Optimized cross-slot flow geometry for microfluidic extension rheometry

A precision-machined cross-slot flow geometry with a shape that has been optimized by numerical simulation of the fluid kinematics is fabricated and used to measure the extensional viscosity of a dilute polymer solution. Full-field birefringence microscopy is used to monitor the evolution and growth of macromolecular anisotropy along the stagnation point streamline, and we observe the formation of a strong and uniform birefringent strand when the dimensionless flow strength exceeds a critical Weissenberg number Wicrit 0:5. Birefringence and bulk pressure drop measurements provide self consistent estimates of the planar extensional viscosity of the fluid over a wide range of deformation rates (26 s1 "_ 435 s1) and are also in close agreement with numerical simulations performed by using a finitely extensible nonlinear elastic dumbbell model

Intravital placenta imaging reveals microcirculatory dynamics impact on sequestration and phagocytosis of Plasmodium-infected erythrocytes

Malaria in pregnancy is exquisitely aggressive, causing a range of adverse maternal and fetal outcomes prominently linked to Plasmodium-infected erythrocyte cytoadherence to fetal trophoblast. To elucidate the physiopathology of infected erythrocytes (IE) sequestration in the placenta we devised an experimental system for intravital placental examination of P. berghei-infected mice. BALB/c females were mated to C57Bl/6 CFP+ male mice and infected with GFP+ P. berghei IE, and at gestational day 18, placentas were exposed for time-lapse imaging acquisition under two-photon microscopy. Real-time images and quantitative measurements revealed that trophoblast conformational changes transiently restrain blood flow in the mouse placental labyrinth. The complex dynamics of placental microcirculation promotes IE accumulation in maternal blood spaces with low blood flow and allows the establishment of stable IE-trophoblast contacts. Further, we show that the fate of sequestered IE includes engulfment by both macrophagic and trophoblastic fetal-derived cells. These findings reinforce the current paradigm that IE interact with the trophoblast and provide definitive evidence on two novel pathogenesis mechanisms: (1) trophoblast layer controls placental microcirculation promoting IE sequestration; and (2) fetal-derived placental cells engulf sequestered IE

Rap1 binding and a lipid-dependent helix in talin F1 domain promote integrin activation in tandem.

Rap1 GTPases bind effectors, such as RIAM, to enable talin1 to induce integrin activation. In addition, Rap1 binds directly to the talin1 F0 domain (F0); however, this interaction makes a limited contribution to integrin activation in CHO cells or platelets. Here, we show that talin1 F1 domain (F1) contains a previously undetected Rap1-binding site of similar affinity to that in F0. A structure-guided point mutant (R118E) in F1, which blocks Rap1 binding, abolishes the capacity of Rap1 to potentiate talin1-induced integrin activation. The capacity of F1 to mediate Rap1-dependent integrin activation depends on a unique loop in F1 that has a propensity to form a helix upon binding to membrane lipids. Basic membrane-facing residues of this helix are critical, as charge-reversal mutations led to dramatic suppression of talin1-dependent activation. Thus, a novel Rap1-binding site and a transient lipid-dependent helix in F1 work in tandem to enable a direct Rap1-talin1 interaction to cause integrin activation

Stabilization by Fusion to the C-terminus of Hyperthermophile Sulfolobus tokodaii RNase HI: A Possibility of Protein Stabilization Tag

RNase HI from the hyperthermophile Sulfolobus tokodaii (Sto-RNase HI) is stabilized by its C-terminal residues. In this work, the stabilization effect of the Sto-RNase HI C-terminal residues was investigated in detail by thermodynamic measurements of the stability of variants lacking the disulfide bond (C58/145A), or the six C-terminal residues (ΔC6) and by structural analysis of ΔC6. The results showed that the C-terminal does not affect overall structure and stabilization is caused by local interactions of the C-terminal, suggesting that the C-terminal residues could be used as a “stabilization tag.” The Sto-RNase HI C-terminal residues (-IGCIILT) were introduced as a tag on three proteins. Each chimeric protein was more stable than its wild-type protein. These results suggested the possibility of a simple stabilization technique using a stabilization tag such as Sto-RNase HI C-terminal residues

Spleen plays a major role in DLL4-driven acute T-cell lymphoblastic leukemia.

The Notch pathway is highly active in almost all patients with T-cell acute lymphoblastic leukemia (T-ALL), but the implication of Notch ligands in T-ALL remains underexplored. Methods: We used a genetic mouse model of Notch ligand delta like 4 (DLL4)-driven T-ALL and performed thymectomies and splenectomies in those animals. We also used several patient-derived T-ALL (PDTALL) models, including one with DLL4 expression on the membrane and we treated PDTALL cells in vitro and in vivo with demcizumab, a blocking antibody against human DLL4 currently being tested in clinical trials in patients with solid cancer. Results: We show that surgical removal of the spleen abrogated T-ALL development in our preclinical DLL4-driven T-ALL mouse model. Mechanistically, we found that the spleen, and not the thymus, promoted the accumulation of circulating CD4+CD8+ T cells before T-ALL onset, suggesting that DLL4-driven T-ALL derives from these cells. Then, we identified a small subset of T-ALL patients showing higher levels of DLL4 expression. Moreover, in mice xenografted with a DLL4-positive PDTALL model, treatment with demcizumab had the same therapeutic effect as global Notch pathway inhibition using the potent γ-secretase inhibitor dibenzazepine. This result demonstrates that, in this PDTALL model, Notch pathway activity depends on DLL4 signaling, thus validating our preclinical mouse model. Conclusion: DLL4 expression in human leukemic cells can be a source of Notch activity in T-ALL, and the spleen plays a major role in a genetic mouse model of DLL4-driven T-ALL.We thank Drs. Susan Schwab, Dan Littman, Sherif Ibrahim, Angel Pellicer, Susanne Tranguch and Adolfo Ferrando for helpful discussions and/or critically comments on the manuscript. Elisabetta Andermarcher professionally edited the manuscript. We are indebted to Dr. M. Yan (Genentech) for the anti-DLL4 antibody for cytometry. We are also in debt with Christopher Murriel from Oncomed who provided the therapeutic murine anti-DLL4 antibody and demcizumab (anti-human DLL4 antibody). We thank the NYU School of Medicine Flow Cytometry Core facility, particularly Dr. Peter Lopez, Keith Kobylarz and Michael Gregory, and also the NYU School of Medicine Confocal imaging facility, particularly Yan Deng. We also thank Henry Alexandre Michaud for his great help with the FACS analysis of PDTALL cells. We thank Nelly Pirot and the rest of members of the IRCM IHC platform for their fantastic work. M.M. is supported by a contract from Fondation ARC. The NYU Cancer Institute Center Support Grant partially funded this core through grant NIH/NCI 5 P30CA16087-31. Work in JJL's laboratory is supported by the NIH/NIAID, National Multiple Sclerosis Society, and the Helmsley Charitable Trust. Work in AM's laboratory is supported by the Fondation ARC (PJA 20131200405), the European Commission (CIG631431), the Institute de Cancer de Montpellier Fondation, and the Institut National du Cancer (INCa_9257 and INCa-DGOS-Inserm 12553).S

In vitro anti-tumour activity of α-galactosylceramide-stimulated human invariant Vα24+NKT cells against melanoma

α-galactosylceramide (KRN 7000, α-GalCer) has shown potent in vivo anti-tumour activity in mice, including against melanoma and the highly specific effect of inducing proliferation and activation of human Vα24+NKT-cells. We hypothesized that human Vα24+NKT-cells activated by α-GalCer might exhibit anti-tumour activity against human melanoma. To investigate this, Vα24+NKT-cells were generated from the peripheral blood of patients with melanoma after stimulation with α-GalCer pulsed monocyte-derived dendritic cells (Mo-DCs). Vα24+NKT-cells did not exhibit cytolytic activity against the primary autologous or allogeneic melanoma cell lines tested. However, proliferation of the melanoma cell lines was markedly suppressed by co-culture with activated Vα24+NKT-cells (mean ± SD inhibition of proliferation 63.9 ± 1.3%). Culture supernatants of activated Vα24+NKT-cell cultures stimulated with α-GalCer pulsed Mo-DCs exhibited similar antiproliferative activities against melanoma cells, indicating that the majority of the inhibitory effects were due to soluble mediators rather than direct cell-to-cell interactions. This effect was predominantly due to release of IFN-γ, and to a lesser extent IL-12. Other cytokines, including IL-4 and IL-10, were released but these cytokines had less antiproliferative effects. These in vitro results show that Vα24+NKT-cells stimulated by α-GalCer-pulsed Mo-DCs have anti-tumour activities against human melanoma through antiproliferative effects exerted by soluble mediators rather than cytolytic effects as observed against some other tumours. Induction of local cytokine release by activated Vα24+NKT-cells may contribute to clinical anti-tumour effects of α-GalCer. © 2001 Cancer Research Campaign http://www.bjcancer.co

Binding Properties and Stability of the Ras-Association Domain of Rap1-GTP Interacting Adapter Molecule (RIAM)

The Rap1-GTP interacting adapter protein (RIAM) is an important protein in Rap1-mediated integrin activation. By binding to both Rap1 GTPase and talin, RIAM recruits talin to the cell membrane, thus facilitating talin-dependent integrin activation. In this article, we studied the role of the RIAM Ras-association (RA) and pleckstrin-homology (PH) domains in the interaction with Rap1. We found that the RA domain was sufficient for GTP-dependent interaction with Rap1B, and the addition of the PH domain did not change the binding affinity. We also detected GTP-independent interaction of Rap1B with the N-terminus of RIAM. In addition, we found that the PH domain stabilized the RA domain both in vitro and in cells

What Role Do Annelid Neoblasts Play? A Comparison of the Regeneration Patterns in a Neoblast-Bearing and a Neoblast-Lacking Enchytraeid Oligochaete

The term ‘neoblast’ was originally coined for a particular type of cell that had been observed during annelid regeneration, but is now used to describe the pluripotent/totipotent stem cells that are indispensable for planarian regeneration. Despite having the same name, however, planarian and annelid neoblasts are morphologically and functionally distinct, and many annelid species that lack neoblasts can nonetheless substantially regenerate. To further elucidate the functions of the annelid neoblasts, a comparison was made between the regeneration patterns of two enchytraeid oligochaetes, Enchytraeus japonensis and Enchytraeus buchholzi, which possess and lack neoblasts, respectively. In E. japonensis, which can reproduce asexually by fragmentation and subsequent regeneration, neoblasts are present in all segments except for the eight anterior-most segments including the seven head-specific segments, and all body fragments containing neoblasts can regenerate a complete head and a complete tail, irrespective of the region of the body from which they were originally derived. In E. japonensis, therefore, no antero-posterior gradient of regeneration ability exists in the trunk region. However, when amputation was carried out within the head region, where neoblasts are absent, the number of regenerated segments was found to be dependent on the level of amputation along the body axis. In E. buchholzi, which reproduces only sexually and lacks neoblasts in all segments, complete heads were never regenerated and incomplete (hypomeric) heads could be regenerated only from the anterior region of the body. Such an antero-posterior gradient of regeneration ability was observed for both the anterior and posterior regeneration in the whole body of E. buchholzi. These results indicate that the presence of neoblasts correlates with the absence of an antero-posterior gradient of regeneration ability along the body axis, and suggest that the annelid neoblasts are more essential for efficient asexual reproduction than for the regeneration of missing body parts