Background Heterozygous human mutations of NKX2-5 are highly penetrant and associated with varied congenital heart defects. The heterozygous knockout of murine Nkx2-5, in contrast, manifests less profound cardiac malformations, with low disease penetrance. We sought to study this apparent discrepancy between human and mouse genetics. Since missense mutations in the NKX2-5 homeodomain (DNA binding domain) are the most frequently reported type of human mutation, we replicated this genetic defect in a murine knock-in model. Methods and Results We generated a murine model in a 129/Sv genetic background by knocking-in an Nkx2-5 homeodomain missense mutation previously identified in humans. The mutation was located at homeodomain position 52Arg→Gly (R52G). All the heterozygous neonatal Nkx2-5+/R52G mice demonstrated a prominent trabecular layer in the ventricular wall, so called noncompaction, along with diverse cardiac anomalies, including atrioventricular septal defects, Ebstein’s malformation of the tricuspid valve, and perimembranous and/or muscular ventricular septal defects. In addition, P10 Nkx2-5+/R52G mice demonstrated atrial septal anomalies, with significant increase in the size of the inter-atrial communication and fossa ovalis, and decrease in the length of the flap valve compared to control Nkx2-5+/+ or Nkx2-5+/− mice. Conclusion The results of our study demonstrate that heterozygous missense mutation in the murine Nkx2-5 homeodomain (R52G) are highly penetrant, and result in pleiotropic cardiac effects. Thus, in contrast to heterozygous Nkx2-5 knockout mice, the effects of the heterozygous knock-in mimic findings in humans with heterozygous missense mutation in NKX2-5 homeodomain.

Herein we describe an association between activation of inflammatory pathways following transient hypoxia and the appearance of the multidrug resistant bacteria Staphylococcus simulans in the fetal brain. Reduction of maternal arterial oxygen tension by 50% over 30 min resulted in a subseiuent significant over-expression of genes associated with immune responses 24 h later in the fetal brain. The activated genes were consistent with stimulation by bacterial lipopolysaccharide; an influx of macrophages and appearance of live bacteria were found in these fetal brains. S. simulans was the predominant bacterial species in fetal brain after hypoxia, but was found in placenta of all animals. Strains of S. simulans from the placenta and fetal brain were equally highly resistant to multiple antibiotics including methicillin and had identical genome sequences. These results suggest that bacteria from the placenta invade the fetal brain after maternal hypoxia.

Transient hypoxia in pregnancy stimulates a physiological reflex response that redistributes blood flow and defends oxygen delivery to the fetal brain. We designed the present experiment to test the hypotheses that transient hypoxia produces damage of the cerebral cortex and that ketamine, an antagonist of NMDA receptors and a known anti‐inflammatory agent, reduces the damage. Late gestation, chronically catheterized fetal sheep were subjected to a 30‐min period of ventilatory hypoxia that decreased fetal PaO2 from 17 ± 1 to 10 ± 1 mmHg, or normoxia (PaO2 17 ± 1 mmHg), with or without pretreatment (10 min before hypoxia/normoxia) with ketamine (3 mg/kg, i.v.). One day (24 h) after hypoxia/normoxia, fetal cerebral cortex was removed and mRNA extracted for transcriptomics and systems biology analysis (n = 3–5 per group). Hypoxia stimulated a transcriptomic response consistent with a reduction in cellular metabolism and an increase in inflammation. Ketamine pretreatment reduced both of these responses. The inflammation response modeled with transcriptomic systems biology was validated by immunohistochemistry and showed increased abundance of microglia/macrophages after hypoxia in the cerebral cortical tissue that ketamine significantly reduced. We conclude that transient hypoxia produces inflammation of the fetal cerebral cortex and that ketamine, in a standard clinical dose, reduces the inflammation response.

Key pointsr The fetus responds to decreases in arterial partial pressure of oxygen by redirecting the blood flow mainly to the brain and the heart, at a cost to other peripheral organs like the kidneys.r Renal hypoxia and ischaemia stimulate inflammatory and apoptotic responses. r Ketamine, an NMDA receptor antagonist, is able to reduce renal immune and inflammatory gene expressions stimulated by hypoxia.r Ketamine may have therapeutic potential for protection against ischaemic renal damage in fetuses subjected to acute hypoxia.Abstract Acute fetal hypoxia is a form of fetal stress that stimulates renal vasoconstriction and ischaemia as a consequence of the physiological redistribution of combined ventricular output. Because of the potential ischaemia-reperfusion injury to the kidney, we hypothesized that it would respond to hypoxia with an increase in the expression of inflammatory genes, and that ketamine (an N-methyl-D-aspartate receptor antagonist) would reduce or block this response. Hypoxia was induced for 30 min in chronically catheterized fetal sheep (125 ± 3 days), with or without ketamine (3 mg kg −1 ) administered intravenously to the fetus 10 min prior to hypoxia. Gene expression in fetal kidney cortex collected 24 h after the onset of hypoxia was analysed using ovine Agilent 15.5k array and validated with qPCR and immunohistochemistry in four groups of ewes: normoxic control, normoxia + ketamine, hypoxic control and hypoxia + ketamine (n = 3-4 per group). Significant differences in gene expression between groups were determined with t-statistics using the limma package for R (P ࣘ 0.05). Enriched biological processes for the 427 upregulated genes were immune and inflammatory responses and for the 946 downregulated genes were metabolic processes. Ketamine countered the effects of hypoxia on upregulated immune/inflammatory responses as well as the downregulated metabolic responses. We conclude that our transcriptomics modelling predicts that hypoxia activates inflammatory pathways and reduces metabolism in the fetal kidney cortex, and ketamine blocks or ameliorates this response. The results suggest that ketamine may have therapeutic potential for protection from ischaemic renal damage.

Our objectives were to evaluate the effects of 12 exogenous fibrolytic enzyme products (EFE) on ruminal in vitro neutral detergent fiber digestibility (NDFD) and preingestive hydrolysis of a 4-wk regrowth of bermudagrass haylage (BH), to examine the accuracy of predicting NDFD with EFE activity measures, and to examine the protein composition of the most and least effective EFE at increasing NDFD. In experiment 1, effects of 12 EFE on NDFD of BH were tested. Enzymes were applied in quadruplicate to culture tubes containing ground BH. The suspension was incubated for 24 h at 25 °C before addition of rumen fluid media and further incubation for 24 h at 39 °C. The experiment was repeated twice. In addition, regression relationships between EFE activity measures and NDFD were examined. Compared with the values for the control, 9 EFE-treated substrates had greater NDFD (37.8 to 40.4 vs. 35.6%), 6 had greater total VFA concentration (59.1 to 61.2 vs. 55.4 mM), and 4 had lower acetate-to-propionate ratios (3.03 to 3.16 vs. 3.24). In experiment 2, EFE effects on preingestive fiber hydrolysis were evaluated by incubating enzyme-treated and untreated bermudagrass suspensions in quadruplicate for 24 h at 25 °C and examining fiber hydrolysis measures. Compared with values for the control, 3 EFE reduced neutral detergent fiber concentration (62.8 to 63.7 vs. 67.3%), 10 increased release of water-soluble carbohydrates (26.8 to 58.5 vs. 22.8 mg/g), and 8 increased release of ferulic acid (210 to 391 vs. 198 μg/g). Regression analyses revealed that enzyme activities accurately [coefficient of determination (R(2)) = 0.98] predicted preingestive hydrolysis measures (water-soluble carbohydrates, ferulic acid), moderately (R(2) = 0.47) predicted neutral detergent fiber hydrolysis, but poorly (R(2) ≤ 0.1) predicted dry matter and NDFD. In experiment 3, proteomic tools were used to examine the protein composition of the most and least effective EFE at improving NDFD. Relative to the least effective, the most effective EFE at increasing NDFD contained 10 times more endoglucanase III, 17 times more acetylxylan esterase with a cellulose-binding domain 1, 33 times more xylanase III, 25 times more β-xylosidase, and 7.7 times more polysaccharide monooxygenase with cellulose-binding domain 1 and 3 times more swollenin. The most effective EFE had a much greater quantity of fibrolytic enzymes and key proteins necessary for hemicellulose and lignocellulase deconstruction. This study identified several EFE that increased the NDFD and in vitro fermentation of 4-wk BH and revealed why some EFE are more effective than others.

Our objectives were to evaluate the effects of the dose rates of 5 Trichoderma reesei and Aspergillus oryzae exogenous fibrolytic enzymes (EFE; 1A, 2A, 11C, 13D, and 15D) on in vitro digestibility, fermentation characteristics, and preingestive hydrolysis of bermudagrass haylage and to identify the optimal dose of each EFE for subsequent in vitro and in vivo studies. In experiment 1, EFE were diluted in citrate-phosphate buffer (pH 6) and applied in quadruplicate in each of 2 runs at 0× (control), 0.5×, 1×, 2×, and 3×; where 1× was the respective manufacturer-recommended dose (2.25, 2.25, 10, 15, and 15g of EFE/kg of dry matter). The suspension was incubated for 24h at 25°C and for a further 24h at 39°C after the addition of ruminal fluid. In experiment 2, a similar approach to that in experiment 1 was used to evaluate simulated preingestive effects, except that sodium azide (0.02% wt/vol) was added to the EFE solution. The suspension was incubated for 24h at 25°C and then 15mL of water was added before filtration to extract water-soluble compounds. For both experiments, data for each enzyme were analyzed separately as a completely randomized block design with a model that included effects of EFE dose, run, and their interaction. In experiment 1, increasing the EFE dose rate nonlinearly increased the DM digestibility of 1A, 2A, 11C, and 13D and the neutral detergent fiber digestibility (NDFD) of 1A, 2A, 11C, and 13D. Optimal doses of 1A, 2A, 11C, 13D, and 15D, as indicated by the greatest increases in NDFD at the lowest dose tested, were 2×, 2×, 1×, 0.5×, and 0.5×, respectively. Increasing the dose rate of 2A, 11C, and 13D nonlinearly increased concentrations of total volatile fatty acids and propionate (mM), decreased their acetate-to-propionate ratios and linearly decreased those of samples treated with 1A and 15D. In experiment 2, increasing the dose rate of each EFE nonlinearly decreased concentrations of netural detergent fiber; also, increasing the dose rate of 1A, 2A, 11C, and 1D nonlinearly increased concentrations of water-soluble carbohydrates and free ferulic acid (μg/g). Application of increasing doses of the EFE increased NDF hydrolysis, NDFD, and ruminal fluid fermentation of bermudagrass haylage, but the optimal dose varied with the EFE.

Wood CE, Rabaglino MB, Richards E, Denslow N, Zarate MA, Chang EI, Keller-Wood M. Transcriptomics of the fetal hypothalamic response to brachiocephalic occlusion and estradiol treatment. Physiol Genomics 46: 523-532, 2014. First published May 13, 2014 doi:10.1152 doi:10. /physiolgenomics.00186.2013 is a wellknown modulator of fetal neuroendocrine activity and has been proposed as a critical endocrine signal readying the fetus for birth and postnatal life. To investigate the modulatory role of E2 on fetal stress responsiveness and the response of the fetal brain to asphyxic stress, we subjected chronically catheterized fetal sheep to a transient (10 min) brachiocephalic artery occlusion (BCO) or sham occlusion. Half of the fetuses received subcutaneous pellets that increased plasma E2 concentrations within the physiological range. Hypothalamic mRNA was analyzed using the Agilent 8x15k ovine array (019921), processed and annotated as previously reported by our laboratory. Analysis of the data by ANOVA revealed that E2 differentially regulated (DR) 561 genes, and BCO DR 894 genes compared with control and E2ϩBCO DR 1,153 genes compared with BCO alone (all P Ͻ 0.05). E2 upregulated epigenetic pathways and downregulated local steroid biosynthesis but did not significantly involve genes known to directly respond to the estrogen receptor. Brachiocephalic occlusion upregulated kinase pathways as well as genes associated with lymphocyte infiltration into the brain and downregulated neuropeptide synthesis. E2 upregulated immune-and apoptosis-related pathways after BCO and reduced kinase and epigenetic pathway responses to the BCO. Responses to BCO are different from responses to hypoxic hypoxia suggesting that mechanisms of responses to these two forms of brain hypoxia are distinct. We conclude that cerebral ischemia caused by BCO might stimulate lymphocyte infiltration into the brain and that this response appears to be modified by estradiol. cortisol; fetal heart; mitochondria; metabolism; late gestation DURING THE TRANSITION FROM intra-to extrauterine life, birth asphyxia is a common cause of cerebral ischemia (29), commonly resulting in the need for resuscitation of the neonate. Disturbances in cerebral blood flow can result from various manipulations in the neonatal intensive care unit, including hypocarbia secondary to mechanical ventilation (18), continuous positive airway pressure (16, 42), and extracorporeal membrane oxygenation (7). Reduction of cerebral blood flow in the neonate compromises oxygen delivery or, if severe, produces ischemia/reperfusion injury (10) and results in morbidity, including intraventricular hemorrhage (6) and long-term cognitive deficit (14).We have investigated the cardiovascular and endocrine responses to cerebral hypoperfusion in late-gestation chronically catheterized fetal sheep (31,38,39), in this animal model of late fetal development. This model allows us to investigate responses to stress in utero and better understand the native homeostatic mechanisms that promote survival af...

The physiological response to hypoxia in the fetus has been extensively studied with regard to redistribution of fetal combined ventricular output and sparing of oxygen delivery to fetal brain and heart. Previously, we have shown that the fetal brain is capable of mounting changes in gene expression that are consistent with tissue inflammation. The present study was designed to use transcriptomics and systems biology modeling to test the hypothesis that ketamine reduces or prevents the upregulation of inflammation-related pathways in hypothalamus and hippocampus after transient hypoxic hypoxia. Chronically catheterized fetal sheep (122 ± 5 days gestation) were subjected to 30 min hypoxia (relative reduction in PaO2∼50%) caused by infusion of nitrogen into the inspired gas of the pregnant ewe. RNA was isolated from fetal hypothalamus and hippocampus collected 24 h after hypoxia, and was analyzed for gene expression using the Agilent 15.5 k ovine microarray. Ketamine, injected 10 min prior to hypoxia, reduced the cerebral immune response activation to the hypoxia in both brain regions. Genes both upregulated by hypoxia and downregulated by ketamine after hypoxia were significantly associated with gene ontology terms and KEGG pathways that are, themselves, associated with the tissue response to exposure to bacteria. We conclude that the results are consistent with interruption of the cellular response to bacteria by ketamine.