ISG15-GRAIL1-CD3 axis influences survival of esophageal adenocarcinoma patients.

Immunosuppression is a common feature of esophageal adenocarcinoma (EAC) and has been linked to poor overall survival (OS). We hypothesized that upstream factors might negatively influence CD3 levels and T-cell activity, thus promoting immunosuppression and worse survival. We used clinical data and patient samples of those who progressed from Barrett's (BE) to dysplasia to EAC, investigated gene (RNAseq), protein (tissue microarray) expression and performed cell biology studies to delineate a pathway impacting CD3 protein stability that might influence EAC outcome. We show that the loss of both CD3-ε expression and CD3+ T-cell number are correlated with worse OS in EAC. The GRAIL (gene related to anergy in lymphocytes) isoform 1 (GRAIL1), which is the prominent isoform in EACs, degrades (ε, γ, δ) CD3s and inactivates T-cells. In contrast, isoform 2 (GRAIL2), which is reduced in EACs, stabilizes CD3s. Further, GRAIL1 mediated CD3 degradation is facilitated by interferon stimulated gene 15 (ISG15), a ubiquitin-like protein. Consequently, either the overexpression of a ligase-dead GRAIL1, ISG15 knockdown, or the overexpression of a conjugation-defective ISG15-LRAA mutant can increase CD3 levels. Together, we identified that an ISG15→GRAIL1→mutant p53 amplification loop negatively influencing CD3 levels and T-cell activity, thus promoting immunosuppression in EAC.

Factors affecting timing of surgery following neoadjuvant chemoradiation for esophageal cancer.

Background: Neoadjuvant chemoradiation with esophagectomy is standard management for locally advanced esophageal cancer. Studies have shown that surgical timing following chemoradiation is important for minimizing postoperative complications, however in practice timing is often variable and delayed. Although postoperative impact of surgical timing has been studied, less is known about factors associated with delays. Materials and methods: A retrospective review was performed for 96 patients with esophageal cancer who underwent chemoradiation then esophagectomy between 2018 and 2020 at a single institution. Univariable and stepwise multivariable analyses were used to assess association between social (demographics, insurance) and clinical variables (pre-operative weight, comorbidities, prior cardiothoracic surgery, smoking history, disease staging) with time to surgery (≤8 weeks "on-time" vs. >8 weeks "delayed"). Results: Fifty-one patients underwent esophagectomy within 8 weeks of chemoradiation; 45 had a delayed operation. Univariate analysis showed the following characteristics were significantly different between on-time and delayed groups: weight loss within 3 months of surgery (3.9 ± 5.1 kg vs. 1.5 ± 3.6 kg; P = 0.009), prior cardiovascular disease (29% vs. 49%; P = 0.05), prior cardiothoracic surgery (4% vs. 22%; P = 0.01), history of ever smoked (69% vs. 87%; P = 0.04), absent nodal metastasis on pathology (57% vs. 82%; P = 0.008). Multivariate analysis demonstrated that prior cardiothoracic surgery (OR 8.924, 95%CI 1.67-47.60; P = 0.01) and absent nodal metastasis (OR 4.186, 95%CI 1.50-11.72; P = 0.006) were associated with delayed surgery. Conclusions: Delayed esophagectomy following chemoradiotherapy is associated with prior cardiothoracic surgery and absent nodal metastasis. Further investigations should focus on understanding how these factors contribute to delays to guide treatment planning and mitigate sources of outcome disparities.

Dynamics of a Fermi Gas Quenched to Unitarity.

We present an experimental study of a two component Fermi gas following an interaction quench into the superfluid phase. Starting with a weakly attractive gas in the normal phase, interactions are ramped to unitarity at a range of rates and we measure the subsequent dynamics as the gas approaches equilibrium. Both the formation and condensation of fermion pairs are mapped via measurements of the pair momentum distribution and can take place on very different timescales, depending on the adiabaticity of the quench. The contact parameter is seen to respond very quickly to changes in the interaction strength, indicating that short-range correlations, based on the occupation of high-momentum modes, evolve far more rapidly than the correlations in low-momentum modes necessary for pair condensation.

Immune determinants of Barrett's progression to esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) develops from Barrett's esophagus (BE), a chronic inflammatory state that can progress through a series of transformative dysplastic states before tumor development. While molecular and genetic changes of EAC tumors have been studied, immune microenvironment changes during Barrett's progression to EAC remain poorly understood. In this study, we identify potential immunologic changes that can occur during BE-to-EAC progression. RNA sequencing (RNA-Seq) analysis on tissue samples from EAC patients undergoing surgical resection demonstrated that a subset of chemokines and cytokines, most notably IL6 and CXCL8, increased during BE progression to EAC. xCell deconvolution analysis investigating immune cell population changes demonstrated that the largest changes in expression during BE progression occurred in M2 macrophages, pro-B cells, and eosinophils. Multiplex immunohistochemical staining of tissue microarrays showed increased immune cell populations during Barrett's progression to high-grade dysplasia. In contrast, EAC tumor sections were relatively immune poor, with a rise in PD-L1 expression and loss of CD8+ T cells. These data demonstrate that the EAC microenvironment is characterized by poor cytotoxic effector cell infiltration and increased immune inhibitory signaling. These findings suggest an immunosuppressive microenvironment, highlighting the need for further studies to explore immune modulatory therapy in EAC.

Publisher Correction: G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.

The adhesion G-protein-coupled receptor (GPCR) latrophilin 3 (ADGRL3) has been associated with increased risk of attention deficit hyperactivity disorder (ADHD) and substance use in human genetic studies. Knockdown in multiple species leads to hyperlocomotion and altered dopamine signaling. Thus, ADGRL3 is a potential target for treatment of neuropsychiatric disorders that involve dopamine dysfunction, but its basic signaling properties are poorly understood. Identification of adhesion GPCR signaling partners has been limited by a lack of tools to acutely activate these receptors in living cells. Here, we design a novel acute activation strategy to characterize ADGRL3 signaling by engineering a receptor construct in which we could trigger acute activation enzymatically. Using this assay, we found that ADGRL3 signals through G12/G13 and Gq, with G12/13 the most robustly activated. Gα12/13 is a new player in ADGRL3 biology, opening up unexplored roles for ADGRL3 in the brain. Our methodological advancements should be broadly useful in adhesion GPCR research.

High-Frequency Sound in a Unitary Fermi Gas.

We present an experimental and theoretical study of the phonon mode in a unitary Fermi gas. Using two-photon Bragg spectroscopy, we measure excitation spectra at a momentum of approximately half the Fermi momentum, both above and below the superfluid critical temperature T_{c}. Below T_{c}, the dominant excitation is the Bogoliubov-Anderson (BA) phonon mode, driven by gradients in the phase of the superfluid order parameter. The temperature dependence of the BA phonon is consistent with a theoretical model based on the quasiparticle random phase approximation in which the dominant damping mechanism is via collisions with thermally excited quasiparticles. As the temperature is increased above T_{c}, the phonon evolves into a strongly damped collisional mode, accompanied by an abrupt increase in spectral width. Our study reveals strong similarities between sound propagation in the unitary Fermi gas and bosonic liquid helium.

Contact and Sum Rules in a Near-Uniform Fermi Gas at Unitarity.

We present an experimental study of the high-energy excitation spectra of unitary Fermi gases. Using focused beam Bragg spectroscopy, we locally probe atoms in the central region of a harmonically trapped cloud where the density is nearly uniform, enabling measurements of the dynamic structure factor for a range of temperatures both below and above the superfluid transition. Applying sum rules to the measured Bragg spectra, we resolve the characteristic behavior of the universal contact parameter, C, across the superfluid transition. We also employ a recent theoretical result for the kinetic (second-moment) sum rule to obtain the internal energy of gases at unitarity.

Quantum Anomaly and 2D-3D Crossover in Strongly Interacting Fermi Gases.

We present an experimental investigation of collective oscillations in harmonically trapped Fermi gases through the crossover from two to three dimensions. Specifically, we measure the frequency of the radial monopole oscillation or breathing mode in highly oblate gases with tunable interactions. The breathing mode frequency is set by the adiabatic compressibility and probes the thermodynamic equation of state. In 2D, a dynamical scaling symmetry for atoms interacting via a δ potential predicts the breathing mode to occur at exactly twice the harmonic confinement frequency. However, a renormalized quantum treatment introduces a new length scale which breaks this classical scale invariance resulting in a so-called quantum anomaly. Our measurements deep in the 2D regime lie above the scale-invariant prediction for a range of interaction strengths providing evidence for the quantum anomaly and signifying the breakdown of an elementary δ-potential model of atomic interactions. By varying the atom number we can tune the chemical potential and see the breathing mode frequency evolve smoothly between the 2D to 3D thermodynamic limits.

Thermodynamics of an Attractive 2D Fermi Gas.

Thermodynamic properties of matter are conveniently expressed as functional relations between variables known as equations of state. Here we experimentally determine the compressibility, density, and pressure equations of state for an attractive 2D Fermi gas in the normal phase as a function of temperature and interaction strength. In 2D, interacting gases exhibit qualitatively different features to those found in 3D. This is evident in the normalized density equation of state, which peaks at intermediate densities corresponding to the crossover from classical to quantum behavior.

Role for myosin-V motor proteins in the selective delivery of Kv channel isoforms to the membrane surface of cardiac myocytes.

RATIONALE: Kv1.5 (KCNA5) mediates the ultra-rapid delayed rectifier current that controls atrial action potential duration. Given its atrial-specific expression and alterations in human atrial fibrillation, Kv1.5 has emerged as a promising target for the treatment of atrial fibrillation. A necessary step in the development of novel agents that selectively modulate trafficking pathways is the identification of the cellular machinery controlling Kv1.5 surface density, of which little is yet known. OBJECTIVE: To investigate the role of the unconventional myosin-V (MYO5A and MYO5B) motors in determining the cell surface density of Kv1.5. METHODS AND RESULTS: Western blot analysis showed MYO5A and MYO5B expression in the heart, whereas disruption of endogenous motors selectively reduced IKur current in adult rat cardiomyocytes. Dominant negative constructs and short hairpin RNA silencing demonstrated a role for MYO5A and MYO5B in the surface trafficking of Kv1.5 and connexin-43 but not potassium voltage-gated channel, subfamily H (eag-related), member 2 (KCNH2). Live-cell imaging of Kv1.5-GFP and retrospective labeling of phalloidin demonstrated motility of Kv1.5 vesicles on actin tracts. MYO5A participated in anterograde trafficking, whereas MYO5B regulated postendocytic recycling. Overexpression of mutant motors revealed a selective role for Rab11 in coupling MYO5B to Kv1.5 recycling. CONCLUSIONS: MYO5A and MYO5B control functionally distinct steps in the surface trafficking of Kv1.5. These isoform-specific trafficking pathways determine Kv1.5-encoded IKur in myocytes to regulate repolarizing current and, consequently, cardiac excitability. Therapeutic strategies that manipulate Kv1.5 selective trafficking pathways may prove useful in the treatment of arrhythmias.

Pharmacologic characterization of a kinetic in vitro human co-culture angiogenesis model using clinically relevant compounds.

Angiogenesis, the formation of new vessels from preexisting vessels, involves multiple cell types acting in concert to cause endothelial cell proliferation, migration, and differentiation into microvascular arrays. Under pathologic conditions, microenvironment changes result in altered blood vessel production. Historically, in vitro angiogenesis assays study individual aspects of the process and tend to be variable, difficult to quantify, and limited in clinical relevance. Here, we describe a kinetic, quantitative, co-culture angiogenesis model and demonstrate its relevance to in vivo pharmacology. Similar to in vivo angiogenesis, a co-culture of human umbilical vein endothelial cells with normal human dermal fibroblasts remains sensitive to multiple cytokines, resulting in a concentration-dependent stimulation of tube formation over time. Treatment with axitinib, a selective vascular endothelial growth factor (VEGF) antagonist, inhibited VEGF-mediated tube length and branch point formation and was selective for inhibiting VEGF over basic fibroblast growth factor (bFGF), similar to previous studies. Conversely, an FGFR-1 selective compound, PD-161570, was more potent at inhibiting bFGF-mediated angiogenesis. These results demonstrate the cytokine dynamics, selective pharmacology, and translational application of this model system. Finally, combining quantitative angiogenic biology with kinetic, live-content imaging highlights the importance of using validated in vitro models in drug discovery research.

Development and characterization of a high-throughput in vitro cord formation model insensitive to VEGF inhibition.

BACKGROUND: Anti-VEGF therapy reduces tumor blood vessels, however, some vessels always remain. These VEGF insensitive vessels may help support continued tumor growth and metastases. Many in vitro assays examining multiple steps of the angiogenic process have been described, but the majority of these assays are sensitive to VEGF inhibition. There has been little focus on the development of high-throughput, in vitro assays to model the vessels that are insensitive to VEGF inhibition. METHODS: Here, we describe a fixed end-point and kinetic, high-throughput stem cell co-culture model of cord formation. RESULTS: In this system, cords develop within 24 hours, at which point they begin to lose sensitivity to VEGF inhibitors, bevacizumab, and ramucirumab. Consistent with the hypothesis that other angiogenic factors maintain VEGF-independent vessels, pharmacologic intervention with a broad spectrum anti-angiogenic antagonist (suramin), a vascular disrupting agent (combretastatin), or a combination of VEGF and Notch pathway inhibitors reduced the established networks. In addition, we used our in vitro approach to develop an in vivo co-implant vasculogenesis model that connects with the endogenous vasculature to form functional blood vessels. Similar to the in vitro system, over time these vessels become insensitive to VEGF inhibition. CONCLUSION: Together, these models may be used to identify novel drugs targeting tumor vessels that are not sensitive to VEGF inhibition.

Gene therapy rescues cilia defects and restores olfactory function in a mammalian ciliopathy model.

Cilia are evolutionarily conserved microtubule-based organelles that are crucial for diverse biological functions, including motility, cell signaling and sensory perception. In humans, alterations in the formation and function of cilia manifest clinically as ciliopathies, a growing class of pleiotropic genetic disorders. Despite the substantial progress that has been made in identifying genes that cause ciliopathies, therapies for these disorders are not yet available to patients. Although mice with a hypomorphic mutation in the intraflagellar transport protein IFT88 (Ift88Tg737Rpw mice, also known as ORPK mice)5 have been well studied, the relevance of IFT88 mutations to human pathology is unknown. We show that a mutation in IFT88 causes a hitherto unknown human ciliopathy. In vivo complementation assays in zebrafish and mIMCD3 cells show the pathogenicity of this newly discovered allele. We further show that ORPK mice are functionally anosmic as a result of the loss of cilia on their olfactory sensory neurons (OSNs). Notably, adenoviral-mediated expression of IFT88 in mature, fully differentiated OSNs of ORPK mice is sufficient to restore ciliary structures and rescue olfactory function. These studies are the first to use in vivo therapeutic treatment to reestablish cilia in a mammalian ciliopathy. More broadly, our studies indicate that gene therapy is a viable option for cellular and functional rescue of the complex ciliary organelle in established differentiated cells.

Young adults with intellectual disability transitioning from school to post-school: a literature review framed within the ICF.

PURPOSE: The purpose of this review was to describe literature relating to transition for young people with an intellectual disability and identify gaps within the current knowledge base. METHOD: A narrative literature review was undertaken. Searches of databases Medline, CINAHL, PsycINFO, ERIC, ISI Web of Science and ProQuest 500 International provided relevant research articles. The search terms used were intellectual disability, transition, employment, and ICF as well as other terms derived from the ICF. Manual searches of reference lists identified additional studies. Furthermore, government websites were searched for relevant reports and policies. RESULTS: Transition literature was explored by ICF domains; body functions and structures, activity and participation and contextual factors. Studies were identified in some but not all areas and included literature describing self-determination and participation in leisure activities for those with mild intellectual disability. However, significant gaps were found particularly for those with severe intellectual disability. CONCLUSIONS: The ICF is a useful tool in framing a review of transition literature for young people with intellectual disability due to the complexity and multi-faceted nature of transition. The important influence of environmental factors including family systems, post-school services and access to transport were highlighted as having considerable impacts on transition outcomes.

Temperature dependence of the universal contact parameter in a unitary Fermi gas.

The contact I, introduced by Tan, has emerged as a key parameter characterizing universal properties of strongly interacting Fermi gases. For ultracold Fermi gases near a Feshbach resonance, the contact depends upon two quantities: the interaction parameter 1/(k(F)a), where k(F) is the Fermi wave vector and a is the s-wave scattering length, and the temperature T/T(F), where T(F) is the Fermi temperature. We present the first measurements of the temperature dependence of the contact in a unitary Fermi gas using Bragg spectroscopy. The contact is seen to follow the predicted decay with temperature and shows how pair-correlations at high momentum persist well above the superfluid transition temperature.

Crossover from 2D to 3D in a weakly interacting Fermi gas.

We have studied the transition from two to three dimensions in a low temperature weakly interacting 6Li Fermi gas. Below a critical atom number N(2D) only the lowest transverse vibrational state of a highly anisotropic oblate trapping potential is occupied and the gas is two dimensional. Above N(2D) the Fermi gas enters the quasi-2D regime where shell structure associated with the filling of individual transverse oscillator states is apparent. This dimensional crossover is demonstrated through measurements of the cloud size and aspect ratio versus atom number.

Universal behavior of pair correlations in a strongly interacting Fermi gas.

We show that short-range pair correlations in a strongly interacting Fermi gas follow a simple universal law described by Tan's relations. This is achieved through measurements of the static structure factor which displays a universal scaling proportional to the ratio of Tan's contact to the momentum C/q. Bragg spectroscopy of ultracold 6Li atoms from a periodic optical potential is used to measure the structure factor for a wide range of momenta and interaction strengths, providing broad confirmation of this universal law. We calibrate our Bragg spectra using the f-sum rule, which is found to improve the accuracy of the structure factor measurement.

Single molecule imaging reveals differences in microtubule track selection between Kinesin motors.

Cells generate diverse microtubule populations by polymerization of a common alpha/beta-tubulin building block. How microtubule associated proteins translate microtubule heterogeneity into specific cellular functions is not clear. We evaluated the ability of kinesin motors involved in vesicle transport to read microtubule heterogeneity by using single molecule imaging in live cells. We show that individual Kinesin-1 motors move preferentially on a subset of microtubules in COS cells, identified as the stable microtubules marked by post-translational modifications. In contrast, individual Kinesin-2 (KIF17) and Kinesin-3 (KIF1A) motors do not select subsets of microtubules. Surprisingly, KIF17 and KIF1A motors that overtake the plus ends of growing microtubules do not fall off but rather track with the growing tip. Selection of microtubule tracks restricts Kinesin-1 transport of VSVG vesicles to stable microtubules in COS cells whereas KIF17 transport of Kv1.5 vesicles is not restricted to specific microtubules in HL-1 myocytes. These results indicate that kinesin families can be distinguished by their ability to recognize microtubule heterogeneity. Furthermore, this property enables kinesin motors to segregate membrane trafficking events between stable and dynamic microtubule populations.

The voltage-gated Na+ channel beta3 subunit does not mediate trans homophilic cell adhesion or associate with the cell adhesion molecule contactin.

Voltage-gated Na(+) channel (VGSC) beta1 and beta2 subunits are multifunctional, serving as both channel modulators and cell adhesion molecules (CAMs). The purpose of this study was to determine whether VGSC beta3 subunits function as CAMs. The beta3 extracellular domain is highly homologous to beta1, suggesting that beta3 may also be a functional CAM. We investigated the trans homophilic cell adhesive properties of beta3, its association with the beta1-interacting CAM contactin, as well as its ability to interact with the cytoskeletal protein ankyrin. Our results demonstrate that, unlike beta1, beta3 does not participate in trans homophilic cell-cell adhesion or associate with contactin. Further, beta3 does not associate with ankyrin(G) in a heterologous system. Previous studies have shown that beta3 interacts with the CAM neurofascin-186 but not with VGSC beta1. Taken together, these findings suggest that, although beta1 and beta3 exhibit similar channel modulatory properties in heterologous systems, these subunits differ with regard to their homophilic and heterophilic CAM binding profiles.