1,720,972 research outputs found
Combined ion conductance and fluorescence confocal microscopy for biological cell membrane transport studies
No full textOptical visualization of nanoscale morphological changes taking place in living biological cells during such important processes as endo- and exocytosis is challenging due to the low refractive index of lipid membranes. In this paper we summarize and discuss advances in the powerful combination of two complementary live imaging techniques, ion conductance and fluorescence confocal microscopy, that allows cell membrane topography to be related with molecular-specific fluorescence at high spatial and temporal resolution. We demonstrate the feasibility of the use of ion conductance microscopy to image apical plasma membrane of mouse embryo trophoblast outgrowth cells at a resolution sufficient to depict single endocytic pits. This opens the possibility to study individual endocytic events in embryo trophoblast outgrowth cells where endocytosis plays a crucial role during early stages of embryo development
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
Characterisation of impairments in endocytosis induced by Centronuclear myopathy and Charcot-Marie-Tooth peripheral neuropathy
Get PDFDynamin-2 is a ubiquitous mechano-GTPase that is involved in various secretory pathway
stages. Dynamin-2 scissions budding vesicles from the plasma membrane during endocytosis
and is additionally implicated in a wide range of cellular processes. It is also seen in the
development of new vesicles from the Golgi network, vesicle trafficking, fusion processes, and
microtubule regulation as well as actin cytoskeleton dynamics. Two inherited neuromuscular
diseases have been linked with more than 20 mutations found in the dynamin-2 gene over the
past 20 years in Charcot–Marie–Tooth neuropathy and centronuclear myopathy in a tissue
specific manner. The majority of these mutations are bundled in the pleckstrin homology
domain. However, there are no specific mutations correlated with both diseases, indicating
they have different effects on the role of dynamin-2 in various tissues. I am trying to investigate
how the mutations associated with the disease affect the function of Dynamin-2, during vesicle
trafficking and endocytic processes. Current research has characterised the biochemical
properties of DNM2 mutants, but none of these properties have been linked to cell function.
However, this research cannot explain contradictory findings made using fluorescently labelled
cargo uptake. The development of correlative Scanning Ion Conductance Microscopy
combined with laser fluorescent confocal microscopy have allowed for live cell topographical
imaging directly correlated with fluorescently labelled cargo or proteins. We have used this
method to investigate the effect dynamin-2 mutations have on clathrin mediated endocytosis
and caveolae mediated endocytosis.
Formation of calthrin coated pits, their scission and internalisation are dependent on dynamin
2. Transmission electron microscopy was used to characterise the impact of dynamin 2
mutations on populations of calthrin coated pits and their distributions on the cell membrane
and the different stages of clathrin mediated endocytosis. Fibroblasts were selected and
surveyed along the cell membrane to characterize clathrin coated pits which have a distinct
coat and are less than 200nm in diameter were analyzed, p.R465W-DNM2 fibroblasts, control
human skin fibroblasts and p.R522H-DNM2 fibroblasts were examined. A substantial reduction
in the number of mature, omega and internalized pits were observed in p.R465W mutant cells
when compared with healthy controls. This indicated that the mutation in dynamin 2 for this
cell line may affect the pit formation process. Overall fewer clathrin coated pits were observed
in both mutant cell lines compared with the controls. Also since both mutant cell lines have
different mutations, the counts between these mutant cell lines was calculated and it was
found that there was a significant difference observed between the number of fully
internalized coated pits between p.R465W and p.R522H suggesting that the mutation in
p.R522H may not suppress vesicle formation in this cell line.
Furthermore, qPCR experimentations were carried out to ascertain the level of RNA expression
between the cell lines. This showed there was greater expression of dynamin 2 in p.R522H
compared with control cells whilst there was a lower expression of dynamin 2 in p.S619L
compared to control. Overall despite the mutations, DNM2 is expressed, therefore it could be
the function that is being affected. In conjunction with this, western blot was carried to
ascertain the level of caveolin and clathrin protein found in the mutant cells compared with
the control. As this could explain the difference in the number of clathrin coated pits observed
between the controls and the mutant cells. It was found that there was more clathrin and
caveolin in the mutant cell lines.
In order to ascertain the effect dynamin 2 mutations, have on cargo sequestration, a bulk uptake assay was carried out. Transferrin and cholera toxin B were used as uptake agents, there was a lower uptake of cholera toxin B in mutant cell lines compared with the control cell.
This suggests that the although caveolin protein is being produced and dynamin 2 RNA is also
being expressed the cargo is not being endocytosed, this could be because the caveolae
structures are docked at the cell membrane as seen in the TEM and are unable to endocytose
cargo effectively. Therefore, cholera toxin B could be up taken by an alternative mechanism.
While lower uptake of transferrin by p.R465W and more so p.R522H however in TEM we
observe fewer clathrin coated pits for p.R465W than p.R522H, this also suggests that some
transferrin uptake maybe taking place by an alternative mechanism.
One such alternative mechanism could be the uptake through membrane ruffles, which also
utilise dynamin. Dynamin is known to directly interact with actin and modulate actin dynamics,
one of the key components which make up the cytoskeleton. However, it has not been well
characterised if mutations in DNM2 alter the surface morphology. Therefore, experiments
were carried out to measure the roughness of the cell membranes as well as cell stiffness by
SICM in cell lines from healthy subject and individuals with DNM2 mutations, for which
preliminary data is being obtained.
In this study I have shown that there is an adverse effect on clathrin coated pit lifetime and
depth when mutations are present in dynamin-2. Scanning ion conductance microscopy was
used to visualise the clathrin mediated endocytosis pit formation. COS-7 cell lines and human
skin fibroblasts (controls, p.R465W, p.R522H, p.S619L) were transfected with lipofectamine
(life technologies) 24 hours prior to scanning, in dishes with >80% cell confluence.
The characterisation of mutant human skin fibroblasts have shown there is a difference in pit
visualisation, maturation, progression and internalisation when compared with the wild type.
In conclusion it has been established that mutations in dynamin 2 affect clathrin coated pit
formation adversely. As well as reducing the amount of cargo that can be taken up by cells.
Mutations in dynamin 2 also affect caveolae endocytosis as the pits become arrested at the
cell membrane.Open Acces
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
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
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Development of scanning ion conductance microscopy for biomedical applications
No full textEThOS - Electronic Theses Online ServiceGBUnited Kingdo
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