Suchergebnisse

In this work a novel taxon-specific probe to detect Cladosporium was designed in silico. Probe specificity was evaluated experimentally using various model microorganisms belonging to different phylum and taxa. ; 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 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). Marina González-Pérez acknowledges FCT for the economic support through the post-doctoral grant SFRH/BPD/100754/2014.

Dekkera bruxellensis is the main reason for spoilage in the wine industry. It renders the products unacceptable leading to large economic losses. Fluorescence In Situ Hybridisation (FISH) technique has the potential for allowing its specific detection. Nevertheless, some experimental difficulties can be encountered when FISH technique is applied in the wine environment (e.g. matrix and cells autofluorescence, fluorophore inadequate selection and probes low specificity to the target organisms). An easy and fast in-suspension RNA-FISH procedure was applied for the first time for identifying D. bruxellensis in wine. A previously designed RNA-FISH probe to detect D. bruxellensis (26S D. brux.5.1) was used and the matrix and cells fluorescence interferences, the influence of three fluorophores in FISH performance and the probe specificity were evaluated. The results revealed that to apply RNA-FISH technique in the wine environment a red-emitting fluorophore should be used. Good probe performance and specificity was achieved with 25% of formamide. The resulting RNA-FISH protocol was applied in wine samples artificially inoculated with D. bruxellensis. This spoilage microorganism was detected in wine at cell densities lower than those associated with phenolic off-flavours. Thus, the RNA-FISH procedure described in this work represents an advancement to facilitate early detection of the most dangerous wine spoilage yeast and, consequently, to reduce the economic losses caused by this yeast to the wine industry. ; This work was co-financed by Foundation for Science and Technology (FCT) and the European Union through the European Regional Development Fund ALENTEJO 2020 through the projects PTDC/BBB-IMG/0046/2014 and ALT20-03-0145-FEDER-000015, respectively. Marina González-Pérez acknowledges FCT for the economic support through the post-doctoral grant SFRH/BPD/100754/2014.

Various non-invasive RNA-FISH methodologies were tested in this work. They seem to be good alternatives for analyzing the potential biodeteriogenic microorganisms thriving in CH objects. ; 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 through the post-doctoral grant SFRH/BPD/100754/2014. 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).

The only resource to systematically review current experimental methods, this handy reference enables researchers to select the best solution for their experimental problems. For each method covered, the book provides step-by-step protocols, illustrated by typical research applications. After an initial section on probing the lipid bilayer, the text moves on to discuss probing proteins -- including membrane proteins -- and nucleic acids. The first single publication to incorporate chemical markers, fluorescent probes and genetic tags allows a well-informed comparison of different solutions for.

In this work a novel taxon-specific probe to detect Cladosporium was designed in silico. Probe specificity was evaluated experimentally using various model microorganisms belonging to different phylum and taxa. ; FCT – Fundação para a Ciência e a Tecnologia through the project "MICROTECH-ART- Microorganisms Thriving on and Endamaging Cultural Heritage -an Analytical Rapid Tool-" and post-doctoral grant SFRH/BPD/100754/2014. (PTDC/BBB-IMG/0046/2014). 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).

The suitability of using a novel coumarin TFP ester for the synthesis of fluorescent FISH probes with a single fluorophore was investigated in this work. ; 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 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).Marina González-Pérez acknowledges FCT for the economic support through the post-doctoral grant SFRH/BPD/100754/2014.

This design involves having students create actual companies and focuses all classroom attention on the interpersonal processes of managing a small group. This article describes modifications made to Nirenberg's model that were stimulated by feedback from our culturally diverse student body. Our modifications and innovations include: (a) providing "thought starters" to help students evaluate their course experiences, (b) changing the method of selecting group leaders, (c) requiring a business plan for the project, (d) requiring teams to develop a performance appraisal and feedback system for assessing members, (e) adding the use of textbooks, and (f) using several team-building activities during the semester.

Lactobacillus species constitute one of the dominant and beneficial bacteria in our body and are used in developed countries as a microbial adjuvant. Identification of these probiotic bacteria is traditionally performed by culture-based techniques. However, such methods are very time-consuming and can give inaccurate results, especially when Lactobacillus is present in mixed bacterial complex communities. Our study aimed to accurately identify Lactobacillus spp. using a novel Peptide Nucleic Acid (PNA) Fluorescence In Situ Hibridization (FISH) probe. The probe (Lac663) was tested on 36 strains belonging to different Lactobacillus species and on 20 strains of other bacterial species. The sensitivity and specificity of the method were 100% (95% confidence interval (CI), 88.0 to 100.0%) and 95.0% (95% CI, 73.1 to 99.7%), respectively. Additionally, we tested the applicability of the method on milk samples added with Lactobacillus strains at probiotic range concentrations and others taxonomically related bacteria, as well as pathogenic bacteria. The Lac663 probe bound exclusively to Lactobacillus strains and the described PNA-FISH method was capable of directly quantifying Lactobacillus spp. in concentrations at which these potential probiotic bacteria are considered to have an effective benefit on human health. ; This work was supported by European Union funds (FEDER/COMPETE) and by national funds (FCT) under the project with reference FCOMP-01-0124-FEDER-008991 (PTDC/BIA-MIC/098228/2008. Antonio Machado acknowledges the FCT individual fellowship-SFRH/BD/62375/2009. However, it is important to refer that FCT did not have any role in the design of the experiments or manuscript ...

Background: Bacterial vaginosis (BV) is a common vaginal infection occurring in women of reproductive age. It is widely accepted that the microbial switch from normal microflora to BV is characterized by a decrease in vaginal colonization by Lactobacillus species together with an increase of Gardnerella vaginalis and other anaerobes. Our goal was to develop and optimize a novel Peptide Nucleic Acid (PNA) Fluorescence in situ Hybridization assay (PNA FISH) for the detection of Lactobacillus spp. and G. vaginalis in mixed samples. Results: Therefore, we evaluated and validated two specific PNA probes by using 36 representative Lactobacillus strains, 22 representative G. vaginalis strains and 27 other taxonomically related or pathogenic bacterial strains commonly found in vaginal samples. The probes were also tested at different concentrations of G. vaginalis and Lactobacillus species in vitro, in the presence of a HeLa cell line. Specificity and sensitivity of the PNA probes were found to be 98.0% (95% confidence interval (CI), from 87.8 to 99.9%) and 100% (95% CI, from 88.0 to 100.0%), for Lactobacillus spp.; and 100% (95% CI, from 92.8 to 100%) and 100% (95% CI, from 81.5 to 100.0%) for G. vaginalis. Moreover, the probes were evaluated in mixed samples mimicking women with BV or normal vaginal microflora, demonstrating efficiency and applicability of our PNA FISH. Conclusions: This quick method accurately detects Lactobacillus spp. and G. vaginalis species in mixed samples, thus enabling efficient evaluation of the two bacterial groups, most frequently encountered in the vagina. ; This work was supported by European Union funds (FEDER/COMPETE) and by national funds (FCT) under the project with reference FCOMP-01-0124-FEDER-008991 (PTDC/BIA-MIC/098228/2008). AM acknowledges the FCT individual fellowship - ...

Bacterial vaginosis (BV) is one of most common vaginal infections. However, its diagnosis by classical methods reveals low specificity. Our goal was to evaluate the accuracy diagnosis of 150 vaginal samples with research gold standard methods and our Peptide Nucleic Acid (PNA) probes by Fluorescence in situHybridization (FISH) methodology. Also, we described the first PNA-FISH methodology for BV diagnosis, which provides results in approximately 3 h. The results showed a sensitivity of 84.6% (95% confidence interval (CI), from 64.3 to 95.0%) and a specificity of 97.6% (95% CI [92.699.4%]), demonstrating the higher specificity of the PNA-FISH method and showing false positive results in BV diagnosis commonly obtained by the classical methods. This methodology combines the specificity of PNA probes for Lactobacillus species and G. vaginalis visualization and the calculation of the microscopic field by Nugent score, allowing a trustful evaluation of the bacteria present in vaginal microflora and avoiding the occurrence of misleading diagnostics. Therefore, the PNA-FISH methodology represents a valuable alternative for BV diagnosis. ; This work was supported by European Union funds (FEDER/COMPETE) and by national funds (FCT) under the project FCOMP-01-0124-FEDER-008991 (PTDC/BIA-MIC/098228/2008). Antonio Machado was funded by the FCT individual fellowship SFRH/BD/62375/2009. Other fundings came from the FCT Strategic Project PEst-OE/EQB/LA0023/2013, the Project NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2-O Novo Norte), QREN, FEDER, and the project FCOMP-01-0124-FEDER-008991 (RECI/BBB-EBI/0179/2012). Nuno Cerca is an Investigador FCT (Portuguese national funds). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the ...

Fluorescence in situ hybridization (FISH) has been the most important technique in plant cytogenetic research. However, application of FISH techniques in most plant species has been hindered by the lack of cloned probes that generate distinct signals on chromosomes. We have recently developed a procedure to develop FISH probes by selecting a large number of oligonucleotides (oligos) derived from single copy DNA sequences. The bioinformatically selected oligos are then massively synthesized de novo in parallel, amplified, and labeled as FISH probes. The "oligo-FISH probes" designed from conserved DNA sequences generate distinct FISH signals on related plant species that have diverged for more than 10 million years. We have conducted oligo-FISH in several distantly related species in the Solanaceae family. This comparative FISH mapping effort has revealed karyotype evolution and discovered species-specific chromosomal translocations in several Solanum species.

Background: International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection. Methodology/Principal Findings: This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S), cytochrome b (cyt b), and cytochrome oxidase subunit I (COI) for the identification of 50 European marine fish species by combining techniques of "DNA barcoding" and microarrays. In a DNA barcoding approach, neighbour Joining (NJ) phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the "position of label" effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridization experiments (<90%) renders the DNA barcoding marker as rather unsuitable for this high-throughput technology. Conclusions/Significance: Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products. © 2010 Kochzius et al. ; SCOPUS: ar.j ...