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Many transmembrane receptors have a desensitized state, in which they are unable to respond to external stimuli. The family of microbial rhodopsin proteins includes one such group of receptors, whose inactive or dark-adapted (DA) state is established in the prolonged absence of light. Here, we present high-resolution crystal structures of the ground (light-adapted) and DA states of Archaerhodopsin-3 (AR3), solved to 1.1 Å and 1.3 Å resolution respectively. We observe significant differences between the two states in the dynamics of water molecules that are coupled via H-bonds to the retinal Schiff Base. Supporting QM/MM calculations reveal how the DA state permits a thermodynamic equilibrium between retinal isomers to be established, and how this same change is prevented in the ground state in the absence of light. We suggest that the different arrangement of internal water networks in AR3 is responsible for the faster photocycle kinetics compared to homologs. Archaerhodopsin-3 (AR3) mutants are commonly used in optogenetics for neuron silencing and membrane voltage sensing. High-resolution crystal structures show that desensitization of the AR3 photoreceptor occurs when internal hydrogen-bonded water networks are modified in response to changes in chromophore isomerization.

Deciphering breast cancer treatment resistance remains hindered by the lack of models that can successfully capture the four-dimensional dynamics of the tumor microenvironment. Here, we show that microextrusion bioprinting can reproducibly generate distinct cancer and stromal compartments integrating cells relevant to human pathology. Our findings unveil the functional maturation of this millimeter-sized model, showcasing the development of a hypoxic cancer core and an increased surface proliferation. Maturation was also driven by the presence of cancer-associated fibroblasts (CAF) that induced elevated microvascular-like structures complexity. Such modulation was concomitant to extracellular matrix remodeling, with high levels of collagen and matricellular proteins deposition by CAF, simultaneously increasing tumor stiffness and recapitulating breast cancer fibrotic development. Importantly, our bioprinted model faithfully reproduced response to treatment, further modulated by CAF. Notably, CAF played a protective role for cancer cells against radiotherapy, facilitating increased paracrine communications. This model holds promise as a platform to decipher interactions within the microenvironment and evaluate stroma-targeted drugs in a context relevant to human pathology. [Display omitted] •The bioprinted model further replicates the three-dimensional structure of breast cancer, integrating relevant stromal cells.•CAFs influence model maturation by remodeling ECM, increasing microvascular structures and enhancing stiffness, mimicking fibrosis.•The bioprinted model shows that CAFs protect cancer cells from radiotherapy via paracrine signals, offering insights for stroma-targeted therapies.

In plants, MADS domain transcription factors act as central regulators of diverse developmental pathways. In Arabidopsis thaliana, one of the most central members of this family is SEPALLATA3 ( SEP3), which is involved in many aspects of plant reproduction, including floralmeristem and floral organ development. SEP3 has been shown to form homo and heterooligomeric complexes with other MADS domain transcription factors through its intervening ( I) and keratin-like ( K) domains. SEP3 function depends on its ability to form specific protein-protein complexes; however, the atomic level determinants of oligomerization are poorly understood. Here, we report the 2.5 angstrom crystal structure of a small portion of the intervening and the complete keratin-like domain of SEP3. The domains form two amphipathic alpha helices separated by a rigid kink, which prevents intramolecular association and presents separate dimerization and tetramerization interfaces comprising predominantly hydrophobic patches. Mutations to the tetramerization interface demonstrate the importance of highly conserved hydrophobic residues for tetramer stability. Atomic force microscopy was used to show SEP3-DNA interactions and the role of oligomerization in DNA binding and conformation. Based on these data, the oligomerization patterns of the larger family of MADS domain transcription factors can be predicted and manipulated based on the primary sequence.

Many transmembrane receptors have a desensitized state, in which they are unable to respond to external stimuli. The family of microbial rhodopsin proteins includes one such group of receptors, whose inactive or dark-adapted (DA) state is established in the prolonged absence of light. Here, we present high-resolution crystal structures of the ground (light-adapted) and DA states of Archaerhodopsin-3 (AR3), solved to 1.1 Å and 1.3 Å resolution respectively. We observe significant differences between the two states in the dynamics of water molecules that are coupled via H-bonds to the retinal Schiff Base. Supporting QM/MM calculations reveal how the DA state permits a thermodynamic equilibrium between retinal isomers to be established, and how this same change is prevented in the ground state in the absence of light. We suggest that the different arrangement of internal water networks in AR3 is responsible for the faster photocycle kinetics compared to homologs.

The pathogenicity of Staphylococcus aureus relies on the secretion of various toxins, including phenol-soluble modulins α3 (PSMα3), which play key roles in invasion and infection through its cytolytic activities. Recent controversies have focused on whether PSMα3 propensity to self-assemble into unique amyloid-like cross-α structures would underlie its high cytotoxicity. Here, by integrating Thioflavin T fluorescence, electron microscopy, and cell-based assays, we demonstrate that early soluble entities formed upon fibrillation - rather than mature fibrils - drive PSMα3 cytotoxicity under near-physiological conditions. This mirrors the behavior of neurodegenerative disease related peptides, thus pointing towards a general mechanism of toxicity by amyloid forming peptides. Importantly, we elucidate the critical role of serum in this process: lipoproteins inhibit the formation of oligomeric and fibrillar entities, both in a time- and concentration-dependent manner, thereby drastically reducing cytotoxicity. We further reveal the uptake of such soluble entities in cells, where membrane interactions likely initiate and trigger cell death. These findings reconcile conflicting interpretations of PSMα3 toxicity, highlighting the importance of the cellular environment in shaping peptide aggregation and biological function. By targeting such key virulence determinants, e.g. though the rational design of aggregation inhibitors, this study could open novel avenues in the quest for novel drugs against S. aureus infections.Competing Interest StatementThe authors have declared no competing interest.Funder Information DeclaredEuropean Research Council, 101162069European Union, 101064573Université de Bordeaux, https://ror.org/057qpr032, MISTICBasque Government, 449IT1720-22

The virulence of Staphylococcus aureus, a multi-drug resistant pathogen, notably depends on the expression of the phenol soluble modulins α3 (PSMα3) peptides, able to self-assemble into amyloid-like cross-α fibrils. Despite remarkable advances evidencing the crucial, yet insufficient, role of fibrils in PSMα3 cytotoxic activities towards host cells, the relationship between its molecular structures, assembly propensities, and modes of action remains an open intriguing problem. In this study, combining Atomic Force Microscopy (AFM) imaging and infrared spectroscopy, we first demonstrated in vitro that the charge provided by the N-terminal capping of PSMα3 alters its interactions with model membranes of controlled lipid composition, without compromising its fibrillation kinetics or morphology. N-formylation eventually dictates PSMα3 - membrane binding via electrostatic interactions with the lipid head groups. Furthermore, PSMα3 insertion within the lipid bilayer is favoured by hydrophobic interactions with the lipid acyl chains, only in the fluid-phase of membranes, and not in the gel-like ordered domains. Strikingly, our real-time AFM imaging emphasizes how intermediate protofibrillar entities, formed along PSMα3 self-assembly and promoted at the membrane interface, likely disrupt membrane integrity via peptide accumulation, and subsequent membrane thinning in a peptide concentration and lipid-dependent manner. Overall, our multiscale and multimodal approach sheds new light on the key roles of N-formylation and intermediate self-assembling entities, rather than mature fibrils, in dictating deleterious interactions of PSMα3 with specific membrane lipids, likely underscoring its ultimate cellular toxicity in vivo, and in turn S. aureus pathogenesis.Competing Interest StatementThe authors have declared no competing interest.

Nuclear pore complexes (NPCs) are the only gateways between the nucleus and cytoplasm in eukaryotic cells. They restrict free diffusion to molecules below 5 nm while facilitating the active transport of selected cargoes, sometimes as large as the pore itself. This versatility implies an important pore plasticity. Recently, cryo-EM and AI-based protein modeling revealed with acute precision how most NPC constituents are arranged. But the basket, a fish trap-like structure capping the nucleoplasmic side of the pore, remains the missing piece in this puzzle. Here by Atomic Force Microscopy (AFM) coupled to Single Molecule Localization Microscopy (SMLM) we revealed that the basket is very soft and explores a large conformational landscape: apart from its canonical shape, it dives into the central pore channel or opens, with filaments reaching to the pore sides. Our observations enlighten how this structure can adapt and let morphologically diverse cargoes shuttling through NPCs. Competing Interest Statement The authors have declared no competing interest.

Non-contact hamstring injuries (HSIs) commonly occur during the late swing phase of sprinting, when muscle–tendon units (MTUs) approach maximum length. Although sagittal-plane pelvis, hip and knee angles are often used as surrogate measures of overall hamstring lengthening, their predictive validity remains uncertain. This study investigated whether peak three-dimensional lower limb joint angles predict length change in the biarticular hamstring MTUs from peak hip flexion through to toe off (0–100 %) in fourteen intermediate-level male soccer players sprinting at maximal speed (8.56 ± 0.47 m·s−1). Participant-specific musculoskeletal models were used to compute MTU lengths for the biceps femoris long head (BFlh), semimembranosus (SM), and semitendinosus (ST). To account for inter-subject temporal variability and enable accurate point-to-point comparisons across trials, dynamic time warping was applied for non-linear temporal registration. Statistical parametric mapping regression was used to assess associations between pelvis, hip and knee peak joint angles (sagittal, frontal, transverse) and length change of BFlh, SM, and ST. Peak sagittal angles were poor predictors while peak frontal pelvis angle was negatively associated with MTU length change during late swing (BFlh peak |r| = −0.371; SM peak |r| = −0.460). Frontal hip adduction was negatively associated with MTU length change from peak hip flexion to early stance (peak |r| −0.39 to −0.46). Internal hip rotation was associated with SM and ST lengthening (peak |r| = 0.51) from late swing to early stance. Knee extension angles showed no significant associations. These findings suggest caution when using single-plane joint angles as isolated indicators of hamstring MTU length.

Myotonic Dystrophy type 1 (DM1) is one of the most heterogeneous hereditary disease in terms of age of onset, clinical manifestations, and severity, challenging both medical management and clinical trials. The CTG expansion size is the main factor determining the age of onset although no factor can finely predict phenotype and prognosis. Differences between males and females have not been specifically reported. Our aim is to study gender impact on DM1 phenotype and severity. We first performed cross-sectional analysis of main multiorgan clinical parameters in 1409 adult DM1 patients (>18 y) from the DM-Scope nationwide registry and observed different patterns in males and females. Then, we assessed gender impact on social and economic domains using the AFM-Téléthon DM1 survey (n = 970), and morbidity and mortality using the French National Health Service Database (n = 3301). Men more frequently had (1) severe muscular disability with marked myotonia, muscle weakness, cardiac, and respiratory involvement; (2) developmental abnormalities with facial dysmorphism and cognitive impairment inferred from low educational levels and work in specialized environments; and (3) lonely life. Alternatively, women more frequently had cataracts, dysphagia, digestive tract dysfunction, incontinence, thyroid disorder and obesity. Most differences were out of proportion to those observed in the general population. Compared to women, males were more affected in their social and economic life. In addition, they were more frequently hospitalized for cardiac problems, and had a higher mortality rate. Gender is a previously unrecognized factor influencing DM1 clinical profile and severity of the disease, with worse socio-economic consequences of the disease and higher morbidity and mortality in males. Gender should be considered in the design of both stratified medical management and clinical trials.

We created neutral antimalarial prodrugs that deliver bisthiazolium compounds with antimalarial activity in the nanomolar range. These drugs primarily affect early intraerythrocytic stages through rapid, nonreversible cytotoxicity. The compounds are suitable for both parenteral and oral use and plasma promotes rapid conversion of the prodrug into the drug. We demonstrate that very low doses offer protection in a murine model of malaria. The drugs show great potential for curing high parasitemia with short-course treatments. Oral administration of the TE3 prodrug completely cures Plasmodium cynomolgi infection in rhesus monkeys. The drugs specifically accumulate inside infected erythrocytes, block phosphatidylcholine biosynthesis, and interact with hemozoin. To our knowledge, this class of compounds represents one of the most potent antimalarials tested to date. These unique properties signal a promising future for this class of antimalarial.