Antibiotic resistance can evolve through sequential accumulation of multiple mutations1. To study such gradual evolution, we developed a selection device, the morbidostat, which continuously monitors bacterial growth and dynamically regulates drug concentrations such that the evolving population is constantly challenged. We analyzed evolutionary trajectories of Escherichia coli populations towards resistance to chloramphenicol, doxycycline, and trimethoprim. Over a period of ~20 days, resistance levels increased dramatically, with parallel populations showing similar phenotypic trajectories. Whole-genome sequencing revealed both drug-specific and drug-general genetic changes. Chloramphenicol and doxycycline resistance evolved through diverse combinations of mutations in genes involved in translation, transcription, and transport2. In contrast, trimethoprim resistance evolved in a stepwise manner1,3, through mutations restricted to the target enzyme dihydrofolate reductase (DHFR)4,5. Sequencing DHFR over time revealed that parallel populations not only evolved similar mutations, but also acquired them in similar order6. Uncovering such recurrent genotypic pathways may help the spread of resistance.
Acquired abdominal aortic aneurysms are usually associated with a mural thrombus through which blood continues to flow. Some early data suggest that aneurysmal evolution correlates with the biological activity of the thrombus. Our hypothesis was therefore that the thrombus could adsorb blood components and store, release, and participate in the activation of proteases involved in aneurysmal evolution. For this purpose, we have explored both the metalloproteinase and fibrinolytic systems in the thrombus and the wall of human aneurysms. We have first investigated blood clot formation and lysis in vitro. Spontaneous clotting induces a release of promatrix metalloproteinase (pro-MMP)-9 into the serum that was fourfold higher than in paired control plasma (P < 0.001). Fibrinolysis progressively released more MMP-9 in a time-dependent manner (P < 0.01). After selective isolation, we demonstrated that polymorphonuclear leukocytes are the main source of MMP-9 release during clot formation. Protease content was then analyzed in 35 mural thrombi and walls of human abdominal aortic aneurysms sampled during surgical repair. In 15 aneurysms, the liquid phase at the interface between the thrombus and the wall was sampled separately. Both thrombus and wall contained MMP-2 and MMP-9 but the ratio MMP-9/MMP-2 was higher in the thrombus than in the wall. The liquid interface also contained active MMP-9. Immunohistochemistry of the thrombus confirmed these findings, showing the presence of polymorphonuclear leukocytes at the luminal pole of the thrombus, co-localizing with MMP-9 storage. In contrast, MMP-3 and MMP-7 were only present in the aneurysmal wall. Plasminogen was present in the mural thrombus but plasmin activity was present in both thrombus and wall. In the liquid interface, plasmin-alpha(2)-anti-plasmin complexes were detected demonstrating in vivo the activation of plasminogen. In contrast, u-PA and t-PA were detectable only in the wall, suggesting that plasminogen present in the thrombus could be activated by factors secreted by the arterial wall. This was demonstrated in vitro, in which co-incubation of thrombus and wall extracts generated plasmin in the presence of a fibrin matrix and activated MMPs. In conclusion, our study strongly suggests that the mural thrombus, by trapping polymorphonuclear leukocytes and adsorbing plasma components could act as a source of proteases in aneurysms that may play a critical role in enlargement and rupture.
A predisposition for thoracic aortic aneurysms leading to acute aortic dissections can be inherited in families in an autosomal dominant manner. Genome-wide linkage analysis of two large unrelated families with thoracic aortic disease, followed by whole exome sequencing of affected relatives, identified causative mutations in TGFB2. These mutations, a frameshift mutation in exon 6 and a nonsense mutation in exon 4, segregated with disease with a combined LOD score of 7.7. Sanger sequencing of 276 probands from families with inherited thoracic aortic disease identified two additional TGFB2 mutations. TGFB2 encodes the transforming growth factor beta-2 (TGF-β2) and the mutations are predicted to cause haploinsufficiency for TGFB2, but aortic tissue from cases paradoxically shows increased TGF-β2 expression and immunostaining. Thus, haploinsufficiency of TGFB2 predisposes to thoracic aortic disease, suggesting the initial pathway driving disease is decreased cellular TGF-β2 levels leading to a secondary increase in TGF-β2 production in the diseased aorta.
A colony of 26 chimpanzees given a fruit and vegetable diet of very low Na and high K intake were maintained in long-standing, socially stable small groups for three years. Half of them had salt added progressively to their diet during 20 months. This addition of salt within the human dietetic range caused a highly significant rise in systolic, mean and diastolic blood pressure. The change reversed completely by six months after cessation of salt. The effect of salt differed between chimpanzees, some having a large blood pressure rise and others small or no rise. These results in the species phylogenetically closest to humans bear directly on causation of human hypertension, particularly in relation to migration of preliterate people, with low Na diet, to a Western urban lifestyle with increased salt intake. The hedonic liking for salt and avid ingestion was apt during human prehistory involving hunter-gatherer-scavenger existence in the interior of continents with a scarcity of salt, but is maladaptive in urban technological life with salt cheap and freely available.
Hemodynamic overload of the atria is an important pathogenic factor of fibrosis; MMP-7 appears to be involved in the early stage of this tissue remodeling process.
Vascular smooth muscle cells (vSMCs) play a crucial role in both the pathogenesis of Aneurysms and Dissections of the ascending thoracic aorta (TAAD) in humans and in the associated adaptive compensatory responses, since thrombosis and inflammatory processes are absent in the majority of cases. Aneurysms and dissections share numerous characteristics, including aetiologies and histopathological alterations: vSMC disappearance, medial areas of mucoid degeneration, and extracellular matrix (ECM) breakdown. Three aetiologies predominate in TAAD in humans: (i) genetic causes in heritable familial forms, (ii) an association with bicuspid aortic valves, and (iii) a sporadic degenerative form linked to the aortic aging process. Genetic forms include mutations in vSMC genes encoding for molecules of the ECM or the TGF-β pathways, or participating in vSMC tone. On the other hand, aneurysms and dissections, whatever their aetiologies, are characterized by an increase in wall permeability leading to transmural advection of plasma proteins which could interact with vSMCs and ECM components. In this context, blood-borne plasminogen appears to play an important role, because its outward convection through the wall is increased in TAAD, and it could be converted to active plasmin at the vSMC membrane. Active plasmin can induce vSMC disappearance, proteolysis of adhesive proteins, activation of MMPs and release of TGF-β from its ECM storage sites. Conversely, vSMCs could respond to aneurysmal biomechanical and proteolytic injury by an epigenetic phenotypic switch, including constitutional overexpression and nuclear translocation of Smad2 and an increase in antiprotease and ECM protein synthesis. In contrast, such an epigenetic phenomenon is not observed in dissections. In this context, dysfunction of proteins involved in vSMC tone are interesting to study, particularly in interaction with plasma protein transport through the wall and TGF-β activation, to establish the relationship between these dysfunctions and ECM proteolysis.
Development and progression of acquired abdominal aortic aneurysms (AAAs) involve proteolytic activity. In the present study, we investigate the distribution of fibrinolytic system components within mural thrombi of human AAAs. 20 mural thrombi and the remaining AAA walls were dissected. The luminal, intermediate and abluminal thrombus layers, and media and adventitia were separately incubated in cell culture medium. Conditioned media were then analysed for plasminogen activators (PAs), plasminogen activator inhibitor-1 (PAI-1), free-plasmin, plasmin alpha(2)-antiplasmin complexes (PAPs) and D-dimers release. In parallel, PA and PAI-1 mRNA expression analysis was performed by RT-PCR. The study was completed by immunohistochemical localization of these components in AAA, ex vivo functional imaging using (99m)Tc-aprotinin as a ligand and measurement of PAP and D-dimer plasma levels. All fibrinolytic system components were present in each aneurysmal layer. However, the mural thrombus was the main source of active serine-protease release. Interestingly, the luminal layer of the thrombus released greater amounts of PAPs and D-dimers. This paralleled the preferential immunolocalization of plasminogen and PAs, and the (99m)Tc-aprotinin scintigraphic signal observed in the luminal pole of the thrombus. In contrast, mRNA expression analysis showed an exclusive synthesis of tPA and PAI-1 within the wall, whereas uPA mRNA was also expressed within the thrombus. Taken together, these results suggest that the increased plasma concentrations of PAPs and D-dimers found in AAA patients are related to mural thrombus proteolytic activity, thus explaining their known link with AAA progression. Components of the fibrinolytic system could also represent a target for functional imaging of thrombus activities in AAA.
Atherosclerosis is a chronic disease that affects medium and large arteries. This process originates from the interaction between cells of the arterial wall, lipoproteins and inflammatory cells, leading to the development of complex lesions or plaques that protrude into the arterial lumen. Plaque rupture and thrombosis result in acute clinical complications such as myocardial infarction and stroke. Owing to the heterogeneous cellular composition of the plaques, a proteomic analysis of the whole lesion is not appropriate. Therefore, we have studied the proteins secreted by human carotid atherosclerotic plaques, obtained by endarterectomy. Normal artery segments and different regions of the surgical pieces (noncomplicated plaque, complicated plaque with thrombus) were cultured in protein-free medium and the secreted proteins (supernatants) analyzed by two-dimensional gel electrophoresis. Normal artery segments secreted a moderate number of proteins (42 spots). However in the two-dimensional (2-D) gels (pH 3-10) of segments bearing a plaque, the number of spots increased markedly (154). The number of spots also increased (202) in the 2-D gels of artery segments with a ruptured plaque and thrombus. Thus, the more complicated the lesion, the higher the number of secreted proteins, suggesting the production of specific proteins relating to the complexity of the atherosclerotic lesion.
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