Differentiation is accompanied by a progressive loss in transcriptional memory.

BACKGROUND: Cell differentiation requires the integration of two opposite processes, a stabilizing cellular memory, especially at the transcriptional scale, and a burst of gene expression variability which follows the differentiation induction. Therefore, the actual capacity of a cell to undergo phenotypic change during a differentiation process relies upon a modification in this balance which favors change-inducing gene expression variability. However, there are no experimental data providing insight on how fast the transcriptomes of identical cells would diverge on the scale of the very first two cell divisions during the differentiation process. RESULTS: In order to quantitatively address this question, we developed different experimental methods to recover the transcriptomes of related cells, after one and two divisions, while preserving the information about their lineage at the scale of a single cell division. We analyzed the transcriptomes of related cells from two differentiation biological systems (human CD34+ cells and T2EC chicken primary erythrocytic progenitors) using two different single-cell transcriptomics technologies (scRT-qPCR and scRNA-seq). CONCLUSIONS: We identified that the gene transcription profiles of differentiating sister cells are more similar to each other than to those of non-related cells of the same type, sharing the same environment and undergoing similar biological processes. More importantly, we observed greater discrepancies between differentiating sister cells than between self-renewing sister cells. Furthermore, a progressive increase in this divergence from first generation to second generation was observed when comparing differentiating cousin cells to self renewing cousin cells. Our results are in favor of a gradual erasure of transcriptional memory during the differentiation process.

Fungal Colonization of the Airways of Patients with Cystic Fibrosis: the Role of the Environmental Reservoirs.

Filamentous fungi frequently colonize the airways of patients with cystic fibrosis and may cause severe diseases, such as the allergic bronchopulmonary aspergillosis. The most common filamentous fungi capable to chronically colonize the respiratory tract of the patients are Aspergillus fumigatus and Scedosporium species. Defining the treatment strategy may be challenging, the number of available drugs being limited and some of the causative agents being multiresistant microorganisms. The knowledge of the fungal niches in the outdoor and indoor environment is needed for understanding the origin of the contamination of the patients. In light of the abundance of some of the causative molds in compost, agricultural and flower fields, occupational activities related to such environments should be discouraged for patients with cystic fibrosis (CF). In addition, the microbiological monitoring of their indoor environment, including analysis of air and dust on surfaces, is essential to propose preventive measures aiming to reduce the exposure to environmental molds. Nevertheless, some specific niches were also identified in the indoor environment, in relation with humidity which favors the growth of thermotolerant molds. Potted plants were reported as indoor reservoirs for Scedosporium species. Likewise, Exophiala dermatitidis may be spread in the kitchen via dishwashers. However, genotype studies are still required to establish the link between dishwashers and colonization of the airways of CF patients by this black yeast. Moreover, as nothing is known regarding the other filamentous fungi associated with CF, further studies should be conducted to identify other potential specific niches in the habitat.

An image-guided microfluidic system for single-cell lineage tracking.

Cell lineage tracking is a long-standing and unresolved problem in biology. Microfluidic technologies have the potential to address this problem, by virtue of their ability to manipulate and process single-cells in a rapid, controllable and efficient manner. Indeed, when coupled with traditional imaging approaches, microfluidic systems allow the experimentalist to follow single-cell divisions over time. Herein, we present a valve-based microfluidic system able to probe the decision-making processes of single-cells, by tracking their lineage over multiple generations. The system operates by trapping single-cells within growth chambers, allowing the trapped cells to grow and divide, isolating sister cells after a user-defined number of divisions and finally extracting them for downstream transcriptome analysis. The platform incorporates multiple cell manipulation operations, image processing-based automation for cell loading and growth monitoring, reagent addition and device washing. To demonstrate the efficacy of the microfluidic workflow, 6C2 (chicken erythroleukemia) and T2EC (primary chicken erythrocytic progenitors) cells are tracked inside the microfluidic device over two generations, with a cell viability rate in excess of 90%. Sister cells are successfully isolated after division and extracted within a 500 nL volume, which was demonstrated to be compatible with downstream single-cell RNA sequencing analysis.

Deciphering the Role of PIG1 and DHN-Melanin in Scedosporium apiospermum Conidia.

Scedosporium apiospermum is a saprophytic filamentous fungus involved in human infections, of which the virulence factors that contribute to pathogenesis are still poorly characterized. In particular, little is known about the specific role of dihydroxynaphtalene (DHN)-melanin, located on the external layer of the conidia cell wall. We previously identified a transcription factor, PIG1, which may be involved in DHN-melanin biosynthesis. To elucidate the role of PIG1 and DHN-melanin in S. apiospermum, a CRISPR-Cas9-mediated PIG1 deletion was carried out from two parental strains to evaluate its impact on melanin biosynthesis, conidia cell-wall assembly, and resistance to stress, including the ability to survive macrophage engulfment. ΔPIG1 mutants did not produce melanin and showed a disorganized and thinner cell wall, resulting in a lower survival rate when exposed to oxidizing conditions, or high temperature. The absence of melanin increased the exposure of antigenic patterns on the conidia surface. PIG1 regulates the melanization of S. apiospermum conidia, and is involved in the survival to environmental injuries and to the host immune response, that might participate in virulence. Moreover, a transcriptomic analysis was performed to explain the observed aberrant septate conidia morphology and found differentially expressed genes, underlining the pleiotropic function of PIG1.

Lignin-Modifying Enzymes in Scedosporium Species.

Scedosporium species are usually soil saprophytes but some members of the genus such as S. apiospermum and S. aurantiacum have been regularly reported as causing human respiratory infections, particularly in patients with cystic fibrosis (CF). Because of their low sensitivity to almost all available antifungal drugs, a better understanding of the pathogenic mechanisms of these fungi is mandatory. Likewise, identification of the origin of the contamination of patients with CF may be helpful to propose prophylactic measures. In this aim, environmental studies were conducted demonstrating that Scedosporium species are abundant in human-made environments and associated with nutrient-rich substrates. Although their natural habitat remains unknown, there is accumulated evidence to consider them as wood-decaying fungi. This study aimed to demonstrate the ability of these fungi to utilize lignocellulose compounds, especially lignin, as a carbon source. First, the lignolytic properties of Scedosporium species were confirmed by cultural methods, and biochemical assays suggested the involvement of peroxidases and oxidases as lignin-modifying enzymes. Scedosporium genomes were then screened using tBLASTn searches. Fifteen candidate genes were identified, including four peroxidase and seven oxidase genes, and some of them were shown, by real-time PCR experiments, to be overexpressed in lignin-containing medium, thus confirming their involvement in lignin degradation.

Evidence for close molecular proximity between reverting and undifferentiated cells.

BACKGROUND: According to Waddington's epigenetic landscape concept, the differentiation process can be illustrated by a cell akin to a ball rolling down from the top of a hill (proliferation state) and crossing furrows before stopping in basins or "attractor states" to reach its stable differentiated state. However, it is now clear that some committed cells can retain a certain degree of plasticity and reacquire phenotypical characteristics of a more pluripotent cell state. In line with this dynamic model, we have previously shown that differentiating cells (chicken erythrocytic progenitors (T2EC)) retain for 24 h the ability to self-renew when transferred back in self-renewal conditions. Despite those intriguing and promising results, the underlying molecular state of those "reverting" cells remains unexplored. The aim of the present study was therefore to molecularly characterize the T2EC reversion process by combining advanced statistical tools to make the most of single-cell transcriptomic data. For this purpose, T2EC, initially maintained in a self-renewal medium (0H), were induced to differentiate for 24H (24H differentiating cells); then, a part of these cells was transferred back to the self-renewal medium (48H reverting cells) and the other part was maintained in the differentiation medium for another 24H (48H differentiating cells). For each time point, cell transcriptomes were generated using scRT-qPCR and scRNAseq. RESULTS: Our results showed a strong overlap between 0H and 48H reverting cells when applying dimensional reduction. Moreover, the statistical comparison of cell distributions and differential expression analysis indicated no significant differences between these two cell groups. Interestingly, gene pattern distributions highlighted that, while 48H reverting cells have gene expression pattern more similar to 0H cells, they are not completely identical, which suggest that for some genes a longer delay may be required for the cells to fully recover. Finally, sparse PLS (sparse partial least square) analysis showed that only the expression of 3 genes discriminates 48H reverting and 0H cells. CONCLUSIONS: Altogether, we show that reverting cells return to an earlier molecular state almost identical to undifferentiated cells and demonstrate a previously undocumented physiological and molecular plasticity during the differentiation process, which most likely results from the dynamic behavior of the underlying molecular network.

Draft Genome Sequence of the Environmental Fungus Scedosporium dehoogii.

Today, the genus Scedosporium comprises at least ten species with four of them, Scedosporium apiospermum, Scedosporium boydii, Scedosporium aurantiacum and Scedosporium minutisporum capable of colonizing the lungs of patients with cystic fibrosis. Scedosporium dehoogii, which is also common in the soil, has never been reported as causing human pulmonary infections. Here we report the first genome sequence for S. dehoogii, an invaluable resource to understand the genetic bases of pathogenesis in the genus Scedosporium.

The Glycosylphosphatidylinositol-Anchored Superoxide Dismutase of Scedosporium apiospermum Protects the Conidia from Oxidative Stress.

Scedosporium species are common fungal pathogens in patients with cystic fibrosis (CF). To colonize the CF lungs, fungi must cope with the host immune response, especially the reactive oxygen species (ROS) released by phagocytic cells. To this aim, pathogens have developed various antioxidant systems, including superoxide dismutases (SODs) which constitute the first-line protection against oxidative stress. Interestingly, one of the S. apiospermum SOD-encoding genes (SODD gene) exhibits a glycosylphosphatidylinositol (GPI) anchor-binding site and encodes a conidial-specific surface SOD. In this study, a SODDΔ mutant was engineered from a non-homologous end joining-deficient strain (KU70Δ) of S. apiospermum. Compared to its parent strain, the double mutant KU70Δ/SODDΔ exhibited increased susceptibility to various oxidizing agents and triazole antifungals. In addition, the loss of SodD resulted in an increased intracellular killing of the conidia by M1 macrophages derived from human blood monocytes, suggesting the involvement of this superoxide dismutase in the evasion to the host defenses. Nevertheless, one cannot disregard an indirect role of the enzyme in the synthesis or assembly of the cell wall components since transmission electron microscopic analysis revealed a thickening of the inner cell wall layer of the conidia. Further studies are needed to confirm the role of this enzyme in the pathogenesis of Scedosporium infections, including the production of a recombinant protein and study of its protective effect against the infection in a mouse model of scedosporiosis.

Lower Funneling Pathways in Scedosporium Species.

Lignin, a natural polyaromatic macromolecule, represents an essential component of the lignocellulose biomass. Due to its complexity, the natural degradation of this molecule by microorganisms still remains largely misunderstood. Extracellular oxidative degradation is followed by intracellular metabolic degradation of conserved aromatic intermediate compounds (protocatechuate, catechol, hydroxyquinol, and gentisic acid) that are used as carbon and energy sources. The lower funneling pathways are characterized by the opening of the aromatic ring of these molecules through dioxygenases, leading to degradation products that finally enter into the tricarboxylic acid (TCA) cycle. In order to better understand the adaptation mechanisms of Scedosporium species to their environment, these specific catabolism pathways were studied. Genes encoding ring-cleaving dioxygenases were identified in Scedosporium genomes by sequence homology, and a bioinformatic analysis of the organization of the corresponding gene clusters was performed. In addition, these predictions were confirmed by evaluation of the expression level of the genes of the gentisic acid cluster. When the fungus was cultivated in the presence of lignin or gentisic acid as sole carbon source, experiments revealed that the genes of the gentisic acid cluster were markedly overexpressed in the two Scedosporium species analyzed (Scedosporium apiospermum and Scedosporium aurantiacum). Only the gene encoding a membrane transporter was not overexpressed in the gentisic acid-containing medium. Together, these data suggest the involvement of the lower funneling pathways in Scedosporium adaptation to their environment.

Multilocus Sequence Typing Reveals Extensive Genetic Diversity of the Emerging Fungal Pathogen Scedosporium aurantiacum.

Scedosporium spp. are the second most prevalent filamentous fungi after Aspergillus spp. recovered from cystic fibrosis (CF) patients in various regions of the world. Although invasive infection is uncommon prior to lung transplantation, fungal colonization may be a risk factor for invasive disease with attendant high mortality post-transplantation. Abundant in the environment, Scedosporium aurantiacum has emerged as an important fungal pathogen in a range of clinical settings. To investigate the population genetic structure of S. aurantiacum, a MultiLocus Sequence Typing (MLST) scheme was developed, screening 24 genetic loci for polymorphisms on a tester strain set. The six most polymorphic loci were selected to form the S. aurantiacum MLST scheme: actin (ACT), calmodulin (CAL), elongation factor-1α (EF1α), RNA polymerase subunit II (RPB2), manganese superoxide dismutase (SOD2), and ß-tubulin (TUB). Among 188 global clinical, veterinary, and environmental strains, 5 to 18 variable sites per locus were revealed, resulting in 8 to 23 alleles per locus. MLST analysis observed a markedly high genetic diversity, reflected by 159 unique sequence types. Network analysis revealed a separation between Australian and non-Australian strains. Phylogenetic analysis showed two major clusters, indicating correlation with geographic origin. Linkage disequilibrium analysis revealed evidence of recombination. There was no clustering according to the source of the strains: clinical, veterinary, or environmental. The high diversity, especially amongst the Australian strains, suggests that S. aurantiacum may have originated within the Australian continent and was subsequently dispersed to other regions, as shown by the close phylogenetic relationships between some of the Australian sequence types and those found in other parts of the world. The MLST data are accessible at http://mlst.mycologylab.org. This is a joined publication of the ISHAM/ECMM working groups on "Scedosporium/Pseudallescheria Infections" and "Fungal Respiratory Infections in Cystic Fibrosis".

The quiescent fraction of chronic myeloid leukemic stem cells depends on BMPR1B, Stat3 and BMP4-niche signals to persist in patients in remission.

Chronic myelogenous leukemia arises from the transformation of hematopoietic stem cells by the BCR-ABL oncogene. Though transformed cells are predominantly BCR-ABL-dependent and sensitive to tyrosine kinase inhibitor treatment, some BMPR1B+ leukemic stem cells are treatment-insensitive and rely, among others, on the bone morphogenetic protein (BMP) pathway for their survival via a BMP4 autocrine loop. Here, we further studied the involvement of BMP signaling in favoring residual leukemic stem cell persistence in the bone marrow of patients having achieved remission under treatment. We demonstrate by single-cell RNA-Seq analysis that a sub-fraction of surviving BMPR1B+ leukemic stem cells are co-enriched in BMP signaling, quiescence and stem cell signatures, without modulation of the canonical BMP target genes, but enrichment in actors of the Jak2/Stat3 signaling pathway. Indeed, based on a new model of persisting CD34+CD38- leukemic stem cells, we show that BMPR1B+ cells display co-activated Smad1/5/8 and Stat3 pathways. Interestingly, we reveal that only the BMPR1B+ cells adhering to stromal cells display a quiescent status. Surprisingly, this quiescence is induced by treatment, while non-adherent BMPR1B+ cells treated with tyrosine kinase inhibitors continued to proliferate. The subsequent targeting of BMPR1B and Jak2 pathways decreased quiescent leukemic stem cells by promoting their cell cycle re-entry and differentiation. Moreover, while Jak2-inhibitors alone increased BMP4 production by mesenchymal cells, the addition of the newly described BMPR1B inhibitor (E6201) impaired BMP4-mediated production by stromal cells. Altogether, our data demonstrate that targeting both BMPR1B and Jak2/Stat3 efficiently impacts persisting and dormant leukemic stem cells hidden in their bone marrow microenvironment.

Scedosporium species in soils from various biomes in Northwestern Morocco.

Scedosporium species are opportunistic pathogens causing various infections, including disseminated infections in severely immunocompromised patients. Preventive measures aiming to reduce the risk of exposure to these fungi require a better knowledge on their ecology and on the sources of contamination of the patients. In this context, 99 soil samples from the Rabat-Sale-Kenitra and Fez-Meknes regions in Morocco were analyzed. Samples were inoculated on the highly selective Scedo-Select III culture medium, and seven chemical parameters of the soils were measured. Scedosporium species were detected in 48 of the samples, with the highest density in soils from wastewater treatment plants and landfills, followed by those from roadsides and polluted riverbanks, thus confirming the impact of human activities on their ecology. Scedosporium apiospermum was the most common species, followed by S. boydii and S. aurantiacum. Analysis of the chemical parameters of the soils revealed the presence of Scedosporium species was mainly associated with a moderate electrical conductivity, a pH range of 7.0 to 7.6, a nutrient-rich content and a moderate phosphorus amount. Thereby, these results demonstrated the relatively high occurrence of Scedosporium in Morocco and highlighted the impact of phosphorus content on their ecology.

Drugs modulating stochastic gene expression affect the erythroid differentiation process.

To better understand the mechanisms behind cells decision-making to differentiate, we assessed the influence of stochastic gene expression (SGE) modulation on the erythroid differentiation process. It has been suggested that stochastic gene expression has a role in cell fate decision-making which is revealed by single-cell analyses but studies dedicated to demonstrate the consistency of this link are still lacking. Recent observations showed that SGE significantly increased during differentiation and a few showed that an increase of the level of SGE is accompanied by an increase in the differentiation process. However, a consistent relation in both increasing and decreasing directions has never been shown in the same cellular system. Such demonstration would require to be able to experimentally manipulate simultaneously the level of SGE and cell differentiation in order to observe if cell behavior matches with the current theory. We identified three drugs that modulate SGE in primary erythroid progenitor cells. Both Artemisinin and Indomethacin decreased SGE and reduced the amount of differentiated cells. On the contrary, a third component called MB-3 simultaneously increased the level of SGE and the amount of differentiated cells. We then used a dynamical modelling approach which confirmed that differentiation rates were indeed affected by the drug treatment. Using single-cell analysis and modeling tools, we provide experimental evidence that, in a physiologically relevant cellular system, SGE is linked to differentiation.

Erythroid differentiation displays a peak of energy consumption concomitant with glycolytic metabolism rearrangements.

Our previous single-cell based gene expression analysis pointed out significant variations of LDHA level during erythroid differentiation. Deeper investigations highlighted that a metabolic switch occurred along differentiation of erythroid cells. More precisely we showed that self-renewing progenitors relied mostly upon lactate-productive glycolysis, and required LDHA activity, whereas differentiating cells, mainly involved mitochondrial oxidative phosphorylation (OXPHOS). These metabolic rearrangements were coming along with a particular temporary event, occurring within the first 24h of erythroid differentiation. The activity of glycolytic metabolism and OXPHOS rose jointly with oxgene consumption dedicated to ATP production at 12-24h of the differentiation process before lactate-productive glycolysis sharply fall down and energy needs decline. Finally, we demonstrated that the metabolic switch mediated through LDHA drop and OXPHOS upkeep might be necessary for erythroid differentiation. We also discuss the possibility that metabolism, gene expression and epigenetics could act together in a circular manner as a driving force for differentiation.

WASABI: a dynamic iterative framework for gene regulatory network inference.

BACKGROUND: Inference of gene regulatory networks from gene expression data has been a long-standing and notoriously difficult task in systems biology. Recently, single-cell transcriptomic data have been massively used for gene regulatory network inference, with both successes and limitations. RESULTS: In the present work we propose an iterative algorithm called WASABI, dedicated to inferring a causal dynamical network from time-stamped single-cell data, which tackles some of the limitations associated with current approaches. We first introduce the concept of waves, which posits that the information provided by an external stimulus will affect genes one-by-one through a cascade, like waves spreading through a network. This concept allows us to infer the network one gene at a time, after genes have been ordered regarding their time of regulation. We then demonstrate the ability of WASABI to correctly infer small networks, which have been simulated in silico using a mechanistic model consisting of coupled piecewise-deterministic Markov processes for the proper description of gene expression at the single-cell level. We finally apply WASABI on in vitro generated data on an avian model of erythroid differentiation. The structure of the resulting gene regulatory network sheds a new light on the molecular mechanisms controlling this process. In particular, we find no evidence for hub genes and a much more distributed network structure than expected. Interestingly, we find that a majority of genes are under the direct control of the differentiation-inducing stimulus. CONCLUSIONS: Together, these results demonstrate WASABI versatility and ability to tackle some general gene regulatory networks inference issues. It is our hope that WASABI will prove useful in helping biologists to fully exploit the power of time-stamped single-cell data.

Developing collaborative works for faster progress on fungal respiratory infections in cystic fibrosis.

Cystic fibrosis (CF) is the major genetic inherited disease in Caucasian populations. The respiratory tract of CF patients displays a sticky viscous mucus, which allows for the entrapment of airborne bacteria and fungal spores and provides a suitable environment for growth of microorganisms, including numerous yeast and filamentous fungal species. As a consequence, respiratory infections are the major cause of morbidity and mortality in this clinical context. Although bacteria remain the most common agents of these infections, fungal respiratory infections have emerged as an important cause of disease. Therefore, the International Society for Human and Animal Mycology (ISHAM) has launched a working group on Fungal respiratory infections in Cystic Fibrosis (Fri-CF) in October 2006, which was subsequently approved by the European Confederation of Medical Mycology (ECMM). Meetings of this working group, comprising both clinicians and mycologists involved in the follow-up of CF patients, as well as basic scientists interested in the fungal species involved, provided the opportunity to initiate collaborative works aimed to improve our knowledge on these infections to assist clinicians in patient management. The current review highlights the outcomes of some of these collaborative works in clinical surveillance, pathogenesis and treatment, giving special emphasis to standardization of culture procedures, improvement of species identification methods including the development of nonculture-based diagnostic methods, microbiome studies and identification of new biological markers, and the description of genotyping studies aiming to differentiate transient carriage and chronic colonization of the airways. The review also reports on the breakthrough in sequencing the genomes of the main Scedosporium species as basis for a better understanding of the pathogenic mechanisms of these fungi, and discusses treatment options of infections caused by multidrug resistant microorganisms, such as Scedosporium and Lomentospora species and members of the Rasamsonia argillacea species complex.

Scedosporium and Lomentospora: an updated overview of underrated opportunists.

Species of Scedosporium and Lomentospora are considered as emerging opportunists, affecting immunosuppressed and otherwise debilitated patients, although classically they are known from causing trauma-associated infections in healthy individuals. Clinical manifestations range from local infection to pulmonary colonization and severe invasive disease, in which mortality rates may be over 80%. These unacceptably high rates are due to the clinical status of patients, diagnostic difficulties, and to intrinsic antifungal resistance of these fungi. In consequence, several consortia have been founded to increase research efforts on these orphan fungi. The current review presents recent findings and summarizes the most relevant points, including the Scedosporium/Lomentospora taxonomy, environmental distribution, epidemiology, pathology, virulence factors, immunology, diagnostic methods, and therapeutic strategies.

Automated cell cycle and cell size measurements for single-cell gene expression studies.

OBJECTIVES: Recent rise of single-cell studies revealed the importance of understanding the role of cell-to-cell variability, especially at the transcriptomic level. One of the numerous sources of cell-to-cell variation in gene expression is the heterogeneity in cell proliferation state. In order to identify how cell cycle and cell size influences gene expression variability at the single-cell level, we provide an universal and automatic toxic-free label method, compatible with single-cell high-throughput RT-qPCR. The method consists of isolating cells after a double-stained, analyzing their morphological parameters and performing a transcriptomic analysis on the same identified cells. RESULTS: This led to an unbiased gene expression analysis and could be also used for improving single-cell tracking and imaging when combined with cell isolation. As an application for this technique, we showed that cell-to-cell variability in chicken erythroid progenitors was negligibly influenced by cell size nor cell cycle.

Gene Disruption in Scedosporium aurantiacum: Proof of Concept with the Disruption of SODC Gene Encoding a Cytosolic Cu,Zn-Superoxide Dismutase.

Scedosporium species are opportunistic pathogens responsible for a large variety of infections in humans. An increasing occurrence was observed in patients with underlying conditions such as immunosuppression or cystic fibrosis. Indeed, the genus Scedosporium ranks the second among the filamentous fungi colonizing the respiratory tracts of the CF patients. To date, there is very scarce information on the pathogenic mechanisms, at least in part because of the limited genetic tools available. In the present study, we successfully developed an efficient transformation and targeted gene disruption approach on the species Scedosporium aurantiacum. The disruption cassette was constructed using double-joint PCR procedure, and resistance to hygromycin B as the selection marker. This proof of concept was performed on the functional gene SODC encoding the Cu,Zn-superoxide dismutase. Disruption of the SODC gene improved susceptibility of the fungus to oxidative stress. This technical advance should open new research areas and help to better understand the biology of Scedosporium species.