The unique regulation of iron-sulfur cluster biogenesis in a Gram-positive bacterium

Iron-sulfur clusters function as cofactors of a wide range of proteins, with diverse molecular roles in both prokaryotic and eukaryotic cells. Dedicated machineries assemble the clusters and deliver them to the final acceptor molecules in a tightly regulated process. In the prototypical Gram-negative bacterium Escherichia coli, the two existing iron-sulfur cluster assembly systems, iron-sulfur cluster (ISC) and sulfur assimilation (SUF) pathways, are closely interconnected. The ISC pathway regulator, IscR, is a transcription factor of the helix-turn-helix type that can coordinate a [2Fe-2S] cluster. Redox conditions and iron or sulfur availability modulate the ligation status of the labile IscR cluster, which in turn determines a switch in DNA sequence specificity of the regulator: cluster-containing IscR can bind to a family of gene promoters (type-1) whereas the clusterless form recognizes only a second group of sequences (type-2). However, iron-sulfur cluster biogenesis in Gram-positive bacteria is not so well characterized, and most organisms of this group display only one of the iron-sulfur cluster assembly systems. A notable exception is the unique Gram-positive dissimilatory metal reducing bacterium Thermincola potens, where genes from both systems could be identified, albeit with a diverging organization from that of Gram-negative bacteria. We demonstrated that one of these genes encodes a functional IscR homolog and is likely involved in the regulation of iron-sulfur cluster biogenesis in T. potens. Structural and biochemical characterization of T. potens and E. coli IscR revealed a strikingly similar architecture and unveiled an unforeseen conservation of the unique mechanism of sequence discrimination characteristic of this distinctive group of transcription regulators.We thank Jorge Vieira for help with Automatic Detection of Positively Selected Sites. We acknowledge the European Synchrotron Radiation Facility (ESRF) for provision of synchrotron radiation facilities and thank the ESRF staff for help with data collection. Microscale thermophoresis data collection was carried out at the Campus Science Support Facilities Protein Technologies Facility (www.csf.ac.at). This work was funded by Fundo Europeu de Desenvolvimento Regional through the Operational Competitiveness Programme-COMPETE and by national funds through Fundacao para a Ciencia e a Tecnologia under project FCOMP-01-0124-FEDER-028116 (PTDC/BBB - BEP/2127/2012) and PhD Fellowship SFRH/BD/66461/2009 (to J.A.S.). The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (Grant Agreement 283570)

Effectiveness factor for immobilized biocatalysts: two substratestwo products reactions

Immobilized enzymes are being increasingly used as biocatalysts in numerous processes to obtain high-value products for the pharmaceutical, flavour and fragrance industries (Gandhi et al., 2000). The major advantages of immobilization include the increase in enzyme stability, the possibility of enzyme reutilization and the easy separation of the biocatalysts from the reaction mixture. However, it is necessary to account for mass transfer limitations that, under some conditions, may arise in these systems (Gómez et al., 2003; Jeison et al., 2003). These resistances comprise the effects of intraparticle diffusion and external mass-transfer. Given the complexity of the kinetics of multisubstrate enzyme reactions, reactor modelling studies that account for mass-transfer phenomena are so far limited to single-substrate ones (Gómez et al., 2003). To compare the observed reaction rate with the reaction rate in the absence of mass-transfer limitations, an overall effectiveness factor is usually calculated (Gómez et al., 2003; Jeison et al., 2003). In this work, a model is developed to calculate the overall effectiveness factor for immobilized enzymes that carry out irreversible two substrates-two products reactions following kinetic mechanisms such as the Ternary Complex or the Ping-Pong Bi-Bi with inhibition by the second substrate. The model has two dimensionless parameters for each substrate – Thiele modulus (reaction/intraparticle diffusion), Biot number (film diffusion/intraparticle diffusion) – and one related to the reaction kinetics. Their influence on the effectiveness factor is analysed. The results obtained can be applied in the design and simulation of enzymatic reactors

Molecular fingerprints for a novel enzyme family in actinobacteria with glucosamine kinase activity

Actinobacteria have long been the main source of antibiotics, secondary metabolites with tightly controlled biosynthesis by environmental and physiological factors. Phosphorylation of exogenous glucosamine has been suggested as a mechanism for incorporation of this extracellular material into secondary metabolite biosynthesis, but experimental evidence of specific glucosamine kinases in Actinobacteria is lacking. Here, we present the molecular fingerprints for the identification of a unique family of actinobacterial glucosamine kinases. Structural and biochemical studies on a distinctive kinase from the soil bacterium Streptacidiphilus jiangxiensis unveiled its preference for glucosamine and provided structural evidence of a phosphoryl transfer to this substrate. Conservation of glucosamine-contacting residues across a large number of uncharacterized actinobacterial proteins unveiled a specific glucosamine binding sequence motif. This family of kinases and their genetic context may represent the missing link for the incorporation of environmental glucosamine into the antibiotic biosynthesis pathways in Actinobacteria and can be explored to enhance antibiotic production. IMPORTANCE The discovery of novel enzymes involved in antibiotic biosynthesis pathways is currently a topic of utmost importance. The high levels of antibiotic resistance detected worldwide threaten our ability to combat infections and other 20th-century medical achievements, namely, organ transplantation or cancer chemotherapy. We have identified and characterized a unique family of enzymes capable of phosphorylating glucosamine to glucosamine-6-phosphate, a crucial molecule directly involved in the activation of antibiotic production pathways in Actinobacteria, nature’s main source of antimicrobials. The consensus sequence identified for these glucosamine kinases will help establish a molecular fingerprint to reveal yet-uncharacterized sequences in antibiotic producers, which should have an important impact in biotechnological and biomedical applications, including the enhancement and optimization of antibiotic production.We acknowledge the European Synchrotron Radiation Facility (Grenoble, France) for provision of synchrotron radiation facilities and thank their staff for help with data collection. Part of these experiments were performed at beamline BL13-XALOC of ALBA Synchrotron (Cerdanyola del Vallès, Spain), with the collaboration of ALBA staff and CALIPSOplus (grant 730872) funding. The support of the X-ray Crystallography Scientific Platform of i3S (Porto, Portugal) is also acknowledged. We thank Pedro Lamosa from CERMAX, ITQB-NOVA (Oeiras, Portugal), for acquiring and interpreting the NMR data. This work was supported by the Structured Program on Bioengineered Therapies for Infectious Diseases and Tissue Regeneration (Norte-01-0145-FEDER-‏000‏012), funded by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through Fundo Europeu de Desenvolvimento Regional (FEDER) and by FEDER through the COMPETE 2020-Operational Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT-Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of project Institute for Research and Innovation in Health Sciences (POCI-01-0145-FEDER-007274) and also by grants UID/NEU/04539/2019 and POCI-01-0145-FEDER-029221. D.N.-C. acknowledges the European Regional Development Fund (CENTRO-01-0145-FEDER-000012-ealthyAging2020) for a research fellowship and FCT for Ph‏D f‏ellowship SFRH/BD/117777/2016

Purification, crystallization and preliminary X-ray diffraction analysis of the seryl-tRNA synthetase from Candida albicans

The seryl-tRNA synthetase (SerRS) from Candida albicans exists naturally as two isoforms resulting from ambiguity in the natural genetic code. Both enzymes were crystallized by the sitting-drop vapour-diffusion method using 3.2-3.4 M ammonium sulfate as precipitant. The crystals belonged to the hexagonal space group P6(1)22 and contained one monomer per asymmetric unit, despite the synthetase existing as a homodimer (with a molecular weight of ∼116 kDa) in solution. Diffraction data were collected to 2.0 Å resolution at a synchrotron source and the crystal structures of unliganded SerRS and of its complexes with ATP and with a seryl-adenylate analogue were solved by molecular replacement. The structure of C. albicans SerRS represents the first reported structure of a eukaryotic cytoplasmic SerRS.publishe

Natural based reusable materials for microfluidic substrates: The silk road towards sustainable portable analytical systems

Portable analytical systems are versatile tools for application in areas including biomedicine, biosecurity, food safety and environmental monitoring. This work contributes to the increasing demand for low-cost, environmentally friendly substrates for portable analytical systems by using natural Bombyx mori cocoons. Further, silk fibroin is also extracted from these cocoons and electrospun into oriented and randomly oriented fiber substrates. Oxygen plasma treatment is applied to improve their hydrophilicity. Fiber morphology, mechanical properties, porosity, thermal characteristics and surface contact angle are extensively characterized and the ability of the samples for passive capillary flows demonstrated. Plasma treated pressed cocoons show superhydrophilicity, capillary flow rates of 44.8 ± 3.75 mm.min-1, and high mechanical resistance with Young's modulus values up to 592.13 ± 19.83 MPa. The developed materials are used as substrates for the colorimetric quantification of three commonly scrutinized clinical analytes. Hydrophobic barriers are first wax-printed on all samples with a proper design and albumin assays are performed on all substrates. Further assays for uric acid and glucose quantification are successfully accomplished on the pressed cocoons after a simple in between washing step, with overall high coefficient of determination, proving the suitability of the developed materials as low-cost, sustainable and reusable microfluidic substrates.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under strategic funding UID/FIS/04650/2020, UIDB/04436/2020, UIDP/04436/2020 and project PTDC/EMD-EMD/28159/2017 (POCI-01–0145-FEDER-028159). The authors also thank FCT for financial support under grants SFRH/BD/140698/2018 (R.B. P.), 2020.09218.BD (A.S.M.), 2020.04163.CEECIND (C.R.) and 2020.02304.CEECIND (V.F.C.). Finally, the authors acknowledge fund ing by Spanish State Research Agency (AEI) and the European Regional Development Fund (ERFD) through the project PID2019–106099RB C43/AEI/10.13039/501100011033 and from the Basque Government Industry Departments under the ELKARTEK program. Finally, the au thors also thank Dr. J. Borges and Prof. F. Vaz for experimental support.info:eu-repo/semantics/publishedVersio

Analysis of a vegetable oil performance in a milling process by MQL Lubrication

In this work, we carried out a comparison between the dry machining of an aluminum block with conventional cutting oil and a block with vegetable oil. The two oils had different flow rates. Using the Taguchi method, it was possible to determine the matrices for optimizing the best parameters for each group of tests. Then, we studied the utility of using vegetable oil as a cutting lubricant. We found that the vegetable oil studied in this work had good properties in terms of reducing cutting temperatures but was less effective than conventional cutting oil in reducing the surface roughness of the machined part. Tribological tests were carried out to understand the influence of the selected lubricants in reducing friction and wear. After the sliding experiments, which were performed without lubrication in the presence of the same lubricants that were used in the machining tests and in the presence of distilled water, we concluded that vegetable oil has satisfactory lubricating properties that are similar to those of the conventional cutting fluid, indicating a potential for consideration as an effective alternative to the conventional cutting fluid, with economic, environmental, and health advantages.Financial support was provided by Portugal’s national funding FCT/MCTES (PIDDAC) to Centro de Investigação de Montanha (CIMO) (UIDB/00690/2020 and UIDP/00690/2020) and SusTEC (LA/P/0007/2020). Additionally, this work was partially supported by Portuguese FCT, under the reference projects UIDB/04077/2020, UIDB/00532/2020 and UIDB/04436/2020. This research was also partially funded by EXPL2021CIMO_01. Inês Afonso acknowledges the financial support of CIMO through EXPL2021CIMO_01

Production of esters by biocatalysed transesterification in supercritical CO2 and Hexane

The development of sustainable processes that use renewable raw materials and minimize chemical and energetic waste has attracted considerable attention and represents a great challenge to both academic researchers and industrial experts. The use of enzymes to catalyse chemical transformations can constitute a more sustainable alternative to some traditional chemical processes and more than 100 biotransformations are already operated at an industrial scale [1]. Lipases usually operate in mild conditions and can catalyse both hydrolytic and synthetic reactions, depending on the surrounding medium. The hydrolysis of esters can be performed in water, but the reverse production reactions are not favoured in this medium, and are usually performed in organic solvents. Supercritical CO2 can constitute a more sustainable alternative to organic solvents as a reaction medium, provided that it does not have a direct adverse effect on the enzyme's active site or significantly reduce its activity. Decyl acetate was chosen as a model compound and its production by a transesterification reaction catalysed by Novozym 435 (immobilized Candida Antarctica Lipase B) was studied in both hexane and supercritical CO2. A comparative analysis between these two alternatives was performed, focusing on the differences on the enzyme's catalytic activity, solubilities of the substrates and mass transfer rates; which significantly affect the outcome of the reaction process and its productivity and provide information on when such solvents can be used. [1] Straathof, A.J.J., Panke, S., Schmid, A. The production of fine chemicals by biotransformations. Current Opinion in Biotechnology, 2002, 13(6), 548-556

Systemic inhibition of nitric oxide synthesis in non-diabetic individuals produces a significant deterioration in glucose tolerance by increasing insulin clearance and inhibiting insulin secretion

Funding Information: Source of funding The study received partial support from FTC (Fundação para a Ciência e a Tecnologia - Portuguese Foundation for Science and Technology) under the project PTDC/DTP-EPI/0207/2012. Other funds came from resources made available by other funded studies.Aims/hypothesis: Endogenous NO inhibits insulin release in isolated beta cells and insulin-degrading enzyme activity in hepatocytes, while NO release from endothelial cells has been suggested to enhance insulin action. We assessed the overall effect of systemic inhibition of endogenous NO synthesis on glucose homeostasis in humans. Methods: Twenty-four non-diabetic volunteers underwent two hyperglycaemic (+7 mmol/l) clamps with either saline or L-NG-nitroarginine methyl ester (l-NAME, at rates of 2.5, 5, 10 and 20 μg min-1 kg-1) infusion. Another five volunteers underwent an OGTT with either saline or l-NAME (20 μg min-1 kg-1) infusion. Blood pressure and heart rate were measured to monitor NO blockade; during the OGTT, endothelial function was assessed by peripheral arterial tonometry and insulin secretion by C-peptide deconvolution and insulin secretion modelling. Results: Compared with saline, l-NAME at the highest dose raised mean blood pressure (+20 ± 2 mmHg), depressed heart rate (-12 ± 2 bpm) and increased insulin clearance (+50%). First-phase insulin secretion was impaired, but insulin sensitivity (M/I index) was unchanged. During the OGTT, l-NAME raised 2 h plasma glucose by 1.8 mmol/l (p < 0.01), doubled insulin clearance and impaired beta cell glucose sensitivity while depressing endothelial function. Conclusions/interpretation: In humans, systemic NO blockade titrated to increase blood pressure and induce endothelial dysfunction does not affect insulin action but significantly impairs glucose tolerance by increasing plasma insulin clearance and depressing insulin secretion, namely first-phase and beta cell glucose sensitivity.publishersversionpublishe

Recent developments on the thermal properties, stability and applications of nanofluids in machining, solar energy and biomedicine

In this review work, the recent progress made in the use of nanofluids (NFs) applied in three specific areas will be presented: machining, solar energy, and biomedical engineering. Within this context, the discussions will be guided by emphasizing the thermal and stability properties of these fluids. In machining, NFs play a prominent role in the processes of turning, milling, drilling, and grinding, being responsible for their optimization as well as improving the useful life of the tools and reducing costs. In the solar energy field, NFs have been used in the thermal management of the panels, controlling and homogenizing the operating temperature of these systems. In the biomedical area, the advantages of using NFs come from the treatment of cancer cells, the development of vaccines before the improvement of diagnostic imaging, and many others. In all lines of research mentioned in this study, the main parameters that have limited or encouraged the use of these fluids are also identified and debated. Finally, the discussions presented in this review will inspire and guide researchers in developing new techniques to improve the applications of NFs in several fields.This research was partially funded by the Portuguese national funds of FCT/MCTES (PIDDAC) through the base funding from the following research units: UIDB/00690/2020 (CIMO), UIDB/04077/2020 (MEtRICs), and UIDB/00532/2020 (CEFT). The authors are also grateful for the funding of ANI and CIMO through the projects POCI-01-02B7-FEDER-069844 and CMFPE3- EXPL2021CIMO_01, respectively. The authors also acknowledge partial financial support from the project NORTE-01-0145-FEDER-030171 (PTDC/EMD-EMD/30171/2017), PTDC/EME-TED/7801/ 2020 and EXPL/EME-EME/0732/2021 funded by the NORTE 2020 Portugal Regional Operational Programme, under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) and by Fundação para a Ciência e Tecnologia (FCT)

Comparação da produção de poliedroses de SfMNPV em células de inseto SF9 e SF21.

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