A Yeast Modular Cloning (MoClo) Toolkit Expansion for Optimization of Heterologous Protein Secretion and Surface Display in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is an attractive host for the expression of secreted proteins in a biotechnology context. Unfortunately, many heterologous proteins fail to enter, or efficiently progress through, the secretory pathway, resulting in poor yields. Similarly, yeast surface display has become a widely used technique in protein engineering but achieving sufficient levels of surface expression of recombinant proteins is often challenging. Signal peptides (SPs) and translational fusion partners (TFPs) can be used to direct heterologous proteins through the yeast secretory pathway, however, selection of the optimal secretion promoting sequence is largely a process of trial and error. The yeast modular cloning (MoClo) toolkit utilizes type IIS restriction enzymes to facilitate an efficient assembly of expression vectors from standardized parts. We have expanded this toolkit to enable the efficient incorporation of a panel of 16 well-characterized SPs and TFPs and five surface display anchor proteins into S. cerevisiae expression cassettes. The secretion promoting signals are validated by using five different proteins of interest. Comparison of intracellular and secreted protein levels reveals the optimal secretion promoting sequence for each individual protein. Large, protein of interest-specific variations in secretion efficiency are observed. SP sequences are also used with the five surface display anchors, and the combination of SP and anchor protein proves critical for efficient surface display. These observations highlight the value of the described panel of MoClo compatible parts to allow facile screening of SPs and TFPs and anchor proteins for optimal secretion and/or surface display of a given protein of interest in S. cerevisiae.

A rapid improvement event: progesterone prescribing in prevention of miscarriage.

A rapid improvement event (RIE) is a standard operational excellence technique that uses team-based problem solving to improve processes. In this study, a RIE was undertaken to improve progesterone prescribing rates for those with a history of miscarriage experiencing vaginal bleeding in early pregnancy. This was on the basis of a recent change in guidelines regarding prescribing in these instances. NICE guidelines changed in November 2021 after Cochrane meta-analysis and the PRISM (Progesterone in Spontaneous Miscarriage) randomised control trial demonstrated a higher incidence of live births in those prescribed vaginal micronised progesterone for threatened miscarriage, when compared with those not prescribed it.A RIE involves a team approach and a standard sequence of events allowing analysis and improvement of a process. Analysis in the form of audit revealed a low progesterone prescribing rate for eligible patients in our unit. Dissection of this problem into its elements revealed a low level of staff knowledge regarding the change in guidelines and a lack of confidence in prescription of progesterone. A plan of actionable events to improve prescribing rates was devised. The updated guidance and local recommendations on appropriate micronised progesterone formulations were presented at hospital Grand Rounds with multidisciplinary attendance. Infographics were displayed in areas visible to stakeholders within the hospital and on the hospital's social media pages. The validity of these educational measures to improve the process was reaudited after 3 months.Progesterone prescribing improved by 48%. Those comfortable with prescribing as per the new guidelines improved from 43% to 78%. A RIE proved to be an effective and efficient approach to collaboration, decision-making and action.

Patient perspectives of bedside teaching in an obstetrics, Gynaecology and neonatology hospital.

BACKGROUND: Osler taught doctors to "have no teaching without a patient for a text, and the best teaching is that taught by the patient himself". Bedside teaching (BST) facilitates clinical practice of skills, teaches empathy, instils confidence and builds on patient-doctor relationships. However, its use has declined dramatically due to concerns regarding privacy and autonomy. Most of the research in this area concentrates on medical student or academic opinion of BST using survey based methods. This qualitative study aimed to explore the patient's experiences and opinions of BST. METHODS: With ethical approval a qualitative study was conducted using semi-structured interviews which were examined using Thematic Analysis. Patients who had participated in a BST tutorial were invited to participate and gave written consent after discussion with a study researcher. RESULTS: Twenty-two patients were interviewed (obstetrics ante-natal [n = 10], obstetrics post-natal [n = 5] and gynaecology [n = 7]) ranging from ages 24-80 yrs. Four major themes were identified, with 11 sub-themes. The major themes included (i) Professional Mannerisms (ii) Privacy and Personal Wellbeing (iii) Quality of Patient Experience of BST and (iv) Clinical Experience and Learning Importance. The reaction of patients toward teaching at the bedside was altruistic and positive, with importance placed on learning. CONCLUSION: This research supports the concept of patient focused learning, and can reassure faculty that patients largely support its continuation as an integral component in education. Future research aims to extend this assessment to other patient groups with the aim of learning from and improving their experience.