iBB

Intrinsically disordered proteins/regions (IDPs/IDRs) are widespread in the human proteome and play critical roles in distinct biological processes and dysfunction. Contrary to the classical “structure–function” paradigm, IDPs lack stable secondary/tertiary structures under physiological conditions, and instead adopt a dynamic ensemble of multiple conformations. Moreover, several neurodegenerative disorders are associated with the pathological self-assembly of neuronal IDPs, including tau (Alzheimer’s disease), α-synuclein (Parkinson’s disease), and huntingtin exon 1 (Huntington’s disease). Therefore, there is an emerging medical interest in understanding their physical and structural features. However, their characterization is inherently challenging by traditional ensemble and time-averaging methodologies. A recent review co-authored by Ana Melo from BSIRG-iBB, and published in Frontiers in Molecular Neurosciences the advantages of employing cutting-edge single-molecule fluorescence techniques are discussed to characterize the conformational ensemble of neurodegeneration-promoting IDPs under normal and disease conditions to obtain insights into their gain- or loss-of-function.

In a paper recently published in the journal Antibiotics, a series of cyclam- and cyclen-derived salts were designed and studied as antibacterials by the research groups headed by JH Leitão (BSRG) and AM Martins and LG Alves (CQE). The compounds were designed specifically to gain insights into their structure and antibacterial activity towards Staphylococcus aureus and Escherichia coli, used respectively, as Gram-positive and Gram-negative model organisms. The newly synthesized compounds are monosubstituted and trans-disubstituted tetraazamacrocycles that display benzyl, methylbenzyl, trifluoromethylbenzyl, or trifluoroethylbenzyl substituents appended on the nitrogen atoms of the macrocyclic ring. The results obtained show that the chemical nature, polarity, and substitution patterns of the benzyl groups, as well as the number of pendant arms, are critical parameters for the antibacterial activity of the cyclam-based salts. The paper was published as part of the journal Special Issue New Insights into Antibacterial Compounds: From Synthesis and Discovery to Molecular Mechanisms of Action, guest-edited by Jorge H. Leitão.

The synthesis of hydroxide [Ag(OH)L] (L =I VL, VL, VIL, VIIL), oxide [{AgL}2}(μ-O)] (L = IL, IIL, IIIL, VL, VIL) or chloride [AgIIL]Cl, [Ag(VIL)2]Cl complexes, obtained from reactions of mono- or bicamphorimine derivatives with Ag(OAc) or AgCl, appeared in a recent publication on the journal Antibiotics, resulting from a collaborative research involving the iBB members Jorge H. Leitão, Nuno Mira, Ana Rego and Sílvia Sousa; M. Fernanda Carvalho from CQE; and Fernanda Marques from C2TN. The new complexes were characterized by spectroscopic (NMR, FTIR) and elemental analysis. X-ray photoelectron spectroscopy (XPS), ESI mass spectra and conductivity measurements were undertaken to corroborate formulations. The antimicrobial activity of complexes and some ligands were evaluated towards Candida albicans and Candida glabrata, and strains of the bacterial species Escherichia coli, Burkholderia contaminans, Pseudomonas aeruginosa and Staphylococcus aureus based on the Minimum Inhibitory Concentrations (MIC). A significant feature of these redesigned complexes is their ability to sensitize C. albicans, a trait that was not found for the previously investigated [Ag(NO3)L] complexes. The MIC values of the complexes towards bacteria were in the range of those of [Ag(NO3)L] and well above those of the precursors Ag(OAc) or AgCl. The activity of the complexes towards normal fibroblasts V79 was evaluated by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Results showed that the complexes have a significant cytotoxicity.

The mechanisms underlying the adsorption of monoclonal antibodies (mAbs) towards phenylboronate (PB) ligands have been elucidated by Flow Microcalorimetry. The knowledge acquired BERG-iBB researchers, in collaboration with Cristina Dias-Cabral's group from CICS-UBI, suggest how to increase the salt tolerance of the bond between ligand and biomolecule by manipulating the environmental conditions.It was demonstrated that such feature can be accomplished either by using mobile phase modulators that promote the specific cis-diol interactions between PB ligands and mAbs or by diminishing the non-specific interactions involved in the adsorptive process. The research team believes that these findings could contribute towards the effective integration of boronate chromatography in the current mAb downstream processing platform. The paper was published in Biotechnology Journal.

The YEASTRACT+ information system (http://YEASTRACT-PLUS.org/) is a wide-scope computational tool for the analysis and prediction of transcription regulatory associations at the gene and genomic levels in 10 yeast species of biotechnological or human health relevance. A large team led by Isabel Sá-Correia, Miguel Teixeira, from iBB, and Pedro Monteiro, from INESC-ID, has just released this new web-portal in the 2020 Nucleic Acids Research Database Issue and is compromised to continue its efforts to expand this resource, which is currently supported within the context of the Biodata.pt/Elixir.pt Research Infrastructure.

The synthesis, characterization and antibacterial activity of eleven new complexes of general formula [Ag(NO3)(L-Y)2] corresponding to Ag(I) camphorimine complexes and a camphor sulfonylimine complex were reported in a recent publication in Journal of Inorganic Biochemistry, resulting from collaborative work of JH Leitão from BSRG and MF Carvalho from CQE. The study was performed to gain insights into structure/antimicrobial activity relationships. The lipophilicity and polarity which are important parameters concerning the biological activity of the complexes are also high dependent of the characteristics of the camphor ligands. The redox properties of the complexes studied by cyclic voltammetry showed that their reduction potentials are essentially independent of their electronic and steric properties. The antibacterial activity of all the complexes, against Gram-positive (S. aureus Newman) and Gram-negative (Escherichia coli ATCC25922, Pseudomonas aeruginosa 477, Burkholderia contaminans IST408) strains was evaluated through calculation of MIC values. Results show that complexes with camphor imine ligands that combine high lipophilicity with low dipolar moment exhibit enhanced antibacterial activity. The ability to establish hydrogen bonding emerged as an important contribution to the antibacterial activity of the camphor sulphonylimine complex.

Aquimarina is a recently described bacterial genus of increasing research interest due to its high metabolic versatility and emerging role in the cycling of organic matter in marine ecosystems. Using comprehensive functional and comparative genomics, Sandra Godinho Silva, Tina Keller-Costa and Rodrigo Costa from BSRG-iBB revealed a previously underestimated number of gene clusters involved in the biosynthesis of natural products across several Aquimarina species, suggesting they are a promising target for the discovery of new bioactive compounds. The study, published in Environmental Microbiology, results from an extensive analysis of all the 26 Aquimarina genomes available to-date and uncovers 928 secondary metabolite biosynthetic gene clusters (BGCs) present in these genomes. Polyketide synthases, terpene synthases and non‐ribosomal peptide synthetases ranked as the most frequent BGCs encoding drug‐like candidates.

The establishment of novel, simpler and improved purification processes is extremely important to make antibody-based treatment options more accessible and with a lower economic burden to healthcare systems and patients. In this context, the development of microfluidic tools for the optimization of chromatography operating conditions can offer advantages such as low consumption of molecules, fast generation of results and ability to automate and multiplex the assays. In a collaboration between INESC-MN and iBB, researchers have recently reported a novel microfluidics-based methodology to perform rapid and multiplexed screening of various chromatography ligands relative to their ability to bind different target molecules in solution. The work was published in the Biotechnology Journal and is expected to contribute to the fields of separation sciences and analytical chemistry and inspire new developments within the purification of biopharmaceuticals or other molecules.

Photodiodes are on the rise. In the past decade many efforts have been made to deliver cheaper, more flexible, and increasingly better performing photodiode devices. The demand for enhanced optoelectronics led to the development of new inorganic, organic, and hybrid materials. A tutorial review published in Journal of Materials NanoScience by Rita Pires and Vasco Bonifácio from BSIRG-iBB describes in detail the working principles, the characterization, fabrication (types and architectures) and major applications of photodiodes. Recent applications, mainly in imaging, sensing and healthcare are also presented.

The organic fraction of municipal solid waste (OFMSW) accounts for approximately 30-40% of MSW in Europe. BERG-iBB researchers developed a process that uses this type of waste as a raw material or the production of the biodegradable biopolymer P(3HB). In a first step the complex carbohydrates in the waste are hydrolysed into simple monosaccharides. The hydrolysate is then used as a carbon source to feed and induce the bacterium Burkholderia sacchari to produce P(3HB). In order to overcome nutritional deficiencies and attain a significant polymer accumulation (58% g polymer/g CDW) the C/N ratio was adjusted and the hydrolysate was supplemented with minerals. This work demonstrates that an easily accessible waste can be transformed into valuable biodegradable bioplastics. The work was published in Bioresource Technology.

Photo details: municipal waste by OpenIDUser2, GFDL. 

Pathogenic infections in plant systems are on the rise, making their early detection in the field an important asset in order to avoid propagation amongst the rest of the crops. One biomarker of interest for this detection is Azelaic Acid (AzA). Based on this, a microfluidic device capable of detecting low concentrations of the acid was developed by Eduardo Brás under the supervision of Prof. João Pedro Conde (INESC-MN) and Prof. Pedro Fernandes (iBB), with the collaboration of Prof. Ana Margarida Fortes (FCUL). The results obtained using real samples as well as a description of the system, which exploits the inhibitory effect of AzA on the activity of the enzyme tyrosinase was published in the journal Analyst.

Photo details: Samir Budimčić - Own work, CC BY-SA 2.5, Wikimedia.

During crude oil extraction, the reduction in temperature and pressure results in the precipitation of paraffin wax, which may accumulate inside production tubes, pipelines, and also in tankers during petroleum transportation. Few bacterial strains are able to degrade this type of solid substrates. In a paper, published  in Biotechnology Journal, Carlos Rodrigues and Carla CCR de Carvalho (BERG-iBB) evaluate the biodegradation of paraffin by Rhodococcus erythropolis cells. The cells could grow as a thick biofilm over the solid substrate and rapidly degrade paraffin when it was supplied in the form of microparticles. The phenotypic adaptations allowing the cells to degrade the 20-40 chain hydrocarbons are discussed in the paper.

Many metal nanoparticles exhibit plasmonic properties that can be explored in different applications. The synthesis of these nanoparticles often leads to a heterogeneous mixture in terms of sizes and shapes that needs to be fractionated to yield samples with narrow plasmon resonances. A simple method based on sucrose density gradient centrifugation has been proposed by BERG-iBB researchers and colleagues from Centro de Química Estrutural and Universidade de Aveiro for the fractionation of colloidal silver nanotriangles. The method affords particle fractions with surface plasmon resonances spanning from red to infrared spectral ranges that could be used to tune optical properties for plasmonic applications. The work is part of the PhD thesis of Rui Oliveira Silva and was published in Nanomaterials.

Bacterial cell envelope plays a central role in cell physiology and the alteration of surface properties can implicate the variation of phenotypes that play a crucial role in the pathogenesis of infectious diseases, such as the resistance to antibiotics and other environmental stresses, biofilm formation, persistence of infection. However, the influence that Burkholderia cenocepacia adaptive evolution during long-term respiratory infection in cystic fibrosis (CF) patients has on cell wall morphology and mechanical properties is poorly understood. This study, just published in the journal Scientific Reports, has examined cell wall morphology and mechanical properties of three sequential B. cenocepacia clonal variants by Atomic Force Microscopy (AFM). Results reinforce the concept of the occurrence of phenotypic variation and adaptive evolution during chronic infection, also at the level of cell size, form, envelope topography and physical properties. This research work, coordinated by Prof. Isabel Sá-Correia from iBB-BSRG and IST, was the result of a national collaboration with the team of Dr. Mário S. Rodrigues from BioISI, Faculty of Sciences, Universidade de Lisboa, having the IST PhD student of the BIOTECnico program Amir Hassan, from iBB-BSRG, as first author of the publication.

The effective removal from textile industry wastewaters of the hazards of azo dyes and their partial degradation metabolites is severely hampered by the difficulties in cultivating microbial populations able to mineralize these recalcitrant pollutants in treatment bioreactors. In recent work applying the novel aerobic granular sludge (AGS) technology to this challenge, researchers Rita DG Franca, Nídia D Lourenço and Helena M Pinheiro (BERG-iBB) and M Conceição Oliveira (CQE) used liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to identify the colourless metabolites produced by AGS from azo dye Acid Red 14. Metabolite profiles under different bioreaction conditions provided insights into the associated biodegradation pathways. This work was published in ACS Sustainable Chemistry & Engineering.

The development of bioprocesses for industrial applications is often hampered by the use of time-consuming systems needed to find and characterize new biocatalysts. In particular, transaminases for the synthesis of optically pure chiral amines usually require multi-enzymatic and/or expensive high throughput equipment. In a recent published paper, Carlos JC Rodrigues (BERG-iBB), João M Sanches (LARSyS, ISR), and Carla CCR de Carvalho (BERG-iBB), developed an automatic time-lapse imaging system allowing both the identification of the most promising transaminases, and the determination of their kinetic parameters. The developed method reduces the time required for detection and quantification of specific transaminase activity, thus contributing to the success of biocatalyst application in industrial processes.

Bioreactors that provide different biophysical stimuli have been used in tissue engineering approaches aimed at enhancing the quality of the cartilage tissue generated. However, such systems are often highly complex, costly and not very versatile. In a recent study published in Biotechnology Journal, researchers from SCERG-iBB working with colleagues from the Polytechnic Institute of Leiria and Rensselaer Polytechnic Institute (USA) developed a novel, cost-effective and customizable perfusion bioreactor fabricated by additive manufacturing (AM) to study of the effect of fluid flow on the chondrogenic differentiation of human bone-marrow mesenchymal stem/stromal cells (hBMSCs) in 3D porous poly (ε-caprolactone) (PCL) scaffolds. Results suggest that the chondrogenic differentiation of hBMSCs was enhanced in cell-scaffold constructs cultured under perfusion and highlights the potential of customizable AM platforms for developing more reliable in vitro models and improved personalized cartilage repair strategies.

Bacteria of the Burkholderia cepacia complex (Bcc) are ubiquitous multidrug resistant organisms and opportunistic pathogens capable of causing life threatening lung infections among cystic fibrosis (CF) patients. No effective therapies are available to eradicate Bcc bacteria from CF patients, as these organisms are inherently resistant to most clinically available antimicrobials. In a collaborative research work headed by BSRG members JH Leitão and SA Sousa, in collaboration with the CBMA (UMinho) team headed by Pedro Santos, and the Cystic Fibrosis center (HSM) headed by MDs C Barreto and L Pereira, an immunoproteomics approach was used to identify Bcc proteins that stimulate the humoral immune response of the CF host. The work was published in the journal New Biotechnology and reports for the first time the identification of 19 proteins as immunogenic. Ten proteins were predicted as extracytoplasmic, 9 of them being conserved in Bcc genomes. The immunogenic Bcc extracytoplasmic proteins are potential targets for development of novel therapeutic strategies and diagnostic tools to protect patients against the onset of chronic Bcc lung infections.

Burkholderia cepacia complex (Bcc) bacteria can adapt to the hostile lung environment of cystic fibrosis (CF) patients leading to chronic infection. While it is unclear why different Bcc species/strains differ in their pathogenic potential, lipopolysaccharides (LPS) are considered a major virulence factor. The O-antigen (OAg) component of LPS is believed to modulate host-pathogen interaction and to be under selective pressure. A study recently published in Frontiers in Cellular and Infection Microbiology was performed to understand whether OAg loss can be considered a general phenomenon that affects immune evasion favoring chronic infection. A systematic retrospective and longitudinal screening was performed based on a collection of 357 isolates involving 21 different Bcc strains of six/seven Bcc species/lineages isolated from CF patients. These isolates were recovered from 1995 to 2016 from 19 CF patients under surveillance at Hospital de Santa Maria over the duration of chronic infection (ranging from 1.2 to 15.2 years). B. cenocepacia and B. multivorans showed a tendency to lose the OAg along chronic infection, with the switch frequency increasing with the duration of infection and level of lung function deterioration. For the first time, this study shows that the rarely found species B. cepacia and B. contaminans keep the OAg even during infections that last for 10 and 15 years. This research, coordinated by Prof. Isabel Sá-Correia and with the BIOTECnico PhD student Amir Hassan as first author, reinforces the relevance attributed to OAg-expression switch suggesting marked differences in the various Bcc species.

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.

Bacterial trans-acyltransferase polyketide synthases (trans-AT PKSs) are complex enzymes encoding the biosynthesis of a large diversity of polyketides, which are secondary metabolites usually possessing antitumoral and antibacterial bioactivities. Trans-AT PKSs are often found in difficult-to-cultivate and understudied microorganisms, making the assessment of their biosynthetic components and genome-guided natural product identification challenging. In a multidisciplinary effort involving researchers from ETH Zürich, Jena University, Institut Pasteur, and BSRG-iBB, a bio- and chemoinformatics web application (TransATor) was created to enable de novo structural prediction of trans-AT PKS-derived polyketides from sequence data, and tested as a structure elucidation-aid in the description of new polyketides from unusual bacterial sources. The results were communicated in Nature Chemical Biology.

Photo details: Aquimarina cultures, the source of the novel polyketide cuniculene. Credits: Patrícia Paula.

Acinetobacter baumannii is an important nosocomial pathogen resistant to many antibiotics. The relevance of a bacteriophage capsular depolymerase as a therapeutic agent against A. baumannii has now been unveiled by a collaborative work between researchers from BSRG-iBB (Dalila Mil-Homens, Andreia Pimenta, Arsénio M. Fialho) and the group of Joana Azeredo from the University of Minho. The greater wax moth Galleria mellonella and mice were used as animal models to address the therapeutic efficacy of the depolymerase against the infection. Results show that the enzyme makes bacterial cells fully susceptible to the host complement system killing effect. The depolymerase characterized here fits the new trend of alternative antibacterial agents needed against multidrug resistant pathogens. The work was published in Appl. Environ. Microbiol.