Quantitative proteomic (iTRAQ) analysis of 1st trimester maternal plasma samples in pregnancies at risk for preeclampsia.

A current major obstacle is that no reliable screening markers exist to detect pregnancies at risk for preeclampsia. Quantitative proteomic analysis employing isobaric labelling (iTRAQ) has been suggested to be suitable for the detection of potential plasma biomarkers, a feature we recently verified in analysis of pregnancies with Down syndrome foetuses. We have now examined whether this approach could yield biomarkers to screen pregnancies at risk for preeclampsia. In our study, we used maternal plasma samples obtained at 12 weeks of gestation, six from women who subsequently developed preeclampsia and six with uncomplicated deliveries. In our analysis, we observed elevations in 10 proteins out of 64 proteins in the preeclampsia study group when compared to the healthy control group. These proteins included clusterin, fibrinogen, fibronectin, and angiotensinogen, increased levels of which are known to be associated with preeclampsia. An elevation in the immune-modulatory molecule, galectin 3 binding protein, was also noted. Our pilot study, therefore, indicates that quantitative proteomic iTRAQ analysis could be a useful tool for the detection of new preeclampsia screening markers.

Microarray screening for novel preeclampsia biomarker candidates.

INTRODUCTION: Our aim was to identify novel biomarker candidates for the near-term prediction of preeclampsia in a homogenous collective. In this study, we screened at the genome-wide level for gene expression in placental villous tissue from patients with severe preeclampsia in comparison to normal healthy pregnancies. MATERIAL AND METHODS: Total RNA was extracted from placental villous tissue from 9 preeclamptic patients and 7 normotensive controls after scheduled cesarean sections. After sample pooling, gene expression analysis was performed using six Affymetrix Human Gene 1.0 ST arrays, followed by quantitative RT-PCR and validation of selected markers in the serum of patients at the protein level. RESULTS: In total, 896 significantly differentially expressed genes were identified (p ≤ 0.05). After restricting these to molecules present in the circulation, 9 upregulated and 5 downregulated genes were selected. Four of them (ß-hCG, HTRA4, LHB1, all upregulated; and NOX4, downregulated) were validated by quantitative real-time RT-PCR. Finally, the maternal plasma protein levels of 2 of these genes (LHB and ß-hCG) were confirmed to be significantly different between preeclampsia cases and controls. DISCUSSION: We identified 14 potential new biomarker candidates for preeclampsia and validated 4 of them by quantitative RT-PCR and 2 of them with subsequent serum protein analyses. Further studies will assess the optimal marker combination for the imminent prediction of impending preeclampsia.

Determination of fetal chromosome aberrations from fetal DNA in maternal blood: has the challenge finally been met?

The analysis of cell-free fetal nucleic acids in maternal blood for prenatal diagnosis has been transformed by several recent profound technology developments. The most noteworthy of these are 'digital PCR' and 'next-generation sequencing' (NGS), which might finally deliver the long-sought goal of noninvasive detection of fetal aneuploidy. Recent data, however, indicate that NGS might even be able to offer a much more detailed appraisal of the fetal genome, including paternal and maternal inheritance of point mutations for mendelian disorders such as ß-thalassaemia. Although these developments are very exciting, in their current form they are still too complex and costly, and will need to be simplified considerably for their optimal translation to the clinic. In this regard, targeted NGS does appear to be a step in the right direction, although this should be seen in the context of ongoing progress with the isolation of fetal cells and with proteomic screening markers.

Detection of increased amounts of cell-free fetal DNA with short PCR amplicons.

AIM: A digital PCR approach has recently been suggested to detect greater amounts of cell-free fetal DNA in maternal plasma than conventional real-time quantitative PCR (qPCR). Because the digital qPCR approach uses shorter PCR amplicons than the real-time qPCR assay, we investigated whether a real-time qPCR assay appropriately modified for such short amplicons would improve the detection of cell-free fetal DNA. METHOD: We developed a novel universal-template (UT) real-time qPCR assay that was specific for the DYS14 sequence on Y chromosome and had a short amplicon size of 50 bp. We examined this "short" assay with 50 maternal plasma samples and compared the results with those for a conventional real-time qPCR assay of the same locus but with a longer amplicon (84 bp). RESULTS: Qualitatively, both assays detected male cell-free fetal DNA with the same specificity and detection capability. Quantitatively, however, the new UT real-time qPCR assay for shorter amplicons detected, on average, almost 1.6-fold more cell-free fetal DNA than the conventional real-time qPCR assay with longer amplicons. CONCLUSIONS: The use of short PCR amplicons improves the detection of cell-free fetal DNA. This feature may prove useful in attempts to detect cell-free fetal DNA under conditions in which the amount of template is low, such as in samples obtained early in pregnancy.

Quantitative proteomics analysis of maternal plasma in Down syndrome pregnancies using isobaric tagging reagent (iTRAQ).

Currently no specific biomarkers exist for the screening of pregnancies at risk for Down syndrome (DS). Since a quantitative proteomic approach with isobaric labelling (iTRAQ) has recently been suggested to be highly suitable for the discovery of novel plasma biomarkers, we have now used this method to examine for potential quantitative changes in the plasma proteome of the pregnancies bearing DS fetuses in comparison to normal healthy babies. In our study, we used plasma from six women with DS pregnancies and six with uncomplicated pregnancies care were taken to match cases and controls for gestational and maternal age, as these could be a confounder. In our quantitative proteomics analysis we were able to detect 178 proteins using iTRAQ labelling in conjunction with 4800 MALDI TOF/TOF. Amongst these we observed changes in betaHCG, a known screening marker for DS, indicating that our assay was functional. We found a number of elevated proteins Ig lambda chain C region, serum amyloid P-component, amyloid beta A4, and under expressed proteins like gamma-actin and titin in DS pregnancies. These proteins are also found in the sera of patients with Alzheimer disease, which share similar pathologies of DS. Our study therefore indicates that the iTRAQ labelling approach may be indeed useful for the detection of novel biomarkers.

Proteomic technologies for prenatal diagnostics: advances and challenges ahead.

Proteomics-based identification of biomarkers for fetal abnormalities in maternal plasma, amniotic fluid and reproductive fluids has made significant progress in the past 5 years. This is attributed mainly to advances in various technology platforms associated with mass spectrometry-based techniques. As these techniques are highly sensitive and require only small quantities of body fluids, it is hoped that they will pave the way for the development of effective noninvasive approaches, without subjecting the developing fetus to the same degree of harm as current invasive procedures (e.g., amniocentesis). It is possible that these developments will include same-day analyses, thereby permitting rapid intervention when necessary. To date, a host of body fluids, such as maternal serum and plasma, amniotic fluid, cervical fluid, vaginal fluid, urine, saliva or fetal material, such as placental trophoblast, fetal membranes or cord blood, have been used successfully in the quest to develop markers for a number of pregnancy-related pathologies. In the current review update we focus on the emergence of proteomics as a major platform technology in studying various types of fetal conditions and developing markers for pregnancy-related disorders, such fetal aneuploidy, preterm birth, preeclampsia, intra-amniotic infection and fetal stress. Should the development of these markers be successful, then it is to be envisaged that proteomic approaches will become standard of care for a number of disease conditions associated with feto-maternal health.

Arbuscular mycorrhizae fungi a potential eco-friendly tool for sustainable agriculture under changing climatic conditions/ in biotic and abiotic stress conditions

Los hongos micorrízicos arbusculares (HMA) son biotrofos obligados que viven en asociación simbiótica con las raíces de las plantas. Se encuentran entre los microorganismos del suelo más extendidos que proporcionan a la planta huésped nutrientes y protección contra patógenos. Las prácticas agrícolas modernas, como la labranza frecuente, el alto empleo de fertilización inorgánica pesticidas junto con condiciones climáticas cambiantes debido al calentamiento global, tienen enormes impactos en la colonización de los HMA, la interacción con las plantas y la productividad de los cultivos. Los HMA afectan positivamente la tolerancia de las plantas al estrés biótico y abiótico, a los ecosistemas severos y sus patógenos al alterar la estructura de las raíces, la exudación, la microflora de la rizosfera, la producción de antifúngicos y antibacterianos, y al competir con los patógenos por la absorción de nutrientes. Por lo tanto, juegan un papel importante en el crecimiento, la productividad y la calidad de las plantas. Además, el efecto de un fungicida varía según su modo de acción y las especies de HMA asociadas, lo que sugiere que estos hongos tienen un gran potencial como herramienta para la agricultura sostenible ecológica en el actual escenario de calentamiento global.

Arbuscular Mycorrhizal Fungi (AMF) are obligate biotrophs living in symbiotic association with roots of plants. They are among the most widespread soil microorganisms that provide the host plant with nutrients and pathogen protection. Modern farming practices like frequent tillage, high input inorganic fertilization and pesticide along changing climatic conditions due to global warming, have huge impacts on AMF colonization, interaction with plants and on crop productivity. AMF positively affect the plant tolerance to biotic and abiotic stresses, harsh ecosystems and plant pathogens by altering root structure, exudation, rhizosphere microflora, production of antifungals, antibacterials, and competing with pathogens for nutrient uptake. Thus, it plays a significant role in plant growth, productivity and quality. Further, the effect of a fungicide is varied depending on its mode of action and the associated AMF species, suggesting that these fungi have a strong potential as a tool for eco-friendly sustainable farming in the present scenario of global warming.

Noninvasive prenatal diagnosis of fetal aneuploidies and Mendelian disorders: new innovative strategies.

The application of recent technical developments, such as digital PCR or shot-gun sequencing, for the analysis of cell-free fetal DNA, have indicated that the long-sought goal of the noninvasive detection of Down syndrome may finally be attained. Although these methods are still cumbersome and not high throughput, they provide a paradigm shift in prenatal diagnosis, as they could effectively pronounce the end of invasive procedures, such as amniocentesis or chorionic villous sampling for the detection of such fetal anomalies. However, it remains to be determined how suitable these approaches are for the detection of more subtle fetal genetic alterations, such as those involved in hereditary Mendelian disorders (e.g., thalassemia and cystic fibrosis). New technical developments, such as microfluidics and reliable automated scanning microscopes, have indicated that it may be possible to efficiently retrieve and examine circulating fetal cells. As these contain the entire genomic complement of the fetus, future developments may include the noninvasive determination of the fetal karyotype.