Biotechnologie et Signalisation Cellulaire

Viruses transmitted by the obligate biotrophic parasite Polymyxa graminis cause substantial losses in agriculture worldwide. A better knowledge of the infection process may lead to the identification of novel strategies for virus control. The coat protein readthrough (CP-RT) protein or its homologs, present in all P. graminis-transmitted viruses, are transmission factors of the viruses. To better characterize CP-RT function, we modified an infectious Japanese soil-borne wheat mosaic virus (JSBWMV) cDNA clone to express fluorescent protein-tagged CP-RT and analyzed the protein during infection in the model host plant Nicotiana benthamiana. We found that JSBWMV CP-RT localized to and self-interacted at the endoplasmic reticulum (ER) network and in small cortical particles during infection. The particles moved along the ER network and associated with sub-ER sites, most likely ER import sites, as indicated by co-localization with Sar1-GTP:GFP and AtSYP72:GFP. Particles were still present after reabsorption of the Golgi into the ER network after Brefeldin A treatment, thus they were not an intrinsic part of the Golgi-ER compartment. In natural CP-RT deletion mutants, the localization to the ER network was lost, while localization to the particles was maintained. Consistent with earlier data our findings from transmission experiments in hydroponic barley culture indicate that an intact JSBWMV CP-RT may be important for virus transmission by the vector, as more wild type CP-RT forms compared to mutant CP-RT forms accumulated in newly infected plants. ER-association of CP-RT may thus be important for virus transmission and early infection processes.

Photodynamic therapy (PDT) is a promising strategy forhead andneck squamous cell carcinoma (HNSCC), but the immune consequencesof tumor cell death remain incompletely understood. We compared tworuthenium­(II) polypyridine photosensitizers (PSs) in HNSCC modelsand found that both were potently phototoxic (nanomolar IC50s), triggered diverse cell death pathways (including autophagy andferroptosis), and promoted hallmark danger signals of immunogeniccell death (ICD). Strikingly, only one PS induced apoptosis and strongendoplasmic reticulum (ER) stress, yet paradoxically led to immunetolerance in vivo. Conversely, the PS that did notinduce apoptotic cell death with milder stress responses resultedin a better antitumor immunity in vivo. These unexpectedfindings challenge the prevailing view that PDT-triggered apoptosisand ER stress are essential for ICD. Our study underscores the complexityof PDT-induced cell death balance and immunogenic signals and highlightsthe need to redefine ICD-inducing criteria for the rational designof next-generation PSs.

The use of plant-based remedies in traditional medecine to combat bacterial infections has been scientifically studied. As part of an ongoing research for the discovery of new therapeutic options to treat bacterial infections, Ammoniacum gum resin from Ferula communis L. roots was explored. Ammoniacum appeared in many traditional formulations of Arab, Indian and Western medicines and its use has been reported throughout the centuries. This present study aimed to further explore this gum-resin using a molecular-networking-guided method for the accelerated discovery of new antibacterial compounds. HPLC-PDA-HRMS/MS molecular-networking-based dereplication strategy highlighted the presence of known sesquiterpene coumarins among potential new derivatives. By comparison of MS/MS fragmentation spectra between known and unknown compounds, a hydroxycinnamic sesquiterpene coumarin structure was predicted as a potential new compound. Mass-guided isolation followed by structural elucidation allowed us to corroborate the predicted structure of this new derivative identified as o-coumaric-ω-hydroxyferulenol (10). The new derivative showed MIC90 values of 1.84-6.17μM against cutaneous Gram-positive bacteria with a bactericidal effect measured by MBC/MCI ratio evaluation and flow cytometry methodology. Furthermore, the new compound showed no in vitro cytotoxicity against eukaryotic cells. This study confirms the interest to analyse with modern techniques ancient traditional remedies in the exploration of new bioactive compounds with therapeutical potential.

GPCR associated sorting protein 1 (GPRASP1) interacts with numerous GPCRs including the Beta2-adrenoceptor (B2AR) and has been proposed to be involved in adaptations associated with chronic stimulation of those receptors. In clinic, long acting B2AR agonists (LABAs) such as formoterol are used in the treatment of asthma as potent bronchodilators but with adverse side effects including the development of tolerance and airway hyperresponsiveness upon chronic administration. Here, we investigated the role of GPRASP1 on B2AR activity and on B2AR agonists-associated side effects in vitro and in vivo. To this purpose, we set-up a model of chronic formoterol administration in mouse leading to B2AR down-regulation as well as to the development of airway hyperreactivity and bronchodilator tolerance and studied the phenotype of GPRASP1 knockout animals. We show in cells that GPRASP1 expression has no impact on agonist-induced B2AR down-regulation but strongly modulate B2AR-associated signalling. Moreover, wild-type mice chronically treated with formoterol developed airway hyperresponsiveness to methacholine and bronchodilator tolerance to formoterol that were absent in GPRASP1 KO mice while B2AR down-regulation was similar in both genotypes. These adverse side effects were correlated with an increase in the number of cells and in collagen levels in the lungs of wild-type but not of GPRASP1 KO mice. Collectively, our data show that GPRASP1 is critically involved in adaptations to chronic activation of B2AR that leads to lung tissue remodelling, development of bronchial hyperresponsiveness and bronchodilator tolerance to B2AR agonist formoterol and could therefore represent an interesting target to limit side effects associated with LABAs.

ABSTRACT The rising prevalence of bacterial infections, antibiotic resistance, and emerging pathogens underscores the urgent need for innovative diagnostic approaches. Aptamers, short nucleic acid sequences with high specificity and affinity for their targets, are promising candidates for diagnostic applications due to their ability to detect a wide range of pathogens. In this study, we present a fluorescent aptamer-based bioimaging approach for detecting Pseudomonas aeruginosa , a multidrug-resistant pathogen of significant clinical concern. Conjugated with fluorescent dye, the detection efficacy of the F23 aptamer was evaluated on 15 different Gram-negative and Gram-positive bacteria, including fixed and lived cells, as well as homogeneous and heterogeneous population. To quantify microscopy images, we developed an automated, open-access identification software using ImageJ. Its high sensitivity provides a robust platform for accurately quantifying bacteria labeled with aptamers and potentially other fluorescent ligands. For instance, it successfully detected 1122 P. aeruginosa cells labeled with aptamer F23 out of a total of 1123 P. aeruginosa cells in a single image. With almost 200,000 analyzed bacteria and an exceptionally clear signal-to-noise ratio, we demonstrated that the F23 aptamer effectively detects various reference and clinical strains of Pseudomonas aeruginosa , while failing to detect Gram-positive Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus epidermidis and Corynebacterium striatum , as well as Gram-negative Klebsiella pneumonia, Acinetobacter baumannii , and Escherichia coli . The F23 aptamer is therefore a promising tool to distinguish Pseudomonas aeruginosa from different strains of the skin microbiota. However, our quantitative method also revealed partial labeling to other bacterial cells, highlighting the issue of refining aptamer selection to improve selectivity.

Abstract. In recent decades, three major volcanic eruptions of different intensity have occurred (Mount Agung in 1963, El Chichón in 1982 and Mount Pinatubo in 1991), with reported climate impacts on seasonal to decadal timescales that could have been potentially predicted with accurate and timely estimates of the associated stratospheric aerosol loads. The Decadal Climate Prediction Project component C (DCPP-C) includes a protocol to investigate the impact of volcanic aerosols on the climate experienced during the years that followed those eruptions through the use of decadal predictions. The interest of conducting this exercise with climate predictions is that, thanks to the initialisation, they start from the observed climate conditions at the time of the eruptions, which helps to disentangle the climatic changes due to the initial conditions and internal variability from the volcanic forcing. The protocol consists of repeating the retrospective predictions that are initialised just before the last three major volcanic eruptions but without the inclusion of their volcanic forcing, which are then compared with the baseline predictions to disentangle the simulated volcanic effects upon climate. We present the results from six Coupled Model Intercomparison Project Phase 6 (CMIP6) decadal prediction systems. These systems show strong agreement in predicting the well-known post-volcanic radiative effects following the three eruptions, which induce a long-lasting cooling in the ocean. Furthermore, the multi-model multi-eruption composite is consistent with previous work reporting an acceleration of the Northern Hemisphere polar vortex and the development of El Niño conditions the first year after the eruption, followed by a strengthening of the Atlantic Meridional Overturning Circulation the subsequent years. Our analysis reveals that all these dynamical responses are both model- and eruption-dependent. A novel aspect of this study is that we also assess whether the volcanic forcing improves the realism of the predictions. Comparing the predicted surface temperature anomalies in the two sets of hindcasts (with and without volcanic forcing) with observations we show that, overall, including the volcanic forcing results in better predictions. The volcanic forcing is found to be particularly relevant for reproducing the observed sea surface temperature (SST) variability in the North Atlantic Ocean following the 1991 eruption of Pinatubo.

Here, we demonstrate that the direct binding of p53 on the IL-18 promoter region regulates its gene expression. However, the presence of E6 and E7 from human papillomavirus type 38 impairs this mechanism via a new inhibitory complex formed by DNA methyltransferase 1 (DNMT1)/PKR/ΔNp73α, which binds to the region formerly occupied by p53 in primary keratinocytes.

Abstract Chromobodies made of nanobodies fused to fluorescent proteins are powerful tools for targeting and tracing intracellular proteins in living cells. Typically, this is achieved by transfecting plasmids encoding the chromobodies. However, an excess of unbound chromobody relative to the endogenous antigen can result in high background fluorescence in live cell imaging. Here, we overcome this problem by using mRNA encoding chromobodies. Our approach allows one to precisely control the amount of chromobody expressed inside the cell by adjusting the amount of transfected mRNA. To challenge our method, we evaluate three chromobodies targeting intracellular proteins of different abundance and cellular localization, namely lamin A/C, Dnmt1 and actin. We demonstrate that expression of chromobodies in living cells by transfection of tuned amounts of the corresponding mRNAs allows the accurate tracking of their cellular targets by time-lapse fluorescence microscopy.