Multiplexed single-cell analysis of organoid signaling networks

Organoids are biomimetic tissue models comprising multiple cell types and cell states. Post-translational modification (PTM) signaling networks control cellular phenotypes and are frequently dysregulated in diseases such as cancer. Although signaling networks vary across cell types, there are limited techniques to study cell type–specific PTMs in heterocellular organoids. Here, we present a multiplexed mass cytometry (MC) protocol for single-cell analysis of PTM signaling and cell states in organoids and organoids co-cultured with fibroblasts and leukocytes. We describe how thiol-reactive organoid barcoding in situ (TOBis) enables 35-plex and 126-plex single-cell comparison of organoid cultures and provide a cytometry by time of flight (CyTOF) signaling analysis pipeline (CyGNAL) for computing cell type–specific PTM signaling networks. The TOBis MC protocol takes ~3 d from organoid fixation to data acquisition and can generate single-cell data for >40 antibodies from millions of cells across 126 organoid cultures in a single MC run

Cell-type-specific signaling networks in heterocellular organoids

Despite the widespread adoption of organoids as biomimetic tissue models, methods to comprehensively analyze cell-type-specific post-translational modification (PTM) signaling networks in organoids are absent. Here, we report multivariate single-cell analysis of such networks in organoids and organoid cocultures. Simultaneous analysis by mass cytometry of 28 PTMs in >1 million single cells derived from small intestinal organoids reveals cell-type- and cell-state-specific signaling networks in stem, Paneth, enteroendocrine, tuft and goblet cells, as well as enterocytes. Integrating single-cell PTM analysis with thiol-reactive organoid barcoding in situ (TOBis) enables high-throughput comparison of signaling networks between organoid cultures. Cell-type-specific PTM analysis of colorectal cancer organoid cocultures reveals that shApc, KrasG12D and Trp53R172H cell-autonomously mimic signaling states normally induced by stromal fibroblasts and macrophages. These results demonstrate how standard mass cytometry workflows can be modified to perform high-throughput multivariate cell-type-specific signaling analysis of healthy and cancerous organoids

Abnormal LDIflare but Normal Quantitative Sensory Testing and Dermal Nerve Fiber Density in Patients with Painful Diabetic Neuropathy

OBJECTIVE—Abnormal small nerve fiber function may be an early feature of diabetic neuropathy and may also underlie painful symptoms. Methods for assessing small-fiber damage include quantitative sensory testing (QST) and determining intraepidermal nerve fiber density. We recently described a reproducible physiological technique, the LDIflare, which assesses small-fiber function and thus may reflect early dysfunction before structural damage. The value of this technique in painful neuropathy was assessed by comparing it with QST and dermal nerve fiber density (NFD)

Decision Support Intervention for people with advanced dementia residing in a nursing home: A study protocol for an International advance care planning intervention (mySupport study)

Background Where it has been determined that a resident in a nursing home living with dementia loses decisional capacity, nursing home staff must deliver care that is in the person's best interests. Ideally, decisions should be made involving those close to the person, typically a family carer and health and social care providers. The aim of the Family Carer Decisional Support intervention is to inform family carers on end-of-life care options for a person living with advanced dementia and enable them to contribute to advance care planning. This implementation study proposes to; 1) adopt and apply the intervention internationally; and, 2) train nursing home staff to deliver the family carer decision support intervention. Methods This study will employ a multiple case study design to allow an understanding of the implementation process and to identify the factors which determine how well the intervention will work as intended. We will enrol nursing homes from each country (Canada n = 2 Republic of Ireland = 2, three regions in the UK n = 2 each, The Netherlands n = 2, Italy n = 2 and the Czech Republic n = 2) to reflect the range of characteristics in each national and local context. The RE-AIM (reach, effectiveness, adoption, implementation, maintenance) framework will guide the evaluation of implementation of the training and information resources. Our mixed methods study design has three phases to (1) establish knowledge about the context of implementation, (2) participant baseline information and measures and (3) follow up evaluation. Discussion The use of a multiple case study design will enable evaluation of the intervention in different national, regional, cultural, clinical, social and organisational contexts, and we anticipate collecting rich and in-depth data. While it is hoped that the intervention resources will impact on policy and practice in the nursing homes that are recruited to the study, the development of implementation guidelines will ensure impact on wider national policy and practice. It is our aim that the resources will be sustainable beyond the duration of the study and this will enable the resources to have a longstanding relevance for future advance care planning practice for staff, family carers and residents with advanced dementia

Diabetic Neuropathies: Update on Definitions, Diagnostic Criteria, Estimation of Severity, and Treatments

Preceding the joint meeting of the 19th annual Diabetic Neuropathy Study Group of the European Association for the Study of Diabetes (NEURODIAB) and the 8th International Symposium on Diabetic Neuropathy in Toronto, Canada, 13–18 October 2009, expert panels were convened to provide updates on classification, definitions, diagnostic criteria, and treatments of diabetic peripheral neuropathies (DPNs), autonomic neuropathy, painful DPNs, and structural alterations in DPNs

Interaction of hyperalgesia and sensory loss in complex regional pain syndrome type I (CRPS I)

Sensory abnormalities are a key feature of Complex Regional Pain Syndrome (CRPS). In order to characterise these changes in patients suffering from acute or chronic CRPS I, we used Quantitative Sensory Testing (QST) in comparison to an age and gender matched control group

Polygenic burden in focal and generalized epilepsies

Rare genetic variants can cause epilepsy, and genetic testing has been widely adopted for severe, paediatric-onset epilepsies. The phenotypic consequences of common genetic risk burden for epilepsies and their potential future clinical applications have not yet been determined. Using polygenic risk scores (PRS) from a European-ancestry genome-wide association study in generalized and focal epilepsy, we quantified common genetic burden in patients with generalized epilepsy (GE-PRS) or focal epilepsy (FE-PRS) from two independent non-Finnish European cohorts (Epi25 Consortium, n = 5705; Cleveland Clinic Epilepsy Center, n = 620; both compared to 20 435 controls). One Finnish-ancestry population isolate (Finnish-ancestry Epi25, n = 449; compared to 1559 controls), two European-ancestry biobanks (UK Biobank, n = 383 656; Vanderbilt biorepository, n = 49 494), and one Japaneseancestry biobank (BioBank Japan, n = 168 680) were used for additional replications. Across 8386 patients with epilepsy and 622 212 population controls, we found and replicated significantly higher GE-PRS in patients with generalized epilepsy of European-ancestry compared to patients with focal epilepsy (Epi25: P = 1.64 710-15; Cleveland: P = 2.85 710-4; Finnish-ancestry Epi25: P = 1.80 710-4) or population controls (Epi25: P = 2.35 710-70; Cleveland: P = 1.43 710-7; Finnish-ancestry Epi25: P = 3.11 710-4; UK Biobank and Vanderbilt biorepository meta-analysis: P = 7.99 710-4). FE-PRS were significantly higher in patients with focal epilepsy compared to controls in the non-Finnish, non-biobank cohorts (Epi25: P = 5.74 710-19; Cleveland: P = 1.69 710-6). European ancestry-derived PRS did not predict generalized epilepsy or focal epilepsy in Japanese-ancestry individuals. Finally, we observed a significant 4.6-fold and a 4.5-fold enrichment of patients with generalized epilepsy compared to controls in the top 0.5% highest GE-PRS of the two non-Finnish European cohorts (Epi25: P = 2.60 710-15; Cleveland: P = 1.39 710-2). We conclude that common variant risk associated with epilepsy is significantly enriched in multiple cohorts of patients with epilepsy compared to controls-in particular for generalized epilepsy. As sample sizes and PRS accuracy continue to increase with further common variant discovery, PRS could complement established clinical biomarkers and augment genetic testing for patient classification, comorbidity research, and potentially targeted treatment