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As Klebsiella pneumoniae (KP) has acquired high levels of resistance to multiple antibiotics, it is considered a worldwide pathogen of concern, and substitutes for traditional antibiotics are urgently needed. 3-Phenyllactic acid (PLA) has been reported to have antimicrobial activity against food-borne bacteria. However, there was no experiment evidence for the exact antibacterial effect and mechanism of PLA kills pathogenic KP. In this study, the Oxford cup method indicated that PLA is effective to KP with a minimum inhibitory concentration of 2.5 mg/mL. Furthermore, PLA inhibited the growth and biofilm formation of in a time- and concentration-dependent manner. In vivo, PLA could significantly increase the survival rate of infected mice and reduce the pathological tissue damage. The antibacterial mode of PLA against KP was further explored. Firstly, scanning electron microscopy illustrated the disruption of cellular ultrastructure caused by PLA. Secondly, measurement of leaked alkaline phosphatase demonstrated that PLA disrupted the cell wall integrity of KP and flow cytometry analysis with propidium iodide staining suggested that PLA damaged the cell membrane integrity. Finally, the results of fluorescence spectroscopy and agarose gel electrophoresis demonstrated that PLA bound to genomic DNA and initiated its degradation. The anti-KP mode of action of PLA was attributed to the destruction of the cell wall, membrane, and genomic DNA binding. These findings suggest that PLA has great potential applications as antibiotic substitutes in feed additives against KP infection in animals.

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An efficient method of genomic DNA extraction that provides high quality and yield is a crucial pre-requisite and limiting factor in plant genetic analysis. However, pure genomic DNA can be challenging to obtain from some plant species due to their sugar and secondary metabolite contents. Lippia alba is an important aromatic and medicinal plant, chemically characterized by the presence of tannins, flavonoids, anthocyanins, and essential oils, which interfere with the extraction of pure genomic DNA. In this scenario, optimizing the extraction methods and minimizing the effects of these compounds are necessary. This study compares six plant DNA extraction protocols based on the CTAB method. The quality and quantity of DNA samples obtained were determined by physical appearance by electrophoresis in agarose gels and spectrophotometry. The results highlight the difficulty in obtaining pure and clear bands for all tested methods, except for the polyvinylpyrrolidone (PVP)-based protocol created by our team, which was the better option for obtaining high-quality genomic DNA of L. alba. We conclude that adding PVP-40 into DNA extraction buffers can optimize the DNA extraction of L. alba and indicate this protocol for DNA extraction from other aromatic plants.

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Among bioluminescent beetles of the Elateroidea superfamily, Phengodidae is the third largest family, with 244 bioluminescent species distributed only in the Americas, but is still the least studied from the phylogenetic and evolutionary points of view. The railroad worm Phrixothrix hirtus is an essential biological model and symbolic species due to its bicolor bioluminescence, being the only organism that produces true red light among bioluminescent terrestrial species. Here, we performed partial genome assembly of P. hirtus, combining short and long reads generated with Illumina sequencing, providing the first source of genomic information and a framework for comparative analyses of the bioluminescent system in Elateroidea. This is the largest genome described in the Elateroidea superfamily, with an estimated size of ∼3.4 Gb, displaying 32 % GC content, and 67 % transposable elements. Comparative genomic analyses showed a positive selection of genes and gene family expansion events of growth and morphogenesis gene products, which could be associated with the atypical anatomical development and morphogenesis found in paedomorphic females and underdeveloped males. We also observed gene family expansion among distinct odorant-binding receptors, which could be associated with the pheromone communication system typical of these beetles, and retrotransposable elements. Common genes putatively regulating bioluminescence production and control, including two luciferase genes corresponding to lateral lanterns green-emitting and head lanterns red-emitting luciferases with 7 exons and 6 introns, and genes potentially involved in luciferin biosynthesis were found, indicating that there are no clear differences about the presence or absence of gene families associated with bioluminescence in Elateroidea.

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In this work, we report the construction of a novel electrochemical device for molecular diagnosis of hepatitis B virus in the blood plasma of infected patients, using graphite electrodes functionalized with poly(4-aminophenol) and sensitized with a specific DNA probe. The recognition of genomic DNA was evaluated by electrochemical techniques (DPV and EIS) and scanning electron microscopy. The genosensor was efficient in detecting genomic DNA with a linear range from 1.176 to 4.825 µg mL-1 and detection limit of 35.69 ng mL-1 (4.63 IU ml-1 or 25.93 copies.ml-1), which is better than the 10.00 IU ml-1 limit of reference method, real-time PCR, used in point of care. EIS analysis shows that the genosensor resistance increased exponentially with the concentration of the genomic DNA target. This novel platform has advantages to its applicability in real samples, such as good sensitivity, selectivity, low sample volume, and fast assay time (36 min), thus interesting for application in the diagnosis of hepatitis B virus in blood plasma. Also, the ease of synthesis of the low-cost polymer by electrosynthesis directly on the electrode surface allows the translation of the platform to portable devices.

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RESUMEN Objetivos: Determinar el efecto citotóxico y genotóxico in vitro del extracto crudo y etanólico del rizoma de Curcuma longa L. Materiales y métodos: El efecto citotóxico fue evaluado utilizando líneas celulares DU-145, HT-29, 3T3 BALB/c. Se hallaron los porcentajes de crecimiento en 48 horas y se determinó la concentración inhibitoria 50 (CI50). El efecto genotóxico en el ADN genómico humano se determinó mediante el método Tomasevich. Resultados: El extracto crudo produjo una CI50 de 12,98 ± 0,21 μg/mL para la línea celular tumoral HT-29, que es inferior a DU-145 con una CI50 de 36,77 ± 9,12 μg/mL; el extracto etanólico presentó una CI50 de 13,24 ± 0,77 y 20,54 ± 2,58 µg/mL para ambas líneas celulares, respectivamente; el compuesto estándar curcumina presentó una CI50 de 3,96 ± 0,60 y 13,94 ± 2,79 μg/mL, respectivamente. El extracto crudo a concentraciones de 50 y 100 mg/mL fragmentó entre el 40% a 95% de ADN genómico humano; mientras que, a 200 mg/mL, la fragmentación fue mayor al 95%. El extracto etanólico a todas las concentraciones no fragmentó el ADN. La curcumina a 200 mg/mL fragmentó menos del 5% de ADN genómico humano. Conclusiones: Los extractos crudo y etanólico de Curcuma longa L. demuestran efecto citotóxico in vitro diferencial para la línea celular tumoral humana DU-145 y HT29 semejante al compuesto estándar curcumina. El extracto crudo de Curcuma longa L. presenta una potente actividad genotóxica in vitro frente al ADN genómico humano, esta actividad está ausente en el extracto etanólico.

ABSTRACT Objectives: To determine the in vitro cytotoxic and genotoxic effect of the crude and ethanolic extract from the Curcuma longa L. rhizome. Materials and methods: The cytotoxic effect was evaluated using DU-145, HT-29, 3T3 BALB/c cell lines. The growth percentages in 48 hours; and the half maximal inhibitory concentration (IC50) were determined. The genotoxic effect on human genomic DNA was determined using the Tomasevich method. Results: Crude extract produced an IC50 of 12.98 ± 0.21 μg/mL for the HT-29 tumor cell line, which is lower than the value obtained for DU-145, with an IC50 of 36.77 ± 9.12 μg/mL. The ethanolic extract presented an IC50 of 13.24 ± 0.77 and 20.54 ± 2.58 μg/mL for both cell lines, respectively; the curcumin standard compound presented an IC50 of 3.96 ± 0.60 and 13.94 ± 2.79 μg/mL, respectively. Crude extract concentrations of 50 and 100 mg/mL fragmented between 40% to 95% of human genomic DNA; while at 200 mg/mL, fragmentation was greater than 95%. The ethanolic extract at all concentrations did not fragment the DNA. Curcumin at 200 mg/mL fragmented less than 5% of human genomic DNA. Conclusions: The crude and ethanolic extracts of Curcuma longa L. demonstrate different in vitro cytotoxic effects for the human tumor cell lines DU-145 and HT-29; similar to the standard curcumin compound. The crude extract of Curcuma longa L. shows a potent genotoxic in vitro activity against human genomic DNA; this type of effect is not produced by the ethanolic extract.

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Prolificacy is a desirable trait for genetic improvement of sheep flocks, since it holds the potential to improve productivity. Animals carrying single-nucleotide polymorphisms (SNPs) in genes associated with this trait can be identified and employed to increase prolificacy in flocks. In this study, we report a diagnostic method based on quantitative PCR and high-resolution melting curves to detect different SNPs in the prolificacy-associated gene growth differentiation factor 9 (GDF9). The diagnostic method was validated using artificial sequences representing known SNPs in GDF9, then applied to a real flock comprising four breeds and admixed animals (n = 306). Five different SNPs were identified in this flock, as was a low or null frequency of occurrence of SNPs positively associated with prolificacy. This indicates a need to implement a breeding strategy for recovering or reintroducing such SNPs. Our method provides a genotyping strategy for identifying individuals with SNPs of interest for prolificacy, which will help producers plan a breeding strategy for this trait. This method can be adapted and expanded for the diagnosis of other traits of interest.

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Molecular detection of Sarcocystis spp. in tissue samples can be useful for experimental and diagnostic purposes. However, the parasite spreads unevenly through tissues, forming tissue cysts, and the cystic wall is an obstacle in DNA extraction protocols. Therefore, adequate sampling and effective disruption of the cysts are essential to improve the accuracy of DNA detection by PCR. The aims of this study were to evaluate the suitability of four protocols for DNA extraction from cysts of Sarcocystis spp. present in bovine myocardium samples or after their harvest in phosphate-buffered saline (PBS) solution as well as determine the effects of single or multiple sampling on the accuracy of molecular diagnosis of sarcocystosis in cattle hearts. Cysts and myocardium samples from nine bovine hearts were randomly distributed to four DNA extraction protocols: kit, kit with modification, DNAzol, and cetyl-trimethyl ammonium bromide (CTAB). Samples were submitted to DNA extraction and PCR as replicates of each heart (simplicate, duplicate, and triplicate), and the probability of a true positive diagnostic was calculated. Among the protocols tested, the kit with modification was determined to be the most suitable for DNA extraction from cysts in PBS solution (92.6 % of DNA detection by PCR); DNAzol resulted in higher DNA detection frequency from bovine myocardium samples (48.1 %). Multiple sampling improved the molecular diagnosis of Sarcocystis spp. infection in cattle hearts, increasing at 22.2 % the rate of true positive diagnostic.

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Background More than 200 Scyphozoa species have been described, but few have been properly studied regarding their chemical and genetic characteristics.Catostylus tagi, an edible Scyphozoa and the sole European Catostylidae, occurs in summer at Tagus and Sado estuaries. Neither a systematic comparison between the two Catostyluscommunities nor a chemical approach on their nematocytes had been carried out yet.Methods In order to achieve these purposes, optimisation of DNA extraction and of histochemical staining procedures were developed.Catostylus specimens from Tagus and Sado estuaries were compared by ribosomal 18S, 28S, and ITS1 partial sequencing. The morphochemistry of nematocytes was studied by optical and electronic microscopy.Results Macroscopic and molecular results indicated that both communities belong to the same species, C. tagi. The hematoxylin and eosin staining allowed the visualisation of nematocyst genesis and indicated a basic character for the macromolecules on the shaft of euryteles and on the tubule of isorhizae and birhopaloids. By Massons trichrome procedure, the basic properties of the tubules were confirmed and a collagenous profile for the toxins was suggested. Results of the alcian blue staining showed that the outer membrane of nematocyte may consist of macromolecules with acidic polysaccharides, consistent with NOWA and nematogalectin glycoproteins detected in Hydra, but also with poly-gamma-glutamate complex, chitin-like polysaccharides and hyaluronic acids. Through the von Kossa assays, calcium was detected; its position suggested interactions with polysaccharides of the membrane, with proteins of the contractile system or with both.Conclusions The optimisation of sample preparation for DNA extraction may facilitate further studies on little known jellyfish species. The improvement of the smear procedure simplified the use of stained reactions in zooplankton. Moreover, it was shown that good slide images might be acquired manually. The development of specific reactions, with traditional dyes and others, can give important contributions to clarify the chemical nature of the components of nematocytes. The characterisation of nematocyst toxins by staining tests is a goal to achieve.(AU)

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BACKGROUND: More than 200 Scyphozoa species have been described, but few have been properly studied regarding their chemical and genetic characteristics. Catostylus tagi, an edible Scyphozoa and the sole European Catostylidae, occurs in summer at Tagus and Sado estuaries. Neither a systematic comparison between the two Catostylus communities nor a chemical approach on their nematocytes had been carried out yet. METHODS: In order to achieve these purposes, optimisation of DNA extraction and of histochemical staining procedures were developed. Catostylus specimens from Tagus and Sado estuaries were compared by ribosomal 18S, 28S, and ITS1 partial sequencing. The morphochemistry of nematocytes was studied by optical and electronic microscopy. RESULTS: Macroscopic and molecular results indicated that both communities belong to the same species, C. tagi. The hematoxylin and eosin staining allowed the visualisation of nematocyst genesis and indicated a basic character for the macromolecules on the shaft of euryteles and on the tubule of isorhizae and birhopaloids. By Masson's trichrome procedure, the basic properties of the tubules were confirmed and a collagenous profile for the toxins was suggested. Results of the alcian blue staining showed that the outer membrane of nematocyte may consist of macromolecules with acidic polysaccharides, consistent with NOWA and nematogalectin glycoproteins detected in Hydra, but also with poly-gamma-glutamate complex, chitin-like polysaccharides and hyaluronic acids. Through the von Kossa assays, calcium was detected; its position suggested interactions with polysaccharides of the membrane, with proteins of the contractile system or with both. CONCLUSIONS: The optimisation of sample preparation for DNA extraction may facilitate further studies on little known jellyfish species. The improvement of the smear procedure simplified the use of stained reactions in zooplankton. Moreover, it was shown that good slide images might be acquired manually. The development of specific reactions, with traditional dyes and others, can give important contributions to clarify the chemical nature of the components of nematocytes. The characterisation of nematocyst toxins by staining tests is a goal to achieve.

RESUMO

PURPOSE: The aim of this clinical investigation was to identify and quantify the microbial species adhering to toothbrush bristles after controlled brushing and storage in different antimicrobial agents. METHODS: Sixteen healthy participants were enrolled in this study and randomly submitted to 4 interventions in a cross-over design: brushing and toothbrush storage in (I) Periogard/(II) Periobio (Chlorhexidine gluconate 0.12%), (III) Cepacol (cetylpyridinium chloride 0.05%) and (IV) distilled water (positive control). Thirty-eight bacterial species including putative pathogens and 5 Candida spp. were assessed by Checkerboard DNA-DNA hybridization. RESULTS: The results of the study have shown a striking reduction of the total microbial counts, including bacteria and Candida spp., on the toothbrush bristles after storage in cetylpyridinium chloride 0.05% (p < 0.0001). Chlorhexidine gluconate 0.12% showed no differences on the total bacterial count when compared to distilled water (p > 0.05). Cetylpyridinium chloride solution also presented the lowest genome counts and frequency of detection for individual target species; distilled water showed the highest individual genome counts (p < 0.05). Potential pathogenic species were recorded in moderate to high levels for chlorhexidine gluconate and distilled water. CONCLUSION: Cetylpyridinium chloride 0.05% was the most effective storage solution in the reduction of total and individual microbial counts, including pathogenic species.

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Background More than 200 Scyphozoa species have been described, but few have been properly studied regarding their chemical and genetic characteristics.Catostylus tagi, an edible Scyphozoa and the sole European Catostylidae, occurs in summer at Tagus and Sado estuaries. Neither a systematic comparison between the two Catostyluscommunities nor a chemical approach on their nematocytes had been carried out yet.Methods In order to achieve these purposes, optimisation of DNA extraction and of histochemical staining procedures were developed.Catostylus specimens from Tagus and Sado estuaries were compared by ribosomal 18S, 28S, and ITS1 partial sequencing. The morphochemistry of nematocytes was studied by optical and electronic microscopy.Results Macroscopic and molecular results indicated that both communities belong to the same species, C. tagi. The hematoxylin and eosin staining allowed the visualisation of nematocyst genesis and indicated a basic character for the macromolecules on the shaft of euryteles and on the tubule of isorhizae and birhopaloids. By Masson's trichrome procedure, the basic properties of the tubules were confirmed and a collagenous profile for the toxins was suggested. Results of the alcian blue staining showed that the outer membrane of nematocyte may consist of macromolecules with acidic polysaccharides, consistent with NOWA and nematogalectin glycoproteins detected in Hydra, but also with poly-gamma-glutamate complex, chitin-like polysaccharides and hyaluronic acids. Through the von Kossa assays, calcium was detected; its position suggested interactions with polysaccharides of the membrane, with proteins of the contractile system or with both.Conclusions The optimisation of sample preparation for DNA extraction may facilitate further studies on little known jellyfish species. The improvement of the smear procedure simplified the use of stained reactions in zooplankton. Moreover, it was shown that good slide images might be acquired manually. The development of specific reactions, with traditional dyes and others, can give important contributions to clarify the chemical nature of the components of nematocytes. The characterisation of nematocyst toxins by staining tests is a goal to achieve.(AU)

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Background More than 200 Scyphozoa species have been described, but few have been properly studied regarding their chemical and genetic characteristics.Catostylus tagi, an edible Scyphozoa and the sole European Catostylidae, occurs in summer at Tagus and Sado estuaries. Neither a systematic comparison between the two Catostyluscommunities nor a chemical approach on their nematocytes had been carried out yet.Methods In order to achieve these purposes, optimisation of DNA extraction and of histochemical staining procedures were developed.Catostylus specimens from Tagus and Sado estuaries were compared by ribosomal 18S, 28S, and ITS1 partial sequencing. The morphochemistry of nematocytes was studied by optical and electronic microscopy.Results Macroscopic and molecular results indicated that both communities belong to the same species, C. tagi. The hematoxylin and eosin staining allowed the visualisation of nematocyst genesis and indicated a basic character for the macromolecules on the shaft of euryteles and on the tubule of isorhizae and birhopaloids. By Massons trichrome procedure, the basic properties of the tubules were confirmed and a collagenous profile for the toxins was suggested. Results of the alcian blue staining showed that the outer membrane of nematocyte may consist of macromolecules with acidic polysaccharides, consistent with NOWA and nematogalectin glycoproteins detected in Hydra, but also with poly-gamma-glutamate complex, chitin-like polysaccharides and hyaluronic acids. Through the von Kossa assays, calcium was detected; its position suggested interactions with polysaccharides of the membrane, with proteins of the contractile system or with both.Conclusions The optimisation of sample preparation for DNA extraction may facilitate further studies on little known jellyfish species. The improvement of the smear procedure simplified the use of stained reactions in zooplankton. Moreover, it was shown that good slide images might be acquired manually. The development of specific reactions, with traditional dyes and others, can give important contributions to clarify the chemical nature of the components of nematocytes. The characterisation of nematocyst toxins by staining tests is a goal to achieve.

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Several extraction methods of genomic DNA for identification and characterization of genetic diversity in different plant species are routinely applied during molecular analysis. However, the presence of undesirable compounds such as polyphenols and polysaccharides is one of the biggest problems faced during the isolation and purification of high quality DNA in plants. Therefore, achievement of fast and accurate methods for DNA extraction is crucial in order to produce pure samples. Leaves of strawberry genotypes (Fragaria ananassa) have high contents of polysaccharides and polyphenols which increase the sample viscosity and decrease the DNA quality, interfering with the PCR performance. Thereby, in this study we evaluated the quality and amount of genomic DNA extracted from young leaves of strawberry after tissue lyophilization and maceration in presence of polivinilpirrolidone (PVP). The CTAB method was used as reference procedure and it was modified to improve the DNA extraction. The modifications consisted of tissue lyophilization overnight until it was completely freeze-dried and addition of PVP during the tissue maceration in liquid nitrogen. The results showed the efficiency and reliability of the modified method compared to the unmodified method, indicating that combination of lyophilization and PVP improve the quality and amount of the DNA extracted from strawberry leaves.

Vários métodos de extração de DNA genômico para a identificação e caracterização da diversidade genética em diferentes espécies de plantas são rotineiramente aplicados durante a análise molecular. Entretanto, a presença de compostos indesejáveis, tais como polifenóis e polissacarídeos, é um dos maiores problemas que ocorrem durante o isolamento e purificação de DNA de alta qualidade em plantas. Dessa forma, o sucesso no desenvolvimento de métodos de extração de DNA rápidos e acurados é crucial para produzir amostras puras. Folhas de genótipos de morangueiro (Fragaria ananassa) têm elevado conteúdo de polissacarídeos e polifenóis que aumentam a viscosidade da amostra e reduzem a qualidade do DNA, interferindo no desempenho da PCR. Neste estudo, avaliamos a qualidade e a quantidade de DNA genômico extraído de folhas jovens de morangueiro após a liofilização do tecido e a maceração na presença de polivinilpirrolidona (PVP). O método CTAB foi utilizado como procedimento de referência e foi modificado para melhorar a extração do DNA. As modificações consistiram na liofilização do tecido a baixa temperatura até que ele tivesse sido desidratado completamente, associada à adição de PVP durante a maceração do tecido no nitrogênio líquido. Os resultados demonstraram a eficiência e a confiabilidade do método modificado comparado ao método não modificado, indicando que a combinação da liofilização com PVP melhora a qualidade e quantidade do DNA extraído de folhas de morangueiro.

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Several extraction methods of genomic DNA for identification and characterization of genetic diversity in different plant species are routinely applied during molecular analysis. However, the presence of undesirable compounds such as polyphenols and polysaccharides is one of the biggest problems faced during the isolation and purification of high quality DNA in plants. Therefore, achievement of fast and accurate methods for DNA extraction is crucial in order to produce pure samples. Leaves of strawberry genotypes (Fragaria ananassa) have high contents of polysaccharides and polyphenols which increase the sample viscosity and decrease the DNA quality, interfering with the PCR performance. Thereby, in this study we evaluated the quality and amount of genomic DNA extracted from young leaves of strawberry after tissue lyophilization and maceration in presence of polivinilpirrolidone (PVP). The CTAB method was used as reference procedure and it was modified to improve the DNA extraction. The modifications consisted of tissue lyophilization overnight until it was completely freeze-dried and addition of PVP during the tissue maceration in liquid nitrogen. The results showed the efficiency and reliability of the modified method compared to the unmodified method, indicating that combination of lyophilization and PVP improve the quality and amount of the DNA extracted from strawberry leaves.

Vários métodos de extração de DNA genômico para a identificação e caracterização da diversidade genética em diferentes espécies de plantas são rotineiramente aplicados durante a análise molecular. Entretanto, a presença de compostos indesejáveis, tais como polifenóis e polissacarídeos, é um dos maiores problemas que ocorrem durante o isolamento e purificação de DNA de alta qualidade em plantas. Dessa forma, o sucesso no desenvolvimento de métodos de extração de DNA rápidos e acurados é crucial para produzir amostras puras. Folhas de genótipos de morangueiro (Fragaria ananassa) têm elevado conteúdo de polissacarídeos e polifenóis que aumentam a viscosidade da amostra e reduzem a qualidade do DNA, interferindo no desempenho da PCR. Neste estudo, avaliamos a qualidade e a quantidade de DNA genômico extraído de folhas jovens de morangueiro após a liofilização do tecido e a maceração na presença de polivinilpirrolidona (PVP). O método CTAB foi utilizado como procedimento de referência e foi modificado para melhorar a extração do DNA. As modificações consistiram na liofilização do tecido a baixa temperatura até que ele tivesse sido desidratado completamente, associada à adição de PVP durante a maceração do tecido no nitrogênio líquido. Os resultados demonstraram a eficiência e a confiabilidade do método modificado comparado ao método não modificado, indicando que a combinação da liofilização com PVP melhora a qualidade e quantidade do DNA extraído de folhas de morangueiro.

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Genomic DNA (gDNA) extraction allows genetic material accessible to apply molecular techniques for diagnostic and/or genomic characterization. The main goal of this work was to analyze comparatively three manual methods of genomic DNA extraction - DNAzol, FTA and Silica - evaluating quality and yield of the nucleic acids obtained.[...](AU)

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Genomic DNA (gDNA) extraction allows genetic material accessible to apply molecular techniques for diagnostic and/or genomic characterization. The main goal of this work was to analyze comparatively three manual methods of genomic DNA extraction - DNAzol, FTA and Silica - evaluating quality and yield of the nucleic acids obtained.[...]

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Several extraction methods of genomic DNA for identification and characterization of genetic diversity in different plant species are routinely applied during molecular analysis. However, the presence of undesirable compounds such as polyphenols and polysaccharides is one of the biggest problems faced during the isolation and purification of high quality DNA in plants. Therefore, achievement of fast and accurate methods for DNA extraction is crucial in order to produce pure samples. Leaves of strawberry genotypes (Fragaria ananassa) have high contents of polysaccharides and polyphenols which increase the sample viscosity and decrease the DNA quality, interfering with the PCR performance. Thereby, in this study we evaluated the quality and amount of genomic DNA extracted from young leaves of strawberry after tissue lyophilization and maceration in presence of polivinilpirrolidone (PVP). The CTAB method was used as reference procedure and it was modified to improve the DNA extraction. The modifications consisted of tissue lyophilization overnight until it was completely freeze-dried and addition of PVP during the tissue maceration in liquid nitrogen. The results showed the efficiency and reliability of the modified method compared to the unmodified method, indicating that combination of lyophilization and PVP improve the quality and amount of the DNA extracted from strawberry leaves.

Vários métodos de extração de DNA genômico para a identificação e caracterização da diversidade genética em diferentes espécies de plantas são rotineiramente aplicados durante a análise molecular. Entretanto, a presença de compostos indesejáveis, tais como polifenóis e polissacarídeos, é um dos maiores problemas que ocorrem durante o isolamento e purificação de DNA de alta qualidade em plantas. Dessa forma, o sucesso no desenvolvimento de métodos de extração de DNA rápidos e acurados é crucial para produzir amostras puras. Folhas de genótipos de morangueiro (Fragaria ananassa) têm elevado conteúdo de polissacarídeos e polifenóis que aumentam a viscosidade da amostra e reduzem a qualidade do DNA, interferindo no desempenho da PCR. Neste estudo, avaliamos a qualidade e a quantidade de DNA genômico extraído de folhas jovens de morangueiro após a liofilização do tecido e a maceração na presença de polivinilpirrolidona (PVP). O método CTAB foi utilizado como procedimento de referência e foi modificado para melhorar a extração do DNA. As modificações consistiram na liofilização do tecido a baixa temperatura até que ele tivesse sido desidratado completamente, associada à adição de PVP durante a maceração do tecido no nitrogênio líquido. Os resultados demonstraram a eficiência e a confiabilidade do método modificado comparado ao método não modificado, indicando que a combinação da liofilização com PVP melhora a qualidade e quantidade do DNA extraído de folhas de morangueiro.

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BACKGROUND: Fetal lead exposure is associated with adverse pregnancy outcomes and developmental and cognitive deficits; however, the mechanism(s) by which lead-induced toxicity occurs remains unknown. Epigenetic fetal programming via DNA methylation may provide a pathway by which environmental lead exposure can influence disease susceptibility. OBJECTIVE: This study was designed to determine whether prenatal lead exposure is associated with alterations in genomic methylation of leukocyte DNA levels from umbilical cord samples. METHODS: We measured genomic DNA methylation, as assessed by Alu and LINE-1 (long interspersed nuclear element-1) methylation via pyrosequencing, on 103 umbilical cord blood samples from the biorepository of the Early Life Exposures in Mexico to Environmental Toxicants (ELEMENT) study group. Prenatal lead exposure had been assessed by measuring maternal bone lead levels at the mid-tibial shaft and the patella using a spot-source (109)Cd K-shell X-ray fluorescence instrument. RESULTS: We found an inverse dose-response relationship in which quartiles of patella lead correlated with cord LINE-1 methylation (p for trend = 0.01) and and tibia lead correlated with Alu methylation (p for trend = 0.05). In mixed effects regression models, maternal tibia lead was negatively associated with umbilical cord genomic DNA methylation of Alu (beta= -0.027; p = 0.01). We found no associations between cord blood lead and cord genomic DNA methylation. CONCLUSIONS: Prenatal lead exposure is inversely associated with genomic DNA methylation in cord blood. These data suggest that the epigenome of the developing fetus can be influenced by maternal cumulative lead burden, which may influence long-term epigenetic programming and disease susceptibility throughout the life course.

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This study describes a rapid procedure for the isolation of genomic DNA from Staphylococcus aureus that yielded a good amount of high quality DNA for the amplification of staphylococcal enterotoxins genes (A, B, C, D, and E) and the TSST-1 gene as well as enzymatic restriction (HaeIII) from environmental isolates. With this method, it was possible to detect these genes in a sample containing as little as 10(5) cells with positive PCR reactions obtained from approximately 10pg of DNA in a final reaction volume of 25µl.

Descreve-se um procedimento rápido para extração de DNA genômico de isolados de Staphylococcus aureus capaz de produzir DNA estafilocócico em qualidade e quantidade suficiente para a amplificação de genes que codificam enterotoxinas estafilocócicas (A - E) e para TSST-1 e restrição enzimática (HaeIII) de isolados ambientais. O método proposto foi capaz de detectar esses genes em um produto de extração contendo tanto quanto 10(5) células, e reações positivas de PCR foram obtidas de aproximadamente 10pg de DNA.

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This study describes a rapid procedure for the isolation of genomic DNA from Staphylococcus aureus that yielded a good amount of high quality DNA for the amplification of staphylococcal enterotoxins genes (A, B, C, D, and E) and the TSST-1 gene as well as enzymatic restriction (HaeIII) from environmental isolates. With this method, it was possible to detect these genes in a sample containing as little as 10(5) cells with positive PCR reactions obtained from approximately 10pg of DNA in a final reaction volume of 25µl.(AU)

Descreve-se um procedimento rápido para extração de DNA genômico de isolados de Staphylococcus aureus capaz de produzir DNA estafilocócico em qualidade e quantidade suficiente para a amplificação de genes que codificam enterotoxinas estafilocócicas (A - E) e para TSST-1 e restrição enzimática (HaeIII) de isolados ambientais. O método proposto foi capaz de detectar esses genes em um produto de extração contendo tanto quanto 10(5) células, e reações positivas de PCR foram obtidas de aproximadamente 10pg de DNA.(AU)

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