Linking Human Diseases to Animal Models Using Ontology-Based Phenotype Annotation

A novel method for quantifying the similarity between phenotypes by the use of ontologies can be used to search for candidate genes, pathway members, and human disease models on the basis of phenotypes alone

AAV Exploits Subcellular Stress Associated with Inflammation, Endoplasmic Reticulum Expansion, and Misfolded Proteins in Models of Cystic Fibrosis

Barriers to infection act at multiple levels to prevent viruses, bacteria, and parasites from commandeering host cells for their own purposes. An intriguing hypothesis is that if a cell experiences stress, such as that elicited by inflammation, endoplasmic reticulum (ER) expansion, or misfolded proteins, then subcellular barriers will be less effective at preventing viral infection. Here we have used models of cystic fibrosis (CF) to test whether subcellular stress increases susceptibility to adeno-associated virus (AAV) infection. In human airway epithelium cultured at an air/liquid interface, physiological conditions of subcellular stress and ER expansion were mimicked using supernatant from mucopurulent material derived from CF lungs. Using this inflammatory stimulus to recapitulate stress found in diseased airways, we demonstrated that AAV infection was significantly enhanced. Since over 90% of CF cases are associated with a misfolded variant of Cystic Fibrosis Transmembrane Conductance Regulator (ΔF508-CFTR), we then explored whether the presence of misfolded proteins could independently increase susceptibility to AAV infection. In these models, AAV was an order of magnitude more efficient at transducing cells expressing ΔF508-CFTR than in cells expressing wild-type CFTR. Rescue of misfolded ΔF508-CFTR under low temperature conditions restored viral transduction efficiency to that demonstrated in controls, suggesting effects related to protein misfolding were responsible for increasing susceptibility to infection. By testing other CFTR mutants, G551D, D572N, and 1410X, we have shown this phenomenon is common to other misfolded proteins and not related to loss of CFTR activity. The presence of misfolded proteins did not affect cell surface attachment of virus or influence expression levels from promoter transgene cassettes in plasmid transfection studies, indicating exploitation occurs at the level of virion trafficking or processing. Thus, we surmised that factors enlisted to process misfolded proteins such as ΔF508-CFTR in the secretory pathway also act to restrict viral infection. In line with this hypothesis, we found that AAV trafficked to the microtubule organizing center and localized near Golgi/ER transport proteins. Moreover, AAV infection efficiency could be modulated with siRNA-mediated knockdown of proteins involved in processing ΔF508-CFTR or sorting retrograde cargo from the Golgi and ER (calnexin, KDEL-R, β-COP, and PSMB3). In summary, our data support a model where AAV exploits a compromised secretory system and, importantly, underscore the gravity with which a stressed subcellular environment, under internal or external insults, can impact infection efficiency

The Myosin Va Head Domain Binds to the Neurofilament-L Rod and Modulates Endoplasmic Reticulum (ER) Content and Distribution within Axons

The neurofilament light subunit (NF-L) binds to myosin Va (Myo Va) in neurons but the sites of interaction and functional significance are not clear. We show by deletion analysis that motor domain of Myo Va binds to the NF-L rod domain that forms the NF backbone. Loss of NF-L and Myo Va binding from axons significantly reduces the axonal content of ER, and redistributes ER to the periphery of axon. Our data are consistent with a novel function for NFs as a scaffold in axons for maintaining the content and proper distribution of vesicular organelles, mediated in part by Myo Va. Based on observations that the Myo Va motor domain binds to intermediate filament (IF) proteins of several classes, Myo Va interactions with IFs may serve similar roles in organizing organelle topography in different cell types

Regulation of Hemolysin Expression and Virulence of Staphylococcus aureus by a Serine/Threonine Kinase and Phosphatase

Exotoxins, including the hemolysins known as the alpha (α) and beta (β) toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1) were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1) increased expression. Transcription of the hla gene encoding α toxin was decreased in a Δstp1 mutant strain and increased in a Δstk1 strain. Microarray analysis of a Δstk1 mutant revealed increased transcription of additional exotoxins. A Δstp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Δstk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU), serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE) and a hypothetical protein (NWMN_1123) were present in the wild type and not in the Δstk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence

Mutation in Archain 1, a Subunit of COPI Coatomer Complex, Causes Diluted Coat Color and Purkinje Cell Degeneration

Intracellular trafficking is critical for delivering molecules and organelles to their proper destinations to carry out normal cellular functions. Disruption of intracellular trafficking has been implicated in the pathogenesis of various neurodegenerative disorders. In addition, a number of genes involved in vesicle/organelle trafficking are also essential for pigmentation, and loss of those genes is often associated with mouse coat-color dilution and human hypopigmentary disorders. Hence, we postulated that screening for mouse mutants with both neurological defects and coat-color dilution will help identify additional factors associated with intracellular trafficking in neuronal cells. In this study, we characterized a mouse mutant with a unique N-ethyl-N-nitrosourea (ENU)–induced mutation, named nur17. nur17 mutant mice exhibit both coat-color dilution and ataxia due to Purkinje cell degeneration in the cerebellum. By positional cloning, we identified that the nur17 mouse carries a T-to-C missense mutation in archain 1 (Arcn1) gene which encodes the δ subunit of the coat protein I (COPI) complex required for intracellular trafficking. Consistent with this function, we found that intracellular trafficking is disrupted in nur17 melanocytes. Moreover, the nur17 mutation leads to common characteristics of neurodegenerative disorders such as abnormal protein accumulation, ER stress, and neurofibrillary tangles. Our study documents for the first time the physiological consequences of the impairment of the ARCN1 function in the whole animal and demonstrates a direct association between ARCN1 and neurodegeneration

Monitoring Urban Tree Cover Using Object-Based Image Analysis and Public Domain Remotely Sensed Data

Urban forest ecosystems provide a range of social and ecological services, but due to the heterogeneity of these canopies their spatial extent is difficult to quantify and monitor. Traditional per-pixel classification methods have been used to map urban canopies, however, such techniques are not generally appropriate for assessing these highly variable landscapes. Landsat imagery has historically been used for per-pixel driven land use/land cover (LULC) classifications, but the spatial resolution limits our ability to map small urban features. In such cases, hyperspatial resolution imagery such as aerial or satellite imagery with a resolution of 1 meter or below is preferred. Object-based image analysis (OBIA) allows for use of additional variables such as texture, shape, context, and other cognitive information provided by the image analyst to segment and classify image features, and thus, improve classifications. As part of this research we created LULC classifications for a pilot study area in Seattle, WA, USA, using OBIA techniques and freely available public aerial photography. We analyzed the differences in accuracies which can be achieved with OBIA using multispectral and true-color imagery. We also compared our results to a satellite based OBIA LULC and discussed the implications of per-pixel driven vs. OBIA-driven field sampling campaigns. We demonstrated that the OBIA approach can generate good and repeatable LULC classifications suitable for tree cover assessment in urban areas. Another important finding is that spectral content appeared to be more important than spatial detail of hyperspatial data when it comes to an OBIA-driven LULC

Developing a Flexible Learning Activity on Biodiversity and Spatial Scale Concepts Using Open-Access Vegetation Datasets from the National Ecological Observatory Network

Biodiversity is a complex, yet essential, concept for undergraduate students in ecology and other natural sciences to grasp. As beginner scientists, students must learn to recognize, describe, and interpret patterns of biodiversity across various spatial scales and understand their relationships with ecological processes and human influences. It is also increasingly important for undergraduate programs in ecology and related disciplines to provide students with experiences working with large ecological datasets to develop students’ data science skills and their ability to consider how ecological processes that operate at broader spatial scales (macroscale) affect local ecosystems. To support the goals of improving student understanding of macroscale ecology and biodiversity at multiple spatial scales, we formed an interdisciplinary team that included grant personnel, scientists, and faculty from ecology and spatial sciences to design a flexible learning activity to teach macroscale biodiversity concepts using large datasets from the National Ecological Observatory Network (NEON). We piloted this learning activity in six courses enrolling a total of 109 students, ranging from midlevel ecology and GIS/remote sensing courses, to upper-level conservation biology. Using our classroom experiences and a pre/postassessment framework, we evaluated whether our learning activity resulted in increased student understanding of macroscale ecology and biodiversity concepts and increased familiarity with analysis techniques, software programs, and large spatio-ecological datasets. Overall, results suggest that our learning activity improved student understanding of biological diversity, biodiversity metrics, and patterns of biodiversity across several spatial scales. Participating faculty reflected on what went well and what would benefit from changes, and we offer suggestions for implementation of the learning activity based on this feedback. This learning activity introduced students to macroscale ecology and built student skills in working with big data (i.e., large datasets) and performing basic quantitative analyses, skills that are essential for the next generation of ecologists

Clinical Outcomes with Rapid Detection of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Isolates from Routine Blood Cultures

Staphylococcus aureus is a common cause of bacteremia, with a substantial impact on morbidity and mortality. Because of increasing rates of methicillin-resistant Staphylococcus aureus, vancomycin has become the standard empirical therapy. However, beta-lactam antibiotics remain the best treatment choice for methicillin-susceptible strains. Placing patients quickly on the optimal therapy is one goal of antimicrobial stewardship. This retrospective, observational, single-center study compared 33 control patients utilizing only traditional full-susceptibility methodology to 22 case patients utilizing rapid methodology with CHROMagar medium to detect and differentiate methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains hours before full susceptibilities were reported. The time to targeted therapy was statistically significantly different between control patients (mean, 56.5 ± 13.6 h) and case patients (44.3 ± 17.9 h) (P = 0.006). Intensive care unit status, time of day results emerged, and patient age did not make a difference in time to targeted therapy, either singly or in combination. Neither length of stay (P = 0.61) nor survival (P = 1.0) was statistically significantly different. Rapid testing yielded a significant result, with a difference of 12.2 h to targeted therapy. However, there is still room for improvement, as the difference in time to susceptibility test result between the full traditional methodology and CHROMagar was even larger (26.5 h). This study supports the hypothesis that rapid testing plays a role in antimicrobial stewardship by getting patients on targeted therapy faster