Protein crystallogenesis represents a key step in X-ray crystallography studies that employ co-crystallization and ligand soaking for investigating ligand binding to proteins. Co-crystallization is a method that enables the precise determination of binding positions, although it necessitates a significant degree of optimization. The utilization of microseeding can facilitate a reduction in sample requirements and accelerate the co-crystallization process. Ligand soaking is the preferred method due to its simplicity; however, it requires careful control of soaking conditions to ensure the successful integration of the ligands. This research protocol details the procedures for co-crystallization and soaking to achieve protein-ligand complex formation, which is essential for advancing drug discovery. Additionally, a simple protocol for demonstrating soaking for educational purposes is described.
- Klíčová slova
- advanced crystallization, co‐crystallization, crystal soaking, crystallization protocol, microseeding,
- MeSH
- konformace proteinů MeSH
- krystalizace metody MeSH
- krystalografie rentgenová metody MeSH
- ligandy MeSH
- proteiny * chemie metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- ligandy MeSH
- proteiny * MeSH
In the first days of life, the newborns' intestinal microbiota develops simultaneously with the intestinal gut barrier and follows intestinal immunity. The mode of delivery shows significant impact on microbial development and, thus, the initiation of the tryptophan catabolism pathway. Further antibiotics (ATB) treatment of mothers before or during delivery affects the microbial and tryptophan metabolite composition of stool of the caesarean- and vaginal-delivered newborns. The determination of microbiome and levels of tryptophan microbial metabolites in meconium and stool can characterize intestinal colonization of a newborn. From 134 samples from the Central European Longitudinal Studies of Parents and Children: The Next Generation (CELSPAC: TNG) cohort study, 16S rRNA gene sequencing was performed, and microbial tryptophan metabolites were quantified using ultra-high-performance liquid chromatography with triple-quadrupole mass spectrometry. Microbial diversity and concentrations of tryptophan metabolites were significantly higher in stool compared to meconium. Treatment of mothers with ATB before or during delivery affects metabolite composition and microbial diversity in stool of vaginal- and caesarean-delivered newborns. Correlation of microbial and metabolite composition shows significant positive correlations of indol-3-lactic acid, N-acetyl-tryptophan and indol-3-acetic acid with Bifidobacterium, Bacteroides and Peptoclostridium. The positive effect of vaginal delivery on newborns' microbiome development is degraded when mother is treated with ATB before or during delivery. KEY POINTS: • Antibiotic treatment diminishes the positive effects of vaginal delivery. • Antibiotic treatment affects metabolite and microbial composition in newborns. • Bifidobacterium and Peptoclostridium could be the producer of indole-lactic acid.
- Klíčová slova
- Caesarean delivery, Kynurenine, Microbiome, Stool, Tryptophan catabolites, Vaginal delivery,
- MeSH
- antibakteriální látky * MeSH
- Bifidobacterium metabolismus růst a vývoj MeSH
- feces * mikrobiologie chemie MeSH
- indoly metabolismus MeSH
- kyseliny indoloctové metabolismus MeSH
- lidé MeSH
- longitudinální studie MeSH
- mekonium * mikrobiologie chemie MeSH
- novorozenec MeSH
- RNA ribozomální 16S * genetika MeSH
- střevní mikroflóra * účinky léků MeSH
- tryptofan * metabolismus MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- lidé MeSH
- novorozenec MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antibakteriální látky * MeSH
- indole-3-lactic acid MeSH Prohlížeč
- indoleacetic acid MeSH Prohlížeč
- indoly MeSH
- kyseliny indoloctové MeSH
- RNA ribozomální 16S * MeSH
- tryptofan * MeSH
