Genome Editing and Induced Pluripotent Stem Cell Technologies for Personalized Study of Cardiovascular Diseases

PURPOSE OF REVIEW: The goal of this review is to highlight the potential of induced pluripotent stem cell (iPSC)-based modeling as a tool for studying human cardiovascular diseases. We present some of the current cardiovascular disease models utilizing genome editing and patient-derived iPSCs. RECENT FINDINGS: The incorporation of genome-editing and iPSC technologies provides an innovative research platform, providing novel insight into human cardiovascular disease at molecular, cellular, and functional level. In addition, genome editing in diseased iPSC lines holds potential for personalized regenerative therapies. The study of human cardiovascular disease has been revolutionized by cellular reprogramming and genome editing discoveries. These exceptional technologies provide an opportunity to generate human cell cardiovascular disease models and enable therapeutic strategy development in a dish. We anticipate these technologies to improve our understanding of cardiovascular disease pathophysiology leading to optimal treatment for heart diseases in the future

Virtual reality and consciousness inference in dreaming

This article explores the notion that the brain is genetically endowed with an innate virtual reality generator that – through experience-dependent plasticity – becomes a generative or predictive model of the world. This model, which is most clearly revealed in rapid eye movement (REM) sleep dreaming, may provide the theater for conscious experience. Functional neuroimaging evidence for brain activations that are time-locked to rapid eye movements (REMs) endorses the view that waking consciousness emerges from REM sleep – and dreaming lays the foundations for waking perception. In this view, the brain is equipped with a virtual model of the world that generates predictions of its sensations. This model is continually updated and entrained by sensory prediction errors in wakefulness to ensure veridical perception, but not in dreaming. In contrast, dreaming plays an essential role in maintaining and enhancing the capacity to model the world by minimizing model complexity and thereby maximizing both statistical and thermodynamic efficiency. This perspective suggests that consciousness corresponds to the embodied process of inference, realized through the generation of virtual realities (in both sleep and wakefulness). In short, our premise or hypothesis is that the waking brain engages with the world to predict the causes of sensations, while in sleep the brain’s generative model is actively refined so that it generates more efficient predictions during waking. We review the evidence in support of this hypothesis – evidence that grounds consciousness in biophysical computations whose neuronal and neurochemical infrastructure has been disclosed by sleep research

Investigating pediatric disorders with induced pluripotent stem cells

The study of disease pathophysiology has long relied on model systems, including animal models and cultured cells. In 2006, Shinya Yamanaka achieved a breakthrough by reprogramming somatic cells into induced pluripotent stem cells (iPSCs). This revolutionary discovery provided new opportunities for disease modeling and therapeutic intervention. With established protocols, investigators can generate iPSC lines from patient blood, urine, and tissue samples. These iPSCs retain ability to differentiate into every human cell type. Advances in differentiation and organogenesis move cellular in vitro modeling to a multicellular model capable of recapitulating physiology and disease. Here, we discuss limitations of traditional animal and tissue culture models, as well as the application of iPSC models. We highlight various techniques, including reprogramming strategies, directed differentiation, tissue engineering, organoid developments, and genome editing. We extensively summarize current established iPSC disease models that utilize these techniques. Confluence of these technologies will advance our understanding of pediatric diseases and help usher in new personalized therapies for patients

HIV-1 Clade B and C Isolates Exhibit Differential Replication: Relevance to Macrophage-Mediated Neurotoxicity

HIV-associated neurocognitive disorders (HAND) continue to be a consequence of HIV-1 infection among clade B-infected individuals. In contrast, the incidence of severe neurological impairment is lower among clade C-infected patients in regions of Sub-Saharan Africa and India. Biological aspects such as replication, cytopathicity, inflammatory response, and neurotoxicity unique to each clade influence neuropathogenicity and ultimately affect the clinical outcome of the disease. We hypothesize that productive infection by clade C isolates leads to macrophagemediated neurotoxicity, although to a lesser extent than clade B isolates. Using a panel of primary isolates of clades B and C we demonstrated that clade B has higher replication efficiency in monocyte-derived macrophages (MDM) through reverse transcriptase activity assay and HIV-1 p24 antigen ELISA. To test the neurotoxicity of clades B and C, we used an in vitro neurotoxicity model. Conditioned medium from clade B-infected MDM was neurotoxic to rat and human neuron cultures. In contrast, clade C isolates mediated neurotoxicity when a higher initial viral titer was used for MDM infection. Furthermore, neurotoxicity mediated by isolates of both clades correlated with virus replication in MDM. Together, these results suggest that in comparison to clade B, primary isolates of clade C have slower replication kinetics in primary MDM, leading to lower levels of macrophage-mediated neurotoxicity. Elucidating the differences in replication and macrophage-mediated neurotoxicity between isolates of HIV-1 clades B and C will provide important insights needed to clarify the disparity seen in HAND incidence

Molecular Determinants of HIV-1 Subtype C Coreceptor Transition from R5 to R5X4

The molecular mechanism(s) underlying transition from CCR5 to CXCR4 usage of subtype C viruses remain largely unknown. We previously identified a subtype C HIV-1 infected child whose virus demonstrated CXCR4 usage along with CCR5 upon longitudinal follow-up. Here we delineated the molecular determinants of Env involved in expanded coreceptor usage. Residue changes in three positions of Env V3 domain are critical for the dual-tropic phenotype. These include: substitution of arginine at position 11, MG or LG insertion between positions 13 and 14, and substitution of threonine at the position immediately downstream of the GPGQ crown. Introducing these mutations into V3 region of another R5 virus also conferred dual tropism. Molecular modeling of V3 revealed a possible structural basis for the dual-tropic phenotype. Determining what defines a subtype C X4 virus will lead to a better understanding of subtype C HIV-1 pathogenesis, and will provide important information relevant to anti-retroviral therapy

Hypoplastic Left Heart Syndrome Sequencing Reveals a Novel NOTCH1 Mutation in a Family with Single Ventricle Defects

Hypoplastic left heart syndrome (HLHS) has been associated with germline mutations in 12 candidate genes and a recurrent somatic mutation in HAND1 gene. Using targeted and whole exome sequencing (WES) of heart tissue samples from HLHS patients, we sought to estimate the prevalence of somatic and germline mutations associated with HLHS. We performed Sanger sequencing of the HAND1 gene on 14 ventricular (9 LV and 5 RV) samples obtained from HLHS patients, and WES of 4 LV, 2 aortic, and 4 matched PBMC samples, analyzing for sequence discrepancy. We also screened for mutations in the 12 candidate genes implicated in HLHS. We found no somatic mutations in our HLHS cohort. However, we detected a novel germline frameshift/stop-gain mutation in NOTCH1 in a HLHS patient with a family history of both HLHS and hypoplastic right heart syndrome (HRHS). Our study, involving one of the first familial cases of single ventricle defects linked to a specific mutation, strengthens the association of NOTCH1 mutations with HLHS and suggests that the two morphologically distinct single ventricle conditions, HLHS and HRHS, may share a common molecular and cellular etiology. Finally, somatic mutations in the LV are an unlikely contributor to HLHS