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Modulazione allosterica del recettore dell'ormone follicolo-stimolante (FSHR) mediata da VHH
No full textL’ormone follicolo-stimolante (FSH) è una gonadotropina che svolge un ruolo fondamentale nella riproduzione umana e fa parte dell'asse endocrino ipotalamo-ipofisi-gonadi. Il recettore di FSH (FSHR) è accoppiato a proteina G (GPCR) ed espresso in cellule della granulosa ovariche durante la fase follicolare del ciclo mestruale. FSHR media l’attivazione di una complessa rete di vie di trasduzione del segnale che modulano la steroidogenesi, segnali mitogenici e apoptosi. Tra queste, l’adenosina monofosfato ciclica (cAMP)/proteina chinasi A (PKA) e il trafficking del recettore mediato dalle β-arrestine sono tra le pathway più studiate. I contraccettivi ormonali contengono progestinici ed estrogeni, e contrastano la produzione di FSH. Tuttavia, tali molecole possono indurre effetti collaterali indesiderati.
In questo studio, vengono caratterizzati cinque nanoanticorpi (VHH) anti-FSHR per valutarne la capacità di modulare allostericamente il recettore. Tre di questi VHH (A) bersagliano lo stesso epitopo localizzato sul recettore e sono stati prodotti in tre formati strutturali differenti, a seconda della presenza del frammento cristallizabile (Fc) e della regione “cerniera” (Hinge). Essi sono denominati VHH A, VHH A-Fc e VHH A-Hinge-Fc. Altri due (B e C) bersagliano epitopi differenti e sono stati prodotti nel formato VHH B-Hinge-Fc e VHH C-Hinge-Fc. I VHH potrebbero agire da modulatori allosterici positivi (PAM), negativi (NAM) o biased (BAM), con lo scopo di bloccare i segnali proliferativi cAMP- e β-arrestina 2-dipendenti, senza tuttavia impattare sulla steroidogenesi. Un prodotto avente tali caratteristiche dovrebbe essere in grado di bloccare l’ovulazione senza dare effetti indesiderati a livello metabolico.
Gli esperimenti sono stati condotti utilizzando la linea cellulare tumorale della granulosa umana (KGN) e cellule primarie umane della granulosa luteinizzate (hGLC). Le cellule sono state trattate con concentrazioni crescenti di FSH, in presenza o meno di diverse dosi di nanoanticorpo. Utilizzando metodiche quali Bioluminescence Resonance Energy Transfer (BRET), Homogeneous Time Resolved Fluorescence (HTRF), Western blotting e saggio con 3-[4,5-dimetiltiazol-2-il]-2,5-difeniltetrazolio (MTT), ho valutato l'accumulo intracellulare di cAMP, il reclutamento di β-arrestina 2, la produzione di estradiolo (E2) e progesterone (P4), il clivaggio della pro-caspasi 3 e la vitalità cellulare.
Nessuno dei VHH ha ridotto la vitalità cellulare in cellule KGN o hGLC, né ha indotto apoptosi mediata da caspasi 3 da cellule KGN. Inoltre, in cellule KGN, VHH A, VHH A-Fc e VHH A-Hinge-Fc hanno modulato la produzione intracellulare di cAMP mediata da FSH. VHH A-Fc ha agito come NAM, mentre gli altri due nanoanticorpi hanno funzionato come PAM. Al contrario, l'impatto sull'accumulo di cAMP nelle hGLC è stato meno prominente rispetto alla linea cellulare KGN. VHH B-Hinge-Fc e VHH C-Hinge-Fc hanno agito entrambi come NAM, in tutti i modelli cellulari utilizzati. In particolare, VHH B-Hinge-Fc è risultato essere il più efficace rispetto a tutti i nanoanticorpi testati. Solo VHH A-Fc ha ridotto il legame tra FSHR e β-arrestina 2, agendo come NAM sull'interazione della molecola con il recettore. I risultati ottenuti in hGLC dimostrano che nessuno degli anticorpi ha impattato la produzione di E2 e P4. Infine, la specificità di legame di VHH B-Hinge-Fc è stata testata in tessuto ovarico umano, mediante metodica di immunofluorescenza, confermando l'espressione del recettore in cellule della granulosa.
In conclusione, i risultati evidenziano che i nanoanticorpi testati potrebbero fornire nuove opportunità farmacologiche per lo sviluppo di contraccettivi non ormonali, agendo da modulatori allosterici BAM di FSHR.Follicle-stimulating hormone (FSH) is a gonadotropin that plays a pivotal role in human reproduction and belongs to the hypothalamic-pituitary-gonadal endocrine axis. The FSH receptor (FSHR) is a G protein-coupled receptor (GPCR) expressed in ovarian granulosa cells during the follicular phase of menstrual cycle. FSHR mediates the activation of a complex network of signaling pathways that modulate steroidogenesis, mitogenic signals, and apoptosis. Among these, cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and receptor trafficking mediated by β-arrestins are among the most studied pathways. Hormonal contraceptives contain progestins and estrogens, and they counteract FSH production. However, such molecules may induce undesirable side effects.
In this study, five anti-FSHR nanobodies (VHHs) were characterized to evaluate their ability to allosterically modulate the receptor. Three of these VHHs (A) target the same epitope localized on the receptor and were produced in three different structural formats, depending on the presence of the crystallizable fragment (Fc) and the hinge region. They are named VHH A, VHH A-Fc, and VHH A-Hinge-Fc. Two others (B and C) target different epitopes and were produced in the VHH B-Hinge-Fc and VHH C-Hinge-Fc formats. The VHHs may act as positive (PAM), negative (NAM), or biased (BAM) allosteric modulators, aiming to block cAMP- and β-arrestin 2-dependent proliferative signals without impacting steroidogenesis. A product with these characteristics should be able to block ovulation without leading to undesired metabolic effects.
Experiments were performed using the human granulosa tumor cell line (KGN) and primary human granulosa lutein cells (hGLC). Cells were treated with increasing concentrations of FSH, with or without various doses of nanobody. Using techniques such as bioluminescence resonance energy transfer (BRET), homogeneous time-resolved fluorescence (HTRF), Western blotting, and the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT) assay, I evaluated intracellular cAMP accumulation, β-arrestin 2 recruitment, estradiol (E2) and progesterone (P4) production, pro-caspase 3 cleavage, and cell viability.
None of the VHHs reduced cell viability in KGN or hGLC cells, nor did they induce caspase 3-mediated apoptosis in KGN cells. Moreover, in KGN cells, VHH A, VHH A-Fc, and VHH A-Hinge-Fc modulated FSH-mediated intracellular cAMP production. VHH A-Fc acted as a NAM, while the other two nanobodies functioned as PAMs. In contrast, the impact on cAMP accumulation in hGLC was less prominent compared to the KGN cell line. VHH B-Hinge-Fc and VHH C-Hinge-Fc both acted as NAMs in all cell models used. Notably, VHH B-Hinge-Fc proved to be the most effective among all nanobodies tested. Only VHH A-Fc reduced the binding between FSHR and β-arrestin 2, acting as a NAM on the interaction between the molecule and the receptor. The results obtained in hGLC demonstrated that none of the antibodies impacted E2 and P4 production. Finally, the binding specificity of VHH B-Hinge-Fc was tested in human ovarian tissue using immunofluorescence, confirming receptor expression in granulosa cells.
In conclusion, the results highlight that the tested nanobodies could provide new pharmacological opportunities for the development of non-hormonal contraceptives, acting as BAM allosteric modulators of FSHR
In vitro analysis of FSHR internalization and signalling mediated by two different FSH preparations
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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
Luteinizing hormone (LH)- and choriogonadotropin (hCG)-induced internalization of the receptor (LHCGR) is responsible for hormone-specific signaling
No full textVariations 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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