1,720,966 research outputs found
Epiretinal membranes: cell receptors involved in cell migration and their interaction with the extracellular matrix
No full textEpiretinal membranes (ERMs) are fibro-cellular membranes that form at the vitreoretinal interface, potentially causing vision impairment, particularly when they exhibit a contractile behavior, leading to macular puckers. They are classified in idiopathic (iERM) and secondary membranes and are known to be composed by different cell types and extracellular matrix (ECM). Numerous studies have characterized ERM cells identifying Müller cells, astrocytes, retinal pigmented epithelium (RPE) cells, hyalocytes and myofibroblasts. In the ECM, the presence of collagen protein family and of some matrix associated proteins such as fibrillin and tenascin has been reported. Nevertheless, there is still a lack of information on ERM membrane composition and formation, i.e. ECM components such as proteoglycans (PGs) and glycosaminoglycans (GAGs) and on how the cells migrate to reach the vitreoretinal interface. These are the two aspects on which this thesis work is focused on. In a first bioinformatics analysis on previously reported iERM-associated proteins, we identified cluster of differentiation 44 (CD44) as a central molecule in ERM development. Since CD44 is reported to interact with the glycoprotein podoplanin (PDPN), we added this protein to our network; PDPN entered the net mainly thanks to transcription factors (TFs), among which we selected the ones involved in fibrotic, inflammatory or trans-differentiation processes. These TFs were SRY-box transcription factor 2 (SOX2), yes associated protein 1 (YAP1) and signal transducer and activator of transcription 3 (STAT3), that are also all linked to high mobility group box 1 (HMGB1), a protein itself involved in inflammatory response. To test if these bioinformatic-highlighted proteins were present in ERMs, we collected 45 ERM samples from patients undergoing surgery. We performed both immunofluorescence and real time PCR (RT-qPCR) experiments and determined that PDPN and CD44 are expressed in all the samples, regardless of their idiopathic or secondary nature. Moreover, regardless of their glial, epithelial or mesenchymal origin, all cell types showed PDPN and CD44 expression in the plasma membrane. We also detected SOX2, YAP1 and STAT3 mainly located in cells cytoplasm and faintly in the nuclei. Since CD44 is a hyaluronic acid (HA) receptor we investigated HA localization and distribution in ERMs samples, together with some PGs that were previously reported and detected in proteomic analysis, namely decorin (DCN), perlecan (PLC), collagen XVIII/endostatin and agrin (AGR). We found PLC and collagen XVIII in all the sample tested with a reticular or lamellar distribution, often co-localizing with collagen IV and laminin. DCN and PRELP were detected in almost all the samples preferentially co-localizing each other and avoiding PLC+/collagen XVIII+ regions. AGR was the least represented PG (50%) and formed basement membrane-like structures, preferentially co-localizing with collagen XVIII. We also investigated these PGs in ERM associated internal limiting membranes (ILMs), comparing them with a “reference ILM sample” of a 23 years old patient without ERM. ILMs stained positively for PLC and collagen XVIII extensively on the retinal side, and a thin rim of fluorescence was also detectable on the vitreal side. AGR displayed a diffuse immunofluorescence extending also in ILM bulk. HA was detected in all the samples and preferentially did not co-localize with PGs, showing an immunostaining also in the cytosol of ERM cells. Since we detected CD44 and HA and HA is one of the constituents of the eye vitreous, we decided to investigate HA effects on Müller cells, which represent one cell type found in ERMs. For this reason, we stimulated a human immortalized Müller cell line, MIO-M1, with 10ng/ml of HA, alone or in combination with 10ng/ml of TGFβ2, which is a cytokine involved in ERM formation. We then investigated by RT-qPCR experiments the expression of genes that could uncover a trans-differentiation process of MIO-M1 cells toward the mesenchymal phenotype associated with ECM production. Results showed that TGFβ2 and TGFβ2/HA treatments cause a significant increase in mRNA levels of mesenchymal markers (ACTA2, TAGLN), along with a significant decrease in nervous/glial markers (GFAP, NES), indicating mesenchymal transition. In agreement, we found an increase in mRNA levels of different ECM components, namely collagen I e IV, XVIII and fibronectin. MIO-M1 stimulation caused also PDPN up-regulation and CD44 down-regulation. Given their localization on the plasma membrane and their already known role in cell migration in other contexts, we investigated if their expression could be related to migration necessary for cell reaching the vitreoretinal interface and for ERM formation. To this end we performed a scratch test on MIO-M1 cell line in presence of HA and TGFβ2 and we found that HA accelerated MIO-M1 migration rate. In conclusion, we demonstrated the presence of PDPN and CD44 in ERM samples, regardless of their idiopathic or secondary nature. We observed that the ECM of ERM is composed of different types of PGs, which tend to form two co-localizing groups: PLC and collagen XVIII on one side, and PRELP and DCN on the other; AGR instead showed a basement-membrane localization. Furthermore, we demonstrated the widespread presence of HA in ERM, which appeared also able to enhance the migration rate of the MIO-M1 cell line. PDPN and CD44 were localized in the cytoplasmic protrusions of MIO-M1 cells, suggesting that these cells may utilize these two molecules in the migration process. However, under the influence of TGFβ2, we observed a reduction of the MIO-M1 migration rate, the acquisition of mesenchymal markers, an increase in PDPN and a decrease in CD44 mRNA levels, leading to the hypothesis that under TGFβ2 influence the cells, already migrated, begin to produce ECM proteins resulting in ERM formation. Although some questions remain unresolved, this work shed light on the composition of the ERMs ECM and on potential molecules involved in the complex mechanisms underlying the formation of these membranes
Radiological anatomy of the trochlear spine and associated bony structures around the superior oblique tendon: a CT-based study
Full text linkThe superior oblique muscle tendon is known to bend in the anterior orbit around a cartilaginous trochlea. The site where the tendon bends is frequently interested by the presence of a small depression, the trochlear fovea, and/or the trochlear spine. Exact topography, size and frequency of these items are still undetermined. For this purpose, we studied 120 orbits of individuals that underwent computed tomography for pathologies not involving the anterior orbit. We detected the presence of the trochlear spine in 10% of orbits and we determined its location and size. We also observed the presence of two tubercles (TT1 and TT2), with distinct positions relative to the tendon. TT1 was present in 5% of orbits and was located on the same spot of the spine differing from the latter only for its morphology. TT2 lied in a more advanced position and it was rarer (1,67% of orbits). The spine and the first type of tubercle were located above and behind the tendon reflection; the second tubercle lied below and ahead of the tendon reflection. A distinct trochlear fovea was detected in 25.83% of orbits and lied 3.42 ± 0.97 mm behind the orbital rim. Fovea diameters were 4.16 ± 1.08 mm × 3.84 ± 0.97 mm. In conclusion we demonstrate that in the anterior orbit a bony process is present in at least 15% or orbits. It is a note of interest for strabismus surgery when it is necessary to intervene on the superior oblique muscle or when it is needed access to the medial orbital wall
Preclinical Application of Computer-Aided High-Frequency Ultrasound (HFUS) Imaging: A Preliminary Report on the In Vivo Characterization of Hepatic Steatosis Progression in Mouse Models
No full text: Metabolic dysfunction-associated steatotic liver disease (MASLD) is one of the most common chronic liver disorders worldwide and can lead to inflammation, fibrosis, and liver cancer. To better understand the impact of an unbalanced hypercaloric diet on liver phenotype in impaired autophagy, the study compared C57BL/6J wild type (WT) and MAPK15-ERK8 knockout (KO) male mice with C57BL/6J background fed for 17 weeks with "Western-type" (WD) or standard diet (SD). Liver features were monitored in vivo by high-frequency ultrasound (HFUS) using a semi-quantitative and parametric assessment of pathological changes in the parenchyma complemented by computer-aided diagnosis (CAD) methods. Liver histology was considered the reference standard. WD induced liver steatosis in both genotypes, although KO mice showed more pronounced dietary effects than WT mice. Overall, HFUS reliably detected steatosis-related parenchymal changes over time in the two mouse genotypes examined, consistent with histology. Furthermore, this study demonstrated the feasibility of extracting quantitative features from conventional B-mode ultrasound images of the liver in murine models at early clinical stages of MASLD using a computationally efficient and vendor-independent CAD method. This approach may contribute to the non-invasive characterization of genetically engineered mouse models of MASLD according to the principles of replacement, reduction, and refinement (3Rs), with interesting translational implications
Alteration of Immunoregulatory Patterns and Survival Advantage of Key Cell Types in Food Allergic Children
Full text linkAll allergic responses to food indicate the failure of immunological tolerance, but it is unclear why cow’s milk and egg (CME) allergies resolve more readily than reactivity to peanuts (PN). We sought to identify differences between PN and CME allergies through constitutive immune status and responses to cognate and non-cognate food antigens. Children with confirmed allergy to CME (n = 6) and PN (n = 18) and non-allergic (NA) (n = 8) controls were studied. Constitutive secretion of cytokines was tested in plasma and unstimulated mononuclear cell (PBMNC) cultures. Blood dendritic cell (DC) subsets were analyzed alongside changes in phenotypes and soluble molecules in allergen-stimulated MNC cultures with or without cytokine neutralization. We observed that in allergic children, constitutively high plasma levels IL-1β, IL-2, IL-4, IL-5 and IL-10 but less IL-12p70 than in non-allergic children was accompanied by the spontaneous secretion of sCD23, IL-1β, IL-2, IL-4, IL-5, IL-10, IL-12p70, IFN-γ and TNF-α in MNC cultures. Furthermore, blood DC subset counts differed in food allergy. Antigen-presenting cell phenotypic abnormalities were accompanied by higher B and T cell percentages with more Bcl-2 within CD69+ subsets. Cells were generally refractory to antigenic stimulation in vitro, but IL-4 neutralization led to CD152 downregulation by CD4+ T cells from PN allergic children responding to PN allergens. Canonical discriminant analyses segregated non-allergic and allergic children by their cytokine secretion patterns, revealing differences and areas of overlap between PN and CME allergies. Despite an absence of recent allergen exposure, indication of in vivo activation, in vitro responses independent of challenging antigen and the presence of unusual costimulatory molecules suggest dysregulated immunity in food allergy. Most importantly, higher Bcl-2 content within key effector cells implies survival advantage with the potential to mount abnormal responses that may give rise to the manifestations of allergy. Here, we put forward the hypothesis that the lack of apoptosis of key immune cell types might be central to the development of food allergic reactions
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
Development and validation of derivatization-based LC-MS/MS method for quantification of short-chain fatty acids in human, rat, and mouse plasma
No full textShort-chain fatty acids (SCFAs), the end products of gut microbial fermentation of dietary fibers and non-digestible polysaccharides, act as a link between the microbiome, immune system, and inflammatory processes. The importance of accurately quantifying SCFAs in plasma has recently emerged to understand their biological role. In this work, a sensitive and reproducible LC-MS/MS
method is reported for SCFAs quantification in three different matrices such as human, rat and mouse plasma via derivatization, using as derivatizing agent O-benzylhydroxylamine (O-BHA), coupled with liquid-liquid extraction. First, the instrumental parameters of the mass spectrometer and then the
chromatographic conditions were optimized using previously SCFAs derivatives synthetized and used as standards. After that, the best conditions for derivatization and extraction from plasma were studied and a series of determinations were performed on human, rat, and mouse plasma aliquots to validate the overall method (derivatization, extraction, and LC-MS/MS determination). The method showed good performance in terms of recovery (> 80%), precision (RSD <14%), accuracy (RE <±10%) and sensitivity (LOQ of 0.01 μM for acetic, butyric, propionic and isobutyric acid) in all plasma samples. The method thus developed and validated was applied to the quantification of major
SCFAs in adult and aged mice, germ-free mice and in germ-free recipient mice subjected to faecal transplant from adult and aged donors. Results highlighted how plasma concentrations of SCFAs are correlated with age further highlighting the importance of developing a method that is reliable for the quantification of SCFAs to study their biological rol
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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