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Le protestantisme libéral est un courant libéral du protestantisme qui met l'accent sur la lecture critique des textes bibliques et « le souci de se « libérer » des contraintes du dogme et de l'institution, ainsi que des pesanteurs sociologiques qui, fatalement, ont tendance à les figer et à les rigidifier ».

Il naît de la lignée théologique qui part de Friedrich Schleiermacher (1768-1834) et Adolf von Harnack (1851-1930), et en particulier de la révolution exégétique du , où Albrecht Ritschl (1822-1889) et Ernst Troeltsch (1865-1923) s'illustrèrent dans le renouvellement de la lecture de la Bible.

Parmi ses représentants en France, on compte Charles Wagner, Wilfred Monod, André Gounelle, l'Oratoire du Louvre ou le Foyer de l'Âme.

Origines
Les écrits de plusieurs théologiens ont préparé le mouvement libéral
 Heinrich Paulus pour Das Leben Jesu als Grundlage einer reinen Geschichte des Urchristentums, en 1828.
 David Strauss Le Christ de la foi et le Jésus de l'histoire, en 1865
 Ferdinand Christian Baur pour Kritische Untersuchungen über die kanonischen Evangelien, ihr Verhältniss zu einander, ihren Charakter und Ursprung, en 1847

La liberté d'expression naît à la suite de la publication des ouvrages signalés ci-dessus mais dont les sources intègrent des courants de pensée autochtones issus de la philosophie des Lumières et de l'Encyclopédie, et parfois, remontant à la Réforme.

Caractéristiques

En se distinguant par sa volonté de sortir des dogmes et de la lecture littérale des textes, le protestantisme libéral critique les régulations orthodoxes des croyances et des pratiques, les appareils ecclésiastiques et leur pouvoir normatif. Il confesse volontiers l'universalité du salut du fait d'une perception plutôt optimiste de l'homme et de la civilisation.

La Bible est considérée comme pouvant être relue et interprétée en fonction des époques ; le protestantisme libéral s'est ainsi démarqué par son soutien à la bénédiction des unions homosexuelles ou au droit à l'avortement.

Il soutient le dialogue interreligieux et du pluralisme, ainsi que du dialogue de la religion avec la culture. Ses représentants sont généralement impliqués dans l'œcuménisme, avec une ouverture aux autres confessions, y compris dans les cultes.

Son rejet du dogmatisme et de son imposition par des institutions en fait aussi un défenseur de la laïcité : l'une des premières théories de la séparation de l'Église et de l'État fut formulée dans le livre de Sébastien Castellion Contre le Libelle de Monsieur Calvin puis reformulée par Alexandre Vinet quand il était en Belgique. Il a été en France un des artisans de la loi de 1905, avec Wilfred Monod, Athanase Coquerel, tandis que Félix Pécaut, Charles Wagner et Ferdinand Buisson intervenaient dans la construction de l'école laïque.

Par pays

En Suisse

Ésaïe Gasc (1748-1813)
Ésaïe Gasc est genevois. Nommé professeur à la faculté de théologie protestante de Montauban en 1809, il prend ses fonctions en 1810. Il appartient à la première génération de professeurs de la Faculté de théologie protestante de Montauban , ouverte sur décision de Napoléon en 1809. Sa réfutation du dogme trinitaire provoquent des controverses, il est attaqué par des pasteurs protestants du Midi, et par Daniel Encontre, qui prendra sa succession à Montauban. .

Jean-Jacques Caton Chenevière (1783-1871)

 six Essais théologiques (1831) : le premier de ces Essais s'en prenait au « système théologique de la trinité ». Le quatrième portait l'un sur la notion de rédemption, ce qui était l'occasion de remettre en cause le dogme de la divinité de Jésus. Voir l'article de Marc Chenevière dans le colloque Genève protestante en 1831

Théologiens et pasteurs :

 Henry Babel (1923-2019)
 Laurent Gagnebin
 Pierre-Olivier Léchot
 Bernard Reymond
 Charles Rittmeyer (1918-2002)

En France

 Albert Schweitzer (1875-1965) conteste l'omnipotence et l'omniscience de Jésus.

 Wilfred Monod (1867-1937)
 André-Numa Bertrand (1876-1946)
 Albert Réville
 Jean Réville
 Georges Marchal
 Élie Lauriol

La laïcité
Des personnalités telles que Ferdinand Buisson et Félix Pécaut ont joué un rôle central par leur volonté de créer une religion moderne, laïque, certes d'inspiration protestante, mais ouverte à toutes les voies spirituelles.

En Belgique

 Temple protestant de Liège-Marcellis
 L'Église protestante de Bruxelles (Chapelle royale)
 L'Eglise protestante de Verviers-Hodimont
 L'Eglise protestante luthérienne du Pays d'Arlon (congrégation reconnue par la FLM)

En Grande-Bretagne

 le débat autour Honest to God de John Arthur Thomas Robinson, évêque de Woolwitch
 le débat autour du Guide to debate about God de David Edward Jenkins

Aux États-Unis

Le débat (entre autres) autour du théisme par l'évêque épiscopalien J. S. Spong et ses nombreux ouvrages, dont : Pour un christianisme d'avenir (Traduction de l'américain aux éditions Karthala 2019).

Notes et références

Bibliographie
 .
 Patrick Cabanel, Le Dieu de la République.
 .
 Félix Pécaut. De l'avenir du théisme chrétien considéré comme religion, rééd. Théolib 2007
 Pierre-Jean Ruff, Le protestantisme libéral, Nouvelle édition : Théolib 2005.
 Pierre-Yves Ruff, "Le Parti-pris du Livre".
 Charles Wagner, "l'Évangile et la Vie".
 Marie-Claire Weber-Lefeuvre, Étude des Évangiles. Suivi de Les Évangiles et l'écologie, L'Harmattan 2006 - collection « Chrétiens autrement ».
 Marie-Claire Weber-Lefeuvre, Interroger sa foi. Du calvinisme au judéo-christianisme libéral, Préface de P. J. Ruff., Edilivre 2013 - 261 p.-
 .

Pour approfondir

Articles connexes
 Libéralisme théologique
 Théologie du process de Alfred North Whitehead et John B. Cobb
 Évangile et Liberté, mensuel francophone du protestantisme libéral.
 Jean-Marie de Bourqueney
 Gilles Castelnau
 Sébastien Castellion
 John B. Cobb
 Laurent Gagnebin
 André Gounelle
 Charles Hartshorne
 John Hick
 Pierre-Olivier Léchot
 Raphaël Picon, théologien libéral
 Bernard Reymond
 Charles Rittmeyer réduit les Évangiles aux paroles de Jésus et constate leur similitude avec les conceptions des francs-maçons (en Suisse)
 John Arthur Thomas Robinson
 Louis-Auguste Sabatier
 Friedrich Schleiermacher
 John Shelby Spong
 Paul Tillich
 Ernst Troeltsch
 Adolph von Harnack
 Charles Wagner
 James Woody

Bibliographie complémentaire
 Le protestantisme libéral : une théologie aux limites, dossier de la revue ThéoRèmes, 8 | 2016.
 Le protestantisme libéral par André Gounelle in Évangile et Liberté


Aaron Klug (natus Želvae in Lituania die 11 Augusti 1926; mortuus die 20 Novembris 2018) fuit chemicus et physicus biologiae, in Africa Australi apud universitates Witwatersrand et Civitatis Capitis moxque in Anglia in Collegio Trinitatis apud Cantabrigienses eruditus. Klug anno 1982 Nobelianum Chemiae Praemium accepit studii crystallographiae microscopii electrici. Klug est ab anno 1969 sodalis Regalis Societatis Londinii, cuius praeses (1995-2000) etiam fuerat.
Novissima mutatio die 22 Novembris 2018 hora 14:52 facta.


text:
Kazimierz Mitera (ur. 16 listopada 1897 w Skołyszynie, zm. 17 listopada 1936 w Krakowie) – polski malarz, pedagog i krytyk sztuki.

Życiorys
Urodził się jako syn nauczycieli Adolfa i Walerii z domu Hnatowicz. Po wybuchu I wojny światowej wstąpił do Legionów Polskich w 1914 i służył w szeregach 1 pułku ułanów Legionów Polskich. Od 1915 był żołnierzem armii austriackiej. Podczas mobilizacji w listopadzie 1918 wstąpił do Batalionu Akademickiego i walczył w obronie Lwowa i Przemyśla. Następnie został członkiem załogi pociągu pancernego "Smok", z którą uczestniczył w walkach pod Borysławiem i Lwowem. 21 lutego 1919 został pojmany i uwięziony przez Ukraińców, został uwolniony po dwóch miesiącach. Został przeniesiony do batalionu kolejowego z którym brał udział w wojnie polsko-bolszewickiej, a po zakończeniu działań wojennych uczestniczył w odbudowie linii kolejowych na Kresach Wschodnich. Działał również w organizacjach zajmujących się repatriacją Polaków z Syberii i centralnej Rosji. Po 1922 studiował równocześnie na krakowskiej Akademii Sztuk Pięknych i na Wydziale Historii Sztuki Uniwersytetu Jagiellońskiego. Był współzałożycielem Związku Zawodowego Polskich Artystów Plastyków, piastował funkcję redaktora naczelnego "Głosu Plastyków", w którym publikował również felietony własnego autorstwa. Pochłonięty działalnością społeczną nie uczestniczył w zbyt wielu wystawach, jego dorobek artystyczny stanowią nieliczne pejzaże, portrety i martwe natury, ale także wystrój malarski kościoła św. Katarzyny w Toruniu. Zmarł w wielu 39 lat w Krakowie, spoczywa na Cmentarzu Podgórskim (kw. XXXV, rząd płn., msc. 11).

Jego braćmi byli Stanisław (1890-1915, doktor historii, harcerz i nauczyciel, poległy w bitwie pod Kostiuchnówką, pośmiertnie odznaczony Orderem Virtuti Militari) i Zygmunt (1903-1940, doktor inżynier geofizyki stosowanej oficer Wojska Polskiego, ofiara zbrodni katyńskiej.

Przypisy

Bibliografia
 Wacława Milewska, Maria Zientara "Sztuka Legionów Polskich i jej twórcy, 1914-1918" Wydawnictwo "Arkady" 1999

Linki zewnętrzne

 Muzeum Historii Fotografii, Zygmunt Grodziński, Kazimierz Mitera, Bolesław Skarżyński, Jan Zimowski 20.03 - 31.05.1999 Krakowianie - uczestnicy wojny polsko-bolszewickiej 1919-1920
 Prosto z Mostu : tygodnik literacko-artystyczny / [red. i wyd. Stanisław Piasecki. R.2 (1936) nr 48]

Absolwenci Uniwersytetu Jagiellońskiego
Absolwenci Akademii Sztuk Pięknych im. Jana Matejki w Krakowie
Ludzie urodzeni w Skołyszynie
Orlęta lwowskie (1918–1919)
Pochowani na Nowym Cmentarzu Podgórskim
Polscy malarze
Uczestnicy wojny polsko-bolszewickiej (strona polska)
Urodzeni w 1897
Załogi polskich pociągów pancernych w wojnie polsko-ukraińskiej
Zmarli w 1936
Żołnierze I Brygady Legionów Polskich

redpajama_set_name: RedPajamaWikipedia


Diabetic kidney disease (DKD) is associated with oxidative stress and mitochondrial injury. Myo-inositol oxygenase (MIOX), a tubular-specific enzyme, modulates redox imbalance and apoptosis in tubular cells in diabetes, but these mechanisms remain unclear. We investigated the role of MIOX in perturbation of mitochondrial quality control, including mitochondrial dynamics and autophagy/mitophagy, under high-glucose (HG) ambience or a diabetic state. HK-2 or LLC-PK1 cells subjected to HG exhibited an upregulation of MIOX accompanied by mitochondrial fragmentation and depolarization, inhibition of autophagy/mitophagy, and altered expression of mitochondrial dynamic and mitophagic proteins. Furthermore, dysfunctional mitochondria accumulated in the cytoplasm, which coincided with increased reactive oxygen species generation, Bax activation, cytochrome C release, and apoptosis. Overexpression of MIOX in LLC-PK1 cells enhanced the effects of HG, whereas MIOX siRNA or d-glucarate, an inhibitor of MIOX, partially reversed these perturbations. Moreover, decreasing the expression of MIOX under HG ambience increased PTEN-induced putative kinase 1 expression and the dependent mitofusin-2–Parkin interaction. In tubules of diabetic mice, increased MIOX expression and mitochondrial fragmentation and defective autophagy were observed. Dietary supplementation of d-glucarate in diabetic mice decreased MIOX expression, attenuated tubular damage, and improved renal functions. Notably, d-glucarate administration also partially attenuated mitochondrial fragmentation, oxidative stress, and apoptosis and restored autophagy/mitophagy in the tubular cells of these mice. These results suggest a novel mechanism linking MIOX to impaired mitochondrial quality control during tubular injury in the pathogenesis of DKD and suggest d-glucarate as a potential therapeutic agent for the amelioration of DKD.
Mitochondria are dynamic organelles that constantly undergo fusion and fission cycles to maintain a balance of cellular reticular network and mitochondrial turnover.10 Their dynamics are regulated by profission proteins (Drp1 and Fis1) and profusion mediators (Mfn1/2 and OPA1). During this process, the stress-induced dysfunctional mitochondria undergo fragmentation and membrane depolarization, and then, they are engulfed and degraded by the autophagosomes—a process known as mitophagy.11 In the mitochondrial lifecycle, mitochondrial fusion, mitochondrial fission, and mitophagy form an integrated quality control axis that prevents damaged mitochondria from accumulating within the cell, generation of reactive oxygen species (ROS), buildup of mutated mtDNA, and release of apoptogenic proteins (i.e., Cytochrome C). Thus, the cell avoids undergoing oxidative stress, mitochondrial outer membrane (MOM) permeabilization, and apoptosis, and such quality control maintains a population of healthy mitochondria.12,13 In kidneys, because of high energy demand in the mitochondrial-enriched tubular compartment, it is likely that mitochondrial dysfunctions in the diabetic state could contribute to cell injury, proteinuria, and loss of renal functions.4,14 However, the mechanism of mitochondrial damage and the dynamic regulation in states of hyperglycemia in tubular pathobiology are not clearly defined and remain to be investigated.
Myo-inositol oxygenase (MIOX) is a tubular-specific enzyme that catabolizes myo-inositol to d-glucuronate, which enters into the pentose phosphate pathway.15,16 Other than its role to channel glucose intermediaries, studies have indicated upregulation of MIOX and its relevance in the pathogenesis of tubular injury in DKD.15,16 Previously, we had shown that the upregulation of MIOX during hyperglycemia could modulate redox imbalance by transcriptional mechanisms and also, conceivably, induce mitochondrial-dependent apoptosis, which inevitably may lead to tubulointerstitial injury and fibrosis.16–18 With respect to the amelioration of such an injury, d-glucaric acid, an anticancer drug, may serve as a potential pharmacologic inhibitor of MIOX.19 Interestingly, a derivative of this drug is believed to have a renoprotective effect in DKD by a yet to be defined mechanism.20 Also, the mechanism by which MIOX induces mitochondrial dysfunctions during tubular injury and whether its pharmacologic inhibition ameliorates DKD are unknown.
In this study, we showed the effect of MIOX on mitochondrial quality control in tubules in DKD and highlighted the mechanism by which MIOX perturbs the mitochondrial homeostasis. Activated MIOX during hyperglycemia induces mitochondrial fragmentation and depolarization but meanwhile, inhibits autophagic removal of damaged mitochondria. The surveillance of quality control was noted to be disrupted, and accumulated dysfunctional organelles generated excessive ROS and initiated apoptotic cascade, leading to tubular injury. Importantly, inhibition of MIOX by d-glucarate normalized regulation of the mitochondrial lifecycle, and thus, it could serve as a potential therapeutic agent for the amelioration of tubulopathy in DKD.
Confocal microscopy delineated mitochondrial morphology in HK2 cells after Mitotracker red staining. Under low-glucose (LG; 5 mM) ambience, mitochondria exhibited a filamentous shape, whereas they transformed into short rods after high-glucose (HG; 30 mM) treatment for 24 hours (Figure 1A, a). Immunoblot analyses revealed upregulation of the mitochondrial profission proteins Drp1 and Fis1, whereas the MOM profusion protein Mfn2 was downregulated. Interestingly, expression of OPA1, a protein regulating fusion of the mitochondrial inner membrane, was not obviously altered under HG ambience (Figure 1B). LC3 was used as an autophagy marker and the immunofluorescence performed by using LC3 antibody yielded punctate green fluorescence demarcating the autophagosomes (Figure 1A, b). After HG treatment, the number of autophagosomes as well as their intensity of fluorescence were reduced (Figure 1A, b). During autophagy, LC3-I is known to convert to LC3-II.21 Expression of converted LC3-II was decreased under HG ambience by immunoblot analyses, but no significant change was seen in Autophagy-Related Gene 5 (Atg5) (Figure 1C). In addition, expression of mitophagy-related proteins PTEN-induced putative kinase 1 (Pink1) and Parkin was decreased (Figure 1C). Cytosolic and mitochondrial ROS generations were increased notably by HG treatment, and they are indicated by H2-DCFDA (Figure 1A, green) and MitoSox (Figure 1A, red) staining, respectively (Figure 1A, c and d). An increase in apoptosis was also observed in cells subjected to HG, which was assessed by the terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick end-labeling (TUNEL) procedure (Figure 1A, e). The apoptosis was accompanied with upregulation of Bax and Cytochrome C (Figure 1D). Notably, the level of the tubular-specific enzyme MIOX was increased under HG ambience (Figure 1, A, f and D). Quantitative analyses confirmed that HG induced mitochondrial fragmentation in a time-dependent manner, concurring with increased intracellular ROS production and apoptosis after HG treatment (Figure 1, E, G, and H). Autophagy was marginally increased initially, but it was reduced after 6 hours under HG ambience (Figure 1F).
Effect of HG ambience on mitochondrial dynamics, autophagy, ROS production, and apoptosis in renal proximal tubular cells. (A, a) Under LG (5 mM) ambience, mitochondria exhibited a filamentous network, whereas they transformed into short rods after HG (30 mM) treatment for 24 hours. (B) Western blot analyses revealed upregulation of mitochondrial profission proteins Drp1 and Fis1; profusion protein Mfn2 was downregulated, whereas another fusion mediator OPA1 was not obviously altered. (A, b) By immunofluorescence microscopy, LC3, an autophagy marker, yielded an intense punctate green fluorescence highlighting the cellular autophagosomes, which was reduced under HG ambience. (C) By immunoblotting, expression of converted LC3-II was found to be decreased under HG ambience, whereas that of Atg5 was unaltered. Expression of mitophagy-related proteins, Pink1 and Parkin, was decreased. (A, c and d) Intracellular and mitochondrial ROS generation was increased by HG treatment, which was detected by H2-DCFDA (green) and MitoSox (red) staining. (A, e) By TUNEL procedure, an increase in apoptosis was observed in HK2 cells subjected to HG treatment. (D) The apoptosis was associated with upregulation of Bax and Cytochrome C. (A, f and D) The expression of MIOX, a tubular-specific enzyme, was also concomitantly increased. (E–H) Quantitative analyses confirmed HG-induced mitochondrial fragmentation in HK2 cells in a time-dependent manner, concurring with increased intracellular ROS production and apoptosis after HG treatment. Interestingly, autophagy was reduced only after 6 hours after the HG treatment. D-Glu, D-glucose. *P<0.05 compared with the LG (5 mM) group.
To determine whether MIOX upregulation has any effect on mitochondrial fusion and fission processes, we used an MIOX stably transfected LLC-PK1 cell line that is available in our laboratory. The cells overexpressing MIOX have higher percentages of fragmented mitochondria under LG or HG ambience (Figure 2, A, a and b and C). This was accompanied with dose-dependent increased expressions of Drp1 and Fis1 under HG ambience, whereas the levels of Mfn2 were reduced compared with control cells under similar conditions (Figure 2, A, c and d and B). Cells transfected with MIOX and subjected to LG had Drp1 and Mfn2 fluorescence intensities similar to that of control cells treated with HG (Figure 2A, c and d). Notably, activated Drp1 was translocated to mitochondria in a dose-dependent manner, and this process was further accentuated in cells overexpressing MIOX (Figure 2B). In addition, a loss of mitochondrial voltage potential (ΔΨm) was seen under HG ambiance, which was assessed by TMRE staining, and it was further attenuated by MIOX overexpression (Figure 2, A, e and D).
Accelerating effect of MIOX on the altered mitochondrial dynamics, associated proteins, and mitochondrial membrane potential under HG ambience. (A, a and b and C) MIOX overexpressing LLC-PK1 cells had higher percentages of fragmented mitochondria under LG or HG ambience. (A, c and d) Under LG ambience, MIOX-transfected cells had Drp1 and Mfn2 fluorescence intensity similar to the control cells treated with HG. (B) A dose-dependent increase in the expression of Drp1 and Fis1 was seen under HG ambience, whereas the expression of Mfn2 was slightly reduced. Translocation of Drp1 to mitochondria was also seen in a dose-dependent manner, and it was accentuated in MIOX overexpressing cells. (A, e and D) This was associated with a decrease in TMRE staining, indicating loss of mitochondrial voltage potential (ΔΨm). Con, control.
During fission, the daughter mitochondria may undergo uneven division, and the ones with significant loss of membrane potential (ΔΨm) are likely to be segregated and eliminated by mitophagy, whereas healthy daughter mitochondria with increased voltage potential can undergo fusion to maintain mitochondrial homeostasis. These dynamic events lead to the maintenance of a purified mitochondrial pool and efficient cellular processes.22 In view of this dynamic interplay, the effect of MIOX overexpression on both autophagy and mitophagy was assessed. LC3 staining was used to mark autophagosome, whereas a combination of LC3 and Mitotracker red staining was used to delineate mitophagy. A reduction of autophagy was noted in cells overexpressing MIOX under LG or HG ambience compared with the control cells (Figure 3A, a). This was accompanied with increased mitochondrial fission, and the merged photographs indicated an associated reduction of mitophagy (Figure 3A, b and c). The decrease of autophagy was confirmed after enumeration of cells with punctate LC3 appearance (Figure 3C). Western blot analyses also indicated a reduced expression of autophagy-associated proteins (LC3-II and Atg5), and this decrease was dose dependent in cells subjected to HG treatment. The LC3-II expression was further attenuated by MIOX overexpression, especially under HG ambience, whereas Atg5 expression was largely unaffected (Figure 3B). In addition, expression of mitophagy-related proteins, Pink1 and Parkin, was reduced in a dose-dependent manner under HG ambience. Notably, their expression was not significantly changed by MIOX under LG but was inhibited by MIOX overexpression after HG treatment (Figure 3, A, d and B).
Accelerating effect of MIOX on autophagy, mitophagy, and their associated protein expression under HG ambience. (A, a–c) Fluorescent punctate LC3 staining (green) was reduced in MIOX overexpressing cells in both LG and HG ambience, and this was associated with altered Mitored staining (red) and decreased LC3 and Mitored merged dots (yellow), indicating increased mitochondrial fission and decreased autophagy and mitophagy. (B) Western blot analyses revealed a reduced expression of LC3-II and Atg5 in a dose-dependent manner in cells subjected to HG treatment, and their expression was further attenuated by MIOX overexpression, whereas Atg5 expression was not obviously affected. (C) Quantification also indicated reduced cell percentage with punctate LC3 staining. (A, d and B) The expression of mitophagy-related proteins, Pink1 and Parkin, was also reduced under HG ambience, and their expression was further suppressed by MIOX overexpression when subjected to HG. Con, control.
Failure of mitochondrial quality control and accumulation of damaged mitochondria may inevitably lead to oxidative stress and cell death. In view of this information, the effect of MIOX on ROS production and mitochondrial-dependent apoptosis was investigated. Both the intracellular and mitochondrial ROS levels were increased under HG ambience (Figure 4A). The cells overexpressing MIOX exhibited relatively higher intensity of staining with both H2-DCFDA and MitoSox probes, and statistics attested to these observations (Figure 4, A and B). For assessment of apoptogenic proteins Cytochrome C and Bax, double immunofluorescence was performed. Under LG ambience, cells with MIOX overexpression had higher degrees of fluorescence and altered distribution of both Cytochrome C (Figure 4C, green) and Bax (Figure 4C, red) as well as an increased number of apoptotic nuclei (Figure 4C, blue) (4′,6-diamidino-2-phenylindole [DAPI]), suggesting that MIOX alone may induce Cytochrome C and Bax redistribution that leads to subsequent apoptosis (Figure 4C, columns 1 and 2). While under HG ambience, MIOX further enhanced their redistribution and apoptosis (Figure 4C, columns 3 and 4). Western blot analyses clearly delineated the correlative changes that cytosolic Bax activated and translocated to mitochondria after HG treatment, whereas intramitochondrial Cytochrome C was released into cytosol. These changes were further enhanced in cells overexpressing MIOX, leading to accentuated apoptosis (Figure 4, D and E).
Accelerating effect of MIOX on ROS production, Cytochrome C release, Bax mitochondrial translocation, and apoptosis under HG ambience. (A and B) Both intracellular and mitochondrial ROS levels were increased under HG ambience, which was measured by H2-DCFDA and MitoSox staining, respectively. The MIOX overexpressing cells exhibited relatively strong staining with both H2-DCFDA and MitoSox. *P<0.05 compared with the H2-DCFDA-stained LG group. #P<0.05 compared with the MitoSox-stained LG group. (C) The MIOX overexpressing cells had (columns 1 and 2) a higher degree of fluorescence, altered distribution of Cytochrome C (green) and Bax (red), and an increased number of apoptotic nuclei (blue; DAPI), whereas they were enhanced (columns 3 and 4) after HG treatment. (D and E) Western blot analyses indicated translocation of Bax into the mitochondria and that of Cytochrome C into the cytosol in a dose-dependent manner in cells subjected to HG treatment, which is exacerbated by MIOX overexpression, leading to increased apoptosis. Con, control; Cyt.C, Cytochrome C; cyto, cytosol; mito, mitochondria.
MIOX expression was inhibited in HK2 and LLC-PK1 cells using MIOX siRNA and an inhibitor of MIOX, d-glucaric acid. The drug has been reported to suppress MIOX in tubular cells.19 Under HG conditions, cells transfected with MIOX siRNA or incubated with 10 mM d-glucaric acid had increased autophagy and mitophagy, which was evidenced by the increased number of punctate LC3 dots and Pink1 upregulation. At the same time, there was an attenuation of mitochondrial fragmentation and altered expression of mitochondrial-shaping proteins (Figure 5, A, a–c, B, C, and F). These changes were accompanied by reduced mitochondrial ROS production, decreased expression of Bax and Cytochrome C, and apoptosis compared with cells transfected with empty vector (Figure 5, A, d, D, and F). These observations indicated that MIOX inhibition could normalize the mitochondrial quality surveillance by attenuating mitochondrial fragmentation, generation of ROS, and apoptosis while accentuating mitophagy under HG ambience. To further explore the possible mechanisms by which MIOX disrupts the mitochondrial quality control axis and induces cell injury, we investigated two conceivable pathways that may relate to Pink1 regulation and MIOX-induced oxidative stress. As shown in Figure 5, A–F, cells transfected with Pink1 pcDNA plasmid and subjected to HG had reduced mitochondrial fragmentation and Drp1 levels but increased Mfn2 expression together with increased autophagy and mitophagy and decreased ROS generation and apoptosis. These cellular and biochemical changes are similar to those seen with MIOX knockdown. Interestingly, Pink1 overexpression had little effect on MIOX expression, whereas MIOX inhibition upregulated Pink1 expression compared with cells treated with HG (Figure 5F), suggesting that MIOX may serve as an upstream factor that negatively modulates Pink1 activity. In addition, cells pretreated with the ROS scavenger N-acetylcysteine (NAC) and then subjected to HG showed similar recovery of mitochondrial integrity and reduced apoptosis. Because MIOX has been shown to induce redox imbalance under HG ambience,18 this would indicate another conceivable pathway through which MIOX may disrupt mitochondrial quality surveillance. Furthermore, Pink1 has been recently reported to facilitate and enhance the protein–protein interaction between Parkin and Mfn2 on the MOM, which could induce activation of mitophagy.23 The coimmunoprecipitation experiments of this study indicated that, in tubular cells, HG inhibited the Parkin–Mfn2 interaction compared with LG (Figure 5G). Knockdown of MIOX under HG ambience upregulated Pink1 expression and increased the Parkin–Mfn2 interaction as well, whereas overexpression of Pink1 had similar and more prominent effects on such an interaction, which thereby enhance the process of mitophagy. This suggested that the negative effect of MIOX on mitophagy under HG ambience may be through the downregulation of Pink1 and consequentially, the reduced Parkin–Mfn2 interaction. These in vitro data indicated a key role of mitochondrial quality control in the maintenance of their homeostasis and cellular integrity, where MIOX may be critically involved in such pathobiologic processes. In view of this contention, we examined if inhibition of MIOX in vivo has a therapeutic potential in amelioration of tubulopathy in diabetic mice.
Attenuation of mitochondrial fragmentation, ROS, and apoptosis but increased autophagy and mitophagy after inhibition of MIOX under HG ambience. (A, a–c, B, C, and F) Under HG, cells transfected with MIOX siRNA or incubated with d-glucaric acid had increased expression of punctate LC3 and Pink1, indicative of increased autophagy/mitophagy, as well as decreased mitochondrial fragmentation and altered expression of mitochondrial shaping proteins. (A, d and D–F) This was accompanied with decreased mitochondrial ROS production, reduced expression of Bax and Cytochrome C, and apoptosis compared with empty vector-transfected cells treated with HG. MIOX may modulate mitochondrial quality control through two different mechanisms (i.e., Pink1 pathway and MIOX-induced oxidative stress). (A, columns 5 and 6 and F) Pink1 pcDNA-transfected cells had reduced mitochondrial fragmentation and Drp1 levels but increased Mfn2 expression together with increased autophagy and mitophagy and decreased ROS generation and apoptosis. Similarly, the cells pretreated with ROS scavenger N-acetylcysteine (NAC) and subjected to HG showed partial recovery of mitochondrial integrity and reduced apoptosis. (E and F) These changes were similar to those seen with MIOX inhibition, whereas MIOX has been shown to induce ROS and inhibit Pink1 expression. (G–I) The coimmunoprecipitation experiments indicated that, in tubular cells, HG inhibited the Parkin–Mfn2 interaction, whereas knockdown of MIOX upregulated Pink1 expression and increased the Parkin–Mfn2 interaction. The overexpression of Pink1 had similar and more prominent effects on such interactions. Cyt.C, Cytochrome C; D-Glu, D-glucose; IP, immunoprecipitation; WB, Western blot. *P<0.05 compared with HG plus the empty vector group.
d-glucarate, the calcium salt of d-glucaric acid, was added to the mouse dietary chow (1% or 2% [wt/wt]) to inhibit MIOX. Four groups of mice (n=8 per group) were assigned: control CD1, streptozotocin (STZ)-induced diabetic, and STZ+1% or STZ+2% d-glucarate dietary supplementation groups. d-glucarate treatment was initiated after 1 week of STZ injection and continued for another 8 weeks. During this time frame, body weight, blood glucose, serum creatinine, and urinary albumin-to-creatinine ratio (ACR) were monitored. Compared with controls, the STZ group had higher blood glucose levels and increased in serum creatinine and urinary ACR values but reduced body weight (Figure 6, A–D). The diabetic mice receiving d-glucarate had a smaller reduction in body weight and slightly reduced blood glucose as well as notably decreased serum creatinine and urinary ACR compared with the STZ group (Figure 6, A–D). Interestingly, mice with 2% d-glucarate treatment had better preservation of renal function than those receiving 1% d-glucarate. Morphology studies revealed increased periodic acid–Schiff-positive tubular brush border staining, which is indicative of protein excretion, notable tubular epithelial disruption, hypertrophy of glomeruli, and increase of mesangial matrices in the STZ group. The diabetic mice with drug treatment showed notable improvement largely in the tubular compartment with less cellular disruption (Figure 6E). Similar tubular alterations as well as changes in mitochondria morphology were observed after Toluidine blue staining of EPON-embedded sections (Figure 6F). In the control group, the mitochondria appeared cylindrical, elongated, and well organized. However, the organelles were fragmented and scattered in tubules as well in their lumens in the STZ group. There was partial recovery in mitochondrial morphology along with improvement in tubular cell integrity in the STZ and d-glucarate groups. Quantitative analyses with damage scores ranging from zero to three indicated that both the STZ+1% and STZ+2% d-glucarate groups had slightly reduced glomerular damage but notable decrease of tubular damage compared with the STZ group (Figure 6, H and I). Concurrently, the MIOX expression and activity (Figure 6, G and J) were markedly increased in proximal tubules in the STZ group compared with controls and notably attenuated in STZ-treated mice administered d-glucarate.
Ameliorative effect of d-glucarate on renal functional and morphologic characteristics by modulation of MIOX expression in STZ-induced diabetes in mice. (A–D) The STZ-treated group had higher blood glucose levels, an increase in serum creatinine and urinary ACR values, and reduced body weight compared with the controls. In general, the diabetic mice receiving 2% d-glucarate had better preservation of renal functions than those receiving 1% d-glucarate. (E) Increased periodic acid–Schiff (PAS)-positive tubular brush border staining, tubular epithelial disruption, hypertrophy of glomeruli, and mesangial matrix numbers were observed in the STZ group. Treatment of diabetic mice with d-glucarate had notable improvement (largely in the tubular compartment) with less cellular disruption. (F) In the tubules of the control group after Toluidine blue staining, the mitochondria appeared cylindrical, elongated, and well organized. The mitochondria were fragmented and scattered in the STZ group. A partial recovery in mitochondrial morphology along with improvement in tubular cell integrity was seen after 1% or 2% d-glucarate administration. (H and I) Quantitative analyses with damage scores ranging from zero to three indicated that d-glucarate treatment resulted in amelioration of changes in glomeruli and tubules after STZ induction of diabetes. (G and J) Concurrently, MIOX expression and activity were decreased in diabetic mice receiving d-glucarate. DG, d-glucarate. *P<0.05 compared with the STZ group; #P<0.05 compared with the control group.
To confirm alterations of mitochondrial morphology as observed by Toluidine blue staining, expression of mitochondrial-shaping proteins and organelle ultrastructural changes were assessed. The mitochondrial profission proteins Drp1 and Fis1, expressed mainly in renal tubules by immunohistochemistry, were upregulated in the STZ group, but their in situ expression was reduced to a certain extent in STZ+1% or STZ+2% d-glucarate groups (Figure 7A, a and b). Conversely, Mfn2 levels were notably decreased in tubules of the STZ group but partially restored in some of the proximal tubules after d-glucarate treatment (Figure 7A, c). These results suggested a restoration of disrupted mitochondrial dynamics by d-glucarate in tubules of diabetic mice. Next, status of mitochondrial morphology was assessed by electron microscopy. In the control group, the majority of the tubular cells had elongated cylindrical-shaped mitochondria (marked by the asterisks in Figure 7, B and F). In the STZ group, >50% of the tubular cells exhibited fragmented mitochondria; the mitochondrial morphology was partially restored by d-glucarate supplementation, and the percentage of tubular cells having fragmented mitochondria was reduced. High-magnification electron microscopy (Figure 7, C–E) revealed elongated mitochondria with organized cristae in the control group (Figure 7C). However, in the STZ group, most mitochondria exhibited rod or sphere shapes and had cristolysis with focal disruption of the inner mitochondrial membranes (Figure 7D). These changes in the diabetic group were partly restored after administration of d-glucarate, and they showed the morphologic features of the mitochondrial fusion–fission process (the fragmented mitochondria in the hyperglycemic state underwent refusion to form elongated mitochondria after d-glucarate treatment) (Figure 7, E and F).
Restoration of altered tubular mitochondrial morphology in diabetic mice after administration of d-glucarate. (A) The mitochondrial profission proteins Drp1 and Fis1, expressed mainly in renal tubules, were upregulated in the STZ group, but their expression was reduced after administration of d-glucarate. Conversely, expression of mitochondrial profusion protein Mfn2 was decreased in the STZ group but partially restored in some of the proximal tubules after drug treatment. (B and F) By electron microscopy, the majority of the tubular cells had mitochondria with elongated cylindrical shape (yellow asterisks) in the control group, whereas in the STZ group, about 50% of the tubular cells had fragmented mitochondria, and their morphology was partially restored by d-glucarate treatment. (C) Higher magnification electron micrographs showed elongated mitochondria with organized cristae in the control group. (D) In the STZ group, mitochondria showed a spherical shape and had undergone cristolysis (matrix vacuoles). (E) These changes were negated after d-glucarate treatment, and mitochondria undergoing fusion are seen. (F) The mitochondrial changes were quantified and included in bar graphs. DG, d-glucarate; EM, electron microscopy. *P<0.05 compared with the STZ group; #P<0.05 compared with the control group.
Various morphologic studies were performed to investigate the changes of autophagy and mitophagy in STZ-induced diabetic mice and the effect of MIOX inhibitor on these pathobiologic events. Frozen kidney sections from four groups were stained with LC3. A basal degree of autophagy, which was indicated by punctate LC3 appearance in renal tubules, was seen in the control group. The LC3 staining was greatly reduced in tubules from the STZ group, and the formation of autophagosomes was partially restored or enhanced after d-glucarate treatment (Figure 8A). The morphometric analyses showed that the number of autophagosomes per tubule in the STZ and d-glucarate groups was much higher than that in the STZ group and comparable with or higher than that in the control group (Figure 8B). Meanwhile, Atg5 and Pink1 showed parallel changes in the tubules of mice from four groups, and their in situ expression was reduced in the STZ group but partially restored after d-glucarate treatment (Figure 8C). The status of autophagic vacuoles, including autophagosome and autolysosome, was evaluated by electron microscopy. Autophagic vacuoles were reduced in tubular cells in the STZ group compared with the controls, whereas they were partially restored in tubules from mice treated with 1% d-glucarate and even enhanced in the 2% drug-treated group (Figure 8, D and H). High-magnification electron micrographs depicted a typical morphology of autophagosome containing fragmented cellular organelles (Figure 8E). An autophagosome-engulfing mitochondria, indicative of mitophagy (Figure 8F), and an autolysosome (Figure 8G) were readily seen in tubular cells of d-glucarate–treated groups, suggesting restoration of autophagy and mitophagy.
Restorative effect of d-glucarate on autophagy and mitophagy in STZ-induced diabetic mice. (A) The basal level of autophagy, indicated by punctate LC3 staining, was seen in the renal tubules of the control group, which disappeared in the tubules of the STZ group, whereas they were restored after d-glucarate treatment. (B) Morphometric analyses confirmed these observations. (C) The expressions of Atg5 and Pink1 exhibited parallel reduction and restoration in the tubules of mice from four groups. (D and H) By electron microscopy, autophagic vacuoles seen in the control group were greatly reduced in proximal tubular cells in the STZ group, whereas they were restored in tubules from mice treated with d-glucarate. (E) Higher magnification electron micrographs revealed a typical morphology of an autophagosome containing fragmented cellular organelles, (F) a mitophagosome, and (G) an autolysosome in tubular cells after d-glucarate treatment. DG, d-glucarate; EM, electron microscopy. *P<0.05 compared with the STZ group; #P<0.05 compared with the control group.
Expression of one key apoptogenic protein, Bax, was found to be increased in tubules of the STZ group, and it was partially inhibited by d-glucarate treatment in diabetic mice (Figure 9A). Similarly, Cytochrome C, which is normally confined to the mitochondrial matrix in the control group, was seen released into the cytosol in the STZ group with concomitant upregulation of MIOX (Figure 9B). However, lesser degrees of Cytochrome C redistribution together with the suppressed expression of MIOX were noted in tubules of the STZ mice treated with d-glucarate. The renal tissues of the STZ group subjected to DHE or TUNEL staining showed concomitant increases of oxidative stress and apoptosis, both of which were largely reduced with d-glucarate treatment, especially in tubules (Figure 9C). These observations suggest that d-glucarate, by inhibiting MIOX, exerts antioxidant and antiapoptotic effects in the renal tubular compartment of diabetic mice.
Reduction in the expression of apoptogenic proteins, ROS production, and apoptosis in STZ-induced diabetic mice after d-glucarate administration. (A) Expression of Bax was found to be increased in tubules of the STZ group, which were partially inhibited by d-glucarate treatment in diabetic mice. (B) Similarly, Cytochrome C, normally confined to the mitochondria, was seen diffusely distributed in the cytosol in the STZ group with concomitant upregulation of MIOX, and this redistribution was suppressed with the d-glucarate treatment. Their concomitant changes were readily seen in the merged (MIOX and Cyt.C) images. (C) An increase in DHE and TUNEL staining, indicative of increased oxidative stress and apoptosis, respectively, was observed in the STZ group, and both were greatly reduced with the d-glucarate treatment, especially in tubules. Con, control; Cyt.C, Cytochrome C; DG, d-glucarate; G, glomerulus. *P<0.05 compared with the STZ group; #P<0.05 compared with the control group.
Glomerular lesions have been the major focus of investigation in DKD; however, in recent years, the importance of specific tubular changes has also been emphasized. The features of tubulointerstitial histopathology include inflammation and apoptosis, which are not necessarily secondary to glomerular lesions.7,24–26 In addition, tubular injury alone is believed to be closely correlate with clinical manifestations of DKD, including the extent albumin excretion and its degradation in tubules. The latter may serve as a primary key site for the development of DKD, because tubular injury has been described to be a better predictor in the progression of the renal disease.3,4 Thus, the dominant glomerulocentric view of DKD has partly shifted to tubules, and the injury has been described as diabetic tubulopathy.3 In this study, notable changes in the tubules were also observed in STZ-induced diabetic mice (Figures 6 and 7). This brings up an important question of whether attenuation of tubular damage by therapeutic approaches can delay or reverse DKD progression? Our results (Figure 6) indicate that d-glucarate, an MIOX inhibitor, not only ameliorates diabetic tubular injury but also, improves the renal functions, which are reflected by decreased serum creatinine levels and urinary ACR. Interestingly, d-glucarate also attenuated glomerular damage to some extent, suggesting a possible link between tubular injury and glomerular burden during the development of DKD. Similarly, recent studies by Lin et al.26,27 showed that inhibition of TLR4 in the tubules of diabetic mice led to reduced albuminuria, BUN, glomerular hypertrophy, and glomerulosclerosis in addition to attenuation of tubulointerstitial injury. In this regard, the approach targeting the tubular compartment with respect to mitochondrial homeostasis can be beneficial for the overall amelioration of DKD.
Mitochondria are abundantly present in tubular cells to meet high metabolic energy demand, such that they play a pivotal role in the pathogenesis of acute or chronic tubular injury.10,28,29 Recent studies have delineated an integrated regulation of the mitochondrial lifecycle.11,22 The dynamic nature of mitochondria enables them to constantly undergo fusion and fission cycles, whereas the selective autophagic removal of damaged organelles (mitophagy) purifies the mitochondrial pool, all of which form an integrated quality control axis to maintain mitochondrial homeostasis. This concept of mitochondrial quality control is intricately related to mitochondrial dynamics and mitophagy, and it has been investigated in various diseases,12,30–32 but studies on mitochondrial quality control in kidney diseases, including DKD, are somewhat limited. A few reports have indicated a pivotal role of mitochondrial dynamics in the homeostasis of different kidney cells during DKD and AKI.8,28 However, autophagy is believed to exert a cytoprotective effect during kidney injury, such as in AKI, DKD, and obesity, where the basal rate of autophagy may increase initially but because of its relative decrease, subsequently, the podocytes and tubular cells get readily damaged.33–36 In addition, elimination of damaged mitochondria by mitophagy was reported to be relatively deficient or decreased in the cardiac musculature of patients with diabetes.37 Similarly, two mitophagy regulators, Pink1 and Parkin (the genes that are relevant in the pathogenesis of Parkinson disease), were found to be downregulated in cardiac and skeletal muscles in diabetic states.37,38 In this study, we observed that most of the mitochondria within the injured tubules in STZ-induced diabetic mice were fragmented and had a randomly disorganized cellular distribution (Figures 6F and 7B). At the same time, the cellular autophagic vacuoles and mitophagy were reduced in tubules, indicating less efficient removal of damaged mitochondria and a compromised surveillance mechanism. Importantly, increased mitochondrial fragmentation and reduced autophagy or mitophagy under HG ambience were observed in tubular cells in both our in vitro and in vivo studies (Figures 2, 3, 7, and 8). These two cellular events together indicated an impaired mitochondrial quality control, which may inevitably result in excessive ROS generation and initiation of the mitochondrial apoptotic cascade (Figures 4 and 9).
Next, the question needs to be addressed of what caused the mitochondrial quality control failure to rescue the damaged mitochondria. It is conceivable that MIOX may have interfered with the mitochondrial self-rescuing process. MIOX, a tubular enzyme, is upregulated during HG ambience, and its critical involvement in the pathogenesis of DKD has been shown.15–18 During hyperglycemia, MIOX induces redox imbalance and mitochondrial dysfunction that could result in tubular oxidative injury and apoptosis.17,18 In view of this contention, the underlying mechanisms by which MIOX may cause mitochondrial dysfunction and tubular cell injury were delineated. As shown in Figure 10, under HG ambience, MIOX is upregulated in tubular cells,16–18 and then, it modulates mitochondrial dynamics through two conceivable pathways. In the first pathway, MIOX increases intracellular ROS generation through transcriptional mechanisms,18 which would then activate cytosolic Drp1 and promote its mitochondrial translocation, leading to mitochondrial fragmentation.10,39 In the second pathway, elevated MIOX levels would suppress Pink1 expression, which has a negative regulatory effect on Drp1 activity, such that the Pink1 deficiency would increase Drp1-dependent mitochondrial fragmentation.40 In other words, excessive ROS generation and paucity of Pink1, both induced by MIOX, lead to Drp1 activation and translocation to mitochondria. Subsequently, Drp1 interacts with Fis1 on the MOM and causing mitochondrial fragmentation. Excessive fragmented mitochondria are depolarized and lose viability for the fusion process, but the quality control of mitochondria allows these injured organelles to be recycled by autophagosomes through mitophagy. However, under HG ambience, activated MIOX inhibited Pink1 and conceivably, prevented its downstream Parkin translocation to mitochondria, thus blocking subsequent interaction between Parkin and Mfn2 on MOM. Eventually, these events led to a compromise in the process of mitophagy. As a result, the accumulation of fragmented and damaged mitochondria triggered ROS overproduction as well as the mitochondrial apoptotic pathway—a process by which the apoptogenic Bax translocates and inserts into mitochondria, whereas Cytochrome C is released into the cytosol, leading to Caspase 9 activation and eventually, apoptosis. Conversely, inhibition of MIOX by d-glucarate restores mitochondrial quality surveillance by preventing mitochondrial damage and cellular apoptosis, thereby attenuating diabetes-induced tubular damage.
Schematic diagram depicting the conceivable cellular events by which MIOX disrupts mitochondrial integrity under HG ambience, which results in renal tubular cell injury. Under HG ambience, MIOX is upregulated in tubular cells, which then modulate mitochondrial dynamics through two conceivable pathways. (1) MIOX increases intracellular ROS generation through transcriptional mechanisms, which promote Drp1 activation and mitochondrial translocation.10,18,39 (2) Upregulated MIOX reduces Pink1 expression, which has a negative regulatory effect on Drp1 activity, such that Pink1 inhibition would increase Drp1-dependent mitochondrial fragmentation.40 In this regard, Drp1 is activated and translocates to MOM through these possible MIOX-mediated pathways, where it interacts with Fis1, which would lead to mitochondrial fragmentation. Excessively fragmented mitochondria are depolarized and lose viability for the fusion process, but the surveillance of mitochondrial quality control allows these injured organelles to be recycled by mitophagy. However, under HG, both autophagy and mitophagy are suppressed, partly because of the activation of MIOX, which inhibited Pink1 and conceivably, prevented subsequent Parkin translocation into mitochondria, thus blocking the interaction between Parkin and Mfn2 on MOM that eventually resulted in a compromise in the process of mitophagy. As a result of insufficient autophagic removal of mitochondria, the accumulation of fragmented and damaged mitochondria triggered ROS overproduction as well as the mitochondrial apoptotic pathway, by which the apoptogenic Bax translocates and inserts into mitochondria, whereas Cytochrome C is released into the cytosol, leading to apoptosis. Interestingly, pharmacologic inhibition of MIOX by d-glucarate restores mitochondrial quality control machinery by preventing mitochondrial damage and cell apoptosis, thereby attenuating hyperglycemia-induced tubular damage. Cyt.C, Cytochrome C.
Of note, d-glucarate is a natural compound, and it is present in substantial amounts in certain fruits and vegetables. Daily supplementation of d-glucarate has some key beneficial effects (i.e., detoxification of noxious substances and anticancer regimens).41–43 The primary mechanisms of action of d-glucarate may be through its ability to enhance detoxification of toxins and carcinogens as well as its antioxidative potential,44,45 which may be linked to the inhibition of MIOX. In addition, a derivative of d-glucaric acid, d-saccharic acid 1, 4-lactone, has been recently reported to be protective in both diabetes and its renovascular complications in rats.20,46 Likewise, we elucidated an important therapeutic effect of d-glucarate, which was through the inhibition of MIOX and thus, normalization of mitochondrial quality control, reduction of oxidative stress, and apoptosis in tubular cells with improvement in renal functions. Despite our extensive investigatory efforts, there are still certain issues that remain unresolved. For instance, the mechanism by which MIOX modulates Pink1 and downstream Parkin remains to be delineated. Also, whether MIOX plays a direct role in the regulation of mitochondrial dynamics and functions is worthy of future studies.
In conclusion, this study shows the effect of MIOX on mitochondrial quality control disruption during DKD using in vitro and in vivo models. MIOX upregulation under HG ambience induces mitochondrial fragmentation and depolarization; meanwhile, it inhibits selective autophagic removal of damaged mitochondria through Pink1-dependent Mfn2–Parkin interaction. The net effect of these events would be disruption in the surveillance of mitochondrial quality control, accumulation of dysfunctional organelles, generation of ROS, and apoptosis, leading to tubular injury and DKD progression. Interestingly, the fact that d-glucarate could normalize mitochondrial homoeostasis by inhibition of MIOX would suggest a therapeutic potential of d-glucarate in the amelioration of DKD.
Renal proximal tubular epithelial cell lines HK-2 (human) and LLC-PK1 (porcine) were used.17,18 They were maintained in media containing 5–30 mM d-glucose. For overexpression of MIOX and Pink1, cells were transfected with pcDNA3.1 or pcDNA-DEST47 plasmids. For gene disruption, cells were treated with MIOX siRNA using Lipofectamine 2000 reagent (Life Technologies). To pharmacologically inhibit MIOX, d-glucaric acid (Sigma-Aldrich) was added to the culture media and cotreated with various concentrations of glucose. N-acetylcysteine (Sigma-Aldrich) was added for 2 hours before glucose treatment to scavenge the ROS.
MIOX plasmid construct and MIOX overexpressing cell lines were generated in our laboratory.16,17 A porcine MIOX cDNA was generated by RT-PCR and cloned into pcDNA3.1 (Life Technologies). The plasmid constructs and empty vector (control) were then transfected into LLC-PK1 cells, and stable transfectants were selected by growing cells in the presence of Geneticin and maintained.
For Western blot analyses, whole-cell lysate, cytoplasmic, or mitochondrial extracts of treated cells were subjected to 5%–15% SDS-PAGE. The fractionated proteins were transferred onto nitrocellulose membranes, which were then incubated with various primary antibodies. The anti-MIOX was previously generated in our laboratory.18 Other antibodies included anti-Drp1, anti-Fis1, anti-Bax, anti-Cytochrome C, anti-Pink1, anti-Parkin (Santa Cruz Biotechnology), anti-LC3B (Cell Signaling Technology), anti-OPA1 and anti-Mfn2 (Abcam, Inc.), and anti-Atg5 (Gene Tex). After overnight incubation, the membranes were immersed in a solution containing individual secondary antibodies (Santa Cruz Biotechnology). Membrane blots were then used for developing autoradiograms using the ECL system (Amersham Biosciences).
For coimmunoprecipitation, cell lysates from various experiments were used. They were incubated with respective rabbit polyclonal antibodies followed by the addition of protein A-Sepharose CL-4B, and incubation was extended for 12 hours. The antigen–Ab–protein A complex was pelleted and washed with RIPA buffer. The pellet was suspended in lysis buffer and subjected to SDS-PAGE for Western blot analyses.
Cells were fixed with paraformaldehyde, permeabilized, blocked with BSA, and incubated with primary antibodies. After washing with PBS, they were reincubated with secondary antibodies conjugated with Alexa Fluor 488 or 594 (Life Technologies). Cells were counterstained with DAPI to delineate the nuclei and examined by confocal microscopy.
For kidney tissue immunofluorescence, 4-µm-thick cryostat sections were prepared, mounted on glass slides, air dried, and rehydrated with PBS. Similarly, 3-µm-thick paraffin-embedded sections were prepared, dewaxed, and rehydrated. After blocking with BSA, sections were incubated with primary antibodies and then, Alexa Fluor-conjugated secondary antibodies.
To assess the extent of apoptosis, TUNEL staining was performed on cells plated on coverslips and paraffin-embedded kidney sections using an In Situ Cell Death Detection Kit (Roche Applied Science).7 Ten random fields of cells (approximately 200 cells per group) were counted to determine the percentage of cells undergoing apoptosis.
After various treatments, the live cells were incubated with Mitotracker dye (Life Technologies) and examined by confocal microscopy. Also, their morphology was assessed after paraformaldehyde fixation and detergent permeabilization. For each treated group, 10 fields of cells were randomly selected (>100 cells/group) to delineate the shape of mitochondria.
To assess perturbations in the mitochondrial membrane potential (ΔΨm), the cells were stained with TMRE (Life Technologies) and examined by confocal microscopy.7 Average intensity of fluorescence from 10 randomly selected fields was measured using ImageJ software (National Institutes of Health, Bethesda, MD) to evaluate ΔΨm.
For autophagy/mitophagy assessment, cells incubated with Mitotracker red dye were fixed and permeabilized. They were successively incubated with rabbit polyclonal LC3 antibody and secondary antibodies conjugated with Alexa Fluor. After counterstaining with DAPI, cells were examined. Ten random fields of cells (>100 cells/group) were evaluated to calculate the percentage of cells with punctate LC3.
To assess intracellular and mitochondrial ROS production, cells were incubated with H2-DCFDA or MitoSOX (Life Technologies), respectively, and examined by confocal microscopy. Intracellular ROS production in kidney tissues was assessed using 4-µm-thick cryostat sections from various experimental groups and stained with DHE (Sigma-Aldrich).47 For both cells and tissue sections, 20 randomly selected fields were photographed, and mean fluorescence intensity was calculated.
Eight-week-old ICR male mice (Harlan Co.) were used and divided into four groups (n=8): control, STZ-induced diabetic, and diabetic with 1% or 2% d-glucarate supplement. d-glucarate (1% or 2% [wt/wt]) was mixed with dietary chow.42 A diabetic state was induced by daily intraperitoneal injection of STZ (50 mg/kg body wt; Sigma-Aldrich) for 5 days. One week after the injection, mice with blood glucose levels>250 mg/dl were selected for dietary supplementation and euthanized after 2 months. All animal procedures used in this study were approved by the Animal Care and Use Committee of Northwestern University.
Bimonthly body weight, blood, serum, and urine collections were carried out. Blood glucose was measured using the Roche Accu-Chek Advantage Meter. Serum and urine creatinine and urine albumin were assessed using the QuantiChrom Creatinine and BCG Albumin Assay Kits (Bioassay Systems). Urinary ACR (milligrams per gram) was calculated as follows: urine albumin (milligrams per deciliter)/urine creatinine (grams per deciliter).
Three-micrometer-thick paraffin-embedded kidney sections from four groups of mice were dewaxed in xylene and rehydrated, and then, they were successively incubated with various primary antibodies and anti-rabbit IgG conjugated with peroxidase (ImmPRESS kit; Vector Labs). Sections were treated with diaminobenzidine or NovaRed substrate (Vector Labs) followed by counterstaining with Mayer hematoxylin and examined.
MIOX activity in kidney tissues was determined as previously described.16,18 Briefly, cortices were homogenized in a buffer (20 mM sodium acetate [pH 6.0], 2 mM l-cysteine, 1 mM ferrous ammonium sulfate, 1 mM glutathione, and 1 mM PMSF). The homogenate was centrifuged, and the supernatants were saved for measuring protein concentration and MIOX activity by the Orcinol method.
Renal cortices were minced into 1-mm3 pieces and processed for electron microscopy.7 After surveying the Toluidine blue-stained EPON-embedded sections, thin sections were prepared for electron microscopy to delineate the extent of mitochondrial fragmentation, autophagy, and mitophagy in tubules.28,50 Mitochondria having length>2 µm were considered filamentous, and those with length<1 µm and spherical configuration were designated as fragmented.
The data were expressed as means±SEMs. Comparison of the results between two groups was evaluated by one-way ANOVA. P value<0.05 was considered statistically significant.
This study was supported by National Institutes of Health Grant DK60635.
Myocardial redox status, mitophagy and cardioprotection: a potential way to amend diabetic heart?


You are here: Home / Whats New? / News and Events / New Erin Twiggs RIPPED muscle video added!
New Clips of Alli Schmohl's Massive Arms & SUPER RIPPED Erin Twiggs!
Alli Schmohl – the Ultimate Female Muscle Beefcake! Rock Solid, Shredded Monstar Muscle!


"Darlene Turner" has no idea who you are or where you live and certainly doesn't have a "son (Kenneth" who wants to learn Chinese.
Her aim is to send you a large (fake) cheque. She will probably then overpay you and ask you to forward some of the money to somewhere else. At which point you will find out that the original cheque was a fake and be out of pocket.
He will be staying there for one months.
problem about the lessons,my caregiver lives very close to the Area.
So there is no problem for the lesson OK my caregiver will be bringing him to your location for the lessons and you can teach my Him Anywhere around you if that is OK by you so i will like you to teach my Son the best of you when he get to the USA for the lessons.
I will like you to email me with your schedule for the lessons,I will like you to email me with the name on the check and Full mailing address where the check will be mailed to and including your Home and Cell phone number because my attorney that want to issue out the check is leaving the State by this week okay and also Kenneth is a beginner lessons learning, Awaiting your response asap.
Also the lessons will commence by 17th December Until 29th December 2011 is this okay by you?
I will be awaiting to read from you.
« Greeting From Mr Anthony Owusu	» PLEASE KINDLY HELP ME!!!


This sweet greeting card of little girl and her puppy taken from a vintage photograph can be customized with a two- to three-word greeting (ie Happy Birthday, Thank You, Thinking of You, etc. in your favorite typeface (try our Font Previewer). Text block is available in nine colors to coordinate with Colored Envelopes as shown. Leave inside blank or add your own greeting (optional). Available as single card or in packets of 4.


Yesterday, Treyarch Studios has announced for Call of Duty: Black Ops 4 '2XP New Year's Event' which live until new year on January 1st.
🎉 2XP in Multiplayer and Zombies!
🎉 2X Weapon XP in Multiplayer and Zombies!
Yes, the 2XP event is now live until January 1st, presents numbers of XP all around the battle royale. And one more surprise from this new year's event is, the zombies are back!
Well, previously we have acknowledged for the disappearing of zombies in the game, but now, Treyarch has brought them back along with the addition of new permanent zombie spawn. I guess you better prepare for this return of zombies as they are now more deadly and horrified in order to make your life miserable (in the game)!
2XP + 2X Weapon XP in MP and Zombies + 2X Merits this weekend!
Now are you ready to grind and unlock some great in-game stuff rewards?


text:
Langland's French Song
This essay is part of the forum on 'Langland and the French Tradition', edited by R. D. Perry and Elizaveta Strakhov. It is a sustained engagement with one instance of L's use of French: the refrain at the end of the prologue, in which distracted diggers 'dryueþ forþ þe longe day with "Dieu saue dame Emme"' (B.Prol.225). Long thought by critics to be a snippet of a popular song, Perry argues instead that it is a synthetic everysong, a bit of verse designed to sonically resemble popular song but that has no independent existence outside of L's poem. This occasions some reflection on the critical category of the 'popular'. Perry argues further that such an understanding of the song has a wide variety of ramifications. First, it posits the limits of source study by showing that there are moments in L's poem that seem like they would have a source but do not. Second, it demonstrates that L has the capacity to create convincing French verse with a line that would be at home in the writings of his French contemporaries. Third, it denotes a willingness on L's part to incorporate different French forms into his verse, that what he does later in the poem to French romance is done here with French song. Fourth and finally, that the incorporation of this French form is part of L's broader concern with idleness and the production of poetry.
Yearbook of Langland Studies, 30 (2016), 277–96.
Perry, R. D.

redpajama_set_name: RedPajamaCommonCrawl


Lower Similkameen Indian Band and BC Wildfire Service planning prescribed burns.
Feb. 6, 2019 11:25 a.m.
It's been a few years in the making but a prescribed burn is slated to take place later this year in the Crater Mountain area.
In total 680-hectares is expected to be burned in several phases. The first phase will be 192 hectares in size.
The prescribed burns are part of a partnership between the BC Wildfire Service and the Lower Similkameen Indian Band.
"The project is intended to provide a level of community protection from wildfire, restore the ecosystem, including critical bighorn sheep habitat, and enhance Indigenous cultural values in the area," a release stated.
Project managers' will be on-hand at the open house on Thurs., Feb. 7 from 6 p.m. to 8 p.m.
Google maps Approximate location of Crater Mountain near Keremeos where a prescribed burn will take place later this year.


Lily and Leon are a Washington State couple planning a wedding in Maui, but they also wanted to have a wedding ceremony in Lily's hometown of Vancouver, BC. So they planned for a wedding at the Law Courts Inn in downtown Vancouver and designed it with a very unique twist. They planned a small and intimate wedding ceremony with only a few guests and Lily put in a ton of work to design the ultimate pink wedding. I knew how beautiful this was going to be when I first met with Lily and she pulled out her iPad inspiration board to show me their "pink" vision for the wedding. It was creative, vibrant, splendid, and of course, all pink! She later admitted that her design was so opposite her personality. She describes herself as somewhat of a "tom-boy", so she wanted a wedding that would look somewhat unexpected…and boy did she pull it off. Her design did not disappoint! She made the cake herself as well as the "broach" bouquet (yes – hand-made completely out of broaches). They brought in stylish ghost chairs, a pink table cloth, flowers by Garden Party Flowers, a white chocolate poodle, and pink (rose) Moet Champagne, other modern vintage and touches of bling, and of course a pink cotton candy machine from Frankie's Candy Bar. Bailey was on hand to help with the day of coordination and to help with setting up Lily's amazing wedding design work – Can't believe she did it all herself. Lily is such an amazing talent. Above are just a few previews from their pink Vancouver wedding!
LOVE all the pink! 🙂 I used to have a Barbie that had a similar dress! Love it!
I love it! Pink is 100% not my colour at all, but this is so striking, sophisticated and lovely! Absolutely stunning.
Wow! Amazing! I love her dress and all the pink details – and especially the little poodle! Pink perfection!
This wedding is so absolutely perfect. The poodle was just the cherry on top. LOVE!
I love the pink dress. You rarely see wedding dresses that are not white in England. Nice work.
Wow. The details are incredible. Beautiful photos too!
Like her pink original dress!It is great!
She had a wonderful motif color – pink! It's very feminine though she said she is somewhat "tom-boy".


Skilled Salesperson or Full Time Prospector?
If you accepted the place as an Insurance coverage Gross sales Agent, did you see that as a option to additionally change into an expert in your area? Did you might have pictures of incomes an above common earnings, residing an above common life and connecting with movers and shakers?Gross sales is an expert place. An excellent agent will and will earn as a lot as an expert in lots of different fields. Insurance coverage Gross sales Brokers ought to maintain their heads up and be pleased with their occupation.I'm wondering why so many brokers will not be in a position to do this? What causes an agent to really feel like they're doing numerous work and but receiving little to no pay for it?Right here is likely one of the causes; as an alternative of working as an expert gross sales agent, they work as skilled prospectors.
What's an expert prospector? It's an agent who spends 80-90% of their time searching for that subsequent appointment. Drawback is, none of us receives a commission to prospect; we receives a commission to promote. So how achieve this many brokers fall into this lure? Take into account the next;1) Opposite to in style perception the profitable agent doesn't spend the vast majority of his time prospecting. He isn't on the telephone making 100's of telephone calls a day; he isn't bodily stuffing and mailing 100's of letters per week. He isn't exterior knocking on doorways, strolling the pavement and making an attempt to persuade somebody to purchase from him whereas he's going broke.2) What skilled brokers do is notice they're paid to promote. You solely become profitable while you flip in an software that will get accepted. Prospecting is a way to an finish however not the top. In the event you focus your entire consideration on prospecting I can virtually assure you'll not final lengthy on this enterprise.three) Prospecting is essential. However how do you go about doing it? By working smarter and never tougher. How a lot talent does it take to stuff a letter into an envelope and mail it? To move out flyers on automobiles in a parking zone or within the doorways of the properties in a neighborhood? A few of these jobs will be performed by a highschool scholar.four) Professionals have discovered that they receives a commission essentially the most by doing their occupation. Docs rent employees to attract blood, test your insurance coverage, gather your charge for the go to, set appointments, and many others. Docs did not spend all that money and time at school to do jobs that they will rent another person to do. As an expert gross sales agent you're going to get paid essentially the most while you do what you might be licensed to do; promote insurance coverage.
I do know that while you first begin off your finances could be very restricted and it's important to do these items as a result of nobody else will. You could not be capable of pay for these companies. But when I might give you a bit of recommendation; focus in your strengths (promoting insurance coverage) and employees to your weak point. Mentally place your self to carry on assist to do the issues that it's essential to have performed however shouldn't be doing your self.Focus first on promoting. Work out how you can use different accessible assets to prospect.


text:
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We are evolving from a product company to a lifestyle experiential company: Sanjay Behl, Raymond
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At the first edition of exchange4media's music conference—Music Inc., popular music artist Aditya Prateek Singh Sisodia, popularly known as Badshah opened up in a candid interview with noted Film Critic and author Anupama Chopra about his musical journey and what it takes to become a musicpreneur.
Wizcraft to kickoff Arijit Singh's 10-city tour from Kolkata on 24 Dec '17
Singing sensation Arijit Singh is all set to woo audiences in Kolkata as he kick-starts the 1st ever 10 city India tour on 24th December at New Town Eco Park, 6:30 pm onwards. This Christmas Eve unwind and soak in the festive extravaganza as Royal Stag Mega Music Arijit Singh Live in Concert- MTV India Tour Powered by Wizcraft marks its 10 - city footprint with The City Of Joy.
MTV partners Wizcraft and TM Talent Management for 10 city concert with Arijit Singh
MTV, the brand that introduced India to music television and India's Leading Media and Entertainment Company- Wizcraft International have come together to present the first-ever Royal Stag Mega Music Arijit Singh Live in Concert - The MTV India tour. The concert series, an initiative of GiMA Concerts, is slated to begin on Arijit's home-turf, Kolkata on December 24th and will be held across major cities right up till March 2018.
Droom bags title sponsorship of startup reality show MTV Dropout
From Bill Gates to Sachin Tendulkar, people who have become some of the most iconic figures in our country have achieved what they have only because they had strong vision, resilience and courage to back their passion in achieving that vision. In keeping with the same spirit, Droom, India's pioneering online automobile transactional marketplace has now become the title sponsor for MTV Dropout, India's first start-up reality show that seeks to find out India's next big entrepreneur.
Pepsi MTV Indies and BlueFROG present the second edition of Ribbit


Once upon a time, wicker loveseats served the very important purpose of keeping two individuals appropriately separated while they discreetly conveyed their feelings for one another. Earlier than that, however, these "mini" couches were considered just roomy enough for a lady to sit comfortably surrounded by her voluminous skirts. Today, those old-fashioned sentiments have transitioned beautifully into our collection of wicker seating for two.
Spanning many design eras and inspired by popular originals, all of our rattan loveseats are brand new and are handcrafted utilizing premium grade natural materials. Rattan core fibers and cane are always in fashion along with grasses and rope crafted in tough fibers from the banana family. Sustainably harvested, each featured piece is sure to be an heirloom favorite.
Traditionalists will fall in love with the many coastal-inspired loveseats available. Some feature simple woven patterns while others are more elaborate in their displays of spindling and latticework, which are fine examples of early 1900s furnishings found among the wealthy. From the Art Deco period, subtle diamond patterns inset into loveseat backs and around skirting are also in evidence. Some seating is formal in modular and curved profiles while other pieces are more relaxed with such amenities as magazine racks and cup holders built into the arms.
Finish choices among these classics begin with cottage hues that include white, whitewash and shades of brown. For those wanting to brighten up sunrooms or interior spaces, a palette of vivid shades is also available. These include greens and yellows while more dramatic reds and blacks will certainly make an impact. In between, subtler blues are among the varied neutral selections.
For exotic touches, rattan cane always delivers its island appeal in solid crafting that offers warm natural hues and characteristic markings. Styles vary from relaxed to more formal and from traditional to contemporary, but each has that unique appeal that will turn any spot into an inviting everyday getaway. Open spindling and choices in loose or dense wicker patterns are appealing appointments inspired by faraway places and ocean breezes.
With many of our wicker and cane loveseats, other accent pieces are also available. Side and coffee tables along with arm chairs and accompanying ottomans are available for groupings in smaller areas. For larger spaces, adding a matched sofa is also an option.
In any style, the loveseat or wicker settee is an eternal favorite because of its size, versatility and charm.


Why are more people entering forensic mental health services?
A CAMH study explores the reasons behind the growing number of people involved in the criminal justice system who need treatment for mental illness — and provides insights to inform policy and clinical care.
As part of a unique study collaboration between CAMH and Saugeen First Nation, CAMH's mobile research lab is providing a hub to gather knowledge that will inform the development of a community wellness strategy.
"Like many communities, Saugeen First Nation experiences issues with mental health, substance use and violence," says Ningwakwe George, Field Coordinator on the project and member of Saugeen First Nations. "What affects one, affects all. We feel this, as a community, and we are endeavouring to address it as a community.
Parked next to the James Mason Memorial Culture and Recreation Centre in Saugeen First Nation, the mobile lab is equipped with meeting and interview rooms, as well as computer stations for people to complete an online survey. Until February 2019, four research staff from Saugeen are interviewing community members and providing assistance in filling out the survey. The interviews and surveys are focused on people's experiences related to mental health and substance use, as well as any supports, services, and/or programs they have tried to access.
This project was approved by Band Council and has a local Community Advisory Circle that oversees the research and project activities. The research findings from approximately 30 interviews and 200 to 300 surveys will be shared back to community members of Saugeen First Nation in the spring of 2019.
"At this point, community members will be part of a process to review key research findings to determine a priority area and develop a community mental wellness strategy for and by people who can relate to the priority area," says Melody Morton Ninomiya, CAMH Project Scientist. This approach is known as participatory action research, where members of the community use arts-based methods to determine what is best for the community.
Saugeen First Nation is one of five First Nation communities participating in a larger project on community mental wellness strategies. Two communities have already identified their priority areas, including men's mental health and youth depression and anxiety, while others are in the early stages of creating a Community Advisory Circle and reviewing interview and survey questions. The project is funded by the Ontario Ministry of Health and Long- term Care's Health System Research Fund and is led by co-Principal Investigators Samantha Wells, Julie George, and Renee Linklater at CAMH.
To find out more about this project, please contact Ningwakwe George, Field Coordinator in Saugeen First Nation (226-378-5416 or ningwakwegeorge@camh.ca) or Melody Morton-Ninomiya, Project Scientist (226-378-3891 or melody.mortonninomiya@camh.ca).


39 134 What's next for Nintendo?
Have an original character? Post, discuss, develop them here. Maybe we can see them featured in a game one day.
Discuss them here! If you think a specific game needs its own forum, suggest it.
Discuss our GameGrabber line of video capture devices and signal converters.
Discuss our line of monitors dedicated to gaming.
What's a forum without one of these?
Our members have made a total of 24,794 posts in 24,575 threads.


text:
Johnny Appleseed Day
Yearly Date: Always March 11th or September 26th
"An apple a day keeps anyone away, if you throw it hard enough."
Famous People Born in March
March 1, 1994 – Justin Bieber, Canadian singer-songwriter, dancer
March 2, 1779- Joel Roberts Poinsett, American Ambassador to Mexico brought Poinsettias to America.
March 2, 1904 – Theodore Seuss Geisel, "Dr. Seuss"
March 2, 1917- Desi Arnaz, "Ricky Ricardo" on "I Love Lucy"
March 2, 1931- Mikhail Gorbachev, Leader of USSR
March 2, 1950- Karen Carpenter, singer "We've Only Just Begun"
March 2, 1962- Jon Bon Jovi, singer, actor
March 3, 1847- Alexander Graham Bell, invented the telephone
March 3, 1920 – James Doohan, actor, "Scotty" on TV Series and movies "Star Trek"
March 3, 1962- Jackie Joiner-Kersee, Olympic gold medalist
March 4, 1888- Knute Rockne, Notre Dame's "Fighting Irish" football coach
March 5, 1908- Rex Harrison, actor
March 5, 1936- Dean Stockwell, actor
March 5, 1958- Andy Gibb, member of "Beegees" band
March 5, 1989- Jake Lloyd, actor "Anakin Skywalker" in "Star Wars: Phantom Menace"
March 6, 1475- Michelangelo, Renaissance painter
March 6, 1906- Lou Costello, comedian, of Abbott and Costello
March 6, 1923- Ed McMahon, "Johnny Carson Tonight Show"
March 6, 1926- Alan Greenspan, Chairman of Federal Reserve
March 6, 1945- Rob Reiner, actor, director
March 6, 1959- Tom Arnold, actor
March 6, 1972- Shaquille O'Neal, NBA star
March 7, 1934- Willard Scott, NBC weatherman
March 8, 1945- Mickey Dolenz, singer, actor, "Mickey" of the "Monkees"
March 9, 1934- Yuri Gagarin, Russian astronaut, first man in space
March 9, 1943- Bobby Fischer, World Chess champion
March 10, 1928- James Earl Ray, assassinated Martin Luther King Jr.
March 10, 1940 Chuck Norris, , martial arts, "Walker" in TV series "Walker, Texas Ranger"
March 10, 1957- Osama Bin Laden, , Al Queda terrorist leader
March 10, 1958- Sharon Stone, , actress, "Basic Instinct"
March 10, 1983- Carrie Underwood, , American Idol winner
March 11, 1903- Lawrence Welk, , bandleader
March 11, 1934- Sam Donaldson, TV Journalist
March 12, 1923- Wally Schirra, astronaut
March 12, 1941- Barbara Feldon, actress
March 12, 1946- Liza Minnelli, actress, singer
March 12, 1948- James Taylor, American singer-songwriter
March 12, 1962- Darryl Strawberry, baseball player
March 13, 1855- Percival Lowell, astronomer
March 13, 1911- L. Ron Hubbard, science fiction writer
March 13, 1939- Neil Sedaka, singer, songwriter
March 14, 1864- Casey Jones, railroad engineer
March 14, 1879- Albert Einstein, physicist
March 14, 1912- Les Brown, and his "Band of Renown"
March 14, 1920- Hank Ketcham, cartoonist, created "Dennis the Menace"
March 14, 1928- Frank Borman, astronaut, airline executive
March 14, 1933- Michael Caine, actor
March 14, 1947- Billy Crystal, actor, comedian
March 15, 1767 – Andrew Jackson, 7th U.S. President(1829-1837), hero of the War of 1812
March 15, 1933- Ruth Bader Ginsburg, Supreme Court Justice
March 15, 1935- Judd Hirsch, actor, TV series "Taxi"
March 15, 1941- Mike Love, singer, musician, Beach Boys
March 15, 1961- Fabio, actor, model
March 15, 1975- Eva Longoria, actress, TV series "Desperate Housewives"
March 16, 1751 – James Madison, 4th U.S President(1809-1817)
March 16, 1822- Rosa Bonheur, French painter
March 16, 1906- Henny Youngman, comedian
March 16, 1912- Pat Nixon, Wife of Richard M. Nixon, First Lady
March 16, 1926- Jerry Lewis, actor, comedian
March 16, 1949- Erik Estrada, actor
March 17, 1902- Bobby Jones, golfer
March 17, 1919- Nat "King" Cole, singer
March 17, 1938- Rudolf Nureyev, Russian ballet dancer
March 17, 1945- Tony Dow, actor, brother "Wallie" on TV series "Leave it to Beaver"
March 17, 1949- Patrick Duffy, actor "Bobby Ewing" on TV Series "Dallas"
March 17, 1951- Kurt Russell, actor
March 17, 1964- Rob Lowe, actor
March 18, 1782- John C. Calhoun, statesman, vice president
March 18, 1837- Grover Cleveland, 22d and 24th U.S. President(1885-1889, 1893-1897)
March 18, 1869- Neville Chamberlain, British Prime Minister
March 18, 1926- Peter Graves, actor
March 18, 1939- Charley Pride, singer
March 18, 1941- Wilson Pickett, singer, songwriter
March 18, 1963- Vanessa Williams, singer, actress
March 18, 1964- Bonnie Blair, Olympic speed skating champion
March 18, 1970- Queen Latifah, rap singer, actress
March 19, 1589- William Bradford, , Pilgrim/Governor of Plymouth Colony
March 19, 1813- David Livingstone, , physician, explorer
March 19, 1848- Wyatt Earp, , Wild West lawman
March 19, 1891- Earl Warren, chief justice of the Supreme Court
March 19, 1906- Adolf Eichmann, Nazi leader
March 19, 1936- Ursula Andress, actress
March 19, 1947- Glenn Close, actress
March 19, 1955- Bruce Willis, actor
March 20, 1811- Napoleon Bonaparte II, Emperor of France and King of Rome
March 20, 1906- Ozzie Nelson, Actor, "The Nelsons"
March 20, 1922- Carl Reiner, actor, comedian
March 20, 1928- Fred Rogers, actor Children's show "Mr. Rogers"
March 20, 1931- Hal Linden, actor, singer
March 20, 1939- Brian Mulroney, Canadian Prime Minister
March 20, 1948- Bobby Orr, hockey player
March 20, 1957- Spike Lee, director, filmmaker
March 21, 1685- Johann Sebastian Bach, , German musical composer
March 21, 1910- Julio Gallo, , California wine vintner
March 21, 1944- Timothy Dalton, , actor
March 21, 1962- Matthew Broderick, actor
March 21, 1962- Rosie O'Donnell, TV personality, actress
March 22, 1887- Chico Marx , of Marx Brothers
March 22, 1913- Karl Malden, actor
March 22, 1919- Werner Klemperner, actor
March 22, 1923- Marcel Marceau, French mime
March 22, 1930- Pat Robertson, , evangelist, presidential candidate
March 22, 1931- William Shatner, , actor James T. Kirk, on Star Trek
March 22, 1934- Orrin Hatch, , senator
March 22, 1943- George Benson, , singer, musician
March 22, 1948- Andrew Lloyd Webber, , English composer
March 22, 1952- Bob Costas, sportscaster
March 22, 1959- Matthew Modine, actor
March 23, 1904- Joan Crawford, Oscar winning actress
March 23, 1900- Erich Fromm, psychoanalyst
March 23, 1912- Werner von Braun, rocket pioneer, scientist
March 23, 1929- Roger Bannister, runner, first person to break 4-minute mile
March 23, 1953- Louie Anderson, comedian, actor
March 23, 1953- Chaka Khan, singer
March 24, 1855- Andrew Mellon, financier, industrialist
March 24, 1874- Harry Houdini, magician
March 24, 1893- George Sisler, baseball player
March 24, 1930- Steve McQueen, actor
March 24, 1970- Lara Flynn Boyle, actress
March 24, 1973- Jim Parsons, American actor, "Sheldon" on TV show "Big Bang Theory"
March 24, 1976- Peyton Manning, Indianapolis Colts All Star Quarterback
March 25, 1871- Gutzon Borglum, sculpted Mt. Rushmore
March 25, 1918- Howard Cosell, American Sportscaster
March 25, 1934- Gloria Steinem, author
March 25, 1942- Aretha Franklin, singer
March 25, 1922- Paul Michael Glaser, actor, director
March 25, 1947- Elton John, English singer, songwriter, musician
March 25, 1965- Sarah Jessica Parker, actress
March 25, 1982- Danica Patrick, American NASCAR race car driver
March 26, 1874- Robert Frost, poet
March 26, 1911- Tennessee Williams, playwright
March 26, 1930- Sandra Day O'Connor, Supreme Court justice
March 26, 1931- Leonard Nimoy, actor, director, "Spock" in "Star Trek"
March 26, 1934- Alan Arkin, actor
March 26, 1939- James Caan, actor
March 26, 1940- Nancy Pelosi, American Democratic party congresswoman, Speaker of the House
March 26, 1944- Diana Ross, singer, actress
March 26, 1950- Martin Short, actor, comedian
March 26, 1960- Marcus Allen, NFL running back, sportscaster
March 26, 1962- John Stockton, basketball player
March 27, 1845- Wilhelm Roentgen, scientist, discovered X-rays
March 27, 1899- Gloria Swanson, actress
March 27, 1931- David Janssen, actor, "The Fugitive"
March 27, 1940- Cale Yarborough, auto racer
March 27, 1963- Randall Cunningham, NFL Quarterback
March 27, 1963- Quentin Tarantino, actor, director
March 27, 1970- Mariah Carey, American singer and actress
March 28, 1899- August Anheuser Busch Jr.,Beer baron
March 28, 1921- Dirk Bogarde, actor
March 28, 1944- Ken Howard, actor
March 28, 1955- Reba McEntire, country western singer
March 28, 1981- Julia Stiles, actress
March 28, 1986- Lady Gaga, American singer, songwriter, actress
March 29, 1790- John Tyler, 10th U.S. President (1841-1845)
March 29, 1867- Cy Young, baseball pitcher
March 29, 1916- Eugene McCarthy, senator, presidential nominee
March 29, 1918- Pearl Bailey, singer, entertainer
March 29, 1918- Sam Walton, founded Wal-Mart
March 29, 1945- Walt Frazier, basketball player
March 29, 1955- Earl Campbell, football player
March 29, 1963- M. C. Hammer, rap singer
March 29, 1964- Elle MacPherson, Australian actress, super model
March 29, 1968- Lucy Lawless, actress
March 29, 1976- Jennifer Capriati, tennis player
March 30, 1853- Vincent van Gogh, Dutch painter
March 30, 1929- Richard Dysart, actor
March 30, 1930- John Astin, actor
March 30, 1937- Warren Beatty, actor, director
March 30, 1945- Eric Clapton, musician, singer, songwriter
March 30, 1957- Paul Reiser, actor
March 30, 1962- M.C. Hammer, rapper
March 30, 1968- Celine Dion, singer
March 31, 1596- René Descartes, philosopher, mathematician
March 31, 1732- Franz Joseph Haydn, composer
March 31, 1811- Robert Wilhelm Eberhard von Bunsen, invented the Bunsen Burner
March 31, 1878- Jack Johnson, first Afro-American boxer to win heavyweight crown
March 31, 1927- Cesar Chavez, labor leader
March 31, 1928- Gordie Howe, hockey player
March 31, 1929- Liz Claiborne, fashion designer
March 31, 1932- John Jakes, author
March 31, 1934- Shirley Jones, actress, singer
March 31, 1935- Herb Albert, musician
March 31, 1935- Richard Chamberlain, actor
March 31, 1943- Christopher Walken, actor
March 31, 1948- Al Gore Jr., vice president , presidential candidate
March 31, 1948- Rhea Perlman, actress


For a year, I have been choked, on grief for the loss of my best friend, on worry for this country, on epilepsy, on depression, on sleep disorder, on stress, you name it.
I was so choked, thatI was near blacking out.
Without realizing it, I forgot what it was like to breathe at all, so much so that I stopped fighting for air.
And I thought that was living.
I forgot what it was like to be the person I've been for forty years. I forgot what it was like to enjoy, to feel, to laugh, to love, to hope, to live!
I was so excited for my Mom to finally hear Melinda sing, but I was just hoping to manage a smile for Melinda. I knew the magic wouldn't be there for me. All the light was gone from the world, all the beauty erased by too much negativity, and bitterness in world events, and no matter how incredible, one voice couldn't hope to combat all the angst and hate of 2016. For me personally, my unsinkable ship of optimism was looking more and more like the Titanic, as health problem after health problem pulled me down.
It was going to break my heart, to listen to her sing, and realize that the last place where magic and beauty might be left in this world, would be empty of it. But you do things for those you love, and so I would go, and I would smile. I just prayed I could bluff good enough for her, so she wouldn't feel bad and think it was her fault if I couldn't love the concert.
Melinda is very much in tune with, and responsive to, her audience, doubly so if she has met you in person. It really will disappoint her if she thinks you aren't enjoying the show. I wanted to warn her, it's not you Melinda, it's me, there is something wrong with me now, there is everything wrong with me now. Instead, I said a quick prayer that whatever was wrong with me would not have any effect on her, and off I went.
And then began the music.
The band grooved on the opening bass line of a Stevie wonder song . Those notes and rhythms beckoned me, but it was like hearing sounds from the depths of sleep, where you have no concept of what it is to be awake. Something was calling to me from that music, something distant and sweetly remembered. I just couldn't identify what it was. And then . . .
The first breath of air after you thought there would be no more, sweet . . powerful . . life-giving!
It was startling, like the first pain-free moment after an agonizing toothache, when you suddenly remember that there is more to the world than pain.
Had I really been foolish enough to think one voice couldn't beat back all the bad in this world?
OH YES IT COULD, if it was the voice of Melinda, if it was a voice so full of love for the music and the people, that all else dissolved in the face of its sincerity.
Hopelessness fled, in the face of scorching vocals and a mighty groove. And then I remembered, the thing that beckoned to me was feeling! It was life!
And when she reminded me that no matter the adversity, there was hope, with "A Change is Gonna Come," I gave her a standing ovation . . . all by myself!
Well, a Melinda show isn't a Melinda show without a backup, right? In truth, I was trying to get my Mom to stand up, in hopes that since we were in the front row, if everyone saw us standing, they would stand too.
It didn't work out that way, but Melinda appreciated it enough that she said hi to me from the stage!
I was clapping for the song, for the power of its lyrics, and certainly above all, for Melinda.
But I was also standing and clapping as a Thank You to her, because finally, after more than a year, there was something that made me want to stand up and cheer. So instead of wanting to do it, I did it! If you know me, you know that my feeling about drawing attention to myself is the same as what flies feel about flypaper.
So this was a double shock in that regard. But artists offer you a piece of their soul when they give you their art. They do so, at risk of criticism and maybe worse, apathy. Well by God, that song made me feel something! It deserved a standing ovation, so that's what it received!
As if we needed more proof that "girl can SANG", during "I'm a Woman," she came down off the stage, walked around, and hugged me, while still singing! How do you sing and hug at the same time? I would fall on my face if I tried it!
There was an intermission, and when Melinda returned to the stage, my Mom told me she had changed into a beautiful blue and gold dress. Mom said it reminded her of something Egyptian, and that gave me some lovely imagery to go along with the sounds.
Now there were silky soul-stirring vocals, ballads like "Somewhere Over the Rainbow,", "Home", and "Midnight Train to Georgia." I could literally FEEL the music stripping away all the darkness. It was no cliché!
I floated on that sea of sound, wishing I could stay in it forever. And then she sang "Toxic," a Postmodern Jukebox torch song that blew me away! Please do yourself a favor, and google "Toxic by Melinda Doolittle, youtube." It will be well worth the extra step for you to hear the song.
The concert ended with Gloria Gaynor's "I Will survive," one of my alltime Melinda favorites.
It made me want to dance!
Y'all, I dance like a blind white girl, so it would have been at least a misdemeanor for this to occur in public, but the fact that I even wanted to dance was a miracle.
After the show, Melinda came over, gave me a hug, and took pictures. She was as gracious and humble as always, and this is her most endearing quality, of many. She introduced me to her band, and I left that auditorium a different person than when I walked in.
With your songs, you restored me to myself.
You let me breathe, and feel, and hope, and live again.
I will never again doubt the power, or the magic, of your music.