The BB’s team Diana M. C. Marques, Madalena Jabouille, Afonso Gusmão, Paola Sanjuan Alberte, and Frederico Ferreira, recently reported in npj Sicence of Food (Nature NPJ), the development of novel (bio)inks for producing cultured seafood products by 3D bioprinting.
The study introduces novel (bio)inks: (i) κ-CAM bioinks (κ-carrageenan, alginate, and methylcellulose) compatible with seabass cells; and (ii) mFAT inks, plant-based fat inks containing microalgae for enhanced organoleptic properties.
This research demonstrates that the algae- and plant-based multi-material 3D bioprinting process can be used for the fabrication of cultured seafood products with enhanced organoleptic properties (such as sea-like smell and flavor).
A 3D-printed calamari was fabricated to showcase the potential in the manufacturing of complex structures.
Link to the publication: https://lnkd.in/dx5XNjy2

Abstract

Infections caused by the opportunist pathogens Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc) are of particular concern due to their resistance to multiple drugs (MDR). These pathogens can lead to severe infections in patients with cystic fibrosis (CF) and those who are immunocompromised. Chronic lung infections caused by these bacteria remain problematic to CF patients because of their difficult eradication using the currently available treatments. Moreover, these infections are associated with a faster decline in lung function, increased morbidity and mortality rates. So, currently, novel treatment approaches are being pursued to decrease the use of antibiotics. With this aim, passive immunotherapy has been revisited for MDR pathogens infection prevention and/or treatment. In particular, monoclonal antibodies (mAbs) have been studied as neutralizers of bacterial virulence factors, being regarded as alternative or complement to antibiotic therapy, resulting in faster infection resolution and shorter stays in intensive care units (ICU) as well as reduction of morbidity, mortality and health care costs. However, their use has some drawbacks, such as the large size of mAbs that can lead to inaccessibility to some epitopes and relatively high production costs. Single-domain antibodies (sdAbs) offer several advantages, including smaller size, a larger number of accessible epitopes, relatively low production costs and improved robustness. Recently, immunoconjugates using mAbs or sdAbs as scaffolds have been explored as potential therapeutics for infectious diseases and as alternatives to antibiotics, directing unspecific antimicrobials to the pathogens. One group of antimicrobials extensively studied are the antimicrobial peptides (AMPs) that have the advantage of being abundant in nature. However, several drawbacks have limited their use as antimicrobials, such as their low stability, low specificity, and off-target toxicity. To overcome these limitations, antibody conjugates with AMPs have been studied and shown to increase the stability and specificity of the antimicrobial agent. However, this therapeutic approach against Bcc and P. aeruginosa infections is poorly explored. Surface-exposed antigens are important pathogenicity-related molecules involved in the interactions between hosts and microorganisms, being useful as targets for the development of therapeutic antibodies. Recently, our research group identified the protein BCAL2645 from B. cenocepacia and their orthologues in P. aeruginosa and B. multivorans as a potential target for the development of new therapeutic antibodies that interfere with host-pathogen interactions and neutralize the infections caused by these bacteria. In the present proposal, we aim to develop a sdAb anti-BCAL2645 with the ability to target and neutralize P. aeruginosa and Bcc infections. Additionally, to study this sdAb as a scaffold to generate an antibody-drug conjugate (ADC) combined with a synthetic antimicrobial peptide. The directed cytotoxicity of the new ADC against P. aeruginosa and Bcc infections will be assessed. The research team of the project has all the different expertise required for the achievement of each task delivery proposed. The BSRG/iBB research team has long-term experience in Bcc and P. aeruginosa bacteria biology and their interaction with the host. Lately, the BSRG/iBB research team has been focusing their research on the identification and study of bacterial targets for the development of immunotherapeutic approaches against these bacterial species. Dr. Frederico Aires da Silva (CIISA-FMV) will reinforce the project team with expertise in sdAb and ADC production, purification and characterization. Dr. Marilia Mateus (2BRG/iBB) will reinforce the project with expertise in protein overexpression and purification methods. In summary, this proposal is expected to contribute to the development of a new immunotherapeutic approach using sdAbs as a scaffold to generate ADCs combined with a synthetic antimicrobial peptide to combat earlier established Bcc and/or P. aeruginosa infections, allowing the improvement of their quality of life and lifetime expectancy. This project is aligned with the “One Health” approach from WHO and the United Nations Sustainable Development Goal 3 “Ensure healthy lives and promote well-being for all at all ages”.

TEAM

Sílvia A. Sousa (PI)

Frederico Aires da Silva (CIISA-FMV),

Marilia Mateus (iBB),

Jorge H. Leitão (iBB)

Jeremias Muazeia (iBB)

ABSTRACT

The emergence and global dissemination of antimicrobial resistant pathogens is one of the most serious global public health threats in the 21st century. Due to the limited success in developing novel antibiotics, alternative therapeutic approaches such as antisense antimicrobial therapies are being explored as promising strategies to address bacterial resistance. To achieve this goal, it will be crucial to understand how bacteria regulate gene expression and resist antimicrobial treatments. This involves accessing their regulatory networks, in which small noncoding RNAs (sRNAs) play a prominent role.

Bacterial sRNAs, as an important class of posttranscriptional regulators, play critical roles in influencing various biological processes, including antibiotic resistance. These regulators enable rapid responses to fluctuating environmental conditions and antibiotic exposure by altering mRNA accessibility or degradation. While several sRNAs in certain pathogens have been demonstrated to influence intrinsic antibiotic resistance mechanisms by affecting processes such as antibiotic uptake, cell wall synthesis, drug efflux, and biofilm formation, our understanding of riboregulatory sRNAs and their specific pathways in other pathogens, such as those within the Burkholderia cepacia complex (Bcc), is still limited.

Bcc bacteria are a group of opportunistic pathogens that are particularly feared among the cystic fibrosis (CF) community due to their resistance to multiple antibiotics, and the unpredictable outcome of infections, ranging from asymptomatic cases to the cepacia syndrome, a fast and fatal necrotizing pneumonia often associated with septicemia. Currently there is no evidence of an effective eradication protocol for Bcc bacteria, and some combinations of antibiotics are becoming less effective against these pathogens.

Recently, it was demonstrated that the B. cenocepacia susceptibility to antimicrobials is modulated by the acidic pH and the CF nutritional environment, suggesting that Bcc resistance to antibiotics is dependent on gene expression regulation.

Therefore, this exploratory project aims to identify and characterize the regulatory sRNAs involved in Bcc antibiotic resistance under clinically relevant conditions, and to assess their regulatory networks to find potential targets for RNA-based antimicrobial strategies against Bcc infections.

To achieve this goal, we will leverage the expertise and experience of team members in the identification and characterization of sRNAs from B. cenocepacia, as well as in the study of the targetome of three important RNA-binding proteins in these bacteria: the chaperones Hfq, Hfq2, and ProQ.

To identify sRNAs that modulate antibiotic resistance in B. cenocepacia, the expression profile of the 264 sRNAs already identified in B. cenocepacia will be accessed under conditions that mimic the acidic CF nutritional environment and with subinhibitory concentrations of clinically relevant antibiotics. The differentially expressed sRNAs, whose predicted targets include genes involved in antimicrobial resistance and are conserved only among B. cenocepacia or among Bcc species, will be selected. These sRNAs will be overexpressed and silenced in B. cenocepacia using appropriate plasmids, and the effect of this silencing or overexpression on B. cenocepacia resistance to certain antibiotics will be tested in vitro using minimum inhibitory concentration assays (MICs). The sRNAs that exhibit an effect on the intrinsic drug resistance of B. cenocepacia will be selected, and the global transcriptomic changes induced by their altered expression will be assessed using RNA-sequencing.

The data obtained will enable the prediction of the differentially expressed targets of each sRNA, as well as the exploration of their regulatory networks under the various conditions tested. This knowledge will be crucial for the subsequent development of an antisense therapy against Bcc infections. The impact of manipulating the selected sRNAs on the efficacy of antibiotic treatments will finally be assessed in vivo using the larva of the greater wax moth Galleria mellonella.

Overall, this project will not only contribute to identifying sRNAs involved in Bcc resistance to antibiotics, but also to deciphering the antibiotic-responsive sRNA networks. This will provide an opportunity to enhance the efficacy of existing antibiotics against Bcc bacteria by silencing resistance genes in combined therapy, as well as to improve antibiotic efficacy by manipulating the levels of sRNAs involved in resistance. The rational development of novel strategies to combat infections caused by Bcc bacteria in patients suffering from Cystic Fibrosis and other diseases is aligned with the 2030 Agenda and its sustainable development goals for health.

TEAM

Joana Feliciano (PI)

Jorge Leitão 

Gonçalo Matos

Dalila Mil-Homens (ITQB NOVA)

iBB’s team Catarina Jones, Marta Carvalho, Paola Sanjuan-Alberte, Teresa Esteves and Frederico Ferreira, recently reported in ACS Applied Materials And Interfaces a nanobioelectronic system designed to target cancer cells’ bioelectrical properties. This system combines piezoelectric barium titanate nanoparticles with a conducting poly(3,4-ethylenedioxythiophene) shell (PEDOT), functioning as nanoantennas that convert ultrasound-induced mechanical signals into electrical signals.

This system demonstrated effective and selective targeting of cancer cells, significantly reducing the viability of MCF-7 and MDA-MB-231 cells— only when activated by ultrasound —while leaving healthy cells unharmed. The goal of this approach was to re-engineer the membrane potential of cancer cells, based on the hypothesis that membrane potential and cell bioelectricity regulate tumorigenic features. Further investigation revealed that the nanoparticles induced membrane hyperpolarization, which increased intracellular reactive oxygen species and calcium levels. This disruption of bioelectric signaling may suppress tumorigenic properties, ultimately leading to cancer cell death and cell cycle arrest.

This work serves as a proof of concept, demonstrating that cancer cell bioelectricity can be harnessed as a potential therapeutic target through wireless and non-invasive methods. It underscores the importance of further interdisciplinary research—spanning both engineering and biological perspectives—to better understand the role of bioelectricity.

Link to paper: https://pubs.acs.org/doi/10.1021/acsami.4c12387#

In this week´s “Meet the… PeX“, we introduce you the project: Engineering advanced colorectal cancer ecosystems to evaluate tumour-microbiome dynamics in vitro, lead by Paola Sanjuan Alberte.

Abstract:

Colorectal cancer (CRC) remains a leading cause of cancer-related deaths, with increasing incidence and mortality rates despite significant progress has been done in screening and treatment in the last decades. The role of the gut microbiota has recently been highlighted as one of the key players in CRC initiation and progression as certain microbial metabolites promote carcinogenesis or resistance to therapeutics. Such influence of the gut microbiota on CRC, underscores the need for innovative research models accounting for this, which is currently neglected.
Therefore, in order to advance CRC research, there is a pressing need to develop more physiologically relevant models in vitro that accurately represents the tumour microenvironment and the complex relationship between CRC and gut microbiota. Despite the increasing evidence of the role of microbial metabolites and specific bacteria in CRC development, current in vitro models fail to mimic these dynamics. This is in part due to the lack of models available that recapitulate in high detail the heterogenicity of gut microbiota.
In BIOMIMIC-CRC we propose the development of advanced in vitro platforms that combine CRC with highly representative models of healthy and pathological gut microbiota provided by our industrial partner, Bac3Gel, using bioprinting and microfluidic technologies. These platforms aim to replicate the complex dynamics between CRC and gut microbiota for its potential applications in drug screening and personalised medicine to elucidate the CRC-microbiota interactions.
In order to reach that aim, three main objectives have been established in BIOMIMIC-CRC which are directly related to the tasks of the project:
1. Development of healthy and pathological models of gut microbiota to evaluate the impact of specific microbial populations on CRC lines in static 2D conditions.
2. Formulation of bioinks and 3D bioprinting of bi-layered micropillars containing gut microbiota and CRC cells for the assessment of their dynamics in a more structurally accurate context and;
3. Development of a CRC-on-chip replicating the biophysical stimuli of the colon to provide a bio-inspired miniaturised platform for CRC research.
This multidisciplinary approach, combining tissue engineering, microbiology, and microfluidics, is expected to have significant scientific, technological, and societal impacts, from advancing CRC research to developing high-throughput systems for drug screening applications with potential to optimise treatment strategies and reduce healthcare burdens. Additionally, the strategy proposed in BIOMIMIC-CRC has the potential to be translated to other systems where an interaction between microbiota and mammalian cells occurs.
The team of BIOMIMIC-CRC is composed by a multidisciplinary team of young and motivated researchers, committed to the development of more accessible and physiologically relevant models that have the potential to propel CRC research.

TEAM

Paola Sanjuan Alberte (PI) – iBB
Daniela Pacheco (Bac3Gel)
Natalia Suarez (Bac3Gel)
Ana Carina Manjua (TUE)
Kristin Schueler (iBB)
Carlos Carreira (INESC.MN)
Duarte Almeida (iBB)
Margarida Domingues (iBB)

The bacterial strain Aliivibrio sp. EL57 was featured last week in the BlueTreasure edition of the Portuguese Blue Biobank.

This strain is part of a fascinating group of bioluminescent bacteria that can glow in the dark when they reach high population densities. This ability to recognize the population density is driven by quorum sensing, a mechanism that allows the bacteria to detect and respond to their surroundings.

Aliivibrio sp. EL57 was isolated from the octocoral Eunicella labiata Thomson, 1927, sampled off the coast of Faro, Portugal.

It is a valuable part of the MicroEcoEvo collection (Microbial Ecology and Evolution Research Group), housed at the Institute for Bioengineering and Biosciences (iBB), Instituto Superior Técnico.

Isolation and Strain Curation: Tina Keller-Costa and Rodrigo Costa
📸Tina Keller-Costa and Matilde Marques