189 research outputs found
Diet-derived microRNAs: unicorn or silver bullet?
No full textIn ancient lore, a bullet cast from silver is the only effective weapon against monsters. The uptake of active diet-derived microRNAs (miRNAs) in consumers may be the silver bullet long sought after in nutrition and oral therapeutics. However, the majority of scientists consider the transfer and regulation of consumer’s gene activity by these diet-derived miRNAs to be a fantasy akin to spotting a unicorn. Nevertheless, groups like Dr. Chen-Yu Zhang’s lab in Nanjing University have stockpiled breathtaking amounts of data to shoot down these naysayers. Meanwhile, Dr. Ken Witwer at John Hopkins has steadfastly cautioned the field to beware of fallacies caused by contamination, technical artifacts, and confirmation bias. Here, Dr. Witwer and Dr. Zhang share their realities of dietary miRNAs by answering five questions related to this controversial field
High-resolution characterization of HIV and extracellular vesicles by STORM-FISHing: combining dSTORM and smFISH
No full textOver the last two decades, advancements in optical microscopy have led to the development of a variety of super-resolution microscopy (SRM) methods, each with the capacity to examine subcellular structures at high resolution, including the virions of exogenous viruses such as human immunodeficiency virus (HIV) and host extracellular vesicles (EVs). Accurately identifying and characterizing complete virions within complex biological samples is essential for advancing our understanding of the structural diversity of virions and virus-like particles. However, few approaches take advantage of the detailed structural insights that SRM can offer to characterize free-floating virions and multiple types of molecules associated with them. In this study, we evaluate STORM-FISH, a dual-labeling technique that combines antibody-based HIV protein labeling and smFISH-based HIV RNA labeling with dSTORM for accurate, high-resolution characterization of HIV virions. Using smFISH probes specific to BaL HIV vRNA, we observed high specificity for virion labeling in dSTORM imaging, with minimal levels of binding in control samples (DCL4 plant probes and non-BaL HIV strains). While individual labeling of viral proteins (gp120) alone resulted in considerable non-specific binding to host-derived EVs, the combined use of gp120 antibodies and BaL smFISH probes allowed clear identification of complete BaL HIV virions, as demonstrated by distinct populations of double-positive (BaL smFISH+/gp120+) and triple-positive (BaL smFISH+/gp120+/CD63+) particles. Interestingly, the smFISH probes sometimes localized externally to gp120+ particles, raising questions about the origin of this potential external vRNA. The high-resolution imaging capabilities of STORM-FISH allow for detailed analysis of virion structure, protein localization, and component orientation, facilitating deeper insights into the structural dynamics of HIV. Our results validate STORM-FISH as an effective technique for dual protein/RNA labeling of free-floating virions in dSTORM microscopy and could translate well into furthering virological research and characterization of therapeutic extracellular vesicles or virus-mediated gene delivery systems
High-resolution characterization of HIV and extracellular vesicles by STORM-FISHing: combining dSTORM and smFISH
No full textOver the last two decades, advancements in optical microscopy have led to the development of a variety of super-resolution microscopy (SRM) methods, each with the capacity to examine subcellular structures at high resolution, including the virions of exogenous viruses such as human immunodeficiency virus (HIV) and host extracellular vesicles (EVs). Accurately identifying and characterizing complete virions within complex biological samples is essential for advancing our understanding of the structural diversity of virions and virus-like particles. However, few approaches take advantage of the detailed structural insights that SRM can offer to characterize free-floating virions and multiple types of molecules associated with them. In this study, we evaluate STORM-FISH, a dual-labeling technique that combines antibody-based HIV protein labeling and smFISH-based HIV RNA labeling with dSTORM for accurate, high-resolution characterization of HIV virions. Using smFISH probes specific to BaL HIV vRNA, we observed high specificity for virion labeling in dSTORM imaging, with minimal levels of binding in control samples (DCL4 plant probes and non-BaL HIV strains). While individual labeling of viral proteins (gp120) alone resulted in considerable non-specific binding to host-derived EVs, the combined use of gp120 antibodies and BaL smFISH probes allowed clear identification of complete BaL HIV virions, as demonstrated by distinct populations of double-positive (BaL smFISH+/gp120+) and triple-positive (BaL smFISH+/gp120+/CD63+) particles. Interestingly, the smFISH probes sometimes localized externally to gp120+ particles, raising questions about the origin of this potential external vRNA. The high-resolution imaging capabilities of STORM-FISH allow for detailed analysis of virion structure, protein localization, and component orientation, facilitating deeper insights into the structural dynamics of HIV. Our results validate STORM-FISH as an effective technique for dual protein/RNA labeling of free-floating virions in dSTORM microscopy and could translate well into furthering virological research and characterization of therapeutic extracellular vesicles or virus-mediated gene delivery systems
RNA Landscapes of Brain and Brain-Derived Extracellular Vesicles in Simian Immunodeficiency Virus Infection and Central Nervous System Pathology
No full textThis is a pre-copyedited, author-produced version of an article accepted for publication in The Journal of Infectious Diseases following peer review. The version of record Yiyao Huang, Ahmed Abdelgawad, Andrey Turchinovich, Suzanne Queen, Celina Monteiro Abreu, Xianming Zhu, Mona Batish, Lei Zheng, Kenneth W Witwer, RNA Landscapes of Brain and Brain-Derived Extracellular Vesicles in Simian Immunodeficiency Virus Infection and Central Nervous System Pathology, The Journal of Infectious Diseases, 2023;, jiad563, https://doi.org/10.1093/infdis/jiad563 is available online at: https://doi.org/10.1093/infdis/jiad563. © The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: [email protected]. This article will be embargoed until 12/11/2024.Background
Brain tissue-derived extracellular vesicles (bdEVs) act locally in the central nervous system (CNS) and may indicate molecular mechanisms in human immunodeficiency virus (HIV) CNS pathology. Using brain homogenate (BH) and bdEVs from a simian immunodeficiency virus (SIV) model of HIV disease, we identified RNA networks in SIV infection and neuroinflammation.
Methods
Postmortem occipital cortex samples were obtained from uninfected controls and SIV-infected subjects (acute and chronic phases with or without CNS pathology [SIV encephalitis]). bdEVs were separated and characterized per international consensus guidelines. RNAs from bdEVs and BH were sequenced and quantitative polymerase chain reaction (qPCR)-amplified to detect levels of small RNAs (sRNAs, including microRNAs [miRNAs]) and longer RNAs including messenger RNAs (mRNAs) and circular RNAs (circRNAs).
Results
Dysregulated RNAs in BH and bdEVs were identified in acute and chronic infection with pathology groups, including mRNAs, miRNAs, and circRNAs. Most dysregulated mRNAs in bdEVs reflected dysregulation in source BH. These mRNAs are disproportionately involved in inflammation and immune responses. Based on target prediction, several circRNAs that were differentially abundant in source tissue might be responsible for specific differences in sRNA levels in bdEVs during SIV infection.
Conclusions
RNA profiling of bdEVs and source tissues reveals potential regulatory networks in SIV infection and SIV-related CNS pathology.This work was supported by the National Institutes of Health (NIH), National Institute on Drug Abuse (grant numbers DA040385 and DA047807 to K. W. W.); by 2 pilot grants awarded to Y. H. through the Johns Hopkins National Institute of Mental Health (NIMH) Center (supported by National Institute of Mental Health MH075673) and the Johns Hopkins University Center for AIDS Research (supported by National Institutes of Health/National Institute of Allergy and Infectious Diseases P30AI094189-01A1); and the National Science Foundation (grant number 2244127 to M. B.). The Witwer laboratory is also supported in part by National Cancer Institute/Common Fund (grant number CA241694); National Institute of Allergy and Infectious Diseases (grant number AI144997); National Institute of Mental Health (NIMH) (grant number MH118164); and the Johns Hopkins University Richman Family Precision Medicine Center of Excellence in Alzheimer’s Disease. Samples used in this study were derived in part from research supported by National Institutes of Health (NIH) (grant number U42OD013117 to Johns Hopkins pigtailed macaque breeding colony); National Institute of Neurological Disorders and Stroke (grant number NS089482 to Joseph L. Mankowski); and National Institute of Mental Health (NIMH) (grant number MH070306 to Janice E. Clements)
Advances in Extracellular Vesicle Research Over the Past Decade: Source and Isolation Method are Connected with Cargo and Function
No full textThe evolution of extracellular vesicle (EV) research has introduced nanotechnology into biomedical cell communication science while recognizing what is formerly considered cell "dust" as constituting an entirely new universe of cell signaling particles. To display the global EV research landscape, a systematic review of 20 364 original research articles selected from all 40 684 EV-related records identified in PubMed 2013-2022 is performed. Machine-learning is used to categorize the high-dimensional data and further dissected significant associations between EV source, isolation method, cargo, and function. Unexpected correlations between these four categories indicate prevalent experimental strategies based on cargo connectivity with function of interest being associated with certain EV sources or isolation strategies. Conceptually relevant association of size-based EV isolation with protein cargo and uptake function will guide strategic conclusions enhancing future EV research and product development. Based on this study, an open-source database is built to facilitate further analysis with conventional or AI tools to identify additional causative associations of interest.A total of 20 364 original extracellular vesicle (EV) research articles for the decade 2013-2022 are analyzed for the presence or absence of 36 selected parameters in the four categories EV source, isolation, cargo, and function. The results are displayed in machine-learning-based 2D landscapes and further dissected by correlation analysis to identify conceptually relevant associations and draw strategic conclusions. imag
Witwer, K.W.; et al. Correction: miRNA Profiles of Monocyte-Lineage Cells Are Consistent with Complicated Roles in HIV-1 Restriction. Viruses 2012, 4, 1844-1864.
No full textA funding designation in our 2012 Viruses publication, doi: 10.3390/v4101844 (Viruses 2012, 4, 1844-1864), contained an incorrect digit. On page 1860, (Acknowledgments), U19 support was incorrectly listed as AI076113. The correct designation is AI096113
EXPLORING THE ROLE OF PTCH53: A HIGH CONFIDENCE P53 TARGET GENE
No full textThe tumor suppressor gene TP53 is mutated in over 50% of cancers. Despite being an object of study for decades, many of its target genes remain uncharacterized, particularly in non-cancer derived models. PTCH53, alternatively known as PTCHD4, is a recently identified target gene of p53. Induced to the same degree as the canonical p53 target gene CDKN1A upon induction by a variety of cellular stressors, PTCH53’s primary cellular function remains elusive. Sharing 25% homology with PTCH1, the canonical hedgehog receptor and tumor suppressor, PTCH53 is a member of the patched-domain-containing (PTCHD) family of proteins. Several PTCHD family members have been linked to non-neoplastic diseases, including autism spectrum disorder (ASD). Despite the relevance of these genes to human disease, the biochemical functions of their encoded proteins are poorly understood. Characterization of the PTCHD family and related proteins remains difficult due to limited reagents and the family’s complex 12-pass transmembrane structure.
In the studies described here, I present evidence that PTCH53 may play a role in cellular stress responses in concert with p53. The PTCH53 and TP53 loci were targeted in a human immortalized retinal pigment epithelial cell line known as hTERT-RPE1 by CRISPR/Cas9 gene editing. Cells with disrupted PTCH53 alleles showed increased p53 protein stabilization, both basally as well as upon induction with radiation, DNA damaging drugs, and a small molecule agonist of p53 activity. Interestingly, despite the increased levels of p53 protein present in these cells, p53 target gene induction was broadly muted. Furthermore, this muted transcriptional program allowed the cells to gain radioresistance, and a growth advantage within culture over the parental line, a phenotype also exhibited in T cells isolated from the Ptch53-/- mouse.
Additionally, I present preliminary data that suggest that PTCH53 deficiency causes the activation of the kynurenine pathway of tryptophan catabolism. Dysregulation of this immunomodulatory metabolic pathway has been implicated in a number of inflammatory diseases and cancers. Genetic ablation of PTCH53 in the hTERT-RPE1 cell line led to significant alterations in several pathway-related metabolites and related transcripts, suggesting a novel role for PTCH53 in the regulation of this critical pathway
PROFILING EXTRACELLULAR VESICLES IN CIGARETTE SMOKE-INDUCED BLOOD-BRAIN BARRIER DAMAGE AND ESTABLISHING AN INDUCED PLURIPOTENT STEM CELL VASCULAR MODEL
No full textCigarette smoke exposure adversely affects neurovascular health and is linked to disorders such as stroke, vascular dementia, and Alzheimer's disease. In this thesis, we use cigarette smoke extract (CSE) and cell culture models to assess the contributions of cigarette smoke exposure and extracellular vesicles (EVs) to blood-brain barrier (BBB) damage. Specifically, we investigate the viability and morphology of brain microvascular endothelial cells (BMECs) under exposure to different concentrations of CSE, as well as BBB marker proteins including tight junction marker ZO-1, cytoskeletal marker VE-cad, and glucose transporter GLUT-1. Additionally, EVs were collected from CSE-treated BMECs, characterized, and used to treat BMECs to gain insight into how EVs affect the BBB. Lastly, we establish a model of endothelial cell-pericyte interactions. Induced pluripotent stem cells (iPSCs) were differentiated into endothelial cells (CD31+) and pericytes (NG2+) and combined into a three-dimensional model. EVs from iPSC-derived endothelial cells (iECs) were then introduced into the model to investigate endothelial cell communication with pericytes
Alternative miRNAs? Human sequences misidentified as plant miRNAs in plant studies and in human plasma [version 1; referees: 2 approved]
No full textBackground: A 2017 study reported that “Plant miRNAs found in human circulating system provide evidences of cross kingdom RNAi”. Analysis of two human blood plasma sequencing datasets was said to provide evidence for uptake of plant miRNAs into human plasma. The results were also purportedly inconsistent with contamination. Methods: Sequences from public datasets and miRNA databases were compared with results downloaded from the website of the reporting journal. Results: Only one putative plant miRNA (“peu-MIR2910) mapped consistently above background, and this sequence is found with 100% identity in a human rRNA. Several other rarer but consistently mapped putative plant miRNAs also have 100% or near 100% matches to human transcripts or genomic sequences, and some do not appear to map to plant genomes at all. Conclusions: Reanalysis of public data suggests that dietary plant xenomiR uptake is not supported, but instead confirms previous findings that detection of rare plant miRNAs in mammalian sequencing datasets is artifactual. Some putative plant miRNAs, including MIR2910 and MIR2911, may represent human sequence contamination or other artifacts in plant studies, emphasizing the need for rigorous controls and data filtering strategies when assessing possible xenomiRNAs
Hypothetical Plant-Mammal Small RNA CommunicationCommunication : Packaging and Stoichiometry
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