Probing prothrombin structure by limited proteolysis

Prothrombin, or coagulation factor II, is a multidomain zymogen precursor of thrombin that undergoes an allosteric equilibrium between two alternative conformations, open and closed, that react differently with the physiological activator prothrombinase. Specifically, the dominant closed form promotes cleavage at R320 and initiates activation along the meizothrombin pathway, whilst the open form promotes cleavage at R271 and initiates activation along the alternative prethrombin-2 pathway. Here we report how key structural features of prothrombin can be monitored by limited proteolysis with chymotrypsin that attacks W468 in the flexible autolysis loop of the protease domain in the open but not the closed form. Perturbation of prothrombin by selective removal of its constituent Gla domain, kringles and linkers reveals their long-range communication and supports a scenario where stabilization of the open form switches the pathway of activation from meizothrombin to prethrombin-2. We also identify R296 in the A chain of the protease domain as a critical link between the allosteric open-closed equilibrium and exposure of the sites of cleavage at R271 and R320. These findings reveal important new details on the molecular basis of prothrombin functio

Interplay between conformational selection and zymogen activation

Trypsin-like proteases are synthesized as zymogens and activated through a mechanism that folds the active site for efficient binding and catalysis. Ligand binding to the active site is therefore a valuable source of information on the changes that accompany zymogen activation. Using the physiologically relevant transition of the clotting zymogen prothrombin to the mature protease thrombin, we show that the mechanism of ligand recognition follows selection within a pre-existing ensemble of conformations with the active site accessible (E) or inaccessible (E) to binding. Prothrombin exists mainly in the Econformational ensemble and conversion to thrombin produces two dominant changes: a progressive shift toward the E conformational ensemble triggered by removal of the auxiliary domains upon cleavage at R271 and a drastic drop of the rate of ligand dissociation from the active site triggered by cleavage at R320. Together, these effects produce a significant (700-fold) increase in binding affinity. Limited proteolysis reveals how the E-E equilibrium shifts during prothrombin activation and influences exposure of the sites of cleavage at R271 and R320. These new findings on the molecular underpinnings of prothrombin activation are relevant to other zymogens with modular assembly involved in blood coagulation, complement and fibrinolysis

Urban design strategies for the upcycling of urban infrastructure residual pockets: 3D city modelling from open data and low-cost rapid mapping tools

This paper deals with the 3D City Modelling specific procedure developed as a tool to support strategies for urban regeneration, within the framework of the B-ROAD research project. The B-ROAD research project, whose acronym stands for Below the Road, is developing urban design strategies for upcycling urban infrastructure residual pockets. The B-ROAD’s methodology is conceived as research by design as it is carried out by creating pilot scenarios, disclosing the latent and still unexpressed potential of these wasted areas and displaying their potential transformations, to turn them into precious resources for the contemporary city. The 3D City Modelling of the study area has proved to be essential and strategic yet often complex and critical as most of the spatial and architectural features of B-ROAD spaces, as well as their potential, cannot be detected nor represented through the traditional means of representation of urbanised land, as aerial survey-based representations, or GIS. Likewise, traditional, or even cutting-edge, survey techniques that can be used to acquire missing data are often costly and time-consuming, thus making it hardly impossible to achieve the purpose of extensive and deep knowledge of such a vast area. Thus, 3D City Modelling aimed at examining spaces and providing a final representation of pilot scenarios has been a crucial stage requiring a specific in-depth study

Cryo-EM structure of the prothrombin-prothrombinase complex

The intrinsic and extrinsic pathways of the coagulation cascade converge to a common step where the prothrombinase complex, comprising the enzyme factor Xa (fXa), the cofactor fVa, Ca2+ and phospholipids, activates the zymogen prothrombin to the protease thrombin. The reaction entails cleavage at 2 sites, R271 and R320, generating the intermediates prethrombin 2 and meizothrombin, respectively. The molecular basis of these interactions that are central to hemostasis remains elusive. We solved 2 cryogenic electron microscopy (cryo-EM) structures of the fVa-fXa complex, 1 free on nanodiscs at 5.3-Å resolution and the other bound to prothrombin at near atomic 4.1-Å resolution. In the prothrombin-fVa-fXa complex, the Gla domains of fXa and prothrombin align on a plane with the C1 and C2 domains of fVa for interaction with membranes. Prothrombin and fXa emerge from this plane in curved conformations that bring their protease domains in contact with each other against the A2 domain of fVa. The 672ESTVMATRKMHDRLEPEDEE691 segment of the A2 domain closes on the protease domain of fXa like a lid to fix orientation of the active site. The 696YDYQNRL702 segment binds to prothrombin and establishes the pathway of activation by sequestering R271 against D697 and directing R320 toward the active site of fXa. The cryo-EM structure provides a molecular view of prothrombin activation along the meizothrombin pathway and suggests a mechanism for cleavage at the alternative R271 site. The findings advance our basic knowledge of a key step of coagulation and bear broad relevance to other interactions in the blood

Fault location on multiterminal transmission lines using only two terminals data

Um novo método de localização de faltas em linhas de transmissão continuamente transpostas e com múltiplas derivações é apresentado neste artigo. O mesmo é baseado nos fasores de tensão e corrente, pré-falta e pós-falta, medidos em regime permanente nos terminais local e remoto, para determinar as informações de falta, tais como: o trecho de ocorrência, a distância de falta, ou seja, o ponto de ocorrência, e a resistência. A principal caracterstica deste novo método é o processo de varredura da rede, onde a falta é localizada dentro de cada um dos trechos da linha, buscando-se minimizar por meio de uma função objetivo, as diferenças entre as tensões e correntes medidas e calculadas nos terminais local e remoto. O erro calculado pela função objetivo, no caso de a falta estar em um determinado trecho, é comparado com o erro obtido para o trecho anterior, sendo o trecho com o maior erro descartado. No final do processo de varredura, o método de localização de faltas tem como resultado o trecho de ocorrência, a distância e a resistência da falta. Os resultados das simulações mostram a eficiência e a precisão do método proposto frente a variação da distância, resistência e tipo da falta.A new multiterminal continuously transposed transmission lines fault location method is presented in this paper, which it is based on steady-state pre-fault and post-fault voltage and current measured phasors at local and remote terminals to determine the fault information, which are: the section occurrence, the distance, or the occurrence point, and resistance. The main characteristic of this new method is the process of networkscanning, where the fault is located within each line section, trying to minimize by means of an objective function, the differences between the voltages and currents measured and calculated in local and remote terminals. The the error calculated by the goal function, in case of the fault to be in a certain section, is compared with the error obtained for the previous section, so the section with the largest error is dismissed. At the end of the scanning process, the method of fault location presents the section of fault occurrence, distance and resistance. The results obtained from the simulations show the efficiency and accuracy of the proposed method for the fault distance, resistance and type variation

Neural correlates of psychodynamic and non-psychodynamic therapies in different clinical populations through fMRI: a meta-analysis and systematic review

BACKGROUND: The COVID-19 pandemic has exacerbated the ongoing crisis in psychiatric and psychological care, contributing to what we have identified as a new psychological and psychiatric pandemic. Psychotherapy is an effective method for easing the psychological suffering experienced also by the various impacts of COVID-19. This treatment can be examined from a neurological perspective, through the application of brain imaging techniques. Specifically, the meta-analysis of imaging studies can aid in expanding researchers' understanding of the many beneficial applications of psychotherapy. OBJECTIVES: We examined the functional brain changes accompanying different mental disorders with functional Magnetic Resonance Imaging (fMRI), through a meta-analysis, and systematic review in order to better understand the general neural mechanism involved in psychotherapy and the potential neural difference between psychodynamic and non-psychodynamic approaches. DATA SOURCES: The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were employed for our systematic review and meta-analysis. We conducted a computer-based literature search, following the Population, Intervention, Comparison and Outcomes (PICO) approach, to retrieve all published articles in English regarding the above-described topics from PubMed (MEDLINE), Scopus, and Web of Science. STUDY ELIGIBILITY CRITERIA, PARTICIPANTS, AND INTERVENTIONS: We combined terms related to psychotherapy and fMRI: (“psychotherapy” [All Fields] OR “psychotherapy” [MeSH Terms] OR “psychotherapy” [All Fields] OR “psychotherapies” [All Fields] OR “psychotherapy s” [All Fields]) AND (“magnetic resonance imaging” [MeSH Terms]) OR (“magnetic”[All Fields] AND “resonance”[All Fields] AND “imaging”[All Fields]) OR (“magnetic resonance imaging”[All Fields] OR “fmri”[All Fields]). We considered (1) whole brain fMRI studies; (2) studies in which participants have been involved in a clinical trial with psychotherapy sessions, with pre/post fMRI; (3) fMRI results presented in coordinate-based (x, y, and z) in MNI or Talairach space; (4) presence of neuropsychiatric patients. The exclusion criteria were: (1) systematic review or meta-analysis; (2) behavioral study; (3) single-case MRI or fMRI study; and (4) other imaging techniques (i.e., PET, SPECT) or EEG. RESULTS: After duplicates removal and assessment of the content of each published study, we included 38 sources. The map including all studies that assessed longitudinal differences in brain activity showed two homogeneous clusters in the left inferior frontal gyrus, and caudally involving the anterior insular cortex (p < 0.0001, corr.). Similarly, studies that assessed psychotherapy-related longitudinal changes using emotional or cognitive tasks (TASK map) showed a left-sided homogeneity in the anterior insula (p < 0.000) extending to Broca's area of the inferior frontal gyrus (p < 0.0001) and the superior frontal gyrus (p < 0.0001). Studies that applied psychodynamic psychotherapy showed Family-Wise Error (FWE) cluster-corrected (p < 0.05) homogeneity values in the right superior and inferior frontal gyri, with a small cluster in the putamen. No FWE-corrected homogeneity foci were observed for Mindful- based and cognitive behavioral therapy psychotherapy. In both pre- and post-therapy results, studies showed two bilateral clusters in the dorsal anterior insulae (p = 0.00001 and p = 0.00003, respectively) and involvement of the medial superior frontal gyrus (p = 0.0002). LIMITATIONS: Subjective experiences, such as an individual's response to therapy, are intrinsically challenging to quantify as objective, factual realities. Brain changes observed both pre- and post-therapy could be related to other factors, not necessary to the specific treatment received. Therapeutic modalities and study designs are generally heterogeneous. Differences exist in sample characteristics, such as the specificity of the disorder and number and duration of sessions. Moreover, the sample size is relatively small, particularly due to the paucity of studies in this field and the little contribution of PDT. CONCLUSIONS AND IMPLICATIONS OF KEY FINDINGS: All psychological interventions seem to influence the brain from a functional point of view, showing their efficacy from a neurological perspective. Frontal, prefrontal regions, insular cortex, superior and inferior frontal gyrus, and putamen seem involved in these neural changes, with the psychodynamic more linked to the latter three regions

Studies on the Basis for the Properties of Fibrin Produced from Fibrinogen-Containing γ′ Chains

Human fibrinogen 1 is homodimeric with respect to its γ chains (`γA-γA\u27), whereas fibrinogen 2 molecules each contain one γA (γA1-411V) and one γ\u27 chain, which differ by containing a unique C-terminal sequence from γ\u27408 to 427L that binds thrombin and factor XIII. We investigated the structural and functional features of these fibrins and made several observations. First, thrombin-treated fibrinogen 2 produced finer, more branched clot networks than did fibrin 1. These known differences in network structure were attributable to delayed release of fibrinopeptide (FP) A from fibrinogen 2 by thrombin, which in turn was likely caused by allosteric changes at the thrombin catalytic site induced by thrombin exosite 2 binding to the γ\u27 chains. Second, cross-linking of fibrin γ chains was virtually the same for both types of fibrin. Third, the acceleratory effect of fibrin on thrombin-mediated XIII activation was more prominent with fibrin 1 than with fibrin 2, and this was also attributable to allosteric changes at the catalytic site induced by thrombin binding to γ\u27 chains. Fourth, fibrinolysis of fibrin 2 was delayed compared with fibrin 1. Altogether, differences between the structure and function of fibrins 1 and 2 are attributable to the effects of thrombin binding to γ\u27 chains