Unité de Glycobiologie Structurale et Fonctionnelle

The sialylated Lewis antigens sLea and sLex are terminal glycans that are crucial for adhesive and signalling functions during cancer metastasis. As high affinity ligands for E-, P-, and L-selectins, these epitopes allow tumor cells to arrest, evade immune cells and extravasate in the vascular microenvironment, mimicking leukocyte trafficking. This review provides a detailed analysis of sLea/x biosynthesis, structure, function and their role in metastatic progression. We focus on the differential roles of glycoprotein versus glycolipid associated sLea/x, their glycosyltransferase mediated synthesis and the spatial trafficking mechanisms that govern their surface expression. We also highlight the carrier scaffolds and glyco-enzyme hierarchies that present the ligand across different cancers, mucins, integrins and glycosphingolipids. A section is dedicated to how exosome bound sLea/x primes pre-metastatic niche and immune modulation, a new perspective on glycan mediated systemic signaling. We also compile validated tumor specific profiles of sLea/x across several cancer types, linking structural expression to metastatic phenotypes. Additionally, we discuss emerging strategies targeting sLea/x pathways from glycosylation inhibitors to selectin blocking therapeutics and the translational challenges and opportunities. Overall, this synthesis shows the importance of sLea and sLex in metastasis and lays the foundation for their use as biomarkers and therapeutic targets in precision oncology.

Chemical biology has reshaped the ability to investigate complex biological systems at the molecular level. In this context, chemical reporters have become important tools for labeling and tracking biomolecules in living systems with spatial and temporal precision. In plant biology, they provide an alternative to genetic approaches and allow the study of dynamic processes in species or organs that are not easily accessible. Through the use of click and bioorthogonal chemistry, small‐molecule probes can be metabolically incorporated into specific molecular scaffolds such as sugars, monolignols, amino acids, and lipids. These probes make it possible to follow events like glycosylation, lignification, lipid turnover, or protein synthesis in living plant tissues. This review presents an overview of current chemical reporter strategies, from molecular design and synthetic considerations to their application in plant imaging. Herein, how these tools have contributed to the development of plant chemical biology by enabling precise and modular investigations of plant structure and metabolism is described. Herein, it is also examined how chemical reporters have entered interdisciplinary contexts, including collaborations between science and the arts. By converting molecular‐level information into visual and sensory formats, these approaches open new perspectives for research, education, and communication across scientific and creative disciplines.

ABSTRACTMassive sampling with AlphaFold2 has become a widely used approach in protein structure prediction. Here we present the MassiveFold CASP16‐CAPRI dataset, a systematic, large‐scale sampling of both monomeric and multimeric protein targets. By exploiting maximal parallelization, we produced up to 8040 models per target and shared them with the community for collaborative selection and scoring. This collective effort minimizes redundant computation and environmental impact, while granting resource‐limited groups ‐ especially those focused on scoring ‐ access to high quality structures. In our analysis, we define an interface‐difficulty classification based on DockQ metrics, showing that massive sampling yields the greatest gains on most of the challenging interfaces. Crucially, this classification can be predicted from the median ipTM scores of a routine AF2 run, enabling users to selectively deploy massive sampling only when it is most needed. Combined with a reduction of the massive sampling from 8040 to 2475 predictions, such targeted strategies dramatically cut computation time and resource use with minimal loss of accuracy. Finally, we underscore the persistent challenge of choosing optimal models from massive sampling datasets, emphasizing the need for more robust scoring methods. The MassiveFold datasets, together with AlphaFold ranking scores and CASP and CAPRI assessment metrics, are publicly available at https://github.com/GBLille/CASP16‐CAPRI_MassiveFold_Data to accelerate further progress in protein structure prediction and assembly modeling.

ObjectivesThe study of seaweed cell walls, including their metabolism and composition, is crucial to monitor and understand their adaptation to climate change. Microscopy-based techniques that facilitate studies of seaweed cell walls in situ, including staining and immunolabelling, exist but have significant limitations, including that only a few monoclonal antibodies have been developed towards seaweed cell wall components. Furthermore, not all seaweed cell wall components have been described fully. This makes in situ studies focused on the metabolism of seaweed cell walls particularly challenging. Here, we present a method for labelling seaweed cell walls by incorporating chemical reporters in muro, followed by their association with fluorophores by click chemistry.MethodsTwo different species of seaweed, representing different groups, i.e. the red seaweed Phycodrys rubens and the green seaweed Ulva spp., were selected for their abundance on the Irish coast and because they have relatively thin tissues, a feature likely to facilitate uptake and labelling using monosaccharide reporters. We selected three different activated sugars (fucose, galactose and glucose analogues) on the basis that they are major components of seaweed polysaccharides. Small sections of the seaweeds were incubated with the activated sugars, and their uptake and incorporation were visualized by attachment to the fluorescent probe AF488 and imaged using confocal microscopy.ResultsAfter incubation with the activated sugars, the seaweed cell walls, i.e. the contours of the cells and, to a lesser extent, some cell organelles, fluoresced at 517 nm (emission wavelength of AF488), suggesting that the seaweed had incorporated the activated sugars. Fiji software was used to remove non-specific fluorescence (autofluorescence of the seaweed and non-specific binding of the fluorochrome), with the final images suggesting good and specific incorporation of the fluorochrome by the seaweeds. More interestingly, the fluorescence was associated primarily with the cell walls, implying that the activated sugars were incorporated predominantly into cell wall components (most probably either polysaccharides or glycoproteins).ConclusionTo date, bioorthogonal click chemistry has not been applied to seaweeds, but it represents a useful tool for phycologists to gain a better understanding of seaweed cell wall composition and dynamics, for example, throughout the seaweed life cycle or in the face of stresses (biotic or abiotic), including those resulting from climate change.

(1→3)-ß-d-glucans (BDG), major cell wall components of most pathogenic fungi, are useful for the diagnosis of invasive fungal diseases (IFD) due to their high negative predictive value. For several years, a number of BDG detection tests are commercially available, including Fungitell Assay (FA) and Wako assay, and more recently Fungitell STAT (STAT) unit test. Our aim was to compare the performance of the two Fungitell assays for IFD diagnosis. Sera from 90 patients with Pneumocystis jirovecii pneumonia (PJP, n=30), candidemia (n=30), and invasive pulmonary aspergillosis (IPA, n=30), 30 patients colonized by P. jirovecii, and 70 healthy controls (women followed-up during pregnancy) were analyzed retrospectively. STAT and FA assays were performed according to manufacturer's instructions. The overall level of agreement between two Fungitell assays was excellent (weighted Cohen's kappa=0.87 [95% CI: 0.80-0.94]). BDG rates were significantly higher in candidemia, IPA and PJP than in healthy controls (P<.0001). BDG rates were also significantly higher in PJP than for P. jirovecii colonization (P<.0001). Area Under the Curve (AUC) of STAT (0.92) was higher than FA (0.86) for IFD diagnosis. Using optimized positivity thresholds for IFD diagnosis (94 pg/ml and 0.86 for FA and STAT, respectively), sensitivities and specificities were 78.9% and 90% for FA and 85.6% and 88.6% for STAT, respectively. AUC of FA and STAT were higher for PJP diagnosis than for candidemia and IPA diagnosis. Compared to FA, STAT assay represents an interesting option for emergency IFD diagnosis and for small care centers.

Blue rings (BRs) are anomalies in tree rings revealed by a double staining technique. They have been thought to reflect reduced cell wall lignification during ephemeral end-of-growing-season cold periods, often following significant volcanic eruptions. BRs have been distinguished visually according to qualitative descriptions and are considered a promising proxy for cold extremes at the intra-seasonal scale. However, reduced cell wall lignin content has never been successfully measured in BRs, limiting understanding of their relationship with thermal (or other) conditions. Further, difference in lignin content between BRs and ‘standard’ rings has not been confirmed independently of the double staining method. Here, using confocal microscopy of samples of Scots pine, we show that BRs are indeed associated with marked reduction of lignin in latewood cells. We also identify subtle variations in lignin across rings that are not well resolved by the double staining method. We draw on these findings to propose a new framework to visualise and measure lignin changes in tree rings. Our findings emphasise the potential of BRs as a paleoproxy of intraseasonal climate variability.

In hereditary papillary renal cell carcinoma (HPRCC), the hepatocyte growth factor receptor (MET) receptor tyrosine kinase (RTK) mutations recorded to date are located in the kinase domain and lead to constitutive MET activation. This contrasts with MET mutations identified in non-small cell lung cancer (NSCLC), which lead to exon 14 skipping and deletion of a regulatory domain: in this latter case, the mutated receptor still requires ligand stimulation. Sequencing of MET in samples from 158 HPRCC and 2808 NSCLC patients revealed ten uncharacterized mutations. Four of these, all found in HPRCC and leading to amino acid substitutions in the N-lobe of the MET kinase, proved able to induce cell transformation, which was further enhanced by hepatocyte growth factor (HGF) stimulation: His1086Leu, Ile1102Thr, Leu1130Ser and Cis1125Gly. Similar to the variant resulting in MET exon 14 skipping, the two N-lobe MET variants His1086Leu and Ile1102Thr were found to require stimulation by HGF in order to strongly activate downstream signaling pathways and epithelial cell motility. The Ile1102Thr mutation also displayed transforming potential, promoting tumor growth in a xenograft model. In addition, the N-lobe-mutated MET variants were found to trigger a common HGF-stimulation-dependent transcriptional program, consistent with an observed increase in cell motility and invasion. Altogether, this functional characterization revealed that N-lobe variants still require ligand stimulation, in contrast to other RTK variants. This suggests that HGF expression in the tumor microenvironment is important for tumor growth. The sensitivity of these variants to MET inhibitors opens the way for use of targeted therapies for patients harboring the corresponding mutations.

BackgroundAnemonefish association with sea anemones is a prime example of mutualistic symbiosis. These fish live inside the sea anemone, benefitting from the protection of its toxic nematocysts, and in return, protect the anemone from its own predators. How anemonefish manage to avoid their host toxic stings remains unclear. One hypothesis suggests that low levels of sialic acids in anemonefish mucus prevent nematocyst discharge.ResultsThis study verified four predictions: (i) anemonefish mucus has lower sialic acid levels than non-symbiotic damselfish; (ii) this reduction is specific to mucus; (iii) during development, sialic acid levels inversely correlate with protection; (iv) sea anemone mucus has minimal sialic acids.ConclusionsWe conclude that anemonefish regulates the level of sialic acids in their mucus to avoid nematocyst discharge. We also highlight several genes implicated in sialic acid removal that could explain the protection mechanisms in place. This mechanism, potentially used by Dascyllus trimaculatus juveniles, suggests a convergent strategy for mutualistic associations with sea anemones.