Project description:Platform chemicals and polymer precursors can be produced via enzymatic pathways starting from lignocellulosic waste materials. The hemicellulose fraction of lignocellulose contains aldopentose sugars, such as D-xylose and L-arabinose, which can be enzymatically converted into various biobased products by microbial non-phosphorylated oxidative pathways. The Weimberg and Dahms pathways convert pentose sugars into α-ketoglutarate, or pyruvate and glycolaldehyde, respectively, which then serve as precursors for further conversion into a wide range of industrial products. In this review, we summarize the known three-dimensional structures of the enzymes involved in oxidative non-phosphorylative pathways of pentose catabolism. Key structural features and reaction mechanisms of a diverse set of enzymes responsible for the catalytic steps in the reactions are analysed and discussed.
Project description:Melanoma is an aggressive cancer that utilizes multiple signaling pathways, including those that involve oncogenes, proto-oncogenes, and tumor suppressors. It has been suggested that melanoma formation requires cross-talk of the PI3K/Akt/mTOR and Ras-ERK pathways. This pathway cross-talk has been associated with aggressiveness, drug resistance, and metastasis; thus, simultaneous targeting of components of the different pathways involved in melanoma may aid in therapy. We have previously reported that bacterial cyclodipeptides (CDPs) are cytotoxic to HeLa cells and inhibit Akt phosphorylation. Here, we show that CDPs decreased melanoma size and tumor formation in a subcutaneous xenografted mouse melanoma model. In fact, CDPs accelerated death of B16-F0 murine melanoma cells. In mice, antitumor effect was improved by treatment with CDPs using cyclodextrins as drug vehicle. In tumors, CDPs caused nuclear fragmentation and changed the expression of the Bcl-2 and Ki67 apoptotic markers and promoted restoration of hyperactivation of the PI3K/Akt/mTOR pathway. Additionally, elements of several signaling pathways such as the Ras-ERK, PI3K/JNK/PKA, p27Kip1/CDK1/survivin, MAPK, HIF-1, epithelial-mesenchymal transition, and cancer stem cell pathways were also modified by treatment of xenografted melanoma mice with CDPs. The findings indicate that the multiple signaling pathways implicated in aggressiveness of the murine B16-F0 melanoma line are targeted by the bacterial CDPs. Molecular modeling of CDPs with protein kinases involved in neoplastic processes suggested that these compounds could indeed interact with the active site of the enzymes. The results suggest that CDPs may be considered as potential antineoplastic drugs, interfering with multiple pathways involved in tumor formation and progression.
Project description:The actinobacterium Rhodococcus jostii RHA1 grows on a remarkable variety of aromatic compounds and has been studied for applications ranging from the degradation of polychlorinated biphenyls to the valorization of lignin, an underutilized component of biomass. In RHA1, the catabolism of two classes of lignin-derived compounds, alkylphenols and alkylguaiacols, involves a phylogenetically distinct extradiol dioxygenase, AphC, previously misannotated as BphC, an enzyme involved in biphenyl catabolism. To better understand the role of AphC in RHA1 catabolism, we first showed that purified AphC had highest apparent specificity for 4-propylcatechol (kcat/KM ∼106 M-1 s-1), and its apparent specificity for 4-alkylated substrates followed the trend for alkylguaiacols: propyl > ethyl > methyl > phenyl > unsubstituted. We also show AphC only poorly cleaved 3-phenylcatechol, the preferred substrate of BphC. Moreover, AphC and BphC cleaved 3-phenylcatechol and 4-phenylcatechol with different regiospecificities, likely due to the substrates' binding mode. A crystallographic structure of the AphC·4-ethylcatechol binary complex to 1.59 Å resolution revealed that the catechol is bound to the active site iron in a bidentate manner and that the substrate's alkyl side chain is accommodated by a hydrophobic pocket. Finally, we show RHA1 grows on a mixture of 4-ethylguaiacol and guaiacol, simultaneously catabolizing these substrates through meta-cleavage and ortho-cleavage pathways, respectively, suggesting that the specificity of AphC helps to prevent the routing of catechol through the Aph pathway. Overall, this study contributes to our understanding of the bacterial catabolism of aromatic compounds derived from lignin, and the determinants of specificity in extradiol dioxygenases.
Project description:Cell-cell interactions are important to numerous biological systems, including tissue microenvironments, the immune system, and cancer. However, current methods for studying cell combinations and interactions are limited in scalability, allowing just hundreds to thousands of multi-cell assays per experiment; this limited throughput makes it difficult to characterize interactions at biologically relevant scales. Here, we describe a new paradigm in cell interaction profiling that allows accurate grouping of cells and characterization of their interactions for tens to hundreds of thousands of combinations. Our approach leverages high throughput droplet microfluidics to construct multicellular combinations in a deterministic process that allows inclusion of programmed reagent mixtures and beads. The combination droplets are compatible with common manipulation and measurement techniques, including imaging, barcode-based genomics, and sorting. We demonstrate the approach by using it to enrich for CAR-T cells that activate upon incubation with target cells, a bottleneck in the therapeutic T cell engineering pipeline. The speed and control of our approach should enable valuable cell interaction studies.
Project description:Hydrogen/deuterium exchange (HDX) methods for studying protein dynamics would benefit from millisecond-scale incubations to probe intrinsically disordered proteins, highly dynamic regions and conformation changes. Here we investigate droplet microfluidics for rapid mixing to trigger D2O labelling, uniform incubations and rapid droplet merging for acid quenching in advance of mass spectrometry. A surfactant-free merging approach combining expansion elements for synchronised droplet collision proved robust. The high diffusive flux of D2O and protons enable microsecond mixing to trigger and arrest D2O labelling, respectively, affording the possibility of single millisecond incubations. Millisecond droplet HDX processors were used to measure forward exchange and demonstrate that D2O labelling is the rate-limiting step, in essence defining 10 milliseconds as the minimum practical incubation time. With the ability to access millisecond time scales the fast dynamics of calmodulin, a model of calcium-triggered allostery with rapid conformational switching, was investigated. Calcium binding increases D2O accessibility to the linker region, reporting the flexibility which enables the scissor-like motion of calmodulin for capturing proteins. The millisecond precision of droplet microfluidic HDX paves the way to advance our understanding of protein structural dynamics.
Project description:The mucus layer covering the skin of fish has several roles, including protection against pathogens and mechanical damage. While the mucus layers of various bony fish species have been investigated, the composition and glycan profiles of shark skin mucus remain relatively unexplored. In this pilot study, we aimed to explore the structure and composition of shark skin mucus through histological analysis and glycan profiling. Histological examination of skin samples from Atlantic spiny dogfish (Squalus acanthias) sharks and chain catsharks (Scyliorhinus retifer) revealed distinct mucin-producing cells and a mucus layer, indicating the presence of a functional mucus layer similar to bony fish mucus albeit thinner. Glycan profiling using liquid chromatography-electrospray ionization tandem mass spectrometry unveiled a diverse repertoire of mostly O-glycans in the mucus of the two sharks as well as little skate (Leucoraja erinacea). Elasmobranch glycans differ significantly from bony fish, especially in being more sulfated, and some bear resemblance to human glycans, such as gastric mucin O-glycans and H blood group-type glycans. This study contributes to the concept of shark skin having unique properties and provides a foundation for further research into the functional roles and potential biomedical implications of shark skin mucus glycans.
Project description:Droplet microfluidics-based iX-seq uncovers cross-feeding interactions between Phascolarctobacterium faecium and Eubacterium limosum in the gut microbiota
Project description:Mucus forms a critical barrier against enteric pathogens like Salmonella enterica serovar Typhimurium. While in vivo studies indicate that secreted, gel-forming mucins and specifically Core 3 glycosylation are protective against S. Typhimurium, the molecular mechanisms involved remain unclear. Here, we measure gene expression changes in Salmonella enterica serovar Typhimurium LT2 following growth in SPI-1 inducing medium (LB + 0.3M NaCl) with or without purified MUC2 (0.1%, w/v), MUC2 glycans (0.1%, w/v), a pool of monosaccharide components comprised of D-galactose, D-GalNAc, D-GlcNAc, D-fuc, and Neu5Ac (0.1%, w/v), or specific individual mucin sugars, namely N-acetyl galactosamine (GalNAc) (0.2%, w/v) and N-acetyl glucosamine (GlcNAc) (0.2%, w/v). Notably, we find MUC2, MUC2 glycans, and to a lesser extent,D-GalNAc and D-GlcNAc downregulate SPI-1 gene expression.
Project description:Although gut microbiomes are generally symbiotic or commensal, some of microbiomes become pathogenic under certain circumstances, which is one of key processes of pathogenesis. However, the factors involved in these complex gut-microbe interactions are largely unknown. Here we show that bacterial nucleoside catabolism using gut luminal uridine is required to boost inter-bacterial communications and gut pathogenesis in Drosophila. We found that uridine-derived uracil is required for DUOX-dependent ROS generation on the host side, whereas uridine-derived ribose induces quorum sensing and virulence gene expression on the bacterial side. Importantly, genetic ablation of bacterial nucleoside catabolism is sufficient to block the commensal-to-pathogen transition in vivo. Furthermore, we found that major commensal bacteria lack functional nucleoside catabolism, which is required to achieve gut-microbe symbiosis. The discovery of a novel role of bacterial nucleoside catabolism will greatly help to better understand the molecular mechanism of the commensal-to-pathogen transition in different contexts of host-microbe interactions.