determining the grade of glioma is a critical step in choosing patients' treatment plans in clinical practices. The pathological diagnosis of patient's glioma samples requires extensive staining and imaging procedures, which are expensive and time-consuming. Current advanced uniform-width-constriction-channel-based microfluidics have proven to be effective in distinguishing cancer cells from normal tissues, such as breast cancer, ovarian cancer, prostate cancer, etc. However, the uniform-width-constriction channels can result in low yields on glioma cells with irregular morphologies and high heterogeneity. In this research, we presented an innovative cyclic conical constricted (CCC) microfluidic device to better differentiate glioma cells from normal glial cells. Compared with the widely used uniform-width-constriction microchannels, the new CCC configuration forces single cells to deform gradually and obtains the biophysical attributes from each deformation. The human-derived glioma cell lines U-87 and U-251, as well as the human-derived normal glial astrocyte cell line HA-1800 were selected as the proof of concept. The results showed that CCC channels can effectively obtain the biomechanical characteristics of different 12-25 μm glial cell lines. The patient glioma samples with WHO grades II, III, and IV were tested by CCC channels and compared between Elastic Net (ENet) and Lasso analysis. The results demonstrated that CCC channels and the ENet can successfully select critical biomechanical parameters to differentiate the grades of single-glioma cells. This CCC device can be potentially further applied to the extensive family of brain tumors at the single-cell level.

A simplified protocol to induce hypoxia in a standard incubator: A focus on retinal cells

Hypoxia chambers have traditionally been used to induce hypoxia in cell cultures. Cellular responses to hypoxia can also be mimicked with the use of chemicals such as cobalt chloride (CoCl2), which stabilizes hypoxia-inducible factor alpha-subunit proteins. In studies of ocular cells using primary cells and cell lines, such as Müller glial cell (MGC) lines, photoreceptor cell lines, retinal pigment epithelial (RPE) cell lines and retinoblastoma cell lines oxygen levels employed in hypoxia chambers range typically between 0.2% and 5% oxygen. For chemical induction of hypoxic response in these cells, the CoCl2 concentrations used typically range from 100 to 600 μM. Here, we describe simplified protocols for stabilizing cellular hypoxia-inducible factor-1α (HIF-1α) in cell culture using either a hypoxia chamber or CoCl2. In addition, we also provide a detailed methodology to confirm hypoxia induction by the assessment of protein levels of HIF-1α, which accumulates in response to hypoxic conditions. Furthermore, we provide a summary of conditions applied in previous studies of ocular cells.

Defining super-enhancers by highly ranked histone H4 multi-acetylation levels identifies transcription factors associated with glioblastoma stem-like properties

BackgroundSuper-enhancers (SEs), which activate genes involved in cell-type specificity, have mainly been defined as genomic regions with top-ranked enrichment(s) of histone H3 with acetylated K27 (H3K27ac) and/or transcription coactivator(s) including a bromodomain and extra-terminal domain (BET) family protein, BRD4. However, BRD4 preferentially binds to multi-acetylated histone H4, typically with acetylated K5 and K8 (H4K5acK8ac), leading us to hypothesize that SEs should be defined by high H4K5acK8ac enrichment at least as well as by that of H3K27ac.ResultsHere, we conducted genome-wide profiling of H4K5acK8ac and H3K27ac, BRD4 binding, and the transcriptome by using a BET inhibitor, JQ1, in three human glial cell lines. When SEs were defined as having the top ranks for H4K5acK8ac or H3K27ac signal, 43% of H4K5acK8ac-ranked SEs were distinct from H3K27ac-ranked SEs in a glioblastoma stem-like cell (GSC) line. CRISPR-Cas9–mediated deletion of the H4K5acK8ac-preferred SEs associated with MYCN and NFIC decreased the stem-like properties in GSCs.ConclusionsCollectively, our data highlights H4K5acK8ac’s utility for identifying genes regulating cell-type specificity.

CRISPR/sgRNA-directed synergistic activation mediator (SAM) as a therapeutic tool for Parkinson´s disease

Parkinson`s disease (PD) is the second most prevalent neurodegenerative disease, and different gene therapy strategies have been used as experimental treatments. As a proof-of-concept for the treatment of PD, we used SAM, a CRISPR gene activation system, to activate the endogenous tyrosine hydroxylase gene (th) of astrocytes to produce dopamine (DA) in the striatum of 6-OHDA-lesioned rats. Potential sgRNAs within the rat th promoter region were tested, and the expression of the Th protein was determined in the C6 glial cell line. Employing pseudo-lentivirus, the SAM complex and the selected sgRNA were transferred into cultures of rat astrocytes, and gene expression and Th protein synthesis were ascertained; furthermore, DA release into the culture medium was determined by HPLC. The DA-producing astrocytes were implanted into the striatum of 6-OHDA hemiparkinsonian rats. We observed motor behavior improvement in the lesioned rats that received DA-astrocytes compared to lesioned rats receiving astrocytes that did not produce DA. Our data indicate that the SAM-induced expression of the astrocyte´s endogenous th gene can generate DA-producing astrocytes that effectively reduce the motor asymmetry induced by the lesion.