Ana Machado

Impact of copper on the diversity, abundance and transcription of nitrite and nitrous oxide reductase genes in an urban European estuary

In this study, the effects of increasing copper (Cu) concentrations on the denitrification pathway and on the diversity of a denitrifier community and gene expression were evaluated in intertidal sandy sediments of the Douro River estuary (north-west Portugal). The results demonstrated that sediment denitrification rates were highly affected by Cu; almost complete inhibition (85%) of the process was observed in sediments amended with 60 μg Cu g(-1) wet wt sediment. Moreover, the addition of Cu stimulated the accumulation of both N(2)O and NO(2)(-) and inhibited the rate of NO(3)(-) uptake. Further, the amendment with even the lowest Cu concentration (4 μg Cu g(-1) wet wt sediment) yielded a drastic decrease in the abundance of nirK, nirS and nosZ (between 79% and 81%) assessed by means of real-time PCR. In agreement, reverse transcription-PCR-denaturing gradient gel electrophoresis analysis of nirK, nirS and nosZ transcripts showed a progressive decrease in the diversity of the transcription products of these genes with an increase of the Cu concentration.

An efficient protocol for the detection of chromosomal abnormalities in spontaneous miscarriages or foetal deaths

OBJECTIVE Characterization of chromosomal abnormalities in 232 spontaneous miscarriages or foetal deaths using both classical and molecular cytogenetics. STUDY DESIGN Chromosomal abnormalities are responsible for 40-50% of all early pregnancy losses. Conventional cytogenetics is associated with 10-40% of culture failure. Comparative genomic hybridization (CGH) is a DNA-based technique that screens chromosome imbalances in the whole genome and may overcome this problem, although additional methods are required to distinguish between different ploidies, mosaicisms and maternal cell contamination. For a full characterization of chromosomal aberrations in 232 spontaneous miscarriages or foetal deaths we applied a sequential protocol that uses conventional cytogenetics, plus CGH and touch fluorescence in situ hybridization (Touch FISH). RESULTS Successful karyotyping was obtained in 173/232 (74.6%) of the cases, 66/173 (38.2%) of which had an abnormal chromosomal complement. CGH and Touch FISH analyses revealed another 19 abnormal cases in the 63 failures of culture. Overall there were 85/233 (36.6%) cases with an abnormal chromosomal complement, with examples from all three trimesters. Comparing cases, with or without chromosomal abnormalities, no statistical differences were found between women with one or recurrent miscarriages. On the contrary, significant differences were found comparing mean maternal ages or mean gestational ages, in cases with or without chromosomes abnormalities. CONCLUSION Adopting this sequential protocol, chromosomal complement information was available even in cases with culture failure.

Prevalence of antibiotic resistance in bacteria isolated from drinking well water available in Guinea-Bissau (West Africa)

The dissemination of antibiotic-resistant bacteria and the spread of antibiotic resistance genes are a major public health concern worldwide, being even proposed as emerging contaminants. The aquatic environment is a recognized reservoir of antibiotic resistant bacteria, and antibiotic resistance genes have been recently detected in drinking water. In this study, the water quality and the prevalence of antibiotic resistance of heterotrophic culturable bacteria were characterized seasonally in wells that serve the population of Guinea-Bissau (West Africa) as the sole source of water for drinking and other domestic proposes. The results revealed that well water was unfit for human consumption independently of the season, owing to high acidity and heavy fecal contamination. Moreover, potentially pathogenic bacteria, which showed resistance to the most prescribed antibiotics in Guinea-Bissau, were isolated from well water, posing an additional health risk. Our results suggest that well water not only fosters the transmission of potential pathogenic bacteria, but also represents an important reservoir for the proliferation of antibiotic resistant bacteria, that can aggravate the potential to cause disease in a very vulnerable population that has no other alternative but to consume such water.

The 17β-hydroxysteroid dehydrogenase 4: Gender-specific and seasonal gene expression in the liver of brown trout (Salmo trutta f. fario)

Previously, it was documented that liver peroxisomes display seasonal size changes in the adult Salmo trutta fario, especially in females (and negatively correlated with ovary maturation). It was then hypothesized that decreases in peroxisome size could be paralleled by changes in peroxisomal beta-oxidation and estradiol catabolism actions. The 17beta-hydroxysteroid dehydrogenase 4 has been portrayed as playing an important role in both processes. To elucidate its function in the described peroxisomal pattern, we isolated the cDNA and predicted the protein sequence of the enzyme in that species. The seasonal gene expression pattern in both genders was addressed through quantitative PCR. Fish sampling was in post-spawning period, early and advanced gonad maturation, and pre-spawning. Males did not vary seasonally. As to females, a seasonal pattern was evidenced according to our previous hypothesis. We suggest that the decreased levels observed during vitellogenesis are related to lipid needs for ovary maturation, and, additionally, with the need of modulating estradiol titers.

Cloning and expression analysis of the 17β hydroxysteroid dehydrogenase type 12 (HSD17B12) in the neogastropod Nucella lapillus.

HSD17B12 is a member of the hydroxysteroid dehydrogenase superfamily, a multifunctional group of enzymes involved in the metabolism of steroids, retinoids, bile and fatty acids. Whether the main role of HSD17B12 in mammals is in steroid or fatty acid metabolism is a subject of intense debate. In mollusks it has been shown that an HSD17B12 orthologue can convert estrone into estradiol in vitro, although its primary in vivo function remains unknown. To gain insight into its role in gastropods, we provide here the first cloning of Hsd17b12 in Nucella lapillus and its detailed tissue distribution through quantitative PCR. Furthermore, given that the endocrine disruptor tributyltin (TBT) has been reported to unbalance steroid and lipid levels in gastropods, we tested its impact in on NlHsd17b12 transcript expression. Our results show that NlHsd17b12 is ubiquitously expressed in all tissues analyzed, with higher levels in organs with high metabolic rates, such as kidney and digestive gland, a pattern consistent with an involvement in lipid metabolism. Exposure to TBT chloride at 100 ng Sn/L caused a decrease in NlHsd17b12 mRNA levels in digestive gland, after one and two months, while no effect was observed in gonads. Overall, these results suggest that in mollusks, as in mammals, this enzyme is likely to be involved in lipid metabolism, and emphasize the need to perform more detailed studies on its in vivo function, in order to understand its physiological role and the biological impact of its disruption by pollutants such as TBT.

Revisiting Amniotic Band Sequence: A Wide Spectrum of Manifestations

Objective: To describe the spectrum of clinical deformities related to amniotic band sequence (ABS), ranging from mild to severe. Methods: Retrospective evaluation of 9 cases of ABS managed during a 4-year period (February 2006 to April 2010) in a tertiary hospital. Results: The median gestational age at prenatal diagnosis was 15 weeks (range 11-26). Only 2 patients had clinical prenatal evidence of amnion rupture. In the other 5 cases the diagnosis of ABS was established prenatally based on fetal structural abnormalities. These abnormalities included: (1) upper limb defects: syndactyly, amputation at the level of phalanges or first metacarpal or forearm; (2) inferior extremities defects: tallus equinovarus and flexed knee, and constriction ring; (3) umbilical cord strangulation, and (4) acrania. Conclusion: ABS is a polymorphous syndrome and recognizing its various manifestations can be of help in prenatal identification.

Sequential injection methodology for carbon speciation in bathing waters

A sequential injection method (SIA) for carbon speciation in inland bathing waters was developed comprising, in a single manifold, the determination of dissolved inorganic carbon (DIC), free dissolved carbon dioxide (CO2), total carbon (TC), dissolved organic carbon and alkalinity. The determination of DIC, CO2 and TC was based on colour change of bromothymol blue (660 nm) after CO2 diffusion through a hydrophobic membrane placed in a gas diffusion unit (GDU). For the DIC determination, an in-line acidification prior to the GDU was performed and, for the TC determination, an in-line UV photo-oxidation of the sample prior to GDU ensured the conversion of all carbon forms into CO2. Dissolved organic carbon (DOC) was determined by subtracting the obtained DIC value from the TC obtained value. The determination of alkalinity was based on the spectrophotometric measurement of bromocresol green colour change (611 nm) after reaction with acetic acid. The developed SIA method enabled the determination of DIC (0.24-3.5 mg C L(-1)), CO2 (1.0-10 mg C L(-1)), TC (0.50-4.0 mg C L(-1)) and alkalinity (1.2-4.7 mg C L(-1) and 4.7-19 mg C L(-1)) with limits of detection of: 9.5 μg C L(-1), 20 μg C L(-1), 0.21 mg C L(-1), 0.32 mg C L(-1), respectively. The SIA system was effectively applied to inland bathing waters and the results showed good agreement with reference procedures.

Methanethiol accumulation exacerbates release of N2O during denitrification in estuarine sediments and bacterial cultures

Microbes play critical roles in the biogeochemical cycling of nitrogen and sulfur in aquatic environments. Here we investigated the interaction between the naturally occurring organic sulfur compound methanethiol (MeSH) and the final step of the denitrification pathway, the reduction of nitrous oxide (N2 O) to dinitrogen (N2 ) gas, in sediment slurries from the temperate Douro and Ave estuaries (NW Portugal) and in pure cultures of the marine bacterium Ruegeria pomeroyi. Sediment slurries and cell suspensions were amended with a range of concentrations of either MeSH (0-120 µM) or methionine (0-5 mM), a known precursor of MeSH. MeSH or methionine additions caused N2 O to accumulate and this accumulation was linearly related to MeSH concentrations in both coastal sediments (R(2)  = 0.7-0.9, P < 0.05) and R. pomeroyi cell suspensions (R(2)  = 0.9, P < 0.01). Our results suggest that MeSH inhibits the final step of denitrification resulting in N2 O accumulation.

Diversity and dynamics of the Vibrio community in well water used for drinking in Guinea-Bissau (West Africa)

Bacteria of the genus Vibrio are ubiquitous in aquatic environments and can be found either in culturable or in a viable but nonculturable (VBNC) state. The genus comprises many pathogenic species accountable for water and food-borne diseases that prove to be fatal, especially in developing countries, as in Guinea-Bissau (West Africa), where cholera is endemic. In order to ascertain the abundance and structure of Vibrio spp. community in well waters that serve as the sole source of water for the population, quantitative polymerase chain reaction (qPCR), PCR-denaturant gradient gel electrophoresis (DGGE), and cloning approaches were used. Results suggest that Vibrio spp. were present throughout the year in acidic, freshwater wells with a seasonal community composition shift. Vibrio spp. abundance was in accordance with the abundance found in coastal environments. Sequences closely related to pathogenic Vibrio species were retrieved from well water revealing exposure of the population to such pathogens. pH, ammonium, and turbidity, regulated by the rain pattern, seem to be the variables that contributed mostly to the shaping and selection of the Vibrio spp. community. These results reinforce the evidence for water monitoring with culture-independent methods and the clear need to create/recover water infrastructures and a proper water resources management in West African countries with similar environmental conditions.

Water bags as a potential vehicle for transmitting disease in a West African capital, Bissau

BACKGROUND Street vendors of chilled packaged water have an increasing role in meeting the drinking water demand of people on the move in developing nations. Hygienic conditions can be questionable, and water quality screening scarce or non-existent. METHODS In order to ascertain the quality of the packaged water sold by street vendors in Bissau, the capital of the Western African country Guinea-Bissau, water bags were screened in 2011 and during the 2012 cholera outbreak for key physical, chemical and microbiological parameters. RESULTS Water used to fill the hand-filled hand-tied bags originated from communal tap water and melted ice. All samples (n=36) were microbiologically contaminated, although levels showed a pronounced variability (e.g. 7-493 372 cfu 250 ml(-1) for Escherichia coli). In 2012, the fecal contamination levels increased (p<0.05), and Vibrio cholerae was detected in all water bags obtained from the neighborhood where the outbreak started. CONCLUSION Findings showed that all packaged water samples were unfit for human consumption and during the 2012 cholera outbreak represented a potential vehicle for the spread of the disease. The design of measures to decrease the risk associated to the consumption of highly contaminated chilled water is clearly required.