Repurposing of Drugs as Novel Influenza Inhibitors From Clinical Gene Expression Infection Signatures

Influenza virus infections remain a major and recurrent public health burden. The intrinsic ever-evolving nature of this virus, the suboptimal efficacy of current influenza inactivated vaccines, as well as the emergence of resistance against a limited antiviral arsenal, highlight the critical need for novel therapeutic approaches. In this context, the aim of this study was to develop and validate an innovative strategy for drug repurposing as host-targeted inhibitors of influenza viruses and the rapid evaluation of the most promising candidates in Phase II clinical trials. We exploited in vivo global transcriptomic signatures of infection directly obtained from a patient cohort to determine a shortlist of already marketed drugs with newly identified, host-targeted inhibitory properties against influenza virus. The antiviral potential of selected repurposing candidates was further evaluated in vitro, in vivo, and ex vivo. Our strategy allowed the selection of a shortlist of 35 high potential candidates out of a rationalized computational screening of 1,309 FDA-approved bioactive molecules, 31 of which were validated for their significant in vitro antiviral activity. Our in vivo and ex vivo results highlight diltiazem, a calcium channel blocker currently used in the treatment of hypertension, as a promising option for the treatment of influenza infections. Additionally, transcriptomic signature analysis further revealed the so far undescribed capacity of diltiazem to modulate the expression of specific genes related to the host antiviral response and cholesterol metabolism. Finally, combination treatment with diltiazem and virus-targeted oseltamivir neuraminidase inhibitor further increased antiviral efficacy, prompting rapid authorization for the initiation of a Phase II clinical trial. This original, host-targeted, drug repurposing strategy constitutes an effective and highly reactive process for the rapid identification of novel anti-infectious drugs, with potential major implications for the management of antimicrobial resistance and the rapid response to future epidemic or pandemic (re)emerging diseases for which we are still disarmed

Natural Single-Nucleosome Epi-Polymorphisms in Yeast

Epigenomes commonly refer to the sequence of presence/absence of specific epigenetic marks along eukaryotic chromatin. Complete histone-borne epigenomes have now been described at single-nucleosome resolution from various organisms, tissues, developmental stages, or diseases, yet their intra-species natural variation has never been investigated. We describe here that the epigenomic sequence of histone H3 acetylation at Lysine 14 (H3K14ac) differs greatly between two unrelated strains of the yeast Saccharomyces cerevisiae. Using single-nucleosome chromatin immunoprecipitation and mapping, we interrogated 58,694 nucleosomes and found that 5,442 of them differed in their level of H3K14 acetylation, at a false discovery rate (FDR) of 0.0001. These Single Nucleosome Epi-Polymorphisms (SNEPs) were enriched at regulatory sites and conserved non-coding DNA sequences. Surprisingly, higher acetylation in one strain did not imply higher expression of the relevant gene. However, SNEPs were enriched in genes of high transcriptional variability and one SNEP was associated with the strength of gene activation upon stimulation. Our observations suggest a high level of inter-individual epigenomic variation in natural populations, with essential questions on the origin of this diversity and its relevance to gene x environment interactions

Capturing a Single Cell

International audienceA major problem encountered in genomic and proteomic studies arises from the heterogeneous nature of different tissue. Analysis of a pure cell population is essential for correlating relevant molecular signatures in diseased and disease-free cells. During the last 30 years this challenge has led to the development of different technologies able to isolate cells of interest. Laser capture microdissection (LCM) is the last available technology using the precision of a laser beam to isolate single cells from complex tissue. In this chapter we will review the different technologies available and some applications

The microglial reaction signature revealed by RNAseq from individual mice

International audienceMicroglial cells have a double life as the immune cells of the brain in times of stress but have also specific physiological functions in homeostatic conditions. In pathological contexts, microglia undergo a phenotypic switch called “reaction” that promotes the initiation and the propagation of neuro‐inflammation. Reaction is complex, molecularly heterogeneous and still poorly characterized, leading to the concept that microglial reactivity might be too diverse to be molecularly defined. However, it remains unknown whether reactive microglia from different pathological contexts share a common molecular signature. Using improved flow cytometry and RNAseq approaches we studied, with higher statistical power, the remodeling of microglia transcriptome in a mouse model of sepsis. Through bioinformatic comparison of our results with published datasets, we defined the microglial reactome as a set of genes discriminating reactive from homeostatic microglia. Ultimately, we identified a subset of 86 genes deregulated in both acute and neurodegenerative conditions. Our data provide a new comprehensive resource that includes functional analysis and specific molecular markers of microglial reaction which represent new tools for its unambiguous characterization

Deregulation of miR-183 and KIAA0101 in Aggressive and Malignant Pituitary Tumors

Changes in microRNAs (miRNAs) expression in many types of cancer suggest that they may be involved in crucial steps during tumor progression. Indeed, miRNAs deregulation has been described in pituitary tumorigenesis, but few studies have described their role in pituitary tumor progression toward aggressiveness and malignancy. To assess the role of miRNAs within the hierarchical cascade of events in prolactin (PRL) tumors during progression, we used an integrative genomic approach to associate clinical-pathological features, global miRNA expression, and transcriptomic profiles of the same human tumors. We describe the specific down-regulation of one principal miRNA, miR-183, in the 8 aggressive (A, grade 2b) compared to the 18 non-aggressive (NA, grades 1a, 2a) PRL tumors. We demonstrate that it acts as an anti-proliferative gene by directly targeting KIAA0101, which is involved in cell cycle activation and inhibition of p53-p21-mediated cell cycle arrest. Moreover, we show that miR-183 and KIAA0101 expression significantly correlate with the main markers of pituitary tumors aggressiveness, Ki-67 and p53. These results confirm the activation of proliferation in aggressive and malignant PRL tumors compared to non-aggressive ones. Importantly, these data also demonstrate the ability of such an integrative genomic strategy, applied in the same human tumors, to identify the molecular mechanisms responsible for tumoral progression even from a small cohort of patients

Sequencing the Genome of Indian Flying Fox, Natural Reservoir of Nipah Virus, Using Hybrid Assembly and Conservative Secondary Scaffolding

International audienceIndian fruit bats, flying fox Pteropus medius was identified as an asymptomatic natural host of recently emerged Nipah virus, which is known to induce a severe infectious disease in humans. The absence of P. medius genome sequence presents an important obstacle for further studies of virus–host interactions and better understanding of mechanisms of zoonotic viral emergence. Generation of the high-quality genome sequence is often linked to a considerable effort associated to elevated costs. Although secondary scaffolding methods have reduced sequencing expenses, they imply the development of new tools for the integration of different data sources to achieve more reliable sequencing results. We initially sequenced the P. medius genome using the combination of Illumina paired-end and Nanopore sequencing, with a depth of 57.4x and 6.1x, respectively. Then, we introduced the novel scaff2link software to integrate multiple sources of information for secondary scaffolding, allowing to remove the association with discordant information among two sources. Different quality metrics were next produced to validate the benefits from secondary scaffolding. The P. medius genome, assembled by this method, has a length of 1,985 Mb and consists of 33,613 contigs and 16,113 scaffolds with an NG50 of 19 Mb. At least 22.5% of the assembled sequences is covered by interspersed repeats already described in other species and 19,823 coding genes are annotated. Phylogenetic analysis demonstrated the clustering of P. medius genome with two other Pteropus bat species, P. alecto and P. vampyrus, for which genome sequences are currently available. SARS-CoV entry receptor ACE2 sequence of P. medius was 82.7% identical with ACE2 of Rhinolophus sinicus bats, thought to be the natural host of SARS-CoV. Altogether, our results confirm that a lower depth of sequencing is enough to obtain a valuable genome sequence, using secondary scaffolding approaches and demonstrate the benefits of the scaff2link application. The genome sequence is now available to the scientific community to (i) proceed with further genomic analysis of P. medius, (ii) to characterize the underlying mechanism allowing Nipah virus maintenance and perpetuation in its bat host, and (iii) to monitor their evolutionary pathways toward a better understanding of bats’ ability to control viral infections

Repurposing of Drugs as Novel Influenza Inhibitors From Clinical Gene Expression Infection Signatures

Influenza virus infections remain a major and recurrent public health burden. The intrinsic ever-evolving nature of this virus, the suboptimal efficacy of current influenza inactivated vaccines, as well as the emergence of resistance against a limited antiviral arsenal, highlight the critical need for novel therapeutic approaches. In this context, the aim of this study was to develop and validate an innovative strategy for drug repurposing as host-targeted inhibitors of influenza viruses and the rapid evaluation of the most promising candidates in Phase II clinical trials. We exploited in vivo global transcriptomic signatures of infection directly obtained from a patient cohort to determine a shortlist of already marketed drugs with newly identified, host-targeted inhibitory properties against influenza virus. The antiviral potential of selected repurposing candidates was further evaluated in vitro, in vivo, and ex vivo. Our strategy allowed the selection of a shortlist of 35 high potential candidates out of a rationalized computational screening of 1,309 FDA-approved bioactive molecules, 31 of which were validated for their significant in vitro antiviral activity. Our in vivo and ex vivo results highlight diltiazem, a calcium channel blocker currently used in the treatment of hypertension, as a promising option for the treatment of influenza infections. Additionally, transcriptomic signature analysis further revealed the so far undescribed capacity of diltiazem to modulate the expression of specific genes related to the host antiviral response and cholesterol metabolism. Finally, combination treatment with diltiazem and virus-targeted oseltamivir neuraminidase inhibitor further increased antiviral efficacy, prompting rapid authorization for the initiation of a Phase II clinical trial. This original, host-targeted, drug repurposing strategy constitutes an effective and highly reactive process for the rapid identificatio