Mnn2 gene affects drug resistance in Candida glabratas biofilms

Eurobiofilms 2017 - 5th European Congress on Microbial BiofilmsInfections caused by Candida species have increased worldwide substantially over the latest decades, and are a significant cause of morbidity and mortality, mostly among critically ill patients. Candida glabrata is the second most common Candida responsible for these infections in the USA and the third in Europe, and is characterized by a high antifungal resistance. The goal of this study was to understand the role of mannans in C. glabrata biofilms and in biofilm cells resistance to antifungal drugs (fluconazole - Flu, amphotericin B - AmB, caspofungin - Csf and micafungin - Mcf)This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norteinfo:eu-repo/semantics/publishedVersio

Purification, identification and characterisation of the active site of a Seprase-like activity from bovine serum

The study and identification for the first time of a soluble form of a Seprase activity from bovine serum is presented. To date, this activity has only been reported to be an integral membrane protease but has been known to shed from its membrane. The activity was purified 30,197-fold to homogeneity, using a combination of column chromatographies, from bovine serum. Inhibition by DFP, resulting in an IC50 of 100nM confirms classification as a serine protease. The protease after separation and visualisation by native PAGE was subjected to tryptic digestion and the subsequent peptides sequenced. Each peptide sequenced was found to be present in the primary structure of Seprase/Fibroblast Activation Protein (FAP), a serine gelatinase specific for proline-containing peptides and macromolecules. Substrate specificity studies using kinetic, RP-HPLC and LC-MS analysis of synthetic peptides suggest that this peptidase has an extended substrate-binding region in addition to the primary specificity site S1. This analysis revealed at least five subsites to be involved in enzyme-substrate binding, 1 with the smallest peptide cleaved being a tetrapeptide. A proline residue in position P1 was absolutely necessary therefore showing high primary substrate specificity for the Pro-X bond, while a preference for a hydrophobic residue at the C-terminal end of the scissile bond (P1ʹ′) was evident. The enzyme also showed complete insensitivity to the prolyl oligopeptidase specific inhibitors, JTP-4819, Fmoc-Ala-pyrrCN and Z-Phe-Pro-BT. To date, no physiological substrate has clearly been defined for this protease but its ability to effectively degrade gelatin suggests a candidate protein substrate in vivo and a possible role in extracellular matrix protein degradation

Molecularly Engineered Self-Assembling Membranes for Cell-Mediated Degradation

The use of peptide engineering to develop self-assembling membranes that are responsive to cellular enzyme activities is reported. The membranes are obtained by combining hyaluronan (HA) and a rationally designed peptide amphiphile (PA) containing a proteolytic domain (GPQGIWGQ octapeptide) sensitive to matrix metalloproteinase-1 (MMP-1). Insertion of an octapeptide in a typical PA structure does not disturb its self-assembly into fibrillar nanostructures neither the ability to form membranes with HA. In vitro enzymatic degradation with hyaluronidase and MMP-1 shows that membranes containing the MMP-1 substrate exhibit enhanced enzymatic degradation, compared with control membranes (absence of MMP-1 cleavable peptide or containing a MMP-1 insensitive sequence), being completely degraded after 7 days. Cell viability and proliferation is minimally affected by the enzymatically cleavable functionality of the membrane, but the presence of MMP-1 cleavable sequence does stimulate the secretion of MMP-1 by fibroblasts and interfere with matrix deposition, particularly the deposition of collagen. By showing cell-responsiveness to biochemical signals presented on self-assembling membranes, this study highlights the ability of modulating certain cellular activities through matrix engineering. This concept can be further explored to understand the cellular remodeling process and as a strategy to develop artificial matrices with more biomimetic degradation for tissue engineering applications.This work was funded by the European Regional Development Fund (ERDF) through the Operational Competitiveness Programme "COMPETE" (FCOMP-01-0124-FEDER-014758) and national funds through the Portuguese Foundation for Science and Technology (FCT) under the project PTDC/EBB-BIO/114523/2009. The authors also thank a start-up grant provided by the School of Engineering and Materials Science at QMUL. D.S.F. gratefully acknowledges FCT for the PhD scholarship (SFRH/BD/44977/2008)

Seprase: An overview of an important matrix serine protease

Seprase or Fibroblast Activation Protein (FAP) is an integral membrane serine peptidase, which has been shown to have gelatinase activity. Seprase has a dual function in tumour progression. The proteolytic activity of Seprase has been shown to promote cell invasiveness towards the ECM and also to support tumour growth and proliferation. Seprase appears to act as a proteolytically active 170-kDa dimer, consisting of two 97- kDa subunits. It is a member of the group type II integral serine proteases, which includes dipeptidyl peptidase IV (DPPIV/CD26) and related type II transmembrane prolyl serine peptidases, which exert their mechanisms of action on the cell surface. DPPIV and Seprase exhibit multiple functions due to their abilities to form complexes with each other and to interact with other membrane-associated molecules. Localisation of these protease complexes at cell surface protrusions, called invadopodia, may have a prominent role in processing soluble factors and in the degradation of extracellular matrix components that are essential to the cellular migration and matrix invasion that occur during tumour invasion, metastasis and angiogenesis

Streetsport: Supporting and facilitating the development of enhanced graduate attributes.

Streetsport is a programme that aims to exercise social innovation by reducing instances of youth crime and anti-social behaviour; whilst promoting health and wellbeing through sport, physical activity and creative endeavour. As a vehicle for delivery the initiative facilitates work based educational experiences that are embedded within disadvantaged communities; supporting the development of enhanced graduate attributes by way of collaborative teaching and learning support. Adopting a collaborative partnership model, the programme brings together multiple courses, students and stakeholders to work within communities resulting in activities that react and respond to local needs, interests and demand

Vacunación por elección contra COVID-19 por la comunidad mexicana

The COVID-19 pandemic is the defining global health crisis of our time, it is the greatest challenge that has been faced in a long time. This is why health organizations work hard to find a vaccine that reduces the impact of this virus. Objective. Describe the opinion of the public about the COVID-19 vaccine to generate an overview of the community\u27s knowledge, the impact it will have, as well as the preventive health areas that will support the community to be convinced of applying the vaccine. Method. A descriptive, cross-sectional observational study was carried out by applying a survey in Google forms to men and women from the states of Hidalgo, Guerrero, Puebla and Mexico to 150 people with an age range of 15-80 years. Results. 29.33% would not apply the vaccine against COVID-19 mainly due to fear of adverse consequences and lack of knowledge, on the other hand 70.67% indicated that they would apply it because they trust the advances of science in the area Of the health. Conclusion. It is necessary to implement more knowledge in the Mexican population to generate interest in health advances, as is the vaccine for covid-19, otherwise the results will be negative.La pandemia por COVID-19 es la crisis de salud mundial que define nuestro tiempo, es el mayor desafío que se ha enfrentado en mucho tiempo. Es por esto que las organizaciones de salud trabajan duro para encontrar una vacuna que reduzca el impacto de este virus. Objetivo. Describir la opinión del público sobre la vacuna COVID-19 para generar un panorama del conocimiento de la comunidad, del impacto que esta tendrá, así como de las áreas de salud preventiva que servirán de apoyo para que la comunidad esté convencida de aplicarse la vacuna. Método. Se realizó un estudio observacional descriptivo de carácter transversal mediante la aplicación de una encuesta en Google forms a hombres y mujeres de los estados de Hidalgo, Guerrero, Puebla y México a 150 personas con un rango de edad de 15-80 años. Resultados. El 29.33% no se se aplicaría la vacuna contra COVID-19 principalmente por el miedo a consecuencias adversas y la falta de conocimiento, en cambio un 70.67% indicó que si se la aplicaría debido a que confían en los avances de la ciencia en el área de la salud. Conclusión. Es necesario implementar más conocimiento en la población mexicana para generar interés sobre los avances de la salud, así como lo es la vacuna para el covid-19, de lo contrario los resultados serán negativos

Structural and Functional Diversity of the Microbial Kinome

The eukaryotic protein kinase (ePK) domain mediates the majority of signaling and coordination of complex events in eukaryotes. By contrast, most bacterial signaling is thought to occur through structurally unrelated histidine kinases, though some ePK-like kinases (ELKs) and small molecule kinases are known in bacteria. Our analysis of the Global Ocean Sampling (GOS) dataset reveals that ELKs are as prevalent as histidine kinases and may play an equally important role in prokaryotic behavior. By combining GOS and public databases, we show that the ePK is just one subset of a diverse superfamily of enzymes built on a common protein kinase–like (PKL) fold. We explored this huge phylogenetic and functional space to cast light on the ancient evolution of this superfamily, its mechanistic core, and the structural basis for its observed diversity. We cataloged 27,677 ePKs and 18,699 ELKs, and classified them into 20 highly distinct families whose known members suggest regulatory functions. GOS data more than tripled the count of ELK sequences and enabled the discovery of novel families and classification and analysis of all ELKs. Comparison between and within families revealed ten key residues that are highly conserved across families. However, all but one of the ten residues has been eliminated in one family or another, indicating great functional plasticity. We show that loss of a catalytic lysine in two families is compensated by distinct mechanisms both involving other key motifs. This diverse superfamily serves as a model for further structural and functional analysis of enzyme evolution

Insights into the Complex Formed by Matrix Metalloproteinase-2 and Alloxan Inhibitors: Molecular Dynamics Simulations and Free Energy Calculations

Matrix metalloproteinases (MMP) are well-known biological targets implicated in tumour progression, homeostatic regulation, innate immunity, impaired delivery of pro-apoptotic ligands, and the release and cleavage of cell-surface receptors. Hence, the development of potent and selective inhibitors targeting these enzymes continues to be eagerly sought. In this paper, a number of alloxan-based compounds, initially conceived to bias other therapeutically relevant enzymes, were rationally modified and successfully repurposed to inhibit MMP-2 (also named gelatinase A) in the nanomolar range. Importantly, the alloxan core makes its debut as zinc binding group since it ensures a stable tetrahedral coordination of the catalytic zinc ion in concert with the three histidines of the HExxHxxGxxH metzincin signature motif, further stabilized by a hydrogen bond with the glutamate residue belonging to the same motif. The molecular decoration of the alloxan core with a biphenyl privileged structure allowed to sample the deep S1′ specificity pocket of MMP-2 and to relate the high affinity towards this enzyme with the chance of forming a hydrogen bond network with the backbone of Leu116 and Asn147 and the side chains of Tyr144, Thr145 and Arg149 at the bottom of the pocket. The effect of even slight structural changes in determining the interaction at the S1′ subsite of MMP-2 as well as the nature and strength of the binding is elucidated via molecular dynamics simulations and free energy calculations. Among the herein presented compounds, the highest affinity (pIC50 = 7.06) is found for BAM, a compound exhibiting also selectivity (>20) towards MMP-2, as compared to MMP-9, the other member of the gelatinases