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.

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.

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.

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.

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.

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.

The Challenges of Implementing Artificial Intelligence into Surgical Practice.

BACKGROUND: Artificial intelligence is touted as the future of medicine. Classical algorithms for the detection of common bile duct stones (CBD) have had poor clinical uptake due to low accuracy. This study explores the challenges of developing and implementing a machine-learning model for the prediction of CBD stones in patients presenting with acute biliary disease (ABD). METHODS: All patients presenting acutely to Christchurch Hospital over a two-year period with ABD were retrospectively identified. Clinical data points including lab test results, demographics and ethnicity were recorded. Several statistical techniques were utilised to develop a machine-learning model. Issues with data collection, quality, interpretation and barriers to implementation were identified and highlighted. RESULTS: Issues with patient identification, coding accuracy, and implementation were encountered. In total, 1315 patients met inclusion criteria. Incorrect international classification of disease 10 (ICD-10) coding was noted in 36% (137/382) of patients recorded as having CBD stones. Patients with CBD stones were significantly older and had higher aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin and gamma-glutamyl transferase (GGT) levels (p < 0.001). The no information rate was 81% (1070/1315 patients). The optimum model developed was the gradient boosted model with a PPV of 67%, NPV of 87%, sensitivity of 37% and a specificity of 96% for common bile duct stones. CONCLUSION: This paper highlights the utility of machine learning in predicting CBD stones. Accuracy is limited by current data and issues do exist around both the ethics and practicality of implementation. Regardless, machine learning represents a promising new paradigm for surgical practice.

Necessity is the mother of invention: how the COVID-19 pandemic could change medical student placements for the better.

COVID-19 led to the widespread withdrawal of face-to-face hospital-based clinical placements, with many medical schools switching to online learning. This precipitated concern about potential negative impact on clinical and interprofessional skill acquisition. To overcome this problem, we piloted a 12-week COVID-19 safe face-to-face clinical placement for 16 medical students at the Hospital for Tropical Diseases, London, during the first wave of the COVID-19 pandemic. COVID-19 infection control measures necessitated that students remained in 'social bubbles' for placement duration. This facilitated an apprenticeship-style teaching approach, integrating students into the clinical team for placement duration. Team-based learning was adopted to develop and deliver content. Teaching comprised weekly seminars, experiential ward-based attachments and participation in quality improvement and research projects. The taught content was evaluated through qualitative feedback, reflective practice, and pre-apprenticeship and post-apprenticeship confidence questionnaires across 17 domains. Students' confidence improved in 14 of 17 domains (p<0.05). Reflective practice indicated that students valued the apprenticeship model, preferring the longer clinical attachment to existent shorter, fragmented clinical placements. Students described improved critical thinking, group cohesion, teamwork, self-confidence, self-worth and communication skills. This article describes a framework for the safe and effective delivery of a longer face-to-face apprenticeship-based clinical placement during an infectious disease pandemic. Longer apprenticeship-style attachments have hidden benefits to general professional training, which should be explored by medical schools both during the COVID-19 pandemic and, possibly, for any future clinical placements.

Evaluating Supplemental Samples in Longitudinal Research: Replacement and Refreshment Approaches.

Despite the wide application of longitudinal studies, they are often plagued by missing data and attrition. The majority of methodological approaches focus on participant retention or modern missing data analysis procedures. This paper, however, takes a new approach by examining how researchers may supplement the sample with additional participants. First, refreshment samples use the same selection criteria as the initial study. Second, replacement samples identify auxiliary variables that may help explain patterns of missingness and select new participants based on those characteristics. A simulation study compares these two strategies for a linear growth model with five measurement occasions. Overall, the results suggest that refreshment samples lead to less relative bias, greater relative efficiency, and more acceptable coverage rates than replacement samples or not supplementing the missing participants in any way. Refreshment samples also have high statistical power. The comparative strengths of the refreshment approach are further illustrated through a real data example. These findings have implications for assessing change over time when researching at-risk samples with high levels of permanent attrition.

On Standardizing Within-Person Effects: Potential Problems of Global Standardization.

Person-mean centering has been recommended for disaggregating between-person and within-person effects when modeling time-varying predictors. Multilevel modeling textbooks recommended global standardization for standardizing fixed effects. An aim of this study is to evaluate whether and when person-mean centering followed by global standardization can accurately estimate fixed-effects within-person relations (the estimand of interest in this study) in multilevel modeling. We analytically derived that global standardization generally yields inconsistent (asymptotically biased) estimates for the estimand when between-person differences in within-person standard deviations exist and the average within-person relation is nonzero. Alternatively, a person-mean-SD standardization (P-S) approach yields consistent estimates. Our simulation results further revealed (1) how misleading the results from global standardization were under various circumstances and (2) the P-S approach had accurate estimates and satisfactory coverage rates of fixed-effects within-person relations when the number of occasions is 30 or more (in many conditions, performance was satisfactory with 10 or 20 occasions). A daily diary data example, focused on emotional complexity, was used to empirically illustrate the approaches. Researchers should choose standardization approaches based on theoretical considerations and should clearly describe the purpose and procedure of standardization in research articles.

Sample-Size Planning for More Accurate Statistical Power: A Method Adjusting Sample Effect Sizes for Publication Bias and Uncertainty.

The sample size necessary to obtain a desired level of statistical power depends in part on the population value of the effect size, which is, by definition, unknown. A common approach to sample-size planning uses the sample effect size from a prior study as an estimate of the population value of the effect to be detected in the future study. Although this strategy is intuitively appealing, effect-size estimates, taken at face value, are typically not accurate estimates of the population effect size because of publication bias and uncertainty. We show that the use of this approach often results in underpowered studies, sometimes to an alarming degree. We present an alternative approach that adjusts sample effect sizes for bias and uncertainty, and we demonstrate its effectiveness for several experimental designs. Furthermore, we discuss an open-source R package, BUCSS, and user-friendly Web applications that we have made available to researchers so that they can easily implement our suggested methods.

Addressing the "Replication Crisis": Using Original Studies to Design Replication Studies with Appropriate Statistical Power.

Psychology is undergoing a replication crisis. The discussion surrounding this crisis has centered on mistrust of previous findings. Researchers planning replication studies often use the original study sample effect size as the basis for sample size planning. However, this strategy ignores uncertainty and publication bias in estimated effect sizes, resulting in overly optimistic calculations. A psychologist who intends to obtain power of .80 in the replication study, and performs calculations accordingly, may have an actual power lower than .80. We performed simulations to reveal the magnitude of the difference between actual and intended power based on common sample size planning strategies and assessed the performance of methods that aim to correct for effect size uncertainty and/or bias. Our results imply that even if original studies reflect actual phenomena and were conducted in the absence of questionable research practices, popular approaches to designing replication studies may result in a low success rate, especially if the original study is underpowered. Methods correcting for bias and/or uncertainty generally had higher actual power, but were not a panacea for an underpowered original study. Thus, it becomes imperative that 1) original studies are adequately powered and 2) replication studies are designed with methods that are more likely to yield the intended level of power.

Severe alkalosis and hypokalemia with stanozolol misuse.

Stanozolol is a popular androgenic anabolic steroid, used by body builders and athletes for physical performance enhancement. There are few data on its potential adverse effects and no documented cases of it causing severe electrolyte imbalance. Here, we report a patient presenting to a tertiary care emergency department with reduced conscious level, profound hypokalemia, and severe metabolic alkalosis, resulting from stanozolol misuse. This is the first such case reported.

Lipofuscin accumulates in ganglionic neurons in chronic equine dysautonomia.

Lipofuscin is a complex mixture of highly oxidized, cross-linked macromolecules that accumulates in neurons with age and some neurodegenerative diseases. Equine dysautonomia (ED) is a polyneuropathy that mainly affects autonomic and enteric nervous systems, resulting in alimentary tract dysfunction. Our main aim was to determine whether neuronal lipofuscin increased with increasing duration of ED. We investigated the prevalence of lipofuscin in cranial cervical ganglia of horses with acute (AED), subacute (SED), and chronic ED (CED), young controls (of similar age to ED cases), and aged controls (n = 8 per group). We used Schmorl stain for histologic detection of lipofuscin and assessed its accumulation in neurons using image analysis software. The percentage of neurons positive for lipofuscin increased with age in individual groups and all groups combined (p < 0.001). There were fewer positive neurons in AED and SED compared to aged controls (p < 0.001) and more in CED than AED cases (p = 0.042) and young controls (p = 0.012). We found a strong positive correlation between percentage positive neurons and percentage positive area of the neuron containing lipofuscin for combined groups (p < 0.001). Although neuronal lipofuscin increased in cranial cervical ganglion in CED cases, it remains to be determined whether this is a cause or consequence of neuronal degeneration.

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.

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.

The Role of Activator Protein-1 Complex in Diabetes-Associated Atherosclerosis: Insights From Single-Cell RNA Sequencing.

Despite advances in treatment, atherosclerotic cardiovascular disease 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 used 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 (HAECs) and human THP-1-derived macrophages. We then performed a preclinical study using an activator protein-1 (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 individuals with diabetes compared with those without diabetes. 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, the inhibition of which treats the underlying pathobiology of atherosclerosis in diabetes.