1,720,960 research outputs found
Measuring pH in insulin secretory granules by phasor-based fluorescence lifetime imaging of a genetically encoded sensor
Get PDFIt is widely accepted that the pH of insulin granules is acidic, and that its active regulation during granule maturation plays a role in the process of insulin secretion by β-cells. Yet, a calibrated measurement of the absolute granule pH with organelle specificity is still lacking. To tackle this issue, we use the genetically encoded E1GFP pH reporter inserted into the C-peptide of proinsulin and expressed in Insulinoma 1E cells. Following verification of correct targeting of the E1GFP reporter in the insulin granules, phasor-based Fluorescence Lifetime Imaging Microscopy (FLIM) is applied to obtain a calibrated and probe-concentration-independent measurement of insulin-granule pH. Our results confirm the acidic nature of insulin granules under maintenance cell culture conditions, with an average luminal pH of ~5.8, and show that acidity is actively maintained, as evidenced by its near-neutralization upon treatment with the vacuolar H+-ATPase inhibitor concanamycin. Additionally, by ..
Spatiotemporal analysis of glucagon secretory granule dynamics
No full textThe secretion of insulin and glucagon by pancreatic β and α cells, respectively, is critical for glucose homeostasis. While the insulin granule dynamics is well-characterized, the intracellular behavior of glucagon secretory granules (GSG) remains poorly understood. Here, we analyze the mobility of GSGs in αTC1-9 cells and insulin secretory granules (ISG) in INS-1E cells using spatiotemporal correlation spectroscopy and single-particle tracking (SPT), with a focus on the role of the cytoskeleton in regulating their transport. Under basal conditions, SPT classification reveals that GSGs predominantly exhibit diffusive motion (57.6 ± 10%), with smaller fractions categorized as almost immobile (35.8 ± 10.6%) or drifted (6.6 ± 3%), closely resembling ISGs dynamics. By disrupting microtubules, we confirmed their role as active tracks for directed granule transport in both cell types. Upon exposure to their respective secretory stimuli—high glucose for β cells and low glucose for α cells—both granule populations underwent a comparable shift toward increased diffusive and drifted motions. Treatment with the actin depolymerizing agent Latrunculin-B reproduced this stimulatory effect in INS-1E cells but not in αTC1-9 cells, suggesting that despite their overall similarity in granule behavior under physiological conditions, α and β cells may rely on partially distinct mechanisms to engage the cytoskeletal network
Meccanismi molecolari coinvolti nella crescita indotta da stimoli meccanici
No full textI neuroni sono cellule altamente polarizzate, in grado di percepire e rispondere a stimoli chimici e a forze provenienti dall’ambiente esterno (esogene). Queste forze sono in grado di stimolare la crescita assonale. Un metodo per generare forze esogene è basato sull’uso di nanoparticelle magnetiche (MNP) di ossido di ferro, che vengono naturalmente internalizzate dai neuroni. È possibile, infatti, generare forze meccaniche su neuroni marcati con MNP attraverso l’applicazione di campi magnetici esterni. Con l’impiego di questa metodologia, ho studiato in neuroni ippocampali di topo le modifiche indotte a livello nucleare, locale e nella maturazione sinaptica dall’applicazione di forze meccaniche. A livello nucleare ho indagato le differenze morfologiche. Per i fenomeni locali (traduzione locale e trasporto assonale) ho studiato la relazione tra il trasporto dei granuli di RNA e gli endosomi tardivi, poiché studi in letteratura suggeriscono il ruolo di endosomi tardivi come delle piattaforme per la sintesi di nuove proteine. Dalle analisi condotte ho osservato un aumento significativo nella frequenza di co-localizzazioni nei neuroni soggetti a stretching meccanico. Infine, la maturazione sinaptica è stata valutata con diversi approcci e a diversi timepoint. I dati raccolti suggeriscono che lo stimolo meccanico acceleri il processo di maturazione sinaptica.
I risultati ottenuti aprono nuove interessanti strade nella comprensione dei meccanismi alla base della crescita assonale indotta da forze esogene suggerendo lo sviluppo di nuovi target terapeutici, utili per guidare e potenziare i fenomeni di rigenerazione nervosa a seguito di lesione o malattia
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
Get PDF“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
Get PDFWe 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
Unveiling nanoscale optical signatures of cytokine-induced β-cell dysfunction
Get PDFAbstract Pro-inflammatory cytokines contribute to β-cell failure in both Type-1 and Type-2 Diabetes. Data collected so far allowed to dissect the genomic, transcriptomic, proteomic and biochemical landscape underlying cytokine-induced β-cell progression through dysfunction. Yet, no report thus far complemented such molecular information with the direct optical nanoscopy of the β-cell subcellular environment. Here we tackle this issue in Insulinoma 1E (INS-1E) β-cells by label-free fluorescence lifetime imaging microscopy (FLIM) and fluorescence-based super resolution imaging by expansion microscopy (ExM). It is found that 24-h exposure to IL-1β and IFN-γ is associated with a neat modification of the FLIM signature of cell autofluorescence due to the increase of either enzyme-bound NAD(P)H molecules and of oxidized lipid species. At the same time, ExM-based direct imaging unveils neat alteration of mitochondrial morphology (i.e. ~ 80% increase of mitochondrial circularity), marked degranulation (i.e. ~ 40% loss of insulin granules, with mis-localization of the surviving pool), appearance of F-actin-positive membrane blebs and an hitherto unknown extensive fragmentation of the microtubules network (e.g. ~ 37% reduction in the number of branches). Reported observations provide an optical-microscopy framework to interpret the amount of molecular information collected so far on β-cell dysfunction and pave the way to future ex-vivo and in-vivo investigations
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe 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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