Implementation of a Practical Teaching Course on Protein Engineering

Simple Summary Proteins are the workhorses of the cell. With different combinations of the 20 common amino acids and some modifications of these amino acids, proteins have evolved with a staggering array of new functions and capabilities due to Protein Engineering techniques. The practical course presented was offered to undergraduate bioengineering and chemical students at the Faculty of Engineering of the University of Porto (Portugal) and consists of sequential laboratory sessions to learn the basic skills related to the expression and purification of recombinant proteins in bacterial hosts. These experiments were successfully applied by students as all working groups were able to isolate a model recombinant protein (the enhanced green fluorescent protein) from a cell lysate containing a mixture of proteins and other biomolecules produced by an Escherichia coli strain and evaluate the performance of the extraction and purification procedures they learned. Protein Engineering is a highly evolved field of engineering aimed at developing proteins for specific industrial, medical, and research applications. Here, we present a practical teaching course to demonstrate fundamental techniques used to express, purify and analyze a recombinant protein produced in Escherichia coli-the enhanced green fluorescent protein (eGFP). The methodologies used for eGFP production were introduced sequentially over six laboratory sessions and included (i) bacterial growth, (ii) sonication (for cell lysis), (iii) affinity chromatography and dialysis (for eGFP purification), (iv) bicinchoninic acid (BCA) and fluorometry assays for total protein and eGFP quantification, respectively, and (v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for qualitative analysis. All groups were able to isolate the eGFP from the cell lysate with purity levels up to 72%. Additionally, a mass balance analysis performed by the students showed that eGFP yields up to 46% were achieved at the end of the purification process following the adopted procedures. A sensitivity analysis was performed to pinpoint the most critical steps of the downstream processing

Susceptibility Amplitude Ratios Near a Lifshitz Point

The susceptibility amplitude ratio in the neighborhood of a uniaxial Lifshitz point is calculated at one-loop level using field-theoretic and $\epsilon_{L}$-expansion methods. We use the Schwinger parametrization of the propagator in order to split the quadratic and quartic part of the momenta, as well as a new special symmetry point suitable for renormalization purposes. For a cubic lattice (d = 3), we find the result $\frac{C_{+}}{C_{-}} = 3.85$.Comment: 7 pages, late

Recent advances in antimicrobial surfaces for urinary catheters

Although urinary catheters (UCs) are one of the most used medical devices, they are related to a high incidence of urinary tract infections resulting from microbial colonization and consequent biofilm development on UC surfaces. Currently, a panoply of antimicrobial and antifouling surfaces is available to solve this longstanding problem. However, despite their high performance, these surfaces are still far from clinical application. In this current opinion article, we evaluate and critically discuss the antimicrobial performance and applicability of UC surfaces with different antibiofilm mechanisms. It is our opinion that either killing or anti-adhesive coatings are promising in controlling infection development. However, most of them are more effective in delaying microbial adhesion rather than preventing it

Hospital-acquired pneumonia is more frequent and lethal in stroke patients: A nationwide 4-year study

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Increased intraspecies diversity in Escherichia coli biofilms promotes cellular growth at the expense of matrix production

Intraspecies diversity in biofilm communities is associated with enhanced survival and growth of the individual biofilm populations. Studies on the subject are scarce, namely, when more than three strains are present. Hence, in this study, the influence of intraspecies diversity in biofilm populations composed of up to six different Escherichia coli strains isolated from urine was evaluated in conditions mimicking the ones observed in urinary tract infections and catheter-associated urinary tract infections. In general, with the increasing number of strains in a biofilm, an increase in cell cultivability and a decrease in matrix production were observed. For instance, single-strain biofilms produced an average of 73.1 µg·cm-2 of extracellular polymeric substances (EPS), while six strains biofilms produced 19.9 µg·cm-2. Hence, it appears that increased genotypic diversity in a biofilm leads E. coli to direct energy towards the production of its offspring, in detriment of the production of public goods (i.e., matrix components). Apart from ecological implications, these results can be explored as another strategy to reduce the biofilm burden, as a decrease in EPS matrix production may render these intraspecies biofilms more sensitive to antimicrobial agents.This work was financially supported by Base Funding—UIDB/00511/2020 of the Laboratory for Process Engineering, Environment, Biotechnology and Energy—LEPABE—funded by national funds through the FCT/MCTES (PIDDAC); Project POCI-01-0145-FEDER-030431 (CLASInVivo) and project POCI-01-0145-FEDER-029841 (POLY-PREVENTT), funded by FEDER funds through COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI) and by national funds (PIDDAC) through FCT/MCTES; Strategic funding of UIDB/04469/2020 of the Centre of Biological Engineering–CEB–funded by national funds through the FCT; Project BeMundus Brazil Europe/Erasmus Mundus scholarship granted by BM13DF0014

A synthetic biology approach to engineer "therapeutic" bacteria

The high incidence and mortality of solid tumors like breast cancer makes the development of novel therapeutic agents a high priority. Curcumin, a natural substance from the rhizome of Curcuma longa, has captured the attention of the scientific community. Pre-clinical trials and extensive research has demonstrated its ability to prevent cancer. Indeed, curcumin has been shown to target critical genes involved in angiogenesis, apoptosis, cell cycle and metastasis, and consequently to inhibit cell growth. Currently, the clinical use of curcumin is mainly limited by its poor bioavailability which implies repetitive oral doses in order to achieve the therapeutic concentrations inside the cell. The idea of the present work is to design a strategy that could link the common technique used to treat solid tumors (ultrasound) with the therapeutic effects of curcumin. The plan is to use the temperature increase (consequence of ultrasound treatment) to trigger the in situ expression of curcumin by engineered bacteria. Escherichia coli was chosen as the model organism in which the genes involved in the curcumin pathway will be cloned. Those genes (4-coumarate: CoA ligase, diketide-CoA synthase and curcumin synthase) were successfully cloned under the control of a temperature sensitive promoter (dnaK). The proof-of-concept that the dnaK promoter can be induced by a temperature increase, leading to the expression of the 3 necessary genes, is currently being tested, using several biochemical assays. Moreover, several knockouts (KO) of specific genes from the E. coli K-12 MG1655 genome were performed in order to maximize the production of curcumin. The deletion strategy, as well as the definition of the non-essential genes to be KO, was determined in silico. This strategy included one single KO (gnd gene) and the multiple KO of five non-essential genes for aerobic growth (fumA, fumB, fumC, ccmA and argO) and serA gene for anaerobic growth. After optimizing the genes expression under the control of the temperature inducible promoter, the several KO will be transformed with this construction to confirm the improvement of curcumin production

A new picture of the Lifshitz critical behavior

New field theoretic renormalization group methods are developed to describe in a unified fashion the critical exponents of an m-fold Lifshitz point at the two-loop order in the anisotropic (m not equal to d) and isotropic (m=d close to 8) situations. The general theory is illustrated for the N-vector phi^4 model describing a d-dimensional system. A new regularization and renormalization procedure is presented for both types of Lifshitz behavior. The anisotropic cases are formulated with two independent renormalization group transformations. The description of the isotropic behavior requires only one type of renormalization group transformation. We point out the conceptual advantages implicit in this picture and show how this framework is related to other previous renormalization group treatments for the Lifshitz problem. The Feynman diagrams of arbitrary loop-order can be performed analytically provided these integrals are considered to be homogeneous functions of the external momenta scales. The anisotropic universality class (N,d,m) reduces easily to the Ising-like (N,d) when m=0. We show that the isotropic universality class (N,m) when m is close to 8 cannot be obtained from the anisotropic one in the limit d --> m near 8. The exponents for the uniaxial case d=3, N=m=1 are in good agreement with recent Monte Carlo simulations for the ANNNI model.Comment: 48 pages, no figures, two typos fixe

Oriented immobilization of Pep19-2.5 on antifouling brushes suppresses the development of Staphylococcus aureus biofilms

Bacterial colonization of indwelling medical devices poses a danger to the patient and is a tremendous economic burden that costs billions of dollars to the healthcare system. Thus, it is essential to develop an effective mechanism that prevents the attachment of bacteria to the surface in combination with bactericidal strategies to kill them in direct contact. In this work, we combine the repellent/antifouling properties of polymer brushes with the antimicrobial activity of the synthetic peptide Pep19-2.5 and test its efficacy to inhibit Staphylococcus aureus biofilm formation. To tackle this, we utilized hierarchical polymer brushes, where the bottom block provides an effective barrier against adhesion while the top block provides functional groups for the immobilization of active molecules. Further, these polymer brushes were decorated with dibenzocyclooctine (DBCO)-functionalized Pep19-2.5 using strain-promoted alkyne-azide cycloaddition (SPAAC). This click chemistry proceeds very fast and does not require any catalyst, which is crucial for biomedical applications. The obtained coating was not only able to decrease the number of freely planktonic bacteria in the surrounding media (by 52.5%) but also inhibit the development of S. aureus biofilm by reducing the number of total, viable, and viable but non-culturable (VBNC) cells (up to 58%, 66%, and 70% respectively) and reduce the biovolume and thickness. Conversely, this coating does not exert any cytotoxic effect on Normal Human Dermal Fibroblasts (NHDF) cells. Thus, the combination of hierarchical polymer brushes with Pep19-2.5 is a promising approach to fight medical biofilms without affecting surrounding tissues

Production and Characterization of Graphene Oxide Surfaces against Uropathogens

Graphene and its functionalized derivatives have been increasingly applied in the biomedical field, particularly in the production of antimicrobial and anti-adhesive surfaces. This study aimed to evaluate the performance of graphene oxide (GO)/polydimethylsiloxane (PDMS) composites against Staphylococcus aureus and Pseudomonas aeruginosa biofilms. GO/PDMS composites containing different GO loadings (1, 3, and 5 wt.%) were synthesized and characterized regarding their morphology, roughness, and hydrophobicity, and tested for their ability to inhibit biofilm formation under conditions that mimic urinary tract environments. Biofilm formation was assessed by determining the number of total and culturable cells. Additionally, the antibacterial mechanisms of action of GO were investigated for the tested uropathogens. Results indicated that the surfaces containing GO had greater roughness and increased hydrophobicity than PDMS. Biofilm analysis showed that the 1 wt.% GO/PDMS composite was the most effective in reducing S. aureus biofilm formation. In opposition, P. aeruginosa biofilms were not inhibited by any of the synthesized composites. Furthermore, 1% (w/v) GO increased the membrane permeability, metabolic activity, and endogenous reactive oxygen species (ROS) synthesis in S. aureus. Altogether, these results suggest that GO/PDMS composites are promising materials for application in urinary catheters, although further investigation is required.</p

Selección de proveedor de servicios logísticos: alineación entre criterios e indicadores

Este artículo tiene por objetivo identificar los criterios de selección de proveedores de servicios logísticos, sus respectivos indicadores de desempeño y su alineación en los procesos de selección y evaluación de proveedores en el sector de comercio minorista. Se realizó un estudio de múltiples casos en un operador logístico y en cuatro consignatarios, por medio de entrevistas, informes de indicadores de desempeño y observaciones directas. Los resultados muestran que son pocos los criterios comunes; el “costo de flete” es usado por todas las empresas, y los criterios relacionados a la calidad de los servicios prestados no son muy gestionados. Además, existe poca alineación entre criterios e indicadores, lo que apunta a que las empresas no adoptan mecanismos de evaluación de los proveedores de servicios logísticos.This article aims to identify the criteria for selecting logistics service providers, their performance indicators and their alignment in the selection and evaluation processes for suppliers in the retail sector. A study of multiple cases was carried out in a logistics operator and four consignees through interviews, reports of performance indicators and direct observations. General results show that few common criteria are considered; the “cost of freight” is used by all companies; and criteria related to the quality of the services provided are not often managed. Moreover, there is little alignment between criteria and indicators, which suggests that companies do not adopt evaluation mechanisms for logistics service providers.Este artigo tem como objetivo identificar os critérios de seleção de fornecedores de serviços logísticos, seus respectivos indicadores de desempenho e seu alinhamento nos processos de seleção e avaliação de fornecedores no setor varejista. Realizou-se um estudo de múltiplos casos num operador logístico e em quatro consignatários por meio de entrevistas, relatórios de indicadores de desempenho e observações diretas. Os resultados mostram que são poucos os critérios comuns; o “custo do frete” é usado por todas as empresas, e os critérios relacionados com a qualidade dos serviços prestados não são muito gerenciados. Além disso, existe pouco alinhamento entre critérios e indicadores, o que indica que as empresas não adotam mecanismos de avaliação dos fornecedores de serviços logísticos