Publications by authors named "Hongjun Song"

231 Publications

High-Affinity Chimeric Antigen Receptor With Cross-Reactive scFv to Clinically Relevant EGFR Oncogenic Isoforms.

Front Oncol 2021 10;11:664236. Epub 2021 Sep 10.

Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States.

Tumor heterogeneity is a key reason for therapeutic failure and tumor recurrence in glioblastoma (GBM). Our chimeric antigen receptor (CAR) T cell (2173 CAR T cells) clinical trial (NCT02209376) against epidermal growth factor receptor (EGFR) variant III (EGFRvIII) demonstrated successful trafficking of T cells across the blood-brain barrier into GBM active tumor sites. However, CAR T cell infiltration was associated only with a selective loss of EGFRvIII+ tumor, demonstrating little to no effect on EGFRvIII tumor cells. Post-CAR T-treated tumor specimens showed continued presence of EGFR amplification and oncogenic EGFR extracellular domain (ECD) missense mutations, despite loss of EGFRvIII. To address tumor escape, we generated an EGFR-specific CAR by fusing monoclonal antibody (mAb) 806 to a 4-1BB co-stimulatory domain. The resulting construct was compared to 2173 CAR T cells in GBM, using and models. 806 CAR T cells specifically lysed tumor cells and secreted cytokines in response to amplified EGFR, EGFRvIII, and EGFR-ECD mutations in U87MG cells, GBM neurosphere-derived cell lines, and patient-derived GBM organoids. 806 CAR T cells did not lyse fetal brain astrocytes or primary keratinocytes to a significant degree. They also exhibited superior antitumor activity when compared to 2173 CAR T cells. The broad specificity of 806 CAR T cells to EGFR alterations gives us the potential to target multiple clones within a tumor and reduce opportunities for tumor escape antigen loss.
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http://dx.doi.org/10.3389/fonc.2021.664236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8461175PMC
September 2021

Applications of Brain Organoids for Infectious Diseases.

J Mol Biol 2021 Sep 15:167243. Epub 2021 Sep 15.

Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Developmental and Cell Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Psychiatry, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address: https://twitter.com/UPenn_Ming.

Brain organoids are self-organized three-dimensional aggregates generated from pluripotent stem cells. They exhibit complex cell diversities and organized architectures that resemble human brain development ranging from neural tube formation, neuroepithelium differentiation, neurogenesis and gliogenesis, to neural circuit formation. Rapid advancements in brain organoid culture technologies have allowed researchers to generate more accurate models of human brain development and neurological diseases. These models also allow for direct investigation of pathological processes associated with infectious diseases affecting the nervous system. In this review, we first briefly summarize recent advancements in brain organoid methodologies and neurodevelopmental processes that can be effectively modeled by brain organoids. We then focus on applications of brain organoids to investigate the pathogenesis of neurotropic viral infection. Finally, we discuss limitations of the current brain organoid methodologies as well as applications of other organ specific organoids in the infectious disease research.
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http://dx.doi.org/10.1016/j.jmb.2021.167243DOI Listing
September 2021

CYFIP1 Dosages Exhibit Divergent Behavioral Impact via Diametric Regulation of NMDA Receptor Complex Translation in Mouse Models of Psychiatric Disorders.

Biol Psychiatry 2021 May 5. Epub 2021 May 5.

Department of Neuroscience, Mahoney Institute for Neurosciences, Perelman School for Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; Department of Cell and Developmental Biology, Perelman School for Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; Institute for Regenerative Medicine, Perelman School for Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; Department of Psychiatry, Perelman School for Medicine, University of Pennsylvania, Philadelphia, Pennsylvania. Electronic address:

Background: Gene dosage imbalance caused by copy number variations (CNVs) is a prominent contributor to brain disorders. In particular, 15q11.2 CNV duplications and deletions have been associated with autism spectrum disorder and schizophrenia, respectively. The mechanism underlying these diametric contributions remains unclear.

Methods: We established both loss-of-function and gain-of-function mouse models of Cyfip1, one of four genes within 15q11.2 CNVs. To assess the functional consequences of altered CYFIP1 levels, we performed systematic investigations on behavioral, electrophysiological, and biochemical phenotypes in both mouse models. In addition, we utilized RNA immunoprecipitation sequencing (RIP-seq) analysis to reveal molecular targets of CYFIP1 in vivo.

Results: Cyfip1 loss-of-function and gain-of function mouse models exhibited distinct and shared behavioral abnormalities related to autism spectrum disorder and schizophrenia. RIP-seq analysis identified messenger RNA targets of CYFIP1 in vivo, including postsynaptic NMDA receptor (NMDAR) complex components. In addition, these mouse models showed diametric changes in levels of postsynaptic NMDAR complex components at synapses because of dysregulated protein translation, resulting in bidirectional alteration of NMDAR-mediated signaling. Importantly, pharmacological balancing of NMDAR signaling in these mouse models with diametric Cyfip1 dosages rescues behavioral abnormalities.

Conclusions: CYFIP1 regulates protein translation of NMDAR and associated complex components at synapses to maintain normal synaptic functions and behaviors. Our integrated analyses provide insight into how gene dosage imbalance caused by CNVs may contribute to divergent neuropsychiatric disorders.
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http://dx.doi.org/10.1016/j.biopsych.2021.04.023DOI Listing
May 2021

Novel Treatment for Glioblastoma Delivered by a Radiation Responsive and Radiopaque Hydrogel.

ACS Biomater Sci Eng 2021 07 23;7(7):3209-3220. Epub 2021 Jun 23.

Department of Radiology, University of Pennsylvania, 3400 Spruce Street, 1 Silverstein Philadelphia, Pennsylvania 19104, United States.

Successful treatment of glioblastoma (GBM) is hampered by primary tumor recurrence after surgical resection and poor prognosis, despite adjuvant radiotherapy and chemotherapy. In search of improved outcomes for this disease, quisinostat appeared as a lead compound in drug screening. A delivery system was devised for this drug and to exploit current clinical methodology: an injectable hydrogel, loaded with both the quisinostat drug and radiopaque gold nanoparticles (AuNP) as contrast agent, that can release these payloads as a response to radiation. This hydrogel grants high local drug concentrations, overcoming issues with current standards of care. Significant hydrogel degradation and quisinostat release were observed due to the radiation trigger, providing high in vitro anticancer activity. In vivo, the combination of radiotherapy and the radiation-induced delivery of quisinostat from the hydrogel, successfully inhibited tumor growth in a mice model bearing xenografted human GBM tumors with a total response rate of 67%. Long-term tolerability was observed after intratumoral injection of the quisinostat loaded hydrogel. The AuNP payload enabled precise image-guided radiation delivery and the monitoring of hydrogel degradation using computed tomography (CT). These exciting results highlight this hydrogel as a versatile imageable drug delivery platform that can be activated simultaneously to radiation therapy and potentially offers improved treatment for GBM.
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http://dx.doi.org/10.1021/acsbiomaterials.1c00385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8411482PMC
July 2021

Seq-ing out cell types across the isocortex and hippocampal formation.

Cell 2021 Jun;184(12):3083-3085

Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; The Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

A central quest in neuroscience is to gain a holistic understanding of all cell types in the brain. In this issue of Cell, Yao et al. establish a molecular architectural view of cell types across the entire adult mouse isocortex and hippocampal formation and reveal surprising similarities of cell types in these two brain regions.
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http://dx.doi.org/10.1016/j.cell.2021.05.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8370710PMC
June 2021

Generation of hypothalamic arcuate organoids from human induced pluripotent stem cells.

Cell Stem Cell 2021 09 6;28(9):1657-1670.e10. Epub 2021 May 6.

Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Psychiatry, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Human brain organoids represent remarkable platforms for recapitulating features of human brain development and diseases. Existing organoid models do not resolve fine brain subregions, such as different nuclei in the hypothalamus. We report the generation of arcuate organoids (ARCOs) from human induced pluripotent stem cells (iPSCs) to model the development of the human hypothalamic arcuate nucleus. Single-cell RNA sequencing of ARCOs revealed significant molecular heterogeneity underlying different arcuate cell types, and machine learning-aided analysis based on the neonatal human hypothalamus single-nucleus transcriptome further showed a human arcuate nucleus molecular signature. We also explored ARCOs generated from Prader-Willi syndrome (PWS) patient iPSCs. These organoids exhibit aberrant differentiation and transcriptomic dysregulation similar to postnatal hypothalamus of PWS patients, indicative of cellular differentiation deficits and exacerbated inflammatory responses. Thus, patient iPSC-derived ARCOs represent a promising experimental model for investigating nucleus-specific features and disease-relevant mechanisms during early human arcuate development.
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http://dx.doi.org/10.1016/j.stem.2021.04.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8419002PMC
September 2021

Ontogeny of adult neural stem cells in the mammalian brain.

Curr Top Dev Biol 2021 17;142:67-98. Epub 2020 Dec 17.

Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States; Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States; Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA, United States; The Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States. Electronic address:

Neural stem cells (NSCs) persist into adulthood in the subgranular zone (SGZ) of the dentate gyrus in the hippocampus and in the ventricular-subventricular zone (V-SVZ) of the lateral ventricles, where they generate new neurons and glia cells that contribute to neural plasticity. A better understanding of the developmental process that enables NSCs to persist beyond development will provide insight into factors that determine the size and properties of the adult NSC pool and thus the capacity for life-long neurogenesis in the adult mammalian brain. We review current knowledge regarding the developmental origins of adult NSCs and the developmental process by which embryonic NSCs transition into their adult form. We also discuss potential mechanisms that might regulate proper establishment of the adult NSC pool, and propose future directions of research that will be key to unraveling how NSCs transform to establish the adult NSC pool in the mammalian brain.
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http://dx.doi.org/10.1016/bs.ctdb.2020.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8363052PMC
December 2020

Building the brain from scratch: Engineering region-specific brain organoids from human stem cells to study neural development and disease.

Curr Top Dev Biol 2021 18;142:477-530. Epub 2021 Feb 18.

Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States; Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States; Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA, United States; Department of Psychiatry, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States. Electronic address:

Human brain development is an intricate process that involves precisely timed coordination of cell proliferation, fate specification, neuronal differentiation, migration, and integration of diverse cell types. Understanding of these fundamental processes, however, has been largely constrained by limited access to fetal brain tissue and the inability to prospectively study neurodevelopment in humans at the molecular, cellular and system levels. Although non-human model organisms have provided important insights into mechanisms underlying brain development, these systems do not fully recapitulate many human-specific features that often relate to disease. To address these challenges, human brain organoids, self-assembled three-dimensional neural aggregates, have been engineered from human pluripotent stem cells to model the architecture and cellular diversity of the developing human brain. Recent advancements in neural induction and regional patterning using small molecules and growth factors have yielded protocols for generating brain organoids that recapitulate the structure and neuronal composition of distinct brain regions. Here, we first provide an overview of early mammalian brain development with an emphasis on molecular cues that guide region specification. We then focus on recent efforts in generating human brain organoids that model the development of specific brain regions and highlight endeavors to enhance the cellular complexity to better mimic the in vivo developing human brain. We also provide examples of how organoid models have enhanced our understanding of human neurological diseases and conclude by discussing limitations of brain organoids with our perspectives on future advancements to maximize their potential.
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http://dx.doi.org/10.1016/bs.ctdb.2020.12.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8363060PMC
February 2021

Application of niclosamide and analogs as small molecule inhibitors of Zika virus and SARS-CoV-2 infection.

Bioorg Med Chem Lett 2021 05 6;40:127906. Epub 2021 Mar 6.

National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Bethesda, MD 20892-3370, USA. Electronic address:

Zika virus has emerged as a potential threat to human health globally. A previous drug repurposing screen identified the approved anthelminthic drug niclosamide as a small molecule inhibitor of Zika virus infection. However, as antihelminthic drugs are generally designed to have low absorption when dosed orally, the very limited bioavailability of niclosamide will likely hinder its potential direct repurposing as an antiviral medication. Here, we conducted SAR studies focusing on the anilide and salicylic acid regions of niclosamide to improve physicochemical properties such as microsomal metabolic stability, permeability and solubility. We found that the 5-bromo substitution in the salicylic acid region retains potency while providing better drug-like properties. Other modifications in the anilide region with 2'-OMe and 2'-H substitutions were also advantageous. We found that the 4'-NO substituent can be replaced with a 4'-CN or 4'-CF substituents. Together, these modifications provide a basis for optimizing the structure of niclosamide to improve systemic exposure for application of niclosamide analogs as drug lead candidates for treating Zika and other viral infections. Indeed, key analogs were also able to rescue cells from the cytopathic effect of SARS-CoV-2 infection, indicating relevance for therapeutic strategies targeting the COVID-19 pandemic.
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http://dx.doi.org/10.1016/j.bmcl.2021.127906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7936759PMC
May 2021

Pharmacological rescue in patient iPSC and mouse models with a rare DISC1 mutation.

Nat Commun 2021 03 3;12(1):1398. Epub 2021 Mar 3.

Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School for Medicine, University of Pennsylvania, Philadelphia, PA, USA.

We previously identified a causal link between a rare patient mutation in DISC1 (disrupted-in-schizophrenia 1) and synaptic deficits in cortical neurons differentiated from isogenic patient-derived induced pluripotent stem cells (iPSCs). Here we find that transcripts related to phosphodiesterase 4 (PDE4) signaling are significantly elevated in human cortical neurons differentiated from iPSCs with the DISC1 mutation and that inhibition of PDE4 or activation of the cAMP signaling pathway functionally rescues synaptic deficits. We further generated a knock-in mouse line harboring the same patient mutation in the Disc1 gene. Heterozygous Disc1 mutant mice exhibit elevated levels of PDE4s and synaptic abnormalities in the brain, and social and cognitive behavioral deficits. Pharmacological inhibition of the PDE4 signaling pathway rescues these synaptic, social and cognitive behavioral abnormalities. Our study shows that patient-derived isogenic iPSC and humanized mouse disease models are integral and complementary for translational studies with a better understanding of underlying molecular mechanisms.
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http://dx.doi.org/10.1038/s41467-021-21713-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7930023PMC
March 2021

An Integrated Systems Biology Approach Identifies the Proteasome as A Critical Host Machinery for ZIKV and DENV Replication.

Genomics Proteomics Bioinformatics 2021 02 19;19(1):108-122. Epub 2021 Feb 19.

Department of Pharmacology & Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA. Electronic address:

The Zika virus (ZIKV) and dengue virus (DENV) flaviviruses exhibit similar replicative processes but have distinct clinical outcomes. A systematic understanding of virus-host protein-protein interaction networks can reveal cellular pathways critical to viral replication and disease pathogenesis. Here we employed three independent systems biology approaches toward this goal. First, protein array analysis of direct interactions between individual ZIKV/DENV viral proteins and 20,240 human proteins revealed multiple conserved cellular pathways and protein complexes, including proteasome complexes. Second, an RNAi screen of 10,415 druggable genes identified the host proteins required for ZIKV infection and uncovered that proteasome proteins were crucial in this process. Third, high-throughput screening of 6016 bioactive compounds for ZIKV inhibition yielded 134 effective compounds, including six proteasome inhibitors that suppress both ZIKV and DENV replication. Integrative analyses of these orthogonal datasets pinpoint proteasomes as critical host machinery for ZIKV/DENV replication. Our study provides multi-omics datasets for further studies of flavivirus-host interactions, disease pathogenesis, and new drug targets.
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http://dx.doi.org/10.1016/j.gpb.2020.06.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8498969PMC
February 2021

Decoding neuronal composition and ontogeny of individual hypothalamic nuclei.

Neuron 2021 04 17;109(7):1150-1167.e6. Epub 2021 Feb 17.

Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; The Epigenetic Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

The hypothalamus plays crucial roles in regulating endocrine, autonomic, and behavioral functions via its diverse nuclei and neuronal subtypes. The developmental mechanisms underlying ontogenetic establishment of different hypothalamic nuclei and generation of neuronal diversity remain largely unknown. Here, we show that combinatorial T-box 3 (TBX3), orthopedia homeobox (OTP), and distal-less homeobox (DLX) expression delineates all arcuate nucleus (Arc) neurons and defines four distinct subpopulations, whereas combinatorial NKX2.1/SF1 and OTP/DLX expression identifies ventromedial hypothalamus (VMH) and tuberal nucleus (TuN) neuronal subpopulations, respectively. Developmental analysis indicates that all four Arc subpopulations are mosaically and simultaneously generated from embryonic Arc progenitors, whereas glutamatergic VMH neurons and GABAergic TuN neurons are sequentially generated from common embryonic VMH progenitors. Moreover, clonal lineage-tracing analysis reveals that diverse lineages from multipotent radial glia progenitors orchestrate Arc and VMH-TuN establishment. Together, our study reveals cellular mechanisms underlying generation and organization of diverse neuronal subtypes and ontogenetic establishment of individual nuclei in the mammalian hypothalamus.
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http://dx.doi.org/10.1016/j.neuron.2021.01.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8035319PMC
April 2021

An all-to-all approach to the identification of sequence-specific readers for epigenetic DNA modifications on cytosine.

Nat Commun 2021 02 4;12(1):795. Epub 2021 Feb 4.

Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Epigenetic modifications of DNA play important roles in many biological processes. Identifying readers of these epigenetic marks is a critical step towards understanding the underlying mechanisms. Here, we present an all-to-all approach, dubbed digital affinity profiling via proximity ligation (DAPPL), to simultaneously profile human TF-DNA interactions using mixtures of random DNA libraries carrying different epigenetic modifications (i.e., 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine) on CpG dinucleotides. Many proteins that recognize consensus sequences carrying these modifications in symmetric and/or hemi-modified forms are identified. We further demonstrate that the modifications in different sequence contexts could either enhance or suppress TF binding activity. Moreover, many modifications can affect TF binding specificity. Furthermore, symmetric modifications show a stronger effect in either enhancing or suppressing TF-DNA interactions than hemi-modifications. Finally, in vivo evidence suggests that USF1 and USF2 might regulate transcription via hydroxymethylcytosine-binding activity in weak enhancers in human embryonic stem cells.
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http://dx.doi.org/10.1038/s41467-021-20950-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862700PMC
February 2021

Zika Virus-Induced Neuronal Apoptosis via Increased Mitochondrial Fragmentation.

Front Microbiol 2020 23;11:598203. Epub 2020 Dec 23.

National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, United States.

The 2015 to 2016 outbreak of Zika virus (ZIKV) infections in the Americas coincided with a dramatic increase in neurodevelopmental abnormalities, including fetal microcephaly, in newborns born to infected women. In this study, we observed mitochondrial fragmentation and disrupted mitochondrial membrane potential after 24 h of ZIKV infection in human neural stem cells and the SNB-19 glioblastoma cell line. The severity of these changes correlated with the amount of ZIKV proteins expressed in infected cells. ZIKV infection also decreased the levels of mitofusin 2, which modulates mitochondria fusion. Mitochondrial division inhibitor 1 (Mdivi-1), a small molecule inhibiting mitochondria fission, ameliorated mitochondria disruptions and reduced cell death in ZIKV-infected cells. Collectively, this study suggests that abnormal mitochondrial fragmentation contributes to ZIKV-induced neuronal cell death; rebalancing mitochondrial dynamics of fission-fusion could be a therapeutic strategy for drug development to treat ZIKV-mediated neuronal apoptosis.
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http://dx.doi.org/10.3389/fmicb.2020.598203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7785723PMC
December 2020

Differential Timing and Coordination of Neurogenesis and Astrogenesis in Developing Mouse Hippocampal Subregions.

Brain Sci 2020 Nov 26;10(12). Epub 2020 Nov 26.

Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Neocortical development has been extensively studied and therefore is the basis of our understanding of mammalian brain development. One fundamental principle of neocortical development is that neurogenesis and gliogenesis are temporally segregated processes. However, it is unclear how neurogenesis and gliogenesis are coordinated in non-neocortical regions of the cerebral cortex, such as the hippocampus, also known as the archicortex. Here, we show that the timing of neurogenesis and astrogenesis in the Cornu Ammonis (CA) 1 and CA3 regions of mouse hippocampus mirrors that of the neocortex; neurogenesis occurs embryonically, followed by astrogenesis during early postnatal development. In contrast, we find that neurogenesis in the dentate gyrus begins embryonically but is a protracted process which peaks neonatally and continues at low levels postnatally. As a result, astrogenesis, which occurs during early postnatal development, overlaps with the process of neurogenesis in the dentate gyrus. During all stages, neurogenesis overwhelms astrogenesis in the dentate gyrus. In addition, we find that the timing of peak astrogenesis varies by hippocampal subregion. Together, our results show differential timing and coordination of neurogenesis and astrogenesis in developing mouse hippocampal subregions and suggest that neurogenesis and gliogenesis occur simultaneously during dentate gyrus development, challenging the conventional principle that neurogenesis and gliogenesis are temporally separated processes.
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http://dx.doi.org/10.3390/brainsci10120909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760658PMC
November 2020

Novel gene-specific translation mechanism of dysregulated, chronic inflammation reveals promising, multifaceted COVID-19 therapeutics.

bioRxiv 2020 Nov 16. Epub 2020 Nov 16.

Hyperinflammation and lymphopenia provoked by SARS-CoV-2-activated macrophages contribute to the high mortality of Coronavirus Disease 2019 (COVID-19) patients. Thus, defining host pathways aberrantly activated in patient macrophages is critical for developing effective therapeutics. We discovered that G9a, a histone methyltransferase that is overexpressed in COVID-19 patients with high viral load, activates translation of specific genes that induce hyperinflammation and impairment of T cell function or lymphopenia. This noncanonical, pro-translation activity of G9a contrasts with its canonical epigenetic function. In endotoxin-tolerant (ET) macrophages that mimic conditions which render patients with pre-existing chronic inflammatory diseases vulnerable to severe symptoms, our chemoproteomic approach with a biotinylated inhibitor of G9a identified multiple G9a-associated translation regulatory pathways that were upregulated by SARS-CoV-2 infection. Further, quantitative translatome analysis of ET macrophages treated progressively with the G9a inhibitor profiled G9a-translated proteins that unite the networks associated with viral replication and the SARS-CoV-2-induced host response in severe patients. Accordingly, inhibition of G9a-associated pathways produced multifaceted, systematic effects, namely, restoration of T cell function, mitigation of hyperinflammation, and suppression of viral replication. Importantly, as a host-directed mechanism, this G9a-targeted, combined therapeutics is refractory to emerging antiviral-resistant mutants of SARS-CoV-2, or any virus, that hijacks host responses.
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http://dx.doi.org/10.1101/2020.11.14.382416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685324PMC
November 2020

Modeling Human Cytomegalovirus-Induced Microcephaly in Human iPSC-Derived Brain Organoids.

Cell Rep Med 2020 Apr 25;1(1):100002. Epub 2020 Mar 25.

Division of Stem Cell Biology Research, Department of Developmental and Stem Cell Biology, Beckman Research Institute of City of Hope, 1500 E. Duarte Road, Duarte, CA 91010, USA.

Although congenital infection by human cytomegalovirus (HCMV) is well recognized as a leading cause of neurodevelopmental defects, HCMV neuropathogenesis remains poorly understood. A major challenge for investigating HCMV-induced abnormal brain development is the strict CMV species specificity, which prevents the use of animal models to directly study brain defects caused by HCMV. We show that infection of human-induced pluripotent-stem-cell-derived brain organoids by a "clinical-like" HCMV strain results in reduced brain organoid growth, impaired formation of cortical layers, and abnormal calcium signaling and neural network activity. Moreover, we show that the impeded brain organoid development caused by HCMV can be prevented by neutralizing antibodies (NAbs) that recognize the HCMV pentamer complex. These results demonstrate in a three-dimensional cellular biosystem that HCMV can impair the development and function of the human brain and provide insights into the potential capacity of NAbs to mitigate brain defects resulted from HCMV infection.
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http://dx.doi.org/10.1016/j.xcrm.2020.100002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7659592PMC
April 2020

Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR T cell testing.

Nat Protoc 2020 12 9;15(12):4000-4033. Epub 2020 Nov 9.

Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Glioblastoma tumors exhibit extensive inter- and intratumoral heterogeneity, which has contributed to the poor outcomes of numerous clinical trials and continues to complicate the development of effective therapeutic strategies. Most in vitro models do not preserve the cellular and mutational diversity of parent tumors and often require a lengthy generation time with variable efficiency. Here, we describe detailed procedures for generating glioblastoma organoids (GBOs) from surgically resected patient tumor tissue using a chemically defined medium without cell dissociation. By preserving cell-cell interactions and minimizing clonal selection, GBOs maintain the cellular heterogeneity of parent tumors. We include details of how to passage and cryopreserve GBOs for continued use, biobanking and long-term recovery. In addition, we describe procedures for investigating patient-specific responses to immunotherapies by co-culturing GBOs with chimeric antigen receptor (CAR) T cells. It takes ~2-4 weeks to generate GBOs and 5-7 d to perform CAR T cell co-culture using this protocol. Competence with human cell culture, tissue processing, immunohistology and microscopy is required for optimal results.
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http://dx.doi.org/10.1038/s41596-020-0402-9DOI Listing
December 2020

The epitranscriptome in stem cell biology and neural development.

Neurobiol Dis 2020 12 13;146:105139. Epub 2020 Oct 13.

Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School for Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; The Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

The blossoming field of epitranscriptomics has recently garnered attention across many fields by findings that chemical modifications on RNA have immense biological consequences. Methylation of nucleotides in RNA, including N6-methyladenosine (mA), 2-O-dimethyladenosine (mA), N1-methyladenosine (mA), 5-methylcytosine (mC), and isomerization of uracil to pseudouridine (Ψ), have the potential to alter RNA processing events and contribute to developmental processes and different diseases. Though the abundance and roles of some RNA modifications remain contentious, the epitranscriptome is thought to be especially relevant in stem cell biology and neurobiology. In particular, mA occurs at the highest levels in the brain and plays major roles in embryonic stem cell differentiation, brain development, and neurodevelopmental disorders. However, studies in these areas have reported conflicting results on epitranscriptomic regulation of stem cell pluripotency and mechanisms in neural development. In this review we provide an overview of the current understanding of several RNA modifications and disentangle the various findings on epitranscriptomic regulation of stem cell biology and neural development.
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http://dx.doi.org/10.1016/j.nbd.2020.105139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686257PMC
December 2020

Human Pluripotent Stem Cell-Derived Neural Cells and Brain Organoids Reveal SARS-CoV-2 Neurotropism Predominates in Choroid Plexus Epithelium.

Cell Stem Cell 2020 12 21;27(6):937-950.e9. Epub 2020 Sep 21.

Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Psychiatry, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Neurological complications are common in patients with COVID-19. Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal pathogen of COVID-19, has been detected in some patient brains, its ability to infect brain cells and impact their function is not well understood. Here, we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found that neurons and astrocytes were sparsely infected, but choroid plexus epithelial cells underwent robust infection. We optimized a protocol to generate choroid plexus organoids from hiPSCs and showed that productive SARS-CoV-2 infection of these organoids is associated with increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our findings provide evidence for selective SARS-CoV-2 neurotropism and support the use of hiPSC-derived brain organoids as a platform to investigate SARS-CoV-2 infection susceptibility of brain cells, mechanisms of virus-induced brain dysfunction, and treatment strategies.
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http://dx.doi.org/10.1016/j.stem.2020.09.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7505550PMC
December 2020

HIF-1α-Mediated Telomerase Reverse Transcriptase Activation Inducing Autophagy Through Mammalian Target of Rapamycin Promotes Papillary Thyroid Carcinoma Progression During Hypoxia Stress.

Thyroid 2021 02 9;31(2):233-246. Epub 2020 Sep 9.

Department of Nuclear Medicine, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.

It is important to properly understand the molecular mechanisms of aggressive tumors among papillary thyroid carcinomas (PTCs) that are often the most indolent. Hypoxia inducible factor-1α (HIF-1α), induced by hypoxia, plays pivotal roles in the development and metastasis of the many tumors, including PTCs. Upregulation of telomerase reverse transcriptase (TERT) activity is found in highly invasive PTCs. Further, previous studies have reported that autophagy serves as a protective mechanism to facilitate PTC cell survival. We, therefore, hypothesized that there was a link between HIF-1α, TERT, and autophagy in promoting PTC progression. Immunohistochemistry staining was conducted to evaluate the expressions of HIF-1α, TERT, and autophagy marker, LC3-II, in matched PTC tumors and corresponding nontumor tissues. Two PTC cell lines (TPC-1 and BCPAP) were used in subsequent cytological function studies. Cell viability, proliferation, apoptosis, migration, and invasion were assessed during hypoxia, genetic enhancement and inhibition of TERT, and chemical and genetic inhibition of autophagy. The protein expression levels of the corresponding biomarkers were determined by Western blotting, and autophagy flow was detected. We characterized the molecular mechanism of PTC cell progression. The protein expression levels of HIF-1α, TERT, and LC3-II were upregulated in PTCs and were significantly correlated with high tumor-node-metastasis stage. Further, an study indicated that HIF-1α induced by hypoxia functioned as a transcriptional activator by binding with sequences potentially located in the TERT promoter and was positively correlated with the malignant behavior of PTC cell lines. Overexpression of TERT inhibited the kinase activity of mammalian target of rapamycin (mTOR), resulting in the activation of autophagy. Functionally, TERT-induced autophagy provided a survival advantage to PTC cells during hypoxia stress. We identified a novel molecular mechanism involving the HIF-1α/TERT axis, which promoted PTC progression by inducing autophagy through mTOR during hypoxia stress. These findings may provide a basis for the new treatment of aggressive PTCs.
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http://dx.doi.org/10.1089/thy.2020.0023DOI Listing
February 2021

Human Pluripotent Stem Cell-Derived Neural Cells and Brain Organoids Reveal SARS-CoV-2 Neurotropism.

bioRxiv 2020 Jul 28. Epub 2020 Jul 28.

Neurological complications are common in patients with COVID-19. While SARS-CoV-2, the causal pathogen of COVID-19, has been detected in some patient brains, its ability to infect brain cells and impact their function are not well understood, and experimental models using human brain cells are urgently needed. Here we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found modest numbers of infected neurons and astrocytes, but greater infection of choroid plexus epithelial cells. We optimized a protocol to generate choroid plexus organoids from hiPSCs, which revealed productive SARS-CoV-2 infection that leads to increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our results provide evidence for SARS-CoV-2 neurotropism and support use of hiPSC-derived brain organoids as a platform to investigate the cellular susceptibility, disease mechanisms, and treatment strategies for SARS-CoV-2 infection.
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http://dx.doi.org/10.1101/2020.07.28.225151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402032PMC
July 2020

Robust Hi-C Maps of Enhancer-Promoter Interactions Reveal the Function of Non-coding Genome in Neural Development and Diseases.

Mol Cell 2020 08 26;79(3):521-534.e15. Epub 2020 Jun 26.

Department of Genetics and Genome Sciences, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA; Department of Computer and Data Sciences, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44106, USA. Electronic address:

Genome-wide mapping of chromatin interactions at high resolution remains experimentally and computationally challenging. Here we used a low-input "easy Hi-C" protocol to map the 3D genome architecture in human neurogenesis and brain tissues and also demonstrated that a rigorous Hi-C bias-correction pipeline (HiCorr) can significantly improve the sensitivity and robustness of Hi-C loop identification at sub-TAD level, especially the enhancer-promoter (E-P) interactions. We used HiCorr to compare the high-resolution maps of chromatin interactions from 10 tissue or cell types with a focus on neurogenesis and brain tissues. We found that dynamic chromatin loops are better hallmarks for cellular differentiation than compartment switching. HiCorr allowed direct observation of cell-type- and differentiation-specific E-P aggregates spanning large neighborhoods, suggesting a mechanism that stabilizes enhancer contacts during development. Interestingly, we concluded that Hi-C loop outperforms eQTL in explaining neurological GWAS results, revealing a unique value of high-resolution 3D genome maps in elucidating the disease etiology.
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http://dx.doi.org/10.1016/j.molcel.2020.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415676PMC
August 2020

Using Two- and Three-Dimensional Human iPSC Culture Systems to Model Psychiatric Disorders.

Adv Neurobiol 2020 ;25:237-257

Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Psychiatric disorders are among the most challenging human diseases to understand at a mechanistic level due to the heterogeneity of symptoms within established diagnostic categories, the general absence of focal pathology, and the genetic complexity inherent in these mostly polygenic disorders. Each of these features presents unique challenges to disease modeling for biological discovery, drug development, or improved diagnostics. In addition, live human neural tissue has been largely inaccessible to experimentation, leaving gaps in our knowledge derived from animal models that cannot fully recapitulate the features of the disease, indirect measures of brain function in human patients, and from analyses of postmortem tissue that can be confounded by comorbid conditions and medication history.
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http://dx.doi.org/10.1007/978-3-030-45493-7_9DOI Listing
November 2020

Modeling neurological disorders using brain organoids.

Semin Cell Dev Biol 2021 03 17;111:4-14. Epub 2020 Jun 17.

Department of Neuroscience and Mahoney Institute for Neurosciences, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA; Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA; Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA; Department of Psychiatry, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA. Electronic address:

Neurological disorders are challenging to study given the complexity and species-specific features of the organ system. Brain organoids are three dimensional structured aggregates of neural tissue that are generated by self-organization and differentiation from pluripotent stem cells under optimized culture conditions. These brain organoids exhibit similar features of structural organization and cell type diversity as the developing human brain, creating opportunities to recapitulate disease phenotypes that are not otherwise accessible. Here we review the initial attempt in the field to apply brain organoid models for the study of many different types of human neurological disorders across a wide range of etiologies and pathophysiologies. Forthcoming advancements in both brain organoid technology as well as analytical methods have significant potentials to advance the understanding of neurological disorders and to uncover opportunities for meaningful therapeutic intervention.
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http://dx.doi.org/10.1016/j.semcdb.2020.05.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7738381PMC
March 2021

An anomalous bi-macroalgal bloom caused by Ulva and Sargassum seaweeds during spring to summer of 2017 in the western Yellow Sea, China.

Harmful Algae 2020 03 28;93:101760. Epub 2020 Feb 28.

Jiangsu Marine Fisheries Research Institute, Nantong, 226007, China.

An unprecedented bi-macroalgal bloom caused by Ulva prolifera and Sargassum horneri occurred from spring to summer of 2017 in the western Yellow Sea (YS) of China, where annual large-scale green tides have prevailed for a decade. The distinct genesis and blooming dynamics of the two seaweed species were detected and described. Unlike the consistent raft-origin of the floating Ulva biomass, the massive pelagic S. horneri was derived from multiple sources (residual seaweeds from the previous winter bloom and those drifting from offshore water in the south). The scale of the green tide in 2017 was found smaller than the previous four years. We then discussed a number of hypotheses attributing to this reduction, including reduced epiphytic green algae from aquaculture rafts and the influences of the massive pelagic S. horneri. However, further research is needed to identify the origin of the pelagic S. horneri in the western YS and any affiliations with the benthic populations, and to elucidate the interactions of this species with the annual green tides and the ensuing consequences.
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http://dx.doi.org/10.1016/j.hal.2020.101760DOI Listing
March 2020

Sliced Human Cortical Organoids for Modeling Distinct Cortical Layer Formation.

Cell Stem Cell 2020 05 5;26(5):766-781.e9. Epub 2020 Mar 5.

Department of Neuroscience and Mahoney Institute for Neurosciences, University of Pennsylvania, Philadelphia, PA 19104, USA; Neuroscience Graduate Program, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Psychiatry, University of Pennsylvania, Philadelphia, PA 19104, USA; Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Human brain organoids provide unique platforms for modeling development and diseases by recapitulating the architecture of the embryonic brain. However, current organoid methods are limited by interior hypoxia and cell death due to insufficient surface diffusion, preventing generation of architecture resembling late developmental stages. Here, we report the sliced neocortical organoid (SNO) system, which bypasses the diffusion limit to prevent cell death over long-term cultures. This method leads to sustained neurogenesis and formation of an expanded cortical plate that establishes distinct upper and deep cortical layers for neurons and astrocytes, resembling the third trimester embryonic human neocortex. Using the SNO system, we further identify a critical role of WNT/β-catenin signaling in regulating human cortical neuron subtype fate specification, which is disrupted by a psychiatric-disorder-associated genetic mutation in patient induced pluripotent stem cell (iPSC)-derived SNOs. These results demonstrate the utility of SNOs for investigating previously inaccessible human-specific, late-stage cortical development and disease-relevant mechanisms.
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http://dx.doi.org/10.1016/j.stem.2020.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7366517PMC
May 2020

Persistent Cyfip1 Expression Is Required to Maintain the Adult Subventricular Zone Neurogenic Niche.

J Neurosci 2020 03 27;40(10):2015-2024. Epub 2020 Jan 27.

Department of Neurology,

Neural stem cells (NSCs) persist throughout life in the subventricular zone (SVZ) neurogenic niche of the lateral ventricles as Type B1 cells in adult mice. Maintaining this population of NSCs depends on the balance between quiescence and self-renewing or self-depleting cell divisions. Interactions between B1 cells and the surrounding niche are important in regulating this balance, but the mechanisms governing these processes have not been fully elucidated. The cytoplasmic FMRP-interacting protein (Cyfip1) regulates apical-basal polarity in the embryonic brain. Loss of Cyfip1 during embryonic development in mice disrupts the embryonic niche and affects cortical neurogenesis. However, a direct role for Cyfip1 in the regulation of adult NSCs has not been established. Here, we demonstrate that Cyfip1 expression is preferentially localized to B1 cells in the adult mouse SVZ. Loss of in the embryonic mouse brain results in altered adult SVZ architecture and expansion of the adult B1 cell population at the ventricular surface. Furthermore, acute deletion of in adult NSCs results in a rapid change in adherens junction proteins as well as increased proliferation and number of B1 cells at the ventricular surface. Together, these data indicate that Cyfip1 plays a critical role in the formation and maintenance of the adult SVZ niche; furthermore, deletion of unleashes the capacity of adult B1 cells for symmetric renewal to increase the adult NSC pool. Neural stem cells (NSCs) persist in the subventricular zone of the lateral ventricles in adult mammals, and the size of this population is determined by the balance between quiescence and self-depleting or renewing cell division. The mechanisms regulating these processes are not fully understood. This study establishes that the cytoplasmic FMRP interacting protein 1 (Cyfip1) regulates NSC fate decisions in the adult subventricular zone and adult NSCs that are quiescent or typically undergo self-depleting divisions retain the ability to self-renew. These results contribute to our understanding of how adult NSCs are regulated throughout life and has potential implications for human brain disorders.
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http://dx.doi.org/10.1523/JNEUROSCI.2249-19.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7055134PMC
March 2020
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