Chk1 Suppresses a Caspase-2 Apoptotic Response to DNA Damage that Bypasses p53, Bcl-2, and Caspase-3

Evasion of DNA damage-induced cell death, via mutation of the p53 tumor suppressor or overexpression of prosurvival Bcl-2 family proteins, is a key step toward malignant transformation and therapeutic resistance. We report that depletion or acute inhibition of checkpoint kinase 1 (Chk1) is sufficient to restore ?-radiation-induced apoptosis in p53 mutant zebrafish embryos. Surprisingly, caspase-3 is not activated prior to DNA fragmentation, in contrast to classical intrinsic or extrinsic apoptosis. Rather, an alternative apoptotic program is engaged that cell autonomously requires atm (ataxia telangiectasia mutated), atr (ATM and Rad3-related) and caspase-2, and is not affected by p53 loss or overexpression of bcl-2/xl. Similarly, Chk1 inhibitor-treated human tumor cells hyperactivate ATM, ATR, and caspase-2 after ?-radiation and trigger a caspase-2-dependent apoptotic program that bypasses p53 deficiency and excess Bcl-2. The evolutionarily conserved "Chk1-suppressed" pathway defines a novel apoptotic process, whose responsiveness to Chk1 inhibitors and insensitivity to p53 and BCL2 alterations have important implications for cancer therapy. © 2008 Elsevier Inc. All rights reserved.

Pressure drop of gas–liquid Taylor flow in round micro-capillaries for low to intermediate Reynolds numbers

In this paper, a model is presented that describes the pressure drop of gas-liquid Taylor flow in round capillaries with a channel diameter typically less than 1 mm. The analysis of Bretherton (J Fluid Mech 10:166-188, 1961) for the pressure drop over a single gas bubble for vanishing liquid film thickness is extended to include a non-negligible liquid film thickness using the analysis of Aussillous and Qu,r, (Phys Fluids 12(10):2367-2371, 2000). This result is combined with the Hagen-Poiseuille equation for liquid flow using a mass balance-based Taylor flow model previously developed by the authors (Warnier et al. in Chem Eng J 135S:S153-S158, 2007). The model presented in this paper includes the effect of the liquid slug length on the pressure drop similar to the model of Kreutzer et al. (AIChE J 51(9):2428-2440, 2005). Additionally, the gas bubble velocity is taken into account, thereby increasing the accuracy of the pressure drop predictions compared to those of the model of Kreutzer et al. Experimental data were obtained for nitrogen-water Taylor flow in a round glass channel with an inner diameter of 250 mu m. The capillary number Ca (gl) varied between 2.3 x 10(-3) and 8.8 x 10(-3) and the Reynolds number Re (gl) varied between 41 and 159. The presented model describes the experimental results with an accuracy of +/- 4% of the measured values.

Autophagic bulk sequestration of cytosolic cargo is independent of LC3, but requires GABARAPs

LC3, a mammalian homologue of yeast Atg8, is assumed to play an important part in bulk sequestration and degradation of cytoplasm (macroautophagy), and is widely used as an indicator of this process. To critically examine its role, we followed the autophagic flux of LC3 in rat hepatocytes during conditions of maximal macroautophagic activity (amino acid depletion), combined with analyses of macroautophagic cargo sequestration, measured as transfer of the cytosolic protein lactate dehydrogenase (LDH) to sedimentable organelles. To accurately determine LC3 turnover we developed a quantitative immunoblotting procedure that corrects for differential immunoreactivity of cytosolic and membrane-associated LC3 forms, and we included cycloheximide to block influx of newly synthesized LC3. As expected, LC3 was initially degraded by the autophagic-lysosomal pathway, but, surprisingly, autophagic LC3-flux ceased after ~2h. In contrast, macroautophagic cargo flux was well maintained, and density gradient analysis showed that sequestered LDH partly accumulated in LC3-free autophagic vacuoles. Hepatocytic macroautophagy could thus proceed independently of LC3. Silencing of either of the two mammalian Atg8 subfamilies in LNCaP prostate cancer cells exposed to macroautophagy-inducing conditions (starvation or the mTOR-inhibitor Torin1) confirmed that macroautophagic sequestration did not require the LC3 subfamily, but, intriguingly, we found the GABARAP subfamily to be essential.

Modulation of intracellular calcium homeostasis blocks autophagosome formation

Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stress-the calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca (2+)-ATPase inhibitor thapsigargin (TG)-potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.

Studying Gender in Conference Talks -- data from the 223rd meeting of the American Astronomical Society

We present a study on the gender balance, in speakers and attendees, at the recent major astronomical conference, the American Astronomical Society meeting 223, in Washington, DC. We conducted an informal survey, yielding over 300 responses by volunteers at the meeting. Each response included gender data about a single talk given at the meeting, recording the gender of the speaker and all question-askers. In total, 225 individual AAS talks were sampled. We analyze basic statistical properties of this sample. We find that the gender ratio of the speakers closely matched the gender ratio of the conference attendees. The audience asked an average of 2.8 questions per talk. Talks given by women had a slightly higher number of questions asked (3.2$\pm$0.2) than talks given by men (2.6$\pm$0.1). The most significant result from this study is that while the gender ratio of speakers very closely mirrors that of conference attendees, women are under-represented in the question-asker category. We interpret this to be an age-effect, as senior scientists may be more likely to ask questions, and are more commonly men. A strong dependence on the gender of session chairs is found, whereby women ask disproportionately fewer questions in sessions chaired by men. While our results point to laudable progress in gender-balanced speaker selection, we believe future surveys of this kind would help ensure that collaboration at such meetings is as inclusive as possible.

Analgesic exposure in pregnant rats affects fetal germ cell development with inter-generational reproductive consequences

Analgesics which affect prostaglandin (PG) pathways are used by most pregnant women. As germ cells (GC) undergo developmental and epigenetic changes in fetal life and are PG targets, we investigated if exposure of pregnant rats to analgesics (indomethacin or acetaminophen) affected GC development and reproductive function in resulting offspring (F1) or in the F2 generation. Exposure to either analgesic reduced F1 fetal GC number in both sexes and altered the tempo of fetal GC development sex-dependently, with delayed meiotic entry in oogonia but accelerated GC differentiation in males. These effects persisted in adult F1 females as reduced ovarian and litter size, whereas F1 males recovered normal GC numbers and fertility by adulthood. F2 offspring deriving from an analgesic-exposed F1 parent also exhibited sex-specific changes. F2 males exhibited normal reproductive development whereas F2 females had smaller ovaries and reduced follicle numbers during puberty/adulthood; as similar changes were found for F2 offspring of analgesic-exposed F1 fathers or mothers, we interpret this as potentially indicating an analgesic-induced change to GC in F1. Assuming our results are translatable to humans, they raise concerns that analgesic use in pregnancy could potentially affect fertility of resulting daughters and grand-daughters.