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Spatial and temporal single-cell profiling of RNA compartmentalization in neurons with nanotweezers.
No full textEmerging techniques for mapping mRNAs within the subcellular compartments of live cells hold great promise for advancing our understanding of the spatial distribution of transcripts and enabling the study of single-cell dynamics in health and disease. This is particularly critical for polarized cells, such as neurons, where mRNA compartmentalization is essential for regulating gene expression, and defects in these localization mechanisms are linked to numerous neurological disorders. However, many subcellular analysis techniques require a compromise between subcellular precision, live-cell measurements, and nondestructive access to single cells in their native microenvironment. To overcome these challenges, we employ a single-cell technology that we have recently developed, the nanotweezer, which features a nanoscale footprint (∼100 nm), avoids cytoplasmic fluid aspiration, and enables rapid RNA isolation from living cells with minimal invasiveness. Using this tool, we investigate single-cell mRNA compartmentalization in the soma and dendrites of hippocampal neurons at different stages of neuronal development. By combining precise targeting with sequential sampling, we track changes in mRNA abundance at dendritic spine regions of the same neuron, both before and after stimulation. This minimally invasive approach enables time-resolved, subcellular gene expression profiling of the same single cell. This could provide critical insights into polarized cells and advance our understanding of biological processes and complex diseases
Robust proteome profiling of cysteine-reactive fragments using label-free chemoproteomics.
No full textIdentifying pharmacological probes for human proteins represents a key opportunity to accelerate the discovery of new therapeutics. High-content screening approaches to expand the ligandable proteome offer the potential to expedite the discovery of novel chemical probes to study protein function. Screening libraries of reactive fragments by chemoproteomics offers a compelling approach to ligand discovery, however, optimising sample throughput, proteomic depth, and data reproducibility remains a key challenge. We report a versatile, label-free quantification proteomics platform for competitive profiling of cysteine-reactive fragments against the native proteome. This high-throughput platform combines SP4 plate-based sample preparation with rapid chromatographic gradients. Data-independent acquisition performed on a Bruker timsTOF Pro 2 consistently identified ~23,000 cysteine sites per run, with a total of ~32,000 cysteine sites profiled in HEK293T and Jurkat lysate. Crucially, this depth in cysteinome coverage is met with high data completeness, enabling robust identification of liganded proteins. In this study, 80 reactive fragments were screened in two cell lines identifying >400 ligand-protein interactions. Hits were validated through concentration-response experiments and the platform was utilised for hit expansion and live cell experiments. This label-free platform represents a significant step forward in high-throughput proteomics to evaluate ligandability of cysteines across the human proteome
June2024_predictions, SAG 100nM down
No full textDifferentiation to test predictions. June 2024. 100 nM SAG for 24h followed by 0 nM SAG. </p
Assessing contrast sensitivity function in CRB1-retinopathies: Exploring child-friendly measures of visual function.
No full textPURPOSE: Mutations affecting the CRB1 gene can result in a range of retinal phenotypes, including early onset severe retinal dystrophy/Leber congenital amaurosis (EOSRD/LCA), retinitis pigmentosa, cone-rod dystrophy (CORD), and macular dystrophy (MD). As research into treatment strategies advances towards clinical translation, there is a need to establish reliable outcome metrics. This study explores the contrast sensitivity function (CSF) across different spatial frequencies in individuals with CRB1-retinopathies using the child-friendly PopCSF test, an iPad-based "gamified" assessment. METHODS: Prospective cross-sectional study of 20 patients with molecularly confirmed biallelic CRB1 pathogenic variants from Moorfields Eye Hospital, London, UK, was conducted. Best-corrected visual acuity (BCVA), contrast sensitivity using the Pelli-Robson chart, and the PopCSF test were performed. RESULTS: Of the 20 CRB1 patients, seven had EOSRD/LCA, three had CORD, and 10 had MD. There was no statistically significant difference between the mean BCVA between phenotypes (P = 0.066). However, a significant difference was found between groups in the mean letter log contrast sensitivity (logCS) and area under the contrast sensitivity function (AUCSF) with P = 0.047 and P </p
Obesity, white adipose tissue and cancer.
No full textWhite adipose tissue (WAT) is crucial for whole-body energy homeostasis and plays an important role in metabolic and hormonal regulation. While healthy WAT undergoes controlled expansion and contraction to meet the body's requirements, dysfunctional WAT in conditions like obesity is characterized by excessive tissue expansion, alterations in lipid homeostasis, inflammation, hypoxia, and fibrosis. Obesity is strongly associated with an increased risk of numerous cancers, with obesity-induced WAT dysfunction influencing cancer development through various mechanisms involving both systemic and local interactions between adipose tissue and tumors. Unhealthy obese WAT affects circulating levels of free fatty acids and factors like leptin, adiponectin, and insulin, altering systemic lipid metabolism and inducing inflammation that supports tumor growth. Similar mechanisms are observed locally in an adipose-rich tumor microenvironment (TME), where WAT cells can also trigger extracellular matrix remodeling, thereby enhancing the TME's ability to promote tumor growth. Moreover, tumors reciprocally interact with WAT, creating a bidirectional communication that further enhances tumorigenesis. This review focuses on the complex interplay between obesity, WAT dysfunction, and primary tumor growth, highlighting potential targets for therapeutic intervention
Compromised epigenetic robustness in cancer: fueling evolution, exposing weakness.
No full textThe complex network of proteins that regulate chromatin and DNA methylation landscapes is often disrupted in cancer. Clonal and subclonal mutations targeting a wide range of molecular functions are frequently observed across cancer types, and emerging evidence suggests that loss of robust epigenetic control promotes both cancer initiation and evolution, independently of context-specific effects. Here, we review how diverse genetic alterations that destabilize the epigenetic regulatory network (ERN) may converge into common phenotypes. We also discuss the implications of altered network topology and systemic epigenetic disorder for the evolution, vulnerability, and therapeutic resistance of cancers
Two (AgI3I)4L4 cages elucidate the rules for silver-cluster vertex design
No full textNo description supplie
EVOLVE Questionnaire
No full textThis questionnaire was designed for the EVOLVE-HBV study at the Africa Health Research Institute (AHRI) in KwaZulu Natal, South Africa. This is a project to study hepatitis B virus (HBV) infection and liver disease in a rural population. The questionnaire is a brief tool to collect data about risk factors for liver disease including family history, diet, medications (including traditional medicines), alcohol and smoking. We ask a question about barriers to seeking diagnosis and care, and a question about knowledge of HBV as a cause of liver cancer. We have introduced preliminary questions on mental health to allow us to collect pilot data in this domain.</p
Evaluation of Hepatitis B core related antigen (HBcrAg) as a biomarker in cohorts from the United Kingdom and South Africa.
No full textOBJECTIVES: We set out to evaluate Hepatitis B core related antigen (HBcrAg) as a proxy for hepatitis B (HBV) viral load (VL) and liver disease in two different population settings. METHODS: We undertook a cross-sectional retrospective observational study using samples and data from adults living with chronic HBV infection from the United Kingdom (UK, n=142) and South Africa (SA, n=211). We assessed HBcrAg distribution, relationship with other biomarkers, and risk stratification performance using point of care test (POCT) thresholds. RESULTS: SA and UK cohorts differed by ethnicity, HIV coinfection, HBeAg-positivity and proportion with HBV VL >200,000 IU/ml (all p200,000 IU/ml in the UK cohort, compared to 94% sensitive and 86% specific in the SA population. CONCLUSIONS: HBcrAg correlated with VL, but less so with liver disease. Use of this biomarker needs tailoring for use in diverse populations
Neutrophils drive vascular occlusion, tumour necrosis and metastasis.
No full textTumour necrosis is associated with poor prognosis in cancer1,2 and is thought to occur passively when tumour growth outpaces nutrient supply. Here we report, however, that neutrophils actively induce tumour necrosis. In multiple cancer mouse models, we found a tumour-elicited Ly6GHighLy6CLow neutrophil population that was unable to extravasate in response to inflammatory challenges but formed neutrophil extracellular traps (NETs) more efficiently than classical Ly6GHighLy6CHigh neutrophils. The presence of these 'vascular-restricted' neutrophils correlated with the appearance of a 'pleomorphic' necrotic architecture in mice. In tumours with pleomorphic necrosis, we found intravascular aggregates of neutrophils and NETs that caused occlusion of the tumour vasculature, driving hypoxia and necrosis of downstream vascular beds. Furthermore, we found that cancer cells adjacent to these necrotic regions (that is, in 'perinecrotic' areas) underwent epithelial-to-mesenchymal transition, explaining the paradoxical metastasis-enhancing effect of tumour necrosis. Blocking NET formation genetically or pharmacologically reduced the extent of tumour necrosis and lung metastasis. Thus, by showing that NETs drive vascular occlusion, pleomorphic necrosis and metastasis, we demonstrate that tumour necrosis is not necessarily a passive byproduct of tumour growth and that it can be blocked to reduce metastatic spread.</p