Efficient genome editing in erythroid cells unveils novel MYB target genes and regulatory functions.

Targeted genome editing holds great promise in biology. However, efficient genome modification, including gene knock-in (KI), remains an unattained goal in multiple cell types and loci due to poor transfection efficiencies and low target genes expression, impeding the positive selection of recombined cells. Here, we describe a genome editing approach to achieve efficient gene targeting using hard to transfect erythroid cell lines. We demonstrate robust fluorescent protein KI efficiency in low expressed transcription factor (TF) genes (e.g., Myb or Zeb1). We further show the ability to target two independent loci in individual cells, exemplified by MYB-GFP and NuMA-Cherry double KI, allowing multicolor labeling of regulatory factors at physiological endogenous levels. Our KI tagging approach allowed us to perform genome-wide TF analysis at increased signal-to-noise ratios, and highlighted previously unidentified MYB target genes and pathways. Overall, we establish a versatile CRISPR-Cas9-based platform, offering attractive opportunities for the dissection of the erythroid differentiation process.

Var∣Decrypt: a novel and user-friendly tool to explore and prioritize variants in whole-exome sequencing data.

BACKGROUND: High-throughput sequencing (HTS) offers unprecedented opportunities for the discovery of causative gene variants in multiple human disorders including cancers, and has revolutionized clinical diagnostics. However, despite more than a decade of use of HTS-based assays, extracting relevant functional information from whole-exome sequencing (WES) data remains challenging, especially for non-specialists lacking in-depth bioinformatic skills. RESULTS: To address this limitation, we developed Var∣Decrypt, a web-based tool designed to greatly facilitate WES data browsing and analysis. Var∣Decrypt offers a wide range of gene and variant filtering possibilities, clustering and enrichment tools, providing an efficient way to derive patient-specific functional information and to prioritize gene variants for functional analyses. We applied Var∣Decrypt on WES datasets of 10 acute erythroid leukemia patients, a rare and aggressive form of leukemia, and recovered known disease oncogenes in addition to novel putative drivers. We additionally validated the performance of Var∣Decrypt using an independent dataset of ~ 90 multiple myeloma WES, recapitulating the identified deregulated genes and pathways, showing the general applicability and versatility of Var∣Decrypt for WES analysis. CONCLUSION: Despite years of use of WES in human health for diagnosis and discovery of disease drivers, WES data analysis still remains a complex task requiring advanced bioinformatic skills. In that context, there is a need for user-friendly all-in-one dedicated tools for data analysis, to allow biologists and clinicians to extract relevant biological information from patient datasets. Here, we provide Var∣Decrypt (trial version accessible here: https://vardecrypt.com/app/vardecrypt ), a simple and intuitive Rshiny application created to fill this gap. Source code and detailed user tutorial are available at https://gitlab.com/mohammadsalma/vardecrypt .

High-throughput methods for the analysis of transcription factors and chromatin modifications: Low input, single cell and spatial genomic technologies.

Genome-wide analysis of transcription factors and epigenomic features is instrumental to shed light on DNA-templated regulatory processes such as transcription, cellular differentiation or to monitor cellular responses to environmental cues. Two decades of technological developments have led to a rich set of approaches progressively pushing the limits of epigenetic profiling towards single cells. More recently, disruptive technologies using innovative biochemistry came into play. Assays such as CUT&RUN, CUT&Tag and variations thereof show considerable potential to survey multiple TFs or histone modifications in parallel from a single experiment and in native conditions. These are in the path to become the dominant assays for genome-wide analysis of TFs and chromatin modifications in bulk, single-cell, and spatial genomic applications. The principles together with pros and cons are discussed.

ONTdeCIPHER: an amplicon-based nanopore sequencing pipeline for tracking pathogen variants.

MOTIVATION: Amplicon-based nanopore sequencing is increasingly used for molecular surveillance during epidemics (e.g. ZIKA, EBOLA) or pandemics (e.g. SARS-CoV-2). However, there is still a lack of versatile and easy-to-use tools that allow users with minimal bioinformatics skills to perform the main steps of downstream analysis, from quality testing to SNPs effect to phylogenetic analysis. RESULTS: Here, we present ONTdeCIPHER, an amplicon-based Oxford Nanopore Technology sequencing pipeline to analyze the genetic diversity of SARS-CoV-2 and other pathogens. Our pipeline integrates 13 bioinformatics tools. With a single command line and a simple configuration file, users can pre-process their data and obtain the sequencing statistics, reconstruct the consensus genome, identify variants and their effects for each viral isolate, infer lineage and, finally perform multi-sequence alignments and phylogenetic analyses. AVAILABILITY AND IMPLEMENTATION: ONTdeCIPHER is available at https://github.com/emiracherif/ONTdeCIPHER. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

PPARγ agonists promote the resolution of myelofibrosis in preclinical models.

Myelofibrosis (MF) is a non-BCR-ABL myeloproliferative neoplasm associated with poor outcomes. Current treatment has little effect on the natural history of the disease. MF results from complex interactions between (a) the malignant clone, (b) an inflammatory context, and (c) remodeling of the bone marrow (BM) microenvironment. Each of these points is a potential target of PPARγ activation. Here, we demonstrated the therapeutic potential of PPARγ agonists in resolving MF in 3 mouse models. We showed that PPARγ agonists reduce myeloproliferation, modulate inflammation, and protect the BM stroma in vitro and ex vivo. Activation of PPARγ constitutes a relevant therapeutic target in MF, and our data support the possibility of using PPARγ agonists in clinical practice.

p53 activation during ribosome biogenesis regulates normal erythroid differentiation.

The role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q- syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis showed that ribosome biogenesis is abruptly interrupted by the decline in ribosomal DNA transcription and the collapse of ribosomal protein neosynthesis. Its premature arrest by the RNA Pol I inhibitor CX-5461 targeted the proliferation of immature erythroblasts. p53 was activated spontaneously or in response to CX-5461, concomitant to ribosome biogenesis arrest, and drove a transcriptional program in which genes involved in cell cycle-arrested, negative regulation of apoptosis, and DNA damage response were upregulated. RNA Pol I transcriptional stress resulted in nucleolar disruption and activation of the ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation is crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold downregulation of ribosome biogenesis could be prematurely reached and, together with pathological p53 activation, prevents a normal expansion of erythroid progenitors.

Cryopreservation of Date Palm Pro-Embryonic Masses Using the D Cryo-plate Technique.

In this chapter, we describe a cryopreservation (liquid nitrogen, -196 °C) protocol developed for long-term storage of date palm pro-embryonic masses (PEMs), which uses the recently established D cryo-plate technique. Clumps of PEMs (3-5 mm in size) were dissected from PEM cultures and placed on pretreatment medium containing 171 g/L sucrose for 3 days. Clumps were placed in the wells of aluminum cryo-plates in which they were made to adhere using droplets of 3% calcium alginate. PEMs were treated for 20 min with a loading solution containing 184 g/L glycerol and 136.8 g/L sucrose. They were then dehydrated for 90-120 min in the air current of a laminar airflow cabinet and immersed directly in liquid nitrogen. For rewarming, the cryo-plates holding the PEMs were immersed for 15 min in an unloading solution containing 410.4 g/L sucrose. The PEMs were then detached from the cryo-plates, placed for 3 days in the dark on posttreatment medium containing 102.6 g/L sucrose, and transferred on recovery medium under light conditions. Using this protocol, 74.6 and 95.8% recovery were achieved with the PEMs of the two cultivars tested, Sukkari and Sultany.

Viable But Not Culturable (VBNC) state of Brettanomyces bruxellensis in wine: New insights on molecular basis of VBNC behaviour using a transcriptomic approach.

The spoilage potential of Brettanomyces bruxellensis in wine is strongly connected with the aptitude of this yeast to enter in a Viable But Non Culturable (VBNC) state when exposed to the harsh wine conditions. In this work, we characterized the VBNC behaviour of seven strains of B. bruxellensis representing a regional intraspecific biodiversity, reporting conclusive evidence for the assessment of VBNC as a strain-dependent character. The VBNC behaviour was monitored by fluorescein diacetate staining/flow cytometry for eleven days after addition of 0.4, 0.6, 0.8, 1 and 1.2 mg/L of molecular SO2 (entrance in the VBNC state) and after SO2 removal (exit from the VBNC state). Furthermore, one representative strain was selected and RNA-seq analysis performed after exposure to 1.2 mg/L SO2 and during the recovery phase. 30 and 1634 genes were identified as differentially expressed following VBNC entrance and 'resuscitation', respectively. The results reported strongly suggested that the entrance in the SO2-induced VBNC state in B. bruxellensis is associated with both, sulfite toxicity and oxidative stress response, confirming the crucial role of genes/proteins involved in redox cell homeostasis. Among the genes induced during recovery, the expression of genes involved in carbohydrate metabolism and encoding heat shock proteins, as well as enriched categories including amino acid transport and transporter activity was observed. The evidences of a general repression of genes involved in DNA replication suggest the occurrence of a true resuscitation of cell rather than a simple regrowth.

Effect of the successive steps of a cryopreservation protocol on the structural integrity of Rubia akane Nakai hairy roots.

In this work, we studied the impact of the successive steps of the droplet-vitrification protocol technique employed for cryopreservation of Rubia akane hairy roots on the features of cortical, pericycle and endoderm cells of apical and central root segments, using histology techniques and combining qualitative and quantitative observations. In apical segments, plasmolysis (22-71 %, depending on cell type) was observed only after the loading treatment and did not increase after the following steps of the protocol. By contrast, in central segments, plasmolysis (39-45 %) was already observed after the sucrose pretreatment; it increased to 54-68 %, depending on cell type, after the loading treatment, but no further changes were noted after treatment with the vitrification solution. After liquid nitrogen exposure and unloading treatment, deplasmolysis was more rapid in apical segments, with cortical and pericycle cells having retrieved their original features. In central segments, only cortical cells had retrieved their original features and endoderm and pericycle cells were still highly plasmolysed. Nuclei were more strongly impacted by the cryopreservation protocol in central segments, where they displayed a highly condensed nucleoplasm from the loading treatment onwards and had not retrieved their original aspect after the unloading treatment. By contrast, nuclei had a much less condensed nucleoplasm in cells of apical segments, and they had retrieved their original aspect after the unloading treatment.

Characterization of the Viable but Nonculturable (VBNC) State in Saccharomyces cerevisiae.

The Viable But Non Culturable (VBNC) state has been thoroughly studied in bacteria. In contrast, it has received much less attention in other microorganisms. However, it has been suggested that various yeast species occurring in wine may enter in VBNC following sulfite stress.In order to provide conclusive evidences for the existence of a VBNC state in yeast, the ability of Saccharomyces cerevisiae to enter into a VBNC state by applying sulfite stress was investigated. Viable populations were monitored by flow cytometry while culturable populations were followed by plating on culture medium. Twenty-four hours after the application of the stress, the comparison between the culturable population and the viable population demonstrated the presence of viable cells that were non culturable. In addition, removal of the stress by increasing the pH of the medium at different time intervals into the VBNC state allowed the VBNC S. cerevisiae cells to "resuscitate". The similarity between the cell cycle profiles of VBNC cells and cells exiting the VBNC state together with the generation rate of cells exiting VBNC state demonstrated the absence of cellular multiplication during the exit from the VBNC state. This provides evidence of a true VBNC state. To get further insight into the molecular mechanism pertaining to the VBNC state, we studied the involvement of the SSU1 gene, encoding a sulfite pump in S. cerevisiae. The physiological behavior of wild-type S. cerevisiae was compared to those of a recombinant strain overexpressing SSU1 and null Δssu1 mutant. Our results demonstrated that the SSU1 gene is only implicated in the first stages of sulfite resistance but not per se in the VBNC phenotype. Our study clearly demonstrated the existence of an SO2-induced VBNC state in S. cerevisiae and that the stress removal allows the "resuscitation" of VBNC cells during the VBNC state.

Cytofluorometric detection of wine lactic acid bacteria: application of malolactic fermentation to the monitoring.

In this study we report for the first time a rapid, efficient and cost-effective method for the enumeration of lactic acid bacteria (LAB) in wine. Indeed, up to now, detection of LAB in wine, especially red wine, was not possible. Wines contain debris that cannot be separated from bacteria using flow cytometry (FCM). Furthermore, the dyes tested in previous reports did not allow an efficient staining of bacteria. Using FCM and a combination of BOX/PI dyes, we were able to count bacteria in wines. The study was performed in wine inoculated with Oenococcus oeni (10(6) CFU ml(-1)) stained with either FDA or BOX/PI and analyzed by FCM during the malolactic fermentation (MLF). The analysis show a strong correlation between the numbers of BOX/PI-stained cells determined by FCM and the cell numbers determined by plate counts (red wine: R (2) ≥ 0.97, white wine R (2) ≥ 0.965). On the other hand, we found that the enumeration of O. oeni labeled with FDA was only possible in white wine (R (2) ≥ 0.97). Viable yeast and LAB populations can be rapidly discriminated and quantified in simultaneous malolactic-alcoholic wine fermentations using BOX/PI and scatter parameters in a one single measurement. This rapid procedure is therefore a suitable method for monitoring O. oeni populations during winemaking, offers a detection limit of <10(4) CFU ml(-1) and can be considered a useful method for investigating the dynamics of microbial growth in wine and applied for microbiological quality control in wineries.