Genetic analysis of the flowering date and number of petals in rose

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Genetic differentiation among Parastichopus regalis populations in the Western Mediterranean Sea: potential effects from its fishery and current connectivity

Parastichopus regalis (Cuvier, 1817) is the most expensive seafood product on the Catalonian market (NE Spain), with prices at approximately 130 €/Kg (fresh weight). Despite its ecological and economic importance, biological and genetic information on this sea cucumber species is scarce. Here, we provide both the first insight on the genetic structure of P. regalis using sequences of cytochrome oxidase I (COI) and 16S genes and a morphological description of its population. Individual sea cucumbers were collected in six locations along the Spanish Mediterranean coast, including an area under fishery pressure (Catalonia). We found high haplotype diversity and low nucleotide diversity for both genes, with higher levels of genetic diversity observed in the COI gene. The population pairwise fixation index (FST), AMOVA and correspondence analysis (CA) based on the COI gene revealed significant genetic differentiation among some locations. However, further analysis using nuclear markers (e.g., microsatellites) is necessary to corroborate these results. Moreover, the genetic and morphological data may indicate fishery effects on the Catalonian population with a decrease in the size and weight averages and lower genetic diversity compared with locations that lack fishery pressure. For the appropriate management of this species, we suggest the following: 1) accurately assessing the stock status along the Spanish coasts; 2) studying the reproductive cycle of this target species and the establishment of a closed fishery season according to the reproductive cycle; and 3) establishing protected areas (i.e., not take zones) to conserve healthy populations and favour recruitment in the nearby areas.

West versus East Mediterranean Sea: origin and genetic differentiation of the sea cucumber Holothuria polii

We studied the genetic structure of the sea cucumber Holothuria (Roweothuria) polii (Delle Chiaje 1823) by analysing the mitochondrial DNA variation in two fragments of cytochrome oxidase I (COI) and 16S genes. Individuals were collected in seven locations along the Mediterranean Sea, which cover a wide range of the species distribution. We found high haplotype diversity for COI and moderate diversity for 16S, and low nucleotide diversity for both genes. Our results for the COI gene showed many recent and exclusive haplotypes with few mutational changes, suggesting recent or ongoing population expansion. The Western and Eastern Mediterranean populations exhibited slight but significant genetic differentiation (COI gene) with higher genetic diversity in the East. The most ancient haplotype was not present in the westernmost sampling location (SE Spain). The oldest expansion time was observed in Turkey, corresponding to mid-Pleistocene. Turkey had also the highest genetic diversity (number of total and exclusive haplotypes, polymorphisms, haplotype and nucleotide diversity). This suggests that this region could be the origin of the subsequent colonizations through the Mediterranean Sea, a hypothesis that should be assessed with nuclear markers in future research.

Estudo da associação dos genes GGR, GGPPS2 e GGPPS6 em Arabidopsis thaliana L. (Brassicaceae) a compostos alelopáticos

Dissertação (mestrado)—Universidade de Brasília, Departamento de Botânica, Programa de Pós-Graduação em Botânica, 2016.

Detecção de genes de enterotoxinas e da toxina-1 da Síndrome do Choque Tóxico de Staphylococcus aureus isolados de mastites no Distrito Federal e Entorno

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, Programa de Pós-Graduação em Saúde Animal, 2016.

Swine influenza virus PA and neuraminidase gene reassortment into human H1N1iInfluenza Virus is associated with an altered pathogenic phenotype linked to increased MIP-2 expression

Swine are susceptible to infection by both avian and human influenza viruses, and this feature is thought to contribute to novel reassortant influenza viruses. In this study, the influenza virus reassortment rate in swine and human cells was determined. Coinfection of swine cells with 2009 pandemic H1N1 virus (huH1N1) and an endemic swine H1N2 (A/swine/Illinois/02860/09) virus (swH1N2) resulted in a 23% reassortment rate that was independent of α2,3- or α2,6-sialic acid distribution on the cells. The reassortants had altered pathogenic phenotypes linked to introduction of the swine virus PA and neuraminidase (NA) into huH1N1. In mice, the huH1N1 PA and NA mediated increased MIP-2 expression early postinfection, resulting in substantial pulmonary neutrophilia with enhanced lung pathology and disease. The findings support the notion that swine are a mixing vessel for influenza virus reassortants independent of sialic acid distribution. These results show the potential for continued reassortment of the 2009 pandemic H1N1 virus with endemic swine viruses and for reassortants to have increased pathogenicity linked to the swine virus NA and PA genes which are associated with increased pulmonary neutrophil trafficking that is related to MIP-2 expression. IMPORTANCE: Influenza A viruses can change rapidly via reassortment to create a novel virus, and reassortment can result in possible pandemics. Reassortments among subtypes from avian and human viruses led to the 1957 (H2N2 subtype) and 1968 (H3N2 subtype) human influenza pandemics. Recent analyses of circulating isolates have shown that multiple genes can be recombined from human, avian, and swine influenza viruses, leading to triple reassortants. Understanding the factors that can affect influenza A virus reassortment is needed for the establishment of disease intervention strategies that may reduce or preclude pandemics. The findings from this study show that swine cells provide a mixing vessel for influenza virus reassortment independent of differential sialic acid distribution. The findings also establish that circulating neuraminidase (NA) and PA genes could alter the pathogenic phenotype of the pandemic H1N1 virus, resulting in enhanced disease. The identification of such factors provides a framework for pandemic modeling and surveillance.

Epigenetic regulation of neurodevelopmental genes in response to in utero exposure to phthalate plastic chemicals: how can we delineate causal effects?

Accumulating evidence, from animal models and human observational studies, implicates the in utero (and early postnatal) environment in the 'programming' of risk for a variety of adverse outcomes and health trajectories. The modern environment is replete with man-made compounds such as plastic product chemicals (PPC), including phenols and phthalates. Evidence from several human cohorts implicates exposure to these chemicals in adverse offspring neurodevelopment, though a direct causal relationship has not been firmly established. In this review we consider a potential causal pathway that encompasses epigenetic human variation, and how we might test this mechanistic hypothesis in human studies. In the first part of this report we outline how PPCs induce epigenetic change, focusing on the brain derived neurotrophic factor (BDNF) gene, a key regulator of neurodevelopment. Further, we discuss the role of the epigenetics of BDNF and other genes in neurodevelopment and the emerging human evidence of an association between phthalate exposure and adverse offspring neurodevelopment. We discuss aspects of epidemiological and molecular study design and analysis that could be employed to strengthen the level of human evidence to infer causality. We undertake this using an exemplar recent research example: maternal prenatal smoking, linked to methylation change at the aryl hydrocarbon receptor repressor (AHRR) gene at birth, now shown to mediate some of the effects of maternal smoking on birth weight. Characterizing the relationship between the modern environment and the human molecular pathways underpinning its impact on early development is paramount to understanding the public health significance of modern day chemical exposures.

The expression of genes encoding enzymes regulating fat metabolism is affected by maternal nutrition when lambs are fed algae high in omega-3

The current study investigated the effects of the supplementation of lambs with algae on the expression of genes that direct the accumulation of long chain omega-3 polyunsaturated fatty acids (LCn-3PUFA). The nutrition of the lamb's dam around mating was also considered. The dams were fed with either silage (SLG) or oat/cottonseed (OAT) based diets for six weeks prior to and, three weeks following conception. mRNA levels of FADS1, FADS2, CPT1, SCD, ACC and fad2 were measured in the liver, muscle and subcutaneous fat from lambs fed a control diet consisting of oat and lupin grains and chopped lucerne (CTRL) or the CTRL diet with algae (DHAgold™) added at 1.92% DM (ALG). The lambs were 117.4±2.4 days of age and weighed 37.3±3.2 kg at the beginning of the experimental period, and six weeks later they were slaughtered at 160.4±2.4 days of age and a final live weight of 50.1±4.4 kg. The expression of FADS1 in liver tissue was not affected (P>0.05) by the interaction between dam nutrition and algae supplementation, however it was higher (P<0.05) when lambs received the ALG diet compared with the CTRL and when their dams were fed SLG compared with OAT diet. The expression of FADS1 in muscle was negatively correlated (P<0.05) with the concentration of 20:4n-6, 20:5n-3 and 22:4n-6. The expression of FADS1, FADS2, SCD and ACC genes in lamb muscle was differentially affected by dam nutrition with the highest levels for the SLG+ALG treatment (P<0.05) compared with other treatments. The expression of SCD gene was not affected (P>0.05) by algae supplementation, but it was higher (P<0.05) when dams were fed SLG compared with OAT, however ACC was not affected (P>0.05).

A Livestock-Associated, Multidrug-Resistant, Methicillin-Resistant Staphylococcus aureus Clonal Complex 97 Lineage Spreading in Dairy Cattle and Pigs in Italy

Pandemic methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 97 (CC97) lineages originated from livestock-to-human host jumps. In recent years, CC97 has become one of the major MRSA lineages detected in Italian farmed animals. The aim of this study was to characterize and analyze differences in MRSA and methicillin-susceptible S. aureus (MSSA) mainly of swine and bovine origins. Forty-seven CC97 isolates, 35 MRSA isolates, and 6 MSSA isolates from different Italian pig and cattle holdings; 5 pig MRSA isolates from Germany; and 1 human MSSA isolate from Spain were characterized by macrorestriction pulsed-field gel electrophoresis (PFGE) analysis, multilocus sequence typing (MLST), spa typing, staphylococcal cassette chromosome mec (SCCmec) typing, and antimicrobial resistance pattern analysis. Virulence and resistance genes were investigated by PCR and microarray analysis. Most of the isolates were of SCCmec type V (SCCmec V), except for two German MRSA isolates (SCCmec III). Five main clusters were identified by PFGE, with the German isolates (clusters I and II) showing 60.5% similarity with the Italian isolates, most of which (68.1%) grouped into cluster V. All CC97 isolates were Panton-Valentine leukocidin (PVL) negative, and a few (n = 7) tested positive for sak or scn. All MRSA isolates were multidrug resistant (MDR), and the main features were erm(B)- or erm(C)-mediated (n = 18) macrolide-lincosamide-streptogramin B resistance, vga(A)-mediated (n = 37) pleuromutilin resistance, fluoroquinolone resistance (n = 33), tet(K) in 32/37 tet(M)-positive isolates, and blaZ in almost all MRSA isolates. Few host-associated differences were detected among CC97 MRSA isolates: their extensive MDR nature in both pigs and dairy cattle may be a consequence of a spillback from pigs of a MRSA lineage that originated in cattle as MSSA and needs further investigation. Measures should be implemented at the farm level to prevent spillover to humans in intensive farming areas.

Livestock-Associated Methicillin Resistant and Methicillin Susceptible Staphylococcus aureus Sequence Type (CC)1 in European Farmed Animals: High Genetic Relatedness of Isolates from Italian Cattle Herds and Humans

Methicillin-resistant Staphylococcus aureus (MRSA) Sequence Type (ST)1, Clonal Complex(CC)1, SCCmec V is one of the major Livestock-Associated (LA-) lineages in pig farming industry in Italy and is associated with pigs in other European countries. Recently, it has been increasingly detected in Italian dairy cattle herds. The aim of this study was to analyse the differences between ST1 MRSA and methicillin-susceptible S. aureus (MSSA) from cattle and pig herds in Italy and Europe and human isolates. Sixty-tree animal isolates from different holdings and 20 human isolates were characterized by pulsed-field gel electrophoresis (PFGE), spa-typing, SCCmec typing, and by micro-array analysis for several virulence, antimicrobial resistance, and strain/host-specific marker genes. Three major PFGE clusters were detected. The bovine isolates shared a high (≥90% to 100%) similarity with human isolates and carried the same SCCmec type IVa. They often showed genetic features typical of human adaptation or present in human-associated CC1: Immune evasion cluster (IEC) genes sak and scn, or sea; sat and aphA3-mediated aminoglycoside resistance. Contrary, typical markers of porcine origin in Italy and Spain, like erm(A) mediated macrolide-lincosamide-streptograminB, and of vga(A)-mediated pleuromutilin resistance were always absent in human and bovine isolates. Most of ST(CC)1 MRSA from dairy cattle were multidrug-resistant and contained virulence and immunomodulatory genes associated with full capability of colonizing humans. As such, these strains may represent a greater human hazard than the porcine strains. The zoonotic capacity of CC1 LA-MRSA from livestock must be taken seriously and measures should be implemented at farm-level to prevent spill-over.

Characterization of some bacterial strains isolated from animal clinical materials and identified as Corynebacterium xerosis by molecular biological techniques

Eighteen Corynebacterium xerosis strains isolated from different animal clinical specimens were subjected to phenotypic and molecular genetic studies. On the basis of the results of the biochemical characterization, the strains were tentatively identified as C. xerosis. Phylogenetic analysis based on comparative analysis of the sequences of 16S rRNA and rpoB genes revealed that the 18 strains were highly related to C. xerosis, C. amycolatum, C. freneyi, and C. hansenii. There was a good concordance between 16S rRNA and partial rpoB gene sequencing results, although partial rpoB gene sequencing allowed better differentiation of C. xerosis. Alternatively, C. xerosis was also differentiated from C. freneyi and C. amycolatum by restriction fragment length polymorphism analysis of the 16S-23S rRNA gene intergenic spacer region. Phenotypic characterization indicated that besides acid production from D-turanose and 5-ketogluconate, 90% of the strains were able to reduce nitrate. The absence of the fatty acids C(14:0), C(15:0), C(16:1)omega 7c, and C(17:1)omega 8c can also facilitate the differentiation of C. xerosis from closely related species. The results of the present investigation demonstrated that for reliable identification of C. xerosis strains from clinical samples, a combination of phenotypic and molecular-biology-based identification techniques is necessary.

Identification of differentially expressed genes in the heart precursor cells of the chick embryo

Genetic evidence has implicated several genes as being critical for heart development. However, the inducers of these genes as well as their targets and pathways they are involved with, remain largely unknown. Previous studies in the avian embryo showed that at HH4 Cerberus (cCer) transcripts are detected in the anterior endomesoderm including the heart precursor cells and later in the left lateral plate mesoderm. We have identified a promoter element of chick cCer able to drive EGFP expression in a population of cells that consistently exit from the anterior primitive streak region, from as early as stage HH3+, and that later will populate the heart. Using this promoter element as a tool allowed us to identify novel genes previously not known to potentially play a role in heart development. In order to identify and study genes expressed and involved in the correct development and differentiation of the vertebrate heart precursor cell (HPC) lineages, a differential screening using Affymetrix GeneChip system technologies was performed. Remarkably, this screening led to the identification of more than 700 transcripts differentially expressed in the heart forming regions (HFR). Bioinformatic tools allowed us to filter the large amount of data generated from this approach and to select a few transcripts for in vivo validation. Whole-mount in situ hybridization and sectioning of selected genes showed heart and vascular expression patterns for these transcripts during early chick development. We have developed an effective strategy to specifically identify genes that are differentially expressed in the HPC lineages. Within this set we have identified several genes that are expressed in the heart, blood and vascular lineages, which are likely to play a role in their development. These genes are potential candidates for future functional studies on early embryonic patterning.

Unconjugated bile acids influence expression of circadian genes: a potential mechanism for microbe-host crosstalk

Disruptions to circadian rhythm in mice and humans have been associated with an increased risk of obesity and metabolic syndrome. The gut microbiota is known to be essential for the maintenance of circadian rhythm in the host suggesting a role for microbe-host interactions in the regulation of the peripheral circadian clock. Previous work suggested a role for gut bacterial bile salt hydrolase (BSH) activity in the regulation of host circadian gene expression. Here we demonstrate that unconjugated bile acids, known to be generated through the BSH activity of the gut microbiota, are potentially chronobiological regulators of host circadian gene expression. We utilised a synchronised Caco-2 epithelial colorectal cell model and demonstrated that unconjugated bile acids, but not the equivalent tauro-conjugated bile salts, enhance the expression levels of genes involved in circadian rhythm. In addition oral administration of mice with unconjugated bile acids significantly altered expression levels of circadian clock genes in the ileum and colon as well as the liver with significant changes to expression of hepatic regulators of circadian rhythm (including Dbp) and associated genes (Per2, Per3 and Cry2). The data demonstrate a potential mechanism for microbe-host crosstalk that significantly impacts upon host circadian gene expression. Disruptions to circadian rhythm in mice and humans have been associated with an increased risk of obesity and metabolic syndrome. The gut microbiota is known to be essential for the maintenance of circadian rhythm in the host suggesting a role for microbe-host interactions in the regulation of the peripheral circadian clock. Previous work suggested a role for gut bacterial bile salt hydrolase (BSH) activity in the regulation of host circadian gene expression. Here we demonstrate that unconjugated bile acids, known to be generated through the BSH activity of the gut microbiota, are potentially chronobiological regulators of host circadian gene expression. We utilised a synchronised Caco-2 epithelial colorectal cell model and demonstrated that unconjugated bile acids, but not the equivalent tauro-conjugated bile salts, enhance the expression levels of genes involved in circadian rhythm. In addition oral administration of mice with unconjugated bile acids significantly altered expression levels of circadian clock genes in the ileum and colon as well as the liver with significant changes to expression of hepatic regulators of circadian rhythm (including Dbp) and associated genes (Per2, Per3 and Cry2). The data demonstrate a potential mechanism for microbe-host crosstalk that significantly impacts upon host circadian gene expression.

Development of molecular approaches to assess the diversity and abundance of marine nematodes assemblages for assessment of Good Environmental Status of the estuarine and marine ecosystems

In Europe, the concerns with the status of marine ecosystems have increased, and the Marine Directive has as main goal the achievement of Good Environmental Status (GES) of EU marine waters by 2020. Molecular tools are seen as promising and emerging approaches to improve ecosystem monitoring, and have led ecology into a new era, representing perhaps the most source of innovation in marine monitoring techniques. Benthic nematodes are considered ideal organisms to be used as biological indicator of natural and anthropogenic disturbances in aquatic ecosystems underpinning monitoring programmes on the ecological quality of marine ecosystems, very useful to assess the GES of the marine environment. dT-RFLP (directed Terminal-Restriction Fragment Length Polymorphism) allows to assess the diversity of nematode communities, but also allows studying the functioning of the ecosystem, and combined with relative real-time PCR (qPCR), provides a high-throughput semi-quantitative characterization of nematode communities. These characteristics make the two molecular tools good descriptors for the good environmental status assessment. The main aim of this study is to develop and optimize the dT-RFLP and qPCR in Mira estuary (SW coast, Portugal). A molecular phylogenetic analysis of marine and estuarine nematodes is being performed combining morphological and molecular analysis to evaluate the diversity of free-living marine nematodes in Mira estuary. After morphological identification, barcoding of 18S rDNA and COI genes are being determined for each nematode species morphologically identified. So far we generated 40 new sequences belonging to 32 different genus and 17 families, and the study has shown a good degree of concordance between traditional morphology-based identification and DNA sequences. These results will improve the assessment of marine nematode diversity and contribute to a more robust nematode taxonomy. The DNA sequences are being used to develop the dT-RFLP with the ability to easily process large sample numbers (hundreds and thousands), rather than typical of classical taxonomic or low throughput molecular analyses. A preliminary study showed that the digest enzymes used in dT-RFLP for terrestrial assemblages separated poorly the marine nematodes at taxonomic level for functional group analysis. A new digest combination was designed using the software tool DRAT (Directed Terminal Restriction Analysis Tool) to distinguished marine nematode taxa. Several solutions were provided by DRAT and tested empirically to select the solution that cuts most efficiently. A combination of three enzymes and a single digest showed to be the best solution to separate the different clusters. Parallel to this, another tool is being developed to estimate the population size (qPCR). An improvement in qPCR estimation of gene copy number using an artificial reference is being performed for marine nematodes communities to quantify the abundance. Once developed, it is proposed to validate both methodologies by determining the spatial and temporal variability of benthic nematodes assemblages across different environments. The application of these high-throughput molecular approaches for benthic nematodes will improve sample throughput and their implementation more efficient and faster as indicator of ecological status of marine ecosystems.

Expression of rice genes homologous of Arabidopsis genes previously related to drought tolerance.

The identification and validation of candidate genes related to traits of interest is a time consuming and expensive process and the homology among genes from different species can facilitate the identification of genes of the target species from the genomic information of a model species. This study aimed to quantify the expression of homologous rice genes previously related to drought tolerance in Arabidopsis. Five genes (CPK6, PLDa, GluR2, CesA8, and EIN2) were identified in rice by the homology of the amino acid sequence between rice and Arabidopsis. The genotypes Douradão (drought tolerant) and Primavera (drought susceptible) were subjected to a water deficit experiment, and subsequently evaluated for gene expression by qPCR for the five homologous and Lsi1 genes. The qPCR analysis clearly showed that the five homologous genes were expressed in rice, which is an indication that these genes could preserve their function in rice as a response to drought. In Douradão, of the five homologous genes, all but OsGluR2 displayed an increase in the average expression in drought treatment when compared to the control, while in Primavera, the average expression of the five genes did not differ between the control and drought treatment. In Douradão, the OsPLDa1, which showed the higher expression level in drought in relation to the control (10.82), significantly increased the gene expression in the leaf and root tissues as a response to drought, in both vegetative and reproductive stages, whereas in Primavera, this gene was suppressed in both tissues and stages under drought. Therefore, the OsPLDa1 gene was the most important in relation to drought response and is an interesting candidate for further studies in developing rice cultivars that are more tolerant to this stress.