1,720,962 research outputs found
Oligomerization of ZFYVE27 (Protrudin) is necessary to promote neurite extension.
No full textZFYVE27 (Protrudin) was originally identified as an interacting partner of spastin, which is most frequently mutated in hereditary spastic paraplegia. ZFYVE27 is a novel member of FYVE family, which is implicated in the formation of neurite extensions by promoting directional membrane trafficking in neurons. Now, through a yeast two-hybrid screen, we have identified that ZFYVE27 interacts with itself and the core interaction region resides within the third hydrophobic region (HR3) of the protein. We confirmed the ZFYVE27's self-interaction in the mammalian cells by co-immunoprecipitation and co-localization studies. To decipher the oligomeric nature of ZFYVE27, we performed sucrose gradient centrifugation and showed that ZFYVE27 oligomerizes into dimer/tetramer forms. Sub-cellular fractionation and Triton X-114 membrane phase separation analysis indicated that ZFYVE27 is a peripheral membrane protein. Furthermore, ZFYVE27 also binds to phosphatidylinositol 3-phosphate lipid moiety. Interestingly, cells expressing ZFYVE27(ΔHR3) failed to produce protrusions instead caused swelling of cell soma. When ZFYVE27(ΔHR3) was co-expressed with wild-type ZFYVE27 (ZFYVE27(WT)), it exerted a dominant negative effect on ZFYVE27(WT) as the cells co-expressing both proteins were also unable to induce protrusions and showed cytoplasmic swelling. Altogether, it is evident that a functionally active form of oligomer is crucial for ZFYVE27 ability to promote neurite extensions
Studying synaptopathies using Mecp2 transgenic mouse models
Get PDFThe MECP2 gene is located in the Xq28, which encodes for methyl-CpG binding protein 2 (MeCP2). Mutations in MECP2 gene is the primary cause of postnatal neurodevelopmental disorder, Rett syndrome (RTT) (loss-of-function). In contrast the over expression of MECP2 leads to duplication syndrome (gain-of-function). In both cases, gain and loss-of function of MECP2 leads to neurological disorders.
In the first part of the thesis, we attempted to characterize the pathomechanism associated with human R270X mutation in MECP2, which is one of the most frequent mutation in RTT cohorts. The R270X mutation is located within the transcriptional repression domain- nuclear localization signals (TRD-NLS) of MeCP2 protein. This nonsense mutation exhibits severe phenotype with higher mortality rate as compared to the other mutations in RTT cohorts. To evaluate the molecular and functional role of R270X mutation, we generated a Mecp2R270_EGFP transgenic mouse by BAC recombineering technology, which is equivalent to human R270X mutation. The Mecp2R270_EGFP transgenic mouse showed similar pattern of expression as compared to the endogenous MeCP2. Further, we bred the Mecp2R270_EGFP transgenic male mouse with Mecp2 knockout female to obtain Mecp2R270 knockin (KI) mouse. Phenotypical evaluation revealed no neurological impairment in Mecp2R270_EGFP transgenic mice, but KI mice were positive for hindlimb clasping. In addition, we observed no difference in neurite length, area of soma and spine density between wildtype and Mecp2R270_EGFP neurons, but detected reduced neurite length, soma size and spine number in KI neurons. From our experimental data, we conclude that MeCP2R270_EGFP protein has a deleterious effect on neuronal morphology and alters the spine density.
In the second part of the thesis, we generated a Mecp2 transgenic mouse model to determine the MECP2 dosage effect on duplication syndrome. The generated Mecp2WT_EGFP transgenic mice mildly overexpressed MeCP2 (~1.5X) (MeCP2 transgenic with endogenous) From our extensive behavioral analyzes, we observed increased aggressiveness and higher seizures in Mecp2WT_EGFP transgenic mice propensity after treatment with epileptogenic compound pentylenetetrazole (PTZ). Furthermore, induction of Mecp2WT_EGFP transgenic cultured neurons with PTZ caused increase calcium amplitude with a higher frequency. Additionally, the ex vivo and in vivo evaluation of neuronal parameters of hippocampal neurons revealed an increased area of soma, reduced tertiary branching sites and increased spine density in Mecp2WT_EGFP transgenic mouse. Collectively, our results suggest that mild MeCP2 overexpression in mice leads to epileptic seizures as a first symptom. Furthermore, precise MeCP2 dosage is necessary for proper neurodevelopment in mice.
In the third part of the thesis, we reported that the MeCP2 expression is not restricted only in the neurons but also expressed in astrocytes. Recent studies highlighted the role of MeCP2 in astrocytes. The co-cultured assay system with Mecp2 null astrocytes together with wild type neurons displayed shorter and small number of dendrites. These findings suggested the unknown secreted factor(s) from Mecp2 deficient astrocytes cause toxic effect to the neighboring neurons in non-cell-autonomous manner. In order to determine and characterize the MeCP2 targets in astrocytes, we performed proteomic analysis and characterized ~5000 different proteins in astrocytes with three independent technical repeats. Our proteomic analysis revealed 69 up-regulated and 29 down-regulated proteins in knockin (KI) and 50 up-regulated and 32 down-regulated proteins in knockout (KO), when compared to wildtype astocytes. The identified Mecp2 target proteins from proteomic analysis revealed that MeCP2 is involved in cell adhesions through desmosomal proteins (Dsp and Pkp1), regulation of reactive oxygen species (ROS) (Txn2) and glutamate homeostasis via Slc25a18 in astrocytes. We could validate the differential expression of Mecp2 target proteins such as Dsp and Pkp1 in Mecp2 knockout astrocytes compared to wild type astrocytes through Western blot analysis. After validating the proteomic data, in future an ex vivo assay can be established to test the effect of non-cell autonomous astrocytes factors identified through proteomics approach. Establishing the role of glial dysfunction in RTT pathogenesis could provide a new avenue for understanding RTT and will assist us in developing novel therapy for this disease
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
Mild overexpression of Mecp2 in mice causes a higher susceptibility toward seizures
No full textAn intriguing finding about the gene encoding methyl-CpG binding protein 2 (MeCP2) is that the loss-of-function mutations cause Rett syndrome and duplication (gain-of-function) of MECP2 leads to another neurological disorder termed MECP2 duplication syndrome. To ensure proper neurodevelopment, a precise regulation of MeCP2 expression is critical, and any gain or loss of MeCP2 over a narrow threshold level may lead to postnatal neurological impairment. To evaluate MeCP2 dosage effects, we generated Mecp2(WT_EGFP) transgenic (TG) mouse in which MeCP2 (endogenous plus TG) is mildly overexpressed (approximately 1.5×). The TG MeCP2(WT_EGFP) fusion protein is functionally active, as cross breeding of these mice with Mecp2 knockout mice led to alleviation of major phenotypes in the null mutant mice, including premature lethality. To characterize the Mecp2(WT_EGFP) mouse model, we performed an extensive battery of behavioral tests, which revealed that these mice manifest increased aggressiveness and higher pentylenetetrazole (PTZ)-induced seizure propensity. Evaluation of neuronal parameters revealed a reduction in the number of tertiary branching sites and increased spine density in Mecp2(WT_EGFP) transgenic (TG) neurons. Treatment of TG neurons with epileptogenic compound-PTZ led to a marked increase in amplitude and frequency of calcium spikes. Based on our ex vivo and in vivo data, we conclude that epileptic seizures are manifested as the first symptom when MeCP2 is mildly overexpressed in mice
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
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