An IMiD-inducible degron provides reversible regulation for chimeric antigen receptor expression and activity.

The recent development of successful CAR (chimeric antigen receptor) T cell therapies has been accompanied by a need to better control potentially fatal toxicities that can arise from adverse immune reactions. Here we present a ligand-controlled CAR system, based on the IKZF3 ZF2 ß-hairpin IMiD-inducible degron, which allows for the reversible control of expression levels of type I membrane proteins, including CARs. Testing this system in an established mouse xenotransplantation model for acute lymphoblastic leukemia, we validate the ability of the CAR19-degron to target and kill CD19-positive cells displaying complete control/clearance of the tumor. We also demonstrate that the activity of CAR19-degron can be regulated in vivo when dosing a US Food and Drug Administration-approved drug, lenalidomide.

Nonlinear dynamics of confined liquid systems with interfaces subject to forced vibrations.

A review is presented of the dynamic behavior of confined fluid systems with interfaces under monochromatic mechanical forcing, emphasizing the associated spatio-temporal structure of the fluid response. At low viscosity, vibrations significantly affect dynamics and always produce viscous mean flows, which are coupled to the primary oscillating flow and evolve on a very slow timescale. Thus, unlike the primary oscillating flow, mean flows may easily interact with the surface rheology, which generates dynamics that usually exhibit a much slower timescale than that of typical gravity-capillary waves. The review is made with an eye to the typical experimental devices used to measure surface properties, which usually consist of periodically forced, symmetric fluid systems with interfaces. The current theoretical description of these systems ignores the fluid mechanics, which could play a larger role than presently assumed.

R-Spondin potentiates Wnt/ß-catenin signaling through orphan receptors LGR4 and LGR5.

The Wnt/ß-catenin signaling pathbway controls many important biological processes. R-Spondin (RSPO) proteins are a family of secreted molecules that strongly potentiate Wnt/ß-catenin signaling, however, the molecular mechanism of RSPO action is not yet fully understood. We performed an unbiased siRNA screen to identify molecules specifically required for RSPO, but not Wnt, induced ß-catenin signaling. From this screen, we identified LGR4, then an orphan G protein-coupled receptor (GPCR), as the cognate receptor of RSPO. Depletion of LGR4 completely abolished RSPO-induced ß-catenin signaling. The loss of LGR4 could be compensated by overexpression of LGR5, suggesting that LGR4 and LGR5 are functional homologs. We further demonstrated that RSPO binds to the extracellular domain of LGR4 and LGR5, and that overexpression of LGR4 strongly sensitizes cells to RSPO-activated ß-catenin signaling. Supporting the physiological significance of RSPO-LGR4 interaction, Lgr4-/- crypt cultures failed to grow in RSPO-containing intestinal crypt culture medium. No coupling between LGR4 and heterotrimeric G proteins could be detected in RSPO-treated cells, suggesting that LGR4 mediates RSPO signaling through a novel mechanism. Identification of LGR4 and its relative LGR5, an adult stem cell marker, as the receptors of RSPO will facilitate the further characterization of these receptor/ligand pairs in regenerative medicine applications.

USP4 is regulated by AKT phosphorylation and directly deubiquitylates TGF-ß type I receptor.

The stability and membrane localization of the transforming growth factor-ß (TGF-ß) type I receptor (TßRI) determines the levels of TGF-ß signalling. TßRI is targeted for ubiquitylation-mediated degradation by the SMAD7-SMURF2 complex. Here we performed a genome-wide gain-of-function screen and identified ubiquitin-specific protease (USP) 4 as a strong inducer of TGF-ß signalling. USP4 was found to directly interact with TßRI and act as a deubiquitylating enzyme, thereby controlling TßRI levels at the plasma membrane. Depletion of USP4 mitigates TGF-ß-induced epithelial to mesenchymal transition and metastasis. Importantly, AKT (also known as protein kinase B), which has been associated with poor prognosis in breast cancer, directly associates with and phosphorylates USP4. AKT-mediated phosphorylation relocates nuclear USP4 to the cytoplasm and membrane and is required for maintaining its protein stability. Moreover, AKT-induced breast cancer cell migration was inhibited by USP4 depletion and TßRI kinase inhibition. Our results uncover USP4 as an important determinant for crosstalk between TGF-ß and AKT signalling pathways.

RNF12 controls embryonic stem cell fate and morphogenesis in zebrafish embryos by targeting Smad7 for degradation.

TGF-ß members are of key importance during embryogenesis and tissue homeostasis. Smad7 is a potent antagonist of TGF-ß family/Smad-mediated responses, but the regulation of Smad7 activity is not well understood. We identified the RING domain-containing E3 ligase RNF12 as a critical component of TGF-ß signaling. Depletion of RNF12 dramatically reduced TGF-ß/Smad-induced effects in mammalian cells, whereas ectopic expression of RNF12 strongly enhanced these responses. RNF12 specifically binds to Smad7 and induces its polyubiquitination and degradation. Smad7 levels were increased in RNF12-deficient mouse embryonic stem cells, resulting in mitigation of both BMP-mediated repression of neural induction and activin-induced anterior mesoderm formation. RNF12 also antagonized Smad7 during Nodal-dependent and BMP-dependent signaling and morphogenic events in early zebrafish embryos. The gastrulation defects induced by ectopic and depleted Smad7 were rescued in part by RNF12 gain and loss of function, respectively. These findings demonstrate that RNF12 plays a critical role in TGF-ß family signaling.

Weakly nonlinear analysis of impulsively-forced Faraday waves.

Parametrically-excited surface waves, forced by a repeating sequence of delta-function impulses, are considered within the framework of the Zhang-Viñals model [W. Zhang and J. Viñals, J. Fluid Mech. 336, 301 (1997)]. With impulsive forcing, the linear stability analysis can be carried out exactly and leads to an implicit equation for the neutral stability curves. As noted previously [J. Bechhoefer and B. Johnson, Am. J. Phys. 64, 1482 (1996)], in the simplest case of N=2 equally-spaced impulses per period (which alternate up and down) there are only subharmonic modes of instability. The familiar situation of alternating subharmonic and harmonic resonance tongues emerges only if an asymmetry in the spacing between the impulses is introduced. We extend the linear analysis for N=2 impulses per period to the weakly nonlinear regime, where we determine the leading order nonlinear saturation of one-dimensional standing waves as a function of forcing strength. Specifically, an analytic expression for the cubic Landau coefficient in the bifurcation equation is derived as a function of the dimensionless spacing between the two impulses and the fluid parameters that appear in the Zhang-Viñals model. As the capillary parameter is varied, one finds a parameter regime of wave amplitude suppression, which is due to a familiar 1:2 spatiotemporal resonance between the subharmonic mode of instability and a damped harmonic mode. This resonance occurs for impulsive forcing even when harmonic resonance tongues are absent from the neutral stability curves. The strength of this resonance feature can be tuned by varying the spacing between the impulses. This finding is interpreted in terms of a recent symmetry-based analysis of multifrequency forced Faraday waves [J. Porter, C. M. Topaz, and M. Silber, Phys. Lett. 93, 034502 (2004); C. M. Topaz, J. Porter, and M. Silber, Phys. Rev. E 70, 066206 (2004)].

Pattern control via multifrequency parametric forcing.

We use symmetry considerations to investigate control of a class of resonant three-wave interactions relevant to pattern formation in weakly damped, parametrically forced systems near onset. We classify and tabulate the most important damped, resonant modes and determine how the corresponding resonant triad interactions depend on the forcing parameters. The relative phase of the forcing terms may be used to enhance or suppress the nonlinear interactions. We compare our symmetry-based predictions with numerical and experimental results for Faraday waves. Our results suggest how to design multifrequency forcing functions that favor chosen patterns in the lab.

Tutoring deaf students in higher education: a comparison of baccalaureate and sub-baccalaureate student perceptions.

Seventy-three deaf college students completed a survey examining perceptions about tutoring outcomes and emphases, characteristics of tutors, and responsibilities associated with learning through tutoring. The comparisons revealed that while baccalaureate and sub-baccalaureate students have many similar perceptions about tutoring, there are also some striking differences. In particular, as compared to the sub-baccalaureate students, baccalaureate students have a stronger preference for focusing on course content and for working with tutors who actively involve them during the tutoring sessions. In addition, baccalaureate students prefer to decide the focus of the tutoring themselves while sub-baccalaureate students tend to leave the decision to the tutor. The results of the analyses with three scales measuring perceptions of tutoring dimensions are summarized and recommendations for the selection and preparation of tutors, as well as for future research, are provided.

Multifrequency control of Faraday wave patterns.

We show how pattern formation in Faraday waves may be manipulated by varying the harmonic content of the periodic forcing function. Our approach relies on the crucial influence of resonant triad interactions coupling pairs of critical standing wave modes with damped, spatiotemporally resonant modes. Under the assumption of weak damping and forcing, we perform a symmetry-based analysis that reveals the damped modes most relevant for pattern selection, and how the strength of the corresponding triad interactions depends on the forcing frequencies, amplitudes, and phases. In many cases, the further assumption of Hamiltonian structure in the inviscid limit determines whether the given triad interaction has an enhancing or suppressing effect on related patterns. Surprisingly, even for forcing functions with arbitrarily many frequency components, there are at most five frequencies that affect each of the important triad interactions at leading order. The relative phases of those forcing components play a key role, sometimes making the difference between an enhancing and suppressing effect. In numerical examples, we examine the validity of our results for larger values of the damping and forcing. Finally, we apply our findings to one-dimensional periodic patterns obtained with impulsive forcing and to two-dimensional superlattice patterns and quasipatterns obtained with multifrequency forcing.

Broken symmetries and pattern formation in two-frequency forced faraday waves.

We exploit the approximate (broken) symmetries of time translation, time reversal, and Hamiltonian structure to obtain general scaling laws governing the process of pattern formation in weakly damped Faraday waves. Using explicit parameter symmetries we determine, for the case of two-frequency forcing, how the strength of observed three-wave interactions depends on the frequency ratio and on the relative phase of the two driving terms. These symmetry-based predictions are verified for numerically calculated coefficients, and help explain the results of recent experiments.