Nanopore Sequencing for Microbial Keratitis
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ABSTRACT:
PROVIDER: PRJEB33660 | ENA |
REPOSITORIES: ENA
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Project description:The goal of this project was to analyze virulence factor expression during microbial keratitis and to determine the impact of the Rcs system regulator GumB on the transcriptional profile
2021-06-01 | GSE155486 | GEO
Project description:MicroRNAs (miRNAs) are small, stable non-coding RNA molecules with regulatory function and marked tissue specificity that post-transcriptionally regulate gene expression, however their role in fungal keratitis remain unknown. Our purpose was to identify the miRNAs in human cornea from fungal keratitis patients and understand their key role in regulation of pathogenesis. Corneal samples from normal cadaver (n=3) and fungal keratitis (n=5) patients were pooled separately and total RNA was extracted. Deep sequencing was done using Illumina HiSeq1000 platform to identify miRNA profile. We identified seventy five differentially expressed miRNAs in fungal keratitis corneas. Select miRNAs were validated by real-time RT-PCR (Q-PCR). We predicted their role in regulating target genes in several pathways by combining miRNA target genes and pathway analysis, and mRNA expression of select target genes were further analysed by Q-PCR. MiR-21-5p, miR-223-3p, miR-146b-5p, miR-155-5p, miR-511-5p were found to be involved in inflammatory and immune responses, regulating Toll like receptor signaling pathways, which is of particular interest. MiR-451a with an increased expression in keratitis may have a role in wound healing by targeting Macrophage Migration Inhibitory Factor (MIF). Further, we highlighted that Neurotrophin signaling pathway may play a role in wound healing process. One novel miRNA was also detected in cornea. In conclusion, several miRNAs with high expression in fungal keratitis corneas point towards their role in regulation of pathogenesis. Further insights in understanding miRNAs role in wound healing and inflammation may help design new therapeutic strategies. Control and fungal keratitis, human corneal miRNA profiles were generated using IlluminaHiseq1000 platform
2016-07-28 | E-GEOD-64843 | biostudies-arrayexpress
Project description:MicroRNAs (miRNAs) are small, stable non-coding RNA molecules with regulatory function and marked tissue specificity that post-transcriptionally regulate gene expression, however their role in fungal keratitis remain unknown. Our purpose was to identify the miRNAs in human cornea from fungal keratitis patients and understand their key role in regulation of pathogenesis. Corneal samples from normal cadaver (n=3) and fungal keratitis (n=5) patients were pooled separately and total RNA was extracted. Deep sequencing was done using Illumina HiSeq1000 platform to identify miRNA profile. We identified seventy five differentially expressed miRNAs in fungal keratitis corneas. Select miRNAs were validated by real-time RT-PCR (Q-PCR). We predicted their role in regulating target genes in several pathways by combining miRNA target genes and pathway analysis, and mRNA expression of select target genes were further analysed by Q-PCR. MiR-21-5p, miR-223-3p, miR-146b-5p, miR-155-5p, miR-511-5p were found to be involved in inflammatory and immune responses, regulating Toll like receptor signaling pathways, which is of particular interest. MiR-451a with an increased expression in keratitis may have a role in wound healing by targeting Macrophage Migration Inhibitory Factor (MIF). Further, we highlighted that Neurotrophin signaling pathway may play a role in wound healing process. One novel miRNA was also detected in cornea. In conclusion, several miRNAs with high expression in fungal keratitis corneas point towards their role in regulation of pathogenesis. Further insights in understanding miRNAs role in wound healing and inflammation may help design new therapeutic strategies.
2016-07-28 | GSE64843 | GEO
Project description:PURPOSE Corneal infections are a leading cause of visual impairment and blindness worldwide. Here we applied high-resolution transcriptomic profiling to assess the general and pathogen-specific molecular and cellular mechanisms during human corneal infection. METHODS Clinical diagnoses of herpes simplex virus (HSV) (n=5) and bacterial/fungal (n=5) keratitis were confirmed by histology. Healthy corneas (n=7) and keratoconus (n=4) samples served as controls. Formalin-fixed, paraffin-embedded (FFPE) human corneal specimens were analyzed using the 3' RNA sequencing method Massive Analysis of cDNA Ends (MACE RNA-seq). The cellular host response was investigated using comprehensive bioinformatic deconvolution (xCell) analyses and by integration with single cell RNA-seq data. RESULTS Our analysis identified 216 and 561 genes, that were specifically overexpressed in viral or bacterial/fungal keratitis, respectively, and allowed to distinguish the two etiologies. The virus-specific host response was driven by adaptive immunity and associated molecular signaling pathways, whereas the bacterial/fungal-specific host response mainly involved innate immunity signaling pathways and cell types. We identified several molecular mechanisms involved in the host response to infectious keratitis, including CXCL9, CXCR3, and MMP9 for viral, and S100A8/A9, MMP9, and the IL17 pathway for bacterial/fungal keratitis. CONCLUSIONS High-resolution molecular profiling provides new insights into the human corneal host response to viral and bacterial/fungal infection. Pathogen-specific molecular profiles may provide the foundation for novel diagnostic biomarker and therapeutic approaches that target inflammation-induced damage to corneal host cells with the goal to improve the outcome of infectious keratitis.
2024-01-30 | GSE241715 | GEO
Project description:Microbial keratitis is a major cause of blindness worldwide. An excessive host inflammatory response can occur even after adequate antimicrobial treatment. This results in tissue damage with corneal thinning and even perforation, which may require corneal transplantation. In this study we investigated the pathways involved in the pathophysiology of this disease by comparing the human transcriptome profile of tissue from culture-proven bacterial and fungal keratitis (n=7 and n=8 respectively) with normal non-infected cadaveric corneal tissue (C, n=12) using Illumina HT12 v4 microarrays. The causative organisms were Streptococcus pneumoniae (n=6) and Pseudomonas aeruginosa (n=1) for bacterial keratitis (BK). Fungal keratitis (FK) was caused by Fusarium sp. (n=5), Aspergillus sp. (n=2, A. flavus and terreus) and Lasiodiplodia sp. (n=1). Differential expression (DE) analysis revealed 2310 significantly altered probes in the BK v C comparison, and 1813 probes for FK v C. The most highly upregulated gene in both comparisons was MMP9 with fold changes (FC) of 64 (fdr-adjusted p<6 x10-11) for FK v C and 89 for BK v C (fdr-adjusted p<4 x10-11) respectively. Network co-expression analyses revealed the defense response, inflammatory response and extracellular matrix mechanisms to be the main functional pathways involved. Microarray results were validated by performing real-time quantitative PCR (RTqPCR) for 46 DE genes using RNA extracted from the same samples. There was a high correlation between log2 FC values from microarray and RTqPCR. Further studies are needed to evaluate the most highly differentially expressed genes as possible biomarkers of disease progression or therapeutic targets.
2014-10-31 | GSE58291 | GEO
Project description:Tuberous sclerosis complex (TSC) is a relatively common autosomal dominant disorder characterized by multiple dysplastic organ lesions and neuropsychiatric symptoms, caused by loss-of-function mutation of either TSC1 or TSC2. Target-capture full-length double-stranded cDNA sequencing using long-read sequencer Nanopore (Nanopore Long-read Target Sequencing) revealed that the various kinds of the TSC1 and TSC2 full-length transcripts and the novel intron retention transcripts of TSC2 in TSC patient. Our results indicate that the Nanopore Long-read Target Sequencing is useful for the detection of mutations and confers information on the full-length alternative splicing transcripts for the genetic diagnosis.
2023-09-04 | GSE241410 | GEO
Project description:Microbial keratitis is a major cause of blindness worldwide. An excessive host inflammatory response can occur even after adequate antimicrobial treatment. This results in tissue damage with corneal thinning and even perforation, which may require corneal transplantation. In this study we investigated the pathways involved in the pathophysiology of this disease by comparing the human transcriptome profile of tissue from culture-proven bacterial and fungal keratitis (n=7 and n=8 respectively) with normal non-infected cadaveric corneal tissue (C, n=12) using Illumina HT12 v4 microarrays. The causative organisms were Streptococcus pneumoniae (n=6) and Pseudomonas aeruginosa (n=1) for bacterial keratitis (BK). Fungal keratitis (FK) was caused by Fusarium sp. (n=5), Aspergillus sp. (n=2, A. flavus and terreus) and Lasiodiplodia sp. (n=1). Differential expression (DE) analysis revealed 2310 significantly altered probes in the BK v C comparison, and 1813 probes for FK v C. The most highly upregulated gene in both comparisons was MMP9 with fold changes (FC) of 64 (fdr-adjusted p<6 x10-11) for FK v C and 89 for BK v C (fdr-adjusted p<4 x10-11) respectively. Network co-expression analyses revealed the defense response, inflammatory response and extracellular matrix mechanisms to be the main functional pathways involved. Microarray results were validated by performing real-time quantitative PCR (RTqPCR) for 46 DE genes using RNA extracted from the same samples. There was a high correlation between log2 FC values from microarray and RTqPCR. Further studies are needed to evaluate the most highly differentially expressed genes as possible biomarkers of disease progression or therapeutic targets. Case - control study design. Corneal ulcer tissue from 8 bacterial and 9 fungal ulcers was excised at the time of corneal transplantation surgery and immediately preserved in RNALater. Non-infected corneal tissue from 13 cadaver corneas were the control tissue. Transcriptome profile generated using Illumina HT12 v4 beadchips. Differential expression analysis was performed with pairwise comparisons: bacterial ulcers versus controls, fungal ulcers versus controls and bacterial versus fungal ulcers. Microarray results validated with RTqPCR.
2014-10-31 | E-GEOD-58291 | biostudies-arrayexpress
Project description:Evaluation of Full-Length Nanopore 16S Sequencing for Detection of Pathogens in Microbial Keratitis
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Project description:P. aeruginosa isolates from keratitis and healthy conjunctival sacs were obtained. The transcriptome profile of P. aeruginosa was characterized by a high throughput RNA-seq strategy using the Illumina HiSeq 2500 platform. The DEGs were analyzed with DESeq and validated through quantitative real-time polymerase chain reaction (PCR) and with experimental mice.
2022-08-24 | GSE209539 | GEO
Project description:Nanopore sequencing of K562 human cell line using the Oxford Nanopore GridION and R9.4.1 version chemistry
2021-09-20 | GSE173688 | GEO