By informing timely targeted treatments, rapid whole-genome sequencing can improve the outcomes of seriously ill children with genetic diseases, particularly infants in neonatal and pediatric intensive care units (ICUs). The need for highly qualified professionals to decipher results, however, precludes widespread implementation. We describe a platform for population-scale, provisional diagnosis of genetic diseases with automated phenotyping and interpretation. Genome sequencing was expedited by bead-based genome library preparation directly from blood samples and sequencing of paired 100-nt reads in 15.5 hours. Clinical natural language processing (CNLP) automatically extracted children’s deep phenomes from electronic health records with 80% precision and 93% recall. In 101 children with 105 genetic diseases, a mean of 4.3 CNLP-extracted phenotypic features matched the expected phenotypic features of those diseases, compared with a match of 0.9 phenotypic features used in manual interpretation. We automated provisional diagnosis by combining the ranking of the similarity of a patient’s CNLP phenome with respect to the expected phenotypic features of all genetic diseases, together with the ranking of the pathogenicity of all of the patient’s genomic variants. Automated, retrospective diagnoses concurred well with expert manual interpretation (97% recall and 99% precision in 95 children with 97 genetic diseases). Prospectively, our platform correctly diagnosed three of seven seriously ill ICU infants (100% precision and recall) with a mean time saving of 22:19 hours. In each case, the diagnosis affected treatment. Genome sequencing with automated phenotyping and interpretation in a median of 20:10 hours may increase adoption in ICUs and, thereby, timely implementation of precise treatments.

Although transforming growth factor-beta (TGF-␤) signaling negatively regulates branching morphogenesis in early lung development, few studies to date have addressed the role of this family of growth factors during late lung development. We describe here that the expression, tissue localization, and activity of components of the TGF-␤ signaling machinery are dynamically regulated during late lung development in the mouse and human. Pronounced changes in the expression and localization of the TGF-␤ receptors Acvrl1, Tgfbr1, Tgfbr2, Tgfbr3, and endoglin, and the intracellular messengers Smad2, Smad3, Smad4, Smad6, and Smad7 were noted as mouse and human lungs progressed through the canalicular, saccular, and alveolar stages of development. TGF-␤ signaling, assessed by phosphorylation of Smad2, was detected in the vascular and airway smooth muscle, as well as the alveolar and airway epithelium throughout late lung development. These data suggest that active TGF-␤ signaling is required for normal late lung development. Developmental Dynamics 237:259 -269, 2008.

Breathing-disordered states, such as in obstructive sleep apnea, which are cyclical in nature, have been postulated to induce neurocognitive morbidity in both pediatric and adult populations. The oscillatory nature of intermittent hypoxia, especially when chronic, may mimic the paradigm of ischemia-reperfusion in that tissues and cells are exposed to episodes of low and high O(2) and this may lead to oxidant stress. Therefore, we decided to explore the potential contribution of oxidant stress in our intermittent hypoxia/hypercapnia animal model and the role that mitochondria might play in this stress. Neonatal mice were exposed to intermittent hypoxia/hypercapnia for 10 days and 2 wk. Combined intermittent hypoxia/hypercapnia led to a marked increase in apoptotic cell death in the cerebral cortex. Oxygen consumption studies in isolated mitochondria from intermittent hypoxia/hypercapnia-exposed brains demonstrated significant reductions in both state 4 and state 3 respiratory activities by approximately 60% and 75%, respectively. Electron paramagnetic resonance spectroscopy registered a significant increase in superoxide production during nonphosphorylating state 4 by 37%, although superoxide leakage during state 3 did not increase upon treatment. Neuronal superoxide-specific dihydroethidium oxidation was also greater in exposed animals. These studies indicate that intermittent hypoxia/hypercapnia leads to oxidative stress due to mitochondrial response within the mouse central nervous system.

Bacteria of the genusT he genus Shigella comprises 4 species, including Shigella flexneri, which is comprised of 14 different serotypes and subserotypes (2). S. flexneri strain M90T Sm is commonly used in the laboratory to understand the molecular basis of shigellosis (1,5,9).Two amplification-free libraries were constructed from 5 g of M90T Sm genomic DNA each, and the genome was sequenced using a 2-by-110-base paired-end strategy on the Illumina HiSeq2000 platform. Libraries were constructed using the NEBNext DNA sample prep master mix set 1 (NEB; part number E6040L) and barcoded in an overnight ligation. Libraries were size selected from 450 bp to 500 bp, quantified by quantitative PCR (qPCR) (Kapa BioSystems), pooled, requantified, and clustered using cBot on a single lane with TruSeq PE cluster kit v2 chemistry. A total of 129,937,458 single-end reads were filtered by demultiplexing using two indexes (AGTCGATC/CAGTCTGA) and the signal purity filter. The resulting 73,325,436 reads were trimmed to 54 bp, the point at which the mean quality score dropped below 30 (Illumina 1.5ϩ phred quality score), using the Fastx toolkit (version 0.0.13) (http://hannonlab.cshl.edu/fastx_toolkit/index .html). Half of the preprocessed reads (34,231,825 reads) were mapped onto the chromosomal sequence of S. flexneri serotype 5b strain 8401 (GenBank accession no. NC_008258) (6) using the Burrows-Wheeler aligner (BWA) (version 0.5.9) (3). A total of 28,876,862 reads (84.3%) were successfully mapped to a depth of 340ϫ. Single nucleotide polymorphism (SNP) calls and consensus sequence generation were performed using the pileup function in SAMtools (version 0.1.16) (4). The integrated genomics viewer (IGV) (version 1.5) (7) was used to examine regions of low coverage (0ϫ to 160ϫ) and/or low quality. A total of 39 positions within the single-copy region of the genome were verified by Sanger sequencing. The remaining 119 gaps were within multicopy regions, such as insertion sequences (IS), ribosomal DNAs, and the tufA gene. The GϩC content was calculated using Artemis (version 13.0) (8).The sequence of the S. flexneri M90T Sm genome covers 99.9% of the 4,574,284-bp chromosome of S. flexneri 8401 (6). A total of 1,433 SNPs and 176 indels were identified. About one-third of the SNPs (543 sites) cause nonsynonymous substitutions in S. flexneri M90T Sm. Twenty-one indels in M90T Sm cause frameshifts, and 9 indels correct 9 frame-shifted genes in S. flexneri 8401. A tandem repeat of tRNAs (tRNA-Lys and tRNA-Val) and one hypothetical protein that both do not exist in S. flexneri 8401 were found in M90T Sm. The M90T Sm genome contains 3,676 open reading frames (ORFs) (excluding IS ORFs), 99 tRNA genes, and 22 rRNA genes. The sequence has an average GϩC content of 50.9% in its entirety with a GϩC content of 52% within coding genes.The S. flexneri M90T Sm genome will enhance ongoing wholegenome comparisons across all S. flexneri serotypes for a fuller understanding of the evolution of S. flexneri and provides accurate sequence information need...

Lung development is a highly orchestrated process characterized by timed expression and activation of growth factor and protease/antiprotease systems. This interplay is essential in regulating vasculogenesis, alveolarization, and epithelial to mesenchymal transition during lung development. Alterations in the proteolytic/antiproteolytic balance of the lung have been associated with several respiratory diseases characterized by changes in the lung extracellular matrix (ECM). Here, we characterized the expression pattern of matrix metalloproteases (MMP) and their inhibitors, the tissue inhibitors of metalloproteases (TIMP), in human and mouse lung development. Using MMP/TIMP expression arrays, RT-PCR, Western Blotting, and ELISA analyses, we demonstrate that fetal human lung is characterized by a dominant proteolytic profile with high MMP-2 and little TIMP-3 expression. Adult human lung, in contrast, exhibits a more anti-proteolytic profile with decreased MMP-2 and increased TIMP-3 expression. MMP-14, MMP-20, TIMP-1, and TIMP-2 were constitutively expressed, irrespective of the developmental stage. Similar results were obtained using mouse lungs of different developmental stages, with the addition that in mouse lung, TIMP-2 and TIMP-3 were upregulated as lung development progressed. Exposure of neonatal mice to chronic hypoxia (10% O2), a stimulus that leads to an arrest of lung development, resulted in upregulation of MMP-2 with a concomitant downregulation of TIMP-2. These results provide a comprehensive analysis of MMP and TIMP expression during human and mouse lung development. MMP-2, TIMP-2, and TIMP-3 may be key regulatory enzymes during lung development, possibly through their complex action on ECM components, membrane receptor ectodomain shedding, and growth factor bioactivity.

Severe hypoxia can lead to injury and mortality in vertebrate or invertebrate organisms. Our research is focused on understanding the molecular mechanisms that lead to injury or adaptation to hypoxic stress using Drosophila as a model system. In this current study, we employed the UAS–Gal4 system to dissect the protective role of Hsp70 in specific tissues in vivo under severe hypoxia. In contrast to an over-expression in tissues like muscles, heart and brain, we found that over-expression of Hsp70 in hemocytes of flies provides a remarkable survival benefit to flies exposed to severe hypoxia for days. Furthermore, these flies were not only tolerant to severe hypoxia but also to other stresses such as oxidant stress (e.g., paraquat feeding or hyperoxia). Interestingly we observed that the better survival with Hsp70 over-expression in hemocytes under hypoxia or oxidant stress is causally linked to reactive oxygen species (ROS) reduction in whole flies. We also show that hemocytes are a major source of ROS generation, leading to injury during hypoxia and their elimination results in a better survival under hypoxia. Hence, our study identified a protective role of Hsp70 in Drosophila hemocytes which is linked to ROS reduction in the whole flies and thus helps in their remarkable survival during oxidant or hypoxic stress.

Despite the deleterious effects associated with elevated carbon dioxide (CO(2)) or hypercapnia, it has been hypothesized that CO(2) can protect the lung from injury. However, the effects of chronic hypercapnia on the neonatal lung are unknown. Hence, we investigated the effect of chronic hypercapnia on neonatal mouse lung to identify genes that could potentially contribute to hypercapnia-mediated lung protection. Newborn mouse litters were exposed to 8% CO(2), 12% CO(2), or room air for 2 wk. Lungs were excised and analyzed for morphometric alterations. The alveolar walls of CO(2)-exposed mice appeared thinner than those of controls. Analyses of gene expression differences by microarrays revealed that genes from a variety of functional categories were differentially expressed following hypercapnia treatment, including those encoding growth factors, chemokines, cytokines, and endopeptidases. In particular and of major interest, the expression level of genes encoding surfactant proteins A and D, as well as chloride channel calcium-activated 3, were significantly increased, but the expression of WNT1-inducible signaling pathway protein 2 was significantly decreased. The significant changes in gene expression occurred mostly at 8% CO(2), but only a few at 12% CO(2). Our results lead us to conclude that 1) there are a number of gene families that may contribute to hypercapnia-mediated lung protection; 2) the upregulation of surfactant proteins A and D may play a role as anti-inflammatory or antioxidant agents; and 3) the effects of CO(2) seem to depend on the level to which the lung is exposed.

The Epic electronic health record (EHR) platform supports structured data entry systems (SDES), which allow developers, with input from users, to create highly customized patient-record templates in order to maximize data completeness and to standardize structure. There are many potential advantages of using discrete data fields in the EHR to capture data for secondary analysis and epidemiological research, but direct data acquisition from clinicians remains one of the largest obstacles to leveraging the EHR for secondary use. Physician resistance to SDES is multifactorial. A 35-item questionnaire based on Unified Theory of Acceptance and Use of Technology, was used to measure attitudes, facilitation, and potential incentives for adopting SDES for clinical documentation among 25 pediatric specialty physicians and surgeons. Statistical analysis included chi-square for categorical data as well as independent sample t-tests and analysis of variance for continuous variables. Mean scores of the nine constructs demonstrated primarily positive physician attitudes toward SDES, while the surgeons were neutral. Those under 40 were more likely to respond that facilitating conditions for structured entry existed as compared to the two older age groups (p = .02). Pediatric surgeons were significantly less positive than specialty physicians about SDES effects on Performance (p = .01) and the effect of Social Influence (p = .02); but in more agreement that use of forms was voluntary (p = .02). Attitudinal differences likely reflect medical training, clinical practice workflows, and division specific practices. Identified resistance indicate efforts to increase SDES adoption should be discipline-targeted rather than a uniform approach.