mTOR complex 2 - akt signaling is physically and functionally at mam

The target of rapamycin (TOR) is a conserved protein kinase and a central controller of growth. TOR can be part of two structurally and functionally distinct complexes, termed TOR complex 1 and TOR complex 2. Mammalian TOR complex 2 (mTORC2) is composed of mTOR, Rictor, Sin1 and mLST8. Both mTORC1 and mTORC2 are activated by growth factors. The mechanism via which growth factors regulate mTORC2 has been elusive until recently. mTORC2 binds ribosomes in a growth factor stimulated manner and this association is required for mTORC2 activity. mTOR complex 2 functions include control of spatial cell growth and metabolism and thus, mTORC2 deregulation has been linked to various disorders including cancer and diabetes. mTORC2 phosphorylates and thereby activates the AGC kinase family member Akt (PKB). Akt has many different targets and functions, not all of which depend on mTORC2 mediated Akt phosphorylation. In order to gain a better understanding of mTORC2 function, we asked where mTORC2 signaling is localized. A number of studies localized mTORC2, functionally or physically, either to the endoplasmic reticulum (ER) or to mitochondria.We investigated whether these seemingly unrelated observations concerning mTORC2 localization, might be the consequence of mTORC2 signaling at MAM. MAM or mitochondria-associated ER membrane is a quasi-synaptic subdomain between the ER and mitochondria. MAM plays a crucial role in the regulation of mitochondrial metabolism and cell survival by gating both the calcium flux and phospholipid trafficking between the ER and mitochondria. First, we analyzed mTORC2 subcellular localization. mTORC2 is localized to the ER adjacent to mitochondria and mTORC2 can be biochemically isolated from MAM structures. mTOR complex 2 interacts with the IP3R-Grp75-VDAC1 complex, a tether that connects ER and mitochondria at MAM. Insulin stimulates mTORC2 localization to MAM and mTORC2 interaction with the IP3R-Grp75-VDAC1 complex. MAM localization of mTORC2 depends on mTORC2-ribosome interaction. Next we investigated the function of mTORC2 at MAM. Rictor (mTORC2) knockout causes a decrease in MAM formation. Growth factors stimulate MAM formation via mTORC2 and the Akt substrate PACS2, a MAM resident protein. As expected for MAM deficient cells, mTORC2 disruption changes the calcium flux from the ER to mitochondria at MAM. Furthermore, we observe a reduction of Akt mediated phosphorylation of the MAM calcium channel IP3R upon Rictor knockout. Thus, mTORC2 signaling at MAM controls MAM mediated calcium release via the Akt targets PACS2 and IP3R. Since MAM disruption and calcium signaling both affect mitochondrial metabolism, we proceeded by analyzing the mitochondrial physiology of mTORC2 deficient cells. Rictor knockout cells exhibit a disruption of VDAC1-HK2 binding, caused by a lack of Akt mediated phosphorylation of HK2 at MAM. This, together with the defect in MAM, induces an increase in basal respiration, mitochondrial inner membrane potential, and ATP production in the mTORC2 deficient cells, culminating in apoptosis. Thus, mTORC2 at MAM appears to control several aspects of mitochondrial physiology. These findings emphasize the role of MAM as a signaling hub that controls cell physiology. By identifying the integral role of mTORC2 at the core of this platform, our results provide new insights on the mechanisms that regulate growth and metabolism. These observations may offer new therapeutic strategies against mTORC2 and MAM driven diseases such as diabetes, Alzheimer’s and cancer

Physeal Distraction for Joint Preservation in Malignant Metaphyseal Bone Tumors in Children

Background: Physeal distraction facilitates metaphyseal bone tumor resection in children and preserves the adjacent joint. The technique was first described by Cañadell. Tumor resection procedures allowing limb-sparing reconstruction have been used increasingly in recent years without compromising oncologic principles. Questions/purposes: We report our results with Cañadell's technique by assessing tumor control, functional outcome, and complications. Methods: Six consecutive children with primary malignant metaphyseal bone tumors underwent physeal distraction as a part of tumor resection. Tumor location was the distal femur in four patients, the proximal humerus in one patient, and the proximal tibia in one patient. The functional outcome was evaluated after a minimum of 18months (median, 62months; range, 18-136months) using the Musculoskeletal Tumor Society (MSTS) score and the Toronto Extremity Salvage Score (TESS). Results: At latest followup, five patients were alive and disease-free and one had died from metastatic disease. All tumor resections resulted in local control; there were no local recurrencies. The mean MSTS score was 79% (range, 53%-97%) and corresponding mean TESS was 83% (range, 71%-92%). In one case, postoperative infection required amputation of the proximal lower leg. All physeal distractions were successful except for one patient in whom distraction resulted in rupturing into the tumor. This situation was salvaged by transepiphyseal resection. Conclusions: We consider Cañadell's technique a useful tool in the armamentarium to treat children with malignant tumors that are in close proximity to an open physis. Level of Evidence: Level IV, therapeutic study. See Guidelines for Authors for a complete description of levels of evidenc

Syrosingopine sensitizes cancer cells to killing by metformin

We report that the anticancer activity of the widely used diabetic drug metformin is strongly potentiated by syrosingopine. Synthetic lethality elicited by combining the two drugs is synergistic and specific to transformed cells. This effect is unrelated to syrosingopine's known role as an inhibitor of the vesicular monoamine transporters. Syrosingopine binds to the glycolytic enzyme α-enolase in vitro, and the expression of the γ-enolase isoform correlates with nonresponsiveness to the drug combination. Syrosingopine sensitized cancer cells to metformin and its more potent derivative phenformin far below the individual toxic threshold of each compound. Thus, combining syrosingopine and codrugs is a promising therapeutic strategy for clinical application for the treatment of cancer

Gender Sorting at the Application Interface

We document gender sorting of candidates into gender-typed jobs at the point of initial application to a company. At this step of the hiring process, the firm has implemented a policy whereby organizational screeners’ discretion has been eliminated such that there is no opportunity for contact between hiring agents and applicants. Thus, the job choices studied here offer unique insight as they are uncontaminated by screeners’ steering of candidates toward gender-typed jobs. Even in the absence of steering, we find clear patterns of gendered job choices that line up with gender stereotypes of job roles. Moreover, these gendered patterns recur both within individuals and within race groups. Comparing our findings to the pattern of job sorting in the external local labor market, we find that supply-side factors do not fully account for the levels job sex segregation observed in the open labor market. Although probably not the entire story, we show clear evidence that supply-side sorting processes are important factors contributing to job sex segregation

Longitudinal Research on Aging Drivers (LongROAD): study design and methods

Background: As an important indicator of mobility, driving confers a host of social and health benefits to older adults. Despite the importance of safe mobility as the population ages, longitudinal data are lacking about the natural history and determinants of driving safety in older adults. Methods: The Longitudinal Research on Aging Drivers (LongROAD) project is a multisite prospective cohort study designed to generate empirical data for understanding the role of medical, behavioral, environmental and technological factors in driving safety during the process of aging. Results: A total of 2990 active drivers aged 65–79 years at baseline have been recruited through primary care clinics or health care systems in five study sites located in California, Colorado, Maryland, Michigan, and New York. Consented participants were assessed at baseline with standardized research protocols and instruments, including vehicle inspection, functional performance tests, and “brown-bag review” of medications. The primary vehicle of each participant was instrumented with a small data collection device that records detailed driving data whenever the vehicle is operating and detects when a participant is driving. Annual follow-up is being conducted for up to three years with a telephone questionnaire at 12 and 36 months and in-person assessment at 24 months. Medical records are reviewed annually to collect information on clinical diagnoses and healthcare utilization. Driving records, including crashes and violations, are collected annually from state motor vehicle departments. Pilot testing was conducted on 56 volunteers during March–May 2015. Recruitment and enrollment were completed between July 2015 and March 2017. Conclusions: Results of the LongROAD project will generate much-needed evidence for formulating public policy and developing intervention programs to maintain safe mobility while ensuring well-being for older adults

Control of dynamic cell behaviors during angiogenesis and anastomosis by Rasip1

Organ morphogenesis is driven by a wealth of tightly orchestrated cellular behaviors, which ensure proper organ assembly and function. Many of these cell activities involve cell-cell interactions and remodeling of the F-actin cytoskeleton. Here, we analyze the requirement for Rasip1 (Ras-interacting protein 1), an endothelial-specific regulator of junctional dynamics, during blood vessel formation. Phenotype analysis of rasip1 mutants in zebrafish embryos reveals distinct functions of Rasip1 during sprouting angiogenesis, anastomosis and lumen formation. During angiogenic sprouting, loss of Rasip1 causes cell pairing defects due to a destabilization of tricellular junctions, indicating that stable tricellular junctions are essential to maintain multicellular organization within the sprout. During anastomosis, Rasip1 is required to establish a stable apical membrane compartment; rasip1 mutants display ectopic, reticulated junctions and the apical compartment is frequently collapsed. Loss of Ccm1 and Heg1 function mimics the junctional defects of rasip1 mutants. Furthermore, downregulation of ccm1 and heg1 leads to a delocalization of Rasip1 at cell junctions, indicating that junctional tethering of Rasip1 is required for its function in junction formation and stabilization during sprouting angiogenesis

Next-generation plasmids for transgenesis in zebrafish and beyond

Transgenesis is an essential technique for any genetic model. Tol2-based transgenesis paired with Gateway-compatible vector collections has transformed zebrafish transgenesis with an accessible, modular system. Here, we established several next-generation transgenesis tools for zebrafish and other species to expand and enhance transgenic applications. To facilitate gene-regulatory element testing, we generated Gateway middle entry vectors harboring the small mouse beta-globin minimal promoter coupled to several fluorophores, CreERT2, and Gal4. To extend the color spectrum for transgenic applications, we established middle entry vectors encoding the bright, blue-fluorescent protein mCerulean and mApple as an alternative red fluorophore. We present a series of p2A peptide-based 3' vectors with different fluorophores and subcellular localizations to co-label cells expressing proteins of interest. Lastly, we established Tol2 destination vectors carrying the zebrafish exorh promoter driving different fluorophores as a pineal gland-specific transgenesis marker active prior to hatching and through adulthood. exorh-based reporters and transgenesis markers also drive specific pineal gland expression in the eye-less cavefish (Astyanax). Together, our vectors provide versatile reagents for transgenesis applications in zebrafish, cavefish, and other models

Next-generation plasmids for transgenesis in zebrafish and beyond

Transgenesis is an essential technique for any genetic model. Tol2-based transgenesis paired with Gateway-compatible vector collections has transformed zebrafish transgenesis with an accessible, modular system. Here, we established several next-generation transgenesis tools for zebrafish and other species to expand and enhance transgenic applications. To facilitate gene-regulatory element testing, we generated Gateway middle entry vectors harboring the small mouse betaglobin minimal promoter coupled to several fluorophores, CreERT2, and Gal4. To extend the color spectrum for transgenic applications, we established middle entry vectors encoding the bright, blue-fluorescent protein Cerulean and mApple as an alternative red fluorophore. We present a series of p2A peptide-based 3' vectors with different fluorophores and subcellular localizations to co-label cells expressing proteins of interest. Lastly, we established Tol2 destination vectors carrying the zebrafish exorh promoter driving different fluorophores as a pineal gland-specific transgenesis marker active prior to hatching and through adulthood. exorh-based reporters and transgenesis markers also drive specific pineal gland expression in the eye-less cavefish (Astyanax). Together, our vectors provide versatile reagents for transgenesis applications in zebrafish, cavefish, and other models

Late 1920s film theory and criticism as a test-case for Benjamin’s generalizations on the experiential effects of editing

This article investigates Walter Benjamin’s influential generalization that the effects of cinema are akin to the hyper-stimulating experience of modernity. More specifically, I focus on his oft-cited 1935/36 claim that all editing elicits shock-like disruption. First, I propose a more detailed articulation of the experience of modernity understood as hyper-stimulation and call for distinguishing between at least two of its subsets: the experience of speed and dynamism, on the one hand, and the experience of shock/disruption, on the other. Then I turn to classical film theory of the late 1920s to demonstrate the existence of contemporary views on editing alternative to Benjamin’s. For instance, whereas classical Soviet and Weimar theorists relate the experience of speed and dynamism to both Soviet and classical Hollywood style editing, they reserve the experience of shock/disruption for Soviet montage. In order to resolve the conceptual disagreement between these theorists, on the one hand, and Benjamin, on the other, I turn to late 1920s Weimar film criticism. I demonstrate that, contrary to Benjamin’s generalizations about the disruptive and shock-like nature of all editing, and in line with other theorists’ accounts, different editing practices were regularly distinguished by comparison to at least two distinct hyper-stimulation subsets: speed and dynamism, and shock-like disruption. In other words, contemporaries regularly distinguished between Soviet montage and classical Hollywood editing patterns on the basis of experiential effects alone. On the basis of contemporary reviews of city symphonies, I conclude with a proposal for distinguishing a third subset – confusion. This is an original manuscript / preprint of an article published by Taylor & Francis in Early Popular Visual Culture on 02 Aug 2016 available online: https://doi.org/10.1080/17460654.2016.1199322