ASU Scholarship Showcase

Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications.

Background: Postmortem memento photography has emerged in Western hospitals as part of compassionate bereavement care for parents facing perinatal death. Many parents endorse this psychosocial intervention, yet implementation varies greatly and little research on parents’ specific needs guides health care professionals. Parents are in crisis and vulnerable after the death of their child, thus best practice is crucial. This study contributes 104 parents’ experiences and opinions toward the understanding of best practice in perinatal bereavement photography.

Methods: Parents who experienced the perinatal death of their child were recruited from U.S.-based bereavement organizations and social media sites. Volunteers completed an anonymous internet survey with open- and closed-ended questions. Direct recommendations and pertinent statements regarding the process of postmortem photography were analyzed for thematic content in keeping with conventional content analysis. Recurrent themes and sub-themes were counted to identify response patterns.

Results: Of 93 parents with pictures, 92 endorsed them. Of 11 without pictures, nine wanted them. Parents made a variety of recommendations regarding appropriate psychosocial support, the consent process, obstacles to photography, logistics of photography, and material aspects of photographs themselves. Overall, parents wanted many pictures and much variety. Some wanted professional photography while others wanted support for taking their own pictures. Parents wanted guidance from staff who respected their particular needs. Many said decisions were difficult during their crisis. Parents who were initially resistant expressed current appreciation for pictures or expressed regret that they had not participated. Parents recommended that professionals strongly encourage parents to create memento photos despite parents’ initial reservations. Persistent cultural reasons against photography emerged in one case. Quotes by parents illuminate themes and enable respondents to speak directly to health care professionals.

Conclusions: Parents overwhelmingly support postmortem bereavement photography when conducted sensitively, even if imperfectly executed. Providers significantly influence parents during their crises; mindful, patient-centered care with appropriate respect for difference is necessary. Providers must understand the importance of postmortem photographs to parents who have limited opportunity to capture memories of their child. Hospitals should provide education and support for this important psychosocial intervention.

Background: Osteosarcoma is one of the most common bone cancers in children. Most patients with metastatic osteosarcoma die of pulmonary disease and limited curative therapeutic options exist for such patients. We have previously shown that PD-1 limits the efficacy of CTL to mediate immune control of metastatic osteosarcoma in the K7M2 mouse model of pulmonary metastatic disease and that blockade of PD-1/PD-L1 interactions can partially improve survival outcomes by enhancing the function of osteosarcoma-specific CTL. However, PD-1/PD-L1 blockade-treated mice eventually succumb to disease due to selection of PD-L1 mAb-resistant tumor cells. We investigated the mechanism of tumor cell resistance after blockade, and additional combinational therapies to combat resistance.

Methods: We used an implantable model of metastatic osteosarcoma, and evaluated survival using a Log-rank test. Cellular analysis of the tumor was done post-mortem with flow cytometry staining, and evaluated using a T-test to compare treatment groups.

Results: We show here that T cells infiltrating PD-L1 antibody-resistant tumors upregulate additional inhibitory receptors, notably CTLA-4, which impair their ability to mediate tumor rejection. Based on these results we have tested combination immunotherapy with α-CTLA-4 and α-PD-L1 antibody blockade in the K7M2 mouse model of metastatic osteosarcoma and show that this results in complete control of tumors in a majority of mice as well as immunity to further tumor inoculation.

Conclusions: Thus, combinational immunotherapy approaches to block additional inhibitory pathways in patients with metastatic osteosarcoma may provide new strategies to enhance tumor clearance and resistance to disease.

Background: A growing body of research recommends controlling alcohol availability to reduce harm. Various common approaches, however, provide dramatically different pictures of the physical availability of alcohol. This limits our understanding of the distribution of alcohol access, the causes and consequences of this distribution, and how best to reduce harm. The aim of this study is to introduce both a gravity potential measure of access to alcohol outlets, comparing its strengths and weaknesses to other popular approaches, and an empirically-derived taxonomy of neighborhoods based on the type of alcohol access they exhibit.

Methods: We obtained geospatial data on Seattle, including the location of 2402 alcohol outlets, United States Census Bureau estimates on 567 block groups, and a comprehensive street network. We used exploratory spatial data analysis and employed a measure of inter-rater agreement to capture differences in our taxonomy of alcohol availability measures.

Results: Significant statistical and spatial variability exists between measures of alcohol access, and these differences have meaningful practical implications. In particular, standard measures of outlet density (e.g., spatial, per capita, roadway miles) can lead to biased estimates of physical availability that over-emphasize the influence of the control variables. Employing a gravity potential approach provides a more balanced, geographically-sensitive measure of access to alcohol outlets.

Conclusions: Accurately measuring the physical availability of alcohol is critical for understanding the causes and consequences of its distribution and for developing effective evidence-based policy to manage the alcohol outlet licensing process. A gravity potential model provides a superior measure of alcohol access, and the alcohol access-based taxonomy a helpful evidence-based heuristic for scholars and local policymakers.

Current research on criminal case processing typically examines a single decision-making point, so drawing reliable conclusions about the impact that factors such as defendants’ race or ethnicity exert across successive stages of the justice system is difficult. Using data from the New York County District Attorney's Office that tracks 185,275 diverse criminal cases, this study assesses racial and ethnic disparity for multiple discretionary points of prosecution and sentencing. Findings from multivariate logistic regression analyses demonstrate that the effects of race and ethnicity vary by discretionary point and offense category. Black and Latino defendants were more likely than White defendants to be detained, to receive a custodial plea offer, and to be incarcerated—and they received especially punitive outcomes for person offenses—but were more likely to benefit from case dismissals. The findings for Asian defendants were less consistent but suggest they were the least likely to be detained, to receive custodial offers, and to be incarcerated. These findings are discussed in the context of contemporary theoretical perspectives on racial bias and cumulative disadvantage in the justice system.

Contemporary vaccine development relies less on empirical methods of vaccine construction, and now employs a powerful array of precise engineering strategies to construct immunogenic live vaccines. In this review, we will survey various engineering techniques used to create attenuated vaccines, with an emphasis on recent advances and insights. We will further explore the adaptation of attenuated strains to create multivalent vaccine platforms for immunization against multiple unrelated pathogens. These carrier vaccines are engineered to deliver sufficient levels of protective antigens to appropriate lymphoid inductive sites to elicit both carrier-specific and foreign antigen-specific immunity. Although many of these technologies were originally developed for use in Salmonella vaccines, application of the essential logic of these approaches will be extended to development of other enteric vaccines where possible. A central theme driving our discussion will stress that the ultimate success of an engineered vaccine rests on achieving the proper balance between attenuation and immunogenicity. Achieving this balance will avoid over-activation of inflammatory responses, which results in unacceptable reactogenicity, but will retain sufficient metabolic fitness to enable the live vaccine to reach deep tissue inductive sites and trigger protective immunity. The breadth of examples presented herein will clearly demonstrate that genetic engineering offers the potential for rapidly propelling vaccine development forward into novel applications and therapies which will significantly expand the role of vaccines in public health.

Background: The current influenza vaccines are effective against seasonal influenza, but cannot be manufactured in a timely manner for a sudden pandemic or to be cost-effective to immunize huge flocks of birds. We propose a novel influenza vaccine composing a bacterial carrier and a plasmid cargo. In the immunized subjects, the bacterial carrier invades and releases its cargo into host cells where the plasmid expresses viral RNAs and proteins for reconstitution of attenuated influenza virus. Here we aimed to construct a mouse Poll-driven plasmid for efficient production of influenza virus. Results: A plasmid was constructed to express all influenza viral RNAs and proteins. This all-in-one plasmid resulted in 10(5)-10(6) 50 % tissue culture infective dose (TCID50)/mL of influenza A virus in baby hamster kidney (BHK-21) cells on the third day post-transfection, and also reconstituted influenza virus in Madin-Darby canine kidney (MDCK) and Chinese hamster ovary (CHO) cells. A 6-unit plasmid was constructed by deleting the HA and NA cassettes from the all-in-one plasmid. Cotransfection of BHK-21 cells with the 6-unit plasmid and the two other plasmids encoding the HA or NA genes resulted in influenza virus titers similar to those produced by the 1-plasmid method. Conclusions: An all-in-one plasmid and a 3-plasmid murine Poll-driven reverse genetics systems were developed, and efficiently reconstituted influenza virus in BHK-21 cells. The all-in-one plasmid may serve as a tool to determine the factors inhibiting virus generation from a large size plasmid. In addition, we recommend a simple and robust "1 + 2" approach to generate influenza vaccine seed virus.