Prenatal diagnosis for CF using High Resolution Melting Analysis and simultaneous haplotype analysis through QF-PCR

AbstractBackgroundHigh Resolution Melting (HRM) Analysis is a validated, robust, low-cost, high throughput CF screening method. Here, we report the development and retrospective evaluation of the diagnostic value of a novel multiplex HRM, genotyping and haplotyping method for CF prenatal diagnosis (generic HRM/haplotyping).Methods80 study samples from 20 carrier couples referred for PND (whole blood in EDTA and CVS or amniotic fluid) were genotyped retrospectively using the suggested protocol.ResultsAll DNA samples (variable sources, extraction methods and unknown concentrations) were successfully amplified by the 1st and 2nd round PCR. The Se, Sp, NPV and PPV for the generic HRM/haplotyping method are calculated at 100%.ConclusionsThis generic protocol for PND using HRM, facilitates the simultaneous analysis of DNA samples from various sources in a fast, robust and efficient way. It can be easily adapted and applied for any genetic condition

Genome stability of bovine in vivo-conceived cleavage-stage embryos is higher compared to in vitro-produced embryos.

STUDY QUESTION Is the rate and nature of chromosome instability (CIN) similar between bovine in vivo-derived and in vitro-cultured cleavage-stage embryos? SUMMARY ANSWER There is a major difference regarding chromosome stability of in vivo-derived and in vitro-cultured embryos, as CIN is significantly lower in in vivo-derived cleavage-stage embryos compared to in vitro-cultured embryos. WHAT IS KNOWN ALREADY CIN is common during in vitro embryogenesis and is associated with early embryonic loss in humans, but the stability of in vivo-conceived cleavage-stage embryos remains largely unknown. STUDY DESIGN, SIZE, DURATION Because human in vivo preimplantation embryos are not accessible, bovine (Bos taurus) embryos were used to study CIN in vivo. Five young, healthy, cycling Holstein Friesian heifers were used to analyze single blastomeres of in vivo embryos, in vitro embryos produced by ovum pick up with ovarian stimulation (OPU-IVF), and in vitro embryos produced from in vitro matured oocytes retrieved without ovarian stimulation (IVM-IVF). PARTICIPANTS/MATERIALS, SETTING, METHODS Single blastomeres were isolated from embryos, whole-genome amplified and hybridized on Illumina BovineHD BeadChip arrays together with the bulk DNA from the donor cows (mothers) and the bull (father). DNA was also obtained from the parents of the bull and from the parents of the cows (paternal and maternal grandparents, respectively). Subsequently, genome-wide haplotyping and copy-number profiling was applied to investigate the genomic architecture of 171 single bovine blastomeres of 16 in vivo, 13 OPU-IVF and 13 IVM-IVF embryos. MAIN RESULTS AND THE ROLE OF CHANCE The genomic stability of single blastomeres in both of the in vitro-cultured embryo cohorts was severely compromised (P < 0.0001), and the frequency of whole chromosome or segmental aberrations was higher in embryos produced in vitro than in embryos derived in vivo. Only 18.8% of in vivo-derived embryos contained at least one blastomere with chromosomal anomalies, compared to 69.2% of OPU-IVF embryos (P < 0.01) and 84.6% of IVM-IVF embryos (P < 0.001). LARGE SCALE DATA Genotyping data obtained in this study has been submitted to NCBI Gene Expression Omnibus (GEO; accession number GSE95358) LIMITATIONS REASONS FOR CAUTION There were two main limitations of the study. First, animal models may not always reflect the nature of human embryogenesis, although the use of an animal model to investigate CIN was unavoidable in our study. Second, a limited number of embryos were obtained, therefore more studies are warranted to corroborate the findings. WIDER IMPLICATIONS OF THE FINDINGS Although CIN is also present in in vivo-developed embryos, in vitro procedures exacerbate chromosomal abnormalities during early embryo development. Hence, the present study highlights that IVF treatment compromises embryo viability and should be applied with care. Additionally, our results encourage to refine and improve in vitro culture conditions and assisted reproduction technologies. STUDY FUNDING/COMPETING INTEREST(S) The study was funded by the Agency for Innovation by Science and Technology (IWT) (TBM-090878 to J.R.V. and T.V.), the Research Foundation Flanders (FWO; G.A093.11 N to T.V. and J.R.V. and G.0392.14 N to A.V.S. and J.R.V.), the European Union's FP7 Marie Curie Industry-Academia Partnerships and Pathways (IAPP, SARM, EU324509 to J.R.V., T.V., O.T, A.D., A.S. and A.K.) and Horizon 2020 innovation programme (WIDENLIFE, 692065 to J.R.V., O.T., T.V., A.K. and A.S.). M.Z.E., J.R.V. and T.V. are co-inventors on a patent application ZL913096-PCT/EP2014/068315-WO/2015/028576 (‘Haplotyping and copy-number typing using polymorphic variant allelic frequencies’), licensed to Cartagenia (Agilent Technologies

Genome-wide haplotyping embryos developing from 0PN and 1PN zygotes increases transferrable embryos in PGT-M.

STUDY QUESTION Can genome-wide haplotyping increase success following preimplantation genetic testing for a monogenic disorder (PGT-M) by including zygotes with absence of pronuclei (0PN) or the presence of only one pronucleus (1PN)? SUMMARY ANSWER Genome-wide haplotyping 0PNs and 1PNs increases the number of PGT-M cycles reaching embryo transfer (ET) by 81% and the live-birth rate by 75%. WHAT IS KNOWN ALREADY Although a significant subset of 0PN and 1PN zygotes can develop into balanced, diploid and developmentally competent embryos, they are usually discarded because parental diploidy detection is not part of the routine work-up of PGT-M. STUDY DESIGN, SIZE, DURATION This prospective cohort study evaluated the pronuclear number in 2229 zygotes from 2337 injected metaphase II (MII) oocytes in 268 cycles. PGT-M for 0PN and 1PN embryos developing into Day 5/6 blastocysts with adequate quality for vitrification was performed in 42 of the 268 cycles (15.7%). In these 42 cycles, we genome-wide haplotyped 216 good quality embryos corresponding to 49 0PNs, 15 1PNs and 152 2PNs. The reported outcomes include parental contribution to embryonic ploidy, embryonic aneuploidy, genetic diagnosis for the monogenic disorder, cycles reaching ETs, pregnancy and live birth rates (LBR) for unaffected offspring. PARTICIPANTS/MATERIALS, SETTING, METHODS Blastomere DNA was whole-genome amplified and hybridized on the Illumina Human CytoSNP12V2.1.1 BeadChip arrays. Subsequently, genome-wide haplotyping and copy-number profiling was applied to investigate the embryonic genome architecture. Bi-parental, unaffected embryos were transferred regardless of their initial zygotic PN score. MAIN RESULTS AND THE ROLE OF CHANCE A staggering 75.51% of 0PN and 42.86% of 1PN blastocysts are diploid bi-parental allowing accurate genetic diagnosis for the monogenic disorder. In total, 31% (13/42) of the PGT-M cycles reached ET or could repeat ET with an unaffected 0PN or 1PN embryo. The LBR per initiated cycle increased from 9.52 to 16.67%. LIMITATIONS, REASONS FOR CAUTION The clinical efficacy of the routine inclusion of 0PN and 1PN zygotes in PGT-M cycles should be confirmed in larger cohorts from multicenter studies. WIDER IMPLICATIONS OF THE FINDINGS Genome-wide haplotyping allows the inclusion of 0PN and 1PN embryos and subsequently increases the cycles reaching ET following PGT-M and potentially PGT for aneuploidy (PGT-A) and chromosomal structural rearrangements (PGT-SR). Establishing measures of clinical efficacy could lead to an update of the ESHRE guidelines which advise against the use of these zygotes. STUDY FUNDING/COMPETING INTEREST(S) SymBioSys (PFV/10/016 and C1/018 to J.R.V. and T.V.), the Horizon 2020 WIDENLIFE: 692065 to J.R.V., T.V., E.D., A.D. and M.Z.E. M.Z.E., T.V. and J.R.V. co-invented haplarithmisis (‘Haplotyping and copy-number typing using polymorphic variant allelic frequencies’), which has been licensed to Agilent Technologies. H.M. is fully supported by the (FWO) (ZKD1543-ASP/16). The authors have no competing interests to declare

How can zygotes segregate entire parental genomes into distinct blastomeres? The zygote metaphase revisited

Zygote cytokinesis produces two symmetric blastomeres, which contain one copy of each parental genome. Contrary to this dogma, we recently discovered that mammalian zygotes can spontaneously segregate entire parental genomes into different blastomeres and coined this novel form of genome segregation heterogoneic division. The molecular mechanisms underlying the emergence of blastomeres with different parental genomes during the first mitotic cycle remain to be elucidated. Here, we speculate on which parental genome asymmetries could provide a mechanistic foundation for these remarkable zygote divisions. In reviewing the field and considering our findings, we revisit the architecture of the first zygotic metaphase by invoking asymmetric interactions between the mitotic spindle and the parental kinetochores. We also speculate on how asynchronous parental cell cycles can be a source of heterogoneic zygote divisions through the formation of parental genome private spindles.status: publishe

Investigation of the cell-free DNA clinical significance in various pathological conditions

Circulating nucleic acids have been detected in the plasma of normal and diseased individuals since 1948 (cell free DNA, cell free RNA) and although the exact mechanisms that underlie the release of nucleic acid molecules in the peripheral circulation are not yet fully understood, cell death -through apoptosis and necrosis- is considered the major contributor of their rise in plasma according to findings reported mainly in cancer studies. Elevated cell free DNA levels may serve as an indicator of clinical severity in various pathological conditions such as autoimmune diseases, tissue injury and stroke and they have been correlated with the degree of tissue damage and the severity of post traumatic organ failure thus granting this new biomarker a potential prognostic value for risk stratification of trauma patients. The objective of this study was to investigate the clinical significance of cell-free DNA kinetics in patients with an acute myocardial infarction (AMI), in acute and chronic inflammation in exhaustive exercise and in juvenile rheumatoid arthritis. For the purposes of the present study, we developed a sensitive, reproducible and reliable quantitative real- time PCR (q RT-PCR) to measure cell-free DNA extracted from plasma. The method’s reproducibility and accuracy were assessed by calculating the interassay and intrassay coefficient of variation (CV%). Serial cell-free DNA level determinations were performed by quantitative Real-Time PCR in 47 AMI patients once daily during hospitalization (235 samples) and once in 100 healthy subjects (to establish the normal reference range). In addition, blood samples were obtained from 15 male athletes, participating in the ultradistance foot race of the 246 Km “Spartathlon”, in order to measure plasma interleukin-6 (IL-6), the serum inflammatory markers C-reactive protein (CRP) and serum amyloid A (SAA), and the tissue cell-free plasma DNA, before, at the end, and 48 h after the end of the race. Moreover, cell-free plasma DNA was measured along with C-reactive protein (CRP), creatine kinase (CK), and uric acid (UA) in 17 recreationally trained men participating in a 12-week resistance training regimen (8 resistance multi-joint exercises selected to stress the entire musculature). Finally, cell-free DNA was measured in children who have been diagnosed with juvenile arthritis upon diagnosis and after therapy administration. Statistical analysis of the measurements showed that cell-free DNA concentrations are significantly higher in patients throughout hospitalization compared to healthy subject levels (P<0.001). The median maximum cell-free DNA concentration was 3.5-fold higher (Mann Whitney P=0.0035) in 20/47patients with complicated post AMI course (1719.7, range 117.32-4996212.1 GenEq/ml plasma) compared with 27/47 patients without complications (492.9, range 56.43-4715.15 GenEq/ml plasma). Substantial differences exist between cell-free DNA concentrations measured on tpre (the day before the complication) and tc (the day the complication occurred) as well as tpost (the day after the complication) in group I whereby cell-free DNA rises significantly in tc and remains elevated in tpost (tpre vs. tc, 2.445 vs. 2.965, P=0.0171 and tpre vs. tpost 2.445 vs. 2.913, P=0.023). In addition, cell-free DNA could serve as a prognostic marker in heart failure (LVEF<40%). In the ‘Spartathlon’ paradigm of exhaustive exercise, IL-6, CRP, SAA and free plasma DNA levels markedly increased (by 8000-, 152- 108- and 10-fold, respectively) over the baseline at the end of the race. Cell-free DNA concentrations were elevated in AMI patients compared to healthy subjects, rise significantly when complications occur and have a potential clinical value in monitoring patient progress during hospitalization. Our findings suggest that the kinetics of cell-free DNA released in the peripheral circulation reflect the damage of the myocardium during the post AMI period. In the chronic inflammation model, cell-free DNA concentrations correlated with training volume whereas CRP, UA and CPK did not. Statistical analysis of the data obtained from the juvenile arthritis patients showed that significant differences exist between the patient and healthy control group. Cell-free DNA concentrations were elevated in AMI patients compared to healthy subjects, rise significantly when complications occur and have a potential clinical value in monitoring patient progress during hospitalization. This study also demonstrates that, after chronic excessive resistance exercise, plasma DNA concentrations increase in proportion to training load, suggesting that cell-free DNA may be a sensitive marker for overtraining-induced inflammation. In conclusion, cell-free DNA should be further studied to reveal its potential clinical use as a marker of the inflammatory processes, which play a role in the development of heart failure and death.Ο κυτταρικός θάνατος (απόπτωση/νέκρωση) οδηγεί στην απελευθέρωση νουκλεϊκών οξέων (ελεύθερο DNA/ελεύθερο RNA) στην περιφερική κυκλοφορία του ατόμου. Τα ελεύθερα νουκλεϊκά οξέα έχουν ανιχνευθεί στο πλάσμα υγιών καθώς και ασθενών και προέρχονται από κύτταρα που καταστρέφονται στα πλαίσια της ομοιόστασης και της νόσου, αντίστοιχα. Τα επίπεδα του ελεύθερου DNA στο πλάσμα έχουν συσχετισθεί με τη βαρύτητα και την πρόγνωση διαφόρων παθολογικών καταστάσεων και μπορεί να αποτελέσουν δείκτη παρακολούθησης της ανταπόκρισης των ασθενών στη φαρμακευτική αγωγή, όπως για παράδειγμα στους ασθενείς με καρκίνο. Στόχο της παρούσας ερευνητικής προσπάθειας αποτέλεσε η διερεύνηση της κλινικής αξίας των επιπέδων του ελεύθερου DNA στο οξύ έμφραγμα του μυοκαρδίου, στην οξεία και χρόνια φλεγμονή και στην πρωτοδιάγνωστη νεανική αρθρίτιδα. Για το σκοπό αυτό, προτυποποίηθηκε μια ευαίσθητη και αξιόπιστη ποσοτική PCR πραγματικού χρόνου στο σύστημα LightCycler (Roche) για τον ποσοτικό προσδιορισμό του ελεύθερου DNA, το οποίο απομονώθηκε από δείγματα πλάσματος που συλλέχθηκαν στα πλαίσια της παρούσας μελέτης. Αρχικά, προσδιορίστηκε η αξιοπιστία και η επαναληψιμότητα της μεθόδου (intra/inter- assay variation), ώστε να διαπιστωθεί η καταλληλότητά της για εφαρμογή στη συγκεκριμένη μελέτη. Στη συνέχεια, μετρήθηκε το ελεύθερο DNA στο πλάσμα 100 υγιών ατόμων προκειμένου να εξακριβωθεί το εύρος των φυσιολογικών τιμών. Έπειτα η μέθοδος εφαρμόστηκε για να μετρηθούν 235 δείγματα πλάσματος από 47 ασθενείς με οξύ έμφραγμα του μυοκαρδίου (ΟΕΜ) ως πρώτη εκδήλωση της στεφανιαίας νόσου καθώς και 15 δείγματα από αθλητές του «Σπάρταθλου» αγώνα δρόμου, 17 εθελοντών αθλούμενων και 39 παιδιών με πρωτοδιάγνωστη νεανική αρθρίτιδα. Στην ομάδα των καρδιοπαθών, πραγματοποιήθηκε διαδοχική συλλογή δειγμάτων περιφερικού αίματος κατά τη διάρκεια της νοσηλείας (1 δείγμα/ημέρα νοσηλείας), προκειμένου να απομονωθεί το ελεύθερο DNA. Παράλληλα, συλλέχθηκε ορός για τη μέτρηση της CPK και της cTnI (1 δείγμα/ημέρα νοσηλείας) και προσδιορίστηκε το κλάσμα εξώθησης αριστερής κοιλίας (LVEF) την 1η μετεμφραγματική ημέρα και κατά την έξοδο από τη νοσηλεία. Στην ομάδα των Σπαρταθλητών, έγινε λήψη ενός δείγματος περιφερικού αίματος πριν την έναρξη του αγώνα, κατά τον τερματισμό και 48 ώρες μετά τη λήξη του αγώνα, με στόχο την απομόνωση ελεύθερου DNA από το πλάσμα. Ταυτόχρονα, σε αυτή την ομάδα ατόμων, ελήφθησαν δείγματα ορού για τον προσδιορισμό των επιπέδων των περιφερικών παραγόντων φλεγμονής (IL-6, CRP και SAA). Οι εθελοντικά αθλούμενοι, υποβλήθηκαν σε ένα πρωτόκολλο πολυ-αρθρικών ασκήσεων αντιστάσεως μέσω προοδευτικά αυξανόμενου προπονητικού όγκου κατά τη διάρκεια τεσσάρων προπονητικών περιόδων (Τ1 έως Τ4). Η συλλογή των δειγμάτων περιφερικού αίματος, με στόχο την απομόνωση του ελεύθερου DNA, πραγματοποιήθηκε 96 ώρες μετά από την περίοδο ηρεμίας (Το) και μετά από κάθε προπονητική περίοδο. Ταυτόχρονα, σε αυτή την ομάδα ατόμων ελήφθησαν δείγματα ορού για τη μέτρηση της CRP, της CPK και του UA. Η στατιστική επεξεργασία των αποτελεσμάτων έδειξε πως το ελεύθερο DNA είναι σημαντικά αυξημένο στους ασθενείς με ΟΕΜ καθ’ όλη τη διάρκεια της νοσηλείας τους σε σχέση με τα επίπεδα που μετρήθηκαν στα υγιή άτομα (P<0.001). Επίσης, τα επίπεδα του υπό μελέτη δείκτη είναι σημαντικά υψηλότερα στους ασθενείς με επιπλοκές κατά τη νοσηλεία (1719.7, range 117.32-4996212.1 GenEq/ml plasma) σε σχέση με τα επίπεδα αυτών που είχαν ομαλή μετεμφραγματική πορεία έως την έξοδο από τη νοσηλεία (492.9,εύρος 56.43-4715.15 GenEq/ml plasma). Στην ομάδα των ασθενών με επιπλοκές, η συγκέντρωση του ελεύθερου DNA είναι υψηλότερη μεταξύ του ελεύθερου DNA της ημέρας μετά την επιπλοκή σε σχέση με τα επίπεδα που μετρήθηκαν την προηγούμενη και την επόμενη ημέρα (tpre vs. tc, 2.445 vs. 2.965, Ρ= 0.0171 and tpre vs. tpost 2.445 vs. 2.913, P=0.023). Επίσης, το ελεύθερο DNA φαίνεται πως έχει προγνωστική αξία στην περίπτωση της καρδιακής ανεπάρκειας (LVEF<40%). Στους αθλητές του «Σπάρταθλου» αγώνα, η στατιστική ανάλυση των μετρήσεων έδειξε πως το ελεύθερο DNA μπορεί να αποτελέσει αξιόπιστο δείκτη της οξείας φλεγμονής, καθώς μεταβάλλεται ανάλογα με τις πρωτεΐνες απόκρισης οξείας φάσης CRP και SAA. Στην περίπτωση των εθελοντικά αθλούμενων, στο μοντέλο της χρόνιας φλεγμονής, το ελεύθερο DNA φαίνεται να είναι πιο ευαίσθητος δείκτης της χρόνιας επιβάρυνσης καθώς μεταβάλλεται ανάλογα με τον προπονητικό όγκο. Αντιθέτως, οι μεταβολές της CRP και του UA δεν είναι ανάλογες του προοδευτικά αυξανόμενου προπονητικού όγκου. Στην ομάδα των παιδιών με πρωτοδιάγνωστη νεανική αρθρίτιδα διαπιστώθηκαν σημαντικές διαφορές στα επίπεδα του ελεύθερου DNA μεταξύ των ασθενών και των υγιών παιδιών. Ωστόσο, στα παιδιά που προσήλθαν για επανεκτίμηση (έπειτα από θεραπεία), οι συγκεντρώσεις του ελεύθερου DNA είναι χαμηλότερες από αυτές της πρώτης μέτρησης (θέση πρώτης διάγνωσης). Συνεπώς, η ανάλυση των μετρήσεων έδειξε πως το ελεύθερο DNA μπορεί να αποτελέσει αξιόπιστο δείκτη της παρακολούθησης της ενδονοσοκομειακής πορείας των ασθενών με ΟΕΜ καθώς επίσης να χρησιμοποιηθεί και σαν δείκτης παρακολούθησης της έκτασης της φλεγμονής που προκαλείται από υπερπροπόνηση στους αθλητές. Τα δεδομένα της παρούσας εργασίας αποτελούν ισχυρές ενδείξεις για την αξία του υπό μελέτη δείκτη. Απαιτείται όμως μελέτη με μεγαλύτερου αριθμού δειγμάτων, ώστε να αναδειχθεί περαιτέρω η κλινική χρήση του ελεύθερου DNA

Zygotes segregate entire parental genomes in distinct blastomere lineages causing cleavage-stage chimerism and mixoploidy

Dramatic genome dynamics, such as chromosome instability, contribute to the remarkable genomic heterogeneity among the blastomeres comprising a single embryo during human preimplantation development. This heterogeneity, when compatible with life, manifests as constitutional mosaicism, chimerism, and mixoploidy in live-born individuals. Chimerism and mixoploidy are defined by the presence of cell lineages with different parental genomes or different ploidy states in a single individual, respectively. Our knowledge of their mechanistic origin results from indirect observations, often when the cell lineages have been subject to rigorous selective pressure during development. Here, we applied haplarithmisis to infer the haplotypes and the copy number of parental genomes in 116 single blastomeres comprising entire preimplantation bovine embryos (n = 23) following in vitro fertilization. We not only demonstrate that chromosome instability is conserved between bovine and human cleavage embryos, but we also discovered that zygotes can spontaneously segregate entire parental genomes into different cell lineages during the first post-zygotic cleavage division. Parental genome segregation was not exclusively triggered by abnormal fertilizations leading to triploid zygotes, but also normally fertilized zygotes can spontaneously segregate entire parental genomes into different cell lineages during cleavage of the zygote. We coin the term "heterogoneic division" to indicate the events leading to noncanonical zygotic cytokinesis, segregating the parental genomes into distinct cell lineages. Persistence of those cell lines during development is a likely cause of chimerism and mixoploidy in mammals

A Study on the Clustering of Extra Virgin Olive Oils Extracted from Cultivars Growing in Four Ionian Islands (Greece) by Multivariate Analysis of Their Phenolic Profile, Antioxidant Activity and Genetic Markers

Background: The phenolic fraction of extra virgin olive oil (EVOO) has disease preventive and health-promoting properties which are supported by numerous studies. As such, EVOO is defined as a functional food. The aim of the present study was to characterize the phenolic profile of olive oil from cultivars farmed in the Ionian Islands (Zakynthos, Kefalonia, Lefkada, and Kerkyra) and to investigate the association of phenols to antioxidant activity, which is central to its functionality. Furthermore, the study investigates whether multivariate analyses on the concentration of individual biophenolic compounds and genetic population diversity could classify the olive oil samples based on their geographic origin. Methods: Phenols were determined in 103 samples from different Ionian Island tree populations by 1H nuclear magnetic resonance (NMR), and sample antioxidant activity was measured by their capacity to reduce the free radical 2,2-diphenyl-1-picrylhydrazyl) (DPPH). Genetic diversity was measured by estimating Nei&rsquo;s population genetic distance using 15 reproducible bands from random amplified polymorphic DNA (RAPD) genotyping. Results: Principal component analysis (PCA) of the secoiridoid concentrations clustered samples according to cultivar. Clustering based on genetic distances is not concordant with phenolic clustering. A cultivar effect was also demonstrated in the association between the concentration of individual phenols with DPPH reducing activity. Conclusions: Taken together, the study shows that the olive oil phenolic content defines &ldquo;cultivar-specific phenolic profiles&rdquo; and that environmental factors other than agronomic conditions contribute more to phenotype variance than genetics

RASSF1A in maternal plasma as a molecular marker of preeclampsia

Objectives This study aimed to quantitate cell free (cf) and cell free fetal (cff) DNA in maternal plasma by determining RASSF1A levels before and after enzyme digestion in women who subsequently developed preeclampsia (PE) and compare them with uncomplicated pregnancies. Methods Twenty-four samples from pregnant women who developed PE and 48 samples from women with uncomplicated pregnancies were analysed. Blood samples were obtained at 11-13 weeks. cfDNA was determined by quantifying RASSF1A using qRT-PCR. A second qRT-PCR was performed following methylation-sensitive enzyme digestion by BstUI, to quantitate hypermethylated RASSF1A sequences of fetal origin. ACTB gene was used as control to confirm complete enzyme digestion. Results cfDNA and cffDNA levels were significantly increased in women who developed PE as compared with uncomplicated pregnancies (median cfDNA: 9402 vs 2698, median cffDNA: 934.5 vs 62, respectively). Following operating characteristic curve analysis, cut-off values of 7486.q/mL for cfDNA and 512.q/mL for cffDNA were chosen, which provided a sensitivity of 75% and 100% and specificity of 98% and 100%, respectively, to identify women at risk for PE. Conclusions The study demonstrates potential use of cfDNA and cffDNA in maternal plasma as markers for the early prediction of women at risk for PE. (C) 2013 John Wiley &amp; Sons, Ltd

Pharmahuasca and DMT Rescue ROS Production and Differentially Expressed Genes Observed after Predator and Psychosocial Stress: Relevance to Human PTSD

Post-traumatic stress disorder (PTSD) is associated with cognitive deficits, oxidative stress, and inflammation. Animal models have recapitulated features of PTSD, but no comparative RNA sequencing analysis of differentially expressed genes (DEGs) in the brain between PTSD and animal models of traumatic stress has been carried out. We compared DEGs from the prefrontal cortex (PFC) of an established stress model to DEGs from the dorsolateral PFC (dlPFC) of humans. We observed a significant enrichment of rat DEGs in human PTSD and identified 20 overlapping DEGs, of which 17 (85%) are directionally concordant. ,-dimethyltryptamine (DMT) is a known indirect antioxidant, anti-inflammatory, and neuroprotective compound with antidepressant and plasticity-facilitating effects. We tested the capacity of DMT, the monoamine oxidase inhibitor (MAOI) harmaline, and pharmahuasca (DMT + harmaline) to reduce reactive oxygen species (ROS) production and inflammatory gene expression and to modulate neuroplasticity-related gene expression in the model. We administered DMT (2 mg/kg IP), harmaline (1.5 mg/kg IP), pharmahuasca, or vehicle every other day for 5 days, following a 30 day stress regiment. We measured ROS production in the PFC and hippocampus (HC) by electron paramagnetic resonance spectroscopy and sequenced total mRNA in the PFC. We also performed assays to measure the affinity and efficacy of DMT and harmaline at 5HTR compared to 5-HT. DMT and pharmahuasca reduced ROS production in the PFC and HC, while harmaline had mixed effects. Treatments normalized 9, 12, and 14 overlapping DEGs, and pathway analysis implicated that genes were involved in ROS production, inflammation, growth factor signaling, neurotransmission, and neuroplasticity

Zygotes segregate entire parental genomes in distinct blastomere lineages causing cleavage-stage chimerism and mixoploidy

Dramatic genome dynamics, such as chromosome instability, contribute to the remarkable genomic heterogeneity among the blastomeres comprising a single embryo during human preimplantation development. This heterogeneity, when compatible with life, manifests as constitutional mosaicism, chimerism, and mixoploidy in live-born individuals. Chimerism and mixoploidy are defined by the presence of cell lineages with different parental genomes or different ploidy states in a single individual, respectively. Our knowledge of their mechanistic origin results from indirect observations, often when the cell lineages have been subject to rigorous selective pressure during development. Here, we applied haplarithmisis to infer the haplotypes and the copy number of parental genomes in 116 single blastomeres comprising entire preimplantation bovine embryos (n = 23) following in vitro fertilization. We not only demonstrate that chromosome instability is conserved between bovine and human cleavage embryos, but we also discovered that zygotes can spontaneously segregate entire parental genomes into different cell lineages during the first post-zygotic cleavage division. Parental genome segregation was not exclusively triggered by abnormal fertilizations leading to triploid zygotes, but also normally fertilized zygotes can spontaneously segregate entire parental genomes into different cell lineages during cleavage of the zygote. We coin the term "heterogoneic division" to indicate the events leading to noncanonical zygotic cytokinesis, segregating the parental genomes into distinct cell lineages. Persistence of those cell lines during development is a likely cause of chimerism and mixoploidy in mammals.status: publishe