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The preparation of Agrobacterium tumefaciens cultures with strains encoding proteins intended for therapeutic or industrial purposes is an important activity prior to treatment of plants for transient expression of valuable protein products. The rising demand for biologic products such as these underscores the expansion of molecular pharming and warrants the need to produce transformed plants at an industrial scale. This requires large quantities of A. tumefaciens culture, which is challenging using traditional growth methods (e.g., shake flask). To overcome this limitation, we investigate the use of bioreactors as an alternative to shake flasks to meet production demands. Here, we observe differences in bacterial growth among the tested parameters and define conditions for consistent bacterial culturing between shake flask and bioreactor. Quantitative proteomic profiling of cultures from each growth condition defines unique growth-specific responses in bacterial protein abundance and highlights the functional roles of these proteins, which may influence bacterial processes important for effective agroinfiltration and transformation. Overall, our study establishes and optimizes comparable growth conditions for shake flask versus bioreactors and provides novel insights into fundamental biological processes of A. tumefaciens influenced by such growth conditions.

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.

ObjectivesThe 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.MethodsAll 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.ResultsWe 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).ConclusionsNo 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.

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.

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.

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.

Introduction: While a majority of patients presenting to the emergency department (ED) are discharged home without need for inpatient hospitalization, many require outpatient follow-up. Currently, outpatient referrals from our ED are made via a complex and error-prone series of manual steps which have the potential to be unreliable and negatively impact quality of care. We sought to perform a current state analysis of our outpatient referral processes across the hospital’s specialties. Methods: We conducted a retrospective health records review at a tertiary academic centre (>160,000 ED visits/year) from January 1 to January 7, 2015. All consecutive outpatient consultation requests triggered by an ED physician were identified and included for chart review. All cases were subsequently followed up to 11 months. A single reviewer extracted data on demographics, actual referral attendance rates, incomplete referrals, return ED visits, and time intervals. The top 3 and bottom 3 performing services were identified for further analysis of their outpatient referral mechanisms and processes. We present descriptive statistics. Results: A total of 251 outpatient referrals to a broad range of specialty services were identified during the study period. 216 (86.1%) of patients attended the intended appointment, while 35 (13.9%) of referrals were incomplete at 11 months post index ED visit. The overall median time to successful outpatient follow-up appointments was 8.5 days [IQR=3.8-24.2]. 8 (3.2%) patients had a return ED visit for a related complaint prior to being seen at their outpatient appointment. The top 3 performers were Ophthalmology [Median=1.0 day, IQR=0.0-1.0, Incomplete=2.8%], Plastic Surgery [Median=5.0 days, IQR=2.8-6.0, Incomplete=7.7%], and Orthopedics [Median=8.0 days, IQR=7.0-10.0, Incomplete=0.0%]. The bottom 3 performers were Dermatology [Median=52days, IQR=41.5-92.5, Incomplete=25.0%], Neurology [Median=40.0 days, IQR= 2.5-43.5, Incomplete=56.3%], and Urology [Median=14.0 days, IQR=10.5-48.0, Incomplete=33.3%]. Conclusion: We found a tremendous range of variability in both the waiting times and actual reliability of outpatient referral processes from the ED. Future phases of this project will focus on examining specific processes of the top and bottom performing specialties in order to improve and standardize all outpatient referrals.