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Institute for Bioengineering and Biosciences
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Cellulose-based LFA for Detection of Cystatin C

Cellulose-based LFA for Detection of Cystatin C | iBB | Scoop.it

The relevance and impact of Lateral Flow Assays (LFA) as point-of-care devices has been amply demonstrated with the current SARS-CoV-2 pandemic. However, and despite their usefulness, drawbacks like low sensitivity, low specificity, and lack of quantitation are often associated with LFAs. In a paper published in Scientific Reports, Miguel Prazeres from BERG-IST and colleagues from the Indian Institute of Technology (Madras) propose a new LFA architecture that combines nitrocellulose strips with layered cellulose, ZZ-CBM3 fusions and fluorescently labeled Fab fragments. The system was successfully applied to the the quantitative, fluorescence-based detection of the kidney biomarker cystatin C. The work was funded by project CBM-X.

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Magnetic Particles for the Purification of DNA Scaffolds for Biomanufacturing DNA-Origami Nanostructures

Magnetic Particles for the Purification of DNA Scaffolds for Biomanufacturing DNA-Origami Nanostructures | iBB | Scoop.it

Asymmetric PCR (aPCR) is often used to generate single-stranded DNA (ssDNA) scaffolds, which can then be assembled into nanoobjects by the DNA-origami technique. The scaffolds are usually purified by agarose gel extraction, a laborious, time consuming, limited, and non-scalable technique that presents low recovery yields, delivers low-quality products. To overcome such pitfalls, Ana Silva-Santos, Rui Silva, Sara Rosa and Miguel Prazeres from BERG-iBB, in collaboration with Pedro Paulo from CQE developed a simple, fast, and potentially scalable affinity-based method comprising magnetic particles. Specifically, scaffolds were synthesized by aPCR and purifed using magnetic particles functionalized with a 20 nt oligonucleotide complementary to the 3′ end of the target. The purified scaffolds were used to assemble 31 and 63 bp edge length tetrahedra with short oligonucleotides and thermal annealing, demonstrating the potential of affinity-based magnetic beads in the production of DNA-origami nanostructures. The work was published in ACS Applied Nanomaterials.

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Functionalization of Cellulose Hydrogels with Carbohydrate-Binding Module Fusions

Functionalization of Cellulose Hydrogels with Carbohydrate-Binding Module Fusions | iBB | Scoop.it

Materials with novel and enhanced functionalities can be obtained by modifying cellulose with a range of biomolecules. In a recent paper published in Materials, Mariana Barbosa, Hélvio Simões and Miguel Prazeres from BERG-iBB describe the modification of cellulose hydrogels using bi-functional biomolecular constructs based on carbohydrate binding modules (CBMs). The binding of CBM fusions, the capture of antibodies and the grafting of oligonucleotides onto hydrogels was successfully demonstrated. The CBM-based platform could contribute significantly to the development of advanced medical diagnostic sensors or specialized biomaterials, among others. The work was funded by project CBM-X.

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GPCR Screening Assays: Methods and Protocols

GPCR Screening Assays: Methods and Protocols | iBB | Scoop.it

The book G Protein-Coupled Receptor Screening Assays: Methods and Protocols, edited by Sofia Martins from INESC-MN and Miguel Prazeres from BERG-iBB, has just been published. This fully updated edition targets not only those assays directly involved in the discovery of GPCR-active compounds but also those involved in cell-based experiments designed to study physiological responses. Whether coming from academia or industry, or being an experienced researcher or a newcomer to the field, the reader will find accessible methods and protocols that cover the latest developments on receptor purification, molecular biology, recombinant engineering, and analytical techniques that enable the real time monitoring of the complex GPCR signaling cascade and identification of potential drug targets. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

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Minicircle‐based Expression of VEGF in Mesenchymal Stromal Cells from Human Tissues

Minicircle‐based Expression of VEGF in Mesenchymal Stromal Cells from Human Tissues | iBB | Scoop.it

Mesenchymal stromal cells (MSC) have been exploited for the treatment of ischemic diseases given their angiogenic potential. A recent study published in the Journal of Gene Medicine by BERG and SCERG researchers compares the angiogenic potential of MSC obtained from bone marrow (BM), adipose tissue (AT) and umbilical cord matrix (UCM) that were genetically modified with VEGF‐encoding minicircle vectors. Transfected cells displayed higher in vitro angiogenic potential than non‐transfected controls, as demonstrated by functional in vitro assays, but no significant differences were observed among cells from different sources.

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mRNA Vaccine Manufacturing: Challenges and Bottlenecks

mRNA Vaccine Manufacturing: Challenges and Bottlenecks | iBB | Scoop.it

Vaccines are one of the most important tools in public health and play an important role in infectious diseases control. mRNA vaccines are reaching the stoplight as a new alternative to conventional vaccines due to their precision, safe profile and flexible manufacturing. In fact, the first Covid-19 vaccines to receive regulatory approval are based on mRNA technology. In a joint review just published in Vaccine, Sara Sousa Rosa, Miguel Prazeres, Ana Azevedo from BERG-iBB, together with Marco Marques from University College London, describe the current state-of-art of mRNA vaccines, focusing on the challenges and bottlenecks of manufacturing that need to be addressed to turn this new vaccination technology into an effective, fast and cost-effective response to emerging health crises.

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Six iBB Researchers are Among the Most Cited Top Scientists in the World

Six iBB Researchers are Among the Most Cited Top Scientists in the World | iBB | Scoop.it

Six iBB researchers are among the most cited top scientists at the top 2% of their areas. Portugal is represented in this list compiled by Stanford University with 385 scientists affiliated to national institutions. The rankings are based upon a researcher’s citations for both a single year (2019) and cumulative across their careers. Titled “Updated science-wide author databases of standardized citation indicators,” the list uses algorithms that quantify and systematically rank individuals into consistent scientific fields. The scores are provided  with and without self-citations to lessen the impact of self-citations or the use of citation farms (small clusters of researchers massively citing each other’s work). Ranked within the careerlong citation impact list are these iBB researchers: Joaquim Cabral, Isabel Sá-Correia, Mário Berberan Santos, Miguel Prazeres, Carla Carvalho, also along with Carina Crucho in the
2017 single-year citations list.

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iBB and TechnoPhage Collaborate on Bacteriophage Purification

iBB and TechnoPhage Collaborate on Bacteriophage Purification | iBB | Scoop.it

Phages offer an alternative solution to antibiotic–based strategies in the fight against multidrug-resistant bacteria. The implementation of phage therapy must be accompanied by the development of phage biomanufacturing processes. Within the framework of a collaboration with the company Technophage and of the Phage4BacID project (LISBOA-01-0247-FEDER-033603), BERG-iBB researchers led by Ana Azevedo have contributed extensively with analytical data for the refinement of bacteriophage purification techniques. Of particular note, the know-how in chromatographic methods was key to to support not only the project itself, but also the scientific information that integrated Tecnophage's successfull submission of an Investigational New Drug (IND) application to the US Food and Drug Administration for the company's proprietary product TP-102, which targets diabetic foot infections. Clinical trials are expected to start soon.

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Monitoring Proteolytic Activity in Real-Time

Monitoring Proteolytic Activity in Real-Time | iBB | Scoop.it

Proteases play a pivotal role in biological processes, from digestion, cell proliferation and differentiation to fertility. Deregulation of protease metabolism can result in several pathological conditions (i.e., cancer, neurodegenerative disorders, etc). Monitoring proteolytic activity in real-time can ultimately help us understand the role of such enzymes within a certain condition. This may contribute to an early diagnosis, the follow-up of disease progression or the development of protease-targetted treatments (e.g. the current HIV treatment). In a recent paper published in Trends in Biochemical Sciences, Rui Oliveira-Silva and colleagues from BERG, CQE and University of Aveiro review the main approaches used to develop biosensors for monitoring proteolytic activity. A comparison of the advantages and disadvantages of each approach is provided along with a discussion of their importance and promising opportunities for the integration of such sensors into Personalized Medicine.

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Assembly of Dyes at the Tips of Gold Nanorods Enhances Fluorescence Emission

Assembly of Dyes at the Tips of Gold Nanorods Enhances Fluorescence Emission | iBB | Scoop.it

The conjugation of dye-labelled DNA oligonucleotides with gold nanorods has been widely explored for the development of multifunctional fluorescent nanoprobes. In a recent publication in Nanoscale, BERG-iBB researchers in collaboration with colleagues from Centro de Química Estrutural and Centro de Ciências do Mar show that the functionalization route is crucial to achieve enhanced emission in dye nano-assemblies based on gold nanorods. By using a tip-selective approach for thiol attachment of dye molecules onto gold nanorods, it was possible to effectively increase the emission by more than 10-fold relatively to that of free dye. The results could improve significantly the performance of dye nano-assemblies for imaging or sensing applications.The work is part of the PhD thesis of David Botequim, which is supervised by Pedro Paulo (CQE) and Miguel Prazeres (BERG-iBB).

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Featured Photo: Illustration of DNA Origami Nanotetrahedron

Featured Photo: Illustration of DNA Origami Nanotetrahedron | iBB | Scoop.it

Description: Illustration of a DNA origami nanotetrahedron created with David Goodsell’s Illustrate software.

 

Context: BIOTecnico PhD student Ana Rita Santos, under the supervision of BERG-iBB researcher Miguel Prazeres is developing processes to produce and purify DNA scaffolds and DNA origami nano-objects.

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Teaching Protein Structure With Tangible 3D Models

Teaching Protein Structure With Tangible 3D Models | iBB | Scoop.it

Engaging with undergraduate students is an important part of ithe activities of many BB members. In a context of a pedagogical innovation project funded by IST and lead by Miguel Prazeres from BERG-iBB, 1st yr students of biological engineering learn the basics of protein structure by creating tangible 3D models.

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Cover of Biotechnology Journal Highlights iBB's Work on Lactic Acid Bacteria

Cover of Biotechnology Journal Highlights iBB's Work on Lactic Acid Bacteria | iBB | Scoop.it

BERG-iBB researchers led by Gabriel Monteiro are engineering lactic acid bacteria and plasmid vectors in order to develop a flexible platform for biomolecule production. The cover image of the August issue of Biotechnology Journal highlights a recent contribution of the group. The work is part of the FCT-funded project LactoSynt.

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A CBM-Hexapeptide Fusion Confers Antimicrobial Properties to Cellulose

A CBM-Hexapeptide Fusion Confers Antimicrobial Properties to Cellulose | iBB | Scoop.it

A new strategy to modify cellulose with the short antimicrobial hexapeptide MP196 (RWRWRW-NH2) is proposed by BERG and BSIRG researchers that uses fusions of Cys-terminated derivatives of MP196 and a carbohydrate binding module (CBM). CBM3-MP196-modified cellulose hydrogels displayed antibacterial activity that was significantly higher when compared with controls. This versatile concept offers a toolbox for the functionalization of different cellulose materials with a broad choice in peptides. the paper was published in Acta Biomaterialia. The work was funded by project CBM-X.

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Determination of Recombination Efficiency in Minicircle Production by Real-time PCR

Determination of Recombination Efficiency in Minicircle Production by Real-time PCR | iBB | Scoop.it

The determination of the recombination efficiency and the monitoring of parental plasmids (PP), minicircles (MC) and miniplasmid (MP) species during processing and in the final product are critical aspects of minicircle manufacturing. A paper published in Analytical Biochemistry by BERG-iBB researchers Cláudia Alves, Miguel Prazeres and Gabriel Monteiro describes a real-time PCR method for the specific identification of PP, MP or MC that uses sets of primers specific for each species. The results presented indicate that the method can be applied for the determination of recombination efficiency in pure DNA samples.

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Monitoring Intracellular Calcium in Response to GPCR Activation Using Microfluidic Assays

Monitoring Intracellular Calcium in Response to GPCR Activation Using Microfluidic Assays | iBB | Scoop.it

Microfluidic strategies combined with transduction and electronic integration have the promise of enabling miniaturized, combinatorial assays at higher speeds and lower costs, while at the same time mimicking the local chemical concentrations and force fields of the cellular in vivo environment. In A chapter just published in the book "G Protein-Coupled Receptor Screening Assays: Methods and Protocols", researchers from BERG-iBB and INESC-MN introduce a microfluidic structure with hydrodynamic cell traps and a culture volume in the nanoliter range (50 nL), to quantitatively evaluate the transient calcium response of the endogenous Muscarinic type 1 receptor (M1) in HEK 293 T cells. The microfluidic fabrication protocol is described as well as a methodology to monitor the cell response in real time, after stimulation with M1 agonists (e.g., carbachol) and antagonists (e.g., Pirenzepin).

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Cláudia Alves to Defend PhD Thesis in Biotechnology and Biosciences

Cláudia Alves to Defend PhD Thesis in Biotechnology and Biosciences | iBB | Scoop.it

Cláudia Patrícia Almeida Alves will be defending her PhD thesis in Biotechnology and Biosciences at Instituto Superior Técnico, Friday the 14th of May 2021, at 14:00 am (https://videoconf-colibri.zoom.us/j/82471896554). During the last years, and under the supervision of Gabriel Monteiro and Miguel Prazeres from iBB, Cláudia engineered Escherichia coli strains for the production of minicircle vectors by resolvase-catalyzed in vivo recombination. The title of her thesis is “Escherichia coli Genome Engineering For Improved Minicircle Production". Yo can check more about Cláudia's work here.

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Manufacturing of Bacteriophages for Therapeutic Applications

Manufacturing of Bacteriophages for Therapeutic Applications | iBB | Scoop.it

The use of bacteriophages, or simply phages, as therapeutic agents offer is expected to reduce antibiotic use and mitigate the emergence of multidrug-resistant bacteria. The development of phage therapies should be accompanied by the improvement of phage biomanufacturing processes. In a review published in Biotechnology Advances, Jorge João, João Lampreia, Miguel Prazeres and Ana Azevedo from BERG-iBB showcase current processes used for the manufacturing of phages and discuss future alternatives. On the production side, key factors (e.g. bacterial physiological state, phage infection, operation parameters) are described alongside with the different bioreactor operation modes. Traditional methods used in the initial phage isolation steps are then described followed by the presentation of current state-of-the-art purification approaches. Continuous purification of phages is finally presented as a future biomanufacturing trend.

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Cellulose-Based Lateral Flow Immunoassay for the Fluorescent Detection of Troponin

Cellulose-Based Lateral Flow Immunoassay for the Fluorescent Detection of Troponin | iBB | Scoop.it

Lateral flow assays (LFA) are the dominant segment in the Point-Of-Care testing market. Analytical strips made of nitrocellulose (NC) are the heart of a LFA. However, NC is not necessarily the best matrix due to the inability to control the orientation of proteins, inconsistencies in flow rates, lot-to-lot variability, flammable nature, sensitivity to humidity, and an inherent brittleness. In a paper published in Biosensors, Miguel Prazeres from BERG-iBB with colleagues from the Indian Institute of Technology show that cellulose paper possesses the adequate characteristics to replace NC in a LFA for the quantitative, fluorescence-based detection of cardiac troponin I. It is further shown that the layering of carbon nanofibers favor the generation of more intense immunofluorescence signals. The results open up the possibility for companies to develop specialized cellulose strips for LFA. The work was funded by project CBM-X.

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Minicircle Biopharmaceuticals: An Overview of Purification Strategies

Minicircle Biopharmaceuticals: An Overview of Purification Strategies | iBB | Scoop.it

Minicircles are non-viral delivery vectors with promising features for biopharmaceutical applications. These vectors are obtained in vivo in Escherichia coli by the intramolecular recombination of a parental plasmid, which generates the minicircle of interest and an unwanted miniplasmid. Several strategies have been proposed over the years to meet the challenge of purifying and obtaining high quality minicircles. The characteristics of the strain and parental plasmid used have a high impact and strongly affect the purification strategy that can be applied. In a review published in Frontiers in Chemical Engineering, Cláudia Alves, Miguel Prazeres and Gabriel Monteiro from BERG-IST summarize the different methods developed so far, focusing not only on the purification method itself but also on its dependence on the upstream production strategy used.

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David Botequim to Defend PhD Thesis in Biotechnology and Biosciences

David Botequim to Defend PhD Thesis in Biotechnology and Biosciences | iBB | Scoop.it

David José Roxo Botequim will be defending his PhD thesis in Biotechnology and Biosciences at Instituto Superior Técnico, Wednesday the 18th of November 2020, at 10:30 am (https://videoconf-colibri.zoom.us/j/89168693131). During the last years, and under the supervision of Pedro Paulo from CQE and Miguel Prazeres from BERG-iBB, David studied fluorescently-labeled DNA probes conjugated onto gold nanoparticles for the improved detection of nucleic acids. The title of his thesis is “Fluorescently-labeled DNA Probes Functionalized onto Gold Nanorods for Signal Enhancement in Nucleic Acids’ Detection”.

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Hélvio Simões Joins CBM-X Team as Junior Researcher

Hélvio Simões Joins CBM-X Team as Junior Researcher | iBB | Scoop.it

Hélvio Simões has joined the team of the FCT-funded project "CBM-X: Biorecognition as a Tool for the Functionalization of Cellulose-based Materials with Biomolecules and Nanostructures". During the next year, Hélvio will focus on the development of bi-functional biomolecular constructs combining Carbohydrate Binding Modules (CBMs) and oligonucleotide strands for nucleic acid detection.

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Ana Margarida Rosa to Defend PhD Thesis in Biotechnology and Biosciences

Ana Margarida Rosa to Defend PhD Thesis in Biotechnology and Biosciences | iBB | Scoop.it

Ana Margarida Rosa will be defending her PhD thesis in Biotechnology and Biosciences on Wednesday the 29th january 2020 (10:30 H, amphitheater PA-3, Mathematics Building). During the last years, and under the supervision of Miguel Prazeres from BERG-iBB and Pedro Paulo from CQE-IST, Ana developed a cellulose-based system for the colorimetric detection of nucleic acids by exploring the recognition of biotin-labeled DNA hybrids by anti-biotin antibodies immobilized through ZZ-CBM chimeric proteins. These fusions combine carbohydrate-binding modules (CBM) with affinity to cellulose and double Z domains (ZZ) that bind to IgG antibodies. The title of the thesis is “DNA recognition system on bioactive paper using fusions of carbohydrate-binding modules and IgG-binding Z domains”.

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Featured Photo: CRISPR/Cas9 Engineering of E. coli for Minicircle Production

Featured Photo: CRISPR/Cas9 Engineering of E. coli for Minicircle Production | iBB | Scoop.it

Description: plate for screening CRISPR modifications, featured photo by Cláudia Alves, Copyright BERG-iBB and University of Florida 2019.

 

Context: BIOTecnico PhD student Cláudia Alves, under the supervision of BERG-iBB researchers Gabriel Monteiro and Miguel Prazeres and in collaboration with Christopher Reisch from the University of Florida, is using CRISPR/Cas9 to create mutations in E. coli that improve minicircle production.

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Back Cover of Biotechnology Journal Highlights iBB's Work on Functionalization of Cellulose-based Materials

Back Cover of Biotechnology Journal Highlights iBB's Work on Functionalization of Cellulose-based Materials | iBB | Scoop.it

BERG-iBB researchers led by Miguel Prazeres are developing biomolecular constructs for the precise modification of cellulose matrices with nanostructures and/or biomolecules. The back cover image of the August issue of Biotechnology Journal highlights a recent contribution of the group. The work is part of the FCT-funded project CBM-X.

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