Simulation-based equation of state of the hard disk fluid and prediction of higher-order virial coefficients

We present new molecular dynamics results for the pressure of the pure hard disk fluid up to the hexatic transition (about reduced density 0.9). The data combined with the known virial coefficients (up to $B_{10}$) are used to build an equation of state, to estimate higher-order virial coefficients, and also to obtain a better value of $B_{10}$. Finite size effects are discussed in detail. The ``van der Waals-like'' loop reported in literature in the vicinity of the fluid/hexatic transition is explained by suppressed density fluctuations in the canonical ensemble. The inflection point on the pressure-density dependence is predicted by the equation of state even if the hexatic phase simulation data are not considered.Comment: 9 pages, 3 figures, presented at The Seventh Liblice Conference on the Statistical Mechanics of Liquids (Lednice, Czech Republic, June 11--16, 2006

Increased Oxidative Stress Induces Apoptosis in Human Cystic Fibrosis Cells

Oxidative stress results in deleterious cell function in pathologies associated with inflammation. Here, we investigated the generation of superoxide anion as well as the anti-oxidant defense systems related to the isoforms of superoxide dismutases (SOD) in cystic fibrosis (CF) cells. Pro-apoptotic agents induced apoptosis in CF but not in control cells that was reduced by treatment with SOD mimetic. These effects were associated with increased superoxide anion production, sensitive to the inhibition of IκB-α phosphorylation, in pancreatic but not tracheal CF cells, and reduced upon inhibition of either mitochondrial complex I or NADPH oxidase. CF cells exhibited reduced expression, but not activity, of both Mn-SOD and Cu/Zn-SOD when compared to control cells. Although, expression of EC-SOD was similar in normal and CF cells, its activity was reduced in CF cells. We provide evidence that high levels of oxidative stress are associated with increased apoptosis in CFTR-mutated cells, the sources being different depending on the cell type. These observations underscore a reduced anti-oxidant defense mechanism, at least in part, via diminished EC-SOD activity and regulation of Cu/Zn-SOD and Mn-SOD expressions. These data point to new therapeutic possibilities in targeting anti-oxidant pathways to reduce oxidative stress and apoptosis in CF cells

Cell–substrate adhesion drives Scar/WAVE activation and phosphorylation by a Ste20-family kinase, which controls pseudopod lifetime

The Scar/WAVE complex is the principal catalyst of pseudopod and lamellipod formation. Here we show that Scar/WAVE’s proline-rich domain is polyphosphorylated after the complex is activated. Blocking Scar/WAVE activation stops phosphorylation in both Dictyostelium and mammalian cells, implying that phosphorylation modulates pseudopods after they have been formed, rather than controlling whether they are initiated. Unexpectedly, phosphorylation is not promoted by chemotactic signaling but is greatly stimulated by cell:substrate adhesion and diminished when cells deadhere. Phosphorylation-deficient or phosphomimetic Scar/WAVE mutants are both normally functional and rescue the phenotype of knockout cells, demonstrating that phosphorylation is dispensable for activation and actin regulation. However, pseudopods and patches of phosphorylation-deficient Scar/WAVE last substantially longer in mutants, altering the dynamics and size of pseudopods and lamellipods and thus changing migration speed. Scar/WAVE phosphorylation does not require ERK2 in Dictyostelium or mammalian cells. However, the MAPKKK homologue SepA contributes substantially—sepA mutants have less steady-state phosphorylation, which does not increase in response to adhesion. The mutants also behave similarly to cells expressing phosphorylation-deficient Scar, with longer-lived pseudopods and patches of Scar recruitment. We conclude that pseudopod engagement with substratum is more important than extracellular signals at regulating Scar/WAVE’s activity and that phosphorylation acts as a pseudopod timer by promoting Scar/WAVE turnover

Pulmonary vein reconnection and repeat ablation characteristics following cryoballoon‐compared to radiofrequency‐based pulmonary vein isolation

Background: Despite advances in efficacy and safety of pulmonary vein isolation (PVI), atrial fibrillation (AF) recurrence after PVI remains common. PV‐reconnection is the main finding during repeat PVI procedures performed to treat recurrent AF. Objective: To analyze pulmonary vein (PV) reconnection patterns during repeat ablation procedures in a large cohort of consecutive patients undergoing radio frequency or cryoballoon‐based PVI. Methods: Retrospective analysis of PV‐reconnection patterns and analysis of re‐ablation strategies in consecutive index RF‐ and CB‐based PVI and their respective re‐ablation procedures during concomitant usage of both energy sources at a single high‐volume center in Germany. Results: A total of 610 first (06/2015–10/2022) and 133 s (01/2016–11/2022) repeat ablation procedures after 363 (60%) RF‐ and 247 (40%) CB‐based index PVIs between 01/2015 and 12/2021 were analyzed. PV‐reconnection was found in 509/610 (83%) patients at first and 74/133 (56%) patients at second repeat procedure. 465 of 968 (48%) initially via CB isolated PVs were reconnected at first re‐ablation but 796 of 1422 initially RF‐isolated PV (56%) were reconnected (OR: 0.73 [95% CI: 0.62–0.86]; p < .001). This was driven by fewer reconnections of the left PVs (LSPV: OR: 0.60 [95% CI: 0.42–0.86]; p = .005 and LSPV: 0.67 [0.47–0.95]; p = .026). PV‐reconnection was more likely after longer, RF‐based index PVI and in older females. Repeat procedures were shorter after CB‐compared to after RF‐PVI. Conclusions: Reconnection remains the most common reason for repeat AF ablation procedures after PVI. Our data suggest to preferentially use of the cryoballoon during index PVI, especially in older women

Pulmonary vein reconnection and repeat ablation characteristics following cryoballoon‐compared to radiofrequency‐based pulmonary vein isolation

Background: Despite advances in efficacy and safety of pulmonary vein isolation (PVI), atrial fibrillation (AF) recurrence after PVI remains common. PV‐reconnection is the main finding during repeat PVI procedures performed to treat recurrent AF. Objective: To analyze pulmonary vein (PV) reconnection patterns during repeat ablation procedures in a large cohort of consecutive patients undergoing radio frequency or cryoballoon‐based PVI. Methods: Retrospective analysis of PV‐reconnection patterns and analysis of re‐ablation strategies in consecutive index RF‐ and CB‐based PVI and their respective re‐ablation procedures during concomitant usage of both energy sources at a single high‐volume center in Germany. Results: A total of 610 first (06/2015–10/2022) and 133 s (01/2016–11/2022) repeat ablation procedures after 363 (60%) RF‐ and 247 (40%) CB‐based index PVIs between 01/2015 and 12/2021 were analyzed. PV‐reconnection was found in 509/610 (83%) patients at first and 74/133 (56%) patients at second repeat procedure. 465 of 968 (48%) initially via CB isolated PVs were reconnected at first re‐ablation but 796 of 1422 initially RF‐isolated PV (56%) were reconnected (OR: 0.73 [95% CI: 0.62–0.86]; p < .001). This was driven by fewer reconnections of the left PVs (LSPV: OR: 0.60 [95% CI: 0.42–0.86]; p = .005 and LSPV: 0.67 [0.47–0.95]; p = .026). PV‐reconnection was more likely after longer, RF‐based index PVI and in older females. Repeat procedures were shorter after CB‐compared to after RF‐PVI. Conclusions: Reconnection remains the most common reason for repeat AF ablation procedures after PVI. Our data suggest to preferentially use of the cryoballoon during index PVI, especially in older women

SMER28 attenuates PI3K/mTOR signaling by direct inhibition of PI3K p110 delta

SMER28 (Small molecule enhancer of Rapamycin 28) is an autophagy-inducing compound functioning by a hitherto unknown mechanism. Here, we confirm its autophagy-inducing effect by assessing classical autophagy-related parameters. Interestingly, we also discovered several additional effects of SMER28, including growth retardation and reduced G1 to S phase progression. Most strikingly, SMER28 treatment led to a complete arrest of receptor tyrosine kinase signaling, and, consequently, growth factor-induced cell scattering and dorsal ruffle formation. This coincided with a dramatic reduction in phosphorylation patterns of PI3K downstream effectors. Consistently, SMER28 directly inhibited PI3Kδ and to a lesser extent p110γ. The biological relevance of our observations was underscored by SMER28 interfering with InlB-mediated host cell entry of Listeria monocytogenes, which requires signaling through the prominent receptor tyrosine kinase c-Met. This effect was signaling-specific, since entry of unrelated, gram-negative Salmonella Typhimurium was not inhibited. Lastly, in B cell lymphoma cells, which predominantly depend on tonic signaling through PI3Kδ, apoptosis upon SMER28 treatment is profound in comparison to non-hematopoietic cells. This indicates SMER28 as a possible drug candidate for the treatment of diseases that derive from aberrant PI3Kδ activity

Scar/WAVE drives actin protrusions independently of its VCA domain using proline-rich domains

Cell migration requires the constant modification of cellular shape by reorganization of the actin cytoskeleton. Fine-tuning of this process is critical to ensure new actin filaments are formed only at specific times and in defined regions of the cell. The Scar/WAVE complex is the main catalyst of pseudopod and lamellipodium formation during cell migration. It is a pentameric complex highly conserved through eukaryotic evolution and composed of Scar/WAVE, Abi, Nap1/NCKAP1, Pir121/CYFIP, and HSPC300/Brk1. Its function is usually attributed to activation of the Arp2/3 complex through Scar/WAVE’s VCA domain, while other parts of the complex are expected to mediate spatial-temporal regulation and have no direct role in actin polymerization. Here, we show in both B16-F1 mouse melanoma and Dictyostelium discoideum cells that Scar/WAVE without its VCA domain still induces the formation of morphologically normal, actin-rich protrusions, extending at comparable speeds despite a drastic reduction of Arp2/3 recruitment. However, the proline-rich regions in Scar/WAVE and Abi subunits are essential, though either is sufficient for the generation of actin protrusions in B16-F1 cells. We further demonstrate that N-WASP can compensate for the absence of Scar/WAVE’s VCA domain and induce lamellipodia formation, but it still requires an intact WAVE complex, even if without its VCA domain. We conclude that the Scar/WAVE complex does more than directly activating Arp2/3, with proline-rich domains playing a central role in promoting actin protrusions. This implies a broader function for the Scar/WAVE complex, concentrating and simultaneously activating many actin-regulating proteins as a lamellipodium-producing core

Microtubules as Platforms for Assaying Actin Polymerization In Vivo

The actin cytoskeleton is continuously remodeled through cycles of actin filament assembly and disassembly. Filaments are born through nucleation and shaped into supramolecular structures with various essential functions. These range from contractile and protrusive assemblies in muscle and non-muscle cells to actin filament comets propelling vesicles or pathogens through the cytosol. Although nucleation has been extensively studied using purified proteins in vitro, dissection of the process in cells is complicated by the abundance and molecular complexity of actin filament arrays. We here describe the ectopic nucleation of actin filaments on the surface of microtubules, free of endogenous actin and interfering membrane or lipid. All major mechanisms of actin filament nucleation were recapitulated, including filament assembly induced by Arp2/3 complex, formin and Spir. This novel approach allows systematic dissection of actin nucleation in the cytosol of live cells, its genetic re-engineering as well as screening for new modifiers of the process

The Arp2/3 complex is crucial for colonisation of the mouse skin by melanoblasts

The Arp2/3 complex is essential for the assembly of branched filamentous actin, but its role in physiology and development is surprisingly little understood. Melanoblasts deriving from the neural crest migrate along the developing embryo and traverse the dermis to reach the epidermis, colonising the skin and eventually homing within the hair follicles. We have previously established that Rac1 and Cdc42 direct melanoblast migration in vivo. We hypothesised that the Arp2/3 complex might be the main downstream effector of these small GTPases. Arp3 depletion in the melanocyte lineage results in severe pigmentation defects in dorsal and ventral regions of the mouse skin. Arp3 null melanoblasts demonstrate proliferation and migration defects and fail to elongate as their wild-type counterparts. Conditional deletion of Arp3 in primary melanocytes causes improper proliferation, spreading, migration and adhesion to extracellular matrix. Collectively, our results suggest that the Arp2/3 complex is absolutely indispensable in the melanocyte lineage in mouse development, and indicate a significant role in developmental processes that require tight regulation of actin-mediated motility