Neural Tube Defect-causing Teratogens Affect Tissue Mechanical Properties and Cytoskeletal Morphology in Axolotl Embryos

The teratogenic drugs cytochalasin B and valproic acid have been shown to alter F-actin polymerization, an effect that is crucial in forming microfilaments. Microfilaments form important cytoskeletal structures that maintain the structural integrity of the cell, cause cell motility and cell migration. Microfilament alterations are known to cause neural tube defects such as spina bifida and anencephaly (Walmod et al., 1999). We here aim to show that disruption of microfilaments by cytochalasin B and valproic acid affects the tensile properties of the tissue. Biomechanics is an interdisciplinary field that allows mechanical concepts to help us understand embryo development. This project used a novel tissue stretching device that measures the tensile properties of neural and epidermal tissue. The instrument used a pair of cantilevered wires to which the specimen was glued. This device stretched the mid-neural and -lateral tissue anterior-posterior (AP) and medio-lateral (ML) unidirectionally. The tensile properties of the tissue were determined by Resultant Young’s Modulus that depends on the true stress and true strain in the tissue sample. The experiment was conducted at a strain rate of 50%. Axolotl embryos were treated with 5ug/mL and 2.5ug/mL cytochalasin B and 5mM valproic acid at stage 13 (early neurula) for an hour, washed, and allowed to develop to stage 15 before it was used in the uniaxial tissue stretcher. Changes in the F-actin filaments were analysed by phalloidin staining and viewed under a confocal microscope. The tests show that disruption of microfilaments by cytochalasin B increases the stiffness of the dorsal-tissue by as much as 101% for CB-treated tissues stretched in the AP direction and 298% when stretched in the ML direction. VA-treated neural plate tissue showed a stiffness increase of 278% when stretched in the AP direction and 319%, when stretched in the ML direction. Changes in the F-actin filaments are quantified by phalloidin staining viewed with confocal microscopy. These findings indicate that direction-dependent mechanical forces in the tissue are contributing factors in closure of the neural tube in axolotl embryos

Non-invasive prenatal determination of fetal RhD genotyping from maternal plasma: a preliminary study in Pakistan

Objectives: To determine the accuracy of the non-invasive pre-natal real-time polymerase chain reaction based fetal RhD genotyping from maternal plasma. Study Design: Cross-sectional study. Place And Duration Of Study: Juma Health Sciences Research Laboratory, The Aga Khan University Hospital, Karachi, from July to December 2008. Methodology: Cell-free plasma DNA from 21 D-negative women with D-positive spouse between 20-39 weeks of gestation was tested for the presence of exon 5 region of RhD gene using real-time polymerase chain reaction. b-globin was employed as the house-keeping gene. Sensitivity and specificity of the real-time PCR-based non-invasive fetal RhD genotyping was obtained by calculating proportion of the D-positive fetuses that were D-positive at birth as well. Results: Of the 21 D-negative women 13 and 8 neonates were determined to be D-positive and D-negative, respectively, by serologic studies on cord blood samples at birth. RhD status was correctly determined in 17 of 21 cases. There were three false-positive and one false-negative results. The sensitivity and specificity of the assay was 92.3% (95% CI: 62.1, 99.6) and 62.5% (95% CI: 25.9, 89.8), respectively. The positive and negative predictive value of the assay was 80% (95% CI: 51.4, 94.7) and 83.3% (36.5, 99.1), respectively. Conclusion: These preliminary results demonstrate the feasibility of non-invasive pre-natal diagnosis of fetal RhD status of D-negative mothers in Pakistan

Frequent hypomethylation of PTGS2 gene promoter in human term placenta

Background: Gene expression profiles of several tumor suppressor genes are regulated by the methylation and demethylation of their promoters. Here, we aim to identify and quantify the methylation status of four tumor suppressor genes from placentas at term and compare them with the maternal white-blood-cells. Methods: In order to achieve this objective, DNA enriched from twenty placentas at term and maternal white blood cells was bisulfite-converted and amplified using quantitative real-time methyl-light polymerase chain reaction for the four-genes studied (RASSF1A, APC, RAR-beta, and PTGS2). Results: Among the four genes examined, RASSF1A, APC and RAR-beta promoter regions were hypermethylated in all the placental samples compared with maternal WBCs. Strikingly, PTGS2 was found to be hypomethylated in the placentas compared to the maternal cells. Conclusion: Since placental DNA represents fetal methylation profile and it is an established fact that there is certain amount of cell free circulating DNA in human plasma/serum, these data strongly suggest that hypermethylation of RASSF1A, APC and RAR-beta can be used as gender independent biomarkers to distinctly identify placental DNA in maternal blood. In addition, this is the first report which demonstrates hypomethylation of PTGS2 locus which may have important clinical implications e.g. placental abnormalities