RESUMO
It was found that Argentatins A and B triterpenoids make up approximately 20-30 % of the waste resin produced from the industrial processes to isolate rubber from P. argentatum. We have developed an efficient protocol for synthesizing cycloartane-16ß-ol derivatives by opening the oxepane ring of argentatin B acetate (2) with BF3-OEt2. Although three new cycloartenol derivatives showed high cytotoxicity against PC-3 and HCT-15 cancer cell lines, nevertheless, the best results were obtained for (16ß,24R) -(16,24-epoxy-cycloartan-2(1H)-ylidene) acetate (14), compound with intact oxepane ring. These results indicate that the substituents in the argentatin nucleus and a side chain account for the cytotoxic activity. However, according to the selectivity index (SI), 14 did not show selectivity activity to cancer cell lines over the HaCat noncancerous cell line. The compound 3ß,16ß-Dihydroxy-cycloartan-24-one (5), synthesized by oxepane opening, demonstrated high cytotoxic activity to cancer cell lines and showed a remarkable selectivity to cancer cell lines over the noncancerous ones. These results suggest that 5 could lead to the development of new anticancer compounds.
Assuntos
Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Relação Estrutura-Atividade , Proliferação de Células/efeitos dos fármacos , Triterpenos/farmacologia , Triterpenos/química , Triterpenos/síntese química , Tetrazóis/farmacologia , Tetrazóis/síntese química , Tetrazóis/química , Estrutura Molecular , Relação Dose-Resposta a Droga , Sobrevivência Celular/efeitos dos fármacosRESUMO
BACKGROUND: Left ventricular assist devices (LVADs) are implantable pumps that act as a life support therapy for patients with severe heart failure. Despite improving the survival rate, LVAD therapy can carry major complications. Particularly, the flow distortion introduced by the LVAD in the left ventricle (LV) may induce thrombus formation. While previous works have used numerical models to study the impact of multiple variables in the intra-LV stagnation regions, a comprehensive validation analysis has never been executed. The main goal of this work is to present a model of the LV-LVAD system and to design and follow a verification, validation and uncertainty quantification (VVUQ) plan based on the ASME V&V40 and V&V20 standards to ensure credible predictions. METHODS: The experiment used to validate the simulation is the SDSU cardiac simulator, a bench mock-up of the cardiovascular system that allows mimicking multiple operation conditions for the heart-LVAD system. The numerical model is based on Alya, the BSC's in-house platform for numerical modelling. Alya solves the Navier-Stokes equation with an Arbitrary Lagrangian-Eulerian (ALE) formulation in a deformable ventricle and includes pressure-driven valves, a 0D Windkessel model for the arterial output and a LVAD boundary condition modeled through a dynamic pressure-flow performance curve. The designed VVUQ plan involves: (a) a risk analysis and the associated credibility goals; (b) a verification stage to ensure correctness in the numerical solution procedure; (c) a sensitivity analysis to quantify the impact of the inputs on the four quantities of interest (QoIs) (average aortic root flow [Formula: see text], maximum aortic root flow [Formula: see text], average LVAD flow [Formula: see text], and maximum LVAD flow [Formula: see text]); (d) an uncertainty quantification using six validation experiments that include extreme operating conditions. RESULTS: Numerical code verification tests ensured correctness of the solution procedure and numerical calculation verification showed a grid convergence index (GCI)95% <3.3%. The total Sobol indices obtained during the sensitivity analysis demonstrated that the ejection fraction, the heart rate, and the pump performance curve coefficients are the most impactful inputs for the analysed QoIs. The Minkowski norm is used as validation metric for the uncertainty quantification. It shows that the midpoint cases have more accurate results when compared to the extreme cases. The total computational cost of the simulations was above 100 [core-years] executed in around three weeks time span in Marenostrum IV supercomputer. CONCLUSIONS: This work details a novel numerical model for the LV-LVAD system, that is supported by the design and execution of a VVUQ plan created following recognised international standards. We present a methodology demonstrating that stringent VVUQ according to ASME standards is feasible but computationally expensive.
Assuntos
Insuficiência Cardíaca , Coração Auxiliar , Simulação por Computador , Insuficiência Cardíaca/cirurgia , Ventrículos do Coração , Coração Auxiliar/efeitos adversos , Hemodinâmica , Humanos , IncertezaRESUMO
The cytotoxic activity of combinations of masticadienonic (AMD) or 3αOH-hydroxy-masticadienonic (3αOH-AMD) acids with cisplatin (CDDP) was evaluated against PC3 prostate and HCT116 colon cancer cell lines. Combinations A (half the IC50 value), B (IC50 value), and C (twice the IC50 value) were tested at a 1 : 1 ratio. All AMD plus CDDP combinations demonstrated increased cytotoxic effect, as determined by the sulforhodamine B test, in both cell types. The best combination was B, which showed 93 % and 91 % inhibition of the proliferation of PC3 and HCT116 cells, respectively. It also increased apoptosis in the PC3â cell lines, as evaluated by flow cytometry. However, inâ vivo tests showed no additional activity from the AMD plus CDDP combinations. These results showed that the increased cytotoxic activity of the combinations inâ vitro did not reflect inâ vivo tests. All combinations of 3αOH-AMD plus CDDP exerted antagonistic effects in both cell types.
RESUMO
Understanding early amyloidogenesis is key to rationally develop therapeutic strategies. Tau protein forms well-characterized pathological deposits but its aggregation mechanism is still poorly understood. Using single-molecule force spectroscopy based on a mechanical protection strategy, we studied the conformational landscape of the monomeric tau repeat domain (tau-RD244-368 ). We found two sets of conformational states, whose frequency is influenced by mutations and the chemical context. While pathological mutations Δ280K and P301L and a pro-amyloidogenic milieu favored expanded conformations and destabilized local structures, an anti-amyloidogenic environment promoted a compact ensemble, including a conformer whose topology might mask two amyloidogenic segments. Our results reveal that to initiate aggregation, monomeric tau-RD244-368 decreases its polymorphism adopting expanded conformations. This could account for the distinct structures found in vitro and across tauopathies.
Assuntos
Tauopatias , Proteínas tau , Humanos , Proteínas tau/metabolismo , Tauopatias/genética , Tauopatias/metabolismo , Tauopatias/patologia , Conformação Molecular , MutaçãoRESUMO
BACKGROUND: The life course accumulation of overt and subclinical myocardial dysfunction contributes to older age mortality, frailty, disability and loss of independence. The Medical Research Council National Survey of Health and Development (NSHD) is the world's longest running continued surveillance birth cohort providing a unique opportunity to understand life course determinants of myocardial dysfunction as part of MyoFit46-the cardiac sub-study of the NSHD. METHODS: We aim to recruit 550 NSHD participants of approximately 75 years+ to undertake high-density surface electrocardiographic imaging (ECGI) and stress perfusion cardiovascular magnetic resonance (CMR). Through comprehensive myocardial tissue characterization and 4-dimensional flow we hope to better understand the burden of clinical and subclinical cardiovascular disease. Supercomputers will be used to combine the multi-scale ECGI and CMR datasets per participant. Rarely available, prospectively collected whole-of-life data on exposures, traditional risk factors and multimorbidity will be studied to identify risk trajectories, critical change periods, mediators and cumulative impacts on the myocardium. DISCUSSION: By combining well curated, prospectively acquired longitudinal data of the NSHD with novel CMR-ECGI data and sharing these results and associated pipelines with the CMR community, MyoFit46 seeks to transform our understanding of how early, mid and later-life risk factor trajectories interact to determine the state of cardiovascular health in older age. TRIAL REGISTRATION: Prospectively registered on ClinicalTrials.gov with trial ID: 19/LO/1774 Multimorbidity Life-Course Approach to Myocardial Health- A Cardiac Sub-Study of the MCRC National Survey of Health and Development (NSHD).
Assuntos
Doenças Cardiovasculares , Imageamento por Ressonância Magnética , Idoso , Doenças Cardiovasculares/diagnóstico por imagem , Doenças Cardiovasculares/epidemiologia , Inquéritos Epidemiológicos , Coração , Humanos , MiocárdioRESUMO
BACKGROUND: Amyloids are ordered, insoluble protein aggregates, characterized by a cross-ß sheet quaternary structure in which molecules in a ß-strand conformation are stacked along the filament axis via intermolecular interactions. While amyloids are typically associated with pathological conditions, functional amyloids have also been identified and are present in a wide variety of organisms ranging from bacteria to humans. The cytoplasmic polyadenylation element-binding (CPEB) prion-like protein is an mRNA-binding translation regulator, whose neuronal isoforms undergo activity-dependent aggregation, a process that has emerged as a plausible biochemical substrate for memory maintenance. CPEB aggregation is driven by prion-like domains (PLD) that are divergent in sequence across species, and it remains unknown whether such divergent PLDs follow a similar aggregating assembly pathway. Here, we describe the amyloid-like features of the neuronal Aplysia CPEB (ApCPEB) PLD and compare them to those of the Drosophila ortholog, Orb2 PLD. RESULTS: Using in vitro single-molecule and bulk biophysical methods, we find transient oligomers and mature amyloid-like filaments that suggest similarities in the late stages of the assembly pathway for both ApCPEB and Orb2 PLDs. However, while prior to aggregation the Orb2 PLD monomer remains mainly as a random coil in solution, ApCPEB PLD adopts a diversity of conformations comprising α-helical structures that evolve to coiled-coil species, indicating structural differences at the beginning of their amyloid assembly pathways. CONCLUSION: Our results indicate that divergent PLDs of CPEB proteins from different species retain the ability to form a generic amyloid-like fold through different assembly mechanisms.
Assuntos
Amiloide/metabolismo , Aplysia/metabolismo , Príons/metabolismo , Animais , Aplysia/química , Poliadenilação , Príons/químicaRESUMO
Dendritic cells are antigen-presenting cells, which identify and process pathogens to subsequently activate specific T lymphocytes. To regulate the immune responses, DCs have to mature by the recognition of TLR ligands, TNFα or IFNγ. These ligands have been used as adjuvants to activate DCs in situ or in vitro, with toxic effects. It has been shown that some molecules affect the immune system, e.g., Masticadienonic acid (MDA) and 3α-hydroxy masticadienoic acid (3α-OH MDA) triterpenes naturally occurring in several medicinal plants, since they activate the nitric oxide synthase in macrophages and induce T lymphocyte proliferation. The DCs maturation induced by MDA or 3a-OH MDA was determined by incubating these cells with MDA or 3α-OH MDA, and their phenotype was afterwards analyzed. The results showed that only 3α-OH MDA was able to induce DCs maturation. When mice with melanoma were inoculated with DCs/3α-OH MDA, a decreased tumor growth rate was observed along with an extended cell death area within tumors compared to mice treated with DCs incubated with MDA. In conclusion, it is proposed that 3α-OH MDA may be an immunostimulant molecule. Conversely, it is proposed that MDA may be a molecule with anti-inflammatory properties.
Assuntos
Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Imunomodulação/efeitos dos fármacos , Triterpenos/química , Triterpenos/farmacologia , Animais , Biomarcadores , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Imunofenotipagem , Camundongos , Estrutura Molecular , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
AIMS: Develop, calibrate and evaluate with clinical data a human electromechanical modelling and simulation framework for multiscale, mechanistic investigations in healthy and post-myocardial infarction (MI) conditions, from ionic to clinical biomarkers. METHODS AND RESULTS: Human healthy and post-MI electromechanical simulations were conducted with a novel biventricular model, calibrated and evaluated with experimental and clinical data, including torso/biventricular anatomy from clinical magnetic resonance, state-of-the-art human-based membrane kinetics, excitation-contraction and active tension models, and orthotropic electromechanical coupling. Electromechanical remodelling of the infarct/ischaemic region and the border zone were simulated for ischaemic, acute, and chronic states in a fully transmural anterior infarct and a subendocardial anterior infarct. The results were compared with clinical electrocardiogram and left ventricular ejection fraction (LVEF) data at similar states. Healthy model simulations show LVEF 63%, with 11% peak systolic wall thickening, QRS duration and QT interval of 100 ms and 330 ms. LVEF in ischaemic, acute, and chronic post-MI states were 56%, 51%, and 52%, respectively. In linking the three post-MI simulations, it was apparent that elevated resting potential due to hyperkalaemia in the infarcted region led to ST-segment elevation, while a large repolarization gradient corresponded to T-wave inversion. Mechanically, the chronic stiffening of the infarct region had the benefit of improving systolic function by reducing infarct bulging at the expense of reducing diastolic function by inhibiting inflation. CONCLUSION: Our human-based multiscale modelling and simulation framework enables mechanistic investigations into patho-physiological electrophysiological and mechanical behaviour and can serve as testbed to guide the optimization of pharmacological and electrical therapies.
Assuntos
Infarto do Miocárdio , Função Ventricular Esquerda , Eletrocardiografia , Humanos , Infarto do Miocárdio/complicações , Volume Sistólico , SístoleRESUMO
Antimicrobial resistance is one of the current public health challenges to be solved. The World Health Organization (WHO) has urgently called for the development of strategies to expand the increasingly limited antimicrobial arsenal. The development of anti-virulence therapies is a viable option to counteract bacterial infections with the possibility of reducing the generation of resistance. Here we report on the chemical structures of pyrrolidones DEXT 1-4 (previously identified as furan derivatives) and their anti-virulence activity on Pseudomonas aeruginosa strains. DEXT 1-4 were shown to inhibit biofilm formation, swarming motility, and secretion of ExoU and ExoT effector proteins. Also, the anti-pathogenic property of DEXT-3 alone or in combination with furanone C-30 (quorum sensing inhibitor) or MBX-1641 (type III secretion system inhibitor) was analyzed in a model of necrosis induced by P. aeruginosa PA14. All treatments reduced necrosis; however, only the combination of C-30 50 µM with DEXT-3 100 µM showed significant inhibition of bacterial growth in the inoculation area and systemic dispersion. In conclusion, pyrrolidones DEXT 1-4 are chemical structures capable of reducing the pathogenicity of P. aeruginosa and with the potential for the development of anti-virulence combination therapies.
Assuntos
Antibacterianos , Furanos , Hidrocarbonetos Halogenados , Infecções por Pseudomonas , Pseudomonas aeruginosa , Pirrolidinonas , Sistemas de Secreção Tipo III/antagonistas & inibidores , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Furanos/química , Furanos/farmacologia , Humanos , Hidrocarbonetos Halogenados/química , Hidrocarbonetos Halogenados/farmacologia , Camundongos , Necrose , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidade , Pirrolidinonas/química , Pirrolidinonas/farmacologia , Percepção de Quorum/efeitos dos fármacos , Sistemas de Secreção Tipo III/metabolismo , Fatores de Virulência/metabolismoRESUMO
Animal-borne video recordings from blue whales in the open ocean show that remoras preferentially adhere to specific regions on the surface of the whale. Using empirical and computational fluid dynamics analyses, we show that remora attachment was specific to regions of separating flow and wakes caused by surface features on the whale. Adhesion at these locations offers remoras drag reduction of up to 71-84% compared with the freestream. Remoras were observed to move freely along the surface of the whale using skimming and sliding behaviors. Skimming provided drag reduction as high as 50-72% at some locations for some remora sizes, but little to none was available in regions where few to no remoras were observed. Experimental work suggests that the Venturi effect may help remoras stay near the whale while skimming. Understanding the flow environment around a swimming blue whale will inform the placement of biosensor tags to increase attachment time for extended ecological monitoring.
Assuntos
Balaenoptera , Perciformes , Animais , Peixes , Hidrodinâmica , NataçãoRESUMO
Parthenium argentatum (Gray), commonly known as guayule, has been used to obtain natural rubber since the beginning of the 20th century. Additionally, the so called "resin" is a waste product derived from the industrial process. The cycloartane-type triterpene Argentatin A (AA) is one of the main constituents of the industrial waste resin. In this study we evaluated the AA anticancer activity both in vitro and in vivo in the HCT116 colon cancer cells. The apoptosis promotion of AA was assessed by the annexin V/propidium iodide (PI) assay. The senescence was evaluated for SA-ß-galactosidase, and PCNA was used as a marker of proliferation. Its antitumor activity was evaluated using a xenograft mouse model. The results indicated that AA-induced apoptosis in HCT-116 cells and was positively stained for SA-ß-galactosidase. In the xenografted mice test, the administration of AA at the dose of 250 mg/kg three times a week for 21 days reduced tumor growth by 78.1%. A comparable tumor reduction was achieved with cisplatin at the dose of 2 mg/kg administered three times a week for 21 days. However, nude mice treated with AA did not lose weight, as they did remarkably when treated with cisplatin. Furthermore, the animals treated with AA showed similar blood profiles as the healthy control group. These data indicate the low toxicity of AA compared to that shown by cisplatin.
Assuntos
Antineoplásicos/administração & dosagem , Triterpenos/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Imuno-Histoquímica , Camundongos , Estrutura Molecular , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , beta-Galactosidase/metabolismoRESUMO
Cellulosomes are bacterial protein complexes that bind and efficiently degrade lignocellulosic substrates. These are formed by multimodular scaffolding proteins known as scaffoldins, which comprise cohesin modules capable of binding dockerin-bearing enzymes and usually a carbohydrate-binding module that anchors the system to a substrate. It has been suggested that cellulosomes bound to the bacterial cell surface might be exposed to significant mechanical forces. Accordingly, the mechanical properties of these anchored cellulosomes may be important to understand and improve cellulosome function. Here we used single-molecule force spectroscopy to study the mechanical properties of selected cohesin modules from scaffoldins of different cellulosomes. We found that cohesins located in the region connecting the cell and the substrate are more robust than those located outside these two anchoring points. This observation applies to cohesins from primary scaffoldins (i.e. those that directly bind dockerin-bearing enzymes) from different cellulosomes despite their sequence differences. Furthermore, we also found that cohesin nanomechanics (specifically, mechanostability and the position of the mechanical clamp of cohesin) are not significantly affected by other cellulosomal components, including linkers between cohesins, multiple cohesin repeats, and dockerin binding. Finally, we also found that cohesins (from both the connecting and external regions) have poor refolding efficiency but similar refolding rates, suggesting that the high mechanostability of connecting cohesins may be an evolutionarily conserved trait selected to minimize the occurrence of cohesin unfolding, which could irreversibly damage the cellulosome. We conclude that cohesin mechanostability is a major determinant of the overall mechanical stability of the cellulosome.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Celulossomas/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Membrana/metabolismo , Fenômenos Biomecânicos , Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , Clostridium thermocellum/metabolismo , Cinética , Microscopia de Força Atômica/métodos , Simulação de Dinâmica Molecular , Ligação Proteica , Redobramento de Proteína , Estabilidade Proteica , CoesinasRESUMO
Transactive Response DNA-Binding Protein of 43â¯kDa (TDP-43) is an essential human protein implicated in Amyotrophic Lateral Sclerosis (ALS) and common dementias. Its C-terminal disordered region, composed of residues 264-414 includes a hydrophobic segment (residues 320-340), which drives physiological liquid/liquid phase separation and a Q/N-rich segment (residues 341-357), which is essential for pathological amyloid formation. Due to TDP-43's relevance for pathology, identifying inhibitors and characterizing their mechanism of action are important pharmacological goals. The Polyglutamine Binding Peptide 1 (QBP1), whose minimal active core is the octapeptide WGWWPGIF, strongly inhibits the aggregation of polyQ-containing amyloidogenic proteins such as Huntingtin. Rather promiscuous, this inhibitor also blocks the aggregation of other glutamine containing amyloidogenic proteins, but not Aß, and its mechanism of action remains unknown. Using a series of spectroscopic assays and biochemical tests, we establish that QBP1 binds and inhibits amyloid formation by TDP-43's Q/N-rich region. NMR spectroscopic data evince that the aromatic rings of QBP1 accept hydrogen bonds from the HN groups of the Asn and Gln to block amyloidogenesis. This mechanism of blockage may be general to polyphenol amyloid inhibitors.
Assuntos
Amiloide/biossíntese , Proteínas de Ligação a DNA/antagonistas & inibidores , Oligopeptídeos/fisiologia , Sequência de Aminoácidos , Proteínas de Ligação a DNA/metabolismo , Fluorescência , Humanos , Oligopeptídeos/químicaRESUMO
Amyloids are ordered protein aggregates that are typically associated with neurodegenerative diseases and cognitive impairment. By contrast, the amyloid-like state of the neuronal RNA binding protein Orb2 in Drosophila was recently implicated in memory consolidation, but it remains unclear what features of this functional amyloid-like protein give rise to such diametrically opposed behaviour. Here, using an array of biophysical, cell biological and behavioural assays we have characterized the structural features of Orb2 from the monomer to the amyloid state. Surprisingly, we find that Orb2 shares many structural traits with pathological amyloids, including the intermediate toxic oligomeric species, which can be sequestered in vivo in hetero-oligomers by pathological amyloids. However, unlike pathological amyloids, Orb2 rapidly forms amyloids and its toxic intermediates are extremely transient, indicating that kinetic parameters differentiate this functional amyloid from pathological amyloids. We also observed that a well-known anti-amyloidogenic peptide interferes with long-term memory in Drosophila. These results provide structural insights into how the amyloid-like state of the Orb2 protein can stabilize memory and be nontoxic. They also provide insight into how amyloid-based diseases may affect memory processes.
Assuntos
Proteínas Amiloidogênicas/metabolismo , Proteínas de Drosophila/metabolismo , Consolidação da Memória , Fatores de Transcrição/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Animais , Células COS , Chlorocebus aethiops , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Masculino , Mutação , Oligopeptídeos , Estrutura Terciária de Proteína , Fatores de Transcrição/química , Fatores de Transcrição/genética , Leveduras , Fatores de Poliadenilação e Clivagem de mRNA/química , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
AIMS: Myocardial infarction (MI) alters cardiac fibre organization with unknown consequences on ventricular arrhythmia. We used diffusion tensor imaging (DTI) of three-dimensional (3D) cardiac fibres and scar reconstructions to identify the main parameters associated with ventricular arrhythmia inducibility and ventricular tachycardia (VT) features after MI. METHODS AND RESULTS: Twelve pigs with established MI and three controls underwent invasive electrophysiological characterization of ventricular arrhythmia inducibility and VT features. Animal-specific 3D scar and myocardial fibre distribution were obtained from ex vivo high-resolution contrast-enhanced T1 mapping and DTI sequences. Diffusion tensor imaging-derived parameters significantly different between healthy and scarring myocardium, scar volumes, and left ventricular ejection fraction (LVEF) were included for arrhythmia risk stratification and correlation analyses with VT features. Ventricular fibrillation (VF) was the only inducible arrhythmia in 4 out of 12 infarcted pigs and all controls. Ventricular tachycardia was also inducible in the remaining eight pigs during programmed ventricular stimulation. A DTI-based 3D fibre disorganization index (FDI) showed higher disorganization within dense scar regions of VF-only inducible pigs compared with VT inducible animals (FDI: 0.36; 0.36-0.37 vs. 0.32; 0.26-0.33, respectively, P = 0.0485). Ventricular fibrillation induction required lower programmed stimulation aggressiveness in VF-only inducible pigs than VT inducible and control animals. Neither LVEF nor scar volumes differentiated between VF and VT inducible animals. Re-entrant VT circuits were localized within areas of highly disorganized fibres. Moreover, the FDI within heterogeneous scar regions was associated with the median VT cycle length per animal (R2 = 0.5320). CONCLUSION: The amount of scar-related cardiac fibre disorganization in DTI sequences is a promising approach for ventricular arrhythmia stratification after MI.
Assuntos
Cicatriz , Imagem de Tensor de Difusão/métodos , Coração/fisiopatologia , Imageamento por Ressonância Magnética/métodos , Infarto do Miocárdio/complicações , Miocárdio/patologia , Taquicardia Ventricular , Animais , Cicatriz/diagnóstico por imagem , Cicatriz/patologia , Cicatriz/fisiopatologia , Medição de Risco , Suínos , Taquicardia Ventricular/diagnóstico , Taquicardia Ventricular/etiologia , Taquicardia Ventricular/fisiopatologiaRESUMO
AIMS: We aimed to study the differences in biventricular scar characterization using bipolar voltage mapping compared with state-of-the-art in vivo delayed gadolinium-enhanced cardiac magnetic resonance (LGE-CMR) imaging and ex vivo T1 mapping. METHODS AND RESULTS: Ten pigs with established myocardial infarction (MI) underwent in vivo scar characterization using LGE-CMR imaging and high-density voltage mapping of both ventricles using a 3.5-mm tip catheter. Ex vivo post-contrast T1 mapping provided a high-resolution reference. Voltage maps were registered onto the left and right ventricular (LV and RV) endocardium, and epicardium of CMR-based geometries to compare voltage-derived scars with surface-projected 3D scars. Voltage-derived scar tissue of the LV endocardium and the epicardium resembled surface projections of 3D in vivo and ex vivo CMR-derived scars using 1-mm of surface projection distance. The thinner wall of the RV was especially sensitive to lower resolution in vivo LGE-CMR images, in which differences between normalized low bipolar voltage areas and CMR-derived scar areas did not decrease below a median of 8.84% [interquartile range (IQR) (3.58, 12.70%)]. Overall, voltage-derived scars and surface scar projections from in vivo LGE-CMR sequences showed larger normalized scar areas than high-resolution ex vivo images [12.87% (4.59, 27.15%), 18.51% (11.25, 24.61%), and 9.30% (3.84, 19.59%), respectively], despite having used optimized surface projection distances. Importantly, 43.02% (36.54, 48.72%) of voltage-derived scar areas from the LV endocardium were classified as non-enhanced healthy myocardium using ex vivo CMR imaging. CONCLUSION: In vivo LGE-CMR sequences and high-density voltage mapping using a conventional linear catheter fail to provide accurate characterization of post-MI scar, limiting the specificity of voltage-based strategies and imaging-guided procedures.
Assuntos
Potenciais de Ação , Arritmias Cardíacas/diagnóstico , Cicatriz/diagnóstico por imagem , Técnicas Eletrofisiológicas Cardíacas , Sistema de Condução Cardíaco/fisiopatologia , Imageamento por Ressonância Magnética , Infarto do Miocárdio/diagnóstico por imagem , Miocárdio/patologia , Animais , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/fisiopatologia , Cicatriz/etiologia , Cicatriz/patologia , Cicatriz/fisiopatologia , Meios de Contraste/administração & dosagem , Modelos Animais de Doenças , Frequência Cardíaca , Masculino , Meglumina/administração & dosagem , Infarto do Miocárdio/complicações , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Compostos Organometálicos/administração & dosagem , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Sus scrofaRESUMO
Bacterial cellulases are drawing increased attention as a means to obtain plentiful chemical feedstocks and fuels from renewable lignocellulosic biomass sources. Certain bacteria deploy a large extracellular multi-protein complex, called the cellulosome, to degrade cellulose. Scaffoldin, a key non-catalytic cellulosome component, is a large protein containing a cellulose-specific carbohydrate-binding module and several cohesin modules which bind and organize the hydrolytic enzymes. Despite the importance of the structure and protein/protein interactions of the cohesin module in the cellulosome, its structure in solution has remained unknown to date. Here, we report the backbone 1H, 13C and 15N NMR assignments of the Cohesin module 5 from the highly stable and active cellulosome from Clostridium thermocellum. These data reveal that this module adopts a tightly packed, well folded and rigid structure in solution. Furthermore, since in scaffoldin, the cohesin modules are connected by linkers we have also characterized the conformation of a representative linker segment using NMR spectroscopy. Analysis of its chemical shift values revealed that this linker is rather stiff and tends to adopt extended conformations. This suggests that the scaffoldin linkers act to minimize interactions between cohesin modules. These results pave the way towards solution studies on cohesin/dockerin's fascinating dual-binding mode.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ciclo Celular/química , Celobiose/química , Proteínas Cromossômicas não Histona/química , Clostridium thermocellum/química , Complexos Multiproteicos/química , Proteínas de Bactérias/genética , Proteínas de Ciclo Celular/genética , Celobiose/genética , Proteínas Cromossômicas não Histona/genética , Clostridium thermocellum/genética , Complexos Multiproteicos/genética , Ressonância Magnética Nuclear Biomolecular , Estrutura Quaternária de Proteína , CoesinasRESUMO
We combine experimental and theoretical methods to assess the effect of a set of point mutations on c7A, a highly mechanostable type I cohesin module from scaffoldin CipA from Clostridium thermocellum. We propose a novel robust and computationally expedient theoretical method to determine the effects of point mutations on protein structure and stability. We use all-atom simulations to predict structural shifts with respect to the native protein and then analyze the mutants using a coarse-grained model. We examine transitions in contacts between residues and find that changes in the contact map usually involve a non-local component that can extend up to 50 Å. We have identified mutations that may lead to a substantial increase in mechanical and thermodynamic stabilities by making systematic substitutions into alanine and phenylalanine in c7A. Experimental measurements of the mechanical stability and circular dichroism data agree qualitatively with the predictions provided the thermal stability is calculated using only the contacts within the secondary structures.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Genéticos , Mutação Puntual , Alanina/química , Alanina/genética , Substituição de Aminoácidos , Clostridium thermocellum/genética , Simulação de Dinâmica Molecular , Fenilalanina/química , Fenilalanina/genética , Domínios Proteicos , Estabilidade Proteica , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
In plants, the presence and distribution of specialized metabolites during the early stages of development are not documented enough, even though their biosynthesis is one of the most important strategies for survival. In this study, five alkaloids and four acetogenins were detected in Annona muricata L. during early development seedling, including three phases of root emergence and three of seedling formation. Hexane and alkaloid extracts were obtained from each organ, which were analyzed in a gas-mass chromatograph and in a high-performance liquid chromatograph coupled with a photodiode array UV detector (HPLC-DAD). This research shows the presence of the acetogenins cis-uvarimicin IV, mosinone, muricina B, and cis-annonacin-10-one, as well as of the alkaloids reticuline, coreximine, anonaine, asimilobine, and nornuciferine, both groups with a variable organ-specific distribution, related with the formation of organs and tissues.
Assuntos
Acetogeninas/isolamento & purificação , Alcaloides/isolamento & purificação , Annona/metabolismo , Raízes de Plantas/metabolismo , Plântula/metabolismo , Acetogeninas/química , Acetogeninas/classificação , Alcaloides/química , Alcaloides/classificação , Annona/química , Annona/crescimento & desenvolvimento , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , Especificidade de Órgãos , Desenvolvimento Vegetal/fisiologia , Extratos Vegetais/química , Raízes de Plantas/química , Raízes de Plantas/crescimento & desenvolvimento , Plântula/química , Plântula/crescimento & desenvolvimentoRESUMO
The triterpenes have been constituted as a group of interesting molecules as possible antitumor agents. Despite several of them not presenting a potent cytotoxic activity in vitro against cancer cells, in vivo in xenotransplant tumors studies, they show promising results. Based on the above considerations, we investigated the antitumor activity of both masticadienonic (MDA) and 3α-OH masticadienoic (3α-OH MDA) acids in a mouse prostate cancer xenograft model. Immunohistochemical assays were used to evaluate the decrease in the expression of the Proliferating Cell Nuclear Antigen (PCNA) and the Ki-67 induced by MDA and 3α-OH MDA. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to demonstrate the fragmentation of DNA. Our results showed that the two triterpenes inhibited tumor growth, had anti-proliferative effect in vivo and induced cell death by apoptosis. Collectively, our data suggested that the antitumor mechanism of MDA and 3α-OH MDA involves several molecular targets related to cell proliferation and apoptosis.