The biosynthesis of the lincosamide antibiotics lincomycin A and celesticetin involves the pyridoxal-5'-phosphate (PLP)-dependent enzymes LmbF and CcbF, which are responsible for bifurcation of the biosynthetic pathways. Despite recognizing the same S-glycosyl-L-cysteine structure of the substrates, LmbF catalyses thiol formation through β-elimination, whereas CcbF produces S-acetaldehyde through decarboxylation-coupled oxidative deamination. The structural basis for the diversification mechanism remains largely unexplored. Here we conduct structure-function analyses of LmbF and CcbF. X-ray crystal structures, docking and molecular dynamics simulations reveal that active-site aromatic residues play important roles in controlling the substrate binding mode and the reaction outcome. Furthermore, the reaction selectivity and oxygen-utilization of LmbF and CcbF were rationally engineered through structure- and calculation-based mutagenesis. Thus, the catalytic function of CcbF was switched to that of LmbF, and, remarkably, both LmbF and CcbF variants gained the oxidative-amidation activity to produce an unnatural S-acetamide derivative of lincosamide.
Azo dyes are used as coloring agent in textile industries at larger scale. As a result, large quantity of dye-enriched waste water is generated which subsequently poses environmental problems. Biological tool involving bacteria having azoreductase enzyme has proved to be more effective and efficient in dye effluent treatment. Current work focuses on Staphylococcus caprae (S. caprae) for degradation and decolorization of Reactive Red-195 (RR-195) azo dye. For this purpose, factors such as pH, temperature, inoculums, carbon and nitrogen sources, and dye concentrations have been optimized for maximum decolorization and degradation. S. caprae (4 mg/mL) efficiently resulted into 90% decolorization of RR-195 dye under static condition at 100 μg/mL concentration, 30 °C and pH 7.0 at a 12-h contact period. FTIR analysis has revealed the formation of new functional groups in the treated dye such as O-H stretch at 3370 cm-1, C-H band stretching at 2928 cm-1, and new band at 1608 cm-1 which specify the degradation of aromatic ring, 1382 and 1118 cm-1 represents desulfonated peaks. Biodegraded metabolites of RR-195 dye such as phenol, 3, 5-di-tert-butylphenol, and phthalic acid have been identified respectively that find industrial applications. Phytotoxicity test has shown non-toxic effects of treated dye on germination of Vigna radiata and Triticum aestivum seeds. Further, antibiotic diffusion assay has confirmed the biosafety of S. caprae.
- MeSH
- Azo Compounds * metabolism toxicity MeSH
- Coloring Agents * metabolism MeSH
- Biodegradation, Environmental * MeSH
- Water Pollutants, Chemical metabolism MeSH
- Nitrogen metabolism MeSH
- Hydrogen-Ion Concentration MeSH
- Wastewater * microbiology chemistry MeSH
- Industrial Waste MeSH
- Staphylococcus capitis metabolism isolation & purification MeSH
- Staphylococcus metabolism MeSH
- Temperature MeSH
- Textiles MeSH
- Textile Industry MeSH
- Carbon metabolism MeSH
- Publication type
- Journal Article MeSH
Super- and low-shedding phenomena have been observed in genetically homogeneous hosts infected by a single bacterial strain. To decipher the mechanisms underlying these phenotypes, we conducted an experiment with chicks infected with Salmonella Enteritidis in a non-sterile isolator, which prevents bacterial transmission between animals while allowing the development of the gut microbiota. We investigated the impact of four commensal bacteria called Mix4, inoculated at hatching, on chicken systemic immune response and intestinal microbiota composition and functions, before and after Salmonella infection. Our results revealed that these phenotypes were not linked to changes in cell invasion capacity of bacteria during infection. Mix4 inoculation had both short- and long-term effects on immune response and microbiota and promoted the low-shedder phenotype. Kinetic analysis revealed that Mix4 activated immune response from day 4, which modified the microbiota on day 6. This change promotes a more fermentative microbiota, using the aromatic compounds degradation pathway, which inhibited Salmonella colonization by day 11 and beyond. In contrast, control animals exhibited a delayed TNF-driven pro-inflammatory response and developed a microbiota using anaerobic respiration, which facilitates Salmonella colonization and growth. This strategy offers promising opportunities to strengthen the barrier effect against Salmonella and possibly other pathogens.
- MeSH
- Bacteria * classification genetics isolation & purification MeSH
- Chickens * microbiology immunology MeSH
- Poultry Diseases * microbiology immunology prevention & control MeSH
- Salmonella enteritidis * immunology growth & development MeSH
- Salmonella Infections, Animal * microbiology immunology prevention & control MeSH
- Gastrointestinal Microbiome * MeSH
- Symbiosis MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
The hedgehog signaling pathway plays an important role in vertebrate embryonic development, tissue homeostasis, and tumorigenesis. Constitutive activation of Hh signaling in various human tumors leads to GLI-mediated transcription and tumor progression. Based on the preliminary screening of a large library of known triterpenes that exhibited interesting Hh inhibitory activity, we designed and synthesized a new series of triterpenoid analogues containing aromatic heterocyclic substituents at position C-2 to enhance their interference with Hh signaling. In this study, we evaluated the effect of 15 synthesized triterpenoids on cell proliferation and Hh pathway activity in relevant cancer cell lines. Among these compounds, two derivatives, 11a and 11b, both featuring a furan ring at position C-2, demonstrated potent inhibitory effects on proliferation and induced cell death in nonsmall cell lung cancer (NSCLC) and prostate cancer cell lines exhibiting hyper-activated Hh signaling. Moreover, these compounds significantly reduced GLI-mediated transcription in cell-based reporter assays. Detailed immunoblot analyses revealed that compounds 11a and 11b decreased the expression of endogenous GLI1 protein and its target genes associated with tumor progression and proliferation, such as Cyclin D1, N-Myc, and Bcl-2, in A549 and DU-145 cancer cells. These findings suggest that the antiproliferative effects of 11a and 11b are mediated through inhibition of the Hh signaling pathway and are promising candidates for the development of new anticancer therapies targeting Hh-dependent tumors.
- Publication type
- Journal Article MeSH
Lens epithelium-derived growth factor p75 (LEDGF/p75), member of the hepatoma-derived growth-factor-related protein (HRP) family, is a transcriptional co-activator and involved in several pathologies including HIV infection and malignancies such as MLL-rearranged leukemia. LEDGF/p75 acts by tethering proteins to the chromatin through its integrase binding domain. This chromatin interaction occurs between the PWWP domain of LEDGF/p75 and nucleosomes carrying a di- or trimethylation mark on histone H3 Lys36 (H3K36me2/3). Our aim is to rationally devise small molecule drugs capable of inhibiting such interaction. To bootstrap this development, we resorted to X-ray crystallography-based fragment screening (FBS-X). Given that the LEDGF PWWP domain crystals were not suitable for FBS-X, we employed crystals of the closely related PWWP domain of paralog HRP-2. As a result, as many as 68 diverse fragment hits were identified, providing a detailed sampling of the H3K36me2/3 pocket pharmacophore. Subsequent structure-guided fragment expansion in three directions yielded multiple compound series binding to the pocket, as verified through X-ray crystallography, nuclear magnetic resonance and differential scanning fluorimetry. Our best compounds have double-digit micromolar affinity and optimally sample the interactions available in the pocket, judging by the Kd-based ligand efficiency exceeding 0.5 kcal/mol per non-hydrogen atom. Beyond π-stacking within the aromatic cage of the pocket and hydrogen bonding, the best compounds engage in a σ-hole interaction between a halogen atom and a conserved water buried deep in the pocket. Notably, the binding pocket in LEDGF PWWP is considerably smaller compared to the related PWWP1 domains of NSD2 and NSD3 which feature an additional subpocket and for which nanomolar affinity compounds have been developed recently. The absence of this subpocket in LEDGF PWWP limits the attainable affinity. Additionally, these structural differences in the H3K36me2/3 pocket across the PWWP domain family translate into a distinct selectivity of the compounds we developed. Our top-ranked compounds are interacting with both homologous LEDGF and HRP-2 PWWP domains, yet they showed no affinity for the NSD2 PWWP1 and BRPF2 PWWP domains which belong to other PWWP domain subfamilies. Nevertheless, our developed compound series provide a strong foundation for future drug discovery targeting the LEDGF PWWP domain as they can further be explored through combinatorial chemistry. Given that the affinity of H3K36me2/3 nucleosomes to LEDGF/p75 is driven by interactions within the pocket as well as with the DNA-binding residues, we suggest that future compound development should target the latter region as well. Beyond drug discovery, our compounds can be employed to devise tool compounds to investigate the mechanism of LEDGF/p75 in epigenetic regulation.
- MeSH
- Small Molecule Libraries chemistry pharmacology chemical synthesis MeSH
- Crystallography, X-Ray MeSH
- Humans MeSH
- Intercellular Signaling Peptides and Proteins metabolism chemistry MeSH
- Models, Molecular MeSH
- Molecular Structure MeSH
- Protein Domains MeSH
- Drug Design * MeSH
- Dose-Response Relationship, Drug MeSH
- Structure-Activity Relationship MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Aloisie (Aloysia Paláu) jsou aromatické keře či polokeře zčeledi sporýšovitých (Verbenaceae), pocházející z Jižní, Střední a Severní Ameriky a sekundárně rozšířené v mnoha oblastech světa. K nejznámějším a nejvyužívanějším druhům patří aloisie citronová, též trojlistá (Aloysia citrodora Paláu). Aloisie se hojně využívají v tradiční medicíně k léčbě zažívacích potíží, průjmu, zvracení, revmatismu, astmatu, bronchitidy, kardiovaskulárních onemocnění, epilepsie, infekcí, kožních onemocnění, chřipky, horečky, malárie, ale také k léčbě psychických poruch, jako jsou úzkost, deprese nebo nespavost.
loisies (Aloysia Paláu) are aromatic shrubs or semi-shrubs of the Verbenaceae family, native to South, Central and North America and widely distributed in many parts of the world. Well-known is especially lemon verbena (Aloysia citrodora Paláu). Aloisies are widely used in traditional medicine to treat indigestion, diarrhea, vomiting, rheumatism, asthma, bronchitis, cardiovascular diseases, epilepsy, infections, skin diseases, flu, fever, malaria, but also to treat mental disorders, such as anxiety, depression or insomnia.
- Keywords
- aloisie citronová, verbaskosid,
- MeSH
- Phytotherapy methods MeSH
- Humans MeSH
- Nervous System drug effects MeSH
- Randomized Controlled Trials as Topic MeSH
- Medicine, Traditional * MeSH
- Verbenaceae * chemistry classification MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
BACKGROUND: Specific toxic compounds, such as polycyclic aromatic hydrocarbons (PAHs) and their metabolites, may affect the inhalation injury (INHI) grade, patients' status, and prognosis for recovery. This pilot prospective study aimed to: i) evaluate the suitability of bronchoalveolar lavage (BAL) for determination of PAHs in the LRT and of urine for determination of hydroxylated metabolites (OH-PAHs) in patients with INHI, ii) describe the dynamic changes in the levels of these toxic compounds, and iii) correlate these findings with clinical variables of the patients with INHI. METHODS: The BAL and urine samples from 10 patients with INHI were obtained on Days 1, 3, 5, 7, and 14 of hospitalization, if possible, and PAHs (BAL) and OH-PAHs (urine) were analyzed using chromatographic methods (GC-MS and HPLC). RESULTS: Concentrations of analyzed PAHs were in most cases and time points below the limit of quantification in BAL samples. Nine OH-PAHs were detected in the urine samples; however, their concentrations sharply decreased within the first three days of the hospitalization. On Day 14, the total amount of OH-PAHs in urine was higher in surviving patients with High-grade INHI (≥3) than in those with Low-grade INHI (<3, p = 0.032). Finally, a significant correlation between certain OH-PAHs and clinical variables (AST/ALT, TBSA, ABSI) from Day 1 of the hospitalization was observed (p<0.05). CONCLUSIONS: BAL samples are not suitable for the analysis of PAHs. However, the OH-PAHs levels in urine can be measured reliably and were correlated with several clinical variables. Moreover, High-grade INHI was associated with higher total concentrations of OH-PAHs in urine.
- MeSH
- Bronchoalveolar Lavage Fluid * chemistry MeSH
- Adult MeSH
- Hospitalization * MeSH
- Middle Aged MeSH
- Humans MeSH
- Pilot Projects MeSH
- Polycyclic Aromatic Hydrocarbons * urine analysis MeSH
- Prospective Studies MeSH
- Aged MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Environmental compartments polluted with animal charcoal from the skin and hide cottage industries are rich in toxic heavy metals and diverse hydrocarbon classes, some of which are carcinogenic, mutagenic, and genotoxic, and thus require a bio-based eco-benign decommission strategies. A shotgun metagenomic approach was used to decipher the microbiome, hydrocarbon degradation genes, and heavy metal resistome of a microbial consortium (FN8) from an animal-charcoal polluted site enriched with fluorene. Structurally, the FN8 microbial consortium consists of 26 phyla, 53 classes, 119 orders, 245 families, 620 genera, and 1021 species. The dominant phylum, class, order, family, genus, and species in the consortium are Proteobacteria (51.37%), Gammaproteobacteria (39.01%), Bacillales (18.09%), Microbulbiferaceae (11.65%), Microbulbifer (12.21%), and Microbulbifer sp. A4B17 (19.65%), respectively. The microbial consortium degraded 57.56% (28.78 mg/L) and 87.14% (43.57 mg/L) of the initial fluorene concentration in 14 and 21 days. Functional annotation of the protein sequences (ORFs) of the FN8 metagenome using the KEGG GhostKOALA, KofamKOALA, NCBI's conserved domain database, and BacMet revealed the detection of hydrocarbon degradation genes for benzoate, aminobenzoate, polycyclic aromatic hydrocarbons (PAHs), chlorocyclohexane/chlorobenzene, chloroalkane/chloroalkene, toluene, xylene, styrene, naphthalene, nitrotoluene, and several others. The annotation also revealed putative genes for the transport, uptake, efflux, and regulation of heavy metals such as arsenic, cadmium, chromium, mercury, nickel, copper, zinc, and several others. Findings from this study have established that members of the FN8 consortium are well-adapted and imbued with requisite gene sets and could be a potential bioresource for on-site depuration of animal charcoal polluted sites.
- MeSH
- Biodegradation, Environmental MeSH
- Charcoal MeSH
- Fluorenes MeSH
- Gammaproteobacteria * MeSH
- Soil Pollutants * analysis MeSH
- Humans MeSH
- Microbiota * genetics MeSH
- Polycyclic Aromatic Hydrocarbons * metabolism MeSH
- Soil MeSH
- Soil Microbiology MeSH
- Metals, Heavy * MeSH
- Hydrocarbons MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
This study investigates the potential relationship between exposure to polycyclic aromatic hydrocarbons (PAHs), specifically monohydroxylated metabolites (OH-PAHs), in urine, and the prevalence of respiratory diseases in 2-year-old children residing in two locations within the Czech Republic - České Budějovice (control location) and the historically contaminated mining district of Most. Despite current air quality and lifestyle similarities between the two cities, our research aims to uncover potential long-term health effects, building upon previous data indicating distinctive patterns in the Most population. A total of 248 urine samples were analysed for the presence of 11 OH-PAHs. Employing liquid-liquid extraction with ethyl acetate and clean-up through dispersive solid-phase extraction, instrumental analysis was conducted using ultra-high performance liquid chromatography coupled with tandem mass spectrometry. The incidence of respiratory diseases was assessed through questionnaires administered by paediatricians. The concentrations of OH-PAHs were elevated in urine samples from 2-year-olds in Most compared to those from České Budějovice. The incidence of respiratory diseases showed statistically significant higher levels of OH-PAHs in children from Most, together with a higher incidence of influenza. This association underlines the impact of environmental PAH exposure on children's respiratory health. It suggests that elevated urinary OH-PAH levels indicate an increased risk of developing respiratory diseases in the affected population. Further studies are needed to clarify the possible long-term health effects and to contribute to sound public health strategies.
- MeSH
- Humans MeSH
- Respiratory Tract Diseases epidemiology urine MeSH
- Pilot Projects MeSH
- Polycyclic Aromatic Hydrocarbons * urine MeSH
- Child, Preschool MeSH
- Environmental Exposure adverse effects analysis MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Child, Preschool MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czech Republic MeSH
