Melanoma cells repress Desmoglein 1 in keratinocytes to promote tumor cell migration.

Melanoma is an aggressive cancer typically arising from transformation of melanocytes residing in the basal layer of the epidermis, where they are in direct contact with surrounding keratinocytes. The role of keratinocytes in shaping the melanoma tumor microenvironment remains understudied. We previously showed that temporary loss of the keratinocyte-specific cadherin, Desmoglein 1 (Dsg1), controls paracrine signaling between normal melanocytes and keratinocytes to stimulate the protective tanning response. Here, we provide evidence that melanoma cells hijack this intercellular communication by secreting factors that keep Dsg1 expression low in the surrounding keratinocytes, which in turn generate their own paracrine signals that enhance melanoma spread through CXCL1/CXCR2 signaling. Evidence suggests a model whereby paracrine signaling from melanoma cells increases levels of the transcriptional repressor Slug, and consequently decreases expression of the Dsg1 transcriptional activator Grhl1. Together, these data support the idea that paracrine crosstalk between melanoma cells and keratinocytes resulting in chronic keratinocyte Dsg1 reduction contributes to melanoma cell movement associated with tumor progression.

PP2A-B55alpha controls keratinocyte adhesion through dephosphorylation of the Desmoplakin C-terminus.

Critical for the maintenance of epidermal integrity and function are attachments between intermediate filaments (IF) and intercellular junctions called desmosomes. The desmosomal cytoplasmic plaque protein desmoplakin (DP) is essential for anchoring IF to the junction. DP-IF interactions are regulated by a phospho-regulatory motif within the DP C-terminus controlling keratinocyte intercellular adhesion. Here we identify the protein phosphatase 2A (PP2A)-B55α holoenzyme as the major serine/threonine phosphatase regulating DP's C-terminus and consequent intercellular adhesion. Using a combination of chemical and genetic approaches, we show that the PP2A-B55α holoenzyme interacts with DP at intercellular membranes in 2D- and 3D- epidermal models and human skin samples. Our experiments demonstrate that PP2A-B55α regulates the phosphorylation status of junctional DP and is required for maintaining strong desmosome-mediated intercellular adhesion. These data identify PP2A-B55α as part of a regulatory module capable of tuning intercellular adhesion strength and a candidate disease target in desmosome-related disorders of the skin and heart.

A detailed kinetic model of glycolysis in Plasmodium falciparum-infected red blood cells for antimalarial drug target identification.

Upon infection by the malaria parasite Plasmodium falciparum, the glycolytic rate of a red blood cell increases up to 100-fold, possibly contributing to lactic acidosis and hypoglycemia in patients with severe malaria. This dramatic increase in glucose uptake and metabolism was correctly predicted by a newly constructed detailed enzyme kinetic model of glucose metabolism in the trophozoite-infected red blood cell. Subsequently, we expanded the model to simulate an infected red blood cell culture, including the different asexual blood-stage forms of the malaria parasite. The model simulations were in good agreement with experimental data, for which the measured parasitic volume was an important parameter. Upon further analysis of the model, we identified glucose transport as a drug target that would specifically affect infected red blood cells, which was confirmed experimentally with inhibitor titrations. This model can be a first step in constructing a whole-body model for glucose metabolism in malaria patients to evaluate the contribution of the parasite's metabolism to the disease state.

Transcriptional profiling of rare acantholytic disorders suggests common mechanisms of pathogenesis.

Darier, Hailey-Hailey, and Grover diseases are rare acantholytic skin diseases. While these diseases have different underlying causes, they share defects in cell-cell adhesion in the epidermis and desmosome organization. To better understand the underlying mechanisms leading to disease in these conditions, we performed RNA-seq on lesional skin samples from patients. The transcriptomic profiles of Darier, Hailey-Hailey, and Grover diseases were found to share a remarkable overlap, which did not extend to other common inflammatory skin diseases. Analysis of enriched pathways showed a shared increase in keratinocyte differentiation, and a decrease in cell adhesion and actin organization pathways in Darier, Hailey-Hailey, and Grover diseases. Direct comparison to atopic dermatitis and psoriasis showed that the downregulation in actin organization pathways was a unique feature in the acantholytic skin diseases. Furthermore, upstream regulator analysis suggested that a decrease in SRF/MRTF activity was responsible for the downregulation of actin organization pathways. Staining for MRTFA in lesional skin samples showed a decrease in nuclear MRTFA in patient skin compared with normal skin. These findings highlight the significant level of similarity in the transcriptome of Darier, Hailey-Hailey, and Grover diseases, and identify decreases in actin organization pathways as a unique signature present in these conditions.

Improving transitions between clinical placements.

BACKGROUND: Students regularly transition between clinical learning environments as they rotate through their clinical placements. These transitions are stressful for learners, as they must navigate unfamiliar policies, people and physical spaces. It is important to reduce cognitive overload at the start of each placement through appropriate inductions. Our governance processes found there was significant variation between induction processes at our affiliated teaching-hospital sites: our aim was to optimise and standardise these. APPROACH: We opted for induction websites for each of our affiliated hospital sites, as these could be dynamically updated and quality assured. Our websites were informed by a conceptual framework of the clinical learning environment and the theory of sociomateriality. We co-produced them with students and other stakeholders through iterative evaluation and improvement cycles. EVALUATION: To elicit end-user analysis, we conducted three focus groups with 19 students. We used the technology acceptance model to inform our topic guide and coding categories. Students reported that the websites were useful, easy to use, and fulfilled a significant unmet need. IMPLICATIONS: Induction websites can be optimised through the involvement of a range of stakeholders and the application of theory. They can be pushed to students before each new placement and used to scaffold in-person inductions. Further research is needed to explore the wider impacts of improved site inductions on participation and engagement with clinical learning opportunities and on student satisfaction and experience.

Polarity signaling balances epithelial contractility and mechanical resistance.

Epithelia maintain a functional barrier during tissue turnover while facing varying mechanical stress. This maintenance requires both dynamic cell rearrangements driven by actomyosin-linked intercellular adherens junctions and ability to adapt to and resist extrinsic mechanical forces enabled by keratin filament-linked desmosomes. How these two systems crosstalk to coordinate cellular movement and mechanical resilience is not known. Here we show that in stratifying epithelia the polarity protein aPKCλ controls the reorganization from stress fibers to cortical actomyosin during differentiation and upward movement of cells. Without aPKC, stress fibers are retained resulting in increased contractile prestress. This aberrant stress is counterbalanced by reorganization and bundling of keratins, thereby increasing mechanical resilience. Inhibiting contractility in aPKCλ-/- cells restores normal cortical keratin networks but also normalizes resilience. Consistently, increasing contractile stress is sufficient to induce keratin bundling and enhance resilience, mimicking aPKC loss. In conclusion, our data indicate that keratins sense the contractile stress state of stratified epithelia and balance increased contractility by mounting a protective response to maintain tissue integrity.

MicroRNA-205 promotes hair regeneration by modulating mechanical properties of hair follicle stem cells.

Stiffness and actomyosin contractility are intrinsic mechanical properties of animal cells required for the shaping of tissues. However, whether tissue stem cells (SCs) and progenitors located within SC niche have different mechanical properties that modulate their size and function remains unclear. Here, we show that hair follicle SCs in the bulge are stiff with high actomyosin contractility and resistant to size change, whereas hair germ (HG) progenitors are soft and periodically enlarge and contract during quiescence. During activation of hair follicle growth, HGs reduce contraction and more frequently enlarge, a process that is associated with weakening of the actomyosin network, nuclear YAP accumulation, and cell cycle reentry. Induction of miR-205, a novel regulator of the actomyosin cytoskeleton, reduces actomyosin contractility and activates hair regeneration in young and old mice. This study reveals the control of tissue SC size and activities by spatiotemporally compartmentalized mechanical properties and demonstrates the possibility to stimulate tissue regeneration by fine-tuning cell mechanics.

Factors underpinning an improved menopausal experience in the workplace for doctors: a UK-based qualitative study.

OBJECTIVES: A recent British Medical Association survey revealed that very few National Health Service (NHS) doctors felt comfortable discussing symptoms with their managers, and many feel unable to make changes to their working lives to accommodate their menopause. An improved menopausal experience (IME) in the workplace has been associated with increased job satisfaction, increased economic participation and reduced absenteeism. Currently, existing literature fails to explore menopausal doctors' experiences and none factors in non-menopausal colleagues' perspectives. This qualitative study aims to determine the factors underpinning an IME for UK doctors. DESIGN: Qualitative study using semistructured interviews and thematic analysis. PARTICIPANTS: Menopausal doctors (n=21) and non-menopausal (n=20) doctors including men. SETTING: General practices and hospitals in the UK. RESULTS: Four overarching themes underpinning an IME were identified: menopausal knowledge and awareness, openness to discussion, organisational culture, and supported personal autonomy. The levels of knowledge held by menopausal participants themselves, their colleagues and their superiors were identified as crucial in determining menopausal experiences. Likewise, the ability to openly discuss menopause was also identified as an important factor. The NHS culture, gender dynamics and an adopted superhero mentality-where doctors feel compelled to prioritise work over personal well-being-further impacted under the umbrella of Organisational culture. Personal autonomy at work was considered important in improving menopausal experiences at work for doctors. The superhero mentality, lack of organisational support and a lack of open discussion were identified as novel themes not found in current literature, particularly in the healthcare context. CONCLUSIONS: This study highlights that doctors' factors underpinning an IME in the workplace are comparable to other sectors. The potential benefits of an IME for doctors in the NHS are considerable. NHS leaders can address these challenges by using pre-existing training materials and resources for their employees if menopausal doctors are to feel supported and retained.

Desmosome-anchored intermediate filaments facilitate tension-sensitive RhoA signaling for epithelial homeostasis.

Epithelia are subject to diverse forms of mechanical stress during development and post-embryonic life. They possess multiple mechanisms to preserve tissue integrity against tensile forces, which characteristically involve specialized cell-cell adhesion junctions coupled to the cytoskeleton. Desmosomes connect to intermediate filaments (IF) via desmoplakin (DP)1,2, while the E-cadherin complex links to the actomyosin cytoskeleton in adherens junctions (AJ)3. These distinct adhesion-cytoskeleton systems support different strategies to preserve epithelial integrity, especially against tensile stress. IFs coupled to desmosomes can passively respond to tension by strain-stiffening4-10, whereas for AJs a variety of mechanotransduction mechanisms associated with the E-cadherin apparatus itself11,12, or proximate to the junctions13, can modulate the activity of its associated actomyosin cytoskeleton by cell signaling. We now report a pathway where these systems collaborate for active tension-sensing and epithelial homeostasis. We found that DP was necessary for epithelia to activate RhoA at AJ on tensile stimulation, an effect that required its capacity to couple IF to desmosomes. DP exerted this effect by facilitating the association of Myosin VI with E-cadherin, the mechanosensor for the tension-sensitive RhoA pathway at AJ12. This connection between the DP-IF system and AJ-based tension-sensing promoted epithelial resilience when contractile tension was increased. It further facilitated epithelial homeostasis by allowing apoptotic cells to be eliminated by apical extrusion. Thus, active responses to tensile stress in epithelial monolayers reflect an integrated response of the IF- and actomyosin-based cell-cell adhesion systems.

Partners in academic endeavour: Characterising student engagement across internationally excellent medical schools.

There is increasing interest in how student engagement can be enhanced in medical schools: not just engagement with learning but with broader academic practices such as curriculum development, research, organisational leadership, and community involvement. To foster evidence-based practice, it is important to understand how institutions from diverse sociocultural contexts achieve excellence in student engagement.We analysed 11 successful applications for an international award in student engagement and interviewed nine key informants from five medical schools across four continents, characterising how and why student engagement was fostered at these institutions.Document analysis revealed considerable consensus on the core practices of student engagement, as well as innovative and creative practices often in response to local strengths and challenges. The interviews uncovered the importance of an authentic partnership culture between students and faculty which sustained mutually beneficial enhancements across multiple domains. Faculty promoted, welcomed, and acted on student inputs, and students reported greater willingness to participate if they could see the benefits. These combined to create self-perpetuating virtuous cycles of academic endeavour. Successful strategies included having participatory values actively reinforced by senior leadership, engagement activities that are driven by both students and staff, and focusing on strategies with reciprocal benefits for all stakeholders.

Crosstalk in skin: Loss of desmoglein 1 in keratinocytes inhibits BRAFV600E-induced cellular senescence in human melanocytes.

Melanoma arises from transformation of melanocytes in the basal layer of the epidermis where they are surrounded by keratinocytes, with which they interact through cell contact and paracrine communication. Considerable effort has been devoted to determining how the accumulation of oncogene and tumor suppressor gene mutations in melanocytes drive melanoma development. However, the extent to which alterations in keratinocytes that occur in the developing tumor niche serve as extrinsic drivers of melanoma initiation and progression is poorly understood. We recently identified the keratinocyte-specific cadherin, desmoglein 1 (Dsg1), as an important mediator of keratinocyte:melanoma cell crosstalk, demonstrating that its chronic loss, which can occur through melanoma cell-dependent paracrine signaling, promotes behaviors that mimic a malignant phenotype. Here we address the extent to which Dsg1 loss affects early steps in melanomagenesis. RNA-Seq analysis revealed that paracrine signals from Dsg1-deficient keratinocytes mediate a transcriptional switch from a differentiated to undifferentiated cell state in melanocytes expressing BRAFV600E, a driver mutation commonly present in both melanoma and benign nevi and reported to cause growth arrest and oncogene-induced senescence (OIS). Of ~220 differentially expressed genes in BRAFV600E cells treated with Dsg1-deficient conditioned media (CM), the laminin superfamily member NTN4/Netrin-4, which inhibits senescence in endothelial cells, stood out. Indeed, while BRAFV600E melanocytes treated with Dsg1-deficient CM showed signs of senescence bypass as assessed by increased senescence-associated ß-galactosidase activity and decreased p16, knockdown of NTN4 reversed these effects. These results suggest that Dsg1 loss in keratinocytes provides an extrinsic signal to push melanocytes towards oncogenic transformation once an initial mutation has been introduced.

Meeting report - Desmosome dysfunction and disease: Alpine desmosome disease meeting.

Desmosome diseases are caused by dysfunction of desmosomes, which anchor intermediate filaments (IFs) at sites of cell-cell adhesion. For many decades, the focus of attention has been on the role of actin filament-associated adherens junctions in development and disease, especially cancer. However, interference with the function of desmosomes, their molecular constituents or their attachments to IFs has now emerged as a major contributor to a variety of diseases affecting different tissues and organs including skin, heart and the digestive tract. The first Alpine desmosome disease meeting (ADDM) held in Grainau, Germany, in October 2022 brought together international researchers from the basic sciences with clinical experts from diverse fields to share and discuss their ideas and concepts on desmosome function and dysfunction in the different cell types involved in desmosome diseases. Besides the prototypic desmosomal diseases pemphigus and arrhythmogenic cardiomyopathy, the role of desmosome dysfunction in inflammatory bowel diseases and eosinophilic esophagitis was discussed.

Epidermal stratification requires retromer-mediated desmoglein-1 recycling.

Sorting transmembrane cargo is essential for tissue development and homeostasis. However, mechanisms of intracellular trafficking in stratified epidermis are poorly understood. Here, we identify an interaction between the retromer endosomal trafficking component, VPS35, and the desmosomal cadherin, desmoglein-1 (Dsg1). Dsg1 is specifically expressed in stratified epidermis and, when properly localized on the plasma membrane of basal keratinocytes, promotes stratification. We show that the retromer drives Dsg1 recycling from the endo-lysosomal system to the plasma membrane to support human keratinocyte stratification. The retromer-enhancing chaperone, R55, promotes the membrane localization of Dsg1 and a trafficking-deficient mutant associated with a severe inflammatory skin disorder, enhancing its ability to promote stratification. In the absence of Dsg1, retromer association with and expression of the glucose transporter GLUT1 increases, exposing a potential link between Dsg1 deficiency and epidermal metabolism. Our work provides evidence for retromer function in epidermal regeneration, identifying it as a potential therapeutic target.

Organotypic Human Skin Cultures Incorporating Primary Melanocytes.

Three-dimensional (3D) human organotypic skin cultures provide a physiologically relevant model that recapitulates in vivo skin features. Most commonly, organotypic skin cultures are created by seeding isolated epidermal keratinocytes onto a collagen/fibroblast plug and lifting to an air liquid interface. These conditions are sufficient to drive stratification and differentiation of the keratinocytes to form an epidermal-like sheet with remarkable similarities to human epidermis. Coupled with genetic or pharmacological treatments, these cultures provide a powerful tool for elucidating keratinocyte biology. Recent focus has been placed on increasing the utility of organotypic skin cultures by incorporating other cell types that are present in the skin, such as melanocytes, immune cells, and other cells. Here we describe a step-by-step protocol for the isolation of neonatal human epidermal keratinocytes and melanocytes from foreskins, and the creation of organotypic skin cultures that include both cell types. We also describe methods that can be used to assess melanocyte behavior in these organotypic cultures, including methods for whole mount staining, measurement of melanocyte dendricity, staining for pigment, and 5-bromo-2'-deoxyuridine (BrdU) labeling for identification of proliferating cells. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of primary cells Alternate Protocol: Isolation of primary cells using differential trypsinization Basic Protocol 2: Organotypic culture protocol Support Protocol 1: Culture and maintenance of NHEKs and melanocytes Support Protocol 2: Lentiviral transduction of melanocytes Support Protocol 3: Retroviral transduction of NHEKs Support Protocol 4: Whole mount immunostaining protocol Support Protocol 5: Measuring melanocyte dendricity Support Protocol 6: Fontana-Masson staining protocol Support Protocol 7: BrdU labeling and staining.

The Relationships Between Childhood Abuse and Neglect, Sub-clinical Symptoms of Psychosis and Self-harm in a Non-clinical Community Sample.

There is now substantial evidence that childhood adverse events are a significant risk factor for symptoms of psychosis in both clinical and community samples. Both childhood trauma and positive symptoms of psychosis are associated with an increased risk of self-harming behaviours. Therefore the current study aimed to consider the relationship between retrospective reports of childhood adversity, sub-clinical positive symptoms of psychosis and self-harm in a non-clinical community sample. The study employed a cross-sectional survey design, distributed online. Participants were asked to complete psychometric assessments relating to: demographic characteristics including past-year substance misuse; childhood adversity; sub-clinical symptoms of psychosis (delusions and hallucinations) and self-harming behaviours. The results found that, after controlling for substance misuse, childhood adversity predicted significant variance in sub-clinical delusions and hallucinations in the general population. Both symptoms of psychosis and childhood adversity increased the risk of self-harming behaviours. Positive symptoms partially mediated the relationship between early adversity and self-harming behaviours. For some people, the sequelae of early adversity including sub-clinical delusions and hallucinations may increase the risk of self-harming behaviours. Future research would benefit from considering the role of dissociation in these relationships and the affective impact of pseudo-psychotic experiences. Practitioners should consider the impact of childhood adversity, unusual perceptual experiences and distorted beliefs when working with people who self-harm. The current research was limited by the cross-sectional survey design and non-random sampling methodology.

The Desmosome-Keratin Scaffold Integrates ErbB Family and Mechanical Signaling to Polarize Epidermal Structure and Function.

While classic cadherin-actin connections in adherens junctions (AJs) have ancient origins, intermediate filament (IF) linkages with desmosomal cadherins arose in vertebrate organisms. In this mini-review, we discuss how overlaying the IF-desmosome network onto the existing cadherin-actin network provided new opportunities to coordinate tissue mechanics with the positioning and function of chemical signaling mediators in the ErbB family of receptor tyrosine kinases. We focus in particular on the complex multi-layered outer covering of the skin, the epidermis, which serves essential barrier and stress sensing/responding functions in terrestrial vertebrates. We will review emerging data showing that desmosome-IF connections, AJ-actin interactions, ErbB family members, and membrane tension are all polarized across the multiple layers of the regenerating epidermis. Importantly, their integration generates differentiation-specific roles in each layer of the epidermis that dictate the form and function of the tissue. In the basal layer, the onset of the differentiation-specific desmosomal cadherin desmoglein 1 (Dsg1) dials down EGFR signaling while working with classic cadherins to remodel cortical actin cytoskeleton and decrease membrane tension to promote cell delamination. In the upper layers, Dsg1 and E-cadherin cooperate to maintain high tension and tune EGFR and ErbB2 activity to create the essential tight junction barrier. Our final outlook discusses the emerging appreciation that the desmosome-IF scaffold not only creates the architecture required for skin's physical barrier but also creates an immune barrier that keeps inflammation in check.

"Concrete ways we can make a difference": A multi-centre, multi-professional evaluation of sustainability in quality improvement education.

Quality improvement (QI) projects are a mandatory part of postgraduate medical training in the UK and graduating medical students must be competent in QI theory. We evaluated an educational toolkit that links concepts of sustainable healthcare with established quality improvement methodologies (the SusQI approach, available at www.susqi.org). The SusQI approach was implemented across a range of undergraduate and postgraduate nursing and medical education contexts. Educational strategies included guided online learning, live interactive webinars, small group activities and scaffolded project work. The evaluation strategy was informed by theories of academic motivation, educational value within communities of practice and behaviour change. A simultaneous nested design was tested using a mixed methods survey with input from learners and teachers. 177 survey responses were analysed to quantify and compare self-rated impacts of teaching across different audiences. Qualitative data were inductively coded into themes that were categorised according to above theoretical frameworks. Participants felt that this was 'time well spent' and many described transformative impacts that guided their daily professional practice beyond learning about QI. We suggest that meaningful space is found within both undergraduate and postgraduate healthcare curricula for SusQI, as a way of engaging and motivating learners to contribute to the creation of a sustainable healthcare system.

Plectin pulls it together, coupling the cortical actin and intermediate filament cytoskeletons.

The integration of cytoskeletal/adhesive networks is critical to epithelial mechanobiology. In this issue, Prechova et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202105146) demonstrate that the cytolinker protein plectin is essential for the construction of a cortical cytoskeletal architecture required for epithelial tensional homeostasis.

"More than just a medical student": a mixed methods exploration of a structured volunteering programme for undergraduate medical students.

BACKGROUND: As a result of the COVID-19 pandemic Imperial College School of Medicine developed a structured volunteering programme involving 398 medical students, across eight teaching hospitals. This case study aims to explore the relationship between the processes, context, participant experiences and impacts of the programme so that lessons can be learned for future emergencies and service-learning programmes. METHODS: Using an illuminative approach to evaluation we invited all volunteers and supervisors to complete a mixed-methods survey. This explored differences in experience across demographics and contextual factors, correlations between aspects of induction, supervision and overall experience, and reviewed the impacts of the programme. Quantitative responses were statistically analysed and qualitative reflections were thematically coded to triangulate and explain quantitative findings. Follow up interviews were carried out to check back findings and co-create conclusions. RESULTS: We received responses from 61 students and 17 supervisors. Student participants described predominantly altruistic motivations and transformational changes to their professional identity driven by feeling included, having responsibility, and engaging in authentic workplace-based learning afforded by freedom from the assessed curriculum. They reported new perspectives on their future professional role within the multidisciplinary team and the value of workplace-based learning. They reported increases in wellbeing and self-esteem related to feeling included and valued, and positively contributing to service provision at a time of need. Significantly higher overall satisfaction was associated with a personalised induction, active supervision, earlier stage of training, and male gender. Gender-related differences were not explained through our data but have been reported elsewhere and warrant further study. The duration, intensity and type of role that volunteers performed was similar across demographics and did not appear to modulate their overall experience. CONCLUSIONS: Whilst acknowledging the uniqueness of emergency volunteering and the survey response rate of 15% of volunteers, we suggest the features of a successful service-learning programme include: a learner-centred induction, regular contact with engaged and appreciative supervisors, and roles where students feel valued. Programmes in similar settings may find that service learning is most impactful earlier in medical students' training and that students with altruistic motivations and meaningful work may flourish without formal outcomes and assessments.