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1.  Correlation between initial neonatal and early childhood outcomes following preterm birth 
Neonatal diagnoses are often used as surrogate endpoints for longer-term outcomes. We sought to characterize the correlation between neonatal diagnoses and early childhood neurodevelopment.
We conducted secondary analysis of a multicenter randomized controlled trial of antenatal magnesium sulfate vs placebo administered to women at imminent risk for delivery <32.0 weeks to prevent death and cerebral palsy in their offspring. Singletons and twins delivering 23.0–33.9 weeks who survived to hospital discharge and had 2-year-old outcome data were included. Those surviving to age 2 years were assessed by trained physicians and the Bayley II Scales of Infant Development Mental Development and Psychomotor Development Indices. Neonatal diagnoses at the time of each baby’s initial hospital discharge were examined singly and in combination to determine those most predictive of childhood neurodevelopmental impairment, defined as a childhood diagnosis of moderate/severe cerebral palsy and/or Bayley scores >2 SD below the mean. Data were analyzed by multiple regression models and area under receiver operating characteristic curves.
A total of 1771 children met criteria. Children were delivered at a mean of 29.4 weeks’ gestation. In all, 459 (25.9%) had neuro-developmental impairment. In models controlling for gestational age at delivery, maternal education, maternal race, tobacco/alcohol/drug use during pregnancy, randomization to magnesium, fetal sex, and chorioamnionitis, individual neonatal morbidities were moderately predictive of childhood neurodevelopmental impairment (best model area under receiver operating characteristic curve, 0.68; 95% confidence interval, 0.65–0.71). Combinations of 2, 3, and 4 morbidities did not improve the prediction of neurodevelopmental impairment.
Approximately 1 in 4 previously preterm children had neurodevelopmental impairment at age 2 years. Prediction of childhood outcomes from neonatal diagnoses remains imperfect.
PMCID: PMC4270007  PMID: 24793722
neonatal outcomes; neurodevelopment; prematurity
2.  High-Throughput Genome Editing and Phenotyping Facilitated by High Resolution Melting Curve Analysis 
PLoS ONE  2014;9(12):e114632.
With the goal to generate and characterize the phenotypes of null alleles in all genes within an organism and the recent advances in custom nucleases, genome editing limitations have moved from mutation generation to mutation detection. We previously demonstrated that High Resolution Melting (HRM) analysis is a rapid and efficient means of genotyping known zebrafish mutants. Here we establish optimized conditions for HRM based detection of novel mutant alleles. Using these conditions, we demonstrate that HRM is highly efficient at mutation detection across multiple genome editing platforms (ZFNs, TALENs, and CRISPRs); we observed nuclease generated HRM positive targeting in 1 of 6 (16%) open pool derived ZFNs, 14 of 23 (60%) TALENs, and 58 of 77 (75%) CRISPR nucleases. Successful targeting, based on HRM of G0 embryos correlates well with successful germline transmission (46 of 47 nucleases); yet, surprisingly mutations in the somatic tail DNA weakly correlate with mutations in the germline F1 progeny DNA. This suggests that analysis of G0 tail DNA is a good indicator of the efficiency of the nuclease, but not necessarily a good indicator of germline alleles that will be present in the F1s. However, we demonstrate that small amplicon HRM curve profiles of F1 progeny DNA can be used to differentiate between specific mutant alleles, facilitating rare allele identification and isolation; and that HRM is a powerful technique for screening possible off-target mutations that may be generated by the nucleases. Our data suggest that micro-homology based alternative NHEJ repair is primarily utilized in the generation of CRISPR mutant alleles and allows us to predict likelihood of generating a null allele. Lastly, we demonstrate that HRM can be used to quickly distinguish genotype-phenotype correlations within F1 embryos derived from G0 intercrosses. Together these data indicate that custom nucleases, in conjunction with the ease and speed of HRM, will facilitate future high-throughput mutation generation and analysis needed to establish mutants in all genes of an organism.
PMCID: PMC4263700  PMID: 25503746
3.  Incidence, management and outcomes of cardiovascular insufficiency in critically ill term and late preterm newborn infants 
American journal of perinatology  2014;31(11):947-956.
To characterize the incidence, management and short term outcomes of cardiovascular insufficiency (CVI) in mechanically ventilated newborns, evaluating 4 separate pre-specified definitions.
Study Design
Multicenter, prospective cohort study of infants ≥34 weeks gestational age (GA) and on mechanical ventilation during the first 72 hours. CVI was prospectively defined as either (1) mean arterial pressure (MAP)
Of 647 who met inclusion criteria, 419 (65%) met ≥1 definition of CVI. Of these, 98% received fluid boluses, 36% inotropes and 17% corticosteroids. Of treated infants, 46% did not have CVI as defined by a MAP < GA ± signs of inadequate perfusion. Inotrope therapy was associated with increased mortality (11.1% vs. 1.3%; P < 0.05).
More than half of the infants met at least one definition of CVI. However, almost half of the treated infants met none of the definitions. Inotropic therapy was associated with increased mortality. These findings can help guide the design of future studies of CVI in newborns.
PMCID: PMC4127379  PMID: 24515617
blood pressure; cardiovascular insufficiency; mechanical ventilation; inotrope; fluid bolus; glucocorticoid; outcomes; newborn
Objective: Chronic neurological deficits are a significant complication of preterm birth. Magnesium supplementation has been suggested to have neuroprotective function in the developing brain. Our objective was to determine whether higher neonatal serum magnesium levels were associated with better long-term neurodevelopmental outcomes in very-low birth weight infants.
Study Design: A retrospective cohort of 75 preterm infants (<1500 g, gestational age <27 weeks) had follow-up for the outcomes of abnormal motor exam and for epilepsy. Average total serum magnesium level in the neonate during the period of prematurity was the main independent variable assessed, tested using a Wilcoxon rank-sum test.
Results: Higher average serum magnesium level was associated with a statistically significant decreased risk for abnormal motor exam (p = 0.037). A lower risk for epilepsy in the group with higher magnesium level did not reach statistical significance (p = 0.06).
Conclusion: This study demonstrates a correlation between higher neonatal magnesium levels and decreased risk for long-term abnormal motor exam. Larger studies are needed to evaluate the hypothesis that higher neonatal magnesium levels can improve long-term neurodevelopmental outcomes.
PMCID: PMC4220726  PMID: 25414842
magnesium; prematurity; neurological; VLBW; neuroprotection; neonate
To determine if current retinopathy of prematurity screening guidelines1 adequately identify treatable ROP in a contemporary cohort of extremely low gestation infants.
Study Design
Data from the Surfactant, Positive Pressure, and Pulse Oximetry Randomized Trial were used. Inborn infants 24 0/7 to 27 6/7 weeks gestational age with consent prior to delivery were enrolled in 2005-2009. Severe retinopathy of prematurity (Type 1 retinopathy of prematurity or treatment with laser, cryotherapy, or bevacizumab) or death was the primary outcome for the randomized trial. Examinations followed then current American Academy of Pediatrics (AAP) screening recommendations, beginning by 31-33 weeks postmenstrual age.2,3
1316 infants were enrolled in the trial. 997 of the 1121 who survived to first eye exam had final retinopathy of prematurity outcome determined. 137 (14% of 997) met criteria for severe retinopathy of prematurity and 128 (93%) of those had sufficient data (without missing or delayed exams) to determine age of onset of severe retinopathy of prematurity. Postmenstrual age at onset was 32.1 to 53.1 wks. In this referral center cohort, 1.4% (14/997) developed severe retinopathy of prematurity after discharge.
Our contemporary data support the 2013 AAP screening guidelines for ROP for infants 24 0/7 to 27 6/7 weeks gestational age.1 Some infants do not meet treatment criteria until after discharge home. Post-discharge follow-up of infants who are still at risk for severe ROP is crucial for timely detection and treatment.
PMCID: PMC3969774  PMID: 24503911
extremely premature infant
To examine changes in arterial blood pressure (ABP) after birth in extremely preterm infants.
Study Design
Prospective observational study of infants 230/7 – 266/7 weeks gestational age (GA). Antihypotensive therapy use and ABP measurements were recorded for the first 24 hours.
A cohort of 367 infants had 18,709 ABP measurements recorded. ABP decreased for the first three hours, reached a nadir at 4 – 5 hours, then increased at an average rate of 0.2 mmHg / hour. The rise in ABP from hour 4 – 24 was similar for untreated infants (n=164) and infants given any antihypotensive therapy (n=203), a fluid bolus (n=135), or dopamine (n=92). GA specific trends were similar. ABP tended to be lower as GA decreased, but varied widely at each GA.
Arterial blood pressure increased spontaneously over the first 24 postnatal hours for extremely preterm infants. The rate of rise in ABP did not change with antihypotensive therapy.
PMCID: PMC3982788  PMID: 24503912
Antihypotensive therapy; fluid bolus; dopamine
Pediatric research  2013;75(4):507-516.
Short-term high-frequency nasal ventilation (HFNV) of preterm neonates provides acceptable gas exchange compared to endotracheal intubation and intermittent mandatory ventilation (IMV). Whether long-term HFNV will provide acceptable gas exchange is unknown. We hypothesized that HFNV for up to 21d would lead to acceptable gas exchange at lower inspired oxygen (O2) levels and airway pressures compared to intubation and IMV.
Preterm lambs were exposed to antenatal steroids, and treated with perinatal surfactant and postnatal caffeine. Lambs were intubated and resuscitated by IMV. At ~3h of age, half of the lambs were switched to non-invasive HFNV. Support was for 3d or 21d. By design, PaO2 and PaCO2 were not different between groups.
At 3d (n=5) and 21d (n=4) of HFNV, fractional inspired O2 (FiO2), peak inspiratory pressure, mean airway, intra-tracheal, and positive end-expiratory pressures, oxygenation index, and Alveolar-arterial gradient were significantly lower than matched periods of intubation and IMV. PaO2/FiO2 ratio was significantly higher at 3d and 21d of HFNV compared to matched intubation and IMV. HFNV led to better alveolarization at 3d and 21d.
Long-term HFNV provides acceptable gas exchange at lower inspired O2 levels and respiratory pressures compared to intubation and IMV.
PMCID: PMC3961520  PMID: 24378898
We describe the localization of the golgin GMAP210 and the intraflagellar protein IFT88 in the Golgi of spermatids and the participation of these two proteins in the development of the acrosome-acroplaxome complex, the head-tail coupling apparatus (HTCA) and the spermatid tail. Immunocytochemical experiments show that GMAP210 predominates in the cis-Golgi whereas IFT88 prevails in the trans-Golgi network. Both proteins co-localize in proacrosomal vesicles, along acrosome membranes, the HTCA and the developing tail. IFT88 persists in the acrosome-acroplaxome region of the sperm head whereas GMAP210 is not longer seen there. Spermatids of the Ift88 mouse mutant display abnormal head shaping and are tail-less. GMAP210 is visualized in the Ift88 mutant during acrosome-acroplaxome biogenesis. However, GMAP210–stained vesicles, mitochondria and outer dense fiber material build up in the manchette region and fail to reach the abortive tail stump in the mutant. In vitro disruption of the spermatid Golgi and microtubules with Brefeldin-A and nocodazole blocks the progression of GMAP210- and IFT88-stained proacrosomal vesicles to the acrosome-acroplaxome complex but F-actin distribution in the acroplaxome is not affected. We provide the first evidence that IFT88 is present in the Golgi of spermatids, that the microtubule-associated golgin GMAP210 and IFT88 participate in acrosome, HTCA and tail biogenesis, and that defective intramanchette transport of cargos disrupts spermatid tail development.
PMCID: PMC4175411  PMID: 21337470
acrosome; acroplaxome; microtubules; F-actin; intramanchette transport; intraflagellar transport
PLoS ONE  2014;9(9):e106576.
It has been known for decades that neurons throughout the brain possess solitary, immotile, microtubule based appendages called primary cilia. Only recently have studies tried to address the functions of these cilia and our current understanding remains poor. To determine if neuronal cilia have a role in behavior we specifically disrupted ciliogenesis in the cortex and hippocampus of mice through conditional deletion of the Intraflagellar Transport 88 (Ift88) gene. The effects on learning and memory were analyzed using both Morris Water Maze and fear conditioning paradigms. In comparison to wild type controls, cilia mutants displayed deficits in aversive learning and memory and novel object recognition. Furthermore, hippocampal neurons from mutants displayed an altered paired-pulse response, suggesting that loss of IFT88 can alter synaptic properties. A variety of other behavioral tests showed no significant differences between conditional cilia mutants and controls. This type of conditional allele approach could be used to distinguish which behavioral features of ciliopathies arise due to defects in neural development and which result from altered cell physiology. Ultimately, this could lead to an improved understanding of the basis for the cognitive deficits associated with human cilia disorders such as Bardet-Biedl syndrome, and possibly more common ailments including depression and schizophrenia.
PMCID: PMC4153651  PMID: 25184295
The New England journal of medicine  2012;367(26):2495-2504.
Previous results from our trial of early treatment with continuous positive airway pressure (CPAP) versus early surfactant treatment in infants showed no significant difference in the outcome of death or bronchopulmonary dysplasia. A lower (vs. higher) target range of oxygen saturation was associated with a lower rate of severe retinopathy but higher mortality. We now report longer-term results from our prespecified hypotheses.
Using a 2-by-2 factorial design, we randomly assigned infants born between 24 weeks 0 days and 27 weeks 6 days of gestation to early CPAP with a limited ventilation strategy or early surfactant administration and to lower or higher target ranges of oxygen saturation (85 to 89% or 91 to 95%). The primary composite outcome for the longer-term analysis was death before assessment at 18 to 22 months or neurodevelopmental impairment at 18 to 22 months of corrected age.
The primary outcome was determined for 1234 of 1316 enrolled infants (93.8%); 990 of the 1058 surviving infants (93.6%) were evaluated at 18 to 22 months of corrected age. Death or neurodevelopmental impairment occurred in 27.9% of the infants in the CPAP group (173 of 621 infants), versus 29.9% of those in the surfactant group (183 of 613) (relative risk, 0.93; 95% confidence interval [CI], 0.78 to 1.10; P = 0.38), and in 30.2% of the infants in the lower-oxygen-saturation group (185 of 612), versus 27.5% of those in the higher-oxygen-saturation group (171 of 622) (relative risk, 1.12; 95% CI, 0.94 to 1.32; P = 0.21). Mortality was increased with the lower-oxygen-saturation target (22.1%, vs. 18.2% with the higher-oxygen-saturation target; relative risk, 1.25; 95% CI, 1.00 to 1.55; P = 0.046).
We found no significant differences in the composite outcome of death or neurodevelopmental impairment among extremely premature infants randomly assigned to early CPAP or early surfactant administration and to a lower or higher target range of oxygen saturation. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and the National Heart, Lung, and Blood Institute; SUPPORT number, NCT00233324.)
PMCID: PMC4140695  PMID: 23268664
Developmental biology  2011;360(1):66-76.
Tumor necrosis factor alpha receptor 3 interacting protein 1 (Traf3ip1), also known as MIPT3, was initially characterized through its interactions with tubulin, actin, TNFR-associated factor-3 (Traf3), IL-13R1, and DISC1. It functions as an inhibitor of IL-13-mediated phosphorylation of Stat6 and in sequestration of Traf3 and DISC1 to the cytoskeleton. Studies of the Traf3ip1 homologs in C. elegans (DYF-11), Zebrafish (elipsa), and Chlamydomonas (IFT54) revealed that the protein localizes to the cilium and is required for ciliogenesis. Similar localization data has now been reported for mammalian Traf3ip1. This raises the possibility that Traf3ip1 has an evolutionarily conserved role in mammalian ciliogenesis in addition to its previously indicated functions. To evaluate this possibility, a Traf3ip1 mutant mouse line was generated. Traf3ip1mutant cells are unable to form cilia. Homozygous Traf3ip1 mutant mice are not viable and have both neural developmental defects and polydactyly, phenotypes typical of mouse mutants with ciliary assembly defects. Furthermore, in Traf3ip1 mutants the hedgehog pathway is disrupted, as evidenced by abnormal dorsal-ventral neural tube patterning and diminished expression of a hedgehog reporter. Analysis of the canonical Wnt pathway indicates that it was largely unaffected; however, specific domains in the pharyngeal arches have elevated levels of reporter activity. Interestingly, Traf3ip1 mutant embryos and cells failed to show alterations in IL-13 signaling, one of the pathways associated with its initial discovery. Novel phenotypes observed in Traf3ip1 mutant cells include elevated cytosolic levels of acetylated microtubules and a marked increase in cell size in culture. The enlarged Traf3ip1 mutant cell size was associated with elevated basal mTor pathway activity. Taken together, these data demonstrate that Traf3ip1 function is highly conserved in ciliogenesis and is important for proper regulation of a number of essential developmental and cellular pathways. The Traf3ip1 mutant mouse and cell lines will provide valuable resources to assess cilia function in mammalian development and also serve as a tool to explore the potential connections between cilia and cytoskeletal dynamics, mTor regulation, and cell volume control.
PMCID: PMC4059607  PMID: 21945076
primary cilia; IFT; Traf3ip1; MIPT3
Pediatrics  2013;131(6):e1865-e1873.
To investigate the relationships among blood pressure (BP) values, antihypotensive therapies, and in-hospital outcomes to identify a BP threshold below which antihypotensive therapies may be beneficial.
Prospective observational study of infants 230/7 to 266/7 weeks’ gestational age. Hourly BP values and antihypotensive therapy use in the first 24 hours were recorded. Low BP was investigated by using 15 definitions. Outcomes were examined by using regression analysis controlling for gestational age, the number of low BP values, and illness severity.
Of 367 infants enrolled, 203 (55%) received at least 1 antihypotensive therapy. Treated infants were more likely to have low BP by any definition (P < .001), but for the 15 definitions of low BP investigated, therapy was not prescribed to 3% to 49% of infants with low BP and, paradoxically, was administered to 28% to 41% of infants without low BP. Treated infants were more likely than untreated infants to develop severe retinopathy of prematurity (15% vs 8%, P = .03) or severe intraventricular hemorrhage (22% vs 11%, P < .01) and less likely to survive (67% vs 78%, P = .02). However, with regression analysis, there were no significant differences between groups in survival or in-hospital morbidity rates.
Factors other than BP contributed to the decision to use antihypotensive therapies. Infant outcomes were not improved with antihypotensive therapy for any of the 15 definitions of low BP investigated.
PMCID: PMC3666108  PMID: 23650301
extremely preterm infant; antihypotensive therapy; blood pressure; hypotension
Cytoskeleton (Hoboken, N.J.)  2012;70(1):24-31.
Disruption of the primary cilium is associated with a growing number of human diseases collectively termed ciliopathies. Ciliopathies present with a broad range of clinical features consistent with the near ubiquitous nature of the organelle and its role in diverse signaling pathways throughout development and adult homeostasis. The clinical features associated with cilia dysfunction can include such phenotypes as polycystic kidneys, skeletal abnormalities, blindness, anosmia, and obesity. Although the clinical relevance of the primary cilium is evident, the effects that cilia dysfunction has on the cell and how this contributes to disease remains poorly understood. Here, we show that loss of ciliogenesis genes such as Ift88 and Kif3a lead to increases in post-translational modifications on cytosolic microtubules. This effect was observed in cilia mutant kidney cells grown in vitro and in vivo in cystic kidneys. The hyper-acetylation of microtubules resulting from cilia loss is associated with both altered microtubule stability and increased α-tubulin acetyl-transferase activity. Intriguingly, the effect on microtubules was also evident in renal samples from patients with autosomal recessive polycystic kidneys. These findings indicate that altered microtubule post-translational modifications may influence some of the phenotypes observed in ciliopathies.
PMCID: PMC3552319  PMID: 23124988
cilia; tubulin; acetylation; microtubules; Polycystic Kidney Disease
Congenital diaphragmatic hernia (CDH) is a developmental defect of the diaphragm that causes high newborn mortality. Isolated or non-syndromic CDH is considered a multifactorial disease, with strong evidence implicating genetic factors. As low heritability has been reported in isolated CDH, family-based genetic methods have yet to identify the genetic factors associated with the defect. Using the Utah Population Database, we identified distantly related patients from several extended families with a high incidence of isolated CDH. Using high-density genotyping, seven patients were analyzed by homozygosity exclusion rare allele mapping (HERAM) and phased haplotype sharing (HapShare), two methods we developed to map shared chromosome regions. Our patient cohort shared three regions not previously associated with CDH, i.e. 2q11.2-q12.1, 4p13 and 7q11.2, and two regions previously involved in CDH, i.e. 8p23.1 and 15q26.2. The latter regions contain GATA4 and NR2F2, two genes implicated in diaphragm formation in mice. Interestingly, three patients shared the 8p23.1 locus and one of them also harbored the 15q26.2 segment. No coding variants were identified in GATA4 or NR2F2, but a rare shared variant was found in intron 1 of GATA4. This work shows the role of heritability in isolated CDH. Our family-based strategy uncovers new chromosomal regions possibly associated with disease, and suggests that non-coding variants of GATA4 and NR2F2 may contribute to the development of isolated CDH. This approach could speed up the discovery of the genes and regulatory elements causing multifactorial diseases, such as isolated CDH.
PMCID: PMC3507422  PMID: 23165927
congenital diaphragmatic hernia; Utah population database; shared segment analysis; GATA4; NR2F2
Cilia  2013;2:8.
Cilia are found on nearly every cell type in the mammalian body, and have been historically classified as either motile or immotile. Motile cilia are important for fluid and cellular movement; however, the roles of non-motile or primary cilia in most tissues remain unknown. Several genetic syndromes, called the ciliopathies, are associated with defects in cilia structure or function and have a wide range of clinical presentations. Much of what we know about the formation and maintenance of cilia comes from model systems like C. elegans and Chalmydomonas. Studies of mammalian cilia in live tissues have been hampered by difficulty visualizing them.
To facilitate analyses of mammalian cilia function we generated an inducible CiliaGFP mouse by targeting mouse cDNA encoding a cilia-localized protein somatostatin receptor 3 fused to GFP (Sstr3::GFP) into the ROSA26 locus. In this system, Sstr3::GFP is expressed from the ubiquitous ROSA26 promoter after Cre mediated deletion of an upstream Neo cassette flanked by lox P sites. Fluorescent cilia labeling was observed in a variety of live tissues and after fixation. Both cell-type specific and temporally regulated cilia labeling were obtained using multiple Cre lines. The analysis of renal cilia in anesthetized live mice demonstrates that cilia commonly lay nearly parallel to the apical surface of the tubule. In contrast, in more deeply anesthetized mice the cilia display a synchronized, repetitive oscillation that ceases upon death, suggesting a relationship to heart beat, blood pressure or glomerular filtration.
The ability to visualize cilia in live samples within the CiliaGFP mouse will greatly aid studies of ciliary function. This mouse will be useful for in vivo genetic and pharmacological screens to assess pathways regulating cilia motility, signaling, assembly, trafficking, resorption and length control and to study cilia regulated physiology in relation to ciliopathy phenotypes.
PMCID: PMC3700774  PMID: 23819925
Somatostatin receptor 3; ROSA26 locus; Inducible transgene; In vivo cilia labeling; Intravital microscopy
The Journal of Pediatrics  2012;161(1):65-69.e1.
To assess the feasibility of a randomized placebo controlled trial (RCT) of blood pressure (BP) management for extremely preterm infants.
Study design
This was a prospective pilot RCT of infants 230/7 – 266/7 weeks gestation who had protocol-defined low BP in the first 24 postnatal hours. Enrolled infants were administered a study infusion (dopamine or placebo) and a study syringe medication (hydrocortisone or placebo).
Of the 366 infants screened, 119 (33%) had low BP, 58 (16%) met all entry criteria, and 10 (3%) were enrolled. 161 (44%) infants were ineligible because they received early indomethacin. Only 17% of eligible infants were enrolled. Problems with consent included insufficient time, parent unavailability, and physician unwillingness to enroll critically ill infants. Two infants were withdrawn from the study due to the potential risk of intestinal perforation with simultaneous administration of hydrocortisone and indomethacin.
This pilot RCT was not feasible due to low eligibility and consent rates. An RCT of BP management for extremely preterm infants may require a waiver of consent for research in emergency care. The frequent use of early indomethacin and the associated risk of intestinal perforation when used with hydrocortisone may limit future investigations to only inotropic medications.
PMCID: PMC3357442  PMID: 22336574
Extremely preterm infant; hypotension; hydrocortisone; dopamine; informed consent
Pediatric research  2009;65(4):397-402.
Administration of retinoic acid, the active metabolite of vitamin A, is linked to the stimulation of nephrogenesis. The aim of this study was to determine whether early postnatal administration of retinoic acid (RA) could enhance ongoing nephrogenesis in a baboon model of premature birth. Unbiased stereological methods were employed to estimate kidney volume, renal corpuscle volume and nephron number. The percentage of abnormal glomeruli and the number of glomerular generations were also determined in the kidneys of preterm control (n=6) and preterm +RA (n=6) animals that received 500 μg/kg/day of all-trans retinoic acid following premature delivery. There was no significant difference between the preterm control and the preterm +RA groups in kidney size, nephron number (preterm control: 329,924 ± 41,752; preterm +RA: 354,041 ± 52,095; p = 0.59), renal corpuscle volume, number of glomerular generations, or the percentage of abnormal glomeruli. The proportion of abnormal glomeruli did not appear to be linked to any elements of postnatal care examined. The results of this study indicate that early postnatal administration of retinoic acid is unable to stimulate nephrogenesis in the kidney of the preterm baboon. Encouragingly, it does not appear to have any adverse effects on kidney development.
PMCID: PMC3633555  PMID: 19092718
Vitamin A; Preterm Birth; Papio hamadryas; Kidney; Nephron
PLoS ONE  2013;8(4):e60981.
Cytosolic carboxypeptidase 1 (CCP1) is a metallopeptidase that removes C-terminal and side-chain glutamates from tubulin. The Purkinje cell degeneration (pcd) mouse lacks CCP1 due to a mutation. Previously, elevated levels of peptides derived from cytosolic and mitochondrial proteins were found in adult pcd mouse brain, raising the possibility that CCP1 functions in the degradation of intracellular peptides. To test this hypothesis, we used a quantitative peptidomics technique to compare peptide levels in wild-type and pcd mice, examining adult heart, spleen, and brain, and presymptomatic 3 week-old amygdala and cerebellum. Contrary to adult mouse brain, young pcd brain and adult heart and spleen did not show a large increase in levels of intracellular peptides. Unexpectedly, levels of peptides derived from secretory pathway proteins were altered in adult pcd mouse brain. The pattern of changes for the intracellular and secretory pathway peptides in pcd mice was generally similar to the pattern observed in mice lacking primary cilia. Collectively, these results suggest that intracellular peptide accumulation in adult pcd mouse brain is a secondary effect and is not due to a role of CCP1 in peptide turnover.
PMCID: PMC3620535  PMID: 23593366
Pediatrics  2012;129(3):480-484.
The Surfactant Positive Airway Pressure and Pulse Oximetry Randomized Trial (SUPPORT) antenatal consent study demonstrated that mothers of infants enrolled in the SUPPORT trial had significantly different demographics and exposure to antenatal steroids compared with mothers of eligible, but not enrolled infants. The objective of this analysis was to compare the outcomes of bronchopulmonary dysplasia, severe retinopathy of prematurity, severe intraventricular hemorrhage or periventricular leukomalacia (IVH/PVL), death, and death/severe IVH/PVL for infants enrolled in SUPPORT in comparison with eligible, but not enrolled infants.
Perinatal characteristics and neonatal outcomes were compared for enrolled and eligible but not enrolled infants in bivariate analyses. Models were created to test the effect of enrollment in SUPPORT on outcomes, controlling for perinatal characteristics.
There were 1316 infants enrolled in SUPPORT; 3053 infants were eligible, but not enrolled. In unadjusted analyses, enrolled infants had significantly lower rates of death before discharge, severe IVH/PVL, death/severe IVH/PVL (all < 0.001), and bronchopulmonary dysplasia (P = .003) in comparison with eligible, but not enrolled infants. The rate of severe retinopathy of prematurity was not significantly different. After adjustment for perinatal factors, enrollment in the trial was not a significant predictor of any of the tested clinical outcomes.
The results of this analysis demonstrate significant outcome differences between enrolled and eligible but not enrolled infants in a trial using antenatal consent, which were likely due to enrollment bias resulting from the antenatal consent process. Additional research and regulatory review need to be conducted to ensure that large moderate-risk trials that require antenatal consent can be conducted in such a way as to ensure the generalizability of results.
PMCID: PMC3289530  PMID: 22371462
antenatal steroids; clinical research/trials; informed consent; neonatal
Cilia  2012;1:20.
Clusterin associated protein 1 (CLUAP1) was initially characterized as a protein that interacts with clusterin, and whose gene is frequently upregulated in colon cancer. Although the consequences of these observations remain unclear, research of CLUAP1 homologs in C. elegans and zebrafish indicates that it is needed for cilia assembly and maintenance in these models. To begin evaluating whether Cluap1 has an evolutionarily conserved role in cilia in mammalian systems and to explore the association of Cluap1 with disease pathogenesis and developmental abnormalities, we generated Cluap1 mutant mice.
Cluap1 mutant embryos were generated and examined for gross morphological and anatomical defects using light microscopy. Reverse transcription PCR, β-galactosidase staining assays, and immunofluorescence analysis were used to determine the expression of the gene and localization of the protein in vivo and in cultured cell lines. We also used immunofluorescence analysis and qRT-PCR to examine defects in the Sonic hedgehog signaling pathway in mutant embryos.
Cluap1 mutant embryos die in mid-gestation, indicating that it is necessary for proper development. Mutant phenotypes include a failure of embryonic turning, an enlarged pericardial sac, and defects in neural tube development. Consistent with the diverse phenotypes, Cluap1 is widely expressed. Furthermore, the Cluap1 protein localizes to primary cilia, and mutant embryos were found to lack cilia at embryonic day 9.5. The phenotypes observed in Cluap1 mutant mice are indicative of defects in Sonic hedgehog signaling. This was confirmed by analyzing hedgehog signaling activity in Cluap1 mutants, which revealed that the pathway is repressed.
These data indicate that the function of Cluap1 is evolutionarily conserved with regard to ciliogenesis. Further, the results implicate mammalian Cluap1 as a key regulator of hedgehog signaling and as an intraflagellar transport B complex protein. Future studies on mammalian Cluap1 utilizing this mouse model may provide insights into the role for Cluap1 in intraflagellar transport and the association with colon cancer and cystic kidney disorders.
PMCID: PMC3556011  PMID: 23351563
Intraflagellar transport; Sonic hedgehog; Clusterin associated protein 1; IFT complex B
Human Molecular Genetics  2011;20(15):2942-2954.
A spectrum of complex oligogenic disorders called the ciliopathies have been connected to dysfunction of cilia. Among the ciliopathies are Nephronophthisis (NPHP), characterized by cystic kidney disease and retinal degeneration, and Meckel–Gruber syndrome (MKS), a gestational lethal condition with skeletal abnormalities, cystic kidneys and CNS malformation. Mutations in multiple genes have been identified in NPHP and MKS patients, and an unexpected finding has been that mutations within the same gene can cause either disorder. Further, there is minimal genotype–phenotype correlation and despite recessive inheritance, numerous patients were identified as having a single heterozygous mutation. This has made it difficult to determine the significance of these mutations on disease pathogenesis and led to the hypothesis that clinical presentation in an individual will be determined by genetic interactions between mutations in multiple cilia-related genes. Here we utilize Caenorhabditis elegans and cilia-associated behavioral and morphologic assays to evaluate the pathogenic potential of eight previously reported human NPHP4 missense mutations. We assess the impact of these mutations on C. elegans NPHP-4 function, localization and evaluate potential interactions with mutations in MKS complex genes, mksr-2 and mksr-1. Six out of eight nphp-4 mutations analyzed alter ciliary function, and three of these modify the severity of the phenotypes caused by disruption of mksr-2 and mksr-1. Collectively, our studies demonstrate the utility of C. elegans as a tool to assess the pathogenicity of mutations in ciliopathy genes and provide insights into the complex genetic interactions contributing to the diversity of phenotypes associated with cilia disorders.
PMCID: PMC3131040  PMID: 21546380
Rationale: Benefits of identifying risk factors for bronchopulmonary dysplasia in extremely premature infants include providing prognostic information, identifying infants likely to benefit from preventive strategies, and stratifying infants for clinical trial enrollment.
Objectives: To identify risk factors for bronchopulmonary dysplasia, and the competing outcome of death, by postnatal day; to identify which risk factors improve prediction; and to develop a Web-based estimator using readily available clinical information to predict risk of bronchopulmonary dysplasia or death.
Methods: We assessed infants of 23–30 weeks' gestation born in 17 centers of the Eunice Kennedy Shriver National Institute of Child Health and Human Development Neonatal Research Network and enrolled in the Neonatal Research Network Benchmarking Trial from 2000–2004.
Measurements and Main Results: Bronchopulmonary dysplasia was defined as a categorical variable (none, mild, moderate, or severe). We developed and validated models for bronchopulmonary dysplasia risk at six postnatal ages using gestational age, birth weight, race and ethnicity, sex, respiratory support, and FiO2, and examined the models using a C statistic (area under the curve). A total of 3,636 infants were eligible for this study. Prediction improved with advancing postnatal age, increasing from a C statistic of 0.793 on Day 1 to a maximum of 0.854 on Day 28. On Postnatal Days 1 and 3, gestational age best improved outcome prediction; on Postnatal Days 7, 14, 21, and 28, type of respiratory support did so. A Web-based model providing predicted estimates for bronchopulmonary dysplasia by postnatal day is available at
Conclusions: The probability of bronchopulmonary dysplasia in extremely premature infants can be determined accurately using a limited amount of readily available clinical information.
PMCID: PMC3136997  PMID: 21471086
bronchopulmonary dysplasia; prematurity; low-birth-weight infant
Circulation research  2011;108(9):1093-1101.
Primary cilia are cellular protrusions which serve as mechanosensors for fluid flow. In endothelial cells (EC) they function in transducing local blood flow information into functional responses, like nitric oxide production and initiation of gene expression. Cilia are present on EC in areas of low or disturbed flow and absent in areas of high flow. In the embryonic heart high flow regime applies to the endocardial cushion area, and the absence of cilia here coincides with the process of endothelial-to-mesenchymal transition (EndoMT).
In this study we investigate the role of the primary cilium in defining the responses of EC to fluid shear stress and in EndoMT.
Methods and Results
Non-ciliated mouse embryonic EC with a mutation in Tg737/Ift88 were used to compare the response to fluid shear stress to that of ciliated EC. In vitro, non-ciliated EC undergo shear-induced EndoMT which is accompanied by downregulation of Klf4. This Tgfβ/Alk5 dependent transformation is prevented by blocking Tgfβ signaling, overexpression of Klf4, or rescue of the primary cilium. In the hearts of Tg737orpk/orpk embryos Tgfβ/Alk5 signaling was activated in areas in which EC would normally be ciliated, but now lack cilia due to the mutation. In these areas EC show increased Smad2 phosphorylation and expression of αSMA.
This study demonstrates the central role of primary cilia in rendering EC prone to shear-induced activation of Tgfβ/Alk5 signaling and EndoMT, and thereby provides a functional link between primary cilia and flow related endothelial performance.
PMCID: PMC3094764  PMID: 21393577
cilia; shear stress; endothelial cells; Tg737orpk/orpk; EndoMT; Tgfβ/Alk5; Klf4
Pediatric research  2011;69(3):212-216.
Ibuprofen-induced ductus closure improves pulmonary mechanics and increases alveolar surface area in premature baboons compared with baboons with a persistent patent ductus arteriosus (PDA). Ibuprofen-treatment has no effect on the expression of genes that regulate pulmonary inflammation but does increase the expression of alpha-ENaC (the transepithelial sodium channel that is critical for alveolar water clearance). Although ligation eliminates the PDA, it does not improve pulmonary mechanics or increase alveolar surface area. We used preterm baboons (delivered at 67% of term gestation and ventilated for 14 days) to study whether the lack of beneficial effects, following PDA ligation, might be due to alterations in pulmonary gene expression. We found no differences in Ventilation or Oxygenation Indices between animals that were ligated (n=7) on day of life 6 and those that had a persistent PDA (n=12) during the entire 14 days study. In contrast with no intervention, PDA ligation produced a significant increase in the expression of genes involved with pulmonary inflammation (COX-2, TNF-alpha, and CD14), and a significant decrease in alpha-ENaC sodium channel expression. We speculate that these changes may decrease the rate of alveolar fluid clearance and contribute to the lack of improvement in pulmonary mechanics after PDA ligation.
PMCID: PMC3065199  PMID: 21131894
American journal of obstetrics and gynecology  2010;204(2):177.e8-177.e14.
Estrogen receptors are present within the fetal brain suggesting that estrogens may exert an influence on cerebral development. Loss of placentally-derived estrogen in preterm birth may impair development.
Study Design
Baboons were delivered at 125 days of gestation (term~185 days), randomly allocated to receive estradiol (n=10) or placebo (n=8) and ventilated for 14 days. Brains were assessed for developmental and neuropathological parameters.
Body and brain weights were not different between groups but the brain/body weight ratio was increased (p<0.05) in estradiol-treated animals. There were no differences (p>0.05) between groups in any neuropathological measure in either the forebrain or cerebellum. There were no intraventricular hemorrhages; one estradiol animal displayed ectactic vessels in the subarachnoid space.
Brief postnatal estradiol administration to primates does not pose an increased risk of injury or impaired brain development.
PMCID: PMC3032810  PMID: 21074139
Postnatal estradiol; premature delivery; brain injury; brain development; baboon

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