ABSTRACT Stem-cell-derived β-like-cells (SCβ-cells) provide a promising platform for diabetes modelling and cell replacement therapy, but their incomplete functional maturation remains a challenge. Here, we compared immature SCβ-cells to human primary β-cells utilizing a multi-omic, single-cell framework integrating patch-clamp electrophysiology with scRNA sequencing (patch-seq), regulatory network inference, and functional phenotyping. Despite low insulin secretion and reduced insulin content, SCβ-cells displayed larger Na + and Ca 2+ currents and depolarization-induced exocytosis. Ultrastructural and metabolic profiling revealed immature insulin granules, altered mitochondrial morphology, elevated basal respiration and proton leak, and diminished spare respiratory capacity and glucose-responsive metabolism. Patch-seq linked exocytotic activity in SCβ-cells to oxidative phosphorylation and MYC target programs, consistent with incomplete terminal differentiation, whereas SCβ-cells expressing higher levels of mature identity markers showed reduced ion channel hyperactivity. Multi-omics profiling showed that electrophysiological features in SCβ-cells were embedded in transcriptional programs distinct from those of primary β-cells and other endocrine cells. Network control theory nominated SREBP1, an endoplasmic reticulum tethered transcription factor regulating cholesterol and lipid homeostasis, as a promising candidate involved in this immature state. Inhibition of cholesterol trafficking increased SREBF1 expression and shifted metabolic and transcriptional features towards a more mature β-like state. These data identify potential targets and pathways that can be leveraged to improve SCβ-cell maturation and validate cholesterol and lipid homeostasis through the SREBP1 axis as one such candidate.

Epidemiological studies consistently report associations between circulating concentrations of persistent organic pollutants and increased type 2 diabetes risk. Measures of pollutant concentrations in pancreas are limited; given the role of the endocrine pancreas in diabetes pathogenesis, this is an important gap in the literature. Additionally, no studies have correlated pollutant concentrations with direct measures of human beta cell function. We measure concentrations from 3 pollutant classes-dioxins/furans, polychlorinated biphenyls, and organochlorine pesticides-in pancreas and peripancreatic adipose tissue biopsies obtained from 31 human organ donors. We show that pollutants are consistently detected in human pancreas, and for some analytes, at higher concentrations than in adipose. We next assess correlations between pollutant concentrations and systemic indicators of diabetes risk (body mass index, age, and haemoglobin A1c) and direct measures of beta cell function in isolated islets from the same 31 donors. Pancreas polychlorinated biphenyls and organochlorine pesticides positively correlate with body mass index, age and basal insulin secretion but negatively correlate with stimulation index (ratio of insulin secretion under high glucose / low glucose conditions). Pancreas dioxins/furans positively correlate with fatty acid- and amino acid-stimulated insulin secretion. These data indicate that lipophilic pollutants accumulate in human pancreas and positively correlate with surrogate markers of diabetes risk.

Type 1 diabetes (T1D) is characterized by the autoimmune destruction of most insulin-producing β-cells, along with dysregulated glucagon secretion from pancreatic α-cells. We conducted an integrated analysis that combines electrophysiological and transcriptomic profiling, along with machine learning, of islet cells from T1D donors to investigate the mechanisms underlying their dysfunction. Surviving β-cells exhibit altered electrophysiological properties and transcriptomic signatures indicative of increased antigen presentation, metabolic reprogramming, and impaired protein translation. In α-cells, we observed hyper-responsiveness and increased exocytosis, which are associated with upregulated immune signaling, disrupted transcription factor localization and lysosome homeostasis, as well as dysregulation of mTORC1 complex signaling. Notably, key genetic risk signals for T1D were enriched in transcripts related to α-cell dysfunction, including MHC class I which were closely linked with α-cell dysfunction. Our data provide novel insights into the molecular underpinnings of islet cell dysfunction in T1D, highlighting pathways that may be leveraged to preserve residual β-cell function and modulate α-cell activity. These findings underscore the complex interplay between immune signaling, metabolic stress, and cellular identity in shaping islet cell phenotypes in T1D. ### Competing Interest Statement The authors have declared no competing interest.

AIMS/HYPOTHESIS:Diabetes is associated with the dysfunction of glucagon-producing pancreatic islet alpha cells, although the underlying mechanisms regulating glucagon secretion and alpha cell dysfunction remain unclear. While insulin secretion from pancreatic beta cells has long been known to be controlled partly by intracellular phospholipid signalling, very little is known about the role of phospholipids in glucagon secretion. Using patch-clamp electrophysiology and single-cell RNA sequencing, we previously found that expression of PIP4P2 (encoding TMEM55A, a lipid phosphatase that dephosphorylates phosphatidylinositol-4,5-bisphosphate [PIP2] to phosphatidylinositol-5-phosphate [PI5P]) correlates with alpha cell function. We hypothesise that TMEM55A is involved in glucagon secretion and aim to validate the role of TMEM55A and its potential signalling molecules in alpha cell function and glucagon secretion. METHODS:Correlation analysis was generated from the data in www.humanislets.com . Human islets were isolated at the Alberta Diabetes Institute IsletCore. Electrical recordings were performed on dispersed human or mouse islets with scrambled siRNA or si-PIP4P2 (si-Pip4p2 for mouse) transfection. Glucagon secretion was measured using an islet perfusion system with intact mouse islets. TMEM55A activity was measured using an in vitro on-beads phosphatase assay and live-cell imaging. GTPase activity was measured using an active GTPase pull-down assay. Confocal microscopy was used to quantify F-actin intensity using primary alpha cells and alphaTC1-9 cell lines after chemical treatment. RESULTS:TMEM55A regulated alpha cell exocytosis and glucagon secretion. TMEM55A knockdown in both human and mouse alpha cells reduced exocytosis at low glucose levels and this was rescued by the direct reintroduction of PI5P. PI5P, instead of PIP2 increased the glucagon secretion using intact mouse islets. This did not occur through an effect on Ca2+ channel activity but through a remodelling of cortical F-actin dependent on TMEM55A lipid phosphatase activity, which occurred in response to oxidative stress. TMEM55A- and PI5P-induced F-actin remodelling depends on the inactivation of GTPase and RhoA, instead of Ras-related C3 botulinum toxin substrate 1 or CDC42. CONCLUSIONS/INTERPRETATION:We reveal a novel pathway by which TMEM55A regulates alpha cell exocytosis by controlling intracellular PI5P and the F-actin network.

Epidemiological studies consistently report associations between circulating concentrations of persistent organic pollutants (POPs) and increased type 2 diabetes risk. Measures of POP concentrations in pancreas are limited; given the role of the endocrine pancreas in diabetes pathogenesis, this is an important gap in the literature. Additionally, no studies have correlated POP concentrations with direct measures of beta cell function in humans. We hypothesized that lipophilic POPs accumulate in human pancreas and correlate with markers of diabetes risk. To test this hypothesis, we measured POP concentration from 3 chemical classes - dioxins/furans, polychlorinated biphenyls (PCBs), and organochlorine pesticides (OCPs) - in pancreas and peripancreatic adipose tissue biopsies obtained from 31 human organ donors via the Alberta Diabetes Institute IsletCore. Indeed, POPs were consistently detected in human pancreas, and for some pollutants, at higher concentrations than in adipose. We next assessed correlations between POP concentrations and systemic indicators of diabetes risk (BMI, age, and %HbA1c) and direct measures of beta cell function. To this end, we measured insulin secretion in response to numerous secretagogues (i.e. glucose, fatty acids, amino acids, exendin-4, or KCl) in isolated islets from the same 31 donors. Pancreas PCBs and OCPs positively correlated with BMI, age and basal insulin secretion but negatively correlated with stimulation index (ratio of insulin secretion under high glucose / low glucose conditions). In contrast, pancreas dioxins/furans positively correlated with fatty acid- and amino acid-stimulated insulin secretion. These data confirm that lipophilic pollutants accumulate in human pancreas and positively correlate with markers of diabetes risk. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This research was supported by a Canadian Institutes of Health Research (CIHR) Project Grant (#PJT-2018-159590). J.E.B. is supported by an Early Researcher Award from the Ontario Government. E.E.M is supported by a Diabetes Canada End Diabetes Award (OG-3-24-5818-EM). M.P.H. was supported by a CIHR CGS-D award. M.E.A.C. was supported by an NSERC CGS-M and NSERC CGS-D award. J.P. was supported by the Guiding interdisciplinary Research on Womens and girls health and Wellbeing (GROWW) scholarship and Ontario Graduate Scholarship. This work includes data from HumanIslets.com funded by the Canadian Institutes of Health Research, JDRF Canada, and Diabetes Canada (5-SRA-2021-1149-S-B/TG 179092) with data from islets isolated by the Alberta Diabetes Institute IsletCore with the support of the Human Organ Procurement and Exchange program, Trillium Gift of Life Network, BC Transplant, Quebec Transplant, and other Canadian organ procurement organizations. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: the Research Ethics Board at Carleton University (#106701) and the University of Alberta (Pro00013094) gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.

BACKGROUND:Induced pluripotent stem cells (iPSCs) offer the potential to generate autologous iPSC-derived islets (iPSC islets), however, remain limited by scalability and product safety. METHODS:Herein, we report stagewise characterization of cells generated following a bioreactor-based differentiation protocol. Cell characteristics were assessed using flow cytometry, quantitative reverse transcription polymerase chain reaction, patch clamping, functional assessment, and in vivo functional and immunohistochemistry evaluation. Protocol yield and costs are assessed to determine scalability. RESULTS:Differentiation was capable of generating 90.4% PDX1 + /NKX6.1 + pancreatic progenitors and 100% C-peptide + /NKX6.1 + iPSC islet cells. However, 82.1%, 49.6%, and 0.9% of the cells expressed SOX9 (duct), SLC18A1 (enterochromaffin cells), and CDX2 (gut cells), respectively. Explanted grafts contained mature monohormonal islet-like cells, however, CK19 + ductal tissues persist. Using this protocol, semi-planar differentiation using 150 mm plates achieved 5.72 × 10 4 cells/cm 2 (total 8.3 × 10 6 cells), whereas complete suspension differentiation within 100 mL Vertical-Wheel bioreactors significantly increased cell yield to 1.1 × 10 6 cells/mL (total 105.0 × 10 6 cells), reducing costs by 88.8%. CONCLUSIONS:This study offers a scalable suspension-based approach for iPSC islet differentiation within Vertical-Wheel bioreactors with thorough characterization of the ensuing product to enable future protocol comparison and evaluation of approaches for off-target cell elimination. Results suggest that bioreactor-based suspension differentiation protocols may facilitate scalability and clinical implementation of iPSC islet therapies.

Type 1 diabetes (T1D) is characterized by the autoimmune destruction of most insulin-producing β cells, along with dysregulated glucagon secretion from pancreatic α cells. We conducted an integrated analysis that combines electrophysiological and transcriptomic profiling, along with machine learning, of islet cells from T1D donors. The few surviving β cells exhibit altered electrophysiological properties and transcriptomic signatures indicative of increased antigen presentation, metabolic reprogramming, and impaired protein translation. In α cells, we observed hyperresponsiveness and increased exocytosis, which are associated with upregulated immune signaling, disrupted transcription factor localization, and lysosome homeostasis, as well as dysregulation of mTORC1 complex signaling. Notably, key genetic risk signals for T1D were enriched in transcripts related to α cell dysfunction, including MHC class I, which were closely linked with α cell dysfunction. Our data provide what we believe are novel insights into the molecular underpinnings of islet cell dysfunction in T1D, highlighting pathways that may be leveraged to preserve residual β cell function and modulate α cell activity. These findings underscore the complex interplay between immune signaling, metabolic stress, and cellular identity in shaping islet cell phenotypes in T1D.

The list of generative AI tools is ever-expanding, as is the hype around such systems. Many such tools are oriented towards augmenting or complementing the existing work of designers. Tools such as Midjourney and others provide features that easily fit into the long-standing processes, research, and creative work currently done by interface designers. For example, generative AI tools may speed the process of developing and iterating on interface prototypes. However, as happens with all technologies when acquiring broader use, these systems are being creatively repurposed. Of interest to the EduCHI community are those unanticipated uses that serve to contribute to user experience design and research in novel, unexpected ways. This position paper provides an overview of existing generative AI tools, their intended purpose, and their unanticipated uses within the design and research process, as evidenced by a brief review of related literature and selected classroom examples. This position paper aims to pose questions relevant to CHI educators, namely how can these possibilities be woven into existing courses? How might our students contribute to these creative approaches and at what level of maturity shall these tools and techniques be presented alongside more traditional methods? Lastly, we raise ethical questions about the use of these tools and highlight the challenges of using them in the context of design.

Pancreatic β-cells respond to metabolic stress by upregulating insulin secretion, however the underlying mechanisms remain unclear. Here we show, in β-cells from overweight humans without diabetes and mice fed a high-fat diet for 2 days, insulin exocytosis and secretion are enhanced without increased Ca 2+ influx. RNA-seq of sorted β-cells suggests altered metabolic pathways early following high fat diet, where we find increased basal oxygen consumption and proton leak, but a more reduced cytosolic redox state. Increased β-cell exocytosis after 2-day high fat diet is dependent on this reduced intracellular redox state and requires the sentrin-specific SUMO-protease-1. Mice with either pancreas- or β-cell-specific deletion of this fail to up-regulate exocytosis and become rapidly glucose intolerant after 2-day high fat diet. Mechanistically, redox-sensing by the SUMO-protease requires a thiol group at C535 which together with Zn + -binding suppresses basal protease activity and unrestrained β-cell exocytosis, and increases enzyme sensitivity to regulation by redox signals.

Purpose: Competency-based education and creation of profession-based entrustable professional activities are current trends in physical therapist (PT) education. The purpose of this project is to determine the current entry-level PT competencies for cardiovascular & pulmonary (CVP) physical therapy. Methods: The Academy of Cardiovascular & Pulmonary Physical Therapy Competency Task Force was established to update and expand previous competencies. A mixed method approach of Subject Matter Expert (SME) consensus (modified Delphi process) and a broader survey process was used to develop these entry-level competencies. With SME guidance, a modified version of the levels of competence, None-to-Proficient, was adopted based on previous competency work by the American Council of Academic Physical Therapy. Results: Twenty-nine SME members engaged in the consensus process, and 268, 194, 305, and 222 respondents completed the 4 surveys, respectively. Six hundred fifty-one competency items were rated across most Commission on the Accreditation of Physical Therapy Education Standard 7 Curriculum areas, following the patient–client management model. Conclusions: These competencies establish specific expected levels of knowledge, skills, and behaviors essential for entry-level CVP PT practice. Adopting these competencies will help lay the groundwork for future competency-based PT education and aid in the development of the physical therapy profession's core entrustable professional activities.

Racial and ethnic underrepresentation in aging research and need for effective recruitment strategies is well documented. A Community Research Liaison (CRL) role created under a NIA award is demonstrating the value of embedding staff from underrepresented communities in the research infrastructure. The role and qualifications for a CRL were developed with individuals from an African American community. The CRL hired from this community is engaging in outreach to understand barriers to and discuss the value of research with community members. Through the CRL, community input is being translated into changes in recruitment methods that reflect the concerns, interests and needs of potential African American participants. This case study reports on recruitment outcomes for a study on markers of cognitive decline in older adults requiring 2 hours in an MRI, 5 for testing, and 3 for travel. Recruitment data are compared 6 months before and 3 months after the CRL was engaged. In the 6 months prior to the CRL’s involvement, 309 individuals passed an initial screening and 156 were found potentially eligible after a second screening. Five (3.2%) of the 156 were African American. Of these, one was found ineligible, one was lost to follow-up and the 3 participated in the study. In the first 3 months after the CRL was engaged, 35 were referred to the study after recruitment by the CRL. All were African American, 15 (43%) were eligible and enrolled, 9 (26%) were ineligible, and 9 (26%) are still in the screening process. The case study demonstrates the promise of a CRL who is a member of an African American community in increasing African American participation in research. The CRL dramatically increased the number of older African Americans recruited despite potential participation barriers. The increase was due in large part to the CRL’s understanding of and recognition as a trusted member of the community and to changes she recommended to promotional materials and logistics based on community feedback. Data on recruitment effectiveness are continuing to be evaluated across a range of aging research studies and clinical trials in which the CRL is engaged.

Background and Purpose. Competency-based education (CBE) is an emerging topic within physical therapy (PT). It has emerged to assure all stakeholders that physical therapist education program graduates are proficient in the requisite knowledge, skills, and behaviors (KSBs) essential for entry-level practice. Competencies have existed within cardiovascular and pulmonary (CVP) PT since 1980, updated in 2008, and most recently updated in 2022. This article discusses how individuals should apply the 2022 CVP competencies to clinical practice and education. Position and Rationale. The 2022 CVP competencies were developed using a modified mixed-method Delphi approach. These competencies set a level of proficiency for KSBs used within entry-level CVP PT practice following the patient–client management model. The position put forward in this article describes how and why multiple stakeholder groups should apply these entry-level competencies specifically for graduates of physical therapist education programs who are entering practice (entry level). The competencies provide a more detailed description of expected proficiency for entry-level CVP PT practice than currently available documents. These competencies may form the basis for developing entrustable professional activities (EPAs). Discussion and Conclusion. The establishment of entry-level competencies is essential for use by multiple stakeholders to inform physical therapist curriculum, provide clinical instructors with a reference for expected levels of proficiency during final student clinical experiences, guide content on the Federation of State Boards of PT national licensure examination, and prepare employers to provide needed continued professional development, based on the clinical environment. These competencies lend themselves to the future development of EPAs in the PT profession for CVP PT.

Designated a pandemic in March 2020, the spread of severe acute respiratory syndrome virus 2 (SARS-CoV2), the virus responsible for coronavirus disease 2019 (COVID-19), led to new guidelines and restrictions being implemented for individuals, businesses, and societies in efforts to limit the impacts of COVID-19 on personal health and healthcare systems. Here we report the impacts of the COVID-19 pandemic on pancreas processing and islet isolation/distribution outcomes at the Alberta Diabetes Institute IsletCore, a facility specializing in the processing and distribution of human pancreatic islets for research. While the number of organs processed was significantly reduced, organ quality and the function of cellular outputs were minimally impacted during the pandemic when compared to an equivalent period immediately prior. Despite the maintained quality of isolated islets, feedback from recipient groups was more negative. Our findings suggest this is likely due to disrupted distribution which led to increased transit times to recipient labs, particularly those overseas. Thus, to improve overall outcomes in a climate of limited research islet supply, prioritization of tissue recipients based on likely tissue transit times may be needed.

AIMS:1) Can virtual fall risk screens be performed safely? 2) Are older adults able to manage technology to participate in telehealth? 3) Does an algorithm aid in referral appropriate evidence-based (EBP) fall prevention programs?METHODS:An algorithm was piloted using the Zoom platform to screen for falls, to assign to intervention groups, and to guide referral to EBP. Statistical analysis of data included descriptive, parametric, and non-parametric tests.RESULTS:Forty-four participants, aged 55-94 years, were screened. A significant relationship between 30-second chair stand and referral between two programs was found (p<0.05). Spearman correlations revealed statistically significant negative correlation between 30-second chair stand and timed up-and-go (TUG) (r= -0.584; p=0.003). No safety incidents occurred. Ninety-five percent of screened participants managed technology requirements successfully.CONCLUSION:Virtual fall risk screens are feasible and offer clinicians an alternative means to screen and refer older adults for EBP.

In diabetes, glucagon secretion from pancreatic α-cells is dysregulated. We examined α-cells from human donors and mice using combined electrophysiological, transcriptomic, and computational approaches. Rising glucose suppresses α-cell exocytosis by reducing P/Q-type Ca2+ channel activity, and this is disrupted in type 2 diabetes (T2D). Upon high-fat-feeding of mice, α-cells shift towards a ‘β-cell-like’ electrophysiologic profile in concert with an up-regulation of the β-cell Na+ channel isoform Scn9a and indications of impaired α-cell identity. In human α-cells we identify links between cell membrane properties and cell surface signalling receptors, mitochondrial respiratory complex assembly, and cell maturation. Cell type classification using machine learning of electrophysiology data demonstrates a heterogenous loss of ‘electrophysiologic identity’ in α-cells from donors with T2D. Indeed, a sub-set of α-cells with impaired exocytosis is defined by an enrichment in progenitor markers suggesting important links between α-cell maturation state and dysfunction in T2D. Key findings α-cell exocytosis is suppressed by glucose-dependent inhibition of P/Q-type Ca2+ currents Dysfunction of α-cells in type 2 diabetes is associated with a ‘β-cell-like’ electrophysiologic signature Patch-seq links maturation state, the mitochondrial respiratory chain, and cell surface receptor expression to α-cell function α-cell dysfunction occurs preferentially in cells enriched in endocrine lineage markers

Pancreatic β-cells respond to metabolic stress by upregulating insulin secretion, however underlying mechanisms remain unclear. In β-cells from overweight humans without diabetes, and mice fed high-fat diet (HFD) for as little as 2 days, insulin exocytosis and secretion are enhanced without increased Ca2+ influx. β-cell RNA-seq suggests altered metabolic pathways linked to cytosolic redox following HFD. The increased β-cell exocytosis upon HFD is dependent on a shift towards a reduced intracellular redox state, and increased expression of sentrin-specific protease-1 (Senp1). Mechanistically, allosteric binding of Zn2+ at a site that includes C535 suppresses basal SENP1 activity and unrestrained β-cell exocytosis and increases SENP1 sensitivity to activation by redox signals. Mice with pancreas- or β-cell SENP1 deletion fail to up-regulate exocytosis after 2-day HFD and become rapidly glucose intolerant. This highlights a key role for Zn2+-dependent redox signaling via SENP1 in β-cell functional responses to metabolic stress. (Words: 145) Teaser Early after high fat feeding in mice, a cytosolic reducing signal acting via SENP1 increases the capacity of β-cells to secrete insulin.

Identification of the genes and processes mediating genetic association signals for complex diseases represents a major challenge. As many of the genetic signals for type 2 diabetes (T2D) exert their effects through pancreatic islet-cell dysfunction, we performed a genome-wide pooled CRISPR loss-of-function screen in a human pancreatic beta cell line. We assessed the regulation of insulin content as a disease-relevant readout of beta cell function and identified 580 genes influencing this phenotype. Integration with genetic and genomic data provided experimental support for 20 candidate T2D effector transcripts including the autophagy receptor CALCOCO2. Loss of CALCOCO2 was associated with distorted mitochondria, less proinsulin-containing immature granules and accumulation of autophagosomes upon inhibition of late-stage autophagy. Carriers of T2D-associated variants at the CALCOCO2 locus further displayed altered insulin secretion. Our study highlights how cellular screens can augment existing multi-omic efforts to support mechanistic understanding and provide evidence for causal effects at genome-wide association studies loci.

ABSTRACTGenome-wide association studies have identified hundreds of signals for type 2 diabetes (T2D), most of which confer risk through effects on gene expression. We previously identified the transcription factor ZMIZ1 as a probable effector transcript in human islets, but how altered ZMIZ1 expression impacts T2D risk is unknown. We now show that islets from carriers of the T2D-risk alleles have reduced islet insulin content and glucose-stimulated insulin secretion. To elucidate the mechanism for islet-cell dysfunction, we generated β-cell-specific Zmiz1 knockout (Zmiz1βKO) mice. Male and female Zmiz1βKO mice were glucose intolerant with impaired insulin secretion, compared with control littermates. Transcriptomic profiling of Zmiz1βKO islets identified over 500 differentially expressed genes including those involved in β-cell function and maturity which we confirmed at the protein level. After high fat feeding, Zmiz1βKO mice fail to expand β-cell mass and become severely diabetic. Thus, Zmiz1 is required for normal glucose homeostasis and may contribute to T2D risk by maintaining a mature β-cell state and allowing islet mass expansion upon metabolic stress.