Coumarins as Fluorescent Labels of Biomolecules

Important areas such as environmental sciences, medicine, pharmacy, and cellular biology are dependent on very sensitive analytical techniques. One of the most common methodologies used for their bioanalytical purposes is the fluorescent labelling. The synthesis of new fluorophores and the great development of fluorescent-labelling techniques combined with the enormous technological advances in the field of fluorescence microscopy allowed to deepen the structural knowledge of biomolecules. This new organic fluorophores form covalent bonds with the sample to be analyzed, producing stable bioconjugates that show fluorescence in a wide range of wavelengths, depending on the label used. Coumarin derivatives represent one of the most important chemical classes of organic fluorescent materials being one of the most extensively investigated and commercially significant groups of organic fluorescent materials. In this chapter, it is reviewed the use of fluorescent coumarin derivatives and their application to labelling biomolecules. These fluorescent labels allow researchers to study, and understand, biomolecular assemblies that exhibit complex sensitivity and selectivity. Reactive fluorescent coumarin derivatives are actually widely used in labelling biomolecules as peptides, proteins, oligonucleotides, nucleic acids, and carbohydrates, among other biological molecules

detection for yeast and bacteria in wood slabs by RNA FISH

The deterioration of cultural heritage objects and assets (mural paintings, statues, and many other art objects made of wood, stone, paper, ceramic, glass, inter alia) can be caused by microorganisms [1]. One of the most important steps for applying adequate conservation and protection measures is early identification and monitorization of microbial colonization. The conventional culture-based methods used so far have become insufficient to detect/identify the biodeteriogenic agents. Thus, molecular techniques have started to attract considerable interest [2,3]. Our group is focused on detecting and identifying microorganisms that cause biodeterioration on artworks using the RNA-FISH molecular technique [4]. It is a simple, rapid and promising molecular technique enabling the detection, visualization and identification of the viable microorganisms of interest [5,6]. As any other technique, RNA-FISH has its own minimum Limit Of Detection (LOD) and for ensuring the reliability of RNA-FISH analyses, determination of the associated LODs is imperative. Thus, the aim of this work was to determine the LOD for yeast and bacteria in wood slabs by RNA-FISH. Universal probes for targeting eukaryotes (EUK516) and bacteria (EUB338) labeled with Cy3 or Atto-647N dyes were used.This work was co-financed by ALT20-03-0246-FEDER-000004-ALENTEJO 2020 project and by FCT through the project PTDC/BBB-IMG/0046/2014 and grants SFRH/BD/118028/2016 and SFRH/BPD/100754/2014

Combined use of LC–ESI-MS and antifungal tests for rapid identification of bioactive lipopeptides produced by Bacillus amyloliquefaciens CCMI 1051

The strain Bacillus amyloliquefaciens CCMI 1051 used in this study has been isolated in our laboratory from healthy Quercus suber in the south of Portugal and shows high levels of antagonistic properties against filamentous fungi that attack forest products industry due to the production of bioactive peptides. A combined use of LC–ESI-MS and antifungal tests allowed a rapid identification of lipopeptides as active compounds produced. Applying autobiographic methods it was possible to obtain active compounds. LC–ESI-MS, a powerful tool for rapid identification, indicates the presence of lipopeptides and MS2 electrospray ionization showed the partial sequence Tyr–Asn–Pro–Glu in the peptidic portion of some compounds produced. The association of mass spectrometry and chromatography, used in parallel with antifungal tests proved to be an efficient approach for the characterization of active lipopeptides without the need of previous total isolation. This methodology can be employed for screening and optimization the production of antifungal iturinic lipopeptides, showing a great potential for future application

Innovative approaches for immunodetection of proteic binders in art

The characterization and identification of the proteinaceous compounds in a complex and multi-layered painting is crucial for studying the technique used by the artist and for conservation and restoration purposes. These organic compounds, such as animal glues, milk and egg, have a particular importance since they are widely used as binders and adhesives in paintings. Proteins are typically detected by methods like chromatographic and proteomic techniques. However, the immunodetection approach shows to be a powerful method in protein analysis. In this study immunologic techniques, based on enzyme-linked immunosorbent assay (ELISA), were used in order to identify different proteic binders used in easel paintings. The methodology based on indirect ELISA allows the detection of the target antigen in paint models microsamples. These approach is very promising with regard to the possibility of apply these methodology in the detection of proteinaceous binders in real samples

DETECTION OF SPORES AND HYPHAE OF ARTWORKS’ BIODETERIOGENIC FILAMENTOUS FUNGI BY RNA-FISH

Filamentous fungi are a threat to the conservation of Cultural Heritage. Thus, detection and identification of viable filamentous fungi are crucial for applying adequate Safeguard measures. RNA-FISH protocols have been previously applied with this aim in Cultural Heritage samples. However, only hyphae detection was reported in the literature, even if spores and conidia are not only a potential risk to Cultural Heritage but can also be harmful for the health of visitors, curators and restorers. Thus, the aim of this work was to evaluate various permeabilizing strategies for their application in the detection of spores/conidia and hyphae of artworks’ biodeteriogenic filamentous fungi by RNA-FISH. Besides of this, the influence of cell aging on the success of the technique and on the development of fungal autofluorescence (that could hamper the RNA-FISH signal detection) were also investigated. Five common biodeteriogenic filamentous fungi species isolated from biodegradated artworks were used as biological model: Aspergillus niger, Cladosporium sp, Fusarium sp, Penicillium sp. and Exophialia sp. Fungal autofluorescence was only detected in cells harvested from Fusarium sp, and Exophialia sp. old cultures, being aging-dependent. However, it was weak enough to allow autofluorescence/RNA-FISH signals distinction. Thus, autofluorescence was not a limitation for the application of RNA-FISH for detection of the taxa investigated. All the permeabilization strategies tested allowed to detect fungal cells from young cultures by RNA-FISH. However, only the combination of paraformaldehyde with Triton X-100 allowed the detection of conidia/spores and hyphae of old filamentous fungi. All the permeabilization strategies failed in the Aspergillus niger conidia/spores staining, which are known to be particularly difficult to permeabilize. But, even in spite of this, the application of this permeabilization method increased the analytical potential of RNA FISH in Cultural Heritage biodeterioration. Whereas much work is required to validate this RNA-FISH approach for its application in real samples from Cultural Heritage it could represent an important advance for the detection, not only of hyphae but also of spores and conidia of various filamentous fungi taxa by RNA-FISH

Actualização da prevalência de Leishmaniose canina nos concelhos de Setúbal e de Palmela

A leishmaniose constitui uma das doenças tropicais mais negligenciadas em todo o Mundo. Na região Mediterrânica, a doença na forma visceral atinge preferencialmente crianças com idade inferior a três anos e adultos imunocomprometidos. É causada por protozoários da espécie Leishmania infantum. O cão é considerado o principal reservatório peridoméstico para a infecção humana, conferindo grande importância a esta zoonose em termos de Saúde Pública. A leishmaniose também possui elevada importância em Medicina Veterinária, por causar doença grave no cão. Não obstante, embora a infecção por Leishmania infantum esteja amplamente difundida na população canina das áreas endémicas, apenas uma fracção destes cães desenvolve doença clínica. O diagnóstico e tratamento precoces da leishmaniose canina são essenciais, quer para controlar a expansão da doença, quer como parte integrante de um sistema de controlo da leishmaniose visceral humana zoonótica. Estudos realizados na Península de Setúbal na década de 1980 estabeleceram a seroprevalência da doença na população canina em 11,5% (Abranches et al, 1983). Outro estudo, realizado na região de Lisboa entre Dezembro de 2002 e Dezembro de 2003, estimou a prevalência da infecção por L. infantum em 18,4% nos cães domésticos e 21,6% nos cães sem dono (Cortes et al, 2007). Neste trabalho foi efectuada uma actualização da prevalência de leishmaniose canina nos Concelhos de Palmela e Setúbal. Realizaram-se colheitas de sangue periférico a uma amostra aleatória constituída por dois grupos de cães: 100 que se apresentaram à consulta a uma clínica veterinária localizada em Palmela (representando cães com dono) e 83 cães abrigados por uma associação de defesa de animais, situada em Setúbal (considerados sem dono). O rastreio serológico foi efectuado com recurso ao teste de aglutinação directa e os soros com resultados positivos naquela reacção foram também analisados por contra-imunoelectroforese e imunofluorescência indirecta. Também foi feita pesquisa parasitológica por PCR no sangue periférico. Determinou-se a seroprevalência total de leishmaniose de 7,1%, para a população canina estudada (5% no grupo de cães da clínica e 9,6% para o grupo de cães da associação). Utilizando o teste de qui-quadrado, não foram encontradas diferenças significativas entre os dois grupos estudados. Foi preenchido um questionário no acto da colheita das amostras biológicas, para avaliar a importância de determinados factores (género, porte, proveniência, tipo de habitat, uso de repelentes do vector e co-habitação com outros cães) envolvidos na infecção. Embora os testes aplicados sugerissem algumas tendências, nenhum dos parâmetros avaliados se revelou como factor de risco de forma estatisticamente significativa.Leishmaniasis is one of the most neglected tropical diseases worldwide. In the Mediterranean Region, the disease, in the visceral form, is most important in children under three years old and immunocomprised adults. It is caused by a protozoan of the species Leishmania infantum. The dog is considered the main peridomestic reservoir for the human infection, which makes canine leishmaniasis a very important zoonosis as far as Public Health is concerned. Canine leishmaniasis is also highly important in Veterinary Medicine, because it can cause severe disease in canines. Nevertheless, although L. infantum infection is widely distributed in the canine population living in endemic regions, only a small proportion of these dogs will develop clinical disease. Early diagnosis and treatment of canine leishmaniasis are essential to control the spread of the disease and as part of a zoonotic human visceral leishmaniasis control system. In the 1980’s, studies developed at the Setúbal’s Peninsula established the seroprevalence of canine leishmaniasis at 11,5% (Abranches et al, 1983). In another study, developed in Lisbon area between December 2002 and December 2003, determined the prevalence of canine infection by Leishmania infantum as 18,4% for domestic dogs and 21,6% for stray dogs (Cortes et al, 2007). In the present study, the author intended to perform an update in the prevalence of canine leishmaniasis in Setúbal and Palmela regions. Blood was sampled from a random sample made of a hundred dogs that attended a veterinary practice in Palmela (representing dogs with owner), and 83 dogs from an animals’ shelter, located in Setúbal (considered stray dogs). The serum was used to perform direct agglutination test for leishmaniasis. Samples with positive results in this test were also analyzed trough counter-immunoelectrophoresis and indirect immunofluorescence test. Polymerase chain reaction was also preformed in peripheral blood samples. Total seroprevalence was 7,1% for the studied population (5% for the group of dogs from the veterinary practice and 9,6% for the group of dogs from the shelter). Based on chi-square test, there are no statistical significant differences between the studied samples. A quiz was filled, by the time of biological sample collection that allowed the author to evaluate the impact of some factors (such as origin, habitat, application of insecticides effective against the vector of the disease and living with other dogs) involved in this infection process. Although the tests suggested a few tendencies, none of the studied subjects proved to be a risk factor in a statistically significant manner

Species-specific RNA-FISH probes for yeast identification: evaluation of their specificity and performance

The aim of this work was to investigate the possibility of applying the RNA-FISH protocol previously developed by our group for simultaneous analysis of fungi and bacteria using kingdom-specific probes for specific detection of various yeast species using previously published species-specific probes. Their specificity and performance were analysed both in silico and experimentally.FCT through PTDC/BBB-IMG/0046/2014 project and SFRH/BPD/100754/2014 grant and ALT20-03-0246-FEDER-000004-ALENTEJO 2020 projec

Overcoming the limitations to identify Dekkera bruxellensis in wine environment: performance and specificity evaluation of two species-specific RNA-FISH probes.

RNA-Fluorescence In Situ Hybridisation (RNA-FISH) allows the detection and identification of microorganisms in complex matrices in few hours being considered as one of the most powerful techniques for various applications in microbiology. Thus, it has the analytical potential for allowing specific detection of D. bruxellensis. However, some experimental difficulties can hamper its successful application such as: a) the autofluorescence of the sample and its interference with the RNA-FISH probe-conferred fluorescence, and b) the lack of probes with the desired levels of specificity and FISH performance. This is why this study was focused on overcoming the possible experimental drawbacks that can be found in RNA-FISH application for detecting D. bruxellensis in the wine environment and on evaluating the performance and specificity (both in silico and experimentally) of two RNA-FISH probes targeting to D. bruxellensis: a novel probe designed by us and a probe previously designed by other authors, 26S D. brux.5.1 .This work was co-financed by Foundation for Science and Technology (FCT) through the project PTDC/BBB-IMG/0046/2014 and the post-doctoral grant SFRH/BPD/100754/2014 and by ALT20-03-0145-FEDER- 000015 project

Fluorophore´s role on the reliability of microorganism detection by Fluorescence In Situ Hybridisation (FISH).

FISH has been applied in many ecological and phylogenetic studies becoming the method of choice for the direct detection and identification of microorganisms in their natural environments [1]. For reliable results, it is crucial to minimise or avoid background fluorescence and cellular autofluorescence but also to maximise the specific FISH signals obtained. Red-emitting dyes have been usually used for eliminating the problems of non-specific fluorescence interference. Therefore, in this work we evaluated the role of three of these fluorophores (Alexa Fluor 647 (AF647), ATTO 647N and Cy5) on the reliability of the RNAFISH results obtained both with a universal and a specie-specific probe.ALT20-03-0246-FEDER-000004-ALENTEJO 2020 project and FCT through PTDC/BBB-IMG/0046/2014 project and SFRH/BPD/100754/2014 grant

RNA-FISH for investigating Cultural Heritage Microbiology- Autofluorescence of the materials and fluorophores selection.

This work was focused on: i) the investigation of the autofluorescence of various materials that are found forming part of Cultural Heritage objects (parchment, paper, wood, fabric, rock, mortar and plastic among others) by epifluorescence microscopy using fluorescence flter sets targeted at commonly used fluorophores (TRITC/CY5/6-FAM); ii) the detection of RNA-FISH stained microbial cells on the selected materials or in the presence of residual particles.This work was co-financed by FCT – Fundação para a Ciência e a Tecnologia through the project "MICROTECH-ART- Microorganisms Thriving on and Endamaging Cultural Heritage -an Analytical Rapid Tool-" (PTDC/BBB-IMG/0046/2014) and post-doctoral grant SFRH/BPD/100754/2014 and by European Union, European Regional Development Fund ALENTEJO 2020 through the project “HIT3CH - HERCULES Interface for Technology Transfer and Teaming in Cultural Heritage” (ALT20-03-0246-FEDER-000004)