Caffeine inhibits PI3K and mTORC2 in Dictyostelium and differentially affects multiple other cAMP chemoattractant signaling effectors.

Caffeine is commonly used in Dictyostelium to inhibit the synthesis of the chemoattractant cAMP and, therefore, its secretion and the autocrine stimulation of cells, in order to prevent its interference with the study of chemoattractant-induced responses. However, the mechanism through which caffeine inhibits cAMP synthesis in Dictyostelium has not been characterized. Here, we report the effects of caffeine on the cAMP chemoattractant signaling network. We found that caffeine inhibits phosphatidylinositol 3-kinase (PI3K) and mechanistic target of rapamycin complex 2 (mTORC2). Both PI3K and mTORC2 are essential for the chemoattractant-stimulated cAMP production, thereby providing a mechanism for the caffeine-mediated inhibition of cAMP synthesis. Our results also reveal that caffeine treatment of cells leads to an increase in cAMP-induced RasG and Rap1 activation, and inhibition of the PKA, cGMP, MyoII, and ERK1 responses. Finally, we observed that caffeine has opposite effects on F-actin and ERK2 depending on the assay and Dictyostelium strain used, respectively. Altogether, our findings reveal that caffeine considerably affects the cAMP-induced chemotactic signaling pathways in Dictyostelium, most likely acting through multiple targets that include PI3K and mTORC2.

An evidence based conceptual framework for the multifactorial understanding of proximal junctional kyphosis.

Introduction: Adult spinal deformity (ASD) is a debilitating pathology that arises from a variety of etiologies. Spinal fusion surgery is the mainstay of treatment for those who do not achieve symptom relief with conservative interventions. Fusion surgery can be complicated by a secondary deformity termed proximal junctional kyphosis (PJK). Research question: This scoping review evaluates the modern body of literature analyzing risk factors for PJK development and organizes these factors according to a multifactorial framework based on mechanical, tissue or demographic components. Materials and methods: An extensive search of the literature was performed in PubMed and Embase back to the year 2010. Articles were assessed for quality. All risk factors that were evaluated and those that significantly predicted the development of PJK were compiled. The frequency that a risk factor was predictive compared to the number of times it was evaluated was calculated. Results: 150 articles were reviewed. 57.3% of papers were of low quality. 76% of risk factors analyzed were focusing on the mechanical contribution to development of PJK versus only 5% were focusing on the tissue-based contribution. Risk factors that were most frequently predictive compared to how often they were analyzed were Hounsfield Units of vertebrae, UIV disc degeneration, paraspinal muscle cross sectional area and fatty infiltration, ligament augmentation, instrument characteristics, postoperative hip and lower extremity radiographic metrics, and postoperative teriparatide supplementation. Discussion and conclusion: This review finds a multifactorial framework accounting for mechanical, patient and tissue-based risk factors will improve the understanding of PJK development.

Interdisciplinary Approach to Expedited Outpatient Gastrostomy Tube Placement in Head and Neck Cancer Patients: A Single Center Retrospective Study.

RATIONALE AND OBJECTIVE: Treatment for head and neck cancer (HNC) can lead to decreased oral intake which often requires gastrostomy tube (g-tube) placement to provide nutritional support. A multidisciplinary team (MDT) consisting of interventional radiology (IR), HNC oncology and surgery, nutrition, and speech language pathology departments implemented an expedited outpatient g-tube placement pathway to reduce hospital stays and associated costs, initiate feeds sooner, and improve communication between care teams. This single center study investigates differences in complications, time to procedure and costs savings with implementing this pathway. METHODS: 142 patients with HNC who underwent elective image guided g-tube placement by IR from 2015 to 2022 were identified retrospectively. 52 patients underwent the traditional pathway, and 90 patients underwent the expedited pathway. Patient demographics, procedure characteristics, periprocedural costs and 90-day complication rates were collected and compared statistically. RESULTS: The 90-day complication rate was comparable between groups (traditional=32.7%; expedited=22.2%; p-value=0.17). The expedited pathway decreased the time from consult to procedure by 11.1 days (95% CI 7.6 - 14.6; p < 0.001) and decreased charge per procedure by $2940 (95% CI $989-$4891; p < 0.001). CONCLUSION: A MDT for the treatment of patients with HNC successfully provided enteral nutrition support faster, with fewer associated costs, and in a more patient centered approach than previously done at this institution.

The cAMP-induced G protein subunits dissociation monitored in live Dictyostelium cells by BRET reveals two activation rates, a positive effect of caffeine and potential role of microtubules.

To study the dynamics and mechanisms controlling activation of the heterotrimeric G protein Gα2ßγ in Dictyostelium in response to stimulation by the chemoattractant cyclic AMP (cAMP), we monitored the G protein subunit interaction in live cells using bioluminescence resonance energy transfer (BRET). We found that cAMP induces the cAR1-mediated dissociation of the G protein subunits to a similar extent in both undifferentiated and differentiated cells, suggesting that only a small number of cAR1 (as expressed in undifferentiated cells) is necessary to induce the full activation of Gα2ßγ. In addition, we found that treating cells with caffeine increases the potency of cAMP-induced Gα2ßγ activation; and that disrupting the microtubule network but not F-actin inhibits the cAMP-induced dissociation of Gα2ßγ. Thus, microtubules are necessary for efficient cAR1-mediated activation of the heterotrimeric G protein. Finally, kinetics analyses of Gα2ßγ subunit dissociation induced by different cAMP concentrations indicate that there are two distinct rates at which the heterotrimeric G protein subunits dissociate when cells are stimulated with cAMP concentrations above 500 nM versus only one rate at lower cAMP concentrations. Quantitative modeling suggests that the kinetics profile of Gα2ßγ subunit dissociation results from the presence of both uncoupled and G protein pre-coupled cAR1 that have differential affinities for cAMP and, consequently, induce G protein subunit dissociation through different rates. We suggest that these different signaling kinetic profiles may play an important role in initial chemoattractant gradient sensing.

Heterogeneous Responses of Hematopoietic Stem Cells to Inflammatory Stimuli Are Altered with Age.

Long-term hematopoietic stem cells (LT-HSCs) maintain hematopoietic output throughout an animal's lifespan. However, with age, the balance is disrupted, and LT-HSCs produce a myeloid-biased output, resulting in poor immune responses to infectious challenge and the development of myeloid leukemias. Here, we show that young and aged LT-HSCs respond differently to inflammatory stress, such that aged LT-HSCs produce a cell-intrinsic, myeloid-biased expression program. Using single-cell RNA sequencing (scRNA-seq), we identify a myeloid-biased subset within the LT-HSC population (mLT-HSCs) that is prevalent among aged LT-HSCs. We identify CD61 as a marker of mLT-HSCs and show that CD61-high LT-HSCs are uniquely primed to respond to acute inflammatory challenge. We predict that several transcription factors regulate the mLT-HSCs gene program and show that Klf5, Ikzf1, and Stat3 play an important role in age-related inflammatory myeloid bias. We have therefore identified and isolated an LT-HSC subset that regulates myeloid versus lymphoid balance under inflammatory challenge and with age.

Adipocyte-Derived Lipids Mediate Melanoma Progression via FATP Proteins.

Advanced, metastatic melanomas frequently grow in subcutaneous tissues and portend a poor prognosis. Though subcutaneous tissues are largely composed of adipocytes, the mechanisms by which adipocytes influence melanoma are poorly understood. Using in vitro and in vivo models, we find that adipocytes increase proliferation and invasion of adjacent melanoma cells. Additionally, adipocytes directly transfer lipids to melanoma cells, which alters tumor cell metabolism. Adipocyte-derived lipids are transferred to melanoma cells through the FATP/SLC27A family of lipid transporters expressed on the tumor cell surface. Among the six FATP/SLC27A family members, melanomas significantly overexpress FATP1/SLC27A1. Melanocyte-specific FATP1 expression cooperates with BRAFV600E in transgenic zebrafish to accelerate melanoma development, an effect that is similarly seen in mouse xenograft studies. Pharmacologic blockade of FATPs with the small-molecule inhibitor Lipofermata abrogates lipid transport into melanoma cells and reduces melanoma growth and invasion. These data demonstrate that stromal adipocytes can drive melanoma progression through FATP lipid transporters and represent a new target aimed at interrupting adipocyte-melanoma cross-talk.Significance: We demonstrate that stromal adipocytes are donors of lipids that mediate melanoma progression. Adipocyte-derived lipids are taken up by FATP proteins that are aberrantly expressed in melanoma. Inhibition of FATPs decreases melanoma lipid uptake, invasion, and growth. We provide a mechanism for how stromal adipocytes drive tumor progression and demonstrate a novel microenvironmental therapeutic target. Cancer Discov; 8(8); 1006-25. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 899.