scholarly journals Cryptococcus neoformanscan form titan-like cellsin vitroin response to multiple signals that require the activation of several transduction pathways

2017 ◽  
Author(s):  
Nuria Trevijano-Contador ◽  
Suelen A. Rossi ◽  
Haroldo Cesar de Oliveira ◽  
Irene Llorente ◽  
Inês Correia ◽  
...  
Keyword(s):  
Gene Expression ◽  
Quorum Sensing ◽  
Signaling Pathways ◽  
Cryptococcus Neoformans ◽  
Gene Expression Profile ◽  
Cell Body ◽  
Expression Profile ◽  
Cell Formation ◽  
Pathogenic Yeast

ABSTRACTCryptococcus neoformansis an encapsulated pathogenic yeast that can change the size of the cells during infection. In particular, this process can occur by enlarging the size of the capsule without modifying the size of the cell body, or by increasing the diameter of the cell body, which is normally accompanied by an increase of the capsule too. This last process leads to the formation of cells of an abnormal enlarged size denominated titan cells. Previous works characterized titan cell formation during pulmonary infection but research on this topic has been hampered due to the difficulty to obtain themin vitro. In this work, we describein vitroconditions (low nutrient, serum supplemented medium at neutral pH) that promote the transition from regular to titan-like cells. Moreover, addition of azide and static incubation of the cultures in a CO2enriched atmosphere favored cellular enlargement. This transition occurred at low cell densities, suggesting that the process was regulated by quorum sensing molecules and was independent of the cryptococcal serotype/species. Transition to titan-like cell formation was impaired by pharmacological inhibition of PKC and TOR signaling pathways. Mutants affected in capsule synthesis did not form titan-like cells. Analysis of the gene expression profile in titan-like cells indicated that they overexpressed membrane proteins and transporters, being the gene encoding the Cig1 mannoprotein involved in iron uptake from heme groups the gene most differentially expressed compared to cells of regular size. We also investigated the gene expression profile of titan-like cells isolated from mice, and observed that during infection these cells mainly overexpressed genes related to metabolism and respiration. In summary, our work provides a new alternative method to investigate titan cell formation devoid the bioethical problems that involve animal experimentation.AUTHOR SUMMARYCryptococcus neoformansis a fungal pathogen that has a significant incidence in HIV+ patients in particular, in Subsaharian Africa, Asia and South America. This yeast poses an excellent model to investigate fungal virulence because it develops many strategies to adapt to the host and evade the immune response. One of the adaptation mechanisms involves the formation of Titan Cells, which are yeast of an abnormal large size. However, research on these cells has been limited to in vivo studies (mainly in mice) because they were not reproducibly found in vitro. In this work, we describe several conditions that induce the appearance of cells that mimic titan cells, and that we denominated as titan-like cells. The main factor that induced titan-like cells was the addition of serum to nutrient limited media. This has allowed to easily performing new approaches to characterize several signaling pathways involved in their development. We found that the formation of these cells is regulated by quorum sensing molecules, and that pathways such as PKC and TOR kinases regulate the process of cellular enlargement. We have also to perform transcriptomic analysis, which led to the identification of new genes that could be involved in the process. This work will open different research lines that will contribute to the elucidation of the role of these cells during infection and on the development of cryptococcal disease.

scholarly journals The transcription factor Pdr802 regulates Titan cell formation, quorum sensing, and pathogenicity of Cryptococcus neoformans

2020 ◽  
Author(s):  
Julia C. V. Reuwsaat ◽  
Daniel P. Agustinho ◽  
Heryk Motta ◽  
Holly Brown ◽  
Andrew L. Chang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Quorum Sensing ◽  
Cryptococcus Neoformans ◽  
Cell Formation ◽  
Negative Regulator ◽  
Pathogenic Yeast ◽  
Opportunistic Fungal Pathogen ◽  
The Brain

ABSTRACTCryptococcus neoformans is a ubiquitous, opportunistic fungal pathogen that kills almost 200,000 people worldwide each year. It is acquired when mammalian hosts inhale the infectious propagules; these are deposited in the lung and, in the context of immunocompromise, may disseminate to the brain and cause lethal meningoencephalitis. Once inside the host, C. neoformans undergoes a variety of adaptive processes, including secretion of virulence factors, expansion of a polysaccharide capsule that impedes phagocytosis, and the production of giant (Titan) cells. The transcription factor Pdr802 is one regulator of these responses to the host environment. Expression of the corresponding gene is highly induced under host-like conditions in vitro and is critical for C. neoformans dissemination and virulence in a mouse model of infection. Direct targets of Pdr802 include the quorum sensing proteins Pqp1, Opt1 and Liv3; the transcription factors Stb4, Zfc3 and Bzp4, which regulate cryptococcal brain infectivity and capsule thickness; the calcineurin targets Had1 and Crz1, important for cell wall remodeling and C. neoformans virulence; and additional genes related to resistance to host temperature and oxidative stress, and to urease activity. Notably, cryptococci engineered to lack Pdr802 showed a dramatic increase in Titan cells, which are not phagocytosed and have diminished ability to directly cross biological barriers. This explains the limited dissemination of pdr802 mutant cells to the central nervous system and the consequently reduced virulence of this strain. The role of Pdr802 as a negative regulator of Titan cell formation is thus critical for cryptococcal pathogenicity.IMPORTANCEThe pathogenic yeast Cryptococcus neoformans presents a worldwide threat to human health, especially in the context of immunocompromise, and current antifungal therapy is hindered by cost, limited availability, and inadequate efficacy. After the infectious particle is inhaled, C. neoformans initiates a complex transcriptional program that integrates cellular responses and enables adaptation to the host lung environment. Here we describe the role of the transcription factor Pdr802 in the response to host conditions and its impact on C. neoformans virulence. We identified direct targets of Pdr802 and also discovered that it regulates cellular features that influence movement of this pathogen from the lung to the brain, where it causes fatal disease. These findings advance our understanding of a serious disease.

Gene expression profile in monocyte during in vitro mineral fiber degradation

2007 ◽  
Vol 82 (6) ◽  
pp. 355-362 ◽  
Author(s):  
Hermine Dika Nguea ◽  
Aymon de Reydellet ◽  
Patrice Lehuédé ◽  
Alain De Meringo ◽  
Alain Le Faou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Mineral Fiber ◽  
Fiber Degradation

scholarly journals Expression of Genes Related to Germ Cell Lineage and Pluripotency in Single Cells and Colonies of Human Adult Germ Stem Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-17 ◽  
Author(s):  
Sabine Conrad ◽  
Hossein Azizi ◽  
Maryam Hatami ◽  
Mikael Kubista ◽  
Michael Bonin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Adult Stem Cells ◽  
Single Cells ◽  
Human Fibroblasts ◽  
Embryonic Stem ◽  
Human Adult

The aim of this study was to elucidate the molecular status of single human adult germ stem cells (haGSCs) and haGSC colonies, which spontaneously developed from the CD49f MACS and matrix- (collagen−/laminin+ binding-) selected fraction of enriched spermatogonia. Single-cell transcriptional profiling by Fluidigm BioMark system of a long-term cultured haGSCs cluster in comparison to human embryonic stem cells (hESCs) and human fibroblasts (hFibs) revealed that haGSCs showed a characteristic germ- and pluripotency-associated gene expression profile with some similarities to hESCs and with a significant distinction from somatic hFibs. Genome-wide comparisons with microarray analysis confirmed that different haGSC colonies exhibited gene expression heterogeneity with more or less pluripotency. The results of this study confirm that haGSCs are adult stem cells with a specific molecular gene expression profilein vitro, related but not identical to true pluripotent stem cells. Under ES-cell conditions haGSC colonies could be selected and maintained in a partial pluripotent state at the molecular level, which may be related to their cell plasticity and potential to differentiate into cells of all germ layers.

Novel Murine Model To Study Modulation of Genes and Molecular Pathways Induced Following In Vivo Interaction between Multiple Myeloma Cells and Human BM Milieu.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3409-3409
Author(s):  
Paola Neri ◽  
Pierfrancesco Tassone ◽  
Masood Shammas ◽  
Mariateresa Fulciniti ◽  
Yu-Tzu Tai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Growth ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Murine Model ◽  
Cell Signalling

Abstract Interaction between multiple myeloma (MM) cells and the bone marrow (BM) microenvironment plays a critical role in promoting MM cell growth, survival, migration and development of drug resistance. This interaction within the bone marrow milieu is unique and its understanding is important in evaluating effects of novel agents in vitro and in vivo. We here describe a novel murine model that allows us to study the expression changes in vivo in MM cells within the human BM milieu. In this model, the green fluorescent protein (INA-6 GFP+) transduced IL-6-dependent human MM cell line, INA-6, was injected in human bone chip implanted into SCID mice. At different time points the bone chip was retrieved, cells flushed out and GFP+ MM cells were purified by CD138 MACS microbeads. Similar isolation process was used on INA-6 GFP+ cells cultured in vitro and used as control. Total RNA was isolated from these cells and gene expression profile analyzed using the HG-U133 array chip (Affymetrix) and DChip analyzer program. We have identified significant changes in expression of several genes following in vivo interaction between INA-6 and the BM microenvironment. Specifically, we observed up-regulation of genes associated with cytokines (IL-4, IL-8, IGFB 2–5) and chemokines (CCL2, 5, 6, 18, 24, CCR1, 2, 4), implicated in cell-cell signalling. Moreover genes implicated in DNA transcription (V-Fos, V-Jun, V-kit), adhesion (Integrin alpha 2b, 7, cadherin 1 and 11) and cell growth (CDC14, Cyclin G2, ADRA1A) were also up-regulated and genes involved in apoptosis and cell death (p-57, BCL2, TNF1a) were down-regulated. Using the Ingenuity Pathway Analysis the most relevant pathways modulated by the in vivo interaction between MM cells and BMSCs were IL-6, IGF1, TGF-beta and ERK/MAPK-mediated pathways as well as cell-cycle regulation and chemokine signalling. These results are consistent with previously observed in vitro cell signalling studies. Taken together these results highlight the ability of BM microenvironment to modulate the gene expression profile of the MM cells and our ability to in vivo monitor the changes. This model thus provides us with an ability to study in vivo effects of novel agents on expression profile of MM cells in BM milieu, to pre-clinically characterize their activity.

Lenalidomide Differentially Modifies the Genomic Profile and miRNA Expression of Mesenchymal Stromal Cells From Patients with 5q- Syndrome,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3810-3810
Author(s):  
Sandra Muntión ◽  
Carlos Santamaría ◽  
Beatriz Roson ◽  
Carlos Romo ◽  
Olga López-Villar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profile ◽  
Mesenchymal Stromal Cells ◽  
Expression Profile ◽  
Stromal Cells ◽  
Microrna Expression ◽  
Advisory Committees ◽  
Healthy Donors ◽  
Lower Expression

Abstract Abstract 3810 Mesenchymal stromal cells (MSC) are a non-hematopoietic BM cell population considered to be not only the osteoblastic progenitors, but also a key component of the hematopoietic microenvironment. Raaijmakers et al (Nature, 2010) have recently shown that deletion of Dicer1 in MSC-derived osteoprogenitors as well as its target gene SBDS resulted in myelodysplasia (MDS) in a murine model. We have previously confirmed these results in human MSC from MDS patients (ASH 2010, # 397). In a previous paper (Leukemia, 2009) we showed that MSC from 5q- syndrome patients were different from MSC from other types of MDS and could be involved in their development. We have hypothesized that lenalidomide, the standard treatment of 5q- patients could act not only on hematopoietic progenitors but also on the BM microenvironment. For this purpose BM-MSC from healthy donors (HD) (n=7) and 5q- syndrome patients (n=5) were expanded in vitro and treated with 50 uM lenalidomide or its solvent (DMSO) as control. RNA was obtained from MSC and DICER1, DROSHA and SBDS relative gene expression was assessed by real-time PCR using TaqMan® assay as well as several microRNAs with known role in hematopoiesis and immune system regulation. In addition, MSC gene expression profile was studied. Labeled samples were hybridized to affymetrix of oligonucleotide HU 1.OST arrays in 5q- patients (n=4) and compared with MSCs from HD (n=3). For this purpose the ratio lenalidomide-treated sample and its paired DMSO control was calculated and markers with a fold change >1.5 were selected for hierarchical clustering analysis (HCA). MSCs from 5q-syndrome showed lower expression of DICER1 when compared with those from HD (.35 x10−3 vs.20 x10−3 p=0.03) but this expression was recovered when 5q-MSCs were treated with Lenalidomide (0.32 x10−3 p= 0.34). By contrast, no differences in DROSHA expression were observed. In addition, 5q-MSC showed SBDS lower expression than HD-MSC and in both groups the expression increased when they were treated with lenalidomide fig1). When microRNAs were analyzed, we observed a lower microRNA expression in lenalidomide-treated MSC from healthy donors when was compared to paired non-treated cells, especially for miRNA-155 (p=0.028), miRNA-222 (p=0.028),and miRNA-181a (p=0.075; Table 1). By contrast, lenalidomide-treated MSC from MDS showed a trend towards higher microRNA expression in comparison to paired non-treated MSC.Table 1.HD-MSC DMSO vs LENA5q-MSC DMSO vs LENAmiRNA 1460.50 vs 0.30p=0.2490.07 vs 0.10p=0.7miRNA 1500.004 vs 0.0065p=0.60.001 vs 0.006p=0.07miRNA 1550.90 vs 0.58p=0.0280.80 vs 0.96p=0.7miRNA 181a2.47 vs 1,83p=0.0751.66 vs 2.32p=0.07miRNA 22286.2 vs 68.0p=0.02843.2 vs 56.2p=0.07 When the gene expression profile was carried out based in 421 selected probes including 306 known genes, MSC-treated cells from 5q- were separated from HD MSC by HCA (Fig2). We can conclude that Lenalidomide not only acts on HPC from 5q- patients but also on microenvironment by modifying the expression of DICER-1 and SBDS as well as the expression of some microRNAs and genes. Disclosures: San Miguel: Celgene Corp.: Membership on an entity's Board of Directors or advisory committees. del Cañizo:Celgene Corp.: Spanishn Adviory committee.

scholarly journals Gene Expression Profile of the Human Colorectal Carcinoma LoVo Cells Treated With Sporamin and Thapsigargin

2021 ◽  
Vol 11 ◽  
Author(s):  
Chun Yang ◽  
Si-Jia Chen ◽  
Bo-Wen Chen ◽  
Kai-Wen Zhang ◽  
Jing-Jie Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Ipomoea Batatas ◽  
Expression Profiles ◽  
Rna Seq ◽  
Ppi Network ◽  
Illumina Platform

Sporamin, a proteinase inhibitor isolated from the sweet potato (Ipomoea batatas), has shown promising anticancer effect against colorectal cancer (CRC) in vitro and in vivo but its mechanisms of action are poorly understood. In the present study, high throughput RNA sequencing (RNA-seq) technology was applied to explore the transcriptomic changes induced by sporamin in the presence of thapsigargin (TG), a non-12-O-tetradecanolphorbol-13-acetate type cancer promoter, in the LoVo human CRC cells. Cellular total RNA was extracted from the cells after they were treated with vehicle (CTL), 1 μM of thapsigargin (TG), or 1 μM of TG plus 30 μM of sporamin (TGSP) for 24 h. The migratory capacity of the cells was determined by wound healing assay. The gene expression profiles of the cells were determined by RNA-seq on an Illumina platform. GO enrichment analysis, KEGG pathway analysis, protein-protein interaction (PPI) network construction, and transcription factors (TF) prediction were all performed based on the differentially expressed genes (DEGs) across groups with a series of bioinformatics tools. Finally, the effect and potential molecular targets of the sporamin at the transcriptome level were evaluated. Sporamin significantly inhibited the migration of cells induced by TG. Among the 17915 genes detected in RNA-seq, 46 DEGs were attributable to the effect of sporamin. RT-PCR experiment validated that the expression of RGPD2, SULT1A3, and BIVM-ERCC5 were up-regulated while NYP4R, FOXN1, PAK6, and CEACAM20 were down-regulated. Sporamin enhanced the mineral absorption pathway, worm longevity regulating pathway, and pyrimidine metabolism pathway. Two TFs (SMIM11A and ATOH8) were down-regulated by sporamin. HMOX1 (up-regulated) and NME1-NME2 (down-regulated) were the main nodes in a PPI network consisting of 16 DEGs that were modulated by sporamin in the presence of TG. Sporamin could favorably alter the gene expression profile of CRC cells, up-regulating the genes that contribute to the homeostasis of intracellular metal ions and the activities of essential enzymes and DNA damage repairment. More studies are warranted to verify its effect on specific genes and delineate the mechanism of action implicated in the process.

scholarly journals Ion homeostasis and transport are regulated by genes differentially expressed in porcine buccal pouch mucosal cells during long-term culture in vitro – a microarray approach

2018 ◽  
Vol 6 (3) ◽  
pp. 75-82 ◽  
Author(s):  
Artur Bryja ◽  
Marta Dyszkiewicz-Konwińska ◽  
Maurycy Jankowski ◽  
Piotr Celichowski ◽  
Katarzyna Stefańska ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ion Transport ◽  
Gene Expression Profile ◽  
Oral Mucosa ◽  
Expression Profile ◽  
Ion Homeostasis ◽  
Starting Point ◽  
Mucosal Cells

Abstract The oral mucosa is a compound tissue composed of several cells types, including fibroblasts and keratinocytes, that are characterized by different morphology, as well as biochemical and metabolomic properties. The oral mucosal cells are the most important factors mediated between transport and drugs delivery. The changes in cellular ion homeostasis may significantly affect the bioavailability of administrated drugs and their transport across the mucous membrane. Therefore we investigated the expression profile of genes involved in ion transport and homeostasis in porcine buccal pouch mucosal cells. The oral mucosa was separated surgically and isolated enzymatically. The cells were examined during long-term in vitro culture (IVC). The cultured cells were collected at 7, 15 and 30 days of IVC and subsequently transferred to RNA isolation and next, the gene expression profile was measured using Affymetrix microarray assays. In the results, we can extract genes belonging to four ontology groups: “ion homeostasis”, “ion transport”, “metal ion transport”, and “inorganic ion homeostasis”. For TGFB1 and CCL2, we observed up-regulation after 7 days of IVC, down-regulation after 15 days of IVC and upregulation again after 30 days of IVC. The ATP13A3, ATP1B1, CCL8, LYN, STEAP1, PDPN, PTGS2, and SLC5A3genes showed high activity after day 7 of IVC, and in the days 15 and 30 of IVC showed low activity. We showed an expression profile of genes associated with the effects of ion influence on the porcine normal oral mucosal cell development in IVC. These studies may be the starting point for further research into oral diseases and will allow for the comparison of the gene expression profile of normal and disease altered cells.

scholarly journals Titan cells formation inCryptococcus neoformansis finely tuned by environmental conditions and modulated by positive and negative genetic regulators

2017 ◽  
Author(s):  
Benjamin Hommel ◽  
Liliane Mukaremera ◽  
Radames J. B. Cordero ◽  
Carolina Coelho ◽  
Christopher A. Desjardins ◽  
...  
Keyword(s):  
Cell Wall ◽  
Quorum Sensing ◽  
Cryptococcus Neoformans ◽  
Cell Size ◽  
Incubation Medium ◽  
Genetic Factors ◽  
Morphological Changes ◽  
Cell Formation ◽  
Low Ph

AbstractThe pathogenic fungusCryptococcus neoformansexhibits morphological changes in cell size during lung infection, producing both typical size 5 to 7 µm cells and large titan cells (> 10 µm and up to 100 µm). We found and optimizedin vitroconditions that produce titan cells in order to identify the ancestry of titan cells, the environmental determinants, and the key gene regulators of titan cell formation. Titan cells generatedin vitroharbor the main characteristics of titan cells producedin vivoincluding their large cell size (>10 µm), polyploidy with a single nucleus, large vacuole, dense capsule, and thick cell wall. Here we show titan cells derived from the enlargement of progenitor cells in the population independent of yeast growth rate. Change in the incubation medium, hypoxia, nutrient starvation and low pH were the main factors that trigger titan cell formation, while quorum sensing factors like the initial inoculum concentration, pantothenic acid, and the quorum sensing peptide Qsp1p also impacted titan cell formation. Inhibition of ergosterol, protein and nucleic acid biosynthesis altered titan cell formation, as did serum, phospholipids and anti-capsular antibodies in our settings. We explored genetic factors important for titan cell formation using three approaches. Using H99-derivative strains with natural genetic differences, we showed that titan cell formation was dependent onLMP1andSGF29genes. By screening a gene deletion collection, we also confirmed thatGPR4/5-RIM101, andCAC1genes were required to generate titan cells and that thePKR1,TSP2,USV101genes negatively regulated titan cell formation. Furthermore, analysis of spontaneous Pkr1 loss-of-function clinical isolates confirmed the important role of the Pkr1 protein as a negative regulator of titan cell formation. Through development of a standardized and robustin vitroassay, our results provide new insights into titan cell biogenesis with the identification of multiple important factors/pathways.Author SummaryCryptococcus neoformansis a yeast that is capable of morphological change upon interaction with the host. Particularly, in the lungs of infected mice, a subpopulation of yeast enlarges, producing cells up to 100 µm in cell body diameter – referred to as titan cells. Along with their large size, the titan cells have other unique characteristics such as thickened cell wall, dense capsule, polyploidization, large vacuole with peripheral nucleus and cellular organelles. The generation of a large number of such cells outside the lungs of mice has been described but was not reproducible nor standardized. Here we report standardized, reproducible, robust conditions for generation of titan cells and explored the environmental and genetic factors underlying the genesis of these cells. We showed that titan cells were generated upon stresses such as change in the incubation medium, nutrient deprivation, hypoxia and low pH. Using collections of well characterized reference strains and clinical isolates, we validated with our model that the cAMP/PKA/Rim101 pathway is a major genetic determinant of titan cell formation. This study opens the way for a more comprehensive picture of the ontology of morphological changes inCryptococcus neoformansand its impact on pathobiology of this deadly pathogen.

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