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Characterisation of mitochondria in urothelial cells of the urinary bladder in vivo and in vitro
No full textUrotelijske celice so epitelijske celice, ki pokrivajo urogenitalni trakt od ledvičnih kotanj do proksimalne sečnice. V procesu diferenciacije se v površinskih urotelijskih celicah in vivo (t.i. dežnikaste celice) sintetizirajo transmembranski proteini uroplakini, ki se uredijo v urotelijske plake in skupaj s tesnimi stiki predstavljajo molekularno osnovo krvno-urinske pregrade sečnega mehurja. V biosintezni poti urotelijskih plakov sodelujeta endoplazemski retikulum in Golgijev aparat, vloga mitohondrijev v urotelijskih celicah pa je slabo raziskana. Raziskave nakazujejo spremembe v mitohondrijih zaradi patoloških stanj urotelija, vključeni pa naj bi bili tudi v alternativno razgradnjo uroplakinov. Mitohondriji v različno diferenciranih normalnih in spremenjenih urotelijskih celicah še niso bili proučeni. Namen dela je bil proučiti izražanje in razporeditev mitohondrijskih proteinov v modelnih sistemih različno diferenciranih urotelijskih celic in vivo in in vitro. Z uporabo celičnobioloških (prenos western, imunooznačevanje) ter mikroskopskih metod smo ugotovili, da se izražanje in razporeditev mitohondrijskega proteina Tom20 razlikuje med različnimi kulturami urotelijskih celic in vitro in celicami urotelija miši in vivo. V kulturah celic smo opazili, da se mitohondriji razporejajo v tri značilne vzorce, poimenovane fragmentirana, tubularna in mrežasta razporeditev. V kulturi celic neinvazivnega urotelijskega papiloma (RT4) smo opazili večinsko fragmentirano razporeditev mitohondrijev, v kulturi mišično invazivnih rakavih urotelijskih celic (T24) večinsko mrežasto razporeditev mitohondrijev, v kulturi delno diferenciranih normalnih prašičjih urotelijskih celic (PD NPU) večinsko mrežasto in delno tubularno razporeditev mitohondrijev in v kulturi visoko diferenciranih normalnih prašičjih urotelijskih celic (HD NPU) večinsko mrežasto razporeditev mitohondrijev. V nadaljevanju smo primerjali razporeditev mitohondrijev in označevalcev diferenciacije urotelijskih celic ter ugotovili, da razporeditev mitohondrijev korelira s stopnjo diferenciranosti urotelijskih celic. Nižje diferencirane celice (RT4) so imele fragmentirane mitohondrije, višje diferencirane pa mrežaste (PD NPU,
HD NPU). Raziskali smo tudi prisotnost in razporeditev proteinov mitohondrijske dinamike (mitofuzija – MFN2, mitofisija – DRP1) in mitofagije (PARK2, PINK, LC3). V nizko diferenciranih celicah RT4 s fragmentiranimi mitohondriji je bilo prisotnega več proteina DRP1 v primerjavi z MFN2, medtem ko je bilo v višje diferenciranih celicah PD NPU in HD NPU z mrežasto razporejenimi mitohondriji ravno obratno. Proteini mitofagije so mestoma sovpadali z razporeditvijo mitohondrijskih proteinov. Rezultati raziskovalnega dela predstavljajo prve raziskave mitohondrijev v urotelijskih celicah in vitro in in vivo ter nakazujejo vpletenost proteinov mitohondrijske dinamike in mitofagije na razporeditev mitohondrijev v različnih stopnjah diferenciranosti urotelijskih celic. Hkrati pa raziskava odpira nova vprašanja o pomenu različnih razporeditev mitohondrijev v urotelijskih celicah in o morebitni specifični vlogi mitohondrijev v njih.Urothelial cells are epithelial cells that line the urogenital tract from renal pelvis to proximal urethra. During differentiation, the superficial urothelial cells in vivo (called umbrella cells) synthesize transmembrane proteins uroplakins, which are organised in urothelial plaques and, together with the tight junctions, form the molecular basis of the blood-urine barrier of the urinary bladder. It is known that the endoplasmic reticulum and the Golgi apparatus are involved in the biosynthetic pathway of urothelial plaques, however the role of mitochondria in urothelial cells is poorly understood. Studies suggest that mitochondria are altered by pathological conditions and are also involved in the alternative degradation of uroplakins. Mitochondria in differentiated normal and altered urothelial cells have not yet been investigated. The aim of this study was to analyse the expression and distribution of mitochondrial proteins in urothelial cells in vivo and in vitro at different stages of differentiation. Using cell-biology (western blotting, immunolabelling) and microscopic methods, we found that expression and distribution of the mitochondrial protein Tom20 differs between different cultured urothelial cells in vitro and mouse urothelial cells in vivo. In cell cultures, we observed that mitochondria were distributed in three characteristic patterns, called fragmented, tubular and network-like distribution. In the culture of non-invasive urothelial papilloma cells (RT4) we observed a predominantly fragmented mitochondrial distribution, in the culture of muscle-invasive urothelial cancer cells (T24) a predominantly network-like distribution, in the culture of partially differentiated normal porcine urothelial cells (PD NPU) predominantly a network-like and partially tubular distribution of mitochondria and in the culture of highly differentiated normal porcine urothelial cells
(HD NPU) predominantly a network-like distribution of mitochondria. We also compared mitochondrial distribution with markers of urothelial cell differentiation and found that mitochondrial distribution correlates with the degree of urothelial cell differentiation. Low differentiated cells (RT4) had a fragmented mitochondrial distribution, whereas partially differentiated cells showed a network-like distribution (PD NPU, HD NPU). We also analysed the presence and distribution of proteins involved in mitochondrial dynamics (mitofusion – MFN2, mitofission – DRP1) and mitophagy (PARK2, PINK, LC3). In low differentiated RT4 cells with fragmented mitochondrial distribution, there was more DRP1 compared to MFN2, whereas the opposite was true in partially differentiated NPU cell cultures with network-like distribution of mitochondria. The mitophagy proteins occasionally overlapped with the distribution of mitochondrial proteins. Results of this study present the first steps in the research of mitochondria in urothelial cells in vitro and in vivo and suggest that mitochondrial dynamics and mitophagy proteins are involved in the distribution of mitochondria at different stages of urothelial cell differentiation. The study also raises new questions about the significance of mitochondrial distribution in urothelial cells and their possible urothelium-specific role
Encapsulation of doxorubicin prodrug in heat-triggered liposomes overcomes off-target activation for advanced prostate cancer therapy
No full textL-377,202 prodrug consists of doxorubicin (Dox) conjugated to a prostate-specific antigen (PSA) peptide substrate that can be cleaved by enzymatically active PSA at the tumor site. Despite the initial promise in phase I trial, further testing of L-377,202 (herein called Dox-PSA) was ceased due to some degree of non-specific activation and toxicity concerns. To improve safety of Dox-PSA, we encapsulated it into low temperature-sensitive liposomes (LTSL) to bypass systemic activation, while maintaining its biological activity upon controlled release in response to mild hyperthermia (HT). A time-dependent accumulation of activated prodrug in the nuclei of PSA-expressing cells exposed to mild HT was observed, showing that Dox-PSA was efficiently released from the LTSL, cleaved by PSA and entering the cell nucleus as free Dox. Furthermore, we have shown that Dox-PSA loading in LTSL can block its biological activity at 37°C, while the combination with mild HT resulted in augmented cytotoxicity in both 2D and 3D PC models compared to the free Dox-PSA. More importantly, Dox-PSA encapsulation in LTSL prolonged its blood circulation and reduced Dox accumulation in the heart of C4-2B tumor-bearing mice over the free Dox-PSA, thus significantly improving Dox-PSA therapeutic window. Finally, Dox-PSA-loaded LTSL combined with HT significantly delayed tumor growth at a similar rate as mice treated with free Dox-PSA in both solid and metastatic PC tumor models. This indicates this strategy could block the systemic cleavage of Dox-PSA without reducing its efficacy in vivo, which could represent a safer option to treat patients with locally advanced PC. STATEMENT OF SIGNIFICANCE: This study investigates a new tactic to tackle non-specific cleavage of doxorubicin PSA-activatable prodrug (L-377,202) to treat advanced prostate cancer. In the present study, we report a nanoparticle-based approach to overcome the non-specific activation of L-377,202 in the systemic circulation. This includes encapsulating Dox-PSA in low temperature-sensitive liposomes to prevent its premature hydrolysis and non-specific cleavage. This class of liposomes offers payload protection against degradation in plasma, improved pharmacokinetics and tumor targeting, and an efficient and controlled drug release triggered by mild hyperthermia (HT) (∼42°C). We believe that this strategy holds great promise in bypassing any systemic toxicity concerns that could arise from the premature activation of the prodrug whilst simultaneously being able to control the spatiotemporal context of Dox-PSA cleavage and metabolism
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
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
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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