1,721,046 research outputs found
High-resolution characterization of HIV and extracellular vesicles by STORM-FISHing: combining dSTORM and smFISH
Full text linkOver 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
Full text linkOver 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
Advances in Extracellular Vesicle Research Over the Past Decade: Source and Isolation Method are Connected with Cargo and Function
Full text linkThe 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
EXPLORING THE ROLE OF PTCH53: A HIGH CONFIDENCE P53 TARGET GENE
Full text linkThe 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
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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