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Michael Wolf, The exhibition is curated by Dr. Sabine Schnakenberg and will be shown at the House of Photography at the phototriennale. From May 10 through July 22, , great photographic work is at home at Fondazione Stelline with the first Italian retrospective exhibition devoted to Michael Wolf. From May 10 through July 22, , the halls in Corso Magenta 61 will host Life in Cities, the first major exhibition devoted to Michael Wolf ever held in Italy, the German artist and photographer who developed his talent under the guidance of master Otto Steinert at the Folkwang School in Essen and at Berkeley University in California.

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The identification of such factors may help to elucidate mechanisms leading to enhanced cardiac differentiation efficiency in vitro as well as promote regeneration in vivo. We analyzed the role of this interaction for early cardiogenesis in an in vitro model by making use of embryoid body cultures from mouse embryonic stem cells ESCs. In this model, stable FHL2 gain-of-function promoted mesodermal cell formation and cell proliferation while arresting cardiac differentiation in an early cardiogenic mesodermal progenitor state.

This resulted in enhanced cardiogenesis. Collectively, our findings offer mechanistic insight into the early cardiogenic code and may be further exploited to enhance cardiac progenitor cell activity in vitro and in vivo. S tem C ells ;— Temporal control of canonical Wnt signaling is essential for normal gastrulation and subsequently germ layer segregation in the early epiblast [ 4 , 5 ]. Later during development, the canonical Wnts promote cardiogenesis during mesoderm induction, but act as inhibitors of committed cardiac progenitor cells in vivo and in vitro [ 6 , 7 ].

Tight control of Wnt-mediated gene expression is critical for the regulation of cell-specific downstream effectors and essential for cardiac cell development. Although the role of the canonical Wnt pathway in cardiogenesis is now explained in a highly complex multiphasic model, the precise regulation of this pathway remains to be investigated.

Several studies suggest that members of the LIM-only protein family may act as coregulators of tissue-specific gene expression by interacting with different transcription factors. FHL2 was initially identified as downregulated in rhabdomyosarcomas LIM domain protein and characterized for its abundant expression in the human heart [ 10 ].

FHL2 contains a cysteine-rich consensus sequence, which is known as a potent protein-protein interaction motif regulating a variety of cell processes [ 11 ]. Despite the strong expression of FHL2 in the heart, the role of the protein in cardiac cells remains unclear. Notably, FHL2-deficient mice do not show a spontaneous cardiac phenotype [ 13 ]. This may be explained by the role of FHL2 in coupling titin and calcineurin activity to metabolic enzymes [ 16 , 17 ]. Currently, no data are available concerning the transcriptional role of FHL2 in cardiomyocyte development.

To explore the role of this interaction in cardiogenesis, we used embryonic stem cell ESCs and embryonic carcinoma cell P19 embryoid body EB cultures, which can consistently recapitulate the sequences of cardiac genes expression observed in early cardiac development in the mouse embryo [ 18 ].

We observed that stable FHL2 gain-of-function in ESC-EBs promoted mesodermal cell formation and abrogated cardiac differentiation by locking cardiac mesodermal cells in a progenitor state.

Moreover, transient overexpression of FHL2 in PEBs led to enhanced cardiac progenitor cell activity with subsequent augmentation of cardiomyocyte differentiation. Frey, Kiel, Germany. The riboprobes were generated by ClaI digestion of the cmyc- Fhl2 plasmid kindly provided by Dr.

After 5 days, cells were plated on 0. EBs containing beating areas were counted and presented in percent of total EBs.

P19 were transfected with a cmyc- Fhl2 -expressing as well as an empty vector and cultured as previously described [ 22 ]. Aggregates were plated onto 0. Neonatal rat cardiomyocytes NRCMs were prepared as described previously [ 23 ]. Transfection was performed using FuGene Roche according to manufacturer's instruction. ESCs were differentiated on 0. AlexaFluor and AlexaFluorconjugated secondary antibodies ; Invitrogen were used for labeling. Luciferase activity was determined using dual-luciferase reporter assay Promega U.

Respective isotype controls were used. Fluorescence signals were detected with a Calibur flow cytometer BD. Copy numbers were calculated using the iCycler software with a relative standard curve obtained using the log dilutions of gene of interest cDNA. All reactions were run in triplicates and normalized to gapdh.

Primers are listed in supporting information Table S1. Detection was done by immunoblotting using a c-myc Santa Cruz antibody. Protein lysate transfected with empty vector served as control. Whole cell lysates were immunoblotted with respective antibodies to detected protein expression. Densitometric analysis was performed using Adobe Photoshop software.

Differences between experimental groups were analyzed using two-tailed Student's t test or ANOVA test followed by Bonferroni's multiple comparison test. To corroborate the relevance of FHL2 in the cardiovascular system, expression analysis of FHL2 was performed in different adult organs.

In agreement with previous reports, we observed prominent expression of FHL2 protein in adult heart, skeletal muscle, and kidney Fig. Comparative analysis in embryonic, fetal, and postnatal cardiac tissue showed Fhl2 transcript expression as early as embryonic day ED 7.

B : qPCR analysis of Fhl2 in embryonic cardiac developing tissue, early postnatal, and adult mouse heart. Relative mRNA levels were normalized to tumor protein, translationally controlled 1 Tpt1 , a gene expressed with insignificant variation along development and adulthood.

Both proteins were also detected in protein lysates from NRCM cultures. Three positive clones were selected for verification of transgene integration by Southern blotting supporting information Fig. S1 A, S1B ; clone 8 was selected for further analysis. S1 C and Western blot analyses supporting information Fig. A : Schematic representation of the 16 days differentiation protocol for in vitro cardiogenesis using ESCs. Differentiating ESCs were harvested for analysis following 3, 7, and 16 days of differentiation.

DAPI blue was used for nuclear staining. Semiquantitative assessment via densitometry in three independent experiments. Formation of cardiomyocytes can be readily identified and approximated by enumeration of spontaneous contracting EBs. This was further substantiated by the observation of reduced expression of cardiomyocyte-specific genes myosin light chain [Mlc] 2a and cTnt at day 16 of differentiation Fig.

These data suggest an abrogation of the cardiac differentiation program upon stable FHL2 overexpression. A : Schematic representation of the day differentiation protocol for in vitro cardiogenesis. Since FHL2 was reported to be necessary for regulating skeletal myoblast cell populations [ 8 , 25 ], we next analyzed whether FHL2 would also regulate early cardiac gene activation during ESCs differentiation.

At day 7 of differentiation, higher gene expression of early cardiogenic progenitor markers such as insulin growth factor binding protein 5 Igfbp5 , Hand1, Tbx5, and Nkx2. Moreover, a continuously elevated transcript abundance of these markers was detected also at day 16 of ESC-EB culture, despite the anticipated downregulation of Igfbp5 and hand1in differentiating ESC-EBs.

Higher NXK2. Collectively, these results indicate that expression of FHL2 blocks cardiac differentiation by arresting the cells in an early progenitor stage.

A : Expression of the early cardiogenic lineage genes Igfbp5, Hand1, Nkx2. Therefore, we first examined changes in gene expression of differentiation markers at day 3 of ESCs-differentiation. Gene expression analysis at 7 days of differentiation showed significant lower expression of the pluripotent marker Oct4 as well as Brachyury and Mesp1 upon FHL2 gain-of-function supporting information Fig. C : FHL2 overexpression promoted significantly augmented expression of the cell cycling marker Cyclin D1 as shown by qPCR as well as increased Ki67 expression as demonstrated by immunofluorescence microscopy percentage of total DAPI-positive cells following 7 and 16 days of differentiation.

Since FHL2 was reported to have a role in cell cycle regulation in noncardiac cell types [ 26 ], we compared surrogate markers for cell proliferation in FHL2-overexpressing and wild-type ESC-EB cultures.

Collectively, these data indicate that FHL2 stimulates formation and expansion of early mesodermal cells at the expense of endodermal cells. Therefore, we asked whether the prevention of this transcriptional activation after mesodermal specification would result in terminal cardiac differentiation of ESCs overexpressing FHL2.

Furthermore, the observed increase in NKX2. Cells were harvested at day 16 of differentiation for analysis. D : Flow cytometry shows a rescue in upregulation of NKX2. To confirm that transient overexpression of FHL2 during cell commitment would enhance mesodermal cell lineage commitment and subsequently allow for enhanced cardiomyocyte differentiation, we expressed FHL2 transiently in P19 cells Fig. As anticipated, loss in Oct4 expression as well as increased expression of the early cardiogenic transcripts Igfbp5, Tbx5, Nkx2.

Augmented expression of Cyclin D1 and of the proliferation marker KI67 was also found in FHL2-P19 cells in comparison to control cells as shown by qPCR and confocal immunofluorescence analysis, respectively supporting information Fig. S4 D, S4E. Along with this finding, significant loss of mesodermal gene expression Brachyury, Flk1, and Mesp1; supporting information Fig. Transient overexpression of FHL2 enhances cardiogenesis in P19 cells in vitro. A : Schematic representation of differentiation protocol for in vitro cardiogenesis.

P19 cells were differentiated in the presence of Dimethyl sulfoxide DMSO and harvested for analysis following 1, 2, and 10 days of differentiation. Significant loss of the pluripotent marker Oct4 and increase of the early cardiogenic markers Igfbp5, Nkx2.

Representative pictures are depicted. Tight regulation of Wnt signaling is essential for heart development and cardiac homeostasis [ 7 , 33 , 34 ]. Identification of cofactors regulating the Wnt canonical pathway may help to increased cardiac differentiation efficiency.

This is in line with previous reports showing Fhl2 expression in the earliest myocardial progenitor cells of the embryonic cardiac crescent [ 36 ]. Fhl2 expression increased steadily during fetal and postnatal heart development with a peak in the adult heart. Notably, FHL2 protein expression in adult cardiac tissue was particularly high as compared to other organs. Similar observations were reported by Chu et al.

Although these data collectively suggest a role for FHL2 in cardiac development and adult heart homeostasis, genetic deletion of FHL2 did not result in obvious cardiac abnormalities [ 13 ]. In contrast, FHL3 showed low ubiquitous expression during heart development [ 14 ]. Notably, FHL1-deficient mice did not exhibit a cardiovascular phenotype [ 37 ]; FHL3-deficient mice appear to be not available, yet.

We acknowledge that double or even triple knockout models for the three described FHL-family members would be ideal to gain complete insight into their transcriptional role in heart development.

These data suggest the specific role of FHL2 in Wnt canonical transcriptional regulation in the heart. Whether this is the case also for diseased myocardium remains to be evaluated. ESCs can be cultured as EBs and are able to form the germ layers normally formed during gastrulation. Under appropriate conditions, ESCs have a high propensity for in vitro cardiogenesis and thus represent a robust model system for cardiogenesis [ 18 ].

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The identification of such factors may help to elucidate mechanisms leading to enhanced cardiac differentiation efficiency in vitro as well as promote regeneration in vivo. We analyzed the role of this interaction for early cardiogenesis in an in vitro model by making use of embryoid body cultures from mouse embryonic stem cells ESCs.

Either your web browser doesn't support Javascript or it is currently turned off. In the latter case, please turn on Javascript support in your web browser and reload this page. The identification of such factors may help to elucidate mechanisms leading to enhanced cardiac differentiation efficiency in vitro as well as promote regeneration in vivo. We analyzed the role of this interaction for early cardiogenesis in an in vitro model by making use of embryoid body cultures from mouse embryonic stem cells ESCs. In this model, stable FHL2 gain-of-function promoted mesodermal cell formation and cell proliferation while arresting cardiac differentiation in an early cardiogenic mesodermal progenitor state.

Dr. med. Britta Lange- Manchot Fachärztin f. Allgemeinmedizin - Hamburg

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Нахмурившись, Беккер набрал второй номер. И на другом конце сразу же сняли трубку.

Keine Ursache. Беккер вышел в коридор. Нет проблем. А как же проваливай и умри.

Прошло еще несколько минут. Она пыталась не думать о Дэвиде, но безуспешно. С каждым завыванием сирены слова Хейла эхом отдавались в ее мозгу: Я сожалею о Дэвиде Беккере. Сьюзан казалось, что она сходит с ума.

Кто-то записал его, и я подумал, что это гостиница. Я здесь проездом, из Бургоса. Прошу прощения за беспокойство, доброй вам но… - Espere. Подождите! - Сеньор Ролдан был коммерсантом до мозга костей. А вдруг это клиент.

Цепная мутация. Она знала, что цепная мутация представляет собой последовательность программирования, которая сложнейшим образом искажает данные. Это обычное явление для компьютерных вирусов, особенно таких, которые поражают крупные блоки информации.

Из почты Танкадо Сьюзан знала также, что цепные мутации, обнаруженные Чатрукьяном, безвредны: они являются элементом Цифровой крепости. - Когда я впервые увидел эти цепи, сэр, - говорил Чатрукьян, - я подумал, что фильтры системы Сквозь строй неисправны.

Но затем я сделал несколько тестов и обнаружил… - Он остановился, вдруг почувствовав себя не в своей тарелке.

Jun 27, - partners were Otto Dicker and Otto Scheffen its first typewriter. Dr. Thompson's soap powder factories Dr. Willy Manchot,. Sigrid Manchot, Andreas Heinrich Thorbecke and Dr. Ernst Petersen Andreas Lange. –

Код страны - 1. Действительно хорошая новость. ГЛАВА 54 - Пусти. А потом раздался нечеловеческий крик. Это был протяжный вопль ужаса, издаваемый умирающим зверем.

- Он сказал, что на кольце были выгравированы какие-то буквы. - Буквы. - Да, если верить ему - не английские.  - Стратмор приподнял брови, точно ждал объяснений.

На военную информацию. Тайные операции. Джабба покачал головой и бросил взгляд на Сьюзан, которая по-прежнему была где-то далеко, потом посмотрел в глаза директору.

Повсюду разбросаны грязные бумажные полотенца, лужи воды на полу. Старая электрическая сушилка для рук захватана грязными пальцами.

Повзрослев, он начал давать компьютерные уроки, зарабатывать деньги и в конце концов получил стипендию для учебы в Университете Досися. Вскоре слава о фугуся-кисай, гениальном калеке, облетела Токио. Со временем Танкадо прочитал о Пёрл-Харборе и военных преступлениях японцев.

Ненависть к Америке постепенно стихала. Он стал истовым буддистом и забыл детские клятвы о мести; умение прощать было единственным путем, ведущим к просветлению.

Но Беккер не ощутил боли. Неожиданно он оказался на открытом воздухе, по-прежнему сидя на веспе, несущейся по травяному газону. Задняя стенка ангара бесследно исчезла прямо перед. Такси все еще двигалось рядом, тоже въехав на газон. Огромный лист гофрированного металла слетел с капота автомобиля и пролетел прямо у него над головой.

Дэвид Беккер и два оперативных агента тоже пробовали сделать это, сидя в мини-автобусе в Севилье. ГЛАВНАЯ РАЗНИЦА МЕЖДУ ЭЛЕМЕНТАМИ, ОТВЕТСТВЕННЫМИ ЗА ХИРОСИМУ И НАГАСАКИ Соши размышляла вслух: - Элементы, ответственные за Хиросиму и Нагасаки… Пёрл-Харбор. Отказ Хирохито… - Нам нужно число, - повторял Джабба, - а не политические теории.

Comments: 1
  1. Kakora

    I hope, it's OK

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