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Antibiotics cause collateral damage to resident microbes that is associated with various health risks. To date, studies have largely focused on the impacts of antibiotics on large intestinal and fecal microbiota. Here, we employ a gastrointestinal (GI) tract-wide integrated multiomic approach to show that amoxicillin (AMX) treatment reduces bacterial abundance, bile salt hydrolase activity, and unconjugated bile acids in the small intestine (SI). Losses of fatty acids (FAs) and increases in acylcarnitines in the large intestine (LI) correspond with spatially distinct expansions of Proteobacteria. Parasutterella excrementihominis engage in FA biosynthesis in the SI, while multiple Klebsiella species employ FA oxidation during expansion in the LI. We subsequently demonstrate that restoration of unconjugated bile acids can mitigate losses of commensals in the LI while also inhibiting the expansion of Proteobacteria during AMX treatment. These results suggest that the depletion of bile acids and lipids may contribute to AMX-induced dysbiosis in the lower GI tract.
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Mass spectrometry (MS)-based thermal stability assays have recently emerged as one of the most promising solutions for the identification of protein-ligand interactions. Here, we have investigated eight combinations of several recently introduced MS-based advancements, including the Phase-Constrained Spectral Deconvolution Method, Field Asymmetric Ion Mobility Spectrometry, and the implementation of a carrier sample as improved MS-based acquisition approaches for thermal stability assays (iMAATSA). We used intact Jurkat cells treated with a commercially available MEK inhibitor, followed by heat treatment, to prepare a set of unfractionated isobarically-labeled proof-of-concept samples to compare the performance of eight different iMAATSAs. Finally, the best-performing iMAATSA was compared to a conventional approach and evaluated in a fractionation experiment. Improvements of up to 82% and 86% were demonstrated in protein identifications and high-quality melting curves, respectively, over the conventional approach in the proof-of-concept study, while an approximately 12% improvement in melting curve comparisons was achieved in the fractionation experiment.
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The microbiome is essential for host health, and perturbations resulting from antibiotic use can lead to dysbiosis and disease. Diet can be a powerful modulator of microbiome composition and function, with the potential to mitigate the negative effects of antibiotic use. Thus, it is necessary to study the impacts of diet and drug interactions on the gut microbiome. Coffee is a commonly consumed beverage containing many compounds that have the potential to affect the microbiome, including caffeine, polyphenols, and fiber. We supplemented mice with caffeinated and decaffeinated coffee in conjunction with amoxicillin, and used 16S rRNA amplicon sequencing of fecal samples to investigate changes in diversity and composition of the murine fecal microbiome. We found that antibiotics, regardless of coffee supplementation, caused significant disruption to the murine fecal microbiome, enriching for Proteobacteria, Verrucomicrobia, and Bacteroidetes, but reducing Firmicutes. While we found that coffee alone did not have a significant impact on the composition of the fecal microbiome, coffee supplementation did significantly affect relative abundance metrics in mice treated with amoxicillin. After caffeinated coffee supplementation, mice treated with amoxicillin showed a smaller increase in Proteobacteria, specifically of the family Burkholderiaceae. Correspondingly we found that in vitro, Burkholderia cepacia was highly resistant to amoxicillin, and that it was inhibited by concentrations of caffeine and caffeinated coffee comparable to levels of caffeine in murine ceca. Overall, this work shows that coffee, and possibly the caffeine component, can impact both the microbiome and microbiome members during antibiotic exposure.
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Owing to the markedly increased reactivity of amino functional groups versus hydroxyls, the 5'-amino-5'-deoxy nucleoside and nucleotide analogs have proven widely useful in biological, pharmaceutical and genomic applications. However, synthetic procedures leading to these analogs have not been fully explored, which may possibly have limited the scope of their utility. Here we describe the synthesis of the 5'-amino-2',5'-dideoxy analogs of adenosine, cytidine, guanosine, inosine and uridine from their respective naturally occurring nucleosides via the reduction of 5'-azido-2',5'-dideoxy intermediates using the Staudinger reaction, and the high yield conversion of these modified nucleosides and 5'-amino-5'-deoxythymidine to the corresponding 5'-N-triphosphates through reaction with trisodium trimetaphosphate in the presence of tris(hydroxymethyl)aminomethane (Tris). We also show that each of these nucleotide analogs can be efficiently incorporated into DNA by the Klenow fragment of Escherichia coli DNA polymerase I when individually substituted for its naturally occurring counterpart. Mild acid treatment of the resulting DNA generates polynucleotide fragments that arise from specific cleavage at each modified nucleotide, providing a sequence ladder for each base. Because the ladders are generated after the extension, the corresponding products may be manipulated by enzymatic and/or purification processes. The potential utility of this extension-cleavage procedure in genomic sequence analysis is discussed.
Assuntos
DNA Polimerase I/metabolismo , Didesoxinucleosídeos/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Citidina/análogos & derivados , Citidina/metabolismo , DNA/química , DNA/genética , DNA/metabolismo , Didesoxinucleosídeos/síntese química , Guanosina/análogos & derivados , Guanosina/metabolismo , Inosina/análogos & derivados , Inosina/metabolismo , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Análise de Sequência de DNA , Timidina/análogos & derivados , Timidina/metabolismo , Uridina/análogos & derivados , Uridina/metabolismoRESUMO
This paper demonstrates development of electrophoretically mediated micro analysis (EMMA), for screening protein tyrosine phosphatase (PTP) inhibitors in natural extracts. It is demonstrated that capillary electrophoresis (CE) separation of the substrate and the product allows for using the assay in an on-column format to monitor the reaction without typically used fluorogenic substrates. Michaelis-Menten kinetics parameters calculated based on the EMMA results (Km = 1.2-1.5 microM) were in a good agreement (Km = 1.0-1.5 microM) obtained using an off-line CE functional assay (CE FA). EMMA of PTP titrated with different concentrations of ligand demonstrated the peak-shift phenomenon normally seen in affinity capillary electrophoresis. This feature of EMMA gives an indication of the binding affinity of the ligand in addition to its functional activity, providing another dimension in characterization of the protein-inhibitor interaction. It was demonstrated that simultaneous screening of the primary PTP target and a secondary, counter target (PTP-C) using the EMMA format can be used to prioritize hits based on their specificity.
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Fatores Biológicos , Eletroforese Capilar/métodos , Inibidores Enzimáticos/análiseRESUMO
Polycystic kidney disease (PKD) represents a family of genetic disorders characterized by renal cystic growth and progression to kidney failure. No treatment is currently available for people with PKD, although possible therapeutic interventions are emerging. Despite genetic and clinical heterogeneity, PKDs have in common defects of cystic epithelia, including increased proliferation, apoptosis and activation of growth regulatory pathways. Sphingolipids and glycosphingolipids are emerging as major regulators of these cellular processes. We sought to evaluate the therapeutic potential for glycosphingolipid modulation as a new approach to treat PKD. Here we demonstrate that kidney glucosylceramide (GlcCer) and ganglioside GM3 levels are higher in human and mouse PKD tissue as compared to normal tissue, regardless of the causative mutation. Blockade of GlcCer accumulation with the GlcCer synthase inhibitor Genz-123346 effectively inhibits cystogenesis in mouse models orthologous to human autosomal dominant PKD (Pkd1 conditional knockout mice) and nephronophthisis (jck and pcy mice). Molecular analysis in vitro and in vivo indicates that Genz-123346 acts through inhibition of the two key pathways dysregulated in PKD: Akt protein kinase-mammalian target of rapamycin signaling and cell cycle machinery. Taken together, our data suggest that inhibition of GlcCer synthesis represents a new and effective treatment option for PKD.