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.

Throughout the long history of virus-host co-evolution, viruses have developed delicate strategies to facilitate their invasion and replication of their genome, while silencing the host immune responses through various mechanisms. The systematic characterization of viral protein-host interactions would yield invaluable information in the understanding of viral invasion/evasion, diagnosis and therapeutic treatment of a viral infection, and mechanisms of host biology. With more than 2,000 viral genomes sequenced, only a small percent of them are well investigated. The access of these viral open reading frames (ORFs) in a flexible cloning format would greatly facilitate both in vitro and in vivo virus-host interaction studies. However, the overall progress of viral ORF cloning has been slow. To facilitate viral studies, we are releasing the initiation of our panviral proteome collection of 2,035 ORF clones from 830 viral genes in the Gateway® recombinational cloning system. Here, we demonstrate several uses of our viral collection including highly efficient production of viral proteins using human cell-free expression system in vitro, global identification of host targets for rubella virus using Nucleic Acid Programmable Protein Arrays (NAPPA) containing 10,000 unique human proteins, and detection of host serological responses using micro-fluidic multiplexed immunoassays. The studies presented here begin to elucidate host-viral protein interactions with our systemic utilization of viral ORFs, high-throughput cloning, and proteomic technologies. These valuable plasmid resources will be available to the research community to enable continued viral functional studies.

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: Most excess deaths that occur during extreme hot weather events do not have natural heat recorded as an underlying or contributing cause. This study aims to identify the specific individuals who died because of hot weather using only secondary data. A novel approach was developed in which the expected number of deaths was repeatedly sampled from all deaths that occurred during a hot weather event, and compared with deaths during a control period. The deaths were compared with respect to five factors known to be associated with hot weather mortality. Individuals were ranked by their presence in significant models over 100 trials of 10,000 repetitions. Those with the highest rankings were identified as probable excess deaths. Sensitivity analyses were performed on a range of model combinations. These methods were applied to a 2009 hot weather event in greater Vancouver, Canada.

Results: The excess deaths identified were sensitive to differences in model combinations, particularly between univariate and multivariate approaches. One multivariate and one univariate combination were chosen as the best models for further analyses. The individuals identified by multiple combinations suggest that marginalized populations in greater Vancouver are at higher risk of death during hot weather.

Conclusions: This study proposes novel methods for classifying specific deaths as expected or excess during a hot weather event. Further work is needed to evaluate performance of the methods in simulation studies and against clinically identified cases. If confirmed, these methods could be applied to a wide range of populations and events of interest.

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.

The image of “Shostakovich” and the relationships surrounding it in the West during the Cold War can be viewed from several angles. Selected Cold War encounters between the United States and the Soviet Union involving Shostakovich’s music—especially the 1959 New York Philharmonic tour to the USSR—offer insight into three perspectives on Shostakovich symphonies in the Cold War: (1) the direct, (2) the implicit, and (3) the micro/intimate. This heuristic hones our understanding of the various types of relationships cultivated with music during the Cold War, while also widening the discussion of Shostakovich’s symbolic presentation during the conflict.

The Arctic, even more so than other parts of the world, has warmed substantially over the past few decades. Temperature and humidity influence the rate of development, survival and reproduction of pathogens and thus the incidence and prevalence of many infectious diseases. Higher temperatures may also allow infected host species to survive winters in larger numbers, increase the population size and expand their habitat range. The impact of these changes on human disease in the Arctic has not been fully evaluated. There is concern that climate change may shift the geographic and temporal distribution of a range of infectious diseases. Many infectious diseases are climate sensitive, where their emergence in a region is dependent on climate-related ecological changes. Most are zoonotic diseases, and can be spread between humans and animals by arthropod vectors, water, soil, wild or domestic animals. Potentially climate-sensitive zoonotic pathogens of circumpolar concern include Brucella spp., Toxoplasma gondii, Trichinella spp., Clostridium botulinum, Francisella tularensis, Borrelia burgdorferi, Bacillus anthracis, Echinococcus spp., Leptospira spp., Giardia spp., Cryptosporida spp., Coxiella burnetti, rabies virus, West Nile virus, Hantaviruses, and tick-borne encephalitis viruses.

Background: In Africa and Asia, sugarcane is the host of at least seven different virus species in the genus Mastrevirus of the family Geminiviridae. However, with the exception of Sugarcane white streak virus in Barbados, no other sugarcane-infecting mastrevirus has been reported in the New World. Conservation and exchange of sugarcane germplasm using stalk cuttings facilitates the spread of sugarcane-infecting viruses.

Methods: A virion-associated nucleic acids (VANA)-based metagenomics approach was used to detect mastrevirus sequences in 717 sugarcane samples from Florida (USA), Guadeloupe (French West Indies), and Réunion (Mascarene Islands). Contig assembly was performed using CAP3 and sequence searches using BLASTn and BLASTx. Mastrevirus full genomes were enriched from total DNA by rolling circle amplification, cloned and sequenced. Nucleotide and amino acid sequence identities were determined using SDT v1.2. Phylogenetic analyses were conducted using MEGA6 and PHYML3.

Results: We identified a new sugarcane-infecting mastrevirus in six plants sampled from germplasm collections in Florida and Guadeloupe. Full genome sequences were determined and analyzed for three virus isolates from Florida, and three from Guadeloupe. These six genomes share >88% genome-wide pairwise identity with one another and between 89 and 97% identity with a recently identified mastrevirus (KR150789) from a sugarcane plant sampled in China. Sequences similar to these were also identified in sugarcane plants in Réunion.

Conclusions: As these virus isolates share <64% genome-wide identity with all other known mastreviruses, we propose classifying them within a new mastrevirus species named Sugarcane striate virus. This is the first report of sugarcane striate virus (SCStV) in the Western Hemisphere, a virus that most likely originated in Asia. The distribution, vector, and impact of SCStV on sugarcane production remains to be determined.