The role of activator protein-1 (AP-1) complex in diabetes associated atherosclerosis: Insights from single cell RNA sequencing.

Despite advances in the treatment of atherosclerotic cardiovascular disease, it remains the leading cause of death in patients with diabetes. Even when risk factors are mitigated, the disease progresses, and thus newer targets need to be identified that directly inhibit the underlying pathobiology of atherosclerosis in diabetes. A single cell sequencing approach was utilised to distinguish the proatherogenic transcriptional profile in aortic cells in diabetes using a streptozotocin induced-diabetic Apoe-/- mouse model. Human carotid endarterectomy specimens from individuals with and without diabetes were also evaluated via immunohistochemical analysis. Further mechanistic studies were performed in human aortic endothelial cells and human THP-1 derived macrophages. We then performed a preclinical study using an AP-1 inhibitor in a diabetic Apoe-/- mouse model. Single cell RNA sequencing analysis identified the AP-1 complex as a novel target in diabetes-associated atherosclerosis. AP-1 levels were elevated in carotid endarterectomy specimens from diabetic when compared to non-diabetic individuals. AP-1 was validated as a mechanosensitive transcription factor via immunofluorescence staining for regional heterogeneity of endothelial cells of the aortic region exposed to turbulent blood flow and by performing microfluidics experiments in HAECs. AP-1 inhibition with T-5224 blunted endothelial cell activation as assessed by a monocyte adhesion assay and expression of genes relevant to endothelial function. Furthermore, AP-1 inhibition attenuated foam cell formation. Critically, treatment with T-5224 attenuated atherosclerosis development in diabetic Apoe-/- mice. This study has identified the AP-1 complex as a novel target, inhibition of which treats the underlying pathobiology of atherosclerosis in diabetes.

Coping with Tourette's syndrome: a meta-ethnography of individual and family perspectives.

OBJECTIVE: This systematic review and meta-ethnography aimed to examine how children, adults and families cope with Tourette's syndrome (TS). METHODS: A systematic search of four databases was completed in October 2022. Sixteen papers met the inclusion criteria and were synthesised using Noblit and Hare's (1988) meta-ethnographic approach. RESULTS: Three themes were constructed: redefining the self and social identity, controlling the visible presentation of Tourette's syndrome, and challenging the narrative. CONCLUSION: Findings indicate that coping involves the need to integrate TS with identity, to exert control over tics and to challenge the misrepresentations of TS in wider society. A supportive environment provided by parents and friends enables individuals to feel proud that they can control their tics, and this allows for the positive integration of TS into identity. Raising awareness at a societal level through educational campaigns is important when aiming to improve coping with a stigmatised condition. Further research is recommended, for example, to understand how common co-occurring conditions, such as attention deficit hyperactivity disorder, impact coping.

Glucagon-like peptide-1 receptor signaling modifies the extent of diabetic kidney disease through dampening the receptor for advanced glycation end products-induced inflammation.

Glucagon like peptide-1 (GLP-1) is a hormone produced and released by cells of the gastrointestinal tract following meal ingestion. GLP-1 receptor agonists (GLP-1RA) exhibit kidney-protective actions through poorly understood mechanisms. Here we interrogated whether the receptor for advanced glycation end products (RAGE) plays a role in mediating the actions of GLP-1 on inflammation and diabetic kidney disease. Mice with deletion of the GLP-1 receptor displayed an abnormal kidney phenotype that was accelerated by diabetes and improved with co-deletion of RAGE in vivo. Activation of the GLP-1 receptor pathway with liraglutide, an anti-diabetic treatment, downregulated kidney RAGE, reduced the expansion of bone marrow myeloid progenitors, promoted M2-like macrophage polarization and lessened markers of kidney damage in diabetic mice. Single cell transcriptomics revealed that liraglutide induced distinct transcriptional changes in kidney endothelial, proximal tubular, podocyte and macrophage cells, which were dominated by pathways involved in nutrient transport and utilization, redox sensing and the resolution of inflammation. The kidney-protective action of liraglutide was corroborated in a non-diabetic model of chronic kidney disease, the subtotal nephrectomised rat. Thus, our findings identify a novel glucose-independent kidney-protective action of GLP-1-based therapies in diabetic kidney disease and provide a valuable resource for exploring the cell-specific kidney transcriptional response ensuing from pharmacological GLP-1R agonism.

EZH2 inhibitors promote ß-like cell regeneration in young and adult type 1 diabetes donors.

ß-cells are a type of endocrine cell found in pancreatic islets that synthesize, store and release insulin. In type 1 diabetes (T1D), T-cells of the immune system selectively destroy the insulin-producing ß-cells. Destruction of these cells leads to a lifelong dependence on exogenous insulin administration for survival. Consequently, there is an urgent need to identify novel therapies that stimulate ß-cell growth and induce ß-cell function. We and others have shown that pancreatic ductal progenitor cells are a promising source for regenerating ß-cells for T1D owing to their inherent differentiation capacity. Default transcriptional suppression is refractory to exocrine reaction and tightly controls the regenerative potential by the EZH2 methyltransferase. In the present study, we show that transient stimulation of exocrine cells, derived from juvenile and adult T1D donors to the FDA-approved EZH2 inhibitors GSK126 and Tazemetostat (Taz) influence a phenotypic shift towards a ß-like cell identity. The transition from repressed to permissive chromatin states are dependent on bivalent H3K27me3 and H3K4me3 chromatin modification. Targeting EZH2 is fundamental to ß-cell regenerative potential. Reprogrammed pancreatic ductal cells exhibit insulin production and secretion in response to a physiological glucose challenge ex vivo. These pre-clinical studies underscore the potential of small molecule inhibitors as novel modulators of ductal progenitor differentiation and a promising new approach for the restoration of ß-like cell function.

Circulating epigenomic biomarkers correspond with kidney disease susceptibility in high-risk populations with type 2 diabetes mellitus.

AIMS: To investigate epigenomic indices of diabetic kidney disease (DKD) susceptibility among high-risk populations with type 2 diabetes mellitus. METHODS: KDIGO (Kidney Disease: Improving Global Outcomes) clinical guidelines were used to classify people living with or without DKD. Differential gene methylation of DKD was then assessed in a discovery Aboriginal Diabetes Study cohort (PROPHECY, 89 people) and an external independent study from Thailand (THEPTARIN, 128 people). Corresponding mRNA levels were also measured and linked to levels of albuminuria and eGFR. RESULTS: Increased DKD risk was associated with reduced methylation and elevated gene expression in the PROPHECY discovery cohort of Aboriginal Australians and these findings were externally validated in the THEPTARIN diabetes registry of Thai people living with type 2 diabetes mellitus. CONCLUSIONS: Novel epigenomic scores can improve diagnostic performance over clinical modelling using albuminuria and GFR alone and can distinguish DKD susceptibility.

Characterization of K562 cells: uncovering novel chromosomes, assessing transferrin receptor expression, and probing pharmacological therapies.

Human erythroleukemic K562 cells represent the prototypical cell culture model of chronic myeloid leukemia (CML). The cells are pseudo-triploid and positive for the Philadelphia chromosome. Therefore, K562 cells have been widely used for investigating the BCR/ABL1 oncogene and the tyrosine kinase inhibitor, imatinib-mesylate. Further, K562 cells overexpress transferrin receptors (TfR) and have been used as a model for targeting cytotoxic therapies, via receptor-mediated endocytosis. Here, we have characterized K562 cells focusing on the karyotype of cells in prolonged culture, regulation of expression of TfR in wildtype (WT) and doxorubicin-resistant cells, and responses to histone deacetylase inhibition (HDACi). Karyotype analysis indicates novel chromosomes and gene expression analysis suggests a shift of cultured K562 cells away from patient-derived leukemic cells. We confirm the high expression of TfR on K562 cells using immunofluorescence and cell-surface receptor binding radioassays. Importantly, high TfR expression is observed in patient-derived cells, and we highlight the persistent expression of TfR following doxorubicin acquired resistance. Epigenetic analysis indicates that permissive histone acetylation and methylation at the promoter region regulates the transcription of TfR in K562 cells. Finally, we show relatively high expression of HDAC enzymes in K562 cells and demonstrate the chemotoxic effects of HDACi, using the FDA-approved hydroxamic acid, vorinostat. Together with a description of morphology, infrared spectral analysis, and examination of metabolic properties, we provide a comprehensive characterization of K562 cells. Overall, K562 cell culture systems remain widely used for the investigation of novel therapeutics for CML, which is particularly important in cases of imatinib-mesylate resistance.

Supervised smoking facility access, harm reduction practices, and substance use changes during the COVID-19 pandemic: a community-engaged cross-sectional study.

BACKGROUND: The potential public health benefits of supervised smoking facilities (SSFs) are considerable, and yet implementation of SSFs in North America has been slow. We conducted this study to respond to significant knowledge gaps surrounding SSF utilization and to characterize substance use, harm reduction practices, and service utilization following the onset of the COVID-19 pandemic. METHODS: A questionnaire was self-administered at a single site by 175 clients using an outdoor SSF in Vancouver, Canada, between October-December 2020. Questionnaire responses were summarized using descriptive statistics. Multinomial logistic regression techniques were used to examine factors associated with increased SSF utilization. RESULTS: Almost all respondents reported daily substance use (93% daily use of opioids; 74% stimulants). Most used opioids (85%) and/or methamphetamine (66%) on the day of their visit to the SSF. Respondents reported drug use practice changes at the onset of COVID-19 to reduce harm, including using supervised consumption sites, not sharing equipment, accessing medically prescribed alternatives, cleaning supplies and surfaces, and stocking up on harm reduction supplies. Importantly, 45% of SSF clients reported using the SSF more often since the start of COVID-19 with 65.2% reporting daily use of the site. Increased substance use was associated with increased use of the SSF, after controlling for covariates. CONCLUSIONS: Clients of the SSF reported increasing not only their substance use, but also their SSF utilization and harm reduction practices following the onset of COVID-19. Increased scope and scale of SSF services to meet these needs are necessary.

Pharmacological inhibition of human EZH2 can influence a regenerative ß-like cell capacity with in vitro insulin release in pancreatic ductal cells.

BACKGROUND: Therapeutic replacement of pancreatic endocrine ß-cells is key to improving hyperglycaemia caused by insulin-dependent diabetes . Whilst the pool of ductal progenitors, which give rise to the endocrine cells, are active during development, neogenesis of islets is repressed in the human adult. Recent human donor studies have demonstrated the role of EZH2 inhibition in surgically isolated exocrine cells showing reactivation of insulin expression and the influence on the H3K27me3 barrier to ß-cell regeneration. However, those studies fall short on defining the cell type active in transcriptional reactivation events. This study examines the role of the regenerative capacity of human pancreatic ductal cells when stimulated with pharmacological inhibitors of the EZH2 methyltransferase. RESULTS: Human pancreatic ductal epithelial cells were stimulated with the EZH2 inhibitors GSK-126, EPZ6438, and triptolide using a 2- and 7-day protocol to determine their influence on the expression of core endocrine development marker NGN3, as well as ß-cell markers insulin, MAFA, and PDX1. Chromatin immunoprecipitation studies show a close correspondence of pharmacological EZH2 inhibition with reduced H3K27me3 content of the core genes, NGN3, MAFA and PDX1. Consistent with the reduction of H3K27me3 by pharmacological inhibition of EZH2, we observe measurable immunofluorescence staining of insulin protein and glucose-sensitive insulin response. CONCLUSION: The results of this study serve as a proof of concept for a probable source of ß-cell induction from pancreatic ductal cells that are capable of influencing insulin expression. Whilst pharmacological inhibition of EZH2 can stimulate secretion of detectable insulin from ductal progenitor cells, further studies are required to address mechanism and the identity of ductal progenitor cell targets to improve likely methods designed to reduce the burden of insulin-dependent diabetes.

Reduced methylation correlates with diabetic nephropathy risk in type 1 diabetes.

Diabetic nephropathy (DN) is a polygenic disorder with few risk variants showing robust replication in large-scale genome-wide association studies. To understand the role of DNA methylation, it is important to have the prevailing genomic view to distinguish key sequence elements that influence gene expression. This is particularly challenging for DN because genome-wide methylation patterns are poorly defined. While methylation is known to alter gene expression, the importance of this causal relationship is obscured by array-based technologies since coverage outside promoter regions is low. To overcome these challenges, we performed methylation sequencing using leukocytes derived from participants of the Finnish Diabetic Nephropathy (FinnDiane) type 1 diabetes (T1D) study (n = 39) that was subsequently replicated in a larger validation cohort (n = 296). Gene body-related regions made up more than 60% of the methylation differences and emphasized the importance of methylation sequencing. We observed differentially methylated genes associated with DN in 3 independent T1D registries originating from Denmark (n = 445), Hong Kong (n = 107), and Thailand (n = 130). Reduced DNA methylation at CTCF and Pol2B sites was tightly connected with DN pathways that include insulin signaling, lipid metabolism, and fibrosis. To define the pathophysiological significance of these population findings, methylation indices were assessed in human renal cells such as podocytes and proximal convoluted tubule cells. The expression of core genes was associated with reduced methylation, elevated CTCF and Pol2B binding, and the activation of insulin-signaling phosphoproteins in hyperglycemic cells. These experimental observations also closely parallel methylation-mediated regulation in human macrophages and vascular endothelial cells.

Top-down attention control does not imply voluntary attention control for all individuals.

Top-down information is known to play an important role in the control of visual attention. Often, evidence for top-down attention control is also interpreted as evidence for voluntary attention control. However, this latter theoretical interpretation is not warranted because volition is typically defined in terms of a conscious feeling that prior intentions led to a subsequent action, but this aspect of performance has not been assessed in previous studies. Accordingly, the present study used the construct of "agency" within the context of the spatial cuing paradigm to examine the relation between top-down and voluntary attention control. The results of two experiments consistently showed using growth-curve modeling that standard manipulations of top-down information in the spatial cuing paradigm do not have the same effect on all participants. In particular, the present findings showed that a slight majority of individuals (~60%) exhibited the expected pattern in which they reported feeling more agency when they performed visual search with the aid of an informative (arrow or onset) cue than when they performed this task with an uninformative cue or without any cue at all. However, more importantly, these findings also showed that a substantial number of individuals (~40%) exhibited the opposite pattern in which they reported feeling more agency when they performed visual search with an uninformative cue or without any cue at all. We conclude that the relation between top-down and voluntary attention control is not straightforward and must be studied using methods that are sensitive to individual differences. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Sulforaphane prevents and reverses allergic airways disease in mice via anti-inflammatory, antioxidant, and epigenetic mechanisms.

Sulforaphane has been investigated in human pathologies and preclinical models of airway diseases. To provide further mechanistic insights, we explored L-sulforaphane (LSF) in the ovalbumin (OVA)-induced chronic allergic airways murine model, with key hallmarks of asthma. Histological analysis indicated that LSF prevented or reversed OVA-induced epithelial thickening, collagen deposition, goblet cell metaplasia, and inflammation. Well-known antioxidant and anti-inflammatory mechanisms contribute to the beneficial effects of LSF. Fourier transform infrared microspectroscopy revealed altered composition of macromolecules, following OVA sensitization, which were restored by LSF. RNA sequencing in human peripheral blood mononuclear cells highlighted the anti-inflammatory signature of LSF. Findings indicated that LSF may alter gene expression via an epigenetic mechanism which involves regulation of protein acetylation status. LSF resulted in histone and α-tubulin hyperacetylation in vivo, and cellular and enzymatic assays indicated decreased expression and modest histone deacetylase (HDAC) inhibition activity, in comparison with the well-known pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). Molecular modeling confirmed interaction of LSF and LSF metabolites with the catalytic domain of metal-dependent HDAC enzymes. More generally, this study confirmed known mechanisms and identified potential epigenetic pathways accounting for the protective effects and provide support for the potential clinical utility of LSF in allergic airways disease.

Intestinal microbiota regulates diabetes and cancer progression by IL-1ß and NOX4 dependent signaling cascades.

Diabetes changes the host microbiota, a condition known as dysbiosis. Dysbiosis is an important factor for the pathogenesis of diabetes and colorectal cancer (CRC). We aimed at identifying the microbial signature associated with diabetes and CRC; and identifying the signaling mechanism altered by dysbiosis and leading to CRC progression in diabetes. MKR mice that can spontaneously develop type 2 diabetes were used. For CRC induction, another subset of mice was treated with azoxymethane and dextran sulfate sodium. To identify the role of microbiota, microbiota-depleted mice were inoculated with fecal microbial transplant from diabetic and CRC mice. Further, a mouse group was treated with probiotics. At the end of the treatment, 16S rRNA sequencing was performed to identify microbiota in the fecal samples. Blood was collected, and colons were harvested for molecular, anatomical, and histological analysis. Our results show that diabetes is associated with a microbial signature characterized by reduction of butyrate-forming bacteria. This dysbiosis is associated with gastrointestinal complications reflected by a reduction in colon lengths. These changes are reversed upon treatment with probiotics, which rectified the observed dysbiosis. Inoculation of control mice with diabetic or cancer microbiota resulted in the development of increased number of polyps. Our data also show that inflammatory cytokines (mainly interleukin (IL)-1ß) and NADPH oxidase (NOX)4 are over-expressed in the colon tissues of diabetic mice. Collectively our data suggest that diabetes is associated with dysbiosis characterized by lower abundance of butyrate-forming bacteria leading to over-expression of IL-1ß and NOX4 leading to gastrointestinal complications and CRC.

Inhibition of pancreatic EZH2 restores progenitor insulin in T1D donor.

Type 1 diabetes (T1D) is an autoimmune disease that selectively destroys insulin-producing ß-cells in the pancreas. An unmet need in diabetes management, current therapy is focussed on transplantation. While the reprogramming of progenitor cells into functional insulin-producing ß-cells has also been proposed this remains controversial and poorly understood. The challenge is determining why default transcriptional suppression is refractory to exocrine reactivation. After the death of a 13-year-old girl with established insulin-dependent T1D, pancreatic cells were harvested in an effort to restore and understand exocrine competence. The pancreas showed classic silencing of ß-cell progenitor genes with barely detectable insulin (Ins) transcript. GSK126, a highly selective inhibitor of EZH2 methyltransferase activity influenced H3K27me3 chromatin content and transcriptional control resulting in the expression of core ß-cell markers and ductal progenitor genes. GSK126 also reinstated Ins gene expression despite absolute ß-cell destruction. These studies show the refractory nature of chromatin characterises exocrine suppression influencing ß-cell plasticity. Additional regeneration studies are warranted to determine if the approach of this n-of-1 study generalises to a broader T1D population.

Microbial Isolates and Association with Disease Severity and Quality of Life in Individuals with Hidradenitis Suppurativa: An Observational Study.

Introduction: Hidradenitis suppurativa (HS) is a chronic inflammatory disease characterized by recurrent nodules, abscesses, and sinus tracts, resulting in scarring. Patients suffer significantly impacted quality of life (QoL), manifested by anxiety and depression. We describe microbial isolates identified from active sites in adults with HS, and explore associations between organisms isolated, disease severity, and QoL. Methods: Observational study over 2 years. Assessment was made of disease severity using the Hurley staging score and of QoL scores, and subjective assessment. Wound swab samples were obtained from sites of active disease. Descriptive summary statistics and tests of significance were used to analyse the data. Results: Two hundred and twelve patients participated, resulting in 352 episodes, and 501 lesion swab samples. 54% were female and 59% were obese, median age of 37 years. A lower proportion of Gram-negative organisms and more staphylococcal and streptococcal organisms were isolated from sites of disease in individuals with stage 3 disease compared to those with stage 1-2 disease (p = 0.001). We found no association between microbial isolation and QoL. Discussion/Conclusions: Hurley stage 3 disease is associated with more infected lesions than Hurley stage 1-2 disease, but the QoL experienced by patients with HS is determined by factors other than infection.

The EDA-deficient mouse has Zymbal's gland hypoplasia and acute otitis externa.

In mice, rats, dogs and humans, the growth and function of sebaceous glands and eyelid Meibomian glands depend on the ectodysplasin signalling pathway. Mutation of genes encoding the ligand EDA, its transmembrane receptor EDAR and the intracellular signal transducer EDARADD leads to hypohidrotic ectodermal dysplasia, characterised by impaired development of teeth and hair, as well as cutaneous glands. The rodent ear canal has a large auditory sebaceous gland, the Zymbal's gland, the function of which in the health of the ear canal has not been determined. We report that EDA-deficient mice, EDAR-deficient mice and EDARADD-deficient rats have Zymbal's gland hypoplasia. EdaTa mice have 25% prevalence of otitis externa at postnatal day 21 and treatment with agonist anti-EDAR antibodies rescues Zymbal's glands. The aetiopathogenesis of otitis externa involves infection with Gram-positive cocci, and dosing pregnant and lactating EdaTa females and pups with enrofloxacin reduces the prevalence of otitis externa. We infer that the deficit of sebum is the principal factor in predisposition to bacterial infection, and the EdaTa mouse is a potentially useful microbial challenge model for human acute otitis externa.

Seoul Virus Associated with Pet Rats, Scotland, UK, 2019.

We describe a case of hemorrhagic fever with renal syndrome caused by Seoul virus in a woman in Scotland, UK. Whole-genome sequencing showed the virus belonged to a lineage characterized by recent international expansion, probably driven by trade in pet rats.