Transcriptome analysis of 3D model of blood-brain barrier exposed to Borrelia garini
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ABSTRACT: Using RNA sequencing to map differentially expressed genes in the 3D model of the blood-brain barrier ( composed of human brain endothelial cells, human astrocytes, and human pericytes) challenged with Borrelia garini .
Project description:Using RNA sequencing to map differentially expressed genes in the 3D model of the blood-brain barrier ( composed of human brain endothelial cells, human astrocytes, and human pericytes) challenged with WNV.
Project description:Using RNA sequencing to map differentially expressed genes in the 3D model of the blood-brain barrier ( composed of human brain endothelial cells, human astrocytes, and human pericytes) challenged with TBEV.
Project description:Using RNA sequencing to map differentially expressed genes in human brain microvascular endothelial cells challenged with Borrelia burgdorferi or its ligand Erp23.
Project description:Neisseria meningitidis and Borrelia bavariensis are the causative agents of invasive meningococcal disease and Lyme neuroborrelosis. Before infecting the brain parenchyma the bacterial pathogens should cross the human blood-brain barrier (BBB). To understand the response of BBB against the invading pathogens, in vitro BBB spheroid model was generated by co cultivating human brain microvascular endothelial cells, pericytes and astrocytes in low adhesion conditions. Spheroids (N= 36) were infected with either N. meningitidis (6 x 10e4, MOI 1:4) or B bavariensis(1.5 x 10e5, MOI 1:10) for 3h. Two separate control groups of spheroids (N=36) without any infection were included in the study. After 3h of treatment (infection/no infection), 12 spheroids were pooled to form one biological sample and such 3 biological replicates per treatment were sampled for total RNA extraction. Thereafter, 12 cDNA libraries were prepared using QuantSeq FWD 3’mRNA Library Prep Kit (Lexogen). Second strand of cDNA library was synthesized using the UMI Second Strand Synthesis Mix (USS, Lexogen) containing 6 nucleotides long Unique Molecular Identifiers (UMIs). The double stranded cDNA libraries were amplified in 17-19 PCR cycles using i5 Unique Dual Indexing Add-on Kit (Lexogen). Illumina NextSeq 500 was employed to sequence the libraries having a read lengths of single-end 75 bp producing about 10 million reads per library. To carryout gene expression analysis, Bioconductor R-package - DESeq2 v1.20.0 (Love et al., 2014) was used where in, featureCounts tool v1.6.3 (Liao et al., 2013) was used to enumerate the genes. A minimum threshold of adjusted p-value < 0.05 and log2fold-change (log2FC) = ± 1 was set to considered the gene as differentially expressed (DEG).
Project description:Using RNA sequencing to map differentially expressed genes in human brain microvascular endothelial cells challenged with DIII domain of protein E of WNV and DIII domain of protein E of TBEV.
Project description:Using RNA sequencing to map differentially expressed genes in human brain microvascular endothelial cells challenged with Streptococcus pneumoniae or its ligand Adhesion lipoprotein.
Project description:Primary human astrocytes challenged with either 1. Borrelia, 2. Streptococcus, 3. Neisseria, 4. Listeria, 5. E.coli, 6. Klebsiella. RNAseq analysis was performed to study comparative gene expression.
Project description:Using RNA sequencing to map differentially expressed genes in human brain microvascular endothelial cells challenged with Neisseria meningitidis or its ligand MafA .
Project description:Metastatic melanoma is either intrinsically resistant or rapidly acquires resistance to targeted drugs such as MAPK inhibitors (MAPKi). Here, using a drug screen targeting chromatin regulators in patient-derived 3D melanoma cell cultures, we discovered that PARP inhibitors are capable of restoring MAPKi sensitivity. This synergy was found to be independent of DNA damage repair pathways and was effective both in vitro and in vivo in patients-derived xenografts. Strikingly, through integrated transcriptomic, proteomic and epigenomic analysis, we discovered that PARPi induces lysosomal autophagy which was accompanied by enhanced mitochondrial lipid metabolism that, ultimately, increased antigen presentation and sensitivity to T-cell cytotoxicity. Moreover, we also found that PARP inhibitors regulated EMT-like phenotype switching by dampening the mesenchymal phenotype via transcriptomic and epigenetic rearrangements. This, in turn, redirected melanoma cells towards a proliferative and, thus, MAPKi-sensitive state. Our study provides a scientific rational for treating patients with PARPi in combination with MAPKi to annihilate acquired therapy resistance.
Project description:Human ES (H9) cells were directed towards a neuromesodermal progenitor-like cell state and these cells were then subsequently differentiated towards a neural cell fate. Human ES cells (H9) were differentiated into neuromesodermal progenitor-like cells by culturing in Neurobasal/1x N2/1x B27 medium (N2/B27) supplemented with 20 ng/ml bFgf and 3 μM CHIR99021 for 3 days and exposure to dual SMAD inhibition (dSMADi) (Noggin 50 ng/ml and the TGFb receptor type 1 inhibitor SB431542 10 μM) during day 3 (D3). Transcriptome analysis was then carried out following a selection procedure to enrich for NMP-like cells (sD3/NMP-like). This involved use of a hES (H9) cell line engineered with CRISPR-Cas9 to express GFP under the control of the endogenous Nkx1.2 promoter. At the end of day 3 cells were selected for high GFP expression, as high Nkx1.2 transcription is characteristic of NMP cell populations in mouse and chick embryos. These cells were then lysed and RNA extracted for RNASeq. Humans ES cells (H9) differentiated into NMP-like cells as above (without selection) were also allowed to develop further on day 4 (in the presence of dSMADi and Retinoic acid (RA) 100nM, in N2/B27) and then in RA alone until end of day 8 (D8). These cells were then lysed and RNA extracted for RNASeq.