ISSUES IN BIOTECHNOLOGY TEACHING - Teaching of Biotechnology in the Biochemistry Course

Biotechnology is taught in the Biochemistry course of the Faculty of Science of the Lisbon University during the last year of the course divide into two semesters. These two disciplines are optional with other disciplines that the students may choose in order to complete their knowledge in Biochemistry. Both disciplines have two credits, one for theory and the other for laboratory: Applied Biochemistry, comes in the first semester and Immobilized Biocatalysts in the second. Each discipline is taught during 13 weeks. The main goal of these two disciplines in the Biochemistry course is to give the students an applied vision, devoted to the industry, of the Biochemistry they have been taught during the previous three years

Bioactivities of centaurium erythraea (Gentianaceae) decoctions: antioxidant activity, enzyme inhibition and docking studies

Centaurium erythraea is recommended for the treatment of gastrointestinal disorders and to reduce hypercholesterolemia in ethno-medicinal practice. To perform a top-down study that could give some insight into the molecular basis of these bioactivities, decoctions from C. erythraea leaves were prepared and the compounds were identified by liquid chromatography-high resolution tandemmassspectrometry(LC–MS/MS).Secoiridoidsglycosides,likegentiopicrosideandsweroside, and several xanthones, such as di-hydroxy-dimethoxyxanthone, were identified. Following some of the bioactivities previously ascribed to C. erythraea, we have studied its antioxidant capacity and the ability to inhibit acetylcholinesterase (AChE) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Significant antioxidant activities were observed, following three assays: free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) reduction; lipoperoxidation; and NO radical scavengingcapacity. TheAChEandHMGRinhibitoryactivitiesforthedecoctionwerealsomeasured (56% at 500 µg/mL and 48% at 10 µg/mL, respectively). Molecular docking studies indicated that xanthones are better AChE inhibitors than gentiopicroside, while this compound exhibits a better shape complementarity with the HMGR active site than xanthones. To the extent of our knowledge, thisisthefirstreportonAChEandHMGRactivitiesbyC.erythraeadecoctions,inatop-downanalysis, complemented with in silico molecular docking, which aims to understand, at the molecular level, some of the biological effects ascribed to infusions from this plant.info:eu-repo/semantics/publishedVersio

Edible seaweeds extracts: characterization and functional properties for health conditions

Seaweeds are popular foods due to claimed beneficial health effects, but for many there is a lack of scientific evidence. In this study, extracts of the edible seaweeds Arame, Nori, and Fucus are compared. Our approach intends to clarify similarities and differences in the health properties of these seaweeds, thus contributing to target potential applications for each. Additionally, although Arame and Fucus seaweeds are highly explored, information on Nori composition and bioactivities is scarce. The aqueous extracts of the seaweeds were obtained by decoction, then fractionated and characterized according to their composition and biological activity. It was recognized that fractioning the extracts led to bioactivity reduction, suggesting a loss of bioactive compounds synergies. The Arame extract showed the highest antioxidant activity and Nori exhibited the highest potential for acetylcholinesterase inhibition. The identification of the bioactive compounds in the extracts allowed to see that these contained a mixture of phloroglucinol polymers, and it was suggested that Nori's effect on acetylcholinesterase inhibition may be associated with a smaller sized phlorotannins capable of entering the enzyme active site. Overall, these results suggest a promising potential for the use of these seaweed extracts, mainly Arame and Nori, in health improvement and management of diseases, namely those associated to oxidative stress and neurodegeneration.info:eu-repo/semantics/publishedVersio

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Molecular-level changes induced by hydroxycinnamic acid derivatives in HepG2 cell line: comparison with pravastatin

Hydroxycinnamic acid derivatives are an important class of polyphenols found in fruits, vegetables, and medicinal plants and widely consumed in human diet. In the present work, alterations of HepG2 cells biochemical profile under the effect of four hydroxycinnamic acid derivatives (caffeic acid, m-coumaric acid, chlorogenic acid and rosmarinic acid) relatively to the effect of pravastatin, a drug often prescribed to inhibit HMG-CoA reductase enzyme, the regulator enzyme in the cholesterol biosynthesis pathway, were reported. The application of FTIR spectroscopy in combination with multivariate analysis by PCA showed a similarity between pravastatin and the four hydroxycinnamic acid derivatives in metabolite profile modification expressed by various changes in proteins region, the phosphate region which mainly corresponds to nucleic acids as well as in lipids regions. FTIR structural analysis in the amide I region, using resolution enhancement methods, such as second derivative and amide I deconvolution method, revealed significant decrease in alpha-helix/random coil and intermolecular beta-sheet decreased while intramolecular beta-sheet in treated cells showed an increase. It was also noticed that the intracellular cholesterol as well as esterified ingredients such as cholesterol esters in the cell membrane decreased. Moreover, principal component analysis (PCA) of the spectral data showed that the compounds and pravastatin were well separated from untreated cells showing a different mode of action on HepG2 treated cells for each compound.info:eu-repo/semantics/publishedVersio

Estudos de actividade e estabilidade da #alpha#-Quimotripsina em micelas invertidas para sintese de dipeptidos em reactores enzimaticos

The synthesis of the dipeptide derivative acetylphenylalanineleucinamide (AcPheLeuNH_2) in reverse micelles of the cationic surfactant tetradecyltrimethylammonium bromide (TTAB) in heptane/octanol was studied. #alpha#-Chymotrypsin (#alpha#-CT) in the native and the chemically modified forms was used as bio catalyst. The chemical modification was carried out by hydrophilization of the lysine residues. The influence of several parameters (the chain length of the alcohol, used as co-surfactant, the temperature, the buffer molarity and the ph and the substrate concentrations) on the initial reaction velocities of peptide synthesis and co-products was evaluated. The system was optimized for both the native and the modified enzymes. The #alpha#-CT stability in reverse micelles, together with its stability in co-solvents and buffer system was studied. The native enzyme showed higher stability in reverse micellar system. The chemical modification did not increase the enzyme stability in the reverse micellar system but produced an enzyme that once catalysing reactions in the co-solvent or in the buffer systems could stand higher temperatures, comparatively to the native form. The hydrolysis of the peptidic chain and the cleavage of S-S bonds can be counted among the possible mechanisms of deactivation in the buffer and in the co solvent systems. The reaction of peptide synthesis catalysed by #alpha#-CT in reverse micelles, was also carried out in a membrane reactor. The reactor could be operated continuously during at least one week, without a significant loss of enzyme activity. The system allows the production of peptide with simultaneous separation from the reaction by-products, as the former accumulates inside the reactor and the latter pass through the membrane. A kinetic model was developed for both reactors, based on an empirical model obtained from the experimental results. The kinetic model allowed to predict the reaction yields and the reactor productivityAvailable from Fundacao para a Ciencia e a Tecnologia, Servico de Informacao e Documentacao, Av. D. Carlos I, 126, 1249-074 Lisboa, Portugal / FCT - Fundação para o Ciência e a TecnologiaSIGLEPTPortuga

BioMol4Health_Biological chemistry: longevity in a cup of tea

Infusions have been studied on what concerns Alzheimer Disease, digestive process, diet cholesterol absorption and its biosynthesis inhibition. In the first two cases the inhibition of acetylcholinesterase (AChE) has been addressed. In the last two situations, an in vitro intestinal barrier has been simulated and the inhibition of the regulator enzyme (HMGR) in cholesterol biosysnthesis pathway has been studied. AChE has been the target of infusions inhibitory activity as its inhibition has been seen to improve cognition and global functioning1 in AD suffering people and to improve the gastrointestinal motility2. Given to lab animals the compounds presente in the infusions were able to reach the brain and inhibit the enzyme3. The effect of infusions on cholesterol bioavailability pointed out that some infusions were able to reduce cholesterol permeation4 and also to have some inhibitory activity5. Studies have indicated that phenolics are able to modify the cell proteome6. The infusions have also been shown to modify the amount of cholesterol transporter proteins in cell membrane and this maybe one of possible explanations for the reduction in cholesterol transport detected under the effect of infusions, on some people ando n simulated intestinal barrier.info:eu-repo/semantics/publishedVersio

Cholesterol transporter proteins in HepG2 cells can be modulated by phenolic compounds present in Opuntia ficus-indica aqueous solutions

Increased blood cholesterol is a risk factor for atherosclerotic cardiovascular disease. This study tested the hypothesis that phenolic compounds can modulate the level of cholesterol transporters including Niemann-Pick C1-like 1 (NPC1L1) and ATP-binding cassette transporters in liver cells. HepG2 cells, used as a model of hepatocytes, showed a decrease in the abundance of cholesterol transporters comparatively to the control when treated with the Opuntia ficus-indica's cladodes decoction. The decrease was between 13-70%, 25-60%, 9-60% and 23-60% for NPC1L1, ABCA1, ABCG5 and ABCG8 transporters, respectively, when using between 0.15 and 0.35 mg/mL of decoction in the culture medium. FTIR analysis showed changes in the amount of RNA, which may be the cause of the decrease in the level of several proteins. These in vitro results pave the way to a molecular explanation for the decoction of cladodes effect on cholesterol levels as it reduced the membrane cholesterol transporter proteins, NPC1L1, ABCG5/ABCG8 and ABCA1, in HepG2 cells.info:eu-repo/semantics/publishedVersio

Cork processing wastewaters components fractioned by ultrafiltration membranes–studies of antioxidant and antitumoral activity

BACKGROUND: Cork processing wastewater is an environmental problem due to its high content of organic matter, such as sugars and non-biodegradable compounds such as polyphenols (PPs), namely tannins. Membrane technology aimed at valorisation of the wastewaters components, concentrate stream, and simultaneously offer a pre-treatment of the wastewater, permeate stream. In this work, the identification of bioactive PPs was also envisaged. Several fractions of cork processing wastewaters were generated using two cellulose acetate ultrafiltration membranes of 3 kDa and 74 kDa for PPs isolation according to nature and molecular weights. The membranes were prepared by the phase inversion method and fractionation was made in concentration and diafiltration modes. The wastewater and the fractions were analyzed in terms of total organic carbon, total phenols, tannins and total polysaccharides content and the compounds present were identified by FTIR, LC–MS and quantified by HPLC-DAD. RESULTS: Compounds such as quinic, gallic, protocatechuic, brevifolin carboxylic and ellagic acids were identified as the major compounds in cork wastewater. The wastewater and the fractions were tested for antioxidant activity and for capacity to inhibit the proliferation of the growth of human breast carcinoma cell lines, MCF-7. The fractions revealed high antioxidant activity with EC 50 values ranging from 1.174 ± 0.069 to 1.943 ± 0.179 mg/mgDPPH. The fractions demonstrated to be efficient as cell proliferation inhibitors, with values of IC50 ranging from 0.20 ± 0.003 to 0.46 ± 0.02 mg mL-1. CONCLUSION: The process reported demonstrates that PPs compounds can be recovered from cork effluent and further reused as high-value bioactive compounds.info:eu-repo/semantics/publishedVersio

Mechanism of action and the biological activities of Nigella sativa oil components

Nigella sativa seeds oil is consumed because of its health benefits. Although the oil has been studied for its properties and composition, these effects are often associated with its major compound thymoquinone, and less has been reported about the other components' contributions to the pharmacological properties and mechanism of action. The present study aimed to evaluate the oil compounds' antioxidant and anti-tumour activities, and its effect on molecular targets associated with the claimed health effects. A wide a range of biochemical and biophysical techniques were used to understand the biological activities of the oil components. Compounds such as thymoquinone and volatile monoterpenes such as alpha-thujene, alpha-pynene, sabinene and 3-carene were identified by using GC-MS. Similar high antioxidant activities were obtained for the oil and its non-volatile (NV) fraction, with enzymes inhibition and cytotoxic activity against MCF-7 and A375 cells the oil showed a strong effect but not its NV fraction. Analysis of A375 cells' proteome in contact with the oil showed increased expression of proteins POTEF and HSP 90-b suggesting that the oil's antitumor effect was due to apoptosis and cellular stress. FT-IR studies in two model proteins in contact with the oil allowed oil compounds-protein interactions to be recognized and also showed that volatiles stabilized proteins. Therefore, not only thymoquinone but also the volatile monoterpenes were important for the diversity of properties, and therefore modifications in the chemical composition of the oil, as an outcome of storage or even food preparation, may affect its biological activity.info:eu-repo/semantics/publishedVersio