ASU Scholarship Showcase

S-cysteinylated albumin and methionine-oxidized apolipoprotein A-I (apoA-I) have been posed as candidate markers of diseases associated with oxidative stress. Here, a dilute-and-shoot form of LC–electrospray ionization–MS requiring half a microliter of blood plasma was employed to simultaneously quantify the relative abundance of these oxidized proteoforms in samples stored at −80 °C, −20 °C, and room temperature and exposed to multiple freeze-thaw cycles and other adverse conditions in order to assess the possibility that protein oxidation may occur as a result of poor sample storage or handling. Samples from a healthy donor and a participant with poorly controlled type 2 diabetes started at the same low level of protein oxidation and behaved similarly; significant increases in albumin oxidation via S-cysteinylation were found to occur within hours at room temperature and days at −20 °C. Methionine oxidation of apoA-I took place on a longer time scale, setting in after albumin oxidation reached a plateau. Freeze–thaw cycles had a minimal effect on protein oxidation. In matched collections, protein oxidation in serum was the same as that in plasma. Albumin and apoA-I oxidation were not affected by sample headspace or the degree to which vials were sealed. ApoA-I, however, was unexpectedly found to oxidize faster in samples with lower surface-area-to-volume ratios. An initial survey of samples from patients with inflammatory conditions normally associated with elevated oxidative stress-including acute myocardial infarction and prostate cancer—demonstrated a lack of detectable apoA-I oxidation. Albumin S-cysteinylation in these samples was consistent with known but relatively brief exposures to temperatures above −30 °C (the freezing point of blood plasma). Given their properties and ease of analysis, these oxidized proteoforms, once fully validated, may represent the first markers of blood plasma specimen integrity based on direct measurement of oxidative molecular damage that can occur under suboptimal storage conditions.

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: Interaction in the form of cooperation, communication, and friendly competition theoretically precede the development of group cohesion, which often precedes adherence to health promotion programs. The purpose of this manuscript was to explore longitudinal relationships among dimensions of group cohesion and group-interaction variables to inform and improve group-based strategies within programs aimed at promoting physical activity.

Methods: Ethnic minority women completed a group dynamics-based physical activity promotion intervention (N = 103; 73% African American; 27% Hispanic/Latina; mage = 47.89 + 8.17 years; mBMI = 34.43+ 8.07 kg/m[superscript 2]) and assessments of group cohesion and group-interaction variables at baseline, 6 months (post-program), and 12 months (follow-up).

Results: All four dimensions of group cohesion had significant (ps < 0.01) relationships with the group-interaction variables. Competition was a consistently strong predictor of cohesion, while cooperation did not demonstrate consistent patterns of prediction.

Conclusions: Facilitating a sense of friendly competition may increase engagement in physical activity programs by bolstering group cohesion.

Introduction: This study used social network theory to explore the role of social support and social networks in health information–seeking behavior among Korean American (KA) adults.

Methods: A descriptive qualitative study using a web-based online survey was conducted from January 2013 to April 2013 in the U.S. The survey included open-ended questions about health information–seeking experiences in personal social networks and their importance in KA adults. Themes emerging from a constant comparative analysis of the narrative comments by 129 of the 202 respondents were analyzed.

Results: The sample consisted of 129 KA adults, 64.7% female, with a mean age of 33.2 (SD = 7.7). Friends, church members, and family members were the important network connections for KAs to obtain health information. KAs looked for a broad range of health information from social network members, from recommendations and reviews of hospitals/doctors to specific diseases or health conditions. These social networks were regarded as important for KAs because there were no language barriers, social network members had experiences similar to those of other KAs, they felt a sense of belonging with those in their networks, the network connections promoted increased understanding of different health care systems of the U.S. system, and communication with these network connections helped enhance feelings of being physically and mentally healthy.

Conclusions: This study demonstrates the important role that social support and personal social networks perform in the dissemination of health information for a large ethnic population, KAs, who confront distinct cultural challenges when seeking health information in the U.S. Data from this study also illustrate the cultural factors that influence health information acquisition and access to social support for ethnic minorities. This study provides practical insights for professionals in health information services, namely, that social networks can be employed as a channel for disseminating health information to immigrants.

Background: Latino preschoolers (3-5 year old children) have among the highest rates of obesity. Low levels of physical activity (PA) are a risk factor for obesity. Characterizing what Latino parents do to encourage or discourage their preschooler to be physically active can help inform interventions to increase their PA. The objective was therefore to develop and assess the psychometrics of a new instrument: the Preschooler Physical Activity Parenting Practices (PPAPP) among a Latino sample, to assess parenting practices used to encourage or discourage PA among preschool-aged children.

Methods: Cross-sectional study of 240 Latino parents who reported the frequency of using PA parenting practices. 95% of respondents were mothers; 42% had more than a high school education. Child mean age was 4.5 (±0.9) years (52% male). Test-retest reliability was assessed in 20%, 2 weeks later. We assessed the fit of a priori models using Confirmatory factor analyses (CFA). In a separate sub-sample (35%), preschool-aged children wore accelerometers to assess associations with their PA and PPAPP subscales.

Results: The a-priori models showed poor fit to the data. A modified factor structure for encouraging PPAPP had one multiple-item scale: engagement (15 items), and two single-items (have outdoor toys; not enroll in sport-reverse coded). The final factor structure for discouraging PPAPP had 4 subscales: promote inactive transport (3 items), promote screen time (3 items), psychological control (4 items) and restricting for safety (4 items). Test-retest reliability (ICC) for the two scales ranged from 0.56-0.85. Cronbach’s alphas ranged from 0.5-0.9. Several sub-factors correlated in the expected direction with children’s objectively measured PA.

Conclusion: The final models for encouraging and discouraging PPAPP had moderate to good fit, with moderate to excellent test-retest reliabilities. The PPAPP should be further evaluated to better assess its associations with children’s PA and offers a new tool for measuring PPAPP among Latino families with preschool-aged children.

Background: Community engagement has contributed to disease control and elimination in many countries. Community engagement in malaria elimination (ME) on Aneityum Island has been sustained since its introduction in the early 1990s. Capacity developed within this population has led to a health empowered community response. Health Empowerment Theory (HET) can account for the innovative community actions and capacity development efforts taken to realize and sustain meaningful changes in well-being. This study used the HET framework to investigate participant perceptions of ME efforts on the island focusing on two HET elements, personal and social-contextual resources. The purpose of this study was to explore the role of empowerment as a critical element of community engagement.

Methods: Six focus group discussions, ten key informant interviews and 17 in-depth interviews were conducted in July 2012 on Aneityum. Both deductive and inductive approaches to qualitative content analysis were used to identify themes, which were condensed, coded and classified based on the HET elements above.

Results: Awareness and use of personal and social-contextual resources played an important role in ME efforts. Most participants shared their knowledge to prevent malaria reintroduction. Many participants reported their skills needed for behavioral maintenance, problem-solving or leadership. Participants who perceived a threat took preventive actions even in the dry season. Community leaders focused on second generation capacity development. A local health coalition provided ME services. Members of networks were sources of information and assistance. Face-to-face was the preferred method of communication. Barriers to engagement (e.g., financial difficulties, health literacy issues and underdeveloped infrastructure) were minimized through active collaboration and mutual assistance.

Conclusions: In the community engagement continuum, health empowerment develops incrementally overtime as people gain their knowledge and skills, form coalitions and develop collaborative networks (social capital) to make decisions and take action for change. Community engagement, which facilitates local personal and social-contextual resource development, has potential for ME and multilevel empowerment through community-based capacity development processes. These self-empowered communities have written and will continue to write a ‘prescription’ for sustaining high levels of engagement.

Background: Cysteine sulfenic acid (Cys-SOH) plays important roles in the redox regulation of numerous proteins. As a relatively unstable posttranslational protein modification it is difficult to quantify the degree to which any particular protein is modified by Cys-SOH within a complex biological environment. The goal of these studies was to move a step beyond detection and into the relative quantification of Cys-SOH within specific proteins found in a complex biological setting--namely, human plasma.

Results: This report describes the possibilities and limitations of performing such analyses based on the use of thionitrobenzoic acid and dimedone-based probes which are commonly employed to trap Cys-SOH. Results obtained by electrospray ionization-based mass spectrometric immunoassay reveal the optimal type of probe for such analyses as well as the reproducible relative quantification of Cys-SOH within albumin and transthyretin extracted from human plasma--the latter as a protein previously unknown to be modified by Cys-SOH.

Conclusions: The relative quantification of Cys-SOH within specific proteins in a complex biological setting can be accomplished, but several analytical precautions related to trapping, detecting, and quantifying Cys-SOH must be taken into account prior to pursuing its study in such matrices.