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

Structure evolution during deformation of novel polymer systems based on poly(ethylene terephthalate) reinforced with inorganic particles

Inorganic particles have been incorporated into poly(ethylene terephthalate), PET, matrix with the aim of offering some aesthetic effects to the standard transparent beverage packaging. To assess to the effect of different type of inorganic particles (with different interaction with the polymeric matrix) in the deformation behaviour of PET composites, particles such as aluminium, bronze, nanoclay, graphite and mica, were used. Two types of processing methods, ensuring a good mixing, were adopted for the composite preparation: i) melt-blending in an asymmetric batch mini-mixer followed by compression and ii) melt-blending in a twin screw extruder followed by injection moulding. The structural and mechanical behaviours of neat PET and its composites were evaluated during uniaxial stretching at temperatures above the glass transition temperature, Tg, namely at 85, 90, 95, 100 ºC, using a standard tensile stretching machine equipped with an environmental chamber. This study aims at understanding the deformation behaviour of the materials at temperatures found in conventional transformation methods used for packaging. To interpret the structural evolution occurring in the samples, off-line techniques such as DSC, WAXD and SEM were used. A detailed structural evolution was accomplished by applying different stretching ratios to the samples up to a maximum of 5x. Injection moulded samples were also characterized for their mechanical properties at room temperature in order to assess the materials behaviour in service. From all particles studied the PET/Aluminium and PET/Bronze composites are the ones showing the best combination of aesthetic effects, deformation behaviour and final mechanical properties. Comparing to neat PET, their general properties are the same during the deformation process making the new materials suitable for conventional processing techniques. It was found that both particles offer superior molecular mobility to PET at room temperature. In other words, these composites present a higher deformation capacity, however its elastic modulus and tensile strength at yield are slightly reduced

Synthesis and Characterization of Water Stable η3-Allyl Dicarbonyl Complexes of Molybdenum(II)

Trabalho apresentado nas VIII Jornadas CICECO, 18-19 de março 2011, Aveiro, PortugalN/

Molybdenum(II) Diiodo-Tricarbonyl Complexes Containing Nitrogen Donor Ligands as Catalyst Precursors for the Epoxidation of Methyl Oleate

Resumo alargado de "Molybdenum(II) Diiodo-Tricarbonyl Complexes Containing Nitrogen Donor Ligands as Catalyst Precursors for the Epoxidation of Methyl Oleate"Com o apoio RAADRI

Rare Case of Abdominal Aortic and Multiple Visceral Aneurysms in a Pediatric Patient With PIK3CA Mutation and Vasculitis

Abdominal aortic aneurysms (AAA) are most commonly observed in elderly male patients and are particularly rare in children. Among the pediatric population, they are usually diagnosed in the context of connective tissue disorders, genetic mutations, or vasculitis. The same is true of visceral arteries aneurysms. This case report describes the staged management of an 11-year-old patient presenting PIK3CA mutation and a 5.8 cm infrarenal AAA associated with bilateral common iliac arteries and multiple visceral aneurysms, the largest observed in the superior mesenteric artery (SMA = 3.2 cm). After careful evaluation, decision was made to first approach the most life-threatening lesion (the infrarenal AAA due to the large diameter) and the remaining aneurysms in secondary procedures, with special attention to the SMA aneurysm. The patient underwent a staged repair, with the first phase consisting of an aortobi-iliac graft with the distal anastomosis made at the left common iliac artery and right external iliac artery. The right hypogastric artery was ligated. The second procedure consisted of SMA aneurysm repair with a plication technique, as 7 branches were visualized coming off the aneurysm sac. Postoperative pathology analysis of the aortic and SMA aneurysms sac revealed vasculitis with a mixed inflammatory pattern and a COL3A1 gene heterozygote variant. He is currently in his 18th month after the last surgical intervention, receiving immunomodulatory therapy, with a planned follow-up by the interdisciplinary team to monitor the medications’ side effects and the diameter of the remaining visceral aneurysms

Antispasmodic activity of aqueous extracts from Mentha x piperita native from Trás-os-Montes region (Portugal)

The wild mint Mentha x piperita (Mentha rotundifolia Huds.), has been used by the local people in a northern region of Portugal to prepare infusions for treating digestive pain and spasms, as an appetite stimulant and for treating headache and migraine. This suggests that these aqueous extracts have analgesic; antispasmodic and stomachic properties. In the present study the antioxidant potential of aqueous extracts of Mentha x piperita is investigated since natural antioxidants can scavenge the reactive oxygen species, ROS, and thus might attenuate inflammation pathways. The antispasmodic activity was also checked. The antioxidant potential of the extract was evaluated by the DPPH* method, by the quantification of the total phenolic compounds and by characterization of the main phenolic compounds. The antispasmodic effects were investigated by performing pharmacological assays using the distal ileum of guinea pigs. The aqueous extract exhibits antioxidant properties that may be due to its phenolic content. The main phenolic compounds were quinic, caffeic, rosmarinic and chlorogenic acids. The antispasmodic affects are observed after electrical stimulation of segments of distal ileum of guinea pigs and may be attributed, at least, to a alfa1 blockage

Nanoencapsulation of bovine lactoferrin for food and biopharmaceutical applications

Lactoferrin has for long captured the interest of many researchers as a natural compound with a wide variety of uses. Lactoferrin is a monomeric, iron-binding 80 kDa glycoprotein, and appears to be the subfraction of whey with the best documented antiviral, antimicrobial, anticancer and immune modulating/enhancing effects. It belongs to the family of transferrin proteins, and serves to control iron levels in body fluids by sequestering and solubilizing ferric iron. In the present research effort, production of lactoferrin derivatives (starting from a purified commercial extract), encompassing full stabilization of its three-dimensional structure, has been attempted via nanoencapsulation within lipid nanovesicles, integrating a multiple water-in-oil-in-water emulsion. Long-term storage of the multiple nanoemulsions produced did not lead to leaching of protein, thus proving the effectiveness of the encapsulation procedure. Furthermore, lactoferrin nanovesicle derivatives prepared under optimal conditions were successfully employed at lab-scale antimicrobial trials.Financial support from Fundacao Ensino e Cultura Fernando Pessoa (FECFP, Porto, Portugal) and from Fundacao para a Ciencia e a Tecnologia (FCT, Lisbon, Portugal) as pluriannual funding, is gratefully acknowledged. Financial support to Victor M. Balcao, via an Invited Research Scientist fellowship (FAPESP Ref. No. 2011/51077-8) by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP, Sao Paulo, Brazil), is hereby gratefully acknowledged. The authors are also grateful to Dr. Madalena Vieira (affiliated with the IBB) for technical help

Synthesis, Characterization and Catalytic application of Water Stable η3-Allyl Dicarbonyl Complexes of Molybdenum(II)

Trabalho apresentado em EuropaCat X – Catalysis across disciplines, 28 Aug - 02 Sep 2011, Glasgow, ScotlandN/