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Bone ; 127: 635-645, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31390534


Roux's principle of bone functional adaptation postulates that bone tissue, and particularly trabecular bone tissue, responds to mechanical stimuli by adjusting (modeling) its architecture accordingly. Hence, it predicts that the new modeled trabecular structure is mechanically improved (stiffer and stronger) in line with the habitual in vivo loading direction. While previous studies found indirect evidence to support this theory, direct support was so far unattainable. This is attributed to the fact that each trabecular bone is unique, and that trabecular bone tissue tends to be damaged during mechanical testing. Consequently, a unique modeled trabecular structure can be mechanically tested only along one direction and a comparison to other directions for that specific structure is impossible. To address this issue, we have 3D printed 10 replicas of a trabecular structure from a sheep talus cropped along the 3 principal axes of the bone and in line with the principal direction of loading (denoted on-axis model). Next, we have rotated the same cropped trabecular structure in increments of 10° up to 90° to the bone principal direction of loading (denoted off-axis models) and printed 10 replicas of each off-axis model. Finally, all on-axis and off-axis 3D printed replicas were loaded in compression until failure and trabecular structure stiffness and strength were calculated. Contrary to our prediction, and conflicting with Roux's principle of bone functional adaptation, we found that a trabecular structure loaded off-axis tended to have higher stiffness and strength values when compared to the same trabecular structure loaded on-axis. These unexpected results may not disprove Roux's principle of bone functional adaptation, but they do imply that trabecular bone adaptation may serve additional purposes than simply optimizing bone structure to one principal loading scenario and this suggests that we still don't fully understand bone modeling in its entirety.

mBio ; 10(3)2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31239374


HBsAg and HBeAg have gained traction as biomarkers of control and clearance during chronic hepatitis B virus infection (CHB). Improved understanding of the clearance correlates of these proteins could help inform improvements in patient-stratified care and advance insights into the underlying mechanisms of disease control, thus underpinning new cure strategies. We collected electronic clinical data via an electronic pipeline supported by the National Institute for Health Research Health Informatics Collaborative (NIHR HIC), adopting an unbiased approach to the generation of a robust longitudinal data set for adults testing HBsAg positive from a large UK teaching hospital over a 6-year period (2011 to 2016 inclusive). Of 553 individuals with CHB, longitudinal data were available for 319, representing >107,000 weeks of clinical follow-up. Among these 319 individuals, 13 (4%) cleared HBsAg completely. Among these 13, the HBsAg clearance rate in individuals on nucleos(t)ide analogue (NA) therapy (n = 4 [31%]; median clearance time,150 weeks) was similar to that in individuals not on NA therapy (n = 9 [69%]; median clearance time, 157 weeks). Those who cleared HBsAg were significantly older and less likely to be on NA therapy than nonclearers (P = 0.003 and P = 0.001, respectively). Chinese ethnicity was associated with HBeAg positivity (P = 0.025). HBeAg clearance occurred in individuals both on NA therapy (n = 24; median time, 49 weeks) and off NA therapy (n = 19; median time, 52 weeks). Improved insights into the dynamics of these biomarkers can underpin better prognostication and patient-stratified care. Our systematized approach to data collection paves the way for scaling up efforts to harness clinical data to address research questions and support improvements in clinical care.IMPORTANCE Advances in the diagnosis, monitoring, and treatment of hepatitis B virus (HBV) infection are urgently required if we are to meet international targets for elimination by the year 2030. Here we demonstrate how routine clinical data can be harnessed through an unbiased electronic pipeline, showcasing the significant potential for amassing large clinical data sets that can help to inform advances in patient care and provide insights that may help to inform new cure strategies. Our cohort from a large UK hospital includes adults from diverse ethnic groups that have previously been underrepresented in the literature. By tracking two protein biomarkers that are used to monitor chronic HBV infection, we provide new insights into the timelines of HBV clearance, both on and off treatment. These results contribute to improvements in individualized clinical care and may provide important clues into the immune events that underpin disease control.

Antígenos de Superfície da Hepatite B/sangue , Antígenos E da Hepatite B/sangue , Hepatite B Crônica/sangue , Adolescente , Adulto , Idoso , Antivirais/uso terapêutico , Biomarcadores/sangue , Criança , Estudos de Coortes , Feminino , Vírus da Hepatite B , Hepatite B Crônica/tratamento farmacológico , Hospitais/estatística & dados numéricos , Humanos , Estudos Longitudinais , Masculino , Informática Médica , Pessoa de Meia-Idade , Reino Unido , Adulto Jovem
J Biol Chem ; 293(46): 17971-17984, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30275013


The CTX-M ß-lactamases have emerged as the most widespread extended-spectrum ß-lactamases (ESBLs) in Gram-negative bacteria. These enzymes rapidly hydrolyze cefotaxime, but not the related cephalosporin, ceftazidime. ESBL variants have evolved, however, that provide enhanced ceftazidime resistance. We show here that a natural variant at a nonactive site, i.e. second-shell residue N106S, enhances enzyme stability but reduces catalytic efficiency for cefotaxime and ceftazidime and decreases resistance levels. However, when the N106S variant was combined with an active-site variant, D240G, that enhances enzyme catalytic efficiency, but decreases stability, the resultant double mutant exhibited higher resistance levels than predicted on the basis of the phenotypes of each variant. We found that this epistasis is due to compensatory effects, whereby increased stability provided by N106S overrides its cost of decreased catalytic activity. X-ray structures of the variant enzymes in complex with cefotaxime revealed conformational changes in the active-site loop spanning residues 103-106 that were caused by the N106S substitution and relieve steric strain to stabilize the enzyme, but also alter contacts with cefotaxime and thereby reduce catalytic activity. We noted that the 103-106 loop conformation in the N106S-containing variants is different from that of WT CTX-M but nearly identical to that of the non-ESBL, TEM-1 ß-lactamase, having a serine at the 106 position. Therefore, residue 106 may serve as a "switch" that toggles the conformations of the 103-106 loop. When it is serine, the loop is in the non-ESBL, TEM-like conformation, and when it is asparagine, the loop is in a CTX-M-like, cefotaximase-favorable conformation.

Resistência às Cefalosporinas/genética , beta-Lactamases/genética , Substituição de Aminoácidos , Cefotaxima/química , Ceftazidima/química , Cristalografia por Raios X , Estabilidade Enzimática , Epistasia Genética , Escherichia coli/genética , Hidrólise , Cinética , Testes de Sensibilidade Microbiana , Mutagênese Sítio-Dirigida , Conformação Proteica , beta-Lactamases/química
Biochemistry ; 56(27): 3443-3453, 2017 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-28613873


CTX-M ß-lactamases provide resistance against the ß-lactam antibiotic, cefotaxime, but not a related antibiotic, ceftazidime. ß-Lactamases that carry the P167S substitution, however, provide ceftazidime resistance. In this study, CTX-M-14 was used as a model to study the structural changes caused by the P167S mutation that accelerate ceftazidime turnover. X-ray crystallography was used to determine the structures of the P167S apoenzyme along with the structures of the S70G/P167S, E166A/P167S, and E166A mutant enzymes complexed with ceftazidime as well as the E166A/P167S apoenzyme. The S70G and E166A mutations allow capture of the enzyme-substrate complex and the acylated form of ceftazidime, respectively. The results showed a large conformational change in the Ω-loop of the ceftazidime acyl-enzyme complex of the P167S mutant but not in the enzyme-substrate complex, suggesting the change occurs upon acylation. The change results in a larger active site that prevents steric clash between the aminothiazole ring of ceftazidime and the Asn170 residue in the Ω-loop, allowing accommodation of ceftazidime for hydrolysis. In addition, the conformational change was not observed in the E166A/P167S apoenzyme, suggesting the presence of acylated ceftazidime influences the conformational change. Finally, the E166A acyl-enzyme structure with ceftazidime did not exhibit the altered conformation, indicating the P167S substitution is required for the change. Taken together, the results reveal that the P167S substitution and the presence of acylated ceftazidime both drive the structure toward a conformational change in the Ω-loop and that in CTX-M P167S enzymes found in drug-resistant bacteria this will lead to an increased level of ceftazidime hydrolysis.

Antibacterianos/metabolismo , Ceftazidima/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Modelos Moleculares , beta-Lactamases/metabolismo , Acilação , Substituição de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacologia , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Domínio Catalítico , Ceftazidima/química , Ceftazidima/farmacologia , Cefalosporinas/química , Cefalosporinas/metabolismo , Cefalosporinas/farmacologia , Cristalografia por Raios X , Farmacorresistência Bacteriana Múltipla , Estabilidade Enzimática , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Hidrólise , Ligantes , Mutagênese Sítio-Dirigida , Oximas/química , Oximas/metabolismo , Oximas/farmacologia , Mutação Puntual , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , beta-Lactamases/química , beta-Lactamases/genética
Antimicrob Agents Chemother ; 59(11): 6741-8, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26282414


The widespread use of oxyimino-cephalosporin antibiotics drives the evolution of the CTX-M family of ß-lactamases that hydrolyze these drugs and confer antibiotic resistance. Clinically isolated CTX-M enzymes carrying the P167S or D240G active site-associated adaptive mutation have a broadened substrate profile that includes the oxyimino-cephalosporin antibiotic ceftazidime. The D240G substitution is known to reduce the stability of CTX-M-14 ß-lactamase, and the P167S substitution is shown here to also destabilize the enzyme. Proteins are marginally stable entities, and second-site mutations that stabilize the enzyme can offset a loss in stability caused by mutations that enhance enzyme activity. Therefore, the evolution of antibiotic resistance enzymes can be dependent on the acquisition of stabilizing mutations. The A77V substitution is present in CTX-M extended-spectrum ß-lactamases (ESBLs) from a number of clinical isolates, suggesting that it may be important in the evolution of antibiotic resistance in this family of ß-lactamases. In this study, the effects of the A77V substitution in the CTX-M-14 model enzyme were characterized with regard to the kinetic parameters for antibiotic hydrolysis as well as enzyme expression levels in vivo and protein stability in vitro. The A77V substitution has little effect on the kinetics of oxyimino-cephalosporin hydrolysis, but it stabilizes the CTX-M enzyme and compensates for the loss of stability resulting from the P167S and D240G mutations. The acquisition of global stabilizing mutations, such as A77V, is an important feature in ß-lactamase evolution and a common mechanism in protein evolution.

beta-Lactamases/genética , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Ceftazidima/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Evolução Molecular , Mutação
mBio ; 4(4)2013 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-23882012


UNLABELLED: Bacterial DNA is maintained in a supercoiled state controlled by the action of topoisomerases. Alterations in supercoiling affect fundamental cellular processes, including transcription. Here, we show that substitution at position 87 of GyrA of Salmonella influences sensitivity to antibiotics, including nonquinolone drugs, alters global supercoiling, and results in an altered transcriptome with increased expression of stress response pathways. Decreased susceptibility to multiple antibiotics seen with a GyrA Asp87Gly mutant was not a result of increased efflux activity or reduced reactive-oxygen production. These data show that a frequently observed and clinically relevant substitution within GyrA results in altered expression of numerous genes, including those important in bacterial survival of stress, suggesting that GyrA mutants may have a selective advantage under specific conditions. Our findings help contextualize the high rate of quinolone resistance in pathogenic strains of bacteria and may partly explain why such mutant strains are evolutionarily successful. IMPORTANCE: Fluoroquinolones are a powerful group of antibiotics that target bacterial enzymes involved in helping bacteria maintain the conformation of their chromosome. Mutations in the target enzymes allow bacteria to become resistant to these antibiotics, and fluoroquinolone resistance is common. We show here that these mutations also provide protection against a broad range of other antimicrobials by triggering a defensive stress response in the cell. This work suggests that fluoroquinolone resistance mutations may be beneficial under a range of conditions.

DNA Girase/genética , DNA Girase/metabolismo , DNA Super-Helicoidal/metabolismo , Farmacorresistência Bacteriana Múltipla , Salmonella typhimurium/enzimologia , Transcriptoma , Substituição de Aminoácidos , Antibacterianos/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Seleção Genética , Estresse Fisiológico