Live-cell analysis of IMPDH protein levels during yeast colony growth provides insights into the regulation of GTP synthesis.

The purine nucleotides ATP and GTP are made from the common precursor inosine monophosphate (IMP). Maintaining the correct balance of these nucleotides for optimal cell growth is controlled in part by the enzyme IMP dehydrogenase (IMPDH), which catalyzes the first dedicated step of GTP biosynthesis. The regulation of IMPDH mRNA and protein levels in the yeast S. cerevisiae grown in liquid culture has been studied in some detail, but regulation of IMPDH protein under conditions of cellular crowding on a solid substrate has not been examined. Here, we report real-time, live-cell analysis of the accumulation of the Imd2 isoform of IMPDH in yeast cells forming a monolayer colony in a microfluidic device over a 50-hour time course. We observe two distinct phases of increased Imd2 accumulation: a guanine-insensitive phase early in outgrowth and a guanine-sensitive phase later, when cells become crowded. We show that the IMPDH inhibitor mycophenolic acid enhances both phases of increase. Deletion of a transcription attenuator upstream of the mRNA start site that decreases Imd2 mRNA synthesis in the presence of high GTP increases the baseline level of Imd2 protein 10-fold and abolishes guanine-sensitive but not guanine-insensitive induction. Our results suggest that at least two mechanisms of yeast Imd2 regulation exist, the known GTP-dependent attenuation of RNA polymerase II elongation and a GTP concentration-independent pathway that may be controlled by cell growth state. Live-cell analysis of IMPDH protein levels in a growing yeast colony confirms a known mechanism of regulation and provides evidence for an additional mode of regulation. IMPORTANCE: This study used live-cell microscopy to track changes in the level of a key enzyme in GTP nucleotide biosynthesis, inosine monophosphate dehydrogenase (IMPDH), during growth of a brewers yeast colony over 2 days in a microfluidic device. The results show that feedback regulation via transcription attenuation allows cells to adapt to nutrient limitation in the crowded environs of a yeast colony. They also identify a novel mode of regulation of IMPDH level that is not driven by guanine nucleotide availability.

A mixed-methods evaluation of patients' views on primary care multi-disciplinary teams in Scotland.

BACKGROUND: Expanding primary care multi-disciplinary teams (MDTs) was a key component of the 2018 Scottish GP contract, with over 4,700 MDT staff appointed since then. AIM: To explore patients' views on primary care MDT expansion in Scotland. DESIGN AND METHODS: (1) Survey of patients recently consulting a GP in deprived-urban, affluent-urban and remote/rural areas, assessing awareness of five MDT roles and attitudes towards receptionist signposting; (2) 30 individual interviews exploring MDT-care experiences. RESULTS: Of 1,053 survey respondents, most were unaware of the option of MDT rather than GP consultations for three out of five roles (69% unaware of link worker appointments; 68% mental health nurse; 58% pharmacist). Reception signposting was less popular in deprived-urban areas (34% unhappy vs 29% in remote/rural vs 21% affluent-urban; P<0.001), and in patients with multimorbidity (31% unhappy vs 24% in non-multimorbid; P<0.05).Two-thirds of interviewees had multimorbidity and almost all reported positive MDT-care experiences. However, MDT-care was generally seen as a supplement rather than a substitute for GP care. Around half of patients expressed concerns about reception signposting. These patients were more likely to also express concerns about GP access in general. Both of these concerns were more common in deprived-urban areas than in remote/rural or affluent-urban areas. CONCLUSION: MDT-care has expanded in Scotland with limited patient awareness. Although patients understand its potential value, many patients are unhappy with reception signposting to first-contact MDT care, especially those in deprived-urban areas living with multimorbidity. This represents a barrier to the aims of the new GP contract.

Patients' experiences of GP consultations following the introduction of the new GP contract in Scotland: a cross-sectional survey.

BACKGROUND: The new Scottish GP contract commenced in April 2018 with a stated aim of mitigating health inequalities. AIM: To determine the health characteristics and experiences of patients consulting GPs in deprived urban (DU), affluent urban (AU), and remote and rural (RR) areas of Scotland. DESIGN AND SETTING: In 2022, a postal survey of a random sample of adult patients from 12 practices who had consulted a GP within the previous 30 days was undertaken. METHOD: Patient characteristics and consultation experiences in the three areas (DU, AU, RR) were evaluated using validated measures including the Consultation and Relational Empathy (CARE) Measure and Patient Enablement Instrument (PEI). RESULTS: In total, 1053 responses were received. In DU areas, multimorbidity was more common (78% versus 58% AU versus 68% RR, P<0.01), complex presentations (where the consultation addressed both psychosocial and physical problems) were more likely (16% versus 10% AU versus 11% RR, P<0.05), and more consultations were conducted by telephone (42% versus 31% AU versus 31% RR, P<0.01). Patients in DU areas reported lower satisfaction (82% DU completely, very, or fairly satisfied versus 90% AU versus 86% RR, P<0.01), lower perceived GP empathy (mean CARE score 38.9 versus 42.1 AU versus 40.1 RR, P<0.05), lower enablement (mean PEI score 2.6 versus 3.2 AU versus 2.8 RR, P<0.01), and less symptom improvement (P<0.01) than those in AU or RR areas. Face-to-face consultations were associated with significantly higher satisfaction, enablement, and perceived GP empathy than telephone consultations in RR areas (all P<0.05). CONCLUSION: Four years after the start of the new GP contract in Scotland, patients' experiences of GP consultations suggest that the inverse care law persists.

Primary care transformation in Scotland: a qualitative evaluation of the views of patients.

BACKGROUND: The new Scottish GP contract introduced in April 2018 aims to improve quality of care through expansion of the multidisciplinary team (MDT) to enable GPs to spend more time as expert medical generalists with patients with complex needs. AIM: To explore patients' views on the changes in general practice in Scotland since the inception of the new contract. DESIGN AND SETTING: Qualitative study with 30 patients (10 living in urban deprived areas, 10 living in urban affluent/mixed urban areas, and 10 living in remote and rural areas). METHOD: In-depth semi-structured interviews with thematic analysis. RESULTS: Patients were generally unaware of the new GP contract, attributing recent changes in general practice to the COVID-19 pandemic. Ongoing concerns included access to GP consultations (especially face-to-face ones), short consultation length with GPs, and damage to continuity of care and the GP-patient relationship. Most patients spoke positively about consultations with MDT staff but still wanted to see a known GP for health concerns that they considered potentially serious. These issues were especially concerning for patients with multiple complex problems, particularly those from deprived areas. CONCLUSION: Following the introduction of the new Scottish GP contract, patients in this study's sample were accepting of first contact care from the MDT but still wanted continuity of care and longer face-to-face consultations with GPs. These findings suggest that the expert generalist role of the GP is not being adequately supported by the new contract, especially in deprived areas, though further quantitative research is required to confirm this.

Transcription factor localization dynamics and DNA binding drive distinct promoter interpretations.

Environmental information may be encoded in the temporal dynamics of transcription factor (TF) activation and subsequently decoded by gene promoters to enact stimulus-specific gene expression programs. Previous studies of this behavior focused on the encoding and decoding of information in TF nuclear localization dynamics, yet cells control the activity of TFs in myriad ways, including by regulating their ability to bind DNA. Here, we use light-controlled mutants of the yeast TF Msn2 as a model system to investigate how promoter decoding of TF localization dynamics is affected by changes in the ability of the TF to bind DNA. We find that yeast promoters directly decode the light-controlled localization dynamics of Msn2 and that the effects of changing Msn2 affinity on that decoding behavior are highly promoter dependent, illustrating how cells could regulate TF localization dynamics and DNA binding in concert for improved control of gene expression.

Health inequalities, multimorbidity and primary care in Scotland.

Scotland has an ageing population and the widest health inequalities in Western Europe. Multiple health conditions develop ∼10-15 years earlier in deprived areas than in affluent areas. General practice is central to the effective and safe management of such complex multiple health conditions, but the inverse care law has permeated deprived communities ('Deep End' general practices) for the past 50 years. A new, radical, Scottish GP contract was introduced in April 2018, which has a vision to improve quality of care through cluster working and expansion of the multidisciplinary team (MDT), enabling GPs to deliver 'expert generalism' to patients with complex needs. It states a specific intention to address health inequalities and also to support the integration of health and social care. Here, we discuss recent evidence for whether the ambition of the new GP contract, to reduce health inequalities, is being achieved.

Optogenetic Tools for Control of Public Goods in Saccharomyces cerevisiae.

Microorganisms live in dense and diverse communities, with interactions between cells guiding community development and phenotype. The ability to perturb specific intercellular interactions in space and time provides a powerful route to determining the critical interactions and design rules for microbial communities. Approaches using optogenetic tools to modulate these interactions offer promise, as light can be exquisitely controlled in space and time. We report new plasmids for rapid integration of an optogenetic system into Saccharomyces cerevisiae to engineer light control of expression of a gene of interest. In a proof-of-principle study, we demonstrate the ability to control a model cooperative interaction, namely, the expression of the enzyme invertase (SUC2) which allows S. cerevisiae to hydrolyze sucrose and utilize it as a carbon source. We demonstrate that the strength of this cooperative interaction can be tuned in space and time by modulating light intensity and through spatial control of illumination. Spatial control of light allows cooperators and cheaters to be spatially segregated, and we show that the interplay between cooperative and inhibitory interactions in space can lead to pattern formation. Our strategy can be applied to achieve spatiotemporal control of expression of a gene of interest in S. cerevisiae to perturb both intercellular and interspecies interactions. IMPORTANCE Recent advances in microbial ecology have highlighted the importance of intercellular interactions in controlling the development, composition, and resilience of microbial communities. In order to better understand the role of these interactions in governing community development, it is critical to be able to alter them in a controlled manner. Optogenetically controlled interactions offer advantages over static perturbations or chemically controlled interactions, as light can be manipulated in space and time and does not require the addition of nutrients or antibiotics. Here, we report a system for rapidly achieving light control of a gene of interest in the important model organism Saccharomyces cerevisiae and demonstrate that by controlling expression of the enzyme invertase, we can control cooperative interactions. This approach will be useful for understanding intercellular and interspecies interactions in natural and synthetic microbial consortia containing S. cerevisiae and serves as a proof of principle for implementing this approach in other consortia.

Automated calibration of optoPlate LEDs to reduce light dose variation in optogenetic experiments.

Optogenetic systems use light to precisely control and investigate cellular processes. Until recently, there had been few instruments available for applying controlled light doses to cultures of cells. The optoPlate, a programmable array of 192 LEDs, was developed to meet this need. However, LED performance varies, and without calibration there are substantial brightness differences between LEDs on an optoPlate. Here we present a method for calibrating an optoPlate that uses a programmable microscope stage and optical power meter to automatically measure all 192 LEDs of an optoPlate. The resulting brightness measurements are used to calculate calibration values that tune the electrical current supplied to each optoPlate LED to reduce brightness variation in optogenetic experiments.

Biological signal generators: integrating synthetic biology tools and in silico control.

Biological networks sense extracellular stimuli and generate appropriate outputs within the cell that determine cellular response. Biological signal generators are becoming an important tool for understanding how information is transmitted in these networks and controlling network behavior. Signal generators produce well-defined, dynamic, intracellular signals of important network components, such as kinase activity or the concentration of a specific transcription factor. Synthetic biology tools coupled with in silico control have enabled the construction of these sophisticated biological signal generators. Here we review recent advances in biological signal generator construction and their use in systems biology studies. Challenges for constructing signal generators for a wider range of biological networks and generalizing their use are discussed.

Easy calibration of the Light Plate Apparatus for optogenetic experiments.

Optogenetic systems use genetically-encoded light-sensitive proteins to control and study cellular processes. As the number and quality of these systems grows, there is an increasing need for user-friendly and flexible hardware to provide programmed illumination to cultures of cells. One platform which satisfies this need for a variety of optogenetic systems and organisms is the Light Plate Apparatus (LPA), which delivers a controlled light dose to each well of a 24-well plate. Experimental reproducibility requires appropriate calibration to produce accurate light doses within individual wells of the LPA and between LPAs. In this study, we present an easy and accurate method for calibrating the LPA. In particular, we: •developed a 3D printed adaptor and MATLAB code to allow rapid measurement of irradiance produced by the LPA and subsequent calibration•provide appropriate code and methodology for generating a standard curve for each LPA•demonstrate the utility and accuracy of this method between users and LPAs.

Promoting empathy using video-based teaching.

BACKGROUND: Empathy is fundamental to patient-centred care. It has been shown to improve patient satisfaction and therapeutic outcomes. The success of educational interventions in enhancing students' empathy has been mixed, however, and a consensus on the most effective approach is lacking. METHODS: Patients were videoed discussing their hospital experiences and interactions with clinicians. These videos were shown to groups of medical students, who then took part in a facilitated discussion around themes of health care communication and patient experience. The session was delivered to 48 students over seven sessions and evaluated by way of serial attitude questionnaires (the Patient-Practitioner Orientation Scale, PPOS) and written feedback. RESULTS: There was an improvement in PPOS scores amongst participants, with pre- and post-intervention means of 78.8 and 82.0, respectively, indicating an improvement in patient-centred attitudes. Written feedback highlighted the emotional impact of hearing patients' stories, and that the discussion explored issues not covered in previous teaching. Students reported changes in their approach to patients, including introducing themselves more often, and taking measures to make patients feel more at ease on ward rounds. Empathy is fundamentalto patient-centred care DISCUSSION: This study describes the use of patient video interviews to promote empathy in medical students, by stimulating reflective discussion on themes of health care communication and patient experience. Future use of these methods are recommended, although further research would benefit from larger student numbers and the use of objective clinical examination scores to assess behaviour change.

A patient's journey. Mesothelioma.

Professor Kieran Sweeney was diagnosed with malignant mesothelioma at age 57. He describes here his thoughts on his interactions with the health professionals who care for him.

Extracting information from hospital records: what patients think about consent.

BACKGROUND AND OBJECTIVE: There is increasing regulation and concern about the use of material from patients' records. Studies on patients' views have focused on primary care and on use of material for research. This study investigated patients' preferences about whether and how doctors should seek permission for use of specified items of anonymised information from their hospital records for clinical audit, teaching, national data collection and research. METHOD: A specially designed questionnaire sent to recently discharged patients under the care of medical and surgical specialists. RESULTS: 166/316 (53%) patients completed the questionnaires. The percentage of respondents who "definitely wanted" or "preferred" to be asked for permission for use of anonymised information was highest for medical history (21%) and reasons for treatment (20%). The purpose for which information was requested (eg, research, audit) made little difference to the overall percentages (range 10-12%). 21 (13%) patients "definitely wanted" to be asked for permission for use of some item or proposed use of information--most had no preference or preferred not to be asked. The most popular method for asking permission was signing a form while in hospital, rather than by specific requests later. CONCLUSIONS: Most hospital patients have no preference or prefer not to be asked permission for doctors to use information from their records. About 1 in 8 patients would like to be asked for permission, some even for clinical audit of outcomes--although a minority, this could compromise thorough clinical audit. Systems for obtaining permission when patients are admitted to hospital need to be considered. Resolution of uncertainties surrounding legislation on the use of information would be helpful to clinicians.

Revealing implicit understanding through enthymemes: a rhetorical method for the analysis of talk.

BACKGROUND: Everyday talk is characterised by implicit information that the listener is required to infer. We introduce a method of analysis based on theories from semiotics and classical rhetoric that enables the researcher to reveal unspoken beliefs that underlie such talk. OBJECTIVE: This paper aims to provide a detailed description of the process of unveiling the hidden elements of logical inference within narrative. We wish to enable other medical education researchers to utilise this method in their own field of interest. OVERVIEW: We begin by giving an overview of the concepts of the enthymeme in rhetoric and opposition in narrative analysis. We then demonstrate the steps required in order to undertake such analysis by using examples from our own data. Strengths and weaknesses of this approach, including the problem of inference, will be discussed. CONCLUSION: We identify a number of current domains in which the investigation of enthymemes has been fruitful. We suggest that this method might also be utilised successfully within the domain of medical education research and encourage other researchers to consider this approach.

Listening to patients: choice in cardiac rehabilitation.

BACKGROUND: The benefits of cardiac rehabilitation (CR) after myocardial infarction (MI) are increasingly recognised and is recommended in national guidelines. AIMS: To explore patients' experience of MI and to identify the factors which influence the choice patients make given the option of hospital or home-based CR after MI. METHOD: Qualitative study using semi-structured interviews and interpretive phenomenological analysis (IPA). This study was embedded within a randomised trial with preference arms. RESULTS: Seventeen participants were interviewed before their rehabilitation programme. Ten expressed a preference for home-based and seven for hospital-based rehabilitation. Common to both groups was shock and disbelief, which led to a loss of confidence. They expressed a strong desire to make lifestyle changes and looked for specific advice, guidance and support from knowledgeable experts. The hospital-based group had an emphasis on supervision during exercise, needed the camaraderie of a group, were willing to make travel arrangements and believed they lacked self-discipline. The home-based group believed that their CR should fit in with their lives rather than their lives fitting in with the rehabilitation programme and were self-disciplined. They disliked groups and expressed practical concerns. CONCLUSIONS: Understanding the factors that influence patient's choices may help professionals guide them to the most appropriate CR method and hence improve uptake.