Entire hemithorax irradiation for Masaoka stage IVa thymomas

Thymomas are rare neoplasms that have an indolent growth with a preferentially intra-thoracic dissemination pattern. Surgery is currently the standard treatment of thymomas; however radiotherapy is often used in an adjuvant setting due to a high sensitivity of these tumors to such treatment. Postoperative entire hemithoracic irradiation has been used in selected Masaoka stage IVa cases after complete surgical excision of metastatic lesions. In the present article, the authors report three cases of Masaoka stage IVa thymoma that underwent entire hemithorax irradiation after surgical excision of metastatic lesions. The first two patients presented as stage IVa thymomas. The third case consisted of a pleural recurrence of a thymoma. Hemithoracic irradiation with low doses has been used by different authors; the available data shows that it is a well-tolerated treatment that could potentially lead to better loco-regional control and increased overall survival

Redox tuning of the catalytic activity of soluble fumarate reductases from Shewanella

AbstractMany enzymes involved in bioenergetic processes contain chains of redox centers that link the protein surface, where interaction with electron donors or acceptors occurs, to a secluded catalytic site. In numerous cases these redox centers can transfer only single electrons even when they are associated to catalytic sites that perform two-electron chemistry. These chains provide no obvious contribution to enhance chemiosmotic energy conservation, and often have more redox centers than those necessary to hold sufficient electrons to sustain one catalytic turnover of the enzyme. To investigate the role of such a redox chain we analyzed the transient kinetics of fumarate reduction by two flavocytochromes c3 of Shewanella species while these enzymes were being reduced by sodium dithionite. These soluble monomeric proteins contain a chain of four hemes that interact with a flavin adenine dinucleotide (FAD) catalytic center that performs the obligatory two electron–two proton reduction of fumarate to succinate. Our results enabled us to parse the kinetic contribution of each heme towards electron uptake and conduction to the catalytic center, and to determine that the rate of fumarate reduction is modulated by the redox stage of the enzyme, which is defined by the number of reduced centers. In both enzymes the catalytically most competent redox stages are those least prevalent in a quasi-stationary condition of turnover. Furthermore, the electron distribution among the redox centers during turnover suggested how these enzymes can play a role in the switch between respiration of solid and soluble terminal electron acceptors in the anaerobic bioenergetic metabolism of Shewanella

The Importance of Multiheme Cytochromes

Publisher Copyright: Copyright: © 2022 The Author(s)Extracellular electron transfer is a key metabolic process of many organisms that enables them to exchange electrons with extracellular electron donors/acceptors. The discovery of organisms with these abilities and the understanding of their electron transfer processes has become a priority for the scientific and industrial community, given the growing interest on the use of these organisms in sustainable biotechnological processes. For example, in bioelectrochemical systems electrochemical active organisms can exchange electrons with an electrode, allowing the production of energy and added-value compounds, among other processes. In these systems, electrochemical active organisms exchange electrons with an electrode through direct or indirect mechanisms, using, in most cases, multiheme cytochromes. In numerous electroactive organisms, these proteins form a conductive pathway that allows electrons produced from cellular metabolism to be transferred across the cell surface for the reduction of an electrode, or vice-versa. Here, the mechanisms by which the most promising electroactive bacteria perform extracellular electron transfer will be reviewed, emphasizing the proteins involved in these pathways. The ability of some of the organisms to perform bidirectional electron transfer and the pathways used will also be highlighted.publishersversionpublishe

Measuring transverse relaxation in highly paramagnetic systems

The enhancement of nuclear relaxation rates due to the interaction with a paramagnetic center (known as Paramagnetic Relaxation Enhancement) is a powerful source of structural and dynamics information, widely used in structural biology. However, many signals affected by the hyperfine interaction relax faster than the evolution periods of common NMR experiments and therefore they are broadened beyond detection. This gives rise to a so-called blind sphere around the paramagnetic center, which is a major limitation in the use of PREs. Reducing the blind sphere is extremely important in paramagnetic metalloproteins. The identification, characterization, and proper structural restraining of the first coordination sphere of the metal ion(s) and its immediate neighboring regions is key to understand their biological function. The novel HSQC scheme we propose here, that we termed R2-weighted, HSQC-AP, achieves this aim by detecting signals that escaped detection in a conventional HSQC experiment and provides fully reliable R2 values in the range of 1H R2 rates ca. 50–400 s−1. Independently on the type of paramagnetic center and on the size of the molecule, this experiment decreases the radius of the blind sphere and increases the number of detectable PREs. Here, we report the validation of this approach for the case of PioC, a small protein containing a high potential 4Fe-4S cluster in the reduced [Fe4S4]2+ form. The blind sphere was contracted to a minimal extent, enabling the measurement of R2 rates for the cluster coordinating residues.publishersversionpublishe

1H, 13C and 15N assignment of the paramagnetic high potential iron–sulfur protein (HiPIP) PioC from Rhodopseudomonas palustris TIE-1

High potential iron–sulfur proteins (HiPIPs) are a class of small proteins (50–100 aa residues), containing a 4Fe–4S iron–sulfur cluster. The 4Fe–4S cluster shuttles between the oxidation states [Fe4S4]3+/2+, with a positive redox potential in the range (500–50 mV) throughout the different known HiPIPs. Both oxidation states are paramagnetic at room temperature. HiPIPs are electron transfer proteins, isolated from photosynthetic bacteria and usually provide electrons to the photosynthetic reaction-center. PioC, the HIPIP isolated from Rhodopseudomonas palustris TIE-1, is the smallest among all known HiPIPs. Despite their small dimensions, an extensive NMR assignment is only available for two of them, because paramagnetism prevents the straightforward assignment of all resonances. We report here the complete NMR assignment of 1H, 13C and 15N signals for the reduced [Fe4S4]2+ state of the protein. A set of double and triple resonance experiments performed with standardized parameters/datasets provided the assignment of about 72% of the residues. The almost complete resonance assignment (99.5% of backbone and ca. 90% of side chain resonances) was achieved by combining the above information with those obtained using a second set of NMR experiments, in which acquisition and processing parameters, as well as pulse sequences design, were optimized to account for the peculiar features of this paramagnetic protein.publishersversionpublishe

A non-systematic approach

Funding Information: This work benefited from access to CERM/CIRMMP, the Instruct-ERIC Italy centre. Financial support was provided by European EC Horizon 2020 TIMB3 (Project 810856) Instruct-ERIC (PID 4509). This article is based upon work from COST Action CA15133, supported by COST (European Cooperation in Science and Technology) . Fondazione Ente Cassa di Risparmio di Firenze ( CRF 2016 0985 ) is acknowledged for providing fellowship to MI. This work was funded by national funds through FCT– Fundação para a Ciência e a Tecnologia , I.P., Project MOSTMICRO-ITQB with refs UIDB/04612/2020 and UIDP/04612/2020, and Fundação para a Ciência e a Tecnologia (FCT) Portugal is acknowledged for Grant PD/BD/135187/2017 to IBT. Funding Information: This work benefited from access to CERM/CIRMMP, the Instruct-ERIC Italy centre. Financial support was provided by European EC Horizon 2020 TIMB3 (Project 810856) Instruct-ERIC (PID 4509). This article is based upon work from COST Action CA15133, supported by COST (European Cooperation in Science and Technology). Fondazione Ente Cassa di Risparmio di Firenze (CRF 2016 0985) is acknowledged for providing fellowship to MI. This work was funded by national funds through FCT? Funda??o para a Ci?ncia e a Tecnologia, I.P. Project MOSTMICRO-ITQB with refs UIDB/04612/2020 and UIDP/04612/2020, and Funda??o para a Ci?ncia e a Tecnologia (FCT) Portugal is acknowledged for Grant PD/BD/135187/2017 to IBT. Publisher Copyright: © 2020 The Author(s) Copyright: Copyright 2020 Elsevier B.V., All rights reserved.The complete assignment of 1H, 13C and 15N protein signals, which is a straightforward task for diamagnetic proteins provided they are folded, soluble and with a molecular mass below 30,000 Da, often becomes an intractable problem in the presence of a paramagnetic center. Indeed, the hyperfine interaction quenches signal intensity; this prevents the detection of scalar and dipolar connectivities and the sequential assignment of protein regions close to the metal ion(s). However, many experiments can be optimized and novel experiments can be designed to circumvent the problem and to revive coherences invisible in standard experiments. The small HiPIP protein PioC provides an interesting case to address this issue: the prosthetic group is a [Fe4S4]2+ cluster that is bound to the 54 amino acids protein via four cysteine residues. The four cluster-bound cysteine residues adopt different binding conformations and therefore each cysteine is affected by paramagnetic relaxation to different extent. A network of tailored experiments succeeded to obtain the complete resonance assignment of cluster bound residues.publishersversionpublishe

Crystallization and preliminary crystallographic studies of FoxE from Rhodobacter ferrooxidans SW2, an FeII oxidoreductase involved in photoferrotrophy

FoxE is a protein encoded by the foxEYZ operon of Rhodobacter ferrooxidans SW2 that is involved in Fe^II-based anoxygenic photosynthesis (`photoferrotrophy'). It is thought to reside in the periplasm, where it stimulates light-dependent Fe^II oxidation. It contains 259 residues, including two haem c-binding motifs. As no three-dimensional model is available and there is no structure with a similar sequence, crystals of FoxE were produced. They diffracted to 2.44 Å resolution using synchrotron radiation at the Fe edge. The phase problem was solved by SAD using SHELXC/D/E and the experimental maps confirmed the presence of two haems per molecule

Electron transfer process in microbial electrochemical technologies: the role of cell-surface exposed conductive proteins

Electroactive microorganisms have attracted significant interest for the development of novel biotechnological systems of low ecological footprint. These can be used for the sustainable production of energy, bioremediation of metal-contaminated environments and production of added-value products. Currently, almost 100 microorganisms from the Bacterial and Archaeal domains are considered electroactive, given their ability to efficiently interact with electrodes in microbial electrochemical technologies. Cell-surface exposed conductive proteins are key players in the electron transfer between cells and electrodes. Interestingly, it seems that among the electroactive organisms identified so far, these cell-surface proteins fall into one of four groups. In this review, the different types of cell-surface conductive proteins found in electroactive organisms will be overviewed, focusing on their structural and functional properties

NMR and molecular modelling studies on elastase inhibitor-peptides for wound management

Proteases play an important and critical role in the physiological process of wound repair. However, excessive and unregulated release of proteolytic enzymes (e.g., elastase) mediates abnormal degradation of healthy tissues, which leads to inflammatory disorders such as chronic wounds. Thus, it is of therapeutic interest to develop novel synthetic inhibitor-peptides of elastase, which can restore the balance between the free enzyme and the endogenous inhibitors in chronic wounds. In previous works, we have reported two different drug delivery systems to release novel elastase inhibitors to the wound site. In both systems synthetic peptides (KRCCPDTCGIKCL-Pep4 and KRMMPDTMGIKML-Pep4M) based on the primary structure of the endogenous elastase inhibitor, secretory leucocyte protease inhibitor, were used as active material. Phosphorylation of the reported peptides prompts significant structural differences, which reflects in distinct inhibitory capacity towards elastase. These structural modifications were prompted by electrostatic interactions and hydrogen bonds established from the peptide phosphoresidue. The current study was also extended to another synthetic peptide (WCTASVPPQCY-PepBBI) that is based on the reactive loop of another elastase inhibitor, the Bowmen-Birk inhibitor. PepBBI, phosphorylated and non-phosphorylated, displays similar behaviour to Pep4 and Pep4M. The structural modifications reported herein were evaluated by two-dimensional nuclear magnetic resonance and molecular modelling approaches.The authors gratefully acknowledge the financial support of the Portuguese Foundation for Science and Technology (scholarship SFRH/BD/36522/2007 and PEst-OE/EQB/LA0004/2011), FEDER (European Fund for Regional Development)-COMPETE-QREN-EU and the European Project Lidwine - Multifunctional medical textiles for wound (e.g. Decubitus). We acknowledge CERMAX at ITQB-UNL and Rede Nacional de RMN for access to the facilities. Rede Nacional de RMN is supported with funds from FCT, Projecto de Re-equipamento Cientifico contract REDE/1517/RMN/2005, Portugal. Micaelo, N.M. acknowledges the contract research program "Compromisso corn a Ciencia" reference: C2008-UMINHO-CQ-03 and access to the Minho University GRIUM cluster