Hyperactivated Wnt signaling induces synthetic lethal interaction with Rb inactivation by elevating TORC1 activities.

Inactivation of the Rb tumor suppressor can lead to increased cell proliferation or cell death depending on specific cellular context. Therefore, identification of the interacting pathways that modulate the effect of Rb loss will provide novel insights into the roles of Rb in cancer development and promote new therapeutic strategies. Here, we identify a novel synthetic lethal interaction between Rb inactivation and deregulated Wg/Wnt signaling through unbiased genetic screens. We show that a weak allele of axin, which deregulates Wg signaling and increases cell proliferation without obvious effects on cell fate specification, significantly alters metabolic gene expression, causes hypersensitivity to metabolic stress induced by fasting, and induces synergistic apoptosis with mutation of fly Rb ortholog, rbf. Furthermore, hyperactivation of Wg signaling by other components of the Wg pathway also induces synergistic apoptosis with rbf. We show that hyperactivated Wg signaling significantly increases TORC1 activity and induces excessive energy stress with rbf mutation. Inhibition of TORC1 activity significantly suppressed synergistic cell death induced by hyperactivated Wg signaling and rbf inactivation, which is correlated with decreased energy stress and decreased induction of apoptotic regulator expression. Finally the synthetic lethality between Rb and deregulated Wnt signaling is conserved in mammalian cells and that inactivation of Rb and APC induces synergistic cell death through a similar mechanism. These results suggest that elevated TORC1 activity and metabolic stress underpin the evolutionarily conserved synthetic lethal interaction between hyperactivated Wnt signaling and inactivated Rb tumor suppressor.

Accessing new understandings of trauma-informed care with queer birthing women in a rural context.

AIMS AND OBJECTIVES: Participant narratives from a feminist and queer phenomenological study aim to broaden current understandings of trauma. Examining structural marginalisation within perinatal care relationships provides insights into the impact of dominant models of care on queer birthing women. More specifically, validation of queer experience as a key finding from the study offers trauma-informed strategies that reconstruct formerly disempowering perinatal relationships. BACKGROUND: Heteronormativity governs birthing spaces and presents considerable challenges for queer birthing women who may also have an increased risk of trauma due to structurally marginalising processes that create and maintain socially constructed differences. DESIGN: Analysis of the qualitative data was guided by feminist and queer phenomenology. This was well suited to understanding queer women's storied narratives of trauma, including disempowering processes of structural marginalisation. METHODS: Semistructured and conversational interviews were conducted with a purposeful sample of thirteen queer-identified women who had experiences of birthing in rural Nova Scotia, Canada. RESULTS: Validation was identified as meaningful for queer women in the context of perinatal care in rural Nova Scotia. Offering new perspectives on traditional models of assessment provide strategies to create a context of care that reconstructs the birthing space insofar as women at risk do not have to come out as queer in opposition to the expectation of heterosexuality. CONCLUSIONS: Normative practices were found to further the effects of structural marginalisation suggesting that perinatal care providers, including nurses, can challenge dominant models of care and reconstruct the relationality between queer women and formerly disempowering expectations of heteronormativity that govern birthing spaces. RELEVANCE TO CLINICAL PRACTICE: New trauma-informed assessment strategies reconstruct the relationality within historically disempowering perinatal relationships through potentiating difference which avoids retraumatising women with re-experiencing the process of coming out as queer in opposition to the expectation of heterosexuality.

Proteins in the nutrient-sensing and DNA damage checkpoint pathways cooperate to restrain mitotic progression following DNA damage.

Checkpoint pathways regulate genomic integrity in part by blocking anaphase until all chromosomes have been completely replicated, repaired, and correctly aligned on the spindle. In Saccharomyces cerevisiae, DNA damage and mono-oriented or unattached kinetochores trigger checkpoint pathways that bifurcate to regulate both the metaphase to anaphase transition and mitotic exit. The sensor-associated kinase, Mec1, phosphorylates two downstream kinases, Chk1 and Rad53. Activation of Chk1 and Rad53 prevents anaphase and causes inhibition of the mitotic exit network. We have previously shown that the PKA pathway plays a role in blocking securin and Clb2 destruction following DNA damage. Here we show that the Mec1 DNA damage checkpoint regulates phosphorylation of the regulatory (R) subunit of PKA following DNA damage and that the phosphorylated R subunit has a role in restraining mitosis following DNA damage. In addition we found that proteins known to regulate PKA in response to nutrients and stress either by phosphorylation of the R subunit or regulating levels of cAMP are required for the role of PKA in the DNA damage checkpoint. Our data indicate that there is cross-talk between the DNA damage checkpoint and the proteins that integrate nutrient and stress signals to regulate PKA.

The DNA damage checkpoint and PKA pathways converge on APC substrates and Cdc20 to regulate mitotic progression.

The conserved checkpoint kinases Chk1 and Rad53-Dun1 block the metaphase to anaphase transition by the phosphorylation and stabilization of securin, and block the mitotic exit network regulated by the Bfa1-Bub2 complex. However, both chk1 and rad53 mutants are able to exit from mitosis and initiate a new cell cycle, suggesting that both pathways have supporting functions in restraining anaphase and in blocking the inactivation of mitotic cyclin-Cdk1 complexes. Here we find that the cyclic-AMP-dependent protein kinase (PKA) pathway supports Chk1 in the regulation of mitosis by targeting the mitotic inducer Cdc20. Cdc20 is phosphorylated on PKA consensus sites after DNA damage, and this phosphorylation requires the Atr orthologue Mec1 and the PKA catalytic subunits Tpk1 and Tpk2. We show that the inactivation of PKA or expression of phosphorylation-defective Cdc20 proteins accelerates securin and Clb2 destruction in chk1 mutants and is sufficient to remove most of the DNA damage-induced delay. Mutation of the Cdc20 phosphorylation sites permitted the interaction of Cdc20 with Clb2 under conditions that should halt cell cycle progression. These data show that PKA pathways regulate mitotic progression through Cdc20 and support the DNA damage checkpoint pathways in regulating the destruction of Clb2 and securin.

Quantification of Salmonella enterica transfer between tomatoes, soil, and plastic mulch.

Tomatoes have been linked to Salmonella outbreaks in the United States (US). Plasticulture systems, that combine raised beds, plastic mulch, drip irrigation and fumigation, are common in commercial staked fresh tomato production in the US. The US FDA Produce Safety Rule prohibits the distribution of any produce covered by the rule (including fresh market tomatoes) that drops to the ground before harvest. This research was undertaken to better characterize the risks posed by tomatoes that touch plastic mulch or soil immediately before or during harvest. Research was conducted in three states (Florida, Maryland, and Ohio). Each state utilized tomatoes from their state at the point of harvest maturity most common in that state. Each state used indigenous soil and plastic mulch for transfer scenarios. New plastic mulch obtained directly from the application roll and used plastic mulch that had been present on beds for a growing season were evaluated. A five-strain cocktail of Salmonella enterica isolates obtained from tomato outbreaks was used. Mulch (new or used), soil, or tomatoes were spot inoculated with 100 µl of inoculum to obtain a final population of ~6 log CFU/surface. Items were either touched to each other immediately (1-2 s) after inoculation (wet contact) or allowed to dry at ambient temperature for 1 h or 24 h (dry contact). All surfaces remained in brief (1-5 s) or extended (24 h) contact at ambient temperature. Transfer of Salmonella between a tomato and plastic mulch or soil is dependent on contact time, dryness of the inoculum, type of soil, and contact surface. Transfer of Salmonella to and from the mulch and tomatoes for wet and 1 h dry inocula were similar with mean log % transfers varying from 0.7 ± 0.2 to 1.9 ± 0.1. The transfer of Salmonella between soil or plastic mulch to and from tomatoes was dependent on moisture with wet and 1 h dry inocula generally yielding significantly (p < 0.05) higher transfer than the 24 h dry inoculum. Results indicate that harvesting dry tomatoes significantly (p < 0.05) reduces the risk of contamination from soil or mulch contact. Transfer to tomatoes was generally significantly greater (p < 0.05) from new and used plastic mulch than from soil. If contamination and moisture levels are equivalent and contact times are equal to or <24 h before harvest, significantly (p < 0.05) more Salmonella transfers to tomatoes from mulch than from soil. Our findings support that harvesting tomatoes from soil has similar or lower risk than harvesting from plastic mulch.

Queer Phenomenology, the Disruption of Heteronormativity, and Structurally Responsive Care.

Lesbian, gay, bisexual, transgender, and queer (LGBTQ) health disparities persist and reflect larger structural inequities that negatively impact the health of historically marginalized communities. By way of using queer phenomenology, the author analyzes a personal experience that was harmful to her as a lesbian patient who required emergency medical attention. Also a registered nurse, the author draws on her lived experiences to reveal heteronormativity as a prevalent, but largely unacknowledged, source of structural harms for LGBTQ patients. This aims to bring about an appreciation among nurses and other health care professionals to locate themselves within systems of privilege and oppression and gain an awareness on how they might better respond to ongoing structural harms that are disproportionately experienced by vulnerable patient populations.

Compulsory Heterosexuality and Lesbian Invisibility in Nursing.

Lesbian nurses have historically been silenced, but diversity initiatives within professional health programs suggest a need to initiate scholarly discussions that explore heteronormativity as increasing the risk of harm for nonheterosexual nurses as compared to their heterosexual counterparts. Nurses can reflect on relative privilege within the profession to better understand the ways in which normative health practices perpetuate adverse health outcomes among vulnerable patient populations. Nurses from diverse backgrounds, such as this lesbian author, offer insight into how the nursing profession might illuminate relational aspects of privilege. As such, the nursing profession could uncover solutions to systemic vulnerabilities which are inadvertently perpetuated within our profession, the health-care system, and society more broadly. The notion of compulsory heterosexuality is discussed in relation to nursing in this opinion piece with an aim to render structural harms visible that have been obscured by the institutionalization of heteronormativity in health care, to build awareness of the ways in which systemic vulnerabilities are unknowingly perpetuated despite health-care professionals being well-intended in their practice.

Correction to: Vulnerability, Harm, and Compromised Ethics Revealed by the Experiences of Queer Birthing Women in Rural Healthcare.

The following Acknowledgments were omitted in the original publication.

Vulnerability, Harm, and Compromised Ethics Revealed by the Experiences of Queer Birthing Women in Rural Healthcare.

Phenomenological interviews with queer women in rural Nova Scotia reveal significant forms of trauma experienced during labour and birth. Situating the accounts of participants within both phenomenological and intersectional analyses reveals harms enabled by structurally embedded heteronormative and homophobic healthcare practices and policies. Our account illustrates the breadth and depth of harm experienced and outlines how these violate core ethical principles and values in healthcare.

A role for Saccharomyces cerevisiae Chk1p in the response to replication blocks.

Replication blocks and DNA damage incurred during S phase activate the S-phase and intra-S-phase checkpoint responses, respectively, regulated by the Atrp and Chk1p checkpoint kinases in metazoans. In Saccharomyces cerevisiae, these checkpoints are regulated by the Atrp homologue Mec1p and the kinase Rad53p. A conserved role of these checkpoints is to block mitotic progression until DNA replication and repair are completed. In S. cerevisiae, these checkpoints include a transcriptional response regulated by the kinase Dun1p; however, dun1Delta cells are proficient for the S-phase-checkpoint-induced anaphase block. Yeast Chk1p kinase regulates the metaphase-to-anaphase transition in the DNA-damage checkpoint pathway via securin (Pds1p) phosphorylation. However, like Dun1p, yeast Chk1p is not required for the S-phase-checkpoint-induced anaphase block. Here we report that Chk1p has a role in the intra-S-phase checkpoint activated when yeast cells replicate their DNA in the presence of low concentrations of hydroxyurea (HU). Chk1p was modified and Pds1p was transiently phosphorylated in this response. Cells lacking Dun1p were dependent on Chk1p for survival in HU, and chk1Delta dun1Delta cells were defective in the recovery from replication interference caused by transient HU exposure. These studies establish a relationship between the S-phase and DNA-damage checkpoint pathways in S. cerevisiae and suggest that at least in some genetic backgrounds, the Chk1p/securin pathway is required for the recovery from stalled or collapsed replication forks.

Ginsenoside Rh2 induces apoptosis and paraptosis-like cell death in colorectal cancer cells through activation of p53.

Ginsenosides are the main bioactive components in American ginseng, a commonly used herb. In this study, we showed that the ginsenoside Rh2 exhibited significantly more potent cell death activity than the ginsenoside Rg3 in HCT116 and SW480 colorectal cancer cells. Cell death induced by Rh2 is mediated in part by the caspase-dependent apoptosis and in part by the caspase-independent paraptosis, a type of cell death that is characterized by the accumulation of cytoplasmic vacuoles. Treatment of cells with Rh2 activated the p53 pathway and significantly increased the levels of the pro-apoptotic regulator, Bax, while decreasing the levels of anti-apoptosis regulator Bcl-2. Removal of p53 significantly blocked Rh2-induced cell death as well as vacuole formation, suggesting that both types of cell death induced by Rh2 are mediated by p53 activity. Furthermore, we show that Rh2 increased ROS levels and activated the NF-κB survival pathway. Blockage of ROS by NAC or catalase inhibited the activation of NF-κB signaling and enhanced Rh2-induced cell death, suggesting that the anti-cancer effect of Rh2 can be enhanced by antioxidants.

Targeting Rb mutant cancers by inactivating TSC2.

Retinoblastoma (Rb), a tumor suppressor gene, is inactivated in many types of cancer. However little is known about how the loss of Rb function can be targeted in cancer therapies. We have identified that inactivation of TSC2 in Rb mutant cancer cells will induce a synergistic cell death. The synergistic cell death is due to an increase in cellular stress including metabolic, ER, and oxidative stress. Therefore, inactivation of TSC2 and chemothereputics that result in induction of cellular stress may be a novel and effective way to treat cancers containing inactivated Rb.

Stopped for repairs: a new role for nutrient sensing pathways?

In order to prevent division of damaged chromosomes, cells activate a checkpoint to inhibit mitotic progression in order to repair the damaged DNA. Upon detection of DNA damage two downstream checkpoint kinases, Chk1 and Rad53, are activated by the sensor kinase, Mec1, to block the metaphase to anaphase transition and mitotic exit, respectively. Recent data from studies with budding yeast suggested that the DNA damage checkpoint also enlists the cAMP dependent protein kinase (PKA) pathway, which is an integral part of the nutrient sensing mechanism in budding yeast, to inhibit mitosis in response to DNA damage. Genetic and biochemical evidence suggested that the PKA pathway contributes to the inhibition of mitotic progression by mediating the phosphorylation of the APC specificity factor Cdc20. Phosphorylation of Cdc20 assists the activity of the checkpoint pathways in the inhibition of the degradation of mitotic inhibitors securin, Pds1, and the B type cyclin, Clb2, in order to block anaphase and mitotic exit. Cdc20 was phosphorylated following DNA damage in a PKA and Mec1 dependent manner, suggesting PKA activation is dependent on Mec1. Here we discuss possible mechanisms for how PKA activity could be regulated in response to DNA damage and we will also address the implication of these results in evaluating current cancer treatments.