Preimplantation genetic diagnosis for single gene disorders: experience with five single gene disorders

We report our experience of 14 preimplantation genetic diagnosis (PGD) cycles in eight couples carrying five different single gene disorders, during the last 18 months. Diagnoses were performed for myotonic dystrophy (DM), cystic fibrosis (CF) [ΔF508 and exon 4 (621+1 G>T)], fragile X and CF simultaneously, and two disorders for which PGD had not been previously attempted, namely neurofibromatosis type 2 (NF2) and Crouzon syndrome. Diagnoses for single gene disorders were carried out on ideally two blastomeres biopsied from Day 3 embryos. A highly polymorphic marker was included in each diagnosis to control against contamination. For the dominant disorders, where possible, linked polymorphisms provided an additional means of determining the genotype of the embryo hence reducing the risk of misdiagnosis due to allele dropout (ADO). Multiplex fluorescent polymerase chain reaction (F-PCR) was used in all cases, followed by fragment analysis and/or single-stranded conformation polymorphism (SSCP) for genotyping. Embryo transfer was performed in 13 cycles resulting in one biochemical pregnancy for CF, three normal deliveries (a twin and a singleton) and one early miscarriage for DM and a singleton for Crouzon syndrome. In each case the untransferred embryos were used to confirm the diagnoses performed on the biopsied cells. The results were concordant in all cases. The inclusion of a polymorphic marker allowed the detection of extraneous DNA contamination in two cells from one case. Knowing the genotype of the contaminating DNA allowed its origin to be traced. All five pregnancies were obtained from embryos in which two blastomeres were biopsied for the diagnosis. Our data demonstrate the successful strategy of using multiplex PCR to simultaneously amplify the mutation site and a polymorphic locus, fluorescent PCR technology to achieve greater sensitivity, and two-cell biopsy to increase the efficiency and success of diagnoses. Copyright © 2002 John Wiley & Sons, Ltd.     

Genetic and DNA-methylation changes induced by potassium dichromate in Brassica napus L

This study evaluated genetic and DNA methylation alteration induced by potassium dichromate in Brassica napus L. plants. Amplified fragment length polymorphism (AFLP) and selective amplification of polymorphic loci (SAMPL) tests revealed dose-related increases in sequence alterations in plantlets exposed to 10-200 mg/l potassium dichromate. Individual plantlets exposed to chromium under similar conditions showed different AFLP and SAMPL DNA profiles. These observations suggest random DNA mutation in response to potassium dichromate and argue against preferential sites for mutation. DNA methylation changes in response to chromium treatment were also evaluated. Two complementary approaches were applied: (i) immunolabelling, using a monoclonal antibody against 5-methylcytosine; and (ii) methylation-sensitive amplified polymorphism (MSAP). Immunolabelling showed cytosine-hypermethylation in the Brassica napus L. genome when plants were treated with potassium dichromate. MSAP analysis showed extensive methylation changes in CCGG-sequences, with the net result being genome-wide hypermethylation. These results showed a clear DNA alteration in plants as a response to chromium exposure and the effect was dose-dependent. DNA polymorphism detected by different markers supports the effectiveness of the use of these tools for the investigation of environmental toxicology and for evaluating the concentration of pollutants by DNA analysis in plants.     

Public Perception as a Barrier to Introducing Wood in Rivers for Restoration Purposes

Reintroduction of wood in rivers for restoration purposes is now recognized in a positive way by scientists. Nevertheless, the perception of wood in riverscapes is strongly affected by the socio-cultural environment. This cultural influence might explain why wood reintroduction is accepted and promoted in some regions of the world but not in others, despite the demonstrated ecological benefits. From an extensive student perception survey, we show that most of the groups from nine countries in the world considered riverscapes with wood to be less aesthetic, more dangerous, and needing more improvement than riverscapes without wood. By contrast, this way of thinking was not observed in Germany, Sweden, and Oregon (USA), where the first instances of wood reintroduction occurred.     

Fetal echocardiography using transvaginal and transabdominal probes during the first period of pregnancy: A comparative study

The authors compare the diagnostic possibilities of fetal transabdominal echocardiography versus transvaginal echocardiography. A larger diagnostic capacity is verified in different gestation ages with transvaginal probe between the 11th and 14th week of gestation. The results are emphasized by colour Doppler.     

Occurrence of Cryptosporidium oocysts in sewage effluents and correlation with microbial,chemical and physical water variables

Contaminated water are recognized as a potential source of Cryptosporidium oocysts. In Italy studies on the environmentalspread of the parasite are still scarce even if a high frequencyof serological responses could suggest high rates of endemic Cryptosporidium infections. In this investigation theoccurrence of the parasite in water samples from a municipalwastewater treatment plant was monitored with the aim to evaluatepossible correlation between oocysts and bacterial indicators,pathogens and physico-chemical parameters using a multivariateanalysis. Data collected showed high numbers of oocysts, with ahigh percentage of positive samples and a significant correlationwith pH, redox potential and total organic carbon. No correlationwas found between Cryptosporidium concentration and that of the other microrganisms. The results, through a probing statistical analysis, show that none of the selected microrganisms was a reliable predictor of the presence of theparasite.     

Role of the natural and anthropogenic radiative forcings on global warming: evidence from cointegration–VECM analysis

Over the last few years there has been much debate about the hypothesis that anthropogenic emissions of CO₂ and other greenhouse gases increase global temperature permanently. By using recent advances in time series econometrics, this paper tries to answer the question on how human activity affects Earth’s surface temperatures. Bearing in mind this goal, we estimated the long-run cointegration relations between global temperatures and changes in radiative forcings by a set of perturbing factors. We found that the temperature response to a doubling in radiative forcing of anthropogenic greenhouse gases is + 2.94 °C [95 % CI: + 1.91, + 3.97], in perfect accordance with prior research, and that the orthogonalized cumulated effect over a 100 year time period, in response to a unit increase of size of one standard deviation in greenhouse gas radiative forcing, is + 3.86 °C [95 % CI: + 0.03, + 6.54]. Conversely, the amplitude of solar irradiance variability is hardly sufficient to explain observed variations in the Earth’s climate. Our results show that the combined effect of stochastic trends attributable to anthropogenic radiative forcing variations are driving the Earth’s climate system toward an ongoing phase of global warming, and that such long-run movement is unlikely to be transient.     

Life style and genetic variation in teleosts: the case of pelagic (<Emphasis Type="Italic">Aphia minuta</Emphasis>) and benthic (<Emphasis Type="Italic">Gobius niger</Emphasis>) gobies (Perciformes: Gobiidae)

The pattern of genetic variability of two species of Mediterranean gobiids was compared, with reference to their different life history traits (Aphia minuta paedomorphic and pelagic; Gobius niger metamorphosed and benthic). The aim was to evaluate how different life histories can affect the genetic structure in these marine teleosts. The study was carried out on populations of both species sampled in the western Mediterranean and in the Adriatic Sea. Seven restriction endonucleases were used for the RFLP analysis of a mitochondrial DNA segment comprising the NADH dehydrogenase subunits 3, 4L and 4. The results highlighted two different patterns of genetic variation, a weak genetic structure in A. minuta and population subdivision in G. niger. These observations may be explained not only in terms of the different dispersal capabilities of these species, but also considering that A. minuta is an abbreviate iteroparous spawner while G. niger is a protracted iteroparous spawner. Because abbreviate iteroparity is a reproductive strategy selected in stable environments with high resource availability, Pliocene and Pleistocene climate oscillations may have represented factors that negatively influenced the reproductive success of A. minuta, producing demographic fluctuations and bottlenecks, as suggested by the mismatch distribution analysis. The weak genetic structure of A. minuta populations seems to be therefore due to a more recent re-colonization of the Mediterranean basin after a severe population decline, rather than to the high vagility of this pelagic goby.     

First steps in strategic environmental assessment (SEA) adoption for coastal planning in Sicily (South Italy)

The Industrial District (I.D.) of Messina was the first public authority to consider useful the application of the Strategic Environmental Assessment (SEA) procedure for the development of an Industrial Plan regarding a coastal area. At the moment the I.D. includes six areas, but the application of the SEA concerns only the area of Pace del Mela (along the north-eastern coast of Sicily), where a “District of the two Seas” will be created, based on the synergetic development between an industrialized niche (shipbuilding) and touristic development (port and hotels). The area in which this type of intervention will occur is not in use at the moment and it was not possible to collect environmental data. For this reason, in order to describe the environmental features of this territory, we took into consideration a few indicators which could be easily identified, necessary for a post evaluation which considers the Environmental Impact Assessment (EIA) of the industrial and suburban structures to be built.     

Specificity of metal tolerance and use of excluder metallophytes for the phytostabilization of metal polluted soils: the case of Silene paradoxa L.

This work was planned for providing useful information about the use of excluder metallophytes for phytostabilization of soils contaminated also with elements scarcely represented in the metalliferous environment of origin. To this aim, we investigated tolerance and accumulation of several different elements in a metallicolous and a nonmetallicolous population of Silene paradoxa through a hydroponic experiment. S. paradoxa metallicolous population showed increased tolerance not only to all the metals highly represented in the environment of origin but also to some of those scarcely present. Therefore, our results deposed in favor of the occurrence of the co-tolerance phenomenon in S. paradoxa for some elements. Metal accumulation was higher in the roots than in the shoots and lower in the metallicolous population than in the nonmetallicolous one, thus showing tolerance mechanisms to be based largely on metal exclusion. Anyway, the relative contribution of avoidance and of internal tolerance to metal tolerance was shown to be element-dependent. Present data revealed that metallicolous plants can effectively posses metal co-tolerances, which deserve to be investigated; as such, plants can actually represent a precious and exploitable tool also for the phytostabilization of soils contaminated with elements underrepresented in the environment of their origin.     

Human health risk assessment of lead from mining activities at semi-arid locations in the context of total lead exposure

Lead from historical mining and mineral processing activities may pose potential human health risks if materials with high concentrations of bioavailable lead minerals are released to the environment. Since the Joint Expert Committee on Food Additives of Food and Agriculture Organization/World Health Organization withdrew the Provisional Tolerable Weekly Intake of lead in 2011, an alternative method was required for lead exposure assessment. This study evaluated the potential lead hazard to young children (0–7 years) from a historical mining location at a semi-arid area using the U.S. EPA Integrated Exposure Uptake Biokinetic (IEUBK) Model, with selected site-specific input data. This study assessed lead exposure via the inhalation pathway for children living in a location affected by lead mining activities and with specific reference to semi-arid conditions and made comparison with the ingestion pathway by using the physiologically based extraction test for gastro-intestinal simulation. Sensitivity analysis for major IEUBK input parameters was conducted. Three groups of input parameters were classified according to the results of predicted blood concentrations. The modelled lead absorption attributed to the inhalation route was lower than 2 % (mean?±?SE, 0.9 %?±?0.1 %) of all lead intake routes and was demonstrated as a less significant exposure pathway to children’s blood, compared with ingestion. Whilst dermal exposure was negligible, diet and ingestion of soil and dust were the dominant parameters in terms of children’s blood lead prediction. The exposure assessment identified the changing role of dietary intake when house lead loadings varied. Recommendations were also made to conduct comprehensive site-specific human health risk assessment in future studies of lead exposure under a semi-arid climate.