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Alongside its biosynthetic functions, the small GTPase Rab12 negatively regulates mast cell (MC) exocytosis by its interaction with RILP to promote retrograde transport of the MC secretory granules. Given the role of Rab effectors in mediating Rab functions, in this study we used biochemical and in silico tools to decipher Rab12 interactions with its RILP family effectors. We show that Rab12 interacts with RILP, RILP-L1 and RILP-L2 independently of each other, whereby lysine-71, in mouse Rab12, is critical for Rab12 interactions with RILP-L1 or RILP-L2, but is dispensable for the binding of RILP. Focusing on RILP, and relying on molecular dynamics simulations, functional mutational analyses and peptide inhibition assays, we propose a model for the Rab12-RILP complex, consisting of a RILP homodimer and a single molecule of active Rab12, that interacts with the RILP homology domain (RHD) of one RILP monomer and a C-terminal threonine in the other monomer via its switch I and switch II regions. Mutational analyses of RILP RHD also demonstrate its involvement in the regulation of MC secretory granule transport. Jointly, our results provide structural and functional insights into the Rab12-RILP complex on the basis of which new tools could be generated for decoding Rab12 functions.

e14552 Background: Recent advances in the field of cancer immunology have highlighted the importance of the immune system for eliminating tumors. Numerous studies have shown that tumor-infiltrating immune cells such as antigen-presenting cells (APCs), T cells, and natural killer (NK) cells play critical roles in tumor control. However, the inflammatory anti-tumor immune response is counteracted by the induction of immune regulatory mechanisms within the tumor microenvironment (TME). These findings have led to the development of immune-targeted therapies, which are aimed at activating anti-tumor immune signaling pathways and enhancing anti-tumor immune function. While immunotherapies, have revolutionized the treatment of several solid tumors and leukemias, at best response rates remain low at 25%-30%, and a portion of patients eventually develop resistance to therapy leading to disease progression and mortality. Methods: We have previously reported the potent effects of 3-4 daily intravenous infusions of immune modifying poly(lactic- co-glycolic acid) (PLGA) nanoparticles, ONP-302, free from drugs or other bioactive agents, for the amelioration of acute inflammatory diseases by targeting myeloid cells. The present studies describe a novel use for ONP-302 nanoparticles, employing an altered dosing scheme to reprogram myeloid cells resulting in significant enhancement of tumor immunity. The efficacy of ONP-302 nanoparticles at inducing an anti-tumor immune response was evaluated using syngeneic mouse tumor models. Results: ONP-302 infusion decreased tumor growth via the activation of the cGAS/STING pathway within myeloid cells, and subsequently increased NK cell activation via an IL-15-dependent mechanism. Additionally, ONP-302 treatment increased PD-1/PD-L1 expression in the tumor microenvironment, thereby allowing for functionality of anti-PD-1 for treatment in the B16.F10 melanoma tumor model which is normally unresponsive to monotherapy with anti-PD-1. Conclusions: These findings indicate that ONP-302 allows for tumor control via reprogramming myeloid cells via activation of the STING/IL-15/NK cell mechanism, as well as increasing anti-PD-1 response rates.

Secretory granule (SG) transport is a critical step in regulated exocytosis including degranulation of activated mast cells. The latter process results in the release of multiple inflammatory mediators that play key roles in innate immunity, as well as in allergic responses. In this study, we identified the small GTPase Rab12 as a novel regulator of mast cell SG transport, and we provide mechanistic insights into its mode of action. We show that Rab12 is activated in a stimulus-dependent fashion and promotes microtubule-dependent retrograde transport of the SGs in the activated cells. We also show that this minus end transport of the SGs is mediated by the RILP–dynein complex and identify RILP as a novel effector of Rab12. Finally, we show that Rab12 negatively regulates mast cell degranulation. Taken together, our results identify Rab12 as a novel regulator of mast cell responses and disclose for the first time, to our knowledge, the mechanism of retrograde transport of the mast cell SGs.

Cross-sectional.To determine what physical measures are associated with visually assessed quality of movement among patients with patellofemoral pain (PFP).An altered movement pattern has been implicated as a risk factor for PFP. An understanding of physical measures associated with an altered movement pattern could potentially help guide prevention and management efforts in patients with PFP.Seventy-nine (40 women) Israel Defense Forces soldiers referred to physical therapy with a diagnosis of PFP were included. Movement pattern was assessed visually during a lateral step-down test and rated as "good" or "moderate," based on previously established criteria. Weight-bearing and non-weight-bearing ankle dorsiflexion (DF) range of motion (ROM); hip internal and external rotation ROM; and hip abduction, hip external rotation, and knee extension strength were also assessed. Differences in physical measures between those with good versus moderate quality of movement were assessed.Weight-bearing DF ROM was more limited among participants with a moderate quality of movement compared to those with a good quality of movement (P.01). Among men, non-weight-bearing DF ROM was more limited in those with a moderate quality of movement as well (P.01). In addition, quality of movement was associated with weight-bearing DF ROM for both women (r = -0.39, P = .01) and men (r = -0.46, P.01), and with non-weight-bearing DF ROM for men (r = -0.66, P.01). When the subgroup of participants who exhibited more than 25° of non-weight-bearing DF ROM was assessed, those with a good quality of movement displayed greater hip external rotator and knee extensor muscle strength compared with those with a moderate quality of movement (P.01).Ankle DF ROM should be assessed when patients with PFP demonstrate a lower quality of movement during a lateral step-down test. Lower hip muscle strength may be associated with lower quality of movement among patients with relatively greater ankle DF ROM.

ERK1 and ERK2 are highly homologous isoforms that often play redundant roles in regulating cellular functions. We analyzed the spatiotemporal patterns of ERK1 and ERK2 in resting and activated mast cells. Strikingly, we identified distinct pathways for these kinases. ERK1 localized to the cytosol and translocated to the nucleus upon cell activation and kinase phosphorylation. In contrast, ERK2 distributed between the cytosol and near the microtubule organizing center (MTOC) in resting cells and accumulated further at a pericentrosomal region upon cell trigger. Pericentrosomal accumulation of ERK2 was phosphorylation independent, required an intact microtubule network and was significantly enhanced by the overexpression of Neuronal Calcium Sensor-1 (NCS-1). We also identified γ-tubulin and phosphatidylinositol 4 kinaseβ (PI4Kβ), a downstream effector of NCS-1, as novel partner proteins of ERK2. Taken together, our results imply non-redundant functions of ERK1 and ERK2 in mast cells and implicate NCS-1 and PΙ4Κβ as regulators of ERK2 trafficking.

Patients with cancer may suffer from malnutrition due to cachexia, this maybe secondary to treatment, psychological factors and/or gastrointestinal (GI) obstruction. GI obstruction indicates a need for TPN. Does this apply to patients with incurable terminal cancer? How does TPN affect longevity and quality of life in this group of patients? What is the course of TPN treatment compared with patients receiving TPN due to nonmalignant GI failure (NMGIF). The aim of this work was to help define the role of TPN in patients with incurable cancer and GI obstruction. Data of all patients treated by home TPN (HTPN) 2003–2009 were collected prospectively and analyzed. Sixty-eight patients were treated with HTPN, 30 of them for NMGIF. Mean age was 52 years (37–87). Primary sites of cancer were ovary (9), stomach (8) and others (11). Median survival of patients with malignant GI failure (MGIF) was 140 days (20–783) with no difference with regard to age, gender, primary diagnosis, BMI, percentage of weight loss and albumin level. Patients with MGIF and a higher performance score had longer survival. Patients with MGIF suffered significantly higher rates of overall and infectious complications per treatment days [P < 0.001]. TPN for MGIF incurable patients with cancer prolongs survival but at the cost of frequent complications. TPN is indicated in a selected group of MGIF. We do not have the tools to predict in which patients the benefits will outweigh the cost, but we found variables which may assist caregivers to make clinical and empathic decisions individually.