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Effetti del silenziamento e dell'inibizione della proteasi Lon sui mitocondri di cellule tumorali
No full textLon è una proteasi mitocondriale, ATP-dipendente, codificata nel nucleo, che interviene nel ripiegamento delle proteine, degrada le proteine ossidate o danneggiate, prevenendo il loro accumulo, e partecipa al mantenimento dei livelli del DNA mitocondriale (mtDNA). Lon è indotta da diversi fattori di stress e la sua attività ed espressione sono modulate in diverse patologie. Lon è up-regolata in diverse cellule tumorali umane, e la riduzione dell'espressione di questa proteina causa profonde alterazioni del proteoma mitocondriale e delle funzioni dell’organello, portando alla fine alla morte cellulare. L'attività enzimatica di Lon è indotta dall'ATP, e inibita dal triterpenoide 2-ciano-3,12-dioxooleana-1,9-dien-28-oico (CDDO), e dal suo derivato C-28 metil estere (CDDO-Me).
Al fine di chiarire il ruolo della proteasi Lon nelle trasformazione neoplastica, abbiamo analizzato gli effetti della down-regolazione di Lon in un modello cellulare di tumore al colon (cellule tumorali di colon RKO) in cui Lon è stata condizionatamente silenziata mediante RNA interference. Abbiamo studiato l'impatto della down-regolazione di Lon sull'apoptosi cellulare, sui livelli di mtDNA e mtRNA, sulla funzionalità e morfologia mitocondriale. Inoltre, abbiamo valutato gli effetti dell'inibizione farmacologica di Lon, usando CDDO o CDDO-Me. In particolare, abbiamo studiato gli effetti dei triterpenoidi sui mitocondri di tre linee cellulari di tumore (RKO, cellule umane di tumore al colon; HepG2, cellule umane di epatocarcinoma; MCF7, cellule umane di carcinoma mammario). Abbiamo analizzato diversi parametri, quali il potenziale di membrana, la massa, la morfologia, il contenuto ROS e l'attività proteolitica di Lon.
I mitocondri delle cellule in cui Lon è silenziato sono caratterizzate da bassi livelli di trascritti di mtDNA, minori livelli di alcune subunità dei complessi della fosforilazione ossidativa (il complesso I è risultato il più colpito), da una marcata riduzione del tasso di consumo di ossigeno, e dalla ridotta capacità sintetizzare l'ATP. Inoltre, sono stati osservati alti livelli di perossido d’idrogeno e di anione superossido mitocondriale. La rete mitocondriale è apparsa frammentata, con mitocondri eterogenei nella forma e nelle dimensioni, creste dilatate, vacuoli e inclusioni elettrondense. Inoltre, abbiamo osservato che CDDO e CDDO-Me sono potenti fattori di stress per i mitocondri nelle cellule tumorali, poiché inducono livelli più elevati di ROS e alterano la morfologia mitocondriale, mentre tale effetto è risultato molto meno marcato nelle cellule normali non trasformate. In particolare, queste molecole hanno causato una depolarizzazione della membrana mitocondriale e un'alterazione della massa mitocondriale, hanno influenzato i livelli di Lon e quelli di ACO2 e TFAM, due target dell'attività proteolitica di Lon, e hanno indotto apoptosi mediante la via intrinseca. La overespressione di Lon è apparsa in grado di ridurre la morte cellulare per apoptosi nelle cellule trattate con CDDO e CDDO-Me.
In conclusione, Lon può essere considerata una proteina che non induce tumorigenesi di per sé, ma è importante per la sopravvivenza delle cellule tumorali. La sua inibizione farmacologica potrebbe quindi rappresentare una nuova possibile strategia antitumorale.Lon is a nuclear-encoded, mitochondrial ATP-dependent protease that assists protein folding, degrades oxidized or damaged proteins preventing their accumulation, and participates in maintaining mitochondrial DNA (mtDNA) levels. It is induced by different stressors, and its expression and activity are modulated in different pathologies. Lon is up-regulated in several human cancer cells, and its down-regulation causes profound alterations of mitochondrial proteome and function, and cell death. Lon enzymatic activity is activated by ATP, and inhibited by the triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), or by its C-28 methyl ester derivative (CDDO-Me).
In order to clarify the role of Lon protease in cancer cells, we analyzed the effects of Lon down-regulation in RKO colon cancer cells, in which Lon could be conditionally silenced by RNA interference. We studied the impact of Lon down-regulation on cellular apoptosis, mtDNA and mtRNAs levels, mitochondrial functionality and mitochondrial morphology. Furthermore, we evaluated the effects of pharmacological inhibition of Lon, using CDDO or CDDO-Me, on mitochondria of three different cancer cell lines (RKO human colon cancer cells, HepG2 hepatocarcinoma cells and MCF7 breast carcinoma cells). We paid a particular attention to parameters such as membrane potential, mass, morphology, dynamics and ROS content, and Lon proteolytic activity.
Mitochondria of Lon-silenced cells displayed low levels of mtDNA transcripts, reduced levels of several subunits of oxidative phosphorylation complexes (Complex I being the most affected), a marked reduction of oxygen consumption rate, and impaired capability to synthetize ATP. Higher levels of hydrogen peroxide and mitochondrial superoxide anion could also be observed. Mitochondrial network appeared fragmented, with mitochondria heterogeneous in size and shape, dilated cristae, vacuoles and electrondense inclusions. In addition, we found that CDDO and CDDO-Me were potent stressors for mitochondria in cancer cells, rather than in normal non-transformed cells, being able to induce higher levels of ROS and altered mitochondrial morphology. In particular, these compounds caused depolarization of mitochondrial membrane, altered mitochondrial mass, affected the levels of Lon and those of Aco2 and TFAM, two targets of Lon proteolytic activity, and led to intrinsic apoptosis. The overexpression of Lon could reduce apoptotic of cells treated with CDDO and CDDO-Me.
In conclusion, Lon can be considered a protein that does not initiate tumourigenesis per se, but it is important for cancer cell survival. Its pharmacological inhibition could represent a possible new anticancer strategy
Audiogenic seizures selectively activate hippocampal neurons in young mice affected by fragile X syndrome
No full textYoung, but not adult, fragile X mental retardation gene (Fmr1) knockout (KO) mice display audiogenic seizures (AGS) that can be prevented by inhibiting extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation. In order to identify the cerebral regions involved in these phenomena, we characterized the response to AGS in Fmr1 KO mice and wild type (WT) controls at postnatal day (P) 45 and P90. To characterize the diverse response to AGS in various cerebral regions, we evaluated the activity markers FosB/ΔFosB and phosphorylated ERK1/2 (p-ERK1/2). Wild running (100% of tested mice) followed by clonic/tonic seizures (30%) were observed in P45 Fmr1 KO mice, but not in WT mice. In P90 Fmr1 KO mice, wild running was only present in 25% of tested animals. Basal FosB/ΔFosB immunoreactivity was higher (P < 0.01 vs. WT) in the CA1 and subiculum of P45 Fmr1 KO mice. Following the AGS test, FosB/ΔFosB expression consistently increased in most of the analyzed regions in both groups at P45, but not at P90. Interestingly, FosB/ΔFosB immunoreactivity was significantly higher in P45 Fmr1 KO mice in the medial geniculate body (P < 0.05 vs. WT) and CA3 (P < 0.01). Neurons presenting with immunopositivity to p-ERK1/2 were more abundant in the subiculum of Fmr1 KO mice in control condition (P < 0.05 vs. WT, in both age groups). In this region, p-ERK1/2-immunopositive cells significantly decreased (-75%, P < 0.01) in P90 Fmr1 KO mice exposed to the AGS test, but no changes were found in P45 mice or in other brain regions. In both age groups of WT mice, p-ERK1/2-immunopositive cells increased in the subiculum after exposure to the acoustic test. Our findings illustrate that FosB/ΔFosB markers are overexpressed in the medial geniculate body and CA3 in Fmr1 KO mice experiencing AGS, and that p-ERK1/2 is markedly decreased in the subiculum of Fmr1 KO mice resistant to AGS induction. These findings suggest that resilience to AGS is associated with dephosphorylation of p-ERK1/2 in the subiculum of mature Fmr1 KO mice
Resilience to audiogenic seizures is associated with p-ERK1/2 dephosphorylation in the subiculum of a mice model of Fragile X syndrome.
No full textYoung, but not adult, fragile X mental retardation gene (Fmr1) knockout (KO) mice display audiogenic seizures (AGS) that can be prevented by inhibiting extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation. In order to identify the cerebral regions involved in these phenomena, we characterized the response to AGS in Fmr1 KO mice and wild type (WT) controls at postnatal day (P) 45 and P90. To characterize the diverse response to AGS in various cerebral regions, we evaluated the activity markers FosB/ΔFosB and phosphorylated ERK1/2 (p-ERK1/2). Wild running (100% of tested mice) followed by clonic/tonic seizures (30%) were observed in P45 Fmr1 KO mice, but not in WT mice. In P90 Fmr1 KO mice, wild running was only present in 25% of tested animals. Basal FosB/ΔFosB immunoreactivity was higher (P < 0.01 vs. WT) in the CA1 and subiculum of P45 Fmr1 KO mice. Following the AGS test, FosB/ΔFosB expression consistently increased in most of the analyzed regions in both groups at P45, but not at P90. Interestingly, FosB/ΔFosB immunoreactivity was significantly higher in P45 Fmr1 KO mice in the medial geniculate body (P < 0.05 vs. WT) and CA3 (P < 0.01). Neurons presenting with immunopositivity to p-ERK1/2 were more abundant in the subiculum of Fmr1 KO mice in control condition (P < 0.05 vs. WT, in both age groups). In this region, p-ERK1/2-immunopositive cells significantly decreased (-75%, P < 0.01) in P90 Fmr1 KO mice exposed to the AGS test, but no changes were found in P45 mice or in other brain regions. In both age groups of WT mice, p-ERK1/2-immunopositive cells increased in the subiculum after exposure to the acoustic test. Our findings illustrate that FosB/ΔFosB markers are overexpressed in the medial geniculate body and CA3 in Fmr1 KO mice experiencing AGS, and that p-ERK1/2 is markedly decreased in the subiculum of Fmr1 KO mice resistant to AGS induction. These findings suggest that resilience to AGS is associated with dephosphorylation of p-ERK1/2 in the subiculum of mature Fmr1 KO mic
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
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
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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