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It is known that vascular smooth muscle cells (VSMCs) represent a major part of the atherosclerotic plaque. In addition to forming fibrous cap cells that stabilize the atherosclerotic plaque, VSMCs trans -differentiate into macrophage-like cells that exacerbate the necrotic core. Here, we aim to address the question of how VSMCs are selected to perform distinct functions under a similar environmental stress, and how much cellular reprogramming happens during VSMC-to-macrophage-like transformation. Single-cell RNA-Sequencing (scRNA-Seq) analysis, in vitro transcriptional and metabolic studies, and pathological sample examinations were performed to decipher the cellular reprogramming during VSMC-to-macrophage-like cell transformation. By analyzing scRNA-Seq data of the atherosclerotic plaque, VSMC-derived macrophage-like cells were found to undergo a series promotion of lysosome-related and inflammation-related genes. In vitro transcriptional studies further confirmed that suppression of NOTCH signaling is the prerequisite for VSMCs to undergo sufficient genetic and metabolic reprogramming to a macrophage-like state and perform macrophage-like functions, while high-lipid treatment alone only promote VSMCs into a pro-inflammatory state without gain of lysosome-related functions. Mechanistic studies showed that NOTCH inhibition shifted VSMCs into a de-differentiated state by suppressing the developmental program, including key factor Myocd , leading to complete transformation into macrophage-like cells. NOTCH, a signaling mediated by consistent cell contacts, prevents the complete transformation of VSMCs into macrophage-like cells under high lipid stress. We hope our study could offer some insights into the retarded VSMC-to-macrophage-like transformation observed in previous in vitro studies and clarify the cellular reprogramming underlying the VSMC-to-macrophage-like cell transformation. [Display omitted] •Vascular smooth muscle cells (VSMCs) are known to play pleotroic functions during atherosclerosis plaque development. •We found that NOTCH signaling serves as a brake to prevent VSMCs transformation from a pro-inflammatory state to a macrophage-like state by affecting key VSMC phenotypic regulators. •Our results add some insights into the retarded VSMC-to-macrophage-like cell transformation and the pleiotropic functions and related transcriptome profiles of VSMCs during atherosclerosis development. [ABSTRACT FROM AUTHOR]

Humans are exposed to pyrethroid‐based pesticides through agricultural produce. In this study, male Wistar rats were orally treated for 9 to 12 months with a mixture of pyrethroids that is equivalent to one‐fifth (high dose; HD) or one‐twenty fifth (low dose; LD) of the amount of pyrethroids present in the cereals and rice consumed by an average Indian. In rats treated for 9 months, the spermatogenesis‐associated genes Abp, Ar, Cd9, Dax1, Dazap1, Ddx3y, Gdnf, Gfra1, Grth, Inhb, Ovol1, P1, Plzf, Pygo2, Scf, Tgfb1, Tp1, Tp2, and Vim1 were downregulated in both LD and HD groups. In rats treated for 12 months Gdnf, Hsf2, Inhb, Tgfb1, Thy1, and Ybx2 expression was downregulated in both LD and HD groups. Steroidogenesis‐associated genes 17‐β‐Hsd, Gata4, Hmgcr, Hmgcs1, Pde4b, and Tspo gene expression were reduced in both LD‐ and HD‐treated groups treated for 9 months. In 12‐month‐treated rats, Creb1 expression decreased in both LD and HD groups. The epigenetic reprogramming‐associated genes, Dnmt1, Dnmt3a, Dnmt3b, Hdac10, Hp1bp3, Kat3a Kat3b, Mch2ta, Ncoa7, and Sirt1 were downregulated in both HD and LD groups of 9‐months‐treated rats. In rats treated for 12 months, Hdac10, Mch2ta, Ncoa7, and Sirt1 messenger RNA levels decreased in both the HD and LD groups. Thus, we demonstrate that long‐term exposure to a mixture of pyrethroids caused aberrations in the transcriptome of factors involved in sperm production and development. [ABSTRACT FROM AUTHOR]

Cardiovascular disease remains a major cause of deaths globally. Post heart infarction, the most abundant cell type of the heart, fibroblasts, undergo a series of culminating events that lead to fibrotic scar tissue. In many organisms, injury to the heart can be restored, but the adult human heart is unable to efficiently regenerate after ischemic injury. So, the inefficiency of the heart at regenerating on its own after ischemic injury accounts for its reprogramming. Herein, we demonstrate the effect of microRNAs encapsulating poly-(lactic-co-glycolic acid) (PLGA)-polyethylenimine (PEI) nanocarriers for direct reprogramming of cardiac fibroblast to cardiomyocyte-like cells. Dual, cardiac-muscle-specific miRNA (miR-1 and miR-133a) polyplexes were encapsulated in biodegradable PLGA nanospheres. Cytocompatibility of the nanocomplexes were evaluated by various in vitro assays, confirming their safety profile. The change in cardiac fibroblast phenotype to cardiomyocyte was identified by the expression of late-stage signature markers. The PLGA-PEI-miRNA nanocomplex improved the intracellular internalization of cargo, exhibited pH-dependent release of the genetic material, and efficiently reprogrammed cardiac fibroblasts to cardiomyocyte-like cells. This is a first report of development of a nanovector targeting cardiac fibroblast for direct genetic reprogramming. Thus, this nanoscale approach serves as an ideal system for gene delivery and a promising therapeutic strategy for direct reprogramming of the heart. [ABSTRACT FROM AUTHOR]

Huntingtons disease (HD) is one of the most severe hereditaryneurodegenerative disorders caused by CAG repeats expansionin the HTT gene. A recently elaborated technology of geneticreprogramming allows obtaining induced pluripotent stem(iPS) cells from fibroblasts and other differentiated somaticcells. These iPS cells can grow in culture and differentiate inany cell types, including neurons, necessary for studies ofmolecular mechanisms of HD and other neurodegenerativediseases. We obtained, with the use of lentivirus transfection,iPS cells from primary fibroblasts biopsied from three femalepatients with HD (4246 copies of the CAG repeats in themutant allele). The efficiency of reprogramming was approximately0.2%. The embryoid bodies were obtained from someclones of iPS cells, and derivatives of all the three embryo layerswere shown to be formed as a result of spontaneous iPCcells differentiation. At present, our cell lines represent aunique platform for studies of HD. It may be used for establishingan effective system aimed at discoveries of molecularmechanisms undelaying HD and high-throughput search fornovel neuroprotective drugs.

Acquiring live human nervous tissue for research presents ethical and technical constraints. As a result, clinicians and scientists resort to using animal models to investigate human neuronal development and degeneration. However, innate species differences in neurobiology have hindered the translation of disease pathologies and development of therapeutic strategies. The discovery of endogenous neural stem cells (NSCs) and their examination has been critical for neuronal development, degeneration and regeneration. NSCs can exist in different developmental stages, embryonic through adult, and possess the capacity to generate the various cells that make up the nervous system. Importantly, human somatic cells can be obtained non-invasively and genetically reprogrammed into NSCs providing an ethically viable source of stem cells for translational study and potential therapy. Novel methods to generate NSCs of various developmental origins and regional identities are rapidly evolving to provide safer, quicker, and more efficient reprogramming strategies. Reprogrammed NSCs share many molecular and functional attributes with their endogenous NSC counterparts and can be used for in vitro modelling at a large scale. The accessibility to study patient specific NSCs allows the causal inferences of human disease mechanisms that may be unfeasible to model in animals. Despite the novelty of this burgeoning field, the opportunity for translational discoveries in neuronal development and degeneration and for therapeutic applications is unprecedented. This review will highlight the advances in manufacturing NSCs and their translational implications for disease modelling and potential treatment of the nervous system.

Major events in the long history of paradoxical sleep research, such as the crisis of the monoaminergic theory of sleep, as well as subsequent discoveries and theoretical functional hypotheses, are presented from an epistemological and a more general philosophical point of view. [ABSTRACT FROM AUTHOR]

Leaf senescence is not only genetically programmed but also induced by exogenous stress to ensure completion of the plant life cycle, successful reproduction and environmental adaptability. Genetic reprogramming is a major aspect of leaf senescence, and the senescence signaling that follows is controlled by a complex regulatory network. Recent studies suggest that the activity of transcription factors together with epigenetic mechanisms ensures the robustness of this network, with the latter including chromatin remodeling, DNA modification, and RNA-mediated control of transcription factors and other senescence-associated genes. In this review, we provide an overview of the relevant epigenetic mechanisms and summarize recent findings of epigenetic regulators of plant leaf senescence involved in DNA methylation and histone modification along with the functions of small RNAs in this process. [ABSTRACT FROM AUTHOR]

This article explores the idea of a genealogy of posthumanism while stressing the gene in genealogy. Starting with an analysis of Immanuel Kant's essay on the Enlightenment, among other short works of his, this study examines speculation on the improvement of the human race, and consequently the possible shared concerns of Kantian thought and posthumanism. Because the issues in question hover between conceptualities of “destiny”, “progress”, and “man”, this paper examines the past (and future) progress of humanity in terms of evolution and the techno-biological processes of sexual reproduction and gene expression (specifically, genetic reprogramming). In doing so, the present argument aims to better elucidate Kant's writings and, ultimately, determine if or how Kant may be read as prefiguring the posthumanist ethos. [ABSTRACT FROM AUTHOR]

This article explores the idea of a genealogy of posthumanism while stressing the gene in genealogy. Starting with an analysis of Immanuel Kant's essay on the Enlightenment, among other short works of his, this study examines speculation on the improvement of the human race, and consequently the possible shared concerns of Kantian thought and posthumanism. Because the issues in question hover between conceptualities of “destiny”, “progress”, and “man”, this paper examines the past (and future) progress of humanity in terms of evolution and the techno-biological processes of sexual reproduction and gene expression (specifically, genetic reprogramming). In doing so, the present argument aims to better elucidate Kant's writings and, ultimately, determine if or how Kant may be read as prefiguring the posthumanist ethos. [ABSTRACT FROM AUTHOR]

The promise of genetic reprogramming has prompted initiatives to develop banks of induced pluripotent stem cells (iPSCs) from diverse sources. Sentinel assays for pluripotency could maximize available resources for generating iPSCs. Neural rosettes represent a primitive neural tissue that is unique to differentiating PSCs and commonly used to identify derivative neural/stem progenitors. Here, neural rosettes were used as a sentinel assay for pluripotency in selection of candidates to advance to validation assays. Candidate iPSCs were generated from independent populations of amniotic cells with episomal vectors. Phase imaging of living back up cultures showed neural rosettes in 2 of the 5 candidate populations. Rosettes were immunopositive for the Sox1, Sox2, Pax6 and Pax7 transcription factors that govern neural development in the earliest stage of development and for the Isl1/2 and Otx2 transcription factors that are expressed in the dorsal and ventral domains, respectively, of the neural tube in vivo. Dissociation of rosettes produced cultures of differentiation competent neural/stem progenitors that generated immature neurons that were immunopositive for βIII-tubulin and glia that were immunopositive for GFAP. Subsequent validation assays of selected candidates showed induced expression of endogenous pluripotency genes, epigenetic modification of chromatin and formation of teratomas in immunodeficient mice that contained derivatives of the 3 embryonic germ layers. Validated lines were vector-free and maintained a normal karyotype for more than 60 passages. The credibility of rosette assembly as a sentinel assay for PSCs is supported by coordinate loss of nuclear-localized pluripotency factors Oct4 and Nanog in neural rosettes that emerge spontaneously in cultures of self-renewing validated lines. Taken together, these findings demonstrate value in neural rosettes as sentinels for pluripotency and selection of promising candidates for advance to validation assays.

Genetic reprogramming by ectopic expression of transcription factor genes induces the pluripotent state in somatic cells. This technology provides an opportunity to establish pluripotent stem cells for each person, as well as to get better understanding of epigenetic mechanisms controlling cell state. Interestingly, some of the molecular processes that accompany somatic cell reprogramming in vitro are also characteristic for tumor manifestation. Thus, similar 'molecular barriers' that control the stability of epigenetic state exist for both processes of pluripotency induction and malignant transformation. The reprogramming of tumor cells is interesting in two aspects: first, it will determine the contribution of epigenetic changes in carcinogenesis; second, it gives an approach to evaluate tumor stem cells that are supposed to form the entire cell mass of the tumor. This review discusses the key stages of genetic reprogramming, the similarity and difference between the reprogramming process and malignant transformation. [ABSTRACT FROM AUTHOR]

Engineered DNA-binding proteins that can be targeted to specific sites in the genome to manipulate gene expression have enabled many advances in biomedical research. This includes generating tools to study fundamental aspects of gene regulation and the development of a new class of gene therapies that alter the expression of endogenous genes. Designed transcription factors have entered clinical trials for the treatment of human diseases and others are in preclinical development. High-throughput and user-friendly platforms for designing synthetic DNA-binding proteins present innovative methods for deciphering cell biology and designing custom synthetic gene circuits. We review two platforms for designing synthetic transcription factors for manipulating gene expression: Transcription activator-like effectors (TALEs) and the RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. We present an overview of each technology and a guide for designing and assembling custom TALE- and CRISPR/Cas9-based transcription factors. We also discuss characteristics of each platform that are best suited for different applications. [ABSTRACT FROM AUTHOR]

Long Interspersed Nuclear Element‐1 (LINE‐1 or L1) is an autonomous, mobile element within the human genome that transposes via a “copy and paste” mechanism and relies upon L1‐encoded endonuclease and reverse transcriptase (RT) activities to compromise genome integrity. L1 has been implicated in various forms of cancer, but its role in the regulation of the oncogenic phenotype is not understood. The present studies were conducted to evaluate mechanisms of genetic regulatory control in HepG2 cells by human L1, or a D702Y mutant deficient in RT activity, and their influence on cellular phenotype. Forced expression of synthetic L1 ORF1p and ORF2p was associated with formation of cytoplasmic foci and minor association with the nuclear compartment. While de novo L1 mobilizations were only identified in cells expressing wild type L1, and were absent in the D702Y mutant, changes in gene expression profiles involved RT dependent as well as RT independent mechanisms. Synthetic L1 altered the expression of 24 in silico predicted genetic targets; ten of which showed RT‐dependence, ten RT‐independence, and four reciprocal regulatory control by both wild type and RT mutant. Of five targets examined, only VCAM1 and PTPRB colocalized with newly retrotransposed wild type L1. Biological discretization to partition patterns of gene expression into unique frequencies identified adhesion, inflammation, and cellular metabolism as key processes targeted for molecular interference with disruption of epithelial‐to‐mesenchymal programming seen irrespective of the RT phenotype. These findings establish L1 as a key regulator of genome plasticity and EMT via mechanisms independent of RT activity. Highlights: L1 reprograms the HepG2 genome via mechanisms that are independent of reverse transcriptase activity.Biological discretization identified adhesion, inflammation, and metabolism as processes targeted for molecular interference.The findings established L1 as a regulator of genome plasticity via mechanisms not directly coupled to retrotransposition. [ABSTRACT FROM AUTHOR]

Reexpression of the fetally expressed β-myosin heavy chain (β-MHC) gene is a well documented marker of pathological cardiac hypertrophy and normal aging in many experimental models. To gain insights into factors affecting this reexpression of β-MHC within the complex anatomical structure of the heart, we investigated the spatial pattern of its expression at the level of single cells during aging and hypertrophy. We generated mice that express yellow fluorescent protein fused to the N terminus of the β-MHC and examined its expression pattern during normal aging and in mice with hypertrophy induced by constitutive expression of a renin transgene. The localization of fibrosis within the hearts also was determined by using a fluorescent lectin. The results show that reexpression of β-MHC occurs in discrete subsets of myocytes within the subendocardium rather than uniformly throughout the heart, that β-MHC induction is not an obligatory consequence of cellular hypertrophy, and that β-MHC-expressing cells in the normal aging heart and the hypertrophic heart are distributed pre-dominantly in clusters within and surrounding foci of fibrosis. We conclude that β-MHC gene expression in the normal aging adult and hypertrophic mouse heart is a marker of fibrosis rather than of cellular hypertrophy. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) are a potential source for the construction of tissue-engineered vascular grafts. However, how vascular mechanical forces regulate the genetic reprogramming in MSCs is not well understood. Mechanical strain in the vascular wall is anisotropic and mainly in the circumferential direction. We have shown that cyclic uniaxial strain on elastic substrates causes the cells to align perpendicularly to the strain axis, which is different from that in the vascular wall. To simulate the vascular cell alignment and investigate the anisotropic mechanical sensing by MSCs, we used soft lithography to create elastomeric membranes with parallel microgrooves. This topographic pattern kept MSCs aligned parallel to the strain axis, and the cells were subjected to 5% cyclic uniaxial strain (1 Hz) for 2–4 days. DNA microarray analysis revealed global gene expression changes, including an increase in the smooth muscle marker calponin 1, decreases in cartilage matrix markers, and alterations in cell signaling (e.g., down-regulation of the Jagged1 signaling pathway). In addition, uniaxial strain increased MSC proliferation. However, when micropatterning was used to align cells perpendicularly to the axis of mechanical strain, the changes of some genes were diminished, and MSC proliferation was not affected. This study suggests that mechanical strain plays an important role in MSC differentiation and proliferation, and that the effects of mechanotransduction depend on the orientation of cells with respect to the strain axis. The differential cellular responses to the anisotropic mechanical environment have important implications in cardiovascular development, tissue remodeling, and tissue engineering. [ABSTRACT FROM AUTHOR]

Abstract: The hypothesis that intracrine renin–angiotensin system activated during heart failure is part of the tendency of the heart to return to embryological conditions when organogenesis is possible is presented and discussed. The hypothesis proposes that the change in genetic makeup, which is known to occur during heart failure, includes a drastic change of intercellular chemical and electrical communication such as second messengers and other signal molecules which are involved in cell proliferation and growth. The role of angiotensin II, which is a growth factor, reduces cell coupling in the failing heart through the activation of AT1 receptors and intracellular pathways, such as PKC, MAPK family and increment of intracellular calcium, might play a key role in the genetic reprogramming of the failing heart. [Copyright &y& Elsevier]

Neural stem cell (NSC) mediated adult neurogenesis represents the regenerative plasticity of the brain. The functionality of the neurogenic process appears to be operated by neuroblasts, the multipotent immature neuronal population of the adult brain. While neuroblasts have been realized to play a major role in synaptic remodeling and immunogenicity, neurodegenerative disorders have been characterized by failure in the terminal differentiation, maturation, integration and survival of newborn neuroblasts. Advancement in understanding the impaired neuroregenerative process along the neuropathological conditions has currently been limited by lack of an appropriate experimental model of neuroblasts. The genetic reprogramming of somatic cells into pluripotent state offers a potential strategy for the experimental modeling of brain disorders. Thus, the induced pluripotent stem cell (iPSC) based direct reprogramming of somatic cells into neuroblasts would represent a potential tool to understand the regenerative biology of the adult brain. Therefore, this concise article discusses the significance of iPSCs, the functional roles of neuroblasts in the adult brain and provides a research hypothesis for the direct reprogramming of somatic cells into neuroblasts through the co-induction of a potential proneurogenic marker, the doublecortin (DCX) gene along with the Yamanaka factors. The proposed cellular model of adult neurogenesis may provide us with further insights into neuropathogenesis of many neurodegenerative disorders and will provide a potential experimental platform for diagnostic, drug discovery and regenerative therapeutic strategies., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We used 1 H, 13 C HRMAS and genomic analysis to investigate regionally the transition from oxidative to glycolytic phenotype and its relationship with altered gene expression in adjacent biopsies through the brain of rats bearing C6 gliomas. Tumor-bearing animals were anesthetized and infused with a solution of [1- 13 C]-glucose, and small adjacent biopsies were obtained spanning transversally from the contralateral hemisphere (regions I and II), the right and left peritumoral areas (regions III and V, respectively), and the tumor core (region IV). These biopsies were analyzed by 1 H, 13 C HRMAS and by quantitative gene expression techniques. Glycolytic metabolism, as reflected by the [3- 13 C]-lactate content, increased clearly from regions I to IV, recovering partially to physiological levels in region V. In contrast, oxidative metabolism, as reflected by the [4- 13 C]-glutamate labeling, decreased in regions I-IV, recovering partially in region V. This metabolic shift from normal to malignant metabolic phenotype paralleled changes in the expression of HIF1α, HIF2α, HIF3α genes, downstream transporters, and regulatory glycolytic, oxidative, and anaplerotic genes in the same regions. Together, our results indicate that genetic and metabolic alterations occurring in the brain of rats bearing C6 gliomas colocalize in situ and the profile of genetic alterations in every region can be inferred from the metabolomic profiles observed in situ by multinuclear HRMAS.

Genetic reprogramming holds great potential for disease modeling, drug screening, and regenerative medicine. Genetic reprogramming of mammalian cells is typically achieved by forced expression of natural transcription factors that control master gene networks and cell lineage specification. However, in many instances, the natural transcription factors do not induce a sufficiently robust response to completely reprogram cell phenotype. In this study, we demonstrate that protein engineering of the master transcription factor MyoD can enhance the conversion of human dermal fibroblasts and adult stem cells to a skeletal myocyte phenotype. Fusion of potent transcriptional activation domains to MyoD led to increased myogenic gene expression, myofiber formation, cell fusion, and global reprogramming of the myogenic gene network. This work supports a general strategy for synthetically enhancing the direct conversion between cell types that can be applied in both synthetic biology and regenerative medicine.

The promise of genetic reprogramming has prompted initiatives to develop banks of induced pluripotent stem cells (iPSCs) from diverse sources. Sentinel assays for pluripotency could maximize available resources for generating iPSCs. Neural rosettes represent a primitive neural tissue that is unique to differentiating PSCs and commonly used to identify derivative neural/stem progenitors. Here, neural rosettes were used as a sentinel assay for pluripotency in selection of candidates to advance to validation assays. Candidate iPSCs were generated from independent populations of amniotic cells with episomal vectors. Phase imaging of living back up cultures showed neural rosettes in 2 of the 5 candidate populations. Rosettes were immunopositive for the Sox1, Sox2, Pax6 and Pax7 transcription factors that govern neural development in the earliest stage of development and for the Isl1/2 and Otx2 transcription factors that are expressed in the dorsal and ventral domains, respectively, of the neural tube in vivo. Dissociation of rosettes produced cultures of differentiation competent neural/stem progenitors that generated immature neurons that were immunopositive for βIII-tubulin and glia that were immunopositive for GFAP. Subsequent validation assays of selected candidates showed induced expression of endogenous pluripotency genes, epigenetic modification of chromatin and formation of teratomas in immunodeficient mice that contained derivatives of the 3 embryonic germ layers. Validated lines were vector-free and maintained a normal karyotype for more than 60 passages. The credibility of rosette assembly as a sentinel assay for PSCs is supported by coordinate loss of nuclear-localized pluripotency factors Oct4 and Nanog in neural rosettes that emerge spontaneously in cultures of self-renewing validated lines. Taken together, these findings demonstrate value in neural rosettes as sentinels for pluripotency and selection of promising candidates for advance to validation assays.