Project description:<p>The goal of the RSV Bronchiolitis in Early Life (RBEL) study is to determine how specific genetic, biologic, and immunologic characteristics interact to predispose individuals to develop asthma. Participants were carefully recruited by selecting a prospective cohort of 206 infants with severe respiratory syncytial virus (RSV) bronchiolitis who were at substantial risk of developing asthma.</p>
Project description:Respiratory syncytial virus (RSV) is a prevalent pathogen globally, can cause severe disease in older adults, and remains the leading cause of bronchiolitis and pneumonia in the United States for children during their first year of life. Despite its prevalence worldwide, RSV specific pharmacologic interventions remain unavailable for most infected patients. Although vaccines are available for a subset of adults, further investigation of the molecular interactions between RSV and the host remains essential to understanding this prolific pathogen. To aid our understanding of the host response in both RSV infected cells, and uninfected bystanders, we utilized single-cell RNA sequencing.
Project description:Respiratory Syncytial virus (RSV) is the most common cause of childhood viral bronchiolitis and lung injury. Inflammatory responses significantly contribute to lung pathologies during RSV infections and bronchiolitis but the exact mechanisms have not been completely defined. The double-stranded RNA-activated protein kinase (PKR) functions to inhibit viral replication and participates in several signaling pathways associated with innate inflammatory immune responses. Using a functionally defective PKR (PKR-/-) mouse model, we investigated the role of this kinase in early events of RSV-induced inflammation. Our data showed that bronchoalveolar lavage (BAL) fluid of infected PKR-/- mice had significantly lower levels of several innate inflammatory cytokines and chemokines. Histological examinations revealed that there was less lung injury in infected PKR-/- mice as compared to the wild type. A genome-wide analysis showed that several early anti viral and immune regulatory genes were affected by PKR activation. These data suggest that PKR is a signaling molecule for immune responses during RSV infections.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:Although childhood asthma is in part an airway epithelial disorder, the development of the airway epithelium in asthma is not understood. We sought to characterize airway epithelial developmental phenotypes in those with and without recurrent wheeze and the impact of infant infection with respiratory syncytial virus (RSV). Nasal airway epithelial cells (NAECs) were collected at age 2-3 years from an a priori designed nested birth cohort of children from four mutually exclusive groups of wheezers/non-wheezers and RSV-infected/uninfected in the first year of life. NAECs were cultured in air-liquid interface differentiation conditions followed by a combined analysis of single cell RNA sequencing (scRNA-seq) and in vitro infection with respiratory syncytial virus (RSV). NAECs from children with a wheeze phenotype were characterized by abnormal differentiation and basal cell activation of developmental pathways, plasticity in precursor differentiation and a delayed onset of maturation. NAECs from children with wheeze also had increased diversity of currently known RSV receptors and blunted anti-viral immune responses to in vitro infection. The most dramatic changes in differentiation of cultured epithelium were observed in NAECs derived from children that had both wheeze and RSV in the first year of life. Together this suggests that airway epithelium in children with wheeze is developmentally reprogrammed and characterized by increased barrier permeability, decreased antiviral response, and increased RSV receptors, which may predispose to and amplify the effects of RSV infection in infancy and susceptibility to other asthma risk factors that interact with the airway mucosa.
Project description:As the evolution of miRNA genes has been found to be one of the important factors in formation of the modern type of man, we performed a comparative analysis of the evolution of miRNA genes in two archaic hominines, Homo sapiens neanderthalensis and Homo sapiens denisova, and elucidated the expression of their target mRNAs in bain.A comparative analysis of the genomes of primates, including species in the genus Homo, identified a group of miRNA genes having fixed substitutions with important implications for the evolution of Homo sapiens neanderthalensis and Homo sapiens denisova. The mRNAs targeted by miRNAs with mutations specific for Homo sapiens denisova exhibited enhanced expression during postnatal brain development in modern humans. By contrast, the expression of mRNAs targeted by miRNAs bearing variations specific for Homo sapiens neanderthalensis was shown to be enhanced in prenatal brain development.Our results highlight the importance of changes in miRNA gene sequences in the course of Homo sapiens denisova and Homo sapiens neanderthalensis evolution. The genetic alterations of miRNAs regulating the spatiotemporal expression of multiple genes in the prenatal and postnatal brain may contribute to the progressive evolution of brain function, which is consistent with the observations of fine technical and typological properties of tools and decorative items reported from archaeological Denisovan sites. The data also suggest that differential spatial-temporal regulation of gene products promoted by the subspecies-specific mutations in the miRNA genes might have occurred in the brains of Homo sapiens denisova and Homo sapiens neanderthalensis, potentially contributing to the cultural differences between these two archaic hominines.