Bacterial growth-mediated systems remodelling of Nicotiana benthamiana defines unique signatures of target protein production in molecular pharming.

The need for therapeutics to treat a plethora of medical conditions and diseases is on the rise and the demand for alternative approaches to mammalian-based production systems is increasing. Plant-based strategies provide a safe and effective alternative to produce biological drugs but have yet to enter mainstream manufacturing at a competitive level. Limitations associated with batch consistency and target protein production levels are present; however, strategies to overcome these challenges are underway. In this study, we apply state-of-the-art mass spectrometry-based proteomics to define proteome remodelling of the plant following agroinfiltration with bacteria grown under shake flask or bioreactor conditions. We observed distinct signatures of bacterial protein production corresponding to the different growth conditions that directly influence the plant defence responses and target protein production on a temporal axis. Our integration of proteomic profiling with small molecule detection and quantification reveals the fluctuation of secondary metabolite production over time to provide new insight into the complexities of dual system modulation in molecular pharming. Our findings suggest that bioreactor bacterial growth may promote evasion of early plant defence responses towards Agrobacterium tumefaciens (updated nomenclature to Rhizobium radiobacter). Furthermore, we uncover and explore specific targets for genetic manipulation to suppress host defences and increase recombinant protein production in molecular pharming.

Proteomic Profiling of Interplay Between Agrobacterium tumefaciens and Nicotiana benthamiana for Improved Molecular Pharming Outcomes.

Transient expression of recombinant proteins in plants is being used as a platform for production of therapeutic proteins. Benefits of this system include a reduced cost of drug development, rapid delivery of new products to the market, and an ability to provide safe and efficacious medicines for diseases. Although plant-based production systems offer excellent potential for therapeutic protein production, barriers, such as plant host defense response, exist which negatively impact the yield of product. Here we provide a protocol using tandem mass tags and mass spectrometry-based proteomics to quickly and robustly quantify the change in abundance of host defense proteins produced during the production process. These proteins can then become candidates for genetic manipulation to create host plants with reduced plant defenses capable of producing higher therapeutic protein yields.

The emerging role of mass spectrometry-based proteomics in molecular pharming practices.

Molecular pharming relies on the integration of foreign genes into a plant system for production of the desired recombinant protein. The speed, scalability, and lack of contaminating human pathogens highlights plants as an enticing and feasible system to produce diverse protein-based products, including vaccines, antibodies, and enzymes. However, limitations of expression levels, host defense responses, and production irregularities underscore distinct areas for improvement within the molecular pharming pipeline. Within the past five years, mass spectrometry-based proteomics has begun to address these critical areas and show promise in advancing our understanding of the complex biological systems driving molecular pharming. Further, opportunities to leverage comprehensive proteome profiling have surfaced to meet good manufacturing practice regulations and move biopharmaceuticals derived from plants into mainstream production.

A central role for polyprenol reductase in plant dolichol biosynthesis.

Dolichol is an essential polyisoprenoid within the endoplasmic reticulum of all eukaryotes. It serves as a membrane bound anchor onto which N-glycans are assembled prior to being transferred to nascent polypeptides, many of which enter the secretory pathway. Historically, it has been posited that the accumulation of dolichol represents the 'rate-limiting' step in the evolutionary conserved process of N-glycosylation, which ultimately affects the efficacy of approximately one fifth of the entire eukaryotic proteome. Therefore, this study aimed to enhance dolichol accumulation by manipulating the enzymes involved in its biosynthesis using an established Nicotiana benthamiana platform. Co-expression of a Solanum lycopersicum (tomato) cis-prenyltransferase (CPT) and its cognate partner protein, CPT binding protein (CPTBP), that catalyze the antepenultimate step in dolichol biosynthesis led to a 400-fold increase in the levels of long-chain polyprenols but resulted in only modest increases in dolichol accumulation. However, when combined with a newly characterized tomato polyprenol reductase, dolichol biosynthesis was enhanced by approximately 20-fold. We provide further evidence that in the aquatic macrophyte, Lemna gibba, dolichol is derived exclusively from the mevalonic acid (MVA) pathway with little participation from the evolutionary co-adopted non-MVA pathway. Taken together these results indicate that to effectively enhance the in planta accumulation of dolichol, coordinated synthesis and reduction of polyprenol to dolichol, is strictly required.

Examining the Impacts of CO2 Concentration and Genetic Compatibility on Perennial Ryegrass-Epichloë festucae var lolii Interactions.

Perennial ryegrass (Lolium perenne) is the most cultivated cool-season grass worldwide with crucial roles in carbon fixation, turfgrass applications, and fodder for livestock. Lolium perenne forms a mutualism with the strictly vertically transmitted fungal endophyte, Epichloë festucae var lolii. The fungus produces alkaloids that protect the grass from herbivory, as well as conferring protection from drought and nutrient stress. The rising concentration of atmospheric CO2, a proximate cause of climatic change, is known to have many direct and indirect effects on plant growth. There is keen interest in how the nature of this plant-fungal interaction will change with climate change. Lolium perenne is an obligately outcrossing species, meaning that the genetic profile of the host is constantly being reshuffled. Meanwhile, the fungus is asexual implying both a relatively constant genetic profile and the potential for incompatible grass-fungus pairings. In this study, we used a single cultivar, "Alto", of L. perenne. Each plant was infected with one of four strains of the endophyte: AR1, AR37, NEA2, and Lp19 (the "common strain"). We outcrossed the Alto mothers with pollen from a number of individuals from different ryegrass cultivars to create more genetic diversity in the hosts. We collected seed such that we had replicate maternal half-sib families. Seed from each family was randomly allocated into the two levels of the CO2 treatment, 400 and 800 ppm. Elevated CO2 resulted in an c. 18% increase in plant biomass. AR37 produced higher fungal concentrations than other strains; NEA2 produced the lowest fungal concentrations. We did not find evidence of genetic incompatibility between the host plants and the fungal strains. We conducted untargeted metabolomics and quantitative proteomics to investigate the grass-fungus interactions between and within family and treatment groups. We identified a number of changes in both the proteome and metabalome. Taken together, our data set provides new understanding into the intricacy of the interaction between endophyte and host from multiple molecular levels and suggests opportunity to promote plant robustness and survivability in rising CO2 environmental conditions through application of bioprotective epichloid strains.

Relationship between ratings of performance in the simulated and workplace environments among emergency medicine residents.

OBJECTIVES: The Emergency Medicine (EM) Specialty Committee of the Royal College of Physicians and Surgeons of Canada (RCPSC) specifies that resuscitation entrustable professional activities (EPAs) can be assessed in the workplace and simulated environments. However, limited validity evidence for these assessments in either setting exists. We sought to determine if EPA ratings improve over time and whether an association exists between ratings in the workplace v. simulation environment. METHODS: All Foundations EPA1 (F1) assessments were collected for first-year residents (n = 9) in our program during the 2018-2019 academic year. This EPA focuses on initiating and assisting in the resuscitation of critically ill patients. EPA ratings obtained in the workplace and simulation environments were compared using Lin's concordance correlation coefficient (CCC). To determine whether ratings in the two environments differed as residents progressed through training, a within-subjects analysis of variance was conducted with training environment and month as independent variables. RESULTS: We collected 104 workplace and 36 simulation assessments. No correlation was observed between mean EPA ratings in the two environments (CCC(8) = -0.01; p = 0.93). Ratings in both settings improved significantly over time (F(2,16) = 18.8; p < 0.001; η2 = 0.70), from 2.9 ± 1.2 in months 1-4 to 3.5 ± 0.2 in months 9-12. Workplace ratings (3.4 ± 0.1) were consistently higher than simulation ratings (2.9 ± 0.2) (F(2,16) = 7.2; p = 0.028; η2 = 0.47). CONCLUSIONS: No correlation was observed between EPA F1 ratings in the workplace v. simulation environments. Further studies are needed to clarify the conflicting results of our study with others and build an evidence base for the validity of EPA assessments in simulated and workplace environments.

A Health Records Review of Outpatient Referrals from the Emergency Department.

OBJECTIVES: Many patients discharged home from the emergency department (ED) require urgent outpatient consultation with a specialty service. We sought to identify the best- and worst-performing services with regard to time to outpatient consultation, the proportion of patients lost to follow-up, the rate of related return ED visits prior to consultation, and common strategies used by our top-performing clinics. METHODS: We conducted a health records review of The Ottawa Hospital ED visits during four 1-week periods. All consecutive adult outpatient consultation requests were included for chart review and were followed up to 12 months. Outcome measures included demographics, referral attendance rates, incomplete referrals, return ED visits, and time intervals. Services with at least 15 consultation requests were included for data analysis and qualitative mapping of their referral processes. RESULTS: Of the 963 patients who met inclusion criteria, 803 (83.4%) attended their appointment, while 160 (16.6%) were lost to follow-up. The overall median time to successful consultation was 9 days (IQR = 2-27). 92 (9.6%) patients returned to the ED with a related complaint. The top-performing clinics included ophthalmology, orthopedics, and thrombosis (median = 1, 8, 1 days; incomplete consultation = 3%, 4%, 6%; return ED visits = 0%, 6%, 2% respectively). The bottom-performing clinics included otorhinolaryngology, neurology, and gynecology (median = 47, 39, 27 days; incomplete consultation = 50%, 41%, 37%; return ED visits = 11%, 15%, 26%, respectively). Processes incorporated by top-performing clinics included reserving appointment slots for emergency referrals, structured referral forms, and centralized booking. CONCLUSIONS: We found a substantial variability in both the waiting times and reliability of outpatient referrals from the ED. Top-performing clinics incorporate common referral processes.