Human health is threatened by bacterial infections that are increasingly resistant to multiple drugs. A recently emerged strategy consists of disarming pathogenic bacteria by targeting and blocking ...their virulence factors. The type VI secretion system (T6SS) is a widespread secretion nanomachine encoded and employed by pathogenic strains to establish their virulence process during host invasion. Given the conservation of T6SS in several human bacterial pathogens, the discovery of an effective broad-spectrum T6SS virulence blocker represents an attractive target for development of antivirulence therapies. Here, we identified and validated a protein-protein interaction interface, TssK-TssG, as a key factor in the assembly of the T6SS baseplate (BP) complex in the pathogen enteroaggregative Escherichia coli (EAEC).
and biochemical studies revealed that the determinants of the interface are broadly conserved among pathogenic species, suggesting a role for this interface as a target for T6SS inhibition. Based on the high-resolution structure of the TssKFGE wedge complex, we rationally designed a biomimetic cyclic peptide (BCP) that blocks the assembly of the EAEC BP complex and inhibits the function of T6SS in bacterial cultures. Our BCP is the first compound completely designed from prior structural knowledge with anti-T6SS activity that can be used as a model to target human pathogens.
New therapeutic options are urgently needed to fight drug-resistant and life-threatening infections. In contrast to antibiotics that inhibit the growth pathways of bacteria, the antivirulence strategy is a promising approach to disarm pathogens by interfering with bacterial virulence factors without exerting evolutionary pressure. The type VI secretion system (T6SS) is used by many pathogens, including members of the antibiotic-resistant ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), to establish their virulence during the invasion of the human host. Although the T6SS is undoubtedly involved in pathogenesis, strategies targeting this virulence factor are crucially lacking. Here, we used a combination of genetics, microbiology, biochemical, biophysics, and bioinformatics approaches to rationally design a biomimetic peptide that interferes with T6SS assembly and functioning. This study represents a novel proof of concept for an antivirulence strategy which aims to interfere with the assembly of the T6SS.
Persistently hyperphosphorylated Akt contributes to human oncogenesis and resistance to therapy. Triciribine (TCN) phosphate (TCN-P), the active metabolite of the Akt phosphorylation inhibitor TCN, ...is in clinical trials, but the mechanism by which TCN-P inhibits Akt phosphorylation is unknown. Here we show that in vitro, TCN-P inhibits neither Akt activity nor the phosphorylation of Akt S473 and T308 by mammalian target of rapamycin or phosphoinositide-dependent kinase 1. However, in intact cells, TCN inhibits EGF-stimulated Akt recruitment to the plasma membrane and phosphorylation of Akt. Surface plasmon resonance shows that TCN, but not TCN, binds Akt-derived pleckstrin homology (PH) domain (K(D): 690 nM). Furthermore, nuclear magnetic resonance spectroscopy shows that TCN-P, but not TCN, binds to the PH domain in the vicinity of the PIP3-binding pocket. Finally, constitutively active Akt mutants, Akt1-T308D/S473D and myr-Akt1, but not the transforming mutant Akt1-E17K, are resistant to TCN and rescue from its inhibition of proliferation and induction of apoptosis. Thus, the results of our studies indicate that TCN-P binds to the PH domain of Akt and blocks its recruitment to the membrane, and that the subsequent inhibition of Akt phosphorylation contributes to TCN-P antiproliferative and proapoptotic activities, suggesting that this drug may be beneficial to patients whose tumors express persistently phosphorylated Akt.
À partir d’un échantillon de 1 503 personnes, cette étude s’intéresse aux patients des centres d’assistance médicale à la procréation (AMP) pris en charge en France en 2020 afin d’interroger leur ...parcours médical comme leurs expériences de l’infertilité dans la vie professionnelle, intime, familiale et conjugale. Le questionnaire en ligne, composé de 178 questions, offre trois grandes familles de résultats : les domaines dans lesquels l’AMP donne satisfaction à une majorité de personnes ; les points de vigilance ; les résultats les plus frappants, notamment l’invisibilité des jeunes et des hommes parmi les patients, le pessimisme quant aux résultats de leur traitement et la difficile relation au corps pour les femmes. L’ensemble des résultats souligne l’importance d’une conception globale des patients infertiles, hommes et femmes, jeunes et moins jeunes, au-delà de la seule dimension médicale, pour une prise en compte aussi bien psychologique, sociale que professionnelle, sous-tendue par une équipe d’accompagnants élargie.
Exosomes support cell‐to‐cell communication in physiology and disease, including cancer. We currently lack tools, such as small chemicals, capable of modifying exosome composition and activity in a ...specific manner. Building on our previous understanding of how syntenin, and its PDZ partner syndecan (SDC), impact on exosome composition we optimized a small chemical compound targeting the PDZ2 domain of syntenin. In vitro , in tests on MCF‐7 breast carcinoma cells, this compound is non‐toxic and impairs cell proliferation, migration and primary sphere formation. It does not affect the size or the number of secreted particles, yet it decreases the amounts of exosomal syntenin, ALIX and SDC4 while leaving other exosomal markers unaffected. Interestingly, it also blocks the sorting of EpCAM, a bona fide target used for carcinoma exosome immunocapture. Our study highlights the first characterization of a small pharmacological inhibitor of the syntenin‐exosomal pathway, of potential interest for exosome research and oncology.
Abstract
Study question
What are the uses and real-life experiences of patients currently treated in France in medically-assisted reproduction (MAR) centers?
Summary answer
One in four patients is in ...a situation of significant pain in relation to MAR; half are not optimistic about the outcome of their treatment.
What is known already
Internationally, work on the quality of life and impact of treatments has been completed in the field of endometriosis (Culley et al. 2017). In the field of MAR in France, one study underlines an important level of general well-being, with nearly 4 people out of 5 who say they are rather content (Coudrière et al. 2020). A better understanding of this result is however still lacking, especially in respect to whether patients actually believe in the success of their treatments, or how dependent satisfaction is on variables, like age, secondary infertility or the number of years engaged in fertility treatments.
Study design, size, duration
We conducted an online self-administered questionnaire by means of a two-step process: a first survey dedicated to patients’ uses was carried out from January 15, 2020 to February 26, 2000; the following and complementary survey about real-life experience was conducted from March 3, 2020 to April 10, 2020. The initial sample included 1503 people before selecting relevant participants.
Participants/materials, setting, methods
The final sample of 967 patients targets patients that were enrolled in a French procreative medical center at the time of the study. Were excluded from the sample: ex-patients, patients who have not started treatment or are not followed in France, solo and homosexual care patient trajectories. The questionnaire was composed of 178 questions. In the questionnaire dealing with MAR uses, six fields were examined; in the case of real-life experience, eight themes were questioned.
Main results and the role of chance
The study shows that one in four patients suffers significantly in relation to MAR care; nearly half are pessimistic about treatment outcome and 65% claim that MAR prevents full enjoyment of everyday life. Further findings include: MAR is globally satisfying regarding the quality of care provided by practitioners and staff (80%), general administration of patients (77 to 87%), first encounters with staff and center (78 to 91%), quality of dialogue (81%), physical state of the premises (82 to 97%), continuum with professional activity (73%). Where centers/practitioners need to pay attention: one out of two patients wanted more means of communication; time management was an issue; 41% state that infertility prevents entrepreneurial outreach and general success; treatments strongly disrupt work for 83% and reduce performances for 79%; work rights, while known, were not respected in ¼ cases. Discoveries made: young women are not very positive, men are not well-identified in such medical trajectories, women’s body image is completely transformed for 95%, 85.5% of women are depressed by seeing a pregnant woman, and 15% do not think about wanting a child daily.
Limitations, reasons for caution
This research, carried out online, was distributed mainly through a grassroot organization, potentially biasing representativeness. Also, the study finished at the start of the first French confinement. Previous studies show that the general context does not impact results, but this limitation cannot be ignored (Fisher, Bayhem, 2019).
Wider implications of the findings: These findings underline the need for a comprehensive conception of MAR, including more than medical support. It highlights anew the need to better take men into account in future research as well as people in their twenties as knowledge of these categories of people is still lacking.
Trial registration number
Not applicable
Hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B constitutes a target of choice for the development of anti-HCV drugs. Although many small molecules have been identified as allosteric ...inhibitors of NS5B, very few are active in clinical applications. We have screened 17,000 compounds in an enzymatic assay involving the purified NS5B in order to increase the therapeutic arsenal. We hoped to shed some light on the precise mechanism of RNA synthesis. We succeeded in isolating a series of 21 original inhibitors of the RNA synthesis by NS5B. Four of these non-nucleoside inhibitors (NNIs) could be mapped to the known binding site called ‘B’ as judged by the decrease in their inhibition potency when assayed with a ‘B’ site mutant, M423T NS5B. Incidentally, our
in silico model pointed to Y477 as a key residue for inhibitor binding.
In vitro, Y477F mutant loses its sensitivity to the newly discovered inhibitors but is unable to extend primers during the elongation phase. Our results demonstrate that elements of the ‘B’ site are involved in the conformational changes required in the switch between the different RNA synthesis steps and that compounds targeting this site could lock the enzyme in its initiation phase.
ABSTRACT
Free‐ranging wild vertebrates need to cope with natural and anthropogenic stressors that cause short and/or long‐term behavioural and physiological responses. In areas of high human ...disturbance, the use of glucocorticoid (GC) hormones as biomarkers to measure stress responses is an increasingly common tool for understanding how animals cope with human disturbance. We conducted a meta‐analysis to investigate how human disturbances such as habitat conversion, habitat degradation, and ecotourism influence baseline GC hormones of free‐ranging wild vertebrates, and we further test the role of protected areas in reducing the impact of such disturbances on these hormones. A total of 58 studies met the inclusion criteria, providing 152 data points for comparing levels of GC hormones under disturbed and undisturbed conditions. The overall effect size suggests that human disturbance does not cause a consistent increase in levels of GC hormones (Hedges' g = 0.307, 95% CI = −0.062 to 0.677). However, when the data were analysed by disturbance type, living in unprotected areas or in areas with habitat conversion were found to increase GC hormone levels compared to living in protected or undisturbed areas. By contrast, we found no evidence that ecotourism or habitat degradation generates a consistent increase in baseline GC hormone levels. Among taxonomic groups, mammals appeared more sensitive to human disturbance than birds. We advocate the use of GC hormones for inferring major human‐caused contributors to the stress levels of free‐ranging wild vertebrates – although such information needs to be combined with other measures of stress and interpreted in the context of an organism's life history, behaviour, and history of interactions with human disturbance.
Protein-protein interactions are considered as one of the next generation of therapeutic targets. Specific tools thus need to be developed to tackle this challenging chemical space. In an effort to ...derive some common principles from recent successes, we have built 2P2Idb (freely accessible at http://2p2idb.cnrs-mrs.fr), a hand-curated structural database dedicated to protein-protein interactions with known orthosteric modulators. It includes all interactions for which both the protein-protein and protein-ligand complexes have been structurally characterized. A web server provides links to related sites of interest, binding affinity data, pre-calculated structural information about protein-protein interfaces and 3D interactive views through java applets. Comparison of interfaces in 2P2Idb to those of representative datasets of heterodimeric complexes has led to the identification of geometrical parameters and residue properties to assess the druggability of protein-protein complexes. A tool is proposed to calculate a series of biophysical and geometrical parameters that characterize protein-protein interfaces. A large range of descriptors are computed including, buried accessible surface area, gap volume, non-bonded contacts, hydrogen-bonds, atom and residue composition, number of segments and secondary structure contribution. All together the 2P2I database represents a structural source of information for scientists from academic institutions or pharmaceutical industries.
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