High-Throughput Assembly of Compositionally Controlled 3D Cell Communities for Developmental Engineering.

Cell patterning for 3D culture has increased our understanding of how cells interact among themselves and with their environment during tissue morphogenesis. Building cell communities from the bottom up with size and compositional control is invaluable for studies of morphological transitions. Here, we detail Photolithographic DNA-programmed Assembly of Cells (pDPAC). pDPAC uses a photoactive polyacrylamide gel substrate to capture single-stranded DNA on a 2D surface in large-scale, highly resolved patterns using the photomask technology. Cells are then functionalized with a complementary DNA strand, enabling cells to be temporarily adhered to distinct locations only where their complementary strand is patterned. These temporary 2D patterns can be transferred to extracellular matrix hydrogels for 3D culture of cells in biomimetic microenvironments. Use of a polyacrylamide substrate has advantages, including a simpler photolithography workflow, lower non-specific cell adhesion, and lower stiction to ECM hydrogels during release of patterned hydrogels. The protocol is equally applicable to large (cm)-scale patterns and repetitive arrays of smaller-scale cell interaction or migration experiments.

Highly parallel production of designer organoids by mosaic patterning of progenitors.

Organoids derived from human stem cells are a promising approach for disease modeling, regenerative medicine, and fundamental research. However, organoid variability and limited control over morphological outcomes remain as challenges. One open question is the extent to which engineering control over culture conditions can guide organoids to specific compositions. Here, we extend a DNA "velcro" cell patterning approach, precisely controlling the number and ratio of human induced pluripotent stem cell-derived progenitors contributing to nephron progenitor (NP) organoids and mosaic NP/ureteric bud (UB) tip cell organoids within arrays of microwells. We demonstrate long-term control over organoid size and morphology, decoupled from geometric constraints. We then show emergent trends in organoid tissue proportions that depend on initial progenitor cell composition. These include higher nephron and stromal cell representation in mosaic NP/UB organoids vs. NP-only organoids and a "goldilocks" initial cell ratio in mosaic organoids that optimizes the formation of proximal tubule structures.

The potential for using entrustable professional activities in assessing optometric clinical competence.

Qualifications required to practise optometry seek to provide assurance that those completing them have the necessary competencies to be safe and effective clinicians. Often, this assurance is gained via high stakes assessments such as objective structured clinical examinations. This paper introduces entrustable professional activities (EPAs) as a potential alternative approach to assessing optometric clinical competence. Entrustable professional activities are defined in the context of optometric practice and the potential advantages and challenges of using them are outlined.

Social Protection and Foundational Cognitive Skills during Adolescence: Evidence from a Large Public Works Program.

Many low- and middle-income countries have introduced public works programs (PWPs) to fight poverty. This paper provides the first evidence that children from families who benefit from PWPs show increased foundational cognitive skills. The results, based on unique tablet-based data collected as part of a long-standing longitudinal survey, show positive associations between participation in the Productive Safety Net Programme (PSNP) in Ethiopia during childhood with long-term memory and implicit learning, and suggestive evidence for working memory. These associations appear to be strongest for children whose households were still PSNP participants in the year of data collection. Evidence suggests that the association with implicit learning may be operating partially through children's time reallocation away from unpaid labor responsibilities, while the association with long-term memory may in part be due to the program's success in remediating nutritional deficits caused by early-life rainfall shocks.

The Use of Hyaluronidase in Aesthetic Practice: A Comparative Study of Practitioner Usage in Elective and Emergency Situations.

BACKGROUND: Hyaluronic acids (HAs) continue to be the fillers of choice worldwide and their popularity is growing. Adverse events (AEs) are able to be resolved through the use of hyaluronidase (HYAL). However, routine HYAL use has been at issue due to perceived safety issues. OBJECTIVES: There are currently no guidelines on the use of HYAL in aesthetic practice, leading to variability in storage, preparation, skin testing, and beliefs concerning AEs. This manuscript interrogated the use of this agent in daily practice. METHODS: A 39-question survey concerning HYAL practice was completed by 264 healthcare practitioners: 244 from interrogated databases and 20 from the consensus panel. Answers from those in the database were compared to those of the consensus panel. RESULTS: Compared to the database group, the consensus group was more confident in the preparation of HYAL, kept reconstituted HYAL for longer, and was less likely to skin test for HYAL sensitivity and more likely to treat with HYAL in an emergency, even in those with a wasp or bee sting anaphylactic history. Ninety-two percent of all respondents had never observed an acute reaction to HYAL. Just over 1% of respondents had ever observed anaphylaxis. Five percent of practitioners reported longer-term adverse effects, including 3 respondents who reported loss of deep tissues. Consent before injecting HA for the possible requirement of HYAL was always obtained by 74% of practitioners. CONCLUSIONS: Hyaluronidase would appear to be an essential agent for anyone injecting hyaluronic acid filler. However, there is an absence of evidence-based recommendations with respect to the concentration, dosing, and treatment intervals of HYAL, and these should ideally be available.

Nephron progenitors rhythmically alternate between renewal and differentiation phases that synchronize with kidney branching morphogenesis.

The mammalian kidney achieves massive parallelization of function by exponentially duplicating nephron-forming niches during development. Each niche caps a tip of the ureteric bud epithelium (the future urinary collecting duct tree) as it undergoes branching morphogenesis, while nephron progenitors within niches balance self-renewal and differentiation to early nephron cells. Nephron formation rate approximately matches branching rate over a large fraction of mouse gestation, yet the nature of this apparent pace-maker is unknown. Here we correlate spatial transcriptomics data with branching 'life-cycle' to discover rhythmically alternating signatures of nephron progenitor differentiation and renewal across Wnt, Hippo-Yap, retinoic acid (RA), and other pathways. We then find in human stem-cell derived nephron progenitor organoids that Wnt/ß-catenin-induced differentiation is converted to a renewal signal when it temporally overlaps with YAP activation. Similar experiments using RA activation indicate a role in setting nephron progenitor exit from the naive state, the spatial extent of differentiation, and nephron segment bias. Together the data suggest that nephron progenitor interpretation of consistent Wnt/ß-catenin differentiation signaling in the niche may be modified by rhythmic activity in ancillary pathways to set the pace of nephron formation. This would synchronize nephron formation with ureteric bud branching, which creates new sites for nephron condensation. Our data bring temporal resolution to the renewal vs. differentiation balance in the nephrogenic niche and inform new strategies to achieve self-sustaining nephron formation in synthetic human kidney tissues.

Highly-parallel production of designer organoids by mosaic patterning of progenitors.

Human organoids are a promising approach for disease modeling and regenerative medicine. However, organoid variability and limited control over morphological outcomes remain significant challenges. Here we extend a DNA 'velcro' cell patterning approach, precisely controlling the number and ratio of human stem cell-derived progenitors contributing to nephron and mosaic nephron/ureteric bud organoids within arrays of microwells. We demonstrate long-term control over organoid size and morphology, decoupled from geometric constraints.

Community Partners' Perspectives on Partnering With an Academic Research Team to Promote Disability-inclusive Fitness Programming.

BACKGROUND: Community-based fitness programs can support public health by providing access to physical activity opportunities for a vulnerable population with significant barriers. Unfortunately, programs specifically designed for people with disabilities (PWD) and staff training to promote inclusion for PWD in general population programs is limited. The current study aimed to review an on-going partnership that had formed to address this need. OBJECTIVES: The purpose of this study was to assess community partners' experiences with a community-academic partnership designed to implement a fitness program for people with multiple sclerosis and also to promote inclusion for PWD in community-based fitness programming. METHODS: Semi-structured interviews were conducted with six community partners who had been engaged in a formal partnership with the academic institution for 2 or more years to understand partners' experiences and perspectives about the partnership. Interviews were audio/video recorded, transcribed verbatim, and analyzed thematically. RESULTS: Participants described their experiences as falling into four main areas. Pre-partnership experiences (or lack thereof) shaped participants views on entering into academic partnerships. Communication and planning for mutual benefit were key to getting the partnership started. Partners identified challenges and factors for success while they were in the thick of partnership activities. Finally, evaluation allowed for assessment and improvement of the partnership itself and its ultimate goals. CONCLUSIONS: Findings suggest that academic-community partnerships can be ideal for promoting inclusion for PWD and highlight insights that can be used in the development of future partnerships.

Penicillin allergy status and its effect on antibiotic prescribing, patient outcomes and antimicrobial resistance (ALABAMA): protocol for a multicentre, parallel-arm, open-label, randomised pragmatic trial.

INTRODUCTION: Incorrect penicillin allergy records are recognised as an important barrier to the safe treatment of infection and affect an estimated 2.7 million people in England. Penicillin allergy records are associated with worse health outcome and antimicrobial resistance. The ALlergy AntiBiotics And Microbial resistAnce (ALABAMA) trial aims to determine if an intervention package, centred around a penicillin allergy assessment pathway (PAAP) initiated in primary care, is safe and effective in improving patient health outcomes and antibiotic prescribing. METHODS AND ANALYSIS: The ALABAMA trial is a multicentre, parallel-arm, open-label, randomised pragmatic trial with a nested pilot study. Adults (≥18 years) with a penicillin allergy record and who have received antibiotics in the previous 24 months will be eligible for participation. Between 1592 and 2090 participants will be recruited from participating National Health Service general practices in England. Participants will be randomised to either usual care or intervention to undergo a pre-emptive PAAP using a 1:1 allocation ratio. The primary outcome measure is the percentage of treatment response failures within 28 days of an index prescription. 2090 and 1592 participants are estimated to provide 90% and 80% power, respectively, to detect a clinically important absolute difference of 7.9% in primary outcome at 1 year between groups. The trial includes a mixed-methods process evaluation and cost-effectiveness evaluation. ETHICS AND DISSEMINATION: This trial has been approved by London Bridge Research Ethics Committee (ref: 19/LO/0176). It will be conducted in compliance with Good Clinical Practice guidelines according to the Declaration of Helsinki. Informed consent will be obtained from all subjects involved in the study. The primary trial results will be submitted for publication to an international, peer-reviewed journal. TRIAL REGISTRATION: ISRCTN20579216.

Independent control over cell patterning and adhesion on hydrogel substrates for tissue interface mechanobiology.

Tissue boundaries and interfaces are engines of morphogenesis in vivo. However, despite a wealth of micropatterning approaches available to control tissue size, shape, and mechanical environment in vitro, fine-scale spatial control of cell positioning within tissue constructs remains an engineering challenge. To address this, we augment DNA "velcro" technology for selective patterning of ssDNA-labeled cells on mechanically defined photoactive polyacrylamide hydrogels. Hydrogels bearing photopatterned single-stranded DNA (ssDNA) features for cell capture are then co-functionalized with extracellular matrix (ECM) proteins to support subsequent adhesion of patterned tissues. ECM protein co-functionalization does not alter ssDNA pattern fidelity, cell capture, or hydrogel elastic stiffness. This approach enables mechanobiology studies and measurements of signaling activity at dynamic cell interfaces with precise initial patterning. Combining DNA velcro patterning and ECM functionalization provides independent control of initial cell placement, adhesion, and mechanics, constituting a new tool for studying biological interfaces and for programming multicellular interactions in engineered tissues.

Making Sense of Late Tissue Nodules Associated With Hyaluronic Acid Injections.

BACKGROUND: The pathogenesis of delayed-onset tissue nodules (DTNs) due to hyaluronic acid (HA) injections is uncertain. OBJECTIVES: To formulate a rational theory for DTN development and their avoidance and treatment. METHODS: A multidisciplinary and multicountry DTN consensus panel was established, with 20 questions posed and consensus sought. Consensus was set at 75% agreement. RESULTS: Consensus was reached in 16 of 20 questions regarding the pathogenesis of DTNs, forming the basis for a classification and treatment guide. CONCLUSIONS: The group believes that filler, pathogens, and inflammation are all involved in DTNs and that DTNs most likely are infection initiated with a variable immune response. Injected filler may incorporate surface bacteria, either a commensal or a true pathogen, if the skin barrier is altered. The initially high molecular weight HA filler is degraded to low molecular weight HA (LMWHA) at the edge of the filler. Commensals positioned within the filler bolus may be well tolerated until the filler is degraded and the commensal becomes visible to the immune system. LMWHA is particularly inflammatory in the presence of any local bacteria. Commensals may still be tolerated unless the immune system is generally heightened by viremia or vaccination. Systemic pathogenic bacteremia may also interact with the filler peripheral LMWHA, activating Toll-like receptors that induce DTN formation. Given this scenario, attention to practitioner and patient hygiene and early systemic infection treatment deserve attention. Classification and treatment systems were devised by considering each of the 3 factors-filler, inflammation, and infection-separately.

Investigating eye examination-related anxiety in autistic adults.

CLINICAL RELEVANCE: It is important to investigate whether anxiety is a barrier to accessing eye examinations for autistic adults, because existing research suggests this population are more likely to develop ophthalmic abnormalities. BACKGROUND: Anxiety influences healthcare accessibility for autistic people without learning disabilities. Previous qualitative studies by the research team, with a small sample of autistic adults, have indicated several aspects of eyecare services which cause anxiety. Considering the limited existing research suggesting autistic individuals are more likely to develop ophthalmic abnormalities, this study explored whether this population more widely experiences anxiety when accessing eye examinations. METHODS: A total of 322 UK-based autistic adults completed the Optometric Patient Anxiety Scale (OPAS) online, between July and December 2020. Rasch analysis was used to validate this questionnaire for an autistic adult population, and compare optometric anxiety levels to the general population. RESULTS: Item infit (0.77 to 1.39) and outfit (0.78 to 1.33) values, the person separation index (2.64), and item (0.99) and person (0.97) reliability coefficients suggested that all 10 items in the OPAS are useful to assess optometric anxiety in an autistic adult population. Item probability curves confirmed the response scale to be appropriate. A comparison of optometric anxiety between the autistic population in the current study and a general population in previous work found no statistically significant difference. CONCLUSION: The OPAS is a statistically valid tool for use in the autistic adult population. It appears to suggest no significant difference in optometric anxiety between the autistic adult and general population. However, it is possible that it underestimates the true optometric anxiety of autistic adults since the items do not include some of the anxiety provoking factors for this population which have been indicated in previous studies by the research team.

A Conversational, Virtual, Avatar-Led Cognitive Behavioral Therapy App Intervention for Improving the Quality of Life and Mental Health of People With Epilepsy: Protocol for a Randomized Controlled Trial.

BACKGROUND: Epilepsy is a common neurological disorder affecting about 1 in 100 people in the United Kingdom. Many individuals experience a lower quality of life as a result of their epilepsy diagnosis and are more likely to develop mental health problems, such as anxiety and depression. Medical interventions for this client group tend to focus on the treatment of seizures, whereas mental health disorders often remain undiagnosed and untreated. Early identification and treatment of mental health difficulties in people with epilepsy are vital to ensure better outcomes and improvements in quality of life. OBJECTIVE: The aim of this exploratory randomized controlled trial is to evaluate whether an 8-week cognitive behavioral therapy-based intervention delivered through a mobile app-ThinkNinja for Epilepsy-is a clinically effective tool to improve quality of life, mental health, and emotional well-being in a large sample of people with epilepsy and anxiety or comorbid anxiety and depression. METHODS: The study aims to recruit 184 individuals, 18 to 65 years of age, with a self-reported diagnosis of epilepsy and anxiety or comorbid anxiety and depression. Participants will be randomly assigned to the ThinkNinja for Epilepsy app condition (arm A) or the waiting-list control group (arm B). Participants in arm A will receive access to the ThinkNinja for Epilepsy app first. After 8 weeks, participants in arm B will receive the same full access to the ThinkNinja for Epilepsy app as the participants in arm A. This design will allow an initial between-subjects analysis between the two conditions as well as a within-subject analysis including all participants. The primary outcome is participants' quality of life, measured by the 10-item patient-weighted Quality of Life in Epilepsy questionnaire. The secondary outcomes include measures of anxiety, using the 7-item Generalized Anxiety Disorder assessment; depression, using the 9-item Patient Health Questionnaire; medication adherence, using the Medication Adherence Questionnaire; and impression of change, using the Patient Global Impression of Change questionnaire. RESULTS: Recruitment for this study began in March 2022 and was completed in October 2022. We expect data collection to be finalized by May 2023 and study results to be available within 12 months of the final data collection date. Results of the study will be written up as soon as possible thereafter, with the intention of publishing the outcomes in high-quality peer-reviewed journals. CONCLUSIONS: This study aims to determine the clinical efficacy and safety of the ThinkNinja for Epilepsy intervention at improving the quality of life, mental health, and emotional well-being of people with epilepsy. The findings from our study will hopefully contribute to addressing the critical gap in universal provision and accessibility of mental health and emotional well-being support for people with epilepsy. TRIAL REGISTRATION: ISRCTN Registry 16270209 (04/03/2022); https://www.isrctn.com/ISRCTN16270209. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/40261.

Autism-friendly eyecare: Developing recommendations for service providers based on the experiences of autistic adults.

PURPOSE: People with autism face significant barriers when accessing healthcare services. Eye examinations present unique challenges. Accessibility of this healthcare sector for people with autism has not been investigated previously. The aim of this research was to investigate eye examination accessibility for autistic adults and produce recommendations for autism-friendly eyecare. METHODS: Two qualitative studies were conducted. In Study 1, 18 autistic adults took part in focus groups to elicit their eye examination experiences. Transcripts of the recorded discussions were thematically analysed. Study 1 findings were used to design autism-friendly eye examinations for autistic adults. These were conducted in Study 2. Twenty-four autistic adults participated in these examinations, during which they were interviewed about their experience and how it might be improved by reasonable modifications. Audio recordings of the interviews were content analysed. RESULTS: Knowledge of what to expect, in advance of the eye examination, could greatly reduce anxiety. Participants liked the logical structure of the examination, and the interesting instrumentation used. However, the examination and practice environment did include sensory challenges, due to lights, sound and touch. Changes in practice layout, and interacting with multiple staff members, was anxiety provoking. Participants expressed a need for thorough explanations from the optometrist that outlined the significance of each test, and what the patient was expected to do. CONCLUSION: A number of accessiblity barriers were identified. These suggested that UK eye examinations are not very accessible for autistic adults. Barriers began at the point of booking the appointment and continued through to the dispensing of spectacles. These caused anxiety and stress for this population, but could be reduced with easy-to-implement adaptations. Based on the findings, recommendations are presented here for the whole eyecare team which suggest how more autism-friendly eye examinations can be provided.

The evolution of young people's mental health during COVID-19 and the role of food insecurity: Evidence from a four low-and-middle-income-country cohort study.

OBJECTIVES: Provide evidence on how young people's mental health has evolved in Low-and-Middle-Income-Countries (LMICs) during the progression of the COVID-19 pandemic. Identify particularly vulnerable groups who report high and/or continuously high rates of mental health issues. STUDY DESIGN: Longitudinal, observational. METHODS: Two consecutive phone-surveys (August-October and November-December 2020) in Ethiopia, India, Peru and Vietnam interviewed around 9000 participants of a 20-year cohort study who grew up in poverty, now aged 19 and 26. Rates of at least mild anxiety/depression measured by GAD-7/PHQ-8 were each compared across countries; between males/females, and food secure/food insecure households. RESULTS: Overall, rates of at least mild anxiety and mild depression significantly decreased between mid and end-2020 in all countries but Ethiopia as COVID-19 infection rates fell. Females report higher rates of anxiety and depression in all countries but Ethiopia, however the gender gap is closing. Young people in food insecure households have not shown consistent improvements in their rates of anxiety and depression. Food insecure households are poorer, and have significantly more children (p < 0.05) except in Ethiopia. CONCLUSION: Food insecurity is negatively associated with young people's mental health and urgent support targeted towards the most vulnerable should be a priority. Further research into increasing rates of mental health issues in Ethiopia is needed.

Mixed-methods evaluation of a behavioural intervention package to identify and amend incorrect penicillin allergy records in UK general practice.

OBJECTIVES: About 6% of the UK general practice population has a record of a penicillin allergy but fewer than 10% of these are likely to be truly allergic. In the ALABAMA (Allergy Antibiotics and Microbial resistance) feasibility trial, primary care patients with penicillin allergy were randomised to penicillin allergy assessment pathway or usual care to assess the effect on health outcomes. A behavioural intervention package was developed to aid delabelling. This study aimed to investigate patients' and clinicians' views of penicillin allergy testing (PAT). DESIGN: We conducted a mixed-methods process evaluation embedded within the ALABAMA trial, which included a clinician survey, a patient survey (at baseline and follow-up) and semistructured interviews with patients and clinicians. SETTINGS: The study was conducted in primary care, as part of the feasibility stage of the ALABAMA trial. PARTICIPANTS: Patients and primary care clinicians. RESULTS: Clinicians (N=53; 52.2%) were positive about PAT and its potential value but did not have previous experience of referring patients for a PAT and were unsure whether patients would take penicillin after a negative allergy test. Patients (N=36; 46%) were unsure whether they were severely allergic to penicillin and did not fear a severe allergic reaction to penicillin. Clinician interviews showed that they were already aware of the benefit of PAT. Interviews with patients suggested the importance of safety as patients valued having numerous opportunities to address their concerns about safety of the test. CONCLUSIONS: This study highlights the positive effects of the ALABAMA behavioural intervention for both patients and clinicians. TRIAL REGISTRATION NUMBER: NCT04108637; ISRCTN20579216; Pre-results.

Treating Aging Changes of Facial Anatomical Layers with Hyaluronic Acid Fillers.

The aging process affects every anatomical layer of the face. Improved knowledge of how aging occurs in each anatomical layer of the face has helped evolve the facial rejuvenation strategies with HA fillers. Understanding the age-related changes in the anatomical facial layers, including their time of onset and how the changes occur in the different tissue layers, an injector can provide much more targeted and refined HA filler treatments. As fillers' use has increased, there has been a distinct shift away from procedures lifting the skin and SMAS. We can selectively target the anatomical facial layers with HA fillers for more refined and predictable outcomes. An extensive range of HA filler variants is now available. Each filler type is optimized and designed to be injected into specific tissue planes for the best results. Knowing the predictable aging changes in the different tissue layers of the face is crucial as this guides the optimum filler choice. Working knowledge of the individual characteristics of the numerous HA-based products allows for their effective placement in the correct layer. Familiarity with the correct HA product may also help to minimize the downtime and risk of adverse events.