ASU Scholarship Showcase

In vitro models that mimic in vivo host-pathogen interactions are needed to evaluate candidate drugs that inhibit bacterial virulence traits. We established a new approach to study Pseudomonas aeruginosa biofilm susceptibility on biotic surfaces, using a three-dimensional (3-D) lung epithelial cell model. P. aeruginosa formed antibiotic resistant biofilms on 3-D cells without affecting cell viability. The biofilm-inhibitory activity of antibiotics and/or the anti-biofilm peptide DJK-5 were evaluated on 3-D cells compared to a plastic surface, in medium with and without fetal bovine serum (FBS). In both media, aminoglycosides were more efficacious in the 3-D cell model. In serum-free medium, most antibiotics (except polymyxins) showed enhanced efficacy when 3-D cells were present. In medium with FBS, colistin was less efficacious in the 3-D cell model. DJK-5 exerted potent inhibition of P. aeruginosa association with both substrates, only in serum-free medium. DJK-5 showed stronger inhibitory activity against P. aeruginosa associated with plastic compared to 3-D cells. The combined addition of tobramycin and DJK-5 exhibited more potent ability to inhibit P. aeruginosa association with both substrates. In conclusion, lung epithelial cells influence the efficacy of most antimicrobials against P. aeruginosa biofilm formation, which in turn depends on the presence or absence of FBS.

Background: The discovery of genetic associations is an important factor in the understanding of human illness to derive disease pathways. Identifying multiple interacting genetic mutations associated with disease remains challenging in studying the etiology of complex diseases. And although recently new single nucleotide polymorphisms (SNPs) at genes implicated in immune response, cholesterol/lipid metabolism, and cell membrane processes have been confirmed by genome-wide association studies (GWAS) to be associated with late-onset Alzheimer's disease (LOAD), a percentage of AD heritability continues to be unexplained. We try to find other genetic variants that may influence LOAD risk utilizing data mining methods.

Methods: Two different approaches were devised to select SNPs associated with LOAD in a publicly available GWAS data set consisting of three cohorts. In both approaches, single-locus analysis (logistic regression) was conducted to filter the data with a less conservative p-value than the Bonferroni threshold; this resulted in a subset of SNPs used next in multi-locus analysis (random forest (RF)). In the second approach, we took into account prior biological knowledge, and performed sample stratification and linkage disequilibrium (LD) in addition to logistic regression analysis to preselect loci to input into the RF classifier construction step.

Results: The first approach gave 199 SNPs mostly associated with genes in calcium signaling, cell adhesion, endocytosis, immune response, and synaptic function. These SNPs together with APOE and GAB2 SNPs formed a predictive subset for LOAD status with an average error of 9.8% using 10-fold cross validation (CV) in RF modeling. Nineteen variants in LD with ST5, TRPC1, ATG10, ANO3, NDUFA12, and NISCH respectively, genes linked directly or indirectly with neurobiology, were identified with the second approach. These variants were part of a model that included APOE and GAB2 SNPs to predict LOAD risk which produced a 10-fold CV average error of 17.5% in the classification modeling.

Conclusions: With the two proposed approaches, we identified a large subset of SNPs in genes mostly clustered around specific pathways/functions and a smaller set of SNPs, within or in proximity to five genes not previously reported, that may be relevant for the prediction/understanding of AD.

Background: Glioblastoma is the most aggressive primary central nervous tumor and carries a very poor prognosis. Invasion precludes effective treatment and virtually assures tumor recurrence. In the current study, we applied analytical and bioinformatics approaches to identify a set of microRNAs (miRs) from several different human glioblastoma cell lines that exhibit significant differential expression between migratory (edge) and migration-restricted (core) cell populations. The hypothesis of the study is that differential expression of miRs provides an epigenetic mechanism to drive cell migration and invasion.

Results: Our research data comprise gene expression values for a set of 805 human miRs collected from matched pairs of migratory and migration-restricted cell populations from seven different glioblastoma cell lines. We identified 62 down-regulated and 2 up-regulated miRs that exhibit significant differential expression in the migratory (edge) cell population compared to matched migration-restricted (core) cells. We then conducted target prediction and pathway enrichment analysis with these miRs to investigate potential associated gene and pathway targets. Several miRs in the list appear to directly target apoptosis related genes. The analysis identifies a set of genes that are predicted by 3 different algorithms, further emphasizing the potential validity of these miRs to promote glioblastoma.

Conclusions: The results of this study identify a set of miRs with potential for decreased expression in invasive glioblastoma cells. The verification of these miRs and their associated targeted proteins provides new insights for further investigation into therapeutic interventions. The methodological approaches employed here could be applied to the study of other diseases to provide biomedical researchers and clinicians with increased opportunities for therapeutic interventions.

This study presents the first global transcriptional profiling and phenotypic characterization of the major human opportunistic fungal pathogen, Candida albicans, grown in spaceflight conditions. Microarray analysis revealed that C. albicans subjected to short-term spaceflight culture differentially regulated 452 genes compared to synchronous ground controls, which represented 8.3% of the analyzed ORFs. Spaceflight-cultured C. albicans–induced genes involved in cell aggregation (similar to flocculation), which was validated by microscopic and flow cytometry analysis. We also observed enhanced random budding of spaceflight-cultured cells as opposed to bipolar budding patterns for ground samples, in accordance with the gene expression data. Furthermore, genes involved in antifungal agent and stress resistance were differentially regulated in spaceflight, including induction of ABC transporters and members of the major facilitator family, downregulation of ergosterol-encoding genes, and upregulation of genes involved in oxidative stress resistance.

Finally, downregulation of genes involved in actin cytoskeleton was observed. Interestingly, the transcriptional regulator Cap1 and over 30% of the Cap1 regulon was differentially expressed in spaceflight-cultured C. albicans. A potential role for Cap1 in the spaceflight response of C. albicans is suggested, as this regulator is involved in random budding, cell aggregation, and oxidative stress resistance; all related to observed spaceflight-associated changes of C. albicans. While culture of C. albicans in microgravity potentiates a global change in gene expression that could induce a virulence-related phenotype, no increased virulence in a murine intraperitoneal (i.p.) infection model was observed under the conditions of this study. Collectively, our data represent an important basis for the assessment of the risk that commensal flora could play during human spaceflight missions. Furthermore, since the low fluid-shear environment of microgravity is relevant to physical forces encountered by pathogens during the infection process, insights gained from this study could identify novel infectious disease mechanisms, with downstream benefits for the general public.

The probiotic effects of Lactobacillus reuteri have been speculated to partly depend on its capacity to produce the antimicrobial substance reuterin during the reduction of glycerol in the gut. In this study, the potential of this process to protect human intestinal epithelial cells against infection with Salmonella enterica serovar Typhimurium was investigated. We used a three-dimensional (3-D) organotypic model of human colonic epithelium that was previously validated and applied to study interactions between S. Typhimurium and the intestinal epithelium that lead to enteric salmonellosis. Using this model system, we show that L. reuteri protects the intestinal cells against the early stages of Salmonella infection and that this effect is significantly increased when L. reuteri is stimulated to produce reuterin from glycerol. More specifically, the reuterin-containing ferment of L. reuteri caused a reduction in Salmonella adherence and invasion (1 log unit), and intracellular survival (2 log units). In contrast, the L. reuteri ferment without reuterin stimulated growth of the intracellular Salmonella population with 1 log unit. The short-term exposure to reuterin or the reuterin-containing ferment had no observed negative impact on intestinal epithelial cell health. However, long-term exposure (24 h) induced a complete loss of cell-cell contact within the epithelial aggregates and compromised cell viability. Collectively, these results shed light on a potential role for reuterin in inhibiting Salmonella-induced intestinal infections and may support the combined application of glycerol and L. reuteri. While future in vitro and in vivo studies of reuterin on intestinal health should fine-tune our understanding of the mechanistic effects, in particular in the presence of a complex gut microbiota, this the first report of a reuterin effect on the enteric infection process in any mammalian cell type.

Extra-intestinal pathogenic E. coli (ExPEC), including avian pathogenic E. coli (APEC), pose a considerable threat to both human and animal health, with illness causing substantial economic loss. APEC strain χ7122 (O78∶K80∶H9), containing three large plasmids [pChi7122-1 (IncFIB/FIIA-FIC), pChi7122-2 (IncFII), and pChi7122-3 (IncI₂)]; and a small plasmid pChi7122-4 (ColE2-like), has been used for many years as a model strain to study the molecular mechanisms of ExPEC pathogenicity and zoonotic potential. We previously sequenced and characterized the plasmid pChi7122-1 and determined its importance in systemic APEC infection; however the roles of the other pChi7122 plasmids were still ambiguous. Herein we present the sequence of the remaining pChi7122 plasmids, confirming that pChi7122-2 and pChi7122-3 encode an ABC iron transport system (eitABCD) and a putative type IV fimbriae respectively, whereas pChi7122-4 is a cryptic plasmid. New features were also identified, including a gene cluster on pChi7122-2 that is not present in other E. coli strains but is found in Salmonella serovars and is predicted to encode the sugars catabolic pathways. In vitro evaluation of the APEC χ7122 derivative strains with the three large plasmids, either individually or in combinations, provided new insights into the role of plasmids in biofilm formation, bile and acid tolerance, and the interaction of E. coli strains with 3-D cultures of intestinal epithelial cells. In this study, we show that the nature and combinations of plasmids, as well as the background of the host strains, have an effect on these phenomena. Our data reveal new insights into the role of extra-chromosomal sequences in fitness and diversity of ExPEC in their phenotypes.

Strategies are needed to improve repopulation of decellularized lung scaffolds with stromal and functional epithelial cells. We demonstrate that decellularized mouse lungs recellularized in a dynamic low fluid shear suspension bioreactor, termed the rotating wall vessel (RWV), contained more cells with decreased apoptosis, increased proliferation and enhanced levels of total RNA compared to static recellularization conditions. These results were observed with two relevant mouse cell types: bone marrow-derived mesenchymal stromal (stem) cells (MSCs) and alveolar type II cells (C10). In addition, MSCs cultured in decellularized lungs under static but not bioreactor conditions formed multilayered aggregates. Gene expression and immunohistochemical analyses suggested differentiation of MSCs into collagen I-producing fibroblast-like cells in the bioreactor, indicating enhanced potential for remodeling of the decellularized scaffold matrix. In conclusion, dynamic suspension culture is promising for enhancing repopulation of decellularized lungs, and could contribute to remodeling the extracellular matrix of the scaffolds with subsequent effects on differentiation and functionality of inoculated cells.

Background: Obesity is a metabolic disease caused by environmental and genetic factors. However, the epigenetic mechanisms of obesity are incompletely understood. The aim of our study was to investigate the role of skeletal muscle DNA methylation in combination with transcriptomic changes in obesity.

Results: Muscle biopsies were obtained basally from lean (n = 12; BMI = 23.4 ± 0.7 kg/m[superscript 2]) and obese (n = 10; BMI = 32.9 ± 0.7 kg/m[superscript 2]) participants in combination with euglycemic-hyperinsulinemic clamps to assess insulin sensitivity. We performed reduced representation bisulfite sequencing (RRBS) next-generation methylation and microarray analyses on DNA and RNA isolated from vastus lateralis muscle biopsies. There were 13,130 differentially methylated cytosines (DMC; uncorrected P < 0.05) that were altered in the promoter and untranslated (5' and 3'UTR) regions in the obese versus lean analysis. Microarray analysis revealed 99 probes that were significantly (corrected P < 0.05) altered. Of these, 12 genes (encompassing 22 methylation sites) demonstrated a negative relationship between gene expression and DNA methylation. Specifically, sorbin and SH3 domain containing 3 (SORBS3) which codes for the adapter protein vinexin was significantly decreased in gene expression (fold change −1.9) and had nine DMCs that were significantly increased in methylation in obesity (methylation differences ranged from 5.0 to 24.4 %). Moreover, differentially methylated region (DMR) analysis identified a region in the 5'UTR (Chr.8:22,423,530–22,423,569) of SORBS3 that was increased in methylation by 11.2 % in the obese group. The negative relationship observed between DNA methylation and gene expression for SORBS3 was validated by a site-specific sequencing approach, pyrosequencing, and qRT-PCR. Additionally, we performed transcription factor binding analysis and identified a number of transcription factors whose binding to the differentially methylated sites or region may contribute to obesity.

Conclusions: These results demonstrate that obesity alters the epigenome through DNA methylation and highlights novel transcriptomic changes in SORBS3 in skeletal muscle.

Background: Our publication of the BitTorious portal [1] demonstrated the ability to create a privatized distributed data warehouse of sufficient magnitude for real-world bioinformatics studies using minimal changes to the standard BitTorrent tracker protocol. In this second phase, we release a new server-side specification to accept anonymous philantropic storage donations by the general public, wherein a small portion of each user’s local disk may be used for archival of scientific data. We have implementated the server-side announcement and control portions of this BitTorrent extension into v3.0.0 of the BitTorious portal, upon which compatible clients may be built.

Results: Automated test cases for the BitTorious Volunteer extensions have been added to the portal’s v3.0.0 release, supporting validation of the “peer affinity” concept and announcement protocol introduced by this specification. Additionally, a separate reference implementation of affinity calculation has been provided in C++ for informaticians wishing to integrate into libtorrent-based projects.

Conclusions: The BitTorrent “affinity” extensions as provided in the BitTorious portal reference implementation allow data publishers to crowdsource the extreme storage prerequisites for research in “big data” fields. With sufficient awareness and adoption of BitTorious Volunteer-based clients by the general public, the BitTorious portal may be able to provide peta-scale storage resources to the scientific community at relatively insignificant financial cost.

Background: Centralized silos of genomic data are architecturally easier to initially design, develop and deploy than distributed models. However, as interoperability pains in EHR/EMR, HIE and other collaboration-centric life sciences domains have taught us, the core challenge of networking genomics systems is not in the construction of individual silos, but the interoperability of those deployments in a manner embracing the heterogeneous needs, terms and infrastructure of collaborating parties. This article demonstrates the adaptation of BitTorrent to private collaboration networks in an authenticated, authorized and encrypted manner while retaining the same characteristics of standard BitTorrent.

Results: The BitTorious portal was sucessfully used to manage many concurrent domestic Bittorrent clients across the United States: exchanging genomics data payloads in excess of 500GiB using the uTorrent client software on Linux, OSX and Windows platforms. Individual nodes were sporadically interrupted to verify the resilience of the system to outages of a single client node as well as recovery of nodes resuming operation on intermittent Internet connections.

Conclusions: The authorization-based extension of Bittorrent and accompanying BitTorious reference tracker and user management web portal provide a free, standards-based, general purpose and extensible data distribution system for large ‘omics collaborations.