Shotgun metagenomic sequencing on skin microbiome indicates dysbiosis exists prior to the onset of atopic dermatitis.

BACKGROUND: While the microbiome is increasingly seen as a targetable contributor to atopic dermatitis (AD), questions remain as to whether the dysbiosis is secondary to diseased skin or if it predates symptom onset. Previous work has evaluated how the skin microbiome changes with age and established the influence of factors like delivery mode and breastfeeding on global microbiome diversity. However, these studies were unable to identify taxa which predict subsequent AD. METHODS: Skin swab samples were collected from the first week of life for 72 children in the neonatal intensive care unit (NICU) at a single site hospital. Participants were followed for 3 years to determine their health status. We applied shotgun metagenomic sequencing to assess the microbiome differences between 31 children who went on to develop AD and 41 controls. RESULTS: We identified that subsequent development of AD was associated with differential abundance of several bacterial and fungal taxa as well as several metabolic pathways, each of which have been previously associated with active AD. CONCLUSIONS: Our work provides evidence of reproducibility for the previously reported dysbiotic signatures predating AD onset while also expanding prior findings through the first use of metagenomic assessment prior to AD onset. While extrapolation of our findings beyond the pre-term, NICU cohort is limited, our findings add to the evidence that the dysbiosis associated with AD pre-dates disease onset rather than reflect a secondary consequence of skin inflammation.

Genomic and molecular characterization of preterm birth.

Preterm birth (PTB) complications are the leading cause of long-term morbidity and mortality in children. By using whole blood samples, we integrated whole-genome sequencing (WGS), RNA sequencing (RNA-seq), and DNA methylation data for 270 PTB and 521 control families. We analyzed this combined dataset to identify genomic variants associated with PTB and secondary analyses to identify variants associated with very early PTB (VEPTB) as well as other subcategories of disease that may contribute to PTB. We identified differentially expressed genes (DEGs) and methylated genomic loci and performed expression and methylation quantitative trait loci analyses to link genomic variants to these expression and methylation changes. We performed enrichment tests to identify overlaps between new and known PTB candidate gene systems. We identified 160 significant genomic variants associated with PTB-related phenotypes. The most significant variants, DEGs, and differentially methylated loci were associated with VEPTB. Integration of all data types identified a set of 72 candidate biomarker genes for VEPTB, encompassing genes and those previously associated with PTB. Notably, PTB-associated genes RAB31 and RBPJ were identified by all three data types (WGS, RNA-seq, and methylation). Pathways associated with VEPTB include EGFR and prolactin signaling pathways, inflammation- and immunity-related pathways, chemokine signaling, IFN-γ signaling, and Notch1 signaling. Progress in identifying molecular components of a complex disease is aided by integrated analyses of multiple molecular data types and clinical data. With these data, and by stratifying PTB by subphenotype, we have identified associations between VEPTB and the underlying biology.

Mutations in NOTCH1 cause Adams-Oliver syndrome.

Notch signaling determines and reinforces cell fate in bilaterally symmetric multicellular eukaryotes. Despite the involvement of Notch in many key developmental systems, human mutations in Notch signaling components have mainly been described in disorders with vascular and bone effects. Here, we report five heterozygous NOTCH1 variants in unrelated individuals with Adams-Oliver syndrome (AOS), a rare disease with major features of aplasia cutis of the scalp and terminal transverse limb defects. Using whole-genome sequencing in a cohort of 11 families lacking mutations in the four genes with known roles in AOS pathology (ARHGAP31, RBPJ, DOCK6, and EOGT), we found a heterozygous de novo 85 kb deletion spanning the NOTCH1 5' region and three coding variants (c.1285T>C [p.Cys429Arg], c.4487G>A [p.Cys1496Tyr], and c.5965G>A [p.Asp1989Asn]), two of which are de novo, in four unrelated probands. In a fifth family, we identified a heterozygous canonical splice-site variant (c.743-1 G>T) in an affected father and daughter. These variants were not present in 5,077 in-house control genomes or in public databases. In keeping with the prominent developmental role described for Notch1 in mouse vasculature, we observed cardiac and multiple vascular defects in four of the five families. We propose that the limb and scalp defects might also be due to a vasculopathy in NOTCH1-related AOS. Our results suggest that mutations in NOTCH1 are the most common cause of AOS and add to a growing list of human diseases that have a vascular and/or bony component and are caused by alterations in the Notch signaling pathway.

Neuropsychological Outcomes in Extremely Preterm Preschoolers Exposed to Tiered Low Oxygen Targets: An Observational Study.

An observational study of neuropsychological outcomes at preschool age of tiered lowered oxygen (O2) saturation targets in extremely preterm neonates. We studied 111 three-year-olds born <28 weeks' gestational age. Fifty-nine participants born in 2009-2010 during a time-limited quality improvement initiative each received three-tiered stratification of oxygen rates (83-93% until age 32 weeks, 85-95% until age 35 weeks, and 95% after age 35 weeks), the TieredO2 group. Comparisons were made with 52 participants born in 2007-2008 when pre-initiative saturation targets were non-tiered at 89-100%, the Non-tieredO2 group. Neuropsychological domains included general intellectual, executive, attention, language, visuoperceptual, visual-motor, and fine and gross motor functioning. Descriptive and inferential analyses were conducted. Group comparisons were not statistically significant. Descriptively, the TieredO2 group had better general intellectual, executive function, visual-motor, and motor performance and the Non-tieredO2 group had better language performance. Cohen's d and confidence intervals around d were in similar direction and magnitude across measures. A large effect size was found for recall of digits-forward in participants born at 23 and 24 weeks' gestation, d=0.99 and 1.46, respectively. Better TieredO2 outcomes in all domains except language suggests that the tiered oxygen saturation target method is not harmful and merits further investigation through further studies. Benefit in auditory attention appeared greatest in those born at 23 and 24 weeks. Participants in the tiered oxygen saturation group also had fewer ventilation days and a lower incidence of bronchopulmonary dysplasia, perhaps explanatory for these neuropsychological outcomes at age 3.

Expanding the phenotypic spectrum in EP300-related Rubinstein-Taybi syndrome.

Rubinstein-Taybi syndrome (RSTS) can be caused by heterozygous mutations or deletions involving CREBBP or, less commonly, EP300. To date, only 15 patients with EP300 mutations have been clinically described. Frequently reported manifestations in these patients include characteristic facial and limb features, varying degrees of neurocognitive dysfunction, and maternal preeclampsia. Other congenital anomalies are less frequently reported. We describe a child found to have a de novo EP300 mutation (c.4933C>T, predicted to result in p.Arg1645X) through research-based whole-genome sequencing of the family trio. The child's presentation involved dysmorphic features as well as unilateral renal agenesis, a myelomeningocele, and minor genitourinary anomalies. The involvement of congenital anomalies in all 16 clinically described patients with EP300 mutations (25% of which have been identified by "hypothesis free" methods, including microarray, exome, and whole-genome sequencing) is reviewed. In summary, genitourinary anomalies have been identified in 38%, cardiovascular anomalies in 25%, spinal/vertebral anomalies in 19%, other skeletal anomalies in 19%, brain anomalies in 13%, and renal anomalies in 6%. Our patient expands the phenotypic spectrum in EP300-related RSTS; this case demonstrates the evolving practice of clinical genomics related to increasing availability of genomic sequencing methods.

Mesenchymal stromal cells expressing heme oxygenase-1 reverse pulmonary hypertension.

Pulmonary arterial hypertension (PAH) remains a serious disease, and although current treatments may prolong and improve quality of life, search for novel and effective therapies is warranted. Using genetically modified mouse lines, we tested the ability of bone marrow-derived stromal cells (mesenchymal stem cells [MSCs]) to treat chronic hypoxia-induced PAH. Recipient mice were exposed for 5 weeks to normobaric hypoxia (8%-10% O(2)), MSC preparations were delivered through jugular vein injection and their effect on PAH was assessed after two additional weeks in hypoxia. Donor MSCs derived from wild-type (WT) mice or heme oxygenase-1 (HO-1) null mice (Hmox1(KO)) conferred partial protection from PAH when transplanted into WT or Hmox1(KO) recipients, whereas treatment with MSCs isolated from transgenic mice harboring a human HO-1 transgene under the control of surfactant protein C promoter (SH01 line) reversed established disease in WT recipients. SH01-MSC treatment of Hmox1(KO) animals, which develop right ventricular (RV) infarction under prolonged hypoxia, resulted in normal RV systolic pressure, significant reduction of RV hypertrophy and prevention of RV infarction. Donor MSCs isolated from a bitransgenic mouse line with doxycycline-inducible, lung-specific expression of HO-1 exhibited similar therapeutic efficacy only on doxycycline treatment of the recipients. In vitro experiments indicate that potential mechanisms of MSC action include modulation of hypoxia-induced lung inflammation and inhibition of smooth muscle cell proliferation. Cumulatively, our results demonstrate that MSCs ameliorate chronic hypoxia-induced PAH and their efficacy is highly augmented by lung-specific HO-1 expression in the transplanted cells, suggesting an interplay between HO-1-dependent and HO-1-independent protective pathways.

Fecal sphingolipids predict parenteral nutrition-associated cholestasis in the neonatal intensive care unit.

BACKGROUND: Parenteral nutrition-associated cholestasis (PNAC) in the neonatal intensive care unit (NICU) causes significant morbidity and associated healthcare costs. Laboratory detection of PNAC currently relies on elevated serum conjugated bilirubin levels in the aftermath of impaired bile flow. Here, we sought to identify fecal biomarkers, which when integrated with clinical data, would better predict risk for developing PNAC. METHODS: Using untargeted metabolomics in 200 serial stool samples from 60 infants, we applied statistical and machine learning approaches to identify clinical features and metabolic biomarkers with the greatest associative potential for risk of developing PNAC. Stools were collected prospectively from infants receiving PN with soybean oil-based lipid emulsion at a level IV NICU. RESULTS: Low birth weight, extreme prematurity, longer duration of PN, and greater number of antibiotic courses were all risk factors for PNAC (P < 0.05). We identified 78 stool biomarkers with early predictive potential (P < 0.05). From these 78 biomarkers, we further identified 12 sphingomyelin lipids with high association for the development of PNAC in precholestasis stool samples when combined with birth anthropometry. CONCLUSION: We demonstrate the potential for stool metabolomics to enhance early identification of PNAC risk. Earlier detection of high-risk infants would empower proactive mitigation with alterations to PN for at-risk infants and optimization of energy nutrition with PN for infants at lower risk.

Bone marrow stromal cells attenuate lung injury in a murine model of neonatal chronic lung disease.

RATIONALE: Neonatal chronic lung disease, known as bronchopulmonary dysplasia (BPD), remains a serious complication of prematurity despite advances in the treatment of extremely low birth weight infants. OBJECTIVES: Given the reported protective actions of bone marrow stromal cells (BMSCs; mesenchymal stem cells) in models of lung and cardiovascular injury, we tested their therapeutic potential in a murine model of BPD. METHODS: Neonatal mice exposed to hyperoxia (75% O(2)) were injected intravenously on Day 4 with either BMSCs or BMSC-conditioned media (CM) and assessed on Day 14 for lung morphometry, vascular changes associated with pulmonary hypertension, and lung cytokine profile. MEASUREMENTS AND MAIN RESULTS: Injection of BMSCs but not pulmonary artery smooth muscle cells (PASMCs) reduced alveolar loss and lung inflammation, and prevented pulmonary hypertension. Although more donor BMSCs engrafted in hyperoxic lungs compared with normoxic controls, the overall low numbers suggest protective mechanisms other than direct tissue repair. Injection of BMSC-CM had a more pronounced effect than BMSCs, preventing both vessel remodeling and alveolar injury. Treated animals had normal alveolar numbers at Day 14 of hyperoxia and a drastically reduced lung neutrophil and macrophage accumulation compared with PASMC-CM-treated controls. Macrophage stimulating factor 1 and osteopontin, both present at high levels in BMSC-CM, may be involved in this immunomodulation. CONCLUSIONS: BMSCs act in a paracrine manner via the release of immunomodulatory factors to ameliorate the parenchymal and vascular injury of BPD in vivo. Our study suggests that BMSCs and factor(s) they secrete offer new therapeutic approaches for lung diseases currently lacking effective treatment.

Prenatal and Peripartum Exposure to Antibiotics and Cesarean Section Delivery Are Associated with Differences in Diversity and Composition of the Infant Meconium Microbiome.

The meconium microbiome may provide insight into intrauterine and peripartum exposures and the very earliest intestinal pioneering microbes. Prenatal antibiotics have been associated with later obesity in children, which is thought to be driven by microbiome dependent mechanisms. However, there is little data regarding associations of prenatal or peripartum antibiotic exposure, with or without cesarean section (CS), with the features of the meconium microbiome. In this study, 16S ribosomal RNA gene sequencing was performed on bacterial DNA of meconium samples from 105 infants in a birth cohort study. After multivariable adjustment, delivery mode (p = 0.044), prenatal antibiotic use (p = 0.005) and peripartum antibiotic use (p < 0.001) were associated with beta diversity of the infant meconium microbiome. CS (vs. vaginal delivery) and peripartum antibiotics were also associated with greater alpha diversity of the meconium microbiome (Shannon and Simpson, p < 0.05). Meconium from infants born by CS (vs. vaginal delivery) had lower relative abundance of the genus Escherichia (p < 0.001). Prenatal antibiotic use and peripartum antibiotic use (both in the overall analytic sample and when restricting to vaginally delivered infants) were associated with differential abundance of several bacterial taxa in the meconium. Bacterial taxa in the meconium microbiome were also differentially associated with infant excess weight at 12 months of age, however, sample size was limited for this comparison. In conclusion, prenatal and peripartum antibiotic use along with CS delivery were associated with differences in the diversity and composition of the meconium microbiome. Whether or not these differences in the meconium microbiome portend risk for long-term health outcomes warrants further exploration.

Experience with genomic sequencing in pediatric patients with congenital cardiac defects in a large community hospital.

BACKGROUND: Congenital cardiac defects, whether isolated or as part of a larger syndrome, are the most common type of human birth defect occurring on average in about 1% of live births depending on the malformation. As there is an expanding understanding of the underlying molecular mechanisms by which a cardiac defect may occur, there is a need to assess the current rates of diagnosis of cardiac defects by molecular sequencing in a clinical setting. METHODS AND RESULTS: In this report, we evaluated 34 neonatal and pediatric patients born with a cardiac defect and their parents using exomized preexisting whole genome sequencing (WGS) data to model clinically available exon-based tests. Overall, we identified candidate variants in previously reported cardiac-related genes in 35% (12/34) of the probands. These include clearly pathogenic variants in two of 34 patients (6%) and variants of uncertain significance in relevant genes in 10 patients (26%), of these latter 10, 2 segregated with clinically apparent findings in the family trios. CONCLUSIONS: These findings suggest that with current knowledge of the proteins underlying CHD, genomic sequencing can identify the underlying genetic etiology in certain patients; however, this technology currently does not have a high enough yield to be of routine clinical use in the screening of pediatric congenital cardiac defects.

Comparison of Infant Gut and Skin Microbiota, Resistome and Virulome Between Neonatal Intensive Care Unit (NICU) Environments.

Background: There is a growing move to provide care for premature infants in a single family, private room neonatal intensive care unit (NICU) in place of the traditional shared space, open bay NICU. The resultant effect on the developing neonatal microbiota is unknown. Study Design: Stool and groin skin swabs were collected from infants in a shared-space NICU (old NICU) and a single-family room NICU (new NICU) on the same hospital campus. Metagenomic sequencing was performed and data analyzed by CosmosID bioinformatics software package. Results: There were no significant differences between the cohorts in gestational age, length of stay, and delivery mode; infants in the old NICU received significantly more antibiotics (p = 0.03). Differentially abundant antimicrobial resistance genes and virulence associated genes were found between the cohorts in stool and skin, with more differentially abundant antimicrobial resistance genes in the new NICU. The entire bacterial microbiota analyzed to the genus level significantly differed between cohorts in skin (p = 0.0001) but not in stool samples. There was no difference in alpha diversity between the two cohorts. DNA viruses and fungi were detected but did not differ between cohorts. Conclusion: Differences were seen in the resistome and virulome between the two cohorts with more differentially abundant antimicrobial resistance genes in the new NICU. This highlights the influence that different NICU environments can have on the neonatal microbiota. Whether the differences were due to the new NICU being a single-family NICU or located in a newly constructed building warrants exploration. Long term health outcomes from the differences observed must be followed longitudinally.

Pharmacological strategies in the prevention and management of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a disease of complex and multifactorial etiology and a major cause of morbidity in premature infants. Contributing factors include infection, exposure to toxic oxygen levels, and ventilator-induced lung injury, resulting in arrested lung development and impaired lung function. Several preventive and therapeutic strategies have been employed and include lung protective ventilator strategies, pharmacological and nutritional interventions. These strategies target different components and stages of the disease process, and their success has been variable. This review intends to bring together prior and current pharmacological interventions and future therapeutic modalities that appear promising in the prevention and management of BPD. Better understanding of the pathogenesis has given hope for newer treatment options. Newer studies need to be designed to assess the efficacy of combination therapies that target multiple steps of the disease process.

The Microbiome in Necrotizing Enterocolitis: A Case Report in Twins and Minireview.

Our case describes the serial microbiome changes in twins discordant for necrotizing enterocolitis (NEC), who shared similar intrauterine and early environmental exposures. The key findings were that the 2 neonates had distinctly different microbiome compositions from the first stool samples collected. Also, in the twin who developed NEC there was a decrease in bacterial diversity and an increase in Proteobacteria a week before developing any clinical symptoms, suggesting an early role of the intestinal microbiome in the development of NEC. Here we briefly review the literature on the role of the intestinal microbiome in NEC and how a greater understanding of the neonatal microbiome and host interactions may help mitigate this devastating disease.

Diagnosis of D-Bifunctional Protein Deficiency through Whole-Genome Sequencing: Implications for Cost-Effective Care.

D-Bifunctional protein deficiency, caused by recessive mutations in HSD17B4, is a severe disorder of peroxisomal fatty acid oxidation. Nonspecific clinical features may contribute to diagnostic challenges. We describe a newborn female with infantile-onset seizures and nonspecific mild dysmorphisms who underwent extensive genetic workup that resulted in the detection of a novel homozygous mutation (c.302+1_4delGTGA) in the HSD17B4 gene, consistent with a diagnosis of D-bifunctional protein deficiency. By comparing the standard clinical workup to diagnostic analysis performed through research-based whole-genome sequencing (WGS), which independently identified the causative mutation, we demonstrated the ability of genomic sequencing to serve as a timely and cost-effective diagnostic tool for the molecular diagnosis of apparent and occult newborn diseases. As genomic sequencing becomes more available and affordable, we anticipate that WGS and related omics technologies will eventually replace the traditional tiered approach to newborn diagnostic workup.

Endothelin 1 impairs oxygen delivery in livers from LPS-primed animals.

Endothelin 1 (ET-1) is a potent vasoactive peptide that acts at sinusoidal and extrasinusoidal sites in the liver. Sensitivity to ET-1 increases in LPS-primed animals and is associated with impaired liver microcirculation in these animals. We hypothesized that LPS priming leads to an exacerbation in the impaired oxygen delivery in response to intraportal infusion of ET-1. Rats were studied 24 h after LPS injection (1 mg/kg, i.p.). Surface PO2 was determined using a recently developed technology of O2 mapping. The baseline portal pressure was higher in LPS-primed animals (P < 0.05), and increased to'similar magnitude as sham animals after a 10-min infusion of ET-1. The resultant portal pressure remained elevated in LPS compared to sham animals. There was no significant difference in baseline mean arterial pressure, and no significant systemic response to ET-1 in either group. In contrast to the macrohemodynamic, the decrease in tissue surface PO2 in response to ET-1 infusion was potentiated by LPS treatment (increased from baseline levels 33.8+/-9 to 46.8+/-8.3 in sham; 42.3+/-9.1 to 69+/-6.5 gray scale units in LPS; P < 0.01, sham vs. LPS) at end of infusion of ET-1 for 10 min. This indicates tissue hypoxia in response to ET-1, which is exacerbated in livers from LPS-primed animals compared to sham. Frequency distribution analysis showed a shift in mode from lower intensity (higher PO2) to areas with higher fluorescent intensity ranges (lower PO2), indicating areas with shut down in perfusion in LPS-treated animals. In the whole liver, ET-1 suppressed oxygen consumption, and this response was potentiated by LPS pretreatment. We propose that ET-1 impairs oxygen delivery in the liver during endotoxemia, resulting in areas of focal hypoxia. This response is possibly due to potentiated action of ET-1 at both sinusoidal and extrasinusoidal sites in the liver during endotoxemia.

LPS-induced imbalanced expression of hepatic vascular stress genes in cirrhosis: possible mechanism of increased susceptibility to endotoxemia.

Cirrhosis predisposes the liver to secondary stresses such as endotoxemia possibly via dysregulation of the hepatic portal circulation secondary to imbalanced upregulation of vascular stress genes. In this study we determined the effect of cirrhosis on hepatic vasoregulatory gene expression in response to endotoxin (LPS, i.p., 1 mg/kg). Cirrhosis was induced by bile duct ligation (BDL) for 21 days in male Sprague-Dawley rats. Plasma and liver samples were taken 6 h following an injection of LPS for alanine aminotransferase (ALT) assays and RT-PCR analysis of mRNA levels for genes of interest: endothelin (ET-1), its receptors ET(A) and ET(B), endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), and heme oxygenase-1 (HO-1). ALT release increased by 5.5-fold in the BDL animals and 9.9-fold in BDL + LPS compared to sham. ET-1 mRNA was increased by either LPS or BDL treatment alone and increased significantly more in BDL + LPS compared to sham + LPS. mRNA levels for ET(B) receptors showed no change, whereas ETA transcripts decreased in BDL animals compared to sham, with no significant difference between the saline and LPS treatment groups. The resultant increased ratio of ET(B) over ET(A) in BDL animals was reflected functionally in the portal pressure responses to ET(A) and ET(B) agonists ET-1 and IRL-1620 (a specific ETB receptor agonist). The pressor response to ET-1 was attenuated, while the response to IRL-1620 was similar in BDL and sham. eNOS mRNA levels did not increase in response to either BDL or LPS or a combination of both compared to sham. The increase in iNOS mRNA was attenuated in BDL + LPS compared to sham + LPS. HO-1 expression increased significantly in sham + LPS, but failed to increase in BDL + LPS. Taken collectively, significantly greater induction of the constrictor ET-1 over the dilation forces (i.e., eNOS, iNOS, and HO-1) was observed in BDL + LPS. This suggests a compromised ability of the cirrhotic liver to upregulate sufficient dilatory forces to counterbalance the constrictive effect of ET-1 upon a secondary insult of endotoxemia. These results may partly explain the increased susceptibility of cirrhotic livers to injury as a result of endotoxemia.

Remote trauma sensitizes hepatic microcirculation to endothelin via caveolin inhibition of eNOS activity.

This study addresses the microvascular mechanisms by which a remote, mild stress such as blunt trauma sensitizes the liver to injury. Rats received closed femur fracture (FFx), and 24 h later livers were isolated and perfused at a similar starting flow rate for assessment of vascular response to endothelin-1 (ET-1). Sinusoidal volumetric flow (QS), red blood cell velocity (VRBC), and sinusoidal diameter (Ds) were determined by intravital microscopy. Baseline portal resistance in livers from FFx rats was not changed. The FFx group showed a lower baseline VRBC (322.9 +/- 26.4 and 207.3 +/- 17.2 microm/s in sham and FFx,) and QS (28.4 +/- 4.2 and 17.6 +/- 2.1 pL/s in sham and FFx, P < 0.05). ET-1 caused a decrease in the VRBC in sham but no change after FFx. In contrast, Ds was unchanged by ET-1 in sham but decreased in FFx (10.3 +/- 0.4 to 10.7 +/- 0.5 vs. 10.6 +/- 0.4 to 9.0 +/- 0.4 microm at 10 min in sham and FFx groups, P < 0.05). The overall result of these changes was a greater decrease in sinusoidal flow in FFx compared with sham. There was no significant change in mRNA for ET-1, endothelin A (ETA) receptor, or iNOS (inducible nitric oxide synthase) in FFx compared with sham. However, endothelin B (ETB) receptor mRNA and eNOS (endothelial nitric oxide synthase) mRNA were increased in the FFx group (ETB, 54.81 +/- 8.08 in sham vs. 83.28 +/- 8.19 in FFx; eNOS, 56.11 +/- 2.53 in sham vs. 83.31 +/- 5.51 in FFx; P < 0.05) while the levels of these proteins remained unchanged. Caveolin-1 (cav-1) protein levels were elevated in FFx, and coimmunoprecipitation with both ETB and eNOS showed increased associations with these proteins, suggesting a possible inactivation of eNOS. The eNOS activity was also blunted in FFx animals in the presence of increased cav-1 expression. Taken together, these results demonstrate that remote trauma sensitizes the liver to the sinusoidal constrictor effect of ET-1. We propose that this hyperresponsiveness occurs as a result of uncoupling of the ETB receptor from eNOS activity mediated by interaction of eNOS and possibly the ETB receptor with increased caveolin-1. This vascular sensitization that occurs after FFx may contribute to the exacerbation of injury during subsequent stresses.

Potentiated hepatic microcirculatory response to endothelin-1 during polymicrobial sepsis.

We conducted this study to elucidate the role of endothelins (ET-1) in mediating the hepatic microcirculatory dysfunction observed in response to sepsis. Following 24 h of cecal ligation and puncture (CLP), we performed intravital microscopy both in vivo and on isolated perfused livers. Portal resistance increased in response to ET-1 in both sham and septic rats, with no significant difference between the two in either in vivo or in isolated livers. Sinusoidal volumetric flow (Qs) was evaluated using red blood cell velocity (V(RBC)) and sinusoidal diameter (Ds) to determine microvascular hemodynamic integrity. Qs decreased in response to ET-1 in livers from CLP rats compared with sham (P < 0.05, CLP vs. sham) in both in vivo and isolated livers. In vivo infusion of ET-1 resulted in greater constriction of sinusoids in the CLP group compared with sham (P < 0.05), resulting in higher sinusoidal resistance. Microvascular hyper-responsiveness was accompanied by hepatocellular injury in CLP rats, but not in sham rats. RT-PCR was performed to measure mRNA levels of ET-1, its receptors ET(A) and ET(B), inducible and constitutive nitric oxide (NO) synthase (iNOS and eNOS, respectively), and heme oxygenase 1 (HO-1). After CLP, both ET-1 and ET(B) mRNA increased, whereas ET(A) mRNA tended to decrease, although the change was not statistically significant. Livers from CLP rats showed no significant change in levels of eNOS mRNA, but showed a significant increase in iNOS expression (13.5-fold over sham). There was no change in the level of HO-1 mRNA between sham and CLP groups. Taken together, these results suggest that sepsis sensitizes the hepatic microcirculation to ET-1. More importantly, an impaired microcirculatory flow due to ET-1 in sepsis contributes to hepatic injury. Further, localized imbalances between endothelins and NO may mediate the altered microvascular response during sepsis.

Endothelin receptor remodeling induces the portal venous hyper-response to endothelin-1 following endotoxin pretreatment.

The purpose of this study was to determine the changes in endothelin (ET) receptor subtype expression and their functional significance after endotoxin pretreatment. Rats were pretreated with lipopolysaccharide (LPS) or sterile saline (control). After 24 h, liver samples were homogenized and competitive receptor binding assays were performed. There was no significant difference in ET receptor binding affinity between the control and LPS groups. However, the receptor subtype density showed a significant increase in ET(B) receptors in LPS-treated rats, whereas the amount of ET(A) receptors was almost identical between the two groups. In control, almost all ET receptors (95%) were displaced by using combined ET(A) antagonist (BQ-610) and ET(B) agonist (IRL-1620) as competitors, whereas only 80% (P < 0.05 versus control) was displaced in LPS group, raising the possibility of novel type of ET receptor expression. An infusion of ET(B) agonist (Sarafotoxin 6c, S6c) through portal vein in isolated perfused livers produced the same pressure response in both LPS and control groups; however, the portal pressure increase in response to the ET-1, which binds all ET receptors, was significantly potentiated in LPS-treated rats compared to controls. We conclude that altered regulation of ET receptors, in particular, the appearance of ET binding capacity that is not displaced by ET(A) or ET(B) competitors, may explain the hyper-response of the portal venous system to ET-1 during endotoxemia.

Diagnosis of an imprinted-gene syndrome by a novel bioinformatics analysis of whole-genome sequences from a family trio.

Whole-genome sequencing and whole-exome sequencing are becoming more widely applied in clinical medicine to help diagnose rare genetic diseases. Identification of the underlying causative mutations by genome-wide sequencing is greatly facilitated by concurrent analysis of multiple family members, most often the mother-father-proband trio, using bioinformatics pipelines that filter genetic variants by mode of inheritance. However, current pipelines are limited to Mendelian inheritance patterns and do not specifically address disorders caused by mutations in imprinted genes, such as forms of Angelman syndrome and Beckwith-Wiedemann syndrome. Using publicly available tools, we implemented a genetic inheritance search mode to identify imprinted-gene mutations. Application of this search mode to whole-genome sequences from a family trio led to a diagnosis for a proband for whom extensive clinical testing and Mendelian inheritance-based sequence analysis were nondiagnostic. The condition in this patient, IMAGe syndrome, is likely caused by the heterozygous mutation c.832A>G (p.Lys278Glu) in the imprinted gene CDKN1C. The genotypes and disease status of six members of the family are consistent with maternal expression of the gene, and allele-biased expression was confirmed by RNA-Seq for the heterozygotes. This analysis demonstrates that an imprinted-gene search mode is a valuable addition to genome sequence analysis pipelines for identifying disease-causative variants.