In vivo importance of homologous recombination DNA repair for mouse neural stem and progenitor cells

We characterized the in vivo importance of the homologous recombination factor RAD54 for the developing mouse brain cortex in normal conditions or after ionizing radiation exposure. Contrary to numerous homologous recombination genes, Rad54 disruption did not impact the cortical development without exogenous stress, but it dramatically enhanced the radiation sensitivity of neural stem and progenitor cells. This resulted in the death of all cells irradiated during S or G2, whereas the viability of cells irradiated in G1 or G0 was not affected by Rad54 disruption. Apoptosis occurred after long arrests at intra-S and G2/M checkpoints. This concerned every type of neural stem and progenitor cells, showing that the importance of Rad54 for radiation response was linked to the cell cycle phase at the time of irradiation and not to the differentiation state. In the developing brain, RAD54-dependent homologous recombination appeared absolutely required for the repair of damages induced by ionizing radiation during S and G2 phases, but not for the repair of endogenous damages in normal conditions. Altogether our data support the existence of RAD54-dependent and -independent homologous recombination pathways.     

Evidence for Chronic Low-Grade Systemic Inflammation in Individuals with Agoraphobia from a Population-Based Prospective Study

Background

Anxiety disorders have been linked to an increased risk of incident coronary heart disease in which inflammation plays a key pathogenic role. To date, no studies have looked at the association between proinflammatory markers and agoraphobia.

Methods

In a random Swiss population sample of 2890 persons (35-67 years, 53% women), we diagnosed a total of 124 individuals (4.3%) with agoraphobia using a validated semi-structured psychiatric interview. We also assessed socioeconomic status, traditional cardiovascular risk factors (i.e., body mass index, hypertension, blood glucose levels, total cholesterol/high-density lipoprotein-cholesterol ratio), and health behaviors (i.e., smoking, alcohol consumption, and physical activity), and other major psychiatric diseases (other anxiety disorders, major depressive disorder, drug dependence) which were treated as covariates in linear regression models. Circulating levels of inflammatory markers, statistically controlled for the baseline demographic and health-related measures, were determined at a mean follow-up of 5.5 ± 0.4 years (range 4.7 – 8.5).

Results

Individuals with agoraphobia had significantly higher follow-up levels of C-reactive protein (p = 0.007) and tumor-necrosis-factor-α (p = 0.042) as well as lower levels of the cardioprotective marker adiponectin (p = 0.032) than their non-agoraphobic counterparts. Follow-up levels of interleukin (IL)-1β and IL-6 did not significantly differ between the two groups.

Conclusions

Our results suggest an increase in chronic low-grade inflammation in agoraphobia over time. Such a mechanism might link agoraphobia with an increased risk of atherosclerosis and coronary heart disease, and needs to be tested in longitudinal studies.     

Computational Modelling of Metastasis Development in Renal Cell Carcinoma

The biology of the metastatic colonization process remains a poorly understood phenomenon. To improve our knowledge of its dynamics, we conducted a modelling study based on multi-modal data from an orthotopic murine experimental system of metastatic renal cell carcinoma. The standard theory of metastatic colonization usually assumes that secondary tumours, once established at a distant site, grow independently from each other and from the primary tumour. Using a mathematical model that translates this assumption into equations, we challenged this theory against our data that included: 1) dynamics of primary tumour cells in the kidney and metastatic cells in the lungs, retrieved by green fluorescent protein tracking, and 2) magnetic resonance images (MRI) informing on the number and size of macroscopic lesions. Critically, when calibrated on the growth of the primary tumour and total metastatic burden, the predicted theoretical size distributions were not in agreement with the MRI observations. Moreover, tumour expansion only based on proliferation was not able to explain the volume increase of the metastatic lesions. These findings strongly suggested rejection of the standard theory, demonstrating that the time development of the size distribution of metastases could not be explained by independent growth of metastatic foci. This led us to investigate the effect of spatial interactions between merging metastatic tumours on the dynamics of the global metastatic burden. We derived a mathematical model of spatial tumour growth, confronted it with experimental data of single metastatic tumour growth, and used it to provide insights on the dynamics of multiple tumours growing in close vicinity. Together, our results have implications for theories of the metastatic process and suggest that global dynamics of metastasis development is dependent on spatial interactions between metastatic lesions.     

Analyse der schlagauslösenden Bewegungsparameter einer punktförmigen Beuteattrappe bei der Aeschnalarve

Zusammenfassung Bei der Libellenlarve Aeschna cyanea M. werden die einzelnen, für die Auslösung des Fangschlags relevanten Bewegungsparameter einer punktförmigen Beuteattrappe und die wirksamste Kombination dieser Parameter bestimmt.Als Beiz dient der beliebig bewegbare Leuchtpunkt eines Oszillographen, der auf die ebene Mattscheibe eines Versuchsaquariums projiziert wird. Die Schläge der frei beweglichen Larve werden vom Beobachter gezählt oder elektrisch registriert.Die Bahngeschwindigkeit von kontinuierlich gebotenen Bewegungsreizen wirkt sich stark auf die Schlagzahl aus: Die Schläge nehmen von 0,005–2,5 cm/sec zu, nehmen von 5,1 cm/sec an wieder ab und hören bei 41,0 cm/sec ganz auf. Die Veränderung der mittleren Geschwindigkeit von Sinusschwingungen und Zufallsbewegungen bewirkt ähnliche Reaktionskurven wie die Veränderung der gleichmäigen Geschwindigkeit von Dreiecksschwingungen und kreisförmigen Bewegungen; eine gleichförmige Optimalgesohwindigkeit wird jedoch stärker beantwortet als eine periodisch schwankende.Bietet man eine Folge von diskontinuierlichen Bewegungsreizen, die nach einer einmaligen Durchquerung eines begrenzten Feldes der Projektionsfläche verschwinden, so spielt die Dauer einer Einzelbewegung eine Rolle. Um die Schläge voll in Gang zu bringen, müssen eindimensionale Schwingungen 3–6 sec dauern, ein viel intensiver wirkender zweidimensionaler Reiz (Zickzackbewegung) jedoch nur 0,8 sec.Im optimalen, relativ hohen Geschwindigkeitsbereich läßt die Erhöhung der Bewegungsamplitude von 0,25 auf 2,0 cm die Schlagzahl progressiv absinken. Der Vergleich zwischen diesen Amplituden und dem Öffnungswinkel des frontalen, die Beute fixierenden Ommatidienfeldes zeigt, daß der Leuchtpunkt nur bei sehr kleinen Ablenkungen die frontalen Rezeptoren kontinuierlich reizt. — Die Bevorzugung von raschen Bewegungsreizen mit kleiner Amplitude besteht nicht bei Larven, die durch prompte Fixier- und Folgereaktionen den Leuchtpunkt in ihrer frontalen Fixierebene bewahren.Der Vergleich zwischen den 4 in dieser Untersuchung erzeugten Bewegungsmustern (ein- und zweidimensionale Schwingungen, Kreis- und Zufallsbewegungen) zeigt, daß eine Bewegung um so mehr Schläge auslöst, je vollständiger sie auf dicht aneinanderliegenden, zweidimensionalen Bahnen das frontale Ommatidienfeld abtastet.Die optimale Reizkombination (Zickzackbewegung) besteht aus einer kleinen (0,2–0,4 cm) Vertikalschwingung mit optimaler Geschwindigkeit, die sich langsam (0,32 cm/sec) seitlich verschiebt. Dieser Reiz stellt die Verbindung der optimalen Werte aller Bewegungsparameter dar und bewirkt, daß pro Zeiteinheit eine möglichst große Zahl frontaler Rezeptoren mit der optimalen Bahngeschwindigkeit gereizt wird.

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Analysis of the parameters of a moving lightspot which release the predatory strike in dragonfly larvae

Summary This study analyses the predatory strike response of the dragonfly larva Aeschna cyanea M. towards a moving spot of light. Its aim is to determine the single parameters of movement of the spot which release the strike and the most effective combination of these parameters.As the velocity of a continuously moving lightspot is increased the number of strikes rises to a maximum (at 2.5 cm/sec) and then declines to 0 (at 41.0 cm/sec). Both uniform and non uniform velocities give curves of similar shape but different magnitudes.In the presentation of a sequence of discontinuous movements (where the spot moves across a part of the screen and then disappears) the duration of a single movement is important: Unidimensional oscillations must last 3 to 6 sec in order to release predatory strikes; twodimensional zigzag movements, much more effective, need last only 0.8 sec.In the optimal velocity range, increasing the amplitude of a movement from 0.25 to 2.00 cm produces a progressive decrease of the response rate. The comparison between these amplitudes and the size of the field of the frontal ommatidia, which fixate the prey, suggests that the spot stimulates these receptors continuously only when it moves with small amplitudes. — However, this preference for small amplitudes does not exist in those individuals which have rapid fixation- and following-reactions and which thus can track the stimulus.Comparison between the four patterns of movement which have been presented (one- and two-dimensional oscillations, circular and random movements) suggests that the spot releases more strikes the more exactly its movement covers the frontal fixation plane.The most effective stimulus for eliciting strikes was found to be a twodimensional zigzag oscillation. This movement consists of a small (0.2–0.4 cm), rapid (2.5 cm/ sec), vertical oscillation, which progresses slowly (0.3 cm/sec) sideways. This movement combines the optimal values of all parameters and stimulates with the optimal velocity the greatest possible number of frontal receptors in a given time.

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Diese Arbeit wurde in Seewiesen mit Dr. H. C. Howland begonnen und dank der fortwährenden, großzügigen Unterstützung von Herrn Dr. H. Mittelstaedt beendet. Frau L. Dinnendahl fertigte die Zeichnungen an und durchsah das Manuskript, Dr. E. Kramer und Herr P. Heinecke standen mir ständig in technischen Fragen bei, Herr E. Butenandt leistete wertvolle Kritik am Manuskript. — In Genf gewährte mir Prof. J. Piaget vollkommene Freiheit in der Gestaltung meiner Arbeit. — Ihnen allen sei an dieser Stelle herzlichst gedankt.     

Fanconi DNA repair pathway is required for survival and long-term maintenance of neural progenitors

Although brain development abnormalities and brain cancer predisposition have been reported in some Fanconi patients, the possible role of Fanconi DNA repair pathway during neurogenesis is unclear. We thus addressed the role of fanca and fancg, which are involved in the activation of Fanconi pathway, in neural stem and progenitor cells during brain development and adult neurogenesis. Fanca(-/-) and fancg(-/-) mice presented with microcephalies and a decreased neuronal production in developing cortex and adult brain. Apoptosis of embryonic neural progenitors, but not that of postmitotic neurons, was increased in the neocortex of fanca(-/-) and fancg(-/-) mice and was correlated with chromosomal instability. In adult Fanconi mice, we showed a reduced proliferation of neural progenitor cells related to apoptosis and accentuated neural stem cells exhaustion with ageing. In addition, embryonic and adult Fanconi neural stem cells showed a reduced capacity to self-renew in vitro. Our study demonstrates a critical role for Fanconi pathway in neural stem and progenitor cells during developmental and adult neurogenesis.     

A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm

Background  

Intrabodies are defined as antibody molecules which are ectopically expressed inside the cell. Such intrabodies can be used to visualize or inhibit the targeted antigen in living cells. However, most antibody fragments cannot be used as intrabodies because they do not fold under the reducing conditions of the cell cytosol and nucleus.