scholarly journals RNA sequencing identifies global transcriptional changes in peripheral CD4 + cells during active oesophagitis and following epicutaneous immunotherapy in eosinophilic oesophagitis

2021 ◽  
Vol 10 (7) ◽  
Author(s):  
Melanie A Ruffner ◽  
Zhe Zhang ◽  
Kelly Maurer ◽  
Amanda B Muir ◽  
Antonella Cianferoni ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Cd4 Cells ◽  
Eosinophilic Oesophagitis ◽  
Epicutaneous Immunotherapy ◽  
Transcriptional Changes

scholarly journals Transcriptional Changes in Potato Sprouts upon Interaction with Rhizoctonia solani Indicate Pathogen-Induced Interference in the Defence Pathways of Potato

2021 ◽  
Vol 22 (6) ◽  
pp. 3094
Author(s):  
Rita Zrenner ◽  
Bart Verwaaijen ◽  
Franziska Genzel ◽  
Burkhardt Flemer ◽  
Rita Grosch
Keyword(s):  
Rhizoctonia Solani ◽  
Rna Sequencing ◽  
Control Strategies ◽  
Transcriptional Response ◽  
Black Scurf ◽  
Post Inoculation ◽  
Molecular Response ◽  
Resistance Level ◽  
Sequencing Experiment ◽  
Transcriptional Changes

Rhizoctonia solani is the causer of black scurf disease on potatoes and is responsible for high economical losses in global agriculture. In order to increase the limited knowledge of the plants’ molecular response to this pathogen, we inoculated potatoes with R. solani AG3-PT isolate Ben3 and carried out RNA sequencing with total RNA extracted from potato sprouts at three and eight days post inoculation (dpi). In this dual RNA-sequencing experiment, the necrotrophic lifestyle of R. solani AG3-PT during early phases of interaction with its host has already been characterised. Here the potato plants’ comprehensive transcriptional response to inoculation with R. solani AG3 was evaluated for the first time based on significantly different expressed plant genes extracted with DESeq analysis. Overall, 1640 genes were differentially expressed, comparing control (−Rs) and with R. solani AG3-PT isolate Ben3 inoculated plants (+Rs). Genes involved in the production of anti-fungal proteins and secondary metabolites with antifungal properties were significantly up regulated upon inoculation with R. solani. Gene ontology (GO) terms involved in the regulation of hormone levels (i.e., ethylene (ET) and jasmonic acid (JA) at 3 dpi and salicylic acid (SA) and JA response pathways at 8 dpi) were significantly enriched. Contrastingly, the GO term “response to abiotic stimulus” was down regulated at both time points analysed. These results may support future breeding efforts toward the development of cultivars with higher resistance level to black scurf disease or the development of new control strategies.

scholarly journals Differentially Expressed Gene Profile of Acanthamoeba castellanii Induced by an Endosymbiont Legionella pneumophila

2021 ◽  
Vol 59 (1) ◽  
pp. 67-75
Author(s):  
Eun-Kyung Moon ◽  
So-Min Park ◽  
Ki-Back Chu ◽  
Fu-Shi Quan ◽  
Hyun-Hee Kong
Keyword(s):  
Rna Sequencing ◽  
Legionella Pneumophila ◽  
Differentially Expressed Gene ◽  
Acanthamoeba Castellanii ◽  
Opportunistic Pathogen ◽  
Sequencing Analysis ◽  
Sequencing Data ◽  
Oxidoreductase Activity ◽  
Transcriptional Changes ◽  
Upregulated Genes

Legionella pneumophila is an opportunistic pathogen that survives and proliferates within protists such as Acanthamoeba spp. in environment. However, intracellular pathogenic endosymbiosis and its implications within Acanthamoeba spp. remain poorly understood. In this study, RNA sequencing analysis was used to investigate transcriptional changes in A. castellanii in response to L. pneumophila infection. Based on RNA sequencing data, we identified 1,211 upregulated genes and 1,131 downregulated genes in A. castellanii infected with L. pneumophila for 12 hr. After 24 hr, 1,321 upregulated genes and 1,379 downregulated genes were identified. Gene ontology (GO) analysis revealed that L. pneumophila endosymbiosis enhanced hydrolase activity, catalytic activity, and DNA binding while reducing oxidoreductase activity in the molecular function (MF) domain. In particular, multiple genes associated with the GO term ‘integral component of membrane’ were downregulated during endosymbiosis. The endosymbiont also induced differential expression of various methyltransferases and acetyltransferases in A. castellanii. Findings herein are may significantly contribute to understanding endosymbiosis of L. pneumophila within A. castellanii.

scholarly journals RNA sequencing identifies transcriptional changes in the rabbit larynx in response to low humidity challenge

2020 ◽  
Author(s):  
Taylor W Bailey ◽  
Andrea Santos ◽  
Naila Cannes de Nascimento ◽  
M. Preeti Sivasankar ◽  
Abigail Cox
Keyword(s):  
Rna Sequencing ◽  
Protective Factor ◽  
Rabbit Model ◽  
Zealand White Rabbit ◽  
Vocal Folds ◽  
Rna Seq ◽  
Targeted Analysis ◽  
Transcriptional Changes ◽  
Low Humidity

Abstract Background Voice disorders are a worldwide problem impacting human health, particularly for occupational voice users. Avoidance of surface dehydration is commonly prescribed as a protective factor against the development of dysphonia. The available literature inconclusively supports this practice and a biological mechanism for how surface dehydration of the laryngeal tissue affects voice has not been described. In this study, we used an in vivo male New Zealand white rabbit model to elucidate biological changes based on gene expression within the vocal folds from surface dehydration. Surface dehydration was induced by exposure to low humidity air (18.6% ± 4.3%) for 8 hours. Exposure to moderate humidity (43.0% ± 4.3%) served as the control condition. Ilumina-based RNA sequencing was performed and used for transcriptome analysis with validation by RT-qPCR. Results There were 103 genes identified through Cuffdiff with 64 genes meeting significance by both false discovery rate and fold change. Functional annotation enrichment and predicted protein interaction mapping showed enrichment of various loci, including cellular stress and inflammatory response, ciliary function, and keratinocyte development. Eight genes were selected for RT-qPCR validation. Matrix metalloproteinase 12 (MMP12) and macrophage cationic peptide 1 (MCP1) were significantly upregulated and an epithelial chloride channel protein (ECCP) was significantly downregulated after surface dehydration by RNA-Seq and RT-qPCR. Suprabasin (SPBN) and zinc activated cationic channel (ZACN) were marginally, but non-significantly down- and upregulated by RT-qPCR, respectively. Conclusions The data together support the notion that surface dehydration induces physiological changes in the vocal folds and justifies targeted analysis to further explore the underlying biology of compensatory fluid/ion flux and inflammatory mediators in response to airway surface dehydration.

Primary T-Prolymphocytic Leukemia (T-PLL) Cells Are Sensitive To BCL-2 and HDAC Inhibitors: Results From High-Throughput Ex Vivo Drug Testing

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3828-3828
Author(s):  
Emma I Andersson ◽  
Tea Pemovska ◽  
Samuli Eldfors ◽  
Anneli Lauhio ◽  
Paavo Pietarinen ◽  
...  
Keyword(s):  
T Cell ◽  
Rna Sequencing ◽  
High Throughput ◽  
Research Funding ◽  
Drug Sensitivity ◽  
Ex Vivo ◽  
Hdac Inhibitors ◽  
High Sensitivity ◽  
Leukemic Cells ◽  
Cd4 Cells

Abstract Introduction T-PLL is a rare mature post-thymic T-cell neoplasm with an aggressive clinical course and median overall survival of less than one year. Almost 75% of T-PLL cases harbor chromosome 14 translocations involving the T-cell receptor A/D locus resulting in aberrant activation of the proto-oncogenes TCL1A or MTCP1. T-PLL patients are difficult to treat as the leukemic cells are often resistant to most available chemotherapeutic drugs. Due to the rareness and aggressive nature of the disease, large clinical trials are difficult to execute. We therefore aimed to discover novel potential therapeutic targets using a high-throughput ex vivo drug sensitivity and resistance testing (DSRT) platform covering 306 approved and investigational oncology drugs. Methods Primary T-PLL cells were available from two patients. The first patient had a double positive CD4+CD8+CD3+ Vβ.14+ T-cell phenotype (patient 1) and cells underwent DSRT twice during a 5-month time-period (no treatment during that time). The second patient had a CD4+CD3+ phenotype (patient 2) and the cells were assayed once by DSRT. Fresh blood mononuclear cells (MNCs) were separated by Ficoll centrifugation from the patient samples (over 85 % leukemic cells in the MNC fraction) and healthy controls. Cells were seeded in 384-well plates and 306 active substances were tested using a 10,000-fold concentration range resulting in a dose-response curve for each compound. Cell viability was measured after 72 h incubation and differential drug sensitivity scores (DSS), representing leukemia-specific responses, were calculated by comparing patient samples with those obtained from healthy donors. In addition, both exome and RNA sequencing was performed from T-PLL cells (patient 1). Results Both patient samples showed high sensitivity to small molecule BCL2-inhibitors navitoclax (EC50 values 44nM and 10nM) and ABT-199 (EC50 23nM and 20nM) (Fig. 1 and 2). HDAC-inhibitors (quisinostat, belinostat and panobinostat) also showed high sensitivity in both patients in low nM concentrations (EC50 values 1-80nM). As AKT1/mTOR pathway is activated in most T-PLL patients due to the TCL1 oncoprotein, it was interesting to observe that neither of the patient samples showed any response to an AKT1 inhibitor (MK-2206 EC50 values >1000 nM) nor to mTOR inhibitors (temsirolimus and everolimus)(Fig. 1). Furthermore, T-PLL cells were resistant to corticosteroids such as prednisolone and methylprednisolone. To further elucidate the molecular mechanism behind the drug responses, exome and RNA sequencing was performed from T-PLL cells (patient 1). No deletion was found in the ATM gene, but instead a homozygous missense mutation K2413Q was detected. This particular mutation is in the region coding for the FAT domain and while it has not been described earlier in T-PLL, it is in a cancer mutation hotspot region of ATM, suggesting that it is inactivating. No mutations directly linked to the BCL2-family genes were observed. In the RNA sequencing analysis, TCL1A was overexpressed when compared to the healthy CD4+ cells as expected. Similarly, AKT1 was overexpressed. The expression of BCL-2 and BCL-XL did not differ from those observed in healthy CD4+ cells while pro-apoptotic BCL-2 family members BID and BAD were elevated compared to the healthy control. Conclusions Primary T-PLL cells showed sensitivity to BCL-2 and HDAC inhibitors in a systematic high-throughput ex vivo drug sensitivity testing across a range of clinical and investigational drugs. The BCL-2 inhibitor sensitivity was not related to increased BCL-2 expression or activating mutations in the BCL-2 family genes, and further studies are needed to clarify the mechanism of action. However, the results suggest that BCL-2 inhibitors could be a novel promising candidate drug for T-PLL-patients and warrant further clinical development in this group of patients. In contrast, inhibitors of AKT and mTOR, kinases known to be activated by TCL1, showed no efficacy ex vivo in this assay. Disclosures: Porkka: BMS: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau. Mustjoki:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau.

scholarly journals CRISPR-Cas9 Mediated CD45 Knockout Inactivates Src Family Kinases and Impairs Cell Migration in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1907-1907
Author(s):  
Wing Yu Man ◽  
Tiffany Khong ◽  
Andrew Spencer
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Migration ◽  
Disease Progression ◽  
Rna Sequencing ◽  
Molecular Mechanisms ◽  
Src Family Kinases ◽  
Antibody Array ◽  
Transcriptional Changes

Abstract Introduction Multiple myeloma (MM) is a plasma cell malignancy that manifests continuous cell dissemination to multiple bone marrow (BM) niches and extramedullary (EM) sites. However, the molecular mechanisms behind this phenomenon remain elusive. CD45, a receptor tyrosine phosphatase, is an important regulator for T-cell and B-cell signaling pathways. In MM, the loss of CD45 expression has been correlated with earlier disease progression and inferior treatment outcomes. The downstream targets of CD45, Src family kinases (SFK), are associated with cell migration in many malignancies. It is also known to interact with proline-rich kinase (Pyk2) that binds to cytoskeleton-regulating proteins. Our previous in vitro studies demonstrated a 'metastatic' phenotype for CD45 negative MM. We hypothesise that CD45 acts as a marker for disease progression and mediates cell mobility through SFKs. Method CD45 expression in human myeloma cell lines (HMCL) was assessed by flow cytometry. To investigate the functions of CD45 in MM, CRISPR-Cas9 mediated CD45 knockout (CD45KO) models were established from a HMCL, OCI-MY1. The resulting phenotypic and transcriptional changes were identified by immunoblotting, modified Boyden chamber assays, RNA sequencing and Phospho Explorer Antibody Array. SFK inhibitor (Saracatinib), Pyk2 inhibitor (PF573228) and siRNAs were used to validate the role of SFK (predominately Lyn and Fyn) and Pyk2 in migration. Results We first compared 2 pairs of HMCL contemporaneously derived from BM and extramedullary disease (EM) in the same patient: TK1 (BM) and TK2 (EM), and TK17 (EM) and TK18 (BM). Both TK1 and TK18 had higher CD45 expression than their paired HMCL as expected. To avoid confounding intercellular genetic heterogeneity, we generated CD45KO models from OCI-MY1 and the loss of extracellular and intracellular portion of CD45 was confirmed by flow cytometry and immunoblotting. OCI-MY1 CD45KO cells showed significant SFK (Lyn and Fyn) and Pyk2 inactivation as compared to CD45 wild-type (CD45WT) cells. These cells demonstrated a significant reduction in homing capacity towards healthy and MM-patient derived BM stromal cells (reduced to 11.5% and 2.7%, p<0.0001, respectively) compared with the CD45WT cells. Treatment of CD45WT cells with Saracatinib and PF573228 similarly inhibited the homing capacity (47.3%, p<0.0001 and 71.3%, p<0.01 respectively). Moreover, silencing of Lyn and Fyn with siRNAs in CD45WT cells recapitulated the findings seen with the CD45KO cells (76%, p<0.01 and 55%, p<0.001, reduction in homing, respectively). The inactivation of SFK prompted us to investigate the transcriptional changes in the CD45KO cells. RNA sequencing identified differentially expressed migration-related genes in the CD45KO cell, in particular ADAM19, PARVB, AFAP1L2 and ITGAL, thus raising the possibility that dysregulation of these genes led to the observed reduction of homing potential of the CD45KO cells. To further understand the role of CD45 phosphatase activity, we analysed protein phosphorylation profiles in the CD45KO and CD45WT cells. In addition to the recognised inactivation of SFK and Pyk2, the Phospho Explorer Antibody Array also demonstrated reduced phosphorylation of JAK1 (Y1022), TYK2 (Y1054), c-Raf (S259) and MDM2 (S166), and increased phosphorylation of IRS-1 (S794), SHP-2 (Y580), c-Jun (Y170) and STAT6 (T645). We are currently investigating the correlation of these phosphorylation and MM homing mechanisms. Conclusion Our data demonstrate that CD45 plays an important role in regulating MM homing towards BM stroma by modulating SFK and Pyk2 activity with the loss of CD45 expression also resulting in the dysregulation of migration-related genes and widespread phosphorylation changes. Further in vivo studies evaluating the 'metastatic' impact of loss of CD45 expression are in progress. Disclosures No relevant conflicts of interest to declare.

scholarly journals Altered levels of hsromega lncRNAs further enhance Ras signaling during ectopically activated Ras induced R7 differentiation in Drosophila

2017 ◽  
Author(s):  
Mukulika Ray ◽  
Gunjan Singh ◽  
Subhash C. Lakhotia
Keyword(s):  
Signaling Pathway ◽  
Rna Sequencing ◽  
Rna Processing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Ras Signaling ◽  
Transcript Levels ◽  
Cone Cells ◽  
Ras Signaling Pathway ◽  
Transcriptional Changes

AbstractWe exploited the high Ras activity induced differentiation of supernumerary R7 cells in Drosophila eyes to examine if hsrω lncRNAs influence active Ras signaling. Surprisingly, either down- or up-regulation of hsrω lncRNAs in sev-GAL4>RasV12 expressing eye discs resulted in complete pupal lethality and substantially greater increase in R7 photoreceptor number at the expense of cone cells. Enhanced nuclear p-MAPK and presence of sev-GAL4 driven RasV12 bound RafRBDFLAG in cells not expressing the sev-GAL4 driver indicated non-cell autonomous spread of Ras signaling when hsrω levels were co-altered. RNA-sequencing revealed that down-and up-regulation of hsrω transcripts in sev-GAL4>RasV12 expressing eye discs elevated transcripts of positive or negative modulators, respectively, of Ras signaling so that either condition enhances it. Altered hsrω transcript levels in sev-GAL4>RasV12 expressing discs also affected sn/sno/sca RNAs and some other RNA processing transcript levels. Post-transcriptional changes due to the disrupted intra-cellular dynamicity of omega speckle associated hnRNPs and other RNA-binding proteins that follow down- or up-regulation of hsrω lncRNAs appear to be responsible for the further elevated Ras signaling. Cell autonomous and non-autonomous enhancement of Ras signaling by lncRNAs like hsrω has implications for cell signaling during high Ras activity commonly associated with some cancers.HighlightsOur findings highlight roles of hsrω lncRNAs in conditionally modulating the important Ras signaling pathway and provide evidence for cell non-autonomous Ras signaling in Drosophila eye discs.

scholarly journals RNA sequencing identifies transcriptional changes in the rabbit larynx in response to low humidity challenge

2020 ◽  
Author(s):  
Taylor W Bailey ◽  
Andrea Pires dos Santos ◽  
Naila Cannes do Nascimento ◽  
Shaojun Xie ◽  
Jyothi Thimmapuram ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Protective Factor ◽  
Rabbit Model ◽  
Zealand White Rabbit ◽  
Vocal Folds ◽  
Rna Seq ◽  
Targeted Analysis ◽  
Transcriptional Changes ◽  
Low Humidity

Abstract Background: Voice disorders are a worldwide problem impacting human health, particularly for occupational voice users. Avoidance of surface dehydration is commonly prescribed as a protective factor against the development of dysphonia. The available literature inconclusively supports this practice and a biological mechanism for how surface dehydration of the laryngeal tissue affects voice has not been described. In this study, we used an in vivo male New Zealand white rabbit model to elucidate biological changes based on gene expression within the vocal folds from surface dehydration. Surface dehydration was induced by exposure to low humidity air (18.6% + 4.3%) for 8 hours. Exposure to moderate humidity (43.0% + 4.3%) served as the control condition. Ilumina-based RNA sequencing was performed and used for transcriptome analysis with validation by RT-qPCR. Results: There were 103 statistically significant differentially expressed genes identified through Cuffdiff with 61 genes meeting significance by both false discovery rate and fold change. Functional annotation enrichment and predicted protein interaction mapping showed enrichment of various loci, including cellular stress and inflammatory response, ciliary function, and keratinocyte development. Eight genes were selected for RT-qPCR validation. Matrix metalloproteinase 12 (MMP12) and macrophage cationic peptide 1 (MCP1) were significantly upregulated and an epithelial chloride channel protein (ECCP) was significantly downregulated after surface dehydration by RNA-Seq and RT-qPCR. Suprabasin (SPBN) and zinc activated cationic channel (ZACN) were marginally, but non-significantly down- and upregulated as evidenced by RT-qPCR, respectively. Conclusions: The data together support the notion that surface dehydration induces physiological changes in the vocal folds and justifies targeted analysis to further explore the underlying biology of compensatory fluid/ion flux and inflammatory mediators in response to airway surface dehydration.

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