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1.
J Proteome Res ; 22(9): 2890-2899, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-37584946

RESUMO

Phosphoproteomics is nowadays the method of choice to comprehensively identify and quantify thousands of phosphorylated peptides and their associated proteins with the goal of interrogating changes in signal transduction pathways and other cellular processes. One of the most popular software suites to analyze phosphoproteomic data sets is MaxQuant, which converts mass spectrometric raw data into quantitative information on phosphopeptides and proteins. However, despite the increased utilization of phosphoproteomics in biomedical research, simple and user-friendly tools supporting downstream statistical analysis and interpretation of these highly complex outputs are still lacking. We have therefore developed Phospho-Analyst, which─similar to its sibling LFQ-Analyst─is an easy-to-use, interactive web application specifically designed to reproducibly perform differential expression analyses with "one click" and to visualize phosphoproteomic results in a meaningful and practical manner. Furthermore, if quantitative total proteomic information is available for the same samples, Phospho-Analyst automatically normalizes all phosphoproteomic results to underlying protein abundance levels, thereby ensuring that only genuine changes in phosphorylation events are considered. As such, Phospho-Analyst can not only be used by experienced proteomic veterans but also by researchers without any prior knowledge in (phospho)proteomics, statistics, or bioinformatics. Phospho-Analyst, including a detailed manual, is freely available at https://analyst-suites.org/apps/phospho-analyst/.


Assuntos
Proteínas , Proteômica , Proteômica/métodos , Software , Espectrometria de Massas/métodos , Fosforilação
2.
RNA ; 24(5): 704-720, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29440476

RESUMO

Pasteurella multocida is a Gram-negative bacterium responsible for many important animal diseases. While a number of P. multocida virulence factors have been identified, very little is known about how gene expression and protein production is regulated in this organism. Small RNA (sRNA) molecules are critical regulators that act by binding to specific mRNA targets, often in association with the RNA chaperone protein Hfq. In this study, transcriptomic analysis of the P. multocida strain VP161 revealed a putative sRNA with high identity to GcvB from Escherichia coli and Salmonella enterica serovar Typhimurium. High-throughput quantitative liquid proteomics was used to compare the proteomes of the P. multocida VP161 wild-type strain, a gcvB mutant, and a GcvB overexpression strain. These analyses identified 46 proteins that displayed significant differential production after inactivation of gcvB, 36 of which showed increased production. Of the 36 proteins that were repressed by GcvB, 27 were predicted to be involved in amino acid biosynthesis or transport. Bioinformatic analyses of putative P. multocida GcvB target mRNAs identified a strongly conserved 10 nucleotide consensus sequence, 5'-AACACAACAT-3', with the central eight nucleotides identical to the seed binding region present within GcvB mRNA targets in E. coli and S. Typhimurium. Using a defined set of seed region mutants, together with a two-plasmid reporter system that allowed for quantification of sRNA-mRNA interactions, this sequence was confirmed to be critical for the binding of the P. multocida GcvB to the target mRNA, gltA.


Assuntos
Pasteurella multocida/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/metabolismo , Aminoácidos/biossíntese , Proteínas de Bactérias/genética , Sítios de Ligação , Escherichia coli/genética , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Fator Proteico 1 do Hospedeiro/metabolismo , Motivos de Nucleotídeos , Pasteurella multocida/metabolismo , Transporte Proteico/genética , RNA Bacteriano/química , RNA Mensageiro/química , Pequeno RNA não Traduzido/química , Regulon
3.
Int J Antimicrob Agents ; 62(3): 106887, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37315906

RESUMO

OBJECTIVE: Acute exacerbations of biofilm-associated Pseudomonas aeruginosa infections in cystic fibrosis (CF) have limited treatment options. Ceftolozane/tazobactam (alone and with a second antibiotic) has not yet been investigated against hypermutable clinical P. aeruginosa isolates in biofilm growth. This study aimed to evaluate, using an in vitro dynamic biofilm model, ceftolozane/tazobactam alone and in combination with tobramycin at simulated representative lung fluid pharmacokinetics against free-floating (planktonic) and biofilm states of two hypermutable P. aeruginosa epidemic strains (LES-1 and CC274) from adolescents with CF. METHODS: Regimens were intravenous ceftolozane/tazobactam 4.5 g/day continuous infusion, inhaled tobramycin 300 mg 12-hourly, intravenous tobramycin 10 mg/kg 24-hourly, and both ceftolozane/tazobactam-tobramycin combinations. The isolates were susceptible to both antibiotics. Total and less-susceptible free-floating and biofilm bacteria were quantified over 120-168 h. Ceftolozane/tazobactam resistance mechanisms were investigated by whole-genome sequencing. Mechanism-based modelling of bacterial viable counts was performed. RESULTS: Monotherapies of ceftolozane/tazobactam and tobramycin did not sufficiently suppress emergence of less-susceptible subpopulations, although inhaled tobramycin was more effective than intravenous tobramycin. Ceftolozane/tazobactam resistance development was associated with classical (AmpC overexpression plus structural modification) and novel (CpxR mutations) mechanisms depending on the strain. Against both isolates, combination regimens demonstrated synergy and completely suppressed the emergence of ceftolozane/tazobactam and tobramycin less-susceptible free-floating and biofilm bacterial subpopulations. CONCLUSION: Mechanism-based modelling incorporating subpopulation and mechanistic synergy well described the antibacterial effects of all regimens against free-floating and biofilm bacterial states. These findings support further investigation of ceftolozane/tazobactam in combination with tobramycin against biofilm-associated P. aeruginosa infections in adolescents with CF.


Assuntos
Infecções por Pseudomonas , Tobramicina , Humanos , Adolescente , Tobramicina/farmacologia , Tobramicina/uso terapêutico , Pseudomonas aeruginosa , Cefalosporinas/uso terapêutico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Tazobactam/uso terapêutico , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Biofilmes , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana Múltipla
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