Onkolyyttiset pikornavirukset ja echovirus 7:n käyttö viroterapiassa

Echovirus 7 (E-7) on enterovirus, joka kuuluu pikornaviruksiin, jotka ovat pieniä ja vaipattomia viruksia ja joilla on ikosahedraalinen rakenne. E-7-viruksen positiivissäikeisen ja yksijuosteisen RNA-genomin koko on noin 7500 emäsparia. Se käyttää solupinnan reseptorina DAF-molekyyliä, jota ilmentyy useissa solutyypeissä ja erityisesti syöpäso¬luissa. Onkolyyttiset virukset tuhoavat syöpäsoluja viruksen lyyttisen kierron ansiosta ja kohdistuvat spesifisesti soluihin, jotka ilmentävät viruksen käyttämää reseptoria. Rigvir on onkolyyttinen, elävä, geneettisesti muokkaamaton, soluadaptoitu E-7, joka on Latviassa hyväksytty viruslääkkeeksi melanoomaa vastaan. Työssä tutkittiin, miten Rigvir eroaa muista E-7-isolaateista genomiltaan ja infektiokyvyltään. E-7-viruskannat valittiin kokeisiin virusneutralisaatiotestin perusteella. Työn aikana virukset testattiin myös geneettisellä tyypitystestillä. E-7-virukset (Rigvir, E-7 Wallace ja 7 kliinistä isolaattia) infektoitiin kymmeneen eri solulinjaan ja virusinfektiota ja virusten aiheuttamaa solutuhoa seurattiin mikroskooppisesti ja RT-qPCR:n avulla. Pitkä-PCR-menetelmä kehitettiin virusgenomin kloonaamiseksi geenivektoriin. Illumina MiSeq -NGS-genomisekvensointimenetelmällä sekvensoitiin virus-RNA:ta ja pitkä-PCR-tuotteita, ja tällä pyrittiin näkemään isolaattien välisiä eroja genomitasolla. Rigvir ei eronnut infektiokyvyltään muista E-7-viruksista lukuun ottamatta SW480-paksusuolisyöpäsoluja. Yksikään virusisolaateista ei infektoinut rintasyöpäsoluja (MDA-MB-231 ja MCF-7). Kohdunkaulasyöpäsoluja (HeLa Ohio) infektoivat huonosti kaikki paitsi yksi kliinisistä E-7-isolaateista. Tyypityksessä selvisi, että kolme solukokeissa käytetyistä isolaateista oli E-19-tyyppiä, jotka infektoivat hyvin glioomasoluja. Tutkimuksessa todettiin, että E-7:llä on syöpäsoluja tuhoavia ominaisuuksia, mutta tulokset eivät osoittaneet Rigvirin tuhoavan syöpäsoluja paremmin kuin muut E-7-isolaatit. NGS-sekvensointi ajettiin kerran E-7-kannoista eristetyistä vRNA-paloista, joista kaksi saatiin sopimaan verrokkina käytettyyn Wallace-sekvenssiin. Eroja näkyi varsinkin 5’-pään alueella ja noin 6000 bp vastaavalla alueella. Pitkä-PCR-tuotteista ajettaessa saatiin kaikki näytteet sopimaan verrokkiin, ja eroja näkyi odotetusti 5’-pään alueella. PCR-tuotteet toimivat NGS:ssä paremmin kuin RNA-näytteet, mutta RNA-näytteiden käsittely on huomattavasti helpompaa kuin PCR-näytteiden tuottaminen virus-RNA:sta.

Étude de la guérison des plaies cornéennes grâce à la cornée reconstruite par génie tissulaire

La cornée est la couche la plus antérieure de l’oeil et sa transparence permet de laisser passer les ondes lumineuses vers la rétine. Cependant, la localisation de la cornée la prédispose à des blessures chimiques et mécaniques. La guérison des blessures cornéennes est un mécanisme complexe faisant intervenir la mort cellulaire, la migration, la prolifération, la différenciation et le remodelage de la matrice extracellulaire (MEC). Dans cette étude, nous avons utilisé la cornée humaine reconstruite par génie tissulaire composée d’un épithélium et d’un stroma afin d’étudier les mécanismes cellulaires et moléculaires de la guérison des plaies, en particulier le remodelage de la MEC exercé par les métalloprotéinases matricielles (MMPs). Les analyses en profilage génique sur biopuces à ADN nous ont permis de démontrer que l’expression de plusieurs gènes était dérégulée lors de la guérison des plaies dans notre modèle. L’expression des gènes codant pour les MMPs, tel que confirmée en qPCR, est augmentée dans l’épithélium migrant afin de recouvrir la plaie. Les analyses en zymographie sur gel ont démontré que les MMPs étaient converties en leur forme enzymatiquement active au fur et à mesure que la lésion se referme. Par ailleurs, nous avons démontré que l’expression des MMPs par les cellules épithéliales est influencée par la présence des fibroblastes dans le stroma ainsi que par leur sécrétion d’une MEC enrichie en collagènes. De plus, les analyses en spectrométrie de masse ont confirmé que la présence d’un épithélium stratifié est requise pour la synthèse et l’organisation adéquate de la MEC. Enfin, les résultats de ces travaux améliorent nos connaissances des mécanismes cellulaires et moléculaires qui modulent la guérison des plaies cornéennes et pourront certainement mener à des progrès en clinique, notamment au niveau du développement de thérapies visant à traiter les troubles de la cornée.

Assessment of toxigenic fungi in poultry feed

Introduction - Feed supplies the necessary nutrients for the growth of healthy animals, which are a part of the human diet. The presence of toxigenic fungi in animal feed such as Aspergillus spp. may contribute to 1) the loss of nutritional value of feedstuff, since fungi will assimilate the most readily available nutrients present in the feed, and 2) the development of mycotoxicoses and chronic conditions, which can raise economic issues due to animal disease and contamination of animal derived products. Aim of the study - The goal of this work was to evaluate the incidence of Aspergilli, particularly from the Circumdati, Flavi and Fumigati sections, through real-time quantitative PCR (qPCR) in 11 feed samples.

Prevalence of Aspergillus fumigatus complex in waste sorting and incineration plants: an occupational threat

Aspergillus fumigatus is one of the major ubiquitous saprophytic fungi and it is considered one of the fungal species with higher clinical relevance. This study aimed at characterising the prevalence of A. fumigatus complex in one waste-sorting plant and also in one incineration plant. Conventional and molecular methodologies were applied in order to detect its presence. Aspergillus fumigatus complex was the second most frequently found in the air from the waste-sorting plant (16.0%) and from the incineration plant (18.0%). Regarding surfaces, it ranked the third species most frequently found in the waste-sorting plant (13.8%) and the second in the incineration plant (22.3%). In the waste-sorting plant, it was possible to amplify by qPCR DNA from the A. fumigatus complex in all culture-positive sampling sites plus one other sampling site that was negative by culture analysis. Considering the observed fungal load, it is recommended to apply preventive and protective measures in order to avoid or minimise worker's exposure.

Determination of soybean cultivar resistance to soybean cyst nematode with quantitative polymerase chain reaction

Heterodera glycines, the soybean cyst nematode, is the major pathogen of Glycine max (soybean). Effective management of this pathogen is contingent on the use of resistant cultivars, thus screening for resistant cultivars is essential. The purpose of this research was to develop a method to assess infection of soybean roots by H. glycines with real-time quantitative Polymerase Chain Reaction (qPCR), a prelude to differentiation of resistance levels in soybean cultivars. Two experiments were conducted. In the first one, a consistent inoculation method was developed using to provide active second-stage juveniles (J2). Two-day-old soybean roots were infested with 0 and 1000 J2/mL. Twenty-four hours after infestation, the roots were surface sterilized and DNA was extracted with the DNA FastKit (MP Biomedicals, Santa Ana, CA)). For the qPCR assay, primer pair for single copy gene HgSNO, which codes for a protein involved in the production of vitamin B6, was selected for H. glycines DNA amplification within soybean roots. In the second experiment, compatible Lee 74, incompatible Peking and cultivars with different levels of resistance to H. glycines were inoculated with 0 and 1,000 J2/seedlings. Twenty-four hours post inoculation they were transplanted into pasteurized soil. Subsequently they were harvested at 1, 7, 10, 14 and 21 days post inoculation for DNA extraction. With the qPCR assay, the time needed to differentiate highly resistant cultivars from the rest was reduced. Quantification of H. glycines infection by traditional means (numbers of females produced in 30 days) is a time-consuming practice; the qPCR method can replace the traditional one and improve precision in determining infection levels.

Molecular analysis of RNA1 and RNA2 sequences from a betanodavirus isolated from giant grouper (Epinephelus lanceolatus) in Australia

Betanodavirus infections have a significant impact through direct losses and trade restrictions for aquaculture sectors in Australia. The giant grouper, Epinephelus lanceolatus, is a high-value, fast-growing species with significant aquaculture potential. With subacute to chronic mortalities reported from a commercial aquaculture facility in northern Queensland, the viral nervous necrosis in the affected fish was confirmed using a RT-qPCR followed by virus isolation using the SSN-1 cell line. The RNA1 and RNA2 segments were sequenced and nucleotide sequences were compared with betanodavirus sequences from GenBank. Phylogenetic analysis revealed that both these sequences clustered with sequences representing red spotted grouper nervous necrosis virus genotype and showed high sequence identity to virus sequences affecting other grouper species. This is the first report confirming infection by betanodavirus in E. lanceolatus from Australia with successful isolation of the virus in a cell culture system, and analysis of nearly full length RNA1 and RNA2 sequences.

Real-Time PCR Assays for the Detection of Puccinia psidii

Puccinia psidii (Myrtle rust) is an emerging pathogen that has a wide host range in the Myrtaceae family; it continues to show an increase in geographic range and is considered to be a significant threat to Myrtaceae plants worldwide. In this study, we describe the development and validation of three novel real-time polymerase reaction (qPCR) assays using ribosomal DNA and β-tubulin gene sequences to detect P. psidii. All qPCR assays were able to detect P. psidii DNA extracted from urediniospores and from infected plants, including asymptomatic leaf tissues. Depending on the gene target, qPCR was able to detect down to 0.011 pg of P. psidii DNA. The most optimum qPCR assay was shown to be highly specific, repeatable, and reproducible following testing using different qPCR reagents and real-time PCR platforms in different laboratories. In addition, a duplex qPCR assay was developed to allow coamplification of the cytochrome oxidase gene from host plants for use as an internal PCR control. The most optimum qPCR assay proved to be faster and more sensitive than the previously published nested PCR assay and will be particularly useful for high-throughput testing and to detect P. psidii at the early stages of infection, before the development of sporulating rust pustules.

Effects of early nutrition on the wnt and bone morphogenic protein pathways in the developing intestine

Infant formula is consumed by the majority of infants in the United States for at least part of the first year of life. Infant formula lacks many of the bioactive compounds that are naturally occurring in breast milk. Because of this, there has been an increased interest by the companies that manufacture infant formula to include additives that would potentially allow formula to more closely mimic breast milk activity. One such ingredient currently being added to infant formula is prebiotics. Prebiotics are non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth of specific healthful bacteria in the colon. It is speculated that prebiotics replicate the activity of breast milk oligosaccharides, which through the production of butyrate by intestinal microbiota, may interact with the Wnt/BMP pathways. The Wnt/BMP pathways regulate intestinal stem cells, which determine the growth, development and maintenance of the intestine. Therefore, the objective of this study was to explore the effects that the addition of prebiotics to formula have on the regulation of the Wnt/BMP pathways when fed to neonatal piglets, a model commonly used in the study of infant nutrition. Piglets (n=5) were randomized into sow-reared (SR), fed control formula (F), or fed formula with added prebiotics (F+P). Fructooligosaccharides (FOS) (2 g/L) and polydextrose (PDX) (2 g/L) were chosen as the prebiotics for this study, because this combination had been less studied than other combinations. Ileum and ascending colon were collected at 7 and 14 days-of-age. Dry matter content, pH, and short chain fatty acid (SCFA) content was measured. The mRNA expression of β-catenin, sFRP3, sFRP4, frizzled 6, DKK1 (Wnt pathway), gremlin (BMP pathway), TNF-a, HNF-4α and osteopontin (OPN) was measured by RT-qPCR. Piglets fed the F+P diet had greater acetate concentration and lower pH in the ileum at day 14 and in the colon at day 7 and day 14 than F piglets. Butyrate concentrations were highest in SR with F+P not differing from F in ileum at day 14 and colon at day 7 and day 14. Effects of age were seen in all genes, with the exception of OPN, sFRP-3 and sFRP-4. On day 7, no effect of diet was observed in the ileum, however, mRNA expression of DKK1 and frizzled 6 were greater in F+P than SR (p≤0.05). On day 14, gremlin expression was lower and OPN was greater in the ileum of SR piglets compared to F and F+P. Also on day 14, HNF-4α mRNA expression was greater in both ileum and colon of F+P piglets and sFRP3 mRNA expression was greater in the colon than F or SR . In summary, differences were observed between gene expression of F+P and SR piglet intestines, but the supplementation of 2 g/L scFOS and 2 g/L PDX to formula did not shift expression of genes in the Wnt/BMP pathways to be more similar to SR than F. As the Wnt/BMP pathway is known to exist in a gradient along the crypt-villus axis, with Wnt expression dominating in the crypt region and BMP expression dominating in the villi, it was possible that pooling whole tissue reduced our ability to detect treatment effects that would be concentrated in either region. A method was therefore developed to remove intestinal epithelial cells along the villus-to-crypt axis. Twenty-five-day-old F and SR piglets were euthanized and ileal tissue was collected and placed in a dissociation buffer in a shaking water bath. Exfoliated cells were removed at increasing time points from 5 to 100 minutes in order to remove cells along the villus-to-crypt axis. After the final incubation, remaining mucosal tissue was removed using a sterile glass microscope slide and pooled with the final exfoliated cell isolation. After each cell collection, a section of tissue was fixed in formalin for histomorphological examination. Expression of genes in the Wnt/BMP pathways, along with crypt marker genes (CDK5 and v-myb), were measured in both whole ileal tissue, pooled epithelial cells, and separate epithelial cell isolations from the same piglet. The expression of β-catenin, HNF-4α, TNF-α, TGF-β and the crypt marker v-myb matched the expected villus-to-crypt pattern in cells collected after 10 (incubation 1), 30 (incubation 2) and 60 (incubation 3) minutes. However, expression of expression in cells collected after 100 minutes (incubation 4) was variable, which may be due to the fact that crypt cells were not efficiently removed and the presence of unwanted non-epithelial tissue. Gremlin, OPN, DKK1, sFRP3 and sFRP4 expression was not statistically different along the villus-to-crypt axis. Frizzled 6 and CDK5 did not express as we had predicted, with expression highest towards the villi. In summary, the epithelial cell collection method used was not entirely successful. While much of the gene data suggests that cells were removed along the villus-to-crypt axis through the first three incubations, the last incubation, which involved scraping the tissue, removed non-epithelial components of the mucosa, while leaving the crypts intact. In conclusion, the addition of 2 g/L PDX and 2 g/L scFOS did not cause gene expression of the Wnt/BMP pathways to mirror either F or SR expression. New isolation methods to extract cells along the crypt-villus axis should be considered, including the use of a laser capture microdissection. While this combination of prebiotics did not yield the intended effects, future research should be done on other combinations, such as the inclusion of galactooligosaccharides (GOS), which is commonly added to food products including infant formula.

Inibição de linhagens do complexo Fusarium Graminearum por compostos naturais e sintéticos

Quando produtos alimentícios e especiarias são contaminados por micotoxinas é quase impossível detoxificar utilizando processos usuais da indústria de alimentos ou durante o preparo doméstico. Por isso, controlar o crescimento do fungo e a produção de toxinas é uma demanda para garantir a segurança alimentar. Os agrotóxicos são rotineiramente utilizados como estratégia para proteger as plantas de doenças provocadas pela contaminação fúngica. No entanto, eles estão associados a efeitos adversos ao sistema nervoso central e periférico, têm ação imunodepressora e são cancerígenos. Em virtude disso, o objetivo deste trabalho foi estudar a inibição do desenvolvimento, do potencial toxigênico e da expressão gênica de linhagens do Complexo Fusarium graminearum por compostos naturais comparativamente aos fungicidas azoxistrobina e trifloxistrobina. Do farelo de arroz, foram extraídos o γ-orizanol e os ácidos fenólicos (EFF). Das sementes de nim foram extraídos os ácidos fenólicos (EFN), totalizando três extratos naturais. A capacidade antioxidante dos extratos foi verificada pelo consumo do radical livre DPPH• , capacidade de captura do radical ABTS●+, redução do ferro e inibição da oxidação enzimática. Os mecanismos de inibição de três linhagens de F. graminearum foram avaliados através da determinação de compostos estruturais (glicosamina e ergosterol) e da atividade de enzimas do metabolismo primário (α- amilase e proteases). Foram determinadas as micotoxinas de Fusarium: deoxinivalenol (DON), 15 acetildeoxinivalenol (15AcDON), 3 acetildeoxinivalenol (3AcDON), nivalenol (NIV) e zearalenona (ZEA). A expressão dos genes Tri1 e Tri5 foi determinada a fim de verificar se ocorria modificação da expressão gênica nas linhagens do Complexo F. graminearum ocasionada pela presença dos antifúngicos. O EFF apresenta atividade antioxidante destacada em relação aos demais extratos naturais para inibir a iniciação do processo, a propagação do radical livre e a catálise enzimática. A presença dos compostos naturais mostrou efeito promissor como antifúngico para as linhagens, sendo que a concentração necessária para inibir 50% do crescimento radial das colônias (MIC50) foi 0,9 g/kg para γ-orizanol; 0,032 g/kg para EFF e 0,037 g/kg para EFN, portanto, os extratos fenólicos são mais eficazes para inibição de F. graminearum do que o γ-orizanol. Os extratos naturais afetaram as atividades das enzimas α-amilase e proteases. Também ocorreu a redução da formação de componentes estruturais (glicosamina e ergosterol). Os extratos naturais se destacaram pela capacidade de inibição de micotoxinas produzidas pela biomassa fúngica, com destaque para o EFN sobre a produção de DON, 15AcDON, 3AcDON e ZEA. Sendo assim, é possível dizer que há uma relação direta entre a atividade antioxidante na inibição do fungo e na manifestação do seu potencial toxigênico. Além disso, esse estudo contribuiu com a elucidação do mecanismo de ação dos antifúngicos naturais estudados. Ocorre modificação na expressão gênica quando a linhagem é submetida ao tratamento com antifúngico, havendo uma relação direta entre a expressão do gene Tri5 e a produção de DON.

Two novel COLVI long chains in zebrafish that are essential for muscle development

Collagen VI (COLVI), a protein ubiquitously expressed in connective tissues, is crucial for structural integrity, cellular adhesion, migration and survival. Six different genes are recognized in mammalians, encoding six COLVI-chains that assemble as two ‘short’ (α1, α2) and one ‘long’ chain (theoretically any one of α3–6). In humans, defects in the most widely expressed heterotrimer (α123), due to mutations in the COL6A1-3 genes, cause a heterogeneous group of neuromuscular disorders, collectively termed COLVI-related muscle disorders. Little is known about the function(s) of the recently described α4-6 chains and no mutations have been detected yet. In this study, we characterized two novel COLVI long chains in zebrafish that are most homologous to the mammalian α4 chain; therefore, we named the corresponding genes col6a4a and col6a4b. These orthologues represent ancestors of the mammalian Col6a4-6 genes. By in situ hybridization and RT-qPCR, we unveiled a distinctive expression kinetics for col6a4b, compared with the other col6a genes. Using morpholino antisense oligonucleotides targeting col6a4a, col6a4b and col6a2, we modelled partial and complete COLVI deficiency, respectively. All morphant embryos presented altered muscle structure and impaired motility. While apoptosis was not drastically increased, autophagy induction was defective in all morphants. Furthermore, motoneuron axon growth was abnormal in these morphants. Importantly, some phenotypical differences emerged between col6a4a and col6a4b morphants, suggesting only partial functional redundancy. Overall, our results further confirm the importance of COLVI in zebrafish muscle development and may provide important clues for potential human phenotypes associated with deficiency of the recently described COLVI-chains.

Avaliação transcricional de Glutationas -transferases em PEIXE-ZEBRA (Danio rerio) e alterações causadas pela exposição á Microcistina-LR

As Microcistinas são heptapeptídios cíclicos produzidos como metabólitos secundários por diferentes espécies de cianobactérias, sendo relevantes pelo seu potencial hepatotóxico. Peixes apresentam estratégias bioquímicas para detoxificar contaminantes ambientais, incluindo a ativação de enzimas de fase II de biotransformação, que incluem as isoformas de glutationa S-transferase (GST). As GST catalizam a conjugação de glutationa reduzida (GSH) com uma variedade de xenobióticos, incluindo as microcistinas. O presente estudo avaliou os níveis transcricionais de quinze isoformas de GST a fim de identificar isoformas possivelmente envolvidas na detoxificação de contaminantes ambientais como a microcistina-LR (MC-LR) em Danio rerio. A técnica de PCR em tempo real (RT-qPCR) foi utilizada para avaliação dos níveis transcricionais, permitindo análise das GST em diferentes órgãos, abundância e a ativação/repressão das isoformas de GST pela exposição à MC-LR. Foram avaliados os possíveis efeitos causados em brânquia e fígado após exposição por 24 hs às concentrações de 5 µg.L-1 e 50 µg.L-1 de MC-LR. Baseado nos scores de estabilidade para oito genes normalizadores, foram selecionados glicose-6-fosfato desidrogenase (g6pdh), β-actina1 e beta-2-microglobulina (b2m); b2m, alfa-tubulina 1 (tuba) e β- actin1; e tuba, b2m e g6pdh, para normalização dos níveis trancricionais de GST para distribuição órgão-específica, abundância e efeito da MC-LR em brânquia e fígado, respectivamente. A avaliação transcricional da distribuição órgão-específica revelou níveis significativos de gstal e gstk1.1 no fígado; gstp1 e gstp2 em brânquia; mgst3a, gstr1, gstm2, gstm33, gstp1, gstp2 e gstk1.1 no intestino; gstm2, gstm3 e gstal no olho e gstt1a e gsta2.1 no cérebro. Considerando os níveis de transcritos para um dado órgão, gstk1.1, gstal, gstp1 e gstt2 foram mais abundantes nos órgãos de detoxificação, tais como o fígado, brânquias e intestino, enquanto gstt1a e gsta2.1 foram mais abundantes no rim. Em brânquia, gsta2.1 e gstt1b foram reprimidas por 5 µg.L-1 de MC-LR e mgst1.1 foi reprimida em 50 µg.L-1 de MC-LR. No fígado, as isoformas gst2.2 e gstp2 foram reprimidas em ambas as concentrações, gstal foi reprimida em 5 µg.L-1, e gstt1a e gstk1.1 foram reprimidas em 50 µg.L-1 de MC-LR. As isoformas gstal, gstr1, gstp1, mgst3a, gstm1, gstm2 e gstm3 não foram alteradas pela exposição a MC-LR. Os resultados obtidos fornecem informações para a escolha de isoformas específicas de GST possivelmente envolvidas na detoxificação/toxicidade de MC-LR, a serem melhores caracterizadas ao nível protéico e também contribui para a escolha de genes normalizadores a serem utilizados em outros estudos da mesma natureza

Anaerobic oxidation of methane at a marine methane seep in a forearc sediment basin off Sumatra, Indian Ocean

A cold methane seep was discovered in a forearc sediment basin off the island Sumatra, exhibiting a methane-seep adapted microbial community. A defined seep center of activity, like in mud volcanoes, was not discovered. The seep area was rather characterized by a patchy distribution of active spots. The relevance of anaerobic oxidation of methane (AOM) was reflected by C-13-depleted isotopic signatures of dissolved inorganic carbon. The anaerobic conversion of methane to CO2 was confirmed in a C-13-labeling experiment. Methane fueled a vital microbial community with cell numbers of up to 4 x 10(9) cells cm(-3) sediment. The microbial community was analyzed by total cell counting, catalyzed reporter deposition fluorescence in situ hybridization (CARD FISH), quantitative real-time PCR (qPCR), and denaturing gradient gel electrophoresis (DGGE). CARD FISH cell counts and qPCR measurements showed the presence of Bacteria and Archaea, but only small numbers of Eukarya. The archaeal community comprised largely members of ANME-1 and ANME-2. Furthermore, members of the Crenarchaeota were frequently detected in the DGGE analysis. Three major bacterial phylogenetic groups (delta-Proteobacteria, candidate division OP9, and Anaerolineaceae) were abundant across the study area. Several of these sequences were closely related to the genus Desulfococcus of the family Desulfobacteraceae, which is in good agreement with previously described AOM sites. In conclusion, the majority of the microbial community at the seep consisted of AOM-related microorganisms, while the relevance of higher hydrocarbons as microbial substrates was negligible.

THE DISTRIBUTION AND FUNCTION OF DENITRIFICATION GENES: EXPLORING AGRICULTURAL MANAGEMENT AND SOIL CHEMICAL IMPLICATIONS

Denitrification is a microbially-mediated process that converts nitrate (NO3-) to dinitrogen (N2) gas and has implications for soil fertility, climate change, and water quality. Using PCR, qPCR, and T-RFLP, the effects of environmental drivers and land management on the abundance and composition of functional genes were investigated. Environmental variables affecting gene abundance were soil type, soil depth, nitrogen concentrations, soil moisture, and pH, although each gene was unique in its spatial distribution and controlling factors. The inclusion of microbial variables, specifically genotype and gene abundance, improved denitrification models and highlights the benefit of including microbial data in modeling denitrification. Along with some evidence of niche selection, I show that nirS is a good predictor of denitrification enzyme activity (DEA) and N2O:N2 ratio, especially in alkaline and wetland soils. nirK was correlated to N2O production and became a stronger predictor of DEA in acidic soils, indicating that nirK and nirS are not ecologically redundant.

Revisiting molecular diagnostics of Streptococcus pneumoniae

Streptococcus pneumoniae is a human pathobiont that colonizes the nasopharynx. S. pneumoniae is responsible for causing non-invasive and invasive disease such as otitis, pneumonia, meningitis, and sepsis, being a leading cause of infectious diseases worldwide. Due to similarities with closely related species sharing the same niche, it may be a challenge to correctly distinguish S. pneumoniae from its relatives when using only non-culture based methods such as real time PCR (qPCR). In 2007, a molecular method targeting the major autolysin (lytA) of S. pneumoniae by a qPCR assay was proposed by Carvalho and collaborators to identify pneumococcus. Since then, this method has been widely used worldwide. In 2013, the gene encoding for the ABC iron transporter lipoprotein PiaA, was proposed by Trzcinzki and collaborators to be used in parallel with the lytA qPCR assay. However, the presence of lytA gene homologues has been described in closely related species such as S. pseudopneumoniae and S. mitis and the presence of piaA gene is not ubiquitous between S. pneumoniae. The hyaluronate lyase gene (hylA) has been described to be ubiquitous in S. pneumoniae. This gene has not been used so far as a target for the identification of S. pneumoniae. The aims of our study were to evaluate the specificity, sensitivity, positive predicted value (PPV) and negative predicted value (NPV) of the lytA and piaA qPCR methods; design and implement a new assay targeting the hylA gene and evaluate the same parameters above described; analyze the assays independently and the possible combinations to access what is the best approach using qPCR to identify S. pneumoniae. A total of 278 previously characterized strains were tested: 61 S. pseudopneumoniae, 37 Viridans group strains, 30 type strains from other streptococcal species and 150 S. pneumoniae strains. The collection included both carriage and disease isolates. By Mulilocus Sequence Analysis (MLSA) we confirmed that strains of S. pseudopneumoniae could be misidentified as S. pneumoniae when lytA qPCR assay is used. The results showed that as a single target, lytA had the best combination of specificity, sensitivity, PPV and NPV being, 98.5%, 100.0%, 98.7% and 100.0% respectively. The combination of targets with the best values of specificity, sensibility, PPV and NPV were lytA and piaA, with 100.0%, 93.3%, 97.9% and 92.6%, respectively. Nonetheless by MLSA we confirmed that strains of S. pseudopneumoniae could be misidentified as S. pneumoniae and some capsulated (23F, 6B and 11A) and non-capsulated S. pneumoniae were not Identified using this assay. The hylA gene as a single target had the lowest PPV. Nonetheless it was capable to correctly identify all S. pneumoniae.

Extracting DNA from ‘jaws’: high yield and quality from archived tiger shark (Galeocerdo cuvier) skeletal material

Archived specimens are highly valuable sources of DNA for retrospective genetic/genomic analysis. However, often limited effort has been made to evaluate and optimize extraction methods, which may be crucial for downstream applications. Here, we assessed and optimized the usefulness of abundant archived skeletal material from sharks as a source of DNA for temporal genomic studies. Six different methods for DNA extraction, encompassing two different commercial kits and three different protocols, were applied to material, so-called bio-swarf, from contemporary and archived jaws and vertebrae of tiger sharks (Galeocerdo cuvier). Protocols were compared for DNA yield and quality using a qPCR approach. For jaw swarf, all methods provided relatively high DNA yield and quality, while large differences in yield between protocols were observed for vertebrae. Similar results were obtained from samples of white shark (Carcharodon carcharias). Application of the optimized methods to 38 museum and private angler trophy specimens dating back to 1912 yielded sufficient DNA for downstream genomic analysis for 68% of the samples. No clear relationships between age of samples, DNA quality and quantity were observed, likely reflecting different preparation and storage methods for the trophies. Trial sequencing of DNA capture genomic libraries using 20 000 baits revealed that a significant proportion of captured sequences were derived from tiger sharks. This study demonstrates that archived shark jaws and vertebrae are potential high-yield sources of DNA for genomic-scale analysis. It also highlights that even for similar tissue types, a careful evaluation of extraction protocols can vastly improve DNA yield.