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Disruption of deoxyribonucleotide triphosphate biosynthesis leads to RAS proto-oncogene activation and perturbation of mitochondrial metabolism
No full textInternational audiencePerturbation of the deoxyribonucleotide triphosphate (dNTP) pool is recognized for contributing to the mutagenic processes involved in oncogenesis. The RAS gene family encodes well characterized oncoproteins whose structure and function are among the most frequently altered in several cancers. In this work, we show that fluctuation of the dNTP pool induces CG->TA mutations across the whole genome, including RAS gene at codons for glycine 12 and 13, known hotspots in cancers. Cell culture addition of the ribonucleotide reductase inhibitor thymidine increases the mutation frequency in nuclear DNA and leads to disruption of mitochondrial metabolism. Interestingly, this effect is counteracted by the addition of deoxycytidine. Finally, screening for the loss of hydrogen bonds detecting CG->TA transition in RAS gene of 135 patients with colorectal cancer confirmed the clinical relevance of this process. All together, these data demonstrate that fluctuation of intracellular dNTP pool alters the nuclear DNA and mitochondrial metabolism
Harnessing CRISPR interference to resensitize laboratory strains and clinical isolates to last resort antibiotics
No full textInternational audienceThe global race against antimicrobial resistance requires novel antimicrobials that are not only effective in killing specific bacteria, but also minimize the emergence of new resistances. Recently, CRISPR/Cas-based antimicrobials were proposed to address killing specificity with encouraging results. However, the emergence of target sequence mutations triggered by Cas-cleavage was identified as an escape strategy, posing the risk of generating new antibiotic-resistance gene (ARG) variants. Here, we evaluated an antibiotic re-sensitization strategy based on CRISPR interference (CRISPRi), which inhibits gene expression without damaging target DNA. The resistance to four antibiotics, including last resort drugs, was significantly reduced by individual and multi-gene targeting of ARGs in low- to high-copy numbers in recombinant E. coli . Escaper analysis confirmed the absence of mutations in target sequence, corroborating the harmless role of CRISPRi in the selection of new resistances. E. coli clinical isolates carrying ARGs of severe clinical concern were then used to assess the robustness of CRISPRi under different growth conditions. Meropenem, colistin and cefotaxime susceptibility was successfully increased in terms of MIC (up to > 4-fold) and growth delay (up to 11 h) in a medium-dependent fashion. ARG repression also worked in a pathogenic strain grown in human urine, as a demonstration of CRISPRi-mediated re-sensitization in host-mimicking media. This study laid the foundations for further leveraging CRISPRi as antimicrobial agent or research tool to selectively repress ARGs and investigate resistance mechanisms
Correlates of Protection Against Symptomatic COVID-19: The CORSER 5 Case–Control Study
No full textInternational audienceBackground Establishing correlates of protection often requires large cohorts. A rapid and adaptable case–control study design can be used to identify antibody correlates of protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in serum and saliva. Methods We designed a case–control study to compare antibody levels between cases of SARS-CoV-2 infection within 5 days of symptom onset and uninfected controls. Controls were matched on age, number of coronavirus disease 2019 vaccine doses, time since last dose, and past episodes of infection. We quantified anti-SARS-CoV-2 and seasonal coronavirus immunoglobulin (Ig) G in serum and saliva at inclusion, 1 month, and 6 months. Results We included 90 cases and 62 controls between February and September 2022. A boost and decay pattern of serum antibodies was observed in cases at 1 and 6 months, respectively, but not in controls. Anti-SARS-CoV-2 antibody levels were significantly higher in controls at inclusion both in serum (particularly antinucleocapsid IgG: 4.14 times higher compared with cases; 95% CI, 2.46–6.96) and saliva (particularly antispike for Delta variant IgG: 4.89 times higher compared with cases; 95% CI, 2.91–9.89). Saliva antibodies generally outperformed serum antibodies for case/control differentiation. Conclusions In this case–control study, we provided evidence of correlates of protection of anti-SARS-CoV-2 IgG in saliva and serum, with saliva antibodies often outperforming serum. The finding that antibodies in saliva are a better correlate of protection than antibodies in serum may inform vaccine development by highlighting the importance of robust induction of mucosal immune responses. This study design may be used during future epidemics for the prompt assessment of correlates of protection
Immune control of brain physiology
No full textInternational audienceThe peripheral immune system communicates with the brain through complex anatomical routes involving the skull, the brain borders, circumventricular organs and peripheral nerves. These immune-brain communication pathways were classically considered to be dormant under physiological conditions and active only in cases of infection or damage. Yet, peripheral immune cells and signals are key in brain development, function and maintenance. In this Perspective, we propose an alternative framework for understanding the mechanisms of immune-brain communication. During brain development and in homeostasis, these anatomical structures allow selected elements of the peripheral immune system to affect the brain directly or indirectly, within physiological limits. By contrast, in ageing and pathological settings, detrimental peripheral immune signals hijack the existing communication routes or alter their structure. We discuss why a diversity of communication channels is needed and how they work in relation to one another to maintain homeostasis of the brain
Design, Synthesis and Evaluation of Pyrimidinobenzylamide and Pyrimidinothiophenamide Derivatives as Inhibitors of DOT1L and Related Epigenetic Targets DNMT3a, PRMT4 and other HMTs
No full textInternational audienceThe histone methyltransferase DOT1L (DOT1 like, disruptor of telomeric silencing) is responsible of methylation of H3K79, leading to oncogene transcription, and its involved in the development of different types of cancers such as MLL-rearranged leukemia (MLL-r, Myeloid-Lymphoid Leukemia). Inhibitors of DOT1L have a therapeutic potential. Thus, we present herein the in silico based design and the multi-step synthesis of different series of non-nucleosidic compounds that mimic the S-adenosyl-L-methionine (SAM) cofactor and inhibit DOT1L. The compounds incorporate an aminopyrimidine moiety coupled to an functionalized aryl based on the structure of published DOT1L inhibitors that have entered clinical trials (EPZ-5676, pinometostat). Their DOT1L activity was determined and structure-activity relationships (SAR) were established, leading to the identification of key moieties for the development of DOT1L-selective compounds. To determine their specificity, the activity of the compounds was evaluated on other methyltransferases that also use SAM as cofactor, such as DNA MethylTransferases (DNMT) and Histone MethylTransferases (HMT), including the PRC2 complex, G9a, PRMT1, PRMT4 and PRMT5. We identified compound 19d (IC50 = 8.0 µM) as a DNMT3a inhibitor, and 1n (EC50 = 19.0 µM), 1p (EC50 = 4.8 µM) and 19g (EC50 = 11.0 µM) as PRMT4 inhibitors based on the in silico approach that was employed. The in vitro ADMET profile of the compounds matched with the generally accepted lead-like criteria and encouraged the further optimization of these non-nucleosidic hit compounds
Are psychological attitudes towards vaccination an expression of personality? A cross-sectional study on COVID-19 vaccination in France
No full textInternational audienceBackground: The capacity of the 7C model's psychological antecedents, which include confidence in vaccines, complacency, convenience, calculation, collective responsibility, confidence in the wider system, and social conformism, to explain variance in COVID-19 vaccine intentions and behaviours has been documented. However, it remains unclear whether the attitudes represented by the 7C psychological antecedents are specific to vaccination or if they are, in fact, an expression of underlying personality traits.Methods: From February to June 2022, French adults completed self-administered questionnaires assessing COVID-19 vaccination history, the 7C antecedents, and personality traits ("ComCor" and "Cognitiv" studies). Vaccination behaviours were studied through three outcomes: at-least-one-dose vaccination status by 2022 (N = 49,019), up-to-date vaccination status (N = 46,566), and uptake speed of first dose (N = 25,998). Personality traits were evaluated using the French version of the Big Five Inventory (BFI-Fr). Multivariable logistic regressions and Cox models predicting vaccine behaviours were run with the 7C antecedents, both with and without personality traits.Results: Among the 49,019 participants, 95.0% reported receipt of at least one dose and 89.8% were up to date with recommendations. All 7C antecedents were significantly associated with the outcomes. The inclusion of personality traits did not substantially alter the effect estimates of the association between the 7C antecedents and vaccination behaviours, with differences between effect sizes of models with and without personality traits being < 5%.Conclusions: Our results suggest that the 7C psychological antecedents of vaccination are not the mere expression of personality and that their impact on vaccine behaviours is independent of personality traits. As such, the 7C antecedents may be modifiable by appropriate information and vaccine promotion. Trial registration: The "ComCor" study received ethical approval by the Comité de Protection des Personnes Sud Ouest et Outre Mer 1 on 21/09/2020. The addition of the "Cognitiv" questionnaire received ethical approval by the Comité de Protection des Personnes Sud Ouest et Outre Mer 1 on 01/02/2022. The data protection authority Commission Nationale de l'Informatique et des Libertés (CNIL) authorised the processing of data on 21/10/2020. The study is registered with ClinicalTrials.gov under the identifier NCT04607941
Hepatitis E Virus-induced antiviral response by plasmacytoid dendritic cells is modulated by the ORF2 protein
No full textInternational audienceType I and III interferons (IFN-I/III) are critical to protect the host during viral infection. IFN-mediated antiviral responses against hepatitis E virus (HEV) are suppressed and defeated by viral escape mechanisms at play in infected hepatocytes. Here, we studied the anti-HEV function of IFN secreted by plasmacytoid dendritic cells (pDCs), which are specialized producers of IFNs. We showed that pDCs co-cultured with HEV-infected cells secreted IFN in a cell-to-cell contact-dependent manner. Pharmacological inhibitor and antibodies targeting contact proteins revealed that pDC response against HEV required the endosomal nucleic-acid sensor TLR7 and adhesion molecules, such as ICAM-I and α L β 2 -integrin. IFNs secreted by pDCs reduced viral spread. Intriguingly, ORF2, the capsid protein of HEV, can be produced in various forms by the infected cells. During infection, a fraction of the intracellular ORF2 protein localizes into the nucleus while another ORF2 fraction packages viral genomes to produce infectious virions. In parallel, glycosylated forms of ORF2 are also massively secreted by infected cells. Using viral genome expressing ORF2 mutants, we showed that glycosylated ORF2 forms contribute to better recognition of infected cells by pDCs by regulating contacts between infected cells and pDCs. ORF2 forms may thus modulate pDC-mediated anti-HEV response. Together, our results suggest that liver-resident pDCs, which exhibit comparable IFN-producing ability as blood-derived pDCs, may be essential to control HEV replication
No innocent bystanders: pertussis vaccination epitomizes evolutionary parallelisms between Bordetella parapertussis and B. pertussis
No full textABSTRACT Pathogens adapting to the human host and to vaccination-induced immunity may follow parallel evolutionary paths. Bordetella parapertussis ( Bpp ) contributes significantly to the burden of whooping cough (pertussis), shares vaccine antigens with Bordetella pertussis (Bp), and both pathogens are phylogenetically related and ecological competitors. Bp vaccine antigen-coding genes have accumulated variation, including pertactin disruptions, after introduction of acellular vaccines in the 1990s. We aimed to evaluate evolutionary parallelisms in Bpp , even though pertussis vaccines were designed against Bp . We investigated the temporal evolution of Bpp sublineages, by sequencing 242 Bpp isolates collected in France, the USA and Spain between 1937 and 2019, spanning pre-vaccine and two vaccines eras. We estimated the evolutionary rate of Bpp at 2.12×10 −7 substitutions per site·year -1 , with a most recent common ancestor of all sequenced isolates around year 1877, and found that pertactin deficiency in Bpp was driven by 18 disruptive mutations, including deletion prn :ΔG-1895 estimated to have occurred around 1998 and observed in 73.8% (149/202) of post-2007 isolates. In addition, we detected two mutations in the bvg A- fhaB intergenic region (controlling expression of the master transcriptional regulator BvgA and the filamentous hemagglutinin), that became fixed in the early 1900s. Our findings suggest early adaptation of Bpp to humans through modulation of the bvgAS regulon, and a rapid adaptation through the loss of pertactin expression, representing a late evolutionary parallelism concomitant with acellular vaccination against whooping cough. IMPORTANCE Vaccination against Bordetella pertussis ( Bp ) has strongly affected the recent evolution of this main agent of whooping cough. Whether it may have done so co-incidentally on Bordetella parapertussis ( Bpp ), which is genetically and ecologically very similar to Bp, has not been described in detail. Our findings show striking evolutionary parallelisms of Bpp with Bp , including early changes in a critical regulatory region, and strong evidence of adaptation to vaccine-driven population immunity, even though whooping cough vaccines were not designed explicitly against Bpp . The rapid populational loss of pertactin in countries where acellular pertussis vaccines are used may also reduce protection by vaccination against Bpp , the second agent of whooping cough
Constitutive expression of Cas9 and rapamycin-inducible Cre recombinase facilitates conditional genome editing in Plasmodium berghei
No full textInternational audienceAbstract Malaria is caused by protozoan parasites of the genus Plasmodium and remains a global health concern. The parasite has a highly adaptable life cycle comprising successive rounds of asexual replication in a vertebrate host and sexual maturation in the mosquito vector Anopheles . Genetic manipulation of the parasite has been instrumental for deciphering the function of Plasmodium genes. Conventional reverse genetic tools cannot be used to study essential genes of the asexual blood stages, thereby necessitating the development of conditional strategies. Among various such strategies, the rapamycin-inducible dimerisable Cre (DiCre) recombinase system emerged as a powerful approach for conditional editing of essential genes in human-infecting P. falciparum and in the rodent malaria model parasite P. berghei . We previously generated a DiCre-expressing P. berghei line and validated it by conditionally deleting several essential asexual stage genes, revealing their important role also in sporozoites. Another potent tool is the CRISPR/Cas9 technology, which has enabled targeted genome editing with higher accuracy and specificity and greatly advanced genome engineering in Plasmodium spp. Here, we developed new P. berghei parasite lines by integrating the DiCre cassette and a fluorescent marker in parasites constitutively expressing Cas9. Owing to the dual integration of CRISPR/Cas9 and DiCre, these new lines allow unparalleled levels of gene modification and conditional regulation simultaneously. To illustrate the versatility of this new tool, we conditionally knocked out the essential gene encoding the claudin-like apicomplexan micronemal protein (CLAMP) in P. berghei and confirmed the role of CLAMP during invasion of erythrocytes
NEURAL-NETWORK MODEL FOR CHARACTERIZING STOCHASTIC DYNAMIC VARIABILITY OF CELLULAR PARTICLES
No full textInternational audienceParticle dynamics characterization is fundamental for understanding the biophysical laws orchestrating cellular processes. To classify the dynamic behaviors governing biological particles, we develop a neural network model built on geometric descriptors of trajectories. The model infers the stochastic laws governing the trajectory, enabling the detection of a large family of dynamic behaviors, especially within the subdiffusive regime that characterizes cell signaling processes. Finally, we propose a framework to robustly detect dynamic changes in composed trajectories based on the variability of prediction scores on successive sub-trajectories. The method is validated on simulated composed trajectories simulating the activation pathway of receptors CCR5