mRNAs encoding aquaporins are present during murine preimplantation development.

The present study was conducted to investigate the mechanisms underlying fluid movement across the trophectoderm during blastocyst formation by determining whether aquaporins (AQPs) are expressed during early mammalian development. AQPs belong to a family of major intrinsic membrane proteins and function as molecular water channels that allow water to flow rapidly across plasma membranes in the direction of osmotic gradients. Ten different AQPs have been identified to date. Murine preimplantation stage embryos were flushed from the oviducts and uteri of superovulated CD1 mice. Reverse transcription-polymerase chain reaction (RT-PCR) methods employing primer sets designed to amplify conserved sequences of AQPs (1-9) were applied to murine embryo cDNA samples. PCR reactions were conducted for up to 40 cycles involving denaturation of DNA hybrids at 95 degrees C, primer annealing at 52-60 degrees C and extension at 72 degrees C. PCR products were separated on 2% agarose gels and were stained with ethidium bromide. AQP PCR product identity was confirmed by sequence analysis. mRNAs encoding AQPs 1, 3, 5, 6, 7, and 9 were detected in murine embryos from the one-cell stage up to the blastocyst stage. AQP 8 mRNAs were not detected in early cleavage stages but were present in morula and blastocyst stage embryos. The results were confirmed in experimental replicates applied to separate embryo pools of each embryo stage. These results demonstrate that transcripts encoding seven AQP gene products are detectable during murine preimplantation development. These findings predict that AQPs may function as conduits for trophectoderm fluid transport during blastocyst formation

Effect of haemodilution, acidosis, and hypothermia on the activity of recombinant factor VIIa (NovoSeven®)

Background. A range of plasma volume expanders is used clinically, often in settings where haemostasis may already be impaired. The haemostatic agent, recombinant activated factor VII (rFVIIa, NovoSevenw), may be used to improve haemostasis but potential interactions with different volume expanders are poorly understood. Methods. Clot formation was measured by thromboelastography (TEG) using blood from healthy volunteers. In vitro effects of rFVIIa with haemodilution, acidosis, and hypothermia were examined. Conditions were induced by dilution with NaCl (0.9%), lactated Ringer’s solution, albumin 5%, or hydroxyethyl starch (HES) solutions [MW (molecular weight) 130–670 kDa]; by adjusting pH to 6.8 with 1 M HEPES (N-2-hydroxyethylpiperazine-N0-2-ethanesulphonic acid) buffer; or by reducing temperature to 328C. We also studied the effect of low vs high MW HES (MW 200 vs 600 kDa) and rFVIIa on in vivo bleeding time (BT) in rabbits. Results. Haemodilution progressively altered TEG parameters. rFVIIa improved TEG par-ameters in the presence of acidosis, hypothermia or 20 % haemodilution (P,0.05). At 40 % hae-modilution, the rFVIIa effect was diminished particularly with high MW HES. In vivo, rFVIIa shortened the BT (P,0.05) with low but not high MW HES. Conclusions. Efficacy of rFVIIa was affected by the degree of haemodilution and type of volume expander, but not by acidosis or hypothermia

Maternal depression during pregnancy and cord blood DNA methylation: findings from the Avon Longitudinal Study of Parents and Children

Up to 13% of women may experience symptoms of depression during pregnancy or in the postpartum period. Depression during pregnancy has been associated with an increased risk of adverse neurodevelopmental outcomes in the child and epigenetic mechanisms could be one of the biological pathways to explain this association. In 844 mother–child pairs from the Avon Longitudinal Study of Parents and Children, we carried out an epigenome-wide association study (EWAS) to investigate associations between prospectively collected data on maternal depression ascertained by the Edinburgh Postnatal Depression Scale in pregnancy and DNA methylation in the cord blood of newborn offspring. In individual site analysis, we identified two CpG sites associated with maternal depression in the middle part of pregnancy. In our regional analysis, we identified 39 differentially methylated regions (DMRs). Seven DMRs were associated with depression at any time point during pregnancy, 7 associated with depression in mid-pregnancy, 23 were associated with depression in late pregnancy, and 2 DMRs were associated with depression throughout pregnancy. Several of these map to genes associated with psychiatric disease and brain development. We attempted replication in The Generation R Study and could not replicate our results. Although our findings in ALSPAC suggest that maternal depression could be associated with cord blood DNA methylation the results should be viewed as preliminary and hypothesis generating until further replicated in a larger sample

Whole genome analysis reveals aneuploidies in early pregnancy loss in the horse

The first 8 weeks of pregnancy is a critical time, with the majority of pregnancy losses occurring during this period. Abnormal chromosome number (aneuploidy) is a common finding in human miscarriage, yet is rarely reported in domestic animals. Equine early pregnancy loss (EPL) has no diagnosis in over 80% of cases. The aim of this study was to characterise aneuploidies associated with equine EPL. Genomic DNA from clinical cases of spontaneous miscarriage (EPLs; 14–65 days of gestation) and healthy control placentae (various gestational ages) were assessed using a high density genotyping array. Aneuploidy was detected in 12/55 EPLs (21.8%), and 0/15 healthy control placentae. Whole genome sequencing (30X) and digital droplet PCR (ddPCR) validated results. The majority of these aneuploidies have never been reported in live born equines, supporting their embryonic/fetal lethality. Aneuploidies were detected in both placental and fetal compartments. Rodents are currently used to study how maternal ageing impacts aneuploidy risk, however the differences in reproductive biology is a limitation of this model. We present the first evidence of aneuploidy in naturally occurring equine EPLs at a similar rate to human miscarriage. We therefore suggest the horse as an alternative to rodent models to study mechanisms resulting in aneuploid pregnancies

Promising System for Selecting Healthy In Vitro–Fertilized Embryos in Cattle

Conventionally, in vitro–fertilized (IVF) bovine embryos are morphologically evaluated at the time of embryo transfer to select those that are likely to establish a pregnancy. This method is, however, subjective and results in unreliable selection. Here we describe a novel selection system for IVF bovine blastocysts for transfer that traces the development of individual embryos with time-lapse cinematography in our developed microwell culture dish and analyzes embryonic metabolism. The system can noninvasively identify prognostic factors that reflect not only blastocyst qualities detected with histological, cytogenetic, and molecular analysis but also viability after transfer. By assessing a combination of identified prognostic factors—(i) timing of the first cleavage; (ii) number of blastomeres at the end of the first cleavage; (iii) presence or absence of multiple fragments at the end of the first cleavage; (iv) number of blastomeres at the onset of lag-phase, which results in temporary developmental arrest during the fourth or fifth cell cycle; and (v) oxygen consumption at the blastocyst stage—pregnancy success could be accurately predicted (78.9%). The conventional method or individual prognostic factors could not accurately predict pregnancy. No newborn calves showed neonatal overgrowth or death. Our results demonstrate that these five predictors and our system could provide objective and reliable selection of healthy IVF bovine embryos

Sub-Nucleocapsid Nanoparticles: A Nasal Vaccine against Respiratory Syncytial Virus

Background: Bronchiolitis caused by the respiratory syncytial virus (RSV) in infants less than two years old is a growing public health concern worldwide, and there is currently no safe and effective vaccine. A major component of RSV nucleocapsid, the nucleoprotein (N), has been so far poorly explored as a potential vaccine antigen, even though it is a target of protective anti-viral T cell responses and is remarkably conserved between human RSV A and B serotypes. We recently reported a method to produce recombinant N assembling in homogenous rings composed of 10–11 N subunits enclosing a bacterial RNA. These nanoparticles were named sub-nucleocapsid ring structure (N SRS). Methodology and Principal Findings: The vaccine potential of N SRS was evaluated in a well-characterized and widely acknowledged mouse model of RSV infection. BALB/c adult mice were immunized intranasally with N SRS adjuvanted with the detoxified E. coli enterotoxin LT(R192G). Upon RSV challenge, vaccinated mice were largely protected against virus replication in the lungs, with a mild inflammatory lymphocytic and neutrophilic reaction in their airways. Mucosal immunization with N SRS elicited strong local and systemic immunity characterized by high titers of IgG1, IgG2a and IgA anti-N antibodies, antigen-specific CD8+ T cells and IFN-c-producing CD4+ T cells. Conclusions/Significance: This is the first report of using nanoparticles formed by the recombinant nucleocapsid protein as an efficient and safe intra-nasal vaccine against RSV

Measurement of the viscoelastic properties of blood plasma clot formation in response to tissue factor concentration-dependent activation

© 2016, The Author(s). The coagulation of blood plasma in response to activation with a range of tissue factor (TF) concentrations was studied with a quartz crystal microbalance (QCM), where frequency and half width at half maximum (bandwidth) values measured from the conductance spectrum near resonant frequency were used. Continuous measurement of bandwidth along with the frequency allows for an understanding of the dissipative nature of the forming viscoelastic clot, thus providing information on the complex kinetics of the viscoelastic changes occurring during the clot formation process. Using a mathematical model, these changes in frequency and bandwidth have been used to derive novel QCM parameters of effective elasticity, effective mass density and rigidity factor of the viscoelastic layer. The responses of QCM were compared with those from thromboelastography (TEG) under identical conditions. It was demonstrated that the nature of the clot formed, as determined from the QCM parameters, was highly dependent on the rate of clot formation resulting from the TF concentration used for activation. These parameters could also be related to physical clot characteristics such as fibrin fibre diameter and fibre density, as determined by scanning electron microscopic image analysis. The maximum amplitude (MA) as measured by TEG, which purports to relate to clot strength, was unable to detect these differences