Choice of fusion proteins, expression host, and analytics solves difficult-to-produce protein challenges in discovery research.

High quality biological reagents are a prerequisite for pharmacological research. Herein a protein production screening approach, including quality assessment methods, for protein-based discovery research is presented. Trends from 2895 expression constructs representing 253 proteins screened in mammalian and bacterial hosts-91% of which are successfully expressed and purified-are discussed. Mammalian expression combined with the use of solubility-promoting fusion proteins is deemed suitable for most targets. Furthermore, cases utilizing stable cell line generation and choice of fusion protein for higher yield and quality of difficult-to-produce proteins (Leucine-rich repeat-containing G-protein coupled receptor 4 (LGR4) and Neurturin) are presented and discussed. In the case of Neurturin, choice of fusion protein impacted the target binding 80-fold. These results highlight the need for exploration of construct designs and careful Quality Control (QC) of difficult-to-produce protein reagents.

Mobile device: a useful tool to teach inhaler devices to healthcare professionals.

BACKGROUND: Proper inhaler device usage is paramount for control of underlying obstructive airway disease. Hence, education to healthcare professionals who will eventually educate patients need to be done effectively. We developed an application for mobile devices for education on six medical inhaler devices, the metered-dose inhaler (MDI), Turbuhaler, Accuhaler, Breezhaler, Ellipta and Respimat, and studied if there were any difference between the application and the manufacturer's instructions on inhaler technique. The aim of this study is to see if inhaler education via a mobile phone app is comparable to manual instruction for health care professions. METHODS: Participants, who were nursing students, were randomized to learn the inhaler devices via the manufacturer's instruction guide or a mobile device app designed specifically for education on inhaler devices. RESULTS: There were 45 participants in each group. 78% of them were females with a median age of 21 (IQR 3). 67% used an Apple mobile device and the remainder used an Android device. The mobile device showed better total improvement points for the Turbuhaler device (262 vs 287 points; P = 0.02). Participants learning from the manufacturer's guide had a significantly higher total improvement points in the Breezhaler (370 vs 327 points; P < 0.01) and Ellipta (214 vs 174 points; P < 0.01) device. Both interventions showed improvement in total scores for demonstrating the correct usage of all inhaler devices. MDI has the least number of correct steps for both interventions. The participants' reported their mean (SD) self-rated knowledge was significantly higher for those using the app for all devices as compared to those that did not (4.33 (0.68) vs 4.73 (0.42); P = < 0.01). Self-reported confidence level was found to be higher in the mobile app group, but this was not statistically significant. The app was well received and scored of 4.42 of 5 with regards to its quality. CONCLUSION: Using a mobile inhaler app is just as effective to teach inhaler device techniques to healthcare professionals and is likely a more convenient, versatile and important adjunct to learning. TRIAL REGISTRATION: National Healthcare Group Ethics Board (2018/00960).

Multicomponent Plasmid Protects Mice From Spontaneous Autoimmune Diabetes.

Type 1 diabetes is an autoimmune disease in which insulin-secreting ß-cells are destroyed, leading to a life-long dependency on exogenous insulin. There are no approved disease-modifying therapies available, and future immunotherapies would need to avoid generalized immune suppression. We developed a novel plasmid expressing preproinsulin2 and a combination of immune-modulatory cytokines (transforming growth factor-beta-1, interleukin [IL] 10 and IL-2) capable of near-complete prevention of autoimmune diabetes in non-obese diabetic mice. Efficacy depended on preproinsulin2, suggesting antigen-specific tolerization, and on the cytokine combination encoded. Diabetes suppression was achieved following either intramuscular or subcutaneous injections. Intramuscular plasmid treatment promoted increased peripheral levels of endogenous IL-10 and modulated myeloid cell types without inducing global immunosuppression. To prepare for first-in-human studies, the plasmid was modified to allow for selection without the use of antibiotic resistance; this modification had no impact on efficacy. This pre-clinical study demonstrates that this multi-component, plasmid-based antigen-specific immunotherapy holds potential for inducing self-tolerance in persons at risk of developing type 1 diabetes. Importantly, the study also informs on relevant cytokine and immune cell biomarkers that may facilitate clinical trials. This therapy is currently being tested for safety and tolerability in a phase 1 trial (ClinicalTrials.gov Identifier: NCT04279613).

Multicomponent Plasmid Protects Mice From Spontaneous Autoimmune Diabetes.

Type 1 diabetes is an autoimmune disease in which insulin-secreting ß-cells are destroyed, leading to a life-long dependency on exogenous insulin. There are no approved disease-modifying therapies available, and future immunotherapies would need to avoid generalized immune suppression. We developed a novel plasmid expressing preproinsulin2 and a combination of immune-modulatory cytokines (transforming growth factor-beta-1, interleukin [IL] 10 and IL-2) capable of near-complete prevention of autoimmune diabetes in non-obese diabetic mice. Efficacy depended on preproinsulin2, suggesting antigen-specific tolerization, and on the cytokine combination encoded. Diabetes suppression was achieved following either intramuscular or subcutaneous injections. Intramuscular plasmid treatment promoted increased peripheral levels of endogenous IL-10 and modulated myeloid cell types without inducing global immunosuppression. To prepare for first-in-human studies, the plasmid was modified to allow for selection without the use of antibiotic resistance; this modification had no impact on efficacy. This pre-clinical study demonstrates that this multi-component, plasmid-based antigen-specific immunotherapy holds potential for inducing self-tolerance in persons at risk of developing type 1 diabetes. Importantly, the study also informs on relevant cytokine and immune cell biomarkers that may facilitate clinical trials. This therapy is currently being tested for safety and tolerability in a phase 1 trial (ClinicalTrials.gov Identifier: NCT04279613).