BACKGROUND: Innovations in computer hardware and software capabilities have paved the way for advances in molecular modelling techniques and methods, leading to an unprecedented expansion of their potential applications. In contrast to the docking technique, which usually identifies the most stable selector-selectand (SO-SA) complex for each enantiomer, the molecular dynamics (MD) technique enables the consideration of a distribution of the SO-SA complexes based on their energy profile. This approach provides a more truthful representation of the processes occurring within the column. However, benchmark procedures and focused guidelines for computational treatment of enantioselectivity at the molecular level are still missing. RESULTS: Twenty-eight molecular dynamics simulations were performed to study the enantiorecognition mechanisms of seven N-3,5-dinitrobenzoylated α- and β-amino acids (DNB-AAs), occurring with the two quinine- and quinidine-based (QN-AX and QD-AX) chiral stationary phases (CSPs), under polar-ionic conditions. The MD protocol was optimized in terms of box size, simulation run time, and frame recording frequency. Subsequently, all the trajectories were analyzed by calculating both the type and amount of the interactions engaged by the selectands (SAs) with the two chiral selectors (SOs), as well as the conformational and interaction energy profiles of the formed SA-SO associates. All the MDs were in strict agreement with the experimental enantiomeric elution order and allowed to establish (i) that salt-bridge and H-bond interactions play a pivotal role in the enantiorecognition mechanisms, and (ii) that the π-cation and π-π interactions are the discriminant chemical features between the two SOs in ruling the chiral recognition mechanism. SIGNIFICANCE: The results of this work clearly demonstrate the high contribution given by MD simulations in the comprehension of the enantiorecognition mechanism with Cinchona alkaloid-based CSPs. However, from this research endeavor it clearly emerged that the MD protocol optimization is crucial for the quality of the produced results.
In the constant search for new pharmacological compounds, molecular hybridisation is a well-known technique whereby two or more known pharmacophoric subunits are combined to create a new "hybrid" compound. This hybrid is expected to maintain the characteristics of the original compounds whilst demonstrating improvements to their pharmacological action. Accordingly, we report here a series of molecular hybrid compounds based upon eugenol and chloramphenicol pharmacophores. The hybrid compounds were screened for their in vitro antimicrobial potential against Gram-negative and Gram-positive bacteria and also rapidly growing mycobacteria (RGM). The results highlight that the antimicrobial profiles of the hybrid compounds improve in a very clear fashion when moving through the series. The most prominent results were found when comparing the activity of the hybrid compounds against some of the multidrug-resistant clinical isolates of Pseudomonas aeruginosa, methicillin-resistant clinical isolates of Staphylococcus aureus (MRSA) and clinical isolates of rapidly growing mycobacteria.
- MeSH
- Anti-Bacterial Agents therapeutic use MeSH
- Anti-Infective Agents * pharmacology MeSH
- Chloramphenicol pharmacology MeSH
- Eugenol pharmacology MeSH
- Pharmacophore MeSH
- Methicillin-Resistant Staphylococcus aureus * MeSH
- Microbial Sensitivity Tests MeSH
- Staphylococcus aureus MeSH
- Publication type
- Journal Article MeSH
Live biotherapeutic products constitute an emerging therapeutic approach to prevent or treat inflammatory bowel diseases. Lactobacillus acidophilus is a constituent of the human microbiota with probiotic potential, that is illustrated by improvement of intestinal inflammation and antimicrobial activity against several pathogens. In this study, we evaluated the immunomodulatory properties of the L. acidophilus strain BIO5768 at steady state and upon acute inflammation. Supplementation of naïve mice with BIO5768 heightened the transcript level of some IL-17 target genes encoding for protein with microbicidal activity independently of NOD2 signaling. Of these, the BIO5768-induced expression of Angiogenin-4 was blunted in monocolonized mice that are deficient for the receptor of IL-17 (but not for NOD2). Interestingly, priming of bone marrow derived dendritic cells by BIO5768 enhanced their ability to support the secretion of IL-17 by CD4+ T cells. Equally of importance, the production of IL-22 by type 3 innate lymphoid cells is concomitantly heightened in response to BIO5768. When administered alone or in combination with Bifidobacterium animalis spp. lactis BIO5764 and Limosilactobacillus reuteri, BIO5768 was able to alleviate at least partially intestinal inflammation induced by Citrobacter rodentium infection. Furthermore, BIO5768 was also able to improve colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). In conclusion, we identify a new potential probiotic strain for the management of inflammatory bowel diseases, and provide some insights into its IL-17-dependent and independent mode of action.
- MeSH
- Bifidobacterium animalis MeSH
- Enterobacteriaceae Infections therapy MeSH
- Inflammatory Bowel Diseases * therapy MeSH
- Interleukin-17 MeSH
- Colitis * chemically induced therapy microbiology MeSH
- Trinitrobenzenesulfonic Acid adverse effects MeSH
- Lactobacillus acidophilus * MeSH
- Lymphocytes MeSH
- Mice MeSH
- Immunity, Innate * MeSH
- Probiotics * pharmacology therapeutic use MeSH
- Inflammation MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
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x
- MeSH
- Anti-Bacterial Agents classification MeSH
- Antimicrobial Stewardship methods MeSH
- Anemia, Aplastic etiology prevention & control MeSH
- Chloramphenicol * administration & dosage adverse effects therapeutic use toxicity MeSH
- Drug Evaluation methods statistics & numerical data MeSH
- Humans MeSH
- Surveys and Questionnaires MeSH
- Check Tag
- Humans MeSH
- Publication type
- Review MeSH
- Geographicals
- Czech Republic MeSH
The aim and novelty of this paper are found in assessing the influence of inhibitors and antibiotics on intact cell MALDI-TOF mass spectra of the cyanobacterium Synechococcus sp. UPOC S4 and to check the impact on reliability of identification. Defining the limits of this method is important for its use in biology and applied science. The compounds included inhibitors of respiration, glycolysis, citrate cycle, and proteosynthesis. They were used at 1-10 μM concentrations and different periods of up to 3 weeks. Cells were also grown without inhibitors in a microgravity because of expected strong effects. Mass spectra were evaluated using controls and interpreted in terms of differential peaks and their assignment to protein sequences by mass. Antibiotics, azide, and bromopyruvate had the greatest impact. The spectral patterns were markedly altered after a prolonged incubation at higher concentrations, which precluded identification in the database of reference spectra. The incubation in microgravity showed a similar effect. These differences were evident in dendrograms constructed from the spectral data. Enzyme inhibitors affected the spectra to a smaller extent. This study shows that only a long-term presence of antibiotics and strong metabolic inhibitors in the medium at 10-5 M concentrations hinders the correct identification of cyanobacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF).
- MeSH
- Anti-Bacterial Agents toxicity MeSH
- Antimycin A analogs & derivatives toxicity MeSH
- Azides toxicity MeSH
- Cell Respiration drug effects MeSH
- Chloramphenicol toxicity MeSH
- Citric Acid Cycle drug effects MeSH
- Deoxyglucose toxicity MeSH
- Fluoroacetates toxicity MeSH
- Glycolysis drug effects MeSH
- Malonates toxicity MeSH
- Protein Biosynthesis drug effects MeSH
- Pyruvates toxicity MeSH
- Reproducibility of Results MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods MeSH
- Weightlessness MeSH
- Streptomycin toxicity MeSH
- Synechococcus chemistry drug effects isolation & purification metabolism MeSH
- Publication type
- Journal Article MeSH
Alterations in the gut microbiota composition and diversity seem to play a role in the development of chronic diseases, including inflammatory bowel disease (IBD), leading to gut barrier disruption and induction of proinflammatory immune responses. This opens the door for the use of novel health-promoting bacteria. We selected five Parabacteroides distasonis strains isolated from human adult and neonates gut microbiota. We evaluated in vitro their immunomodulation capacities and their ability to reinforce the gut barrier and characterized in vivo their protective effects in an acute murine model of colitis. The in vitro beneficial activities were highly strain dependent: two strains exhibited a potent anti-inflammatory potential and restored the gut barrier while a third strain reinstated the epithelial barrier. While their survival to in vitro gastric conditions was variable, the levels of P. distasonis DNA were higher in the stools of bacteria-treated animals. The strains that were positively scored in vitro displayed a strong ability to rescue mice from colitis. We further showed that two strains primed dendritic cells to induce regulatory T lymphocytes from naïve CD4+ T cells. This study provides better insights on the functionality of commensal bacteria and crucial clues to design live biotherapeutics able to target inflammatory chronic diseases such as IBD.
- MeSH
- Bacteroidetes genetics immunology isolation & purification MeSH
- Caco-2 Cells MeSH
- DNA, Bacterial genetics metabolism MeSH
- Adult MeSH
- Feces microbiology MeSH
- Inflammatory Bowel Diseases immunology microbiology MeSH
- Colitis chemically induced immunology microbiology MeSH
- Real-Time Polymerase Chain Reaction MeSH
- Trinitrobenzenesulfonic Acid adverse effects MeSH
- Humans MeSH
- Disease Models, Animal MeSH
- Mice, Inbred BALB C MeSH
- Mice MeSH
- Infant, Newborn MeSH
- T-Lymphocytes, Regulatory immunology MeSH
- Gastrointestinal Microbiome immunology MeSH
- Intestinal Mucosa immunology MeSH
- Animals MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Mice MeSH
- Infant, Newborn MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Due to the inappropriate use of florfenicol in agricultural practice, florfenicol resistance has become increasingly serious. In this work, we studied the novel florfenicol resistance mechanism of an animal-derived Leclercia adecarboxylata strain R25 with high-level florfenicol resistance. A random genomic DNA library was constructed to screen the novel florfenicol resistance gene. Gene cloning, gene knockout, and complementation combined with the minimum inhibitory concentration (MIC) detection were conducted to determine the function of the resistance-related gene. Sequencing and bioinformatics methods were applied to analyze the structure of the resistance gene-related sequences. Finally, we obtained a regulatory gene of an RND (resistance-nodulation-cell division) system, ramA, that confers resistance to florfenicol and other antibiotics. The ramA-deleted variant (LA-R25ΔramA) decreased the level of resistance against florfenicol and several other antibiotics, while a ramA-complemented strain (pUCP24-prom-ramA/LA-R25ΔramA) restored the drug resistance. The whole-genome sequencing revealed that there were five RND efflux pump genes (mdtABC, acrAB, acrD, acrEF, and acrAB-like) encoded over the chromosome, and ramA located upstream of the acrAB-like genes. The results of this work suggest that ramA confers resistance to florfenicol and other structurally unrelated antibiotics, presumably by regulating the RND efflux pump genes in L. adecarboxylata R25.
- MeSH
- Anti-Bacterial Agents pharmacology MeSH
- Bacterial Proteins genetics metabolism MeSH
- Enterobacteriaceae drug effects genetics metabolism MeSH
- Escherichia coli genetics MeSH
- Gene Knockout Techniques MeSH
- Cloning, Molecular MeSH
- Microbial Sensitivity Tests MeSH
- Drug Resistance, Multiple, Bacterial drug effects genetics MeSH
- RNA, Ribosomal, 16S MeSH
- Whole Genome Sequencing MeSH
- Thiamphenicol analogs & derivatives MeSH
- Trans-Activators genetics MeSH
- Computational Biology MeSH
- Publication type
- Journal Article MeSH
Crohn's disease is linked to a decreased diversity in gut microbiota composition as a potential consequence of an impaired anti-microbial response and an altered polarization of T helper cells. Here, we evaluated the immunomodulatory properties of two potential probiotic strains, namely a Bifidobacterium animalis spp. lactis Bl 5764 and a Lactobacillus reuteri Lr 5454 strains. Both strains improved colitis triggered by either 2,4,6-trinitrobenzenesulfonic acid (TNBS) or Citrobacter rodentium infection in mice. Training of dendritic cells (DC) with Lr 5454 efficiently triggered IL-22 secretion and regulatory T cells induction in vitro, while IL-17A production by CD4+ T lymphocytes was stronger when cultured with DCs that were primed with Bl 5764. This strain was sufficient for significantly inducing expression of antimicrobial peptides in vivo through the Crohn's disease predisposing gene encoding for the nucleotide-binding oligomerization domain, containing protein 2 (NOD2). In contrast, NOD2 was dispensable for the impact on antimicrobial peptide expression in mice that were monocolonized with Lr 5454. In conclusion, our work highlights a differential mode of action of two potential probiotic strains that protect mice against colitis, providing the rational for a personalized supportive preventive therapy by probiotics for individuals that are genetically predisposed to Crohn's disease.
- MeSH
- Anti-Inflammatory Agents, Non-Steroidal pharmacology MeSH
- Bifidobacterium animalis * MeSH
- Citrobacter rodentium pathogenicity MeSH
- Dendritic Cells physiology MeSH
- Enterobacteriaceae Infections microbiology MeSH
- Germ-Free Life MeSH
- Colitis chemically induced microbiology pathology therapy MeSH
- Trinitrobenzenesulfonic Acid toxicity MeSH
- Limosilactobacillus reuteri * MeSH
- Disease Models, Animal MeSH
- Mice, Inbred BALB C MeSH
- Mice, Inbred C57BL MeSH
- Mice, Knockout MeSH
- Probiotics pharmacology MeSH
- Pancreatitis-Associated Proteins genetics MeSH
- T-Lymphocytes, Regulatory physiology MeSH
- Gastrointestinal Microbiome MeSH
- T-Lymphocytes, Helper-Inducer physiology MeSH
- Animals MeSH
- Check Tag
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Modern sugarcane is an unusually complex heteroploid crop, and its genome comprises two or three subgenomes. To reduce the complexity of sugarcane genome research, the ploidy level and number of chromosomes can be reduced using flow chromosome sorting. However, a cell cycle synchronization (CCS) protocol for Saccharum spp. is needed that maximizes the accumulation of metaphase chromosomes. For flow cytometry analysis in this study, we optimized the lysis buffer, hydroxyurea(HU) concentration, HU treatment time and recovery time for sugarcane. We determined the mitotic index by microscopic observation and calculation. We found that WPB buffer was superior to other buffers for preparation of sugarcane nuclei suspensions. The optimal HU treatment was 2 mM for 18 h at 25 °C, 28 °C and 30 °C. Higher recovery treatment temperatures were associated with shorter recovery times (3.5 h, 2.5 h and 1.5 h at 25 °C, 28 °C and 30 °C, respectively). The optimal conditions for treatment with the inhibitor of microtubule polymerization, amiprophos-methyl (APM), were 2.5 μM for 3 h at 25 °C, 28 °C and 30 °C. Meanwhile, preliminary screening of CCS protocols for Badila were used for some main species of genus Saccharum at 25 °C, 28 °C and 30 °C, which showed that the average mitotic index decreased from 25 °C to 30 °C. The optimal sugarcane CCS protocol that yielded a mitotic index of >50% in sugarcane root tips was: 2 mM HU for 18 h, 0.1 X Hoagland's Solution without HU for 3.5 h, and 2.5 μM APM for 3.0 h at 25 °C. The CCS protocol defined in this study should accelerate the development of genomic research and cytobiology research in sugarcane.
- MeSH
- Cell Cycle physiology MeSH
- Time Factors MeSH
- Chromosomes, Plant * metabolism MeSH
- Genome, Plant genetics MeSH
- Genomics methods MeSH
- Hydroxyurea MeSH
- Metaphase MeSH
- Mitotic Index MeSH
- Nitrobenzenes MeSH
- Organothiophosphorus Compounds MeSH
- Flow Cytometry methods MeSH
- Buffers MeSH
- Saccharum cytology genetics MeSH
- Temperature MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
We present the solid-phase synthesis of 1,2-dihydroquinazoline-2-carboxylate derivatives with a quaternary carbon in position 2 and their subsequent cyclization in solution into compounds with unique 3D architectures and pharmacological relevance-spiroquinazolines, namely, 1' H-spiro[pyrrolidine-3,2'-quinazolin]-2-ones and 1' H-spiro[piperidine-3,2'-quinazolin]-2-ones. Acyclic precursors were prepared from commercially available building blocks: protected amino acids (2,4-diaminobutyric acid and ornithine), 2-nitrobenzensulfonyl chlorides and α-bromoacetophenones. The crucial step of the synthesis was a base-mediated tandem reaction including C-arylation followed by cyclization into indazole oxides, and the formation of a 5-membered heterocycle was accomplished by ring expansion into quinazolines. These derivatives were cyclized into spiro compounds in solution after cleavage from the resin.
- MeSH
- Acetophenones chemistry MeSH
- Aminobutyrates chemistry MeSH
- Quinazolines chemical synthesis MeSH
- Cyclization MeSH
- Nitrobenzenes chemistry MeSH
- Ornithine chemistry MeSH
- Piperidines chemical synthesis MeSH
- Pyrrolidines chemical synthesis MeSH
- Spiro Compounds chemical synthesis MeSH
- Solid-Phase Synthesis Techniques methods MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
