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The biopharmaceutical relevance of producing plasmid DNA at large scale has increased steadily over the years due to the development of a growing number of direct and indirect applications. Be it as biological drugs or as starting materials, plasmids are pervasive across the gene and cell therapy industry of today. In a commentary article in Cell & Gene Therapy Insights, Miguel Prazeres provides an overview of the uses of plasmids, discusses manufacturing challenges, and hints at what the future may bring.

Fluorescence microscopy and spectroscopy are routine tools in basic and applied biological sciences. An expanding library of organic fluorophores and fluorescent proteins with radically different properties are available, offering great flexibility to the user of fluorescence methods. Still, a considerable fraction of casual users of fluorescence tools do not follow rational considerations when selecting a probe. In a recently published book chapter, Fábio Fernandes (iBB) and Maria Sarmento (IMM) provide an overview of the most important factors to weight when selecting a fluorophore. A list of different fluorophores and a summary of their properties and labeling strategies is presented as a tool to assist in the process of choosing a fluorescent probe.

Microbes are known to contaminate money. In the project CleanCoin, funded by INCM, Carla C.C.R. de Carvalho (iBB), Telmo Santos (UNIDEMI, UNL) and Maria José Caramujo (cE3c, FCUL, UL), studied the bacterial population present at the surface of coins. In a recently published white paper, the research team and collaborators from INCM, present the results and compare the number of viable bacteria in coins with those found on other objects such as bank cards and mobile phones.

The embryonic development of the human heart under healthy and pathological conditions is still very poorly understood. In a recently published Nature Communications paper, a iBB team led by Margarida Diogo, in collaboration with Perpetua Pinto do Ó at i3S, engineered a unique organ-like model from stem cells that recreates several stages of embryonic development of the human heart. This humanized model opens now the path to study, in a tissue engineering lab, the development of congenital heart diseases, such as coronary vascular diseases and myocardium non-compaction cardiomyopathies, and can be further applied in drug screening and toxicology assays during embryonic heart development. This last topic is particularly relevant since pregnant women normally do not take part in clinical trials, making this human cell-based in vitro platform the only viable alternative to assess developmental cardiotoxicity. The work was a part of the PhD thesis in Bioengineering – MIT Portugal program of Mariana Branco, the first author of the paper, and was funded by the FCT project “Mini-Hearts - Organoid Engineering for Production of 3D Cardiovascular Microtissues from Human Induced Pluripotent Stem Cells for Cardiotoxicity”.  

As current treatment options for cartilage degeneration remain ineffective, tissue engineering has emerged as an exciting approach to create cartilage substitutes. In particular, hydrogels seem to be suitable candidates for this purpose due to their high biocompatibility and customizability, being able to be tailored to fit the biophysical properties of native cartilage. Moreover, these hydrogel matrices can be combined with conductive materials in order to simulate the natural electrochemical properties of articular cartilage. In this recent review published in the journal Gels, iBB researchers Filipe Miguel (MSc student in Biotechnology), Frederico Barbosa (PhD student in Bioengineering), Prof. Frederico Ferreira and Dr. João Silva highlight the most common conductive materials combined with hydrogels and their diverse applications, and discuss the current state of research on the development of electrically conductive hydrogels for cartilage tissue engineering.

Genome sequencing is a powerful tool that contributes to the advancement of drug resistance studies, population dynamics and evolution in fungal pathogens. In a paper just published in Microbial Cell, emerging from a collaboration between Miguel Cacho Teixeira’s team, iBB, and Hiroji Chibana, MMRC, Japan, the analysis of nearly 100 genomes of globally distributed C. glabrata isolates enabled the identification of new effectors of azole and echinocandin resistance. Their genomic landscape was further explored to evaluate genetic variants specific to each drug resistance profile. Interestingly, the data concludes that changes in the most studied drug resistance genes only occur in a fraction of the resistant isolates, an observation that is expected to contribute to design more suitable antifungal therapeutic strategies.

It is usually accepted that only 0.1-1% of marine bacteria may be cultivated under laboratory conditions. This resulted mainly from the observation that only a small fraction of the total bacteria determined by microscopic counts could be enumerated by culture plate techniques, and has been a justification for the widely implementation of cultivation-independent molecular techniques. In a recent published paper, Carlos J.C. Rodrigues and Carla C.C.R. de Carvalho (BERG-iBB) promoted the growth of bacteria from a marine rock pond by culture dependent techniques and compared the results with culture independent methods. The highest efficiency of cultivation was 45% attained in marine agar. The study shows that it is possible to grow more than the traditionally considered 1% of bacteria from a seawater sample using standard agar plate techniques and laboratorial conditions, and to find bacteria not detected by molecular approaches.

Adaptation and tolerance to industrially relevant stress factors involve highly complex and coordinated molecular mechanisms occurring in the yeast cell with repercussions on the performance and economy of bioprocesses. The cell wall is among the players whose biochemical and biophysical properties can be finely tuned as yeast cells encounter different stresses throughout the course of industrial bioprocesses. A new review article from BSRG-iBB provides a critical opinion and a comprehensive view on the current knowledge on the involvement of the cell wall in the adaptive response and tolerance of yeasts to multiple stresses of biotechnological relevance. A few successful attempts to improve stress tolerance through the manipulation of cell wall biosynthetic pathway are also described and the available information for some non-conventional yeast species included. The review article is co-authored by the PhD student of the IST PhD programme in Biotechnology and Biosciences Ricardo Ribeiro and the thesis supervisor, Prof. Isabel Sá-Correia. It was was published in the journal Frontiers in Microbiology (section: Microbial Physiology and Metabolism).

Exploration of yeast diversity for the sustainable production of biofuels, in particular biodiesel, is gaining momentum in recent years. A BSRG-iBB review paper, just published in the Journal of Fungi (Special Issue: Yeasts for a Sustainable Circular Bio-Based Economy), provides an overview of lipid production by oleaginous yeasts focusing on yeast diversity, metabolism, and other microbiological issues related to the toxicity and tolerance to multiple challenging stresses limiting bioprocess performance. Examples gathered from the literature showing the potential of different oleaginous yeasts/process conditions to produce oils for biodiesel from agro-forestry and industrial organic residues are also provided. This publication has as first-author the PhD student Marta N. Mota of the IST PhD programme in Biotechnology and Biosciences and as corresponding author her thesis supervisor, Professor Isabel Sá-Correia. Paula Múgica, from BIOREF—Collaborative Laboratory for Biorefineries, also contributed to the publication during her stay at BSRG-iBB for scientific training and collaboration in the execution of the Move2LowC project.

DNA-origami biomanufacturing relies in many cases on the use of asymmetric PCR (aPCR) to generate 500-3500 base, single-stranded DNA (ssDNA) scaffolds that are then purified by agarose gel extraction, a technique that is laborious, not scalable and presents low recovery yields. In a paper just published in Separation and Purification Technology, Ana Rita Santos and Miguel Prazeres from BERG-iBB, in collaboration with Pedro Paulo from CQE, present alternative methods based on anion-exchange or multimodal chromatography that yield purified scaffolds from aPCR mixtures. The recovered scaffolds were further used to assemble DNA-origami nanostructures. The work indicates that chromatography could play an important role in scaling-up the purification of ssDNA scaffolds for the biomanufacturing DNA-origami nanostructures.

A joint collaborative effort coordinated by Prof Jörn Piel from the ETH Zürich (Switzerland) led to the identification of new antibiotic compounds from Aquimarina sp. strain Aq135 which was cultivated from the marine sponge Ircinia variabilis in the laboratory of Prof Rodrigo Costa from BSRG-iBB. Activity-guided isolation identified novel antibacterial peptides, named aquimarins, featuring a new scaffold with an unusual C-terminal amino group and chlorine moieties. Structure–activity relationship studies with these compounds showed that the synthetically more laborious chlorinations are not required for antibacterial activity but enhance cytotoxicity. In contrast, variants lacking the C-terminal amine were virtually inactive, suggesting diamines similar to the terminal aquimarin residue as candidate building blocks for new peptidomimetic antibiotics. The study was published in Angewandte Chemie.

L-asparaginases have an acknowledge role in food and pharmaceutical industries. Accordingly, efforts to produce highly efficient, selective and stable L-asparaginases are currently underway. One of the strategies used to fulfill this goal involves the production of recombinant. In a recent paper published in Marine Biotechnology, Pedro Fernandes (iBB-BERG) and colleagues from the University of Hormozgan and Tarbiat Modares University in Iran report the production of a novel marine recombinant L‑asparaginase and provide its biochemical and structural characterization. The results obtained suggest the potential of the thermostable recombinant enzyme obtained for practical applications in food and pharmaceutical areas.

A new article provides a genome-based roadmap to facilitate the selection of coral probiotics, also known as Beneficial Microbes for Corals (BMCs), and was just published in mSystems. Probiotics are starting to be investigated as a potential medicine for corals, marine animals that are threatened with extinction by the advance of climate change. Laboratory experiments have already proved the concept that coral probiotics mitigate coral bleaching and reduce coral mortality. This study now employed comparative genomics to identify exclusive BMC traits associated with specific probiotic strains and proposed new BMC mechanisms. The work was led by Prof Raquel Peixoto and her team from KAUST University at the Red Sea and received guidance on genomics of coral-associated bacteria from iBB/DBE researcher Dr Tina Keller-Costa.

Marine bacteria present enzymatic activities that result from extensive evolution necessary for cell survival under the harsh and continuously changing conditions found in the marine environment. Metagenomic techniques and cultivation of not-yet-cultured bacteria, combined with high-throughput screening techniques, have allowed the identification of new biocatalysts and the development of industrial processes. In a recently published paper, Carlos J.C. Rodrigues and Carla C.C.R. de Carvalho (iBB) review the latest studies related to growth of marine bacteria under laboratorial conditions, screening techniques, and bioprocess development.

Metagenomics is an expanding field within microbiology and related disciplines. The number of metagenomes deposited in public databases is exponentially rising, but data mining and interpretation can be challenging due to mis-annotated metadata entries. The new Marine Metagenome Metadata Database (MarineMetagenomeDB) helps researchers to identify marine metagenomes of interest for re-analysis and meta-analysis and is a valuable resource for non-bioinformaticians to find marine metagenome samples with curated metadata. The study, published in Environmental Microbiome, was led by Dr Ulisses Nunes da Rocha and team from the Helmholtz Centre for Environmental Research (Germany), and counted on specialist guidance in marine metadata standardization from iBB/DBE PhD-student Sandra Silva and researchers Tina Keller-Costa and Rodrigo Costa. The user-friendly web app is publicly available at here.

A pandemic can turn the world upside down. We recently had this hard experience, which made us reflect (a lot) on the importance of science and how it is the stronghold of our survival as a species (we have persisted for at least 200 thousand years!). Resistance to antibiotics and antifungals is nothing new and seems to have become commonplace (or despised). It is too serious a problem to be reassured, for several reasons. The evolution of multidrug resistance in bacteria and fungi (proven by genetic sequencing), associated with the disinvestment in new drugs by large pharmaceutical multinationals, converges in a (next) pandemic scenario of hypervirulent bacteria and fungi. Antimicrobial resistance is fundamentally based on the ability that bacteria and fungi have acquired to escape the action of available drugs, using mainly three strategies: enzymatic degradation of drugs, alteration of drug target proteins, and alteration of membrane permeability (expression of efflux pumps). Bearing in mind these strategies, a team of researchers from iBB-IST (led by Prof. Vasco Bonifácio) in collaboration with researchers from CQE-IST and ITQB-NOVA, designed, and produced antimicrobial polyurea pharmadendrimers, a new class of antibiotics and antifungals. These pharmadendrimers have excellent activity (in vitro and in vivo) against multidrug-resistant bacteria and fungi from clinical isolates collected in Portuguese hospitals and clinics. Their administration is safe as they have a very wide therapeutic window, in which they are not cytotoxic and do not cause hemolysis (the main problems of other analogues under investigation, such as antimicrobial peptides). On the other hand, its mechanism of action prevents the development of resistance, an almost impossible mission for bacteria and fungi because it requires a restructuring of their membrane. Antimicrobial pharmadendrimers have been patented, and an international patent application is pending. In an interview to the RTP TV channel, on the program “O Desafio do Superior em Portugal”, Dr. Rita Pires, a researcher on the team, talks about this invention (video). The research work was recently published in a high impact international journal.

The YEASTRACT+ information system is a wide-scope computational tool for the analysis and prediction of transcription regulatory associations at the gene and genomic levels in 11 yeast species of biotechnological or human health relevance. Its newest release, orchestrated by a large team led by Isabel Sá-Correia, Miguel Teixeira, from iBB, and Pedro Monteiro, from INESC-ID, has just described in the 2023 Nucleic Acids Research Database Issue, highlighting the availability of new regulatory-metabolic network modeling tools. These tools are expected to contribute to guide YEASTRACT+ users in the optimization of yeast cell factories of industrial relevance and the identification of new antifungal drug targets, to be used in advanced antifungal therapy. YEASTRACT+ is currently supported within the context of the Biodata.pt/Elixir.pt Research Infrastructure.

During past years, lipid nanoparticles (LNPs) have emerged as promising carriers for RNA delivery, with several clinical trials focusing on both infectious diseases and cancer. More recently, the success of messenger RNA (mRNA) vaccines for the treatment of severe diseases, such as acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is partially justified by the development of LNPs encapsulating mRNA for efficient cytosolic delivery. In a collaborative work with researchers from Hovione Farmaciência SA and INESC-MN, Pedro Fonte from iBB published an invited review paper in Expert Opinion on Drug Delivery addressing the production and formulation of LNPs by using microfluidic devices, the status of mRNA-loaded LNPs therapeutics and explored spray drying process, as a promising dehydration process to enhance LNP stability and provide alternative administration routes. It is foreseen this work may contribute to advances in the field in the upcoming years.

The increasing number of life-threatening infections observed in cancer patients has been ascribed to chemotherapy-induced neutropenia and to invasive medical procedures. In a recent published study, Carla C.C.R. de Carvalho (BERG-iBB) questioned if the infections could also be favored by an increased resistance of bacteria due to the adaptation to antineoplastic agents used in chemotherapy. After exposure to several antineoplastic agents, it was observed that cells of Staphylococcus aureus, Mycobacterium vaccae, Pseudomonas aeruginosa, and Escherichia coli changed the fatty acid profile of their cellular membranes, produced exopolymeric substances, and formed aggregates that adhered to surfaces. Additionally, when exposed to high concentrations of these compounds, a persister sub-population could be identified. After adaptation to antineoplastic agents, the minimum inhibitory concentration (MIC) of several antibiotics increased considerably in the tested strains. This should be a major concern during oncology treatments.

In a perspective article published in Nature Microbiology, experts in the field of symbiosis and microbiome research address the current, acute loss of plant and animal diversity that poses serious threats to the functioning of terrestrial and marine ecosystems, human health, and the development of a sustainable bio-based economy. The potential, and examples of success, of microbiome-based interventions such as probiotics administration and microbiome transplants in improving the health and resilience of a broad range of host organisms towards climate change stressors and the emergence of novel diseases is debated, and a perspective is provided on how such interventions may contribute to the conservation and maintenance of services of man-made and natural ecosystems. The perspective, led by Prof. Raquel Peixoto at King Abdullah University of Science and Technology (KAUST), counted on expert contributions by iBB-BSRG researchers Dr. Rodrigo Costa and Dr. Tina Keller-Costa on microbiome manipulation strategies in aquaculture and coral reef ecosystems. The initiative was launched by a round table headed by the Beneficial Microorganisms for Marine Organisms (BMMO) network during the 15th Symposium on Bacterial Genetics and Ecology (BAGECO15, Lisbon 2019) chaired by Prof. Rodrigo Costa.

Bacterial pathogens belonging to the Vibrio genus such as Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio vulnificus are some of the most common and widespread disease-causing agents in the aquaculture industry, inducing severe losses in fish and shellfish production worldwide. In a Review Article published in Frontiers in Microbiology, iBB researchers Gracinda Sanches Fernandes, Isabel-Sá Correia and Rodrigo Costa performed a meta-analysis of vibriosis outbreaks reported for the economically valuable, model aquaculture fish gilthead seabream (Sparus aurata) in the Mediterranean zone. The study reveals the acquisition of multiple antibiotic resistance traits among pathogenic Vibrio species during the past 25 years. An increase in human infections caused by Vibrio pathogens of fish – either via seafood ingestion or wound colonization - has been documented in the recent literature, and it is believed to result from the synergistic interaction between the expansion of intensive fish farming across coastal ecosystems and global warming. Finally, the authors critically examine the potential, and review cases of success, of alternative methods to antibiotics use to suppress vibrio pathogens in aquaculture, including vaccines, phage therapy and probiotics supplementation. Diverse bacterial symbionts of fish suppress vibrio pathogens in experimental trials, suggesting that the natural fish microbiome may be a reservoir of biological control agents of interest in applied biotechnology.

Helichrysum italicum is a halophyte shrub with bright yellow flowers with a strong curry-like aroma that have been used in the production of cosmetics and pharmaceuticals, for seasoning and flavoring of food, and as natural food preservatives. In a study  recently published in Food Research International, researchers of Universidade Nova de Lisboa, Polytechnic of Leiria, University of Coimbra, University of Lincoln, the Quadram Institute, and Carla C.C.R. de Carvalho (BERG-iBB), characterize the composition, bioactive compounds, and nutritive value of H. italicum flowers. The flowers were found to be a good source of minerals, omega-6 and omega-3 PUFAs, polyphenols, carotenoids, curcuminoids and other bioactive compounds with relevant antibacterial, antifungal and antioxidant properties.

Photo details: Helichrysum italicum, Bj.schoenmakers, CC0, via Wikimedia Commons.

The marine bacterial genus Aquimarina is a promising source of novel natural products. A new study published in Marine Drugs demonstrated widespread ability of Aquimarina species to inhibit growth of human-pathogenic microbes such as Candida glabrata and methicillin-resistant Staphylococcus aureus, as well as Vibrio and other marine bacteria relevant to aquaculture. Metabolomics and genomics analyses of Aquimarina strains indicated the presence of novel polyketides and peptides, including cyclic depsipeptide-related compounds. The study further showed that Aquimarina species possess low-abundance distributions across marine biotopes worldwide. It emphasizes the relevance of this member of the microbial rare biosphere as a promising source of novel natural products, supporting future efforts to isolate new bioactive compounds from Aquimarina. The work was developed within the SymbioReactor project (DGPM | Fundo Azul program) led by Prof Rodrigo Costa and Dr Tina Keller-Costa and was first-authored by PhD student Sandra Silva. It also includes former MSc student Patrícia Paula, Dr Dalila Mil-Homens, Prof Miguel Teixeira and Prof Arsénio Fialho from BSRG-iBB as coauthors.

A transatlantic endeavour involving researchers from BSRG-iBB, CQE (IST) and Université Laval and INRS (Canada) provided the first comprehensive assessment of prokaryotic communities in the late winter ice and its underlying water along a natural salinity gradient in Hudson Bay, a cryo-environment marking the transition between Subarctic and Arctic biomes. The study, published in Science of Total Environment, was led by Prof. Rodrigo Costa, first-authored by MIT-Portugal PhD student Lígia Coelho and co-authored by PhD student Joana Couceiro, Dr Tina Keller-Costa and Profs. Zita Martins and João Canário. The team found sharp shifts in community structure between the ice and underlying water samples at sampling sites with higher salinity, and high abundance of culturable, pigment-producing bacteria in ice. The study suggests that salinity, photosynthesis and dissolved organic matter are main drivers of prokaryotic community structure in the winter ice of Hudson Bay, the ecosystem with the fastest sea ice loss in the Canadian North.