Non-invasive prenatal diagnosis of single-gene disorders from maternal blood

Prenatal diagnosis (PD) is available for pregnancies at risk of monogenic disorders. However, PD requires the use of invasive obstetric techniques for fetal-sample collection and therefore, involves a risk of fetal loss. Circulating fetal DNA in the maternal bloodstream is being used to perform non-invasive prenatal diagnosis (NIPD). NIPD is a challenging discipline because of the biological features of the maternal blood sample. Maternal blood is an unequal mixture of small (and fragmented) amounts of fetal DNA within a wide background of maternal DNA. For this reason, initial NIPD studies have been based on the analysis of specific paternally inherited fetal tracts not present in the maternal genome so as to ensure their fetal origin. Following this strategy, different NIPD studies have been carried out, such as fetal-sex assessment for pregnancies at risk of X-linked disorders, RhD determination, and analysis of single-gene disorders with a paternal origin. The study of the paternal mutation can be used for fetal diagnosis of dominant disorders or to more accurately assess the risk of an affected child in case of recessive diseases. Huntington's disease, cystic fibrosis, or achondroplasia are some examples of diseases studied using NIPD. New technologies are opening NIPD to the analysis of maternally inherited fetal tracts. NIPD of trisomy 21 is the latest study derived from the use of next-generation sequencing (NGS).     

Molecular and structural analysis of C4-specific PEPC isoform from Pennisetum glaucum plays a role in stress adaptation

Phosphoenolpyruvate carboxylase is an ubiquitous cytosolic enzyme that catalyzes the ß-carboxylation of phosphoenolpyruvate (PEP) and is encoded by multigene family in plants. It plays an important role in carbon economy of plants by assimilating CO₂ into organic acids for subsequent C₄ or CAM photosynthesis or to perform several anaplerotic roles in non-photosynthetic tissues. In this study, a cDNA clone encoding for PEPC polypeptide possessing signature motifs characteristic to ZmC₄PEPC was isolated from Pennisetum glaucum (PgPEPC). Deduced amino acid sequence revealed its predicted secondary structure consisting of forty alpha helices and eight beta strands is well conserved among other PEPC homologs irrespective of variation in their primary amino acid sequences. Predicted PgPEPC quartenary structure is a tetramer consisting of a dimer of dimers, which is globally akin to maize PEPC crystal structure with respect to major chain folding wherein catalytically important amino acid residues of active site geometry are conserved. Recombinant PgPEPC protein expressed in E. coli and purified to homogeneity, possessed in vitro ß-carboxylation activity that is determined using a coupled reaction converting PEP into malate. Tetramer is the most active form, however, it exists in various oligomeric forms depending upon the protein concentration, pH, ionic strength of the media and presence of its substrate or effecters. Recombinant PgPEPC protein confers enhanced growth advantage to E. coli under harsh growth conditions in comparison to their respective controls; suggesting that PgPEPC plays a significant role in stress adaptation.     

Making the past serve the present

Botany has been successfully introduced to environmental design students, as a foundation for ecology and horticulture, in the form of extracts from an 18th century text which students are asked to criticise and update. This historical introduction demonstrates something of both the development ofthe subject and the students ' range of knowledge in it; and is a useful prompt for study. It encourages precision in the use of words, and emphasizes the provisional nature of scientific explanations. Suggestions for the use of this approach in other subjects and more advanced studies are made.     

DNA methylation based biomarkers: Practical considerations and applications

A biomarker is a molecular target analyzed in a qualitative or quantitative manner to detect and diagnose the presence of a disease, to predict the outcome and the response to a specific treatment allowing personalized tailoring of patient management. Biomarkers can belong to different types of biochemical molecules such as proteins, DNA, RNA or lipids, whereby protein biomarkers have been the most extensively studied and used, notably in blood-based protein quantification tests or immunohistochemistry. The rise of interest in epigenetic mechanisms has allowed the identification of a new type of biomarker, DNA methylation, which is of great potential for many applications. This stable and heritable covalent modification mostly affects cytosines in the context of a CpG dinucleotide in humans. It can be detected and quantified by a number of technologies including genome-wide screening methods as well as locus- or gene-specific high-resolution analysis in different types of samples such as frozen tissues and FFPE samples, but also in body fluids such as urine, plasma, and serum obtained through non-invasive procedures. In some cases, DNA methylation based biomarkers have proven to be more specific and sensitive than commonly used protein biomarkers, which could clearly justify their use in clinics. However, very few of them are at the moment used in clinics and even less commercial tests are currently available. The objective of this review is to discuss the advantages of DNA methylation as a biomarker, the practical considerations for their development, and their use in disease detection, prediction of outcome or treatment response, through multiple examples mainly focusing on cancer, but also to evoke their potential for complex diseases and prenatal diagnostics.     

Development of a simple and high-throughput method for detecting aflatoxins production in culture media

Aims: To develop a simple, high‐throughput and inexpensive procedure to detect and quantify aflatoxins into the culture media of growing mycelia. Methods and Results: Fungal conidia (Aspergillus flavus) were inoculated into the wells of a microplate containing 200 μl of different formulations of coconut‐derived liquid medium. Time‐dependent production of aflatoxins in the culture media was evaluated by a procedure relying on the UV‐induced fluorescence emission by the toxin, using a microplate reader. These data were validated by comparison with the outputs of a conventional HPLC‐based procedure. Determinations of aflatoxin concentration, according to the fluorimetric procedure, were performed either by withdrawing samples from the plates or by direct ‘in situ’ readings, the latter method reinforcing the high‐throughput feature of the procedure. Fluorescence enhancers (cyclodextrins) did not ameliorate the sensitivity of the procedure to low concentrations of the toxin into the medium. The efficacy of the procedure was also validated by testing the effect on toxin yield of adding an antioxidant agent (α‐lipoic acid) to the medium. Conclusions: We give evidence that our improved procedure is reliable and suitable to analyse aflatoxin accumulation time course in coconut‐derived culture medium. Significance and Impact of the Study: This study shows that our procedure may profitably be used to give insights into the mechanisms of regulation of mycotoxin production and, consequently, to implement different strategies for the containment of aflatoxin contamination of food and feed commodities.     

Human enhancement and sexual dimorphism

I argue that the existence of sexual dimorphism poses a profound challenge to those philosophers who wish to deny the moral significance of the idea of 'normal human capacities' in debates about the ethics of human enhancement. The biological sex of a child will make a much greater difference to their life prospects than many of the genetic variations that the philosophical and bioethical literature has previously been concerned with. It seems, then, that bioethicists should have something to say about the choice between a male and a female embryo. Either, 1) parents have reason to choose boys over girls; (2) parents have reason to choose girls over boys; or, (3) parents have neither reason to choose girls over boys nor reason to choose boys over girls. Embracing either of the first two alternatives has strongly counterintuitive--and arguably morally repugnant--consequences. To motivate the third option we must either make reference to the idea of 'normal human capacities' or argue that parents should consider the interests of society when thinking about what sort of children they should bring into the world - an implication that should be extremely controversial in debates about the 'new eugenics'. I conclude, then, that the idea of 'normal human capacities' is properly crucial to reasoning about the ethics of shaping future persons.     

Interleukin-28B polymorphisms on the SVR in the treatment of naïve chronic hepatitis C with pegylated interferon-α plus ribavirin: A meta-analysis

There are accurate but inconclusive data on the association between single nucleotide polymorphisms (SNPs) of interleukin (IL)-28B and sustained virological response (SVR) in chronic hepatitis C (CHC). This meta-analysis aimed to derive a more precise estimation of the effects of IL-28B SNPs locus (rs12979860 and rs8099917) on SVR in naïve CHC patients receiving pegylated interferon alpha (PEG-IFN-α) plus ribavirin. Literature search was conducted up to June, 2011, in PubMed, EMBASE and Cochrane Database of Systematic Reviews. A total of 36 studies involving 10912 cases with CHC receiving PEG-IFN-α plus ribavirin met the inclusion criteria. Analyses were stratified either by ethnicity or genotype of hepatitis C virus. In genotype 1/4 patients, rs12979860 CC was associated with high SVR in CHC patients (Caucasian: odds ratio (OR), 4.567; 95% confidence interval (CI), 3.826–5.452; Asian: OR, 4.033; 95%CI, 3.050–5.333; African American: OR, 4.297; 95%CI, 2.168–8.515; Hispanics: OR, 4.350; 95%CI, 2.817–6.717) but had no effect in genotype 2/3. In Caucasian (genotype 1/4: OR, 2.542; 95%CI, 2.108–3.065; genotype 2/3: OR, 1.363; 95%CI, 1.020–1.820) and Asian (genotype 1/4: OR, 5.214; 95%CI, 3.694–7.360; genotype 2/3: OR, 1.785; 95%CI, 1.095–2.910), rs8099917 TT was associated with high SVR in both genotype 1/4 and 2/3. Meta-regression showed that in Caucasians with CHC genotype 1/4, gender male might contribute to the effect of rs12979860 on SVR but advanced fibrosis might weaken this effect. Furthermore, in Asians with CHC genotype 1/4, high baseline viral load and advanced fibrosis might also undermine the effect of rs8099917 on SVR. This meta-analysis suggested that IL-28B rs12979860 CC and rs8099917 TT were associated with high SVR rate in CHC genotype 1/4. In CHC genotype 2/3, rs8099917 TT carriers also had higher SVR.