Molecular Phenotyping at Single-Cell Resolution for Cardiovascular Disease.

In this study, we developed, coordinated, and integrated several technologies including novel whole organ imaging, software development to support the very first precise 3D neuroanatomical mapping and molecular phenotyping of the intracardiac nervous system (ICN). While qualitative and gross anatomical descriptions of the anatomy of the ICN have been presented, we here bring forth the first comprehensive atlas at large scale of the entire ICN in rat at a single cell resolution. Our work for the first time provides a novel 3D model to precisely integrate anatomical, functional and molecular data in the 3D digitally reconstructed whole heart with high resolution at the micron scale. This work represents the cutting edge in a long history of attempts to understand the anatomical substrate upon which the neuronal control of cardiac function is built. To our knowledge, there has not yet been a comprehensive histological mapping to generate a neurocardiac atlas at cellular and molecular level for the whole heart of any species. We now display the full extent and the position of neuronal clusters on the base and posterior left atrium, and the distribution of molecular phenotypes in that context. In addition we display in this context distinct molecular phenotypes that are defined along the base-to-apex axis, and the present novel discovery of their phenotypical spatial gradients, have not been previously described. The development of these approaches needed to acquire these data has produced method pipelines which can not only achieve the goals of anatomical and molecular mapping of the heart, but also provide the method pipelines for mapping other organs (e.g., stomach, lung, kidney, and liver).

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MAFLD and Cardiovascular Events: What Does the Evidence Show?

SummaryWe have developed and integrated several technologies including whole-organ imaging and software development to support an initial precise 3D neuroanatomical mapping and molecular phenotyping of the intracardiac nervous system (ICN). While qualitative and gross anatomical descriptions of the anatomy of the ICN have each been pursued, we here bring forth a comprehensive atlas of the entire rat ICN at single-cell resolution. Our work precisely integrates anatomical and molecular data in the 3D digitally reconstructed whole heart with resolution at the micron scale. We now display the full extent and the position of neuronal clusters on the base and posterior left atrium of the rat heart, and the distribution of molecular phenotypes that are defined along the base-to-apex axis, which had not been previously described. The development of these approaches needed for this work has produced method pipelines that provide the means for mapping other organs.

Molecular phenotyping by imaging of intact tissues has been used to reveal 3D molecular and structural coherence in tissue samples using tissue clearing techniques. However, clearing and imaging of cardiac tissue remains challenging for large-scale (>100 mm3) specimens due to sample distortion. Thus, directly assessing tissue microstructural geometric properties confounded by distortion such as cardiac helicity has been limited. To combat sample distortion, we developed a passive CLARITY technique (Pocket CLARITY) that utilizes a permeable cotton mesh pocket to encapsulate the sample to clear large-scale cardiac swine samples with minimal tissue deformation and protein loss. Combined with light sheet auto-fluorescent and scattering microscopy, Pocket CLARITY enabled the characterization of myocardial microstructural helicity of cardiac tissue from control, heart failure, and myocardial infarction in swine. Pocket CLARITY revealed with high fidelity that transmural microstructural helicity of the heart is significantly depressed in cardiovascular disease (CVD), thereby revealing new insights at the tissue level associated with impaired cardiac function.

The Human Explanted Heart Program: A translational bridge for cardiovascular medicine

We report the successful development and translationof high-fieldnuclear magnetic resonance (NMR) based comprehensive lipoprotein analysisto routine benchtop systems. This demonstrates the potential to reimaginepopulation level cardiovascular disease risk analysis and individuallevel screening based on blood sampling. Using a quantitative calibrationapproach, we obtained stable and reproducible results from multiplesites, despite reduced spectral dispersion and sensitivity at lowerfield strengths. Our study shows that 25 out of 28 major lipoproteinparameters, including key cardiometabolic risk markers, were faithfullymeasured using benchtop NMR systems within 15 min. This developmenthas significant implications for making a powerful diagnostic toolwidely available, enhancing the potential for longitudinal personalizedmedicine through molecular phenotyping in the clinic.

Genetic variants are major determinants of susceptibility to disease and clinical outcomes in many diseases, including cardiovascular diseases, in particular cardiomyopathies. Next-generation sequencing approaches have discovered a vast majority of genetic variants in the human genome and pinpointed several gene loci with particular importance in the development of cardiomyopathies. This particular chapter highlights the genetic diversity of cardiomyopathies, focuses on molecular and functional phenotyping of disease cohorts, and summarizes the current key aspects in specific forms of cardiomyopathies, including hypertrophic and dilated cardiomyopathies, storage disease cardiomyopathies, and peripartum cardiomyopathies. Finally, the chapter describes current knowledge about underlying molecular pathways and provides an overview of future next-generation therapeutics targeting cardiomyopathies.

World Arthritis Day 2018 ‐ Perspectives on Rheumatic musculoskeletal diseases

Understanding complex biological systems requires the system-wide characterization of both molecular and cellular features. Existing methods for spatial mapping of biomolecules in intact tissues suffer from information loss caused by degradation and tissue damage. We report a tissue transformation strategy named ‘Stabilization under Harsh conditions via Intramolecular Epoxide Linkages to prevent Degradation’ (SHIELD), which uses a flexible polyepoxide to form controlled intra- and intermolecular crosslink with biomolecules. SHIELD preserved protein fluorescence and antigenicity, transcripts and tissue architecture under a wide range of harsh conditions. We applied SHIELD to interrogate system-level wiring, synaptic architecture, and molecular features of virally labeled neurons and their targets in mouse at single-cell resolution. We also demonstrated rapid three dimensional (3D) phenotyping of core needle biopsies and human brain cells. SHIELD enables rapid, multiscale, integrated molecular phenotyping of both animal and clinical tissues.

Microscopy analysis of tumour samples is commonly performed on fixed, thinly sectioned and protein-labelled tissues. However, these examinations do not reveal the intricate three-dimensional structures of tumours, nor enable the detection of aberrant transcripts. Here, we report a method, which we name DIIFCO (for diagnosing in situ immunofluorescence-labelled cleared oncosamples), for the multimodal volumetric imaging of RNAs and proteins in intact tumour volumes and organoids. We used DIIFCO to spatially profile the expression of diverse coding RNAs and non-coding RNAs at the single-cell resolution in a variety of cancer tissues. Quantitative single-cell analysis revealed spatial niches of cancer stem-like cells, and showed that the niches were present at a higher density in triple-negative breast cancer tissue. The improved molecular phenotyping and histopathological diagnosis of cancers may lead to new insights into the biology of tumours of patients.

Multiplex immunofluorescence (mIF) assays multiple protein biomarkers on a single tissue section. Recently, high-plex CODEX (co-detection by indexing) systems enable simultaneous imaging of 40+ protein biomarkers, unlocking more detailed molecular phenotyping, leading to richer insights into cellular interactions and disease. However, high-plex data can be slower and more costly to collect, limiting its applications, especially in clinical settings. We propose a machine learning framework, 7-UP, that can computationally generate in silico 40-plex CODEX at single-cell resolution from a standard 7-plex mIF panel by leveraging cellular morphology. We demonstrate the usefulness of the imputed biomarkers in accurately classifying cell types and predicting patient survival outcomes. Furthermore, 7-UP's imputations generalize well across samples from different clinical sites and cancer types. 7-UP opens the possibility of in silico CODEX, making insights from high-plex mIF more widely available.

Chapter 12 - Peripheral blood DNA and RNA biomarkers of cardiovascular disease in clinical practice

N-glycosylation signature and its relevance in cardiovascular immunometabolism.

The journey of a thousand miles must begin with a single step. — —Lao-Tzu, Vietnamese philosopher These are the “best of times.” Cardiologists worldwide are fortunate that they can offer much more to their patients today than was possible even a decade ago. Never before has cardiology been on such an exciting trajectory, with such exponential growth of knowledge and the exhilarating promise of harnessing the secrets of the genome to prevent, diagnose, and treat cardiovascular disease and enhance vascular health over the human life course. The future offers the possibility of realizing the dream of practicing personalized preventive cardiovascular medicine. These are also the most challenging of times. Increased life expectancy has generated an unprecedented growth in the older segment of the population, with the accompanying rising burden of aging-associated cardiovascular disorders. Burgeoning medical knowledge and escalating patient expectations push cardiologists to keep abreast of the latest advances in cardiovascular research as never before. We understand now that common genetic variation may predispose to common forms of cardiovascular disease in the community, and rare genetic conditions provide unique pathogenetic insights into these diseases. The sequential steps in the origins of cardiovascular syndromes are beginning to unfold, revealing the stunning complexities involved in the molecular basis of disease. We are beginning to understand how genetic factors interact with environmental influences over an entire lifetime to pattern and remodel function at the molecular, cellular, tissue, and organ levels and to ultimately manifest as subclinical or clinical cardiovascular disease. Molecular phenotyping of diseases is here to stay and does not respect the traditional boundaries of medical specialties, but the range of innovative diagnostic modalities must expand dramatically if we are …