Exploiting the capacity of Labrys Portucalensis strain F11 for biotransformation of fluoroaromatic compounds

Environmental contamination with toxic chemicals of human origin is a threat to ecosystems and public health. Thus, it is extremely important to understand and explore the removal of these contaminants from different environments through biodegradation. The main objective of the work described in this thesis was the exploitation of the potential of Labrys portucalensis F11 – a bacterial strain previously isolated by its ability to degrade fluorobenzene (FB) - for biotransformation of fluorinated aromatic compounds of different complexity. Typically, contaminated environments are not contaminated with a single pollutant but with mixtures of pollutants. In particular the presence of organic compounds and metals in the same ecosystem, or appearing simultaneously in wastewater streams, is very common. In this study, the effect of three metals with different biological importance - iron, copper and silver - on the degradation of FB by strain F11 was evaluated. At a concentration of 1 mM, iron proved to be beneficial for bacterial growth without adversely affecting the biodegradation of up to 2 mM of FB. The presence of 1 mM of copper and silver inhibited the degradation of FB and led to the accumulation of intermediate metabolites, catechol and 4-fluorocatecol suggesting inhibition of the catechol 1,2-dioxygenase, which is a key enzyme of the metabolic pathway of FB. The degradation of compounds with chemical structures similar to FB, namely chlorobenzene (CB) and difluorobenzenes (DFBS), by L. portucalensis was investigated. Strain F11 was able to cometabolise CB in the presence of FB or when previously induced by this compound. Total degradation of 0.5 mM of each substrate was observed when both were added to the culture medium. Strain F11 was capable of degrading CB when the expression of enzymes is induced by FB however CB was not able to induce the enzymes for its own degradation. For DFBS, strain F11 proved to be able to degrade 0.5 mM of 1,3-DFB as sole carbon source, and to degrade 1,4-DFB (0.5 mM) in cometabolism with FB (0.5 mM). Strain F11 was unable to degrade 1,2-DFB and this compound inhibited the degradation of FB. These results reinforce the importance of the nature, number and position of the substituents in the molecule for enzyme expression and subsequently conversion of the target compounds. Fluoxetine (FLX) is a fluorinated chiral drug, which contamination, toxicity and persistence in the environment have been well documented in the past years. L. portucalensis F11 showed to be able to degrade both enantiomers of this compound as the sole carbon source (up to 9 μM) and in the presence of a conventional carbon source, sodium acetate (up to 89 μM of FLX). Degradation extents of at least 80% of total FLX were obtained. At the lowest FLX concentration tested (2 μM) as single carbon source, degradation was complete and fluoride release was stoichiometric. The degradation was shown to be enantioselective, with preferential degradation of R-FLX in relation to S-FLX. The transient formation of norfluoxetine (NFLX) as an intermediary metabolite was detected. With the objective of finding the genes responsible for the expression of FB dioxygenase, a genomic library consisting of 960 clones was constructed from the DNA of L. portucalensis F11. This library can be used for future work, such as the generation and confirmation of sequencing data, for comparative genomic studies or to search for other genes of interest. It is important to note that this strain has shown extraordinary capabilities of degradation of toxic compounds, and as such it would be very interesting to further study the genes that confer these capabilities. A partial nucleotide sequence of the gene cluster involved in FB degradation was determined. Sequencing results revealed the presence of four open reading frames, namely the gene coding for 1,2-catechol dioxygenase, and three genes encoding a ringhydroxylating dioxygenase (alpha and beta subunit of the dioxygenase component and the oxidoreductase component). Alignment of the deduced aminoacid sequences with sequences of others ring-hydoxylating dioxygenases revealed a high degree of similarity (≥80% identity) to the components of (halo)benzoate dioxygenases. The conserved amino acid residues that are involved in cofactor binding were also identified in the protein sequence. Recombinant strains carrying the putative FB dioxygenase genes were tested for expression. The SDS-PAGE analysis revealed that most of the expressed protein was on the pellet fraction and not on the soluble form, which could be due to improper folding of the enzyme components. Decrease of substrate concentration was observed in bioconversion experiments but the product formed was not detected/ identify. Overall, strain F11 revealed to be capable of degrading a vast range of fluorinated compounds with different complexity and as such can be a potential strain to devise biotechnological solutions for biotransformation processes.A contaminação do ambiente com produtos químicos de origem humana constitui uma ameaça para os ecossistemas e para a saúde pública. Deste modo, é extremamente importante compreender e explorar a sua biodegradação. O trabalho descrito nesta tese teve como objectivo principal a investigação do potencial da estirpe Labrys portucalensis F11 - uma bactéria anteriormente isolada pela sua capacidade de degradar fluorobenzeno (FB) - para a biotransformação de compostos aromáticos fluorados de diferente complexidade. Tipicamente, a contaminação do ambiente resulta da presença de uma mistura de poluentes e não apenas da presença de um único contaminante. Deste modo, a coexistência de compostos orgânicos e metais no mesmo ecossistema, ou em águas residuais, é muito comum. Neste estudo foi avaliado o efeito de 3 metais – cobre, ferro e prata, com diferente importância biológica na degradação do FB. Na concentração de 1 mM, o ferro foi benéfico para o crescimento bacteriano sem afectar adversamente a biodegradação de FB até à concentração de 2 mM. Na presença de 1 mM de cobre e de prata verificou-se a inibição da degradação do FB levando à acumulação de dois metabolitos intermediários, o catecol e o 4-fluorocatecol, sugerindo a inibição da catecol 1,2-dioxigenase, uma enzima chave da via metabólica do FB. A degradação de compostos com estruturas químicas semelhantes à do FB, clorobenzeno (CB) e difluorobenzenos (DFBS), foi avaliada. A estirpe F11 foi capaz de cometabolizar o CB na presença do FB, com degradação total de 0,5 mM de cada um dos substratos quando adicionados simultaneamente ou quando previamente induzida pelo FB. No entanto, o CB não induziu as enzimas para a sua própria degradação. Em relação aos DFBs, a estirpe F11 foi eficaz na degradação de 0,5 mM de 1,3-DFB como única fonte de carbono, e na degradação de 1,4-DFB (0,5 mM) em cometabolismo com FB (0,5 mM). No entanto, a estirpe F11 foi ineficaz na degradação do 1,2-DFB, tendo este composto inibido a degradação de FB. Estes resultados reforçam a importância da natureza, número e posição dos substituintes na molécula para a expressão enzimática e consequente conversão de compostos alvo. A fluoxetina (FLX) é um fármaco quiral fluorado, cuja toxicidade, contaminação e persistência no ambiente têm sido bem documentadas. A estirpe F11 mostrou ser capaz de degradar os dois enantiómeros deste composto como única fonte de carbono (até 9 μM) e na presença de acetato de sódio (até 89 mM de FLX). Foram obtidas extensões de degradação de pelo menos 80% de FLX total. Na concentração mais baixa de FLX (2 μM), testada como única fonte de carbono, a degradação foi completa e a libertação de fluoreto foi estequiométrica. A degradação revelou ser enantiosselectiva, com a degradação preferencial de R-FLX em relação a S-FLX, tendo sido detectada a formação de norfluoxetina (NFLX) como metabolito intermediário. Com o objectivo de pesquisar os genes responsáveis pela expressão da FB dioxigenase, foi construída uma biblioteca genómica, constituída por 960 clones, a partir do DNA da estirpe F11 de L. portucalensis. Esta biblioteca poderá ser usada em trabalhos futuros, como a geração e confirmação dos dados da sequenciação, em estudos de genómica comparativa ou na pesquisa de outros genes de interesse. É importante realçar que a estirpe F11 tem demostrado uma capacidade extraordinária na degradação de compostos tóxicos, sendo deste modo, de grande interesse estudar os genes que lhe conferem estas capacidades. Foi determinada uma sequência parcial dos nucleótidos do operão envolvido na degradação do FB. A sequenciação mostrou a existência de 4 genes, nomeadamente, o gene que codifica a 1,2-catecol dioxigenase, e os 3 genes que codificam as 3 subunidades da FB dioxigenase (alfa e beta da componente dioxigenase e oxidoreductase). O alinhamento das sequências de aminoácidos obtidas com as sequências de outras dioxigenases aromáticas revelou um elevado grau de similaridade (≥80%) com os componentes das (halo)benzoato dioxigenases. A sequenciação também permitiu identificar resíduos de aminoácidos conservados que estão envolvidos na ligação de cofactores. Estirpes recombinantes contendo os genes putativos da FB dioxigenase foram testadas para a expressão da enzima. A análise de SDS-PAGE revelou que a maioria da proteína expressa se encontrava na fracção do pellet e não na fracção solúvel, o que pode estar relacionado com uma conformação incorrecta dos componentes enzimáticos. Nas experiências de bioconversão foi observada diminuição da concentração de substrato mas o produto formado não foi detectado / identificado. Em geral, a estirpe F11 revelou ser capaz de degradar uma vasta gama de compostos fluorados com diferente complexidade e, como tal, pode ser considerada uma estirpe com potencial para aplicação e desenvolvimento de soluções biotecnológicas em processos de biotransformação

Biodegradation of 2,4 DCP herbicide by streptomyces collinus isolated from wastewater treatment plant in eastern Algeria

Wastewater treatment plants are the place where most pollutants are transported. 2, 4 DCP is an herbicide widely used in agriculture. This carcinogenic pollutant is very dangerous because it can reach surface waters through runoff and deep waters widely used by humans and animals. Water treatment plants are a reservoir of multiple and varied microorganisms, able to eliminate the toxic effect of many pollutants. The actinobacteria by their impressive metabolic abilities, are among the most appreciated microbial agents in the bioremediation of these hydric sites. In order to evaluate the functionality of the Ibn Ziad station in Constantine, we tested some physicochemical characteristics and the biodiversity of actinobacteria able to tolerate and degrade 2, 4 DCP. Sampling was carried out on raw wastewater, treated water and aeration tank water. The parameters studied were temperature, pH, conductivity, salinity, BOD5, DOC and suspended matters (MES). Actinobacteria were isolated on four selective media, namely AF, modified Czapeck dox, ISP4, Olson. The determination of herbicide biodegradation capacity by these bacteria was tested first on a minimum solid medium supplemented with 50 mg/L of 2, 4-DCP as a single carbon and energy source. Isolates that grew on this medium were cultured in liquid medium in the presence of 50 mg/L of the same pollutant. The degradation kinetics were monitored by HPLC. The best performing isolate was identified by phenotypic and molecular methods. The results show slightly alkaline pH, ambient temperatures. The DOC/BOD5 ratio is less than three, which indicates a slightly biodegradable effluent. While the MES concentration is around 256.7 mg/L. This station shows an important biodiversity of actinobacteria, with 25 isolates, among which 18 are able to live in the presence of 2, 4-DCP. The study of the kinetics of growth and degradation shows a good performance of an isolate, with a degradation rate of 45% after one month of incubation. The polyphasic identification of this bacterium, allows to assign it to the species Streptomyces collinus strain NBRC 12759 16S. These results show that the waters of thisstation are rich in actinobacteria able to degrade the herbicide 2, 4-DCP. These bacteria can be used in the bioremediation of water ecosystems polluted by this phytosanitary productinfo:eu-repo/semantics/publishedVersio

Study of the role of indigenous actinobacteria from activated sludge in the degradation of the fungicide fenhexamid

info:eu-repo/semantics/publishedVersio

Study of the biodegradation of the insecticide alpha-cypemethrin by indigenous actinobacteria isolated from activated sludge

info:eu-repo/semantics/publishedVersio

Biodegradation of 2,4 DCP herbicide by streptomyces collinus isolated from wastewater treatment plant in eastern Algeria

info:eu-repo/semantics/publishedVersio

Computational approaches in antibody-drug conjugate optimization for targeted cancer therapy

WOS: 000444683500007PubMed ID: 30068276Cancer has become one of the main leading causes of morbidity and mortality worldwide. One of the critical drawbacks of current cancer therapeutics has been the lack of the target-selectivity, as these drugs should have an effect exclusively on cancer cells while not perturbing healthy ones. In addition, their mechanism of action should be sufficiently fast to avoid the invasion of neighbouring healthy tissues by cancer cells. The use of conventional chemotherapeutic agents and other traditional therapies, such as surgery and radiotherapy, leads to off-target interactions with serious side effects. In this respect, recently developed target-selective Antibody-Drug Conjugates (ADCs) are more effective than traditional therapies, presumably due to their modular structures that combine many chemical properties simultaneously. In particular, ADCs are made up of three different units: a highly selective Monoclonal antibody (Mab) which is developed against a tumour-associated antigen, the payload (cytotoxic agent), and the linker. The latter should be stable in circulation while allowing the release of the cytotoxic agent in target cells. The modular nature of these drugs provides a platform to manipulate and improve selectivity and the toxicity of these molecules independently from each other. This in turn leads to generation of second-and third-generation ADCs, which have been more effective than the previous ones in terms of either selectivity or toxicity or both. Development of ADCs with improved efficacy requires knowledge at the atomic level regarding the structure and dynamics of the molecule. As such, we reviewed all the most recent computational methods used to attain all-atom description of the structure, energetics and dynamics of these systems. In particular, this includes homology modelling, molecular docking and refinement, atomistic and coarse-grained molecular dynamics simulations, principal component and cross-correlation analysis. The full characterization of the structure-activity relationship devoted to ADCs is critical for antibody-drug conjugate research and development.Fundacao para a Ciencia e a Tecnologia (FCT) Investigator programme [IF/00578/2014]; European Social Fund; Programa Operacional Potencial Humano; Marie Sklodowska-Curie Individual Fellowship MSCA-IF-2015 [MEMBRANEPROT 659826]; European Regional Development Fund (ERDF), through the Centro 2020 Regional Operational Programme [CENTRO-01-0145-FEDER-000008]; European Regional Development Fund (ERDF), COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation; Portuguese national funds via FCT [POCI-01-0145-FEDER-007440]; FCT [FCT-SFRH/BPD/97650/2013]; Fundacao para a Ciencia e Tecnologia (FCT), Portugal [UID/Multi/04349/2013]Irina S. Moreira acknowledges support by the Fundacao para a Ciencia e a Tecnologia (FCT) Investigator programme - IF/00578/2014 (co-financed by European Social Fund and Programa Operacional Potencial Humano), and a Marie Sklodowska-Curie Individual Fellowship MSCA-IF-2015 [MEMBRANEPROT 659826]. This work was also financed by the European Regional Development Fund (ERDF), through the Centro 2020 Regional Operational Programme under project CENTRO-01-0145-FEDER-000008: Brain-Health 2020, and through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation and Portuguese national funds via FCT, under project POCI-01-0145-FEDER-007440. Rita Melo acknowledges support from the FCT (FCT-SFRH/BPD/97650/2013). This work has been partially supported by the Fundacao para a Ciencia e Tecnologia (FCT), Portugal, through the UID/Multi/04349/2013 project in Centre for Nuclear Sciences and Technologies (C2TN)

How many biomarker measurements are needed to predict prognosis in Crohn's disease patients under infliximab?—A prospective study

BackgroundTimely stratification of Crohn's disease (CD) is essential for patients' management. The use of noninvasive accurate biomarkers is key to monitor treatment and to pursue mucosal healing, the ultimate treatment endpoint in CD. ObjectiveWe aimed to evaluate the performance of readily available biomarkers and develop risk matrices to predict CD progression. MethodsData from 289 CD patients receiving infliximab (IFX) maintenance therapy for 2 years was collected; those patients were included in DIRECT, a prospective multicenter observational study. Disease progression was evaluated using two composite outcomes incorporating clinical and drug-related factors, the first including IFX dose and/or frequency adjustments. Univariate and multivariable logistic regressions were used to calculate the odds ratios (OR) and to develop risk matrices. ResultsThe isolated presence of anemia at least once during follow-up was a significant predictor of disease progression (OR 2.436 and 3.396 [p 10.0 mg/L) and fecal calprotectin (FC; >500.0 mu g/g) in at least one visit were also significant predictors, while milder elevations (3.1-10.0 mg/L and 250.1-500.0 mu g/g) were only relevant when detected in at least two visits (consecutive or not). The combination of biomarkers in risk matrices had good ability to predict progression; patients simultaneously presenting anemia, highly elevated CRP and FC at least once had 42%-63% probability of achieving the composite outcomes. ConclusionThe combined evaluation of hemoglobin, CRP, and FC in at least one time point and their incorporation into risk matrices seems to be the optimal strategy for CD management, as data from additional visits did not meaningfully influence the predictions and may delay decision-making.Portuguese Group of Studies in Inflammatory Bowel Disease (GEDII)info:eu-repo/semantics/publishedVersio

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data