ASU Scholarship Showcase

There are many proteomic applications that require large collections of purified protein, but parallel production of large numbers of different proteins remains a very challenging task. To help meet the needs of the scientific community, we have developed a human protein production pipeline. Using high-throughput (HT) methods, we transferred the genes of 31 full-length proteins into three expression vectors, and expressed the collection as N-terminal HaloTag fusion proteins in Escherichia coli and two commercial cell-free (CF) systems, wheat germ extract (WGE) and HeLa cell extract (HCE). Expression was assessed by labeling the fusion proteins specifically and covalently with a fluorescent HaloTag ligand and detecting its fluorescence on a LabChip[superscript ®] GX microfluidic capillary gel electrophoresis instrument. This automated, HT assay provided both qualitative and quantitative assessment of recombinant protein. E. coli was only capable of expressing 20% of the test collection in the supernatant fraction with ≥20 μg yields, whereas CF systems had ≥83% success rates. We purified expressed proteins using an automated HaloTag purification method. We purified 20, 33, and 42% of the test collection from E. coli, WGE, and HCE, respectively, with yields ≥1 μg and ≥90% purity. Based on these observations, we have developed a triage strategy for producing full-length human proteins in these three expression systems.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene output at the post-transcriptional level by targeting degenerate elements primarily in 3′untranslated regions (3′UTRs) of mRNAs. Individual miRNAs can regulate networks of hundreds of genes, yet for the majority of miRNAs few, if any, targets are known. Misexpression of miRNAs is also a major contributor to cancer progression, thus there is a critical need to validate miRNA targets in high-throughput to understand miRNAs' contribution to tumorigenesis. Here we introduce a novel high-throughput assay to detect miRNA targets in 3′UTRs, called Luminescent Identification of Functional Elements in 3′UTRs (3′LIFE). We demonstrate the feasibility of 3′LIFE using a data set of 275 human 3′UTRs and two cancer-relevant miRNAs, let-7c and miR-10b, and compare our results to alternative methods to detect miRNA targets throughout the genome. We identify a large number of novel gene targets for these miRNAs, with only 32% of hits being bioinformatically predicted and 27% directed by non-canonical interactions. Functional analysis of target genes reveals consistent roles for each miRNA as either a tumor suppressor (let-7c) or oncogenic miRNA (miR-10b), and preferentially target multiple genes within regulatory networks, suggesting 3′LIFE is a rapid and sensitive method to detect miRNA targets in high-throughput.

We report a device to fill an array of small chemical reaction chambers (microreactors) with reagent and then seal them using pressurized viscous liquid acting through a flexible membrane. The device enables multiple, independent chemical reactions involving free floating intermediate molecules without interference from neighboring reactions or external environments. The device is validated by protein expressed in situ directly from DNA in a microarray of ~10,000 spots with no diffusion during three hours incubation. Using the device to probe for an autoantibody cancer biomarker in blood serum sample gave five times higher signal to background ratio compared to standard protein microarray expressed on a flat microscope slide. Physical design principles to effectively fill the array of microreactors with reagent and experimental results of alternate methods for sealing the microreactors are presented.

Sera from patients with ovarian cancer contain autoantibodies (AAb) to tumor-derived proteins that are potential biomarkers for early detection. To detect AAb, we probed high-density programmable protein microarrays (NAPPA) expressing 5177 candidate tumor antigens with sera from patients with serous ovarian cancer (n = 34 cases/30 controls) and measured bound IgG. Of these, 741 antigens were selected and probed with an independent set of ovarian cancer sera (n = 60 cases/60 controls). Twelve potential autoantigens were identified with sensitivities ranging from 13 to 22% at >93% specificity. These were retested using a Luminex bead array using 60 cases and 60 controls, with sensitivities ranging from 0 to 31.7% at 95% specificity. Three AAb (p53, PTPRA, and PTGFR) had area under the curve (AUC) levels >60% (p < 0.01), with the partial AUC (SPAUC) over 5 times greater than for a nondiscriminating test (p < 0.01). Using a panel of the top three AAb (p53, PTPRA, and PTGFR), if at least two AAb were positive, then the sensitivity was 23.3% at 98.3% specificity. AAb to at least one of these top three antigens were also detected in 7/20 sera (35%) of patients with low CA 125 levels and 0/15 controls. AAb to p53, PTPRA, and PTGFR are potential biomarkers for the early detection of ovarian cancer.

Background: Mutual policing is an important mechanism for reducing conflict in cooperative groups. In societies of ants, bees, and wasps, mutual policing of worker reproduction can evolve when workers are more closely related to the queen's sons than to the sons of workers or when the costs of worker reproduction lower the inclusive fitness of workers. During colony growth, relatedness within the colony remains the same, but the costs of worker reproduction may change. The costs of worker reproduction are predicted to be greatest in incipient colonies. If the costs associated with worker reproduction outweigh the individual direct benefits to workers, policing mechanisms as found in larger colonies may be absent in incipient colonies.

Results: We investigated policing behavior across colony growth in the ant 'Camponotus floridanus.' In large colonies of this species, worker reproduction is policed by the destruction of worker-laid eggs. We found workers from incipient colonies do not exhibit policing behavior, and instead tolerate all conspecific eggs. The change in policing behavior is consistent with changes in egg surface hydrocarbons, which provide the informational basis for policing; eggs laid by queens from incipient colonies lack the characteristic hydrocarbons on the surface of eggs laid by queens from large colonies, making them chemically indistinguishable from worker-laid eggs. We also tested the response to fertility information in the context of queen tolerance. Workers from incipient colonies attacked foreign queens from large colonies; whereas workers from large colonies tolerated such queens. Workers from both incipient and large colonies attacked foreign queens from incipient colonies.

Conclusions: Our results provide novel insights into the regulation of worker reproduction in social insects at both the proximate and ultimate levels. At the proximate level, our results show that mechanisms of social regulation, such as the response to fertility signals, change dramatically over a colony's life cycle. At the ultimate level, our results emphasize the importance of factors besides relatedness in predicting the level of conflict within a colony. Our results also suggest policing may not be an important regulatory force at every stage of colony development. Changes relating to the life cycle of the colony are sufficient to account for major differences in social regulation in an insect colony. Mechanisms of conflict mediation observed in one phase of a social group's development cannot be generalized to all stages.

Background: Lizards are evolutionarily the most closely related vertebrates to humans that can lose and regrow an entire appendage. Regeneration in lizards involves differential expression of hundreds of genes that regulate wound healing, musculoskeletal development, hormonal response, and embryonic morphogenesis. While microRNAs are able to regulate large groups of genes, their role in lizard regeneration has not been investigated.

Results: MicroRNA sequencing of green anole lizard (Anolis carolinensis) regenerating tail and associated tissues revealed 350 putative novel and 196 known microRNA precursors. Eleven microRNAs were differentially expressed between the regenerating tail tip and base during maximum outgrowth (25 days post autotomy), including miR-133a, miR-133b, and miR-206, which have been reported to regulate regeneration and stem cell proliferation in other model systems. Three putative novel differentially expressed microRNAs were identified in the regenerating tail tip.

Conclusions: Differentially expressed microRNAs were identified in the regenerating lizard tail, including known regulators of stem cell proliferation. The identification of 3 putative novel microRNAs suggests that regulatory networks, either conserved in vertebrates and previously uncharacterized or specific to lizards, are involved in regeneration. These findings suggest that differential regulation of microRNAs may play a role in coordinating the timing and expression of hundreds of genes involved in regeneration.

Background: Tissue-specific RNA plasticity broadly impacts the development, tissue identity and adaptability of all organisms, but changes in composition, expression levels and its impact on gene regulation in different somatic tissues are largely unknown. Here we developed a new method, polyA-tagging and sequencing (PAT-Seq) to isolate high-quality tissue-specific mRNA from Caenorhabditis elegans intestine, pharynx and body muscle tissues and study changes in their tissue-specific transcriptomes and 3’UTRomes.

Results: We have identified thousands of novel genes and isoforms differentially expressed between these three tissues. The intestine transcriptome is expansive, expressing over 30% of C. elegans mRNAs, while muscle transcriptomes are smaller but contain characteristic unique gene signatures. Active promoter regions in all three tissues reveal both known and novel enriched tissue-specific elements, along with putative transcription factors, suggesting novel tissue-specific modes of transcription initiation. We have precisely mapped approximately 20,000 tissue-specific polyadenylation sites and discovered that about 30% of transcripts in somatic cells use alternative polyadenylation in a tissue-specific manner, with their 3’UTR isoforms significantly enriched with microRNA targets.

Conclusions: For the first time, PAT-Seq allowed us to directly study tissue specific gene expression changes in an in vivo setting and compare these changes between three somatic tissues from the same organism at single-base resolution within the same experiment. We pinpoint precise tissue-specific transcriptome rearrangements and for the first time link tissue-specific alternative polyadenylation to miRNA regulation, suggesting novel and unexplored tissue-specific post-transcriptional regulatory networks in somatic cells.

Introduction: Juvenile idiopathic arthritis (JIA) is a heterogeneous disease characterized by chronic joint inflammation of unknown cause in children. JIA is an autoimmune disease and small numbers of autoantibodies have been reported in JIA patients. The identification of antibody markers could improve the existing clinical management of patients.

Methods: A pilot study was performed on the application of a high-throughput platform, the nucleic acid programmable protein array (NAPPA), to assess the levels of antibodies present in the systemic circulation and synovial joint of a small cohort of juvenile arthritis patients. Plasma and synovial fluid from 10 JIA patients was screened for antibodies against 768 proteins on NAPPAs.

Results: Quantitative reproducibility of NAPPAs was demonstrated with > 0.95 intra-array and inter-array correlations. A strong correlation was also observed for the levels of antibodies between plasma and synovial fluid across the study cohort (r = 0.96). Differences in the levels of 18 antibodies were revealed between sample types across all patients. Patients were segregated into two clinical subtypes with distinct antibody signatures by unsupervised hierarchical cluster analysis.

Conclusion: The NAPPAs provide a high-throughput quantitatively reproducible platform to screen for disease-specific autoantibodies at the proteome level on a microscope slide. The strong correlation between the circulating antibody levels and those of the inflamed joint represents a novel finding and provides confidence to use plasma for discovery of autoantibodies in JIA, thus circumventing the challenges associated with joint aspiration. We expect that autoantibody profiling of JIA patients on NAPPAs could yield antibody markers that can act as criteria to stratify patients, predict outcomes and understand disease etiology at the molecular level.

Background: 3′untranslated regions (3′UTRs) are poorly understood portions of eukaryotic mRNAs essential for post-transcriptional gene regulation. Sequence elements in 3′UTRs can be target sites for regulatory molecules such as RNA binding proteins and microRNAs (miRNAs), and these interactions can exert significant control on gene networks. However, many such interactions remain uncharacterized due to a lack of high-throughput (HT) tools to study 3′UTR biology. HT cloning efforts such as the human ORFeome exemplify the potential benefits of genomic repositories for studying human disease, especially in relation to the discovery of biomarkers and targets for therapeutic agents. Currently there are no publicly available human 3′UTR libraries. To address this we have prepared the first version of the human 3′UTRome (h3′UTRome v1) library. The h3′UTRome is produced to a single high quality standard using the same recombinational cloning technology used for the human ORFeome, enabling universal operating methods and high throughput experimentation. The library is thoroughly sequenced and annotated with simple online access to information, and made publicly available through gene repositories at low cost to all scientists with minimal restriction.

Results: The first release of the h3′UTRome library comprises 1,461 human 3′UTRs cloned into Gateway® entry vectors, ready for downstream analyses. It contains 3′UTRs for 985 transcription factors, 156 kinases, 171 RNA binding proteins, and 186 other genes involved in gene regulation and in disease. We demonstrate the feasibility of the h3′UTRome library by screening a panel of 87 3′UTRs for targeting by two miRNAs: let-7c, which is implicated in tumorigenesis, and miR-221, which is implicated in atherosclerosis and heart disease. The panel is enriched with genes involved in the RAS signaling pathway, putative novel targets for the two miRNAs, as well as genes implicated in tumorigenesis and heart disease.

Conclusions: The h3′UTRome v1 library is a modular resource that can be utilized for high-throughput screens to identify regulatory interactions between trans-acting factors and 3′UTRs, Importantly, the library can be customized based on the specifications of the researcher, allowing the systematic study of human 3′UTR biology.

Background: The molecular mechanisms of variations in individual longevity are not well understood, even though longevity can be increased substantially by means of diverse experimental manipulations. One of the factors supposed to be involved in the increase of longevity is a higher stress resistance. To test this hypothesis in a natural system, eusocial insects such as bees or ants are ideally suited. In contrast to most other eusocial insects, ponerine ants show a peculiar life history that comprises the possibility to switch during adult life from a normal worker to a reproductive gamergate, therewith increasing their life expectancy significantly.

Results: We show that increased resistance against major stressors, such as reactive oxygen species and infection accompanies the switch from a life-history trait with normal lifespan to one with a longer life expectancy. A short period of social isolation was sufficient to enhance stress resistance of workers from the ponerine ant species Harpegnathos saltator significantly. All ant groups with increased stress resistances (reproducing gamergates and socially isolated workers) have lower catalase activities and glutathione levels than normal workers. Therewith, these ants resemble the characteristics of the youngest ants in the colony.

Conclusions: Social insects with their specific life history including a switch from normal workers to reproducing gamergates during adult life are well suited for ageing research. The regulation of stress resistance in gamergates seemed to be modified compared to foraging workers in an economic way. Interestingly, a switch towards more stress resistant animals can also be induced by a brief period of social isolation, which may already be associated with a shift to a reproductive trajectory. In Harpegnathos saltator, stress resistances are differently and potentially more economically regulated in reproductive individuals, highlighting the significance of reproduction for an increase in longevity in social insects. As already shown for other organisms with a long lifespan, this trait is not directly coupled to higher levels of enzymatic and non-enzymatic antioxidants.