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- Antibiotic discovery: where have we come from, where do we go?Publication . Ribeiro Da Cunha, Bernardo; Fonseca, Luís P.; Calado, CecíliaGiven the increase in antibiotic-resistant bacteria, alongside the alarmingly low rate of newly approved antibiotics for clinical usage, we are on the verge of not having effective treatments for many common infectious diseases. Historically, antibiotic discovery has been crucial in outpacing resistance and success is closely related to systematic procedures—platforms—that have catalyzed the antibiotic golden age, namely the Waksman platform, followed by the platforms of semi-synthesis and fully synthetic antibiotics. Said platforms resulted in the major antibiotic classes: aminoglycosides, amphenicols, ansamycins, beta-lactams, lipopeptides, diaminopyrimidines, fosfomycins, imidazoles, macrolides, oxazolidinones, streptogramins, polymyxins, sulphonamides, glycopeptides, quinolones and tetracyclines. During the genomics era came the target-based platform, mostly considered a failure due to limitations in translating drugs to the clinic. Therefore, cell-based platforms were re-instituted, and are still of the utmost importance in the fight against infectious diseases. Although the antibiotic pipeline is still lackluster, especially of new classes and novel mechanisms of action, in the post-genomic era, there is an increasingly large set of information available on microbial metabolism. The translation of such knowledge into novel platforms will hopefully result in the discovery of new and better therapeutics, which can sway the war on infectious diseases back in our favor.
- Optimization of a bioassay to evaluate Escherichia coli stress responsesPublication . Ribeiro Da Cunha, Bernardo; Russo, Ana; Fonseca, Luís; Calado, CecíliaDuring the genomics era, great emphasis was placed on sequencing microbial genomes and understanding their global gene expression. Later in the post-genomic era, focus shifted towards building on said information, mainly towards deeper knowledge on the relationship of genomics and transcriptomics with the biomolecular composition of microbes, and ultimately their metabolism. To that end, exploring microbial adaptation, e.g. to adverse conditions, is an integral part of current research, for which Escherichia coli is a favored model organism, as its metabolic pathways share pronounced similarities to mainstream pathways across other microorganisms. As such, E. coli has been extensively studied regarding general stress and specific metabolic responses, inclusively for establishing bioassays for detecting and characterizing the mode of action of antibacterial agents. For that, as an example, the global responses of E. coli to diverse stress conditions has been previously compared by transcriptomic and Fourier-transform infrared (FTIR) spectroscopy analysis, highlighting the importance of combining multiple techniques to profoundly understand the holistic cell response. In this work, a high-throughput FTIR spectroscopy based bioassay was developed for E. coli, and optimized regarding multiple parameters as inoculum growth phase, growth media composition and stress agent exposure time. The approach described allowed to identify the impact of each parameter on the metabolic resolution of the biomolecular fingerprint acquired with FTIR spectroscopy, and consequently the optimized bioassay maximizes the ability of FTIR spectroscopy to specifically characterize the impact of diverse stress agents on the E. coli metabolism.
- Mid-infrared spectroscopy: a groundbreaking tool for monitoring mammalian cells processesPublication . Rosa, Filipa O.P.; Ribeiro Da Cunha, Bernardo; Carmelo, Joana G.; Fernandes-Platzgummer, Ana; da Silva, Claudia; Calado, CecíliaMammalian cells are extensively used in cell biology studies, e.g. as a model system of human pathologies, or as a major source of very high-value biopharmaceuticals (that can be the cells itself or their products e.g. heterologous proteins). As such, it is highly pertinent to develop monitoring methods for mammalian cultivations capable of delivering detailed bioprocess information in a rapid and economic way. It is relevant to acquire information concerning the conventional critical variables (as cell growth, consumption of nutrients, production and consumption of by-products and the bioproduct production), and the cell metabolism towards a better understanding of the culture process and consequently for more efficient optimization and control procedures. In the present work, Mid-infrared (MIR) spectroscopy was evaluated as a monitoring technique enabling the acquisition of said bioprocess information in a simple (single step of dehydration), rapid (minutes), economic (without reagent consumption), label-free and high-throughput mode (using 96-wells microplates). The new method was evaluated across a highly diverse set of mammalian culture processes: The monitoring of ex vivo expansion of human mesenchymal stem/stromal cells (MSC) conducted under diverse culture strategies, where it was possible to accurately predict glucose, lactate and ammonia concentrations. The monitoring of recombinant human embryonic kidney cells producing green fluorescent protein, which enabled the estimation of transfection efficiency and the metabolic impact of protein production on the host cell metabolism. Finally, the monitoring of infected gastric cell lines with Helicobacter pylori, which enabled to identify spectral biomarkers for defining the status of infection (infected vs non infected) and to characterize the infection conducted by virulent strains, usually associated to severe gastric diseases as peptide ulcer and gastric cancer. In resume, high-throughput MIR spectroscopy enabled to adequately monitor diverse mammalian cell cultures, thus allowing to attain meaningful information concerning said bioprocesses, from traditional critical variables of the process, to the metabolic status of mammalian host cells and even to define disease biomarkers in a groundbreaking way.
- Characterization of gastric cells infection by diverse Helicobacter pylori strains through Fourier-transform infrared spectroscopyPublication . Marques, Vanda; Ribeiro Da Cunha, Bernardo; Couto, Andreia; Sampaio, Pedro; Fonseca, Luís P. P.; Aleixo, Sandra; Calado, CecíliaThe infection of Helicobacter pylori, covering 50% of the world-population, leads to diverse gastric diseases as ulcers and cancer along the life-time of the human host. To promote the discovery of biomarkers of bacterial infection, in the present work, Fourier-transform infrared spectra were acquired from adenocarcinoma gastric cells, incubated with H. pylori strains presenting different genotypes concerning the virulent factors cytotoxin associated gene A and vacuolating cytotoxin A. Defined absorbance ratios were evaluated by diverse methods of statistical inference, according to the fulfillment of the tests assumptions. It was possible to define from the gastric cells, diverse absorbance ratios enabling to discriminate: i) The infection; ii) the bacteria genotype; and iii) the gastric disease of the patients from which the bacteria were isolated. These biomarkers could fasten the knowledge of the complex infection process while promoting a platform for a new diagnostic method, rapid but also specific and sensitive towards the diagnosis of both infection and bacterial virulence.
- Development of a high-throughput monitoring technique of bacteria photodynamic inactivationPublication . Ribeiro Da Cunha, Bernardo; Sampaio, Pedro N.; Calado, CecíliaSummary form only given. Bacterial infections and the fight against them have been one of the major concerns of mankind since the dawn of time. During the `golden years' of antibiotic discovery, during the 1940-90s, it was thought that the war against infectious diseases had been won. However currently, due to the drug resistance increase, associated with the inefficiency of discovering new antibiotic classes, infectious diseases are again a major public health concern. A potential alternative to antibiotic treatments may be the antimicrobial photodynamic inactivation (PDI) therapy. To date no indication of antimicrobial PDI resistance development has been reported. However the PDI protocol depends on the bacteria species [1], and in some cases on the bacteria strains, for instance Staphylococcus aureus [2]. Therefore the development of PDI monitoring techniques for diverse bacteria strains is critical in pursuing further understanding of such promising alternative therapy. The present works aims to evaluate Fourier-Transformed-Infra-Red (FT-IR) spectroscopy to monitor the PDI of two model bacteria, a gram-negative (Escherichia coli) and a gram-positive (S. aureus) bacteria. For that a high-throughput FTIR spectroscopic method was implemented as generally described in Scholz et al. [3], using short incubation periods and microliter quantities of the incubation mixture containing the bacteria and the PDI-drug model the known bactericidal tetracationic porphyrin 5,10,15,20-tetrakis (4-N, N, Ntrimethylammoniumphenyl)-porphyrin p-tosylate (TTAP4+). In both bacteria models it was possible to detect, by FTIR-spectroscopy, the drugs effect on the cellular composition either directly on the spectra or on score plots of principal component analysis. Furthermore the technique enabled to infer the effect of PDI on the major cellular biomolecules and metabolic status, for example the turn-over metabolism. In summary bacteria PDI was monitored in an economic, rapid (in minutes- , high-throughput (using microplates with 96 wells) and highly sensitive mode resourcing to FTIR spectroscopy, which could serve has a technological basis for the evaluation of antimicrobial PDI therapies efficiency.
- Towards an automated statistical workflow for biomarker screening in Fourier-transform infrared spectroscopyPublication . Ribeiro Da Cunha, Bernardo; Aleixo, Sandra; Fonseca, Luís P.; Calado, CecíliaThe increasing availability and sensitivity of analytical technologies has resulted in much higher complexity of molecular and cellular data, which biomarkers facilitate the bridge with a given biological state under scrutiny. Importantly, biomarkers are critical in spectroscopic technologies, as is the case of Fourier-transform infrared spectroscopy, for which different approaches to identify biomarkers have been established, for instance univariate statistical hypothesis tests. A workflow for the automatic application of said statistical tests is proposed with the objective of enabling a high throughput screening-approach that ensures statistical robustness, and works in a user-independent manner, thereby reducing analysis bias. The proposed workflow is the first step towards an automated method for biomarker screening, thus it is limited to distinguishing two populations. Firstly, the method used in previous work was improved to ensure the most powerful and accurate statistical tests are applied. Then, the statistical workflow was generalized to a more comprehensive range of data sets of two populations, for instance when sample size is not deemed high (less than thirty observations). Lastly, future work is outlined regarding the extension of the workflow to cases with more than two populations, but also of methods that can increase the pool of putative biomarkers that will then be screened for statistical significance.
- Infection biomarkers based on metabolomicsPublication . Araújo, Rúben; Bento, Luís; Fonseca, Tiago AH; Von Rekowski, Cristiana; Ribeiro Da Cunha, Bernardo; Calado, CecíliaCurrent infection biomarkers are highly limited since they have low capability to predict infection in the presence of confounding processes such as in non-infectious inflammatory processes, low capability to predict disease outcomes and have limited applications to guide and evaluate therapeutic regimes. Therefore, it is critical to discover and develop new and effective clinical infection biomarkers, especially applicable in patients at risk of developing severe illness and critically ill patients. Ideal biomarkers would effectively help physicians with better patient management, leading to a decrease of severe outcomes, personalize therapies, minimize antibiotics overuse and hospitalization time, and significantly improve patient survival. Metabolomics, by providing a direct insight into the functional metabolic outcome of an organism, presents a highly appealing strategy to discover these biomarkers. The present work reviews the desired main characteristics of infection biomarkers, the main metabolomics strategies to discover these biomarkers and the next steps for developing the area towards effective clinical biomarkers.
- Biopharmaceuticals process monitoring based on infrared spectroscopy according to quality by designPublication . Sales, Kevin; Ribeiro Da Cunha, Bernardo; Calado, CecíliaEscherichia coli, the most common host microorganism to produce high value biopharmaceuticals, presents, however a natural variability due to high sensitivity towards small fluctuations of the cultivation environment. Therefore, to speed up the development process and to control this critical manufacturing step it is relevant to develop monitoring techniques allowing simultaneous characterization of the heterologous product synthesis and of the physiologic cell behavior under a variety of culture conditions. In the present work high-throughput mid-infrared (MIR) spectral analysis of dehydrated E. coli cell pellets and in situ near infrared (NIR) spectral analysis of the whole culture broth were compared to monitor the bioproduction of a model plasmid along recombinant Escherichia coli cultures. Good partial least square regression models were built, either based on MIR or on NIR spectral data. Besides being conducted in situ, NIR spectroscopy allowed building regression models as accurate as those obtained from the MIR setup, and even better models for estimating biomass and glycerol. However, MIR spectroscopy allows retrieving valuable biochemical and metabolic information along the cell culture, e.g. lipids, RNA, protein synthesis and turnover metabolism. This information contributed to better understand the complex interrelationships between the recombinant cell metabolism and the bioprocess environment. In resume, since NIR spectroscopy enables the in situ and real time monitoring without the risk of culture contamination, thus represents an appealing tool for control purposes specially at industrial (i.e. large-scale) productions. On the other hand, MIR spectroscopy is better suited for optimization processes, as it enables the analysis of hundreds of samples at ounce, and to acquire off line historical information along industrial production processes. Both monitoring techniques are therefore complementary, empowering optimization and control procedures more efficiently and quicker, according to the new regulatory framework based on Quality by Design.
- Fast identification of off-target liabilities in early antibiotic discovery with Fourier-transform infrared spectroscopyPublication . Ribeiro Da Cunha, Bernardo; Aleixo, Sandra M.; Fonseca, Luís P. P.; Calado, CecíliaStructural modifications of known antibiotic scaffolds have kept the upper hand on resistance, but we are on the verge of not having antibiotics for many common infections. Mechanism-based discovery assays reveal novelty, exclude off-target liabilities, and guide lead optimization. For that, we developed a fast and automatable protocol using high-throughput Fourier-transform infrared spectroscopy (FTIRS). Metabolic fingerprints of Staphylococcus aureus and Escherichia coli exposed to 35 compounds, dissolved in dimethyl sulfoxide (DMSO) or water, were acquired. Our data analysis pipeline identified biomarkers of off-target effects, optimized spectral preprocessing, and identified the top-performing machine learning algorithms for off-target liabilities and mechanism of action (MOA) identification. Spectral bands with known biochemical associations more often yielded more significant biomarkers of off-target liabilities when bacteria were exposed to compounds dissolved in water than DMSO. Highly discriminative models distinguished compounds with predominant off-target effects from antibiotics with well-defined MOA (AUROC > 0.87, AUPR > 0.79, F1 > 0.81), and from the latter predicted their MOA (AUROC > 0.88, AUPR > 0.70, F1 > 0.70). The compound solvent did not affect predictive models. FTIRS is fast, simple, inexpensive, automatable, and highly effective at predicting MOA and off-target liabilities. As such, FTIRS mechanism-based screening assays can be applied for hit discovery and to guide lead optimization during the early stages of antibiotic discovery.
- High-throughput bioassay for mechanism of action determination of antibacterial drugsPublication . Ribeiro Da Cunha, Bernardo; Fonseca, Luís P. P.; Calado, CecíliaWhile the ‘war’ on infectious diseases has been considered won, antibiotic-resistant bacteria are currently responsible for 25,000 death’s yearly in Europe. No new broadspectrum antibiotic has been introduced since the 1960s, and the last new class was discovered in 1986. As the antibiotic pipeline is clearly exhausted, new tools to advance antibiotic research are required. The current work explored Fourier-transform infrared spectroscopy to classify the mechanism of action of 13 antibiotics, acting by 3 distinct Mode-Of-Action (MOA) and belonging to 7 different classes. After optimization of a biological assay and pre-processing techniques, principal component analysis and partial least squares discriminant analysis were applied in a multi-level approach, including the MOA, antibiotic class and ultimately individual antibiotics acting on very specific molecular targets. Overall results indicate that the proposed method presents metabolic resolution to identify antibiotics at three levels of classification (i.e. different MOA, classes and even acting on specific targets). Interestingly, the resolution capacity obtained at these three levels of classification depended on the antibiotic type, which highlights the importance of the multilevel approach taken. Ultimately the present work reinforces the applicability of the method has a metabolic fingerprinting tool for antibiotic discovery.